Chemical warfare agents: biomedical and psychological effects, medical countermeasures and emergency response [Third edition] 9781498769211, 1498769217

Table of contents :
Content: General. History of Chemical Warfare Agents. Chemistry. Toxicokinetics. OPCW- History, Capability and Accomplishments. Stockpiles. Terrorism and Psychological Effects. Syria-Social Media. Domestic Preparedness. Agent Effects. Nerve Agents. Inhalation Toxicicity of Nerve Agents. Low Dose CW - Non Lethal Levels. Vesicants. Cyanise. Lung Irritants. Ricin. Botulism. Riot Control. Screen Smokes. Drugs as Weapons. Protection. Personal Protection Equipment. Medical Pretreatments. Pyridostigmine: History and FDA Approval. Reversible Cholinesterase Inhibitors. Products to Protect Agains CWA's. Collective Protection.

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Chemical Warfare Agents Biomedical and Psychological Effects, Medical Countermeasures, and Emergency Response Third Edition

Chemical Warfare Agents Biomedical and Psychological Effects, Medical Countermeasures, and Emergency Response Third Edition

Edited by

Brian J. Lukey James A. Romano, Jr. Harry Salem

CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2019 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed on acid-free paper International Standard Book Number-13: 978-1-4987-6921-1 (Hardback) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

“Under every peak is a mountain of support.” This book is dedicated to three groups of people who were critical for its development. Family: Our families’ patience, support, and encouragement enabled us to bring together the many aspects of this finished product. Florence Salem, who passed away on February 22, 2010, was Harry’s greatest inspiration and most ardent supporter. Brian Lukey’s family (his wife Marita and three children Brianna, Tyler, and Camille). James Romano’s family (his wife Candy [Grimes] and three grown children). Harry Salem’s family (Jerry, Amy, Joel, Marshall, and Abby Rose) and his new wife and old friend Phyllis Salem and her children Calla Knopman, Martin Knopman, Ronald Knopman, and Pamela Kaplan, and her grandson Jacob Knopman. Chapter authors: The contribution list identifies great colleagues who dedicated enormous efforts to make this book come to fruition, and we thank them immensely. Deceased friends: Several of our good friends and distinguished colleagues have passed away since the last edition. They devoted their lives to the advancement of research that focused on countermeasures to chemical warfare agents. The reputation of these great scientists remains world renowned, and many of their accomplishments are captured in the chapters of this book. We are humbled by their generous devotion to helping mankind best address the past, present, and future chemical warfare agent atrocities. We are honored to have considered them as friends. We are truly proud to be standing on the shoulders of these giants: Doug Cerosoli, Susan DeLeon, Bryan Ballantyne, Margaret Filbert, Frederick Sidell, Bhupendra Doctor, Hendrik Benschop, Edson Albuquerque, and Steven Baskin We especially want to recognize Dr. Satu Somani, a special friend who took the initiative to design, develop, and edit the first edition of this book, on which the second and this third edition were developed. Although Dr. Somani passed away on October 29, 2002, he remains a great source of inspiration to the editors.

Contents Preface.....................................................................................................................................................................................................xi Acknowledgments................................................................................................................................................................................. xiii Editors.....................................................................................................................................................................................................xv Contributors......................................................................................................................................................................................... xvii

Section I General 1. Brief History and Use of Chemical Warfare Agents in Warfare and Terrorism.....................................................................3 Harry Salem, Andrew L. Ternay, Jr., and Jeffery K. Smart 2. Chemistries of Chemical Warfare Agents.................................................................................................................................. 17 Terry J. Henderson, Ilona Petrikovics, Petr Kikilo, Andrew L. Ternay, Jr., and Harry Salem 3. Toxicokinetics of Nerve Agents....................................................................................................................................................39 Marcel J. van der Schans, Hendrik P. Benschop, and Christopher E. Whalley 4. Organization for the Prohibition of Chemical Weapons (OPCW): History, Mission, and Accomplishments....................59 Karen L. Mumy, William R. Howard, Ariel Parker, Jonathan Forman, and Expert Opinion by Gwyn Winfield 5. Chemical Weapons Holdings and Their Internationally Verified Destruction......................................................................71 John Hart and Thomas Stock 6. Syria’s Chemical Disposal Program............................................................................................................................................87 Al Mauroni and Timothy A. Blades 7. The Use of Chemical Warfare Agents during the Syrian Civil War..................................................................................... 103 Arik Eisenkraft and Avshalom Falk 8. U.S. CBRN Homeland Response and Civil Support...............................................................................................................123 Kelley J. Williams and Steven A. Schmitt

Section II  Agent Effects 9. Mustard Vesicants....................................................................................................................................................................... 131 Rama Malaviya, Diane E. Heck, Robert P. Casillas, Jeffrey D. Laskin, and Debra L. Laskin 10. Health and Psychological Effects of Low-Level Exposure to Chemical Warfare Nerve Agents......................................... 145 Carl D. Smith, Kristin J. Heaton, James A. Romano, Jr., Maurice L. Sipos, and John H. McDonough 11. Inhalation Toxicology of Chemical Agents............................................................................................................................... 163 Stanley W. Hulet, Paul A. Dabisch, Robert L. Kristovich, Douglas R. Sommerville, and Robert J. Mioduszewski 12. Cyanides: Toxicology, Clinical Presentation, and Medical Management............................................................................. 181 Gary A. Rockwood, Gennady E. Platoff, Jr., and Harry Salem 13. The Structural Biology, Biochemistry, Toxicology, and Military Use of the Ricin Toxin and the Associated Treatments and Medical Countermeasures for Ricin Exposure...........................................................................................203 Terry J. Henderson, George Emmett, Russell M. Dorsey, Charles B. Millard, Ross D. LeClaire, and Harry Salem

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Contents

14. Medical Countermeasures for Intoxication by Botulinum Neurotoxin.................................................................................227 Michael Adler, Ajay K. Singh, Nizamettin Gul, and Frank J. Lebeda 15. Incapacitating Agents and Technologies: A Review................................................................................................................245 Sidney A. Katz and Harry Salem 16. Screening Smokes: Applications, Toxicology, Clinical Considerations, and Medical Management..................................277 Lawrence A. Bickford and Harry Salem

Section III Protection 17. Personal Protective Equipment (PPE): Practical and Theoretical Considerations.............................................................303 Michael R. Jones 18. Pyridostigmine Protection from Nerve Agent Intoxication: Concept to FDA Approval.....................................................373 Michael Adler, James P. Apland, Sharad S. Deshpande, Irwin Koplovitz, and David E. Lenz 19. Hormesis: Using Preconditioning to Build Biological Shields—A Novel Approach for Enhancing Resilience to Toxic Agents, Traumatic Illness/Injury, and Age-Related Degenerative Diseases..............................................................393 Edward J. Calabrese

Section IV Detection 20. Clinical Detection of Exposure to Chemical Warfare Agents................................................................................................401 Benedict R. Capacio, J. Richard Smith, Robert C. diTargiani, M. Ross Pennington, Richard K. Gordon, Julian R. Haigh, John R. Barr, Brian J. Lukey, and Daniel Noort 21. Biomarkers for Organophosphate Poisoning: Physiological and Pathological Responses..................................................443 Arik Eisenkraft, Avshalom Falk, and Kevin G. McGarry, Jr. 22. Field Sensors: Military and Civilian......................................................................................................................................... 519 Kelley J. Williams and Juan Stevens 23. Application of Genomic, Proteomic, and Metabolomic Technologies to the Development of Countermeasures against Chemical Warfare Agents.............................................................................................................................................523 Jennifer W. Sekowski and James F. Dillman, III 24. Nanomaterial-Based Sensors: Applications in Chemical Warfare Agent Detection and Identification............................ 539 Richard T. Agans, Madeleine C. DeBrosse, Richard L. Salisbury, and Saber M. Hussain

Section V Decontamination 25. Chemical Warfare Agent Decontamination from Skin...........................................................................................................549 Robert P. Chilcott, Brian J. Lukey, Harry F. Slife, Jr., Edward D. Clarkson, Charles G. Hurst, and Ernest H. Braue, Jr. 26. Aircraft Decontamination and Mitigation............................................................................................................................... 559 William T. Greer, Jr., Angela M.G. Theys, William R. Davis, and Kenneth J. Heater

Section VI Treatment 27. Military Chemical Casualty Treatment...................................................................................................................................577 Timothy J. Byrne, Raymond Vazquez, Dan Boehm, Laukton Rimpel, and Charles G. Hurst

Contents

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28. Pre-Hospital and Hospital Response to Toxicological Mass Casualty Events: The Israeli Approach...............................593 Arik Eisenkraft, Avshalom Falk, and Lion Poles 29. An Overview of the NIAID/NIH Chemical Medical Countermeasures Product Research and Development Program................................................................................................................................................................ 615 David T. Yeung, Gennady E. Platoff, Jr., Jill R. Harper, and David A. Jett 30. Medical Management of Chemical Warfare Agents...............................................................................................................627 Stephen A. Pulley 31. Protein-Based Bioscavengers of Organophosphorus Nerve Agents.......................................................................................655 Moshe Goldsmith, Yacov Ashani, Tamara C. Otto, C. Linn Cadieux, and David S. Riddle 32. Oxime Research........................................................................................................................................................................... 681 Jiri Kassa 33. Brain-Penetrating Reactivators of Organophosphate-Inhibited Acetylcholinesterase........................................................697 Janice E. Chambers and Edward C. Meek 34. Nerve Agent–Induced Seizures and Status Epilepticus: Neuroprotective Strategies..........................................................703 Frederic Dorandeu, Karine Thibault, and Nina Dupuis

Section VII  Predictive Modeling 35. Computational Modeling to Predict Human Toxicity.............................................................................................................723 Janet Moser, Douglas R. Sommerville, and George R. Famini 36. Lung-on-a-Chip........................................................................................................................................................................... 749 M. Tyler Nelson, Xinyu Lu, and Shuichi Takayama 37. Body-on-a-Chip for Pharmacology and Toxicology................................................................................................................ 763 Anthony Atala, Mahesh Devarasetty, Steven D. Forsythe, Russell M. Dorsey, Harry Salem, Thomas D. Shupe, Aleksander Skardal, and Shay Soker 38. Animal Models.............................................................................................................................................................................773 Wenli Li and Juergen Pauluhn 39. Real-Time Physiological Data Collection and Analysis in Animal Inhalation Models: Predictive and Diagnostic Implications.................................................................................................................................... 791 Benjamin Wong, Bryan J. McCranor, Rebecca Lewandowski, and Alfred M. Sciuto 40. Contextual Factors Influencing CBRN Leadership Decision Making..................................................................................803 Terry L. Oroszi Index.....................................................................................................................................................................................................809

Preface The first edition of this book, published in early 2001, focused on chemical warfare agent toxicity at low levels. At that time, various review committees suggested that there were data gaps in our information about the low-level toxicity of chemical warfare agents. Epidemiological studies indicated then that more than 120,000 Gulf War veterans were suffering from many unexplained illnesses and were seeking medical care. Among the putative explanations for these illnesses were exposure to nerve agents or pretreatment drugs. Many U.S. and British troops were given pyridostigmine bromide as a pretreatment drug during the 2 weeks of the air and ground war to protect against possible exposure to nerve gas. One of the notable nerve gases suspected to be present during this first Gulf War was sarin. During this period, military personnel were under physical stress; some have argued for evidence of exposure to low levels of sarin. Certain factors such as stress, surroundings, and other chemical agents can interact with the toxicity of chemical warfare agents. Consequently, the first book addressed many of those issues at that time. The second edition, published in 2007, took a much broader look at chemical warfare agent–related work/issues. Many changes had occurred in this area since that first edition, including the September 11 attacks and the Second Gulf War, which have created a sense of urgency to this field. Heightened terrorist threats underscored the need to readdress these issues. We recognized that mustard gas, defoliants, and nerve gases were used in localized wars in the 1960s, 1970s, and 1980s. Chemical warfare agents, primarily categorized as lethal or incapacitating agents, possess the attractive quality of being easy and inexpensive to synthesize on a large scale. A reasonable chemical-industrial setup could be diverted to produce chemical warfare agents. The agents are particularly terrifying because their toxic effects are indiscriminate and thus affect not only military personnel but also the civilian population as a whole. The agents provide a substantial psychological edge to an otherwise weak military

or terrorist group. Therefore, the second edition included epidemiological or clinical studies of exposed or potentially exposed populations, new treatment concepts and products, improved organization of the national response apparatus in the United States to address the potential for chemical warfare agent terrorism, and improved diagnostic tests that enabled rapid diagnosis and treatment. Many additional chemical warfare agent–related, worldwide events have occurred since 2007, which further highlight the risk of chemical warfare agent use, the greatest being chemical warfare agent–related events during the Syrian uprising. It is important to recognize that advances in biotechnology, nanotechnology, genetic engineering, neurobiology, and computer sciences, among others, may not only assist in the proliferation of traditional chemical warfare agents but also stimulate the emergence of nontraditional agents. Advances have also occurred in the delivery systems of these agents. While the use of chemical warfare agents in terrorist activities appears to have been limited, this may not accurately reflect the potential for their future use. Since the second edition, the chemical warfare agent community has worked hard to advance research for protection and treatment, and developed/improved response approaches for individuals and definitive care. Consequently, in addition to updating previous chapters, we added several new chapters that address the Syrian War, chemical destruction, the Organisation for the Prohibition of Chemical Weapons, biomarkers for chemical warfare agent exposure, field sensors, aircraft decontamination, lung/human on a chip, chemical warfare response decision making, and other research advancements. The chapters are written by world experts in their field, and we are very grateful for their contributions. Brian J. Lukey James A. Romano, Jr. Harry Salem

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Acknowledgments The editors wish to thank Lt. Correy Vigil and Donna M. Hoffman for helping with the preparation of this book, and Janet Stein and the Edgewood Chemical Biological Center Library for all their help with obtaining key documents.

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Editors Col. (retired) Brian Lukey and his coeditor, Col. (retired) James Romano, were previously commanders of the U.S. Army Medical Research Institute of Chemical Defense (Aberdeen Proving Ground, Maryland). The Institute is the Department of Defense’s lead laboratory for medical chemical defense research. Its mission is to discover and develop medical countermeasures to chemical warfare agents, to train and educate personnel in the medical management of chemical casualties, and to provide subject matter expertise in developing defense and national policy, and proper crisis management response. Col (retired) Brian J. Lukey, PhD, received his bachelor of science in biology from Northern Kentucky University in 1980. He earned a master’s degree in pharmacology in 1983 and a PhD in toxicology in 1985 from the University of Louisville. In 1990, he earned his master of science administration at Central Michigan University. He was certified as a diplomate in general toxicology by the American Board of Toxicology in 1993. Dr. Lukey is currently a senior civilian toxicologist for the U.S. Air Force, Wright-Patterson Air Force Base. Due to the nature of a military career, he has had numerous assignments in a variety of areas of medical research. These include managing programs directed toward medical chemical warfare defense, forensic toxicology for drugs of abuse, occupational and environmental health analytical procedures, and military operational medicine. He has authored and coauthored more than 45 manuscripts and technical reports, and is the codeveloper of one patent. Dr. Lukey is a highly decorated military officer, having been awarded the prestigious Medical Service Corps Award of Excellence, two Legion of Merits, and multiple Meritorious Service Medals, Army Commendation Medals, and Army Achievement Medals. He is a member of the Order of Military Medical Merit and wears the Expert Field Medical Badge, Airborne Badge, Air Assault Badge, and German Troop Duty Proficiency Badge. He and his wife, Marita, have three children, Brianna, Tyler, and Camille. They reside in Ohio and enjoy camping, fishing, bluegrass music, and family time. James A. Romano, Jr., PhD, was born in Jersey City, New Jersey. He received a bachelor of arts degree in psychology from the College of the Holy Cross in 1966 and a PhD in experimental psychology from Fordham University in 1975. He was certified as a diplomate in general toxicology by the American Board of Toxicology in 1994. Dr. Romano was an instructor of psychology at Manhattan College, Bronx, New York, from 1970 to 1975. Awarded tenure in 1976, from 1976 until 1978, he was an assistant professor of psychology at Manhattan College. He entered the U.S. Army in 1978, and progressed as a scientist and manager into positions of increasingly greater responsibility to include commander, U.S. Army Medical Research Institute of Chemical Defense (2000–2003), Aberdeen Proving Ground, Maryland, and deputy

commander of the U.S. Army Medical Research and Materiel Command, Fort Detrick, Maryland, his position at retirement from the U.S. Army. He then joined Science Applications International Corporation as a senior principal life scientist in the fall of 2006. In his research, he focused on the neurotoxicological effects of chemical warfare agents and medical countermeasures to these agents. Dr. Romano is the author of more than 100 papers in the areas of medical chemical defense and two successful textbooks: Chemical Warfare Agents: Toxicity at Low Levels (CRC Press, 2001) and Pharmacological Perspectives of Toxic Chemicals and Their Antidotes (Springer-Verlag, 2004). He currently serves on the Homeland Security Subcommittee to the Environmental Protection Agency’s Board of Scientific Counselors. Dr. Romano is married to Candy (Grimes) Romano, and they have three grown children. They reside in Middletown, Maryland, which he describes as a far cry from his beloved New York City–Jersey City roots. Harry Salem, PhD, is chief scientist for Life Sciences at the U.S. Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, Maryland. Edgewood Chemical Biological Center is a U.S. Army Research, Development, and Engineering Command Laboratory and is the Army’s principal research and development center for chemical and biological defense technology, engineering, and field operations. Edgewood Chemical Biological Center has achieved major technological advances for the warfighter and for our national defense, with a long and distinguished history of providing the Armed Forces with quality systems and outstanding customer service. Dr. Salem was previously the chief of the Toxicology Division and also the acting senior team leader for Biosciences at Edgewood. Prior to that, he was employed in positions of increasing complexity and responsibility by the pharmaceutical and contract laboratory industries. His research interests and experience include inhalation and general pharmacology and toxicology, and in vitro and molecular toxicology. Dr. Salem received a bachelor of arts degree from the University of Western Ontario in London, Ontario, Canada, a bachelor of science in pharmacy from the University of Michigan in Ann Arbor, and master’s and doctorate degrees in pharmacology from the University of Toronto, Canada. He was certified as a diplomate in general toxicology by the Academy of Toxicological Sciences in 1985. He has served in adjunct positions at the University of Pennsylvania, Temple University, Drexel University, and the University of Maryland, and continues to be active as a visiting professor at Rutgers University. He has been on the editorial boards of several professional journals and has served as editor-in-chief of the Journal of Applied Toxicology for over 20 years. He has published 18 books, including three volumes of the International Encyclopedia of Pharmacology

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xvi and Therapeutics, which he coauthored, as well as over 100 papers in scientific journals and chapters in books. Dr. Salem is an active member of many professional societies and has been elected a Fellow of the New York Academy of Sciences, the American College of Clinical Pharmacology, the American College of Toxicology, and the Academy of Toxicological Sciences, where he served on the Professional Standards Board and Board of Directors. He served as the chair of the Technical Committee of the Inhalation and Respiratory Specialty Section of the Society of Toxicology and the Council of the International Society of Regulatory Toxicology and Pharmacology, and on the Advisory Board of the Rocky Mountain Center for Homeland Defense. Dr. Salem

Editors was awarded the Decoration for Meritorious Civilian Service in 1989 for his contributions to the field of toxicology. In 2001, Dr. Salem was awarded the Society of Toxicology Congressional Science Fellowship, and he served as a science advisor to Congressman Jim Greenwood. He became a Fellow of the Royal Society of Chemistry in 2015, and was president of the Stem Cell Specialty Section of the Society of Toxicology in 2017–2018. He and his wife Phyllis reside in Elkins Park, Pennsylvania, close to his son Jerry and his wife Amy, his three grandchildren Joel, Marshall, and Abby Rose; his wife’s children Colla, Martin, Ron Knopman, and Pamela Kaplan, and her grandson Jacob Knopman.

Contributors Michael Adler Medical Toxicology Division U.S. Army Medical Research Institute of Chemical Defense Aberdeen Proving Ground, Maryland

Timothy J. Byrne Chemical Casualty Care Division U.S. Army Medical Research Institute of Chemical Defense Aberdeen Proving Ground, Maryland

Richard T. Agans Molecular Bioeffects Branch, Bioeffects Division Airman Systems Directorate Wright-Patterson Air Force Base, Ohio

C. Linn Cadieux U.S. Army Medical Research Institute of Chemical Defense Aberdeen Proving Ground, Maryland

James P. Apland Medical Toxicology Division U.S. Army Medical Research Institute of Chemical Defense Aberdeen Proving Ground, Maryland

Edward J. Calabrese Department of Environmental Health Sciences University of Massachusetts Amherst, Massachusetts

Yacov Ashani Department of Biomolecular Sciences Weizmann Institute of Science Rehovot, Israel

Benedict R. Capacio Medical Toxicology Research Division U.S. Army Medical Research Institute of Chemical Defense Aberdeen Proving Ground, Maryland

Anthony Atala Wake Forest Institute for Regenerative Medicine/Wake Forest School of Medicine Winston-Salem, North Carolina John R. Barr Biological Mass Spectrometry Laboratory Centers for Disease Control and Prevention Atlanta, Georgia Hendrik P. Benschop TNO Defense, Security and Safety Rijswijk, The Netherlands Lawrence A. Bickford U.S. Army Combat Capabilities Development Command Chemical Biological Center Aberdeen Proving Ground, Maryland Timothy A. Blades U.S. Army Combat Capabilities Development Command Chemical Biological Center Aberdeen Proving Ground, Maryland Dan Boehm U.S. Army Medical Research Institute of Chemical Defense Aberdeen Proving Ground, Maryland Ernest H. Braue, Jr. Medical Toxicology Branch U.S. Army Medical Research Institute of Chemical Defense Aberdeen Proving Ground, Maryland

Robert P. Casillas Strategic Global Health Security MRIGlobal Kansas City, Missouri Janice E. Chambers Center for Environmental Health Sciences College of Veterinary Medicine Mississippi State University Mississippi State, Mississippi Robert P. Chilcott Research Centre for Topical Drug Delivery and Toxicology University of Hertfordshire Hatfield, United Kingdom Edward D. Clarkson Medical Toxicology Branch U.S. Army Medical Research Institute of Chemical Defense Aberdeen Proving Ground, Maryland Paul A. Dabisch National Biodefense Analysis and Countermeasures Center Fort Detrick, Maryland Gregory Dal-Bo Toxicology and Chemical Risks Branch Institut de recherche biomédicale des armées Brétigny-sur-Orge, France

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xviii William R. Davis METSS Corporation Westerville, Ohio Madeleine C. DeBrosse Molecular Bioeffects Branch, Bioeffects Division Airman Systems Directorate Wright-Patterson Air Force Base, Ohio Sharad S. Deshpande Medical Toxicology Division U.S. Army Medical Research Institute of Chemical Defense Aberdeen Proving Ground, Maryland Mahesh Devarasetty Wake Forest Institute for Regenerative Medicine/Wake Forest School of Medicine Winston-Salem, North Carolina James F. Dillman III Cellular and Molecular Biology Branch U.S. Army Medical Research Institute of Chemical Defense Aberdeen Proving Ground, Maryland

Contributors George Emmett Battelle Memorial Institute Columbus, Ohio and Chemical Security Analysis Center Department of Homeland Security Aberdeen Proving Ground, Maryland Avshalom Falk The Institute for Research in Military Medicine The Hebrew University Faculty of Medicine Jerusalem, Israel George R. Famini Chemical Safety and Security, LLC Forest Hill, Maryland Jonathan Forman Organisation for the Prohibition of Chemical Weapons (OPCW) The Hague, The Netherlands Steven D. Forsythe Wake Forest Institute for Regenerative Medicine Winston-Salem, North Carolina

Robert C. diTargiani Medical Toxicology Research Division U.S. Army Medical Research Institute of Chemical Defense Aberdeen Proving Ground, Maryland

Moshe Goldsmith Department of Biomolecular Sciences Weizmann Institute of Science Rehovot, Israel

Frederic Dorandeu CBRN Defence Division Institut de recherche biomédicale des armées Brétigny-sur-Orge, France

Richard K. Gordon Division of Biochemistry Walter Reed Army Institute of Research Silver Spring, Maryland

and

William T. Greer, Jr. Air Force Research Laboratory Wright-Patterson Air Force Base, Ohio

Ecole du Val-de-Grâce Paris, France Russell M. Dorsey U.S. Army Combat Capabilities Development Command Chemical Biological Center Aberdeen Proving Ground, Maryland Nina Dupuis Toxicology and Chemical Risks Branch Institut de recherche biomédicale des armées Brétigny-sur-Orge, France Arik Eisenkraft The Institute for Research in Military Medicine The Hebrew University Faculty of Medicine National Advisory Committee of the Emergency and Disaster Preparedness Division Israeli Ministry of Health Jerusalem, Israel

Nizamettin Gul Director, Biosecurity U.S. Army Medical Research Institute of Infectious Diseases Fort Detrick, Maryland Julian R. Haigh Division of Biochemistry Walter Reed Army Institute of Research Silver Spring, Maryland Jill R. Harper National Institute of Allergy and Infectious Diseases National Institutes of Health (NIAID/NIH) Bethesda, Maryland John Hart Stockholm International Peace Research Institute (SIPRI) Stockholm, Sweden

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Contributors Kenneth J. Heater METSS Corporation Westerville, Ohio

Sidney A. Katz Rutgers University at Camden Cherry Hill, New Jersey

Kristin J. Heaton Military Performance Division U.S. Army Research Institute of Environmental Medicine Natick, Massachusetts

Petr Kikilo Department of Chemistry and Biochemistry University of Denver Denver, Colorado

Diane E. Heck School of Health Science and Practice New York Medical College Valhalla, New York

Irwin Koplovitz Medical Toxicology Division U.S. Army Medical Research Institute of Chemical Defense Aberdeen Proving Ground, Maryland

Terry J. Henderson U.S. Army Combat Capabilities Development Command Chemical Biological Center Aberdeen Proving Ground, Maryland William R. Howard Medical Service Corps, U.S. Navy Naval Medical Research Unit Dayton Wright-Patterson Air Force Base, Ohio Stanley W. Hulet U.S. Army Combat Capabilities Development Command Chemical Biological Center Aberdeen Proving Ground, Maryland Charles G. Hurst Chemical Casualty Care Division U.S. Army Medical Research Institute of Chemical Defense Aberdeen Proving Ground, Maryland Saber M. Hussain Molecular Bioeffects Branch Bioeffects Division Airman Systems Directorate Wright-Patterson Air Force Base, Ohio David A. Jett National Institute of Neurological Disorders and Stroke National Institutes of Health (NINDS/NIH) Bethesda, Maryland Michael R. Jones Kennedy Health System Cherry Hill, New Jersey Jiri Kassa Department of Toxicology and Military Pharmacy Faculty of Military Health Sciences University of Defence Hradec Králové, Czech Republic

Robert L. Kristovich U.S. Army Combat Capabilities Development Command Chemical Biological Center Aberdeen Proving Ground, Maryland Debra L. Laskin Department of Pharmacology and Toxicology Rutgers University Ernest Mario School of Pharmacy Piscataway, New Jersey Jeffrey D. Laskin Department of Environmental and Occupational Health Rutgers University School of Public Health and Environmental and Occupational Health Sciences Institute Rutgers University Piscataway, New Jersey Frank J. Lebeda Systems Biology Collaboration Center U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland Ross D. LeClaire New Mexico Veterinary Medical Association Placitas, New Mexico and New Mexico Department of Heath Medical Reserve Corps Santa Fe, New Mexico David E. Lenz Medical Toxicology Division U.S. Army Medical Research Institute of Chemical Defense Aberdeen Proving Ground, Maryland

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Contributors

Rebecca Lewandowski Chemical, Biological, Radiological, Nuclear and Explosives (CBRNE) Analytical and Remediation Activity 20th CBRNE Command Aberdeen Proving Ground, Maryland

Janet Moser Battelle Memorial Institute Chemical Security Analysis Center Department of Homeland Security Aberdeen Proving Ground, Maryland

Wenli Li Department of Toxicology Fourth Military Medical University Shaanxi Province, China

Karen L. Mumy Environmental Health Effects Research Naval Medical Research Unit Dayton Wright-Patterson Air Force Base, Ohio

Xinyu Lu Department of Biomedical Engineering Georgia Tech University Atlanta, Georgia

M. Tyler Nelson Air Force Research Laboratory Airman Systems Directorate Molecular Mechanisms Branch Wright-Patterson Air Force Base, Ohio

Brian J. Lukey Aerospace Toxicology Wright-Patterson Air Force Base, Ohio Rama Malaviya Department of Pharmacology and Toxicology Rutgers University Piscataway, New Jersey Al Mauroni USAF Center for Strategic Deterrence Studies Maxwell Air Force Base, Alabama Bryan J. McCranor Biochemistry and Physiology Branch U.S. Army Medical Research Institute of Chemical Defense Aberdeen Proving Ground, Maryland John H. McDonough Pharmacology Branch U.S. Army Medical Research Institute of Chemical Defense Aberdeen Proving Ground, Maryland Kevin G. McGarry, Jr. Health Business Unit, Clinical and Non-Clinical Research/ Medical and Chemical Defense/Biochemistry Battelle Memorial Institute Columbus, Ohio Edward C. Meek Center for Environmental Health Sciences College of Veterinary Medicine Mississippi State University Mississippi State, Mississippi Charles B. Millard Division of Biochemistry Walter Reed Army Institute of Research Silver Spring, Maryland Robert J. Mioduszewski Bel Air, Maryland

Daniel Noort Department of CBRN Protection The Netherlands Organization for Applied Scientific Research (TNO) Rijswijk, The Netherlands Terry L. Oroszi Pharmacology and Toxicology Department Boonshoft School of Medicine Fairborne, Ohio Tamara C. Otto U.S. Army Medical Research Institute of Chemical Defense Aberdeen Proving Ground, Maryland Ariel Parker Molecular Bioeffects Branch Bioeffects Division Airman Systems Directorate Wright-Patterson Air Force Base, Ohio Juergen Pauluhn Covestro Deutschland AG Global Phosgene Steering Group Leverkusen, Germany M. Ross Pennington Medical Toxicology Research Division U.S. Army Medical Research Institute of Chemical Defense Aberdeen Proving Ground, Maryland Ilona Petrikovics Department of Chemistry Sam Houston State University Huntsville, Texas Gennady E. Platoff, Jr. National Institute of Allergy and Infectious Diseases National Institutes of Health (NIAID/NIH) Bethesda, Maryland

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Contributors Lion Poles National Advisory Committee of the Emergency and Disaster Preparedness Division Israeli Ministry of Health and Kaplan Medical Center Rehovot, Israel Stephen A. Pulley Department of Emergency Medicine Philadelphia College of Osteopathic Medicine Philadelphia, Pennsylvania David S. Riddle U.S. Air Force Research Laboratory Wright-Patterson Air Force Base , Ohio Laukton Rimpel U.S. Army Medical Research Institute of Chemical Defense Aberdeen Proving Ground, Maryland Gary A. Rockwood Medical Toxicology Division U.S. Army Medical Research Institute of Chemical Defense Aberdeen Proving Ground, Maryland James A. Romano, Jr. Science Applications International Corporation Frederick, Maryland Harry Salem U.S. Army Combat Capabilities Development Command Aberdeen Proving Ground, Maryland

Thomas D. Shupe Wake Forest Institute for Regenerative Medicine/Wake Forest School of Medicine Winston-Salem, North Carolina Ajay K. Singh Medical Toxicology Division U.S. Army Medical Research Institute of Chemical Defense Aberdeen Proving Ground, Maryland Maurice L. Sipos U.S. Army War College Carlisle, Pennsylvania Aleksander Skardal Wake Forest Institute for Regenerative Medicine/Wake Forest School of Medicine Winston-Salem, North Carolina Harry F. Slife, Jr. U.S. Army Medical Research Institute of Chemical Defense Aberdeen Proving Ground, Maryland Jeffery K. Smart History Office U.S. Army Combat Capabilities Development Command Chemical Biological Center Aberdeen Proving Ground, Maryland Carl D. Smith Military Performance Division U.S. Army Research Institute of Environmental Medicine Natick, Massachusetts

Richard L. Salisbury Molecular Bioeffects Branch Bioeffects Division Airman Systems Directorate Wright-Patterson Air Force Base, Ohio

J. Richard Smith Medical Toxicology Research Division U.S. Army Medical Research Institute of Chemical Defense Aberdeen Proving Ground, Maryland

Steven A. Schmitt Decontamination Section 64th Weapons of Mass Destruction Civil Support Team Rio Rancho, New Mexico

Shay Soker Wake Forest Institute for Regenerative Medicine/Wake Forest School of Medicine Winston-Salem, North Carolina

Alfred M. Sciuto Biochemistry and Physiology Branch U.S. Army Medical Research Institute of Chemical Defense Aberdeen Proving Ground, Maryland

Douglas R. Sommerville U.S. Army Combat Capabilities Development Command Chemical Biological Center Aberdeen Proving Ground, Maryland

Jennifer W. Sekowski U.S. Army Combat Capabilities Development Command Chemical Biological Center Aberdeen Proving Ground, Maryland

Juan Stevens Analytical Science Section 83rd Weapons of Mass Destruction Civil Support Team Helena, Montana

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Contributors

Thomas Stock Dynasafe Environmental Systems GmbH Langenselbold, Germany

Raymond Vazquez U.S. Army Center for Environmental Health Research Fort Detrick, Maryland

Shuichi Takayama Department of Biomedical Engineering Georgia Tech University Atlanta, Georgia

Christopher E. Whalley U.S. Army Combat Capabilities Development Command Chemical Biological Center Aberdeen Proving Ground, Maryland

Andrew L. Ternay, Jr. Rocky Mountain Center for Homeland Defense Denver Research Institute University of Denver Denver, Colorado

Kelley J. Williams Wright State University CBRN Defense Program 64th Weapons of Mass Destruction Civil Support Team Rio Rancho, New Mexico

Angela M.G. Theys METSS Corporation Westerville, Ohio

Gwyn Winfield CBRNe World Winchester, United Kingdom

Karine Thibault Toxicology and Chemical Risks Branch Institut de recherche biomédicale des armées Brétigny-sur-Orge, France Marcel J. van der Schans Department of CBRN Protection The Netherlands Organization for Applied Scientific Research (TNO) Rijswijk, The Netherlands

Benjamin Wong Biochemistry and Physiology Branch U.S. Army Medical Research Institute of Chemical Defense Aberdeen Proving Ground, Maryland David T. Yeung National Institute of Allergy and Infectious Diseases National Institutes of Health (NIAID/NIH) Bethesda, Maryland

Section I

General

1 Brief History and Use of Chemical Warfare Agents in Warfare and Terrorism Harry Salem, Andrew L. Ternay, Jr., and Jeffery K. Smart CONTENTS 1.1 Introduction.....................................................................................................................................................................................3 1.2 Before the Common Era: Chemical Warfare Agents in Ancient Times........................................................................................3 1.3 Chemical Warfare Agents in the Common Era to World War I.....................................................................................................4 1.4 Chemical Warfare Agents Used in World War I (1914–1918)........................................................................................................6 1.5 Chemical Warfare Agents between World War I and World War II..............................................................................................9 1.6 Chemical Warfare Agents in World War II....................................................................................................................................9 1.7 Chemical Warfare Use after World War II...................................................................................................................................10 1.8 Chemical Warfare Agents Used in Terrorism...............................................................................................................................12 1.9 Conclusions...................................................................................................................................................................................13 References...............................................................................................................................................................................................13

1.1 Introduction The term chemical warfare agent was a 20th century military name used to describe a chemical compound that could be used to injure or kill opposing forces on the battlefield. However, the history and use of chemical warfare agents started long before the 20th century. From ancient times to the 19th century, humans identified and used naturally occurring poisons, toxins, diseases, noxious fumes, incapacitants, and incendiaries to achieve military victory over an opponent. Drawing on the knowledge of the past, during World War I, the participants developed, produced, and used large amounts of chemical warfare agents on the battlefield. During the 1920s and 1930s, several countries continued to use chemical warfare agents in various conflicts around the world. The list of potential chemical warfare agents grew during World War II and afterward as advances in chemistry created new, deadlier agents. Following World War II, some countries used chemical warfare agents against both their enemies and their own people. This led terrorist organizations to take note of what became known as weapons of mass destruction and launch attacks against cities and individuals, no longer limiting chemical warfare agent production to governments and use to the battlefield. Despite several treaties to ban their use and the general abhorrence of them, chemical warfare agent attacks continued into the 21st century.

1.2 Before the Common Era: Chemical Warfare Agents in Ancient Times There were many documented examples in ancient times of the use of naturally occurring poisons, toxins, noxious fumes, and incapacitants in battle. One of the earliest examples of such warfare was in the late Stone Age (10,000 BCE). Hunters in southern Africa, known as the San, used poison arrows. They dipped wood, bone, and stone arrow heads into poisons obtained from scorpion and snake venoms, as well as poisonous plants (CBW Info, 2005; Tagate, 2006; Wikipedia, 2007a). In about 2000 BCE, soldiers in India used toxic fumes on the battlefield. Chinese writings from as far back as the seventh century BCE contain hundreds of recipes for the production of poisonous and irritating smokes for use in war, along with numerous accounts of their use. These accounts describe the arsenic-containing “soul-hunting fog” and the use of finely divided lime dispersed into the air (Geiling, 2003; DeNoon, 2004; Tagate, 2006). The Assyrians in 600 BCE contaminated the water supply of their enemies by poisoning their wells with rye ergot (Mauroni, 2003). Solon of Athens used hellebore roots, a purgative that caused diarrhea, to poison the water in an aqueduct leading to the Pleistrus River, around 590 BCE, during the siege of Cirrha. The Cirrhaeans drank the water, developed violent and uncontrollable diarrhea, and were thus quickly defeated (Hemsley, 1987; United Kingdom Ministry of Defence, 1999; Noji, 2001;

3

4 Robey, 2003; Tschanz, 2003; CBW Info, 2005; Tagate, 2006; Wikipedia, 2007a). In the Peloponnesian War between Athens and Sparta in the fifth century BCE, the Spartan forces used noxious smokes and flame unsuccessfully against the Athenian city of Plataea. Later, the Boeotians successfully used noxious smokes and flame during the siege of Delium by placing a lighted mixture of coal, brimstone, and pitch at the end of a hollow wooden tube. Bellows pushed the resulting smoke through the tube and up to the walls of the besieged city, driving the defenders away (Thucydides, 1989). During the fourth century BCE, the Mohist sect in China used bellows to pump smoke from burning balls of mustard and other toxic vegetables into tunnels being dug by a besieging army (Tagate, 2006). In 200 BCE, the Carthaginians spiked wine with mandrake root, a narcotic, to sedate their enemies, feigned a retreat to allow the enemy to capture the wine, and then, when the enemy were sleeping, returned to kill them (Batten, 1960; United Kingdom Ministry of Defence, 1999).

1.3 Chemical Warfare Agents in the Common Era to World War I Around 50 CE, Nero eliminated his enemies with cherry laurel water that contained hydrocyanic acid (Hickman, 1999). Plutarch described irritating smokes in some of his writings around 46–120 CE. In 1000, the Mongols used gas bombs made of sulfur, niter, oil, aconite, powdered charcoal, wax, and resin. These bombs weighed about 5 pounds each. Aconite was a favorite poison, which is derived from the perennial herb of the genus Aconitum. It is in the buttercup family and is also known as monkshood and wolfsbane. Aconite (Aconitum napellus) is an alkaloid acting on the central nervous system, heart, and skin. It first stimulated and then paralyzed the nerves and heart. The effects began with a tingling of the mouth, fingers, and toes and then spread over the entire body surface. Body temperature dropped quickly and was followed by nausea, vomiting, and diarrhea. Fatal doses were marked by intense pain, irregular breathing, and a slowed and irregular heartbeat. Death resulted from heart failure or asphyxiation. Aconite was used as a poison on arrowheads and to taint enemy water supplies and as a poison by Indian courtesans when they applied it as a lipstick as the “Kiss of Death” (PDR Health, 2006). In about 660, Callinicus of Heliopolis invented a weapon called Greek fire, also referred to as Byzantine fire, wildfire, and liquid fire (Figure 1.1). Since its formulation was a carefully guarded military secret, the exact ingredients remain unknown. It probably consisted of naphtha, niter, and sulfur; petroleum and quicklime sulfur; or phosphorus and saltpeter. It may have been ignited by a flame or ignited spontaneously when it came into contact with water. If it was the latter, the active ingredient could have been calcium phosphide, made by heating lime, bones, and charcoal. On contact with water, calcium phosphide released phosphine, which ignited spontaneously. However, Greek fire was also used on land. The ingredients were apparently heated in a cauldron and then pumped out through a siphon or large syringe, known as a siphonarios, mounted on the bow of the ship. Greek fire was also used in hand

Chemical Warfare Agents grenades made of earthenware vessels containing chambers for fluids that when mixed, ignited when the vessel broke on impact with the target. It was used very effectively in naval battles, as it continued burning even under water and was known to the Byzantine enemies as a wet, dark, sticky fire, because it stuck to the unfortunate objects hit and was impossible to extinguish. It was also very effective on land as a counter-force suppression weapon used against besieging forces. Greek fire was first used on the battlefield to repel the Arab siege of Constantinople in 674–677, then at the Battle of Syllaeum, in 717–718, against the Muslims, and later, against the Russian attacks in 941 and 1043. The Byzantines also used Greek fire against the Vikings in 941 and against the Venetians during the fourth Crusade (Waitt, 1942; CBW Info, 2005; Wikipedia, 2007b). Chinese soldiers during the period 960–1279 used arsenical smokes in battle (CNS, 2001). Germans used noxious smokes in 1155. In the fifteenth and sixteenth centuries, the Venetians used poison-filled mortar shells and poison chests to taint wells, crops, and animals (Geiling, 2003). Leonardo da Vinci (1452–1519) described the preparation of Greek fire in his notebooks. His recipes included boiling charcoal of willow, saltpeter, sulfuric acid, sulfur, pitch, frankincense, camphor, and Ethiopian wool together. Liquid varnish, bituminous oil, turpentine, and strong vinegar were then mixed in and dried in the sun or oven. The mixture was formed into balls and sharp spikes were added, leaving an opening for a fuse (Richter, 1970). Leonardo da Vinci also proposed throwing poison powder on enemy ships. For poison, he recommended chalk, fine sulfide of arsenic, and powdered verdigris (basic copper salts) (MacCurdy, 1938; Dogaroiu, 2003). To protect the friendly soldiers, da Vinci described a simple protective mask made of fine cloth dipped in water that covered the nose and mouth. This is the earliest known description of a protective mask (MacCurdy, 1938). During the Thirty Years’ War (1618–1648), stink bomb grenades were used against fortifications. These were made from shredded hoofs, horns, and asafetida mixed with pitch. In 1672, during the siege of the city of Groningen, soldiers belonging to Christoph van Galen, the Bishop of Munster, used flaming projectiles and poisoned fireworks, but without much success (Beebe, 1923). Three years later, in 1675, the French and Germans concluded the Strasbourg Agreement, the first international agreement that banned the use of poison bullets (Hemsley, 1987; Tagate, 2006). During the Crimean War in 1854, British chemist Lyon Playfair proposed filling a hollow shell with cacodyl cyanide ((CH3)2AsCN) for use against Russian ships (Miles, 1957b; Camerman and Trotter, 1963). The enclosed space of a ship would allow the chemical agent to be deadlier. The British military, however, considered chemical warfare inappropriate and thought it similar to poisoning water wells (Browne, 1922). In his letter to the Prince Consort, Playfair responded that “why a poisonous vapor which could kill men without suffering is to be considered illegitimate is incomprehensible.” He further added that “no doubt, in time, chemistry will be used to lessen the suffering of combatants” (Browne, 1922). Other British proposals for chemical weapons during the Crimean War included ammonia shells, sulfur dioxide smoke clouds, and cacodyl oxide shells (Miles, 1957a, 1958a; Hemsley, 1987).

5

Brief History and Use of Chemical Warfare Agents

FIGURE 1.1  Drawing of a fake dragon shooting Greek fire. (U.S. Army, 1918, The Gas Defender, October 15, 1918, p. 9.)

The many suggestions, inventions, and concepts proposed during the American Civil War were among the forerunners of chemicals used on a much larger scale during World War I (Smart, 2004). In 1861, Confederate Private Isham Walker wrote a letter to Secretary of War Lucius Walker proposing the use of poison gas balloons against Fort Pickens and the Federal ships guarding it near Pensacola, Florida. The plan was not accepted (Wiley, 1968). On April 5, 1862, the same day that the Union Army began the siege operations against the extensive fortifications at Yorktown, Virginia, John Doughty of New York City, a school teacher, sent a letter to the War Department suggesting that shells filled with chlorine be shot at the Confederates (Figure 1.2). He envisioned the shells exploding over the Confederate trenches and creating a chlorine cloud that would either disable the defenders or drive them away. He also addressed the moral question of introducing chemical warfare and concluded that it “would very much lessen the sanguinary character of the battlefield and at the same time render conflicts more decisive in their results” (Haydon, 1938). Also in April 1862, shortly after the naval engagement between the USS Monitor and the CSS Virginia

ended in a draw, Commodore L. M. Goldsborough reported a rumor that the Confederates were going to use chloroform as a knockout gas against the USS Monitor to produce insensibility of their crew. A similar suggestion was made by Joseph Lott from Hartford, Connecticut in 1862 to load hand-pumped fire engines with chloroform and spray it on the enemy troops behind their earthworks defending Yorktown, Virginia and Corinth, Mississippi (Thompson and Wainwright, 1918). During the siege of Petersburg, Virginia in 1864, Forrest Shepherd of New Haven sent a letter to President Abraham Lincoln, suggesting mixing sulfuric acid with hydrochloric or muriatic acid to form a dense toxic cloud. Being slightly heavier than the atmosphere and a visible white color, the cloud would conceal the operator while the breeze blew it across enemy lines. Once it hit the enemy lines, it would cause coughing, sneezing, and tearing, which would prevent the enemy from aiming their guns but would not kill them (Miles, 1958b). Also during the siege, Confederate Colonel William Blackford directed that tunnels be dug toward the Union lines to discover Union tunneling operations and, when the enemy tunnels were found, to

FIGURE 1.2  Drawing of John Doughty’s proposed chlorine shell in 1862. (National Archives.)

6 use cartridges of obnoxious smoke to suffocate or drive out the enemy troops in them. The composition of the smoke was not clearly defined, but it could have been generated from gunpowder with a much higher proportion of sulfur to create a sulfur dioxide cloud when burned. Another possibility was that the material was similar to the mixture used in stink bombs, which would contain sulfur, rosin, pitch, asafetida, and horse-hoof raspings, as well as other materials designed to produce nauseating smokes (Blackford, 1945; Miles, 1959). The use of stink shells was also suggested by Confederate Brigadier General William Pendleton in 1864 to break the siege of Petersburg. He wanted a shell that combined an explosive with an offensive gas that would “render the vicinity of each falling shell intolerable” (U.S. War Department, 1891). Also in 1864, Captain E. C. Boynton described a cacodyl glass grenade that combined an incendiary with a toxic gas. He envisioned this for use against wooden ships, since cacodyl (C4H6As3), a heavy, oily liquid, burst into flame on contact with the air and also produced toxic fumes (Boynton, 1864). During the 1899 Boer War, the British used picric acid in their shells. Although the shells were not particularly effective, the Boers protested against their use (Hersh, 1968; Hemsley, 1987; Tintinalli, 2003). At the First Hague Peace Conference in 1899, Article 23(a) banned the use of poisons or poisoned arms and was ratified by the United States. A separate declaration banning asphyxiating gases in shells was rejected by the United States, even though the major European powers all signed it (Taylor and Taylor, 1985). The Russo-Japanese War also saw limited use of toxic chemical weapons. The Japanese used arsenical rag torches against Russian trenches. The torches were pushed toward the enemy by long bamboo poles to create a choking cloud (Chemical Warfare Service, 1939).

1.4 Chemical Warfare Agents Used in World War I (1914–1918) World War I was called the “Chemist’s War,” because it ushered in the beginning of the modern era of chemical warfare. Most of the key chemical warfare agents used during the war, however, were eighteenth- and nineteenth-century discoveries. These included chlorine (1774); hydrogen cyanide (1782); cyanogen chloride (1802); phosgene (1812); mustard agent (1822); and chloropicrin (1848) (Sartori, 1943). Chlorine (Cl2), designated Cl by the United States and Betholite by the French, is the only substance used in its elementary state as a war gas. Chlorine, a greenish-yellow gas with an irritating and disagreeable odor, caused spasm of the larynx muscles, burning of the eyes, nose, and throat, bronchitis, and asphyxiation. Its asphyxiating properties were first recognized by the Swedish chemist Karl Wilhelm Scheele in 1774. Chlorine was the first chemical agent used on a large scale by the Germans in April 1915 (Sartori, 1943; Field Manual 3–11.9, 2005). Although substances containing cyanide were used for centuries as poisons, it was not until 1782 that hydrogen cyanide or hydrocyanic acid (HCN) was isolated and identified by Karl Scheele, who later may have died from cyanide poisoning in a

Chemical Warfare Agents laboratory accident. Hydrogen cyanide, a colorless liquid with a faint odor of bitter almonds, caused faintness, pain in the chest, difficulty in breathing, and ultimately, death. It was also known as prussic acid and was designated AC by the United States and called Vincennite and Manganite by the French. It was reportedly first used by the Austrians in September 1915 (Foulkes, 1934; Baskin and Brewer, 1997; Field Manual 3–11.9, 2005). Cyanogen chloride (CNCl) was discovered by Wurtz and first prepared by Berthollet in 1802. A colorless gas with an irritating odor that immediately attacked the oral–nasal passages, its symptoms were similar to those of hydrogen cyanide. In high concentrations, it eventually caused death. The initial U.S. designation was CC, which was later changed to CK. The French called it Mauguinite and Vitrite. It was first used by the French in October 1916 (Sartori, 1943; Field Manual 3–11.9, 2005). Phosgene (COCl2) or carbonyl chloride, designated CG by the United States, Collongite by the French, and D-Stoff by the Germans, was obtained in 1812 by Humphrey Davy when he exposed a mixture of chlorine and carbon monoxide to sunlight. Phosgene, a colorless gas with an odor like musty hay, attacked the lungs, causing pulmonary edema and eventual death. It was first used by the Germans as a war gas in December 1915 (Sartori, 1943; Field Manual 3–11.9, 2005). Mustard agent or dichloroethyl sulfide (S(CH2CH2)2Cl2) was discovered by Despretz, who obtained it by the reaction of ethylene on sulfur chloride in 1822. It is normally a pale yellow to dark brown oily liquid with an odor like garlic (although the German mustard agent had an odor similar to the mustard seed). As a vesicant, it attacked the eyes and blistered the skin. The United States designated it HS and then later HD, after a purified version was developed in 1944. The French called it Yperite and the Germans Lost. The German name was derived by taking the first two letters of the two Germans Lommel and Steinkopf, who proposed and studied the use of this agent in warfare. The first use of mustard agent by the Germans near Ypres, Belgium in July 1917 marked the beginning of a new phase of chemical warfare and inflicted about 15,000 British casualties in 3 weeks (Prentiss, 1937; Sartori, 1943; Field Manual 3–11.9, 2005). Chloropicrin or trichloronitromethane (CCl3NO2) was prepared in 1848 by Stenhouse and was extensively used in World War I. A pungent, colorless, oily liquid, it caused oral–nasal irritation, coughing, and vomiting. In high dosages, it caused lung damage and pulmonary edema. It was first employed dissolved in sulfuryl chloride by the Russians in 1916 in hand grenades. In Germany it was known as Klop, in France as Aquinite, and as PS in the United States. It was later used as an insecticide and fungicide as well as for eradicating rats from ships (Sartori, 1943). At the beginning of World War I, both sides used munitions filled with irritants such as ethylbromoacetate (CH2BrCOOC2H5), chloroacetone (CH3COCH2Cl, or French Tonite, German A-Stoff), o-dianisidine chlorosulfonate, xylyl bromide (C6H4CH3CH2Br, German T-Stoff), or benzyl bromide (C6H5CH2Br) (Dogaroiu, 2003). Other irritants used in World War I included acrolein (CH2CHCHO, French Papite), bromoacetone (CH3COCH2Br, U.S. BA, German B-Stoff, French Martonite), and bromobenzyl cyanide (C6H5CHBrCN, U.S. CA, British BBC, French Camite) (Salem et  al., 2006). Thus, the first use of chemicals in World War I involved nonlethal tear gases, which were used by both the French and the Germans in late 1914 and early 1915 (Figure 1.3).

7

Brief History and Use of Chemical Warfare Agents

Brass

Steel

Steel

T.N.T bursting charge

Lead container

25 mm

Felt wad

34 mm

555 mm

Paraffin or cement

FIGURE 1.3  The German 150 mm xylyl bromide (T-Stoff) shell. (Army War College (1918), Gas Warfare, Part I, German Methods of Offense.)

Germany was the leader in first using chemical weapons on the battlefield and then introducing or developing new chemical agents to counter new developments in protective equipment. Fritz Haber was the designer behind many of Germany’s chemical weapons. Although he was not a toxicologist, he profoundly influenced the science of chemical toxicology. Haber and colleagues conducted acute inhalation studies in animals with numerous chemical agents thought to be useful in chemical warfare. He also developed Haber’s law, which is usually interpreted to mean that identical products of the concentration of an airborne agent and the duration of exposure will yield similar biological responses (Witschi, 2000). Actually, the product of the concentration (C) of the gas in air in parts per million (ppm) and the duration of the exposure (t) in minutes was referred to as Haber’s constant (Haber, 1986). It was also referred to as the mortality-product, the Haber product W (C × t = W), or the lethal index; the lower the product or the index number, the greater the toxic power (Sartori, 1943). Although Haber’s law was used by toxicologists to define acute inhalation toxicity for toxic chemicals, it was also used for quantitative risks or safety assessments (Rozman and Doull, 2001). Germany’s use of chemical weapons on the battlefield began on October 27, 1914, when Germans fired shells loaded with dianisidine chlorosulfonate, a tear gas, at the British near Neuve Chapelle. This tear gas normally produced violent sneezing. In this case, however, the chemical dispersed so rapidly in the air that the British never knew they had been attacked with gas (Charles, 2005). Following this experiment, the Germans

continued to test other potential chemical weapons. In midDecember 1914, Haber’s assistant was killed while working on an arsenic-containing weapon (cacodyl: (CH3)2As–As(CH3)2). In January 1915, the Germans used xylyl bromide (T-Stoff) against the Allies, but it was so cold that the gas froze and settled in the snow. By the spring of 1915, Haber convinced the German High Command to use chlorine gas and to create a special gas unit, the 35th Pioneer Regiment. This unit included Otto Hahn, Wilhelm Westphal, Erwin Madelung, James Franck, and Gustav Hartz. Three of these were future Nobel Laureates: Hahn, Franck, and Hertz (Charles, 2005). In preparation for the release of chlorine gas, Haber arranged for over 5000 chlorine cylinders to be placed near Ypres, Belgium (Figure 1.4). Only some of the ordinary German soldiers had protective masks made of cotton gauze, while Haber had provided Draeger masks for the Pioneers. While they were waiting for the wind to blow in the right direction, enemy fire hit some of the chlorine cylinders and released their gas. Three German soldiers were reportedly killed, and 50 were injured. On April 22, 1915, a strong wind from the northeast arrived, and at 5 p.m., Haber’s gas troops opened the valves on the 5730 high-pressure steel tanks, containing about 168 tons of chlorine, along a 4-mile frontline. The chlorine drifted southward toward the French and Canadian lines. It formed a yellow–green smoke wall about 50 feet high and 4 miles long. The wind shifted, and the cloud moved to the east toward the trenches occupied by the 45th Algerian Division (French). Those who tried to stay were quickly overcome, retching and gasping for air as they died. The rest fled in panic, stumbling and falling, and throwing away their rifles. The cloud moved on at about 100 feet per minute and opened up a 4-mile wide hole in the Allied front. After 15 minutes, the German troops emerged from their trenches and advanced cautiously. Had the German High Command provided enough reserve troops to sustain the offensive, they might have been able to break through the Allied defensives and capture Ypres. Instead, the German forces gained only about 2 miles of territory. The French soldiers used rudimentary defensive equipment, including wads of cotton that they were supposed to soak in water and hold to their faces. Estimated casualties for the battle ranged from 3000 to 15,000 killed and

FIGURE 1.4  German trench with a gas cylinder ready for discharge. (Army War College (1918), Gas Warfare, Part I, German Methods of Offense.)

8 wounded (McWilliams and Steel, 1985; Landersman, 2003; Charles, 2005; MSN, 2005). Following this attack, the Germans led repeated chlorine gas attacks on the Allies and drove them back almost to Ypres but were unable to capture the objective. The initial attacks caught the Allies completely unprepared. However, shortly after the first attack, the British troops were told to urinate on their handkerchiefs and tie them over their faces for emergency protection. This caused problems for some soldiers when there were multiple gas attack alerts within a short time and they could not produce enough urine. Within a week of the first attack, Emergency Pad Respirators, similar to a scarf, made of cotton waste soaked in sodium carbonate and sodium thiosulfate (hypo), were available for British soldiers. By early 1916, protective masks included a full facepiece with goggles and an exhaust valve and provided adequate protection against most of the chemical agents used on the battlefield. This was the beginning of a competition between the developers of new chemical warfare agents that penetrated masks and the developers of masks to protect the soldiers. The developers of masks were ahead in the competition until the summer of 1917. Mustard agent (HS) was first used by the Germans against the French on July 12, 1917, also near Ypres (Harris and Paxman, 1982; Mitretek, 2005a). The attack led to about 15,000 Allied casualties. Unlike phosgene, which was disseminated as a gas, mustard agent was relatively non-volatile and resembled motor oil. It was persistent and remained on objects and the ground for long periods of time. The introduction of mustard agent on the battlefield created a dilemma for the mask developers. No longer were the oral–nasal passages and the eyes the only areas that needed protection. Mustard agent required full body protection for both soldiers and all animals used on the battlefield for transportation and communication (Figure 1.5). Unprotected soldiers suffered blisters on all exposed skin that appeared hours after the initial exposure. Fewer than 5% of the mustard casualties who reached medical aid stations died, but the average convalescent period was greater than 6 weeks. Mustard agent damaged eyes, lungs, and skin and tied up large amounts of medical resources (Figure 1.6). On March 9, 1918, a German chemical bombardment began between St. Quentin and Ypres, firing half a million shells containing mustard agent and phosgene at the rate of about 700 shells per minute. On that day, a total of 1000 tons of chemicals were used by Germany. The Germans also attacked Salient du Fey on March 9, 1918, where Colonel Douglas MacArthur led the capture of a German machine gun nest and was awarded the Distinguished Service Cross. Two days later, MacArthur was among those gassed by the Germans, and for this he received the Purple Heart. On March 19, 1918, the British launched a preemptive retaliatory strike against the German positions near St. Quentin. They used nearly 85 tons of phosgene and killed 250 German troops. There were attempts late in the war to develop more potent vesicants than mustard agent. In 1918, Associate Professor Winford Lee Lewis left Northwestern University to become director of the Offensive Branch of the Chemical Warfare Service Unit at Catholic University. This unit was called Organic Unit No. 3 and was tasked with developing and producing novel gases containing arsenic. Lewisite (dichloro-(2-chlorovinyl) arsine)

Chemical Warfare Agents

FIGURE 1.5  Rider and horse protected against mustard agent during World War I. (U.S. Army.)

FIGURE 1.6  Sample of mustard agent. (U.S. Army.)

9

Brief History and Use of Chemical Warfare Agents (C2H2AsCl3) was developed based on early research conducted in 1904 at the university by J. A. Nieuwlands. A brown liquid that smelled like geraniums, it was initially designated G-34 or M-1. By the popular press, it was referred to as Methyl or the “Dew of Death.” Like mustard agent, it was also a vesicant that attacked the eyes and blistered the skin, but its effects occurred much more quickly than those of mustard agent. Developed late in the war, it was not used on the battlefield (Anonymous, 1921; Vilensky and Sinish, 2005; Field Manual 3–9.11, 2005). By the end of the war, approximately 91,000 soldiers of all sides had been killed by chemical warfare agents, and 1.2 million had been wounded. Of these casualties, the Russians alone suffered approximately half a million killed and wounded by chemical warfare agents. During the war, participants released over 113,000 tons of chemical warfare agents and fired over 66 million chemical shells.

1.5 Chemical Warfare Agents between World War I and World War II Following the end of World War I, many nations attempted to ban the use of chemical warfare agents. In 1925, 16 of the world’s major nations signed the Geneva protocol pledging never to use chemical warfare agents again. The United States signed the agreement, but the U.S. Senate refused to ratify it due to a growing isolationism sweeping the country and also concerns that the nation needed to continue preparing in case chemical weapons were used again. Despite the Geneva protocol, there were continued incidents of chemical weapon use. During the Rif War (1921–1926) in Spanish-occupied Morocco, Spanish forces reportedly fired gas shells and dropped mustard agent bombs on the Riffians (Anonymous, 1923; Waitt, 1942; SIPRI, 1971). In 1935, Italy used chemical weapons during its invasion of Ethiopia. The Italian military primarily dropped mustard agent in bombs and experimentally sprayed it from airplanes and spread it in powdered form on the ground. In addition, there were reports that the Italians used chlorine and tear gas. Some sources estimated that Ethiopian chemical casualties were 15,000, mostly from mustard agent (Clark, 1959; SIPRI, 1971). Japan used chemical weapons against Chinese forces during their war starting in 1937. Reports indicated that the Japanese used mustard agent, lewisite, phosgene, hydrogen cyanide, and tear gases filled into bombs, shells, and smoke pots (SIPRI, 1971). The Soviet Union also used chemical weapons on its own people during this period, reportedly using them to suppress a massive peasant uprising around Tambov (Wikipedia, 2007a). Until the mid-1930s, phosgene and mustard agent were considered the most dangerous chemical weapons. Both could readily be identified by their unique smells. That changed when Germany discovered nerve agents. The history of nerve agents began on December 23, 1936, when Dr. Gerhard Schrader of I. G. Farben in Germany accidentally isolated ethyl N,N-dimethylphosphoramidocyanidate (C5H11N2O2P) while engaged in his 2-year program to develop new insecticides. The new agent was a colorless to brown liquid with a faintly fruity odor. Controlled animal laboratory studies revealed that

it caused death within 20 minutes of exposure. In January 1937, Schrader and his assistant were the first to experience the effects of the agent on humans. A small drop spilled on a laboratory bench caused both of them to experience miosis and difficulty in breathing, although they both survived. Schrader reported the discovery to the Ministry of War, which was required by the Nazi decree passed in 1935 that required all inventions of military significance to be reported. The chemical was quickly recognized as a new, more deadly chemical warfare agent. The Germans initially designated it as Le-100, later Trilon-83, and finally, Tabun. The United States later designated it GA, for German Agent A, when it became aware of the agent near the end of World War II. The German military designated Tabun a chemical warfare agent and began production at Elberfeld. In 1940, the military moved production to Dyhernfurth (SIPRI, 1971; Harris and Paxman, 1982; MTS, 2005a, 2005b; CBW Info, 2005; Field Manual 3–9.11, 2005). In 1938, Schrader discovered a second potent nerve agent, isopropyl methylphosphonofluoridate (C4H10FO2P). The Germans designated it T-144, later Trilon-46, and finally, Sarin, an acronym created from the last names of the members of the development team: Schrader, Ambrose, Rudriger, and van der Linde. The United States eventually designated it GB, for German Agent B. The agent was a colorless liquid with no known odor. Animal tests indicated that it was 10 times more effective than Tabun (Harris and Paxman, 1982; MTS, 2005a; Field Manual 3–11.9, 2005).

1.6 Chemical Warfare Agents in World War II At the start of World War II, Germany filled bombs, shells, and rockets with Tabun nerve agent. The Germans stockpiled the weapons but never used them on the battlefield. They remained in storage until the end of the war, when the Allies captured them and discovered their existence (Figure 1.7). The reasons

FIGURE 1.7  German Tabun bombs discovered after the defeat of Germany in 1945. (Office of the Chief of Chemical Corps (1947), The History of Captured Enemy Toxic Munitions in the American Zone European Theater, May 1945 to June 1947.)

10 why Germany did not use nerve agents or any other chemical weapons are still debated. One possible explanation was that Adolf Hitler had been injured by mustard agent during World War I and did not want to use chemical agents again. Another possible reason was that by the time nerve agents could have made a difference on the battlefield, Germany had already lost air superiority and risked massive attack against its cities. President Franklin Roosevelt’s pledge in 1943 not to use chemical weapons unless the United States was attacked first probably also helped convince the Germans not to initiate chemical warfare (Landersman, 2003). Still another reason why Germany did not use its nerve agents was that the United States had prepared a large stockpile of mustard agent and phosgene ready to use should Germany or Japan initiate chemical warfare. As part of its preparations, the United States also forward positioned chemical agent stockpiles around the world. These movements were kept secret, but that caused at least one disaster. On December 2, 1943, a German air raid on Bari, Italy sank 16 ships and damaged eight others. Among those destroyed was the SS John Harvey, whose cargo included 100 tons of mustard agent bombs. The John Harvey was one of the first hit, and all those on board who had knowledge of the chemical weapons were killed. The destroyed ship spread a mixture of oil and mustard agent across the harbor. Hospitals were unaware of the mustard contamination and were unprepared to properly treat the patients. As a result, there were over 600 mustard agent casualties, of whom 83 died within a month (Harris and Paxman, 1982; Landersman, 2003). During the war, research continued on both sides to find new chemical warfare agents. In 1944, the Germans developed Soman (pinacolyl methyl phosphonofluoridate) (C7H16FO2P), eventually designated GD, or German Agent D, by the United States. The name Soman may have been derived either from the Greek verb “to sleep” or from Latin “to bludgeon.” It was a colorless liquid with a fruity or camphor odor and was discovered by Dr. Richard Kuhn, a Nobel Laureate, while he was working for the German Army on the pharmacology of Tabun and Sarin (SIPRI, 1971; MTS, 2005a; Field Manual 3–9.11, 2005). Soman combined features of both Sarin and Tabun (CBW Info, 2005), with initial tests showing that Soman was even more toxic than Tabun and Sarin (Harris and Paxman, 1982). The Germans also apparently researched two other nerve agents, later designated GE (ethyl Sarin, C5H12FO2P) and GF (cyclo Sarin, C7H14FO2P). None of these nerve agents were discovered by the Allies until they overran Germany in 1945 (Harris and Paxman, 1982). Although Hitler and the Nazis did not use chemical warfare agents against their declared enemy countries in World War II, they did use them to kill some of their own people, such as the Jews, Gypsies, homosexuals, and others they declared undesirable. The chemical warfare agent of choice was Xyklon B, consisting primarily of hydrogen cyanide, which they pumped into gas chambers at Auschwitz, Mauthausen, and Sachsenhausen to eliminate those they considered undesirables. Over 6 million Jews were eliminated in this way. The entire Nazi war effort could not have survived as long as it did without the support of many American companies, including IBM, General Motors, Ford Motor Company, Alcoa, Woolworth, Brown Brothers Harriman, Chase Manhattan Bank, Standard Oil, Kodak and

Chemical Warfare Agents Coca Cola (Washington, 2015; Koski, 2015; Washington Blog, 2015; Johnsen, 2015). The United States developed a process to purify mustard agent by distillation in 1944 and designated it as HD (C4H8Cl2S) or distilled mustard. The World War I mustard agent, referred to as Levinstein mustard, had a higher percentage of sulfur, which made it less effective and less stable in storage. Distilled mustard had less smell, had a greater blistering capability, and was more stable in storage. The United States and the British also researched mixing different chemicals with mustard agent. This work identified HL (a mixture of mustard agent and lewisite), HQ (sesqui mustard), HT (a mixture that was more persistent and had a lower freezing point), and HV (a thickened mustard agent). Both sides also researched the nitrogen mustard agents. First identified in the 1930s by Kyle Ward, Jr., eventually, three nitrogen mustard agents were produced during the war. HN-1 (C6H13Cl2N), HN-2 (C5H11Cl2N), and HN-3 (C6H12Cl3N) were all similar to mustard agent but had quicker reaction in the eyes. The United States focused on HN-1, while the British concentrated on HN-2 and HN-3. The Germans focused on HN-3 and filled it into shells and rockets (Brophy et al., 1959). Although most of the wartime research on chemical warfare agents was to produce more lethal agents, there was a discovery on a positive note. Following exposure to mustard agent after the Bari Harbor incident, medical researchers noted that the agent changed patients’ white blood cell count. This led to additional research during and after the war, particularly with nitrogen mustards, as an ingredient for what eventually became known as chemotherapy to fight cancer (Admin, 2013; Hazell, 2014). Following the Bari incident, in which soldiers were exposed in the water to both oil and sulfur mustard, two pharmacologists, Drs. Alfred Gilman and Louis Goodman at Yale University, found that nitrogen mustard had antitumor activity in murine lymphoma with less toxicity than sulfur mustard, and they convinced a thoracic surgeon at Yale, Dr. Gustav Lindskog, to administer nitrogen mustard to his patient. Thus, the first recorded treatment of human cancer with a mustard compound occurred in 1943 in a patient with non-Hodgkin’s lymphoma and severe airway obstruction. Marked, but temporary, regression was observed in this and other lymphoma patients. Wartime secrecy associated with the war gas program precluded the publication of these case reports until 1946. Thus, the modern era of cancer chemotherapy was launched (Morrison, 2010). Although World War I was considered the “Chemists War,” it was in World War II that both sides, the Allies and the Nazis, manufactured and distributed amphetamine tablets. These tablets kept the troops awake, increased alertness and aggressiveness, and fought low morale (Reddit).

1.7 Chemical Warfare Use after World War II After the Allies overran Germany in 1945, they discovered the German nerve agent program for the first time. Both the United States and the Soviet Union took the German technology and made it their primary focus for chemical warfare agents. The United States selected Sarin (GB) as a nonpersistent lethal agent to replace phosgene. The initial pilot plant work was accomplished at Edgewood Arsenal, Maryland. The large-scale

Brief History and Use of Chemical Warfare Agents production plant was built at Rocky Mountain Arsenal near Denver, Colorado (Figure 1.8). The arsenal also filled GB into bombs, projectile, and rockets. Within less than a decade, however, a new nerve agent was discovered by the British. The new agent, eventually designated VX (V for venomous), was developed at the ICI Protection Laboratory in 1952. VX (C11H26NO2PS) was both an organophosphate and an organosulfate compound that was immediately toxic to mammals as well as to insects. Its chemical name was O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothiolate. VX was a colorless and odorless liquid. The British chemist Dr. Ranajit Ghosh discovered the agent while originally intending to find a replacement for the insecticide DDT. When it was found to be too lethal to employ as a pesticide, the formula was passed to the Chemical and Biological Weapons Facility at Porton Down. Because the British were already committed to the production of Tabun and Sarin, they passed the compound on to the United States and Canada. Knowledge of the VX project somehow leaked to the Soviets, who developed their own version of VX, which they designated as VR-55. It was later discovered that VR-55 was only a thickened version of Soman. In 1960, the United States completed a VX production plant at Newport, Indiana (Figure 1.9). VX was filled into projectiles, rockets, and a newly designed land mine. The United States never used nerve agents on the battlefield. However, continued testing and long-term storage created dangers that eventually impacted the entire U.S. chemical weapons program. In 1968, VX apparently leaked from a faulty aerial spray tank being tested at Dugway Proving Ground, Utah, and sickened sheep in Skull Valley. Although the Army did not accept the blame, they did pay for the loss of the sheep. In 1969, GB leaked from a Navy bomb being sandblasted at a secret chemical weapons stockpile on the island of Okinawa, injuring 23 soldiers and one civilian. As a direct result of these accidents, President Richard Nixon issued an executive order in 1969 to halt U.S. production of chemical warfare agents (Newby, 1969; CBW Info, 2005; Mitretek, 2005b; Wikipedia, 2007c).

FIGURE 1.8  The U.S. Sarin (GB) nerve agent plant at Rocky Mountain Arsenal, Colorado. (U.S. Army.)

11

FIGURE 1.9  The U.S. VX nerve agent plant at Newport, Indiana. (U.S. Army.)

Following World War II, there were incidents of chemical weapon use around the world. During the Yemen Civil War (1963– 1967), Egypt reportedly used tear gas, mustard agent, and possibly nerve agents against the Yemeni Royalists (SIPRI, 1971; Taylor and Taylor, 1985). The Soviets allegedly used chemical weapons during their invasion of Afghanistan (1979–1989). Intelligence reports indicated that the Soviets used nerve agents, phosgene oxime (CHCl2NO), and tear gas (Haig, 1982). The most significant use of chemical weapons occurred when Iraq used them against Iran during the Iran–Iraq War (1980–1988). The reports indicated that Iraq used extensive amounts of mustard agent and probably nerve agents against the Iranians. Iran may have also retaliated with chemical weapons of its own near the end of the war. International law and the United Nations provided little deterrence against their use, even though Iraq had ratified the 1925 Geneva Protocol against chemical warfare agent use in 1931 (United Nations, 1986; Pringle, 1993; Landersman, 2003). Toward the end of the war, in 1988, Iraq’s military conducted a massive chemical agent attack by aircraft against their own people in Halabja, a city of 45,000 Iraqi Kurds, knowing they could not retaliate. There were approximately 5000 total casualties with 200 fatalities (Landersman, 2003). Libya reportedly used mustard agent against Chad in 1987 (Pringle, 1993). In all these incidents, there was little outcry or objection from the rest of the world, although the United Nations investigated some of the incidents. There was no deterrence, because only one side had chemical weapons in most cases. In addition, the chemical weapons were only marginally effective in their use and did not win the war for the country that used them. The Russians apparently continued to research new nerve agents after World War II. According to public disclosures, the Russians developed a highly toxic binary nerve agent series, designated Novichuk, during the 1980s. In Russian, “Novichok” means “newcomer.” For the Russians, the advantage of having new chemical agents is that they have never been previously used on the battlefield. Thus, the agents may not be banned by treaty, there are no existing detection and warning devices, and the current protective equipment is not effective (Englund, 1992, 1993; Khripunov and Averre, 1999; Wikipedia, 2007c).

12 Following the use of chemical weapons around the world and the Soviet Union’s continuing interest in chemical weapons, the United States decided to again produce nerve agent weapons in 1987 for a retaliatory capability. However, instead of the earlier unitary version, they designed a binary nerve agent (Figure 1.10). The nerve agent GB was broken down into two less-than-lethal precursor chemicals that were stored in separate canisters for loading into artillery shells, bombs, and rockets. Only after firing did the two ingredients mix to form the lethal nerve agent. The U.S. production of binary chemical agents continued until 1990, when the Soviets agreed to end chemical weapons production. This agreement eventually led in 1993 to many countries signing the Chemical Weapons Convention, which banned all chemical weapons development, production, acquisition, stockpiling, transfer, or use and also required the destruction of all existing stockpiles. The United States ratified the Convention in 1997 (Smart, 1997; Field Manual 3–11.9, 2005). This treaty did not end the use of all types of chemical agents. In 2002, after Chechen terrorists took control of a large theater in Moscow, the Russians used an unidentified incapacitant, possibly a derivative of fentanyl, to disable both the terrorists and the hostages. The use of the agent contributed to the end of the hostage situation, but approximately 40 terrorists and 204 hostages died during the rescue attempt. Most of the hostages were reported to have died from the Russian gas use (Wikipedia, 2018). The Syrian government has reportedly used chemical weapons repeatedly during the Syrian civil war, which has been going on for over 5 years. The people have been exposed to over 161 chemical attacks with chlorine, mustard, and Sarin. Most of the attacks were reportedly perpetrated by the Bashar al-Assad government. With the dismantling of the Syrian stockpile, the use of Sarin declined, but it was replaced by the widespread use of “barrel bombs” containing chlorine. During the civil war, almost 500,000 people have been killed and half the country’s population displaced (Shaheen, 2013, 2016; Goldsmith, 2015). Recent examples of possible state government use of chemical weapons include North Korea and Russia. On February 13, 2017, Kim Jong Nam, the half-brother of North Korean leader Kim Jong Un, died suddenly while at the Kuala Lumpur airport in Malaysia. An investigation determined that he died

FIGURE 1.10  The binary nerve agent plant at Pine Bluff Arsenal, Arkansas. (U.S. Army.)

Chemical Warfare Agents from poisoning by VX, which was apparently administered by a female touching him in the airport. North Korea was immediately accused of ordering the attack, but Kim Jong Un denied any responsibility (Berlinger, 2017). On March 4, 2018, a Russian double agent, Sergei Skripal, and his daughter were found unconscious near their home in Salisbury, England. An investigation led to the Russian government being blamed for using its Novichok agent in the attempted assassination. Russia denied the allegations (BBC, 2018a). On June 30, two residents of Amesbury, near Salisbury, also became ill with similar symptoms. One of these, Dawn Sturgess, died from the exposure to what was also identified as a Novichok agent in a perfume bottle. British authorities reported they identified the alleged perpetrators and linked them to Russia. Russia again denied any responsibility (BBC, 2018b).

1.8 Chemical Warfare Agents Used in Terrorism The Federal Bureau of Investigation (FBI) has defined terrorism as the unlawful use of force or violence against persons or property to intimidate or coerce a government, civilian population, or any segment thereof, to furtherance of political or social objectives (FBI, 2003). The first large-scale chemical terrorism incident occurred in the United States in 1982 (Cooke, 2002). Seven relatively young people in the Chicago, Illinois area collapsed suddenly and died after taking Tylenol capsules that had been laced with 65–100 mg cyanide per capsule. The lethal dose of cyanide is approximately 0.5–1.0 mg/kg or about 70 mg for an adult person (Cai, 1998). These were the first victims to die from product tampering. The police believed that the murderer bought or stole the products from the stores, tampered with them, and returned them to the stores. The police speculated that the terrorist could have had a grudge against the producers of Tylenol, society in general, or even the stores where the tainted products were found, and he or she may have even lived in the area of the stores. The perpetrator was never apprehended, even though Johnson and Johnson, the maker of the capsules, offered a $100,000 reward (Kowalski, 1997). As a result of these events, in 1983, Congress passed the Federal Anti-Tampering Act. This legislation made it a federal crime to tamper with food, drugs, cosmetics, and other consumer products. In addition, many manufacturers made their products tamper resistant (Fearful, 2003). Copycat tampering followed in 1986 with Lipton Cup a Soup, Excedrin, and Tylenol again, Sudafed in 1991, and Goody’s Headache Powder in 1992, resulting in deaths. Sporadic tamperings continued in the Chicago vicinity, Detroit, and Tennessee (Kowalski, 1997). After the first World Trade Center bombing in 1993, sodium cyanide was reported to have been in the bomb, but it had burned in the heat of the explosion. It was speculated that had it vaporized, the cyanide would have been dispersed into the North Tower, and all in the Tower would have been killed, whereas only six people died in the explosion (Mylroie, 1994; 1995–96; Phillips, 1994). Not all chemical terrorism incidents involved lethal chemicals. For example, a Palestinian homicide bomber sprinkled an anticoagulant rat poison among the nuts and bolts of the bomb that he exploded on a bus in Jerusalem, Israel. Among the survivors, a 14-year-old girl was bleeding uncontrollably from every one of her puncture wounds. The use of a

Brief History and Use of Chemical Warfare Agents coagulant drug eventually stopped the young victim’s bleeding (Hammer, 2002). Perhaps the most publicized incident involving chemical terrorism occurred in Japan in 1995. Aum Shinrikyo, a Japanese religious doomsday sect, used Sarin against civilians in Japan on June 27, 1994. The sect targeted a dormitory in Matsumoto, where three judges who had ruled against them in a land deal trial lived. The sect attempted to spread Sarin in the open, but the chemical reaction did not work properly, and the wind changed direction. The judges became ill but survived the attack. Seven victims in the neighborhood died that evening, and over 144 civilians were injured. Aum Shinrikyo’s next attempt was to spread Sarin in a closed area. They chose the subway system in Tokyo, Japan on March 20, 1995, where five trains would meet at 8:15 a.m. Members of the sect on the trains pierced plastic bags containing 30% Sarin with sharp-tipped umbrellas and let the Sarin evaporate into a lethal gas cloud. Twelve passengers on the trains died, and about 5500 sought medical treatment (Biema, 1995; Ohbu and Yamashina, 1997). Following the U.S. and allied military invasion of Iraq in 2003, known as Operation Iraqi Freedom, there were reports of Islamic State (IS) terrorists using chemical agents. These incidents included the use of chlorine and mustard agent, mostly against Iraqi civilians but also occasionally against U.S. and Iraqi military targets (Gardner, 2015; CNN, 2016).

1.9 Conclusions Chemicals have been used in warfare since almost the beginning of recorded history. This started out crudely using malodorous materials, irritants, poisonous plants and animals, as well as decaying bodies. Since the birth of chemistry, toxic chemicals have been created specifically for war. Less-than-lethal and lethal chemicals were also developed that incapacitated or killed the enemy without disfiguring or mutilating the body and without affecting or destroying the infrastructure, which appeared to be distinct advantages offered by the use of these chemicals. In asymmetric warfare and terrorism, it was sometimes difficult to recognize or identify the enemy. Because terrorists availed themselves of toxic industrial chemicals and materials that had been transported and already stockpiled, a working knowledge of the chemistry of chemical warfare agents was no longer a necessity. Advances in biotechnology, nanotechnology, genetic engineering, neurobiology, and computer sciences, among others, not only assisted in the proliferation of traditional chemical warfare agents but also stimulated the emergence of nontraditional agents. Advances also occurred in the delivery systems of these agents. Although the use of chemical warfare agents in terrorist activities has been limited, this may not accurately reflect the potential of their future use.

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Brief History and Use of Chemical Warfare Agents Phillips, J.A. (1994). The changing face of Middle Eastern terrorism. Available from URL: www.Heritage.org/research/middleeast/bg1005.cfm (accessed January 2007). Prentiss, A.M. (1937). Chemicals in War. New York: McGraw-Hill, p. 180. Pringle, L. (1993). Chemical and Biological Warfare. Hillside, NJ: Enslow, p. 55, 57. Reddit. (2014). What role did amphetamines have in WWII, by both sides? Available at URL: www.reddit.com/r/AskHistorians/ comments/1omm8m/what_role. Richter, J.P. (1970). The Notebooks of Leonardo da Vinci, Vol. 11. New York: Dover, pp. 280–281. Robey, J. (2003). Bioterror through time. Discovery Channel Series, February 21, 2003. Available at http://dsc.discovery. com/anthology/spotlight/bioterror/history/history.html, p. 3 (accessed January 2007). Rozman, K.K. and Doull, J. (2001). Paracelsus, Haber and Arndt. Toxicology 160: 191–196. Salem, H., Ballantyne, B., and Katz, S.A. (2006). Inhalation toxicology of riot control agents. In: Inhalation Toxicology, 2nd Edition. H. Salem and S.A. Katz, Boca Raton, FL: CRC Press, pp. 485–520. Sartori, M. (1943). The War Gases: Chemistry and Analysis. New York: Van Nostrand, pp. 3, 33, 59, 165, 181, 188, 217. Shaheen, K. (2013). www.theguardian.com/world/2013/sep/17/ sarin-deadly-history-nerve-agent-syria-un. Shaheen, K. (2016). www.thegurdian.com/world/2016/mar/14/syriachemical-weapons-attacks-almost-1500-killed-report-unitednations. SIPRI. (1971). The Rise of CB Weapons. New York: Humanities Press, pp. 70–75, 142, 147, 280–282, 336–341. Smart, J.K. (1997). History of chemical and biological warfare: An American perspective. In: Textbook of Military Medicine: Medical Aspects of Chemical and Biological Warfare. F.R. Sidell, E.T. Takafugi, and D.R. Franz, Eds. Washington, DC: Office of the Surgeon General, Department of the Army, Chapter 2, pp. 9–86. Available from URL: www.usuhs.mil/ cbw/history.htm (accessed January 2007). Smart, J.K. (2004). History notes: Chemical and biological warfare research and development during the Civil War. CBIAC Newsletter 5(2): 3, 11–15. Tagate. (2006). Available at URL: www.tagate.com/wars/history_ of_warfare/chemical_warfare_4.shtml (accessed January 2007). Taylor, L.B. and Taylor, C.L. (1985). Chemical and Biological Warfare. New York: Franklin Watts, pp. 26–27, 41. Thompson, R.M. and Wainwright, R. (1918). Confidential Correspondence of Gustavus Vasa Fox, Assistant Secretary of the Navy, 1861–1865, Vol. 1. New York: De Vinne Press, p. 263. Thucydides. (1989). The Peloponnesian War. D. Grene, Ed. Chicago: The University of Chicago Press, pp. 134, 286–287.

15 Tintinalli, J.E. (2003). Tintinalli’s Emergency Medicine: A Comprehensive Study Guide, 6th Edition. New York: McGraw-Hill. Available from URL: www.accessmedicine. com/content.aspx?aID¼585607 (accessed January 2007). Tschanz, D.W. (2003). “Eye of newt and toe of frog”: Biotoxins in warfare. StrategyPage.com, October 20, 2003. Available from URL: http://216.239.37.104/search?q=cache:Tv-SBKj TGFIJ:www.strategypage.com/articles/biotoxin_files/BI, p. 3 (accessed January 2007). United Kingdom Ministry of Defence. (1999). Defending against the threat of biological and chemical weapons. Outline history of biological and chemical weapons. Available from URL: www.mod.uk/issues.cbw/history.htm, p. 5 (accessed January 2007). United Nations. (1986). Report of the Mission Dispatched by the Secretary-General to Investigate Allegations of the Use of Chemical Weapons in the Conflict Between the Islamic Republic of Iran and Iraq. United Nations Security Council S=17911. U.S. Army. (1918). The Gas Defender, October 15, 1918, Long Island City, NY: Employees of the Gas Defense Plant, p. 9. U.S. War Department. (1891). War of the Rebellion: A Compilation of the Official Records of the Union and Confederate Armies, Series 1, Volume 36, Part 3, 888. Washington, DC: Government Printing Office. Vilensky, J.A. and Sinish, P.R. (2005). Quarterly Journal of Military History MHQ, Spring, 2005. Available from URL: www. historynet.com/wars_conflicts/weaponry/3035881.html (accessed January 2007). Waitt, A.H. (1942). Gas Warfare. New York: Duell, Sloan and Pearce, pp. 7–8, 135. Washington Blog. (2015). Americans supported and lnspired the Nazis. Available at URL: http://washingtonsblog.com/2015/ 03/Nazis-got-ideas-america.html (accessed March 2019). Washington, G. (2015). Americans supported and inspired the Nazis. Available from URL: www.zerohedge.com/news/2015-0329/americans-supported-and-inspired-nazis (accessed April 2015). Wikipedia. (2007a). Chemical warfare. Available from URL: www. en.wikipedia.org/wiki/Chemical_warfare. Wikipedia. (2007b). Greek fire. Available from URL: www.wikipedia.org/wiki/Greek_fire (accessed January 2007). Wikipedia. (2007c). Nerve agent. Available from URL: http:// en.wikipedia.org/wiki/Nerve_agent (accessed January 2007). Wikipedia (2018). Moscow Theatre Hostage Crisis. Available from URL: https://en.wikipedia.org/wik/Moscow_theatre_hostage_ crisis (accessed March 2018). Wiley, B.I. (1968). Drop poison gas from a balloon. Civil War Times Illustrated 7(4): 40–41. Witschi, H. (2000). Fritz Haber: 1868–1934. Toxicological Sciences 55: 1–2. Available from URL: http://toxsci.oxfordjournals.org/ cgi/content/full/55/1/1.

2 Chemistries of Chemical Warfare Agents Terry J. Henderson, Ilona Petrikovics, Petr Kikilo, Andrew L. Ternay, Jr., and Harry Salem CONTENTS 2.1 Introduction................................................................................................................................................................................... 17 2.2 Early Chemical Warfare Agents................................................................................................................................................... 17 2.2.1 Chlorine (Cl2)................................................................................................................................................................... 18 2.2.2 Phosgene (COCl2).............................................................................................................................................................19 2.2.3 Chloropicrin (CCl3NO2)...................................................................................................................................................20 2.2.4 Hydrogen Cyanide (HCN)................................................................................................................................................ 21 2.2.5 Cyanogen Chloride (ClCN)..............................................................................................................................................23 2.2.6 Sulfur Mustard (C4H8Cl2S)...............................................................................................................................................24 2.2.7 CS (C10H5ClN2).................................................................................................................................................................26 2.3 Nerve Agents.................................................................................................................................................................................27 2.3.1 Tabun (C5H11N2O2P).........................................................................................................................................................27 2.3.2 Sarin (C4H10FO2P)............................................................................................................................................................28 2.3.3 Soman (C7H16FO2P)..........................................................................................................................................................29 2.3.4 VX (C11H26NO2PS)...........................................................................................................................................................29 2.4 Incapacitating Agents....................................................................................................................................................................30 2.4.1 BZ (C21H23NO3)................................................................................................................................................................30 2.4.2 Fentanyl (C22H28N2O).......................................................................................................................................................30 2.5 Concluding Remarks.....................................................................................................................................................................32 References...............................................................................................................................................................................................32

2.1 Introduction

2.2 Early Chemical Warfare Agents

This chapter is a broad overview of the chemistries of selected substances that have been considered as chemical warfare agents (CWAs) at one time or another. Biological toxins such as the botulinum neurotoxin from the bacterium Clostridium botulinum or the ricin toxin from the castor oil plant Ricinus communis are not included, because they are often thought of as biological threats, even though they are perhaps best considered as toxic compounds originating from various organisms. Given size limitations and other restrictions, it was possible to mention only a fraction of the work that we might have otherwise included. The substances are initially identified by their most commonly used names, and alternative names are also presented in each specific discussion along with their North Atlantic Treaty Organization (NATO, Brussels, Belgium) code, American Chemical Society’s Chemical Abstracts Service number (CAS#, Columbus, Ohio) and molecular formulas. In this chapter, we focus on reactions that are representative of the compounds or are of special value. Each section includes, as appropriate, some general remarks followed by discussions of physical properties and chemical reactions. With only a very limited number of exceptions, issues of biological reactions and syntheses are intentionally excluded.

The first large-scale development, manufacture, and use of CWAs was during World War I. During the war, the chemicals were deliberately released on the battlefield as slow-moving poisonous gas clouds toward entrenched adversary troops to facilitate their demoralization, injury, and death. The types of weapons employed ranged from disabling chemicals, such as tear agents, to lethal agents, such as phosgene, chlorine, and sulfur mustard. The chemistries of seven such chemical weapons are detailed in this section and are ordered according to their intended militaryspecific use as choking, blood, blister, or tear agents. Choking or pulmonary agents are substances designed to impede a victim’s ability to breathe by causing a build-up of fluids in the lungs, which then leads to suffocation. Blood agents are compounds that are absorbed into the blood and inhibit the ability of blood cells to use and transfer oxygen, ultimately leading to suffocation as with choking agents. Blister agents, or vesicants, cause severe chemical burns, resulting in large, painful water blisters at the affected area. The inhalation of blister agents can lead to blister formation in the respiratory tract, which in extensive cases, can also result in death by suffocation. Finally, tear agents, or lachrymators, are compounds that cause severe eye, respiratory, and skin irritation, pain, bleeding, and even blindness. In 17

18 the eyes, tear agents stimulate the nerves of the lacrimal gland to produce tears. Today, these agents are sometimes used as riot control agents.

2.2.1 Chlorine (Cl2) Other Names and Designations: molecular chlorine; dichlorine; Red Star; NATO code CL; CAS# 7782-50-5. General Remarks: In 1914, the German chemist Fritz Haber proposed the use of chlorine gas against Allied troops to the German High Command in direct contravention of the international agreement prohibiting the use of poisonous weapons known as the Hague Convention of 1907. Haber worked to weaponize the gas shortly thereafter, and on April 22, 1915, he supervised the use of the gas by German troops against French and Algerian forces on the Western front near Ypres, Belgium. On September 25, 1915, the British used chlorine gas against German soldiers in Loos, France. During World War I, neat chlorine, which was also referred to as Red Star, was used as a general choking agent. Chlorine was also mixed with 20% sulfur chloride (SCl2 or S2Cl2) and used for a relatively short period under the name Blue Star, and a mixture of 50% chlorine and 50% phosgene was referred to as White Star (Richter, 1992). Because it is very reactive, chlorine does not occur in nature as Cl2. It normally is produced from compounds that are relatively rich in chlorine, such as sodium chloride. Manufactured today in large quantities for commercial applications, chlorine is now among the top 10 chemicals produced in the United States and considered to be a toxic industrial chemical. Physical Properties: Chlorine (atomic chlorine, Cl) has an atomic number of 17, an electronegativity of 3.2 (Pauling scale), and an atomic mass of 35.46 u. It is the third most electronegative element behind fluorine and oxygen. Throughout this chapter, the term chlorine refers to Cl2 unless otherwise noted. Chlorine is a highly reactive nonmetal (halogen) element appearing as a greenish-yellow gas with a density of 3.2 g/mL at standard temperature and pressure (STP). Solid chlorine melts at –101°C, and the gas liquefies at room temperature at 7.9 atm, or at −34°C at 1 atm. The substance has a water solubility of 0.64 g Cl2/100 g water at room temperature and a pungent odor. Synthesis and Reactivity: Chlorine was first prepared by the Swedish chemist K. W. Scheele in 1774 using the reaction of manganese dioxide with hydrochloric acid. Erroneously, he thought that the chlorine product was a compound containing oxygen and not an element, but it was ultimately identified and named as an element by Sir Humphry Davy in 1810. The reaction of hydrochloric acid with potassium permanganate provides a second convenient route to small quantities of chlorine. Commercial syntheses employ several techniques, including (1) the electrolysis of brine to form chlorine at the anode, (2) the electrolysis of fused sodium chloride, and (3) the electrolysis of hydrochloric acid. The first two techniques are collectively referred to as the chlor-alkali process and are responsible for the consumption of ~50% of the sodium chloride produced in the United States. Chlorine dissolves in water to form “chlorine water.” Only a portion of the chlorine remains intact, while the remainder converts to hypochlorous acid (aqueous HOCl) and hydrochloric

Chemical Warfare Agents acid (aqueous HCl), enhancing the corrosiveness of “moist” chlorine gas. By way of contrast, fluorine reacts with water to form hydrogen fluoride, HF, and molecular oxygen, O2. Most organic functional groups, including those in biomolecules, react with chlorine. Some reactions yield products containing only one chlorine atom, whereas others yield products with two. Slow reactions can be accelerated in some cases by altering the reaction conditions (solvent, temperature, and catalysts). Chlorine reacts by either radical or ionic processes depending on the substrate and the reaction conditions. Radical reactions require some initiator, such as heat, light, or other radicals, and may involve the conversion of molecular chlorine to atomic chlorine; reactions with alkanes most often are radical processes (Ternay, 1979a, 1979b). In a nonreactive solvent, alkenes can readily add chlorine to form vic-dichlorides by either radical or ionic mechanisms. Anti-addition is frequently observed, especially in ionic additions; cyclohexene, for example, affords trans1,2-dichlorocyclohexane. At high temperatures, chlorine can replace an allylic hydrogen by a radical process with preservation of the double bond. For example, propene yields allyl chloride and hydrogen chloride (Carey, 2000a). Alkenes, R 2C=CR 2, can react with chlorine and water to form halohydrins, R 2C(OH)C(Cl)R 2. With alcohol (R′OH) solvents, the product is a haloether, R 2C(OR′)C(Cl)R 2. In the presence of a carboxylic acid, R′CO2H, the products include the corresponding halohydrin esters, R 2C(O2CR′)C(Cl)R 2 (Wagner and Zook, 1965b). In general, aromatic hydrocarbons are less nucleophilic than are simple alkenes. The chlorination of less nucleophilic aromatics, for example, C6H6, requires the use of a Lewis acid catalyst (anhydrous aluminum chloride, for example) and either excess of the liquid aromatic compound or an inert solvent. The product, C6H5Cl, results from a net substitution process. The chlorination (ortho or para) of activated aromatics such as anisole, C6H5OCH3, may not require a catalyst. Both 1° and 2° alcohols are oxidized to carbonyl-containing substances, with the creation of a ketone from a secondary alcohol being typical. The oxidation, accelerated in alkaline solutions, involves a hypochlorite intermediate as indicated here.

In the presence of base and excess chlorine, both α-chlorination and oxidation may occur. For example, a cyclohexanol–water– calcium carbonate mixture reacts to form 2-chlorocyclohexanone (Wagner and Zook, 1965a). Chlorine also reacts with the sulfur in sulfides to form reactive sulfonium salts:

The reaction of chlorine with aqueous ammonia and ammonium salts is noteworthy, as it leads to various chloramines, including unstable (explosive) nitrogen trichloride, NCl3 (Chloramines, 1999);

Chemistries of Chemical Warfare Agents both Faraday and Dulong were injured while working with this substance (Cardillo, 2001). The reaction of chlorine with phosphorus represents a commercial route to PCl3 and PCl5. In turn, this has become an important process in the synthesis of nerve agent precursors (Greenwood and Earnshaw, 1986). Chlorine reacts with many metals, often violently if the metal is finely divided, to form binary compounds such as iron (III) chloride and magnesium chloride, and traces of water may accelerate the reaction. The product of the reaction of chlorine with calcium hydroxide is bleaching powder. Although sometimes shown as a mixed salt, CaCl(OCl), bleaching powder also has been represented as a mixture of Ca(OCl)2, CaCl2, and Ca(OH)2. Chlorine is a more potent oxidant than bromine but less potent than fluorine (Clifford, 1961a). Thus, chlorine is able to oxidize both bromide and iodide ions to Br2 and I2, respectively, whereas fluorine oxidizes chloride, bromide, and iodide to their corresponding halogens. The oxidizing ability of chlorine is the basis for the starch–iodide test for chlorine. In the test, a mixture of potassium iodide, soluble starch, and zinc chloride produces a blue-violet color in the presence of chlorine. Unfortunately, and like many “spot tests,” this test is nonspecific and gives positive results with many oxidants, such as bromine. Trichloroisocyanuric acid, or TCCA (Tilstam and Weinmann, 2002), itself an interesting oxidant, can be prepared in ~75% yield by passing a stream of chlorine into an alkaline solution of cyanuric acid (Monson, 1971):

In contrast to this imide-based synthesis, amides of the type RC(O)NH2 are decarbonylated to primary amines (RNH2) with chlorine in the presence of base. This process, often called the Hofmann reaction, involves an intermediate isocyanate, R–N=C=O (Fieser and Fieser, 1961; Sandler and Karo, 1983). Aromatic oximes, lacking an α-hydrogen, react with chlorine to form intermediates that are converted to nitrile N-oxides with base (nitrile N-oxides are highly reactive species); this is shown here.

2.2.2 Phosgene (COCl2) Other Names and Designations: carbonyl chloride (or dichloride); Collongite; D-Stoff; chloroformyl chloride; NATO code CG (Ryan et al., 1996); CAS# 75-44-5. General Remarks: Used as a choking agent during World War I, phosgene was perhaps the most effective of all CWAs used during the war. It was developed by Fritz Haber to be a more effective CWA than chlorine. Phosgene was first used in World War I by the Germans, but was later used during the war by the French, the Americans, and the British. The first deployment of

19 the compound was at Ypres, Belgium on December 19, 1915, when German soldiers released ~4000 cylinders of phosgene combined with chlorine gas against British troops. Phosgene has rarely been used since World War I but is still responsible for the majority of deaths resulting from chemical warfare. Today, phosgene has an annual U.S. production demand of ~5400 million pounds, with its most important industrial use in the production of isocyanates, R–N=C=O. Physical Properties: Phosgene is a colorless gas at room temperature with a density, relative to air, of 3.4 and a molecular mass of 98.92 g/mol. It has a melting point (mp) of −118°C, a boiling point (bp) of 8°C, and a vapor pressure of ~1200 mm at 20°C. The compound is slightly soluble in water, in which it hydrolyzes, and is rather soluble in most hydrocarbons (hexane and benzene, for instance) and in glacial acetic acid. Phosgene has a distinct odor of newly mown or “musty” hay. It is estimated that the least detectable odor occurs at ~1 ppm and that the lowest concentration affecting the eyes is ~4 ppm. Chemistry: Phosgene can be prepared in the laboratory using a reaction similar to its commercial synthesis. In this gas-phase process, equimolar amounts of chlorine and carbon monoxide are passed over a bed of activated charcoal granules to generate the phosgene product. Also, chloroform produces phosgene when exposed to light in the presence of oxygen or air. Strictly for this reason, chloroform has sometimes been stabilized with traces of ethanol, as it reacts with any phosgene impurity to form ethyl carbonate. Phosgene is the acid dichloride of carbonic acid, HO–C(O)–OH, and like all acid chlorides, it reacts rapidly with water to produce the corresponding acid and hydrogen chloride. Because carbonic acid is unstable, the ultimate products of reaction with water are hydrogen chloride and carbon dioxide. The hydrogen chloride produced dissolves in excess water to form hydrochloric acid. Phosgene is also known to react with alcohol groups in organic compounds. Depending on reaction stoichiometry, phosgene readily reacts with 1° alcohols to produce either a chloroformate or a carbonate:

Each reaction with a 1° alcohol is accompanied by the loss of hydrochloric acid. Phosgene reacts with vic-diols, including sugars, to generate cyclic carbonates (Kawabata et al., 1986):

A similar reaction may occur with proximal diols (Le Boulch et al., 1967) as shown here.

20

Chemical Warfare Agents

A number of commercially important polymers are synthesized from phosgene. Lexan, for instance, is produced by reacting bisphenol A

Some amine N-oxides are reduced by phosgene. Thus, in the presence of base, 2-methylpyridine N-oxide is deoxygenated with phosgene (Ash and Pews, 1981):

with phosgene in the presence of base. As revealed in the following, phosgene also has been used to lactonize a hydroxyacid (Wehrmeister and Robertson, 1968) and to convert nicotinic acid (a carboxylic acid) to its anhydride (Rinderknecht and Gutenstein, 1967).

Aryl nitriles, ArCN, can also react with phosgene. In the presence of hydrochloric acid, for instance, the nitriles react with phosgene to yield triazine compounds (Yanagida et al., 1969):

The reaction of phosgene with benzyl alcohol yields benzyl chloroformate (or carbobenzoxy chloride), ClCO2CH 2C6H 5, useful in protecting the amino groups of amino acids (Ege, 1999). Reactions of phosgene with nucleophiles other than alcohols can produce other carbonic acid derivatives; for example, reaction with anhydrous ammonia yields urea, O=C(NH 2)2. Depending on reaction conditions, aromatic primary amines can react with phosgene to form carbamoyl chlorides (Csuros et al., 1969):

Isocyanate formation from diamines is important for the commercial production of polymer precursors (Khardin and Pershin, 1979). Under the appropriate conditions, proximal diamines are converted to cyclic structures by reaction with phosgene (Marx et al., 1977):

Like chlorine, phosgene can add to alkenes by a free radical process. As indicated in the following reaction, the addition can be initiated using CH3CO2•.

The photolytic cleavage of phosgene is believed to begin with a homolysis to form an acid chloride, RC(O)Cl, and Cl. In turn, the acid chloride rapidly decomposes to form carbon monoxide and a second chlorine atom (Wijnen, 1961). Like other acyl chlorides, phosgene can participate in Friedel–Crafts reactions. A classic example is the synthesis of Michler’s ketone, which is used to produce dyes (Robertson, 1937):

With less reactive aromatic compounds, a stronger Lewis acid may be required, but the monosubstituted acyl chloride can be isolated. Under appropriate conditions, a symmetric, substituted ketone is formed. Thus, benzophenone can be isolated by the reaction with excess benzene as shown (Wilson and Fuller, 1922): The formation of the biotin skeleton is representative of this process, as revealed by the product in the preceding reaction. Pyridine (labeled in the following reaction as Pyr) reacts with phosgene to form a complex containing two pyridine fragments per carbonyl chloride:

2.2.3 Chloropicrin (CCl3NO2) Other Names and Designations: nitrochloroform; trichloronitromethane; nitrotrichloromethane; Aquinite (French during World War I); vomiting gas (British during World War I); Klop

21

Chemistries of Chemical Warfare Agents (German during World War I); NATO codes PS and PK (earlier designation); CAS# 76-06-2. General Remarks: Chloropicrin is unique because it has been used as both a choking and a tear agent. During World War I, it was often used as a vomiting gas, because it could penetrate through protective respirators and force adversary troops to remove them to vomit, thereby exposing themselves to other toxic chemicals. Russian troops were the first to use chloropicrin as a chemical weapon in August 1916, which was quickly followed by British troops using it mixed with chlorine. As mentioned earlier for chlorine, a mixture of 50% chlorine and 50% phosgene was referred to as White Star (Richter, 1992). A mixture of 30% chloropicrin and 70% chlorine was called Yellow Star, which should not be confused with Yellow Cross, a term for sulfur mustard. Green Star was a mixture of chloropicrin (65%) and hydrogen sulfide (35%). Chloropicrin was found to be a useful fumigant by the end of World War I; however, its use as a fumigant (Jackson, 1934) today is extremely rare. Physical Properties: Chloropicrin is a colorless oil with a bp of 112°C, a mp of −69°C, and a pungent, stinging odor that has been described as anise-like. Its vapor pressure is ~20 mm/Hg at 20°C, and its molecular mass is 164 g/mol (Redeman et al., 1948). The oil has a density of 1.66 g/mL and a vapor density of 5.6 relative to that of air. Chloropicrin has low solubility in water (~2 g/L) but is quite soluble in typical organic solvents (chloroform, acetone, ethyl acetate, etc.) and a variety of organic compounds including, for example, benzoic acid and various resins. Although chloropicrin is nonflammable, contact with oxidizing agents may lead to fires or explosions. Exposure to high temperatures can produce toxic gases, including phosgene and carbon monoxide. Electron diffraction (Knudsen et al., 1966) and mass spectrometry (Murty et  al., 2005) studies of chloropicrin have been reported in addition to the vibrational spectra of bromopicrin and chloropicrin (Mason et al., 1959). Chemistry: Chloropicrin’s first reported preparation was by the Scottish scientist John Stenhouse in 1848 and involved the reaction of a chlorinating agent (bleaching powder) with picric acid (sym-trinitrophenol). It is this synthesis that led to the name chloropicrin, even though the ring system present in picric acid is absent in the chloropicrin product:

Another route to this compound involves the chlorination of nitromethane with chlorine in the presence of calcium carbonate (Orvik, 1980), and the nitration of chloroform with nitric acid also has served as a route to chloropicrin on a large scale (Szmant, 1957). The reaction of chlorine gas and aqueous sodium hydroxide with 4-hydroxy-3,5-dinitrobenzoic acid can also be used to prepare chloropicrin (Simandi and Soos, 1986):

Chloropicrin is stable in cold, dilute aqueous sodium hydroxide and does not decompose rapidly in either cold water or cold mineral acids (hydrochloric acid, for example). It slowly decomposes in ethanolic potassium hydroxide, however, and undergoes more rapid decomposition with sodium ethoxide or ethanolic sodium cyanide. Chloropicrin also decomposes slowly in acetone solutions, which is accompanied by a concomitant deposit of ammonium chloride. As with many other highly chlorinated organic compounds, the stability of chloropicrin decreases in the presence of light, as evidenced by the formation of color in its benzene solutions. Slow thermolysis of chloropicrin under reflux produces phosgene and nitrosyl chloride, NOCl, and photolysis of the compound yields similar results (Ashmore and Norrish, 1951; Castro and Belser, 1981). Heating with 20% fuming sulfuric acid rapidly decomposes chloropicrin to also form phosgene and nitrosyl chloride. Chloropicrin is dehalogenated with aqueous sulfite, SO3 –2, to form dichloronitromethane, CHCl2NO2 (Croue and Reckhow, 1989). In the absence of sulfite, the hydrolysis of chloropicrin is slow and rather independent of pH. Chloropicrin can be used to prepare orthocarbonates, C(OR)4, in a process involving four replacements by alkoxide ions (Hennig, 1937):

Similarly, chloropicrin reacts with iodide to form carbon tetraiodide (Kretov and Mehnikov, 1932). Chloropicrin has been shown to react with iron-bearing clay minerals to produce both chloronitromethane and dichloronitromethane (Cervini-Silva et  al., 2000). A microdetermination of the compound in air using a colorimetric procedure has revealed that analyses based on either chloride or nitrite afford similar results (Feinsilver and Oberst, 1953).

2.2.4 Hydrogen Cyanide (HCN) Other Names and Designations: prussic acid; formonitrile; methan(e)nitrile; Blausaure; hydrocyanic acid; carbon hydride nitride; NATO code AC; CAS# 74-90-8. General Remarks: The terms hydrocyanic acid and prussic acid are best limited to aqueous solutions of hydrogen cyanide. Liquid hydrogen cyanide mixed with substances to reduce polymerization (such as cyanogen chloride, for example) was named Zyklon B in Germany. The compound is described as a blood agent owing its toxicity to the liberation of cyanide ion (CN–) under physiological conditions. Hydrogen cyanide was tested as a chemical weapon in World War I and found not to be very effective. France used it in combat in 1916, but this proved to be ineffective due to weather conditions. The gas is lighter than air and rapidly disperses into the atmosphere, in sharp contrast to denser agents such as phosgene or chlorine, which tend to remain at ground level. Compared with such agents, hydrogen cyanide must be present in higher concentrations to be fatal. Collectively, these properties make its use in the field impractical. The United States and Italy used hydrogen cyanide against the Central Powers (Germany, Austria-Hungary, the Ottoman Empire and Bulgaria) in 1918.

22 The importance of cyanides in commerce is demonstrated by the annual U.S. production capacity for hydrogen cyanide, which was estimated to be >1 billion pounds in 2004. Roughly onehalf of the U.S. production is used to prepare adiponitrile for nylon 66. Its other uses include the preparation of acrylonitrile, methyl methacrylate (via acetone cyanohydrin), methionine, and cyanide salts. When required in laboratory settings, it is common for hydrogen cyanide to be used in situ due to its high toxicity. Physical Properties (http://webbook.nist.gov): Hydrogen cyanide typically occurs as a highly toxic gas with a density of 0.94 relative to air and a molecular dipole moment (gas) of 2.95 D (McClellan, 1963). In the linear hydrogen cyanide molecule, the nitrogen atom resides at the more negative end of the molecular dipole (Orville-Thomas, 1966). With a density of 0.687 g/mL at 10°C, liquid hydrogen cyanide has a low bp of 26°C and a freezing point of −13°C. The compound has a closed-up flash point of −18°C and a molecular mass of 27.0 g/mol. Hydrogen cyanide is colorless as both a gas and a liquid and has a bitter, almond-like odor. The odor threshold for hydrogen cyanide is estimated to be 2–5 ppm; however, some individuals are not capable of smelling the compound’s odor, even at lethal concentrations. Estimates suggest that 40%–60% of the population may not be able to detect the compound by odor alone (https://ehs.princeton.edu/). The compound’s rather high vapor pressure of 620 mm/Hg at 22°C ensures that it performs as a nonpersistent CWA. Hydrogen cyanide is soluble in water, ethanol, chloroform, and benzene. Unlike acids such as sulfuric acid and hydrochloric acid, hydrocyanic acid (i.e., aqueous HCN) is a rather weak acid with a pK A = 9.32 (Clifford, 1961b). An aqueous solution of hydrogen cyanide barely turns litmus red. The photoionization of hydrogen cyanide has been reported by Dibeler and Liston (1968), and the absorption spectrum of hydrogen cyanide has been compared with that of deuterium cyanide (Nagate et al., 1981). Chemistry: A typical route to hydrogen cyanide starting from potassium cyanide and dilute sulfuric acid has been outlined by Streitwieser and Heathcock (1976), and another reported synthetic route starts from sodium cyanide and sulfuric acid (Ziegler, 1941). Hydrogen cyanide can be prepared commercially by several routes, including the reaction of ammonia and air with methane. Hydrogen cyanide is not completely stable and is commonly marketed as a stabilized (often with H3PO4), flammable, anhydrous material. While forming salts with strong bases, the compound does not form salts with carbonates. Hydrogen cyanide reacts when heated, or in the presence of base or water, and may polymerize violently when in contact with strong acids (such as sulfuric acid). Once initiated, polymerization can be autocatalytic and, under confined conditions, can lead to an explosion. It is often removed from waste streams by conversion to ammonium thiocyanate in a process involving the scrubbing of waste streams with elemental sulfur in water. There are many well-studied reactions involving hydrogen cyanide (Cyanides in Organic Reactions; A Literature Review, 1962), the most famous of which is its addition to a carbonyl group (aldehyde or ketone); the products of these additions are referred to as cyanohydrins or hydroxynitriles (Ziegler et  al., 1990). An example reaction is provided that demonstrates the formation of a pair of enantiomers from an aldehyde:

Chemical Warfare Agents

Rates for these addition reactions are pH dependent, with maximum rates generally occurring between pH 4 and 5. The substituents around the carbonyl group generally determine the reactant and product concentrations in such equilibria. Cyanohydrins derived from aldehydes are generally more stable than those from ketones (Ternay, 1976). Cyanohydrin formation is the first step in the well-known chain-lengthening sequence known as the Kiliani–Fischer synthesis. For example, the d-arabinose aldopentose ultimately affords both d-glucose and d-mannose by this set of reactions (Carey, 2000b). Cyanohydrin formation is reversible and also occurs in nature, which can present cyanohydrin ingestion hazards. For example, Prunus amygdalus dulcis (sweet almond tree) is a source of an oil used in confectionery; however, almond kernels of Prunus amygdalus amara (bitter almond tree) yield an oil known as oil of bitter almonds (oleum amygdalae amarae) when crushed. On hydrolysis, ≤6% of the latter oil is converted to hydrogen cyanide (http://en.wikipedia.org). Ketones are converted to the corresponding nitrile compound by a sequence beginning with the conversion of the ketone to a tosylhydrazone followed by hydrogen cyanide addition across the C=N bond. Thermolysis of the intermediate 1-cyanohydrazine affords the cyanide in acceptable yields (Buehler and Pearson, 1976):

The Gatterman aldehyde synthesis involves a Friedel–Craftstype reaction on activated aromatic rings to afford aryl aldehydes (Noller, 1966a):

Amides can also be produced from alkenes and cyanide or from either 2° or 3° alcohols and cyanide by the Ritter reaction (Reddy, 2003), as in the case of the preparation of a formamide:

The interaction of methylphosphonocyanidate with hydrogen cyanide or the cyanide ion has also been described (Morozik et al., 2003). Conjugated aldehydes and ketones can add to hydrogen cyanide by a 1,4-addition pathway (conjugate addition or Michael addition) to produce β-cyano carbonyl compounds. As an example, 1-phenylpropenone, C6H5C(O)CH=CH2, can add to hydrogen cyanide to afford 4-oxo-4-phenylbutanenitrile, C6H5C(O) CH2CH2CN, in ~65% yield. In the presence of an appropriate

23

Chemistries of Chemical Warfare Agents catalyst, alkenes can also add to hydrogen cyanide (Arthur et  al., 1954; Jackson and Lovel, 1982). With a cobalt carbonyl catalyst, for instance, 1-propene adds to hydrogen cyanide by Markownikov addition to produce isopropyl cyanide in ~75% yield. Additionally, hydrogen cyanide can add to alkynes in the presence of metal complexes, and the use of a nickel complex can lead to syn addition (Jackson and Lovel, 1983; Jackson et al., 1988). And through a mechanism involving a π-allyl intermediate, hydrogen cyanide can react with conjugated diene compounds. The course of addition is complex and may lead to more than one product (Keim et al., 1982), as shown here.

Epoxide rings can open through reaction with hydrogen cyanide. Thus, ethylene oxide reacts with hydrogen cyanide to form ethylene cyanohydrin (Mowry, 1948). A critical step in one commercial route to acrylonitrile involves the ring opening of ethylene oxide by hydrogen cyanide:

The ring opening of epichlorohydrin with hydrogen cyanide, illustrated in the following reaction, begins a sequence that leads to carnitine (Yamagutchi, 1989):

The ability of cyanide to form complexes with various metals explains its use in extracting gold from rocks in a process referred to as heap leaching. Cyanide ions react readily with iron salts to form iron complexes, such as is found in ferric ferrocyanide (also known as Prussian Blue, Hamburg Blue, Paris Blue, and mineral blue). Cyanide has a stronger affinity for Fe(III) (ferric) ions than for Fe(II) (ferrous) ions, and therefore, Fe(III) has been exploited for use in a therapy for cyanide poisoning (Baskin and Brewer, 1997). Cyanide ions have also been observed to readily complex with zinc (Feeney and Burgen, 1973). A summary of the color and bonding of cyanide complex ions has been reported (Clifford, 1961b), and the infrared spectra of cyanide complex ions have been studied extensively (Rao, 1963; Bowser, 1993). Hydrogen cyanide adds chlorine to form cyanogen chloride, which in turn, trimerizes to cyanuric chloride (see Section 2.2.5). A platinum catalyst has been used in the hydrogenation of hydrogen cyanide to produce methylamine (Barratt and Titley, 1919), and a silver or gold catalyst can be used in the high-temperature oxidation of hydrogen cyanide using oxygen. The products of this latter reaction include cyanic acid, HOCN, and lesser amounts of cyanogen, (CN)2 (Zima, 1959). Sodium formate can be produced by reacting hydrogen cyanide with dilute aqueous sodium hydroxide, and the reaction has served as the basis for an analytical method for measuring cyanide (Doizine et al., 1982). Finally, the cyanide ion is a good nucleophile and is able to cleave various

bonds, including disulfide bonds, as shown in the following reaction (Gould, 1959):

2.2.5 Cyanogen Chloride (ClCN) Other Names and Designations: chlorine cyanide; chlorocyanogen; chlorcyan; NATO codes CK and CC (earlier designation); CAS# 506-60-2. General Remarks: Like hydrogen cyanide, which also forms cyanide in the body, cyanogen chloride has been described as a blood agent. The two compounds have also been thought of as systemic agents or systemic poisons. Cyanogen chloride was used in large quantities by both the French and the British during World War I. Reportedly, France combined hydrocyanic acid with the compound for use as an irritant to force adversary troops to remove their protective respirators, exposing them to other toxic gases. Cyanogen chloride was also combined with arsenic trichloride later during the war. Cyanogen chloride tends to spontaneously polymerize and therefore, was often combined with stabilizers such as sodium pyrophosphate to extend its shelf life. Although the United States maintained 500 and 1000 pound cyanogen chloride bombs during World War II, these were not used during the war. Although cyanogen is a distinct molecule (see the following), the term is used sometimes to describe any substance that forms cyanide in the body. By this extended definition, cyanogen chloride is a cyanogen. Cl–Cl Molecular chlorine

N≡C–Cl Cyanogen chloride

N≡C–C≡N Cyanogen

The correct formula for cyanogen chloride was first established by Gay-Lussac in 1815. The compound can be considered either as the monocyanide of molecular chlorine or as the chloride of cyanogen. Physical Properties: Cyanogen chloride has a low bp at 13°C, a mp at −6°C, and a molecular mass of 61.5 g/mol. At STP, the compound has a vapor density of ~2.1, whereas that of hydrogen cyanide is 0.93, and a density of 1.2 g/mL at 0°C. Cyanogen chloride has a molecular dipole moment (gas) of 2.80 D (McClellan, 1963), and as was the case for hydrogen cyanide, the nitrogen atom of the linear cyanogen chloride molecule resides at the more negative end of the molecular dipole (Orville-Thomas, 1966). A colorless gas at room temperature, cyanogen chloride is often handled as a cylinder of liquefied gas; this volatility renders cyanogen chloride a nonpersistent threat agent. Cyanogen chloride is soluble in water (69 g/L) and in most organic solvents (ethanol, chloroform, or benzene, for instance). Details of the valence shell photoionization of cyanogen halides have been published (Holland and Shaw, 2004), as have the UV absorption spectra of cyanogen halides (King and Richardson, 1966).

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Chemical Warfare Agents

Chemistry: The first reported preparation of cyanogen chloride was by the French chemist Claude Louis Berthollet (1789), but real interest in the compound did not develop until >100 years later. Just after World War I, Jennings and Scott (1919) observed that changes in various conditions influence the stability of impure cyanogen chloride. A larger-scale synthesis of cyanogen chloride appeared shortly after World War I (Price and Green, 1920). A typical synthesis involves the reaction of chlorine with an aqueous solution of sodium cyanide

Some alkyl cyanates can be prepared from the reaction of alkoxide ions with cyanogen chloride:

Cl 2 + NaCN ® ClCN + NaCl

and a related anhydrous procedure also yields the product. After purification, the compound can be safely stored under refrigeration for months (Coleman et al., 1946). Commercially, cyanogen chloride has been produced by the chlorination of aqueous hydrogen cyanide. An unintended synthesis of the compound may occur during the treatment of cyanide-containing waste water with chlorine or hypochlorite; aspects of this issue have been considered by Bailey and Bishop (1973a). Trimethylsilylcyanide has also been used to produce cyanogen chloride (Nachbaur et  al., 1978), and furthermore, the reaction of active nitrogen and chloromethanes (carbon tetrachloride, for instance) has been reported to involve the formation of cyanogen chloride (Sobering and Winkler, 1958) as well. Some of the reactions of cyanogen chloride are rationalized by the addition-elimination process shown in the following reaction (Nu is a nucleophile). For example, diethyl malonate, CH2(CO2C2H5)2, reacts with cyanogen chloride in the presence of sodium ethoxide to produce diethyl cyanomalonate, CH(CN)(CO2C2H5)2. This explains why nucleophilic solvents, such as ethanol or water, eventually lead to the decomposition of cyanogen chloride.

Their stabilities are enhanced with steric bulk around the −OCN fragment, as it inhibits subsequent trimerization (Kauer and Henderson, 1964). The application of gas–solid reactions permits the synthesis of a variety of functionalities from cyanogen chloride (Kaupp et al., 1998), as shown in the following reaction:

ArNH 2 + ClCN + N ( Me )3 ® ArNHCN + N ( Me )3 H+ Cl –

Cyanogen chloride slowly hydrolyzes to form hydrochloric acid and hypochlorous acid; the hydrolysis rate increases in the presence of bases (Bailey and Bishop, 1973b) such as sodium hydroxide, and it can also hydrolyze on reaction with hypochlorite at pH 7–8 (Price et al., 1947). The complete hydrolysis (aqueous sodium hydroxide with heat) of cyanogen chloride produces ammonia, and the detection of ammonia from this hydrolysis was the basis for an early, nonspecific test for cyanogen chloride. The ammonolysis of cyanogen chloride using ethanolic ammonia produces cyanamide, NH2CN (Cloez and Cannizzaro, 1851):

ClCN + 2NH 3 ® NH 2CN + NH 4Cl

Amines can be produced by the addition of cyanogen chloride to substituted alkenes (Smolin, 1955), usually in the presence of sulfuric acid: During storage, cyanogen chloride tends to form the cyclic trimer cyanuric chloride (cyanuryl chloride), (ClCN)3:

Cyanuric chloride, which is less toxic than cyanogen chloride, is of reduced value as a CWA. To minimize the formation of the compound, the French produced an agent for use in World War I referred to as Vitrite (also identified as Vivrite), a mixture containing 70% cyanogen chloride and 30% arsenic trichloride. Cyanogen chloride can also be stabilized by using sodium pyrophosphate, Na4P2O7 (Kleber and Birdsell, 1959). Cyanogen chloride reacts with sodium sulfide, Na2S, to form sodium thiocyanate, NaSCN. An extension of this reaction involves that of thiolate anions, RS−, with cyanogen chloride to produce thiocyanates, RSCN, as illustrated in the following reaction. Similarly, the reaction of phenols produces aromatic cyanates (Kaupp et al., 1998). In such reactions, 3° amines are often used to trap hydrogen chloride.



CH 3CH 2C ( CH 3 ) =CH 2 + ClCN + H 2SO 4 ® CH 3CH 2C ( CH 3 )2 NH 2 ( 23% )



Cyanogen chloride reacts with sulfite ions as well as sulfur dioxide (Bailey and Bishop, 1973b), and in the presence of sulfur trioxide, cyanogen chloride adds to alkenes to produce sulfonyl chlorides (Arlt, 1970). Cyanogen azide, an unstable compound that is explosive when dry, is produced when sodium azide reacts with cyanogen chloride (Noller, 1966b):

A kinetic study of the shock tube decomposition of cyanogen chloride revealed that a set of six reactions are involved in its thermal decomposition (Schofield et al., 1965).

2.2.6 Sulfur Mustard (C4H8Cl2S) Other Names and Designations: 2,2′-dichloroethyl sulfide; bis(2-chloroethyl) sulfide; Lost; S-Lost; Yellow Cross; HS; Yperite; EA 1033 (U.S. Army); NATO code HD (distilled sulfur mustard only); CAS# 505-60-2.

Chemistries of Chemical Warfare Agents General Remarks: Sulfur mustard was introduced during World War I as a blister agent and was sometimes found in a crude form referred to as H. As was the case with phosgene, Fritz Haber developed sulfur mustard in an effort to produce effective chemical weapons for Germany during the war. Sulfur mustard was first used effectively by the German army against British and Canadian soldiers near Ypres, Belgium in July 1917 and later against the French Second Army. It is one member of a class of compounds known as mustards, which generally bear a heteroatom (Z) separated from a leaving group (L) by two atoms; that is, they contain the Z-(CH2)2-L fragment. Sulfur mustard has the structural formula ClCH2CH2SCH2CH2Cl. Nitrogen mustards, where Z = N, have been considered as anticancer drugs as well as CWAs but are not now considered as likely CWAs or terrorist threats; the compounds are not discussed further in this chapter. Physical Properties: Sulfur mustard is a colorless oil with a bp of 227°C, a mp of 14°C, a molecular dipole moment of 1.78 D (hexane), and a molecular mass of 159 g/mol. As a liquid, it is slightly denser than water at 1.27 g/mL at 20°C. The compound is usually encountered as an impure, pale yellow-brown, odoriferous liquid, and its color generally deepens with increasing amounts of impurities. Sulfur mustard has a vapor density of 5.4 relative to air and a vapor pressure of 0.072 mm Hg at 20°C. It is miscible in typical organic solvents (such as carbon tetrachloride, acetone, or chloroform) but has a lower solubility in water at 0.092 g/100 g at 22°C (Somani, 1992; Sidell et al., 1998). Chemistry: Several routes to sulfur mustard have been reported, but the first production of the compound in a reasonable yield is often credited to Meyer (1886). A second route, known as the Levinstein process, produces elemental sulfur as a contaminant, while a route known as the Steinkopf synthesis involves S(CH2CH2OH)2 (Steinkopf et al., 1920). The reactivity of sulfur mustard is dominated by the participation of its heteroatoms. In particular, the compound’s divalent sulfur atom is a good nucleophile and promotes SN-type reactions (Whitman, 1995a). This atom’s β-position relative to two chlorine atom positions that serve as good leaving groups commonly leads to neighboring group participation (Eliel and Wilen, 1994) and therefore, to episulfonium ion formation. This is the result of an internal nucleophilic displacement reaction that is driven by entropic factors. The resulting episulfonium ion possesses substantial small-ring strain and is equally prone to ring opening at either carbon atom position on the ring. Using water as an external nucleophile, repetition eventually affords thiodiglycol, a diol mustard analog. The intermediate hemi-mustard also possesses vesicant properties. The different reactions are summarized here:

25 Structural variations that hinder episulfonium ion formation or sulfur nucleophilicity reduce activity. Thus, S(C(O)CH2Cl)2 is reported to show little vesicant behavior (Sartori, 1939). Sulfur mustard is denser than water, only slightly water soluble, and hydrolyzes very slowly when dissolved in cold water. Therefore, it can remain a health threat for some time when present in bodies of water, even though it ultimately hydrolyzes to the relatively safe compound thiodiglycol. Depending on conditions, the hydrolysis also can produce 1,4-thioxane, O(CH2CH2)2S; 2-(vinylthio)ethanol, CH2=CHSCH2CH2OH; and a variety of other compounds, some in very small quantities (D’Agostino and Provost, 1985). For example, sulfur mustard can react with thiodiglycol to form monosulfonium or disulfonium ions (Yang et al., 1992; Munro et al., 1999):

In cases where external nucleophiles are bonded to linear molecular chains, two consecutive attacks on sulfur mustard can lead to a cross-linking of the chains:

Molecules similar to sulfur mustard with more extensive separation between their sulfur atom position and leaving group atoms (such as Cl(CH2)6S(CH2)6Cl, for example) behave as simple aliphatic halides (or sulfides), simply because the formation of a episulfonium ion containing a three-membered ring is no longer possible. One convenient method for verifying the formation of an episulfonium ion intermediate in such reactions involves using isotopic carbon labeling; because the ion is symmetric, its formation ultimately leads to a nearly 1:1 distribution of the isotopic label at the two carbon atom sites, which is not observed in a direct displacement.

Sulfides, including sulfur mustard, undergo oxidation at their sulfur atom position. Initial oxidation produces a sulfoxide, whereas further oxidation produces a sulfone as shown in the following reaction. Oxidants that can participate in such reactions include hydrogen peroxide, peroxyacids, nitric acid, permanganate ions, ozone, dinitrogen tetroxide, and dichromate ions. Oxides of sulfur mustard are not as volatile as mustard,

26 which might possibly explain why the surfaces of British air raid shelters were sometimes coated with the oxidant calcium hypochlorite, Ca(OCl)2. Episulfonium ion formation is less likely in the sulfoxide of sulfur mustard than in sulfur mustard itself.

Chemical Warfare Agents A study using nuclear magnetic resonance (NMR) spectroscopy and gas chromatography–mass spectrometry revealed that the hydrolysis of sulfur mustard in deuterium oxide displays a halflife of ~7 min at 22°C, increasing to ~24 min in the presence of sodium chloride (Logan and Sartori, 2003), consistent with previously reported results (Bartlett and Swain, 1949). Sulfur mustard has also been shown to react with nucleic acids at the N7-position of guanine (Rao et al., 2002).

2.2.7 CS (C10H5ClN2) Both titanium dioxide and iron oxide particles (anatase TiO2 and ferrihydrite, respectively) have been explored as detoxification materials for sulfur mustard (Kleinhammes et al., 2005; Ohtani et al., 1987) and other CWAs. The titanium dioxide particles were reported to react more rapidly with the blister agent than the iron oxide particles, converting it into nontoxic products with an effectiveness of 99%. In both cases, however, the resulting reaction products were not identified. Claims have been reported that these metal oxides are more reactive against sulfur mustard than nanocrystalline aerogel-prepared magnesium oxide, AP-MgO (Stengl et al., 2005; Kopeer et al., 1999). Chloramine B oxidizes sulfur mustard to its sulfoxide via an intermediate containing a sulfur–nitrogen bond (Whitman, 1995b):

Other Names and Designations: 2-chlorobenzylidene malononitrile; o-chlorobenzylidene-malonitrile; [(2-chlorophenyl)methylene]propanedinitrile; β,β-dicyano-o-chlorostyrene; NATO code CS; CAS# 2698-41-1. General Remarks: CS is a tear agent and is used today exclusively as a riot-control agent. CS gas is an aerosol of a volatile solvent (a substance that dissolves other active substances and easily evaporates) and 2-chlorobenzalmalononitrile, which is a solid compound at room temperature. It was first synthesized by two American chemists, Ben Corson and Roger Stoughton (the designation CS is derived from the first letters of the scientists’ surnames) in 1928, some 10 years following the end of World War I. CS was developed and tested secretly by the British at Porton Down (Wiltshire, England) in the 1950s and 1960s. It was used first on test animals and subsequently on British Army volunteers. CS is one member of a group of compounds that includes, for example, 1-chloroacetophenone (CN) and 10-chloro-5,10-dihydrophenarszine (DM), both of which are illustrated in the following diagram along with CS. These agents rapidly (in seconds to minutes) induce irritation of the eyes and upper respiratory tract.

Strong bases (such as alkoxide) can dehydrochlorinate mustard and its sulfoxide and sulfone, converting one or both of their 2-chloroethyl fragments to vinyl groups:

The didehydrohalogenation of sulfur mustard has been reported to be complete within 1 min at room temperature using ionic ethylene glycol monomethyl ether, CH3OCH2CH2O –, as a base (Beaudry et  al., 1992). The ether compound is a component of the decontaminating solution known as DS2, a mixture of diethylene triamine, ethylene glycol monomethyl ether, and sodium hydroxide. Nanosize particles of calcium oxide have also been shown to dehydrohalogenate sulfur mustard (Wagner et al., 2000b). Although anhydrous sulfur mustard is not a substantial corrosion threat to most metals, its hydrolysis forms hydrochloric acid, which does contribute the corrosive behavior of sulfur mustard.

Physical Properties: CS is a white solid with a molecular mass of 188.6 g/mol, a mp of 95°C, a bp of 313°C, and a vapor pressure at 20°C of ~3.5 × 10 –5 mm/Hg. It has a relatively low water solubility of 52 mg/L at 25°C but is soluble in typical polar organic solvents. Chemistry: The Corson and Stoughton (1928) synthesis of CS involved the condensation of a starting aldehyde with malononitrile and was catalyzed with base. Even uncatalyzed reactions were eventually successful although significantly slower. The following sequence is written to illustrate hydrolysis, but the reverse represents the (uncatalyzed) synthesis of CS and similar α,β-unsaturated dinitriles (Corson and Stoughton, 1928).

27

Chemistries of Chemical Warfare Agents Unlike simple alkenes, CS has a rather rapid rate of hydrolysis (15 min half-life at 25°C), and this is accelerated by the use of dilute, aqueous sodium hydroxide (Brooks et  al., 1976). Aqueous potassium permanganate oxidizes CS and similar α,βunsaturated dinitriles to the corresponding benzoic acids. The thermal decomposition of CS investigated at 300, 500, 700, and 900°C has been reported to produce 14 products, including quinoline, o-dicyanobenzene, 2-chlorobenzaldehyde, 2,2-dicyano 3-(2-chlorophenyl)oxirane, and 2-chlorocinnamonitrile (Wils and Hulst, 1985; Kluchinsky et al., 2002). The higher temperatures produced a greater diversity of decomposition products. Finally, human exposure to CS has been reported to result in an increased excretion of thiocyanate. Along with other data, this suggests that cyanide is released after CS intraperitoneal administration (Frankenberg and Sorbo, 1973).

2.3 Nerve Agents The “nerve gas” era (Black and Harrison, 1996b; Carlsen, 2005) in chemical warfare is rooted deeply in attempts to develop improved insecticides (Ecobichon, 1991b). The resulting organophosphate (OP) compounds have a central phosphorus atom bonded to three heteroatoms, and in each, the tetrahedral phosphorus atom is surrounded by four different groups. Organophosphates used as nerve agents can be divided into two general types: the V and G agents. The common G agents (tabun or GA, sarin or GB, and soman or GD) were developed shortly before or during World War II and possess two oxygen atoms bonded to their central phosphorus atom. Chemically, tabun has a cyano group bonded to its central phosphorus atom, whereas sarin and soman both have a fluorine atom bonded to this phosphorus atom. Their development in Germany led to their being referred to as G agents. The V agent VX was developed in the early 1950s and contains two oxygen atoms bonded to its central phosphorus atom as well as the sulfur atom of its phosphonothiolate group. VX is less volatile and hence more persistent than are the G agents. Among the four nerve agents under discussion, the general order of volatility is sarin > soman > tabun > VX. Most of the recent investigations into the chemistry of nerve agents has focused on their routes to decomposition, decontamination, and destruction (Yang, 1995; Amitai et al., 2006), which has led to the development of new chemical analysis techniques for the various nerve agents (Carrick et  al., 2001; Cody et  al., 2005). Tabun, sarin, and VX exist as a pair of enantiomers; and with two stereogenic centers, soman exists as four stereoisomers. As expected, these sets of stereoisomers have different biological activities. Rate constants for the inhibition of acetylcholinesterase (AChE) enzymatic activity with different stereoisomers of soman, sarin, tabun, and VX have been summarized (Benschop and De Jong, 1988). Nerve agents inhibit AChE by forming an adduct with the enzyme via a serine residue in the enzyme’s active site. The adducts may decompose hydrolytically or, for example, by the action of some oxime compounds, thereby regenerating the enzyme’s native activity. A second reaction type, one in which the enzyme–OP complex undergoes a subsequent reaction, is

usually described as aging (Bencsura et  al., 1995). Once the enzyme–OP complex has aged, it can no longer be regenerated by oxime reactivators. The rate of aging depends on the specific OP, with soman aging extremely rapidly, usually on the order of a few minutes (Berry and Davies, 1966). Due to the implications of treating nerve agent exposure, there have been numerous studies of this aging process (Coult et al., 1966; Bucht and Puu, 1984; Curtil and Masson, 1993; Segall et al., 1993; Shafferman et al., 1997; Kovach, 2004; Worek et al., 2004).

2.3.1 Tabun (C5H11N2O2P) Other Names and Designations: O-ethyl N,N-dimethylphosph­ oramidocyanidate; ethyl N,N-dimethylphosphoramidocyanidate; Le-100; N-Stoff (along with other compounds); Trilon-83; Taboon A (German); EA 1205 (U.S. Army); NATO code GA; CAS# 77-81-6. General Remarks: Tabun was the first compound ever identified as a nerve agent and was discovered by accident in January 1936 by the German chemist Gerhard Schrader. The chemist was experimenting with OPs to create effective insecticides for I.G. Farben (Elberfeld, Germany), a German chemical and pharmaceutical conglomerate. Research demonstrated that in addition to being a potent insecticide, tabun was enormously toxic to humans. A chemical plant for the manufacture of tabun was established in 1939 in Dyhernfurth, Germany (now Brzeg Dolny, Poland), with production beginning in June 1942 (Borkin, 1997) for use in World War II. The plant was captured by Soviet forces near the end of the war, and the Soviet government had the plant dismantled and moved to the Soviet Union soon after. Physical Properties: Tabun is a colorless compound with a somewhat fruity odor that changes with its decomposition. An odor of cyanide (bitter almonds) is apparent with minimal tabun decomposition, whereas a clear dimethylamine (dead fish) odor is apparent with greater decomposition. The molecular mass of Tabun is 162.13 g/mol, and the compound’s vapor pressure is ~0.04 mm Hg at 20°C, the lowest of all the G agents, revealing that tabun could be a relatively persistent threat at lower temperatures. Tabun has a vapor density of 5.6 relative to air, a liquid density of 1.08 g/mL at 25°C, and a mp and bp of −50 and ~240°C, respectively. The compound is three times as soluble in water (~10% at 20°C) as is VX and is also soluble in typical organic solvents (such as ethanol, diethyl ether, and chloroform). As with other nerve agents, the dissolution of tabun into inert solvents such as diethyl ether enhances its stability. Chemistry: Several synthetic routes to tabun have been reported, including one starting from phosphorus trichloride that incorporates the Arbuzov reaction (Kosolapoff, 1950). The acidic hydrolysis of tabun initially produces C2H5OP(OH)(O)CN and dimethylamine (Benes, 1963) and is generally followed by the loss of the cyanide group. The final step is the loss of the ethoxide group fragment, generating phosphoric acid as the ultimate hydrolysis product. Tabun has a half-life of ~7.0 h at pH 4–5, which increases to ~8.5 h at pH 7. The base-induced hydrolysis (and direct water hydrolysis) of tabun has also been investigated (Larsson, 1958a; D’Agostino and Provost, 1992; Sanchez et al., 1993; McNaughton and Brewer, 1994). In contrast to acidic conditions, where the phosphorus–nitrogen bond is cleaved early, the phosphorus–carbon bond is cleaved early under basic conditions.

28 Larsson (1952) reported a spectrophotometric study of tabun hydrolysis as well as a base-induced hydrolysis of the nerve agent (Larsson, 1958a) and estimated a ~1.5 min half-life for tabun at pH 11 and 25°C. Tabun hydrolysis is less than 5% complete after 20 h at pH 8.5. Depending on conditions (pH, reaction times, etc.), tabun hydrolysis products may include cyanide ion, hydrocyanic acid, the monoethyl ester of dimethylphosphoramidic acid, ethanol, dimethylamine, and phosphoric acid. Molecularly, tabun is structured around a stereogenic chiral phosphorus atom and exists as a pair of enantiomers. A gas chromatographic study of these enantiomers using bis[(1R)3-(heptafluorobutyryl)-camphorate]nickel(II) to ensure separation has been reported (Degenhardt et al., 1986); this approach also separated stereoisomers of both sarin and soman. These authors also reported the stereospecific hydrolysis of racemic tabun using phosphorylphosphatases and noted a species (mouse, rat, horse) dependence for the hydrolysis. Dilute solutions of tabun in inert solvents (carbon tetrachloride, for example) exhibit optical stability for months at −25°C. Optically active chemical shift reagents have aided in the assignment of enantiomer signals in 1H NMR spectra of tabun and other nerve agents (Van Den Berg et  al., 1984), and two-dimensional 1H-31P NMR spectroscopy has been used to identify tabun in environmental samples (Albaret et al., 1997).

2.3.2 Sarin (C4H10 FO2P) Other Names and Designations: isopropoxymethylphosphoryl fluoride; isopropylmethylphosphonofluoridate; methylphosphonofluoridic acid, 1-methylethyl ester; Trilon-46; N-Stoff (along with other compounds); T-144; EA 1208 (U.S. Army); NATO code GB; CAS# 107-44-8. General Remarks: Like tabun, sarin was first prepared by Schrader’s group at I.G. Farben in an effort to produce improved insecticides. Initially reported in 1938, it was the most toxic G series nerve agent at that time. In mid-1939, the formula for the agent was passed on to the chemical warfare section of the German Army Weapons Office, which led to an order that sarin be brought into mass production for wartime use. Pilot plants were built, and a high-production facility was under construction, but never finished, by the end of World War II. Physical Properties: Sarin is a colorless, odorless liquid with a molecular mass of 140.1 g/mol, a density of ~1.1 g/mL at 20°C, a mp of −57°C, and a bp of ~147°C. With a vapor pressure of 2.10 mm Hg at 20°C, sarin is the most volatile of the G agents. It has been reported that sarin will evaporate from a sandy surface in ~2 h at 10°C (Sidell et al., 1998). Chemistry: Two early synthetic routes to high-purity sarin preparations were reported by Bryant et al. (1960), one starting from diisopropyl methylphosphonate, CH3P(O)(OCH(CH3))2, and a second from methylphosphonic dichloride, CH3P(O)Cl2, and 2-propanol, CH3CH(OH)CH3. A more recently reported preparative route involves the reaction of a tetraalkoxysilane, (RO)4Si, as a source of the alkoxy fragment in the nerve agent (Black and Harrison, 1996a). At least one synthesis of radioactive 32P sarin has been reported (Reesor et al., 1960), and optically active sarin has been prepared using the enantiomeric sodium salts of O-isopropyl methylphosphonothioic acid. Optically active sarin racemizes in  pH > 10, but in contrast, phosphorus–oxygen bond cleavage is substantial at all other pH values. The former bond cleavage produces 2-mercapto (N,N-diisopropyl)ethylamine and ethyl methylphosphonic acid, whereas the latter generates S-(2-diisopropylaminoethyl) methyl phosphonothioate (Munro et al., 1999), a compound that is nearly as toxic as VX itself and referred to as EA 2192, and ethanol. Cu(II) does not significantly catalyze the hydrolysis of VX (Albizo and Ward, 1988), even though it has been suggested to catalyze the hydrolysis of both thiophosphoric esters and methylphosphonofluoridates (Ketelaar et al., 1956). The hydrolysis of VX with AP-MgO has also been investigated at room temperature for use in CWA decontamination (Wagner et al., 1999). The authors reported that the hydrolysis proceeded exclusively by cleavage at the phosphorus–sulfur bond and, therefore, did not

30 generate the toxic EA 2192 product. VX and other nerve agents typically hydrolyze over time (Yang et al., 1992), which has led to their stabilization for long-term storage by water removal (Henderson, 2002). Although not as extensively-investigated as nerve agent hydrolysis, oxidation is also an important mechanism for nerve agent destruction. For VX in particular, oxidation with gaseous ozone has been shown to lead to a wide variety of products. In general, the most reasonable sites for the initial oxidation of VX are its sulfur or nitrogen atom sites, the former yielding a sulfoxide and the latter affording an amine oxide. Oxidation in polar solvents enhances S-oxide formation, at least in part by stabilizing the adjacent dipolar (sulfur–oxygen and phosphorus–oxygen) bonds. It has been reported that VX oxidizes to “VX N-oxide” before the subsequent oxidation or hydrolysis to O-ethyl methylphosphonate using a variety of oxidants (Cassagne et al., 2001).

2.4 Incapacitating Agents The term incapacitating agent is defined by the U.S. Department of Defense (DOD) as any agent that produces temporary physiological or mental effects, or both, that render individuals incapable of performing their assigned duties. The idea of using chemicals to induce altered states of mind in an adversary dates back to antiquity and includes the use of plants of the nightshade family (Solanaceae), such as the thornapple (Datura stramonium), that contain various combinations of anticholinergic alkaloids. As early as 184 BC, Hannibal’s army used belladonna plants to induce disorientation among enemy troops. In 1672, the Bishop of Muenster used belladonna-containing grenades in his campaigns. And ~300 years later, during the late 1950s and the 1960s, the U.S. Army explored several classes of drugs, as well as noise, microwaves, and photostimulation, and found none to be as promising for use as incapacitating agents as anticholinergic compounds. Stimulants such as cocaine, amphetamines, and nicotine were also tested but did not have the potency to be an airborne threat. Depressants such as barbiturates, opiates, and neuroleptics were similarly found to be impractical for battlefield use. The unpredictable behavior provoked by psychedelic agents, including lysergic acid diethylamide, LSD, and phencyclidine, PCP, led to an early termination of the testing of this particular class of drugs. By the mid-1960s, after a decade of tests, the U.S. Army concluded that the compound BZ was the best candidate for weaponization and deployment. BZ and fentanyl are likely the best understood compounds for use as incapacitating agents.

2.4.1 BZ (C21H23NO3) Other Names and Designations: QNB; 3-quinuclidyl benzilate; benzilic acid, 3-quinuclidinyl ester; 1-azabicyclo[2.2.2]oct-3-yl 2-hydroxy-2,2-diphenylacetate; Substance 78 (Soviet code); EA 2277 (U.S. Army); NATO code BZ; CAS# 6581-06-2. General Remarks: BZ is a synthetic glycolic ester incapacitating agent. Like other such agents, its purpose is to produce temporary disability but not death (Wiener and Nelson, 2004). Indeed, the onset of symptoms may not appear until several hours after exposure. BZ was invented by the Swiss pharmaceutical

Chemical Warfare Agents company F. Hoffmann-La Roche AG (Basel, Switzerland) in 1951 during efforts to develop anti-spasmodic agents for treating gastrointestinal conditions. Interest in the compound for use as a potential CWA almost immediately followed a screening program conducted at the University of Chicago College of Medicine (Chicago, IL; now the Pritzker School of Medicine) and ultimately led to its standardization for use in chemical munitions by the U.S. DOD in 1961. Physical Properties: BZ has a molecular mass of 337.41 g/mol, and with one chiral center, exists as a pair of enantiomers. The racemic mixture is a white solid with a mp of 164–166°C (Whitaker, 1966). The compound also has a low vapor pressure and in the environment, has an estimated half-life of ~1 month at 25°C. BZ has low solubility in water; however, protonation of its nitrogen atom enhances solubility in dilute, aqueous acids, and the compound is soluble in polar organic solvents. With a flash point of 246°C, it has significant thermal stability, but it is slowly hydrolyzed in refluxing aqueous solutions. A single crystal X-ray analysis of the hydrobromide salt has been reported (Meyerhoeffer and Carlstroem, 1969). Chemistry: The transesterification of methyl benzilate with 3-quinuclidinol provides a convenient route to BZ:

Like most bioactive substances that exist as enantiomeric pairs, the BZ enantiomers have different magnitudes of biological activity. The individual enantiomers have been prepared by starting with the enantiomers of 3-quinuclidinol (Rzeszotarski et al., 1988). The R enantiomer exhibits a higher affinity for the muscarinic receptor than does the S enantiomer in the guinea pig ileum (Lambrecht, 1979). Both 18O- and 11C-labeled analogs of BZ have been synthesized (Calvin et al., 1949). The source of the 11C-label was carbon dioxide (Prenant et  al., 1989); however, the 18O-label was introduced as a result of reacting enriched water with quinuclidinone and then reducing the labeled ketone with borohydride (Sniegoski et al., 1989). The synthesis of a number of tritiumlabeled “neurochemicals,” including BZ, has been reported (Bloxsidge et al., 1981). The ester hydrolysis of BZ over a range of pH and temperature conditions has been investigated (Hull et al., 1979). Bromocresol green forms a colored complex with 3-quinuclidinyl esters of hydroxyacetic acids, which has been used in the spectrophotometric analysis of mixtures containing the esters and 3-quinuclidinol (Stan’kopv et al., 1997). The mass spectra for a number of quinuclidine derivatives are available (Vincze et al., 1980). BZ has also been destroyed safely by pyrolysis (Jensen, 1991).

2.4.2 Fentanyl (C22H28N2O) Other Names and Designations: China White; Synthetic Heroin; Drop Dead; Flatline; Lethal Injection; Apache; China Girl; Chinatown; Dance Fever; Great Bear; Poison;

Chemistries of Chemical Warfare Agents Tango & Cash; TNT; N-(1-(2-phenylethyl)-4-piperidinyl)-Nphenylpropanamide; CAS# 437-38-7. General Remarks: Fentanyl was first prepared by Paul Janssen in 1959 under a patent held by his company, Janssen Pharmaceutica (Beerse, Belgium; now part of Johnson & Johnson Pharmaceutical Research and Development, Titusville, NJ). Research into fentanyl as an incapacitating agent began in the 1960s, and by the 1980s, U.S. researchers were testing primates with aerosolized carfentanil, a compound related to fentanyl that is substantially more toxic. Different fentanyl derivatives have been developed by the legitimate pharmaceutical industry by adding various substituents to the fentanyl molecule to modify its potency. This approach has been mimicked by chemists in clandestine laboratories to produce illicit non-pharmaceutical fentanyl derivatives. Physical Properties: Pure preparations of fentanyl and its salts appear as white or off-white granular and crystalline powders. Fentanyl has a molecular mass of 336.471 g/mol, a density of 1.087 g/mL, a flash point of 186°C, and a reported experimental mp of ~86°C. Other physical properties of fentanyl were derived from a thermogravimetric analysis (TGA) of the compound in its free base form (Gupta et al., 2008). Benzoic acid was used as a reference material to calibrate the TGA instrument, and the vapor pressure of fentanyl was determined at different temperatures between 150 and 220°C. Extrapolation of the measured data to 25°C gave a vapor pressure estimate for subcooled fentanyl of (4.6 ± 2.7) × 10 −6 Pa, with the corresponding solid phase vapor pressure calculated at (5.9 ± 4.7) × 10 −7 Pa after taking the enthalpy of fusion into consideration. Both the enthalpy of sublimation at 25°C and the normal bp of fentanyl were also estimated from these data and reported to be 144.6 ± 2.7 kJ/mol and 390.7 ± 4.7°C, respectively. The solubility of fentanyl in water has been found to be very dependent on pH, especially under acidic conditions. At pH 5.5, for example, fentanyl solubility is 75.13 mg/mL at 35°C, and it drops rapidly to 0.74 mg/mL at pH ~7.0 at this same temperature (Roy and Flynn, 1989); this pH dependence is essentially obliterated at pH > 9, where solubility for the compound is ~0.010 mg/mL. The pK A value of fentanyl’s piperidine nitrogen atom has been reported at 8.43 (Meuldermans et al., 1982) and 8.99 (Roy and Flynn, 1989) for temperatures of 25 and 35°C, respectively. Fentanyl is usually very soluble in alcohol solvents such as methanol and ethanol. Chemistry: Fentanyl was originally synthesized from an N-benzyl-4-piperidone precursor and aniline in the presence of p-toluenesulfonic acid (pTsOH) by Janssen Pharmaceutical (Janssen, 1964; Janssen, 1965; Casey et al., 1969):

31 In the 1980s, however, a new synthetic route became available based on the reaction of 4-piperidone hydrochloride with phenethyl bromide via phase-transfer catalysis (PTC) to give N-phenethyl-4-piperidone (NPP), which is then converted to fentanyl in three additional reactions (Zee et al., 1981):

This route is known as the Siegfried method and uses either phenethyl-tosylate or phenethyl bromide to make the NPP precursor; details of the method are easily found on the Internet. Other, less well-known synthetic routes to fentanyl have been outlined by Hsu and Banks (1992). Recently, there has been some interest in the products produced by the thermal decomposition of drugs, including fentanyl (Lambropoulos et al., 1999; Nishikawa et al., 2009; Garg et al., 2010; Wichitnithad et al., 2010; Lindsay et al., 2016). The most common thermal decomposition products for fentanyl include N-phenylpropanamide and styrene, with small amounts of norfentanyl. As an example, the products derived from the analytical pyrolysis of fentanyl hydrochloride using a pyroprobe system under aerobic and anaerobic conditions (Nishikawa et al., 2009) are presented in the following figure:

32 The pyroprobe system reached a maximum temperature of 750°C, and average residence times in the system were 10 s. The pyrolysis of fentanyl hydrochloride generates hydrochloride vapor, which reacts with fentanyl to generate benzyl chloride and phenethyl chloride under aerobic conditions and phenethyl chloride under anaerobic conditions. Other products found under both conditions include high abundances of N-phenylpropanamide, with much smaller abundancies of styrene, pyridine, benzaldehyde, and norfentanyl.

2.5 Concluding Remarks While the use of disabling chemicals as weapons dates back thousands of years, the indisputable dawn of modern chemical warfare was during World War I. The large-scale development and manufacture of toxic chemicals explicitly for use as CWAs was an integral part of the war effort for both Central and Allied Power nations. The major distinguishing characteristics of these CWAs were a very low molecular mass and high volatility. Most of the CWAs were also very reactive in general, which is why several are still produced today in large quantities for use in the commercial manufacture of other compounds. In the United States today, for example, phosgene has an annual production of ~5400 million pounds, and the annual production of hydrogen cyanide is >1 billion pounds for use in the commercial production of nylon 66 and other compounds. It is highly likely that a number of World War I–era compounds will be found in commercial settings well into the future. During World War II, efforts in the development and manufacture of CWAs were refocused sharply on the nerve agents. These compounds are significantly higher in molecular mass than their World War I–era counterparts, and their reactivities were targeted to only one particular purpose; the effective inhibition of the AChE enzyme. Nerve agents have no practical commercial applications, and essentially all of their reported chemistries concern their synthetic routes and routes to their decomposition, decontamination, and destruction (Yang, 1995; Yang, 1999; Amitai et al., 2006). Nerve agents are far too lethal for use in any civilian application; however, closely related OP compounds that are significantly less effective at inhibiting AChE are commonly used today as insecticides. In the ~15 years following World War II, the search by nations on both sides of the Iron Curtain to find an efficient means to temporarily disable adversary troops ultimately led to the selection of anticholinergic compounds as incapacitating agents. In comparison with other CWAs, anticholinergic incapacitating agents such as BZ and fentanyl are considerably higher in molecular mass and structural complexity. Incapacitating agents are best categorized as pharmaceutical compounds, and accordingly, their synthetic routes constitute the vast majority of their reported chemistries. For the specific case of fentanyl, however, there have been recent reports on the products produced by its thermal decomposition (Lambropoulos et al., 1999; Nishikawa et al., 2009; Garg et  al., 2010; Wichitnithad et  al., 2010; Lindsay et  al., 2016). Fentanyl is increasingly becoming a choice for drug abuse, and the recent investigations into its thermal decomposition products result from efforts to identify biomarkers associated with

Chemical Warfare Agents smoking fentanyl powders and fentanyl transdermal patches (Nishikawa et al., 2009). Due to this unfortunate situation, fentanyl has become the most commonly encountered CWA by the civilian population.

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Chemical Warfare Agents Rao, C. N. R. (1963). Chemical Applications of Infrared Spec­ troscopy. New York: Academic Press: 360–361. Rao, M. K.; Bhadury, P. S.; Sharma, M.; Dangi, R. S.; Bhaskar, A. S. B.; Raza, S. K.; Jaiswai, D. K. (2002). A facile methodology for the synthesis and detection of N7-guanine adduct of sulfur mustard as a biomarker. Canadian Journal of Chemistry, 80: 504–509. Reddy, K. L. (2003). An efficient method for the conversion of aromatic and aliphatic nitriles to the corresponding N-tert-butyl amides: A modified Ritter reaction. Tetrahedron Letters, 44: 1453–1455. Redeman, C. E.; Chaikin, S. W.; Fearing, R. B. (1948). The vapor pressure of eleven organic compounds. Journal of the American Chemical Society, 70: 2582–2583. Reesor, J. B.; Perry, B. J.; Sherlock, E. (1960). The synthesis of highly radioactive methylphosphonofluoridate (sarin) containing 32P as a tracer element. Canadian Journal of Chemistry, 38: 1416–1427. Richter, D. (1992). Chemical Soldiers—British Gas Warfare in World War I. Lawrence: University of Kansas Press. Rinderknecht, H.; Gutenstein, M. (1967). Nicotinic anhydride. Organic Synthesis, 47: 89. Robertson, C. R. (1937). Laboratory Practice of Organic Chemistry. New York: The Macmillan Co.: 270–271. Roy, S. D.; Flynn, G. L. (1989). Solubility behavior of narcotic analgesics in aqueous media: Solubilities and dissociation constants of morphine, fentanyl, and sufentanil. Pharmaceutical Research, 6: 147–151. Ryan, T. A.; Ryan, C.; Seddon, E. A.; Seddon, K. R. (1996). Phosgene and Related Carbonyl Compounds. New York: Elsevier. Rzeszotarski, W. J.; McPherson, D. W.; Ferkanyi, J. W.; Kinnier, W. J.; Noronha-Blob, L.; Kirkien-Rzeszotarski, A. J. (1988). Affinity and selectivity of the optical isomers of 3-quiniclidyl benzilate and related muscarinic antagonists. Journal of Medicinal Chemistry, 31: 1463–1466. Sanchez, M. L.; Russell, C. R.; Randolph, C. L. (1993). Chemical Weapons Convention Signature Analysis, DNA-TR 92–73ADB171788. Alexandria, VA: Defense Technical Information Center. Sandler, S. R.; Karo, W. (1983). Organic Functional Group Preparations, Vol. 12-I. New York: Academic Press: 415–417. Sartori, M. (1939). The War Gases. New York: D. Van Nostrand: 216. Schofield, D.; Tsang, W.; Bauer, S. H. (1965). Thermal decomposition of ClCN. Journal of Chemical Physics, 42: 2132–2138. Segall, Y.; Waysbort, D.; Barak, N.; Ariel, N.; Doctor, B. P.; Grunwald, J.; Ashani, Y. (1993). Direct observation and elucidation of the structures of aged and nonaged phosphorylated cholinesterases by 31P NMR spectroscopy. Biochemistry, 32: 13441–13450. Shafferman, A.; Ordentlich, A.; Barak, D.; Stein, D.; Ariel, N.; Velan, B. (1997). Aging of somanylacetylcholinesterase adducts: Facts and models. Biochemical Journal, 324: 996–998. Sidell, F. R.; Patrick, W. C.; Dashiell, T. R. (1998). Jane’s Chem-Bio Handbook. Alexandria, VA: Jane’s Information Group: 92. Simandi, L.; Soos, J. (1986). Preparation of chloropicrin by reaction of 4-hydroxy-3,5-dinitrobenzoates with hypochlorites. HU Patent 1986–3814. Smolin, E. M. (1955). Reactions of cyanogen chloride. II. Amines from cyanogen chloride and olefins. Journal of Organic Chemistry, 20: 295–301.

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3 Toxicokinetics of Nerve Agents Marcel J. van der Schans, Hendrik P. Benschop,* and Christopher E. Whalley CONTENTS 3.1 Introduction...................................................................................................................................................................................39 3.2 Nerve Agent Stereo-Isomers: Chiral Analysis..............................................................................................................................40 3.3 Experimental.................................................................................................................................................................................40 3.4 Intravenous Toxicokinetics of Soman, Sarin, and VX.................................................................................................................42 3.4.1 Soman...............................................................................................................................................................................42 3.4.2 Sarin.................................................................................................................................................................................42 3.4.3 VX....................................................................................................................................................................................43 3.5 Percutaneous Toxicokinetics of VX..............................................................................................................................................48 3.6 Elimination Pathways of Soman, Sarin, and VX..........................................................................................................................49 3.7 Effect of HuBuChE as a Scavenger on the Toxicokinetics of Nerve Agents................................................................................50 3.8 Concluding Remarks.....................................................................................................................................................................55 References...............................................................................................................................................................................................55

3.1 Introduction Toxicokinetic studies, together with toxicodynamic studies of nerve agents, provide a quantitative basis for the design of new strategies against intoxication with nerve agents. There is a long tradition of investigations on the toxicodynamics of nerve agents since their introduction as potential agents of chemical warfare during World War II. These studies have led to strategies in which phosphylated cholinesterase is reactivated with oximes, often in combination with the administration of a central nervous depressant to suppress convulsions and other central effects, and through administration of the muscarinic cholinergic antagonist atropine. Toxicokinetic studies were initiated in the last two decades of the last century. The reasons for the late development were twofold. First, it was assumed that nerve agents react so quickly and are so rapidly degraded that it is impossible to measure these low levels of agent. Second, the expected levels were so low that the analytical capability was not sufficient to detect the nerve agent at relevant levels. With the advent of sophisticated sensitive analytical capabilities using capillary gas chromatography combined with sensitive detectors such as nitrogen phosphorus detection (NPD), flame photometric detection (FPD), and mass selective detection (MSD), it is possible to measure these extremely low levels of nerve agent. Moreover, there was reason to believe that the nerve agents might be more persistent than anticipated. Wolthuis et al. (1981) showed in 1982 that rats initially surviving a challenge with a supra-lethal dose of soman by immediate treatment with atropine and the oxime HI-6 became fatally re-intoxicated 4–6 h later. Hence, soman appeared to

be far more persistent than anticipated. The persistence is even more pronounced in the case of percutaneous VX intoxication, which is caused by slow penetration through the skin and slow enzymatic hydrolysis. Especially in the case of intoxication with VX, toxicokinetic studies are of ultimate importance, because the time period of the intoxication might span several hours, which means that the timing of the antidote administration has to be adapted to the toxicokinetic process. The toxicokinetics of soman and sarin have been thoroughly described by Benschop and De Jong in the first version of this book, Chemical Warfare Agents: Toxicity at Low Levels (Benschop and De Jong, 2001). In the second version of this series, Chemical Warfare Agents: Chemistry, Pharmacology, Toxicology and Therapeutics (Van der Schans et al., 2008), the intravenous (i.v.) toxicokinetics of soman and sarin were presented again together with the toxicokinetics of VX after i.v. administration and percutaneous application. Additionally, the effects of human butyrylcholinesterase (HuBuChE) as a scavenger on the toxicokinetics of nerve agents and the distribution of sarin among tissues were mentioned as a rationale for the elimination pathways of nerve agents. This chapter will present the same subjects as in the second edition, updated with the latest results on the toxicokinetics of VX, mainly obtained after improvement of the analytical methodology. The discussion of the toxicokinetics will be preceded by a section discussing the importance of distinguishing the stereo-isomers of nerve agents. Readers who are interested in the inhalation toxicokinetics of sarin and soman are referred to Chapter 2 in the first edition of the abovementioned CWA book in this series.

* H. P. Benschop passed away in 2013, and he did not review this updated chapter.

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3.2 Nerve Agent Stereo-Isomers: Chiral Analysis The interpretation and understanding of the toxicokinetics of nerve agents would not be possible without taking into consideration that these agents consist of mixtures of stereo-isomers, which are often extremely different in their toxicokinetic and toxicodynamic properties (Benschop and De Jong, 1988). A common feature of these agents is the chirality on the phosphorus atom. (±)-sarin, C(±)P(±)-soman, and (±)-VX consist of an equimolar mixture of P(−)- and P(+)-stereo-isomers. In the case of sarin and soman, the P(−)-stereo-isomers appeared to be far more toxic and persistent in vivo than the P(+)-stereoisomers. In contrast, the differences in rates of inhibition of AChE and lethality between the stereo-isomers of VX are only moderate. In addition, soman consists of four stereo-isomers, denoted as C(+)P(+), C(+)P(−), C(−)P(−), and C(−)P(+), in which C stands for asymmetry in the pinacolyl moiety and P for asymmetry at the phosphorus atom. The enantiomeric pairs C(+)P(+) + C(−)P(−) and C(+)P(−) + C(−)P(+) are present in synthetic C(±)P(±)-soman in a ratio of 45:55, with equal amounts of the two enantiomers within each pair. The separation of the various stereo-isomers became feasible with optically active stationary phases in chromatography. The four isomers of soman can be separated on a ChiraSil-Val column and the isomers of sarin on a Cyclodex column, whereas the isomers of VX can be separated by high-performance liquid chromatography (HPLC) using a Chiracel OD-H column (Benschop et al., 1981; Kientz et al., 1994; Spruit et al., 2001). With the analytical methodology available, it is possible to monitor the progress of the isolation and purification of the stereo-isomers. In the case of soman, synthetic resolution of the pinacolyl alcohol and subsequent synthesis of soman from these stereoisomers yielded C(+)P(±) and C(−)P(±)-soman (Benschop and De Jong, 1988). After incubation with α-chymotrypsin, which binds the P(−) isomers of soman, the P(+) isomers could be isolated. Incubation with rabbit plasma hydrolyzes the C(±)P(+) isomers and therefore, yields the C(±)P(−)-isomers. Similarly, the incubation of sarin with rabbit plasma gave (−)-sarin. The two stereo-isomers of VX are easily obtained synthetically from optically resolved precursors (Benschop, 1975; MehlsenSorensen, 1977). With sufficient amounts of the stereo-isomers available, it is possible to study the affinity for binding with AChE, the rate of hydrolysis, and finally, the lethality of the different isomers (Boter and Van Dijk, 1969; De Jong and Benschop, 1988; Hall et  al., 1977). As expected, the degree of lethality correlated with the binding constant of AChE (see Table 3.1). Apparently, the P(−)-isomers of soman and sarin inhibit AChE with rate constants that are three or four orders of magnitude higher than those of the corresponding P(+)isomers. The determination of the binding constant of the P(+) isomers requires a high concentration of the inhibitor, while it cannot be excluded that small impurities of the P(−) isomer bias the bimolecular reaction. In contrast to sarin and soman, the binding constant of the P(−) isomer of VX is only two orders of magnitude higher than that of the P(+)-isomers. In this case, the LD50 of both isomers could be determined, and it was verified that the P(−) isomer is only eight times more toxic than the P(+)-isomer. These data make clear that it is essential

Chemical Warfare Agents to distinguish the isomers of the nerve agents from each other in toxicokinetic studies, because one is mostly interested in the elimination rate of the toxic compound.

3.3 Experimental Toxicokinetic studies are only interesting if the agents can be determined at levels that are toxicologically relevant. The relevant levels should be related to the binding constant of the agent with AChE. Since nerve agents inhibit AChE with rates up to 108/M/min, it can be derived from this that blood levels down to a few picograms per milliliter can still cause a significant inhibition over a period of hours. Only the most sophisticated gas chromatographic techniques combined with sensitive detection methods such as NPD, FPD, or mass spectrometry (MS) are sensitive enough to fulfill this task. In the period of time when the toxicokinetic studies mentioned in this chapter were performed, only the NPD detector was available (Benschop et  al., 1985; Spruit et  al., 2001). Nowadays, organophosphofluoridates are analyzed by means of mass spectrometry with chemical ionization using ammonia as a reaction gas (Degenhardt et al., 2004; Jakubowski et al., 2004). This ionization mode is efficient for ionization of organophosphofluoridates but also more selective than electron impact ionization, since it is a softer ionization mode. As mentioned in the previous section, it is also essential that the isomers can be distinguished from each other, in view of the extreme difference in toxicity of the isomers. The combination of chirally sensitive analysis was realized by two-dimensional gas chromatography in combination with large volume sample introduction (up to 400 µl) by means of thermal desorption. This allows the detection of low concentrations of nerve agent, that is, in the picograms per milliliter range. The two-dimensional chromatography used the heart cutting technique. The configuration comprises two GC-columns, a pre-column and a second analytical chiral column, which are connected in series. The sample is introduced on the pre-column. After separation on the precolumn, a small fraction of the effluent is injected on the second analytical chiral column. The reason for using two-dimensional chromatography is twofold. First, the combination of two stationary phases creates additional selectivity, which is required for the detection of nerve agents at trace levels in biological samples. Second, the configuration preserves the chiral column, which has a fragile stationary phase that deteriorates with the formation of liquid phases on the column wall and the elution of interfering matrix components. With this configuration, it was possible to detect amounts of soman and sarin down to 1–5 pg while separating all isomers of sarin and soman (Benschop and De Jong, 1987, 1990, 1993). The separation of the VX isomers was successful using HPLC on a Chiracel OD-H column with a mixture of n-hexane:ethanol (95:5) as a mobile phase (Kientz et  al., 1994; Van der Schans et  al., 2003). Detection was accomplished by electrochemical detection, which is possible by oxidation of VX at the sulfur or nitrogen atom. The mobile phase leaving the chiral column was post-column mixed with a 0.1 M potassium perchlorate in ethanol solution to ensure conductivity, which is necessary for electrochemical detection. The method was workable for testing the optical purity of the isomers or analysis of biological samples

41

Toxicokinetics of Nerve Agents TABLE 3.1 Stereoselectivity in Binding to AChE and Acute Lethality of Nerve Agent Stereo-Isomers Rate Constant (M−1/min)a

LD50 Mouse (µg/kg)

C(+)P(−)-soman C(−)P(−)-soman C(+)P(+)-soman C(+)P(+)-soman C(±)P(±)-soman (−)-sarin

2.8 × 108 1.8 × 108 2000c 156b 41c

(+)-sarin (±)-sarin (−)-VX

10 ng/ml). The selectivity of the separation was not sufficient for the analysis of VX at lower concentrations in biological samples. As an alternative, it appeared possible to separate the isomers of VX on the Chiracel OD-H column and fractionate the VX isomers (Van der Schans et al., 2003). Next, the collected samples were off-line analyzed with GC-NPD. VX itself can be analyzed with GC-NPD using a rather straightforward technique (Bonierbale et al., 1996). Because of the low volatility of VX, the sample can be concentrated in a vial with a gentle stream of nitrogen. Typically, a volume of 8 µl VX in hexane can be injected onto the column, while the detection limit of VX was down to 2 pg. The lowest concentration of VX isomers that could be detected using off-line separation of isomers and detection with GC-NPD was 1 ng/mL. Recently, the method was further improved using chiral LC-MS/MS. The combination of chiral-LC-MS had already been published by Smith (2004), albeit at relatively high concentrations of VX. In 2005, we analyzed the isomers of VX on an LC-MS/MS configuration using atmospheric pressure chemical ionization conditions. It appeared possible to analyze the two stereo-isomers down to 100 pg/mL. At the time when the toxicokinetics of VX were studied, this analytical configuration was not yet available, which means that the isomers of VX were measured using the off-line LC-GC-NPD method. In 2008, Reiter et al. (2008) published a method based on chiral LC-MS/MS using a Chiral AGP column, with which the stereoselective analysis of VX enantiomers down to 5 pg/ml could be accomplished. In the same paper, the authors claim the separation of VX enantiomers using gas chromatography with a Hydrodex-β-TBDAc column. However, that analysis took more than 280 min to ensure sufficient resolution, which made this method less suitable for routine use. The sample preparation of the nerve agent samples appeared to be a critical step as well. In vivo, the stereo-isomers of soman and sarin are subject to a rapid process of elimination by binding

to AChE, BuChE, or CaE and enzymatic hydrolysis. It is essential that these processes should be frozen once the bio-sample has been taken for the time period that is required for the further sample preparation. It appears that enzymatic hydrolysis can be sufficiently suppressed by immediate acidification of the sample using an acetate buffer at pH 4. A second essential step is the saturation of available binding sites such as AChE, BuChE, and CaE. The enzymes were blocked by addition of excess of another organophosphate, such as neopentyl sarin. Finally, it appeared that the presence of a significant quantity of fluoride ions from natural sources or from hydrolysis of the nerve agent itself results in regeneration of the organophosphofluoridate, which leads to substantially higher levels of soman or sarin in the samples (Benschop et al., 1985). This complication was effectively suppressed by the addition of aluminum sulfate, which binds the fluoride ions, mostly in the complex [AlF2]+. Next, the stabilized mixture was extracted into ethyl acetate using a solid phase extraction procedure with Nexus or Seppak C18 cartridges. Some experiments described in this chapter refer to the analysis of regenerated sarin from regenerable binding sites. In that case, aluminum sulfate is not added to the sample. On the contrary, the regeneration of sarin is accelerated by the addition of a high concentration (250 mM final concentration) of fluoride ions (Polhuijs et  al., 1997). The regenerated sarin is also extracted into ethyl acetate by solid phase extraction. Next, the sample is analyzed without the use of a chiral column: regeneration with a high concentration of fluoride ions occurs with rapid racemization of the organophosphofluoridate, which means that a chiral analysis is redundant. The isolation of (±)-VX from a biological matrix is even simpler (Van der Schans et al., 2003). The sample is first stabilized by the addition of an excess of organophosphate, for example, neopentyl sarin, made alkaline to deprotonate the nitrogen atom and extracted with a mixture of n-hexane:methanol (95:5). The

42 extract appeared to be clean enough to be analyzed directly with GC-NPD or normal phase LC-MS/MS. Additionally, preconcentration of the sample to a smaller volume was possible by evaporation of the solvent with a gentle stream of nitrogen and reconstitution of the sample in a small volume of n-hexane. Reiter et  al. (2008) used a different sample clean-up method, since they analyzed their samples with reversed phase LC-MS/ MS in an aqueous mobile system. Moreover, they performed the toxicokinetic experiments in swine, which allowed larger blood samples of 2 ml to be drawn. Therefore, the sample preparation procedure should contain a pre-concentration step to reduce the final sample volume, enabling the detection of low concentrations of agent. The method of Reiter et al. is based on protein precipitation and liquid extraction into an organic phase, followed by a second extraction step into the aqueous phase.

3.4 Intravenous Toxicokinetics of Soman, Sarin, and VX 3.4.1 Soman Initial investigations on the toxicokinetics of nerve agents were performed after i.v. administration at dosages corresponding to multiple LD50 values to obtain the toxicokinetic data at 100% bioavailability (Benschop and De Jong, 1987, 1990, 1991; Benschop and Van Helden, 1993). This data can be used in the interpretation of the results from more relevant exposure routes, such as the respiratory and percutaneous route. The animal species selected for the investigations were the rat, guinea pig, and marmoset, with the last species serving as a model for humans. To perform the experiments, the animals were anesthetized, atropinized, and mechanically ventilated through a tracheal cannula. Blood samples were drawn from a carotid cannula. Intravenous injection was performed in the dorsal penis vein or in the vena jugularis. The LD50 values of soman are highly species dependent, since the amount of CaE in blood is species dependent (Maxwell et al., 1987). These enzymes act as scavengers of nerve agents by irreversibly binding to the nerve agent. The enzyme is present in large amounts in the blood of rats, in a significant smaller amount in guinea pigs, and almost absent in the blood of marmosets and humans. Accordingly, the LD50 values decrease in order rat > guinea pig > marmoset. Blood levels of the C(+) P(−) and C(−)P(−)-isomers are shown in Figures 3.1 and 3.2. The relatively non-toxic C(±)P(+) isomers were eliminated from the bloodstream within 5 min, which is mainly caused by the rapid enzymatic hydrolysis of C(±)P(+)-soman. In contrast to the C(±) P(+)-isomers, the C(±)P(−)-isomers can be measured for several hours. Almost all curves were best described with a three-term exponential fit. Areas under the curve have been calculated using the coefficients of these equations. The toxicokinetic data of the i.v. soman experiments are summarized in Table 3.2 (See Figures 3.3 through 3.6.) The derivation of the time period during which acutely toxic levels of soman stereoisomers persist is based, somewhat arbitrarily, on a scenario of intoxication in which an animal resumes spontaneous respiration presumably due to about 5–7% reactivation by oxime (or protection by carbamate) of completely inhibited AChE in the diaphragm. Since the concentration of AChE

Chemical Warfare Agents in the diaphragm of guinea pigs is approximately 2–2.6 nM, this reactivated fraction corresponds to approximately 150–200 pM AChE. Based on a bi-molecular rate constant of AChE inhibition by soman of about 108 M−1/min, it is calculated that this reactivated fraction of AChE can be re-inhibited by 150 pM (approx. 30 pg/ml) soman with a half-life of about 1 h. A concentration of soman an order of magnitude lower can cause only insignificant inhibition. Therefore, it is assumed that 150 pM soman represents the lowest concentration having toxicological relevance. In a more generalized way, it may be reasoned that an area under the curve of 30 pg/ml × 60 = 1.8 ng/min/ml in the last part of the blood level curve is needed for toxicological relevance. The period of time between intoxication and the point on the time axis at which this area begins can be regarded as the period of time in which toxicologically relevant levels of soman are present. Table 3.2 shows that these time periods in rat, guinea pig, and marmoset range between 50 and 100 min after receiving a dose corresponding to 2 × LD50. Based on the earlier mentioned ratio of the C(+)P(−) and C(−) P(−)-isomers of soman, it would be expected that the area under the curve (AUC) of the C(+)P(−)-isomer is 20% higher than that of the C(−)P(−)-isomer. Instead, the AUC of the C(+)P(−)-isomer is often equal to or even lower than that of the C(−)P(−)-isomer. This outcome can be explained by taking into consideration the difference in binding rate constant with CaE, which is 30-fold higher for the C(+)P(−)-isomer (De Jong, 1993, 1996). The relative effect of differential binding of C(+)P(−)- and C(−)P(−)soman with CaE becomes more pronounced at lower doses of soman, because the elimination route of binding to a finite amount of CaE is relatively more important at lower doses of soman. Indeed, it was observed that the differences of the AUC between the two isomers increase with decreasing dose.

3.4.2 Sarin In comparison with soman, little work has been done so far on the toxicokinetics of sarin (Benschop and Van Helden, 1993; Spruit et  al., 2000). To obtain reference data for inhalational studies, the i.v. toxicokinetics of sarin were investigated at doses corresponding to 0.8 × LD50 (19.2 µg/kg) and 2 × LD50. Blood levels of (−)-sarin are shown in Figure 3.7. The (+)-isomer of sarin was not detectable in the blood of the guinea pigs after an i.v. bolus of a dose corresponding to 0.8 × LD50 and 2 × LD50. The half-life of distribution appears to be 0.15 min after a dose of 0.8 × LD50 and 0.98 min after 2 × LD50, whereas the terminal half-lives resulting from these doses are 58 and 389 min, respectively. The calculated AUC values are 15.3 ng/min/ml for 0.8 × LD50 and 109 ng/min/ml for 2 × LD50, indicating non-linearity of the toxicokinetics with the dose, as was also observed for 0.8 × LD50 and 2 × LD50 of C(±)P(±)-soman (Spruit et al., 2000). It can be observed that (−)-sarin is more persistent in the guinea pig than C(±)P(−)-soman at an i.v. dose corresponding to 0.8 × LD50 and 2 × LD50 (see Table 3.3). It is remarkable that the concentrations of (−)-sarin remain rather constant at a relatively low level for a long period of time. Interestingly, we have hardly ever seen the concentrations of either of the P(−)-isomers of soman to persist in the terminal phase. Tentative explanations for the leveling-off or increase of the (−)-sarin concentration in the terminal phase of the toxicokinetics are release from rather non-specific, non-covalent

43

Toxicokinetics of Nerve Agents

Concentration C(+)P(−)-GD (ng/mL)

1000

100

10

1

0.1

0.01

0

50

100

150

200

250

300

350

400

Time (min) FIGURE 3.1  Semi-logarithmic plot of the concentrations in blood of C(+)P(−)-soman (GD) versus time after i.v. administration of 0.8 × LD50 (66 µg/kg, ▴), 3 × LD50 (▪), and 6 × LD50 (♦) of C(±)P(±)-soman to anesthetized, atropinized, and mechanically ventilated rats. (From Benschop, H.P. and De Jong, L.P.A., Neurosci. Biobehav. Rev., 15, 73, 1991. With permission.)

Concentration C(−)P(−)-GD (ng/mL)

1000

100

10

1

0.1

0.01 0

50

100

150

200

250

300

350

400

450

Time (min) FIGURE 3.2  Semi-logarithmic plot of the concentrations in blood of C(−)P(−)-soman (GD) versus time after i.v. administration of 0.8 × LD50 (66 µg/kg, ▴), 3 × LD50 (▪), and 6 × LD50 (♦) of C(±)P(±)-soman to anesthetized, atropinized, and mechanically ventilated rats. (From Benschop, H.P. and De Jong, L.P.A., Neurosci. Biobehav. Rev., 15, 73, 1991. With permission.)

binding sites or reactivation from covalent binding sites induced by endogenous fluoride ions. The latter reaction proceeds more readily in the case of sarin than in the case of soman.

3.4.3 VX Data on the toxicokinetics of (±)-VX is very scarce. However, it can be anticipated that the toxicokinetics of (±)-VX will differ substantially from that of the phosphofluoridates, since • (±)-VX circulates in vivo as a protonated amine (pKa 9.4) (Van der Schans et al., 2003). • (±)-VX is hydrolyzed much more slowly than phosphonofluoridates (Bajgar et al., 1978; Wang et al., 1998). • (±)-VX is probably also metabolized by other routes than hydrolysis; for example, by means of oxidation reactions at nitrogen and/or sulfur and by undefined

anaerobic mechanisms (Fu and Sun, 1989, 1990, Scaife and Campbell, 1959; Wing et al., 1983). • (±)-VX hardly reacts with carboxylesterases (TNOPML, unpublished results), which may imply that the differences in its toxicokinetics between various species, for example, rats, guinea pigs, and marmosets, are much smaller than for phosphonofluoridates. This assumption is also supported by the rather small range of LD50 values for (±)-VX in various species when compared with, for example, soman (Maxwell, 1992; Maxwell et al., 1987). To obtain the basic data, the toxicokinetics of VX were investigated in anesthetized, atropinized, and mechanically ventilated hairless guinea pigs and marmosets after i.v. administration at doses corresponding to 1 × LD50 (28 µg/kg) and 2 × LD50 (Van der Schans et  al., 2003). The LD50 of VX in marmosets

111 233 61 1 1.2 0.10 0.017 40 877

136 253 63 0.55 1.3 0.11 0.011 64 806 317

Parameter

Dose (µg/kg) A (ng/ml) B (ng/ml) C (ng/ml) a (min−1) b (min−1) c (min−1) Terminal half-life (min) AUC (ng/min/ ml) Acute toxic levels until (min)b

68 301 41 0.9 5.0 0.19 0.032 22 308 95

56 259 37 1.1 4.7 0.15 0.042 16 320

C(−) P(−)

3 LD50 C(+) P(−) 18.6 15 5.9 0.57 0.12 6 76

22.7 18 3.9 0.45 0.096 7 81 37

C(−) P(−)

1 LD50 C(+) P(−) 45.4 339 35 2.8 3.8 0.12 0.034 20 458 126

37.1 406 40 9.9 4.3 0.19 0.046 15 520

C(−) P(−)

6 LD50 C(+) P(−) 15.1 318 11 1 3.8 0.19 0.033 21 169 104

C(+) P(−) 12.4 354 15 1.7 3.9 .21 0.042 16.5 228

C(−) P(−)

2 LD50

Guinea pig

6

5.8 10.6

0.95 0.12

5 3.8 0.8

C(−) P(−)

0.8 LD50 C(+) P(−)

16.5 285 30 1.9 3.9 0.27 0.052 13 218 74

13.5 172 22 1.6 3.0 0.22 0.047 15 191

C(−) P(−)

6 LD50 C(+) P(−)

a

5.5 61 9.9 1.8 2.2 0.35 0.073 9.5 81 49

4.5 52 9.1 2.1 2.0 0.30 0.073 9.4 85

C(−) P(−)

2 LD50 C(+) P(−)

Marmoset

Note: The concentration of each isomer at time t (conct) is described by conct = Ae−at + Be−bt + Ce−ct. Calculated on the basis of a 55/45 ratio of the C(+)P(−) + C(−)P(+)-stereoisomers and the C(+)P(+) + C(−)P(−)-stereo-isomers. b After administration of C(±)P(±)-soman. It is assumed that the area under the curve of 1.8 ng/ml for C(±)P(−)-soman is the minimum area with toxicological relevance.

C(−) P(−)

6 LD50 C(+) P(−)

Rat

Toxicokinetic Parametersa of C(+)P(−)-and C(−)P(−)-Soman in Anesthetized, Atropinized, and Mechanically Ventilated Rats, Guinea Pigs, and Marmosets after i.v. Administration

TABLE 3.2

44 Chemical Warfare Agents

45

Toxicokinetics of Nerve Agents

Concentration C(+)P(−)-GD (ng/mL)

1000

100

10

1

0.1

0.01

0

50

100

150

200

Time (min) FIGURE 3.3  Semi-logarithmic plot of the concentrations in blood of C(+)P(−)-soman (GD) versus time after i.v. administration of 0.8 × LD50 (22 µg/kg, ▴), 3 × LD50 (▪), and 6 × LD50 (♦)of C(±)P(±)-soman to anesthetized, atropinized, and mechanically ventilated guinea pigs.

Concentration C(−)P(−)-GD (ng/mL)

1000

100

10

1

0.1

0.01

0

50

100

150

200

Time (min) FIGURE 3.4  Semi-logarithmic plot of the concentrations in blood of C(−)P(−)-soman (GD) versus time after i.v. administration of 0.8 × LD50 (22 µg/kg, ▴), 2 × LD50 (▪), and 6 × LD50 (♦) of C(±)P(±)-soman to anesthetized, atropinized, and mechanically ventilated guinea pigs.

Concentration C(+)P(−)-GD (ng/mL)

1000

100

10

1

0.1

0.01

0

20

40

60

80

100

120

Time (min) FIGURE 3.5  Semi-logarithmic plot of the concentrations in blood of C(+)P(−)-soman (GD) versus time after i.v. administration of 2 × LD50 (20 µg/kg, ▪) and 6 × LD50 (♦) of C(±)P(±)-soman to anesthetized, atropinized, and mechanically ventilated marmosets.

46

Chemical Warfare Agents

Concentration C(−)P(−)-GD (ng/mL)

1000

100

10

1

0.1

0.01

0

20

40

60

80

100

120

Time (min) FIGURE 3.6  Semi-logarithmic plot of the concentrations in blood of C(−)P(−)-soman (GD) versus time after i.v. administration of 2 × LD50 (20 µg/kg, ▪) and 6 × LD50 (♦) of C(±)P(±)-soman to anesthetized, atropinized, and mechanically ventilated marmosets.

TABLE 3.3

Soman

Parameter

0.8 LD50

2 LD50

0.8 LD50

2 LD50

Dose (µg/kg) A (ng/ml) B (ng/ml) C (ng/ml) a (min−1) b (min−1) c (min−1) Terminal half-life (min) AUC (ng/min/ml) Acute toxic levels until (min)a

9.6 35.9 0.09

24 17.1 0.151

4.95 3.75 0.8

27.5 683 27 3.8 3.9 0.21 0.039 18 397 104

4.6 0.012

0.705 0.00178

0.95 0.12

58 15.3 10

389 109 480

5.8 11

Note: The concentration of each isomer at time t (conct) is described by conct = Ae−at + Be−bt + Ce−ct. a After administration of C(±)P(±)-soman or (±)-sarin. It is assumed that the area under the curve of 1.8 ng/min/ml for C(±)P(−)-soman or 6.9 ng/min/ml for (−)-sarin is the minimum area with toxicological relevance.

is not known but is assumed to have the same value as that in the guinea pig because the influence of CaE on the toxicokinetics is expected to be minimal. In the case when the expected (±)-VX levels were higher than 1 ng/ml, the sample was split, and one part was analyzed with the off-line chiral-LC-GC-NPD method. In the case when the expected concentrations of (±)VX were lower than 1 ng/ml, the samples were analyzed with GC-NPD without off-line separation of the stereo-isomers. It appeared that the ratio of the enantiomers (+)-VX/(−)-VX never exceeded the value of 1.5, indicating that the stereoselectivity in the sequestration of the VX isomers is not as pronounced as with sarin and soman. Some in vitro experiments with plasma and liver homogenates from hairless guinea pigs confirmed that the sequestration of the enantiomers is lower than that observed with the G-agents. For lack of better alternatives, we have assumed

10

1

0.1

0.01

0

10

20

30

40

50

60

Time (min)

FIGURE 3.7  Semi-logarithmic plot of the concentrations in blood of (−)sarin (GB) versus time after i.v. administration of 0.8 × LD50 (▪) and 2 × LD50 (♦) of (±)-sarin to anesthetized, atropinized, and mechanically ventilated guinea pigs. (From Spruit, H.E.T. et al., Toxicol. Appl. Pharmacol., 169, 249, 2000. With permission.) 1000 Concentration in blood (ng/mL)

Sarin

Concentration (−)-GB (ng/mL)

100

Toxicokinetic Parameters of (−)-Sarin and C(±)P(−)-Soman after i.v. Administration of 0.8 × LD50 of (±)-Sarin and 0.8 × LD50 of Soman to Anesthetized, Atropinized, and Mechanically Ventilated Guinea Pigs

100

10

1

0.1

0.01

0

100

200

300

400

500

Time (min)

FIGURE 3.8  Semi-logarithmic plot of the nerve agent concentrations in blood versus time after i.v. administration of 2 × LD50 soman (▪) and 2 × LD50 VX (▴) to anesthetized, atropinized. and mechanically ventilated guinea pigs. VX was administered to hairless guinea pigs. (From Van der Schans, M.J. et al., Toxicol. Appl. Pharmacol., 191, 48, 2003. With permission.)

47

Toxicokinetics of Nerve Agents TABLE 3.4 Toxicokinetic Parameters of VX and C(±)P(−)-Soman after i.v. Administration of 1 × LD50 and 2 × LD50 of Soman and VX to Anesthetized, Atropinized, and Mechanically Ventilated Guinea Pigs or Marmosets HGP Parameter

VX

Dose (µg/kg) Dose (nmol/kg) A (ng/ml) B (ng/ml) C (ng/ml) a (min−1) b (min−1) c (min−1) Terminal half-life (min) AUC (ng/min/ml) AUC (nmol/min/ml) Acute toxic levels until (min)a

56 210 77 17 0.48 0.67 0.033 0.0042 165 744 2.8 1153

HGP

Marmoset

GP

Marmoset

VX

VX

Soman

Soman

28 106 48 8.7 0.39 0.71 0.045 0.0071 98 318 1.2 583

28 106 14 12 1.1 0.42 0.039 0.0062 111 504 1.9 841

27.5 151 683 27 3.8 3.9 0.21 0.039 18 397 2.2 104

10 54 113 19 3.9 2.1 0.33 0.073 9.5 166 0.91 49

HGP = Hairless guinea pig, GP = guinea pig. Note: The concentration of each isomer at time t (conct) is described by conct = Ae−at + Be−bt + Ce−ct. a After administration of C(±)P(±)-soman or (±)-VX. It is assumed that the area under the curve of 1.8 ng/min/ml for C(±)P(−)-soman or 0.9 ng/min/ml for (±)-VX is the minimum area with toxicological relevance.

that equal amounts of (+) and (−)-VX are present in all phases of our toxicokinetic experiments and calculated the toxicokinetic parameters for (±)-VX rather than for the individual isomers. The curve of the toxicokinetics of VX in hairless guinea pigs is shown in Figure 3.8. For comparison, analogous data for soman are also given in Figure 3.8. The toxicokinetic data of VX and soman are shown in Table 3.4. The values of the AUC experiments at doses corresponding to 1 × LD50 and 2 × LD50 show that the toxicokinetics are reasonably linear with the dose. Evidently, the detoxification processes in the animal are not saturated with an excess of VX. The outcome is not surprising, since the binding of VX to CaE is very slow, while this route is a major elimination route for the G-agents. The AUCs of the toxicokinetic experiments are summarized in Figure 3.9. This figure shows

clearly the non-linearity of the AUC as function of the dose in the experiments with rats, guinea pigs, and marmosets. The curves with a straight line through the origin pertain to experiments with a minor influence of CaE on the toxicokinetics because of the absence of this enzyme (in the case of marmosets) or a low affinity of this enzyme with the nerve agent (in the case of VX). Figure 3.8 shows also that the elimination rates of the two types of agents differ widely. Both the terminal half-life and the period of time when toxicologically relevant concentrations are present are approximately one order of magnitude longer for VX than for soman. As shown in Figure 3.10, the toxicokinetics of VX in marmosets and guinea pigs at a dose corresponding to 1 × LD50 in the hairless guinea pig reveal that after a short time, the blood 100 Concentration (±)-VX (ng/mL)

AUC [(ng min)/mL)]

2000

1500

1000

10

1

0.1

500

0.01 0

0

50

100

150

200

250

300

Dose (µg/kg)

FIGURE 3.9  AUC as function of the dose of nerve agent after i.v. administration to anesthetized, atropinized, and mechanically ventilated animals. Somanrat (♦), Soman-guinea pig (▪), Soman-marmoset (▴), VX-guinea pig (●).

0

100

200

300

400

500

Time (min)

FIGURE 3.10  Semi-logarithmic plot of the concentrations in blood of VX versus time after i.v. administration of 1 × LD50 to anesthetized, atropinized, and mechanically ventilated hairless guinea pigs (▴) or marmosets (▪). (From Van der Schans, M.J. et al., Toxicol. Appl. Pharmacol., 191, 48, 2003. With permission.)

48

Chemical Warfare Agents

levels in the marmoset are between the levels found in the blood of the guinea pig at dosages corresponding to 1 × LD50 and 2 × LD50. Correspondingly, the AUC and the period of time for which toxicologically relevant concentrations of VX are present for the marmoset are between the values of 1 × LD50 and 2 × LD50 for the guinea pig. Nevertheless, the data shows that the difference between marmoset and guinea pig is not as large, due to the lack of affinity of VX for CaE. In 2008, Reiter et al. (2008) published the results of an i.v. toxicokinetic study of VX in swine. The animals were injected with VX at a dose of 24.6 µg/kg corresponding to 2 × LD50. Blood samples were processed and analyzed by chiral LC-MS/MS. They found a long persistence of both VX enantiomers. After 6 h, VX was still present in blood at levels of approx. 10 ng/ml. They found also that the less toxic isomer, (+)-VX, was more persistent than (−)-VX, which is contrast to the G-agents. The LC-MS/MS method with a Hypercarb column installed instead of the Chiral AGP enabled the analysis of the toxic metabolite EA-2192, that is, VX without the ethyl group (Reiter et al., 2011). This toxic metabolite is also a strong inhibitor of AChE, but it cannot be reactivated by oximes. The formation of this metabolite could be detected in vivo in the blood of swine after i.v. injection with VX. The concentration of EA-2192 was approximately 4–10 times lower than that of VX in the same sample.

3.5 Percutaneous Toxicokinetics of VX Due to the low volatility of VX, the main porte d’entrée for this agent is by penetration through the skin. The toxicokinetics of VX were investigated in hairless guinea pigs after percutaneous application of VX in 2-propanol at a dose corresponding to 1 × LD50 (125 µg/kg) (Chilcott et al., 2003, 2005; Dalton et al., 2006). Blood samples were taken up to 7 h after the application. Unfortunately, it was not possible to take blood samples for a longer period of time for technical reasons. Therefore, the experiments pertain only to the first phase of the toxicokinetic curve, where the levels of VX just start to decrease after increasing for several hours. The concentration of VX in blood versus time after percutaneous application is shown in Figure 3.11.

Evidently, a large variation is observed in the data of the levels of VX in blood. Presumably, this spread is caused by the variation in thickness and permeability of the skin in the various animals (Boutsiouki et al., 2001). The curve through the data points has been drawn by eye. The AUC was estimated at 43 ± 8 ng/min/ml and was calculated as the mean of the individual curves. The bioavailability can be calculated as the ratio of the AUC after percutaneous application and the extrapolated AUC that would have been derived after i.v. injection of the same dose. The bioavailability after percutaneous application appeared to be only 2.5%. In view of the low bioavailability, it is possible that a significant part of the administered dose of VX is still present in depot in the skin. The blood levels of VX build up slowly and reach a maximum level of approximately 140 pg/ml at 6 h after the application of the toxicant. Figure 3.12 shows the degree of AChE inhibition for three individual animals together with the corresponding individual blood levels of VX. A qualitative agreement between the two sets of data was observed, since a rapid decrease of AChE activity corresponds to relatively high levels of VX, and a slow decrease of AChE corresponds to relatively low levels of VX. The experiment in which the most rapid decrease of AChE activity and high levels of VX were observed was not used in the construction of the toxicokinetic curve (Figure 3.11). This result was considered an outlier. As shown in Table 3.4, the period of time when toxicologically relevant levels of VX circulate after i.v. administration is 10–20 h, but this time period might be considerably longer after percutaneous exposure. Moreover, these time periods are considerably longer than observed with G-agents; that is, at most 2 h for an i.v. dose corresponding to 2 × LD50. This difference, in combination with the gradual buildup of the blood levels after percutaneous exposure, may cause specific problems in the diagnosis and treatment of intoxications with V-agents. These findings are in agreement with the description of the symptoms of a victim who was attacked with VX by a member of the AUM Shinrikyo sect (Nozaki et al., 1995). With G-agents, the most important porte d’entrée is the respiratory route, whereby the symptoms occur shortly after exposure. With percutaneous exposure to liquid VX, there will be a certain lag time between exposure and symptoms, which may complicate the

Concentration (±)-VX (pg/mL)

200

100

0

0

50

100

150

200

250

300

350

400

450

Time (min) FIGURE 3.11  Concentration of VX in blood of anesthetized, atropinized, and artificially ventilated guinea pig after percutaneous application of VX at a dose of 125 µg/kg, corresponding to 1 × LD50. (From Van der Schans, M.J. et al., Toxicol. Appl. Pharmacol., 191, 48, 2003. With permission.)

49

Toxicokinetics of Nerve Agents

Percentage inhibition of AChE in blood

100 1375

80

2387

1684

1678 273

257

249

213

165

41

34

45

60 53

40 40

68

105

20

115

85 68

0

0

100

200

300

400

500

Time (min) FIGURE 3.12  Progressive inhibition of AChE of anesthetized, atropinized, and artificially ventilated hairless guinea pigs after percutaneous application of VX at a dose of 125 µg/kg, which corresponds to 1 × LD50. Numbers at the data points correspond to the concentration (pg/ml) of VX in blood in that particular sample. (From Van der Schans, M.J. et al., Toxicol. Appl. Pharmacol., 191, 48, 2003. With permission.)

timely diagnosis. Reiter et al. (2008, 2011) published the results of toxicokinetics of VX after percutaneous application in swine. They exposed the animals to a dose of 189 µg/kg, corresponding to 3 × LD50. Both VX and the toxic metabolite EA-2192 could be detected in blood. The level of VX climbed in 100 min to a level of approx. 9 ng/ml, which remained stable until 500 min after the application. The level of (+)-VX was slightly higher than that for (−)-VX; the ratio (+)-VX:(−)-VX was 1.5:2). The level of EA-2192 was approximately four times lower than that of VX in the same sample. The traditional treatment of nerve agent intoxication with atropine and oximes might be hampered by the rapid pharmacokinetics of the oxime and slow elimination of the VX, which might still be circulating at toxicologically relevant levels when the blood levels of the oxime have decreased to ineffective levels. In that case, oxime therapy must be continued until the VX levels drop below the toxicologically relevant level. Joosen et al. (2010) exposed guinea pigs percutaneously to VX and treated them with atropine, obidoxime, and diazepam with a single shot or repetitively. A single-shot treatment extended the period of physiological decline and death for several hours. Repetitive administration remained effective as long as the treatment was continued. In the same study, the toxicokinetics of VX were measured. In the non-treated animals, the levels of VX in blood increased to more than 2 ng/ml in 3 h, whereafter the animals died. In the treated animals, the level of VX in blood dropped below 0.5 ng/ml but remained stable for the duration of the experiment (12 h). In accordance with the latter scenario, Clarkson et al. (2005) treated VX-intoxicated guinea pigs with repetitive oxime administration and were able to treat the animals up to challenges of 10 × LD50. However, the animals were euthanized at 24 h after the application of the agent. Presumably, this period is too short to judge whether the treatment had been truly effective, because it is possible that animals would have died after that period of time. Interestingly the paper also mentioned a positive effect of pyridostigmine as treatment. The reports in the literature contradict each other. Gordon et al. (1978) reported a positive effect

of pretreatment with carbamates in a subcutaneous challenge of guinea pigs with VX. However, Koplovitz et al. (1992) reported a negative influence of pretreatment with pyridostigmine on the efficacy of treatment with an oxime. It should be mentioned that treatment with scavengers such as BuChE would be more effective in this respect, since these scavengers have a longer half-life than oximes (Sidell and Groff, 1971).

3.6 Elimination Pathways of Soman, Sarin, and VX The rapid decrease of soman levels in blood after i.v. administration or respiratory exposure is due to three processes: distribution to various tissues, spontaneous or enzymatic hydrolysis, and covalent binding (Adie and Tuba, 1958; Adie et  al., 1958; Augustinsson and Heimburger, 1954; Mazur, 1946; Mounter, 1963). It has been established that the toxic C(±)P(−)-isomers react rapidly with covalent binding sites. The less toxic C(±)P(+)isomers are hydrolyzed several orders of magnitude faster than the C(±)P(−) isomers. The low toxicity of the C(±)P(+)-isomers is primarily due to a low intrinsic reactivity toward AChE and rapid hydrolysis. The elimination of VX deviates from that of soman. As observed in the toxicokinetic studies, the elimination of VX proceeds much more slowly than that of the G-agents. Besides the major detoxification route of VX, which leads to the formation of O-ethyl methylphosphonic acid, it cannot be excluded that toxic metabolites will be formed. Possible toxic metabolites are the dealkylated form of VX or “desethyl-VX” and the N-oxide of VX (Fu and Sun, 1989, 1990; Scaife and Campbell, 1959; Wing et al., 1983). Reiter et al. (2011) indeed detected the formation of desethyl-VX (EA-2192) in the blood of swine that were intoxicated with VX. The other metabolite, N-oxide of VX, has not been found in blood samples taken for toxicokinetic experiments. In vitro experiments, in which high concentrations of VX (10 µg/mL) were incubated in plasma or liver homogenate, did not yield any of these toxic metabolites

50 either. However, desethyl-VX (EA-2192) could be detected in reasonable amounts when plasma was derived from blood that was drawn in tubes with EDTA as anticoagulant. EDTA binds metal ions that are essential for the enzymatic hydrolysis of VX, which leads to the formation of O-Ethyl methylphophonic acid (EMPA). When that route is inhibited, the formation of desethyl-VX may increase (Van der Schans et al., 2000). Elimination products such as hydrolyzed organophosphate or covalently bound organophosphate are the biomarkers of choice for detection of exposure, since the persistence of the intact nerve agents, although sufficiently long to interfere with therapeutic measures, is too short for this purpose.* Incubation of inhibited CaE or BuChE with fluoride ions can regenerate the phosphyl moiety from the protein, thus creating the phosphofluoridate, which can be analyzed with gas chromatography. The method can also be used to study the distribution of the nerve agent over the tissues, provided that the nerve agent can be regenerated with fluoride ions, which is the case for sarin and VX but not for soman.† Whalley et  al. (2006) exposed several guinea pigs to sarin through the respiratory route and by subcutaneous administration. Next, the animals were euthanized at different time points, and the amount of regenerable sarin in the tissues was determined. Figure 3.13 shows the amount of sarin that could be regenerated from the tissues after subcutaneous administration of doses corresponding to 0.1 × LD50 and 0.4 × LD50. The figures show that after subcutaneous administration of sarin corresponding to 0.1 × LD50, the greatest quantity of sarin adducts is found in the lungs and only a small portion in the other organs. After administration of sarin at a dose corresponding to 0.4 × LD50, the kidney appears to be another important site for the binding of sarin. The amount of regenerable sarin seems to correlate with the flow of blood through that organ. Intact sarin is supplied by the bloodstream and reacts with the available binding sites. The lungs are the only organ through which 100% of the cardiac output flows, which may be an explanation for the high amount of regenerable sarin in that particular organ. The liver is an organ with an extremely high number of binding sites, but the limited blood flow through that organ causes a moderate amount of bonded sarin. Figure 3.14 shows the amount of regenerable sarin in tissues of guinea pigs after whole-body exposure to sarin at dosages corresponding to 0.1 × LCt50 and 0.4 × LCt50. It is obvious that the amount of regenerable sarin in the eyes is relatively high, because the uptake of sarin takes place directly from the air into the eyes. It cannot be excluded that the amount of measured sarin partly consists of intact sarin that is not necessarily regenerated from binding sites. At a higher exposure level, the amount of regenerable sarin is highest in the lungs. It is remarkable that the amounts of regenerable sarin in the lungs after whole-body exposure or subcutaneous administration at comparable dosages (0.4 × LCt50 or 0.4 × LD50) are roughly the same. It might be expected that the amount of bonded sarin * For a comprehensive review of methods for diagnosis of exposure to chemical warfare agents, see Noort et al. (2002). † Soman cannot be regenerated with fluoride ions, because dealkylation of the pinacolyl group of soman once bonded to BuChE (a process known as aging) prevents the regeneration of soman.

Chemical Warfare Agents on whole-body exposure is higher because sarin is inhaled. However, the absorption of sarin does not occur in the lungs but proceeds most likely in the upper airways rather than in the lungs. In this way, intact sarin has to be transported by blood to the lungs, where it can react with the binding sites in that organ. In 2007, Whalley et  al. (2007) published results of the same experiments using GF instead of GB. Compared with GB, it is remarkable that the levels of regenerated GF were lower than those found after GB exposure (see Figure 3.15). Either GF has a lower affinity for different binding protein sites or the efficacy of the fluoride regeneration method is lower for GF than for GB.

3.7 Effect of HuBuChE as a Scavenger on the Toxicokinetics of Nerve Agents In the previous section, it was discussed that binding is one of the most effective elimination routes for nerve agents. The difference in lethality of various nerve agents in rats, guinea pigs, and marmosets is a result of the presence of different amounts of CaE, which reacts as a scavenger in the blood of these species. Therefore, attention has been paid to pretreatment with highly reactive scavengers, which would intercept or destroy the nerve agent when it enters the bloodstream, before it could reach its target site. It is anticipated that effective scavengers will offer protection against both lethal and incapacitating effects of an acutely toxic dose. In addition, if a scavenger remains in circulation at an effective concentration during a relatively long period of time, the pretreatment will, a fortiori, protect against longterm exposure to low doses of a nerve agent (Benschop et  al., 1998). It is further anticipated that bioscavengers will not induce adverse physiological effects, particularly when bioscavengers of human origin are applied. As early as 1957, Cohen and Warringa (1957) achieved some protection in rats against a lethal subcutaneous dose of diisopropyl phosphorofluoridate and sarin by pretreatment with an enzyme capable of hydrolyzing organophosphates (Millard et al., 1998). In more recent years, the feasibility of using bioscavengers that can rapidly bind nerve agents has been studied. For this purpose, monoclonal antibodies (Brimfield et al., 1985; Lenz et al., 1984), serum AChE (Ashani et al., 1991; Maxwell et al., 1992; Wolfe et al., 1987, 1992) and plasma HuBuChE (Allon et al., 1998; Ashani, 2000; Ashani and Pistinner, 2004; Ashani et al., 1993; Broomfield et al., 1991; Doctor et al., 1993; Maxwell et al., 1999; Raveh et  al., 1993; 1997; Sweeney and Maxwell, 1999, 2003) have been investigated. Very promising results were obtained with HuBuChE as a scavenger. The enzyme is rapidly distributed in laboratory animals, such as mice, rats, guinea pigs, and rhesus monkeys, after i.v. administration, followed by a slow elimination (Allon et al., 1998; Ashani et  al., 1993; Raveh et  al., 1993). In addition, the enzyme is sufficiently absorbed following an intramuscular (i.m.) administration to provide therapeutically significant blood levels over a time period of 10–70 h in laboratory animals, which is a prerequisite for practical application (Allon et  al., 1998; Ashani et al., 1993; Raveh et al., 1993). The peak level in blood after i.m. administration amounted to 50–60% of the concentration obtained immediately after i.v. administration of the same

51

Toxicokinetics of Nerve Agents 10

Eye

9

Lung

8

Kidney

7

Brain Heart

rGb (ng/g)

6

Liver

5

Diaphragm

4 3 2 1 0

120

160

200 Time (min)

240

360

60 Eye Lung

50

Kidney Brain

rGb (ng/g)

40

Heart Liver

30

Diaphragm 20

10

0

120

160

200 Time (min)

240

360

FIGURE 3.13  Concentration (ng/g) of regenerated sarin (GB) in various tissues of guinea pigs after subcutaneous administration of sarin at dosages corresponding to 0.1 × LD50 (4.2 µg/kg, left) and 0.4 × LD50 (16.8 µg/kg, right).

amount of enzyme. Pretreatment with the enzyme resulted not only in an increase in survival of mice, rats, and rhesus monkeys intoxicated (i.v.) with C(±)P(±)-soman or other nerve agents but also in a significant alleviation of postexposure incapacitation. An effective protection of guinea pigs against respiratory exposure to C(±)P(±)-soman has been reported (Allon et al., 1998). Since the efficacy of HuBuChE as a scavenger is based on the inhibitory properties of the challenging agent, it can be expected that such scavengers will be effective against nerve agents having a wide variety of chemical structures. Moreover the stereoselectivity of HuBuChE as scavenger is in favor of the P(−)-isomers, which means that the toxic isomer of the nerve agents is preferentially eliminated. The ultimate goal is to use these scavengers as a pretreatment to protect humans against nerve agent intoxication. Toxicokinetic studies can be an adequate tool for the quantitative description of the protective mechanism, which is needed for registration of the enzyme as a pretreatment drug for application in humans.

The toxicokinetics of soman in anesthetized, atropinized, and artificially ventilated naive and HuBuChE-pretreated guinea pigs were studied (Van der Schans and Langenberg, 2005). Animals were pretreated with HuBuChE by an i.m. administration 24 h before administration of the nerve agent. The enzyme was administered intramuscularly because this route is much more appropriate than i.v. administration for the application of a scavenger in a realistic scenario. The ratio of the dose of HuBuChE to the dose of the nerve agent was chosen on the basis of results reported by Ashani et  al. (1993) or Allon et al. (1998) to obtain sufficient protection in a similar experiment. The dose of HuBuChE was a 0.7-fold fraction of the molar dose of soman that corresponds to 0.7 × 55/182 = 211 nmol HuBuChE/kg. The level of HuBuChE in blood at 24 h after i.m. administration of HuBuChE was approximately 690 nM, which is over an order of magnitude higher than the baseline level of BuChE, which is approximately 20 nM.

52

Chemical Warfare Agents 14 Eye 12

Lung Kidney

10 rGb (ng/g)

Brain Heart

8

Liver 6

Diaphragm

4 2 0

120

160

200

240

360

Time (min) 30 Eye Lung

25

Kidney Brain

rGb (ng/g)

20

Heart Liver

15

Diaphragm 10

5

0

120

160

200

240

360

Time (min) FIGURE 3.14  Concentration (ng/g) of regenerated sarin in various tissues of guinea pigs after respiratory exposure to sarin (GB) at dosages corresponding to 0.1 × LCt50 (0.4 mg/m3 for 60 min, left) or 0.4 × LCt50 (1.6 mg/m3 for 60 min, right).

To study the effect of the scavenger, we compared the toxicokinetic data of the HuBuChE-pretreated and non-pretreated animals. Figure 3.16 shows the concentration–time curves of C(−)P(−)-soman after i.v. administration of soman in naive and HuBuChE-pretreated animals. Evidently, the concentrations of C(−)P(−)-soman in HuBuChE-pretreated animals are much lower than in naive animals. This effect of the scavenger is also clearly reflected in the values of the AUC shown in Table 3.5. The difference in AUC of the P(−)-isomers between naive and HuBuChEpretreated animals is 65.9 − 2.9 = 63 ng/min/mL, which is equal to 0.34 nmol/min/ml. The period of time for which acutely toxic levels of soman exist is reduced from 40 to 1 min. After one injection of soman, only the C(−)P(−) isomer of soman could be detected in the HuBuChE-pretreated guinea pigs.

The C(+)P(−) isomer had completely disappeared from the bloodstream. In a qualitative sense, this result was expected in view of the higher binding constant of C(+)P(−) with BuChE compared with the binding constant of the C(−)P(−) isomer with BuChE. Experiments analogous to those performed with soman were also performed with VX; that is, the effect of pretreatment of HuBuChE at 0.7 × the molar dose of VX corresponding to 2 × LD50 of VX on the toxicokinetics of the agent was investigated. As shown in Figure 3.17, the VX levels in blood of HuBuChEpretreated guinea pigs are somewhat lower. However, this effect seems not to be very large during the first 180 min. A more pronounced difference between the two curves in the time period beyond 200 min is observed, but the contribution of this part to the AUC of the toxicokinetic curve is less than 7%. Table 3.5

53

Toxicokinetics of Nerve Agents

FIGURE 3.15  Concentration (ng/g) of regenerated sarin (GB) or regenerated cyclosarin (GF) in various tissues of guinea pigs after whole-body exposure to GB or GF at dosages corresponding with 0.4 × LCt50 (1.6 mg.m−3 for 60 min, both GB and GF).

Concentration C(−)P(−)-GD (ng/mL)

100

10

1

0.1

0.01

0

10

20

30

40

50

60

Time (min) FIGURE 3.16  Mean concentration ± SEM (ng/ml) of C(−)P(−)-soman (GD) in blood of individual anesthetized, atropinized, and mechanically ventilated naive (♦) and HuBuChE-pretreated (200 nmol/kg, ▪) guinea pigs after i.v. bolus administration of a dose of C(±)P(±)-soman corresponding to 2 × LD50 (55 µg/kg).

shows a summary of the toxicokinetic parameters derived from the blood levels of soman and VX. The decline of the AUC for the summated stereo-isomers of VX on pretreatment with 200 nmol/kg HuBuChE was 744 − 494 = 260 ng/min/ml, which is equal to 0.97 nmol/min/ml. By comparison, the decrease of the AUC of C(±)P(−)-soman in guinea pigs challenged with 55 µg/kg C(±)P(±)-soman was 0.34 nmol/min/ml. The P(+)-isomers of soman were not detected, but it cannot be excluded that these P(+)-isomers consumed a part of the scavenger (Van der Schans and Langenberg, 2005). Although it cannot be excluded that the P(+)-isomers consumed a part of the scavenger, this will not suffice to explain the lower decrease of the AUC for soman than for VX. The AUCs of the VX curves are calculated over a time span of 360 min, while the AUCs of the soman curves are calculated over a time span of 60 min. Table 3.6 shows the AUCs of both experiments expressed in nanomoles per minute per milliliter for different time spans. The

decrease of AUC as a result of HuBuChE pretreatment is larger for VX than for soman. However, the relative decline of the AUC of the soman curve (97% at t = 90 min) is more pronounced than for VX (29% at t = 90 min). The elimination of soman in non-pretreated animals is caused by enzymatic hydrolysis and binding to CaE, whereas the elimination of VX in such animals is driven by slow enzymatic hydrolysis and slow binding to CaE. Additional binding sites such as BuChE will be consumed rapidly. Addition of fast-binding sites to soman will only increase the number of multiple fastbinding sites. It is therefore explicable that the scavenger will not be consumed completely by soman, which means that the effect of the scavenger on the toxicokinetics of soman is less in this case. Table 3.5 shows clearly that the time period in which acutely toxic levels of VX circulate is reduced from 1153 to a still considerable 230 min after the administration of 200 nmol/ kg HuBuChE. HuBuChE acts as a stoichiometric scavenger for

54

Chemical Warfare Agents TABLE 3.5 The Effect of Pretreatment (i.m.) with HuBuChE on the Toxicokinetic Parameters for Soman and VX in Anesthetized, Atropinized, and Artificially Ventilated Guinea Pigs after i.v. Administration Parameter Isomer Dose scavenger (nmol/kg) Dose nerve agent (µg/kg) A (ng/ml) B (ng/ml) C (ng/ml) a (min−1) b (min−1) c (min−1) Terminal half-life (min) AUC (ng/min/ml) Acutely toxic levels until (min)

Soman

Soman

Soman

Soman

VX

VX

C(+)P(−) 0 15.1 2.05 0.658 – 0.277 0.054 – 14.7 19.6

C(−)P(−) 0 12.4 70.0 4.05 0.356 3.11 0.256 0.0445 15.5 46.3 40

C(+)P(−) 200 15.1 – – – – – – – 0 –

C(−)P(−) 200 12.4 1.11 0.274 – 0.815 0.179 – 3.8 2.89 1

(±) 0 56 77 17 0.48 0.67 0.033 0.0042 165 744 1153

(±) 200 56 54.2 11.0 – 0.817 0.0257 – 26.9 494 230

Note: The concentration of each isomer at time t (conct) is described by conct = Ae−at + Be−bt + Ce−ct. It is assumed that the area under the curve of 1.8 ng/min/ml for C(±)P(−)-soman or 0.9 ng/min/ml for (±)-VX is the minimum area with toxicological relevance.

Concentration VX (ng/ml)

100

100

10

1 10

0

5

10 Time (min)

15

20

0.1

0.01 0

100

200

300

400

500

Time (min) FIGURE 3.17  Mean concentration ± SEM (ng/ml) of (±)-VX in blood of individual anesthetized, atropinized, and mechanically ventilated naive (♦) and HuBuChE-pretreated (200 nmol/kg, ▪) guinea pigs after i.v. bolus administration of a dose of (±)-VX corresponding to 2 × LD50 (56 µg/kg). Inset shows the data for the first 20 min plotted on an expanded scale.

TABLE 3.6 AUC (nmol/min/ml) of (±)-VX and C(±)P(−)-Soman in Blood of Naive or HuBuChE-Pretreated Anesthetized, Atropinized, and Mechanically Ventilated Guinea Pigs i.v. Injected with VX (56 µg/kg) or C(±)P(±)-Soman (55 µg/kg) AUC of (±)-VX (nmol/min/ml)

Time (min) 0 0–30 0–60 0–90 0–120 0–180 0–240

Without HuBuChE 0 1.69 2.18 2.39 2.49 2.58 2.62

HuBuChE (200 nmol/kg)

Difference as Result of HuBuChE Treatment

0 1.11 1.51 1.69 1.78 1.84 1.85

0 0.58 0.67 0.70 0.81 0.74 0.77

AUC of C(±)P(−)-Soman (nmol/min/ml)

Without HuBuChE

HuBuChE (200 nmol/kg)

Difference as Result of HuBuChE Treatment

0 0.337 0.356 0.360

0 0.014 0.015 0.015

0 0.32 0.34 0.35

Toxicokinetics of Nerve Agents nerve agents, which means that the efficacy of the protection by HuBuChE depends on the balance between the amount of nerve agent and the amount of scavenger. In these experiments, the amount of HuBuChE was not sufficient to decrease the concentration of VX below the acutely toxic level within a reasonable time window.

3.8 Concluding Remarks With the analytical methodology available, it appeared possible to measure nerve agents at the toxicologically relevant level. This capability made it possible to measure the toxicokinetics of nerve agents after a variety of exposure routes at several doses and to determine the time period for which acutely toxic levels exist. The relevance of such studies has been shown, since the persistence of nerve agent in vivo is longer than anticipated, with the percutaneous exposure to VX being the most remarkable. It is evident that these studies can be used for the development of strategies for the timely administration of antidotes in the case of nerve agent intoxication. For example, the efficacy of HuBuChE as a scavenger for nerve agents was demonstrated by toxicokinetic studies. Analysis of regenerated sarin from binding sites has provided more insight into the elimination pathways of nerve agents. This is especially true for the lungs and the eyes, which appeared to contain many binding sites for nerve agents following wholebody exposure. Both organs are also the porte d’entrée for the agent, and for the eyes, it cannot be excluded that the amount of measured sarin partly consists of intact sarin. Exposure studies at lower levels (occupational exposure) are near the lower limit of what can be reached with regard to toxicokinetics based on in vivo measurement of the intact nerve agent. Ultra-low-level exposure can only be studied after the analysis of fluoride-regenerated nerve agents (Van Helden et al., 2003). With these studies, it is possible to quantify the link between external and internal dosages, which is highly relevant in the area of regulatory toxicology. Eventually, the toxicokinetic data, together with the distribution data, is very useful for the validation of physiologically based pharmacokinetic modeling (PBPK) (Gearhart et al., 1990; Langenberg et al., 1997; Maxwell et al., 1988; Ramsey and Andersen, 1984). These models are needed because the experiments that were discussed in this chapter can never be performed in humans, while extrapolation of the results obtained in these animal experiments to humans is still the ultimate goal of these investigations.

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55 Ashani, Y., Grunwald, J., Raveh, L., Grauer, E., Brandeis, R., Marcus, D. et al., Cholinesterase prophylaxis against organophosphorus poisoning. Final Report Contract DAMD17–90-C-0033, AD-A277-096, 1993. Ashani, Y. and Pistinner, S., Estimation of the upper limit of human butyrylcholinesterase dose required for protection against organophosphates toxicity: A mathematically based toxicokinetic model, Toxicol. Sci., 77, 358–367, 2004. Ashani, Y., Shapira, S., Levy, D., Wolfe, A.D., Doctor, B.P., and Raveh, L., Butyrylcholinesterase and acetylcholinesterase prophylaxis against soman poisoning in mice, Biochem. Pharmacol., 41, 37–41, 1991. Augustinsson, K.-B. and Heimburger, G., The enzymic hydrolysis of organophosphorus compounds. 1. Occurrence of enzymes hydrolyzing tabun, Acta Chem. Scand., 8, 753, 1954. Bajgar, J., Fusek, J., Patocka, J., and Hrdina, V., Detoxication of phosphonothioates and phosphonofluoridates in the rat, Acta Biol. Med. Gerin., 37, 1261, 1978. Benschop, H.P., Absolute configuration of chiral organophosphorus anticholinesterases, Pestic. Biochem. Physiol., 5, 348, 1975. Benschop, H.P., Bijleveld, E.C., Otto, M.F., Degenhardt, C.E.A.M., Van Helden, H.P.M., and De Jong, L.P.A., Stabilization and gas chromatographic analysis of the four stereoisomers of 1,2,2-trimethylpropyl methyl phosphonofluoridate (soman) in rat blood, Anal. Biochem., 151, 242, 1985. Benschop, H.P. and De Jong, L.P.A., Toxicokinetics of the four stereoisomers of soman in the rat, guinea pig and marmoset, Final Report Grant DAMD1785G–5004, NTIS AD–A 199, 573, 1987. Benschop, H.P. and De Jong, L.P.A., Nerve agent stereoisomers: Analysis, isolation and toxicology, Acc. Chem. Res., 21, 368, 1988. Benschop, H.P. and De Jong, L.PA., Toxicokinetic investigations of C(±)P(±)-soman in the rat, guinea pig and marmoset at low doses—Quantification of elimination pathways, Final Report Grant DAMD17877015, NTIS AD–A 226 807, 1990. Benschop, H.P and de Jong, L.P.A., Toxicokinetics of soman: Species variation and stereospecificity in elimination pathways, Neurosci. Biobehav. Rev., 15, 73, 1991. Benschop, H.P. and De Jong, L.P.A., Toxicokinetics of nerve agents, Ch. 2, in Chemical Warfare Agents: Toxicity at Low Levels, Somani, S.M. and Romano, Jr., J.A., eds., CRC Press, 2001. Benschop, H.P., Konings, C.A.G., and De Jong, L.PA., Gas chromatographic separation and identification of the four stereoisomers of 1,2,2-trimethylpropyl methylphosphonofluoridate (soman). Stereospecificity of in vitro “detoxification” reactions, J. Am. Chem. Soc., 103, 4260, 1981. Benschop, H.P., Konings, C.A.G., Van Genderen, J., and De Jong, L.P.A., Isolation, anticholinesterase properties and acute toxicity in mice of the four stereoisomers of soman, Toxicol. Appl. Pharmacol., 90, 61, 1984. Benschop, H.P., Trap, H.C., Spruit, W.E.T., Van der Wiel, H.J., Langenberg, J.P., and De Jong, L.P.A., Low level nose-only exposure to the nerve agent soman: Toxicokinetics of soman stereoisomers and cholinesterase inhibition in atropinized guinea pigs, Toxicol. Appl. Pharmacol., 153, 179–185, 1998. Benschop, H.P. and Van Heiden, H.P.M., Toxicokinetics of inhaled soman and sarin in guinea pigs, Final Report Contract DAMD1790Z0034, NTIS AD–A277 585, 1993. Bonierbale, E., Debordes, L. and Coppet, L., Application of capillary gas chromatography to the study of the hydrolysis of the nerve agent VX in rat plasma. J. Chromatogr., 668, 255–264, 1996.

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Chemical Warfare Agents Fu, F. and Sun, M., Occurrence of (±)-VX oxidase, Chin. J. Pharmacol. Toxicol., 3, 264–270, 1990; Chem. Abstr., 112, 74346v (1989). Fu, F. and Sun, M., Aerobic metabolism of (±)-VX and mixed function oxidases, Acta Pharmacol. Sin. 11, 123–126, 1990; Chem Abstr., 112, 212942q (1990). Gearhart, J.M., Jepson, G.W., Clewell, H.J., Andersen, M.E., and Conolly, R.B., Physiologically based pharmacokinetics and the pharmacodynamic model for inhibition of acetylcholinesterase by diisopropyl fluorophosphate, Toxicol. Appl. Pharmacol, 106, 295, 1990. Gordon, J.J., Leadbeater, L., and Maidment, M.P., The protection of animals against organophosphate poisoning by pretreatment with a carbamate, Toxicol. Appl. Pharmacol., 43, 207–216, 1978. Hall, C.R., Inch, T.D., Inns, R.H., Muir, A.W., Sellers, D.J., and Smith, A.P., Differences between some biological properties of enantiomers of alkyl S-alkyl methylphosphonothioates, J. Pharm. Pharmacol., 29, 574, 1977. Jakubowski, E.M., McGuire, J.M., Evans, R.A., Edwards, J.L., Hulet, S.W., Benton, B.J. et al., Quantitation of fluoride ion released sarin in red blood cell samples by gas chromatography-chemical ionization mass spectrometry using isotope dilution and large-volume injection, J. Anal. Toxicol., 28(5), 357–363, 2004. Joosen, M.J.A., van der Schans, M.J., and van Helden, H.P.M., Percutaneous exposure to the nerve agent VX: Efficacy of combined atropine, obidoxime and diazepam treatment, Chem-Biol. Interact., 188, 255–263, 2010. Kientz, C.E., Langenberg, J.P., and Brinkman, U.A.Th., Microcolumn liquid chromatography with thermionic detection of the enantiomers of O-ethyl S-diisopropylaminoethylmethylphosphonothioate (VX), J. High Resolut. Chromatogr., 17, 95, 1994. Koplovitz, I., Harris, L.W., Anderson, D.R., Lennox, W.J., and Stewart, J.R., Reduction by pyridostigmine pretreatment of the efficacy of atropine and 2-PAM treatment of sarin and VX poisoning in rodents, Fundam. Appl. Toxicol., 18, 102–106, 1992. Langenberg, J.P., Van Dijk, C., Sweeney, R.E., Maxwell, D.M., De Jong, L.P.A., and Benschop, H.P., Development of a physiologically based model for the toxicokinetics of C(±)P(±)-soman in the atropinized guinea pig, Arch. Toxicol., 71, 320–331, 1997. Lenz, D.E., Brimfield, A.A., Hunter, Jr., K.W., Benschop, H.P., De Jong, L.P.A., Van Dijk, C., and Clow, T.R., Studies using a monoclonal antibody against soman, Fundam. Appl. Toxicol., 4, S156S164, 1984. Maxwell, D.M., The specificity of carboxylesterase protection against the toxicity of organophosphorus compounds, Toxicol. Appl. Pharmacol., 114, 306, 1992. Maxwell, D.M., Brecht, K.M., and O’Neill, B.L., The effect of CaE inhibition on interspecies differences in soman toxicity, Toxicol. Lett., 39, 35, 1987. Maxwell, D.M., Castro, C.A., De La Hoz, D., Gentry, M.K., Gold, M.B., Solana, R.P., Wolfe, A.D., and Doctor, B.P., Protection of rhesus monkeys against soman and prevention of performance decrement by pretreatment with acetylcholinesterase, Toxicol. Appl. Pharmacol., 115, 44–49, 1992. Maxwell, D.M., Saxena, A., Gordon, R.K., and Doctor B.P., Improvements in scavenger protection against organophosphorus agents by modification of cholinesterases, Chem-Bio Interactions, 119–120, 419–428, 1999.

Toxicokinetics of Nerve Agents Maxwell, D.M., Vlahacos, C.P., and Lenz, D.E., A pharmacodynamic model for soman in the rat, Toxicol. Lett., 43, 175, 1988. Mazur, A., An enzyme in animal tissues capable of hydrolyzing the phosphorus–fluorine bond of alkyl fluorophosphates, J. Biol. Chem., 164, 271, 1946. Mehlsen-Sorensen, A., (R)-(+)-O-isopropyl-S-(trimethylam­ monioethyl) methylphosphonothioate iodide, Acta Crystal­ logr., Sect. B, B33, 1693, 1977. Millard, C.B., Lockridge, O., and Broomfield, C.A., Organophosphorus acid anhydride hydrolase activity in human butyrylcholinesterase: Synergy results in a somanase, Biochemistry, 37, 237–247, 1998. Mounter, L.A., Metabolism of organophosphorus anticholinesterase agents, in Handbuch der Experimentellen Pharmakologie. Vol. XV Cholinesterases and Anticholinesterase Agents, Koelle, G.B., ed., Springer, Berlin, Chap. 10, 1963. Noort, D., Benschop, H.P., and Black, R.M., Biomonitoring of exposure to chemical warfare agents: A review, Toxicol. Appl. Pharmacol., 184, 116–126, 2002. Nozaki, H., Aikawa, N., Fujishima, S., Suzuki, M., Shinozawa, Y., Hori, S., and Nogawa, S., A case of VX poisoning and the difference from sarin, Lancet, 346, 698–699, 1995. Ordentlich, A., Barak, D., Kronman, C., Benschop, H.P., De Jong, L.PA., Ariel, N., Barak, R., Segall, Y., Velan, B., and Shafferman, A., Exploring the active center of human acety1cholinesterase with stereoisomers of an organophosphorus inhibitor with two chiral centers, Biochemistry, 38, 3055, 1999. Polhuijs, M., Langenberg, J.P., and Benschop, H.P., New method for retrospective detection of exposure to organophosphorus anticholinesterases. Application to alleged sarin victims of Japanese terrorists, Toxicol. Appl. Pharmacol., 146, 156–161, 1997. Ramsey, J.C. and Andersen, M.E., A physiologically based description of the inhalation pharmacokinetics of styrene in rats and humans, Toxicol. Appl. Pharmacol., 73, 159, 1984. Raveh, L., Grauer, E., Grunwald, J., Cohen, E., and Ashani, Y., The stoichiometry of protection against soman and VX toxicity in monkeys pretreated with human butyrylcholinesterase, Toxicol. Appl. Pharmacol., 145, 43–53, 1997. Raveh, L., Grunwald, J., Marcus, D., Papier, Y., Cohen, E., and Ashani, Y., Human butyrylcholinesterase as a general prophylactic antidote for nerve agent toxicity, Biochem. Pharmacol., 45 (12), 2465–2474, 1993. Reiter, G., Mikler, J., Hill, I., Weatherby, K., Thiermann, H., and Worek, F., Chromatographic resolution, characterisation and quantification of VX enantiomers in hemolysed swine blood samples, J. Chromatogr. B, 873, 86–94, 2008. Reiter, G., Mikler, J., Hill, I., Weatherby, K., Thiermann, H., and Worek, F., Simultaneous quantification of VX and its toxic metabolite in blood and plasma samples and its application for in vivo and in vitro toxicological studies, J. Chromatogr. B, 879, 2704–2713, 2011. Scaife, J.F. and Campbell, D.H., The destruction of O,Odiethylaminoethyl phosphorothiolate by liver microsomes, Can. J. Biochem. Physiol., 37, 297–305, 1959. Sidell, F.R. and Groff, W.A., Intramuscular and intravenous administration of small doses of 2-pyridinium aldoxime methochloride to man, J. Pharm. Sciences, 60, 1224–1227, 1971.

57 Smith. J.R., Analysis of the enantiomers of VX using normal-phase chiral liquid chromatography with atmospheric pressure chemical ionization-mass spectrometry, J. Anal. Toxicol., 28(5), 390–392, 2004. Spruit, H.E.T, Langenberg, J.P., Trap, H.C., Van der Wiel, H.J., Helmich, R.B., Van Helden, H.P.M., and Benschop, H.P., Intravenous and inhalation toxicokinetics of sarin stereoisomers in atropinized guinea pigs, Toxicol. Appl. Pharmacol., 169, 249–254, 2000. Spruit, H.E.T., Trap, H.C., Langenberg, J.P., and Benschop, H.P., Bioanalysis of the enantiomers of (±)-sarin using automated thermal cold trap injection combined with two-dimensional gas chromatography, J. Anal. Toxicol., 25, 57–61, 2001. Sweeney, R.E. and Maxwell, D.M., A theoretical model of the competition between hydrolase and carboxylesterase in protection against organophosphorus poisoning, Math. BioSci., 160, 175–190, 1999. Sweeney, R.E. and Maxwell, D.M., A theoretical expression for the protection associated with stoichiometric and catalytic scavengers in a single compartment model of organophosphorus poisoning, Math. BioSci., 181, 133–143, 2003. Van der Schans, M.J., Benschop, H.P., and Whalley, C.E., Toxicokinetics of nerve agents, in Chemical Warfare Agents: Chemistry, Pharmacology, Toxicology, and Therapeutics, ed. Romano, J.A., Lukey, B.J., Salem, H., eds., Boca Raton, FL: CRC Press, chap. 5., 2008. Van der Schans, M.J., Lander, B.J., Van der Wiel, H., Langenberg, J.P., and Benschop, H.P., Toxicokinetics of the nerve agent (±)VX in anesthetized and atropinized hairless guinea pigs and marmosets after intravenous and percutaneous administration, Toxicol. Appl. Pharmacol., 191, 48–62, 2003. Van der Schans, M.J. and Langenberg, J.P., Effect of pretreatment with human butyrylcholinesterase scavengers on the toxicokinetics and binding of nerve agents in guinea pigs and marmosets, Final report Grant DAMD17-00-2-0032, 2005. Van der Schans, M.J., Langenberg, J.P., and Benschop, H.P., Toxicokinetics of O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothioate [(±)-VX] in hairless guinea pigs and marmosets—Identification of metabolic pathways, Final report Contract DAMD17-97-2-7001, 2000. Van Helden, H.P.M., Trap, H.C., Oostdijk, J.P., Kuijpers, W.C., Langenberg, J.P., and Benschop, H.P., Long-term, low-level exposure of guinea pigs and marmosets to sarin vapor in air: Lowest observable effect level, Toxicol. Appl. Pharmacol., 189, 170–179, 2003. Wang, Q., Sun, M., Zhang, H., and Huang, C., Purification and properties of soman-hydrolyzing enzyme from human liver, J. Biochem. Mol. Toxicol., 12(4), 213–217, 1998. Whalley, C.E., Lumley, L.A., McGuire, J.M., Miller, D.B., Robison, C., Muse, W.T. et al., Comparison of cyclosarin (GF) and sarin (GB) toxicokinetics and toxicodynamics following a single inhalation exposure in the guinea pig. Proceedings of the 46th Annual Meeting of the Society of Toxicology, Charlotte, NC, Toxicologist, 96(1), 31, 25–29 March 2007. Whalley, C.E., McGuire, J.M., Jakubowski, E.M., Miller, D.B., Mioduszewski, R.J., Thomson, S.A., Lumley, L.A., McDonough, J.H., and Shih, T.-M., A kinetics of inhaled and parenteral sarin (GB) following single and multiple sub-lethal exposures in the guinea pig, ECBC-TR-XXX, 2006.

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4 Organization for the Prohibition of Chemical Weapons (OPCW): History, Mission, and Accomplishments Karen L. Mumy,* William R. Howard,† Ariel Parker, Jonathan Forman, and Expert Opinion by Gwyn Winfield CONTENTS 4.1 Introduction...................................................................................................................................................................................59 4.2 History Leading to the Genesis of the Chemical Weapons Convention......................................................................................60 4.3 Genesis of the Organization for the Prohibition of Chemical Weapons....................................................................................... 61 4.4 Membership in the OPCW............................................................................................................................................................ 61 4.5 Structure of the OPCW.................................................................................................................................................................62 4.5.1 Conference of States Parties.............................................................................................................................................62 4.5.2 Executive Council.............................................................................................................................................................62 4.5.3 Technical Secretariat........................................................................................................................................................62 4.5.4 Subsidiary Bodies.............................................................................................................................................................62 4.5.5 Committees.......................................................................................................................................................................63 4.6 Global Mission..............................................................................................................................................................................63 4.6.1 Demilitarization...............................................................................................................................................................63 4.6.2 Nonproliferation...............................................................................................................................................................63 4.6.3 Assistance and Protection................................................................................................................................................64 4.7 Accomplishments..........................................................................................................................................................................64 4.7.1 Nobel Peace Prize 2013....................................................................................................................................................64 4.7.2 Syria Inspection................................................................................................................................................................64 4.7.3 Other Noted Accomplishments........................................................................................................................................65 4.8 The Next 20 Years.........................................................................................................................................................................65 4.9 Outside Expert Opinion: Gwyn Winfield.....................................................................................................................................65 Appendix: OPCW Member States as of 2018........................................................................................................................................67 References...............................................................................................................................................................................................68

4.1 Introduction Poisons have been used as warfare agents for thousands of years, with poisonous projectiles used as a combative strategy as far back as the first century BC (Mayor, 2009). While the use of such agents is not a new strategy, well-intended scientific and engineering advances, as well as the rapid evolvement of the chemical industry, have generated new, potent, and large amounts of chemicals capable of being used against humans to cause panic, harm, and death. While the majority of the world’s leaders and population would vehemently denounce the use of chemical weapons, the need for coordinating such a global stance brings into question who has the authority or responsibility and how such practices should be implemented. When it comes to the coordinated movement to rid the world of such chemical weapons, the

answer to the questions of who and how is the Organization for the Prohibition of Chemical Weapons (OPCW). The OPCW exists as an international body currently comprised of 192 member states with the shared mission of eliminating chemical weapons worldwide. The OPCW acts as the implementing body for the arms control treaty known as the Chemical Weapons Convention (CWC), which bans the development, production, storage, and use of chemical weapons. Ultimately, the OPCW strives for global disarmament, security, and stability while promoting economic development. The CWC and OPCW came into existence in the 1990s with the vision of developing verification strategies primarily designed for use with the traditional state actors toward the goal of eliminating chemical weapons across the globe. However, the rise of

* The views expressed in this article reflect the results of research conducted by the author and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the United States government. † I am a military service member or federal/contracted employee of the United States government. This work was prepared as part of my official duties. Title 17 U.S.C. 105 provides that “copyright protection under this title is not available for any work of the United States Government.” Title 17 U.S.C. 101 defines a U.S. government work as work prepared by a military service member or employee of the U.S. government as part of that person’s official duties.

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60 groups and organizations capable of deploying chemical weapons and falling outside state control created the need to adapt the OPCW to the changing milieu. Further, the existence of such groups unfortunately reiterates the continued need for the OPCW. Please note that the official website for the Organization for the Prohibition of Chemical Weapons served as the primary source for information pertaining to the OPCW for this chapter, and information/material not otherwise referenced or considered as historical fact can be found at www.opcw.org.

4.2 History Leading to the Genesis of the Chemical Weapons Convention The perception of chemical warfare as a potentially effective pathway to victory has stood as a serious impediment to the elimination of its threat and employment. Even the great luminary Leonardo da Vinci (1452–1519) is known to have described a method for delivering gaseous arsenicals to enemy ships (Szinicz, 2005). The noted naval officer Alfred Thayer Mahan (1840–1914), who has been referred to as “the most important American strategist of the 19th century” (Keegan, 2009), helped to derail U.S. support for ridding war of chemical weapons by arguing that there were currently no such chemical stockpiles and that the use of weaponized chemicals was, in fact, no more inhumane than any other accepted methods of warfare (Smart, 1997). Likewise, German chemist Fritz Haber, who would receive the 1919 Nobel Prize in Chemistry for the Haber–Bosch process he developed before World War I, was an advocate of the use of chemical warfare and directly oversaw its employment on the fields of Ypres in 1915 (Joy, 1997), arguing that it was a more humane form of warfare (Charles, 2009). This optimistic view had been previously expressed in 1862, when John W. Doughty provided U.S. Secretary of War Edwin Stanton with a plan for artillery shells that could deliver chlorine gas behind enemy lines. The plan included Doughty’s rationalization of what he viewed as the “moral question,” whereby he concluded that the use of chlorine gas would make the battlefield less bloody and lead to more conclusive results (Waitt, 1942). Although a direct response to this letter is not known, President Lincoln signed General Order 100 in 1863, which succinctly banned the use of such poisons (United States, 1899). From this, we may surmise that at least the high command of the United States was not swayed by the potential opportunity for bloodless battlefields and decisive victories through the use of chemical agents. The loathing of the art of chemical warfare, which led to a long history of belligerents seeking special controls through rules of war, has culminated in numerous efforts to rebut such actions. Centuries of attempted agreements and implementation strategies preceded the genesis of the CWC and the OPCW. The Strasbourg Agreement of 1675 represents an early attempt at restricting the use of chemical warfare, whereby France and Germany sought to ban the use of poisoned bullets, making this the first “international” agreement toward the movement (Frischknecht, 2003). It took nearly two centuries for next agreement to come to pass. In 1874, representatives from 15 European states met in Brussels following the Franco-Prussian War (1870–1871) to discuss the overall laws and customs of war. The resulting Brussels Declaration was an international agreement that included the prohibition of the use of poison and poisoned weapons, as well

Chemical Warfare Agents as basic rules regarding the treatment of civilians and prisoners of war and the laws of belligerent occupation. Unfortunately, not all those involved were willing to consider this as a binding convention, and it was therefore not ratified. Regardless, it was still a significant step in the progression toward the effort and helped to lay the groundwork for further discussions and agreements. The turn of the 20th century saw the generation of the third international agreement at a peace conference held in May 1899 in The Hague. Those in attendance at the convention agreed to refrain from using poisonous gas-containing projectiles, and the resulting 1899 Hague Regulations agreement was born and signed in July 1899. The 1899 Hague Regulations were comprised of three treatises (conventions) and three declarations, one of which prohibited the use of projectiles for the spreading of poisonous gases. This declaration was endorsed by all major powers except the United States. There was a second Hague conference, held in 1907, which offered few major advancements over the 1899 Convention. While the 1899 and 1907 Hague Regulations were another step in the right direction, they ultimately failed in their efforts to prevent the use of suffocating gases, evidenced by the use of chemical weapons during World War I, which resulted in a staggering number of casualties and fatalities. The horrors unleashed at Ypres would set the tone for all future debates. The decisive victories did not materialize, with even Fritz Haber conceding that the chemicals deployed in World War I were not effective once the element of surprise had dissipated and enemy troops were prepared for the attacks (Smart, 1997). The chemical warfare practices of World War I spawned another discussion, the 1922 Washington Conference, leading to yet another prohibition on chemical weapons that was never implemented. Three years later, the 1925 Geneva Protocol (the “Protocol for the Prohibition of the Use in War of Asphyxiating, Poisonous or Other Gases, and of Bacteriological Methods of Warfare”) was signed to ban the practice of chemical, as well as biological, methods of warfare (Szinicz, 2005). The 1925 Geneva Protocol became effective in February 1928. Even though many countries were willing to sign the Geneva Protocol, they did so while maintaining that they reserved the discretion to use chemical weapons against countries that either had not joined in the Protocol or used chemical warfare against them. Since the Protocol became effective in 1928, many of these countries have since reneged on their initial reservations and became willing to participate in a complete ban on chemical and biological warfare. It should be recognized that while the 1925 Geneva Protocol prohibited the use of such materials, it did not define the banning of the development, production, or storage of such chemical and biological warfare materials, thereby requiring later agreements to fully cover the full spectrum of prohibited activities: namely, the 1972 Biological Weapons Convention (BWC) and the most successful 1993 Chemical Weapons Convention (CWC). Due to the recognition of the advancements in the fields of chemistry, toxicology, and science in general, or perhaps the memories of the events at Ypres during World War I, the World War II era brought the anticipation and fear of chemical warfare use. Troops were better prepared, just as they still are today, with gas masks and training on how to recognize and mitigate chemical warfare attacks. While chemical weapons were developed and used in inter-war periods, none were used in battles in Europe at the time. Although they were not used in battle, the fate of the

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Organization for the Prohibition of Chemical Weapons (OPCW) chemicals generated and stockpiled for warfare use was still of concern. In the years that followed, many countries began to view chemical warfare through a different lens, in that the weapons were of minimal value or not worth the risk of retaliation. The ban against chemical weapons was gaining more ground. In 1968, a Geneva disarmament conference took place in Sweden, where the ban on both chemical and biological weapons was an agenda item leading to further discussion. Soon thereafter, the subjects of chemical and biological warfare were divided to be dealt with separately, and the Biological Weapons Convention, which prevented the development, production, and stockpiling of biological warfare agents, was ultimately signed in 1972 and became effective in 1975, leaving behind, but setting the stage for a similar treaty for, chemical weapons. Throughout the 1970s, discussions on the issues of chemical weapons continued at the Geneva Conference, and those discussions became more focused, including the need to consider precursors to chemical warfare agents as well as establishing the means by which oversight (e.g., inspections) for the ban against development, production, and stockpiling of chemical weapons would occur. Membership of the Geneva Conference increased to 40 in 1978; the decision was made to develop a Chemical Weapons working group, who continued to elaborate on the draft treaty, and the United States contributed a draft describing verification measures, such as mandatory inspections. Meanwhile, the fire under these activities was fueled by Iraq’s use of chemical weapons against Iran. By the mid-1980s, the chemical industry had been brought into the discussions, and it became clear to all those involved that international verification would be required for success of the ban, and that this would involve inspections of facilities, industrial and military alike. Trial inspections began in the late 1980s. By the early 1990s, there was an impetus to complete the Chemical Weapons Convention, and the United States and Soviet Union had already signed an agreement between the two countries whereby they both agreed not to produce chemical weapons, to reduce their stockpiles by 80%, and to greatly limit their holdings in the future. While this agreement was signed, it never came into force, but it did demonstrate that two powerhouse countries were willing to work together toward the effort. Unfortunately, this was not reflected by all, with a number of Arab countries viewing a ban on chemical weapons as a means toward nuclear disarmament and developing countries questioning the benefit of their participation. Provisions were added to include chemical warfare victim assistance, exemption from routine inspections for certain divisions of the chemical industry, and text regarding obligations for abandoned chemical weapons. Additionally, a promise was made on behalf of a number of developed countries to review export controls and chemical trade barriers. With many hurdles having been crossed, final revisions of the convention were made in 1992 and delivered to the Conference on Disarmament. It was adopted by the Conference, passed to the UN General Assembly, and opened for signature in January 1993. Support was overwhelming, with 130 states having signed the CWC in the first 2 days alone. The CWC was written such that it would enter into force 180 days following the 65th country’s ratification of the Convention. This timeline was initiated when Hungary, as the 65th country, ratified the treaty in late 1996. In the 180 days following Hungary’s ratification, an additional 22 countries ratified the treaty, and the CWC entered into force on April 29, 1997, with 87 states parties.

4.3 Genesis of the Organization for the Prohibition of Chemical Weapons With the CWC having been signed and many states willing to participate, the urgent need for a governing body was obvious. A “Preparatory Commission” was developed for what would become the Organization for the Prohibition of Chemical Weapons (OPCW), and the first meeting of the Commission was held in February 1993 in The Hague, the Netherlands, which became the future seat of the OPCW. The Preparatory Commission’s task was to prepare for the implementation of the CWC, including generating procedures for operations, such as the verification regime, drafting the program and operating budget of the OPCW, and establishing the infrastructure and logistics for the OPCW Secretariat. They accomplished these tasks through two primary working groups, one for administrative responsibilities and one for verification and technical purposes. The Preparatory Commission accomplished a great deal, to include devising solutions to verification issues, setting up the OPCW Laboratory and Equipment Store, developing the plans for recruitment and training of inspectors, constructing draft documents, policies, and regulations, and arranging for the OPCW Headquarters building in The Hague, the Netherlands. Once these items were accomplished, they had to be transferred to the OPCW. Not surprisingly, there were several issues that could not be resolved by the Preparatory Commission, and these were carried over for resolution to the OPCW, which took over from the Commission in 1997, when the CWC entered into force. As of the writing of this chapter in 2018, the OPCW has 192 states parties, recently celebrated its 20th anniversary in 2017, and continues to strive toward the global elimination of chemical weapons.

4.4 Membership in the OPCW Since 1997, the OPCW has been charged with implementing the CWC: verifying the destruction of all chemical weapons; monitoring chemical production to ensure that new weapons do not emerge; aiding and protecting against chemical threats to member states; and fostering international cooperation in realizing the goals of the CWC. A state is able to become a state party (i.e., a member of the OPCW) by any of three separate mechanisms: ratification, accession, or succession. Documentation of ratification or accession can be deposited with the Depositary of the Convention (the Secretary-General of the United Nations), with the treaty entering into force 30 days following the day the ratification or accession was deposited. Given that the OPCW is the implementing body for the CWC, which is of infinite duration, membership in the OPCW does not have an assigned term per se and is only subject to the possibility of withdrawal. Originally, the OPCW was comprised of the 87 states parties that had initially ratified the CWC as international law, with that number climbing to 192 by the writing of this chapter (per the latest Status of Participation report dated October 17, 2015 [OPCW, 2015a]). To say that the organization has impressive participation would be an understatement. The states parties of the OPCW embody ~98% of the world’s population and area as well as 98% of the global chemical industry. The United Nations strongly encourages the remaining

62 states not party to the CWC to join to unify the movement against chemical weapon development, production, storage, and use. Current membership in the OPCW as of 2018 can be found as an Appendix to this chapter.

4.5 Structure of the OPCW To ensure optimal functionality, the OPCW is separated into primary Organs and Subsidiary Bodies, each with a defined role. Primary oversight is provided by the Conference of the States Parties, and the role of each Organ/Body is described later. Please note that the official website of the OPCW served as the primary reference for this information; please see the website for additional information and sources: www.opcw.org.

4.5.1 Conference of States Parties The primary body of the OPCW is the Conference of the States Parties. The Conference is responsible for overseeing the implementation of the CWC and has the authority to act in the promotion and for the purposes of the CWC. The Conference accepts recommendations and takes questions or concerns on anything with regard to the CWC. Each member state contributes one vote to the Conference, which meets annually in The Hague, the Netherlands for 1 week, although special sessions can be organized conditionally. It is the responsibility of the Conference of the States Parties to ensure compliance with the Convention, make decisions with regard to the program, budget, and required financial contributions to be made by the states parties, approve the annual report of the OPCW, elect the Executive Council members, and appoint the directorgeneral. The Conference is also charged with ensuring international cooperation on chemical activity and reviewing the advancements in science and technology that may impact the CWC. The Conference takes decisions on procedural questions by voting at Conference sessions when a quorum is met, as defined by having a majority of the OPCW members present, with each member having one vote. If a consensus is not reached, a 24 hour deferment is instated by the chairman of the Conference to make efforts toward a consensus. If this is still unattainable, the Conference reserves the right to take a decision with a two-thirds majority of members present. The Conference oversees the function of the Executive Council and the Technical Secretariat.

4.5.2 Executive Council The OPCW is steered by an Executive Council of 41 member states elected by the Conference for 2 year terms. The CWC stipulates that the Executive Council be equally represented geographically as well as representing the importance of the chemical industry and political and security interests. It is the right of each State Party to have an opportunity to serve on the Council, with regional groups being represented according to a formula described in the CWC, with Africa and Asia each being allotted nine members, Eastern Europe allotted five members, seven members for Latin America and the Caribbean, and 10 members for Western Europe and other states. One additional state party is designated in rotation from Asia or Latin America and the Caribbean.

Chemical Warfare Agents The Council carries out functions designated by the CWC and any functions delegated to it by the Conference. These tasks include taking measures in situations of non-compliance by a state party and recommending actions to the Conference; considering/submitting the draft OPCW program and budget to the Conference; drafting the OPCW’s report on the CWC implementation status and the Council’s report on performance; and making recommendations regarding the appointment of the director-general. The Council maintains a great deal of executive power regarding the implementation of the CWC, such as agreements regarding assistance and protection against chemical weapons, implementation of verification activities, and other agreements on behalf of the OPCW. The Council is also involved in resolving compliance concerns and dealing with requests for assistance and protection against the use/threat of chemical weapons. It is also provided with special power in circumstances where there may be a dispute between states parties regarding the CWC.

4.5.3 Technical Secretariat The Technical Secretariat assists the Conference of States Parties and the Executive Council while also performing verification duties outlined in the CWC. Its responsibilities include submitting draft budgets to the Council; preparing annual reports on the implementation of the CWC; facilitating communications and verification agreements between member states; information dissemination to the public; and on-site inspections. The Technical Secretariat is comprised of a director-general, as the head and chief administrative officer, and 500 members, who may serve for up to 7 years. The members come from over 80 states parties and make up nine divisions, functioning as inspectors and scientific, technical, and other personnel. Approximately 60% of the Secretariat’s workforce is devoted to the Inspectorate and Verification divisions.

4.5.4 Subsidiary Bodies There are also two required subsidiary bodies, as mandated by the CWC: the Commission for the Settlement of Disputes related to Confidentiality (“Confidentiality Commission”) and the Scientific Advisory Board (SAB). The Confidentiality Commission currently has 20 members appointed by the Conference of the States Parties, with each state party able to nominate one candidate. Nominations are based on competence, integrity, and background relevant to the Confidentiality Commission (such as dispute resolution, specific provisions of the CWC, chemical industry, military or data security, or legal issues). The SAB was established to enable the director-general to seek expert advice on science and technology. Each year, the SAB elects a chair and vice-chair, and the remaining SAB is comprised of 25 subject matter experts from OPCW member states, with each member able to serve a maximum of two consecutive 3 year terms. The inaugural meeting of the SAB took place in 1998, and it meets once or twice annually. The SAB reports to the directorgeneral, who then submits its reports, alongside his or her own response, to the Executive Council. Every five years, the SAB prepares a larger report for submission to the review conference.

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Organization for the Prohibition of Chemical Weapons (OPCW) For each session, the Conference appoints a General Committee to address administrative or organizational issues, such as agenda items and the establishment of additional committees with assignment of tasks. The Conference is also authorized to establish additional subsidiary organs, as necessary, which they have chosen to invoke on occasion.

4.5.5 Committees The Credentials Committee branches directly from the Conference of the States Parties and is appointed at the start of each session of the Conference as proposed by the chairman of the Conference. This 10-member committee examines the credentials of all representatives for that Conference session and makes the decision as to whether the representatives will be permitted to participate in the session. On the OPCW’s inception in 1997, the Committee of the Whole was the main committee of the Conference, for which a chairperson was elected at the start of each session. This committee served as the primary forum for discussion of issues that were not yet ready for a decision to be made. This provided an opportunity for intersessional consultation and discussion in a forum whereby all members in attendance at the session could participate. If/when the Committee of the Whole resolved the issues, it would make recommendations to the Conference. In 1999, this task and responsibility were transferred from the Committee of the Whole to the Council, with participation open to all member states. While the Committee of the Whole no longer functions in the role of intersessional discussion for unresolved issues, it maintains its role during Conference sessions.

4.6 Global Mission Serving in the role of the global watchdog against chemical weapons, the OPCW is the international cooperative that enforces the steps to rid the world of chemical weapons and instead, to use chemistry solely for peaceful purposes, as outlined in the CWC. With representatives from nearly every country in the world, the OPCW’s primary goal is to eliminate chemical weapons worldwide. Tasks toward this goal can be subdivided into the processes of demilitarization; nonproliferation; and assistance and protection. For more than two decades, the OPCW has promoted peaceful chemistry and global unity against chemical weapons for the ultimate goal of contributing “to international security and stability, to general and complete disarmament, and to global economic development.” The OPCW’s programs have four aims toward implementing the CWC: (1) ensure a clear regime for verifying that chemical weapons are destroyed and prevent opportunities for re-emergence, all the while ensuring that national security and proprietary interests remain protected; (2) offer protection and assistance against chemical weapons; (3) encourage only peaceful practices for chemistry; and (4) strive for global membership in the OPCW through cooperation and the building of national capacities.

4.6.1 Demilitarization Demilitarization encompasses the destruction of chemical weapons per the established destruction timeline; maintaining

destruction technologies; and accounting for environmental concerns and provisions during destruction, as outlined in the CWC. These processes, guided by the OPCW and enacted by member states, require the destruction of chemical weapons that each member state owns or possesses (including weapons they may have abandoned in a foreign country) and the destruction of all facilities involved in the production of chemical weapons. The first step in this process is state party declaration of any existing chemical weapons and materials or equipment that could be used toward production, verified by the OPCW through inspection. Following declaration, the state party must also submit a destruction plan and schedule, in accordance with the CWC, and destruction (with verification) is meant to occur within a given timeline. The destruction of chemical weapons will differ based on the type of chemical weapon, such as the chemical fills for bombs, spray tanks, missiles, landmines, and artillery rockets, as categorized by the CWC. States parties are also responsible for the declaration and destruction (or conversion) of chemical weapons production facilities (CWPF). The CWC defines CWPFs as “any equipment or buildings housing such equipment, designed or used since 1946 to produce chemicals for chemical weapons purposes (in amounts exceeding specified thresholds) or to fill such chemicals into munitions or other devices.” As with destruction timelines, there are also conversion timelines and reports that the state party must submit for CWPFs, and this process is also open to inspection by the Secretariat. The OPCW also verifies the irreversibility of the destruction process. The CWC stipulated that the states parties that signed and ratified the treaty in 1997 had to destroy their arsenals by 2007, with extensions to 2012, if necessary. The process of destruction proved difficult, complex, and expensive, and only three states parties were able to complete destruction prior to 2012. Current timelines vary based on the category of chemical weapon, with two of the three categories of chemical weapons being destroyed within 1 year and the remaining category, with the more challenging and explicitly harmful (Schedule 1) chemical warfare agents, being destroyed within 10 years (with a percentage of progress being made within 3, 5, and 7 years).

4.6.2 Nonproliferation Nonproliferation of chemical weapons is an emphasis of the CWC, whereby each state party agrees never to “assist, encourage or induce, in any way, anyone to engage in any activity prohibited” under the CWC. This is meant to ensure that the spirit of the CWC is maintained and chemical weapons will never again be produced. It is important to note that this does not suggest that states parties should not value or use chemistry, or continue to make chemical advancements; however, any chemical, or its precursor, that is developed, produced, acquired, or retained, even if deemed toxic, must not be used for purposes prohibited by the CWC (i.e., chemical warfare). To guarantee this, states parties shall be subject to verification measures to deter any potential mal-intentions and build confidence that states parties are meeting their obligations. Verification measures, carried out by the Technical Secretariat, are performed based on the schedule of chemicals declared by the state party within the first 30 days of the CWC taking effect for that state party. Schedule 1 chemicals are those that have

64 been or may be used as chemical weapons and do not hold value for peaceful purposes; Schedule 2 chemicals are those that are precursors to chemical weapons, or may hold potential for use as chemical weapons, but have many other peaceful or commercial uses (pesticides, flame-retardants, etc.). Schedule 3 chemicals include those that can be used to produce, or can be used as, chemical weapons but are widely used for peaceful purposes, such as those found in paints, plastics, and coatings. In addition to building confidence and ensuring good intentions, inspections aim to verify that the state party’s declarations of chemical weapons and facilities are correct. At the time this chapter was written, the OPCW had performed 248 inspections at Schedule 1 facilities, 657 at Schedule 2 facilities, and 394 at Schedule 3 facilities since 1997. The OPCW had also conducted 1300 inspections since 2000 at discrete organic chemical (i.e., chemicals not specifically included in Schedule 1, 2, or 3) facilities.

4.6.3 Assistance and Protection Member states of the OPCW have agreed to provide assistance and protection to any other member state that may be under the threat of chemical weapons or warfare, and state parties may formally request protection assistance from the OPCW if they believe that chemical weapons have been used against them or perceive a threat in opposition to the CWC. Protection against chemical weapons encompasses physical protection (body or respiratory protection); medical protection (pretreatment, therapy); and detection and decontamination. The Executive Council immediately processes requests, investigates, and generates a report before granting or denying any request. The Technical Secretariat also plays a role in protection programs and will work with assistance requests as directed by the Executive Council. In the case of national program development, the Secretariat disseminates chemical protection information and advises and assists states parties in implementing their programs, including providing training to national first responders. Individual member states can offer aid in the form of equipment and personnel assistance in the event of a chemical weapon threat, and states parties can independently choose to negotiate with one another. States parties, for political or security reasons, may not be able to directly assist one another but may do so through the OPCW, such as through donations to the Voluntary Fund for Assistance. Additionally, the OPCW has developed a “Practical Guide for Medical Management of Chemical Warfare Casualties” to assist medical professionals in their care for chemical warfare victims.

4.7 Accomplishments Since its institution in 1997, membership of the OPCW has expanded to include nearly 98% of the world, facilitating international cooperation in the execution of its duties. The OPCW has overseen significant eradication of chemical weapons worldwide and has established thorough methods of classification, inspection, demilitarization, and disposal of chemical warfare agents.

Chemical Warfare Agents

4.7.1 Nobel Peace Prize 2013 In October 2013, when the organization was only 16 years old, the OPCW’s efforts were rewarded with the Nobel Peace Prize for its work toward the elimination of chemical weapons. At that time, the OPCW had 190 member states, had conducted 2500 inspections, and had verified that >80% of all declared chemical weapons had been destroyed.

4.7.2 Syria Inspection In the same year as the OPCW received the Nobel Peace Prize for its strides toward abolishing chemical warfare, a sarin gas attack took place in Syria that resulted in the death of more than 1000 civilians. This served as a brutal reminder of the impact of chemical weapons and that elimination was not yet complete. Amidst the backdrop of the Syrian Civil War, which had been ongoing for 2.5 years at this point, the Assad regime was suspected of launching sarin on Eastern Ghouta near the Syrian capital Damascus. Allegations of previous chemical attacks by the Assad regime were reported in December 2012 and March 2013, each with casualties that were orders of magnitude lower than in the sarin gas attack in August 2013. Syria was not yet a state party for the OPCW when the alleged chemical attacks were perpetrated, prompting the Syrian government and the international community to request inspections. The United Nations coordinated with the OPCW and the World Health Organization (WHO) to perform inspections in August 2013, just days before the sarin gas attack (Fischer et al., 2016). The Syrian Arab Republic joined the OPCW, and the CWC entered into force for Syria on October 14, 2013. Two days later, the OPCW–United Nations Joint Mission, in effect until September 30, 2014, was created to eliminate all Syrian chemical weapons. The Syrian chemical weapons destruction timeline was first proposed in September 2013 and outlined the initial declaration of chemical weapons by the Syrian Arab Republic by October 27, 2013; initial on-site inspections by the Technical Secretariat were completed by November 2013; the complete destruction of production and mixing/filling equipment had occurred by November 2013; and in 2014, the OPCW had overseen the destruction of 97% of Syria’s declared chemical weapons (Eaves, 2014). Demonstrating the cooperation of member states within the OPCW, fellow members Denmark and Norway were especially helpful, providing transportation of the chemical weapons. The United Kingdom, the People’s Republic of China, and the Russian Federation provided security during transport. The Priority 1 chemical weapons removed from Syria were mainly destroyed at sea via field-deployable hydrolysis thanks to the Department of Defense of the United States, a fellow member, having provided two hydrolysis systems. The Priority 1 chemical weapons were destroyed with 99.9% effectiveness and negligible release into the environment, while the Priority 2 chemicals and post-hydrolysis Priority 1 byproducts were destroyed at land facilities, having been contracted to companies in the fellow member states of Finland, the United States, and Germany, and the United Kingdom’s government destroyed a portion of the chemical stockpile. The latest progress report on the destruction of Syrian chemical weapons, dated September 2017, indicated that 25 out of 27 declared

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Organization for the Prohibition of Chemical Weapons (OPCW) CWPFs were verified as destroyed by the Technical Secretariat during inspections. Sadly, at the time of writing this chapter, the OPCW has deployed a fact-finding mission (FFM) team to the Syrian Arab Republic to investigate the events surrounding the alleged use of chemical weapons in Douma in April 2018, which may have affected 500 people, killing many.

4.7.3 Other Noted Accomplishments The OPCW reported that as of October 2017, over 96% of the world’s chemical agents (69,610 of 72,304 metric tons) have been destroyed with verification. Six states have completed the destruction of their declared chemical weapons, and 91 of 97 of the declared CWPFs have been destroyed or converted for use in peaceful chemistry. The OPCW marked the complete destruction of chemical weapon stockpiles in Libya in January 2018 and the complete destruction of chemical weapon remnants in Iraq in March 2018.

4.8 The Next 20 Years This book goes to press in the year following the commemoration of the OPCW’s 20th anniversary and 20 years in force of the Chemical Weapons Convention (OPCW, 2017a). There are many accomplishments to highlight: more than 96% of declared chemical weapons stockpiles have been verifiably destroyed; only four of the world’s states remain outside the Convention; more than 6000 inspections have been carried out (amounting to more than 890 years of “inspector time”); and a Nobel Peace Prize has been awarded (The Nobel Peace Prize 2013, 2014). Looking ahead, the complete destruction of declared stockpiles is in sight, but the operating environment of the OPCW has changed significantly over its existence, and the Organization’s activities have been progressively shifting from the disarmament of chemical weapons to preventing their re-emergence. Perhaps the most visible sign of the changing operating environment came with the 2013 United Nations–led mission in the Syrian Arab Republic (United Nations Secretary-General, 2013), a mission followed by continued and increasing activities of the Technical Secretariat involving non-routine inspection, verification, and technical assistance activities in Syria, Libya, and Iraq (Timperley and Tang, 2017). The objectives of the Chemical Weapons Convention are guided by its preamble, in which states parties declare their determination “for the sake of all mankind, to exclude completely the possibility of the use of chemical weapons, through the implementation of the provisions of this Convention” (OPCW, 1997). As noted in OPCW’s “Vision Paper,” this determination suggests that OPCW should “contribute, as a treaty-based international organization, to the disarmament of chemical weapons, to preventing their re-emergence, to providing assistance and protection against them, to supporting national implementation of the Convention, and to facilitating peaceful uses of chemistry through verification, capacity development, or engagement activities” (OPCW, 2015b). In November 2018, the Fourth Review Conference of the Chemical Weapons Convention will take place, where the states parties will set strategic directions for the 5 year period leading

up to the Fifth Review Conference in 2023. Looking ahead, there are assumptions that the verified elimination of currently declared chemical weapons stockpiles will remain on track for completion and that the verified elimination of non-stockpile chemical weapons (recovered abandoned and old chemical weapons) will continue. Other issues that the states parties must consider include retaining chemical weapons–related knowledge and expertise, combined with a rapidly deployable surge capacity in support of assistance and protection (OPCW, 2016a), and contingency operations; the possibility that new states parties could join the Convention as chemical weapons possessor states; the increased use or threat of chemical weapons by non-state actors (especially terrorist groups) (OPCW 2015c, 2016b); advances in science and technology that are increasingly driven by transdisciplinary scientific communities, unprecedented global diffusion of scientific knowledge (McKenzie, 2017), and international scientific collaboration (Nature Index, n.d.); as well as the chemical industry integrating new technologies into routine use, requiring a broadening of familiarity with production equipment and processes. The OPCW’s Medium-Term Plan (OPCW, 2016c) and its “Vision Paper” (OPCW, 2015b) provide further details and insights into all of these issues. Lessons learned from the missions in the Syrian Arab Republic (Trapp, 2015; United Nations Secretary General, 2015) and considerations from the OPCW’s open-ended working groups (OEWGs), such as the OEWGs on future priorities (OPCW, 2016d) and terrorism, for example OPCW, 2017b,c), and subsidiary bodies, such as the Scientific Advisory Board (OPCW, 2015d) and the Advisory Board on Education and Outreach (for example, OPCW, 2017d), will provide additional inputs for states parties to consider. In the face of the OPCW’s objectives and its current and future challenges, investments in a widening range of activities related to verification, capacity development, stakeholder engagement (Ballard and Forman, 2016), and the governance of the Organization must be considered (OPCW, 2015b; for an example of viewpoints that do not necessarily represent those of the OPCW, see Foreign & Commonwealth Office, 2015). The OPCW’s “Vision Paper” places verification to ensure confidence in compliance at the heart of the Organization’s work, noting that its methods and practices will need to adapt to changing realities, maintain a viable industry verification regime, be prepared for non-routine inspections, and enhance analytical capabilities (OPCW, 2017e). Verification must also be complemented by capacity development to support the norms of the Convention and promote peaceful and beneficial uses of chemistry. National implementation of the Chemical Weapons Convention and assistance and protection measures against chemical weapons must also remain core business to achieve these goals. Enhancing chemical security will likewise increase in importance (OPCW, 2016e, 2017f). Ensuring that the OPCW remains fit for purpose and leads the way in preventing the re-emergence of chemical weapons requires that all these issues be given due consideration.

4.9 Outside Expert Opinion: Gwyn Winfield Perhaps more for its mission statement than for its accomplishments, the OPCW was awarded the Nobel Peace Prize in

66 October 2013. Later that month, Syria became the 190th member state of the CWC. Earlier that year, the use of chemical weapons in Syria had provided the OPCW with an extremely serious challenge: OPCW inspectors already in Syria at the time of the August 21 attack were then called on to investigate what the OPCW documented to be a sarin gas attack that killed more than 1000 people (Lally, 2013). Although at first glance, it seems ironic that the Nobel Peace prize was awarded to the OPCW in a year when the system was shown to be ineffective at preventing chemical attacks, and the very nation where the attacks occurred joined the CWC, this series of events may very well have provided a further rationale for the relevance of the treaty and the OPCW. Verification of the event as a chemical warfare attack would have been extremely difficult without an agency like the OPCW. Previously, the OPCW had been a chemical warfare agent auditor, and that had worked nicely in a wide variety of countries; from Albania to Russia, missions had been undertaken and delivered with a large amount of success. It was only with the civil war in Syria that the expectations of what the OPCW was supposed to do in such circumstances became shock tested. These events in Syria suddenly reminded people of the need for a roster of experts who can do chemical verification and investigation missions. This should not have been a novel realization, as previous missions in Iraq had shown what could happen to a possessor of chemical warfare agents and the mission that might follow. Iraq had, in reality, only provided a benign idea of the mission of the chemical weapons inspector, and that had been difficult enough. Saddam Hussein used dual-use technology and all the skill a state can use to hide his nefarious activity and did so with a modicum of success. Inspectors were frequently frustrated, and despite protestations of openness, there was always a feeling that something wasn’t right. Yet despite his chicanery, there was an opportunity and a legal framework that gave inspectors the right to enter any institution. This meant, in time, that either the inspectors would find what was being hidden or the United Nations Security Council would grow tired of his obfuscation and do something. The same model does not apply in Syria, where the ongoing civil war means that the Syrian government can claim, with some legitimacy, that some areas are too dangerous for inspectors to visit. Equally, the geo-political climate in the 1990s was such that no-one on the United Nations Security Council wanted to support Saddam, but flash forward 20 years and that is not the case, because client states are back. The Iraq inspectors were, largely, of the old school and came from institutions that had historical experience of what was needed to create a working chemical weapons capability and hide it from their opponents. Some of them, such as the UK’s David Kelly, had also gained experience of how offensive laboratories on both sides of the Iron Curtain (albeit biological ones in Kelly’s case) were set up and hidden. This provided them with the necessary skill to interrogate scientists and bureaucrats and to understand how you can hide capability. By the time we flash forward to Libya or Syria, much of that experience had retired, and institutions had long forgotten their offensive past. Not only did this mean that individuals going through these institutions were unable to partake of the same experience, but thanks to the cessation of the Cold War, there were fewer people going through these centers.

Chemical Warfare Agents This was a consequence of the larger draw down worldwide in offensive and defensive CBRN research driven by budgets and arms control policies. Scientific institutions, such as U.S. Army Edgewood Chemical Biological Center (ECBC), had long been aware of the impact of the peace dividend in the 1990s (see CBRNe World 2014-3, interview with Joseph Wienand) but were still not able to recruit and train inspectors on the off chance they would be needed. All this combined meant that there was simply less talent available to insert into the roster of experts needed for the current mission set. This is not to suggest that there are no highly gifted scientists out there who are able to understand the technology, but what is lacking is individuals who understand the potential methods of obfuscation and are prepared to go into austere, even kinetic, environments to uncover them. This did not escape the notice of the UN Office of Disarmament Affairs, which released its second lessons learned on Syria,* focusing on the type of individual needed for the task. This boiled down to a politician scientist with experience of working in a war zone. This pushed the ideal individual to a rare expert even among the traditional chemical, biological, radiological, and nuclear (CBRN) defense countries and further raised the risk that these individuals will be targeted as potential intelligence operatives by the nation being investigated. When you consider that Ban Ki Moon previously decided not to use UN Security Council nationals, who generally come from countries with well-established CBRN defense programs, in Syrian investigations, it makes these individuals even more elusive. Additionally, the OPCW has anecdotally admitted that there has been some naiveté among the latest generation of recruits. These have been horrified by the length of the mission, the type of hardship that they might face, and the potential risk to their safety and reputation. Improvements in standards of living worldwide have meant that what is expected and what is offered are worlds apart in these missions. This has an impact on recruitment, as talented individuals join the OPCW but then balk at missions when the reality sinks in. The OPCW was founded to safely destroy existing chemical weapons stocks and to try to ensure that states parties didn’t create new ones. Article 9 and 10 missions, the ability to conduct challenge inspections and so on, are tangential to this. The challenge missions that were envisaged were state on state and would likely involve just verification that a weapon had been used in a binary conflict, where the perpetrator of the deed would be obvious. When the possessors and users of chemical weapons include terrorist organizations or military units not under state control, the problem is of a different and largely unplanned-for nature. The legal framework that needs to be agreed to by all member states on how contested use could be proved is simply not there. The 2013 Ake Sellstrom Syrian mission managed to mask this flaw and instead, delivered a “perfect storm”: a team of skilled individuals on the ground in a timely manner that had a strict, scientific mandate. What they delivered was scientifically robust, and while some states parties might not have been happy about what could be surmised about the individuals behind it, the fact

* https://unoda-web.s3-accelerate.amazonaws.com/wp-content/uploads/ assets/publications/more/syrian-ll-report/syrian-ll-report-2015.pdf

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Organization for the Prohibition of Chemical Weapons (OPCW) that the mission offered no conclusions on blame allowed the team’s findings to escape censure and criticism. Subsequent attempts, such as the fact-finding mission (FFM), were not able to repeat the Sellstrom deliverables. States parties saw what could be intimated in a scientifically robust report, and their “lesson learned” was not to let this happen again. Despite the Declaration Assessment Team drawing some overt conclusions about the state of Assad’s chemical weapons obfuscation, no challenge inspection has been demanded by other states parties, and if there can’t be one now, it seems difficult to find a time in the future when there might be. As has happened in non-CBRN UN Security Council resolutions, Russia decided against anything that would negatively impact its client state, and in a closed meeting of the UN Security Council, shut down the Joint Investigative Mechanism (JIM).* Russia, in principle, thinks something like the JIM is a good idea, but it seems unsure what it should look like. The trouble springs from the fact that a permanent JIM, which would sit somewhere between the UN Security Council and the OPCW, would complicate the lives of many nations, especially if agents, such as riot control agents, were being used by state parties to heavyhandedly quell an unruly population. A “quick trigger” team, which would have far-reaching powers to investigate potential usage, would not go down well with many UN Security Council members. The option of re-creating a JIM every time it is mutually agreed that one is needed is also not a popular choice. JIMs run the risk of being involved in whatever geo-strategic politics are currently at play in that conflict and as such, are likely to suffer the same fate as the recent one. To paraphrase Voltaire, if JIM didn’t exist we’d have to invent it, but much like modern metaphysics, we have yet to properly nail down what it should look like. Despite the myriad problems the CWC faces, there is little appetite for its revision. The CWC might be flawed, but even in its current form, it offers the prospect of a world free from the threat of chemical weapons. The existential challenge that the OPCW faces is one of relevance if there has been a breach of the convention. This has been noted by the director-general himself, who lamented that Syria’s behavior had “[flown] in the face of every civilized norm and is in direct violation of the convention.”† Indeed, it could be argued that Syria’s accession to the CWC has proved to be its salvation. While it remained “outside the tent,” there were clear, unilateral options on the table for its disarmament. Once it moved “inside the tent,” there were rules that needed to be followed. Every time a transgression has been assumed, the rule book has been consulted, and all it says is “try again.” The inability of the OPCW to grapple with a rogue member cannot have been missed by states parties that might choose to keep CWA, and this inadequacy breeds opportunity. This combination of a shortage of individuals who have the necessary skill set to perform a near-impossible mission and a strategic disinterest in arms control poses long-term challenges for the OPCW. Yet, the current political climate in both the Middle East and the South China Sea region is likely to remain volatile, and with a lot to play for, it is quite likely that the

interpretation of treaties that don’t satisfy the national interest will be ignored for gains on the ground. The greatest negative outcome of the Obama presidency may be that red lines can be crossed if you have the right friends. This supports our previous contention that the Syrian conflict has made the OPCW more relevant than ever. It is even better for states that decide that it is in their interest to create a chemical strategic deterrent, in the same way as Syria did, to join the OPCWW. It is easier to lie, cheat, and work the system than to have to deal with the kinetic option that might otherwise be coming your way. If Syria does become a trope and multipleagency conflicts become more frequent, then having an agency that is able to arbitrate, legislate, and investigate becomes more essential. Even if these major geo-political issues are resolved, there will still be significant political and technical approaches for developing and implementing destruction programs that must continue to evolve.

* www.un.org/press/en/2017/sc13040.doc.htm † www.armscontrol.org/act/2017-10/features/remarks-opcw-20-adaptingprohibition-regime-address-emerging-challenges

APPENDIX: OPCW MEMBER STATES AS OF 2018 (PER WWW.OPCW.ORG) Afghanistan Albania Algeria Andorra Angola Antigua and Barbuda Argentina Armenia Australia Austria Azerbaijan Bahamas Bahrain Bangladesh Barbados Belarus Belgium

Germany Ghana Greece Grenada Guatemala Guinea Guinea-Bissau Guyana Haiti Holy See Honduras Hungary Iceland India Indonesia Iran Iraq

Belize Benin Bhutan Bolivia Bosnia and Herzegovina Botswana Brazil Brunei Bulgaria Burkina Faso Burundi Cabo Verde Cambodia Cameroon Canada Central African Republic Chad

Ireland Italy Jamaica Japan Jordan Kazakhstan Kenya Kiribati Kuwait Kyrgyzstan Laos Latvia Lebanon Lesotho Liberia Libya

Oman Pakistan Palau Panama Papua New Guinea Paraguay Peru Philippines Poland Qatar Republic of Korea Romania Russia Rwanda Saint Kitts and Nevis Saint Lucia Saint Vincent and the Grenadines Samoa San Marino São Tomé e Príncipe Saudi Arabia Senegal Serbia Seychelles Sierra Leone Singapore Slovakia Slovenia Solomon Islands Somalia South Africa Spain Sri Lanka

Liechtenstein

Sudan (Continued)

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Chemical Warfare Agents

APPENDIX (CONTINUED) Chile China Colombia Comoros Congo Congo-Brazzaville Cook Islands Costa Rica Côte d’Ivoire Croatia Cuba Cyprus Czech Republic Denmark Djibouti Dominica Dominican Republic Ecuador El Salvador

Lithuania Luxembourg Madagascar Malawi Malaysia Maldives Mali Malta Marshall Islands Mauritania Mauritius Mexico Micronesia Moldova Monaco Mongolia Montenegro Morocco Mozambique

Equatorial Guinea Eritrea

Myanmar Namibia

Estonia Ethiopia Fiji Finland Former Yugoslav Republic of Macedonia France Gabon Gambia Georgia

Nauru Nepal Netherlands New Zealand Nicaragua Niger Nigeria Niue Norway

Suriname Swaziland Sweden Switzerland Syria Tajikistan Tanzania Thailand Timor-Leste Togo Tonga Trinidad and Tobago Tunisia Turkey Turkmenistan Tuvalu Uganda Ukraine United Arab Emirates United Kingdom United States of America Uruguay Uzbekistan Vanuatu Venezuela Viet Nam Yemen Zambia Zimbabwe

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Organization for the Prohibition of Chemical Weapons (OPCW) Organization for the Prohibition of Chemical Weapons. 2016b. The Contribution of Article VI to States Parties’ Efforts to Counter Terrorism; S/1387/2016. Organization for the Prohibition of Chemical Weapons, The Hague, The Netherlands, May 19, 2016. Organization for the Prohibition of Chemical Weapons. 2016c. Medium-Term Plan of the Organization for the Prohibition of Chemical Weapons, 2017–2021; EC-83/S/1 C-21/S/1. Organization for the Prohibition of Chemical Weapons, The Hague, The Netherlands, April 8, 2016. Organization for the Prohibition of Chemical Weapons. 2016d. Establishment of an Open-ended Working Group on the Future Priorities of the OPCW; EC-82/DEC.2. Organization for the Prohibition of Chemical Weapons, The Hague, The Netherlands, July 14, 2016. Organization for the Prohibition of Chemical Weapons. 2016e. The OPCW’s Role in the Field of Chemical Security: Discussion Paper; S/1395/2016; Organization for the Prohibition of Chemical Weapons, The Hague, The Netherlands, June 13, 2016. Organization for the Prohibition of Chemical Weapons. 2017a. The OPCW at 20. https://20years.opcw.org/ (accessed July 2017). Organization for the Prohibition of Chemical Weapons. 2017b. Report by H.E. Ms María Teresa Infante Acting Facilitator of the Open-Ended Working Group on Terrorism to the Executive Council at its Eighty-Fifth Session; EC-85/WP.2. Organization for the Prohibition of Chemical Weapons, The Hague, The Netherlands, July 13, 2017. Organization for the Prohibition of Chemical Weapons. 2017c. Report by H.E. Ambassador María Teresa Infante, Facilitator of the Sub-Working Group on Non-State Actors of the Open-Ended Working Group on Terrorism—Summary of Intersessional Work; EC-85/WP.1. Organization for the Prohibition of Chemical Weapons, The Hague, The Netherlands, June 23, 2017. Organization for the Prohibition of Chemical Weapons. 2017d. Report on the Activities of the Advisory Board on Education and Outreach in 2016; EC-84/DG.7 C-22/DG.1. Organization for the Prohibition of Chemical Weapons, The Hague, The Netherlands, January 5, 2017. Access to further information and additional documents of the OPCW Advisory Board on Education and Outreach can be found at www.opcw.org/ about-opcw/subsidiary-bodies/advisory-board-on-educationand-outreach/ (accessed July 2017).

69 Organization for the Prohibition of Chemical Weapons. 2017e. Upgrading the OPCW Chemical Laboratory to a Centre for Chemistry and Technology; S/1512/2017. Organization for the Prohibition of Chemical Weapons, The Hague, The Netherlands, July 10, 2017. Organization for the Prohibition of Chemical Weapons. 2017f. Note by the Technical Secretariat: Needs Assessment and Compilation of Tools, Guidance, and Best Practices on Chemical Safety and Security Management; S/1483/2017. Organization for the Prohibition of Chemical Weapons, The Hague, The Netherlands, March 30, 2017. Smart, J.K. 1997. “History of chemical and biological warfare: An American perspective.” In: Medical Aspects of Chemical and Biological Warfare. Sidell, F.R., Takafuji, E.T., Franz, D.R. (Eds.). Borden Institute, Walter Reed Army Medical Center, Washington DC. Szinicz, L. 2005. “History of chemical and biological warfare agents.” Toxicology, 214, 167–181. The Nobel Peace Prize 2013. 2014. The Nobel Peace Prize 2013. Nobelprize.org. Nobel Media AB. Web. April 15, 2018. Available at: www.nobelprize.org/nobel_prizes/peace/laureates/2013/ (accessed July 2017). Timperley, C. and Tang, C. 2017. Presentation entitled “OPCW Scientific Advisory Board Briefing to States Parties.” 19 October. Available at: https://www.opcw.org/sites/default/ files/documents/SAB/en/SAB26_Chair_Briefing_to_States_ Parties.pdf (accessed February 2019). Trapp, R. 2015. “Lessons learned from the OPCW mission in Syria”, December 16, 2015. Available at: www.opcw.org/fileadmin/ OPCW/PDF/Lessons_learned_from_the_OPCW_Mission_ in_Syria.pdf (accessed July 2017). United Nations Secretary-General. 2013. United Nations Mission to Investigate Allegations of the Use of Chemical Weapons in the Syrian Arab Republic, New York, USA, December 2013. Available at: https://unoda-web.s3.amazonaws.com/wp-content/uploads/2013/12/report.pdf (accessed July 2017). United Nations Secretary-General. 2015. The Secretary-General’s Mechanism for Investigation of Alleged Use of Chemical, Bacteriological (Biological) or Toxin Weapons: A Lessonslearned Exercise for the United Nations Mission in the Syrian Arab Republic. United Nations Secretary General, New York, United States of America. Available at: www.un.org/disarmament/publications/more/syrian-ll-report (accessed July 2017). Waitt, A.H. 1942. Gas Warfare. Duell, Sloan, and Pearce, New York.

5 Chemical Weapons Holdings and Their Internationally Verified Destruction John Hart and Thomas Stock CONTENTS 5.1 Introduction...................................................................................................................................................................................71 5.2 CWC Provisions............................................................................................................................................................................72 5.3 Role of OPCW Verification...........................................................................................................................................................73 5.4 CW and OACW Declarations and Destruction Operations Since EIF of CWC..........................................................................73 5.5 Destruction Technologies Employed............................................................................................................................................ 74 5.6 Verification and Destruction Practice by Country........................................................................................................................76 5.6.1 Albania.............................................................................................................................................................................76 5.6.2 Belgium............................................................................................................................................................................76 5.6.3 China................................................................................................................................................................................78 5.6.4 France...............................................................................................................................................................................78 5.6.5 Germany...........................................................................................................................................................................78 5.6.6 India..................................................................................................................................................................................79 5.6.7 Iraq....................................................................................................................................................................................79 5.6.8 Italy...................................................................................................................................................................................79 5.6.9 Japan.................................................................................................................................................................................79 5.6.10 Libya.................................................................................................................................................................................79 5.6.11 Panama.............................................................................................................................................................................80 5.6.12 Russia................................................................................................................................................................................80 5.6.13 Syria.................................................................................................................................................................................. 81 5.6.14 United States..................................................................................................................................................................... 81 5.6.14.1 Sea-Dumped CW..............................................................................................................................................82 5.7 Implications...................................................................................................................................................................................82 Abbreviations and Acronyms..................................................................................................................................................................83 References...............................................................................................................................................................................................83

5.1 Introduction The large-scale production of chemical weapons (CW) dates back to World War I. The widest variety of chemical compounds stockpiled for use on a large scale is found among the CW agents manufactured during this conflict. At least 40 different compounds were weaponized for use on the battlefield (Manley, 1998). Augustine M. Prentiss of the U.S. Chemical Warfare Service (CWS) estimated that the Austrian-Hungarian Empire, Germany, Italy, France, the United Kingdom, the United States, and Russia together produced 150,000 metric tons of CW agents during the war, of which 25,000 metric tons were left unused when hostilities ended on November 11, 1918 (Prentiss, 1937, p. 661). World War I–era CW continue to be recovered from former Western European battlefields (mainly in Belgium). CW stockpiles were positioned in all major theaters of operation during World War II (Haug, 1997). Japan used chemical (and biological) warfare agents against mainland China. Large-scale

disposal operations were carried out following both World Wars—mainly through burial, open-pit burning, and dumping. After World War II, the Allies dumped approximately 250,000 metric tons (munition body plus chemical agent weight) of German CW into the Baltic Sea (Program Manager for Nonstockpile Chemical Material, 1993, pp. 2–4.). In the final phase of the CWC negotiations in the late 1980s, the political expectation by governments was that the destruction of the existing CW stockpiles would be completed within 10–15 years. It was also understood that old chemical weapons (OCW) recoveries would be a continuing issue for some states parties. The two major possessor states (Russia and the United States) had different levels of departure after the entry into force (EIF) of the CWC. The United States already had an established destruction program and started systematic destruction operations in June 1990 (the first U.S. chemical weapons destruction facility at Johnston Atoll Chemical Agent Disposal System [JACADS] started its destruction operation on June 30, 1990 and completed 71

72 operations on November 29, 2000). Russia, by contrast, developed and implemented its CW destruction program in the 1990s, including the time of the 1998 financial crisis. Several parties to CWC have established destruction programs for OCW that will continue for some years to come. Munitions retrieved from old battlefields or test ranges may be either chemical or conventional rounds. Some chemical munitions may have been filled with other materials (including inert materials) for training purposes. Alternatively, the chemical fill may have since hydrolyzed or degraded. Chemical munitions can usually be distinguished from conventional munitions through the use of an X-ray. More precise information on the composition of the chemical agent may be obtained by bombarding the munition with a radiation source (e.g., via portable isotopic neutron spectroscopy). Other techniques include neutron induced prompt photon spectroscopy (NIPPS), ultrasonic testing (UT), and hydrogen concentration measurement (HCM) (Starostin, 1999). As of December 31, 2016, six state parties (Belgium, France, Germany, Italy, Netherlands, and the United Kingdom) had not completed destruction of their OCW (OPCW, 2017). As of the same date, abandoned chemical weapons (ACW) (confirmed or suspected) had not been destroyed on the territories of two state parties (“Note by the Director-General, Summary of Verification Activities in 2016,” para. 1.1, p. 2). As of December 31, 2016, the CW destruction facilities listed in Table 5.1 were in service or under construction (OPCW, 2017, Table 4, p. 11) (Table 5.1). Based on the categorization of chemical weapons under the CWC into (a) stockpiled CW (produced after January 1, 1946) and (b) old chemical weapons (i.e., essentially CW produced before January 1, 1946), it is possible to generalize the technologies applied to date. For the first category, the main technologies applied are the incineration or neutralization of the agent after drainage from the munition body. The munition bodies are usually thermally decontaminated. In addition, other technologies exist to deal with small numbers of problematic stockpiled CW such as leakers, overpacked munitions, or munitions that cannot be safely transported. These technologies include thermal detonation and explosive destruction solutions. In 2014, in Libya, CW munitions have been destroyed by applying static detonation chamber (SDC) technology in a semimobile plant, which started in late 2013 (OPCW, 2014).

Chemical Warfare Agents The SDC technology was also used at Anniston Chemical Disposal Facility (ANCDF) for the destruction of leakers in overpacks. SDC will also be applied at Blue Grass Chemical Agent-Destruction Pilot Plant (BGCAPP) for the same kind of problematic munitions. Another explosive destruction technology is currently employed at Pueblo Chemical Agent-Destruction Pilot Plant (PCAPP) for the destruction of problematic munitions via the explosive destruction system (EDS). In both applications, most of the agent and the explosives in the munition are destroyed by heat and in the second case, by detonating donor explosives that are wrapped around the munition. The resulting off-gases are processed through an efficient offgas treatment system including secondary combustion to ensure chemical agent destruction. Despite some expectations to the contrary, only two main technologies have been applied in the past 20 years for OCW and are regarded as being mature (see later). For the longer term, existing or newly recovered OCW will be destroyed. Large quantities of chemical munitions have also been dumped and may also be subject to recovery, remediation, and/or destruction operations.

5.2 CWC Provisions The CWC defines a “chemical weapon” as the following, together or separately: “(a) Toxic chemicals and their precursors, except where intended for purposes not prohibited under this Convention, as long as the types and quantities are consistent with such purposes; (b) Munitions and devices, specifically designed to cause death or other harm through the toxic properties of those toxic chemicals specified in subparagraph (a), which would be released as a result of the employment of such munitions and devices; (c) Any equipment specifically designed for use directly in connection with the employment of munitions and devices specified in subparagraph (b)” (CWC, Article II, para. 1). The CWC defines “old chemical weapons” as “(a) Chemical weapons which were produced before 1925; or (b) Chemical weapons produced in the period between 1925 and 1946 that have deteriorated to such [an] extent that they can no longer be used as chemical weapons” (CWC, Article II, para. 5). The CWC defines “abandoned chemical weapons” as “Chemical weapons, including old chemical weapons, abandoned

TABLE 5.1 Current CW Destruction Facilities Rabta Toxic Chemicals Destruction Facility (RTCDF) (closed in 2017), Libya Gesellschaft zur Entsorgung von chemischen Kampfstoffen und Rüstungsaltlasten mbH (GEKA mbH) (destruction operations of Libyan CW completed in January 2018), Germany Kizner (destruction operations completed 2017), Russia Pueblo Chemical Agent-Destruction Pilot Plant (PCAPP), United States Pueblo Chemical Agent-Destruction Pilot Plant Explosive Destruction System (PCAPP-EDS), United States Blue Grass Chemical Agent-Destruction Pilot Plant (BGCAPP), United States Blue Grass Chemical Agent-Destruction Pilot Plant Static Detonation Chamber (BGCAPP-SDC), United States Prototype Detonation Test and Destruction Facility (PDTDF), United States Aberdeen Proving Ground Chemical Transfer Facility (APG/CTF), United States Recovered Chemical Weapons Destruction Facility (RCWDF), United States

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Chemical Weapons Holdings by a State after 1 January 1925 on the territory of another State without the consent of the latter” (CWC, Article II, para. 6). The states parties follow an “order of destruction,” which is partly based on definitions of three chemical weapons “categories.” (On order of destruction, see CWC, Verification Annex, Part III, paras. 15–19. For definitions of chemical weapons “categories” and destruction “phases,” see CWC, Verification Annex, Part III, paras. 16–17. Category 1 comprises “chemical weapons on the basis of Schedule 1 chemicals and their parts and components.” Category 2 comprises “chemical weapons on the basis of all chemicals and their parts and components.” Category 3 comprises “unfilled munitions and devices, and equipment specifically designed for use directly in connection with employment of chemical weapons.”) The Executive Council (EC) of the Organization for the Prohibition of Chemical Weapons (OPCW) reviews national chemical weapons destruction plans and periodic status of destruction updates. The annual Conference of the States Parties (CSP) also reviews and takes decisions concerning chemical weapons destruction programs. States parties need not declare chemical weapons buried on their territories before January 1, 1977 and which remain buried, or chemical weapons that have been dumped at sea before January 1, 1985 (CWC, Article III, para. 2). In practice, the member states have refrained from declaring buried and dumped chemical weapons. However, there have been informal technical consultations since the EIF of the CWC on such weapons. The CWC sets out three broad principles and methods for the destruction of chemical weapons:

1. Chemical weapons shall be destroyed in a manner that is “essentially irreversible.” 2. Each state party shall determine how to destroy its weapons at specifically designated and appropriately designed and equipped facilities (excluding dumping, land burial, and open-pit burning). 3. Chemical weapons destruction facilities shall be constructed in a manner that ensures destruction and that the process can be verified by the OPCW (CWC, Verification Annex, Part IV(A), paras. 12–14). The experience of a 1989 Memorandum of Understanding (MOU) and a 1990 Bilateral Destruction Agreement (BDA) concluded between the former Soviet Union and the United States partly informs the procedures by which possessor states declare their chemical weapons to the OPCW and the processes the OPCW uses to verify the CW destruction. Over the years, Russia and the United States consulted each other on the development of common understandings on the selection and optimization of chemical weapon destruction technologies, including within the framework of the 1992 Nunn– Lugar Cooperative Threat Reduction (CTR) Program and the Global Partnership against the Spread of Weapons and Materials of Mass Destruction. Other states (such as Canada, Germany, Italy, the Netherlands, Sweden, and the United Kingdom) and civil society (such as Green Cross Russia and Global Green USA) played important roles in such efforts, including by supporting

risk assessment and public outreach at Russia’s seven chemical weapons storage facilities.

5.3 Role of OPCW Verification CWC verification principles are partly based on a material balance accounting approach (e.g., material inputs should correspond with material outputs at destruction facilities). The OPCW takes samples at key points, employs seals and tags, evaluates possibilities for diversion within and between chemical weapons storage facilities and chemical weapons destruction facilities, and uses video surveillance. The OPCW also observes the principle of maintaining on-site inspector presence whenever destruction operations are underway. The EC reviews and approves each possessor state’s chemical weapon destruction plan. The EC also reviews the status of destruction by possessor states when it meets (in regular session, this is about four times per year). Clarifications may be sought from the OPCW’s Technical Secretariat or by using the provisions of Article IX on consultations, cooperation, and fact-finding. Since 2013, special ECs have also been convened to focus on verification-related matters arising from the Syria file. The possessor states also typically update the annual CSP on the overall status of their destruction efforts, as well as more ad hoc support measures (such as assistance for the 2016 maritime removal of toxic chemicals and their precursors from Libya to Germany).

5.4 CW and OACW Declarations and Destruction Operations Since EIF of CWC Eight countries have declared CW stockpiles to the OPCW since the CWC entered into force in 1997: Albania, India, Iraq, Libya, Russia, South Korea, Syria, and the United States (OPCW, 2016). The countries that have declared ACW to the OPCW since the EIF of the CWC are China, Italy, and Panama (Panama recently recategorized its holdings as OCW; see Section 5.6.11). Both Ethiopia and Iran have consulted with OPCW to clarify whether old munitions on their territory were abandoned chemical weapons. However, the results of the joint assessments were that the weapons in question were not chemical munitions and did not meet the CWC definition of ACW. Italy declared abandoned chemical weapons—Adamsite— although no abandoning state party (ASP) was identified. In addition, it was not possible for the Technical Secretariat to establish the identity of the ASP or to determine the precise circumstances of the possible abandonment. Therefore, in the absence of a declaration by an ASP, Italy took it on itself to destroy the abandoned chemical weapons located on its territory (OPCW Technical Secretariat Background Paper, 2003, pp. 5 and 38). Sixteen countries have declared OCW to the OPCW since the EIF of the CWC: Australia, Austria, Belgium, Canada, France, Germany, Italy, Japan, the Marshall Islands, the Netherlands, Poland, Russia, Slovenia, Switzerland, the United Kingdom, and the United States (OPCW, September 19. 2017).

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Chemical Warfare Agents

There have also been periodic recoveries of dumped chemical munitions, including some that have washed ashore (see later).

and for VX as

5.5 Destruction Technologies Employed

+ 2SO2 + 2NO

2(ClCH 2 CH 2 )2 S + 13.5O2 = 8CO2 + 6H 2 O + 4HCl + 2SO2

For GB, the incineration process can be shown as 2 ( CH 3 )2 CHO(CH 3 )POF + 13O2 = P2O5 + 8CO2 + 9H 2O + 2HF



2C11H 26 NO2 PS + 39.5O2 = 22CO2 + 26H 2O + P2O5 + 2SO2 + 2NO2

Destruction of Chemical Warfare Agents, 1958–1993 Site



The baseline incineration process employed in the United States to destroy stockpiled chemical weapons is mature. Incineration is a well-developed technology that has been shown to be highly effective. In the baseline system, liquid chemical agent drained from the munitions and storage containers is collected in a storage tank, from which it is fed into a high-temperature liquid incinerator (LIC), where it is burned at a temperature of 1480°C. The LIC is a two-stage refractory-lined incinerator designed to destroy the nerve agents GA, GB, and VX, and mustard gas (H, HD, HT). The drained agent is atomized by a nozzle and mixed with air to sustain combustion. Auxiliary fuel is used to maintain combustion at or above 1400°C with the flue gases being passed to an afterburner maintained at a minimum temperature of 1090°C for 2 s before ducting to the pollution abatement system.

TABLE 5.2

Rocky Mountain Arsenal, United States Rocky Mountain Arsenal, United States Tooele (CAMDS)b, United States JACADSc Johnston Atoll, United States—OVT (operational verification test) data Tooele (CAMDS), United States JACADS Johnston Atoll, United States—OVT data JACADS Johnston Atoll, United States—OVT data DRES Canada DRES Canada DRES Canada DRES Canada Munster, Germany Shikhany, Russia Shikhany, Russia Porton Down, United Kingdom Nancekuke, United Kingdom Runcorn, United Kingdom Iraq (UNSCOM supervised) Iraq (UNSCOM supervised) Iraq (UNSCOM supervised)



or

Between 1958 and 1993, some 14,500 metric tons of mustard and nerve agents have been destroyed by either incineration (I), neutralization (N), or a combination of these two techniques (N/I) (See Table 5.2. Destruction of Chemical Warfare Agents, 1958–1993.) After the EIF of the CWC, in the past 20 years, two general technologies have been applied for the destruction of chemical weapons: (a) thermal treatment by incineration and (b) hydrolysis/neutralization. Incineration is the thermal decomposition and follow-on oxidation at high temperatures of the organic chemicals. For mustard gas, the incineration process can be shown as follows:

2C11H 26 NO2 PS + 38.5O2 = 22CO2 + 26H 2O + P2O5

Agent

Amount (tonnes)a

Method

Date

H GB GB GB

2786 3799 34.5 36

I N I I

1969–1974 1973–1976 1981–1986 1991

VX VX HD H H VX, GA, GB L H, etc. GB, GD, H VR (Russian VX) H GB H H GA GB, GB/GF

7 49 51 700 12 0.3 1.5 70/year 300 30 20 20 6000 500 30 70

I I I N/I I N/I N I N/I N/I I N I I N N

1984 1992 1992 1974–1976 1990–1991 1990–1991 1980– 1980–1990 1980–1990 1970 1967–1968 1958–1960 1992–1993 1992–1993 1992–1993

Source: Pearson, G.S., and Magee, R.S., Pure and Applied Chemistry, 74, 187, 2002. The table is based on Sutherland, R.G., The Challenge of Old Chemical Munitions and Toxic Armament Wastes, Oxford: Oxford University Press, 1997 and National Research Council, Committee on Alternative Chemical Demilitarization Technologies, Alternative Technologies for the Destruction of Chemical Agents and Munitions, National Academy Press, Washington, DC, 1993. a U.S. figures in short tons (2000 lb) converted to tonnes (1000 kg). b CAMDS was a Chemical Agent Munitions Disposal System experimental facility at the Tooele Army Depot, Utah. c This JACADS data is OVT (Operation Verification Test) data obtained prior to the full-scale operation of the Johnston Atoll Chemical Agent Disposal System. UNSCOM: United Nations Special Commission.

75

Chemical Weapons Holdings In the pollution abatement or scrubbing system, the various acidic or acid-forming products from the incineration are reacted with sodium hydroxide in the caustic brine solution involving reactions such as the following: Reaction with HCl:

NaOH + HCl = NaCl + H 2 O

Reaction with HF: NaOH + HF = NaF + H 2 O

Reaction with SO2:

NaOH + SO2 = NaHSO3



NaHSO3 + NaOH = Na 2SO3 + H 2O



O2 + 2Na 2SO3 = 2Na 2SO 4

Reaction with SO3 (if formed rather than SO2):

NaOH + SO3 = NaHSO3



NaHSO3 + NaOH = Na 2SO 4 + H 2 O

The emptied munitions and bulk items are fed into a metal part furnace (MPF), which maintains all metal parts at a temperature of 540°C for at least 15 min. Metal parts for projectiles and bulk items have a residence time of approximately 2 h. Exhaust gases pass to an afterburner maintained at 1090°C and are then ducted to the pollution abatement system. The decontaminated metal parts are discharged and after deformation, shipped to an approved disposal site or sold for scrap. Under these conditions, the incinerators emit no detectable agent in the stack gases. The U.S. Army has deployed proved incineration technology for the destruction of its stockpile. However, the complete conversion of the carbon and hydrogen in organic compounds to carbon dioxide and water is generally not achievable in practice with incineration. Instead, trace amounts of compounds such as dioxins, furans, and other products of incomplete combustion can be generated during the combustion process and must be controlled in an offgas treatment system. This characteristic of the incineration processes has been a source of difficulty in gaining public acceptance for this technology, especially from stakeholders in local communities and environmental interest groups. In the case of the United States, this resulted in the establishment of the Assembled Chemical Weapons Alternatives (ACWA) program, which was tasked with further evaluating non-incineration-based destruction technologies (In 1996, the U.S. Congress established the ACWA program to test and demonstrate alternative technologies to the incineration baseline. The ACWA program oversaw the design and construction of PCAPP and BGCAPP.) The ACWA program adopted a so-called two-stage process: after applying neutralization or hydrolysis in the first step, a second tier of technology, biodegradation and/or supercritical water oxidation, is applied. At PCAPP, the mustard round stockpile is under destruction using neutralization followed by biotreatment. Hot water

is mixed with caustic solution to neutralize the chemical agent, effectively destroying the mustard agent molecules. The product from this process is called hydrolysate and has a high pH, requiring acid to be added to reduce the pH to neutral, making it suitable for digestion by the microbes used in biotreatment. Ordinary sewage treatment bacteria, or microbes, consume the organics in the hydrolysate. The explosive destruction system, or EDS, is applied to destroy overpacked and problematic munitions whose deteriorated physical condition does not easily allow automated processing through the main plant. For the CW nerve agent stockpile in Blue Grass, Kentucky, the destruction technology that will be used is neutralization followed by supercritical water oxidation, known as SCWO (the SCWO process will blend water, fuel, air, and hydrolysate together in a specialized vessel at temperatures and pressure conditions above the critical point of water. The hydrolysate is oxidized to carbon dioxide, water, and salts). Robotic equipment disassembles the munitions, and the nerve agent is drained and separated from the explosive components (energetics). The nerve agent will be mixed vigorously with hot water and sodium hydroxide to destroy, or neutralize, it. The energetics will be neutralized in a similar process. The resulting hydrolysate will be held and tested to ensure chemical agent destruction before proceeding to secondary treatment. At Blue Grass, for a number of rounds containing solidified mustard, which cannot be processed through the normal procedure, the static detonation chamber was chosen to destroy this mustard agent stockpile. In comparing the applied technologies for the destruction of stockpiled CW with the applied ones for the destruction of OACW as well as leaking stockpiled CW, different criteria or better conditions as a point of departure have to be taken into consideration:









1. OCW/ACW holdings are much smaller than those of “regular” stockpiled CW (i.e., produced after January 1, 1946). 2. Recovered OCW pose less of a security risk to the CWC treaty regime as compared with stockpiled CW because of their limited usability. 3. OCW are usually not re-usable for the purpose for which they were manufactured. 4. It is often difficult to differentiate between chemical and conventional munitions when they are recovered from the field. 5. OCW may be corroded or otherwise physically damaged. They often contain widely varying mixtures of various agents, decomposition products, and explosive material, for which it is difficult to model and implement standardized destruction procedures. 6. Explosive charges and fuses (e.g., picric acid) often mean that it is too dangerous to transport the munition. 7. Highly unstable OCW pose major challenges for any transportation and destruction process.

The last 20 years have also shown that two destruction technologies have been proved to be mature for application to OACW

76 which do not entail the disassembly of the CW munitions and separation of the agent from the explosives. These technologies can be summarized as follows: 1. Cold detonation technology. An explosive donor charge is placed around the munitions and is detonated in a containment system, creating conditions (pressure, fireball, and temperature) that allow the destruction of the explosives and most of the agent. The resulting off-gases are treated in an offgas treatment system. Cold detonation technology is/will be applied to OCW in Belgium, China, Japan, and France. Cold detonation technology is currently employed to support (a) explosive destruction systems (EDS), (b) transportable detonation chambers (TDC), and (c) the detonation of ammunition in a vacuum integrated chamber (DAVINCH®). 2. Hot detonation technology. The munitions are transported into a hot detonation chamber (500–550°C), where the temperature will lead to deflagration, detonation, or burning of the explosive filling of the munitions. In addition, the CW agent will be destroyed. The resulting off-gases are treated in an offgas treatment system. Hot detonation technology is (or has been) applied to the destruction of OCW as well as problematic stockpiled munitions in Belgium, China, Germany, Japan, Libya, and the United States. For this technology, the static detonation chamber (SDC) is currently in use. Another method comparable to cold detonation or better-controlled detonation is the EDS system, which is under consideration to be used for recovered chemical munitions. The U.S. EDS is a system to treat munitions regardless of whether or not they are energetically configured. The system consists of an explosion containment vessel mounted on a trailer (Table 5.3). A traditional approach has been taken to the disposal of small numbers of OCW, which typically involves manual or semi-manual dismantlement (“delaboration”) such as cutting, sawing, removal, and agent draining. This is done in accordance with the particular condition and type of CW munition. Specialized, labor-intensive destruction facilities have therefore been established for the handling and disposal of such munitions. Manual dismantlement requires experienced and well-trained explosive ordnance disposal (EOD) experts. Europe has developed a great deal of specialized expertise based on its experience in dealing with very different CW munitions dating back to World War I. Although experienced EOD experts are able to apply labor-intensive techniques, larger quantities of OCW are not treatable in such a way. In the latter case, a more automated and systematized approach is required. The disassembly of ACW/OCW involves the separation of the fuse and explosive charge from the rest of the shell, the draining of the chemical agent, and the final disposal of the destruction products. This is a process that has been carried out routinely in Germany and in the United Kingdom since the early 1980s and in Belgium since the early 1990s. The necessary equipment consists of drilling, milling, or sawing machines. Such equipment is placed in a bunker structure protecting the personnel from the effects of blast, fragments, toxic aerosols, or other vapors. A weakened blow-out wall opposite the work stations of facility personnel lessens the safety risks should an explosion occur during operations.

Chemical Warfare Agents

5.6 Verification and Destruction Practice by Country 5.6.1 Albania Albania’s chemical weapons consisted of approximately 16 metric tons of sulfur mustard filled into canisters. In early 2003, Albania reported to the OPCW that it had discovered the material in November 2002. Albania’s stockpile comprised: 580 canisters of sulfur mustard (13.71 metric tons), 49 canisters of glass containers containing Lewisite (0.97 metric tons), four canisters of sulfur mustard/ Lewisite mixture (0.4 metric tons), 33 canisters of Adamsite (0.33 metric tons), and 80 canisters of chloroacetophenone (1.04 metric tons) (Vucaj, 2007, pp. 6–10). Albanian sulfur mustard was destroyed by a mobile nitrogen pyrolysis followed by incineration technology provided by Germany. Funding support was also provided within the CTR framework.

5.6.2 Belgium Belgium’s old chemical weapons are consolidated and destroyed by the Dienst Opruiming en Vernietiging Ontploffingtuigen (DOVO) at Poelkapelle (Dienst Opruiming en Vernietiging Ontploffingtuigen, n.d.). Belgium, which declared OCW, has to cope with actual recoveries of OCW from World War I, as in the Flanders region, where the frontline in World War I was located and the first use of lethal CW took place, these munitions appear from the ground on a frequent basis. Before the EIF of the CWC, Belgium had in operation a facility based on the principle of disassembly of old chemical weapons, which involves the separation of the fuse and explosive charge from the rest of the shell, the draining of the chemical agent, and the final disposal. Later, in the early 2000s, the controlled detonation chamber technology (CDC), previously known as the Donovan blast chamber or the contained detonation chamber, was applied in a testing program in Belgium for the destruction of World War I munitions for almost 5 years. In total, two different systems were used at Poelkapelle in Belgium to destroy 3200 recovered chemical munitions. (Several versions of the TDC have been used extensively for the destruction of chemical weapons. A TC-10 system and a TC-60 system were used at Poelkapelle in Belgium.) The Belgian Ministry of Defense (MOD) later installed a DAVINCH® system having a 50 kg TNT-equivalent explosion containment capacity at a Belgian military facility at Poelkapelle. By December 2011, over 4000 munitions containing chemical agent had been destroyed. In 2014, the Belgian MOD decided to purchase an SDC to accelerate the destruction of the OCW in storage as well as the regular newly recovered munitions. Munitions containing liquid agents as well as phosgene and other gaseous material can be directly destroyed without any pre-preparation. The plant went officially into operation in April 2017 (DOVO Neemt Nieuwe Detonatiekamer in Gebruik. Focus WTV, 2017; and Dovo neemt nieuwe detonatiekamer in gebruik Vijfduizend gifgasbommen worden eindelijk vernietigd, Gazet van Antwerpen/Metropool Zuid, 2017, pp. 6–7). The applied SDC technology now has accelerated the destruction operations in Belgium

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Chemical Weapons Holdings TABLE 5.3 Overview of Currently Applied Technologies Technology Type Technology Attribute Agent contained in: Agent in munition accessed by: Agent destroyed by:

Typical cycle time (varies with munition) Offgas treatment

Waste streams

Ability to recycle or further treat offgas

Neutralization EDS, Sandia, NSCMP

Explosive Destruction TDC, CH2M HILL

Explosive Destruction DAVINCH®, Kobe Steel

Thermal Destruction SDC, Dynasafe

Sealed cylindrical vessel on truck bed Shaped charges on munition or munition overpack Reaction with reagenta in vessel at 60°C for 1 h followed by 2 h hot water rinse 48 h

Rectangular detonation chamber Donor charge placed around munition or munition overpack Detonation: heat and pressure from controlled detonation at 700–1000°C

Double-walled cylindrical detonation vessel Shaped and donor charges on munition or munition overpack

Spheroid double-walled static kiln

Detonation: shock wave, compression, thermal destruction in fireball at 2000°C

Heating to 550°C resulting in agent decomposition

35–40 minutes

100 minutesb

20–30 minutes

None. Agent and reagent react until agent is destroyed. No offgas produced.

Catalytic oxidizer with maximum temperature of 1095°C; reactive bed filter with hydrated lime or sodium bicarbonate for acid neutralization; carbon adsorption system; ceramic filter and HEPA for particulates Exhaust gas, metal fragments, gravel dust, spent lime, activated carbon. Discharged scrap metal is ≤1 VSL (formerly 3×)

Cold plasma oxidizer (Glid-Arc) at 600°C, 0.5–1.0 s residence time; in-line gas scrubber with NaOH washdown to neutralize the gas; sulfur-impregnated carbon and activated carbon; HEPA filters for particulates

Thermal oxidizer at 1100°C, 2 s residence time; acid scrubber at approx. 80°C; IONEX filter containing HEPA filter, sulfur-impregnated carbon, and activated carbon; baghouse filter and HEPA for particulatesc

Metal fragments, exhaust gas, dust, activated carbon, scrubber condensate water. Discharged scrap metal is ≤1 VSL (formerly 3×)

Metal fragments, scrubbed offgas, dust, salts, activated carbon. Scrap metal suitable for release for unrestricted use (formerly 5×)

None. Has expansion tank for offgas but no ability to recycle

Yes. Can recycle offgas through cold plasma oxidizer after holding and testing in offgas retention tank Fixed facility but vessel can be moved on three flatbed trailers (one each for the outer chamber, the inner chamber, and the lid) plus two trailers for the offgas treatment unit and additional trailers as needed for supporting equipment 99–143 kg, including donor and shaped charges

Yes. If operated in batch mode, offgas in the static kiln can be held at 550°C and tested until agent is not detected Fixed facility but can be moved in 20–25 ISO containers

8 in. projectile, overpacked M55 rocket

8 in. projectile

Liquid neutralent and rinsate, scrap metal (munition fragments). Discharged scrap metal is ≤1 VSLd (formerly 3×) N.A. No gas stream is produced

Transportability

Transportable on one trailer

Transportable on eight trailers, 10 days

Explosive containment capacity, TNT-equivalent Largest munition

5 lb for EDS-2, including shaped charges 155 mm projectile

40 lb including donor charge 210 mm projectile

Heating of munition, followed by deflagration or detonation

5 lb in munition

Source: Board on Army Science and Technology; Division on Engineering and Physical Sciences; National Research Council, Remediation of Buried Chemical Warfare Materiel, Committee on Review of the Conduct of Operations for Remediation of Recovered Chemical Warfare Materiel from Burial Sites, Washington, DC, 2012. HEPA: high-efficiency particulate air filter; ISO: International Organization for Standardization; NaOH, sodium hydroxide; IONEX, a research company; 3X, level of agent decontamination suitable for transport for further processing (obsolete); 5X, level of agent decontamination suitable for commercial release (obsolete); TNT, trinitrotoluene. a Reagent is monoethanolamine for mustard and NaOH for phosgene and other fills. b Based on experience to date of six cycles per 10-hr day. c In this report, IONEX refers to an offgas treatment system that contains particulate filters and activated carbon adsorbers. d VSL (Vapor Screening Level) is a control limit used to clear materials for off-site shipment based on agent concentration in the atmosphere above the packaged waste materials. The numerical value of the VSL can depend on the permit issued by the regulatory authority for the particular facility involved, but it is often set at either the short-term exposure limit (STEL) or the short-term limit (STL), which is numerically the STEL but without the 15-min time component.

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Chemical Warfare Agents

5.6.3 China

5.6.4 France

China and Japan have undertaken joint assessments on the nature and scope of the ACW left behind since the early 1990s, when the countries conducted their first joint investigation of a site (Conference on Disarmament, 1992). Most of the ACW is located in Harbaling Province. China and Japan have continued to jointly determine the nature and scope of Japanese ACWs left in China during World War II. These ACWs have caused more than 2000 casualties since the People’s Republic of China was founded in 1949 (Tang, 2006, slide 4). This joint work is based on two memoranda of understanding concluded by the countries in 1999 and 2012 (Tang, 2006, slide 6). ACWs have been found at more than 90 sites in 18 provinces (Tang, 2006, slide 7). The ACWs in China include chemical projectiles (75, 105, and 150 mm), mortars (90 mm), air bombs (15 and 50 kg), canisters (i.e., small, medium, and large gas pots), and miscellaneous components (i.e., burster tubes, booster tubes, and containers or drums filled with sulfur mustard) (Tang, 2006, slide 8; Satake, 2016, slide 1). Canisters comprise approximately 70.4% of the total recovered ACW items. Shells comprise approximately 21%. Miscellaneous components account for the remaining 8.6% (Satake, 2016, slide 1). Japan has continued to carry out destruction operations in China in 2016–2018 at multiple sites, mainly in central eastern and northeastern regions. Approximately 53,076 ACWs have been recovered, while at least another 330,000 ACWs are yet to be recovered (Satake, 2016, slide 1). The recovered ACWs are stored in 10 warehouses and 15 temporary warehouses. The 330,000 unrecovered ACWs are almost entirely located at a site in Haerbaling about 1300 km northeast of Beijing. The warehouses are at Guangzhou, Haerbaling, Harbin, Jiamusi, Nanjing, Ningan, Qiqihar, Shenyang, Shijiazhuang, and Yichun. Their contents comprise 11,451 ACW items and over 201 tonnes of contaminated material. The temporary warehouses are at Anqing, Bayannaoer, Bei’an, Dalian, Hangzhou, Hulunbeier, Hunchun, Jixi, Liaoyuan, Longjin, Nanning, Nianzishan, Shangzhi, Taiyuan, and Tonghua. Their contents comprise 2505 ACW items, 74 metric tons of partially disposed chemical warfare agents, and over 450 kg of contaminated material (Tang, 2006, slides 10 and 18). Of the 53,076 recovered ACWs, 39,695 had been destroyed as of May 20, 2016 (Satake, 2016, slide 23). As of June 2017, approximately 56,000 ACW-related items have been found at over 90 locations throughout China. Of these, approximately 46,000 items have been verified as destroyed by the OPCW as of May 2017 (OPCW Executive Council and Director-General Review Abandoned Chemical Weapons’ Destruction Progress in China, 21 June 2017). The baseline destruction technology comprises detonation chamber and SDC systems. Additional future technical challenges include (a) the underwater recovery of munitions (Jiamusi, Heilongjiang Province); (b) the irreversible destruction of pretreated agent mixtures (74 metric tons at Liaoyuan, Jilin Province); (c) the disposal of fused munitions recovered at Taiyuan (Shanxi Province); (d) the final deposition of contaminated soil; and (e) the lack of capacity to destroy incidentally discovered munitions using a mobile destruction system (Tang, 2006, slide 30). Japan expressed the hope of completing the destruction of the ACW in China by 2022. As of October 2017, Japan had spent approximately 1.3 billion euro on ACW in China activities (Japan, 2017).

France has yet to begin destroying its OCWs. It has consolidated its OCWs at the Site d’élimination des chargements d’objets identifiés anciens (SECOIA) in Mailly-le-Camp. The facility has the capacity to destroy 42 metric tons per year (which amounts to around 3000 munitions). France has decided to use the DAVINCH® system as its baseline destruction technology. Groupe Séché, a specialist waste recovery and treatment provider, will provide chemical analysis support (French Ministry of Defence, 2016; Séché Environnement).

5.6.5 Germany The military training ground at Munster was, beginning in World War I, the principal experimental and training area for Germany’s chemical weapons efforts. In 1919, approximately 1 million shells (many of which were CW) were scattered about the site when a train carrying munitions exploded, after which the area had the appearance of a moonscape. After World War II, the British took and used Munster for the field testing of CW munitions. After 1947, they started dismantling the production plants by demolition. Most of the munitions at Munster are German, but it also houses significant quantities of munitions produced by other countries during both World Wars. The soil is also contaminated with toxic material such as arsenic, and one can readily uncover munitions in almost any given area on the facility grounds. Currently, the German CW destruction facility consists of three different plants: Munster I, II, and III. Munster I is used primarily for the thermal treatment of material that results from dismantling old CW munitions. Dunnage material as well was used for the destruction of the German “Sprühbüchse,” a kind of HD-filled mine. Munster I is an incineration plant using car bottom furnace technology and has been in operation since 1980. Munster II is primarily used to clean contaminated soil, and Munster III is an SDC into which munitions are directly fed without disassembly. This SDC, in operation since 2007, is still in operation and today, is used for the destruction of commercial explosives and conventional munitions. However, as in Germany the recovery of OCW is still common, the SDC plant is used as a standby capacity for the destruction of these on a regular basis. In Munster II, primarily arsenic is removed from the soil by a physical-chemical soil-washing process. Following treatment of the concentrated remaining material in a plasma furnace system, the arsenic is either embedded into a non-leaching crystalline structure of vitrified glass-slag or precipitated as an arsenate (as salt) from the offgas scrubber system. The quantities of chemical weapons destroyed at GEKA are summarized in Table 5.4 (chemical projectiles filled with HD/DA/phosgene: more than 15,000; total number of HD munitions: more than 7000. Note that in 2010, GEKA completed the destruction of all accumulated chemical munitions; since then, chemical munitions have been destroyed as they are found and recovered). It should be noted that Libyan chemicals transferred to GEKA in 2016 were destroyed in January 2018 (see Section 5.6.10) (Table 5.4). The GEKA facility has also been used to destroy three OCW from Austria (in October 2005 and October 2006, Austria discovered three old chemical weapons). These old chemical weapons

79

Chemical Weapons Holdings TABLE 5.4 CW Destruction Operations at GEKA Year 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2016–2017

Tons (metric) 25 22 16 16 14 0.5 1.1 0.3 0.1 0.9 3.1 c. 500 (Category 2 chemicals from Libya)

were declared to the OPCW in accordance with Art. III, para. 1(b)(i) and Verification Annex, Part IV(B). The EC authorized their transfer to GEKA along with chemicals from Libya and Syria. The destruction of chemicals from Ruwagha was completed in January (Anonymous, 2018).

5.6.6 India India possessed two chemical weapon stockpiles located at Borkhedi and Janouri. These comprised sulfur mustard and artillery shells. The shells were drilled. India completed the destruction of its stockpile in 2009.

5.6.7 Iraq Iraq joined the CWC in 2009 and declared the possession of chemical weapons and five former chemical weapon production facilities left over from the government of Saddam Hussein. As of October 2017, the remaining destruction efforts comprised destroying two bunkers and their contents at the al-Muthanna Complex via encapsulation in concrete (“Opening Statement by the Director-General…” OPCW document C-22/DG.20, 27 Nov. 2017, para. 17, p. 3).

5.6.8 Italy In Italy, a neutralization process using hydrogen peroxide to destroy agents is one of the technologies applied in Civitavecchia, Italy, at the NBC Joint Technical Logistics Center, which is known by its acronym, Ce.T.L.I. NBC. The demilitarization activities focused on stockpiled munitions containing primarily Clark I agent but also a mixture of sulfur mustard and Clark I, chloropicrin, or Adamsite (DM), respectively. An automated plant that went into full operation in 2003 has destroyed thousands of rounds ranging in size from 65 to 155 mm and larger. Rounds were identified using high-energy X-ray analyses (by means of a linear accelerator–type X-ray source capable of penetrating 10 cm of steel). The liquid agents were frozen in the bottom of the round by chilling the round in an upright position. The rounds were opened by unscrewing, drilling, or cutting; after the agent was liquefied by warming to ambient temperature, it was emptied into a container for neutralization. The emptied round was cleaned with concentrated nitric acid and bleach. The plant

destroyed 64 projectiles in an 8 hour workday and used anti-aerosol and activated carbon filters to mitigate air emissions. The Clark I and the blister agent mixtures were destroyed using hydrogen peroxide, producing an acidic, arsenic-containing solution, which was neutralized with lime. In contrast, Adamsite (in the form of hard, solid cylinders) was milled to form a powder. The products of blister agent neutralization and Adamsite milling were mixed with sand, cement, and water. The mixtures were then placed in containers made of reinforced concrete, and the resulting blocks are stored outside on platforms. The rainwater that collects below is checked periodically for the presence of arsenic. Munitions recovered from excavation or retrieved from the sea are also destroyed at Civitavecchia if they are judged to have sufficient integrity to be safely transported (Committee on Review and Evaluation of International Technologies for the Destruction of Non-Stockpile Chemical Materiel, National Research Council, 2006). However, there are still fused OCW in Civitavecchia in storage awaiting destruction. Italy has been granted at least two extensions to its destruction finalization deadline by the OPCW. Italy has reviewed existing destruction technologies and is planning to establish its own capacity and capability to destroy the remaining OCW at Civitavecchia near Rome. (In the defense budget for the fiscal year 2017, it was outlined that one of the planning tasks is “Modernization program of Centro Logistico Interforce NBC by a set of interventions allowing it to dispose all chemical weapons now stocked and/or found within the national territory.”)

5.6.9 Japan Japan has carried out what is probably the most extensive and sustained recovery operation of dumped World War II–era chemical munitions at Kanda Port. In 2004–2013, Japan recovered and destroyed 2968 chemical munitions (mainly 50 kg yellow munitions and 15 kg red munitions) from Kanda Port and connecting channels (an area of 23 km2) (The yellow munitions typically contain 18 liters of Lewisite-sulfur mustard mixture and 2.3 kg of high explosives. The red munitions typically contain 368 g of diphenylchloroarsine/diphenylcyanoarsine (DA/DC) mixture and 1.3 kg of high explosives; Kitamura, 2016, p. 17). In 2016, the country continued to carry out final magnetometer checks for any possible remaining munitions (Kitamura, 2016).

5.6.10 Libya Libya deposited its instrument of ratification to the CWC on January 6, 2004 (Hart and Kile, 2005, pp. 629–648). The CWC entered into force for Libya on February 5, 2004. CW destruction operations began the same year with the destruction of 3563 air bombs (Hoggins, 2016, slide 2). From January to February 2005, Libya carried out the verified destruction of 551 metric tons of Category 2 chemicals (sodium sulfide and sodium fluoride) by cementation (Hoggins, 2016, slide 2). All Category 3 munitions were destroyed in 2004. The destruction of sulfur mustard began in 2010 using a neutralization system purchased from the Italian firm SIPSA (Hoggins, 2016, slide 3). This destruction campaign ran from 2010 to February 2011 and resumed in 2013 (Hoggins, 2016, slide 3). In November 2011, Libya declared further holdings of Category 1 and Category 3 chemical weapons. The previously undeclared munitions were destroyed using a semi-mobile

80 static detonation chamber that operated from November 2013 to January 2014 and continued until April the same year for the canister material (see later). The following items have been destroyed: 517 projectiles, eight 400 kg HD aerial bombs, 39 agent-filled rocket warhead insert tubes, and in a follow-on campaign, 380 agent canisters with agent, agent heels in the same SDC system. Libya destroyed 114 metric tons of isopropanol alcohol by dilution in August 2015. Nineteen metric tons of pinacolyl alcohol was destroyed using an afterburner chamber (at two locations). Libya has made at least three broad requests for international assistance: (1) destruction assistance in-country, (2) out-of-country removal operation in 2016, and (3) environmental remediation at Ruwagha from the EU (baseline study completed in 2017). The destruction operations of the material from Libya at GEKA comprise the following chemicals: 2-chloroethanol (18 metric tons), tributylamine (238 metric tons), phosphorus trichloride (138 metric tons), and thionyl chloride (100 metric tons) (Libya, June 15, 2017. Libya reported to the Executive Council at its 85th session on the progress achieved toward the complete destruction of the remaining chemical weapons stockpile (May 31, 2017). See as well “Report by the Director-General: status of the implementation of the plan for the destruction of Libya’s remaining Category 2 chemical weapons outside the territory of Libya” (see also Section 5.6.5). Libya is currently implementing, with EU financial support, an environmental survey and cleanup project at the Ruwagha site. Environmental problems stem from the incineration and hydrolysis products of pre-2016 sulfur mustard destruction operations (which possibly comprise several metric tons of contaminated salts). In addition, a number of the older tanks filled with phosphorus trichloride and thionyl chloride were badly corroded and probably leaked. Russia has requested the OPCW to report on the discrepancy between declared chemicals at the Ruwagha site and the amounts shipped to GEKA in 2016. These destruction operations were completed in January 2018 (Federal Foreign Office (Germany), “Vernichtung von Restbeständen des libyschen Chemiewaffenprogramms in Deutschland erfolgreich beendet” [Successful completion of the destruction in Germany of the remnants of Libya’s chemical weapon program], Press release, 5  January 2018, www.auswaertiges-amt.de/de/newsroom/libyschechemiewaffen/1210838).

5.6.11 Panama During World War II, the United States operated a chemical weapons test facility on San José Island. During World War II, Panama agreed to lease San José Island to the United States for chemical warfare field tests. The U.S. Army’s San José Division was activated in September 1944 (Brophy and Fisher, 1959, p. 106; Lindsay-Poland, 2003; Johnston, 2003). Panama declared the possession of ACW on its territory in 2002. However, it has since designated the weapons as OCW. In 2017, Panama declared and destroyed eight old chemical weapons—all located on the island. They comprised six M79 1000 lb air bombs believed to have been originally filled with phosgene (CG), one M78 500 lb air bomb believed to have originally been filled with cyanogen chloride (CK), and one M1A1 cylinder that is rusted through and empty. Panama destroyed the munitions in situ through explosive venting during the rainy season. Solids were rinsed with

Chemical Warfare Agents caustic solution and the rinsate collected in containers that met international standards for disposal by a licensed off-site treatment, storage, and disposal facility (TSDF). The explosive components of the munitions were detonated with donor charges and the metal fragments collected, checked for contamination, and recycled (Panama, 16 June 2017. Panama, Concept Plan for the Destruction of Eight Old Chemical Weapons; OPCW, 8 Sep. 2017. Note by the Technical Secretariat: Modifications to the Arrangement with the Republic of Panama Governing Inspections on San José Island, Panama).

5.6.12 Russia Russia’s stockpile comprised 39,967 metric tons of agent (i.e., excluding munition weight) and was stored at: Gorny, Kambarka, Kizner, Leonidovka, Maradykovksy, Pochep, and Shchuchye. Russia’s baseline destruction technology was neutralization for the bulk storage sites material at Gorny and Kambarka. Gorny had sulfur mustard, Lewisite, and sulfur mustard–Lewisite mixtures, while Kambarka had Lewisite. The Lewisite was destroyed using caustic neutralization, while the mixtures were neutralized using a monoethanolamine (MEA) water solution. A limited amount of phosgene stored in bulk was likewise neutralized in caustic solution. The other chemical weapons at the remaining sites largely consisted of filled munitions. GB was neutralized in MEA water solution, while VR-agent (an isomer of U.S. VX) was neutralized using a process based on a proprietary reagent developed by the State Scientific Research Institute for Organic Chemistry and Technology (GosNIIOKhT) called RD-4. Russia and the United States conducted a joint evaluation of the GosNIIOKhT solution (Beletskaya, 1998, pp. 103–112; Sheluchenko and Utkin, 1998, pp. 113–121). For the nerve agents, a two-stage process was applied—neutralizing the agent (Stage 1) and adding the resulting neutralization product to bitumen to form a stabilized mass (Stage 2) that can be safely stored for indefinitely long periods of time. These procedures have been applied to the organophosphorus nerve agents VR (an isomer of VX), GB, and GD and to sulfur mustard agent. The following shows the principal reaction of the nerve agents GB or GD by neutralization with monoethanolamine at 100°C under atmospheric pressure to yield, in the case of GB, isopropyl methylphosphonic acid and 2-aminoethylisopropyl methylphosphonate and, in the case of GD, the corresponding products. This is relevant for Stage 1. O O || || i-PrO–P–F + H2NCH2CH2OH = i-PrO–P–OCH2CH2NH2 + H2NCH2CH2OH.HF | | CH3 CH3

In the second stage, the reaction mass can then be incinerated or reacted with calcium hydroxide and bitumen at 200°C under reduced pressure to form insoluble calcium methylphosphonate and other salts in the bitumen mass and to release ethanolamine and isopropyl alcohol in the case of GB and the corresponding materials for GD (Pearson and Magee, 2002, pp. 187–316). The nerve agent VR (Russian VX) is neutralized by applying a decontamination solution known as RD-4, which contains potassium isobutylate dissolved in isobutanol and

Chemical Weapons Holdings N-methylpyrrolidinone, at 90–95°C under atmospheric pressure to break the P–S bond and yield diisobutyl methyl phosphonate and other products. This is also relevant for Stage 1. O O || || i-BuO-P-SCH2CH2NEt2 + KO-i-Bu + i-BuOH = i-BuO-P-O-i-Bu + KSCH2CH2NEt2 | | CH3 CH3

In the second stage, the reaction mass can be incinerated or reacted with bitumen (without calcium hydroxide) at a temperature that is increased from 130–140°C to about 180°C under reduced pressure to form potassium methyl phosphonate and other salts in the bitumen mass and to release isobutanol and N-methylpyrrolidinone (Pearson and Magee, 2002, pp. 187–316). Russia completed the destruction of its stockpile in September 2017.

5.6.13 Syria General verification by the OPCW of Syria’s chemical weapon declarations began in November 2013. Given the circumstances of Syria’s accession to the CWC, all OPCW verification work has—in some sense—been non-routine. More specifically, the OPCW Declaration Assessment Team (DAT), the OPCW factfinding mission (FFM), and the OPCW–United Nations joint investigative mechanism (JIM) in Syria have sought to clarify Syrian chemical weapon–related matters, including allegations of use. (For details of earlier work carried out under the UN secretary-general’s mechanism to investigate allegations of chemical and/or biological weapon use and the OPCW maritime removal of chemicals from Syria for out-of-country destruction, see Hart, 2015, pp. 582–585; Hart, 2016, pp. 728–739.) As of September 25, 2017, the status of OPCW verification of Syria was as follows:

1. The Secretariat had verified the destruction of 25 of 27 declared chemical weapons production facilities. 2. All chemicals declared by Syria and removed from its territory from 2014 had been destroyed. 3. Consultations were continuing regarding the completeness and correctness of Syria’s declarations to the OPCW. 4. A second round of OPCW inspections of the Barzah and Jamrayah facilities of the Scientific Studies and Research Centre (SSRC) were planned for late 2017. 5. The FFM was continuing to “study all available information” regarding allegations of use of chemical weapons in Syria, including “credible allegations” reported from December 2015 until the end of March 2017 (“Note by the Director-General, Progress in the Elimination of the Syrian Chemical Weapons Programme,” OPCW 25 September 2017).

5.6.14 United States The genesis of the current U.S. chemical weapon stockpile destruction program can be dated to the March 13, 1968 Skull Valley incident, whereby sheep were killed on properties

81 adjacent to Dugway Proving Ground (DPG) (Utah) by VX. This incident prompted the U.S. Congress to consider the safety of U.S. chemical weapons storage, handling, and disposal, including a ban on the cross-state transportation of chemical weapons pending the findings of a National Academy of Sciences (NAS) study, which was issued in 1969. Throughout much of the 1960s, the United States had disposed of chemical weapons by dumping (e.g., Operation Cut Holes and Sink ‘Em [CHASE]) (Linnenbom, 1971; Czul et  al., 1971; Czul et  al., 1972, 1974; Ferer, 1975; Müller, 2016). The NAS recommended that M34 cluster munitions be disassembled at Rocky Mountain Flats Arsenal (RMA) and the GB destroyed chemically by either acid or alkaline hydrolysis, that sulfur mustard located in 1 ton containers be incinerated, and that M-55 “coffins” be disposed of at a U.S. Army facility. The U.S. Army then undertook a series of industrial-scale chemical agent and munition disposal operations, including at RMA (Colorado). In 1981, the U.S. Army concluded that its incineration should be the baseline destruction technology. A number of members of Congress, members of the public, and others opposed incineration. In 1993, U.S. Public Law 102–484 mandated the destruction of the U.S. stockpile and tasked the U.S. Army with evaluating potential non-incineration-based destruction technologies. This resulted in additional evaluations by the U.S. Army, which later decided to employ non-incineration-based technologies for the stockpiles at Blue Grass, Kentucky and Pueblo, Colorado. The U.S. chemical weapons stockpile, totaling 31,278 metric tons of agent, was originally stored at nine locations: JACADS (1,001 metric tons) (destruction operations completed 2003); Edgewood, Maryland (1624 metric tons); Anniston, Alabama (2254 metric tons); Blue Grass, Kentucky (523 metric tons); Newport, Indiana (1269 metric tons); Pine Bluff, Arkansas (3850 metric tons); Pueblo, Colorado (2611 metric tons); Tooele, Utah (13,616 metric tons); and Umatilla, Oregon (3717 metric tons) (Lajoie, 1996). These numbers were adjusted during destruction operations. The final official stockpile total was 30,465 metric tons. As of 2017, the remaining U.S. stockpile was stored at two locations: Blue Grass, Kentucky (BGCAPP) and Pueblo, Colorado (PCAPP) (Table 5.5). The United States will employ explosive destruction technologies to destroy part of the munitions. EDS will be used to destroy problematic mustard munitions at PCAPP (United States of America, Report to the Eighty-fifth Session of the Executive Council on Progress Achieved towards Complete Destruction (31 May 2017), para. 2.2, p. 1). A Static Detonation Chamber (SDC) will be used to destroy problematic mustard munitions at BGCAPP (United States of America, Report to the Eighty-fifth Session of the Executive Council on Progress Achieved towards Complete Destruction (31 May 2017), para. 2.2, p. 1). In the period from January 31, 2017 to May 31, 2017, the United States destroyed 0.005 metric tons of an unknown agent at the Savanna Army Depot Activity (Savanna, Illinois) and 86.6592 metric tons of sulfur mustard agent at the PCAPP (United States of America, Report to the Eighty-fifth Session of the Executive [sic] Council on Progress Achieved towards Complete Destruction (31 May 2017), para. 3, p. 2). As of May 31, 2017, 87% of the BGCAPP SDC construction was complete, while systemization was 80% complete (United

82

Chemical Warfare Agents TABLE 5.5 Remaining Destruction Stockpiles as of May 31, 2017 Site Blue Grass Pueblo Total

Category 1 (mt)

Destroyed Since EIF (mt)

Remaining (mt)

% Destroyed (mt)

475 2371 2846

0.6497894 145.407427 146.057211

475 2225 2700

0.14 6.13

Source: United States of America, Report to the Eighty-fifth Session of the Executive Council on Progress Achieved towards Complete Destruction (31 May 2017). Document EC-85/NAT.3.

States of America, Report to the Eighty-fifth Session of the Executive Council on Progress Achieved towards Complete Destruction (31 May 2017, p. 3). As of May 31, 2017, construction of the BGCAPP was complete, while systemization was 46.5% complete (United States of America, Report to the Eighty-fifth Session of the Executive Council on Progress Achieved towards Complete Destruction (31 May 2017, p. 3).

5.6.14.1 Sea-Dumped CW Completion of fieldwork by the Hawai’i Military Munitions Assessment (HUMMA), which began in 2006, drew to a close in late 2014 (Follet, 2014; Shjegstad et al., 2014). HUMMA identified over 2500 munitions, took samples near 20 munitions, and failed to locate a number of 100 lb sulfur mustard bombs that archival research indicated should be present in the survey area. The exercise was carried out in partial response to U.S. Public Law 109–364, which requires the Department of Defense to determine the possible environmental impact of dumped (including chemical) munitions (Shjegstad et al., 2014; HUMMA project). The United States is scheduled to complete the destruction of its stockpile by 2023 (OPCW Executive Council, February 18, 2016. Document EC-81/NAT.1 cited in OPCW, July 11, 2016. “Report by the Director-General: overall progress with respect to the destruction of the remaining chemical weapons stockpiles.” Document EC-82/DG.21, para. 23).

5.7 Implications The timelines for CW destruction were overly optimistic. They were agreed partly for political, rather than technical, considerations. The timelines were also linked to the EIF of the CWC rather than being based on the accession of a given country. This was done so as to encourage universal treaty membership as soon as the treaty entered into force. In practice, the OPCW has had to adjust destruction timelines according to the exigencies of later accessions (e.g., Libya and Syria) or the discovery of forgotten stockpiles (e.g., Albania). In retrospect, the major CW possessor states also experienced more complex, mainly technical difficulties than were anticipated at the time the CWC negotiations were concluded. For large CW stockpiles, a “full-scale programmatic” approach has been taken. Environmental acceptance with respect to the selected technology, as well as safety of operations and

susceptibility to automation, has been prioritized. From the economic point of view, for the applied destruction technologies, the throughput rate is an important factor. Another key factor is whether the resulting wastes are environmentally acceptable, and that the amounts are minimized. For the technologies used for O/ACW, the approach with respect to the environmental and safety considerations is similar. O/ACW do have distinctive features, such as being fused/ unfused, corrosion issues, filling, and stability. The throughput rates for these technologies are important but not necessarily a key factor. OCW recoveries will continue. It is clear that for a state having smaller quantities, a stationary destruction facility is not an economically viable solution. Therefore, new solutions with respect to mobility are required, such as those adopted in China for the ACW. Japan is currently evaluating possible further sites in China to use its mobile destruction facility approach (Japan, 2017). Sea-dumped munitions are receiving increased attention. Dumping areas are known, and the international acknowledgment that these dumped munitions pose a risk to the environment, in general, and marine flora and fauna, in particular, is increasing. The growth of offshore wind farms, new pipeline laying, harbor enlargements, and the like are activities that will keep the issue of sea-dumped munitions on the political and social agenda. Over the coming years, new or improved technologies have to be selected and applied that take into account environmental safety and economic feasibility for the recovery of such munitions and secondary destruction and/or remediation. It is perhaps advisable to examine the experience gained in the field of destruction of CW and OACW. Technologies associated with oil and gas exploration and cable laying may be usefully reviewed. However, without having the technical and financial resources available to destroy recovered sea-dumped CW material immediately, a state should not perform such recovery operations. Storage of the recovered material is not a practical solution, because it creates more problems that pose further threats to human and environmental safety. Stockpiled CW (i.e., produced after January 1, 1946) are characterized by uniformity of type and storage protocols, which in turn, facilitates risk assessment, safety procedures, and international verification. Non-stockpiled CW and OACW are characterized by a general lack of uniformity of munition type, fill, and condition. Thus, more ad hoc approaches to destruction have been adopted that are less amenable to automation and higher throughput. Principles for CW destruction of continuing relevance include the need to minimize handling, documentation procedures

83

Chemical Weapons Holdings (assessments, monitoring, post-operation lessons-learned studies), and the need to maximize throughput.

POLITICAL AND TECHNICAL APPROACHES • How do changes in science and technology (S&T) affect monitoring, understandings of environmental and human health effects, and destruction-related routines and standard operating procedures? • Approaches for ensuring OPCW institutional capacity in the “post–chemical weapons destruction era” (e.g., understanding of non-standard programs such as an absence of state military stockpiles in relation to CWC provisions). • Approaches to ensuring the safe and secure destruction of new discoveries of old chemical weapons and dumped chemical weapons. • To what extent have technology evaluations and scale up for destruction operations become “routine,” and what, for example, does this imply for dumped conventional and chemical weapons?

As previously mentioned, political and technical approaches for developing and implementing destruction programs continue to evolve. Their longer-term implications should be further evaluated according to the particular context of a given case. It should also be noted that North Korea remains outside the CWC with an expected substantial stockpile of blister and nerve agents partly filled into munitions. In such a case, destruction technologies will be required that can be used to ensure timely destruction under environmentally acceptable conditions and under international verification. Thus, the lessons learned from the destruction undertaking from the last 20 years after the EIF of CWC remain relevant.

DAT DOVO EC EDS EIF EOD EU FFM GEKA MBH HCM HELCOM HUMMA I JACADS LIC MEA MOD MPF MT N NAS NDE NIPPS OCW OPCW OVT PCAPP PCAPP-EDS PDTDF RCWDF

ABBREVIATIONS AND ACRONYMS ACW ACWA APG/CTF ASP BGCAPP BGCAPP-SDC CAMDS CDC CG CHASE CK CSP CW CWC CWDF CWS

abandoned chemical weapon assembled chemical weapons alternatives Aberdeen Proving Ground Chemical Transfer Facility abandoning state party Blue Grass Chemical Agent-Destruction Pilot Plant Blue Grass Chemical Agent-Destruction Pilot Plant Static Detonation Chamber chemical agent munitions disposal system cold detonation chamber/controlled detonation chamber phosgene Cut Holes and Sink ‘em cyanogen chloride Conference of the States Parties chemical warfare/chemical weapon Chemical Weapons Convention chemical weapons destruction facility Chemical Warfare Service

RMA RTCDF SD SDC SSRC TS TSDF UT

declaration assessment team Dienst Opruiming en Vernietiging Ontploffingtuigen Executive Council explosive destruction system entry into force explosive ordnance disposal European Union fact-finding mission Gesellschaft zur Entsorgung von Chemischen Kamptstoffen und Rüstungsaltlasten MBH hydrogen concentration measurement Helsinki Commission Hawai’i Military Munitions Assessment incineration Johnston Atoll Chemical Agent Disposal System liquid incinerator monoethanolamine Ministry of Defense metal parts furnace metric ton neutralization National Academy of Sciences non-destructive evaluation neutron induced prompt photon spectroscopy old chemical weapons Organization for the Prohibition of Chemical Weapons operational verification test Pueblo Chemical Agent-Destruction Pilot Plant Pueblo Chemical Agent-Destruction Pilot Plant Explosive Destruction System Prototype Detonation Test and Destruction Facility recovered chemical weapons destruction facility Rocky Mountain Arsenal Rabta Toxic Chemicals Destruction Facility static detonation static detonation chamber Syria Scientific Research Centre Technical Secretariat treatment, storage and disposal facility ultrasonic testing

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Chemical Weapons Holdings OPCW. July 14, 2016. Draft Report of the OPCW on the Implementation of the Convention on the Prohibition of the Development, Production, Stockpiling and Use of Chemical Weapons and on Their Destruction, OPCW document EC-82/4, C-21/CRP.1. OPCW. July 24, 2017. Report by the Director-General: Status of the Implementation of the Plan for the Destruction of Libya’s Remaining Category 2 Chemical Weapons outside the Territory of Libya, document EC-86/DG.1. OPCW. September 8, 2017. Note by the Technical Secretariat: Modifications to the Arrangement with the Republic of Panama Governing Inspections on San José Island, Panama. Document EC-86/S/4. OPCW. September 19, 2017. Note by the Director-General, Summary of Verification Activities in 2016, OPCW document S/1537/2017. OPCW. September 25, 2017. Note by the Director-General, Progress in the Elimination of the Syrian Chemical Weapons Programme, OPCW document EC-86/DG.23. OPCW. November 27, 2017. Opening Statement by the DirectorGeneral to the Conference of the States Parties at its TwentySecond Session, OPCW document C-22/DG.20. OPCW Executive Council. February 18, 2016. Document EC-81/ NAT.1 cited in OPCW. July 11, 2016. Report by the DirectorGeneral: Overall Progress with Respect to the Destruction of the Remaining Chemical Weapons Stockpiles, Document EC-82/DG.21, para. 23. OPCW Technical Secretariat Background Paper. April 25, 2003. Consolidated Unclassified Verification Implementation Report (April 1997–31 December 2002). Document RC-1/S/6, 5 and 38. Panama. June 16, 2017. Panama, Concept Plan for the Destruction of Eight Old Chemical Weapons, Document EC-85/NAT.2. Pearson, G. S. and Magee, R. S. 2002. Pure and Applied Chemistry, 74:187–316. Prentiss, A. M. 1937. Chemicals in War: A Treatise on Chemical Warfare. New York: McGraw-Hill.

85 Program Manager for Non-stockpile Chemical Material. November 1993. Survey and Analysis Report, U.S. Army. Satake, M. May 25–27, 2016. Abandoned Chemical Weapons Destruction Projects in China. London, http://cwd2016.weebly. com/presentations.html, 1. Presentation at 19th International Chemical Weapons Demilitarisation Conference CWD 2016. Séché Environnement, www.groupe-seche-international.com/. Sheluchenko, V. V. and Utkin, A. 1998. The Role of GosNIIOKhT in the Russian Chemical Weapon Destruction Program. In Chemical Weapon Destruction in Russia: Political, Legal and Technical Aspects, SIPRI Chemical & Biological Warfare Studies, eds. J. Hart and C. D. Miller. Oxford: Oxford University Press. Shjegstad, S., Carton, G. and Edwards, M. 2014. Searching for history. TME Military Engineer, http://themilitaryengineer.com/index.php/tme-articles/tme-magazine-online/ item/333-searching-for-history. Starostin, L. 1999. OPCW Approved Non-Destructive Evaluation (NDE) Techniques in Verification Activities. The Hague, www.opcw.org/fileadmin/OPCW/event_photos/2010/tabletop_exercise_poland_nov2010/TTE_Sessions/Introduction_ OPCW_NDE_TTX_NDE.pdf. Presentation slides. Sutherland, R. G. 1997. The destruction of old and obsolete chemical weapons: Past experience. In The Challenge of Old Chemical Munitions and Toxic Armament Wastes, eds. T. Stock and K. Lohs, 141. Oxford: Oxford University Press. Tang, C. May 25–27, 2016. Destruction of Japanese Abandoned Chemical Weapons Discovered in China: Progress and Challenges. London, http://cwd2016.weebly.com/presentations.html, 4. Presentation at 19th International Chemical Weapons Demilitarisation Conference CWD 2016. United States. June 22, 2017. United States of America, Report to the Eighty-fifth Session of the Executive [sic] Council on Progress Achieved towards Complete Destruction (31 May 2017). Document EC-85/NAT.3. Vucaj, F. May 2007. Albania, Republic of Albania: World Leader in Chemical Disarmament. Chemical Disarmament, vol. 5, 10th anniversary special edition. The Hague: OPCW.

6 Syria’s Chemical Disposal Program Al Mauroni and Timothy A. Blades CONTENTS 6.1 Introduction...................................................................................................................................................................................87 6.2 The Ghouta Attack........................................................................................................................................................................89 6.3 Developing a Response................................................................................................................................................................. 91 6.3.1 Contingency Options........................................................................................................................................................92 6.4 Implementing a Disposal Program...............................................................................................................................................94 6.4.1 Postscript..........................................................................................................................................................................96 6.5 Lessons Learned...........................................................................................................................................................................97 6.6 Conclusions...................................................................................................................................................................................99 References.............................................................................................................................................................................................100

6.1 Introduction This section will provide a summary of the actions taken in regard to dismantling Syria’s chemical weapons stockpile in response to the Syrian government’s attack on the Ghouta urban area near Damascus in 2013. This is not a technical discussion of the disposal process involved or the composition of the Syrian chemical weapons stockpile, details of which have been kept limited due to promises of confidentiality by the Organization for the Prohibition of Chemical Weapons (OPCW). The intent is to inform the reader about the policy challenges inherent in any U.S. government support for a chemical disposal effort in support of regional stability. U.S. technology capabilities play a significant role in achieving the political objective of deterring and rolling back a country’s weapons of mass destruction (WMD) program. Without getting into a deep discussion of Middle East politics, one can identify the key points of Syria’s domestic and foreign policy as a basis for discussing its WMD program. The combination of religious differences, significant oil reserves, and significant arms sales in the region has made the Middle East a very turbulent region. Syria had allied itself with other Arab nations to go to war against Israel on numerous occasions between 1948 and 1982, armed with weaponry largely supplied by the former Soviet Union. As such, Syria’s actions prior to 1991 were often viewed poorly by the U.S. government, who criticized the regime’s record on human rights violations and its support of, in its view, national resistance movements that others would call terrorist groups. President Hafiz al-Assad, who took power in 1970, sought to establish a stronger link with the United States as Soviet support began to dry up in the late 1980s. The Persian Gulf War provided an opportunity for Syria to join the U.S. coalition against Iraq and to provide a “moderate” face of the Arab nations (Moseley, 1991). Syria provided more than 14,000

troops in a significant, if largely symbolic, show of support during that conflict. Syria and Israel worked toward a comprehensive peace agreement through much of the 1990s, which was to include discussions of the return of the Golan Heights to Syria, but were unable to conclude terms amenable to both sides. Hafiz al-Assad’s death in 2000 brought his son Bashar al-Assad into power. While some in the West had hoped that a young, Western-educated leader would open up Syria to Western values and conclude peace talks with Israel, internal domestic challenges, an increasingly interventionist U.S. government policy, and continued turmoil in Lebanon have complicated that goal (Lesch, 2007). However, the new president did reach out to the political leaders of the United Kingdom, France, Iraq, Lebanon, and Turkey between 2001 and 2008. Political dynamics have placed Syria in a very precarious position relative to regional Middle East politics. Its southern neighbor and long-time adversary, Israel, continues to have a dominant conventional military force and is alleged to have a mature nuclear weapons capability (Birch and Jeffrey Smith, 2014). Turkey, a U.S. ally and NATO partner, has had challenged relations with its neighbor Syria over the past decades. Jordan, a past ally, is perhaps closer to the United States in regional security matters than to Syria. Iraq under Saddam Hussein was not a friend of Syria and given the significant U.S. forces in Iraq, remains a source of concern. Significant U.S. military forces in the Mediterranean Sea and throughout the Middle East also pose a perceived threat to Syria, given regional events over the last 15 years (Jouejati, 2009). Syria’s concern about U.S. military forces was not assuaged by being included in the “Axis of Evil” in 2002 and (then) Under Secretary of State John Bolton’s strong language condemning Syria’s “weapons of mass destruction and missile development programs” in 2003 (Bolton, 2003). To put things into perspective, the concept of a Middle Eastern nation developing nuclear, biological, or chemical weapons is

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88 certainly not a novel one. Between 1970 and today, Egypt, Iran, Iraq, Israel, Libya, Saudi Arabia, and Syria have all been suspected of developing unconventional weapons at one time or another. In particular, the combination of unconventional weapons and ballistic missiles has been seen as particularly destabilizing against regional security concerns (Kerr, 2008). Syria’s WMD program may have begun around 1982, after Israel invaded Lebanon and its military forces trounced Syrian military forces there. Given inferior military forces and reduced support from the Soviet Union, President Hafiz al-Assad decided to start a chemical weapons program to enable a form of strategic deterrence against the challenge of superior conventional attacks by Israel (Jouejati, 2009). The 1997 Proliferation: Threat and Response report by the Department of Defense (DOD) suggested that Syria may have started its program in the 1970s, supported by the Soviet Union and Egypt. Syria relied on external chemical industries for many of its chemical precursors, claiming that the intended use was for agriculture. Syria also had a long-time relationship with the former Soviet Union for the purposes of procuring military hardware and military support (Collelo, 1987). A 1983 Central Intelligence Agency (CIA) report stated that the Soviet Union and Czechoslovakia had supplied Syria with chemical agents, delivery systems, and training (CIA, 1983). Nevertheless, its attempt in the 1980s was to develop an indigenous capability not reliant on external sponsors (Nikitin, 2013). Official unclassified U.S. assessments of Syria’s chemical weapons capability in 2013 suggested a stockpile of more than 1000 metric tons of chemical warfare agents and precursors, including mustard agent in “ready-to-use form” and sarin and VX nerve agents in their binary components (“ready-to-mix”). Despite the desire to have a self-sufficient production capability, Syria remained reliant on external economic sources for precursor chemicals. The main delivery systems were thought to be short-range ballistic missiles (Scud variants), 250 kilogram aerial bombs, and BM-21 multiple rocket launchers. While the media gave breathless reports about Syria having “the largest chemical weapons arsenal” in 2012 and 2013 (Nikitin, 2013), one should use the U.S. chemical weapons program (which produced in excess of 30,000 tons at its height) as a metric for perspective. These public statements were released after most other nations with former chemical weapons stockpiles either had completely destroyed their weapons or were in the final steps of destroying them. The traditional view is that Assad developed chemical or biological weapons as a strategic deterrent against Israel’s superior conventional capability. He was concerned that Israeli forces could easily overrun Damascus and overthrow the regime if they had any intention of doing so. Whether Assad would have actually ordered the Syrian military to use chemical weapons against Israel, if that nation attacked Syria, is a separate discussion that requires analysis of Syria’s political will. In addition, there is no evidence that Syria would have ever allowed sub-state groups to obtain chemical weapons from its stockpile, perhaps due to concerns that these groups might use those weapons against Israel or other adversaries and the resulting retaliation might come back against Syria (Jouejati, 2009). When Syrian security forces killed dozens of people in Daraa in March 2011 in an attempt to suppress public demonstrations,

Chemical Warfare Agents it caused a wave of continuing mass protests across the nation. Assad’s attempts to quiet the protests with force led to U.S. and European economic sanctions against Syria and the rise of Syrian opposition groups. Within a year, the internal conflict had claimed estimates of 8000–10,000 dead. In early February 2012, prior to the escalation of chemical incidents in Syria, the Obama administration revealed that they were reviewing military options with the DOD and U.S. Central Command. One of the potential scenarios included using U.S. military forces to secure Syria’s chemical weapons sites (Starr, 2012a,b). Government estimates of between 50,000 and 75,000 military personnel required were floated in the media. While this was cited as a “worst-case scenario,” the prospect of entering a war zone for the purpose of securing the sites while the Syrian civil conflict was ongoing, and possibly without international assistance, was daunting. Other military options, to include bombing the chemical weapons program-related sites, were not seen as feasible due to the possibility of collateral damage caused by the dispersion of chemical agents (Hosenball and Stewart, 2012). On July 23, 2012, the Syrian Foreign Ministry’s spokesperson suggested—for the first time in public—that Syria had an unconventional weapons capability but that these weapons would not be used except in the event of “external aggression” against Syria. He also noted that the Syrian regime saw the opposition forces as being externally funded and driven by foreign parties (Associated Press, 2012). In response to this statement, President Barack Obama spoke from the White House in August 2012 stating that Syria’s use of chemical weapons would be “a red line for us and that there would be enormous consequences if we start seeing movement on the chemical weapons front or the use of chemical weapons” (White House, 2012). He repeated this warning in December at the National Defense University, saying: “The use of chemical weapons is and would be totally unacceptable. And if you make the tragic mistake of using these weapons, there will be consequences, and you will be held accountable.” This was widely interpreted as a threat to use military force. On the same day as the president’s warning, the Syrian Foreign Ministry stated that “Syria has stressed repeatedly that it will not use these types of weapons, if they were available, under any circumstances against its people” (Nikitin, 2012). Reports from Syria suggested otherwise. Table 6.1 notes the alleged chemical attacks in 2012 and leading up to August 2013, as reported by various government agencies. Not all of these attacks were verified regarding the exact source or type of munitions or numbers of casualties. There was often little in the way of formal attribution of the attacks regarding the weapon source and/or the exact agent, in large part due to the inability to obtain clinical or environmental samples taken directly from the victims or the sites of the alleged attacks. In some cases, it may have been chlorine or tear gases. In other cases, it may have been sarin diluted with other chemicals to mask the nature of the attack. No persistent chemical agents were used, perhaps as a deliberate method to avoid attribution. In most if not all of these early cases, the number of casualties was relatively low—they were point attacks using a small number of rounds to hit a hardened position rather than a large-scale area attack involving many munitions. A former Syrian scientist who had left the country stated that the regime was purposefully

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Syria’s Chemical Disposal Program TABLE 6.1 Alleged Chemical Weapons Incidents in Syria (Prior to the Ghouta Attack) Date October 17, 2012 December 23, 2012 Mid-January 2013 March 13, 2013 March 19, 2013 March 24, 2013 April 12–14, 2013 April 13, 2013 April 25, 2013 April 29, 2013 May 14, 2013 May 23, 2013

Location

Type of Attack

Salquin Homs Qusayr Darayya Khan al-Assal (Aleppo), Otaybah Adra Jobar Sheik Maqsood Darayya Sarqueb Qasr Aba Samrah Adra

Lethal, unknown Unknown Unknown Unknown Rockets, sarin Rockets, possibly white phosphorus Sarin Sarin Unknown Helicopter, sarin Unknown Unknown

Source: Data from United Nations, 2013b; Robinson, J.P., Harvard-Sussex Program Occasional Paper No. 4, 2013; Arms Control Association, “Timeline of Syrian chemical weapons activity, 2012–2017,” 2017.

using sarin nerve agent in small quantities to stop insurgent progress around Damascus and to incapacitate those fighting forces rather than to cause mass casualties. By keeping casualties low, the Syrian regime may have been seeking to cast doubt on the insurgents’ claims of chemical weapons use and to test the limit of President Obama’s “red line” (Al Jazeera, 2013). Between April and June 2013, U.S., U.K., French, and Israeli government officials made public statements that they believed the Syrian regime was using chemical weapons, or at the least, sarin nerve agent was being used in attacks against the civilian population (Castillo and Botelho, 2013). Director of National Intelligence James Clapper told Congress in March 2013 that Syria had an advanced chemical warfare program and assessed that the Assad regime “might be prepared to use [chemical weapons] against the Syrian people” and that “groups or individuals in Syria could gain access to [chemical weapons]-related materials” (Clapper, 2013). Ben Rhodes, deputy national security advisor for strategic communications, stated on June 13, 2013 that the U.S. intelligence community “assesses that the Assad regime has used chemical weapons, including the nerve agent sarin, on a small scale against the opposition multiple times in the last year.” The administration’s response to this use was to increase support to the political opposition in Syria (White House, 2013b). The Syrian military forces stepped over that “red line” on August 21, 2013, when they attacked west and east Ghouta with multiple attacks using sarin nerve agent.

6.2 The Ghouta Attack Between 2 and 5 a.m. on August 21, 2013, several surface-tosurface rockets containing sarin nerve agent landed in west and east Ghouta. This geographical region lies south and east of Damascus, including some 60 towns and villages, and hosted about 2 million people prior to the Syrian civil war. The weather that morning had created a temperature inversion in which aerosols and vapors would stay close to the ground, thus maximizing the effects of a chemical attack. The western attack was decidedly the smaller of the two attacks, believed to be a number

of artillery rockets (four to seven) that landed in Moadamiya around 5 a.m. According to Human Rights Watch, these rockets may have been M-14 projectiles, which are 140 mm rockets fired from a BM-14 launcher. The Syrian military is believed to have procured about 200 of these launchers from the former Soviet Union in the late 1960s. The rockets can be armed with high explosive, smoke, or chemical warheads. Each chemical warhead can hold about 2.2 kilograms of sarin. While no evidence of the rocket warhead has been recovered, remnants of the rocket at a few impact sites suggest this was the weapon system used. The range of the weapon system could place the source of the attack as coming from a local Syrian military base, and the clinical analysis of blood and urine samples suggested sarin was used (Human Rights Watch, 2013). The eastern attack against Zamalka was significantly larger, with evidence that at least eight large artillery rockets were used to deliver sarin nerve agent against the population center. These rockets appear to be of a Syrian design referred to as the family of “Volcano” rockets, which can have either a chemical or a high-yield explosive warhead (Higgins, 2014). These rockets were based on the 122 mm artillery rocket (made popular by the Soviet BM-21 Grad multiple rocket launcher), sporting 330 mm stabilizing fins (thus, the press reports and investigation reports referring to a “330 mm” rocket) and a 350 mm wide warhead that may have held up to 50 or 60 liters of sarin. These rockets could have been launched from the Iranian Falaq-2 333 mm rocket launching system, known to be operated by the Syrian military forces. The lack of large impact craters and clinical evaluation of the casualties suggested a chemical, not high explosive, attack (Human Rights Watch, 2014). Estimated casualties from these attacks ranged from 300 to 1400 dead and more than 3600 injured (Doctors without Borders, 2013). This attack surprised many in the national security community. First, the use of an improvised, indigenous artillery rocket within the context of the civil war was not expected. The attack didn’t fit the profile of using former Soviet Union delivery means (e.g., aerial bombs, 122 mm rockets, or Scud missiles). Second, it was a tactical strike aimed at achieving limited gains. Clearly, Assad was not saving chemical weapons for use only

90 as a strategic deterrent against Israeli conventional forces. Given the increased focus from the international community on earlier chemical attacks, it was difficult to understand why Assad would authorize such a high-visibility attack. To be clear, this was not an isolated artillery strike. The Syrian military targeted the same areas with intensive artillery and rocket barrages for the next 4 days, using conventional high explosive rounds. Following government control of the area, buildings were demolished, forcing civilians out of the area. It is unclear whether this was just part of a continued campaign against the insurgents or an attempt to eliminate forensic evidence of the chemical attacks. Following an emergency meeting by the United Nations Security Council, UN Secretary General Ban Ki-moon announced the intent to conduct an investigation of the attack. The Syrian government agreed to allow a UN investigation team, which was already in country investigating other alleged chemical incidents, access to the site of the August 25 attacks. The White House released its assessment on August 30, “assessing with high confidence” that the Syrian government had carried out the chemical weapons attack. Syria did not necessarily violate the 1925 Geneva Protocol in its conduct of this attack, as many interpret the treaty as only prohibiting first use of a chemical weapon against another state party (Graham and LaVera, 2002). It did violate the “international norms” that view chemical weapons use as a taboo issue in the context of contemporary military conflict. Samantha Powers, the U.S. ambassador to the United Nations and a strong vocal proponent of the “responsibility to protect” concept, stated on August 26 that “Assad has used CWs against civilians in violation of [international] norm” (Alman, 2013). The threat was not just to the Syrian people or regional stability, but in the nature of a precedent that might cause further chemical weapons proliferation across the globe, to include attacks against U.S. national security interests. The next day, President Obama stated that he would seek authorization from Congress for the use of force for a limited military strike against Syria. It was not clear whether these strikes would be against Syrian government buildings, its military forces, or sites associated with its chemical weapons program. Certainly, there was reluctance to attack the chemical agent storage sites themselves, an act that could have caused significant civilian casualties as a result of any hazardous releases. The DOD developed plans in coordination with French forces to attack 50 targets in Syria, starting with Navy Arleigh Burke-class destroyers firing Tomahawk cruise missiles and followed by air strikes (Chollet, 2016). This announcement appeared to have caused Assad to pause his chemical weapons attacks (through 2013 and most of 2014, at least). Russian Foreign Minister Sergey Lavrov announced on September 9 that Syria would agree to dismantle its chemical weapons program in return for a hold on U.S. military action against its regime. Shortly following this, Assad formally acceded to the Chemical Weapons Convention, which opened the door to formal actions by the OPCW to monitor and verify the elimination of Syria’s declared chemical weapons program (ACA, 2017). As noted earlier, the OPCW had already arrived in Syria due to the Syrian government’s request to the United Nations for an impartial, independent investigation of an alleged chemical

Chemical Warfare Agents weapons attack by the Syrian insurgents in the Aleppo area on March 19, 2013. An advance team had traveled to Cyprus in April and begun working with various governments on factfinding missions (UN Mission Report, 2013). A UN Mission team traveled to Damascus in August to conduct activities, which were to investigate allegations of chemical weapons use and to visit the sites, as possible. The Ghouta incident forced a refocus of its initial activities, given the large scale and relative location of the attacks. The team spent 2 weeks in Damascus under an agreement with the Syrian government. This was not without peril, as the UN inspectors’ convoys were targeted by sniper fire as they traveled to the incident sites (United Nations News Centre, 2013). The UN team interviewed survivors of the attack, as well as medical responders, and gathered both clinical and environmental samples in the area. The survivors described their symptoms as including shortness of breath, eye irritation, nausea, and vomiting, all consistent signs of nerve agent poisoning. The clinical samples of nearly all of the patients tested positive for sarin nerve agent exposure. The team also were able to examine and photograph the impact sites as well as to collect rocket fragments, which also tested positive for sarin. As a result, the Mission Team concluded that “surface-to-surface rockets containing the nerve agent sarin were used … in the Ghouta area of Damascus” (United Nations, 2013). Notably, this report did not attribute the attack to the Syrian government, although certainly, the circumstantial evidence pointed in that direction. The particular weapon systems used, the military-grade sarin nerve agent involved, the proximity of Syrian government forces, and the target of the attacks would lead one to all but declare the Syrian Arab Republic as the instigator of the attacks. However, given the United Nations’ need to remain impartial and to encourage nation-states to cooperate in its investigations into chemical weapons possession, attribution without hard facts was not in its interest. The U.S., British, and French governments relied on releases of unclassified intelligence assessments to make their public allegations of Syria’s responsibility. Social media was prominent in rapidly identifying the areas under attack and resulting chemical casualties. The French government suggested that the initial casualties were at least 700 based on an inspection of videos online. Human rights observers in the field used satellite phones to filter information at the site. At the same time, skeptics tried to use social media as a tool to cast doubt on the notion of a chemical attack or to suggest that the Syrian insurgents had done it. While not offering attributional evidence, social media still provided a large amount of data to analysts within and without government. Part of the intelligence may have included U.S. monitoring of the communications to Syrian military units that actually conducted the attack. According to one news article, translation issues prevented the White House from realizing that there was going to be a chemical attack on Ghouta. In addition, message traffic within Syria suggests that the Syrian military commanders had not anticipated such a large number of casualties from the limited artillery attack in east Ghouta. It may be that the intent was to continue the trend of small-scale chemical attacks against the insurgents and the civilian population, rather than the respectively larger attack that occurred (Entous et al., 2013).

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Syria’s Chemical Disposal Program On the other hand, it may have been that the attack was a deliberate gambit. If the international community responded weakly to the Ghouta attack, Assad could continue to use sarin in larger amounts than just single point attacks. If the international community objected, then he could offer the option of giving up his chemical weapons in return for security guarantees that the Western nations would not attack him in retaliation. Either way, Assad had an angle to pursue. President Bashar al-Assad disputed the UN findings, claiming that the attack was caused by the Syrian insurgents and that it wouldn’t make sense to use chemical weapons in an area where Syrian military forces were operating. The Russian government supported this argument, suggesting that the Syrian opposition was trying to provoke an international response by causing this attack (BBC, 2013). Interestingly enough, the Syrian government reported alleged nerve agent attacks against its military forces occurring on August 22, 24, and 25, attributing these attacks to the Syrian insurgents. The UN Mission was given environmental and clinical samples that tested positive for sarin, but the impact sites did not lead to any evidence of attribution (UN Mission Report, 2013). There is no evidence that the insurgents had either the capability to manufacture sarin nerve agent in the quantities required for this attack or the military delivery systems to cause the widespread casualties, as suggested by Seymour Hersh and others (Hersh, 2013). Regardless, Russian Foreign Minister Sergey Lavrov, in a number of talks with Secretary of State John Kerry, proposed that securing Syria’s chemical weapons and removing them from the country might be a feasible political solution. This led to a series of technical discussions as to what could be acceptable to the two nations with respect to a Syrian chemical disposal program. After a meeting in Geneva in mid-September, the two announced a framework by which Syria’s chemical weapons would be eliminated. This was not a novel concept initiated by the Russian government. Senator Richard Lugar (Republican, Indiana) had spoken to Russian officials about the possibility of a joint U.S.–Russian effort to eliminate Syria’s chemical stockpiles prior to the Ghouta incident but had been rebuffed. The Russians claimed that they couldn’t support an elimination effort, as Syria was not a signatory to the Chemical Weapons Convention (Herzenhorn, 2012). While it was true that Syria was not a signatory to the treaty, this would not have prevented the U.S. government from using the successful Cooperative Threat Reduction (CTR) program as a basis for eliminating Syrian chemical weapons, assuming the Syrian government agreed to cooperate. It is more probable that the Russian government did not want to see U.S. military

operations against the Syrian government and so came back to this option as a means to forestall U.S. military attacks. The deal rested on an accelerated schedule in which Syria would immediately declare its stockpile and provide full access to all of its chemical weapons sites. OPCW inspectors would complete their initial inspections by November, and all of Syria’s chemical weapons and agents were to be destroyed by June 30, 2014. President Assad sent a letter to the United Nations on September 12, formally acceding to the Chemical Weapons Convention, and by September 20, had submitted an initial disclosure of Syria’s chemical weapons program. In return for Syria’s complying under the Chemical Weapons Convention, the U.S. government would not attack Syria. Technically, Assad had 30 days before the treaty would enter into force with respect to his program. On October 23, the Syrian government formally submitted the initial declaration to the OPCW. The United Nations Security Council passed a resolution on September 27, 2013 to formalize the U.S.–Russian framework, calling for the complete elimination of all chemical weapons material and equipment by the first half of 2014. UNSCR 2118 was the mechanism to allow the OPCW to carry out its verification activities and to establish a fund through which other nations could support the mission (United Nations, 2013). A 9 month deadline was “exactly on the borderline of being technically feasible and utterly insane,” said former State Department official Tom Countryman (Dozier, 2016).

6.3 Developing a Response Syria’s declaration to the OPCW was not made available to the public, but a summary of its declaration was released in later discussions of how the disposal would take place. The declared Syrian chemical weapons program included about 1300 metric tons of chemicals located on more than 20 sites, including more than 40 distinct facilities. While the Russian government was still being cagy about Syrian regime issues, it was open to meetings with U.S. security officials between September and December 2012. These talks included intelligence sharing on the contents and locations of the Syrian chemical weapons sites (Dozier, 2016). Table 6.2 offers a listing of the declared substances and facilities. Interestingly, there were no actual stocks of sarin or VX nerve agents declared, as the Syrian military supposedly used a binary chemical formula to mix those chemical warfare agents immediately prior to using them. Certainly, the Syrian government may have left off some sites that may have had an association with the chemical weapons program, but it

TABLE 6.2 Syria’s Declared Chemical Weapons Program Chemical agents

Chemical sites Chemical weapons

580 metric tons of methylphosphonyl difloride (DF, a precursor for sarin) 20 metric tons of mustard agent 130 metric tons of isopropyl alcohol 310 metric tons of four “other Category 1 industrial chemicals” 260 metric tons of 13 different “Category 2 industrial chemicals” including chloroethylamine, phosphorus trichloride, phosphorus oxychloride, hexamine, hydrogen chloride, and hydrogen fluoride One research and development site, 10 production sites (including 27 production facilities), and 12 storage sites 1230 unfilled munitions (aerial bombs, missile warheads)

92 is unclear whether this is a deliberate deception on behalf of the Syrian government or merely a difference of definition regarding what had to be declared. In developing options for destroying Syria’s chemical weapons, one must go back in time to U.S. government concerns over the possible loss of Syrian government control over its chemical weapons stockpiles to Syrian insurgents, which included the Islamic State (ISIS) and al Qaeda. An alternate concern was that Assad would be pushed by insurgent successes to use chemical weapons in a mass attack. In part, this was due to past concerns related to Libya, as that country’s civil war in 2011 stopped the dismantlement of its chemical weapons program and put the question of the security of its chemical weapons stockpile up for consideration. Although the Libyan National Transition Council assured the OPCW of its intent to secure the sites and continue the destruction of weapons, the concern over a potential for the loss of control of military-grade chemical weapons to sub-state groups remained present. Following the Syrian government’s public disclosure that it did in fact have a chemical weapons program, discussion on U.S. policy options intensified. U.S. military action was an option in the event that Syria’s government might collapse, putting the security of its chemical stockpile sites into question. Previous to the Ghouta attack, in April 2013, Prime Minister Benjamin Netanyahu suggested to Defense Secretary Chuck Hagel that the British, Americans, and Israelis should collaboratively plan to either disable or secure Syria’s chemical weapons sites with special operations forces. This suggestion for collaborative action was not acted on, but the understanding was that something had to be done (Chollet, 2016). The possibility existed that the Syrian regime would fall and that the U.S. military would need to secure and dispose of the chemical stockpile, similarly to what occurred in Iraq after 1991 and 2003. Assuming that a transitional government was taking control but didn’t quite yet have its security forces deployed, a relatively small U.S. military force augmented by chemical weapons specialists could secure two storage sites in Syria until technical forces could dispose of the agents and munitions. This would at least reduce the chance of al Qaeda and ISIS gaining access to tons of chemical weapons. If this plan of action were followed, the military would require a destruction capability to be fielded in theater. U.S. Central Command identified a need in 2012, through the Joint Staff, for a deployable chemical weapons destruction capability. In August 2012, the assistant secretary of defense for nuclear, chemical, and biological defense programs (ASD[NCB]) directed the Defense Threat Reduction Agency (DTRA) to develop options using the CTR program and to support building partner capacity efforts in the region. In response, DTRA created a “Regional Contingency Team” to coordinate the many agencies within DTRA on this issue. DTRA’s mission included hosting a joint force headquarters with the responsibility of organizing missions to eliminate unconventional weapons stockpiles. Between 2004 and 2012, the DOD had had numerous internal discussions on how a “WMD elimination capability” might be formed, whereby U.S. military forces would lead a U.S. government effort to dismantle and dispose of an adversarial nation’s WMD program. Following the ad hoc effort by the DOD to find an active WMD program in Iraq in 2003 (Hersman, 2004), Defense

Chemical Warfare Agents Secretary Donald Rumsfeld and his successor Bob Gates both continued to advocate for an institutionalized force that could locate, exploit, and eliminate unconventional weapons recovered on foreign territory in a future conflict (Dept of Defense, 2006, 2010). After years of debate over what this desired capability was and how it should be formed (Wright and Skattum, 2010), General Robert Kehler, commander of U.S. Strategic Command, formally activated the Standing Joint Force Headquarters for Elimination at DTRA on February 3, 2012. This was a small command and control element designed to be the center of a joint task force that would be composed largely of Army chemical, biological, radiological, and nuclear (CBRN) defense forces and other military elements as required (DTRA, 2012b). However, it would not be the central focus of the DOD’s response to the task of destroying Syria’s chemical weapons. The elimination headquarters was effectively hobbled by a lack of dedicated personnel and an immature operational concept that inherently relied on non-defense government agencies to fulfill roles that were not acknowledged as resourced requirements and had never been practiced (DOD, 2009). In the fall of 2012, Defense Secretary Ash Carter tasked Frank Kendall, the undersecretary for acquisition, technology and logistics (AT&L), to develop technical and policy options for possible contingencies, assess military capabilities, and work on the interagency to address this challenge (Bleek and Kramer, 2016). In addition, the DOD’s Threat Reduction Advisory Committee (TRAC), an advisory group sponsored by the AT&L office, also engaged with the topic of threat reduction and WMD elimination “for cooperative and non-cooperative engagement” with foreign nations (DTRA, 2012). One of the TRAC’s recommendations in late December 2012 was that the Army’s Edgewood ChemicalBiological Center (ECBC) at Aberdeen Proving Ground should conduct a capabilities assessment to identify technologies that would be capable of destroying bulk liquid chemicals in a remote, hostile location (Blades, 2015). In January 2013, the AT&L formally directed the establishment of a Syria Chemical Weapons Senior Integration Group (SIG), composed of senior leaders who would meet every 2 weeks to work on technical and policy issues and to address interagency processes. This SIG would develop working groups as needed to address distinct parts of the program. One of these groups was the technical community responsible for developing a disposal platform. This group was called the Syria WMD Operational Response and Dismantlement (SWORD) team, which included the Joint Program Executive Office for Chemical-Biological Defense, DTRA and its related elements (the STRATCOM Center for Combating WMD and the Standing Joint Force Headquarters for Elimination), and from Aberdeen Proving Ground, ECBC, U.S. Army Chemical Materials Agency, and the 20th CBRNE Command.

6.3.1 Contingency Options The AT&L office told the team that President Obama had directed them to determine how the U.S. government could move the chemical weapons out of Syria as soon as possible and destroy them but was not looking at any particular technology. If no suitable technology existed, they needed to figure out how to manufacture it. There were a number of options under which

Syria’s Chemical Disposal Program this effort could take place, but there were complications in each one. Technically speaking, parties to the Chemical Weapons Convention (to include the United States) were not supposed to accept chemical weapons from another state. There was no exception for disposal operations, as the owning state is supposed to dispose of the agents under its control. If the U.S. government was going to move the chemical weapons, this would require the United Nations to grant an exception to the treaty’s language. Ideally, using the traditional approach, Syria should have taken the lead responsibility to build a disposal facility and under supervision by OPCW inspectors, destroyed the agents and dismantled its facilities. However, Syria claimed it didn’t have the resources to build a disposal facility. The U.S. government was prevented from funding the construction of a Syrian disposal facility, because Syria was an identified state sponsor of terrorism, and the security of disposal operations would be imperiled if built in a war zone. Option 1 then focused on finding a host nation that would either incinerate the chemicals or build an incineration plant for the short-term purpose of destroying the Syrian stockpile. There was a precedent for this—in 2006–2007, DTRA supported Albania’s destruction of 16 tons of bulk chemical agents using an incineration plant that was built and operated using CTR funds. The second option would be to move the chemicals to another state and allow the U.S. government to deploy a disposal system to eliminate the chemical agents. The technical team proposed to develop a deployable neutralization platform based on the technology used at Aberdeen Proving Ground to destroy 1800 tons of mustard and at Newport Army Depot to destroy nearly 1700 tons of VX nerve agent. This technology could also be used to destroy ricin, which Syria was suspected of producing (also a reportable chemical under the Chemical Weapons Convention). This exercise would be a “CTR-like” operation rather than a WMD elimination mission as was planned in 2003 for Iraq. As a historical note, the U.S. Army had previous experience with chemical disposal systems using neutralization technologies, such as the Chemical Agent Munitions Disposal System (CAMDS), used to destroy chemical agents at various locations within the United States between 1979 and 2009. This system had destroyed more than 363,000 pounds of chemical agent and more than 40,000 munitions without adversely affecting the environment or causing harm to its operators (Bryant, 2009). The U.S. Army also had a transportable “explosive destruction system” that was designed to destroy filled chemical munitions, although not in mass (ideal for what were called non-stockpile chemical munitions discovered at former U.S. defense sites). The requirements, then, were to develop a platform for destroying metric tons of toxic chemicals to an efficiency of 99.9%, operating 24 hours a day, 7 days a week, in a form that was easily transported to overseas locations, operating at remote (barebone) sites, and ready to operate at full capacity within 10 days of arriving on site. This meant that the design had to be modular, using proven technology, with a large degree of self-sufficiency, and relatively easy to set up and operate. This led to the prototype of the field deployable hydrolysis system (FDHS), first demonstrated in late June 2013 (see Figure 6.1). The Army planned to develop six additional systems over the next year, allowing options to expand to more than one operating site, as needed.

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FIGURE 6.1  Field deployable hydrolysis system, with reactor skid standing vertical on the left and the hydrolysis skid horizontal on the right.

In April, U.S. Strategic Command, as the DOD’s combatant command advocate for counter-WMD issues, published a Joint Emergent Operational Need statement to officially establish the requirement to build hardware that would address the elimination of bulk chemical warfare materials. The Joint Requirements Oversight Council quickly approved the statement, allowing the Army’s acquisition team to start developing the FDHS. Each system would cost about $4 million to build using a combination of DTRA funds, Army chemical demilitarization program funds, and other Army funds. The first two systems (following the initial prototype) would be ready to go by September 2013. The U.S. government petitioned several nations between September and November 2013 to be the host for a chemical disposal effort, either using their own facilities or allowing the U.S. government to bring a chemical disposal system into their country. These countries included Italy, Jordan, France, Belgium, Norway, Russia, and Germany. France and Belgium had extensive experience in destroying World War I chemical munitions, and Russia had its own significant chemical disposal program. None of these countries were willing to take on the mission, due more to public perception than to the actual safety concerns relating to handling the chemicals (Bleek, 2016). In mid-October 2013, the Albanian ambassador to the United Nations indicated that his government might consider the responsibility of hosting the disposal operations. Not more than a month later, the Albanian prime minister formally rejected the proposition, citing public concerns over environmental and health hazards (The Guardian, 2013). Following the failure to find a host nation for disposal operations, the planning switched to Option 3: removing the chemical weapons to international waters and destroying them while at sea. This would involve putting two FDHS platforms on a U.S. Maritime Administration ship called the M/V Cape Ray. The Cape Ray is a Ready Reserve Force ship that, when activated, falls under the Military Sealift Command, which is a part of U.S. Transportation Command. Its normal mission is to move vehicles across the ocean in a “roll-on, roll-off” configuration. With its cargo space of more than 175,000 square feet, vast open deck space, and high overhead height clearances, it had

94 the right characteristics to hold the two operational systems, the chemicals to be neutralized, and the waste effluent that resulted from the operation. There was one disadvantage: the ship could not carry enough storage tanks to hold all six chemicals categorized as “Category 1” in a single trip, which meant it would have to focus on destroying the mustard agent and DF chemical (Cheung, 2015). It would also require a number of modifications to ensure the safety of the crew and operators, similar to those on U.S.-based chemical disposal facilities, and to safely store and neutralize the chemicals on the ship (McMahon, 2014) (Figure 6.2). This was not the first time that the DOD had tried to destroy chemical agents on a ship. In 1977, the Dutch-owned ship M/T Vulcanus was used as an operating base to incinerate nearly 10,400 metric liters of Agent Orange from Johnston Atoll under “Operation Pacer” (EPA, 1978). However, there were certainly several technical and policy challenges in adopting this particular process in 2013. This particular system was not tested, in as much as the Army knew the process would work in theory, but it had no experience in actually operating this particular system in an operational environment. The system had to be safe to operate, not just for the crew and technical operators on the ship, but also due to the possibility of any spillage into the ocean. While developed under the defense acquisition process, this project was “fast-tracked” and did not go through the formal process of operational testing and evaluation, as nearly all defense programs must. The nations bordering the Mediterranean Sea and Greenpeace in particular were keenly aware of past practices involving the burial of chemical munitions at sea (CNS, 2017). The overall effort had to be transparent to concerned public parties but at the same time, cognizant of the need for security measures to protect the chemicals from being taken by hostile actors. Additionally, the planners had to account for potential bad weather, resupply, and rotating government personnel on and off the ship. The equipment, to include a laboratory, a decontamination station, and a collective protection system, was installed on the Cape Ray in December, and the ship participated in sea trials in early January to prepare for its mission. It departed from Portsmouth, Virginia on January 27, 2014 with a crew of 36 civilian mariners and more than 60 technical specialists, a security

FIGURE 6.2  USS M/V Cape Ray, a 648-foot maritime transportation vessel.

Chemical Warfare Agents team, and other military members. The Cape Ray arrived at U.S. Naval Station Rota, Spain on February 13, 2013 and settled in to wait for Syria to remove its chemical weapons and move them to its port, Latakia. Unfortunately, the Syrian government was behind on its schedule and missed its deadline of February 5 to have all of its chemical weapons and precursor chemicals out of the country. In fact, it would not be until early March that Syria was able to move more than a third of its stockpile to the port and load it onto ships. About this time, members of environmental, public health, nonproliferation, and arms control groups released a public letter to Secretaries Kerry and Hagel to remind the U.S. government that they were watching the disposal process with great interest. The letter emphasized the need for full transparency and outreach to allow engagement with “all stakeholders,” including those advocacy groups who saw themselves as government watchdogs over former and current U.S. government chemical demilitarization efforts (Green Cross, 2014a). The U.S. government did not respond to the letter. In March, there were large public protests in Crete against the plans to destroy the chemical agents at sea (Green Cross, 2014b). These protests, focusing on the potential for environmental damage in the event of an accident, would continue throughout the summer. While the crew was waiting for the completion of the Syrian movement of chemicals out of country, the Cape Ray hosted a “European Media Day” between April 10 and April 12. In addition to the ship captain, the director of operations for U.S. Naval Forces Europe and an OPCW spokesperson provided comments. Reporters were provided access to all parts of the vessel. CNN, al Jazeera, and the BBC provided video broadcasts, while the UK Guardian, Der Spiegel, the Wall Street Journal, Reuters, and others wrote articles. The safety features of the vessel were highlighted as well as the technical equipment that sat in the cargo hold.

6.4 Implementing a Disposal Program Up to the point of operationalizing this effort, it had taken an enormous amount of coordination, not just between the State Department, Department of Transportation, and the DOD but within the DOD—Army technical offices, DTRA and OSD funding, Navy operators, U.S. Transportation Command, U.S. European Command, U.S. Central Command, the United Nations, the OPCW, and defense contractors—and now there was a considerable international element, not to mention the commercial industry’s interest. The OPCW team would train the Syrians on how to safely secure and handle the chemicals as well as oversee the disposal process on the Cape Ray. Russia provided equipment and transportation assets to the Syrian government for moving the chemicals to Latakia. A Danish and a Norwegian ship would take on the chemicals and move them to Italy. Security for the ships would be provided by Chinese, Danish, Norwegian, and Russian warships. Italy would provide the port of Gioia Tauro as a transload point to move the Syrian chemicals to the Cape Ray. Other security support would be provided by Finland, Germany, Italy, the United Kingdom, and Turkey (Blades, 2015). Jordan and Turkey accepted U.S. aid in the form of chemical-biological

Syria’s Chemical Disposal Program defense equipment and training to support border security and crisis response (Bleek, 2016). Industrial facilities in Germany, Finland, the United States, and the United Kingdom would destroy the industrial chemicals and waste effluents. The OPCW had 35 private companies expressing interest in the operation, of which 14 industrial companies provided bids to handle the final disposition of the chemicals. Two companies were awarded contracts (Veolia ES Technical Solutions and Ekokem Riihimaki). The German government offered the services of GEKA MbH, and the U.K. government offered Mexichem for unplanned shipments. Other nations, to include Japan, Canada, and the European Union, contributed funds to the United Nations and the OPCW. Some of the disposal work already had begun in the fall of 2013. The OPCW had verified that 21 of the 23 initially declared production facilities had been shut down, the last two being closer to active fighting and not deemed safe to visit. The Syrian government committed to destroying the stocks of isopropyl alcohol, containers with residual mustard agent, and unfilled chemical munitions by January 31, 2014. But no chemicals had been moved to Latakia by the end of 2013. The first containers sent to the port would come in early January, and the last arrived before the end of June (ACA, 2017). The shipments were behind schedule, with Syria having moved about half by late March, but continued in good faith. Some of the containers had to be repacked due to leaks. All of them had to be inspected and inventoried before being packed in compliance with international rules of transportation of hazardous materials. Moving the chemicals from the port to waiting ships was not a trivial matter. The Cape Ray, because it resembled a U.S. warship (in color, not due to weapon systems) and carried U.S. military personnel, could not directly load the chemicals at Latakia due to diplomatic sensitivities and security concerns. Denmark volunteered its merchant vessel M/V Ark Futura to carry the mustard agent drums and DF to Italy for transloading to the Cape Ray, along with shipping other industrial chemicals to the United Kingdom and Finland. The Norwegian vessel M/V Taiko would similarly carry Category 2 industrial chemicals for disposal to Finland and the United States. The ships were shadowed by military warships throughout the process. There were no security incidents throughout the event. For security purposes, the two ships stationed at Cyprus while the Syrian chemicals were stockpiled at Latakia. The ships docked about 20 times at Latakia to take on their cargo, remaining only a few hours at a time and while all other operations in the port were suspended. The Taiko picked up its Category 1 and 2 industrial chemicals in early June and carried them to the Ekokem waste facility in Finland and the Veolia waste disposal facility at Port Arthur, Texas, completing its travel by July 9 (Anelli and Rouzbahani, 2015). The Ark Futura would eventually hold 224 storage tanks filled with DF and 15 storage tanks of mustard agent, moving these to Gioia Tauro on July 1, 2014, for a one-day transload to the Cape Ray. It also would carry other industrial chemicals to the Ellesmere Port waste facility in the United Kingdom and Finland, completing its mission by July 20. Table 6.3 identifies the disposal facilities and chemicals shipped to each. The Cape Ray’s technical team was able to process the 600 metric tons of chemicals in 42 days, completing its disposal

95 operations by August 17. Crews worked in 12 hour shifts to maintain operations around the clock. The team neutralized the DF first, running both FDHS systems in parallel, before moving on to the mustard agent. As the technical team monitored the hydrolysis process and the flow of agents and effluent from and to the storage tanks, OPCW inspectors watched the process by video cameras and verified that all of the removed chemicals had been destroyed (ECBC, 2014). In the process of neutralizing the 600 metric tons of toxic chemicals, more than 6000 metric tons of waste effluent was created. The Cape Ray transported more than 330 metric tons of HD effluent to Bremerhaven—this was moved to the GEKA waste facility near Hamburg, Germany— and then sailed to the port of HaminaKotka, Finland, to off-load another 5900 metric tons of DF effluent. The Cape Ray returned to Portsmouth on September 17, 2014 and was cleared for unlimited operations in January. As a minor note, without going into detail regarding each facility’s capabilities, the amount of hazardous chemicals treated as a result of the Syrian chemical disposal operation was very small in comparison with the annual treatment of industrial hazardous waste taken on at each facility. For instance, a 2015 Ekokem report identifies about 1500 kilotons of waste treated annually compared with the nearly 6 kilotons treated in this operation (Ekokem, 2015). The Ellesmere Port facility destroys about 100,000 tons of industrial waste each year (Notman, 2014). Asking these facilities to absorb such a small amount of additional hazardous materials was the easy part of the U.S. government’s disposal operation. The only reason for highlighting the disposal technologies used is to elaborate on the long-standing policy debate on how chemical warfare agents are destroyed in bulk. Environmental advocates, such as Green Cross International, Greenpeace, and the Sierra Club, prefer neutralization technologies as alternatives to incineration, because they claim that incineration allows releases of toxins or other hazardous material, even when pollution-abatement systems such as filters are used (Di Justo, 2013). There is no evidence that any incineration plant operated by the U.S. Army to destroy chemical warfare agents has ever resulted in a hazardous chemical agent release that put public citizens or the environment at risk, but these claims by environmental advocates continue. Ideally, incineration is a more feasible technology for destroying chemical agents, because it is agnostic to what’s being fed to the furnace—neutralization technologies are agent specific and have to be carefully controlled to achieve full disposal of the agent. Incineration is a well-researched technology, is less expensive to operate, and creates fewer waste products as compared with neutralization, and contrary to environmental groups’ claims, is relatively safe to operate. Congress directed the U.S. Army to use neutralization technologies at four of its nine chemical disposal sites, two of which dealt with bulk liquid agent stored in ton-containers. The Army had also used neutralization technology in destroying the nerve agent precursor DF in its Nonstockpile Chemical Munitions Program between 2003 and 2006. Both technologies are viable disposal processes today. The Army had investigated neutralization technologies back in the 1970s, given the possibility that neutralization could offer a quick solution to eliminate immediate hazards with ton-containers of sarin nerve agent, for instance. Neutralization through

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Chemical Warfare Agents TABLE 6.3 Disposing of the Waste Products Waste Treatment Facility Ellesmere Port (Veolia), United Kingdom Mexichem, United Kingdom GEKA, Germany Ekokem, Finland Veolia Technical Solutions, United States

Disposal Technology

Chemicals Destroyed

High-temperature incineration Chemical neutralization High-temperature incineration High-temperature incineration High-temperature incineration

180 tons Category 1 and 2 chemicals 7 tons hydrogen fluoride 330 tons HD effluent 5867 tons DF effluent; 320 tons Category 1 and 2 chemicals 60 tons Category 2 chemicals

hydrolysis prevents the release of toxins into the air but creates a much larger waste product than incineration—in this case, the waste product was 10 times the amount of the original targeted chemicals. Neutralization is a more complex process than incineration, in that one has to carefully control the rate of feed of the neutralent and the output of hazardous waste. But the absence of “smokestacks” has been a powerful incentive to green groups and like-minded public citizens, even as the overall costs of operations of neutralization plants have been much higher than comparable costs of incineration. At the last examination, Congress has authorized $10–11 billion to dispose of the last 10% of the U.S. chemical weapons stockpile (specifically, Pueblo and Blue Grass Army Depots), both using neutralization processes (DoD, 2015). It will have cost just short of $25 billion to dispose of the first 90%, and eventually, all of that waste still returns to the earth in some final form, diluted and fed to the oceans or buried in a landfill. The Army’s chemical demilitarization program was highly politicized after its initial decision in the early 1990s to use incineration as a safe, cost-effective, and proven technology, leading to the direction by Congress to consider “alternative technologies” as potential candidates for disposal. Studies by the National Academies were directed to avoid cost and efficiency comparisons between the two different approaches. But with regard to the Syrian chemical disposal effort, while neutralization was the initial treatment process, practically all of the waste effluent was incinerated—or as some in the waste disposal business say today, “detoxified”—in the final deposition of the material.

6.4.1 Postscript By January 4, 2015, the OPCW declared that all of Syria’s declared chemical stockpile was destroyed. The destruction of 12 chemical weapons production facilities (seven aircraft hangers and five underground facilities) in Syria was still under way but scheduled to be completed by late summer. By mid-June, the waste effluents at GEKA and Ekokem had been verified as destroyed, and only 16 tons of hydrogen fluoride remained at the Veolia Port Arthur complex. Veolia reported the final disposal of the hydrogen fluoride to the OPCW in early January 2016, completing the elimination of Syria’s declared chemical stockpile. By mid-year, the OPCW had confirmed that Syria’s declared storage sites were empty and that 24 of the 27 declared production sites had been destroyed (SIPRI, 2017). The cost of the disposal operations is not entirely clear. The initial building and testing of the FDHS prototype cost $10 million (from DTRA), and the other six required $25 million to build (from Army). Funds allocated from the chemical

weapons destruction program amounted to about $60 million. The DOD identified $150 million as funding issued through the CTR program. These funds paid for training, equipment, protective gear, logistics support, and medical countermeasures for the OPCW inspection teams as well as for border control forces in Jordan and Turkey. The OPCW spent approximately $50–55 million for contracts with Ekokem and Veolia. The funds collected by the United Nations Trust Fund from other countries have not been detailed (other than the European Union, which donated about $25 million). Additional costs for operations and maintenance, outfitting and operating the Cape Ray, and providing security for the ongoing operations have not been identified. Total costs could have run up to $400–500 million by one estimate (CSIS, 2014). The question remains: did Syria’s government declare all of its stockpile and production facilities? Given the Syrian military attacks against Syrian insurgents using nerve agent in April 2017 (Dewan and Alkashali, 2017), there is always the possibility that there are undeclared sites where these chemical weapons still exist. In May 2015, OPCW inspectors found traces of precursors required to make sarin and VX in an undeclared military research site. Other analysts suspect that the Syrian regime deliberately failed to account for 10–15% of its stockpile (Loveluck, 2015). In 2016, the European Union noted that “inconsistencies relating to Syria’s chemical weapon declarations had increased” (SIPRI, 2017). Does this mean that the international effort to destroy Syria’s chemical weapons program was a failure? Not necessarily. As a party to the Chemical Weapons Convention, Syria is liable to challenge inspections brought on by other states. If there is credible evidence suggesting that Syria is holding out with a reserve of chemical weapons, any nation could call for the OPCW to inspect and verify the allegation. At the least, Syria’s ability to use military-grade chemical weapons has been significantly degraded. There have been allegations that Syrian military forces continue to use chlorine barrel-bombs in attacks against Syrian civilians and the insurgency (Human Rights Watch, 2014). In 2016, the United Nations specifically identified the Syrian government as responsible for at least two chemical attacks in the past 2 years (in Talmenes in April 2014 and in Sarmin in March 2015) (Gladstone, 2016). On April 4, 2017, a Syrian Su-22 aircraft may have dropped a nerve agent–filled munition in Idlib province, causing more than 80 deaths and hundreds of casualties (BBC, 2017). Why the Syrian military is continuing these chemical attacks is unclear; it may be that the regime is bent on demonstrating that it has not been cowed by the international community’s determined push to dismantle its chemical weapons program.

Syria’s Chemical Disposal Program The use of improvised chemical weapons (the chlorine barrel bombs) is still a violation of the Chemical Weapons Convention, even if Syria’s government does not have a formal weapons program. However, the challenge remains: what should the United States and other nations’ response be to this issue? The U.S. government is already supporting the Syrian insurgents with both financial and military support. Any direct military response needs to be proportional to the threat, which would seem to dismiss any massive bombing campaign against the Syrian regime or its military forces. The Russian and Chinese governments have vetoed a UN Security Council resolution that proposed to punish Syria for using chemical weapons (Sengupta, 2017) and have voted against continuing the UN investigations into Syrian chemical weapons use. Yet at minimum, the U.S. government was able to force Syria to abide by the international treaty to dismantle a significant part of its chemical weapons production capability. That is the main purpose of the Chemical Weapons Convention: to eliminate a nation-state’s chemical weapons program as a method of warfare. However, the Syrian regime, now bolstered by Russian military support, intends to continue its fight to maintain its state security interests by whatever means possible. Diplomatic and military pressure to abide by its treaty obligations should continue. The effort to dismantle Syria’s chemical weapons program, in particular its research, development, production, and storage sites, still offers lessons for future threat reduction efforts.

6.5 Lessons Learned The Nonproliferation Review recently published a collection of articles on WMD elimination, with one chapter summarizing the “lessons from the last quarter-century” (Bleek, 2016). While its coverage was across six case studies (South Africa 1991, former Soviet Union 1990s, Iraq 1990s, Iraq 2003, Libya 2000s, Syria 2014), the “lessons learned” chapter provides a good framework to review the Syria case study. This section will build on that article to provide a slightly different perspective on the topic. One of the first points, and perhaps a fundamental point, is the need to review how the U.S. government defines WMD and WMD elimination. Despite having used the acronym “WMD” for more than 15 years, the U.S. government still has no single, generally accepted definition for this term. As a result, the U.S. government (and other nations) debate what “WMD” means in each new incident, based on whether the argument is about military operations, global disarmament, human rights, counterterrorism, or law enforcement. The very term “WMD” is unhelpful in developing responses to crises involving unconventional weapons. This also applies to the phrase “WMD elimination,” which has recently been phased out and replaced by the phrase “WMD disablement and disposal.” The lack of a common terminology and concept for a “whole of government” operation has challenged the discussion. The Nonproliferation Review identifies six different case studies that form at least four different typologies, depending on whether the decision to eliminate an arsenal was imposed, coerced, or voluntary and whether the dismantlement was external, cooperative, or internally executed. No one seems to agree on exactly what WMD elimination is or should be.

97 Three of the six case studies are (more or less) traditional cooperative threat reduction efforts (the former Soviet Union, Libya, and Syria). One could also add the Albania chemical demilitarization project to that list (not one of the six case studies in the article). This leads to the question: is cooperative threat reduction, a well-established program executed between the State and Defense Departments, a sub-set of WMD elimination, or is WMD elimination, a poorly established, ad hoc effort executed primarily by the DOD, just a coercive form of threat reduction? The point is that in the constant search to define and establish a new “mission area” in the counter-WMD operational concept, it would make more sense for interagency purposes to just eliminate the term “WMD elimination” and use the accepted construct “threat reduction” as the policy objective. The original argument for WMD elimination—originally WMD exploitation—was based on an artificial worst-case scenario postulating that during a major combat operation or as a regime collapsed in on itself, sub-state actors would swoop in to claim WMD program–related materials or technology prior to U.S. military securing these targets. Because U.S. government policy is to prevent uncontrolled and illicit transfers of WMD program–related materials, especially to sub-state groups, the DOD believed that a capability was needed to quickly move U.S. technical specialists into battle zones for the purposes of securing and moving this material. One could not wait for the battle to be over to start dismantling the adversary’s WMD program. The unintended consequence of this proposal was to separate counter-WMD activities from the general military campaign, given most U.S. commanders’ relative unfamiliarity in dealing with these special events. That pessimistic scenario did not play out in Iraq 2003, Libya 2011, or Syria 2014. While in each case, there were sub-state actors present as national chemical stockpiles were being secured and destroyed, the actual operation of securing and disposing of the material was conducted without incident. Sub-state groups remain focused on gaining access to conventional firearms and explosives rather than unconventional weapons. However, this assumption of U.S. forces having to search for WMD stockpiles in the middle of a war zone remains present in the North Korea scenario. In fact, a RAND study suggests that the number of U.S. military personnel required to take on the WMD elimination mission if North Korea collapsed—to seize, secure, and clear 23 large sites—could exceed 188,000 troops. The RAND analysts recommend that the U.S. military fully resource this mission (Bonds, 2014). Given the history of U.S. military priorities in this field, it is highly unlikely that this operational construct will in fact be created. The DOD, and in particular the U.S. Army, had been exploring the idea of a “Joint Task Force for Elimination” since at least 2006, when the Army integrated WMD elimination exercises into the Ulchi Focus Lens exercise that takes place in the Republic of Korea. DTRA fielded a “Joint Elimination Coordination Element” at the time, composed of fewer than 30 personnel, which would eventually be the catalyst for later discussions of a standing Joint Task Force and then a Standing Joint Force Headquarters for Elimination (Barber, 2008). Between 2009 and 2015, there was no appetite within the four services or U.S. Strategic Command to fully outfit and institutionalize this capability.

98 The Army continues to work on this concept, but without a joint component, any future exercise would again be an ad hoc pick-up game. With the failure of the WMD elimination concept to gain traction and the recent disestablishment of the Standing Joint Force Headquarters for Elimination, one must revisit this assumption to determine whether the U.S. government really needs a WMD elimination capability. If one takes away the Jack Bauer ticking clock, a CTR-led program, led by the State Department and supported by the DOD, still makes the most sense as an established, well-regarded—and most importantly, resourced—effort. There is no well-defined process and no dedicated resources for WMD elimination as a result of a failure to institutionalize this activity and the numerous government agencies involved, to include the international community. The many different WMD elimination scenarios work against having a “playbook” or single advocate. Let’s get past the definitional problems: every WMD elimination case study has dealt with one singular type of unconventional weapon—either nuclear, biological, or chemical but (generally speaking) not all at once. The primary focus has been on the WMD material rather than on the people and institutional knowledge within a regime (the exception being the former Soviet Union). Some of the cases required discussions within the United Nations; others were based on bilateral agreements. There is no generic concept and no single government agency that can execute this mission. It will always be a hand-crafted effort because of the unique nature of the situations. Most defense analysts who discuss the Syrian chemical disposal case study agree that this was a unique situation, that the opportunity of a U.S. government having the time to develop a tailored solution to an adversarial government’s WMD program prior to its agreement to disarm, added to an accelerated time schedule and relatively limited (but willing) participation of the host government, will not be seen again. The number of state or sub-state actors with an active chemical weapons program continues to shrink. This being said, it seems fruitless to call for either a defined concept of employment or the establishment of an institutionalized program within any government agency. And yet, this hasn’t stopped the DOD from continuing to push for a generic joint concept that will allow the organization of technical units and acquisition of specialized equipment. The U.S. government does need to develop an institutionalized approach for future disposal cases. One approach is to create a carefully designed and engineered response force based on a well-defined threat. This approach would be costly to maintain, and if the threat turns out to be larger than expected, the response fails. Some advocates in the DOD wanted this, but the resources were never allocated, and partnerships with other needed government agencies were not secured. The other approach is to designate an existing technical agency that has a mix of technical skills and functional abilities but would have to be augmented with additional forces as needed to address the specific threat. Looking not just at the Syria case but at all six elimination cases, the call for a “checklist” or playbook will be difficult if not impossible to create and will not be helpful because of the unique aspects of each mission. There is no “cookie-cutter” approach. The U.S. Army has the technical and operational expertise and continues to maintain an ability to recover chemical warfare material at Aberdeen Proving Ground. DTRA has a long history

Chemical Warfare Agents of executing the CTR program, but the missions and resources are determined based on Congressional direction and not structured for emerging crises. Both have resourcing issues that would have to be addressed. Between the two agencies, they do have the necessary skills and capabilities to address future contingencies as directed. DTRA’s CTR program has the necessary experience and contracting vehicles in place to enable such future contingencies. The Army’s mission to maintain destruction technology and personnel expertise to destroy recovered chemical munitions is a long-term mission. However, if the U.S. Army fails to invest the necessary intellectual energy into examining strategic challenges and developing partnerships with other government agencies, it will not be prepared for future contingencies similar to the Syrian operation. As Dwight Eisenhower once stated, “plans are worthless, but planning is everything.” Obviously, the State Department’s arms control branch and the DOD’s policy and acquisition offices need to guide and foster this capability if it is seen as an absolutely necessary mission. Without advocacy from political appointees, this mission will lack resources and fail in prioritization against other competing programs. The technical community does not have the political clout to advocate for a resource-intensive mission whose future timeline is uncertain. U.S. Strategic Command has turned away from advocating for counter-WMD programs, and it is unclear that U.S. Special Operations Command will advocate for this mission. The combatant commands are operators, focused on near-real-time challenges, and lack the policy focus to champion for these activities. Collaborating with other nations and nongovernmental organizations is difficult but not an impediment to success. But certainly, the State Department and the offices of the Under Secretary of Defense for Policy do, and are expected to, work on complex policy issues. It’s difficult to call for a new program effort that demands long-term, institutional support for counterWMD activities such as this elimination mission. Although counter-WMD activities are a high priority within the U.S. government, they are not routine, and they do not fit neatly into the usual operational tempo of ongoing political–military discussions. It will be difficult, if not impossible, to avoid surprise. The necessary funds and commitment will not be there to ensure a readily applicable platform for every possible contingency. This comes to one last observation. The Syria chemical disposal mission was overseen by the AT&L office and executed largely by a collection of technical experts. The framework for management included a principal-level group and several working groups dominated by the acquisition community, because they had the funding and personnel to meet frequently and to discuss the technical aspects of the mission. The Syria SIG membership was very similar to a National Security Council Interagency Policy Committee. The State Department, OSD policy, Joint Staff, and military operators were not as well funded or staffed to balance the technical community’s size and influence. As a result, there were duplications in effort and challenges in the integration of other defense functions. The operation was run by the technical community, because it had the resources and thus, the implicit authorities to do what it did. One report notes that this imbalance caused tensions, as the effort transitioned from a material development effort to an operational mission without

Syria’s Chemical Disposal Program any change in how the effort was managed. In particular, the Syria chemical disposal mission was not a “named” combatant command operation, and perceptions were that the technical community, in running the operations, overstepped its authorities and responsibilities (Ivancovich et al., 2015). There are certainly advantages to having a “named” combatant command operation, and in particular, there are other vehicles for organizing interagency efforts, such as the formation of a Joint Interagency Task Force or Joint Interagency Coordination Group. National efforts for counter-drug operations and homeland security have traditionally used these vehicles. When the DOD was debating how to operationalize WMD elimination between 2009 and 2012, the leadership rejected the idea of a permanent joint task force because of the constraints of manpower and the cost of maintaining the force at a particular military base. The challenge in asking a geographical combatant command to establish a Joint Interagency Task Force is that their staff lack the necessary expertize in countering WMD operations and the ability to leverage the DOD acquisition process for time-sensitive acquisition needs. If combatant commands continue to shortstaff their counter-WMD offices, a Joint Interagency Task Force for this mission is not a feasible option.

6.6 Conclusions The removal and destruction of Syrian chemical weapons and associated facilities were successful and should be viewed as a great achievement by the U.S. government and the United Nations. Convincing Syria to give up its declared chemical weapons and to act against a significantly accelerated schedule, using a unique technology platform operated at sea, without significant incidents or loss of chemical agents, was a remarkable effort— and a unique one. It would be difficult to say whether this effort was a deliberate, thoughtful development of military capabilities or a process which the U.S. government would necessarily want to use for future nations’ WMD programs. By all accounts, the National Security Council did not have a ready playbook for this issue in 2012–2013, but as President Obama remarked later, he was more concerned with policy results than being graded on style (Chollet, 2016). That said, there are some conclusions that we can take away from this case study. Destroying Syria’s declared chemical weapons program did not stop the civil war, did not stop the indiscriminate killing of civilians, and did not stop the continued use of chlorine barrel bombs after Syria’s accession to the Chemical Weapons Convention. Given that, the total number of chemical casualties within Syria amounts to fewer than 1% of overall civilian casualties measured up to February 2015 (Reuters, 2015). The elimination of Syria’s chemical weapons program was important, however, from the aspect of strengthening the international arms control regime, which has near universality on a global ban of chemical weapons production and use. It was a success in that the liberal international order was able to convince the Syrian regime to cooperate in eliminating one particular class of unconventional weapons, not just improving the region’s stability (perhaps by a small measure, but regardless) but also preventing the possible loss of control of military-grade chemical weapons to sub-state groups.

99 It was possible for the U.S. government and the international community to separate the singular task of eliminating these chemical weapons from the larger context of addressing Assad’s reprehensible actions in Syria. For instance, there was considerable congressional debate as to the extent and role of U.S. military action when President Obama declared the intent to conduct limited strikes against Syrian forces in response to the Ghouta attack (Cohen, 2013). The president clearly stated that the purpose of the strikes was to deter Assad’s regime from continuing to use chemical weapons, not to remove Assad from power (White House, 2013). Two days after that speech, the Assad regime sent a letter to the United Nations stating its intent to accede to the Chemical Weapons Convention. Deterrence works. But even deterrence has to be put in context. To be effective, deterrence has to be seen by the other party as credible, i.e., that there is a capability and willingness to use that capability. The threat of U.S. military strikes in response to continued Syrian chemical attacks that caused mass casualties was seen as credible. It may be that the Syrian regime did not believe that the United States or other nations would use military force in response to its use of chlorine barrel bombs, which caused much more limited casualties. While the Syrian government’s declared chemical weapons program has been dismantled (for the sake of argument, at least most of it has), the international community cannot ban Syria from producing chlorine as a vital part of its industrial capabilities (e.g., water treatment plants). Of course, the continued use of chlorine as a weapon is a violation of the Chemical Weapons Convention, and there are diplomatic processes to address this casual flouting of the treaty. The OPCW should continue its investigations of any allegations of chemical weapons use in Syria as evidence of human rights violations for war crimes trials and present those results to the United Nations for action. It may be that Russia and China will continue to block any attempts to investigate future violations of the treaty or to punish Syria for its draconic measures to enforce security matters within its own borders. This should not diminish the success of the enterprise in taking Syria’s mustard and nerve agents off the table. The future of the DOD’s role in eliminating unconventional weapons in a non-permissive (combat) environment remains an open question. The 2014 DOD Strategy for Countering WMD notes that “DOD may also lead or assist in the disposal of residual adversary WMD capabilities until such time that a civilian or international entity can assume these responsibilities.” It identifies the need to control, defeat, disable, and dispose of WMD as “specialized activities and tasks” (Dept of Defense, 2014). The term elimination is no longer used; instead, the terms disablement and disposal are the key missions. The overall intent is the same, but the effort to split the singular term elimination into two separate parts allows the DOD to distinguish military-led responsibilities to “disable” a nation-state’s WMD program from what one might expect as State Department–led responsibilities under the “disposition” of a nation-state’s WMD program. The Standing Joint Force Headquarters for Elimination was stood down in 2017, commensurate with the U.S. Special Operations Command taking over the role of the DOD lead in developing counter-WMD concept plans from U.S. Strategic Command. This leads to an uncertain future.

100 The National Security Council must formalize this operational process with expectations that State and Defense will play coequal lead roles in future contingencies. At the least, the U.S. Army should be designated as a lead within the DOD with direction to retain a core capability to mobilize—not as a 24/7 firefighter but rather, with the necessary mandate to maintain the operational expertise with the capability to rapidly ramp up and deploy as required. Similarly, DTRA must be allowed the flexibility to use the CTR program in support of these operations as a vehicle to fund future disposal efforts and to coordinate with the interagency and combatant commands on the necessary plans and capabilities required. The U.S. Army may make the decision that large-scale disposal projects should be contracted out, while its military personnel focus on small-scale (less than a ton) field disposal efforts. The Syria disposal effort would certainly support that decision. Certainly, the numbers of future chemical disposal projects, on the scale of Libya or Syria, are few, perhaps not enough to warrant a permanent standing headquarters. In this day and age of global industrial economies, it is clear that the U.S. military is no longer the single omnipotent actor that it used to be in the 1980s. As a result, future plans must enable maintaining the technical expertise within the military while retaining contract vehicles to bring in commercial vendors to support these boutique exercises.

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7 The Use of Chemical Warfare Agents during the Syrian Civil War Arik Eisenkraft and Avshalom Falk CONTENTS 7.1 Introduction................................................................................................................................................................................. 103 7.2 The Syrian Civil War.................................................................................................................................................................. 103 7.2.1 Background..................................................................................................................................................................... 103 7.2.2 Main Course of Events, Belligerents in the Conflict, and International Involvement...................................................104 7.2.3 Medical Facilities and Infrastructure in the Syrian Civil War...................................................................................... 105 7.3 The Use of Chemical Warfare Agents and Other Toxic Chemicals in Syria.............................................................................106 7.3.1 General Perspective........................................................................................................................................................106 7.3.2 Preliminary Uses of Chemical Agents in Syria during the Recent Civil War...............................................................107 7.3.3 The August 2013 Attack in Ghouta, Damascus.............................................................................................................107 7.3.4 UN Investigations of Other Alleged Attacks.................................................................................................................108 7.3.5 Events Following the Ghouta Attack and the OPCW Involvement in Syria.................................................................. 111 7.3.6 The Use of Chemicals, Including Chemical Warfare Agents, Since the 2013 Ghouta Attack...................................... 113 7.4 Social Media in Disaster Medicine: The Syrian Case Study...................................................................................................... 114 7.4.1 Gathering Data............................................................................................................................................................... 114 7.4.2 A Systematic Approach for the Evaluation of Clinical Syndromes............................................................................... 114 7.4.3 Immediate Medical Treatment of the Ghouta Attack Victims as Reflected in Social Media....................................... 115 7.4.4 Lessons Learned: Preparedness and Planning for Humanitarian Support.................................................................... 115 7.4.4.1 The Israeli Civilian CBRN Defense Doctrine’s View of the Ghouta Video Findings................................... 115 7.4.4.2 Humanitarian Support.................................................................................................................................... 116 7.4.5 Future Implications........................................................................................................................................................ 116 7.4.5.1 Pharmacological Intervention......................................................................................................................... 116 7.4.5.2 Antidote Administration................................................................................................................................. 117 7.4.5.3 Respiratory Support........................................................................................................................................ 117 7.4.5.4 Management of Pediatric CWNA Victims..................................................................................................... 117 7.5 Conclusions................................................................................................................................................................................. 117 References............................................................................................................................................................................................. 118

7.1 Introduction

7.2 The Syrian Civil War

On the night of August 21, 2013, Syrian regime forces fired rockets toward the eastern outskirts of the capital city of Damascus. These rockets contained the nerve agent (NA) sarin. This attack resulted in the deaths of at least 1400 civilians, including women and children of all ages, and severely affected thousands more. Although this was followed by a seemingly strict international response leading to the destruction of most of the Syrian chemical arsenal, dozens of small-scale chemical attacks, tactical in nature, followed, and 4 years later, on April 4, 2017, another large-scale attack with sarin and chlorine led to the deaths of about 100 Syrian civilians, injuring 100 more. In this chapter, we will briefly describe the events leading to the 2013 attack and describe what has happened since to the best of our knowledge. We will discuss the importance of social media in covering these events and broaden the scope to issues of planning humanitarian support and future implications.

7.2.1 Background On March 2011, the Arab Spring reached Syria. A limited local protest over socioeconomic hardship evolved into a prolonged large-scale popular revolt against the Syrian regime, soon becoming a bloody and unpredictable civil war (Berti and Paris, 2014; Zisser, 2013a, 2015). As time passed, the struggle became ethnic and even religious in nature, comprising Islamic groups within Syria and volunteers entering the country from the whole Arab and Muslim world to fight the infidel secular regime in Damascus, which is also supported by the Shiite camp in the Middle East, led by Iran (Berti and Paris, 2014; Zisser, 2014a, 2015). The use of force has escalated the crisis and caused worsening violence among the regime and the opposition. It also became clearer that the Syrian president was directly responsible for the use of brutal force, including chemical weapons, against 103

104 his opponents, including civilians, turning the war into a war of extermination designed to annihilate the rebels and their supporters. As a result of this campaign, over 6.1 million Syrians have become displaced, and over 5.4 million have fled as refugees (see www.unhcr.org/syria-emergency.html).

7.2.2 Main Course of Events, Belligerents in the Conflict, and International Involvement Since it erupted in March 2011, the civil war in Syria has seen ups and downs. At times, it appeared that Assad’s fall was days or at most only a few weeks away. At other times, it seemed as if he was only a step away from achieving victory. In retrospect, it is clear that until the Russians and the Iranians arrived in September 2015 to fight on Syrian soil, the war was moving in only one direction—in favor of the rebels and against the regime (Zisser, 2015, 2017). Several phases can be defined in the Syrian civil war:





1. The first phase, from March 2011 to March 2013, can be characterized by the slow and gradual loss of regime assets of both manpower and territory, more in the rural areas and the periphery. The most important achievement of the rebels was conquering the city of ar-Raqqah in the spring of 2013. This city was the gateway to the energy, water, and granary resources of the Jazeera region (Zisser, 2013b). However, despite these losses and Western assessments, the Syrian regime did not fall. 2. Following the fall of ar-Raqqah, a major change in the Syrian regime’s strategy led to the second phase, from May 2013 to June 2014, which can be characterized by improvement in the state of the Syrian regime. The regime declared a war of total destruction against its opponents, aimed at the armed groups on the battlefields and at civilian populations in the rebel-controlled areas, which, as the regime saw it, was providing cover, support, and a source of manpower for the rebels. The regime used all its available weapons, including chemical weapons, and after being caught in the act, narrowly escaped a clash with the West in late 2013. Still embarking on a war of destruction, the regime switched to the use of chemical materials not included in the Chemical Weapons Convention (CWC), although there were several cases in which the use of sarin was evident during 2014–2016 (Gladstone, 2014). Another strategic element was an effort to maintain control of the Syrian heartland instead of dispersing forces throughout the country as it had done before. The third strategic element was increased reliance on foreign volunteers, mainly Hezbollah fighters, joining Alawite volunteers recruited into a new militia framework, coming to the aid of the regular Syrian army forces. At the same time, the rebels failed to unify their ranks and choose an agreed leadership. This was accompanied by Islamic radicalization among their forces, helping Bashar al-Assad, who now appeared as the lesser of two evils (Berti and Paris, 2014; Zisser, 2014b, 2015). 3. The third phase, from June 2014 until September 2015, primarily consisted of rebel success. Large parts of northern Iraq and eastern Syria fell into the hands

Chemical Warfare Agents of the Islamic State, while a group of rebels, mostly Jabhat al-Nusra, succeeded in getting hold of southern Syria and the north of the country (Zisser, 2015). These achievements, a sign of rebel motivation and determination, followed a unification process among them; from hundreds of armed groups without any coordination, fighting each other and their common enemy, to several major groups within a radical Salafi Islamic framework (Zisser, 2015). Another process with influential consequences was the improved coordination of supporting states, namely Saudi Arabia, Qatar, Turkey, and Jordan. Their help included not only financial aid, logistical support, and training of the rebel forces but also efforts to unify the rebel camp. These efforts have introduced for the first time logic and a systematic element within the ranks of the rebels (Einav, 2015; Hubbard, 2014). As a result, the Syrian regime, with the base of its strength so far being the Ba’ath party, the army, and minority groups including Christians, Druze, Alawites, and members of the Sunni middle and upper class in the major cities, suffered from ongoing exhaustion and decline in strength, forcing it to be dependent on paid local militias, Hezbollah fighters, Iraqi and Afghan Shiite volunteers, and external support by Russia and Iran (Berti and Paris, 2014; Fouad, 2012; Lesch, 2012; Zisser, 2013a, 2015). The war remained a set of limited tactical battles throughout the country, with the regime unable to mobilize forces capable of defeating the rebels in any of these fights (Zisser, 2015). 4. The fourth phase, from September 2015 until now, started following the landing of Russian and Iranian forces on Syrian soil to help the Assad regime and ensure his survival, helping to control at least the Syrian coast, on which a large Alawite population lives (Borger, 2015; Zisser, 2015). The “Chechen model” that the Russians employ in Syria, using air and artillery bombardment of large areas, aiming to break the unity and the fighting spirit of the rebels and to drive away the civilian population supporting them, enabled the regime and its allies to take the initiative and seize control of a number of key positions in Syria (Zisser, 2016). The Russians needed a few dozen planes and combat helicopters and tens of thousands of ground forces, including Hezbollah fighters, Shiite volunteers recruited by Iran from throughout the Middle East, and Iranian soldiers. Unlike the rebels, the Russians had a comprehensive strategic view, including the ability to employ air forces and transport troops. This enabled them to win battles one after the other and to achieve decision at specific locations, breaking the momentum of the rebels and eroding their achievements (Yadlin, 2016). In parallel, the failure to act by the Obama administration deprived the rebels of the hope for a victory aided by the Western powers (Washington Post, 2016). However, the Russians understood that the forces they had sent to Syria would not suffice to achieve a quick decision in the war (Zeitun, 2017). As a result of its military successes in the course of 2016, the Syrian regime now controls one-quarter of the area of

105

The Use of Chemical Warfare Agents during the Syrian Civil War the country. The area, known as “Vital Syria” (Suriya al-Mufida), includes all of its important regions: the strip connecting Daraa in the south to the capital city of Damascus, the cities of Hama and Homs in central Syria; Aleppo in the north, and the coastal area—the stronghold of the Alawites. This allows Syrian state institutions to continue and operate. In addition to its military successes, the Syrian regime has managed to achieve a valuable demographic victory, which is the intentional and systematic “ethnic cleansing” of Syria of approximately one-third of its population, the vast majority of whom are Sunni Muslims, from rural areas and the periphery, where the rebellion erupted (Balanche, 2015). Along with the consolidation of the regime in western Syria, the Islamic State has lost many of its strongholds in northern Iraq and eastern Syria, and its attempt to establish a functioning political entity appears to have failed (Zisser, 2017). At the same time, Iran has tried to establish a land corridor from Tehran to Damascus and Beirut through Baghdad and eastern Syria. In June 2017, the Americans repeatedly attacked forces of Shiite militias and the Syrian army, attempting to seize control of parts of the Syrian desert up to the border with Iraq, beyond which pro-Iranian Shiite militias are located, in an attempt to prevent the Iranians from achieving such a territorial contiguity (Cordesman, 2015). Also, the Kurds have proved able to establish an autonomous structure in northern Syria. However, in light of the regional and international circumstances, mainly Turkish and Iranian determination to prevent the establishment of a Kurdish state in Iraq or Syria, it is unclear whether they will be able to continue and maintain this entity in isolation from the Syrian state (Perlov and Lindenstrauss, 2016). The rebels rely on the extensive support of significant portions of the country’s Sunni population, resulting from deeprooted feelings of hatred and vengeance against the Alawite hegemony in the state, the desire for revenge against the regime’s attempt to use force to suppress the rebellion, and religious radicalization among significant segments of the Sunni population in Syria (Zisser, 2017). The rebels are still active in almost all parts of Syria and continue to attack and cause painful blows to the Syrian regime (Al Jazeera, 2017). The Idlib stronghold in northern Syria remains under their control, as do considerable portions of the country’s eastern region. The turn in American policy implemented by the Trump administration has provided them, for the first time in years, with the hope of not only surviving the war but perhaps also receiving substantial Western military aid in their struggle against the regime. However, their weak point remains their inability to unify and develop an effective political and military leadership. To conclude, after seven years of civil war in Syria, and despite ongoing depletion of the regime’s resources, Bashar al-Assad still stands, with government institutions, the military, security services, and external help from Russia, Iran, and Hezbollah forces. Iran and Russia have taken a significant step by sending forces to fight alongside Bashar. The United States, European Union states, Turkey, and Saudi Arabia, on the other side, are

reassessing their position regarding Syria, as they wish to preserve the institutions of the Syrian state to prevent the repetition of the Iraqi scenario. Protest and rebellion continue to burn and may flare up, particularly if the United States decides to deepen its involvement in the crisis; hence, Moscow’s efforts to promote a political settlement in conjunction with Iran, Turkey, and in the future, possibly also Washington, and in this framework establish protected zones (areas of de-escalation) in Syria and perhaps even divide the country into regions of influence among the different regional and international actors. It seems as if in the months and years to come, the war in Syria will continue at low intensity, mostly in the areas between the territories under the regime’s control and the territory under rebel control in the western part of the country. Currently, a resolution of the Syrian crisis through peaceful means, as opposed to a compromise settlement that the Russians are likely to attempt to achieve and which will amount to the almost complete surrender of the rebels, does not appear to be a realistic possibility. The rebels will presumably refuse to integrate into a state system under Assad’s authority, and Assad, for his part, will likely not agree to any arrangement that will endanger the future of his rule and the rule of the dynasty he leads. After 7 years of bloodshed, little remains of the Syria over which Assad and his adversaries have been fighting (Yashiv, 2016); neither of the sides fighting in Syria possesses the ability to defeat its adversaries on its own and bring the war to an end. Between these two sides stand most of the Syrian population who still remain in the country, mainly concerned with their daily struggle to survive and ensure a basic existence for themselves, their families, and their communities. The war in Syria is no longer a war of the Syrians alone. The involvement of foreign forces in the fighting is fueling it and causing it to continue and may also determine its outcome. Not only the fate of Bashar al-Assad, his regime, and the Syrian state hangs in the balance, but also the outcome of the struggle for regional hegemony waged by Iran and the Sunni camp under Turkish and Saudi leadership. Also at stake is the fate of two parallel and contradictory processes that have been initiated by Putin and Trump: restoring Russia and the United States to their former glory in the regional and global arena.

7.2.3 Medical Facilities and Infrastructure in the Syrian Civil War Besides the use of firepower to weaken vast territories and their population, the Syrian regime has imposed total blockades, cutting off supplies, including food, water, and electricity, and preventing free movement of people, merchandise, and medical aid (Zisser, 2014b). The disruption of health services in Syria has become a weapon of war, the worst of its kind since the adoption of the modern Geneva Conventions. So far during the Syrian civil war, the regime has violated medical neutrality and international law through attacks on health care facilities, health care workers, and the wounded as a deliberate effort to punish health care workers and civilians, to deter them from treating the “enemy” or exposing evidence of war crimes (Hampton, 2013; Heisler et al., 2015; MSF, 2016). Evidence shows that hospitals under the Syrian regime have become instruments of repression in which the

106 wounded have been tortured (see also http://bit.ly/13 mL3Me). In some towns and villages, hospitals and pharmacies have been looted, burned down, and destroyed. Many field hospitals and private clinics have been moved several times after being found and attacked. So far, the Syrian government has destroyed hundreds of hospitals and clinics in rebel-controlled areas (Heisler et al., 2015). The loss is beyond infrastructure and buildings; hundreds of doctors, nurses, and other medical personnel have been killed since the civil war started in 2011 (Heisler et al., 2015; Heisler and Baker, 2015; Physicians for Human Rights, 2016). Inevitably, the numbers of people lacking medical care and the lives lost as a result are incremental (Physicians for Human Rights, 2016). One such example is in eastern Aleppo, where more than two-thirds of the hospitals have stopped functioning, and almost all physicians have fled or been killed (Heisler and Baker, 2015). Several organizations, among them Doctors Without Borders (MSF), keep track and report on such attacks on health care workers and facilities (MSF, 2015). Health care workers have been the target of security forces for treating wounded without reporting them to the authorities, as providing medical care to people who are defined as enemies of the regime, whether individuals or whole populations, is defined as an act of hostility, subject to an attack. Not only does being a physician in Syria means risking one’s life to save others, but also family members of physicians have been arrested and jailed by regime forces to pressure physicians to stop treating patients. As many as half of the country’s physicians have fled Syria by now; accurate data are not available, but the varying estimates give similar figures: a PHR source estimated that in 2015, 15,000 doctors (of 29,927 in 2009) had left, and in 2016, a UN source stated that 27,000 of 42,000 doctors had left (Fouad et al., 2017). As a result, there are cases in which medical students treat patients and the wounded. since there are no physicians. The results of these acts of violence include not only lack of medical care to the sick and injured, but also a rise in the imminent threat of outbreaks of potentially life-threatening infectious diseases and illnesses resulting from poor sanitary conditions and shortage of medications and vaccines, malnutrition, and the denial of medical supplies and therapies (Hampton, 2013).

7.3 The Use of Chemical Warfare Agents and Other Toxic Chemicals in Syria 7.3.1 General Perspective By the beginning of the civil war in 2011, Syria had one of the largest and most advanced operational arsenals of chemical weapons in the world, with more than 1000 metric tons of chemical warfare agents (CWAs) and precursors, including “ready-to-use” mustard agent, and sarin and VX nerve agents in their “ready-to-mix” binary components. The main delivery systems were short-range SCUD ballistic missiles, SS-21 missiles, 250 kilogram aerial bombs, and BM-21 multiple rocket launchers (NTI, 2018; Farwell, 2012; Mauroni, 2017). The chemical weapons components and the production sites

Chemical Warfare Agents were dispersed over a large number of sites throughout Syria, and the chemical agents were believed to be already weaponized (Blair, 2012; Rogin, 2012). As the Syrian government was seemingly unable to break and end the civil war and with the opposition slowly gaining more success, the international community became more concerned about Syria’s CWA arsenal, especially about the possibility of such weapons falling into the hands of local jihadist groups (Jerusalem Post, 2012). This led to a close monitoring of the Syrian events by the United States and its allies, including several diplomatic and practical measures: among others, preparations for the possibility of preventing dangerous groups from obtaining components or parts of chemical weapons (Grossman, 2012; Oswald, 2011; Rogin, 2012; Solomon and Barnes, 2012; Whitlock and Morello, 2012). Fearing foreign intervention in the civil war, the Assad regime announced on July 2012 that if Syria were to use chemical weapons, it would do so only against foreign elements. This was the first time the Assad regime had actually admitted possessing chemical weapons (Hubbard and Schemm, 2012). In response to the Syrian spokesperson’s statement, President Barack Obama spoke from the White House in August 2012, stating that Syria’s use of chemical weapons would be “a red line for us and that there would be enormous consequences if we start seeing movement on the chemical weapons front or the use of chemical weapons” (White House, 2012). Later, he repeated this warning, saying: “The use of chemical weapons is and would be totally unacceptable. And if you make the tragic mistake of using these weapons, there will be consequences, and you will be held accountable.” On the same day as the president’s warning, the Syrian Foreign Ministry stated: “Syria has stressed repeatedly that it will not use these types of weapons, if they were available, under any circumstances against its people” (Nikitin et al., 2012). At the same time, numerous reports from Syria detailed alleged chemical attacks in 2012–2013 prior to the infamous August attack. In most cases, these attacks were not verified in terms of the source or type of munitions or numbers of victims, as it was often impossible to get clinical or environmental samples directly from the victims or the attack sites. In the background of all of these events, the possibility existed that the Syrian regime would fall and that the U.S. military would need to secure and dispose of the chemical stockpile, similarly to what had happened in Iraq. A plan was raised for a relatively small U.S. military force augmented with chemical weapons specialists to secure two storage sites in Syria until technical forces could dispose of the agents and munitions. This would at least reduce the chance of al Qaeda and Islamic State (ISIS) gaining access to tons of chemical weapons. If this plan of action were followed, the military would require some form of destruction capability in the theater of operations. U.S. Central Command formally identified the need for a deployable chemical weapons destruction capability in 2012. In August 2012, the assistant secretary of defense for nuclear, chemical, and biological defense programs directed the Defense Threat Reduction Agency (DTRA) to develop options and to support building partner capacity efforts in the region, options and efforts that would be used a few months later.

The Use of Chemical Warfare Agents during the Syrian Civil War

7.3.2 Preliminary Uses of Chemical Agents in Syria during the Recent Civil War In most of these early cases in which chemical agents were used, the number of victims was low. These were tactical attacks, using a small number of ammunitions to hit a specific target, rather than a large-scale area attack. A former Syrian scientist indicated that the regime was purposefully using sarin NA in small quantities to stop rebels’ progress around the capital and to incapacitate those fighting forces rather than to cause mass casualties (Al Jazeera, 2013). It was not until between April and June 2013 that senior U.S., U.K., French, and Israeli government officials made public statements that they believed the Syrian regime was using lethal chemical weapons, including the NA sarin and other incapacitating chemical agents, in several occasions in attacks against the rebels and the civilian population (Castillo and Botelho, 2013; Clapper, 2013; Friedman, 2013, 2014). It was known that Syria had an arsenal of chemical weapons, including artillery shells, aerial bombs, and missiles, filled mainly with sarin but with other CWAs as well. Following events in Syria that year, there were several reports of Syrian forces moving chemical weapons components from base to base along with operational preparations and a state of high alert (Friedman, 2014). These reports lead both the United States and Russia to issue firm warnings to Assad not to use his chemical arsenal (Friedman, 2014; Solomon and Barnes, 2012). As mentioned earlier, President Obama and some senior U.S. administration officials stated that changes in the chemical arsenal would be considered as crossing a “red line,” leading to actual measures against the Assad regime. This “red line” was not further defined. In the meantime, several reports claimed that chemical weapons were being used in practice. For example, on March 19, 2013, 25 civilians were reportedly killed and others wounded in a chemical attack in the town of Khan el Assal in the Aleppo region (Friedman, 2013). The agent used was identified by French, British, and Israeli sources as sarin, yet no other official international declaration was given at the time. Both the Assad government and the rebels accused each other. The official U.S. position was that only preliminary data existed, and more evidence was needed to clearly determine and verify the use of chemical weapons. However, the U.S. administration’s response to this use was to increase support to the political opposition in Syria (White House, 2013a).

7.3.3 The August 2013 Attack in Ghouta, Damascus August 21, 2013, was the turning point. In the early hours of that day, surface-to-surface rockets containing the NA sarin landed in Ghouta, south and east of the Syrian capital Damascus, an area that includes dozens of towns and villages with an estimated two million inhabitants before the civil war started. Weather conditions on that night led to a temperature inversion, whereby vapors and aerosols would stay close to the ground and maximize the effects of dispersing a chemical agent. Eleven neighborhoods controlled by opposition forces were targeted. In a matter of minutes, thousands of civilians were exposed to the deadly vapor. Shocking reports, evidence, and videos began to appear along with long lines of bodies, including women and children, with no signs of trauma. Photographs of casualties were published, in which clinical signs and symptoms of NA poisoning

107 were evident (Eisenkraft et al., 2014; Friedman, 2014; Rosman et al., 2014). Clinical analysis of blood and urine samples proved that sarin was used (Human Rights Watch, 2013). These attacks resulted in an unusually high numbers of casualties, estimated at more than 1400 dead and more than 3600 injured (MSF, 2013; Rosman et al., 2014; White House, 2013b). The Syrian military targeted the same areas with intensive artillery and rocket bombardments for the next few days, using conventional high explosive ammunition, and demolished buildings as well as forcing civilians out of the area. It is unclear whether this was part of a continued campaign against the rebels or an attempt to eradicate forensic evidence of the use of chemical agents. Following an emergency United Nations Security Council meeting, in which the UN Secretary General declared the intent to conduct an investigation of the attack, the Syrian regime agreed to allow a UN Fact-Finding Mission (FFM) team, which was already deployed in Syria investigating other alleged chemical incidents, access to the site of the attacks. The team confirmed that 1400 Syrian citizens were killed as a result of sarin poisoning, which also severely affected thousands more. They stated that they had found traces of sarin and fragments of the rockets used to disperse it (Friedman, 2014; UN, 2013a). Fragments of the rockets at a few impact sites suggested that these were M-14 rockets, as well as larger Syrian-designed “Volcano” artillery rockets targeting civilian population centers, with a range that could trace the source of the attack to a local Syrian military base (Higgins, 2014). The UN mission visited the attack sites in Ghouta on August 26, 2013 (Moadamyiah) and on August 28–29, 2013 (Ein Tarma and Zamalka) (UN, 2013a). The activities of the mission included interviews with survivors and other witnesses, inspection and documentation of munitions and components, collection of environmental samples for analysis, assessment of symptoms through interviews with medical professionals and inspection of medical records (Table 7.1), and collection of biomedical samples for analysis. A total of 30 environmental samples were collected; these included wipes and metal fragments from rockets, soil and surface wipes from sites of impacts and nearby homes, and fomites and clothing from casualties. The samples were analyzed in parallel by Organisation for the Prohibition of Chemical Weapons (OPCW)-designated laboratories; 22 samples were found positive for sarin, the degradation products isopropyl methylphosphonic acid (IMPA) and methylphosphonic acid (MPA), the binary process byproduct diisopropyl methylphosphonic acid (DIMP), and/or the stabilizing additive hexamethylenetetramine (UN, 2013a,b). Five samples were negative, and three had only one chemical found; the most unequivocal were the samples taken from rocket fragments, which were found positive for all chemicals of interest by the two laboratories (UN, 2013a,b). Biomedical samples were collected from 34 survivors; these included 34 blood samples, 14 urine samples, and 3 hair samples (UN, 2013a). The majority of blood and urine samples (84–100% for different attack sites and sample media, and fairly consistent) were positive for sarin based on state-of-the-art biomarkers of exposure (see later), and none of the hair samples were positive. The Ghouta investigation was the only comprehensive investigation that included all the necessary elements of clinical and environmental studies, including visits to the attack sites and full control of the chain of custody of the samples.

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TABLE 7.1 Demographic Characteristics, Clinical Signs, and Treatment of Victims of the Syrian Attack According to YouTube Video Analysis and the United Nations Report Variable

YouTube Video Analysis (n, %)

United Nations Report (n, %)a

48 (37.0) 4 (3.0) 78 (60.0) 130 (100.00)

25 (69.0) 11 (31.0) 0 (0.0)b 36 (100.0)

18 (13.8) 7 (5.4) 63 (48.4) 42 (32.3) 8 (6.1) 1 (0.7) 6 (4.6) 24 (18.5) 69 (53) 53 (40.8)

5 (14.0) 8 (22.0) NA 8 (22.0) NA NA 8 (22.0) 7 (19.0) 22 (61.0) 28 (78.0)

42 (34.6) 21 (16.1) 5 (3.8) 18 (13.8) 16 (12.3) 2 (1.5) 8 (6.1)

NA NA NA NA NA NA NA

Demographic characteristics Men Women Infants Total Clinical signs Miosis Lacrimation Diaphoresis Hypersalivation Muscle fasciculation Urinary incontinence Vomiting Convulsions Dyspnea Loss of consciousness Treatment IV access Oxygen supplementation Nasopharyngeal suctioning Bag valve ventilation Tracheal intubation Mechanical ventilation Chest compression

Source: Adapted with permission from Rosman, Y. et al., Ann Intern Med., 160, 644, 2014. a Data from UN (2013a). b The mean age of the survivor cohort in the UN investigation was 30, ranging from 7 to 68 (UN, 2013a). It is not known how many of them were pediatric; we assume that they were a minority, as only a few could be selected as informants for the mission’s objectives.

7.3.4 UN Investigations of Other Alleged Attacks In addition to Ghouta, the UN mission investigated six allegations of nerve agent attacks that took place between April 19, 2013 and August 25, 2013 out of the 16 allegations of CWA use starting from October 12, 2013 (UN, 2013b). Unlike the Ghouta investigation, these investigations were incomplete, mainly because the mission could not access the attack sites due to security problems. The investigations relied on interviews of witnesses, clinical and epidemiological assessments, and in some cases, obtaining samples without proper control of the chain of custody. On the basis of evidence of varying strength, sarin use was verified in two attacks (Khan el Assal on April 19 and Saraqueb on April 29, 2013), suggested in two (Jobar, August 24 and Ashrafyiah Sahnayia, August 25, 2013), and not verified in two (Sheikh Maqsud, April 13, and Bahharyiah, August 12, 2013) (UN, 2013b). Of special interest is the attack that took place in the town of Saraqueb in the opposition-controlled Idlib region on April 29, 2013. According to witnesses, the attack was performed using

small munitions dropped from helicopters (UN, 2013b). The munitions, suspected to contain tear gas and sarin, fell at three locations, one of them in a courtyard of a family home, where a 52-year-old woman was severely poisoned and her daughterin-law was moderately poisoned. An additional nine casualties (six from the same location, two from another, and one a paramedic poisoned by secondary contamination) were treated for mild symptoms with atropine and released shortly after; there was no information on how many of these had been exposed to nerve agent or tear gas or were “worried well” (UN, 2013b). The older woman arrived at a local hospital unconscious and with marked hypersalivation; cardiopulmonary resuscitation with orotracheal intubation was performed, and she received oxygen and repeated doses of 1 mg atropine. As her condition deteriorated, the treating physicians decided to send her to Turkey. The younger woman, who was pregnant, was disoriented and confused and became unconscious, but recovered after 15–20 minutes of atropine treatment. She later gave birth to a healthy child. The women arrived at a transit hospital on the Turkish border at 18:45 and 19:30, about 2–3 hours after their first admission. The older woman was still unconscious but was extubated and given an oxygen mask; a doctor reported that she had bronchoconstriction and edematous lungs, as judged by wheezing and rale-like sounds. She was put on saline and given 12 doses of atropine; her miosis improved, and she was breathing spontaneously but still unconscious. The younger woman was conscious and could walk, but she had nausea and vomiting, spoke slowly, and was confused. On transfer to Turkey, the older woman was declared dead between 22:30 and 22:45 by a Turkish Rescue Service doctor before arriving at the destination hospital. At the hospital, the body of the woman was decontaminated and placed under guard at the morgue. The cholinesterase activity of blood samples withdrawn from the corpse of the patient was 1084 U/L as compared with the normal range of 5100–11,700 U/L (UN, 2013b). Three months later, on July 14, 2013, an autopsy was performed in the presence of members of the UN FFM. They obtained samples from 12 tissues and organs, of which two sets were transferred for analysis to two OPCW-designated laboratories (UN, 2013b). The results, reported in the UN FFM report, were recently published by investigators of the two laboratories—TNO in the Netherlands and Bündeswehr Institute for Pharmacology and Toxicology in Germany (John et al., 2018). As the body, though well preserved, was autopsied and samples taken nearly 3 months post-mortem, the biomarkers amenable to analysis were stable biotransformation products rather than sarin itself. These and the analytical targets and methods are summarized in Figure  7.1. The analytical processes employed, which are sensitive enough to detect the biochemical biomarkers of exposure at sub-nanomolar levels, were developed mainly for analysis of blood, but this time they were successfully employed for detection of these biomarkers in wide range of organs and tissues (Figure 7.2), with congruent results obtained by both laboratories (John et al., 2018; UN, 2013b). Some unexpected findings, which can be explained and thus do not compromise the forensic value of the evidence, are detailed in the legend of Figure 7.3. The finding of the contaminant DIMP in samples taken from external surfaces of the body further corroborates the forensic evidence obtained by

The Use of Chemical Warfare Agents during the Syrian Civil War

109

FIGURE 7.1  Biological fate of sarin and targets for biomedical verification of exposure/poisoning. Sarin undergoes two major biotransformation processes: hydrolysis and adduct formation. The resulting reaction products are unequivocal biomarkers of exposure, which can be assessed by modern mass spectrometric methods. The main hydrolysis product is O-isopropyl methylphosphonic acid (IMPA), which may be further hydrolyzed to methylphosphonic acid (MPA), which is a general indicator for G-type nerve agents. Diisopropylmethylphosphonate (DIMP) is a byproduct of sarin synthesis, which may be present in the unpurified sarin produced in the binary process and is a marker for contamination of the external body surface; for example, hair, skin, and eyes. Protein adducts are of the following types: (i) phosphonylation of the serine residue in the active site of BuChE; there are two such products (the unaged and the aged, which has lost the isopropyl moiety); both can be obtained for spectroscopic analysis as the phosphonylated nonapeptide FGES*AGAAS by proteolytic digestion of BuChE, purified by immunomagnetic separation, with pepsin; (ii) phosphonylation of the tyrosine-411 residue in human serum albumin. The phosphonylated tyrosine residues can be obtained for analysis by proteolytic digestion with pronase; (iii) release of the sarin molecule from protein adducts by treatment with fluoride ions at acid pH, which is the reverse of the phosphonylation reaction. BuChE, butyrylcholinesterase; CI, chemical ionization; EI, electron ionization; ESI, electrospray ionization; GC, gas chromatography; HR, high resolution; LC, liquid chromatography; MRM, multiple reaction monitoring; MS, mass spectrometry; MS/MS, tandem mass spectrometry. (Reproduced from John, H. et al., Forensic Toxicol., 36, 61, 2018, http:// creativecommons.org/licenses/by/4.0/.)

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FIGURE 7.2  Graphical summary of the analyzed organs of the victim of sarin exposure and detected biomarkers of poisoning. Detection of poison markers using diverse bioanalytical mass spectrometric techniques revealed systemic distribution of the nerve agent sarin. Tissues were analyzed by specialized German and Dutch laboratories on behalf of the Organisation for the Prohibition of Chemical Weapons (OPCW). The biomarkers analyzed are shown in Figure 7.1. Some unusual findings were the following: (i) IMPA, which is normally cleared rapidly in the urine, was present extensively in the victim’s tissues, probably because she died within 24 hours of exposure; (ii) The only BuChE adduct detected was the aged MPA-adduct (see legend to Figure 7.1), indicating rapid post-mortem aging of the IMPA-BuChE; (iii) Fluoride ion–regenerated sarin was detected only in blood samples and not muscle and other cholinesterase-containing tissues. The rapid aging suggested that fluoride-regenerated sarin derived from blood proteins other than cholinesterases. (Reproduced from John, H. et al., Forensic Toxicol., 36, 61, 2018, http://creativecommons.org/licenses/by/4.0/.)

FIGURE 7.3  The field deployable hydrolysis system (FDHS) designed by the U.S. Army Research, Development and Engineering Command (U.S. Army RDECOM) for the destruction of Syrian DF and sulfur mustard. (From Blades, T. Field Deployable Hydrolysis System Design and Development. Presented on July 21, 2015. Available at www.dtic.mil/ndia/2015/CBRN/Blades_CBRN.pdf. With permission.)

The Use of Chemical Warfare Agents during the Syrian Civil War the sarin biomarkers. Besides the forensic evidence, this is one of the first “real-life” demonstrations of the newly developed biomarkers of sarin exposure by laboratories that comply with the high standards required by the OPCW.

7.3.5 Events Following the Ghouta Attack and the OPCW Involvement in Syria The massive sarin attack prompted President Obama to declare that the United States would attack Syria to punish Assad and as a warning not to use chemical weapons again (White House, 2013c). On September 9, 2013, while efforts were being made to gather the attack force and before any attack took place, U.S. Secretary of State John Kerry said in a press conference in London that the Syrian government could prevent the planned attack by putting its chemical arsenal under international supervision (Friedman and Brom, 2013). Following this remark, and with the active support of the Russians, a proposal soon became a program: Syria would admit it had a chemical weapons program and that it would accede to the CWC, eliminating its stockpile of chemical weapons, precursors, and associated production facilities in accordance with a joint U.S.–Russian framework. Syria accepted the plan soon thereafter, probably realizing that the United States would attack without such an agreement. As a result, President Obama announced the suspension of the plans for attack (Lewis, 2013). The CWC is a comprehensive agreement, signed in 1992, banning the development, production, manufacture, storage, and transfer of chemical weapons, containing a control mechanism in the shape of the OPCW and a detailed list of banned substances for development, maintenance, and use. Over the years, the OPCW has destroyed thousands of tons of chemical substances all over the world (OPCW, 2019), and it was its responsibility now to implement and verify the implementation of the agreement with Syria. The schedule for eliminating the Syrian program was considerably accelerated as compared with other chemical disposal plans given international concerns about both the Syrian government’s use of chemical weapons and the potential security risk of losing the said chemical weapons to non-state actors in Syria. Syria had to destroy its chemical weapons as soon as possible, as the OPCW Executive Council determined the order of destruction and verification procedures. Syria had to submit a formal declaration to the OPCW by September 2013, including a list of all its chemical weapons programs, sites, quantities, and types and a general plan for dismantling them (OPCW, 2013a). According to the plan, the OPCW delegation would visit Damascus in early October and by the end of that month would neutralize and eliminate the production, mixing, and filling capacity at the sites declared by the Syrian regime. This first stage was completed on time (OPCW, 2012b). The declared Syrian chemical weapons program included about 1300 metric tons of chemicals and 23 sites that hosted more than 40 distinct facilities (Table 7.2). There were no actual stocks of sarin or VX nerve agents declared, as the Syrian military supposedly used a binary chemical formula to mix those CWAs immediately prior to using them. The Syrian government may have omitted some sites that may have had an association with the chemical weapons program, yet it is still unclear whether this was a deliberate deception. The Syrians

111 were required to submit a detailed plan for the dismantling of the whole chemical arsenal of weapons, precursor materials, and materials by November 15, 2013. On December 18, the OPCW approved their plan, which included defined targets and a timetable: the destruction of the most lethal compounds was scheduled to begin by the end of December 2013 and to be completed by the end of March 2014. The less toxic substances would be destroyed by the end of June 2014. By then, Syria’s chemical weapons arsenal and the infrastructure for production of new materials would be eradicated. Implementing the agreement was regarded as the most challenging operation of the OPCW due to the ongoing civil war and the harsh terrain in which some of the facilities were situated. On top of that, some of the sites had been relocated during the fighting, making it more difficult to guarantee that all existing sites were known. In early 2013, the Syria Chemical Weapons Senior Integration Group (SIG) was established at the U.S. Department of Defense (DOD). It was composed of senior leaders who would meet every 2 weeks to work on technical and policy issues and to address interagency processes. They developed a technical working group responsible for developing a disposal platform. This group was called the Syria WMD Operational Response and Dismantlement (SWORD) team, which included the Joint Program Executive Office for Chemical-Biological Defense, DTRA with its related elements, and from Aberdeen Proving Ground, Edgewood Chemical and Biological Center (ECBC), the U.S. Army Chemical Materials Agency, and the 20th CBRNE Command. Although Syria should have taken the lead responsibility to build a disposal facility and under supervision by OPCW inspectors, destroy the agents and dismantle its facilities, Syria claimed it did not have the resources to build a disposal facility. The U.S. government could not fund the construction of a Syrian disposal facility, because it was an identified state sponsor of terrorism, and the security of disposal operations in a war zone was impossible. One option was finding a host nation that would either incinerate the chemicals or build an incineration plant for the short-term purpose of destroying the Syrian stockpile, similarly to the 2006–2007 DTRA-supported destruction of Albania’s CWAs arsenal. The second option would be to move the chemicals to another state and allow the U.S. government to deploy a disposal system to eliminate the chemical agents. The technical team proposed to develop a deployable neutralization platform based on the technology used at Aberdeen Proving Ground to destroy 1800 tons of mustard and at Newport Army Depot to destroy nearly 1700 tons of VX nerve agent. The U.S. Army had past experience with building chemical disposal systems using neutralization technologies without adversely affecting the environment or causing harm to operators (Bryant, 2009; Mauroni, 2017). Also, this was not the first time that the DOD had destroyed chemical agents on a ship: in 1977, nearly 25,000 drums of the defoliant Agent Orange were incinerated aboard the Dutch-owned ship M/T Vulcanus at Johnston Atoll (Mauroni, 2017). The requirement was to develop a platform for the destruction of toxic chemicals in metric ton quantities to an efficiency of 99.9%, operating 24/7, easily transported to overseas locations, operating at remote sites, and ready to operate at full capacity within 10 days of arriving on site. This meant modular design, relatively easy to set up and operate using proven technology,

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TABLE 7.2 Syria’s Declared Chemical Weapons Program Chemical agents

Chemical sites Chemical weapons

580 metric tons of methylphosphonyl difluoride (DF, a precursor for sarin) 20 metric tons of mustard agent 130 metric tons of isopropyl alcohol 310 metric tons of four “other category 1a industrial chemicals” 260 metric tons of 13 different “category 2a industrial chemicals” including chloroethylamine, phosphorus trichloride, phosphorus oxychloride, hexamine, hydrogen chloride, and hydrogen fluoride 1 research and development, 10 production (including 27 production facilities), and 12 storage sites 1230 unfilled munitions (aerial bombs, missile warheads)

Source: Mauroni, A.J., U.S. Air Force Center for Unconventional Weapons Studies. The Counterproliferation Papers Future Warfare Series No. 58, 2017. With permission. a Category 1 chemical weapons are chemical weapons on the basis of the CWC Schedule 1 chemicals and their parts and components. This definition also includes all the chemicals used in any binary chemical weapon system. Category 2 chemical weapons are chemical weapons on the basis of all other chemicals and their parts and components. Category 3 chemical weapons are unfilled munitions and devices, and equipment specifically designed for use directly in connection with the employment of chemical weapons (Anelli and Rouzbahani, 2015).

with a large degree of self-sufficiency. This led to the prototype of the field deployable hydrolysis system (FDHS), first demonstrated in late June 2013 (Figure 7.3) (Blades, 2015). The U.S. government approached several nations between September and November 2013 to be the host for a chemical disposal effort, either using their own facilities or allowing the U.S. government to bring a chemical disposal system into their country. However, following the failure to find a host nation, the OPCW adopted the U.S. proposal to remove the chemical weapons to international waters and destroy them at sea (Gladstone, 2013; Zanders, 2013). This would involve putting two FDHS platforms on a U.S. Maritime Administration ship called the M/V Cape Ray. The Cape Ray was a Ready Reserve Force ship, part of the U.S. Transportation Command. With its cargo space, it had the right characteristics to hold the two operational systems, the chemicals to be neutralized, and the waste run-off that resulted from the operation. There was one disadvantage: the ship could not carry enough storage tanks to hold all six chemicals categorized as “Category 1” in a single trip, which meant that it would have to focus on destroying the mustard agent and the binary precursor chemical DF (Cheung, 2015; McMahon, 2014). While developed under the defense acquisition process, this project was “fast-tracked” and did not go through the

formal process of operational testing and evaluation, as nearly all defense programs must. The overall effort had to be transparent to concerned public parties but at the same time, cognizant of the need for security measures to protect the chemicals from being taken by hostile actors. Additionally, the planners had to account for potential bad weather, resupply, and rotating personnel on and off the ship. The equipment, to include a laboratory, a decontamination station, and a collective protection system, was installed on the Cape Ray in December 2013, and the ship participated in sea trials in early January to prepare for its mission. It departed from Portsmouth, Virginia in January 2014 with a crew of more than 100 personnel, including civilian mariners, technical specialists, a security team, and other military members. The Cape Ray arrived at U.S. Naval Station Rota, Spain in February 2013, waiting for Syria to remove its chemical weapons into its port in Latakia. Although behind schedule, the Syrian government was able to bring more than a third of its chemical weapons and precursor chemicals stockpile to the port by early March and load it onto ships. An OPCW team trained the Syrians on how to safely secure and handle the chemicals and also oversee the disposal process on the Cape Ray. Russia provided equipment and helped the Syrian government with transportation for moving the chemicals

TABLE 7.3 Key Elements of Preparedness for TMCEs as Reflected in the Syrian Sarin Attack Key Elements and Assumptions OP poisoning is recognized on-site by identifying the specific toxidrome Decontamination is highly important and should be done by protected personnel Large numbers of mildly (or not at all) injured persons Available antidotes that could be given rapidly to a large number of injured persons are needed Sufficient supportive measures to treat large numbers of injured persons are needed To be prepared for such an event, regular training of medical personnel and first responders is needed

Characteristics of the Syrian Event from YouTube Analysis OP poisoning was recognized on-site Decontamination was done rather ineffectively and by personnel without protective equipment, resulting in severely injured first responders Large numbers of dead or severely injured persons Injured persons received IV atropine, which is time-consuming and delays medical treatment Severe shortage of supportive measures, which resulted in patients who were intubated but not connected to a ventilator Experience in treating OP poisoning from previous events probably resulted in swift recognition of the toxidrome and early initiation of treatment as indicated

Source: Adapted with permission from Rosman, Y. et al., Ann Intern Med., 160, 644, 2014. IV: Intravenous; OP: Organophosphorus compounds; TMCE: Toxicological mass casualty event.

The Use of Chemical Warfare Agents during the Syrian Civil War to Latakia. The chemicals were loaded on Danish and Norwegian ships, which moved them to Italy, secured by Chinese, Danish, Norwegian, and Russian warships. The Italian port of Gioia Tauro served as a transload point from which the Syrian chemicals were transported to the Cape Ray. Other security support was provided by Finland, Germany, Italy, the United Kingdom, and Turkey (Anelli and Rouzbahani, 2015; Blades, 2015). Industrial facilities in Germany, Finland, the United States, and the United Kingdom destroyed the industrial chemicals and waste effluents. The OPCW also had two private industrial companies handle the final disposition of the chemicals—Veolia ES Technical Solutions and Ekokem Riihimaki (Anelli and Rouzbahani, 2015). The German government offered the services of GEKA MbH. The disposal work started in the fall of 2013. The OPCW had verified that 21 of the 23 initially declared production facilities had been shut down. The first shipments to the port of Latakia came in early January 2014, and the last shipments before the end of June, a delay of almost 6 months from what had been committed to by the Syrian government. Some of the containers had to be repacked due to leaks. All of them had to be inspected and inventoried before being packed in compliance with international rules of transportation of hazardous materials (Arms Control Association, 2018). The Cape Ray’s technical team was able to process the 600 metric tons of chemicals in 42 days, completing its disposal operations by August 17, 2014. As the technical team monitored the hydrolysis process and the flow of agents and effluent from and to the storage tanks, OPCW inspectors watched the process by video cameras and verified that all of the removed chemicals had been destroyed (ECBC, 2014). In the process of neutralizing the 600 metric tons of toxic chemicals, more than 6,000 metric tons of waste effluent was created. The Cape Ray transported sulfur mustard (HD) effluent to Bremerhaven, which was moved to the GEKA waste facility near Hamburg, Germany, and then sailed to the port of HaminaKotka, Finland. The Cape Ray returned to the United States in September 2014 and was cleared for unlimited operations in January 2015. By that time, the OPCW declared that all of Syria’s declared chemical stockpile had been destroyed. By mid-June, the waste effluents at GEKA and Ekokem had been verified as destroyed. Veolia reported the final disposal of the hydrogen fluoride to the OPCW in early January 2016. There were no security incidents throughout the event.

7.3.6 The Use of Chemicals, Including Chemical Warfare Agents, Since the 2013 Ghouta Attack Analysts suspect that the Syrian regime deliberately failed to account for 10–15% of its stockpile (Loveluck, 2015). As a party to the CWC, Syria is accountable to challenge inspections brought on by other states. If there is credible evidence suggesting that Syria is holding out with a reserve of chemical weapons, any nation can call for the OPCW to inspect and verify the allegation. After the Ghouta attack, Syria’s joining the CWC, and the dismantling of the strategic CWA stockpiles, there have been allegations of chemical attacks by Syrian military forces against Syrian civilians and rebels in the opposition-controlled regions (Human Rights Watch, 2014). The main alleged attacks, subsequently corroborated as chemical by the OPCW FFM, employed chlorine, dispersed by “barrel

113 bombs” dropped from helicopters (OPCW, 2014, 2015). The first cluster of attacks took place between April 14 and August 30, 2014 against villages in north Syria (Talmenes, Al Tamannah, and Kafr Zita) (OPCW, 2014). A second cluster of 15 similar attacks occurred between March 16 and May 20, 2015 in the Idlib region in North Syria, targeting the town of Idlib and six villages in the region (OPCW, 2015). According to the FFM investigation, the numbers of casualties in the first wave were 200 injured and two deaths in Talmenes, and 150, of whom eight died, in El Tamanah (OPCW, 2014). The numbers of casualties in the second wave ranged between 12 and 40 in the different attacks, with two and six deaths in two of them (OPCW, 2015). The casualty numbers and distribution reflect the low lethality of chlorine gas: those who died were mostly women and children exposed to high concentrations of the gas, and a considerable proportion of these were severely injured and expired later on (OPCW, 2014, 2015). In May 2015, OPCW inspectors found traces of precursors required to make sarin and VX in an undeclared military research site (Loveluck, 2015). The most recent major event was a sarin attack on the opposition-controlled village of Khan Seikhoun in the Idlib region on April 4, 2017 (BBC, 2017a; OPCW, 2017). The agent, tentatively identified through the already familiar pattern of signs, symptoms, and high numbers of casualties, was delivered by fixedwing Su-22 aircraft and later verified by an investigation, carried out by the OPCW FFM, on the basis of analyses of biomedical and environmental samples by OPCW-designated laboratories (OPCW, 2017). The death and casualty toll of this attack was high: open sources initially reported 86 killed and 451 injured (BBC, 2017a); the OPCW FFM, surveying five medical facilities, including one across the border, could not reach an accurate casualty count and estimated the numbers as ~100 dead and ~100 surviving casualties (OPCW, 2017). Western states such as the United States and others, backed by their intelligence services, were confident that the Assad regime had conducted the attack as one of its serial violations of its obligation to the CWC; the regime and its allies refuted the allegations and tried to put the blame on the opposition forces using fabricated accounts (BBC, 2017b). The United States retaliated by shelling with Tomahawk cruise missiles the Syrian air base from which the attack was launched (BBC, 2017b). Although the technical and intelligence evidence points clearly to the Assad regime as responsible for the attacks against civilians, the possibility of chemical weapon acquisition and use by non-state elements and terrorist groups involved in the conflict is not unreal, as exemplified by sulfur mustard attacks attributed to ISIS (Ackerman, 2016; Schmitt, 2016). To conclude, the removal and destruction of the Syrian CWA stockpile and related infrastructure was successful and should be viewed as a great achievement by the U.S. government and the UN. Convincing Syria to give up its declared chemical weapons and to act against a significantly accelerated schedule, using a unique form of technology at sea, with no significant incidents or loss of chemical agents, was an outstanding effort. However, eliminating Syria’s declared chemical weapons program did not stop the civil war, and it did not stop the continued use of chemical agents after Syria’s accession to the CWC. The total number of chemical casualties within Syria amounts to less than 1% of overall civilian casualties measured up to February 2015 (Reuters, 2015).

114 The continued use of chlorine as a weapon is a violation of the CWC, and the OPCW should continue its investigations of any such allegations as evidence of human rights violations and present those results to the UN.

7.4 Social Media in Disaster Medicine: The Syrian Case Study 7.4.1 Gathering Data The civil war in Syria is being intensely covered in almost “real time” through social media networks. For the first time, a clinical syndrome, in this case NA poisoning toxidrome, was diagnosed using social media (Rosman et al., 2014). As the U.S. President Barack Obama said in his speech about the sarin attack in Syria, “The world saw thousands of videos, cell phone pictures, and social media accounts from the attack … [of] people who had symptoms of poison gas” (White House, 2013c). We have decided to delineate the clinical presentation and treatment of this mass casualty event of NA poisoning as shown in YouTube videos documenting the attack in Syria. We also compared these findings with the current Israeli paradigm for such an event. We searched for videos uploaded between August 21 and September 15, 2013, on YouTube about the sarin attack in Syria. We used the keywords “Syria,” “sarin,” “NAs,” or “August 21” in English or Arabic. We excluded videos that showed external trauma injuries. Next, we had the videos analyzed separately by chemical, biological, radiological, and nuclear (CBRN) medicine specialists. They were blinded to the exact purpose of the study and to each other’s findings. Blinding was assured by hiding the title of the video clips, by inserting random unrelated yet similar videos of other toxidromes, and by providing a list of clinical findings that were not all related to organophosphate (OP) poisoning. Another physician, also a CBRN medicine expert, settled inconsistencies in the analysis. We included videos that focused on at least one victim for at least 3 seconds, allowing the physicians to get a clinical impression about predefined clinical signs. OP-related clinical signs we looked for included diaphoresis, miosis, lacrimation, excessive salivation, vomiting, muscle fasciculations, urinary incontinence, convulsions, dyspnea, loss of consciousness, and respiratory arrest or death. Findings were marked as “yes” or “no.” We also looked for supportive measures such as intubation, suctioning, opening intravenous (IV) lines, decontamination measures, and other treatments given to casualties. Repeated frames of the same casualties were identified before handling them to the experts so that they would be analyzed only once.

7.4.2 A Systematic Approach for the Evaluation of Clinical Syndromes As mentioned earlier, we tried to identify the cholinergic toxidrome. Injuries were classified as mild if the victims were fully alert, spontaneously breathing with no noticeable respiratory distress, and able to walk; moderately if they were reclining, conscious, and with some degree of respiratory distress; and severe if they were unconscious, convulsing, or with an ineffective breathing pattern. Videos were included in the analysis only if the victims were actively treated or definitely breathing.

Chemical Warfare Agents Victims were classified as deceased if they had no noticeable signs of life or were covered. We calculated κ coefficients to assess interobserver agreement of clinical findings using Excel 2010 (Microsoft, Redmond Washington). Using these methods, we identified and analyzed a total of 210 YouTube video clips, of which 71 were excluded due to repetitions or low quality and 72 were excluded because they showed only fatalities. The remaining 67 videos included 114 minutes of video data, in which 130 victims met the previously described criteria and were included. Most of the victims were children (60%), and only four (3%) were women. One hundred and nineteen (91.5%) of the casualties were defined as moderately to severely injured. The overall interobserver agreement was high (κ = 0.91). Clinical signs and medical interventions are summarized in Table 7.1. The clinical signs observed in the videos and reported by the UN investigation were loss of consciousness, dyspnea, convulsions, vomiting, hypersalivation, and miosis (Table 7.1). The proportions of these signs were roughly similar in the two surveys, although the number of casualties interviewed by the UN mission was smaller (36 vs. 130). The most prevalent signs observed in the two surveys were dyspnea and loss of consciousness, followed by hypersalivation, convulsions, miosis, lacrimation, and vomiting. Signs observed in the video inspections but not reported by the UN mission were diaphoresis, muscle fasciculations, and urinary incontinence. The frequency of diaphoresis was considerable (48.4%). The more detailed toxidromic picture obtained from the video analysis may be partly explained by the real-time character of the evidence in contrast to the retrospective survey by the UN mission. The four striking observations in the video analysis were (i) the high fatality rate of the attack; (ii) a high percentage of infants and young children among the dead victims; (iii) a high percentage of moderately and severely poisoned victims; and (iii) almost a complete lack of mildly affected and “worried well” people. The grave results of the attack can be explained by the high agent payload of the rockets (estimated by the UN mission as 56 liters per warhead), high-quality weapon-grade agent, and nighttime weather conditions favorable for agent dispersal in the air (Rosman et al., 2014; UN, 2013a). The high percentage of children among the injured and killed can be explained by their higher vulnerability due to physiological susceptibility and behavioral characteristics such as dependence on adult assistance (Rotenberg and Newmark, 2003; Sandilands et  al., 2009). The absence of mildly poisoned victims and “worried well” in the “real-time” videos from the Ghouta attack did not match the expectations or basic assumptions, based on the lessons from the 1995 Tokyo subway sarin attack, of such victims, which may greatly hamper the medical response (Markel et al., 2008). This trend was also noted in a later social media study comparing videos from the Ghouta attack with the 1995 Tokyo sarin attack (Reddy and Colman, 2017). In their analysis, the severity distribution for Ghouta was 72% severe, 20% moderate, and 8% mild, compared with 46%, 22%, and 32%, respectively, for Tokyo, reflecting the amounts and quality of agent employed in the two events. The absence of mildly injured persons can be explained away by reporting bias in the social media; however, it is also possible that those with mild injuries either refrained from seeking medical help due to shortage of medical facilities, personnel, or evacuation platforms or preferred to stay (“shelter

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The Use of Chemical Warfare Agents during the Syrian Civil War in place”), as may happen in war zones, where a constantly bombarded population may tend to seek medical help only when it is badly needed (Goldberg et al., 2013; Plakht et al., 2011). The similar severity distribution in the UN mission investigation resulted from deliberate attention to moderately and severely poisoned survivors of the Ghouta attack and other sarin attacks investigated (UN, 2013a,b). In Khan Sheikhoun, the reported distributions of severity at presentation among those arriving alive at some medical facilities included high proportions of mild and moderately injured people (OPCW, 2017). When the numbers of the dead are included, the distributions become closer to that seen in the Ghouta videos. Even though reporting or selection bias was present in the Ghouta material, it had a negligible, if any, impact on the credibility of the grave picture. Anyway, it is important to emphasize the possibility of deliberate description and inclusion bias of the exposed population to gain favorable public opinion. With time, it has become more obvious that this is one of the major strengths of the new media.

7.4.3 Immediate Medical Treatment of the Ghouta Attack Victims as Reflected in Social Media Medical treatment was given in nonmedical facilities in 66 of the 67 videos reviewed by our experts. We saw no standard medical monitoring equipment used to monitor cardiac activity, blood pressure, or pulse oximetry (this was documented in one case only). All medications were given IV with no documented use of autoinjectors. This is in agreement with the fact that we were approached by international nongovernmental organizations (NGOs) seeking opportunities to purchase autoinjectors around the world following the Ghouta attack to be used in these improvised medical facilities. From the soundtrack of the video clips, we learned that the treatment protocol was based on the administration of high doses of atropine. In some cases with respiratory distress, steroids and diuretics were used on top of atropine (Rosman et al., 2014; UN, 2013a). We found no evidence of the use of oximes or benzodiazepines, though there were reports from NGOs describing infrequent use of these important drugs; their use in the management of severe casualties was reported in medical facilities receiving casualties of the Khan Sheikhoun attack of April, 4, 2017 (OPCW, 2017). No ventilators were seen in the provisional care facilities. Some of the movies showed victims that were intubated but not connected to mechanical ventilators and were ventilated with a bag valve device. In a few cases, intubated victims were trying to breathe without ventilator support. Decontamination was performed in 25% of the video clips, in most cases by pouring pots or bottles of tap water on the exposed victims and rubbing their face or chest. In a couple of these films, we noticed the use of a water hose. Full undressing was conducted in only 11 video clips (16.4%). In 10 of the clips, decontamination procedures were carried out inside the medical facility with poor aeration and drainage, which may increase the risk of contamination, and in most of the videos, medical personnel did not wear any CBRN personal protective equipment (PPE). We found only sporadic use of latex gloves and surgical masks, which are not relevant as chemical protection gear. This finding was consistent with repeated reporting on medical staff and first responders becoming casualties through primary

exposure and secondary contamination in Ghouta and in other chemical attacks (UN, 2013a,b), including the Khan Sheikhoun attack (OPCW, 2017).

7.4.4 Lessons Learned: Preparedness and Planning for Humanitarian Support 7.4.4.1 The Israeli Civilian CBRN Defense Doctrine’s View of the Ghouta Video Findings In Israel, the continuous awareness of chemical threats imposed on its civilian population by state or non-state adversaries, which culminated in the SCUD missile attacks from Iraq during the 1991 Gulf War, prompted the development of operational concepts and procurement of means to protect the civilian population and respond to a CWA-based toxic mass casualty event (TMCE) in a war-type or terror-type attack (Markel et al., 2008). The videos of the medical response provided in Ghouta stress several important principles that have been implemented in the doctrine in Israel and in other Western countries. The principles whose implementation, or lack of it, were manifest in the Ghouta event include the following (Eisenkraft et al., 2014): (i) the need to recognize the toxidrome as soon as possible, by clinical recognition of the typical signs and symptoms of NA poisoning, even without any detection equipment; (ii) the need to protect medical staff in close contact with contaminated casualties. This includes both first responders and medical staff in the “hot zone” of the event theatre and at the entrance to the hospitals; (iii) the need to decontaminate the casualties. As decontamination of casualties in the field is time consuming and delays evacuation, the Israeli approach is based on performing decontamination only at the entrance to the hospitals using decontamination showers, while undressing the casualty en route is a practical and efficient means to reduce contamination (Markel et al., 2008); (iv) it is vital to use readily available antidotes stockpiled in advance, preferably autoinjectors that serve as fast, simple, and efficient means of delivery. These should be prepositioned mainly with medical first responders and hospitals and not only in central locations, as it is anticipated that it would take too much time to transport them to the hospitals in the case of an event; (v) quick and simple triage to differentiate the mild cases (which are expected to be the majority) from the severe. The former can be managed by nurses who are allowed to give antidotes based on the toxidrome and without a physician’s order, freeing the physicians to care for the severely poisoned casualties; (vi) simple yet crucial supportive care. Supportive care includes several major elements: (1) oxygen supplementation, (2) ventilation, and (3) suctioning of oropharyngeal secretions. It has been recognized that respiratory distress is the major cause of early death in NA poisoning (Hulse et al., 2014), and respiratory distress was the most frequent sign among casualties in the NA attacks in Syria; (vii) continuous training of first responders and medical personnel to maintain proficiency in the principles and practices of CWNA casualty care and management. The characteristics of the response to the Ghouta attack in view of these stated principles are summarized in Table 7.3 (Rosman et al., 2014). It is important not to underrate the Syrian first responders and medical staff, who did the best they could in those harsh circumstances and severe shortage of essential

116 assets. Recognition of NA poisoning by clinical signs and symptoms was fast, and so was the response in general, probably due to previous experience with chemical attacks in Syria. The deviations from the standard doctrines, seen in the videos and confirmed by other reports, were in antidotal treatment, supportive care, and contamination avoidance measures—decontamination and protection of first responders in the field and caregivers in hospitals. The only antidotal treatment observed was IV atropine; this means that delivery is slow and cumbersome and can be given by qualified personnel only, while the intramuscular autoinjectors are fast and simple to operate, so that they could be operated by lay persons. The lack of other antidotes such as oximes and diazepam stands in contradistinction to the complex pharmacology of NA poisoning, which requires the additional antidotes to ensure the survival of moderately/severely poisoned casualties (Kaur et al., 2014). Ventilation support was given, if at all, in a manner that was not only inefficient but also unsafe. PPE is critically important for first responders and pre-hospital caregivers. It should be stressed that the necessary and adequate protection level for first responders and medical staff is level C or the parallel military CBRN PPE (Markel et al., 2008).

7.4.4.2 Humanitarian Support The Doctors Without Borders (Medecins Sans Frontieres [MSF]) organization is the main international medical relief operating in Syria since the beginning of the crisis (MSF, 2018). It directly operates four medical facilities in northern Syria and provides support in the form of equipment, medicines, and guidance to more than 150 medical facilities all over Syria (MSF, 2017, 2018). On August 21 2013, nearly 3600 casualties displaying neurotoxic symptoms were received in three MSF-supported hospitals in the Damascus region within 3 hours of the attack (MSF, 2013). MSF also reported the delivery of 1600 atropine vials before, plus an additional 7000 vials after, the Ghouta attack (MSF, 2013). After the Khan Sheikhoun attack, MSF reported the supply of various antidotes as well as protective clothing for the medical attendants at emergency rooms (MSF, 2017). In the Marburg hemorrhagic fever epidemic in Angola in 2005, MSF activities, providing basic protective measures and guidance in the community, played a considerable part in containing the epidemic (Roddy et  al., 2007), thus underscoring the importance of work at the community level, which we have identified as an element that should be strengthened. The humanitarian support at the community level should include: (i) development of leadership capable of coordinating the response and guiding the population; (ii) the establishment of trained groups of first responders provided with protective gear; (iii) the provision of antidotes (atropine and oxime autoinjectors) and other pre-hospital lifesupport facilities, mainly respiratory support equipment; (iv) the implementation of simple contamination avoidance procedures for both first responders and civilians, and (v) the organization of a pre-established casualty evacuation network, realizing that evacuation is elementary in this situation. Support plans should include the supply of equipment that is suitable for respiratory support in a contaminated pre-hospital environment along with standard equipment for the treatment of decontaminated casualties at the hospital or medical facility. Equipment for respiratory support should also include airway management equipment

Chemical Warfare Agents such as laryngeal masks, which may be useful as an alternative to endotracheal intubation and can be operated by a PPEwearing caregiver (Talmor, 2008). Oxygen supplementation as well as ventilation should be based on portable equipment; this is relevant not only for the pre-hospital setting but also for inhospital treatment, as hospitals in Syria are under frequent air and artillery attacks that cause heavy damage to infrastructure and life-saving assets (Fouad et  al., 2017; MSF, 2018). This point underscores that humanitarian support is part of the general international effort required to protect the right to neutrality, endowed by international law, of medical professionals and facilities engaged in humanitarian activity and to protect them from intentional attacks (Fouad et al., 2017).

7.4.5 Future Implications TMCEs of the magnitude that occurred in Ghouta in 2013 and more recently in Khan Sheikhoun are challenging not only for a war-ridden community, as in Syria, but also in the relatively peaceful developed world. Events such as the Tokyo underground sarin attack in in 1995 and the 9/11 mega-terror and anthrax letter attacks that hit the United States in 2001 were a reminder of overall vulnerability and the technological, tactical, and organizational gaps in the preparedness for coping with large-scale disasters. The points raised earlier are a reminder to emphasize operational concepts, procedures, and training and to prioritize efforts in the research, development, and procurement of medical and protective materiel. This section is a brief summary of research and development (R&D) directions and needs arising from the lessons of the Syrian chemical attacks; we shall focus mainly on (i) short-range solutions that are based on existing materiel and medicines, and R&D direction, and (ii) needs relevant for pre-hospital care. Important issues and exemplary solutions will be presented.

7.4.5.1 Pharmacological Intervention Atropine, the primary antidote in CWNA/OP poisoning, has several limitations: it is not effective alone, and additional drugs, currently oximes, are required for effectiveness; repeated/escalating doses are required until a clinical response is obtained; furthermore, atropinization, the indicator of clinical response, is reached slowly and may not always occur (Eisenkraft and Falk, 2016a; McDonough and Shih, 2007). In the case of a shortage of atropine and autoinjectors, several substitutes may be considered, such as scopolamine (Dolgin, 2013; Koplovitz and Schulz, 2010; Markel et al., 2008). Another atropine substitute or adjunct is the atropine-like anticholinergic alkaloid anisodamine, which is somewhat less potent but also less toxic, has additional beneficial pharmacological activities, and was shown to be effective in some case reports on OP-poisoned patients who were slow responders to atropine (Eisenkraft and Falk, 2016a; Kaur et al., 2014). Another supportive therapy suggested by us is infusion of Intralipid® emulsion (ILE), which is used today in resuscitation of patients severely poisoned by an overdose of lipophilic drugs (Eisenkraft and Falk, 2016b). As in the case of anisodamine, systemic studies on its role in CWNA poisoning have not been done, but favorable results from its use in some cases of severe OP poisoning and a sound pharmacological rationale

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The Use of Chemical Warfare Agents during the Syrian Civil War make it a possible supportive adjunct in severe CWNA poisoning (Eisenkraft and Falk, 2016b). The need to administer more than one antidote simultaneously in the case of CWNA/OP poisoning was addressed by the use of autoinjectors with a combination of atropine and oxime or with an additional central component (an anticonvulsant or benactyzine). An alternative approach is to explore antidotes with more than one pharmacological activity. The antitussive drug Caramiphen, which has both anticholinergic and antiglutamatergic activities, was shown to be neuroprotective in animal models of OP poisoning and thus a candidate for development as an OP antidote (Apland et al., 2018; Raveh et al., 2014; Schultz et al., 2014). Novel strategies such as bioscavengers, novel reactivators, and neuroprotectants, which are promising in drug discovery and early pre-clinical research, are options for the more distant future (Dolgin, 2013).

7.4.5.2 Antidote Administration The establishment of an IV line under the stressful conditions characteristic of a TMCE is difficult and slow, and wearing PPE adds difficulty. An alternative method used in resuscitation is intraosseous (IO) infusion when intravascular access is unobtainable, as is expected in the case of children or patients in shock. Advanced simulation studies have shown that it can be easily operated by caregivers clad in full protective gear with a high success rate (Vardi et al., 2004), and its efficacy was demonstrated in an animal model study, in which IO administration of midazolam into paraoxon-poisoned pigs was as efficient as IV administration (Eisenkraft et al., 2007).

7.4.5.3 Respiratory Support Respiratory support may be required while the patient is still in a contaminated zone, that is, before and/or in parallel with decontamination. This requires the provision of clean air. The only available way to do this is to use a bag-valve-mask device or a portable ventilator, if available, plus suction of excessive airway secretions (Baker, 1999; Ben Abraham et  al., 2002). Furthermore, positive pressure ventilation requires endotracheal intubation, which is difficult to perform by a PPE-wearing caregiver. A possible solution free of these and other hurdles of positive pressure ventilation in a contaminated environment is non-invasive, negative pressure ventilation by a biphasic extrathoracic cuirass assisted ventilator, which could be easily operated even by non-medical caregivers wearing full PPE (Gur et al., 2005). Pilot studies showed that the ventilator was superior to a bag-valve-mask device in the rescue of lethally paraoxonpoisoned pigs (Gur et al., 2015) and was safe in healthy subjects wearing protective masks (Gur et al., 2017).

7.4.5.4 Management of Pediatric CWNA Victims A considerable proportion of the dead and severely poisoned patients in the Ghouta attack were infants and young children, raising the requirement to meet the needs of pediatric victims in support programs. Procedures for the care of pediatric victims have been developed, based on experience in pediatric critical care from other fields and extrapolation from the better-known toxicity and treatment in adults (Rotenberg and Newmark, 2003).

Many questions, especially the optimal doses and modes of administration of antidotes, are not yet fully settled (Sandilands et  al., 2009). In Israel, the Strategic National Supply (SNS) includes autoinjectors with doses adjusted for infants and children. These autoinjectors are included in the emergency packages in every ambulance and every emergency department throughout the country. The actual experience in treatment of pediatric OP/CWNAs is extremely limited, and so are experimental data, mainly due to the lack of suitable animal models. Models of juvenile animals are being currently developed to test existing and novel therapies (Apland et al., 2018; Miller et al., 2015).

7.5 Conclusions The extensive CWA arsenal in Syria was recognized years before the civil war started in 2011. Despite warning signals and allegations of chemical weapon attacks on opposition-controlled towns by the Syrian government forces, the international response was hesitant in view of the allegedly inconclusive evidence of the use of CWAs against civilians. The turning point was the attack on opposition-controlled quarters in Eastern Ghouta, Damascus with sarin-filled rockets on August 21, 2013. This dramatic mass casualty and mass fatality event aroused an international response that forced Syria to join the CWC, declare its chemical weapon arsenal, production, and storage facilities, and submit to the destruction of materials, weapons, and facilities. The destruction program, facing operational and technical challenges, was carried out successfully with the international cooperation of seven CWC member countries. The demilitarization severely mutilated the Syrian chemical weapon capability but did not annihilate it. The motivation remained, as seen by the chlorine bomb attacks on opposition-controlled towns in northern Syria. Moreover, the sarin attack on the town of Khan Sheikhoun on April 4, 2015, occurring after most of the demilitarization has been completed, indicated that a residual chemical weapon capability remained. Two factors, discussed in this chapter, contributed to the international recognition that led to the eradication of the Syrian chemical weapon program: (i) the investigations of the allegations by the Fact Finding Missions, appointed first by the UN and later, upon Syria’s becoming a CWC member state, by the OPCW, and (ii), the publicity given to these events in the social media. The investigations, with their thorough field, clinical, or forensic laboratory studies employing state-of-the-art methods and novel biomarkers, produced highlevel evidence of chemical weapon use. Although the mandate of the investigations was to prove chemical weapon use and not to identify the perpetrators, the technical and factual evidence points clearly to the Syrian government. The spread of information in the social media was not only the earliest messenger drawing attention to the attacks. The videos, made and posted by eye witnesses, were also a source of valuable medical information and insight when viewed and analyzed by professional experts. The study by Israel Defense Forces (IDF) Medical Corps experts on the basis of YouTube videos taken in Ghouta produced “real-time” information on the severity of the casualties, the characteristic signs and symptoms, and the desperate efforts of the first responders to cope with the consequences of the attack given a severe shortage of medicines, equipment,

118 and properly trained medical personnel. The most striking findings were the absence of protective gear, antidote autoinjectors, and proper respiratory support. Some of the practices employed by the poorly equipped and untrained medical staff were not only inefficient but also harmful, such as poor or no contamination avoidance measures and improper respiratory support, which increased the risk of secondary exposure of caregivers and compromised the condition of critical casualties. This study produced important lessons that should be incorporated in plans for humanitarian support and also in plans for readiness of other communities worldwide to cope with chemical terror and other toxicological mass casualty events. The implications of these events in prioritizing R&D activities are also discussed, presenting solutions that are close at hand and others that are a subject for longer-range activities. The implementation of humanitarian support plans for the Syrian population is a matter of great concern; as of March 2018, the war is still going on, with a grave humanitarian crisis in Eastern Ghouta due to massive shelling and air raids and deliberate efforts by the government to hamper support efforts.

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Chemical Warfare Agents Raveh, L., Eisenkraft, A., Weissman, B.A. 2014. Caramiphen edisylate: an optimal antidote against organophosphate poisoning. Toxicology, 325: 115–124. Reddy, D.S., Colman, E. 2017. A comparative toxidrome analysis of human organophosphate and NA poisonings using social media. Clin Transl Sci., 10: 225–230. Reuters. 2015. Syria death toll now exceeds 210,000: rights group, February 7, 2015. Available at www.reuters.com/article/ us-mideast-crisis-toll-idUSKBN0LB0DY20150207. Roddy, P., Weatherill, D., Jeffs, B., Abaakouk, Z., Dorion, C., Rodriguez-Martinez, J., Palma, P.P., et al. 2007. The Medecins Sans Frontieres intervention in the Marburg hemorrhagic fever epidemic, Uige, Angola, 2005. II. Lessons learned in the community. J Infect Dis., 196(Suppl 2): S162–S167. Rogin, J. 2012. Exclusive: state department quietly warning region on Syrian WMDs. Foreign Policy, February 24, 2012. Available at http://tehcable.foreignpolicy.com/posts/2012/02/24/exclusive_state_department_quietly_warning_region_on_syrian_ wmds. Rosman, Y., Eisenkraft, A., Milk, N., Shiyovich, A., Ophir, N., Shrot, S., Kreiss, Y., Kassirer, M. 2014. Lessons learned from the Syrian sarin attack: evaluation of a clinical syndrome through social media. Ann Intern Med., 160: 644–648. Rotenberg, J.S., Newmark, J. 2003. NA attacks on children: diagnosis and management. Pediatrics, 112: 648–658. Sandilands, E.A., Good, A.M., Bateman, D.N. 2009. The use of atropine in a NA response with specific reference to children: are current guidelines too cautious? Emerg Med J., 26: 690–794. Schmitt, E. 2016. ISIS used chemical arms at least 52 times in Syria and Iraq, report says. New York Times. Available at www. nytimes.com/2016/11/21/world/middleeast/isis-chemicalweapons-syria-iraq-mosul.html. Schultz, M.K., Wright, L.K., de Araujo Furtado, M., Stone, M.F., Moffett, M.C., Kelley, N.R., Bourne, A.R., et  al. 2014. Caramiphen edisylate as adjunct to standard therapy attenuates soman-induced seizures and cognitive deficits in rats. Neurotoxicol Teratol., 44: 89–104. Solomon, J., Barnes, J.E. 2012. US warns Syria on chemical arms. Wall Street Journal, December 3, 2012. Available at http:// online.wsj.com/article/SB100014241278873243559057815773 04242057288.html. Talmor, D. 2008. Airway management during a mass casualty event. Resp Care, 53: 226–230. UN. 2013a. U.N. Mission to Investigate Allegations of the Use of Chemical Weapons in the Syrian Arab Republic: Report on the Alleged Use of Chemical Weapons in the Ghouta Area of Damascus on 21 August 2013. United Nations, September 13, 2013. Available at www.un.org. UN. 2013b. United Nations Mission to Investigate Allegations of the Use of Chemical Weapons in the Syrian Arab Republic. Final Report. United Nations, December 13, 2013. Available at www.un.org/ga/search/view_doc.asp?symbol= S/2013/735. Vardi, A., Berkenstadt, H., Levin, I., Bentencur, A., Ziv, A. 2004. Intraosseous vascular access in the treatment of chemical warfare casualties assessed by advanced simulation: proposed alteration of treatment protocol. Anesth Analg., 98: 1753–1758.

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8 U.S. CBRN Homeland Response and Civil Support Kelley J. Williams and Steven A. Schmitt CONTENTS 8.1 Introduction.................................................................................................................................................................................123 8.2 National Response Framework (NRF).......................................................................................................................................124 8.3 Department of Defense CBRN Response Enterprise (DOD-CRE)...........................................................................................124 8.3.1 WMD Civil Support Teams (WMD-CST)....................................................................................................................124 8.3.2 CBRNE-Enhanced Response Force Packages (CERFPs).............................................................................................125 8.3.3 Homeland Response Force (HRF).................................................................................................................................125 8.3.4 Joint Task Force Civil Support (JTF-CS).......................................................................................................................125 8.3.5 Defense CBRN Response Force (DCRF)......................................................................................................................126 8.3.6 Command and Control CBRN Response Element A and B (C2CRE)..........................................................................126 8.4 Local, Regional, and Federal Response Capabilities.................................................................................................................126 8.4.1 Interagency Operations..................................................................................................................................................126 8.4.2 HazMat Teams................................................................................................................................................................126 8.4.3 FBI WMD Directorate...................................................................................................................................................127 References.............................................................................................................................................................................................127

8.1 Introduction The Defense Against Weapons of Mass Destruction Act of 1996 highlighted the easy availability of weapons of mass destruction (WMD)–related materials and information for the creation of such weapons (Defense Against Weapons of Mass Destruction Act of 1996, 1996). The U.S. government’s foresight of potential threats related to chemical, biological, radiological, nuclear, and high-yield explosives (CBRNE) was vital to the mitigation and response efforts in local, state, and federal incidents. Occasionally, the acronym CBRN is used to describe chemical, biological, radiological, and nuclear materials but excludes high-yield explosives. This chapter summarizes the capabilities and limitations of the primary military and civilian response units that would form a federal response to a chemical agent attack. Within the Department of Defense (DOD), the primary response organization is known as the CBRN Response Enterprise (CRE). By examining the numerous assets and capabilities provided by these agencies, researchers in the field of homeland security can better understand the broad spectrum of responders from the local level to the federal level. In a speech prior to the attack on 9/11, President George W. Bush declared: First, we must prepare our nations against the dangers of a new era. The grave threat from nuclear, biological and chemical weapons has not gone away with the Cold War. It has evolved into many separate threats,

some of them harder to see and harder to answer and the adversaries seeking these tools of terror are less predictable, more diverse. With advance technology, we must confront the threats that come on a missile. With shared intelligence and enforcement, we must confront the threats that come in a shipping container or in a suitcase (2001).

The Department of Homeland Security was established on November 25, 2002 in response to the attacks on September 11, 2001 and marked the most comprehensive government reorganization since the Cold War (Andreas, 2003). Preceding the September 11 attacks, the use of chemical weapons by Iraq against the Kurds and Iranian forces in the 1980s, the discovery of advanced chemical weapons programs in the former Soviet Union, and the Tokyo sarin nerve agent attack in 1995 demonstrated the continued need for defense against WMD and the relevancy of specially trained domestic response capabilities (Tucker, 2007). In the field of WMD and CBRNE response, the terms weapon of mass destruction and CBRNE are not often well delineated. In general, the two terms may be used interchangeably. More specifically, CBRNE are the categories of materials. If CBRNE materials are used to threaten or attack, they are considered WMD (Crimes and Criminal Procedure, 18 U.S.C. §2332a, 2011). For example, there may be an accident involving CBRNE materials that is not a terrorist or criminal attack, such as the large chlorine release from a train car that occurred in Graniteville, SC in 2005 (Cahill, 2005). This was a CBRNE incident but not a WMD event, as the term WMD implies malice.

123

124

8.2 National Response Framework (NRF) Presidential Decision Directive 39 (PDD-39) outlined national anti-terrorism policies and assigned specific missions to federal departments and agencies. While PDD-39 was declassified in 2007, the national security imperatives are presumed to remain relevant. This directive indicated that it is increasingly likely that terrorist groups will possess the knowledge, skills, and abilities to use WMD against Americans (Clinton, 1995). The United States’ response to a WMD attack can be incredibly complex, and the attack is treated as a terrorist attack. The National Response Framework (NRF) is a guide to how the United States will conduct all-hazards response. According to HSPD-5, “the Attorney General has lead responsibility for criminal investigations of terrorist acts or terrorist threats by individuals or groups inside the United States, or directed at U.S. citizens or institutions abroad” (“Management of Domestic Incidents, HSPD-5,” 2003). The Terrorism Incident Law Enforcement and Investigation Annex lists the primary federal agencies which have responsibility for the law enforcement and investigative response, including Department of Defense (DOD) Department of Energy (DOE) Department of Health and Human Services (HHS) Department of Homeland Security (DHS) Department of Justice/Federal Bureau of Investigation (FBI) • Environmental Protection Agency (EPA) (Terrorism Incident Law Enforcement and Investigation Annex, 2004) • • • • •

The National Response Plan (NRP) Base Plan outlines the broad response priorities following a terrorist attack, and the National Incident Management System (NIMS) provides standard command and management structures that apply to response activities. The Incident Command System (ICS) provides guidance for key management concerns during incident response. Under the NRF, the NRP, NIMS, and ICS are cooperative documents, which guide response functions from local to national levels. The United States has a large number of organizations that are capable of supporting a CBRN response. These specialized teams can be primarily found in the DOD, the FBI, the DOS, and the Defense Threat Reduction Agency (DTRA). The NRFcompliant activities would be active during the response to a chemical agent attack; however, some FBI and DOD assets may not necessarily fall within the formal NRF structure and may operate as independent functions within the larger response.

8.3 Department of Defense CBRN Response Enterprise (DOD-CRE) The U.S. Army has clear roles in the mission areas of WMD interdiction, WMD offensive operations, WMD elimination, active defense, passive defense, and consequence management (U.S. Army TRADOC ARCIC, 2009). The DOD has allotted

Chemical Warfare Agents Army and Air Force resources primarily for the homeland CBRN response mission, which are collectively referred to as the CBRN Response Enterprise (CRE) (“Defense Response to CBRN Incidents in the Homeland, CJCSI 3125.01D,” 2015). In 2005, the deputy secretary of defense mandated that the DOD implement policies and procedures consistent with the National Response Plan (“Defense Response to CBRN Incidents in the Homeland, CJCSI 3125.01D,” 2015). The DOD doctrine for operations in CBRN environments is presented in Joint Publication 3-11 (JP 3-11). The CRE does not maintain explosive ordnance disposal (EOD) capabilities but is able to provide support during a postblast response. While subject to growth and reorganization, the CRE is generally comprised of five types of response groups, which are organized for a rapid, tiered response to domestic WMD incidents and may include support from Joint Task Force Civil Support (JTF-CS) leadership. The quantity and type of these DOD organizations are as follows: • 57 WMD Civil Support Teams (WMD-CST) • 17 CBRNE-Enhanced Response Force Packages (CERFP) • 10 Homeland Response Force (HRF) • 2 Defense CBRN Response Force (DCRF) • 2 Command and Control CBRN Response Elements (C2CRE A/B) The DOD dedicates over 18,000 personnel to the CRE across these five deployable forces. During a WMD response involving a chemical incident, there is a low probability that the bulk of these resources would be used. Due to the limited physical destruction and small affected area associated with anticipated chemical attacks, it is possible that the bulk of a response could involve patient extraction, medical, and decontamination resources.

8.3.1 WMD Civil Support Teams (WMD-CST) The WMD-CSTs are the lead element of the National Guard response force and an integral element of the DOD’s overall CBRNE program strategy to augment civil authorities in the event of an incident involving WMD or the effects of natural disasters in the United States (“U.S. Army Posture Statement,” 2009). WMD-CSTs are designed and trained to provide initial assessment of incidents and advise and assist the incident commander and other response organizations as established by 10 USC §12310. The mission of the WMD-CST program is to support civil authorities during domestic CBRN incidents by identifying CBRN agents/substances, assessing current and projected consequences, advising on response measures, and assisting with requests for additional support. The first 10 WMD-CSTs were formed under Title 10 USC, Section 12310, and the remaining 47 teams were authorized under National Defense Authorization Act (NDAA) 2007 and NDAA 2013 (Section 1435). WMD-CSTs have made great contributions in many real-world incidents, have trained thousands of first responders in WMD response operations, and provide standby support at hundreds of large events each year. Major operations include supporting the World Trade Center response after the September 11, 2001 attacks, Space Shuttle Columbia recovery operations, and numerous

125

U.S. CBRN Homeland Response and Civil Support other national events, including the Republican and Democratic National Conventions, the G8 Summit, the winter Olympics, the Pentagon 9-11 dedication ceremony, and presidential inaugurations. Most recently, WMD-CSTs were identified as a lead response element to support the growing opioid crisis. There are 57 WMD-CSTs distributed across the 50 states and territories. Each team consists of 22 full-time Army and Air National Guard personnel (1254 personnel in total). As operational units within the National Guard, the WMD-CSTs are uniquely poised to respond to WMD incidents. They often train and respond with civilian first responders and maintain working relationships with key emergency management stakeholders in their state or territory. WMD-CSTs offer a unique capability, in that they are often the only Type-1 HazMat team in a state, meaning they are the only resource capable of dealing with WMD chemical and biological hazards (Federal Emergency Management Agency, 2005). Each WMD-CST has six functional elements: Command, Operations, Communications, Decontamination, Medical/Analytical, and Survey. Most personnel serve several roles and are highly trained in their areas of emphasis. The WMD-CST Survey section specializes in hazard identification and analysis in a hot zone environment. The Survey section uses all of the field sensor technology described in Chapter X. The WMD-CST Analytical section offers a special capability to perform field confirmatory analysis of samples, and each team maintains International Organization for Standardization (ISO) 17025 accreditation as an analytical laboratory. WMD-CSTs operate under three response management plan (RMP) cycles, which determine their required response time, varying from 1 to 72 hours after initial notification of a validated mission. At least six geographically dispersed teams are always maintained on the shortest response cycle, which rotates each month. WMD-CSTs are expected to be the first DOD asset to respond to a WMD incident. As a small unit, the WMD-CSTs cannot sustain regular operations beyond approximately 72 hours without sustainment support. In the planning for largescale incidents, WMD-CSTs will receive follow-on support from a CERFP and other CRE elements as needed (GAO, 2016).

8.3.2 CBRNE-Enhanced Response Force Packages (CERFPs) There are 17 CERFPs, which places at least one in each Federal Emergency Management Agency (FEMA) region: New York, Massachusetts, Pennsylvania, West Virginia, Colorado, California, Texas, Illinois, Missouri, Florida, Hawaii, Washington, Virginia, Ohio, Georgia, Minnesota, and Nebraska (“U.S. Army Posture Statement,” 2009). Each team consists of 203 traditional and full-time Army National Guard personnel (3451 personnel in total). Unlike WMD-CSTs, CERFPs are not dedicated operational units within the National Guard but are comprised of existing Army and Air National Guard units. CERFPs must be ready to deploy within 6 hours of notification following a catastrophic WMD incident. As noted in the 2009 Army Posture Statement, the CERFPs are a key element of the DOD’s overall program to provide support to civil authorities in the event of a domestic WMD incident. CERFPs are designed to fill the 6 to 72 hour response gap in our

nation’s ability to provide mass casualty patient decontamination, medical triage, and treatment and extraction from a contaminated environment. Each CERFP consists of a command and control element, an engineer company, a chemical company, an Air National Guard medical group, and an Air National Guard fatality search and recovery element. The search and extraction function is assigned to an Army or Air National Guard engineering unit, the decontamination element is from an Army National Guard chemical company, the medical element is from an Air National Guard medical group, and the fatality search and recovery element is from the Air National Guard. These organizations maintain their original missions but are given additional training and equipment that builds on existing skills to accomplish the CERFP mission. (“U.S. Army Posture Statement,” 2009)

The CERFPs were authorized under Joint Requirements Oversight Council Memorandum (JROCM) 162-06 and NDAA 2006. CERFPs are trained to receive incident information from WMD-CSTs to support their own operations. Large incidents may include multiple CERFPs, which would then fall under the operational control of a Homeland Response Force.

8.3.3 Homeland Response Force (HRF) Homeland Response Forces are groups of existing National Guard units who are designated with an HRF mission. There are 10 HRFs, which places one in each FEMA region: New York, Massachusetts, Pennsylvania, Utah, California, Texas, Missouri, Washington, Ohio, and Georgia (“U.S. Army Posture Statement,” 2009). Each team consists of 583 traditional and fulltime Army National Guard personnel (5830 personnel in total). HRF personnel are divided among command and control, communications, casualty assistance, medical triage/stabilization, decontamination, search and extraction, and fatality search and recovery capabilities. For Army personnel, the HRF mission is secondary to their Army National Guard wartime mission. The HRF mission is the primary mission for the Air National Guard. HRFs must be ready to deploy within 6 to 12 hours of notification following a catastrophic WMD incident. The HRFs operate alongside other National Guard–sourced CBRN Response Enterprise forces, including WMD-CSTs and CERFPs, as well as federally controlled elements of the enterprise, including DCRFs, C2CREs, and follow-on forces, when necessary.

8.3.4 Joint Task Force Civil Support (JTF-CS) JTF-CS is a standing joint task force and subordinate command of U.S. Army Northern (USARNORTH). JTF-CS leads command and control (C2) operations for the CRE in support of FEMA during the consequence management phase of a large CBRN situation. DOD receives mission authority for JTF-CS operations through the Stafford Act (Robert T. Stafford Disaster Relief and Emergency Assistance Act, 48 U.S.C. § 5121 et seq., 1974). The Stafford Act authorizes the president to provide disaster and emergency assistance to state and

126 local governments on receipt of a request from a governor. The deployment of JTF-CS, at the direction of the USNORTHCOM commander and on the authority of the secretary of defense, would occur only after a governor requests federal assistance from the president and the president issues a Presidential Disaster Declaration (JTF-CS, 2018).

8.3.5 Defense CBRN Response Force (DCRF) The DCRF consists of two scalable force packages of approximately 5200 active duty and reserve service members from all DOD services. The DCRF is a mission assumed by existing active duty forces and is not a permanent resource of troops and equipment. DCRF elements must be ready to deploy within 24 hours to support local, state, tribal, and federal agencies in the event of a large-scale CBRN incident. Each DCRF is comprised of various types of military units from across the country, is focused solely on domestic response, and receives command and control from JTF-CS (JTF-CS Public Affairs Staff, 2018). DCRF-designated units must undergo specialized CBRN training and successfully complete field training exercises, emergency deployment readiness exercises (EDRE), and validation exercises. Units may spend up to 9 months preparing for the DCRF mission and maintain the role for a year before returning to their typical military roles (Dolasinski, 2018). The various DCRF elements are organized, trained, and equipped to provide: CBRN hazard detection, identification, and assessments; command and control of subordinate DOD forces; logistical and transportation functions; and engineering, medical, search and rescue, and decontamination operations (GAO, 2016). These functions would be integrated with existing WMD-CST, CERFP, and HRF activities.

8.3.6 Command and Control CBRN Response Element A and B (C2CRE) Each C2CRE consists of approximately 1500 service members from the Active Duty and Army Reserves (C2CRE-A) and Army National Guard (C2CRE-B). C2CREs can respond to an all-hazards incident within 96 hours of notification and are not restricted to CBRN incidents. C2CREs would not be the first federal forces to respond to an incident: they could either reinforce the Defense CBRN Response Force (DCRF) or even respond to a separate incident. The C2CREs are comprised of a headquarters element (TF-51), five task forces (operations, aviation, sustainment, special troops, and medical), and an Initial Response Force (IRF). The IRF specializes in decontamination, technical search and extraction, and medical triage, which enables the C2CRE to immediately project life-saving capabilities (Wilcox, 2012).

8.4 Local, Regional, and Federal Response Capabilities 8.4.1 Interagency Operations WMD incidents will inevitably involve responders from local, state, and federal organizations. These interagency operations present the potential for massive challenges in communication,

Chemical Warfare Agents coordination, and command authority. During a WMD chemical agent response, the challenging questions are likely to include • • • •

Who is the incident commander? Is this an ongoing attack? Are we evacuating anyone in the affected area? Are the available responders trained to operate in personal protective equipment? • Do they have access to that equipment?

Knowing the roles and skills of all parties in advance can reduce confusion in a real incident, whether it’s a terrorist attack or an accident (FBI News, 2014). WMD response exercises that involve local first responders, specialized HazMat teams (including National Guard WMD-CSTs), and federal assets (FBI units and military CRE elements) are common ways by which the multitude of response units are able to determine who will assume responsibility for different aspects of the response as well as how the response will be commanded and controlled. Since each response will pose unique challenges, the leadership, experience, and creativity of WMD responders are needed to supplement the NIMS framework and apply ICS principles.

8.4.2 HazMat Teams The first responders to a chemical incident are typically law enforcement, fire, or emergency medical services personnel. Once a chemical hazard is identified, local HazMat teams may be called on to respond and mitigate the chemical threat. HazMat teams are a typed resource under FEMA designation. Highlights of each typed HazMat entry team include • Type 3 HazMat entry team: Presumptive testing and identification of chemical substances; evidence collection of known industrial chemicals; liquid splash-protective personal protective equipment (PPE) • Type 2 HazMat entry team: Presumptive testing and identification of unknown industrial chemicals; evidence collection of known industrial chemicals; vaporprotective PPE • Type 1 HazMat entry team: Presumptive testing, identification, and evidence collection of known or suspected WMD substances and unknown industrial chemicals; vapor-protective PPE (FEMA, 2005) HazMat teams possess specialized training, PPE, and hazardous material identification equipment (air monitoring and point/ standoff detection), which allow them to perform a certain set of tasks during a WMD incident response. HazMat teams generally adhere to the guidance of National Fire Protection Association (NFPA) 472: Competence of responders to HazMat/WMD Incidents. NFPA 472 identifies the minimum competencies required for HazMat/WMD responders and is necessary for a risk-based response. The U.S. Occupational Safety and Health Administration (OSHA) provides guidance to public and private entities that are responsible for hazmat emergency and

U.S. CBRN Homeland Response and Civil Support post-emergency response operations, through 29 CFR 1910.120: Hazardous Waste Operations and Emergency Response. The U.S. Environmental Protection Agency (EPA) also provides comparable regulations through 40 CFR 311. It is important to recognize that although originally promulgated to improve hazmat safety, they are also applicable to incidents involving WMD threats and agents. Both the OSHA 1910.120 and EPA 40 CFR Part 311 regulations provide specific requirements for the skills and competencies required for personnel who will respond to or work at hazardous materials incidents. (Noll, 2008)

Local firefighters and HazMat responders are likely to be the first to encounter hazards of a WMD chemical incident. For this reason, it is imperative that these personnel conduct awareness training and hazard mitigation for WMD incidents. Additionally, local responders should work closely with regional HazMat teams when available and become familiar with the National Guard WMD-CST in their state. WMD-CSTs are required to conduct training and exercises with civilian responders and are experienced in ICS operations and integrating with the first responder community.

8.4.3 FBI WMD Directorate The FBI’s WMD Directorate (WMDD) began on July 26, 2006. Its purpose is to ensure that proper policies, protocols, and resources are established and used to minimize and mitigate hazards related to a WMD incident. The WMDD proactively seeks out and relies on intelligence to drive preparedness, countermeasures, and investigations designed to keep WMD threats from becoming reality. This works in conjunction with the FBI’s need to produce statutory intelligence, investigations, and crisis response obligations (FBI, 2016). During a known or suspected WMD incident response, the FBI assumes jurisdiction and will oversee all response, sampling, and mitigation operations. During a WMD response, military resources, such as the CRE, would serve a subordinate role to accomplish incident objectives set forth by the FBI.

REFERENCES Andreas, P. (2003). Redrawing the line: Borders and security in the twenty-first century. International Security, 28(2), 78–111. https://doi.org/10.1162/016228803322761973. Bush, G. W. (2001). Remarks by the President to the Troops and Personnel. Retrieved February 12, 2018, from ht t ps://georgewbush-wh itehouse.a rch ives.gov/news/ releases/20010213-1.html. Cahill, J. (2005). Tokyo, Graniteville, and Innocent Children. Retrieved February 12, 2018, from www.domesticpreparedness. com/healthcare/tokyo--granitville--and-innocent-children/. Clinton, B. (1995). U.S. Policy on Counterterrorism PDD/NSC39. National Security Council and National Security Council Records Management Office. Retrieved December 20, 2017, from https://clinton.presidentiallibraries.us/items/show/12755. Crimes and Criminal Procedure, 18 U.S.C. §2332a (2011). Retrieved from www.gpo.gov/fdsys/pkg/USCODE-2011-title18/pdf/ USCODE-2011-title18-partI-chap113B-sec2332a.pdf.

127 Defense Against Weapons of Mass Destruction Act of 1996, 50 USC § 2301 (1996). Retrieved December 19, 2017, from www.congress.gov/104/bills/hr3730/BILLS-104hr3730ih.pdf. Defense Response to CBRN Incidents in the Homeland, CJCSI 3125.01D (2015). Dolasinski, A. (2018). Fort Bragg soldiers are training for a nuclear nightmare. Retrieved March 3, 2018, from www.fayobserver.com/ news/20180228/fort-bragg-soldiers-are-training-for-nuclearnightmare. FBI News (2014). FBI Worst-Case Exercise Tests Response to Chemical Attack. Retrieved January 1, 2017, from www.fbi. gov/news/stories/fbi-worst-case-exercise-tests-response-tochemical-attack. FBI (2016). Identifying the Vulnerabilities. Retrieved January 1, 2017, from www.fbi.gov/news/stories/weapons-of-mass-destruction -directorate-marks-10-years. Federal Emergency Management Agency (2005). Typed Resource Definitions (FEMA 508-8). Retrieved from www.fema.gov/ nims/mutual_aid.shtm. FEMA. (2005). FEMA 508-4 Typed Resource Definitions: Fire and Hazardous Materials Resources. Retrieved December 30, 2017, from www.fema.gov/pdf/emergency/nims/fire_haz_ mat.pdf. GAO (2016). Defense Civil Support: GAO-16-599. Retrieved November 12, 2017, from www.gao.gov/assets/680/678054. pdf. JTF-CS. (2018). About JTF-CS. Retrieved March 3, 2018, from www.jtfcs.northcom.mil/About/. JTF-CS Public Affairs Staff (2018). JTF-CS Conducts Crisis Response Exercise. Retrieved March 3, 2018, from www. jtfcs.nor thcom.m il / Media / News/ News-A r ticle-View/ Article/1424970/jtf-cs-conducts-crisis-response-exercise/. Management of Domestic Incidents, HSPD-5 (2003). Noll, G. G. (2008). NFPA 472: Developing a Competency-Based Hazmat/WMD Emergency Responder Training Program. Retrieved March 13, 2018, from www.fireengineering.com/ articles/print/volume-161/issue-4/features/nfpa-472-developing-a-competency-based-hazmat-wmd-emergency-respondertraining-program.html. Stafford, R. T. (1974). Disaster Relief and Emergency Assistance Act, 48 U.S.C. § 5121 et seq. Retrieved from http://uscode. house.gov/view.xhtml?path=/prelim@title42/chapter68& edition=prelim. Terrorism Incident Law Enforcement and Investigation Annex (2004). In National Response Plan. Retrieved from www. fema.gov/media-library-data/20130726-1825-25045-5502/ terrorism_incident_law_enforcement___investigation_ annex_2004.pdf. Tucker, J. B. (2007). War of Nerves: Chemical Warfare from World War I to al-Qaeda. New York: Anchor Books. U.S. Army Posture Statement (2009). Retrieved March 1, 2018, from www.army.mil/aps/09/information_papers/national_guard_ chemical_biological.html. U.S. Army TRADOC ARCIC (2009). TRADOC Pam 525-7-19: CCP for CWMD. Fort Monroe, VA. Retrieved from http:// adminpubs.tradoc.army.mil/pamphlets/TP525-7-19.pdf. Wilcox, D. L. (2012). Army North’s Task Force 51: Ready When It Counts. Retrieved March 3, 2018, from http://smallwarsjournal. com/blog/army-north’s-task-force-51-ready-when-it-counts.

Section II

Agent Effects

9 Mustard Vesicants Rama Malaviya, Diane E. Heck, Robert P. Casillas, Jeffrey D. Laskin, and Debra L. Laskin CONTENTS 9.1 Background................................................................................................................................................................................. 131 9.2 Mustards...................................................................................................................................................................................... 131 9.2.1 Physical and Chemical Properties of Mustards............................................................................................................. 131 9.2.2 Toxicity of Mustards....................................................................................................................................................... 132 9.2.3 Mechanisms of Mustard Toxicity................................................................................................................................... 132 9.2.3.1 Cytotoxicity and Cell Death........................................................................................................................... 133 9.2.3.2 Inflammatory Cells and Mediators................................................................................................................. 133 9.2.3.3 Oxidative Stress..............................................................................................................................................134 9.3 Effects of Mustards on the Respiratory Tract.............................................................................................................................134 9.3.1 Human Exposure............................................................................................................................................................134 9.3.1.1 Acute Effects................................................................................................................................................... 135 9.3.1.2 Long-Term Effects.......................................................................................................................................... 135 9.3.2 Animal Studies............................................................................................................................................................... 135 9.4 Potential Targets for Therapeutic Intervention...........................................................................................................................136 9.5 Conclusions................................................................................................................................................................................. 137 Acknowledgments................................................................................................................................................................................. 137 References............................................................................................................................................................................................. 137

9.1 Background Chemical warfare agents, readily produced from commonly available reagents, have been used in combat since World War I. The fatalities and casualties caused by these agents are a source of constant psychological terror. Extensive production and stockpiling by different countries has also proven hazardous to civilians due to accidental exposures (Dacre and Goldman, 1996; Davis and Aspera, 2001; Weibrecht et  al., 2012). Although the Chemical Weapons Convention (CWC), an arms control treaty that bans the production, stockpiling, and use of chemical weapons, has mandated the demilitarization of current stockpiles, chemical warfare agents are being used in isolated incidents and remain a threat. Vesicants, including sulfur mustard, are cytotoxic blistering agents that cause chemical burns when in contact with the body (Graef et  al., 1948). Chemical casualties and incapacitating injuries to the lung, skin, and eyes during World War I were mainly due to sulfur mustard (Smith et  al., 1995). Since then, there have been several incidents of mustard use, with the most recent being the Iran–Iraq war in the 1980s and Syria in attacks on insurgents and civilians in 2017 and 2018. The severity of injuries varies depending on the route of exposure, proximity to the source of mustard, and environmental conditions (Graham and Schoneboom, 2013). Both acute and long-term toxicity have been noted following exposure to the mustards. This chapter

summarizes the toxic effects of mustard exposure on the lung, with a focus on mechanisms of injury and disease pathogenesis, and potential therapeutics.

9.2 Mustards Sulfur mustard (2, 2¢-dichloroethyl sulfide) and nitrogen mustard (bis (2-chloroethyl) methylamine) are bifunctional alkylating agents. Although never used in combat, nitrogen mustard was developed for this purpose during World War II. It is mainly used for clinical applications as a cancer chemotherapeutic agent. However, as nitrogen mustard is closely related chemically to sulfur mustard and is potent in causing debilitating injury to target organs; it has been classified as a high-priority chemical threat agent (Anslow et al., 1947; Graef et al., 1948).

9.2.1 Physical and Chemical Properties of Mustards Mustards are stable, pale yellow to dark brown, oily liquids with a mustard or garlicky odor. They readily mix with organic solvents but are poorly soluble (0.06–0.07% at 10–20°C) in water (Dacre and Goldman, 1996; Kehe et  al., 2008). In aqueous or alkaline solutions, nucleophilic substitution of chlorine atoms by hydroxyl groups results in the formation of thiodiglycol (TDG)

131

132

Chemical Warfare Agents

and thiodiglycol sulfoxide. Due to its easy absorption into food, porous surfaces, paints, and rubber objects, coupled with its poor volatility (900 mg/m3 at 25 °C), sulfur mustard liquid tends to persist, contaminating surfaces for extended periods (Dacre and Goldman, 1996; Rosenblatt et  al., 1975). Moreover, mustard vapors are much denser than air and therefore, stick to the ground, saturating the terrain for long durations. In this regard, snow samples from regions in Norway were found to be contaminated even 2 weeks after mustard exposure (Johnsen and Blanch, 1984). In warmer climates, however, mustard can vaporize, saturating the air.

9.2.2 Toxicity of Mustards LD50 values for sulfur mustard and nitrogen mustard administered by different routes are presented in Table 9.1. It appears that in animals, the LD50 values are influenced by the solvent used to dissolve mustard (Anslow et al., 1947, 1948; Dacre and Goldman, 1996; Graef et al., 1948). Among the species tested, the lowest LD50 values have been observed in rats. Mustards cause debilitating injury to the respiratory tract, eyes, and skin at low to moderate doses but are lethal at high doses (Anslow et al., 1947; Saladi et al., 2006; Smith et al., 1995). Systemic toxicity is also observed as a consequence of absorption into the circulation and distribution throughout the body (Dacre and Goldman, 1996). Depending on the dose and route of exposure, symptoms of injury may in some cases be delayed (Graef et  al., 1948). Acute exposure to mustards is associated with salivation, lacrimation, respiratory-, gastric- and cardiac distress, and blistering of skin (Aasted et al., 1985; Anslow et al., 1948; Dacre and Goldman, 1996; Kehe et al., 2009). Neurologic disturbances including agitation, confusion, and ataxia have also

been noted, along with conjunctivitis, headache, nausea, vomiting, diarrhea, and cachexia (Dacre and Goldman, 1996; Graef et  al., 1948; Kehe et  al., 2009). Injury to hematopoietic tissues results in leukopenia and immunodepletion, which in some cases leads to death due to infections (Alexander, 1947; Beattie and Howells, 1954; Kehe et al., 2009; Philips, 1950). Studies on the clearance of mustard from the body suggest relatively slow excretion. In humans, non-hydrolyzed sulfur mustard (extracted and derivatized) has been detected in brain, fat, and skin, particularly in subcutaneous fat, even several days after exposure (Drasch et al., 1987; Kehe et al., 2008). The mustard metabolites thiodiglycol (TDG) and 1,1′-sulfonylbis [2-(methylthio)] ethane have been reported to persist in urine up to 10 days postexposure (Graham and Schoneboom, 2013). Additionally, blood protein adducts, although decreased in concentration over time, were detected after 40 days. Levels of TDG and protein adducts in blister fluid were 20 ng/ml and 68 pg/mg, respectively, 2 days postexposure, and these remained unchanged for up to 6 days (Graham and Schoneboom, 2013). Sulfur mustard itself has not been identified in blister fluid (Hurst et al., 2008).

9.2.3 Mechanisms of Mustard Toxicity Due to its lipophilic nature, sulfur mustard rapidly penetrate tissues and cells. The initial reaction involves intramolecular cyclization to form an electrophilic ethylene sulfonium intermediate that can bind to a large number of biological molecules, including sulfhydryl, carboxyl, and aliphatic amino groups as well as heterocyclic nitrogen atoms (Giuliani et  al., 1994; Kehe et  al., 2008; Shakarjian et  al., 2010; Smith et  al., 1995, 1998). This results in alkylation and cross-linking of nucleic acids, proteins, lipids, and other membrane components (Papirmeister et  al.,

TABLE 9.1 LD50 of Mustards in Various Animal Species Route

Inhalation

Intravenous

Cutaneous

Subcutaneous

mg-min/m3

mg/kg

mg/kg

mg/kg

Mouse

860–1380

8.6a

92

26a

Rat

800–1512

0.7a 3.3b

18

3.2–9.0a 5.2b 8.5c

Rabbit

1025

2.7a 3.6–4.8b

100

Monkey Human

800 1500

Oral

References

mg/kg

Sulfur Mustard

17

Anslow et al. (1948), Dacre and Goldman (1996), Watson and Griffin (1992) Anslow et al. (1948), Dacre and Goldman (1996), Watson and Griffin (1992) Anslow et al. (1948), Dacre and Goldman (1996), Graef et al. (1948), Watson and Griffin (1992)

100

0.7

Watson and Griffin (1992) U.S. Army Soldier and Biological Chemical Command: Material Safety Data Sheet, Mustard Gas. Available at www.castleviewuk.com. Accessed 5 August 2016; Sidell et al. (1997), Watson and Griffin (1992)

Nitrogen Mustard Mouse Rat Rabbit Monkey Human

2d 1.1d 1.6d

29–35 14–22 12–15 50 ~75

Solvents used: aPropylene glycol; bneat; c95% alcohol; dsaline.

2.6 1.9

10–20 10

Anslow et al. (1948), Graef et al. (1948) Anslow et al. (1948), Graef et al. (1948) Anslow et al. (1948), Graef et al. (1948) Graef et al. (1948) Balali-Mood and Hefazi (2005)

133

Mustard Vesicants 1991; Shakarjian et al., 2010). Since sulfur mustard is a bifunctional alkylating agent, it can form monofunctional, as well as intra- and intermolecular cross-links. Thus, DNA alkylation at the N7-position of guanine or the N3-position of adenine results in monofunctional adducts, whereas alkylation on two adjacent N7-positions of guanines on the same or on opposite strands of DNA, leads to the formation of intra- and intermolecular bifunctional adducts, respectively (Brookes and Lawley, 1961; Fidder et al., 1994; Kehe and Szinicz, 2005; Ludlum et al., 1994; Mol et  al., 1993; Papirmeister et  al., 1985). In mammalian cells, approximately 65% and 17% of the DNA alkylation products are monofunctional adducts on the N7-position of guanine and the N3-position of adenine, respectively, and approximately 17% are N7-position guanine bifunctional cross-links (Shakarjian et al., 2010). About 25% of the DNA cross-links are between complementary strands, whereas the rest are intra-strand (Walker, 1971). Evidence suggests that mustard-induced DNA cross-linking is not solely responsible for DNA breaks (Papirmeister et  al., 1985). In fact, the monofunctional alkylation of purines also renders DNA sensitive to endonucleases, which induce DNA breaks. DNA breaks and adduct formation are associated with the activation of ataxia telangiectasia–mutated (ATM) and ataxia telangiectasia–related (ATR) protein kinases (Hurley and Bunz, 2007; Jowsey et al., 2009). These protein kinases phosphorylate a wide range of target proteins involved in transcriptional regulation, cell cycle progression, and DNA repair (Dillman et  al., 2005; Rosenthal et al., 1998). Specifically, p53 responsive genes including cyclin G1 and MDM2 are upregulated after sulfur mustard exposure along with bax, EphA2, IEX1/IER3, genes regulating apoptosis, and the transcription factor, ATF3. Increases in metallothionein and Snk following mustard exposure may contribute to cell injury and mitosis (Burns et  al., 2003; Dillman et al., 2005; Fan and Cherian, 2002).

9.2.3.1 Cytotoxicity and Cell Death Cutaneous vesication is associated with epidermal edema and basal cell degenerative changes, including cell death (Joseph et al., 2011; Mol et al., 2009; Monteiro-Riviere and Inman, 1997; Petrali and Oglesby-Megee, 1997; Smith et al., 1998). The epithelial layers of the cornea and lung are also sensitive to mustard and demonstrate morphological and biochemical changes congruent with apoptosis and necrosis (McNutt et al., 2012a, 2012b; Mishra et  al., 2012; Pohl et  al., 2009; Ray et  al., 2008, 2010). Exposure of keratinocytes to sulfur mustard has been reported to induce p53 expression and caspase-3 activation, whereas levels of bcl-2, a suppressor of apoptosis, decrease (Rosenthal et al., 1998, 2000). Increased p53 and decreased bcl-2 expression has also been observed in the epidermis of mustard-induced cutaneous lesions in weanling pigs (Smith et al., 1997). Both death receptor (caspase-8) and mitochondrial (caspase-9) pathways of apoptosis are activated in keratinocytes following mustard exposure, leading to caspase-3 activation (Joseph et  al., 2011; Mol et  al., 2009; Rosenthal et  al., 2003). Pretreatment of epidermal cells with specific caspase inhibitors, or expression of dominant-negative Fas-associated death domain (FADD), suppresses mustardinduced markers of apoptosis. Human alveolar and bronchial epithelial cells also undergo apoptosis after exposure to sulfur or nitrogen mustard (Emmler et al., 2007; Lindsay and Hambrook,

1997; Pohl et  al., 2009; Sourdeval et  al., 2006; Steinritz et  al., 2007). The presence of apoptotic cells in the pulmonary epithelia and increased expression of apoptosis-related genes have also been described in rodents exposed to sulfur mustard (Dillman et  al., 2005; Malaviya et  al., 2010, 2015a; Mishra et  al., 2012). This is associated with caspase-3, caspase-8, and caspase-9 activation, suggesting the importance of the death receptor and mitochondrial pathways in mediating mustard-induced apoptosis (Malaviya et al., 2010; Ray et al., 2008, 2010). As observed in keratinocytes, apoptosis in pulmonary epithelial cells is also associated with increases in p53 and FADD, bax activation, bcl-2 downregulation, and cytochrome c release, which contribute to the activation of caspases (Jowsey and Blain, 2014; Malaviya et al., 2010; Pohl et al., 2009; Sourdeval et al., 2006). The observation that these effects are prominent in bronchial epithelium that has detached from the basement membrane suggests cell detachment–dependent apoptosis or anoikis, a process typically noted in the skin (Malaviya et al., 2010; Sourdeval et al., 2006). Poly (ADP-ribose) polymerase (PARP)-1, a 113 kDa chromatin-bound enzyme, is upregulated following DNA damage and stimulates DNA repair (Debiak et al., 2009; Pleschke et al., 2000). Extensive activation of PARP-1 is associated with cytotoxicity and necrotic cell death (Berger, 1985; Ethier et al., 2012; Rosenthal et al., 2001). PARP-1 is also known to be a target for proteolytic degradation by the pro-apoptotic enzyme caspase-3 (Kaufmann et al., 1993). Thus, the presence of both intact and cleaved PARP-1 in mustard-exposed lungs also suggests evidence of cell apoptosis (Kehe et al., 2009; Malaviya et al., 2010).

9.2.3.2 Inflammatory Cells and Mediators Exposure to mustard is accompanied by the release of vasoactive and chemotactic mediators, which leads to the accumulation of inflammatory cells at sites of injury (Allon et al., 2009; Calvet et al., 1999a; Malaviya et al., 2010, 2012, 2015b; Sunil et al., 2011b; Tsuruta et al., 1996). The majority of these cells are neutrophils and macrophages, suggesting a potential contribution of phagocyte-derived inflammatory mediators in the pathogenic response to vesicants (Amir et al., 2000; Malaviya et  al., 2010, 2015b; McClintock et  al., 2002; Tsuruta et  al., 1996; Venosa et al., 2016; Wormser et al., 2005). The inflammatory cell infiltrate also includes some CD4+ and CD8+ T cells, along with interleukin (IL)-17-producing cells, which mediate delayed-type hypersensitivity reactions and contribute to the development of fibrosis (Imani et  al., 2016; Mishra et  al., 2010, 2012). A number of inflammatory cytokines and chemokines are upregulated in the skin and lungs of humans and animals following exposure to vesicants. These include IL-1, IL-2, IL-4, IL-6, IL-8, IL-13, IL-17, IL-23, CCL-2 (monocyte chemoattractant protein [MCP]-1), CCL3 (macrophage inflammatory protein [MIP]-1α), CCL11 (eotaxin), chemokine (C-X-C motif) ligand 1 (CXCL1) (Gro-α), tumor necrosis factor (TNF)α, interferon (IFN)-γ, transforming growth factor (TGF)β, and connective tissue growth factor (Anderson et al., 2009; Ekstrand-Hammarstrom et al., 2011; Imani et al., 2016; Malaviya et al., 2010, 2015b; Mishra et al., 2010; Mishra et al., 2012; Sabourin et al., 2002; Tsuruta et al., 1996; Wormser et al., 2005). In vitro studies in keratinocytes, small airway, or bronchial/tracheal epithelial cell cultures confirm upregulation of

134 these proinflammatory molecules following mustard stimulation (Arroyo et  al., 2004; Dillman et  al., 2004; Gao et  al., 2007; Jowsey and Blain, 2014; Karacsonyi et  al., 2009; Pohl et al., 2009). Studies in mice lacking genes for cytokines, chemokines, or their receptors, such as TNFR1 or CXCR2, provide evidence for a role of these mediators in mustard-induced injury (Milatovic et al., 2003; Sunil et al., 2011a). Mustard-induced injury is also associated with increased expression of enzymes mediating the production of eicosanoids, including prostaglandins and leukotrienes (Black et  al., 2010; Dacre and Goldman, 1996; Lefkowitz and Smith, 2002; Malaviya et al., 2015a; Venosa et al., 2015). Moreover, loss of cyclooxygenase (COX)-2 or pharmacologic inhibition of cyclooxygenase reduces mustard-induced damage (Babin et  al., 2000; Wormser et al., 2004). Mustard toxicity is also associated with increases in matrix metalloproteinases (MMPs), which degrade extracellular matrix components and promote epithelial cell detachment from the basement membrane, a process contributing to inflammation and tissue remodeling (Sabourin et al., 2002). Increased levels of MMP-9 following mustard exposure are observed in skin cocultures, bronchial epithelial cells, bronchoalveolar lavage, and lung tissue (Anderson et  al., 2009; Calvet et  al., 1999b; Guignabert et  al., 2005; Malaviya et  al., 2010; Ries et  al., 2009). In this regard, treatment of animals with protease inhibitors has been shown to blunt the inflammatory response and damage associated with mustard-induced injury. Several signaling pathways, including ERK1/2, JNK, and p38 MAP kinases, and activating protein (AP)-1, have been identified as mediators of the inflammatory response following mustard injury (Black et al., 2010; Dillman et al., 2004; Mukhopadhyay et al., 2008; Pal et al., 2009).

9.2.3.3 Oxidative Stress Tissue damage induced by mustards is associated with oxidative stress, which is characterized by an imbalance between the generation and detoxification of reactive oxygen and nitrogen species (Laskin et al., 2010b). In vitro and in vivo findings in humans and animals indicate that sulfur mustard and related analogs reduce levels of antioxidants, including glutathione (GSH), superoxide dismutase (SOD), and catalase, which are involved in the detoxification of reactive oxygen species (ROS) (Das et al., 2003; Gould et al., 2009; Kumar et al., 2001; Papirmeister et al., 1991; Pohanka et  al., 2011; Shohrati et  al., 2010; Tewari-Singh et al., 2011). Mustards also modify thioredoxin and thioredoxin reductase in lung epithelial cells, potentially exacerbating oxidative stress and tissue injury (Jan et  al., 2010, 2015). Consistent with this notion, markers of oxidative stress, including malondialdehyde, 8-hydroxydeoxyguanosine, 4-hydroxynonenal, and heme oxygenase (HO)-1, are increased in both skin and lung after exposure of animals to mustards (Kumar et al., 2001; Malaviya et  al., 2015b; Mukherjee et  al., 2009; O’Neill et  al., 2010; Pal et  al., 2009). Antioxidants, including GSH; the GSH prodrug N-acetylcysteine (NAC); Trolox, a water-soluble analog of α-tocopherol (vitamin E); catalase; resveratrol; and quercetin, have been shown to reduce oxidative stress and ameliorate tissue injury induced by sulfur mustard and its analogs (Das et al., 2003; Kumar et al., 2001; McClintock et al., 2002; Shohrati et al., 2014; Tewari-Singh et  al., 2011). Similarly, tocopherols, delivered via liposomes, alone, or in combination with NAC, block

Chemical Warfare Agents mustard-induced inflammatory cell and cytokine accumulation, the generation of collagen, and overall lung injury (Hoesel et al., 2008; Mukhopadhyay et al., 2009; Wigenstam et al., 2009). In humans exposed to sulfur mustard, NAC has also been reported to improve clinical outcomes (Ghanei et al., 2008). Accumulating evidence suggests that reactive nitrogen species are also important in the pathogenic response to mustards. Nitric oxide is generated by inflammatory macrophages largely via an inducible form of the enzyme, nitric oxide synthase (iNOS), which catalyzes oxidation of the guanidino nitrogen atom of arginine (Laskin et  al., 2010a). Once generated, nitric oxide readily reacts with superoxide anion, forming peroxynitrite, a relatively long-lived cytotoxic oxidant. Nitric oxide and peroxynitrite oxidize and covalently modify membrane lipids, thiols, proteins, and DNA, inducing cytotoxicity and perpetuating inflammation (Korkmaz et al., 2006, 2008; Tang and Loke, 2012; Zouki et al., 2001). The expression of iNOS is upregulated in phagocytic leukocytes and epithelial cells following exposure of rodents to mustards (Malaviya et al., 2010, 2012; Nyska et al., 2001; Ucar et  al., 2007; Yaren et  al., 2007). Moreover, transgenic mice with a targeted disruption of the gene for iNOS are protected from mustard-induced pulmonary toxicity, oxidative stress, and altered lung functioning (Sunil et al., 2012). Ebselen, a peroxynitrite scavenger, and melatonin (a potent anti-oxidant that scavenges both reactive oxygen and nitrogen species) have been reported to reduce lung injury and oxidative stress induced by nitrogen mustard in rodents (Ucar et al., 2007; Yaren et al., 2007); melatonin also decreases pulmonary expression of IL-1β and TNFα. Similarly, transient inhibition of iNOS using aminoguanidine attenuates nitrogen mustard–induced oxidative stress and inflammation in rats during the acute phase of lung injury (Malaviya et al., 2012; Yaren et al., 2007).

9.3 Effects of Mustards on the Respiratory Tract Although exposure to mustards generally occurs through multiple routes, most mortality and morbidity are due to inhalation of mustards and pulmonary toxicity (Ghanei et al., 2005; Keyser et  al., 2014; Papirmeister et  al., 1991; Rall and Pechura, 1993; Sohrabpour, 1984). Acute pulmonary edema is thought to cause most of the mortality observed in victims (Freitag et al., 1991). The erosion of exposed mucosal membranes associated with mustard inhalation causes occlusion of airways, leading to severe obstructions and choking (Eisenmenger et al., 1991; Sohrabpour, 1984). Hemorrhagic inflammation of terminal airways compromises local defense mechanisms, rendering the victims susceptible to pathogenic infections and sepsis. Over time, lung injury and inflammation continue to progress, causing alterations in lung function and debilitating respiratory diseases, which bring about significant deterioration in physical activity (Graham and Schoneboom, 2013).

9.3.1 Human Exposure Incidents of war-related, accidental, or occupational exposure to mustard have been reported (Dacre and Goldman, 1996). However, as stated earlier, the severity of exposure is affected by multiple factors, including proximity to the vesicant source,

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Mustard Vesicants duration of exposure, environmental conditions (i.e., temperature, humidity, wind, terrain), and the use of protective gear. Therefore, the symptoms may appear immediately after exposure or may be delayed for hours or days. During the Iran–Iraq war, the higher mortality (14%) in Iranian soldiers when compared with World War I victims (3%) was attributed to higher ambient temperatures in the Middle East and greater vaporization of sulfur mustard (Hurst and Smith, 2008). Higher numbers of casualties in the Bari Harbor incident were due to intoxication of victims by an oily mustard gas slick that coated the surface of the water for several days (Dacre and Goldman, 1996). Occupational exposure to mustard in munitions factories is associated with chronic respiratory symptoms in workers, including chronic bronchitis, lower FEV1/FVC ratio, bronchiectasis, and emphysema, and increased incidence of mortality due to recurrent infections, including influenza and pneumonia (BalaliMood and Hefazi, 2005; Easton et al., 1988; Manning et al., 1981; Nishimoto et al., 1970, 1987; Tang and Loke, 2012).

9.3.1.1 Acute Effects The pulmonary effects of sulfur mustard are dependent on respiration rate and are usually evident after exposure to 100–500 mgminute/m3 (Rosemond et al., 2003). Cough, sneezing, hoarseness, sore throat, mucus discharge from nose and throat, loss of smell and taste, and irritation of the nasal mucosa are some of the early symptoms of mustard inhalation (Graham and Schoneboom, 2013; Kehe et al., 2008, 2009; Momeni and Aminjavaheri, 1994; Sohrabpour, 1984). Exposure to higher doses can result in pulmonary edema, damage to the pharynx leading to an inability to speak, tachypnea, and tachycardia (Kehe and Szinicz, 2005; Momeni et  al., 1992; Sohrabpour, 1984). Bronchoscopy in a soldier exposed to mustard gas revealed severely reddened and swollen tracheobronchial mucosa and injury to terminal airways (Freitag et  al., 1991). More severe mustard inhalation causes necrosis of the respiratory epithelium leading to epithelial sloughing and pseudomembrane formation (Ghabili et  al., 2010; Rancourt et al., 2013). This was associated with collapse of the right lung in one sulfur mustard victim (Kehe et al., 2008; Koch, 1921). Acute lung edema, lung inflammation, and moist rales have been reported within the first few days following exposure to hot liquid containing 2–3% nitrogen mustard (Wang and Xia, 2007). Bronchoscopy in this patient also revealed mucosal edema and partial obstruction of the airway. Expulsion of dead mucus tissue during cough in this patient further confirmed epithelial membrane sloughing and pseudomembrane formation (Wang and Xia, 2007).

9.3.1.2 Long-Term Effects Mustard-induced pulmonary injury is progressive in nature (Aghanouri et al., 2004; Hefazi et al., 2005; Malaviya et al., 2012; Namazi et al., 2009). Shortness of breath and productive cough, commonly observed during acute pulmonary injury, can lead to expectoration of large amounts of purulent mucus several months after exposure (Freitag et al., 1991). In these patients, the bronchial tree shows evidence of pus, and victims suffer antibioticresistant recurrent chronic respiratory infections (Freitag et al., 1991; Momeni and Aminjavaheri, 1994). This is associated with

reduced lung compliance and decreased capacity for oxygenation and carbon monoxide diffusion, leading to severe hypoxemia and hypercapnia (Freitag et  al., 1991). Subsequently, an increased incidence of airway hyperreactivity, chronic bronchitis, bronchiolitis, bronchiectasis, tracheal stenosis, lung fibrosis, and acute respiratory distress syndrome with high mortality is observed (Emad and Rezaian, 1997; Freitag et  al., 1991; Ghanei et  al., 2004; Ghasemi et al., 2013; Saber et al., 2012; Sohrabpour, 1984). At 10 years after a single high-dose sulfur mustard exposure, the diagnoses in victims included chronic bronchitis (59%), asthma (11%), bronchiectasis (9%), large airway narrowing due to scarring (10%), and pulmonary fibrosis (12%) (Emad and Rezaian, 1997; Tang and Loke, 2012; Weinberger et al., 2016). The severity of pulmonary fibrosis in these veterans was correlated significantly with the carbon monoxide diffusion capacity of the lung (Emad and Rezaian, 1997). Upper and lower respiratory tract complications of mustard poisoning 20–25 years after exposure in war veterans include dysphonia (79%), postnasal dripping (42%), lower larynx position (30%), limitation of vocal cords (26%), larynx mucosa inflammation (15%), chronic obstructive respiratory disease (84%), bronchiectasis (44%), and fibrosis (8%) (Balali-Mood et  al., 2011). In accord with these findings, high-resolution lung computed tomography scans in victims 18–23 years after exposure showed significant air trapping and parenchymal attenuation patterns (Idani et  al., 2012). In veterans followed for 20 years after sulfur mustard exposure, lung autopsy also show evidence of chronic bronchitis, bronchiolitis obliterans, fibrosis, infections and tuberculosis, and malignant cellular infiltration (Ghanei et al., 2004; Rowell et al., 2009; Taghaddosinejad et  al., 2011). Among factory workers, a considerably higher incidence of emphysema (56% vs. 21%), bronchiectasis (44% vs. 19%), centrilobular nodules (33% vs. 12%), and bronchial wall thickening (33% vs. 7%) has been noted in individuals directly involved in manufacturing sulfur mustard, compared with non-sulfur mustard–manufacturing workers (Nishimura et al., 2016). Sulfur mustard exposure is also associated with the development of cancer; acceleration in the age range for the onset of lung cancer has also been attributed to mustard exposure (Doi et al., 2011; Hosseini-khalili et al., 2009).

9.3.2 Animal Studies Lung injury and respiratory effects similar to those described in humans have also been observed in experimental animals. Neutrophil infiltration into the lung and replacement of mucosal bronchial epithelium by stratified squamous epithelium has been described in nonhuman primates 2 weeks after mustard exposure (Mishra et al., 2012). In dogs, early signs of mustard inhalation include frequent cough, salivation, nasal discharge, and irregular and labored breathing that is prominent about 8 hours after exposure (Winternitz and Finney, 1920). Autopsy of dogs that died 1–3 days after mustard exposure mustard showed injury to the mucosal epithelium of the trachea and bronchi, resulting in pseudomembranes consisting of necrotic epithelium, cell debris, fibrin, mucus, and leukocytes. Atelectasis was noted, along with marked congestion of the lung. The alveoli showed thickening of septal walls, hemorrhage, and an accumulation of inflammatory cells. Evidence of necrotizing bronchopneumonia was observed in dogs that died between 2 and 10 days following

136 exposure. Dogs exposed to very low levels of sulfur mustard recovered following several days of respiratory stress. In a few of these animals, localized ulceration of trachea, tracheal stenosis, or minute areas of remodeling in the lung were noted 1–5 weeks after exposure (Tang and Loke, 2012; Winternitz and Finney, 1920). Inhalation exposure of rabbits to sulfur mustard also produces nasal irritation, nasal secretion, and mild inflammation of the anterior nares, pharynx, larynx, and trachea (Dacre and Goldman, 1996; Warthin and Weller, 1919). Cough, rales, and laryngeal and tracheal edema along with necrosis of mucosa resulting in diphtheritic membranes were observed at relatively higher concentrations of sulfur mustard. Severe sulfur mustard exposure was associated with edema, emphysema, hemorrhagic atelectasis, and epithelial necroses of smaller bronchioles. However, animals die of the complications secondary to mustard exposure, including purulent bronchopneumonia or diphtheritic or purulent laryngitis (Dacre and Goldman, 1996; Tang and Loke, 2012; Warthin and Weller, 1919). Lung injury has been studied at the cellular and molecular level in rodents using sulfur mustard, nitrogen mustard, or the half mustard 2-chloroethyl ethyl sulfide (CEES). Upper airway changes, including focal attenuation of tracheal epithelium, detachment of epithelium from the mucosa, loss of cilia, and accumulation of fibrin and cell debris in the lumen, have been observed after exposure of rodents to mustards (Anderson et  al., 2009; Calvet et  al., 1994, 1996; Guignabert et  al., 2005; Malaviya et al., 2010, 2012; van Helden et al., 2004). The bronchus also showed evidence of focal ulceration and luminal fibrin plug entrapment of necrotic inflammatory cells and bronchial epithelial cells, which extend to the alveolar regions (Anderson et  al., 2009; Malaviya et  al., 2010, 2012; Veress et  al., 2010). In accord with epithelial cell injury, increased cell and protein content is observed in bronchoalveolar lavage fluid (Allon et al., 2009; Malaviya et  al., 2010, 2012, 2015a; Sunil et  al., 2011b). Mustards induce multifocal hyperplasia in the lung parenchyma, as well as patchy mild thickening of alveolar septa. This is associated with increased numbers of macrophages, neutrophils, and mononuclear cells in the tissue. In addition, perivascular and peribronchial edema, hyperplasia and hypertrophy of goblet cells, blood vessel hemorrhage, fibrin deposits, bronchiolization of alveolar walls, and luminal accumulation of cellular debris have been reported, along with atelectasis and edema of the lung lobes (Anderson et al., 2009; Malaviya et al., 2010, 2012, 2015a). Shortly after mustard exposure, alterations in tidal volume, respiratory frequency, peak inspiratory and expiratory pressures, and airway hyperreactivity are noted; these effects persist for several days (Calvet et al., 1994, 1999a; Sunil et al., 2011b; van Helden et al., 2004; Vijayaraghavan, 1997). Mustard induced lung injury is also associated with increases in reactive oxygen and nitrogen species, eicosanoids, proinflammatory cytokines (TNFα, IL-1, IL-2, IL-6, IFN-γ), profibrotic cytokines and growth factors (TGFβ, IL-13, connective tissue growth factor), and proinflammatory chemokines (IL-8, CCL-2, CCL3, CCL11, and CXCL1) (Anderson et al., 2009; Malaviya et al., 2010, 2012; Mishra et  al., 2012; Tang and Loke, 2012; Venosa et  al., 2016; Wigenstam et al., 2009). Markers of oxidative stress, including malondialdehyde, nitrite-nitrates, superoxide dismutases (SOD), heme oxygenase-1, and iNOS, are upregulated in the lung, while glutathione levels are reduced (Allon et  al., 2009; Das et  al.,

Chemical Warfare Agents 2003; Malaviya et  al., 2016; Shohrati et  al., 2010; Sunil et  al., 2011b; Ucar et al., 2007; Yaren et al., 2007). Increases in MMP-9, gelatinase, caspases, the autophagy marker LC3B, proliferating cell nuclear antigen (PCNA), PARP-1, and phospho-H2A.X have also been reported following pulmonary exposure to mustards (Anderson et  al., 2009; Calvet et  al., 1999b; Guignabert et  al., 2005; Malaviya et  al., 2010; Venosa et  al., 2016). As observed in humans, lung injury is progressive in animals. Thus, 14 days after mustard exposure, the tracheal epithelium in guinea pigs is disorganized with decreased cell density (Calvet et al., 1994). Airway hyperreactivity to substance P and histamine is noted, consistent with the onset of asthma-like symptoms in human victims of sulfur mustard exposure (Calvet et  al., 1994; Emad and Rezaian, 1997; Hefazi et al., 2005). By 28 days post sulfur mustard in rodents, multiple areas of fibrosis containing collagen fibers are observed around airways and bronchioles, along with erythrophagocytosis, fibroplasia, squamous metaplasia of the bronchial wall, and emphysema-like changes in the alveolar tissue (Hoesel et al., 2008; Malaviya et al., 2012; Mukherjee et al., 2009; Venosa et al., 2016). Fibrin and collagen deposition also increase in the lung, leading to a collapse of alveolar structures and the appearance of honeycombing (Malaviya et  al., 2012; Mukherjee et al., 2009). The chronic phase of vesicant-induced respiratory injury (2–4 weeks in rodents) is also characterized by a predominance of enlarged foamy macrophages occluding the alveoli, lymphocytes, and IL-17+ cells, with marked increases in TGFβ and myofibroblast proliferation (Malaviya et al., 2012, 2015b; Mishra et al., 2012; Venosa et al., 2016).

9.4 Potential Targets for Therapeutic Intervention Recent advances in understanding the mechanisms of mustardinduced toxicity have led to the development of several strategies to mitigate its pathogenic effects. These include targeting oxidative and nitrosative stress, proteases, and inflammatory and fibrogenic mediators. Antioxidants including NAC, GSH, tocopherol, and liposomes containing NAC or NAC and tocopherol decrease the production of ROS, upregulate antioxidants, and reduce inflammation associated with vesicant exposure (Hoesel et  al., 2008; Kumar et al., 2001; Mukherjee et al., 2009; Mukhopadhyay et  al., 2009; Panahi et  al., 2017; Razavi et  al., 2013; Shohrati et al., 2008; Tewari-Singh et al., 2011). The peroxynitrite scavenger ebselen, and melatonin, which scavenges both reactive oxygen and reactive nitrogen species, reduce mustard–induced lung injury in rodents (Ucar et al., 2007; Yaren et al., 2007). Transient inhibition of iNOS using aminoguanidine has also been reported to reduce mustard–induced oxidative stress and inflammation (Malaviya et al., 2012; Yaren et al., 2007). Doxycycline, a nonspecific MMP inhibitor, which also downregulates iNOS, and inhibits nitric oxide production, reduces inflammatory cytokine release, and scavenges ROS, has been effective experimentally in reducing mustard-induced injury in skin, eye, and lung (Amin et al., 1996; Guignabert et al., 2005; Raza et al., 2006; Shakarjian et al., 2006, 2010; Tewari-Singh et al., 2012; Wasil et al., 1988; Weinberger et al., 2016). Systemic and topical anti-inflammatory drugs have shown some beneficial effects in alleviating the symptoms of mustard

Mustard Vesicants injury. These include dexamethasone, diclofenac, and curcumin (Panahi et al., 2015; Weinberger et al., 2016; Wigenstam et al., 2009, 2012). Treatment of rodents with anti-TNFα antibody has also been reported to mitigate mustard-induced acute lung injury, as well as fibrogenesis (Malaviya et  al., 2015b, 2018). Some other potential drugs with anti-inflammatory characteristics including pentoxifylline, fluticasone propionate, salmetrol, beclomethasone, silibinin, and valproic acid have also been reported to be effective in alleviating symptoms of mustard injury experimentally (Ghanei et  al., 2007; Sunil et  al., 2014; Tewari-Singh et  al., 2012; Venosa et  al., 2017). Cyclosporin A has been reported to be beneficial in the management of mustard-induced ocular injury (Jadidi et  al., 2014, 2015), and immunosuppressive treatment reportedly improves chronic symptoms of mustard-induced lung injury (Ghanei et al., 2006). Other therapies, including calcium channel blockers, transient receptor potential (TRP) channel inhibitors, tissue plasminogen activator, tissue factor pathway inhibitor (TFPI), and bone marrow-derived mesenchymal stem cells (MSCs), are also under investigation (Rancourt et  al., 2013; Veress et  al., 2015; Weinberger et al., 2016).

9.5 Conclusions Mustards are blistering agents that cause debilitating damage to the respiratory tract, skin, and eyes. In cases of severe exposure, mortality can occur due to secondary infections or airway obstruction. The toxicity of mustards is thought to be mediated in part by the alkylation of nucleic acids and proteins, which leads to cytotoxicity, oxidative stress, injury, and inflammation. Both acute and long-term pathologic consequences have been observed in humans, as well as animal models, following a single mustard exposure. Experimental animal models of mustard-induced lung injury have been helpful in delineating mechanisms of toxicity and in identifying potential therapeutics. Targeting reactive oxygen and nitrogen species, TNFα and proteases are some potentially useful strategies. Further understanding of mechanisms of mustard-induced injury and mechanisms that cells employ to protect against toxicity will help in the development of efficacious clinical strategies to treat acute and chronic manifestations of vesicant injury in humans.

ACKNOWLEDGMENTS This work was supported by National Institute of Health Grants U54AR055073, R01ES004738, and P30ES005022.

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10 Health and Psychological Effects of Low-Level Exposure to Chemical Warfare Nerve Agents Carl D. Smith, Kristin J. Heaton, James A. Romano, Jr., Maurice L. Sipos, and John H. McDonough CONTENTS 10.1 Introduction................................................................................................................................................................................. 145 10.2 Health Effects of Low-Level Exposures in Humans.................................................................................................................. 146 10.2.1 1990–1991 Gulf War and Gulf War Illness.................................................................................................................... 146 10.2.2 Chemical Warfare Nerve Agent Use in Terrorist Attacks............................................................................................. 148 10.2.3 Occupational Exposures................................................................................................................................................. 149 10.2.4 Human Laboratory Experiments....................................................................................................................................150 10.3 Effects of Low-Level CWNA Exposures in Animals................................................................................................................ 151 10.3.1 Acute Exposures in Animals.......................................................................................................................................... 151 10.3.2 Repeated Exposures in Animals.................................................................................................................................... 152 10.3.3 Pesticide Exposures in Animals.....................................................................................................................................154 10.4 Conclusions................................................................................................................................................................................. 155 Disclaimer............................................................................................................................................................................................. 155 References............................................................................................................................................................................................. 155

10.1 Introduction As recently demonstrated by the suspected use of chemical warfare nerve agents (CWNA) in Syria (John et al., 2018) and by the assassination of Kim Jong-nam using VX (Paddock and SangHun, 2017), the potential for attack with CWNAs remains a real threat to both military and civilian populations. As discussed throughout this chapter, moderate-to-high doses of CWNAs can be fatal; however, exposure to lower doses can generate longlasting deleterious effects on physical and psychological health among survivors. Since the previous edition of this chapter, numerous studies have expanded our understanding of these effects. In humans, new reports have emerged that explore the role of CWNAs in Gulf War Illness. In animals, experiments on both the acute and repeated effects of low-level CWNA exposure continue to reveal the consequences of such exposure for physiological function and behavioral performance. CWNAs are highly toxic organophosphorus (OP) compounds that are chemically related to some insecticides (e.g., parathion, malathion). The five most common CWNAs are tabun (o-ethyl N, N-dimethyl phosphoramidocyanidate; military designation = GA), sarin (isopropyl methyl phosphonofluoridate; military designation = GB), soman (pinacolyl methyl phosphono­ fluoridate; military designation = GD), cyclosarin (cyclohexyl methylphosphonofluoridate; military designation = GF), and VX (o-ethyl S-2-N, N-diisopropylaminoethyl methyl phosphono­ fluoridate). These relatively odorless liquids can be weaponized and delivered as aerosols or fine sprays. CWNAs exert their toxic

effects by inhibiting the cholinesterase (ChE) family of enzymes, including acetylcholinesterase (AChE; E.C.3.1.1.7), a critically important central nervous system (CNS) and peripheral nervous system (PNS) enzyme that hydrolyzes the neurotransmitter acetylcholine (ACh). Although CWNAs can inhibit other esterases, their potency and specificity for inhibiting AChE account for their exceptionally high toxicity. For example, the rate constants for AChE inhibition by GA, GB, GD, or VX are two to three orders of magnitude greater than for the more commonly known OP compounds such as diisopropylfluorophosphate (DFP), paraoxon, or methylparaoxon (Gray and Dawson, 1987). Likewise, the rate constants for AChE inhibition by CWNAs are also two to five times greater than for trypsin (E.C.3.4.21.4), chymotrypsin (E.C.3.4.21.1), or carboxylesterase (E.C.3.1.1.1; Maxwell and Doctor, 1992), indicative of selective inhibition of this enzyme. Several excellent references provide discussion of the history, chemistry, physiochemical properties, pharmacology, and toxicology of CWNAs (Koelle, 1963; Marrs et al., 1996; Sidell, 1992; Somani et al., 1992; Taylor, 2001), the pertinent details of which are briefly summarized here. CWNAs bind to the active site of the AChE enzyme and prevent it from hydrolyzing ACh. The enzyme is irreversibly inhibited, and the return of esterase activity depends on the synthesis of new enzyme (approximately 1–3% per day in humans). All CWNAs are highly lipophilic and readily penetrate the CNS. Acetylcholine is the neurotransmitter at the neuromuscular junction of skeletal muscle, the preganglionic nerves of the autonomic nervous system, and the postganglionic parasympathetic nerves, as well as muscarinic

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146 and nicotinic cholinergic synapses within the CNS. Following CWNA exposure and the inhibition of approximately > 40% of the AChE enzyme pool, levels of ACh rapidly increase at the various effector sites, resulting in continuous overstimulation. Cholinergic hyperstimulation in the CNS and PNS following CWNA exposure leads to classic toxic signs of OP poisoning. The signs include miosis (constriction of the pupils), increased tracheobronchial secretions, bronchial constriction, laryngospasms, increased sweating, urinary and fecal incontinence, muscle fasciculations, tremors, convulsions/seizures of CNS origin, and loss of respiratory drive from the CNS. Although less potent than CWNAs, OP compounds provide frequent real-world exposure cohorts due to their common use in household and agricultural settings. For example, numerous reports have detailed the neurobehavioral effects of these OP compounds on chemical and agricultural workers and on laboratory animals. The relative prominence and severity of signs of OP poisoning depend highly on the route and degree of exposure. For example, ocular and respiratory effects occur rapidly and are most prominent following vapor exposure, whereas localized sweating, muscle fasciculations, and gastrointestinal disturbances are most prominent following liquid percutaneous exposures and usually develop in a more protracted fashion. The acute lethal effects of CWNA exposure are generally attributed to respiratory failure caused by a combination of central and peripheral effects that are further complicated by copious secretions, muscle fasciculations, and convulsions. CWNAs are particularly effective due to their toxicity and rapid action, producing catastrophic consequences (Sidell, 1974). In some cases, thousands of tons of these agents have been synthesized for military use. Consequently, it is not surprising that major medical research efforts have focused on developing effective lifesaving medical countermeasures, including therapeutic interventions, pretreatments, and treatment strategies using anticonvulsant drugs, to prevent long-term changes in CNS function following a moderate to severe CWNA intoxication (McCarren and McDonough, 2016). Toxicologists often differentiate chemical exposure levels based on the dose and the duration of exposure. For example, duration of exposure is often categorized as acute, subacute, subchronic, and chronic. In light of today’s interest in “longterm, low-level” exposures to CWNAs, it is useful to clarify these terms. Acute exposure is defined as exposure to a chemical for less than 24 h. When referring to an acute inhalation exposure, the most frequently used exposure duration is 4 h. Subacute exposure refers to an exposure of 1 month or less, subchronic for 1–3 months, and chronic for more than 3 months. With regard to exposure level, definitions of “low-level exposure” appear to range from nonlethal exposure to “subtoxic” (defined by DeMenti, 1999 as no clinical signs) to “subclinical” (defined by DeMenti, 1999 as no clinical signs and no significant depression of ChE). According to these definitions, then, exposure is any contact with a chemical that may induce a biochemical effect. Each definition suffers from arbitrariness, and we see no way around this. For the purpose of this review, we will attempt to characterize the effects of CWNA exposures according to the temporal definitions and clinical severity described here. Furthermore, when pertinent, we will include information about the route of exposure, which can significantly influence the outcome of CWNA exposures.

Chemical Warfare Agents

10.2 Health Effects of Low-Level Exposures in Humans 10.2.1 1990–1991 Gulf War and Gulf War Illness Approximately 697,000 U.S. military service members participated in the Gulf War (GW) from August 1990 to June 1991. Nearly one-third of those service members have reported lingering adverse health and performance-related symptoms in the years following their deployments. Collectively these symptoms are known as Gulf War Illness (GWI) (IOM, 2010; White et al., 2016). Symptoms associated with GWI include cognitive impairments, significant physical and mental fatigue, chronic muscle and joint pain, dermatological issues, gastrointestinal problems, and respiratory difficulties. Due to concerns regarding a high incidence of undiagnosed illness among Operation Desert Shield/Operation Desert Storm veterans, a presidential advisory committee (PAC) evaluated the federal government’s outreach, medical care, research, and coordinating activities pertinent to GWI. As part of their analysis, the PAC examined short- and long-term health effects of known GW risk factors, including exposure to chemical and biological weapons, depleted uranium, infectious diseases, environmental factors, and prophylactic treatments for chemical and biological warfare agents, such as pyridostigmine bromide (PB). Among their conclusions, the PAC urged the Department of Defense (DOD) to support additional research efforts concerning possible long-term health effects of low-level exposures to CWNAs such as GB and GF (Presidential Advisory Committee on Gulf War Veterans’ Illnesses, 1996). In March 1991, U.S. forces, deployed in support of Operation Desert Storm, destroyed an Iraqi munitions storage complex at Khamisiyah, Iraq. In 1996, the DOD released reports to the public indicating that the site contained stockpiled CWNAs (including GB and GF) (Directorate for Deployment Health, 1997). During Operation Desert Shield/Desert Storm, the U.S. military used area sampling devices and chemical agent sensors to monitor air quality and to alert U.S. service members to possible chemical weapons attacks. However, the presence and/or extent of exposure to chemical agents at the individual level is unknown. Thus, in June 1996, at the request of a PAC and the National Security Council, the Central Intelligence Agency initiated an effort to model potential chemical warfare agent (CWA) release events during the GW, including detonations of specific bunkers and ammunitions pits located in Khamisiyah, al Muthanna, and Muhammidiyat (Presidential Advisory Committee on Gulf War Veterans’ Illness, 1996). The exposure modeling effort detailed by Bullman et  al. (2005) and Gackstetter et al. (2006) is summarized briefly here. Initial simulations used meteorological data and estimates of atmospheric transport and diffusion across a 4 day period in March 1991 to predict the direction and extent of chemical warfare agent release in the area surrounding these sites. Simulations identified two levels of possible exposure. The first exposure area included a plume wherein exposure levels (nonlethal) were equal to or greater than 1 mg min/m3. At this dose, one would expect individuals to exhibit “first noticeable effects” of AChE inhibition (e.g., miosis, rhinorrhea) at the time of exposure. The second exposure area included a plume wherein exposure levels were

Health and Psychological Effects of Low-Level Nerve Agent Exposure considered a “low-level hazard” (subtoxic) equal to or greater than the general population limit (GPL) of 0.01296 mg min/m3 as defined by the U.S. Army and Centers for Disease Control and Prevention (CDC, 1988). The modeled plume areas were superimposed onto a map of the Khamisiyah region containing the known locations of U.S. military units to identify individual service members who might have been exposed to the plumes. No military units were identified as having been located in the modeled area of “first noticeable effect” (Directorate for Deployment Health, April 2002; Hauschild, 1999). Identification of units located within the estimated “low-level hazard” area was first published on the Internet in 1997 (Directorate for Deployment Health, April 2002). Subsequently, members of those units were sent notification letters from the Office of the Special Assistant for Gulf War Illnesses at DOD. As a result, the Office of the Special Assistant for Gulf War Illnesses notified nearly 98,910 U.S. service members who had served in the region near Khamisiyah of their potential exposure to CWAs. The model was revised in 2000 using updated meteorological information, GB and GF toxicity and exposure data, updated munitions data, and CWA removal practices (Assistant Secretary of Defense, 2002). Based on the revised models, the DOD identified an additional 2942 military persons and notified them of their potential exposure (Directorate for Deployment, April 2002). In 2004, the United States General Accounting Office (GAO, 2004) reviewed the modeling results and concluded that neither simulation could reliably predict which, if any, units were exposed to CWNA. Furthermore, they concluded that neither simulation could reliably determine whether destruction of the Iraqi munitions storage complex had resulted in nerve agent release. While the precise etiology of GWI remains unclear, a variety of chemical hazards present in the theater, including the pervasive use of OP pesticides and possible exposures to low levels of GB and GF, have been identified as potential contributing factors. Although theater medical records provided no clinical evidence of GB/GF exposure at the time of the demolition activity (Riddle et al., 2003), findings from animal exposure studies (described in Section 10.3) and research involving survivors of the Japan GB attacks (described in Section 10.2.2) support the idea that low-level exposure to OPs can have long-term adverse effects on health and performance. While Khamisiyah represents one of the most frequently cited sources of potential CWA exposures among GW veterans, other exposures may have occurred during the GW, including the potential release of mustard gas during demolition activities (Haley and Tuite, 2013; Office of the Special Assistant for Gulf War Illnesses, 1996). Several articles reviewing the health of GW veterans have focused specifically on the effects of toxicants on the CNS (RACGWI, 2014; White et al., 2016). Given limited exposure-related data from the field, much of the research exploring potential links between low-level GB/ GF exposure and GWI has used binary exposure categories (“exposed” versus “not exposed”) or symptom reporting to characterize presumptive GB/GF exposures. Such categorization is based on either exposure notification status (e.g., a notification letter from the DOD) or subjective self-report (e.g., reported frequency of hearing chemical alarms in theater). In some investigations, subtle neurocognitive deficits (e.g., in short-term memory,

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episodic memory, and attention) have been observed (Cooper et al., 2016; Haley et al., 1997; Odegard et al., 2013; White et al., 2001). Epidemiological studies also have found that GW veterans who were potentially exposed to CWNAs had greater risk of hospitalization due to circulatory diseases (Smith et  al., 2003), a higher incidence of brain cancer deaths (Barth et  al., 2009; Bullman et al., 2005), and significantly higher rates of repeated seizures, neuralgia/neuritis, and stroke (Kang et  al., 2009). In contrast, other studies reported no differences in self-reported medical diagnoses and health symptoms (McCauley et al., 2002) or hospitalizations (Gray et  al., 1999; McCauley et  al., 2002) between “exposed” and “unexposed” GW veterans. Studies examining possible neuroanatomical correlates of observed behavioral symptoms associated with low-level GB/ GF exposure have used a wide range of brain imaging and neurophysiological approaches. For example, proton magnetic resonance spectroscopy (MRS) studies have noted significantly lower N-acetylaspartate (NAA) to creatine (Cr) ratios in the basal ganglia and brainstem (Haley et  al., 2000) and in the hippocampus (Menon et al., 2004) of GW veterans reporting adverse health symptoms compared with healthy volunteers. These differences potentially reflect impaired neuronal function in these brain regions. However, visual inspection of brain scans revealed insignificant differences between symptomatic GW veterans and healthy controls based on structural magnetic resonance imaging (MRI) and no evidence of abnormality on single photon emission computed tomography scans (Haley et al., 1997). It is important to note that similar findings have been seen in proton MRS studies involving patients with neurologic disorders, where a reduction in brain NAA was observed in the absence of abnormalities using other imaging modalities (Lu et al., 1996). In contrast, Weiner and colleagues (2011) found no significant differences in NAA and NAA metabolites in the pons and basal ganglia between symptomatic and asymptomatic GW veterans. Nevertheless, evidence of subtle but persistent CNS impairment following low-level GB/GF exposure suggests that neuroanatomical correlates of such exposures might also be subtle and not readily apparent by simple visual inspection of brain scans. Despite lingering concerns regarding the accuracy of the Khamisiyah plume models, a number of researchers have used GB/GF estimates generated by these models to explore the relationship between presumptive CWNA exposure levels and the health status of GW veterans. One such study reported significant decrements in manual dexterity and visuospatial functions associated with higher estimated levels of potential GB/GF exposure (Proctor et al., 2006). In another study, veterans potentially “exposed” to GB/GF did not differ significantly in volumetric measurements of discrete brain tissues compared with their “unexposed” counterparts (Heaton et  al., 2007). In contrast, a linear trend analysis based on the Khamisiyah plume models revealed a significant association between higher levels of estimated GB/GF exposure and reduced white matter. Moreover, higher estimated GB/GF exposure levels were associated with increased right and left lateral ventricle volumes, suggesting subtle but persistent CNS pathology in GW veterans potentially exposed to low levels of GB/GF. Similarly, Chao and colleagues reported a reduction in total brain gray matter volume, white matter volume, total hippocampal volume, and hippocampal subfield volume, and white matter microstructural changes (Chao et al.,

148 2010, 2011, 2014, 2015) as measured by diffusion tensor imaging in “exposed” GW veterans compared with matched, “not exposed” GW veterans based on the Khamisiyah plume model. In their 2010 study, Chao and colleagues reported that observed reduced white matter volume was associated with decreased performance on tests of executive and visuospatial functions. In contrast, Chao and colleagues saw no differences in performance using a different sample of “exposed” and “not exposed” GW veterans in their 2011 report. Finally, an inverse association was noted between GW veterans’ self-reports of how frequently they heard chemical alarms sound during their deployments and total cortical gray matter after controlling for demographics, health status, and other concurrent deployment-related exposures associated with hearing chemical alarms (Chao et  al., 2016). Regional brain volumes, however, were not significantly associated with estimated GB/GF exposure levels based on the predicted Khamisiyah plume or with GWI status.

10.2.2 Chemical Warfare Nerve Agent Use in Terrorist Attacks CWNAs have been used against civilians in a number of cases in recent history. Arguably, the most extensively documented and studied of these cases include the 1994 and 1995 Matsumoto and Tokyo GB attacks in Japan. Approximately 600 people in the vicinity of the Matsumoto district court and 5500 in the Tokyo subway were exposed in these attacks, leaving seven and 12 dead, respectively (for review, see Yanagisawa et al., 2006). Survivors present in the vicinity of the attack reported a range of symptoms from severe (seizures, respiratory distress, darkened/ blurred vision) to mild (miosis, rhinorrhea) to no observable symptoms. As defined in Yanagisawa et  al. (2006), numerous individuals exposed to GB were described as being “slightly affected,” such that they reported symptoms but did not seek medical treatment. The most common symptoms among this group included runny nose, headaches, shortness of breath, and coughing. All reported symptoms appear to have resolved within 3 weeks of the attack. This cohort may be particularly relevant to the immediate and long-term consequences of an acute “low-level” CWNA exposure. Depending on the location, survivors were monitored up to 10 years following the attacks (Yanagisawa et al., 2006). Several studies reported on the neuropsychological effects of GB exposure at approximately 6–8 months following the attack (Murata et  al., 1997; Yokoyama et  al., 1998). Unfortunately, these studies had small sample sizes (n = 18) and only included participants with apparent symptoms that qualified as greater than “slightly affected.” Indeed, all participants required hospitalization, and all but three victims had plasma ChE values below normal values on the day of exposure. Nevertheless, several interesting differences emerged between GB-exposed individuals and healthy controls. GB-exposed individuals scored significantly lower on a digit symbol substitution test and significantly higher on measures of psychiatric symptoms, fatigue, and posttraumatic stress disorder (PTSD) (Yokoyama et  al., 1998). Individuals reporting elevated psychiatric symptoms and fatigue scores also reported increased PTSD symptomatology, but these symptoms were not associated with the degree of ChE inhibition on the day of exposure. Sex differences

Chemical Warfare Agents also emerged, such that GB-exposed females exhibited greater postural sway than males. Finally, the GB-exposed group had significantly longer P300 latencies on event-related brain evoked potentials during an auditory test and longer P100 latencies on brain visual evoked potentials, suggesting that GB produced lasting effects on neural systems regulating cognitive performance and visual function (Yokoyama et al., 1998). Longer-term monitoring of survivors following the attacks suggests lingering effects of the exposure regardless of medical care provided at the time of incident. For example, 1 year following the Matsumoto attacks, surveys of survivors indicated lingering eye strain, fatigue, blurred vision, and weakness, among other symptoms. Fatigue and general weakness was more commonly reported by individuals requiring hospitalization following the attacks compared with those who qualified as “slightly affected.” In addition, significantly lower red blood cell (RBC) AChE activity was correlated with the presence of eye strain and fatigue among respondents referred to a medical provider (Nakajima et al., 1999). One severely affected Matsumoto victim experiencing prolonged seizure activity shortly after the attack continued to exhibit electroencephalographic (EEG) and electrocardiogram abnormalities after 1 year (Sekijima et  al., 1995). At 3 years, some victims still complained of experiencing these symptoms, although with reduced severity and frequency (Nakajima et  al., 1999). Studies of rescue workers and police officers responding to the Tokyo GB attacks up to 7 years later show that they exhibited persistent, significant, dose-dependent changes in psychomotor function (reduced tapping frequency, dominant hand) and memory (e.g., shorter backward digit span) in comparison with a reference group (Miyaki et  al., 2005; Nishiwaki et al., 2001). In addition to the neuropsychological changes attributable to GB exposure alone, many of the Japanese victims were also diagnosed with PTSD (Kawana et  al., 2001; Yanagisawa et  al., 2006). Matsuo et  al. (2003) reported that Tokyo subway GB attack victims with diagnosed PTSD showed significantly greater activation of the prefrontal cortex as assessed by nearinfrared spectroscopy and skin conductance response to stimuli directly related to the exposure incident than did victims without PTSD or controls. Furthermore, Yamasue et al. (2003) reported that victims of the GB attack with PTSD showed a significant reduction in grey matter volume of the left cingulate cortex as assessed by MRI compared with victims of the attack who did not develop PTSD. A similar reduction in anterior cingulate cortex volume has been reported in combat veterans with PTSD (Woodward et al., 2006). It has been hypothesized that the anterior cingulate cortex may become hypofunctional as a consequence of repeated glucocorticoid activation during the exaggerated fear responses of PTSD victims and over time, reduce in volume (Nutt and Malizia, 2004; Shin et al., 2006; Villarreal and King, 2001). Araki et al. (2005) demonstrated that PTSD victims had significantly greater reductions in P300 amplitude than did victims with no PTSD diagnosis. Moreover, within the group of victims diagnosed with PTSD, there was a positive correlation between P300 amplitude and anterior cingulated cortex grey matter density. Some of the neurologic changes seen in GB victims could be confounded with changes in brain function often seen in severe cases of PTSD, thus complicating determinations of longer-term health effects of GB exposures.

Health and Psychological Effects of Low-Level Nerve Agent Exposure

10.2.3 Occupational Exposures Despite examples of CWNAs used in warfare and terrorist attacks, the number of scientific investigations elucidating the physical and neurobehavioral consequences among survivors exposed to low levels of these agents remains relatively limited. Insight into these consequences may be complemented by the extensive literature on OP pesticide exposures in civilian populations. Although not as potent as CWNAs, OP pesticides are used worldwide for numerous agricultural applications and account for millions of chemical poisonings each year (Karalliedde and Senanayake, 1989). Studies examining the effects of OP exposures on human physiology and behavior, as described in the following, generally assess agricultural workers. Neuropsychological assessments have included (but are not limited to) measures of attention, reaction time, memory, and grammatical/mathematical reasoning as well as behavioral measures such as anxiety, depression, and anger. Since many of the studies discussed are historical reports and/or involve workers using a mixture of pesticides, determining exactly which compounds were involved is difficult. Most studies describing occupational OP exposures involve workers assessed during an exposure period or shortly thereafter. One early account reported that pesticide workers with relatively high occupational levels of OP pesticide exposure tended to have increased EEG power within the beta frequencies, primarily in frontal areas of the brain, compared with workers with low occupational levels of OP pesticide exposure. Further, these changes in EEG were associated with poorer performance on a neuropsychological test battery (Korsak and Sato, 1977). Subsequent experiments with sheep dippers (Stephens et  al., 1995), greenhouse workers (Bazylewicz-Walczak et  al., 1999; Roldan-Tapia et al., 2005), cotton farmers (Farahat et al., 2003), and other agricultural workers (Rothlein et al., 2006) revealed numerous deficits in neuropsychological performance and behavioral measures in OP-exposed workers compared with controls. These studies consistently report degradations in measures requiring attentional resources, and those including behavioral assessments (Bazylewicz-Walczak et  al., 1999) indicated elevated levels of reported anxiety, anger, depression, and fatigue. These trends extend to child and adolescent populations as well. For example, Egyptian children and adolescents applying OP pesticides in farming communities performed worse on a battery of neuropsychological tests (Abdel Rasoul et  al., 2008; Rohlman et  al., 2016), as did Hispanic children living in agricultural communities (Lizardi et al., 2008), than controls. Whether these OP pesticide exposures affect younger cohorts to a greater extent than adults is not clear. One report suggested that higher levels of pesticide exposure had more deleterious neuropsychological effects on younger (~10 years old) compared with older participants, although the sample size in this study was small (Eckerman et al., 2007). A larger study examining a similar population of farmers found that cumulative years of exposure, but not age, correlated with poorer performance (Rohlman et  al., 2007). Although these studies support the conclusion that OP pesticides at least transiently degrade neuropsychological performance, not all reports show a significant effect between OP-exposed individuals and controls (Ames et al., 1995; Daniell et al., 1992; Maizlish et  al., 1987; Rodnitsky, 1975). For example, recently,

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chemical workers routinely exposed to chlorpyrifos did not have impaired neurobehavioral performance despite higher urinary levels of chlorpyrifos metabolites and significantly decreased plasma BuChE (Berent et al., 2014). In addition, studies investigating OP exposures among American famers (Starks et  al., 2012) and Thai children living in farming communities (Fiedler et al., 2015) also did not detect differences in neuropsychological performance compared with control cohorts. Numerous studies suggest that OP pesticide exposures can result in longer-term health and performance outcomes following a delay between the exposure and testing. Individuals experiencing acute OP pesticide poisoning performed worse 9 years later on various neuropsychological tests and mood assessments despite matching controls on neurological and blood chemistry exams (Savage et  al., 1988). These results are congruent with other reports of degraded performance following an OP poisoning at 2 years (Rosenstock et al., 1991; Wesseling et al., 2002) and up to 12 years after the event (Steenland et al., 1994), although a third cohort reporting impairment at 7 weeks postexposure did not differ from controls at 2 years (Delgado et  al., 2004). Wesseling et al. (2002) reported that banana workers poisoned by OP compounds exhibited a more robust decrement in neuropsychological performance compared with workers poisoned by n-methyl carbamate, suggesting that OP compounds have more toxic long-term consequences than other pesticides. It is important to note that individuals in these studies experienced an acute OP exposure severe enough to warrant an examination from a physician and/or hospitalization. Relatively few studies have examined longer-term postexposure neuropsychological outcomes in healthy workers handling OP compounds. The deleterious effects of OP pesticide exposure are not limited to civilian occupational exposures. Independently of the CWNA incident described in Section 10.2.1, soldiers working in Iraq during the GW were exposed to OP pesticides. Indeed, results from a DOD pesticide health risk assessment conducted from 1997 to 1998 and summarized in the Environmental Exposure Report—Pesticides (EER) suggest that nearly 41,000 GW veterans were likely overexposed to OP and carbamate pesticides (Winkenwerder, 2003). These were individuals enlisted in positions that required pesticide application; however, the report also noted that some military personnel not involved in pesticide application were also exposed to OP compounds at levels exceeding concern for toxicity based on Environmental Protection Agency guidelines at that time. Within the general military population, specific sources of concern for exposure included fly baits, pest strips, sprayed liquids, and powders. Specific sources among military pesticide applicators included pesticide fogs and prisoner delousing. Of the 12 pesticides identified in the report, eight were OP or carbamate pesticides. Carbamates, such as OPs, inhibit AChE. A significant source of carbamate exposure during the GW was PB, administered in pill form before or during deployment, which was used as a prophylactic against possible CWA attack (Cook et al., 2002; Golomb, 2008; Sullivan et al., 2003; White et al., 2001). Research suggests that the use of PB in GW veterans is associated with degraded cognitive performance, specifically in the area of executive system functioning and with meeting criteria for GWI in particular groups of GW veterans (Steele et al., 2012; Sullivan et al., 2003). Sullivan et al. (2018) conducted an epidemiological study involving 159 GW-deployed

150 preventative medicine personnel with varying levels of pesticide and PB exposure to evaluate the relationship between pesticide and PB exposures and neurocognitive test performance. Results indicated that veterans with both high pesticide and high PB exposure performed significantly worse on neurocognitive assessment (slower information processing speed, greater errors in attention, worse visual memory performance, and more mood complaints) than those with high levels of either pesticide or PB exposure or low exposures to both pesticide and PB. Moreover, a stepwise regression analysis indicated that exposure to pest strips was associated with slower reaction times and attentional errors, while exposure to fly bait and delousing agents was predictive of elevated mood complaints. Although the majority of occupational exposures involve OP pesticides, a small number of reports describe accidental CWNA exposures in occupational settings. Duffy et al. (1979) performed an analysis of EEGs of manufacturer workers exposed to GB at doses that produced clinical signs and symptoms of exposure and reduced RBC AChE at least 25% below the individual’s preexposure baseline. The exposed group included a maximally exposed subgroup of workers who had experienced three or more such exposures previously. When studied at least 1 year following exposure, the exposed group (the maximally exposed subgroup in particular) displayed elevated amounts of spectral energy in high-frequency beta activity. Visual inspection of the EEGs showed decreased amounts of alpha (9–12 Hz) activity along with increased amounts of slow activity (0–8 Hz, delta and theta) and an increased amount of “nonspecific” abnormalities in the EEG background. Finally, the exposed workers displayed increased amounts of rapid eye movement sleep. Although the functional consequences of these EEG changes were not established, this group reportedly had a high incidence of self-reported memory disturbances and difficulty maintaining alertness and appropriate focusing of attention (Metcalf and Holmes, 1969). These findings are consistent with another published summary of 209 acute poisonings by GB, GD, or VX in Russian nerve agent production facilities (Yanno and Musiychuk, 1997). Twentyeight percent of the victims required hospitalization ranging from a few days to a few months, exhibiting long-term symptoms including memory loss, weakness, sleep disorders, and seizure activity. They noted that the CNS symptoms were most prominent and persistent following relatively high-dose accidental GD poisoning, confirming observations made by Sidell (1974) and Lekov et al. (1966). Although symptomatic exposures to CWNA in these occupational settings reveal long-term health effects, an extensive overview of these exposures suggests that asymptomatic exposures likely do not (Marrs et  al., 1996; Sidell, 1996). This is the same conclusion reached by the National Academy of Sciences, which stated that while there may be subtle long-term EEG changes, the clinical significance and functional relevance of such changes had not been demonstrated (National Academy of Sciences, 1982). In summary, studies of occupational pesticide and CWNA exposures provide valuable insights into the effects of lower-level OP exposures on acute and longer-term health and performance outcomes. Studies of pesticide workers indicate an increased prevalence of neurologic symptoms and changes in neurobehavioral performance indicative of mild cognitive and psychomotor dysfunction. These effects appear to be more prevalent during the

Chemical Warfare Agents period of exposure but may also persist over time. Total cumulative exposure may be a greater risk factor for poor performance than any other covariant. Studies have shown fairly consistent decrements in visuomotor speed, as indexed by the digit symbol substitution test and trail making tests, and tests of selective attention and memory (digit span). Many of these same neurobehavioral functions were also reported to be long-term effects of OP pesticide poisoning (Delgado et al., 2004; Rosenstock et al., 1991;Savage et al., 1988; Steenland et al., 1994; Wesseling et al., 2002).

10.2.4 Human Laboratory Experiments In the early 1980s, the Committee on Toxicology, National Academy of Sciences conducted a review of chronic health effects of low-level exposure to CWNAs. This review examined available reports from the soldier-volunteer test program of the Army Chemical Center, Aberdeen Proving Ground (formerly Edgewood Arsenal, MD; National Academy of Sciences, 1982). Soldier-volunteers participated in this test program from 1958 to 1975, during which time 15 anticholinesterases (anti-ChEs) were tested on approximately 1400 subjects, with the majority of testing occurring in the 1950s and 1960s. The National Academy of Sciences found that mortality data compiled in 1981 did not indicate significant differences in mortality rates among soldiervolunteers when compared with soldiers outside the testing program. There was no clear indication of long-lasting CNS effects and no evidence for mutagenicity, carcinogenicity, male reproductive dysfunction, or cataractogenic effects. These findings remained the prevailing scientific opinion regarding longer-term health risks of CWA exposures until the appearance of GWI in the early years following the 1990–1991 Gulf War. Similarly, the United Kingdom’s Ministry of Defence (2006) published a comprehensive review of the exposure to military CWAs in the United Kingdom. Porton Down, the U.K. chemical test center, had maintained a human volunteer program since World War I and conducted numerous human CWNA exposure tests beginning in the late 1940s. Sidell (1996) provides an excellent summary of both U.S. and U.K. reports. The Ministry of Defence (2006) report described three follow-up studies performed to determine whether long-term health effects resulted from voluntary exposure to GB (Technical Notes, 1972, 1973, 1989). All three studies concluded that there was no evidence that exposure to low doses of CWNAs produced adverse health effects. In addition, there were no statistical differences between the number of hospital admissions, out-patient appointments, and incidents of reporting sick or days lost through illness before and after exposure to GB. Nor were there any differences on these measures between those exposed to GB and unexposed control groups of volunteers. Likewise, there was no evidence of long-term psychiatric symptoms or in the type of illnesses that the exposed versus control groups experienced. In another laboratory study of OP exposure involving human volunteers, Grob et al. (1947) described the effects of acute to subacute exposure to the OP pesticide DFP (1–2 mg, intramuscularly [i.m.], daily for up to 7 days) on EEG and psychological parameters. DFP increased EEG potential and frequency, and produced more irregularities in rhythm. The CNS symptoms included excessive dreaming, insomnia, jitteriness and

Health and Psychological Effects of Low-Level Nerve Agent Exposure restlessness, increased tension, emotional lability, subjective tremulousness, nightmares, headache, increased libido, giddiness, drowsiness, paresthesias, mental confusion, and tremor. The EEG changes usually followed the onset of CNS symptoms, both of which were correlated with the depression of RBC ChE to 70% and 60% of original activity, respectively. CNS symptoms disappeared within 1–4 days after exposure, while the EEG changes gradually abated from 8 to 42 days following exposure (average 29 days). Holmes and Gaon (1956) described similar CNS symptoms and EEG changes in workers following acute OP pesticide exposure. They also noted that the more severely exposed individuals or those with multiple exposures tended to display persistent symptoms that included forgetfulness, irritability, and confused thinking, although the duration of these persistent symptoms was never clearly defined. The CNS symptoms and EEG changes described are virtually identical to those reported following exposure to different CWNAs. For example, Grob and Harvey (1953, 1958) noted behavioral and EEG effects in humans following GB exposure that were virtually identical to those reported for DFP. These effects coincided with depressed plasma and RBC ChE activity to approximately 60% and 50% of baseline, respectively, following a single intravenous (i.v.) dose, or 34% and 22% of baseline activity, respectively, following oral administration. For GB, the route of administration clearly affects the rate of ChE inhibition, the rate of ACh increase in the CNS, and the development of symptoms of exposure. In another study, Bowers et al. (1964) evaluated the behavioral and psychological effects of VX exposure in humans. Psychological/behavioral effects were typically evident before the occurrence of physical symptoms. These effects were associated with whole blood ChE inhibitions of > 60%. The acute toxic effects following high-dose exposure (>LD50) to GD (Lekov et  al., 1966; Sidell, 1974), GB (Inoue, 1995; Nakajima et  al., 1998; Sidell, 1974), and VX (Nozaki et al., 1995) in humans are similar to behavioral symptoms following lower CWNA doses (anxiety, psychomotor depression, intellectual impairment, and sleep disturbances). These symptoms appear to dominate the clinical picture in the immediate period following resolution of the acute toxic signs of intoxication and then slowly fade with time, sometimes taking months to fully resolve. Although OP pesticides are comparable substitutes for CWNAs, extrapolation from these exposures to predictions of effects from CWNA should be interpreted with caution. Several phenomena appear to differentiate CWNA exposure from OP pesticide exposure. First, the latency to cholinergic crisis following acute, severe OP pesticide intoxication is generally much longer than for CWNA intoxication (Sidell, 1997). Second, many OP pesticides produce delayed peripheral neuropathy, whereas CWNAs appear to produce polyneuropathy in animals at doses greater than the LD50 —a phenomenon typically seen in the presence of massive pretreatment and therapy with atropine and oxime (Davis et al., 1960). Finally, the “intermediate syndrome,” which involves the onset of muscle paralysis affecting particularly upper-limb muscles, neck flexors, and cranial nerves 24–96 hours after OP or carbamate poisoning (Bird et al., 2016; Vale and Lotti, 2015), has not been described after CWNA exposure in animals or humans (Sidell, 1997).

151

10.3 Effects of Low-Level CWNA Exposures in Animals 10.3.1 Acute Exposures in Animals A number of investigators have studied the potential long-term consequences resulting from acute symptomatic exposure to OP compounds in animals. Early studies established the neurological effects of a single high dose of GB (5 mg/kg, i.v.) on the EEG of rhesus monkeys (Burchfiel et  al., 1976). At this dose, GB produced generalized seizure activity on the EEG that lasted an average of 2.5 h. Twenty-four hours following GB exposure, there was a significant increase in the relative voltage in the beta frequency bands (13–22 Hz = beta-1; 22–50 Hz = beta-2) in the occipital–temporal EEG lead while the animals were awake in darkness that persisted for a year. Functional behavioral tests of other rhesus monkeys exposed to GB under identical conditions revealed no deficits in performance of a previously learned delayed response test 24 h after the exposure (Lattal et  al., 1971). Later experiments reinforced the concept that seizures are a prominent sign of CWNA intoxication following highdose exposure (approximately 0.6 LD50 and higher), and that prolonged seizures can produce both neural and cardiovascular lesions if not promptly treated (McDonough and Shih, 1997). These include behavioral degradations including acquisition in a variety of operant tasks, performance in a serial probe recognition task, acquisition and performance in a variety of maze tasks, and passive avoidance learning (Castro et al., 1991; McDonough and Shih, 1997; McDonough et  al., 1986; Modrow and Jaax, 1989; Raffaele et al., 1987; Raveh et al., 2002, 2003). Invariably, animals with behavioral deficits also had brain lesions in cortical and subcortical limbic structures. Phillippens et al. (1992) clearly demonstrated the relationships between neuropathology and behavioral deficits in a comprehensive study that revealed EEG and performance changes following near-LD50 challenge of GD. In the study, rats previously trained on an active avoidance paradigm were exposed to an LD50 of GD immediately followed by treatment with a low-dose antidotal combination of atropine and diazepam. After recovering motor capacity, the animals were retested and approached baseline performance levels after three test sessions. The researchers observed electrographic correlates of lesions ultimately verified using light microscopy to support a neuroanatomic basis for behavioral deficit. In contrast, animals challenged with an LD50 dose of GD immediately followed by a high-dose combination of atropine and diazepam performed at or near baseline levels on the active avoidance test. The animals were partially protected from electrographic and neuropathologic changes. These findings are similar to most “high-dose” CWNA exposure studies: animals exposed to seizure-inducing CWNA without prompt anticonvulsant treatment develop brain damage and consequent neurobehavioral problems. In contrast, animals that do not develop seizures or whose seizures are treated rapidly and effectively do not suffer brain lesions or concomitant long-term neurobehavioral deficits. In contrast to higher doses of CWNAs that produce clinical signs, Burchfiel et  al. (1976) suggested that a “clinically signfree” dose of GB (1 mg/kg, i.m.) given once per week for 10

152 weeks in nonhuman primates resulted in subtle, but persistent, EEG changes similar to those described following an acute, seizure-inducing dose of GB (5 mg/kg, i.v.). Similarly, Pearce et  al. (1999) tracked behavioral and electrographic changes in marmosets for up to 15 months following a single low-dose exposure to GB (2.5–3 mg/kg, i.m.). At this dose, GB caused 36–67% inhibition of RBC AChE but did not cause acute behavioral signs of intoxication or performance decrements on a series of discrimination tasks immediately following or over a 12 to 15 month period following the exposure. Moreover, there were no significant long-term changes in EEG patterns seen in this study. In contrast, van Helden et al. (2004) reported that vapor exposures to GB at a dose of 0.2 mg/min/m3 were sufficient to modify EEG activity in marmoset monkeys, which is at least an order of magnitude lower than the previously established dose required to produce miosis. Visual examination of the EEG records at 1 year showed that GB-exposed animals had a greater number of bursts within the alpha frequency that resembled sleep spindles than did the controls, suggesting persistent effects of the exposure. Long-lasting electrical activity is not the only neural marker that changes following a single, sublethal exposure to CWNA. Long-term changes are also observed in monoamine activity, as a single exposure to 0.4 LD50 of GB in mice reduced dopamine turnover in the amygdala at 4 weeks and in the frontal cortex at 8 weeks postexposure. Serotonin concentrations were also decreased in the amygdala but were increased in the frontal cortex and caudate nucleus (Oswal et al., 2013). Thus, one sublethal exposure to a CWNA may affect the brain by altering gene expression, monoamine activity, and electrical activity. Genovese and colleagues examined potential long-term behavioral effects in nonhuman primates and rodents following acute exposure to several CWNAs. For example, rhesus and African green monkeys were trained to perform a serial probe recognition task, after which they were exposed to 0.25 LD50 (i.m.) of GB (Genovese et  al., 2007b). The animals were tested for up to 3 months following exposure and showed no decrements or changes in performance despite significant reductions in blood ChE. More recently, Genovese et al. (2011) found that the lowest single percutaneous dose of VX in monkeys that produced observable adverse effects did not alter performance on the serial probe recognition task, whereas higher doses produced transient deficits when animals were treated with CWNA antidotes. In two other studies, rats trained to perform on a food-motivated variable interval 56 s operant task were exposed to varying doses of GF (Genovese et al., 2006) or VX (Genovese et al., 2007a) using a whole-body inhalation exposure system. The rats were tested repeatedly for up to 3 months on the VI 56 task and on a radial arm maze to test spatial memory. GF and VX produced transient, mild CWNA intoxication and transient decreases in VI 56 performance, while only VX affected behavior in the radial arm maze. None of the asymptomatic doses degraded behavioral performance, and no dose produced lasting effects. These findings are largely consistent with 0.53 LC50 of GD (Genovese, 2009a) and GB (Genovese, 2009b), where single inhalational exposures of either agent did not disrupt performance on the operant task or in the radial arm maze (although extending GB exposures to three consecutive days produced transient decrements in operant performance; Genovese et al., 2009b). The authors concluded that doses of CWNA producing acute mild signs of intoxication

Chemical Warfare Agents can produce transient disruptions in performance on a welllearned operant task, but these effects quickly dissipate without long-term effects. They also concluded that doses not eliciting symptoms also did not produce behavioral effects. Acute exposures have physiological effects that extend beyond the CNS. In an extensive series of studies, Kassa and colleagues reported changes in immune function, biochemical parameters, protein and nucleic acid metabolism, and various aspects of neurobehavioral function following single or repeated exposure to GB or GD in mice and rats (Kassa et al., 2003, 2004a, 2004b). For example, they found that rats exposed to a single inhalation exposure of GB (from 0.8 to 2.5 µg/l/60 min) had increased levels of stress markers, decreased DNA synthesis, increased CNS excitability, and impaired spatial discrimination that persisted as long as 3 months after exposure (Kassa et  al., 2004c). The highest dose (2.5 µg/l) produced only mild clinical signs, while lower doses did not. Henderson et al. (2001, 2002) found that rats exposed to single or multiple subclinical doses of GB vapor had decreased immune function that typically recovered over weeks following exposure. Pretreatment of the rats with chlorisondamine, a nicotinic antagonist and ganglionic blocker, attenuated the effects of GB on immune response, implicating the autonomic nervous system in these GB-induced changes (Kalra et  al., 2002). They also reported that there were reductions in serum adrenal corticotrophic hormone and corticosterone levels that were still evident 4 weeks after exposure (Pena-Philippides et al., 2007). Many of GB’s effects on the immunological system may be due to the central nicotinic effects of ACh stimulation as demonstrated by similar changes in immunological function following chronic nicotine exposure (Langley et al., 2004; RazaniBoroujerd et al., 2004).

10.3.2 Repeated Exposures in Animals An original report from Sterri et al. (1980) suggested that animals can tolerate repeated low levels of CWNAs with minimal overt neurobehavioral effects. Blood and brain AChE levels, for example, can be inhibited to  1) and Haber’s rule is shown in Figure 11.3. Extrapolation to other XX% levels is done using Equation 11.2 with TL being substituted for CT and substituting a probit slope (mTL) with respect to TL in place of mC. ten Berge’s findings suggesting that the toxicity of gases was not constant over time had a significant impact on the U.S. Department of Defense’s (DOD) reexamination of nerve agent toxicity estimates in the late 1990s. Investigators (Anthony et al., 2003; Benton et al., 2005, 2006; Dabisch et al., 2008; Hulet et al., 2006a,b, 2007, 2014; Mioduszewski et  al., 2002a, b; Whalley

Haber’s rule (n = 1) Toxic load model (n > 1)

time)

b

Durations (min)

Dose (concentration

a

Cyclosarin Soman VX Sarin Cyclosarin VX Sarin Cyclosarin Soman VX Sarin Cyclosarin Sarin Cyclosarin VX Cyclosarin

Probit Slope

10

1 1

10

100

Exposure time FIGURE 11.3  Comparison of Haber’s rule and toxic load model. If Haber’s rule applies (n = 1), then the dose required to produce a given response is constant relative to exposure time. However, for a toxic load model with n > 1, the dose required to produce a given response increases as the exposure time increases. For a toxic load model with n 0.90 0.892

10 to 240 10 to 180 10 to 180 10 to 180

Mioduszewski et al. (2002a) Anthony et al. (2003) Dabisch et al. (unpublished)a Benton et al. (2006) Hulet et al. (2006a, 2014) Hulet et al. (2014) Hulet et al. (2007)

Ratio calculated for present work based on unpublished lethality and severe effects data collected by Dabisch et al. (2008).

Task Force, 1997). The plume model was cross referenced with known/suspected troop locations over the days following the detonation, leading to a determination that >100,000 U.S. service members had potentially been exposed. In 2000, a revised plume model was developed, which incorporated better estimates of the total number of GB/GF rockets, improved meteorological data, and updated troop locations. Based on medical records of the potentially exposed veterans and available field reports from the exposure area, there was no indication of evident acute clinical signs of nerve agent exposure among the troops at the time of the exposure. However, several follow-up studies have suggested that there are long-term clinical ramifications. Potentially exposed veterans showed an increased risk of brain cancer–related deaths (Bullman et  al., 2005) as well as decreased efficiency on neurobehavioral tasks associated with psychomotor dexterity and visuospatial abilities. Heaton and colleagues (2007) compared magnetic resonance images of brains from 13 potentially exposed veterans and 13 unexposed veterans. A significant association was identified between estimated nerve agent exposure levels and reduced white matter volumes and increased lateral ventricle volumes. Similarly, magnetic resonance imaging (MRI) studies on a cohort of 38 survivors from the 1995 Tokyo subway sarin attack identified a reduction in brain volume in the hippocampus, the insular cortex, and the neighboring white matter (Yamasue et al., 2007) as compared with matched healthy control subjects. Over a series of studies, Chao and her colleagues have identified a number of changes in brain structure and function in cohorts of Gulf War veterans presumably exposed to the Khamisiyah GB/ GF plume as compared with cohorts of matched controls. A group of 40 suspected GB/GF-exposed veterans showed reduced volumes of total gray matter and hippocampus (Chao et al., 2010) as compared with their counterparts. However, no deficits on neurobehavioral measures of attention, memory, visuospatial ability, or dexterity were identified in these veterans. A follow-up study (Chao et al., 2011) using more powerful 4 T MRI and data processing software analyzed images from a separate cohort of 56 suspected GB/GF exposed veterans and identified a decrease in both total gray and white matter volumes. However, the study did not find any evidence of psychomotor slowing in this cohort. A 2014 study (Chao et al., 2014) both replicated the findings from 2010 of reduced hippocampal volume and localized the hippocampal changes to specific subfields. In 2015, Chao and her colleagues

again used powerful MRI imaging and processing techniques to investigate the hypothesis that suspected GB/GF exposed veterans would have evidence of a disruption of the integrity of white matter microstructures as compared with unexposed veterans. The ultimate risk to a soldier for exposure on a battlefield would come from the intentional use of weaponized agents during military operations. Therefore, it is important for operational planning purposes to extrapolate lethality studies performed in animal models to potential human effects at varying toxic endpoints, including lethality and mild, moderate, and severe effects. Most of the studies cited in this text have been used via a meta-analysis of all available data to generate human estimates of toxicity. The values currently accepted by the U.S. Army for the classical nerve agents are listed in Table 11.6 (U.S. Army Field Manual, 2005). As previously noted, the toxicity values from Field Manual 3-11.9 (2005) were developed using experimental data generated by the DOD Low-Level Toxicology Program collected up until the time the Field Manual was published. Subsequently, additional revisions were recommended (values in brackets in Table 11.6) using experimental data collected post 2005. The most important revisions involved several observations from a statistical analysis of the database: • There are no statistically significant differences in the ratios of ECT50 (severe) to LCT50 among the nerve agents. So, all recommended ratios were standardized at 0.80, and minor adjustments were made in previous human estimates for ECT50 (severe) and LCT50 in order to achieve this ratio. • There are no statistically significant differences in the probit slopes for lethality/severe effects among the nerve agents. So, all recommended slopes were standardized at 12. • There are no statistically significant differences in the probit slopes and the toxic load exponents for mild effects among the nerve agents. So, all recommended slopes and toxic load exponents were standardized at 4.5 and 1.4, respectively. It should be emphasized that the toxicity estimates in Table 11.6 are only intended for healthy adult human males (70 kg), since

173

Inhalation Toxicology of Chemical Agents TABLE 11.6 Inhalation Toxicity Estimates for Healthy Humans Exposed to CW Agents from Field Manual 3-11.9 (2005) Nerve Agents

LCT50 (at 2 min) ECT50 (severe effects)a (at 2 min) Probit slope (base 10)b TLEb ECT50 (mild effects)c (at 2 min) Probit slope (base 10) TLE

Tabun (GA)

Sarin (GB)

Soman (GD)

Cyclosarin (GF)

VX

70 [63] 50

35 [33] 25

35 [33] 25

35 [41] 25 [31]

15 [12] 10 [9]

12

12

12

12

6 [12]

1.5 0.40

1.5 0.40

1.25 [1.5] 0.20

1.25 0.20 [0.40]

1 0.10 [0.04]

10 [4.5]

10 [4.5]

10 [4.5]

10 [4.5]

4 [4.5]

1.5 [1.4]

1.5 [1.4]

1.4

1.4

1 [1.4]

Note: CTs are in units of milligrams per minute per cubic meter. The values are for 70 kg healthy males (with a minute volume of 15 liters/min) acutely exposed over the whole body with no protection (i.e. respiratory, percutaneous, or ocular). Values in brackets are updated estimates that were developed at ECBC using toxicity research and modeling performed post publication of FM 3-11.9 (ReutterChristy, S. and Sommerville, DR, Human Toxicity Estimates for Nerve Agents and Recommendations for Revised Human Toxicity Estimates, U.S. Army ECBC, Aberdeen Proving Ground, MD, unpublished work, June 2008). a Severe effects include convulsions, gasping, collapse, or prostration. b Probit slope and TLE are the same for both lethal and severe effects. c Mild effects include miosis, rhinorrhea, lacrimation, and salivation.

the primary focus for DOD research has been to protect military personnel in a CW agent battlefield environment. However, the general civilian population can also be exposed to CW agents due to deliberate (i.e., terrorism) and/or accidental exposures. Such exposures are of particular concern due to the possibility of a CW agent release from the U.S. domestic unitary CW agent stockpile (NRC, 2003). In response to this problem, the NRC developed acute exposure guideline level (AEGL) values for the nerve agents and mustard in 2003. There are three levels of AEGL values: AEGL-1, AEGL-2, and AEGL-3, which roughly correspond to vapor concentrations that will produce in the general population (including susceptible individuals) mild (reversible) effects, severe (long-lasting) effects or impaired ability to escape, and death, respectively. AEGL values are roughly equal to or less than the 1% effect level for the general population. Early results from the DOD Low-Level Program were used in the development of the nerve agent AEGLs (Anthony et al., 2003, 2004; Mioduszewski et al., 2002a, b). However, the AEGLs are not appropriate for casualty assessment purposes (unlike the values in Table 11.6). In a series of three technical reports (Crosier, 2003, 2007; Crosier and Sommerville, 2002), ECBC has developed a mathematical method for the conversion of toxicity estimates from one population basis to another, with Crosier (2007) presenting the final version of the method. The premise of the approach is that mathematically, the approximately normal distribution of log (doses) for a healthy subpopulation is located completely within the distribution formed by the general population (i.e., the smaller bell curve is inside the larger bell curve). This model has been used for modeling non–nerve agent CW agent inhalation lethality by Sommerville (2016) and Sommerville et al. (2009,

2016). Using the Crosier Model, the values in Table 11.6 were converted from a healthy subpopulation to a general population basis—see Table 11.7.

11.9 Inhalation Toxicology of World War I–Era Chemical Agents Of all of the World War I war gases, chlorine, phosgene, and mustard are still considered viable CW agents despite their lethality being considerably less than that of the more modern nerve agents. Lethality is not the sole factor in the selection of a CW agent—other factors include (Smart et al., 2008) • Toxicological properties (dosage required, route of exposure, speed of action, etc.) • Physical properties (physical state, volatility, chemical stability, environmental persistency, etc.) • Logistics (production, availability, and supply) • Delivery system (can it be deployed effectively?) • Objective for use (i.e., to inflict casualties, harass the enemy, terrain denial or restriction) • Availability of countermeasures (medical treatment, protective clothing/equipment, decontamination, etc.) Even modern nerve agents would be next to useless tactically unless the above non-toxicological factors were satisfactorily addressed. It was the extensive chemical industry of World War I Germany that provided a major incentive for their use of CW agents—first chlorine, followed by phosgene, mustard, and other

174

Chemical Warfare Agents TABLE 11.7 Inhalation Toxicity Estimates for the General Human Population Exposed to CW Agents as Extrapolated from Estimates in Field Manual 3-11.9 (2005) Using the Crosier Model Nerve Agents Tabun (GA) LCT50 (at 2 min) ECT50 (severe effects)a (at 2 min) Probit slope (base 10)b TLEb,d ECT50 (mild effects)c (at 2 min) Probit slope (base 10) TLEd

Sarin (GB)

Soman (GD)

Cyclosarin (GF)

VX

55 [50] 40 9

28 [26] 20 9

28 [26] 20 9

28 [33] 20 [25] 9

9.4 [9.5]g 6.3 [7.1]f 4.5 [9]

1.5 0.30 [0.22]e 7.5 [3.5]

1.5 0.30 [0.22]e 7.5 [3.5]

1.25 [1.5] 0.15 [0.11]e 7.5 [3.5]

1.25 0.15 [0.22]f 7.5 [3.5]

1 0.050 [0.022]f 3 [3.5]

1.5 [1.4]

1.5 [1.4]

1.4

1.4

1 [1.4]

Note: CTs are in units of milligrams per minute per cubic meter. The values are for the general population (with the same activity level [light activity] as 70 kg healthy males of Table 11.6) acutely exposed over the whole body with no protection (i.e., respiratory, percutaneous, or ocular). Values in brackets are based on the updated estimates in Table 11.6. The Crosier model was used with the following assumptions: the healthy subpopulation comprises 30% of the total (general) population, and this subpopulation is defined as being healthy enough for military service. The critical Crosier parameters of δT and εT equal 0.900 and 0.744, respectively, for a single truncation subpopulation. a Severe effects include convulsions, gasping, collapse, or prostration. b Probit slope and TLE are the same for both lethal and severe effects. c Mild effects include miosis, rhinorrhea, lacrimation, and salivation. d TLE is the same for both the healthy subpopulation and the general population. e There is no updated healthy human ECT (mild) for this agent, but the healthy human probit slope was updated thereby produc50 ing a new general population ECT50 (mild) value when using the Crosier Model. The ECT50 value in the bracket is based on the updated probit slope value. f Both the healthy human ECT and probit slope values were updated for this agent. These were then both used in the Crosier 50 Model to produce the value in the brackets. g Both the healthy human LCT and probit slope values were updated for this agent. These were then both used in the Crosier 50 Model to produce the value in the brackets.

chemicals. Both chlorine and phosgene are still common industrial chemicals, and they can be readily adapted for use as improvised CW agents (Burklow et  al., 2003), which has been done frequently with chlorine in recent years (BBC Report, 2007; Brooks et al., 2018; Jones et al., 2010; Mahdi, 2007). In contrast, mustard does not have any legitimate industrial applications—it is used solely as a CW agent (thus, it is classified as a Schedule 1 chemical under the 1993 Chemical Warfare Convention). However, mustard is an excellent casualty-producing agent via multiple routes of exposure (inhalation, dermal, ocular), making it the “king” of the World War I war gases. World War I Germany enjoyed about a yearlong monopoly over the Allies on the battlefield use of mustard due to the Germans’ superior chemical production facilities (Hilmas et al., 2008), agent delivery systems, and tactical use doctrine (Heller, 1984). Among the war gases, it inflicted the most casualties (only 2.2% of the total were fatalities) on the American Expeditionary Force in World War I (Gilchrist, 1928) (i.e., phosgene was a distant second, and chlorine was third). Mustard has subsequently been used in other military conflicts, and the U.S. military did not complete the destruction of its main stockpile of bulk mustard liquid at Aberdeen Proving Ground, Maryland, until 2006 (Henemyre-Harris et al., 2008). Civilian, and military, populations have been exposed to these enduring threat materials during military operations as part of the ongoing Syrian civil war. It is therefore important to understand the potential threat to the human population following inhalation exposure to these compounds. Human and animal toxicity data are available for chlorine, phosgene, and mustard, and a meta-analysis was

performed by ECBC to update existing estimates (or propose new values for non-existing estimates) of the human toxicity during (and sometime after) the same time period as the Low-Level Toxicology Program (c. 1998 to 2008). Many of the statistical techniques developed to derive human estimates for nerve agents were used as well for the World War I–era agents. The results for healthy humans are shown in Table 11.8. Corresponding values for the general population (derived using the Crosier model) are shown in Table 11.9.

11.10 Conclusions Exposure to chemical nerve agent atmospheres leads to a myriad of toxic signs that are primarily mediated though acetylcholinesterase inhibition at target tissues. The initial signs of chemical nerve agent vapor and aerosol exposure are attributed to direct effects on target tissues that are most accessible to vapor/aerosols, including the eyes and respiratory tract. Thus, some of the first noticeable effects of nerve agent vapor or aerosol inhalation exposure are miosis, rhinorrhea, and tightness in the chest. Because it is a common finding in exposed individuals, and because it can occur at concentrations much lower than those necessary to cause other signs of toxicity, miosis is often used as a marker of exposure to a nerve agent vapor or aerosol. In a real-world situation, the most common scenarios of exposure would involve the percutaneous or inhalation routes. In the Inhalation Toxicology Laboratory at the ECBC, several methods have been employed to produce nerve agent vapors and aerosols that act via these routes

175

Inhalation Toxicology of Chemical Agents TABLE 11.8 Inhalation Toxicity Estimates for Healthy Humans Exposed to Select World War I–Era CW Agents from Field Manual 3-11.9 (2005) Chlorine LCT50 (at 2 min) ECT50 (severe) (at 2 min)c Probit slope (base 10)g TLEg

None stated [13,500] None stated [1,300]d None stated [8]a None stated [2.75]a

a

Phosgene (CG)

Mustard (HD)

1,500 None stated [250]e None stated [9.5]b 1b

1000 None stated [500]f 6 1.5

b

Note: CTs are in units of milligrams per minute per cubic meter. The values are for 70 kg healthy males (with a minute volume of 15 liters/min) acutely exposed over the whole body with no protection (i.e., respiratory, percutaneous, or ocular). Values in brackets are updated estimates that were developed at ECBC using statistical modeling of historical mammalian data that was performed post publication of FM 3-11.9. a Sommerville et al. (2009). b Development of phosgene estimates used in FM 3-11.9 documented in Sommerville et al. (2016). c Severe inhalation effects include severe respiratory effects (dyspnea, pulmonary edema, lung damage), collapse, or prostration. d Sommerville, DR, Human Toxicity Estimates for Chlorine, U.S. Army ECBC, APG, MD, unpublished work, 2012. Estimate is based on review of human accidental chlorine exposures documented by Shroff et al. (1988). e Sommerville, DR, Human Toxicity Estimates for Phosgene, U.S. Army ECBC, APG, MD, unpublished work, 2012—based on ratios of ECT50 (severe)/LCT50 taken from ECBC-derived ECT50 (severe) and LCT50 estimates based on extrapolations from AEGL-2 and AEGL-3 values, respectively, reported in the phosgene AEGL technical support document (NRC, 2002). f This estimate (developed for the present work) is based on an ECT (severe) to LCT ratio of 0.5 50 50 calculated from two of the several historical (World War I–era) studies (inhalation lethality in dogs) that were used to develop the human lethality estimate for FM 3-11.9 (2005). The endpoint is labored breathing, which always preceded death (though sometimes the animal did recover). It should be noted that in an unprotected individual, the ECT50 (severe ocular) (i.e., blindness) is much lower in value (75—with probit slope of 3 and TLE of 1—FM 3-11.9) than for inhalational severe effects (500). g Probit slope and TLE are the same for both lethal and severe effects.

TABLE 11.9 Inhalation Toxicity Estimates for the General Population Exposed to Select World War I–Era CW Agents from Field Manual 3-11.9 (2005) LCT (at 2 min) ECT50 (severe) (at 2 min)a Probit slope (base 10)b TLEb,c 50

Chlorine

Phosgene (CG)

Mustard (HD)

None stated [9500] None stated [920] None stated [6] None stated [2.75]

1100 None stated [190] None stated [7] 1

630 None stated [310] 4.5 1.5

Note: CTs are in units of milligrams per minute per cubic meter. The values are for the general population (with the same activity level [light activity] as 70 kg healthy males of Table 11.8) acutely exposed over the whole body with no protection (i.e., respiratory, percutaneous, or ocular). Values in brackets are based on the updated estimates in Table 11.8. The Crosier model was used with the following assumptions: the healthy subpopulation comprises 30% of the total (general) population, and this subpopulation is defined as being healthy enough for military service. The critical Crosier parameters of δT and εT equal 0.900 and 0.744, respectively, for a single truncation subpopulation. a Severe inhalation effects include severe respiratory effects (dyspnea, pulmonary edema, lung damage), collapse, or prostration. b Probit slope and TLE are the same for both lethal and severe effects. c TLE is the same for both the healthy subpopulation and the general population.

176 and in that regard, mimic likely exposure scenarios. The challenge for laboratory studies is to safely generate stable vapor or aerosol atmospheres and verify their chamber atmospheric concentration, chemical characterization, and stability throughout the exposure period. The combination of these methods with toxicity studies in multiple animal models has resulted in significantly improved confidence intervals for estimates of human exposure risks to nerve agent atmospheres. This, in turn, provides the basis for defendable criteria for chemical nerve agent detection, protection, decontamination, and countermeasure technologies.

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12 Cyanides: Toxicology, Clinical Presentation, and Medical Management Gary A. Rockwood, Gennady E. Platoff, Jr., and Harry Salem CONTENTS 12.1  12.2  12.3  12.4  12.5  12.6 

Introduction and Background.................................................................................................................................................... 182 Chemical Identities and Physicochemical Properties................................................................................................................ 182 Biological Mechanisms of Acute Toxicity................................................................................................................................. 182 Metabolism and Detoxification of Cyanide............................................................................................................................... 182 Biochemical Sequelae of Cyanide Intoxication......................................................................................................................... 183 Determinants for Cyanide Toxicity............................................................................................................................................ 184 12.6.1  Implications of Detoxification...................................................................................................................................... 184 12.6.2  Rate of Cyanide Absorption......................................................................................................................................... 184 12.6.3  Biodistribution of Cyanide........................................................................................................................................... 184 12.6.4  Miscellaneous Factors.................................................................................................................................................. 185 12.7  Acute Toxicity of Cyanides........................................................................................................................................................ 185 12.7.1  Toxicity to Laboratory Mammals................................................................................................................................. 185 12.7.1.1   Noninhalation Routes of Exposure............................................................................................................... 185 12.7.1.2   Inhalation Route of Exposure....................................................................................................................... 186 12.7.2 Estimates of Acute Human Toxicity............................................................................................................................... 187 12.8  General Considerations on Repeated Exposure Toxicity........................................................................................................... 188 12.9  Specific Organ, Tissue, and Functional Endpoint Toxicity........................................................................................................ 188 12.9.1 Neurotoxicity................................................................................................................................................................ 189 12.9.2 Cardiotoxicity...............................................................................................................................................................190 12.9.3  Vascular Toxicity..........................................................................................................................................................190 12.9.4  Respiratory Effects.......................................................................................................................................................190 12.9.5  Thyroid Gland Toxicity................................................................................................................................................190 12.9.6  Developmental and Reproductive Toxicity.................................................................................................................. 191 12.9.7 Genotoxicity................................................................................................................................................................. 191 12.9.8 Oncogenicity................................................................................................................................................................ 191 12.10  Clinical Toxicology of Human Cyanide Poisoning................................................................................................................... 191 12.10.1  Clinical Presentation..................................................................................................................................................... 191 12.10.2  Investigation and Confirmation of Poisoning............................................................................................................... 191 12.10.3  Management of Acute Cyanide Poisoning...................................................................................................................192 12.10.3.1  First Aid Measures.......................................................................................................................................192 12.10.3.2  General Medical Management....................................................................................................................192 12.10.3.3 Antidotes......................................................................................................................................................192 12.11  Cyanides in Military and Potential Terrorist Settings................................................................................................................194 12.11.1  Chemical Warfare Considerations................................................................................................................................194 12.11.2  Terrorist Implications...................................................................................................................................................194 References............................................................................................................................................................................................. 195

181

182

12.1 Introduction and Background The most commonly available cyanides (CNs) are hydrogen cyanide (HCN), a highly volatile liquid, and sodium, potassium, and calcium cyanides, which are solids. They are widely used industrially and commercially; examples are in manufacturing processes (includes dyes, pigments, chelating agents, various nitriles, monomers, resins, and fibers), case hardening, electroplating, the extraction of precious metals, and fumigation (Allen et al., 2015; Ballantyne, 1988; Ballantyne and Salem, 2005; Homan, 1987). In addition, due to their rapid lethal toxicity, they have been used for suicide, homicide, judicial execution, assassinations, and chemical warfare operations, and there exists a possibility for use by terrorists (Ballantyne, 1987a, 1987b; Ballantyne et al., 2006; Gee, 1987; Pita, 2015; WHO, 2004). Additionally, CN-related toxicity and pathology may result from exposure to man-made and to naturally occurring cyanogens (Ballantyne, 1987b; Brimer, 1988). In addition, HCN is a product of combustion, and inhalation of smoke from fires may cause CN intoxication (Ballantyne, 1987c; Ballantyne and Salem, 2005; Marrs, 2016; Norris and Ballantyne, 1999; Purser, 2016). As discussed later (Section 12.11.2), HCN has been employed by the military in chemical warfare operations because of its lethal and incapacitating effects. In addition, because of their known toxicity and comparatively ready availability, CNs are candidates for use as psychological and lethal agents by terrorist organizations. CN, as a terrorist weapon, has been used in various ways, including the contamination of over-the-counter medication. Additionally, CN has been used for extortion, in state and non-state terrorism, and potentially by jihadi terrorists. A detailed study of incidents with chemical warfare agents (CWAs) linked to al‐Qaeda shows that the nefarious use of CN is of great interest among jihadi terrorists (see Pita, 2015 for review). This chapter discusses the experimental and human clinical toxicology of CNs with particular reference to their potential for application as chemical warfare weapons and use by terrorists.

12.2 Chemical Identities and Physicochemical Properties HCN has the chemical synonyms of formonitrile, prussic acid, and hydrocyanic acid. Some physicochemical properties are compared with those of the sodium, potassium, and calcium salts in Table 12.1. Relevant to the toxicity and hazards of HCN are its liquid state at normal temperature and pressure, poor ionization, low molecular weight (MW), low boiling point, high vapor pressure, low vapor density (0.947 at 318°C), and hence, ready diffusibility. The salts are solids that are readily soluble in water; they ionize; and despite low vapor pressure, they hydrolyze in moist conditions with the liberation of HCN, this being markedly increased under acidic conditions.

12.3 Biological Mechanisms of Acute Toxicity As CNs inhibit many enzyme systems, and since other biochemical and physiological functions may be adversely affected

Chemical Warfare Agents by CNs, the overall mechanism and presentation of CN intoxication may be complex. However, it is generally concluded that the major mechanism of the acute toxic action of CN, which has been extensively investigated, is the inhibition of cytochrome c oxidase, the terminal oxidase of the respiratory chain, resulting in a cytotoxic hypoxia. CN causes intracellular hypoxia by complexing with the ferric iron of mitochondrial cytochrome c oxidase, inhibiting the electron transport chain and oxidative phosphorylation, resulting in anaerobic metabolism with a decrease in adenosine triphosphate (ATP) production and increased lactic acid production (Beasley and Glass, 1998). Tissues having the greatest O2 demand are the most markedly and rapidly affected; these include brain and myocardium. CN binds with both reduced and oxidized forms of the cytochrome a3 component of cytochrome c oxidase (Antonini et al., 1971; Nicholls et al., 1972; Van Burren et al., 1972). The interaction rate of CN with oxidized enzyme is about two orders of magnitude lower than that for the reduced enzyme, suggesting that the disruptive effect of CN on mitochondrial electron transport is at the level of reduced cytochrome a 3 (Yonetani and Ray, 1965). It is probable that CN reacts with the reduced form of cytochrome c oxidase, which may subsequently be converted to an oxidized enzyme–CN complex (Way, 1984). The oxidized form is relatively stable, but in the presence of reducing equivalents, CN can dissociate from the enzyme–inhibitor complex with reactivation of the enzyme (Ballantyne, 1987a). The reversible nature of the inhibition is the basis for the use of certain antidotes, which reactivate the enzyme by depleting intracellular CN. Thus, moving the equilibrium of CN from the intracellular to the plasma compartment is a significant component of the antidotal effects of cyanmethemoglobin (CNMetHb) formation, chelation of CN, or conversion to thiocyanate (SCN) (see Section 12.10.3.3). As CN toxicity is mediated mainly by an intramitochondrial mechanism causing cytotoxic hypoxia, and since CN is sequestered by erythrocytes (Vesey and Wilson, 1978), it is the plasma concentration of CN that is a prime determinant of cytotoxicity (Ballantyne, 1979, 1987a; Vesey, 1976). A major pathophysiological cause for CN-induced lethality is interference with central regulatory mechanisms for breathing, and cardiotoxicity may also be a significant factor (Ballantyne, 1977; Ballantyne and Bright, 1979; Susuki, 1968).

12.4 Metabolism and Detoxification of Cyanide In mammals, the detoxification of CN is rapid; for example, detoxification rates have been estimated at 0.076 mg kg−1 min−1 for guinea pigs (Lendle, 1964) and 0.017 mg kg−1 min−1 in humans (McNamara, 1976). The biodetoxification of CN occurs through several pathways, of which the most important is enzymic conversion of CN to the less acutely toxic thiocyanate (SCN), which is responsible for the conversion of up to 80% of a CN dose. For example, the rat LD50 values for NaCN by the per oral (p.o.) and intraperitoneal (i.p.) routes are, respectively, 5.7 and 4.72 mg kg−1, and the corresponding values for NaSCN are 764 and 540 mg kg−1. Thus, the bioconversion of NaCN to NaSCN is associated with a decrease in acute lethal toxicity of about 120-fold in the rat (Ballantyne, 1984). SCN is excreted renally with a half-life (t1/2) of 2.7 days in healthy human

183

Cyanides TABLE 12.1 Physicochemical Properties of Hydrogen Cyanide and Its Salts Property CAS number Molecular weight Density (g cm−3) Melting point (°C) Vapor pressure (Torr)

HCN

NaCN

KCN

Ca(CN)2

74-90-8 27.04 0.688 (208°C) −13.3 600 (208°C)

143-33-9 49.01

151-50-8 65.12 1.52 (168°C) 634

592-01-8 66.10

563

>350a

Source: Data from Ballantyne, B. and Salem, H., Inhalation Toxicology, Taylor and Francis, Boca Raton, FL, 2005; Homan, E., Clinical and Experimental Toxicology of Cyanides, Wright, Bristol, 1987; Lewis, R.J., Hawley’s Condensed Chemical Dictionary, Von Nostrand Reinhold, New York, NY, 1993. a Decomposes.

subjects (Schulz et al., 1979). Two enzymes responsible for this transulfuration process are thiosulfate–cyanide transulferase (EC 2.8.1.1; rhodanese) and β-mercaptopyruvate–cyanide transulferase (EC 2.8.1.2) (Ballantyne, 1987a; Lang, 1933; Sorbo, 1975). Thiosulfate–cyanide transulferase is a mitochondrial enzyme responsible for the transfer of a sulfane sulfur atom from sulfur donors to sulfur acceptors:

CN - + S2O3- ® SCN - + SO32 -

The basic reaction involves the transfer of sulfane sulfur from the donor (SCN) to the enzyme, forming a persulfide intermediate. The persulfide sulfur is transferred from the enzyme to a nucleophilic receptor (CN) to yield SCN. For most species, this enzyme activity is high in liver, kidney, brain, muscle, and olfactory mucosa (Aminlari et  al., 1994; Dahl, 1989; Himwich and Saunders, 1948). The nasal metabolism of CN may have relevance to the toxicity of inhaled HCN. β-Mercaptopyruvate–cyanide transulfurases are present in blood, liver, and kidney and catalyze the reaction

A third sulfurtransferase, cystathionase (cystathionine γ-lyase; EC 4.4.1.1), which is a cytosolic enzyme, may play a role in CN detoxification in the kidney and rhombencephalon (Wróbel et  al., 2004). A product of the cystathionase reaction, bis(2-amino-2-carboxylethyl)trisulfide (thiocystine) may serve as a sulfur substrate donor for rhodanese. Another reaction product, 3-(thiosulpheno)alanine (thiocysteine), may be an additional link between cystathionase and CN biodetoxification. In addition, cystathionase also functions as a sulfane sulfur carrier. Other, quantitatively more minor, CN biodetoxification pathways are



HS × CH 2 × CO × CO × O- + CN ® SCN - + CH 3 × CO × CO × O-

Since little SCN penetrates the inner mitochondrial membrane, it is generally believed that the thiosulfate sulfurtransferase system may not be the primary detoxification mechanism for CN. A more general concept of the role of sulfur in the detoxification process is that the supply of sulfane sulfur is from a rapidly equilibrating pool of potential sulfane sulfur donors, and these may include per- and polysulfides, thiosulfanates, polythionates, inorganic SCN, and protein-associated elemental sulfur. According to this scheme, the sulfurtransferases catalyze the formation, interconversions, and reactions of compounds containing sulfane sulfur atoms (Westley, 1981; Westley et  al., 1983). It is possible that sulfane sulfur is derived from mercaptopyruvate via β-mercaptopyruvate transulferase, and the various forms of sulfane sulfur are interconverted by thiosulfate sulfurtransferase. The sulfane carrier transporting the sulfur formed is albumin; the sulfur sulfane–albumin complex then reacts with CN. Pharmacokinetic studies indicate that the conversion of CN to SCN is mainly in the central compartment with a volume of distribution approximating that of the blood volume (Way, 1984).



1. Exhalation of HCN and of CO2 from oxidative metabolism (Boxer and Rickards, 1952; Okoh, 1983). Traces of HCN can be detected in expired air of normal humans, but this is not correlated with blood CN. Most HCN in normal breath is derived from the oxidation of SCN by salivary peroxidase in the oropharynx (Lundquist et al., 1988). 2. Reaction with cysteine to produce β-thiocyanoalanine followed by ring closure to 2-aminothiazoline-4-carboxylic acid (ATC) or its tautomer 2-iminothiazoline4-carboxylic acid (Wood and Cooley, 1956). 3. Combination of CN with hydroxocobalamin yields cyanocobalamin, which is then excreted in urine (Boxer and Rickards, 1951; Herbert, 1975).

In addition to these mechanisms, erythrocytes have a high affinity for CN and rapidly sequester CN from plasma (Ballantyne, 1975; Barr, 1966; Schulz, 1984; Vesey et al., 1976). The sequestration of CN by erythrocytes may have a protective function in the detoxification of CN.

12.5 Biochemical Sequelae of Cyanide Intoxication The inhibition of cytochrome c oxidase, and the resultant disturbance of electron transport, results in decreased mitochondrial O2 use and decreased ATP (Olsen and Klein, 1947). Anaerobic metabolism leads to an accumulation of lactic acid and lactate acidosis, and the combination of lactate acidosis and cytotoxic

184 hypoxia causes severe metabolic disturbances, particularly in the central nervous system (CNS), resulting in disturbances of perception and consciousness. The endogenous buffering of lactate leads to a progressive fall in plasma bicarbonate. It has been shown experimentally that in the brains of CN-poisoned mice, there is an increase in lactate, inorganic phosphate, and ADP, with a decrease in ATP, phosphocreatine, glycogen, and glucose (Estler, 1965; Isom et al., 1975). Rats given an intravenous (i.v.) infusion of 4 mg CN kg−1 min−1 had an increase in the tricarboxylic acid intermediates succinate, fumarate, and malate (Hoyer, 1984), indicating disturbance of NAD+ - and FAD+ -dependent redox reactions, including pyruvate oxidation. Lactate acidosis with hyperglycemia has been noted in dogs given KCN (Klimmeck et  al., 1979) and rats given NaCN (Salkowski and Penney, 1994). In rats, Isom et  al. (1975) found that CN increased catabolism of carbohydrate by the pentose phosphate shunt and decreased use by the Embden–Meyerhof–Parnas pathway, the tricarboxylic acid cycle, and the glucuronate pathway. It was suggested that increased catabolism of glucose by the pentose phosphate shunt may produce a source of NADPH that can reduce NAD by a transhydrogenase enzyme and thus, compensate for the aberrant redox state produced by CN intoxication. Such findings indicate that CN can alter carbohydrate metabolism, resulting in increased glycogenolysis and a shunting of glucose to the pentose phosphate pathway by decreasing the rate of glycolysis and inhibition of the tricarboxylic acid cycle.

12.6 Determinants for Cyanide Toxicity Major determinants for the development of CN toxicity are the rate of accumulation and magnitude of free CN at the cellular target sites. These are achieved by the interaction of many factors, which include bioavailability, biodistribution, detoxification, and bioelimination of CN. The major factors are discussed in the following sections.

Chemical Warfare Agents acute exposures to high dosages of CN, there may be swamping of endogenous detoxification mechanisms, resulting in the rapid onset of toxicity. However, in general, the effective detoxification of CN prevents its long-term bioaccumulation. Thus, with an acute exposure to a sublethal dose of CN that produces toxic signs during the absorption phase, after exposure, and as the detoxification process continues, the signs ameliorate as CN is metabolized.

12.6.2 Rate of Cyanide Absorption The dose absorbed through a primary absorption route depends on

1. Physicochemical properties of the materials. HCN is of low MW, is nonionized, and diffuses readily; in contrast, KCN has a higher MW and is ionized and hence, absorbed to a lesser degree. Thus, LD50 values for HCN are numerically smaller than those for KCN by a given route (see Table 12.2). 2. Exposure dose: in particular, the exposure concentration, exposure time, and number and timing of exposures. 3. Route of exposure. For example, HCN is readily absorbed across the pulmonary alveolar membrane, but skin presents a greater barrier to absorption. However, the integrity of the absorbing surface can also be a factor; thus, CNs are more readily absorbed through recently abraded skin than through intact skin (Ballantyne, 1994a). Although the absorbed dose is a primary determinant of the amount of CN available for tissue biodistribution, due to the sequestration of CN by erythrocytes, it is the concentration of free (unbound) CN in plasma and tissue fluids that is the major quantitative determinant of both the latency and the severity of toxicity.

12.6.3 Biodistribution of Cyanide 12.6.1 Implications of Detoxification Since CNs are both quickly absorbed and biodistributed, and due to the mechanism of action by inhibition of mitochondrial cytochrome c oxidase, CNs are rapidly acting compounds in biological systems (although signs of toxicity may be delayed in cases of oral CN exposure). A major determinant of both the latency and the severity of CN toxicity is the balance of the quantitative rates of absorption and endogenous detoxification. The balance is usually such that after the termination of an exposure, bioaccumulation of CN will not occur. However, during exposure, the rate of availability of sulfur substrate is a determinant of the detoxification rate, and a relative decrease in sulfurtransferase detoxification may occur with an increase in available free CN. When the rate of increase of toxicologically available CN is slow, there will be a delay to both time to onset (latency) and progression of toxic effects. Within limits, this relationship between exposure dose and detoxification results in a clear dose–response relationship (Ballantyne, 1987a). With

The biodistribution of CN to various systemic tissues will determine the relative proportions of CN present at detoxification and target tissue or cellular sites. For example, inhaled or percutaneously absorbed CN enters the systemic circulation, and only a small proportion of the absorbed dose will be available for first-pass detoxification, particularly in the liver. In contrast, a high proportion of a p.o. dose will pass through the liver and be available for first-pass detoxification. However, hepatic detoxification processes may be complex, since it has been demonstrated that dietary variations that cause alterations in hepatic sulfurtransferase activity do not correlate with CN toxicity (Rutkowski et al., 1985), and extensive chemical or surgical injury to the liver does not increase the susceptibility of the mouse to CN toxicity (Rutkowski et al., 1986). The influence of route on toxicity is probably due to the relative effects of plasma transulfuration, sequestration by erythrocytes, intracellular macromolecular binding, and the differential distribution to all tissues with a detoxification capacity.

185

Cyanides TABLE 12.2 Acute Lethal Toxicity of HCN and Alkali Salts Given by Various Noninhalation Routes of Exposure LD50 (95% Confidence Limits) Route

Cyanide

Species (Gender)

Intravenous

HCN NaCN KCN NaCN KCN HCN NaCN KCN HCN NaCN KCN HCN HCN NaCN NaCN KCN KCN HCN NaCN KCN HCN HCN NaCN NaCN KCN KCN

Rabbit (F) Rabbit (F) Rabbit (F) Hamster (NS)a Mouse (M) Rabbit (M) Rabbit (M) Rabbit (M) Rabbit (M) Rabbit (M) Rabbit (M) Rat (F) Rat (F) Rat (F) Rat (F) Rat (F) Rat (F) Rabbit (F) Rabbit (F) Rabbit (F) Rabbit (F) Rabbit (F) Rabbit (F) Rabbit (F) Rabbit (F) Rabbit (F)

Subcutaneous Intramuscular

Intraperitoneal Peroral (US)b (S) (US) (S) (US) (S) Transocular Percutaneous (I)c (A) (I) (A) (I) (A)

As mg kg−1

As mmol kg−1

0.59 (0.55–0.65) 1.23 (1.11–1.1.34) 1.89 (1.66–2.13) 7.4 12.0 (10.8–13.3) 1.50 (1.27–1.80) 1.61 (1.38–1.83) 3.06 (2.61–3.63) 1.72 (0.85–2.00) 2.93 (2.72–3.35) 3.60 (2.71–4.10) 4.21 (3.76–4.95) 3.62 (3.08–3.87) 5.72 (5.23–7.08) 5.09 (4.26–5.83) 7.49 (6.68–8.480) 9.69 (8.60–11.30) 1.04 (0.96–1.13) 5.06 (4.44–6.10) 7.87 (6.51–8.96) 6.89 (6.43–7.52) 2.34 (2.02–2.61) 14.6 (13.8–15.4) 11.3 (9.2–12.7) 22.3 (20.4–24.0) 14.3 (13.3–15.1)

0.022 (0.020–0.024) 0.025 (0.023–0.027) 0.029 (0.026–0.033) 0.15 0.25 (0.22–0.27) 0.056 (0.047–0.068) 0.033 (0.028–0.037) 0.047 (0.040–0.056) 0.064 (0.031–0.074) 0.060 (0.056–0.068) 0.055 (0.042–0.063) 0.156 (0.139–0.183) 0.127 (0.114–0.143) 0.117 (0.107–0.144) 0.104 (0.087–0.119) 0.115 (0.103–0.130) 0.149 (0.132–0.174) 0.039 (0.036–0.040) 0.103 (0.091–0.124) 0.121 (0.100–0.138) 0.260 (0.240–0.280) 0.087 (0.077–0.097) 0.30 (0.28–0.31) 0.23 (0.19–0.26) 0.343 (0.314–0.369) 0.220 (0.204–0.232)

Source: Data after Ballantyne, B., Developments in the Science and Practice of Toxicology, Elsevier, Amsterdam, 1983; Ballantyne, B., Cutan. Ocul. Toxicol., 2, 119, 1983b; Ballantyne, B., Clinical and Experimental Toxicology of Cyanides, Wright, Bristol, 1987a; Ballantyne, B., Cutan. Ocul. Toxicol., 13, 249, 1994. a NS = not specified. b US = unstarved; S = starved. c I = intact skin; A = abraded skin.

12.6.4 Miscellaneous Factors Other factors that influence the development of CN toxicity include 1. Diurnal variation in toxicity. The effect of time of dosing on the lethality to mice of i.p. KCN was investigated by Baftis et al. (1981), who used a 12 h light/dark cycle. Mortality peaked at 16 h (83% deaths) and was lowest at 8 h (43% deaths). There was also a circadian rhythm in time to death, with the times being shorter at 20 h and longest at 0.8 h. 2. Age. Fitzgerald (1954) obtained the following LD50 values in mice for s.c. NaCN: adult males, 5 mg kg−1; adult females, 3.5–3.7 mg kg−1; neonatal mice, 2.0–2.5 mg kg−1. 3. Antidotes. The presence of antidotes will clearly influence the development of toxicity. Thus, if CN is bound to methemoglobin (MetHb), chelated, or transulfurated there will be a shift in the equilibrium of

CN from the intracellular to the extracellular compartment and resultant reactivation of cytochrome c oxidase.

12.7 Acute Toxicity of Cyanides 12.7.1 Toxicity to Laboratory Mammals 12.7.1.1 Noninhalation Routes of Exposure A comparison of typical values for the acute (single-dose) lethal toxicity of solutions of CNs by noninhalation routes is given in Table 12.2. The general order of acute lethal toxicity is HCN > NaCN > KCN, which accords with the more diffusible and unionized form of HCN. Some general comments on the various routes follow. Intravenous Toxicity. When expressed on a mass basis (milligrams per kilogram), there are differences in LD50 values between HCN and its alkali salts, but on a molar basis, there is no

186 significant difference in lethal toxicity between HCN and NaCN, while KCN is statistically significantly less lethally toxic. Signs usually appear within 10–30 s and include rapid breathing, ataxia, convulsions, and coma, with death in 2–12 min (Ballantyne, 1983a). If given by slow i.v. injection rather than bolus injection, there will be a greater proportionate detoxification activity, and thus, the latency to signs and lethal threshold for total dose will be increased. Subcutaneous Toxicity. This route has been popular for investigational studies of various types, particularly to assess the effect of therapy given by other routes (DeLeon et al., 2018). Times to onset of signs are usually within minutes, and effects are similar to those seen by the i.v. route. However, as is well known with other chemicals dosed by the s.c. route, absorption may be erratic depending on the tissue stratum into which the injection is given. Intramuscular Toxicity. On a molar basis, HCN and NaCN are both equitoxic and more toxic than KCN. Signs appear within minutes and include rapid breathing weak and uncoordinated movements, ataxia, tremors, convulsions, and respiratory arrest. Postmortem features are few and include alveolar and subpleural hemorrhages and congestion of tracheal mucosa (Ballantyne et al., 1972). Intraperitoneal Toxicity. There are no significant differences in the i.p. acute lethal molar toxicity between HCN, NaCN, and KCN. Time to onset of signs is between 1 and 4 min, and time to death is 1–26 min. Per Oral Toxicity. Molar toxicities for HCN, NaCN, and KCN are similar. For HCN and NaCN, lethal toxicity is somewhat greater for starved than for unstarved animals, but the reverse situation is obtained with KCN (Ballantyne, 1984). Time to onset of signs is around 1–8 min and time to death from 7 to 26 min. When CN is given orally, the gastric environment favors the formation of HCN, which facilitates absorption. In addition, the absorption of HCN across the gastrointestinal mucosa is favored by its weak acidity (with a pKa of 9.2). Rice et al. (2018) recently described a novel oral-consumption model of NaCN ingestion in rats using an assisted drinking technique. Transocular Toxicity. Instillation of HCN and its salts into the inferior conjunctival sac results in the absorption of lethal amounts of CN. HCN is significantly more lethally toxic by this route than either NaCN or KCN. The effect of varying the physical state of local presentation of NaCN to the eye has shown that both solution (10–20%) and solid present a similar hazard (Ballantyne, 1983b). Signs of toxicity appear in the following order: rapid breathing, weak and ataxic movements, convulsions, tonic spasms, irregular and shallow breathing, coma, and cessation of breathing. For HCN, signs develop within 30–60 s, and time to convulsions is from 45 to 90 s. The corresponding times with NaCN and KCN are 2–2.5 min and 2–3 min. Time to death is 3–12 min postinstillation. Thus, following instillation of CN into the conjunctival sac, it is readily absorbed into the systemic circulation. The factors responsible for this are conjunctival hyperemia, drainage through the nasolacrimal duct to the vascular nasal mucosa, and absorption into the systemic circulation with minimal hepatic first-pass detoxification. Local irritant effects on the eye are conjunctival hyperemia and mild chemosis (Ballantyne, 1983b). Percutaneous Toxicity. Early studies demonstrated CN toxicity following percutaneous exposure (Fairley et al., 1934; Walton

Chemical Warfare Agents and Witherspoon, 1926). With intact skin, HCN solution is slightly more lethally toxic than NaCN, which itself is slightly more toxic than KCN. Abrading the skin enhances the percutaneous penetration of CN and increases lethal toxicity. This is particularly marked with HCN solution, for which there was a threefold increase in lethal toxicity. The influence of physical state was investigated with NaCN (Ballantyne, 1994a). Contact of dry solid NaCN with intact skin did not result in the absorption of lethal amounts of CN (LD50 > 200 mg kg−1). However, when dry NaCN was maintained in contact with abraded skin, there was rapid absorption of CN allowing the calculation of a percutaneous (PC) LD50 of 7.7 mg kg−1, which is lower than that resulting from application of NaCN solution to intact skin (14.6 mg kg−1) or to abraded skin (11.3 mg kg−1). Times to onset of signs ranged widely; 5 min–1 h with HCN and 15 min–4 h with NaCN and KCN. Examination of LD50 data by different routes shows the decreasing order of lethal toxicity: HCN: i.v. = i.m. >; transocular >; i.p. >; p.o. >; PC NaCN: i.v. = i.m. >; i.p. >; transocular >; p.o. >; PC KCN: i.v. >; i.m. >; i.p. >; p.o. >; transocular >; PC By different exposure routes, the comparative molar LD50 values for NaCN and KCN are >HCN for i.v., i.m., transocular, and PC =HCN for i.p. and p.o.

12.7.1.2 Inhalation Route of Exposure Due to its low MW, poor ionization, and high diffusion characteristics, HCN is rapidly absorbed following inhalation. Some typical lethality data, expressed as timed LC50 and L(CT)50 values, are given in Table 12.3. These data indicate that over the time period studied, there is a disproportionate relation between the exposure time to produce mortality and the exposure concentration; as the lethal concentration is decreased, the exposure time necessary to cause death increases, but not in proportion. The relationship between exposure time (T), exposure concentration (C), and the inhalation exposure dosage (CT) causing mortality was investigated in detail by Ballantyne (1984, 1987a, 1994b). For short exposures to HCN vapor (a few seconds to a few minutes), as the exposure time required to cause mortality decreases, only short increases in exposure time are needed to give 50% mortality; for example, the exposure time for a 1229 mg m−3 LC50 is 1 min, while that for a 493 mg m−3 LC50 is 5 min. This accords with the fact that under both of these conditions, it is likely that there is saturation of endogenous detoxification processes. However, with exposure times in excess of 5 min, proportionately longer times are required to produce decreases in the LC50; for example, a 173 mg m−3 LC50 requires an exposure time of around 30 min. With higher inspired HCN concentrations, there is a steep concentration gradient across the pulmonary alveolar membrane that facilitates the absorption of HCN, which enters the systemic circulation without a significant degree of firstpass hepatic detoxification and thus, rapidly attains toxic levels. With lower inhaled HCN concentrations, there is a slower rate of

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Cyanides TABLE 12.3 Acute Lethal Inhalation Toxicity of HCN Vapor to Various Species as Timed LC50 and L(CT)50 Values Median Lethal Toxicity (95% Confidence Limits) Species (Gender) Mouse (M) Rabbit (F) Rabbit (F) Rabbit (F) Rat (F) Rat (F) Rat (F) Rat (F) Rat (F)

Exposure Time

LC50 (mg m−3)

L(CT)50 (mg min m−3)

30 min 45 s 5 min 35 min 10 s 1 min 5 min 30 min 60 min

176 (179–260) 2432 (2304–2532) 409 (321–458) 208 (154–276) 3778 (3771–4313) 1129 (664–1471) 493 (372–661) 173 (159–193) 158 (144–174)

5280 (3870–7880) 1824 (1728–1899) 2044 (1603–2288) 7283 (5408–9650) 631 (562–719) 1129 (664–1471) 2463 (1861–3301) 5070 (4690–5497) 9441 (8609–10399)

Source: Data from Ballantyne, B., Proceedings of the Fourth Annual Chemical Defense Bioscience Review, Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD, 1984; Ballantyne, B., Clinical and Experimental Toxicology of Cyanides, Wright, Bristol, 1987a; Ballantyne, B., Toxic Sub. J., 13, 249, 1994; MatijakSchaper, M. and Alarie, Y., J. Combust. Toxicol., 9, 21, 1982.

titration of HCN into the pulmonary circulation and thus, a comparatively longer latency to the onset of signs and death. Typical signs of intoxication with HCN vapor exposure are rapid breathing, weak and ataxic movements, loss of voluntary movements, convulsions, loss of consciousness, decrease in both the rate and the depth of breathing, and breathing irregularities. Necropsy findings are nonspecific and generally consist of congestion of intra-abdominal viscera, pulmonary congestion, and scattered pleural and pulmonary hemorrhages (Ballantyne, 1994b). In a recent study of nose-only HCN exposure in male mice, LC50 values were reported to be 421 and 324 ppm, respectively, using a 10 min continuous exposure model and a 40 min discontinuous exposure model (10 min HCN → 2 min no HCN → 30 min HCN) (DeLeon et al., 2018). Respiratory rates were not reported. Early in an exposure to HCN vapor, there may be an increase in respiratory minute volume, which results in a higher absorbed dose (Purser et al., 1984). An important feature with inhaled HCN is the development of incapacitating effects and impeded mobility (e.g., DeLeon et al., 2018). For example, Purser et al. (1984) found that over a HCN concentration range of 114–175 mg m−3, there was a linear relation between exposure time and the time to development of hyperventilation and subsequent incapacitating effects. The slope was such that doubling the HCN vapor concentration from 112 to 224 mg m−3 reduced the time to incapacitation from 25 to 2 min. Rat studies estimated that the HCN vapor concentration producing incapacitating effects is about 65% of the lethal concentration (Levin et al., 1987).

12.7.2 Estimates of Acute Human Toxicity Estimates of the acute lethal p.o. toxicity of CNs are not too precisely defined because of uncertainties in determining the exact amounts ingested and the absorbed dose. The estimated fatal oral dose for HCN is 50–100 mg as total (DuBois and Geiling, 1959) or 0.7–3.5 mg kg−1 (Hallstrom and Moller, 1945); for KCN, a total dose of 150–250 mg has been suggested (DuBois and Geiling, 1959).

To estimate percutaneous lethal doses, Dugard and Mawdsley (1978) measured CN transport across human epidermis using a diffusion cell technique, and the data obtained allowed absorption for differing conditions. For example, 10% NaCN at pH 11.4 in contact with skin may lead to symptoms within 25 min and death within 1 h. A limited number of industrial exposures to HCN vapor have been documented that allow an estimation of the acute lethal toxicity of HCN vapor to humans. In one case, an individual who had intensive medical management survived a 3 min exposure to approximately 560 mg m−3 (500 ppm) HCN (Bonsall, 1984), but in other cases, 302 mg m−3 (270 ppm) caused immediate death, 203 mg m−3 (181 ppm) was lethal within 1 min, and 151 mg m−3 (135 ppm) was lethal after 30 min (Dudley et  al., 1942). Hall and Rumack (1998) stated that inhalation of high HCN vapor concentrations (200–500 ppm) may cause abrupt loss of consciousness after one or two breaths. Based on available LD50 values in various species, and assuming a detoxification rate of 0.017 mg kg−1 min−1, Moore and Gates (1946) estimated an absorbed lethal inhalation dose for man of HCN at 1.1 mg kg−1. Using this approach, they calculated the timed LC50 values shown in Table 12.4 for a 70 kg man having a breathing rate of 25 L min−1. McNamara (1976) re-evaluated the lethal inhalation toxicity of HCN vapor (Table 12.5). Based on available metabolic rate data, Hilado and Cumming (1977) suggested the following LC50 values for man, which are somewhat lower than those calculated by McNamara (1976): 5 min LC50 of 680 ppm (748 mg m−3) and 30 min LC50 of 200 ppm (220 mg m−3). According to WHO (2004), with exposure to HCN vapor concentrations in the range 120–150 mg m−3, death may occur within 0.5–1.0 h, with 150 mg m−3 being fatal within 30 min; 200 mg m−3 is likely to be fatal after 10 min, and 300 mg m−3 can be immediately fatal. Overall, the various estimates for the human acute lethal inhalation toxicity of HCN vapor suggest that for an exposure period of 5–10 min, a concentration of 500–600 mg m−3 (455–546 ppm) would be fatal, but for a 1 min exposure, the HCN vapor concentration would require to be around 4000 mg m−3 (3640 ppm).

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Chemical Warfare Agents TABLE 12.4 Estimated Human Acute Inhalation Toxicity of HCN Vapor Based on the Moore and Gates (1946) Analysis Calculated Lethal Inhalation Toxicity Time (min) 1 3 10 30 60

As LC50 (mg m−3)

As L(CT)50 (mg min m−3)

4400 1500 504 210 140

4400 4500 5040 6300 8400

Note: Calculated for a 70 kg man with a breathing rate of 25 L min−1 and a detoxification rate for cyanide of 0.017 mg kg−1 min−1.

TABLE 12.5 Estimates of the Acute Inhalation Lethal Toxicity of HCN Vapor after McNamara (1976) Time (min) 0.5 1 3 10 30

LC50 (mg m−3)

L(CT)50 (mg min m−3)

4064 3404 1466 607 688

2032 3404 4400 6072 20,632

Note: Listed as L(CT)50 and timed LC50 values.

The available evidence indicates that HCN vapor can be absorbed across the skin and thus, contribute additively to that resulting from respiratory exposure. Fairley et al. (1934), using guinea pigs and rabbits, demonstrated that exposure to HCN vapor resulted in PC absorption sufficient to cause signs of toxicity and with sustained exposure, to cause mortality. Reports of occupationally exposed workers indicate that symptomatic PC absorption of HCN can occur in humans. For example, employees wearing respiratory protective equipment who were working for 8–10 min in an atmosphere containing about 20,000 ppm HCN vapor (22,400 mg m−3) developed dizziness, weakness, and headache (Drinker, 1932). Two reports of intoxication due to PC HCN vapor absorption, one in a firefighter wearing self-contained breathing apparatus, were described by Steffens (2003). Using diffusion cells, Dugard (1987) studied the in vitro absorption of CN through human skin from solutions of NaCN and HCN vapor. His findings suggest that the rate of absorption of HCN is proportional to the atmospheric concentration of HCN. He calculated that contact of the total body surface (18,500 cm2 for a 70 kg individual) with 1 ppm HCN vapor can result in the absorption of 32 mg CN h−1.

12.8 General Considerations on Repeated Exposure Toxicity Several studies have been undertaken to assess the general (overall) toxic effects of CN in various species. These are considered

briefly in the following and have been discussed in detail in Ballantyne and Salem (2005) and Downey et al. (2015). No significant effects were found for signs, food consumption, body weight, hematology, or histology in dogs fed with NaCN in the diet (2.5 mg kg−1) for 30–32 days (American Cyanamid, 1959). Dogs dosed p.o. with NaCN (up to 6 mg kg−1 day−1) for 15 months had an immediate onset of signs with increased erythrocyte count, decreased blood albumin, and degenerative changes in cerebrocortical neurons and cerebellar Purkinje cells (Herrting et al., 1960). Baboons dosed with KCN (1 mg kg−1) for 9 months developed decreased Hb and mean corpuscular Hb concentrations (Crampton et al., 1979). Male rats dosed with KCN in drinking water for 15 days demonstrated dose-related increases in cytoplasmic resorption vacuoles in the thyroid follicular colloid but no effects on serum T3 or T4. There were hepatocellular cytoplasmic vacuolation and degenerative changes, and vacuolation of renal proximal tubular epithelial cells (Sousa et al., 2002). Feeding KCN in the diet of rabbits (702 ppm) for 40 weeks resulted in increased lactic dehydrogenase activities in serum, liver, and kidney, consistent with a shift to anaerobic metabolism (Okolie and Osagie, 1999). Increases in serum and decreases in liver sorbitol dehydrogenase, alkaline phosphatase, and glutamate– pyruvate transaminase suggested hepatotoxic lesions, and decreased renal alkaline phosphatase with increased serum activity accompanied by increased serum urea and creatinine suggested nephrotoxicity. Histopathologically, there were foci of liver, renal tubular, and glomerular necrosis. It was also demonstrated (Okolie and Osagie, 2000) that aspartate transaminase activity in heart and serum was unaffected, but cardiac and pulmonary alkaline phosphatase was decreased. Histology was normal in pancreas and myocardium, but lungs had foci of edema and necrosis. These findings indicate that long-term p.o. dosing with CN causes hepatorenal and systemic pulmonary injury but not cardiac or pancreatic toxicity. In a multispecies (rat, pig, and goat) subchronic p.o. study with KCN, Soto-Blanco et al. (2001) did not find any biochemical or histological evidence for pancreatic exocrine or endocrine toxicity. In a 13 week study, rats and mice received up to 300 ppm in drinking water (Hébert, 1993), which resulted in a slight reduction in cauda epididymal weights (rats and mice) and reduced numbers of spermatid heads per testis (rats). Rats dosed with up to 500 ppm KCN in drinking water had doserelated decreases in hepatic and cardiac respiration and in cardiac, hepatic, and cerebral ATP concentration (Rickwood et al., 1987). These findings accord with CN producing mitochondrial dysfunction.

12.9 Specific Organ, Tissue, and Functional Endpoint Toxicity Certain specific organ, tissue, and functional endpoint toxic effects may be a direct consequence of CN exposure at the target site; others are due to SCN metabolite exposure, and others are the result of pathophysiological consequences resulting from toxic effects of CN. The more relevant of these for hazard assessment and clinical purposes are discussed briefly in the following sections.

Cyanides

12.9.1 Neurotoxicity Studies in laboratory animals, in vitro models, and clinical investigations on exposed humans have demonstrated a neurotoxic potential for CN, notably for CNS morphological and functional effects; these include convulsions, loss of consciousness, alteration of perception and central control functions, and longer-term degenerative neuropathology. Some experimental and human aspects are briefly summarized in this section. The neurotoxicity of CN is reviewed in detail by Ballantyne and Salem (2005) and Ballantyne et al. (2006). Experimental studies have demonstrated that in CN intoxication, the measured CN concentrations are high in brain (Ballantyne, 1975; Ballantyne and Bright, 1979). CN rapidly equilibrates across the neuronal plasma membrane and then accumulates in the mitochondria and membrane elements of the neuron (Borowitz et al., 1994). Some investigators have stressed the contribution of cytochrome c oxidase inhibition to neurotoxicity. For example, CN produces a highly significant inhibition in cerebral cytochrome oxidase activity (Ballantyne, 1977; Ballantyne and Bright, 1979). Rabbit brain homogenate has a measured I50 for CN-inhibited cytochrome c oxidase of 6.38 mM and a calculated pI50 of 5.20 (Ballantyne, 1977). In vivo measurement of cytochrome c oxidase activity by reflectance spectrometry showed that in the cerebral cortex of rats given sublethal doses of CN, there was a noncumulative, transient, and dose-related inhibition of the respiratory chain (Piantadosi et al., 1983). The decrease in mitochondrial activity was accompanied by increases in regional cerebral HbO2 saturation and blood volume. Further studies (Piantadosi and Sylvia, 1986) demonstrated a dose-related suppression of electroencephalographic (EEG) activity with isoelectric conditions occurring at 50% reduction in cytochrome c oxidase activity. Pretreatment with sodium thiosulfate resulted in a fourfold protection of cerebral cytochrome c oxidase activity from CN-induced redox changes. Cerebral enzyme systems other than cytochrome c oxidase may contribute to central neurotoxic effects. For example, inhibition of glutamate decarboxylase results in depletion of the inhibitory neurotransmitter γ-aminobutyric acid (GABA), which predisposes to convulsions (Tursky and Sajter, 1962). NaCN given i.p. (5–20 mg kg−1) increased glutamic acid concentrations in the cerebellum, striatum, and hippocampus, but higher doses decreased both GABA and glutamic acid (Perrson et al., 1985). Cassel et al. (1991) demonstrated that decreased GABA was associated with increased susceptibility to convulsions, and Yamamoto (1990) demonstrated a 31% decrease in GABA in KCN-dosed mice exhibiting convulsions. The decrease in GABA and the associated convulsions were abolished by dosing with α-ketoglutarate. Patel et  al. (1992) showed that NaCN-mediated cytotoxicity in hippocampal cultures was mediated mainly by activation of N-methyl-d-aspartate (NMDA) receptors. In addition, Yamamoto and Tang (1998) found that exposure of cerebrocortical neurons to KCN or NMDA increased lactate dehydrogenase efflux into extracellular fluid, which was blocked by coexposure to 2-amino-7-phosphonoheptanoic acid (selective inhibitor of NMDA), melatonin (OH and peroxyl scavenger), or NG-nitro-Larginine (nitric oxide synthase inhibitor). These findings suggest that activation of NMDA receptors and nitric oxide synthase and free radical formation may contribute to CN- or NMDA-induced

189 neurotoxicity. Yamamoto and Tang (1996a) found that the incidence of CN-induced convulsions was reduced by preinjection into the cerebral ventricles of carbatapentane (a glutamate release inhibitor) or s.c. melatonin. Predosing with melatonin abolished cerebral lipid peroxidation (Yamamoto and Tang, 1996b). An increase in peroxidized lipid in rat brain cortex slices following incubation with KCN was prevented by omission of Ca++ from the incubating medium or treatment with the Ca++channel blocker diltiazem. The findings suggest that both free radical formation and increased glutamate release may contribute to CN-induced neurotoxicity. Cassel and Persson (1985) demonstrated that NaCN caused dose-related decreases in rat striatum dopamine (DA), and Kanthasamy et  al. (1994) noted that mice dosed with KCN developed central dopaminergic toxicity. Additionally, Kiuchi et  al. (1992) showed that perfusion of the striatum with NaCN produced a transient increase in DA release associated with depletion of ATP. In severe CN intoxication, there is decreased dopaminergic activity in the nigrostriatal area (Rosenberg et al., 1989), and lethal CN doses rapidly decrease striatal DA and increase l-dihydroxyphenylalanine (DOPA) (Cassel and Persson, 1992). Although high CN doses decrease striatal DA and produce neuron damage, low CN doses increase striatal DA but without significant neuronal cytotoxicity (Cassel, 1995). In isolated cellular preparations, ionic disruption results in marked cellular acidosis and accumulation of cytosolic Ca++ (Li and White, 1977; Nieminen et al., 1988), and this may result in disturbance of Ca++-activated lipolytic enzyme activity, peroxidation of membrane phospholipids, changes in transmitter release, and effects on other Ca++-modulating cell-signaling systems. Johnson et al. (1986) found that CN increased whole brain Ca++ and demonstrated that centrally mediated tremors were correlated with changes in brain Ca++; diltiazem prevented the increase in Ca++ and decreased tremors. These and other findings indicate that Ca++ may have a significant effect on CN-mediated neurotoxicity. Several other mechanisms have been implicated in CN neurotoxicity. These include increased blood ammonia and brain amino acids (Yamamoto, 1989, 1993); involvement of caspase-3-like activity (Gunaseker et al., 1999); and uncoupling protein-2 activity (Li et al., 2005). A number of animal studies have provided evidence that acute and repeated exposure to CN produces CNS neuropathological changes. These include gray matter necrosis in dogs (Haymaker et al., 1952); degenerative changes in ganglion cells and Purkinje cells in dogs (Herrting et  al., 1960); degenerative changes in cerebrocortical neurons and cerebellar Purkinje cells in rats (Smith et al., 1963); necrosis of the optic nerve and corpus callosum (Hirano et al., 1967; Lessel, 1971); and leukoencephalopathy (Brierley et al., 1977; Funata et al., 1984). Several human case reports have described the neurological and neuropathological sequelae of acute CN poisoning. Some representative examples of acute and delayed effects are as follows. Finelli (1981) described a 30-year-old male who attempted suicide with CN, and 14 months later developed choreiform movements and dysdiadochokinesis of the left hand. Sixteen years after the incident, mental status was normal, but he was mildly dysarthric and had decreased limb muscle tone and mild athetoid movements in the upper limbs. Computerized axial tomography (CAT) showed bilateral infarction of the globus pallidus and left

190 cerebellar hemisphere. Utti et al. (1985) described an 18-year-old male who swallowed KCN and after 4 months, developed generalized rigidity and bradykinesia with intermittent resting and postural tremor in the arms. Autopsy (after an overdose of imipramine and alcohol) at 18 months after the original poisoning incident revealed lesions in the globus pallidus and widespread lacunae in the striatum. Varnell et al. (1987) described two cases of lethal CN poisoning due to ingestion of adulterated Excedrin capsules. A CAT scan carried out within 3 h showed diffuse cerebral edema with diffuse loss of white–gray discrimination. Borgohain et al. (1955) described a 27-year-old female who attempted suicide with KCN and subsequently developed persistent generalized dystonia. Cranial CAT demonstrated bilateral putaminal lucencies, and magnetic resonance imaging (MRI) showed sharply delineated lesions corresponding to the two putamina. Grandas et al. (1989) described a man who became comatose after ingesting NaCN, and after regaining consciousness, he had reduced speech and loss of balance. During the subsequent years, dystonia and Parkinsonism developed, and CAT revealed lucencies in the putamen and external globus pallidus. Feldman and Feldman (1990) described a 28-year-old man who swallowed KCN and subsequently developed Parkinsonian signs, including micrographia and hypersalivation. Lovecchio et  al. (2006) described cranial CAT scan findings in a 43-year-old man who swallowed CN in a suicide attempt. He collapsed, had a grand mal seizure, became apneic, and died within 24 h despite sodium nitrite–thiosulfate treatment; blood CN was 167 mg dL−1. The CAT scan showed diffuse hypodensity involving the brain and cerebellum with a small fourth ventricle, cisternal space effacement, and abnormal density of the basal ganglia. The authors regarded the findings as consistent with diffuse cerebral and cerebellar edema with impending brainstem herniation. Further details and additional case reports have been given by Ballantyne and Salem (2005), Hantson and Duprez (2006), and Isom and Borowitz (2015).

12.9.2 Cardiotoxicity In experimental acute CN toxicity, high concentrations of CN are found in myocardium (Ballantyne, 1983a, 1984), and there is a significant inhibition of cytochrome oxidase (Ballantyne and Bright, 1979). Direct evidence for cardiotoxic and pathophysiological effects on the myocardium comes from morphological, biochemical, and physiological studies in animals and from human clinical observations (Ballantyne and Salem, 2005; Ballantyne et  al., 2006; Fortin et  al., 2010). Several investigators have discussed ECG changes (Fortin et al., 2010, 2015; Leimdorfer, 1950; Susuki, 1968; Wexler et  al., 1947). Ultrastructural changes in cardiac myocytes have been seen following KCN (Susuki, 1968). O’Flaherty and Thomas (1982) found an increase in cardiospecific creatinine phosphokinase following exposure to HCN vapor. Studies by Baskin et al. (1987) suggested that CN exerts an initial effect on β-adrenergic receptors and reduces myocardial contractility through inhibition of cytochrome oxidase.

12.9.3 Vascular Toxicity On the isolated aorta, CN can cause either contraction or relaxation depending on the CN concentration and the species investigated (Robinson et  al., 1984, 1985a, 1985b).

Chemical Warfare Agents Robinson et  al. (1985b) found that ouabain and verapamil enhanced aortic strip contractions, but atropine, pyrilamine, 2-bromolysergic acid diethylamide, and phentolamine did not alter contractions. In contrast, 4,4′-diisothiocyano-2,2′stilbenedisulfonic acid (DIDS) or chlorpromazine partially reduced strip contractions. These findings may have the following relevance: (1) CN-induced vascular contractions are probably not a consequence of stimulation of muscarinic, serotoninergic, or α-adrenergic receptors and (2) if effects on the coronary arteries are similar to those on the aorta, then hypoxia-induced depolarization could enhance CN-induced coronary artery vasoconstriction. Paulet (1961) found evidence in animal studies for cardiac failure in acute CN intoxication and implicated the following major factors: direct myocardial toxicity, central vagal stimulation, and inhibition of central sympathetic activity. Using intra-aortic injections of NaCN in dogs, Krasney (1971) found that CN produced abrupt increases in cardiac output, cardiac rate, and arterial blood pressure, but systemic vascular resistance was unchanged. These and other findings (Krasney, 1970; Krasney et al., 1966) suggest that the major sites for circulatory responses to CN are outside the sinoaortic reflexogenic zone and probably lie in the CNS.

12.9.4 Respiratory Effects Typical early respiratory signs in acute CN intoxication are tachypnea and hyperpnea, resulting in an increased tidal volume; this clearly may increase the inhaled dose of HCN vapor (Eisenkraft et al., 2015). This is believed to result from stimulation of aortic and carotid chemoreceptors following the accumulation of acid metabolites (Comroe, 1974). Glomus cells, secretory cells in apposition with afferent nerve endings in chemoreceptor zones, probably play a role in the hypoxic transduction process (Ponte and Sadler, 1989; Verna et al., 1975). Additionally, several studies such as infusion of CN into the upper aorta indicate that in addition to the aorticocarotid chemoreceptor stimulation, CN may stimulate ventilation by other mechanisms (Levine, 1975). Depression of respiration by CN may be mediated through the brain, notably the ventral medulla. CN exerts site-specific qualitatively different responses along the neuraxis with respect to respiratory activity; at the ventral medullary surface, it causes respiratory depression, but acting on spinal neurons, it causes increased respiratory motor activity (Haxhiu et  al., 1993). The intermediate ventral medullary areas receive inhibitory influences from carotid chemoreceptors (Carroll et al., 1996).

12.9.5 Thyroid Gland Toxicity Animal studies and clinical observations on occupationally exposed workers indicate that CN, or its detoxification product SCN, may adversely affect thyroid gland function. Most animal studies in which CN is given by the p.o. route show an increase in resorption vacuoles in follicular colloid and sparse colloid, but reported changes in thyroid hormone levels are variable (Kamalu and Agharanya, 1991; Philbrick et al., 1979; Sousa et al., 2002). Several studies in occupationally exposed humans suggest that CN exposure results in impaired thyroid gland function (Banerjee et al., 1997; Blanc et al., 1985). Some investigators believe the effects are mediated by SCN, which inhibits both the

191

Cyanides uptake and the use of iodine by the thyroid gland (Ermans et al., 1972; Fukayama et al., 1992).

12.9.6 Developmental and Reproductive Toxicity Relatively few studies have been conducted. Developmental studies have indicated that CN causes maternal toxicity and is embryofetotoxic and teratogenic (Doherty et  al., 1982; Frakes et al., 1986; Singh, 1982). The limited numbers of reproductive studies that have been undertaken suggest that CN does not produce significant effects on reproductive performance (Hébert, 1993; Tewe and Maner, 1981a,b).

12.9.7 Genotoxicity There is limited information on genetic toxicology studies of CN. In most studies, CN does not cause reverse mutations in Salmonella typhimurium with or without metabolic activation (DeFlora, 1981; Hébert, 1993), and only one study has shown HCN to be marginally mutagenic in S. typhimurium strain TA100 (Kushi et  al., 1983). A DNA repair test in Escherichia coli WP67, CM871, and WP2 with KCN was negative (DeFlora et al., 1984). KCN induced both time- and dose-dependent DNA fragmentation with cytotoxicity in rat thymocytes in vitro. CN also induced DNA damage in hamster kidney cells (BHK-21) in vitro, but unlike with thymocytes, internucleosome DNA fragmentation was not observed (Bhattacharya and Rao, 1997). The pathogenesis of DNA fragmentation was studied using an A549 human epithelial-like lung carcinoma cell line treated with KCN (Vock et  al., 1998). The induction of double-strand breaks by KCN was only observed after cell viability was reduced to less than 60%, indicating that double-strand breaks were the result of extragenomic damage as a secondary effect of high toxicity combined with cytolethality. CN did not induce DNA strand breaks in a culture of mouse lymphoma cells (Garberg et al., 1988).

12.9.8 Oncogenicity In a chronic study with rats given diets containing 0.07 or 0.09 mg kg−1 HCN for 104 weeks, there were no effects on growth rate and no histopathological findings (Howard and Hanzal, 1955). Rats fed diets containing 1.5 g kg−1 KCN for 11.5 months (~30 mg kg−1 day−1) did not show any oncogenic pathology (Philbrick et al., 1979).

12.10 Clinical Toxicology of Human Cyanide Poisoning In general medical practice, acute CN intoxication is a rare, potentially fatal, but treatable condition. It may be encountered in several specific situations: these include smoke inhalation, occupational exposure to CNs, or metabolic release following systemic absorption of laetrile, amygdalin, or cyanogenic glycosides of plant origin and aliphatic nitriles (Ballantyne, 1987c; Beasley and Glass, 1998; Geller et  al., 1991; Hall et  al., 1986; Meyer et al., 1991). Sodium nitroprusside therapy may also result in CN intoxication (Hall and Rumack, 1987).

12.10.1 Clinical Presentation The initial signs and symptoms are generally nonspecific. Their latency to onset, their severity, the number present, and the sequence of appearance depend on a number of factors, which include route of exposure, exposure concentration (dose), duration of exposure, physical mode of presentation of CN, rate of absorption, and total absorbed dose (Ballantyne and Salem, 2005; Ballantyne et  al., 2006). If exposure is to low concentrations (doses), then there may be a slow onset and progressive sequential appearance of signs and symptoms. In contrast, if there is massive exposure, then collapse is usually rapid in onset, and death may follow promptly (Ballantyne and Salem, 2005). Overall, symptoms that may be encountered include weakness, fatigue, headache, anxiety, restlessness, palpitations, confusion, dizziness, vertigo, dyspnea, nausea, nasal irritation (respiratory exposure), and precordial pain. Physical signs may include an initial increase in breathing rate and depth (later becomes slow and gasping), vomiting, diarrhea, facial flushing, transient hypertension followed by hypotension, tachycardia followed by bradycardia, cardiovascular collapse, epistaxis, generalized convulsions, loss of consciousness, urinary and fecal incontinence, cyanosis, areflexia, mydriasis, sluggish or unreactive pupils, apnea, decreased arteriovenous (A-V) O2 difference (visible on retinoscopy), non-cardiogenic pulmonary edema, decerebrate rigidity, and cardiac arrest. Due to the mechanism of toxicity by cytochrome c oxidase inhibition causing a cytotoxic hypoxia, cyanosis is not usually a presenting sign. However, if present, it indicates that a stage of apnea and circulatory collapse has been reached. Initial symptoms and signs are generally nonspecific and may include CNS stimulation (headache, giddiness, and anxiety), hyperpnea, slight hypertension, and palpitations. These early signs may be confused with hyperventilation or simple anxiety. In contrast, massive exposure doses may cause rapid collapse, prompt onset of convulsion and coma, and rapid death. The odor of HCN, likened to that of bitter almonds, has been stated to be an important clinical clue in the recognition of acute CN poisoning. A detection range of 0.5–5.0 ppm has been suggested (Kulig and Ballantyne, 1993). However, it needs to be noted that some individuals are not able to detect the odor of HCN by olfaction. This CN anosmia, present in 2–45% of different ethnic populations, is probably a genetically determined trait (see detailed discussion in Ballantyne and Salem, 2005 and Ballantyne et al., 2006). Late complications of acute CN poisoning may include acute renal failure (Mégarbane and Baud, 2003), rhabdomyolysis (Brivet et al., 1983), CNS degenerative changes and diffuse cerebral edema (Fligner et al., 1987; Varnell et al., 1987), and neuropsychiatric manifestations, including paranoid psychosis (Kales et al., 1997).

12.10.2 Investigation and Confirmation of Poisoning Confirmation of suspected acute CN poisoning should include the following investigations: ECG, plasma lactate, serum electrolytes (with calculation of anion gap), blood glucose, arterial ketone body ratio (AKBR), pulse oximetry, arterial blood gas analysis, chest radiography, and blood CN analysis. Morphological neuroimaging may assist in the early detection of central neurotoxicity

192 (Hantson and Duprez, 2006), with MRI giving better definition than CAT scan. The ECG may show increased T-wave amplitude, shortening of the S-T segment, third-degree heart block, supraventricular or ventricular tachycardias, A-V block, and ischemic myocardial changes (Ballantyne et al., 2006; DeBush and Seidel, 1969; Lee-Jones et al., 1970). Plain chest radiography may demonstrate pulmonary edema. Lactate acidosis is an important biochemical feature of acute CN intoxication and if marked, is accompanied by an elevated anion gap (Baud et al., 1991; Graham et al., 1977; LaPostolle et al., 2006; Peddy et al., 2006). The AKBR (acetoacetate/β-hydroxybutyrate), which reflects the redox state of hepatic mitochondria, is a useful measure for the progress of treatment of CN poisoning (Nakatani et al., 1993). Arterial blood O2 analysis and A-V O2 differences often demonstrate high arterial blood PO2, increased venous blood PO2, and reduced A-V O2 difference. Although most clinical pathology laboratories do not have the capability to undertake rapid quantitative analysis for CN, blood samples should be collected into tightly closed tubes for subsequent analysis. Blood should be collected as soon as possible after intoxication and analyzed promptly to reduce potential artifacts (Ballantyne, 1975, 1976, 1987b; Kulig and Ballantyne, 1993). The confirmation and analysis of CN, CN analogs, thiocyanate, ATCA, and CN–protein adducts in biological matrices and tissues is a valuable tool for forensic, clinical, research, law enforcement, and veterinary purposes (Logue et al., 2010). Methods of analysis include spectrophotometry, fluorescence, chemiluminescence, electrochemistry, gas chromatography (GC), liquid chromatography (LC), flow injection analysis (FIA), capillary electrophoresis (CE), and atomic absorption (AA). There are many factors that influence the choice of which biomarker and/ or analytical technique should be used. Considerations include cellular absorption and detoxification kinetics, sampling and analysis time, sample storage time and conditions, sample matrix, interferences, sensitivity, available instrumentation and equipment, expertise, and cost. Careful sample preparation and storage of biological samples containing CN or its metabolites is a key element in producing accurate results. A significant problem in the analysis of CN and thiocyanate is their interconversion, which can occur during sample preparation and storage and can lead to inaccurate results. Thus, methods have been developed to prevent artificial formation of CN during the storage of collected samples. Recent strides to develop accurate, rapid, and cost-efficient methodologies and technologies have recently been summarized by Jackson and Logue (2017).

12.10.3 Management of Acute Cyanide Poisoning Acute CN intoxication is an acute medical emergency. It is important that those who may encounter a suspect case should seek appropriate advice and guidance, ideally initially from the regional Poison Control Center, who may refer the enquirer to a specialist unit for diagnostic criteria and appropriate treatment, including the necessary antidotal treatment.

12.10.3.1 First Aid Measures First aid should be conducted by appropriately trained personnel who have the necessary background knowledge to understand the basis for primary care. Treating first aid and medical staff should wear protective equipment: ideally, skin protection

Chemical Warfare Agents (including impermeable gloves) and an absorbent filter or preferably, air-supplied self-contained positive pressure breathing (Ballantyne and Salem, 2005). The first aider should ensure that the affected individual is decontaminated (water flushing of skin) and transferred to a clean environment. Contaminated clothing should be kept in isolated double plastic bags. If breathing has stopped or is labored, then artificial ventilation may be required by the Holger–Nielsen method or using a mask with manual ventilation. To avoid secondary intoxication, mouthto-mouth ventilation should not be used. If CN has been swallowed, a dose of activated charcoal (1 g kg−1) may be useful. If breathing is difficult, cylinder O2 should be supplied by mask. If ampules of amyl nitrite are available, and the subject is breathing, a vial should be broken and placed under the nose; this may be repeated every 3–5 min if necessary (details in Ballantyne and Salem, 2005). The vasogenic effect of amyl nitrite may be a factor in its antidotal effect. External cardiac massage may be required if cardiac arrest occurs.

12.10.3.2 General Medical Management A physician should supervise and ensure that the following supportive medical management procedures are conducted:

1. A large-bore i.v. is inserted for therapeutic purposes. 2. There is a patent airway, and ventilation is adequate. This may necessitate employing mechanically assisted ventilation. 3. Clinical experience indicates that the use of O2 may be a valuable adjunct to treatment. Normobaric O2 alone may have minimal effect, but it acts synergistically with other antidotes (Beasley and Glass, 1998; Holland and Koslowski, 1986; Kulig and Ballantyne, 1993; Litowitz, 1987). It is not certain whether hyperbaric oxygen (HBO) offers any clinical advantage over normobaric O2 (Gorman, 1989; Kulig and Ballantyne, 1993; Salkowski and Penney, 1994), but when antidotal treatment is refractory, HBO should be considered as a treatment option (Goodhart, 1994). The AKBR is a useful measure of the efficacy of treatment (Nakatani et al., 1993); it is closely correlated with electron transport and O2 use. During recovery from CN intoxication, as the PvO2 decreases, the AKBR increases. 4. It is important to reverse the acid–base imbalance of lactate acidosis by the i.v. infusion of bicarbonate. 5. Cardiovascular complications may require the administration of atropine, i.v. fluids, and vasopressors. 6. In addition to periodic monitoring of physical signs, blood pressure, and serial measurements of blood chemistry and arterial blood gas analyses, there should be continual monitoring of the ECG and pulse oximetry. However, pulse oximetry may be unreliable following MetHb-inducing antidotes.

12.10.3.3 Antidotes Acute CN poisoning is one of the few chemical-induced intoxications for which specific antidotes are available. Indeed, a large number of antidotes of different structure and mode of action have

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Cyanides been studied, although only a relative few have been approved for clinical use. Since there is variability in the efficacy of different antidotes, and since some may have a high risk:benefit ratio, a decision as to what antidote is to be used should only be taken by a general or emergency room treating physician after consultation with a Poison Control Center. The major antidotes that have been used for the antidotal treatment of human cases of acute CN poisoning are listed in Table 12.6 and briefly reviewed in the following. Detailed discussions on the antidotal treatment are to be found in Marrs (1987, 1988), Meredith et al. (1993), Mégarbane and Baud (2003), Mégarbane et al. (2003), Gracia and Shepherd (2004), Ballantyne and Salem (2005), Ballantyne et al. (2006), and Hall et al. (2015). Sulfur Donors. These enhance the endogenous sulfurtransferase mechanisms for the detoxification of CN to SCN. Sodium thiosulfate is often used in combination with other antidotes having different modes of antidotal action; for example, with sodium nitrite or 4-dimethylaminophenol (4-DMAP). As a generalization, sodium thiosulfate is used as a supplementary treatment on the basis that it is slow acting, possibly due to limited penetration into mitochondria. Recent reports have demonstrated that the sulfur donor dimethyl trisulfide (DMTS) shows promise of being more efficient than sodium thiosulfate in countering CN toxicity (Rockwood et al., 2016) using both KCN injection and HCN inhalation models of CN poisoning in mice (DeLeon et al., 2018). Importantly, DMTS does not require intravenous administration and is efficacious when administered intramuscularly. Sulfanegen, another sulfur donor, has also shown promise as a CN countermeasure (Patterson et al., 2016). MetHb Generators. These are indirectly acting CN antidotes that cause the formation of MetHb, which binds, and sequesters, CN as CNMetHb. MetHb generators include sodium nitrite, 4-DMAP, and p-aminopropiophenone (PAPP). A disadvantage of MetHb generation is the impairment of O2 transport, and a drawback of nitrite MetHb generators is their adverse effects on the cardiovascular system due to induced vasodilation and hypotension. MetHb levels should be kept below 40%. The MetHb generator 4-DMAP probably acts more rapidly in MetHb generation than sodium nitrite (Weger, 1983), but PAPP is slower in antidotal action than 4-DMAP, although both have been experimentally shown to be effective as CN antidotes (Bright and Marrs, 1987). Although there is consensus that these antidotes TABLE 12.6 Antidotes Used Clinically in the Treatment of Human Acute Cyanide Poisoning Antidote

Mode of Antidotal Action

Sodium thiosulfate

a,b

Sodium nitritea,b Stroma-free MetHbc 4-Dimethylaminophenol Dicobalt edetate Hydroxocobalamin (Cyanokit®) a b c

Enhancer of endogenous transulferase activity MetHb generator; vasodilator; elevates nitric oxide Direct formation of CNmetHb MetHb generator Direct binding agent Binding agent with formation of cyanocobalamin

Packaged together as Nithiodote®. FDA approved. MetHb = methemoglobin.

do lead to the formation of MetHb and that this process does counter CN toxicity, there is a growing recognition that this class of antidotes may actually have a different primary mechanism of action. Important work has been done with drugs such as isosorbide dinitrate, which does not lead to the formation of MetHb but is highly effective against CN poisoning (e.g., Lavon, 2015). And while it is clear that those compounds traditionally referred to as MetHb generators/formers (such as sodium nitrite) do indeed lead to the formation of MetHb, and this state will effectively reduce the CN load, it has been suggested that this is likely a secondary mechanism. Peterson and colleagues, as well as Lavon and colleagues, have provided a strong argument that the vasoactive properties and effects on nitric oxide can better explain the rapid onset of countermeasure activity of nitrites and similar “MetHB generators/formers” (Cambal et al., 2011, 2013; Lavon, 2015; Lavon et al., 2017; Pearce et al., 2008). Cobalt Compounds. These act as direct CN-binding agents. Dicobalt edetate has been used in some countries, but there have been reports of severe adverse effects: these have included vomiting, facial edema, urticaria, collapse, chest pains, anaphylactic shock, hypotension, cardiac arrhythmias, and convulsions (Hilmann et al., 1974; Naughton, 1974; Tyrer, 1981). It has been recommended that dicobalt edetate should be used only with clearly established cases of CN poisoning and then, with caution (Meredith et al., 1993; Pontal et al., 1982). Hydroxocobalamin (approved by the Food and Drug Administration [FDA] in 2006 and marketed as Cyanokit®) binds to CN to form cyanocobalamin and does not interfere with O2 transport. Its antidotal efficacy has been confirmed based on animal studies and clinical experience with human acute CN intoxication (Borron and Baud, 1996; Borron et  al., 2006; Dart, 2006; Froyshov et  al., 2006). The recommended use of hydroxocobalamin is based on its demonstrated antidotal action and on its low toxicity (Borron and Baud, 1996; Pontal et  al., 1982). Studies with human volunteers together with postmarketing experience in CN-exposed patients indicate a high safety profile for hydroxocobalamin (Borron et  al., 2005; Forin et  al., 2006; Forsyth et  al., 1993; Uhl et  al., 2006). A few cases of acute allergic reaction have been reported (Branco-Ferreira et  al., 1997; Heyworth-Smith and Hogan, 2002; Uhl et al., 2006; Vidal and Lorenzo, 1998). In some countries, formulations containing several grams of hydroxocobalamin are available. The usual adult antidotal dose is 5 g, and the pediatric dose is 70 mg kg−1 (Ballantyne et al., 2006). Cobinamide has been shown to be an effective intravenous or intramuscular CN countermeasure in a number of different animal models, including rodents, rabbits, and swine (Bebarta et  al., 2014; Brenner et  al., 2010; Chan et  al., 2010). Cobinamide has also shown utility in detecting CN in human blood (Swezey et al., 2013). The current treatment of choice based on efficacy and low therapeutic risk should be hydroxocobalamin, which may be used in combination with sodium thiosulfate. On a global basis and for emergency mass use, we stress the need for an international agreement on the management of CN poisoning, particularly with the antidote of choice for stockpiling. However, emerging countermeasures that can be administered intramuscularly show significant promise, particularly for mass exposure scenarios when intravenous administration is not practical. The criteria for choice of an anti-CN antidote have been given by Ballantyne et al. (2006).

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12.11 Cyanides in Military and Potential Terrorist Settings 12.11.1 Chemical Warfare Considerations A standard military classification of CW agents refers to CNs as lethal blood agents; HCN is coded AC (HMSO, 1987; Maynard, 1999). In the context of CW operational situations, CN is likely to be dispersed atmospherically and would be used for either low-concentration mental and physical incapacitations or highconcentration lethal objectives. For the former, it is well known that exposure to HCN vapor produces a disturbance of consciousness and perception, and combined with muscle weakness and ataxia, this would cause mental and physical incapacitation of troops and a resultant reduction in their ability to conduct military tasks. As a battlefield lethal agent, HCN has been regarded as being of potential strategic usefulness, because it exists as a colorless vapor with effects that have a short latency to onset. As noted earlier, the concentration required to produce lethality within 1–5 min is in the order of 500–4000 mg m−3. HCN was used in World War I by the French, with the first employment of HCN shells being on the Somme on July 1, 1916 (Prentiss, 1937). During World War II, CN was used as an agent of genocide in the German concentration camps (in the form of Zyklon B), and the Japanese allegedly used CN against China. Iraq also supposedly used CN against the Kurds during the 1980s. HCN has an affinity for oxygen and is flammable, and hence, it is not efficient when dispersed by artillery shells (RAMC, 2002). Additionally, due to its low vapor density, low molecular weight, and ready diffusibility, there are problems in achieving lethal concentrations of HCN against unprotected troops in open battlefield conditions. In this respect, HCN acts as a nonpersistent CW hazard. HCN is only strategically useful as a lethal agent for localized and circumscribed circumstances. However, less-than-lethal concentrations of atmospheric HCN cause mental and physical incapacitation, which may be of operational significance by reducing the efficiency and determination of opposing troops. Concentrations of the order of 100–200 ppm would be necessary to induce such incapacitating conditions. Cyanogen bromide and cyanogen chloride were also used in World War I to liberate HCN on the battlefield, but additionally, these halides are strongly irritant to the eyes and other mucosal surfaces. Medical management is as for HCN, but pulmonary irritation may also need attention.

12.11.2 Terrorist Implications A terrorist incident definitionally covers a situation in which there is a desire on the part of the terrorists to produce a physical, chemical, or psychological assault on an organized (law-abiding) society to modify popular attitude, opinion, legislation, or political dictate by the use of procedures designed to make the terrorist motivation fearfully known to the appropriate members or sectors (Ballantyne et al., 2006). The use of chemicals in terrorist operations has been discussed widely and accepted as being likely to occur in some situations. Generically, the chemicals that could be used include irritant and disorientating materials, psychogenic substances, and lethal agents. It is assumed that the majority of terrorists will be attracted to chemicals that are cheap

Chemical Warfare Agents to purchase or synthesize, can be readily obtained or manufactured, are capable of causing mass incapacitation or mortality, are comparatively easy to handle, have high biological activity with short latency to onset, and can be purchased (along with dispersal systems) without arousing a high degree of suspicion. Specific chemicals that have been used, or are considered likely to be used, include organophosphate anticholinesterases (including commercially available pesticides) as in the Tokyo subway attack (Okudera et al., 1997; Simon, 1999), commercially available toxic and irritant materials, CNs, biotoxins, including ricin and botulinum (Tendler and O’Neill, 2005), and pulmonary agents, such as chlorine (Rodriguez-Llanes et al., 2018). Random small-group or individual terrorist activity has included the repacking of medicinal capsules with CN and returning them to the shelves of shops (Brahams, 1991; Centers for Disease Control, 1991), although this has now been made impossible by the use of tamper-proof wrappings. Intramuscular injection of HCN from a concealed syringe was considered as a possibility for assassination (Ballantyne et al., 1972). A notorious example of mass killing with CN was the Jonestown massacre of cult members (Thompson et  al., 1987). There have been several documented accounts of the existence of plans by some international terrorist groups for the possible use of CN in likely major terrorist activities (Ballantyne et  al., 2006; DesLauriers et al., 2006). In the United States, various governmental agencies, including the Centers for Disease Control and the Department of Homeland Security, consider CNs among the most likely agents to be used for chemical terrorism (Khan et al., 2000; NTARC, 2004). Due to its low molecular weight, low vapor density, and diffusibility, HCN vapor is most likely to be most effective when used in enclosed and confined spaces. It could be generated from cylinders of the liquid or from devices (likely to be crude) for mixing NaCN or KCN with acidic fluids, one of which was invented by terrorists for possible use in the New York City subway system (Suskind, 2006). Cyanides are also likely to be used as solid salts or concentrated solutions for the contamination of various domestic, commercial, or other publicly available sources of swallowed materials, such as in pharmaceutical preparations, in bottled drinks, or by injection into food stuffs or food containers. A recent example, as of this writing, occurred during the London, 2012 Summer Olympics. Terrorists, linked to Al Qaeda, were planning a CN attack during the games. The intent was to mix CN with hand cream to maximize dermal exposure. Details of the threat were listed on websites that described formulations and complete instructions to produce the mixture. The websites had links to six individuals associated with alQaeda terrorists using a false identity. One terrorist stated, “It is a good idea and you need to plan well.” Another email stated, “It’s time to prepare for the event, as once again they are interfering with innocent Muslims.” Every available English MI5 agent was deployed during the event to keep the public safe. It was the largest operation since World War II and had counter-espionage agents working 24/7 to focus on preventing a terrorist attack during the games (Allen, 2012; Whitehead, 2012). Other examples include attempts to poison public water supplies by dumping CN into reservoirs are not feasible because of the massive amounts that would be required at source. Therefore, to be effective, CN would need to be introduced into water supplies at points close to the consumer, such as storage tanks.

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Cyanides Preparedness for major chemical terror incidents with CN has been discussed in detail by Ballantyne et  al. (2006), who note the particular need for the following: (1) constant liaison between military, civil, and political “intelligence” agencies to keep informed on the likelihood, nature, possible location, and timing of potential incidents, (2) continually updating on methods likely to be encountered in terrorist actions and appropriately revising methodologies to deal with these, (3) maintaining equipment (including protective equipment) and medical management needs, (4) constant training sessions for all scenarios, (5) having the technology and skills for analytical recognition of threat chemicals, in particular for rapid and on-site use, and (6) ongoing discussions and frequent training exercises between security organizations, police, rescue workers, emergency medical responders, and health-care providers. The U.S. Centers for Disease Control and Prevention (CDC) has prepared strategic recommendations for, and responses to, biological and chemical terrorism (Khan et  al., 2000). They identified the following five main activities that should be undertaken by public health organizations to enhance preparedness for terrorist chemical attacks: 1. Epidemiological capacity should be enhanced for detecting and responding to chemical attacks. 2. Awareness of chemical terrorism should be increased among emergency medical service personnel, police officers, and firefighters. 3. Antidotes should be stockpiled. 4. Bioassays should be developed and provided for the detection and diagnosis of chemical injuries. 5. Educational materials should be prepared to inform the public during and after a chemical attack. In respect of potential CN terrorism, we believe that the following three critical items should be added to the CDC recommendations. First, before antidotes are stockpiled, there should be international agreement on the most appropriate antidote (or combinations) for the treatment of acute CN poisoning. It is our opinion that hydroxocobalamin is the optimum choice. In the context of mass casualty situations with terrorist release of CNs on the public, the chosen antidote should be readily available, effective, easy to administer (even by responders with limited training), and nontoxic, and should not adversely interact with other antidotes (Thompson, 2004). Second, with respect to CN analyses, there is a requirement for portable equipment that is specific and at least semiquantitative, which can be used for on-site reliable bioidentification of CN intoxication. There is also a need for a reliable and sensitive environmental method for the instantaneous measurement of HCN concentrations and ideally, continuous monitoring with automatic warning devices for installation in sites with a potential for HCN attack. Third, educational materials should be made immediately available for distribution to the general population, so that they can be prepared for what to expect in the event of a CN terrorism event. Due to the high pressure with routine duties in health-care facilities, it is considered appropriate that hospitals should appoint a small team, representing relevant specializations, who have the responsibility for the development and organization of

arrangements in the event that there is a terrorist incident in their catchment area. This planning and response team should develop guidelines for dealing with the immediate and medical management of an incident. These guidelines should cover, across the board (chemical, biological, and nuclear), the following: (1) ensuring that the necessary expertise can be summoned promptly, (2) triage procedures, (3) first aid and medical management for all likely situations, (4) ensuring that there are sufficient available supplies of equipment and therapies for use at the incident site and in the hospital area, (5) arranging for decontamination sites and procedures, including in the hospital grounds, (6) ensuring immediate and free lines of communications with other advisory groups (e.g., rescue groups, Poison Control Centers), (7) ensuring that appropriate protective equipment is available for use by staff who could be exposed, and (8) organizing educational and local training sessions, and arranging for practical joint training sessions with local responsible security and rescue services. In the context of the possible use of CN in a terrorist incident, specific considerations and urgent needs are as follows (Ballantyne et al., 2006):

1. A specific, accurate, and reliable portable rapid blood test should be developed to screen for the presence of toxicologically significant concentrations of CN in blood to permit rapid on-site diagnosis. Ideally, the method should differentiate between bound and free CN (Lindsay et al., 2004). 2. Protective equipment is required for those who come into contact with exposed victims and should include impermeable clothing and respiratory equipment (ideally, air supplied). 3. Responders should be made aware of the fact that symptoms, incapacitating signs, and death can occur within seconds to a few minutes after the start of an exposure, and thus, rapid action is required in the context of a well-planned response; this is critical. Speed of recognition of intoxication and appropriate intervention are highly important and lifesaving. 4. First aid and medical management stockpiles for on-site and hospital use should include masks with manual ventilators; oropharyngeal airways; oxygen cylinders with masks; in-date ampoules of amyl nitrite (kept at, or below, 158°C); sodium bicarbonate for i.v. infusion; recommended antidotes (to be administered by a physician). The choice of antidotes should be made in consultation with relevant experts in a Poison Control Center. As noted earlier, there is an urgent need for an international agreement on a rationalized therapeutic approach for acute CN poisoning.

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13 The Structural Biology, Biochemistry, Toxicology, and Military Use of the Ricin Toxin and the Associated Treatments and Medical Countermeasures for Ricin Exposure Terry J. Henderson, George Emmett, Russell M. Dorsey, Charles B. Millard, Ross D. LeClaire, and Harry Salem CONTENTS 13.1 Introduction.................................................................................................................................................................................203 13.2 Structural Biology and Biochemistry of the Ricin Ribosome Inactivating Protein...................................................................204 13.2.1 Crystal Structure of the Ricin Toxin..............................................................................................................................205 13.2.2 Cultivars and Isotoxins of Ricin.....................................................................................................................................206 13.2.3 Cellular Internalization of Ricin....................................................................................................................................207 13.2.4 N-Glycosidase Activity of Ricin....................................................................................................................................207 13.3 Weaponization of the Ricin Toxin..............................................................................................................................................209 13.3.1 The Brief History of Ricin Weaponization....................................................................................................................209 13.3.2 Aerosolization of Ricin.................................................................................................................................................. 210 13.3.3 Polydispersity of Ricin Aerosols.................................................................................................................................... 210 13.4 Inhalation of Aerosolized Ricin.................................................................................................................................................. 211 13.4.1 Aerodynamics of Inhaled Ricin Particles...................................................................................................................... 211 13.4.2 Fate of Aerosolized Ricin in the Respiratory Tract....................................................................................................... 211 13.4.3 Deposition of Aerosol Particles in the Respiratory Tract.............................................................................................. 212 13.4.4 Clearance of Inhaled Particles from the Respiratory Tract........................................................................................... 213 13.5 Signs, Symptoms, and Toxicity of Ricin Exposure.................................................................................................................... 213 13.5.1 Inhalation of Ricin.......................................................................................................................................................... 213 13.5.2 Ingestion and Injection of Ricin..................................................................................................................................... 214 13.6 Diagnosis and Detection of Ricin Exposure............................................................................................................................... 215 13.7 Physical Protection against Ricin............................................................................................................................................... 215 13.8 Inactivation of the Ricin Toxin................................................................................................................................................... 216 13.9 Treatment and Medical Countermeasures for Ricin Exposure................................................................................................... 216 13.9.1 Experimental Vaccines................................................................................................................................................... 216 13.9.1.1 Toxoid Vaccine................................................................................................................................................ 216 13.9.1.2 Ricin Subunit Vaccines................................................................................................................................... 217 13.9.1.3 Neutralizing Epitopes..................................................................................................................................... 218 13.9.2 Experimental Antitoxins................................................................................................................................................ 218 13.9.3 Other Experimental Therapeutics.................................................................................................................................. 219 13.10 Conclusions and Perspectives..................................................................................................................................................... 219 Disclaimer.............................................................................................................................................................................................220 References.............................................................................................................................................................................................220

13.1 Introduction In the wake of recent terrorist-related incidents across the globe, biological agents have become known for the threat they pose to U.S. and Allied militaries as well as the civilian populations they defend. The agents are attractive to both foreign states and terrorists because they are relatively inexpensive to produce and require minimal technical infrastructure. Toward such ends,

aerosolization is recognized to be the most effective means of agent dissemination for inflicting mass casualties. Governments with active offensive biological warfare programs recognized early that pathogens and toxins formulated for protection against environmental stress, sized for optimal pulmonary delivery, and dispersed in a manner that maximizes homogeneity and high-dose exposures were relatively inexpensive to produce and highly effective weapons against military and civilian targets 203

204 (Roy et al., 2010). The governments soon realized that the airborne delivery of appropriately formulated pathogens and toxins was exceedingly difficult to defeat or prevent (Dembek et  al., 2007), largely because they directly target the most susceptible route of entry—the respiratory tract. The biological agents commonly associated with military and terrorist use are aptly classified as biological select agents and toxins (BSATs). Under U.S. law, BSATs are biological agents that have been declared by the U.S. Department of Health and Human Services (HHS) or the U.S. Department of Agriculture to have the potential to pose a severe threat to public health and safety. BSATs include several bacteria, such as Coxiella burnetii, Francisella tularensis, and Yersinia pestis, numerous viruses, such as the coronaviruses, encephalitis viruses, influenza viruses, and viral hemorrhagic fever viruses, and finally, a very large number of biologically derived molecular toxins. These toxins are the metabolic products of living organisms and in contrast to bacteria and viruses, cannot reproduce. A common feature of all such toxins is that minute quantities will exert a pronounced effect on the intended target individuals. Toxins are produced by a vast number of different life forms, ranging from the simplest to the most complex, and each toxin has a distinctive mode of action in conjunction with a characteristic molecular structure and biochemistry. Algal toxins constitute a very diverse group of compounds, ranging from simple ammonia to complex polypeptides and polysaccharides. Dinoflagellates, the causative organisms of red tides and other aquatic blooms, are a source of some potent non-protein toxins such as saxitoxin and tetrodotoxin. The mycotoxins are a wide variety of non-protein substances produced by molds and fungi. Many molds produce more than one toxin, and in several cases, combinations of mycotoxins synergize to enhance toxicity (Weber et al., 2005). Bacteria produce some of the most potent toxins known, including botulinum neurotoxin from Clostridium botulinum and shiga toxin from Shigella dysenteriae. The leaves, roots, beans, or peas from some plants can contain very potent toxins, with ricin from castor beans and abrin from the jequirity pea as the two most recognized examples. Finally, there are a number of toxins produced by animals, including the non-protein batrachotoxin found in certain species of frogs (the poison dart frog) and a wide variety of peptide and proteins from marine snails (the conotoxins in particular) and the venoms of scorpions and snakes. Undoubtedly, the most widely recognized BSAT is the ricin protein toxin from the castor bean plant Ricinus communis. Both the toxin and the plant have contributed to the welfare, prosperity, and interests of humans throughout history. Some of the earliest records contain references to the castor bean plant, which was known and cultivated by the ancient Egyptians. Oil pressed from castor beans was used as a lubricant and fuel for oil lamps, and the beans were sometimes ingested to treat constipation. Even today, castor oil is still commercially produced in many countries across the globe, with ~1 Mt (metric ton, equivalent to 1000 kg or 2205 pounds) of castor beans harvested annually for castor oil production. The oil is currently used in bath oil products, detergents, lubricants, and dyeing agents, and most recently, in the production of biodiesel fuel. The ricin toxin is easily produced from the mash remaining after castor beans have been pressed for extracting castor oil (Wannemacher et  al., 1992). Initially, the ricin protein was known for its ability to aggregate

Chemical Warfare Agents red blood cells and platelets, and later, the field of immunology was founded by the pioneering research of Ehrlich (1891) with the toxin. By introducing small amounts of ricin into mice, Ehrlich was able to induce specific immunity to the toxin. This seminal work demonstrated conclusively that in response to a challenge, certain serum proteins (antibodies) are produced that afford the host protection against the challenge agent. By virtue of its toxicity, availability, and ease of production, ricin was the first BSAT considered for weaponization by the United States (Cookson and Nottingham, 1969) and other governments. Today, ricin is one of the few BSATs ever to have been used successfully in terrorist-related incidents, including the 1978 attack on the Bulgarian exile Georgi Markov (Crompton and Gall, 1980), the 1980 attack on Boris Korczak in a Tysons Corner, Virginia parking lot (Douglas and Livingston, 1987), and the 2008 incident involving Roger Von Bergendorff in Las Vegas, Nevada (Goldman, 2014). There are also over a dozen other documented events that have occurred in the recent past (JMCNS, 2014). Ricin is a HHS select toxin covered under 7 CFR (Title 7 of the U.S. Code of Federal Regulations) Part 331, 9 CFR Part 121, and 42 CFR Part 73. In this review, we discuss the ricin toxin in detail, with special attention focused on its military use and weaponization, the inhalation toxicology of its aerosols, the signs and symptoms associated with exposure, and the development of therapeutics and medical countermeasures for protection against exposure. As an introduction to the toxin, however, we begin by reviewing its structural biology and the biochemistry relevant to its toxicity.

13.2 Structural Biology and Biochemistry of the Ricin Ribosome Inactivating Protein One of the most extensively studied protein families in biology is that of the ribosome-inactivating proteins (RIPs), a large family of protein toxins and related proteins that includes ricin. The expression ribosome-inactivating protein was introduced to designate plant proteins that could inactivate animal ribosomes, but very similar animal ribosome–inactivating proteins were later also found to occur in certain bacteria and fungi (Endo et  al., 1988). Based solely on their quaternary structures, members of the RIP family have been classified into two major types, referred to as types I and II. Type I RIPs are the most numerous and are synthesized as a single chain protein of ~30 kDa. Their type II counterparts, in contrast, are synthesized as larger precursors and contain a ~30 kDa A-chain (RTA) linked by a disulfide bond to a lectin ricin B-chain (RTB) of similar size (Stirpe and Barbieri, 1986). RTA is responsible for the enzymatic activity of the toxin, while RTB binds to a specific carbohydrate receptor on the host cell’s membrane, allowing RTA to cross the membrane and enter the cell interior. RTB contains two carbohydrate-binding sites (Berman et al., 2000; Robertus, 1991) that allow its aggregation with red blood cells and platelets. Type II RIPs were discovered more than a century ago, when Stillmark (1889) found that the beans of the castor plant contained a toxin, which he named ricin. The toxicity of ricin was initially attributed to its agglutination activity with red blood cells and not its ribosome-inactivating activity. Historically, understanding the

Ricin Structural Biology, Biochemistry, Toxicology, Military Use, and Medical Countermeasures ricin toxin was complicated by the presence of a homologous protein also isolated from castor beans, which was later identified as the R. communis agglutinin. The agglutinin shares considerable sequence identity to the ricin toxin but is much less toxic and is a homodimer of two RTA subunits and two RTB subunits. The co-existence of two highly similar proteins from castor beans, one a potent cytotoxin and the other an effective hemagglutinin, came to light due to improved separation methods and the identification of their respective genes (Olsnes et al., 1974; Roberts et al., 1985; Worbs et al., 2011). There is also a rare class of plant RIPs, sometimes referred to as the type III RIPs, which appear to reflect an unusual adaptation in enzyme production and storage. Type III proteins are synthesized as inactive precursor molecules with a polypeptide insert in the active site region of the N-glycosidase domain (Chaudhry et al., 1994; Walsh et al., 1991). Proteolytic processing of the pro-RIP is required to remove the insert and thereby convert these molecules into active N-glycosidases. Once processed, the type III RIPs resemble the non-toxic versions of type I plant RIPs in overall net charge and catalytic activity (Chen et al., 2001; Nielsen and Boston, 2001). Plant RIPs, including all type I toxins and the A chains of type II toxins, are RNA N-glycosidases capable of hydrolyzing the nitrogen–carbon glycosidic bond of a specific adenosine nucleotide located in the sarcin–ricin domain of the largest ribosomal RNA (Bolognesi et al., 2006). RTB binds d-galactose-terminated receptors on animal cell membranes, which facilitates internalization of the enzymatic RTA. To date, more than 50 type I and ~15 type II RIPs have been identified (Stirpe and Barbieri, 1986). While there is some variation at the N- and C-termini of their respective polypeptide chains, the active sites of the type I RIPs and type II RTA RIPs are well conserved, as are their threedimensional structures (Gasperi-Campani et al., 1985). At present, the role of RIPs in plant physiology is not entirely clear. Based on their variable activity toward heterologous and autologous plant ribosomes, several possible roles have been proposed, including anti-viral activity, anti-fungal activity, herbivore defense, a role in the arrest of cellular metabolism during periods of senescence, and finally, a role as storage proteins (Bolognesi et  al., 2002). Conclusive evidence has been discovered that RIPs not only deadenylate ribosomal RNA but also remove adenine nucleotides from DNA and several other polynucleotide substrates.

13.2.1 Crystal Structure of the Ricin Toxin Ricin is a 64–65 kDa type II RIP isolated exclusively from the beans of R. communis. The toxin’s complete 529–amino acid sequence is shown in Figure 13.1, which also includes information about the hydrogen-bonding motifs of its peptide backbone. Further, a cartoon rendering of the ricin protein backbone based on X-ray crystallographic data (Katzin et  al., 1991; Montfort et al., 1987; Rutenber and Robertus, 1991; Rutenber et al., 1991) is presented in Figure 13.2 to illustrate the toxin’s overall molecular architecture. RTA contains considerable secondary structure, while in contrast, RTB is devoid of any regular secondary structural elements, although its backbone contains a significant number of hydrogen-bonding interactions commonly associated with β-sheets and 310-helices. As indicated in Figure 13.2, the key features of RTB are the two galactose-binding sites and two

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bound mannose oligosaccharides. The two chains are linked together by a single disulfide bond between C259 of RTA and C4 of RTB (designated with downward-pointing triangles in Figure 13.1 and yellow bonds in Figure 13.2). There is significantly more X-ray crystallographic data reported on RTA, largely because of investigations into the chain’s catalytic activity. RTA is a 267–amino acid, mushroom-shaped, α/β globular protein subunit whose structure is largely unaffected by the absence of RTB (Weston et al., 1994). The subunit contains eight α-helices and eight β-strands (Westin et al., 1994; Wright and Robertus, 1987) and can be divided into three domains, shown in Figure 13.3 as domains 1, 2, and 3. Domain 1 contains a six-stranded β-sheet with parallel and anti-parallel strands, while domain 2 contains five helices, the longest of which forms the central core of the protein chain. It is kinked near its C-terminus to allow two key residues for enzymatic activity, E177 and R180 (designated with asterisks in Figure 13.1), to reach the active site. Domain 3 contains the amino acids involved in the RTA–RTB interface and contains one α-helix and two β-strands. The location of the active site (red in Figures 13.2 through 13.4) is in a cleft on the RTA surface and is characterized by several conserved amino acid residues. E177 and R180 are critical to the rate-limiting step of the toxin’s catalytic activity and not substrate binding, as mutation of these residues gives a reduction in kcat but not k m. As shown in Figure 13.4, the invariant residues Y80 and Y123 (designated with crosses in Figure 13.1) are principally involved in substrate binding. X-ray crystallography reveals that adenosine is converted to adenine when complexed with RTA, intercalated between the aromatic rings of the two tyrosine residues (Figure 13.4a); the active site lies at the back of a deep pocket on the RTA surface (Figure 13.4b). Recognition of the rRNA substrate contributes substantially to the efficiency of the depurination reaction. Activity is reduced ~10,000-fold when the rRNA proteins are removed (Endo and Tsurugi, 1987), but substrate binding is not reduced. Mutation of S203 (designated with a bold “x” in Figure 13.1) outside the active site to an asparagine reduces RTA activity (Gould et al., 1991), demonstrating clearly that contacts between the rRNA substrate and amino acid residues outside the active site are critical to RTA catalytic activity. There are no known inhibitors of ricin, presumably because of its exceptional affinity for its natural substrate. At 262 amino acid residues, the RTB subunit, sometimes referred to as the ricin-type beta-trefoil lectin, folds into a bilobal, barbell-shaped architecture (Rutenber and Robertus, 1991). Although RTB has no regular secondary structure, it does display several Ω-loops, which are non-regular secondary structures characterized by the peptide backbone following a loop-shaped course (shown in Figure 13.2 as short, poorly formed, blue helices). The folded structure has two homologous domains, each with a galactose-binding site to allow hydrogen bonding to the galactose and N-acetyl galactosamine carbohydrates typically found on cell surfaces. Each domain is comprised of three copies of a primitive 40–amino acid, folding sub-domain packed around a pseudo-threefold-symmetry axis (Rutenber et al., 1987). The subdomains contribute conserved tryptophan, leucine, and isoleucine residues in a compact hydrophobic core, and it is this tight, threefold binding that likely drives folding of the RTB polypeptide and stabilizes the folded protein’s three-dimensional architecture (Rutenber and Robertus, 1991). The amino acid residues of the

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FIGURE 13.1  The complete amino acid sequence of the wild-type ricin toxin with backbone hydrogen-binding information. Amino acid residues are shown with single letter notations, and backbone hydrogen-bonding patterns indicative of α-helices, 310-helices, and β-sheets are shown with red boxes, red- and white-hashed boxes, and blue boxes, respectively, below their corresponding amino acids. Backbone random coil regions are shown with black horizontal lines below the corresponding amino acids. The two cysteine residues forming a disulfide bond between the two chains are designated with downward-pointing triangles (▼). Key residues for enzymatic activity are designated with asterisks (✱), and those principally involved in substrate binding are designated with crosses (✚). The mutation of RTA residue S203, designated with a bold “x” (✖), to an asparagine residue reduces RTA activity (Gould et al., 1991). The two amino acid residues mutated in the RTA variant RiVax® are designated as white letters in red boxes in the amino acid sequence. Notable epitopes on the RTA surface involved in activity-neutralizing interactions with antibodies or T-cells are designated within the amino acid sequence. monoclonal antibody epitopes involving residues 91–108 (Maddaloni et al., 2004), 178 (Maddaloni et al., 2004; Ready et al., 1991), and 66–69 (Maddaloni et al., 2004) are designated with underlined green letters, a white letter in a dark blue box, and underlined violet letters, respectively. T-cell epitopes involving residues 165–175 (Castelletti et al., 2004; Maddaloni et al., 2004) and 175–183 (Castelletti et al., 2004; Tommasi et al., 2001) are designated with underlined dark blue letters and underlined light blue letters, respectively. Residue 175 is common to both T-cell epitopes and is shown as an underlined red letter. (Adapted from Montfort, W. et al., J. Biol. Chem. 262, 5398, 1987; Katzin, B.J. et al., Proteins, 10, 251, 1991; Rutenber, E. and Robertus, J.D., Proteins, 10, 260, 1991; Rutenber, E. et al., Proteins, 10, 240, 1991; Monzingo, A.F. and Robertus, J.D., J. Mol. Biol. 227, 1136, 1992.)

sub-domains collectively account for ~92% of all the RTB amino acids. Galactose binds each domain in a shallow cleft formed by a three-amino acid kink on the bottom and an aromatic ring on the top. At the back of the cleft, an aspartate side chain forms hydrogen bonds to the C-3 and C-4 hydroxyl groups of galactose, and a glutamine side chain bonds to the galactose C-4 alcohol. The mannose oligosaccharides of RTB are able to bind cells that express mannose receptors (Magnusson et  al., 1993), including cells of the reticuloendothelial system (Simmons et al., 1986).

13.2.2 Cultivars and Isotoxins of Ricin A number of varieties, or cultivars, of R. communis have been identified, and while some share a similar morphology, others are somewhat different in appearance. These properties are also found for the beans produced by the different cultivars, where the ricin protein is synthesized and stored. The existence of two

isotoxins of ricin was discovered 40 years ago, when they were first identified as ricin D and E (Mise et al., 1977). The D isotoxin, however, was purified some 13 years before this discovery (Ishiguro et al., 1964) but was not recognized as an isotoxin of ricin at the time. The two isotoxins have slightly different amino acid compositions and isoelectric points that differ by 1.5 pH units, but their toxicities have been reported to be essentially identical by Mise et al. (1977). Other early investigations of the two isotoxins with various murine cell lines, however, made reference to a ~50-fold higher activity for ricin D. The isotoxins were reported to be specific to the large and small castor bean cultivars, with the ricin D isotoxin found in the large bean while both ricin D and E are found in the small bean cultivars. Other researchers have identified the ricin isotoxins as RCLIII and RCLIV, corresponding to ricin D and ricin E, respectively, with RCLI and RCLII referring to two different R. communis agglutinins (Lin and Li, 1980) and not the toxins.

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13.2.3 Cellular Internalization of Ricin

FIGURE 13.2  Cartoon rendering of the ricin backbone in three-dimensional crystallographic space (Katzin et  al., 1991; Montfort et  al., 1987; Monzingo and Robertus, 1992; Rutenber and Robertus, 1991; Rutenber et  al., 1991). Key features of the toxin are illustrated, including the RTA and RTB subunits in gray and blue, respectively, the active site residues in red, the cysteine residues forming the disulfide bond between the two subunits in yellow, and carbohydrates bound to RTB colored by element (oxygen is red, nitrogen is blue, carbon is gray, and hydrogen is not shown). The bound mannose oligosaccharides and the galactose binding sites of RTB are labeled. Figure created with the program USCF Chimera-1.10.1 (Petersen et al., 2004; Sanner et al., 1996) on a computer running Darwin 64 and the Aqua graphical user interface (Apple Inc., Cupertino, CA).

The enzymatic component of ricin is not active until it is released from the native toxin comprised of both subunits. Isolated RTA has enzymatic activity but lacks cell binding and entry capabilities, while isolated RTB may bind to target cells and even block the binding of the native toxin but is not toxic. The entry of ricin into a eukaryotic host cell begins with the reversible binding of its RTB to cell-surface glycolipids and glycoproteins bearing terminal N-acetylgalactosamine or (1→4)-linked galactose (Moya et al., 1985). It has been demonstrated that 106 –108 RTB proteins can bind to a single cell at one time (Sphyris et al., 1995). The ricin proteins are internalized into cells via endocytosis in either coated or smooth (uncoated) pits and vesicles (Moya et al., 1985). Endocytosis can be via clathrin-dependent or independent mechanisms, which are somewhat reliant on the cell type and polarization status (Iversen et al., 2001; Jackman et al., 1994, 1999; Llorente et al., 1998; Moya et al., 1985; Surety et al., 1996). Once in the cytosol, the vesicles fuse with endosomes that can return some of the ricin to the cell surface by exocytosis, or as shown in Figure 13.5, endosomes can fuse with lysosomes that will destroy the toxin proteins (Sandvig and Olsnes, 1979; Sandvig et al., 2004). Alternatively, the endosomes can carry ricin proteins to the Golgi complex and endoplasmic reticulum (ER) by retrograde transport (Sandvig et al., 2004); see Figure 13.5. Ricin reaching the trans-Golgi network can penetrate its membrane and reach the cytosol or in some cases, return to the cell surface (Sandvig et al., 2004). For ricin proteins reaching the ER lumen, their single RTA–RTB disulfide bond will be reduced, and a partial unfolding of RTA will occur (Kornfeld et al., 1991). The unraveling toxin protein will be translocated across the ER membrane via the Sec61p translocon, following the same pathway as that for incorrectly folded proteins targeted for ER-associated degradation. Ricin proteins escaping this degradation may find their way into the cytosol, where these proteins and others crossing the trans-Golgi network membrane into the cytosol can refold into protease-resistant, enzymatically active structures (Lord et  al., 2003). With a Kcat of 1500 m−1, just a single ricin protein entering the cytosol of a living cell can inactivate enough ribosomes to ultimately result in cell death (Endo et al., 1987).

13.2.4  N-Glycosidase Activity of Ricin

FIGURE 13.3  Cartoon rendering of the RTA backbone in three-dimensional crystallographic space (Katzin et  al., 1991; Montfort et  al., 1987; Monzingo and Robertus, 1992; Rutenber and Robertus, 1991; Rutenber et al., 1991) illustrating its three domains (labeled) and active site residues in red. Figure created with the program USCF Chimera-1.10.1; see the legend of Figure 13.2 for more information.

Catalytic RTA recognizes and binds a highly conserved region in the large 28S rRNA referred to as the sarcin/ricin loop (Rajamohan et  al., 2001), a very short stem-loop structure in domain VII of the rRNA molecule ~400 nucleotides from its 3'-end. Within the stem-loop, the ring of a single adenine nucleotide, A4324, becomes intercalated between the aromatic rings of Y80 and Y123 in the catalytic cleft of RTA (see Figure 13.4a) and is hydrolyzed at the carbon–nitrogen glycosidic bond by N-glycosidase action (Endo et al., 1987). Although the detailed mechanism of this event is not known, Monzingo and Robertus (1992) have proposed a mechanism based on their high-resolution X-ray crystallographic data of substrate analogs in the RTA active site. As summarized in Figure 13.6, the mechanism begins with the sarcin–ricin loop substrate binding the RTA active site with the A4324 ring system intercalating between the aromatic rings of Y80 and Y123 (Figure 13.4a). R180 donates a proton to

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a

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a

b FIGURE 13.4  The binding of adenine in the ricin active site. Adenosine is converted to adenine by RTA and (a) is held in the active site by intercalation with the aromatic rings of Y80 and Y123. E177 and R180 are critical for the catalytic activity of the toxin and (b) line the active site located within a deep pocket on the RTA surface. The ricin backbone is represented as a cartoon rendering or solvent-excluded molecular surface in gray with active site amino acid residues shown in red. The structure of adenine is shown without hydrogen atoms and is colored by element (nitrogen is blue and carbon is gray) along with its solvent-excluded molecular surface illustrated with a mesh surface. (The figures were created from the crystallographic coordinates of Carra et al. (2007) using the program USCF Chimera-1.10.1; see the legend of Figure 13.2 for more information.)

N-3 of the A4324 ring system to break the bond between N-9 of the ring system and C-1′ of the A4324 ribose (Figure 13.6a). This bond cleavage leaves cation character on the ribose ring in the form of an oxycarbonium ion and gives anion character to the A4324 ring system (Figure 13.6b); the oxycarbonium ion should have sp2 character at C-1′ (indicated in Figure 13.6b by the partial double-bond character with O-4′). The bond-forming process (represented in the path between Figure 13.6b and c) begins with R180 donating a proton to N-3 of the adenine ring system to increase the basicity of the side chain. This, in turn, acts to pull a proton from a water molecule, creating hydroxide character to assist solvent attack on ribose and ultimately, a neutral ribose (Figure 13.6c). The figure shows the A4324 product with hydrogen at N-3, but this will readily tautomerize in solution to the more stable form with hydrogen at N-9. Site-specific RNA N-glycosidase activity is a feature common to all previously identified type I and II RIP toxins (Endo et al., 1987). The activity prevents the binding of elongation factors EF-1 and EF-2, resulting in the cessation of mRNA translation. Although all RIPs exhibit N-glycosidase activity toward ribosomes, they display marked differences in substrate specificity. Most type I toxins exhibit very broad specificities, whereas type

b FIGURE 13.5  Example of the cellular internalization of ricin. The process involves (a) endocytosis by coated pits and vesicles or smooth pits and vesicles followed by (b) vesicle–endosome fusion. Ricin molecules can then return to the cell surface by exocytosis, or the vesicles may fuse to lysosomes for toxin destruction. (From Audi, J. et al., JAMA, 294, 2342, 2005. With permission.)

II toxins display a preference for animal ribosomes. Ricin, for example, is highly active against mammalian and yeast ribosomes but poorly active, or even inactive, against plant and bacterial ribosomes (Yoshinari et al., 1997). Pokeweed antiviral protein, on the other hand, depurinates ribosomes from plants, bacteria, yeast, and various evolutionally diverse animals (Rajamohan et al., 1999). Both the RIP and the ribosome of an RIP–ribosome complex contribute to the apparent substrate specificity (Kurinov et al., 1999). Because the rRNA sarcin–ricin loop architecture is universally conserved, the broad range of specificities displayed by different RIPs may possibly reside within the associated ribosomal proteins conferring tertiary structure to the ribosome (Kurinov et al., 1999), which either facilitate or hinder access of the RIPs to the sarcin–ricin loop. Vater et  al. (1995) identified

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FIGURE 13.6  Proposed mechanism for RTA enzymatic activity. (a), (b) Bond breaking between the substrate ribose and adenine. Cation character builds on the ribose ring and is stabilized by E177, and anion character on adenine becomes stabilized through the donation of a proton from R180 to the N-3 position of the adenine ring system of the substrate. (b), (c) Bond formation resulting from the attack on the ribose ring by water. R180 activates water for its attack on the ribose ring to give the final products shown in (c). (Adapted from Monzingo, A.F. and Robertus, J.D., J. Mol. Biol., 227, 1136, 1992.)

the rat liver ribosomal proteins L9 and L10e as the binding target of RTA and yeast ribosomal protein L3 as the binding factor for pokeweed antiviral protein. In addition to the  highly specific action on ribosomes, ricin and related RIPs have a less specific action in vitro on single- and double-stranded DNA, RNA substrates releasing multiple adenine nucleotides, and guanine nucleotides in some instances (Wang and Tumer, 1999). RTA also catalyzes the hydrolysis of synthetic oligonucleotides as short as six base pairs, provided that a specific tetra loop of guanosine and adenosine nucleotides is present (Amukele and Schramm, 2004).

13.3 Weaponization of the Ricin Toxin Although many pathogens and toxins cause disease, toxicity, or other deleterious effects, relatively few of the naturally occurring agents can be adapted for use as a biological weapon. The ability to survive the mechanical rigors of large-scale aerosol dissemination is of paramount importance for the weaponization of a biological agent. In the case of BSATs, an “aerosol” is most suitably described as a system of solid or liquid particles of sufficiently small diameter to maintain stability as a suspension in air (Green and Lane, 1957). An aerosol includes both the particles and the suspending gas, which almost always is air. Other important characteristics include availability, ease of production and storage, and lethality and may also include ability to incapacitate through morbidity (DA, 1973). While some who develop BSATs pursue agents of death, the philosophy of others is that the most attractive agents are those that incapacitate an adversary. An ideal agent is one that renders an adversary force defenseless while the disseminating force can be easily protected at the same time. Agent stability after production is another important factor to consider when selecting a BSAT for weaponization. Environmental factors, including temperature, relative humidity, atmospheric pollution, and sunlight, can significantly affect agent viability. The most feasible options for storing agents include their production in a liquid or solid form that is easily stored at room temperature or under refrigeration. The most

desirable option is dry sample storage at room temperature, as this eliminates the need for refrigeration, facilitates particle milling (particle sizing) prior to dissemination, and minimizes environmental effects on dissemination. When all factors are considered, toxins are almost certainly the most suitable biological agents for use as a biological weapon. However, in contrast to agents capable of replication, the use of toxins as biological agents requires that a lethal amount must be injected, ingested, or inhaled. With this in mind, a sizable amount of toxin must be available for dispersion in large-scale attacks, severely limiting the effective toxins to those that are the most lethal and easily produced. Ricin is one of only a handful of toxins that meet these requirements, along with botulinum toxin, staphylococcal enterotoxin B, and the trichothecene mycotoxins. Due principally to its suitability as a biological warfare agent, the ricin toxin has been one of the biological weapons of choice for state-sponsored organizations (Kortepeter and Parker, 1999).

13.3.1 The Brief History of Ricin Weaponization After becoming aware of Germany’s involvement with biological weapons during World War I, the U.S. government initiated its own biological weapons program toward the end of the war, which included the weaponization of the ricin toxin (DA, 1977). The U.S. Bureau of Mines studied the offensive potential of ricin at the American University Experimental Station in Washington, DC, after limited experimental research on animals demonstrated that it was possible to weaponize ricin. Two methods of ricin dispersal were examined at the time: (1) the adherence of ricin to shrapnel and bullets and (2) the production of a ricin dust cloud. Not only was the shrapnel and bullets concept a direct violation of The Hague Convention of 1899 (AP, 2014), but also, the heat generated while firing the coated bullets destroyed a significant amount of the toxin (Williams, 1918). The dust cloud approach, on the other hand, was considered less promising due to the limited amounts of purified ricin on hand together with its less efficient delivery via the respiratory tract. Although both approaches were laboratory tested to some degree, neither was perfected for battlefield use prior to the end of the war. Sometime during the early 1920s, U.S.

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military leaders determined that conducting further research into the use of biological weapons was not an efficient use of resources, and research in the United States on the weaponization of ricin faded. Interest in the weaponization of ricin re-emerged during World War II, when the toxin was given the military designation “W” (and “WA” sometime later). During this time, the British and Canadian governments investigated the use of ricin in cluster bombs (Gupta, 2009). The U.S. military’s interest in ricin was re-invigorated sometime around 1942 as a project of the National Defense Research Committee (OSRD, 1946) and led to chamber and field tests at Dugway Proving Ground, Utah in 1944 (Parker et  al., 1996). Definite plans for mass producing ricin cluster bombs existed at times, and several field trials with different ricin bomblet concepts were conducted. Nevertheless, Allied governments concluded that the use of ricin on the battlefield was no more economical than using the chemical agent phosgene (carbonyl dichloride, military designation “CG”), which was already weaponized at the time. This conclusion, however, was based on comparisons of the final weapons rather than the toxicity of ricin. Interest in developing ricin weapons continued for a short time after World War II but soon diminished when the U.S. Army Chemical Corps initiated a program to weaponize the nerve agent sarin ((RS)-propan-2-yl methylphosphonofluoridate, military designation “GB”). With the exception of one U.S. patent awarded in 1965 for a small bomb that can be used to disperse toxic agents such as ricin in aerosol form (LeTourneau, 1965), there has been no reported research or development of ricin as an offensive weapon since World War II. Present-day research focused on ricin by the U.S. government and its allies is exclusively for defense against the toxin used by adversary forces or terrorists.

13.3.2 Aerosolization of Ricin By a considerable margin, the best option for the use of ricin as an effective military weapon is aerosolization. Research reported by Schep et al. (2009) clearly suggests that the toxicity of ricin particles by inhalation is a function of their aerodynamic equivalent diameter (the diameter of a unit density sphere that would have the identical settling velocity of the particle in the same gas). The aerodynamic equivalent diameter, da, is an indicator of the aerodynamic behavior of a particle, which depends on its overall size, particle shape, and density (Bates et al., 1966) according to the equation 1



 ρ 2 d a = de  p   ρ0 χ 

where de is the equivalent volume diameter (the diameter of a sphere of the same volume as that of an irregular particle) ρp and ρ0 are the particle density and standard particle density (1 g cm−3), respectively χ is the dynamic shape factor (ratio of the resistive force of an irregular particle to that of a spherical particle of the same volume and velocity)

The equation is a simple expression for da derived by neglecting the slip correction and can be used to illustrate the difficulties in preparing ricin aerosols. For an offensive military weapon, the da value of ricin aerosol particles must be of sufficiently low micron size to reach the human lower respiratory tract to cause intoxication (discussed in more detail in Sections 13.4 through 13.4.4). Larger particles >5 μm can be cleared from the upper respiratory tract by a number of different mechanisms (see Section 13.4.4) before ever reaching the lower respiratory tract, and smaller particles 5 μm showed minimal effects when administered an inhaled dose of 20 μg kg−1. If this aerosol contained particles 800 smaller quaternary branches (ICRP, 1972). This branching extends into even smaller airways, where goblet cells producing mucus and secretory glands gradually disappear into the terminal bronchioles. The epithelial lining becomes completely non-ciliated at the terminal bronchioles before they separate into ≤150,000 respiratory

Chemical Warfare Agents bronchioles, which are also non-ciliated (Weibel, 1963). The respiratory bronchioles further divide into ~26 million alveolar ducts (Brain and Valberg, 1974) that are almost entirely lined with alveoli. Collectively, somewhere between 50 million and 100 million alveolar sacs are found in the alveoli of a human adult (Landahl, 1963) to provide 30–100 m2 of gas exchange surface area depending on individual body size and fitness level (Silverman et al., 1951). The alveolar walls are built from a network of reticular and elastic fibers that support fine pulmonary capillaries. Lymphatic channels are found throughout the pleura and septa and constitute a means for clearing insoluble deposited particles. Hilar lymph nodes may become secondary reservoirs of deposited material, causing prolonged release of hazardous inhaled materials to the systemic circulation (Stuart, 1975).

13.4.3 Deposition of Aerosol Particles in the Respiratory Tract Airflow rates for aerosols entering the respiratory tract range from 0 to 120 L m−1 during normal respiration depending on the amount of work performed (Silverman and Billings, 1961). During both inspiration and expiration, particle deposition processes that follow physical laws will occur. The deposition of non-fibrous particles in the respiratory tract results from the three physical processes of impaction, sedimentation, and diffusion (McClellan and Henderson, 1995; Phalen, 2009; Schlesinger, 1985), which

FIGURE 13.8  Anatomical regions of the respiratory tract. Dimensions in millimeters (mm) represent the da values for particles that can penetrate the respiratory tract to the corresponding anatomical region. (From Stuart, B.O., Environ. Health Perspect., 55, 369, 1984. With permission.)

Ricin Structural Biology, Biochemistry, Toxicology, Military Use, and Medical Countermeasures are illustrated in Figure 13.9. The three processes are influenced by (1) the physical and chemical characteristics of the particle, such as its size and shape, density, surface charge, hygroscopicity, solubility, and chemical reactivity, as well as (2) factors specific to the individual, including the respiratory route as a result of nasal or mouth breathing, respiratory rate and tidal volume, and respiratory tract anatomy (Roy et  al., 2010; Stuart, 1984). Impaction is the dominant mechanism for the deposition of larger particles with da > 1–2 μm and usually occurs in the nasopharyngeal and tracheobronchial regions, especially at airway branching points, where bulk airflow is faster and more turbulent. On the other hand, sedimentation becomes the major mechanism for particles with 0.5