Chiral Environmental Pollutants: Analytical Methods, Environmental Implications and Toxicology 3030624552, 9783030624552

Table of contents :
Preface (2nd Edition)
Preface (1st Edition)
References
Acknowledgements
Contents
Chapter 1: Introduction
1.1 General Considerations
1.2 Chirality in a Social and Cultural Context
1.3 Chirality in Living Organisms
1.4 Chirality in Chemistry
1.5 Chirality as Indicator Feature for Living Processes
1.5.1 Chirality as Tools for the Search of Extraterrestrial Life
1.6 General Principles of Chirality in Chemistry
1.6.1 Chiral Environmental Pollutants with a Stereogenic Centre
1.6.2 Environmental Pollutants with Axial Chirality
1.6.3 Asymmetry of Cyclic Environmental Pollutants
1.6.4 Chiral Environmental Pollutants with Two or More Stereogenic Centres
References
Chapter 2: Criteria for the Selection of a Proper Enantiomer-Selective Analytical Method
2.1 Polarity Expressed as Octanol-Water Partitioning Coefficient (KOW)
2.2 Dipole Moment
2.3 Analyte-Stationary Phase Interactions in Chromatography Separation
2.4 Rotation Energy
References
Chapter 3: Enantiomer-Selective High- and Ultra- High-Performance Liquid Chromatography
3.1 Separation Principles
3.1.1 Derivatisation and Mobile-Phase Additives
3.2 Enantiomer-Selective HPLC Columns
3.3 Indirect Methods
3.4 Direct Methods
3.5 The Evolution of Chiral Stationary Phases for Liquid Chromatography
3.6 Other Selection Strategies for Enantioselective Liquid Chromatography
3.6.1 Diffusive Enrichment Through Membranes
3.7 Liquid Chromatography as a Measurement Tool for Chiral Interactions
3.8 Updated Reviews on Enantioselective HPLC/UHPLC
References
Chapter 4: Enantiomer-Selective Electrophoresis and Electrochromatography
4.1 Enantiomer-Selective Capillary Electrophoresis
4.2 Other Experimental Approaches for Enantioselective Capillary Electrophoresis
4.3 Recent Reviews on Enantiomer-Selective Capillary Electrophoresis
References
Chapter 5: Enantiomer-Selective High-Resolution Gas Chromatography (esHRGC)
5.1 The Evolution of Chiral Stationary Phases for Capillary Gas Chromatography
5.2 Enantioselective Multidimensional Capillary Gas Chromatography (MDGC)
5.3 Comprehensive Capillary Gas Chromatography (GCxGC)
5.4 Other Experimental Approaches for Enantioselective Capillary Gas Chromatography
5.5 Recent Reviews on Enantioselective HRGC
References
Chapter 6: Other Methods for the Elucidation of Molecular Structures and Mechanistic Details of Enantiomers
6.1 X-Ray Crystallography
6.2 Nuclear Magnetic Resonance Studies
6.3 Enantiomer-Selective Mass Spectrometry
6.4 Vibrational Circular Dichroism (VCD)
6.5 Recent Reviews
References
Chapter 7: Quality Control and Evaluation Criteria for Enantiomer-Selective Separation Methods in Environmental Sciences
7.1 Enantiomer Distribution in Environmental Samples
7.2 Enantiomeric Ratios or Enantiomeric Fractions?
7.3 Possible Sources of Error in Enantiomer-Selective High-Performance Liquid Chromatography
7.4 Possible Sources of Error in Enantiomer-Selective Capillary Gas Chromatography
References
Chapter 8: Enantiomer-Specific Fate and Behaviour of Chiral Contaminants
8.1 Microbial Transformation of Chiral Environmental Pollutants
8.1.1 Laboratory Experiments
8.1.2 In Situ Investigations in Marine and Limnic Waters
8.2 Transformation/Accumulation of Chiral Xenobiotics in Higher Organisms
8.2.1 Marine and Freshwater Organisms
8.2.1.1 α-Hexachlorocyclohexane
8.2.1.2 Other Chlorinated Pesticides
8.2.1.3 Non-Chlorinated Pesticides
8.2.1.4 Chiral Industrial Chemicals
8.2.2 Terrestrial Ecosystems
8.2.3 Chiral Organochlorines in Soils and Ambient Air
8.2.3.1 Air/Water Gas Exchange Studies in Freshwater Environments
8.2.3.2 Air/Sea Gas Exchange Studies
8.2.4 Currently Used Chiral Non-Halogenated Pesticides
8.2.5 Artificial Fragrances and Personal Care Products
8.2.5.1 Enantiomeric Ratios of ATII in Water and SPMD Samples
8.2.5.2 Enantiomeric Ratios of HHCB, ATII, AHTN and AHDI in Biota Samples
8.2.5.3 Comparison of Tench and Crucian Carp Samples
8.2.5.4 The Transformation Products of HHCB
References
Chapter 9: Source Characterisation and Contamination
9.1 Introduction
9.2 Source Characterisation
9.3 Pesticides
9.4 Polycyclic Aromatic Hydrocarbons
9.5 Polychlorinated Biphenyls
9.6 Plasticisers and Additives
9.7 Phenolic Compounds
9.8 Pharmaceuticals and Personal Care Products
9.9 Endocrine-Disrupting Chemicals
9.10 Conclusion
References
Chapter 10: Chirality in Environmental Toxicity and Fate Assessments
10.1 Introduction
10.2 Toxicity of Chiral Pollutants
10.2.1 Polychlorinated Biphenyls
10.2.2 Hexachlorocyclohexane
10.2.3 Other Chlorinated Pesticides
10.2.4 Organophosphorus Compounds
10.2.5 Polycyclic Aromatic Hydrocarbons
10.2.6 Other Xenobiotics
10.2.7 Drugs and Pharmaceuticals
10.3 Enantioselective Partitioning
10.4 Enantioselective Uptake, Translocation and Metabolism
10.5 Quantitative Assessment of Enantioselective Transformation
10.6 Economic Growth
10.7 Challenges to Risk Assessment
References
Chapter 11: Perspectives
11.1 Introduction
11.2 Challenges and Restrictions of Currently Applied Methods
11.3 Future Directions and Method Requirements
11.4 Regulation and Control
11.5 Impact on Science and Society
References
Index

Citation preview

Roland Kallenborn Heinrich Hühnerfuss Hassan Y. Aboul-Enein Imran Ali

Chiral Environmental Pollutants Analytical Methods, Environmental Implications and Toxicology Second Edition

Chiral Environmental Pollutants

Roland Kallenborn • Heinrich Hühnerfuss • Hassan Y. Aboul-Enein • Imran Ali

Chiral Environmental Pollutants Analytical Methods, Environmental Implications and Toxicology Second Edition

Roland Kallenborn Biotechnology and Food Sciences (KBM) Norwegian University of Life Sciences (NMBU) ÅS, Norway

Heinrich Hühnerfuss Institute of Organic Chemistry University of Hamburg Hamburg, Germany

Hassan Y. Aboul-Enein Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical and Drug Industries Research Division National Research Center Cairo, Egypt

Imran Ali Department of Chemistry College of Sciences Taibah University Al-Madinah Al-Munawarah Kingdom of Saudi Arabia Department of Chemistry Jamia Millia Islamia (A Central University) Jamia Nagar, New Delhi, India

ISBN 978-3-030-62455-2 ISBN 978-3-030-62456-9 https://doi.org/10.1007/978-3-030-62456-9

(eBook)

© Springer Nature Switzerland AG 2001, 2021 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG. The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Roland Kallenborn: I dedicate this work to my family (Mia, Magnus and Berit). Without their continuous and enthusiastic support, this tremendous work would not have been possible. Heinrich Hühnerfuss: Without the extreme patience and continuous support of my wife Erika, my contribution to this book would not have been accomplished. This is gratefully acknowledged. Hassan Y. Aboul Enein: This book is dedicated to my beloved wife Nagla for her unlimited support and encouragement. Imran Ali: This book is dedicated to the memories of my late parents: Basheer Ahmed and Mehmudan Begum.

Preface (2nd Edition)

Twenty years after the first edition of this monograph was completed and published with Springer, encouraged by our families and our publisher, we embarked on the endeavour of evaluating thoroughly the progress made in method development, applications and risk evaluation strategies in the now considered as well-established field of enantioselective trace analysis of chiral organic pollutants and their implication for environmental health. In order to cover a broader and interdisciplinary range of scientific aspects associated with the field of chiral organic pollutants, the author team was extended with two highly experienced scientists with a documented focus on method development, toxicity and pharmacology. A large number of additional relevant chiral pollutants were examined, and a variety of newly developed analytical technologies and strategies were included in our discussions. Furthermore, relevant aspects on environmental toxicology, risk assessment and societal considerations are now included in order to fully cover the strong implication of chirality in pollution research. Consequently, the size of the monograph increased in line with the accumulation of new relevant knowledge on chiral organic pollutants, their fate and environmental implications in ecosystems. The presented update is testimony to the constructive and fruitful collaboration of the author team. The book is written for an academic readership meant to provide updated scientific information on all relevant aspects of chiral organic pollutants. Vinterbro Hamburg Madinah Cairo August 8, 2020

Roland Kallenborn Heinrich Hühnerfuss Imran Ali Hassan Y. Aboul-Enein

vii

Preface (1st Edition)

In 1844, Louis Pasteur was the first to separate two different types of sodium ammonium tartrate [1] which he assumed to be related to each other like two mirror images. Few years later, this phenomenon was denominated chirality (from the Greek word χειρ which means hand) by Lord Kelvin [2] who used the following definition: “I call any geometrical figure or any groups of points chiral and say it has chirality if its image in a plane mirror, ideally realised cannot be brought to coincide with itself” (Lord Kelvin, 1883). Subsequently, a large number of compounds were observed to fulfil these requirements. In biological systems, this phenomenon of asymmetry was already known for quite a long time. Different snail species, for example, are producing mirror image forms; fossils like ammonites exhibit chiral shapes; and last but not least, the human body including feet, hands and ears can be divided into two parts which can be regarded as two non-superimposable mirror images. After the breathtaking scientific findings, awarded with the Nobel Prize in 1957, when two young Chinese-American physicists, Tsung Dao Lee and Chen Ning Yang, proved that the parity of weak interactions is not preserved, enantiomer selective approaches were also applied to elementary and quantum physical processes under certain conditions [3]. The simple principle that may be inferred from symmetry considerations, according to which two structures can be identical but not superimposable, nowadays is a basic research objective in at least four main scientific disciplines: biology, chemistry, physics and mathematics. In the last decade, new trace analytical methods were developed to separate and detect the enantiomers of persistent organic pollutants in a large number of environmental samples at different trophic levels. In 1991, Faller et al. as well as Kallenborn and Hühnerfuss [4, 5] were the first to publish the successful separation of α-HCH in seawater and marine biota samples, respectively. These early publications already showed the potential of this relatively simple and robust enantioselective gas chromatographic method to play a major role in assessing enzymatic transformation processes of anthropogenic and natural organic pollutants in the environment.

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Preface (1st Edition)

However, thus far, no efforts were made to give a comprehensive survey on the state of the art of enantioselective trace analysis and to reflect the future of this promising new field of analytical research. This monograph intends to fill this obvious gap and aims at delivering a comprehensive tool for the information of experienced trace analysts as well as of interested students and non-chemists. The international research on chirality of xenobiotics today mainly focuses on the following two principal questions: Why does chirality play such an outstanding role when biochemical transformation and accumulation processes of chiral organic pollutants in organisms, from bacteria up to human beings, are being discussed? How can chirality as a basic selector for enzymatic processes be documented and proven for organic chemicals, which are degraded and/or accumulated in ultratrace concentrations. These two questions will form the common thread throughout the present survey. We will mainly focus on the trace analytical and ecotoxicological aspects of molecular asymmetry. Since almost the entire research work is centrally related to chiral organic xenobiotics, we will largely confine ourselves to organic compounds, although in general parts of this book theoretical aspects of chiral inorganic substances will also be mentioned. Hamburg Tromsø October 2000

Heinrich Hühnerfuss Roland Kallenborn

References 1. Pasteur L (1848) Memoire sur la relation qui peut exister entre la forme crystalline et la composition chimique, et sur la cause de la polarisation rotatoire. C R Acad Sci 26:535–538 2. Jacques J (1993) The molecule and its double. McGraw-Hill, p 128 3. Gardner M (1990) The ambidextrous universe: mirror asymmetry and time-reversed worlds. Scribner, New York, p 293 4. Faller J, Hühnerfuss H, König WA, Krebber R, Ludwig P (1991) Do marine bacteria degrade alpha-hexachlorocyclohexane stereoselectively? Environ Sci Technol 25:676–678 5. Kallenborn R, Hühnerfuss H, König WA (1991) Enantioselective metabolism of ()-alphahexachlorocyclohexane in organs of the Eider Duck. Angew Chern 103:328–329; Angew Chern Int Ed Engl 130:320–321

Acknowledgements

The authors wish to express their gratitude to all the colleagues who contributed their knowledge, ideas, information and advice to the present endeavour. In particular, the help of the following colleagues is gratefully acknowledged: M. Oehme, C. Wong, T. H. Bidleman, Nicolas Warner, Michael Harjo, Petter Haglund, Martin Schlabach, W. Vetter, Aasim A. Ali, Ivo Havranek, Yngve Stenstrøm, Simen Gjelseth Antonsen, Jens Mortansson Jelstrup Nolsøe, Thomas Ternes, Michael Schlüsener, Christoph Schüth, W.A. König and many more. In addition, we appreciate the opportunity to publish this monograph with Springer Verlag and thank the editors, especially senior editor Sofia Costa, for the administrative support during the realisation of this book. We also thank Stephanie Kolb (Springer Verlag) for her encouragement, help and support during the realisation and the paperwork of our book project. Without the extreme patience and the support of our families, this book would not have been accomplished in its present form. Personal Notes Imran Ali: I express my deep sense of gratitude and warmest felicitations to my wife Seema Imran, who has helped me and supported me while I have carried out this work. My lovely and sweet thanks are also offered to my dearest son, Al-Arsh Basheer Baichain, who has given me freshness and fragrance continuously during the completion of this difficult task. I would also like to acknowledge my other family members and relatives who have helped me, directly and indirectly, during this period. Finally, the administration of the Taibah University, Al-Madinah Al-Munawarah, Saudi Arabia, and Jamia Millia Islamia, New Delhi, India, is also acknowledged. The holiest city of Al-Madinah Al-Munawarah, Saudi Arabia is also acknowledged for allowing us to reside with a pleasant and great time. Roland Kallenborn: I appreciate the idealistic and partial financial support of the Faculty of Chemistry, Biotechnology and Food Sciences (KBM), Norwegian University of Life Sciences (NMBU), as well as the Arctic Technology Department (AT), University Centre in Svalbard (UNIS). xi

Contents

1

2

3

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 General Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Chirality in a Social and Cultural Context . . . . . . . . . . . . . . . . 1.3 Chirality in Living Organisms . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Chirality in Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 Chirality as Indicator Feature for Living Processes . . . . . . . . . 1.5.1 Chirality as Tools for the Search of Extraterrestrial Life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.6 General Principles of Chirality in Chemistry . . . . . . . . . . . . . . 1.6.1 Chiral Environmental Pollutants with a Stereogenic Centre . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.6.2 Environmental Pollutants with Axial Chirality . . . . . . 1.6.3 Asymmetry of Cyclic Environmental Pollutants . . . . . 1.6.4 Chiral Environmental Pollutants with Two or More Stereogenic Centres . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1 1 2 3 4 5

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5 6

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7 9 10

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12 15

Criteria for the Selection of a Proper Enantiomer-Selective Analytical Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Polarity Expressed as Octanol–Water Partitioning Coefficient (KOW) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Dipole Moment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Analyte–Stationary Phase Interactions in Chromatography Separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Rotation Energy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enantiomer-Selective High- and Ultra- High-Performance Liquid Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Separation Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1 Derivatisation and Mobile-Phase Additives . . . . . . . . 3.2 Enantiomer-Selective HPLC Columns . . . . . . . . . . . . . . . . . .

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19 20 21 22 23 23 29 32 32 33 xiii

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3.3 3.4 3.5

Indirect Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Direct Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Evolution of Chiral Stationary Phases for Liquid Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 Other Selection Strategies for Enantioselective Liquid Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6.1 Diffusive Enrichment Through Membranes . . . . . . . . 3.7 Liquid Chromatography as a Measurement Tool for Chiral Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8 Updated Reviews on Enantioselective HPLC/UHPLC . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

5

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45 47 48

Enantiomer-Selective Electrophoresis and Electrochromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Enantiomer-Selective Capillary Electrophoresis . . . . . . . . . . . . . 4.2 Other Experimental Approaches for Enantioselective Capillary Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Recent Reviews on Enantiomer-Selective Capillary Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enantiomer-Selective High-Resolution Gas Chromatography (esHRGC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 The Evolution of Chiral Stationary Phases for Capillary Gas Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Enantioselective Multidimensional Capillary Gas Chromatography (MDGC) . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Comprehensive Capillary Gas Chromatography (GCxGC) . . . . 5.4 Other Experimental Approaches for Enantioselective Capillary Gas Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5 Recent Reviews on Enantioselective HRGC . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other Methods for the Elucidation of Molecular Structures and Mechanistic Details of Enantiomers . . . . . . . . . . . . . . . . . . . 6.1 X-Ray Crystallography . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Nuclear Magnetic Resonance Studies . . . . . . . . . . . . . . . . . . 6.3 Enantiomer-Selective Mass Spectrometry . . . . . . . . . . . . . . . 6.4 Vibrational Circular Dichroism (VCD) . . . . . . . . . . . . . . . . . 6.5 Recent Reviews . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

63 64 68 68 69

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81 82 83

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89 89 90 92 93 93 93

Quality Control and Evaluation Criteria for Enantiomer-Selective Separation Methods in Environmental Sciences . . . . . . . . . . . . . . . 7.1 Enantiomer Distribution in Environmental Samples . . . . . . . . . . 7.2 Enantiomeric Ratios or Enantiomeric Fractions? . . . . . . . . . . . .

97 98 99

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xv

7.3

Possible Sources of Error in Enantiomer-Selective HighPerformance Liquid Chromatography . . . . . . . . . . . . . . . . . . . . 100 7.4 Possible Sources of Error in Enantiomer-Selective Capillary Gas Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 8

Enantiomer-Specific Fate and Behaviour of Chiral Contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1 Microbial Transformation of Chiral Environmental Pollutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1.1 Laboratory Experiments . . . . . . . . . . . . . . . . . . . . . . 8.1.2 In Situ Investigations in Marine and Limnic Waters . . 8.2 Transformation/Accumulation of Chiral Xenobiotics in Higher Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2.1 Marine and Freshwater Organisms . . . . . . . . . . . . . . . 8.2.2 Terrestrial Ecosystems . . . . . . . . . . . . . . . . . . . . . . . 8.2.3 Chiral Organochlorines in Soils and Ambient Air . . . . 8.2.4 Currently Used Chiral Non-Halogenated Pesticides . . . 8.2.5 Artificial Fragrances and Personal Care Products . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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130 130 170 187 206 216 226

9

Source Characterisation and Contamination . . . . . . . . . . . . . . . . . 9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2 Source Characterisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3 Pesticides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4 Polycyclic Aromatic Hydrocarbons . . . . . . . . . . . . . . . . . . . . 9.5 Polychlorinated Biphenyls . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.6 Plasticisers and Additives . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.7 Phenolic Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.8 Pharmaceuticals and Personal Care Products . . . . . . . . . . . . . . 9.9 Endocrine-Disrupting Chemicals . . . . . . . . . . . . . . . . . . . . . . 9.10 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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255 255 255 257 259 260 261 262 264 267 267 268

10

Chirality in Environmental Toxicity and Fate Assessments . . . . . . 10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2 Toxicity of Chiral Pollutants . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.1 Polychlorinated Biphenyls . . . . . . . . . . . . . . . . . . . . 10.2.2 Hexachlorocyclohexane . . . . . . . . . . . . . . . . . . . . . . 10.2.3 Other Chlorinated Pesticides . . . . . . . . . . . . . . . . . . . 10.2.4 Organophosphorus Compounds . . . . . . . . . . . . . . . . . 10.2.5 Polycyclic Aromatic Hydrocarbons . . . . . . . . . . . . . . 10.2.6 Other Xenobiotics . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.7 Drugs and Pharmaceuticals . . . . . . . . . . . . . . . . . . . . 10.3 Enantioselective Partitioning . . . . . . . . . . . . . . . . . . . . . . . . . 10.4 Enantioselective Uptake, Translocation and Metabolism . . . . .

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279 279 280 280 282 284 285 286 287 288 289 290

. 107 . 108 . 108 . 118

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Contents

10.5 Quantitative Assessment of Enantioselective Transformation . . 10.6 Economic Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.7 Challenges to Risk Assessment . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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291 291 292 293

Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 Challenges and Restrictions of Currently Applied Methods . . . 11.3 Future Directions and Method Requirements . . . . . . . . . . . . . . 11.4 Regulation and Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.5 Impact on Science and Society . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . .

307 307 308 309 311 312 313

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319

Chapter 1

Introduction

Scientific knowledge on the three-dimensional structure of complex molecules today is a fundamental prerequisite for many disciplines associated with life sciences. The receptor–target molecule interactions and the specific linkages on the receptor sites play a crucial role in explaining biochemical processes and toxicological mechanisms in the case of hazardous substances (Speranza et al. 2009; Queneau et al. 2011).

1.1

General Considerations

During the past four decades, chemical analytical methods for the determination of enantiomeric signatures have found their way into a broad range of modern scientific environmental applications (Bakhtiar et al. 2001; Kraft and Frater 2001; Liu et al. 2009; Ulrich et al. 2012; Basheer 2017; Zhang et al. 2017), including risk assessment of novel chiral environmental pollutants, investigation of degradation pathways, identification of bioactive substances (biogenic and anthropogenic) and novel environmental toxicity endpoints. This recent expansion of enantiomer-specific research strategies in environmental sciences impressively illustrates the scientific relevance of the stereochemical phenomenon “chirality” as important feature for the elucidation of life processes in modern environmental sciences (Bonner 1998; Cintas 2008; Fuss 2009). Created as an important separation technique in pharmaceutical product development, enantiomer-selective chromatographic separation in combination with highly sensitive and selective detection methods were introduced as tools in environmental chemistry for investigating and assessing the transformation and degradation potential of chiral environmental pollutants in the early 1990s (Hühnerfuss et al. 1993). During the following decades, the principles of enantiomer-specific separation were gradually introduced into a large variety of research fields in the scientific realm of environmental research. The current edition of our book intends to © Springer Nature Switzerland AG 2021 R. Kallenborn et al., Chiral Environmental Pollutants, https://doi.org/10.1007/978-3-030-62456-9_1

1

2

1 Introduction

illustrate the strong scientific potential and versatility of this now well-established research strategy in environmental research.

1.2

Chirality in a Social and Cultural Context

Chirality as asymmetric feature can be found in many facets and artefacts of human culture throughout the history. One of the most prominent symbolic features in a religious context is the swastika (Fig. 1.1), which has its origin from the ancient Armenian astrological maps and later adopted in various shapes by the East-Asian religions. The swastika symbol was also misused by the national socialistic movement in Germany in the period 1930–1945 as their ideological emblem. Please note that the clockwise symbol (swastika) symbolises prosperity and good luck, while the counterclockwise symbol (sauvastika) represents night or tantric aspects of the goddess Kali (the destroyer or evil force in the Hindu religion). This symbolic contrast is in accordance with the asymmetric feature of chirality. Even in the old kingdom of upper and lower Egypt, many examples of burial chambers, be it mural paintings depicting significant events, inscribed stelae or tablets, have been excavated that show an astonishing proximity to our modern approach to chirality. In Fig. 1.2, an example is given of a stele that exhibits Fig. 1.1 Religious and social symbols of swastika (left hand) and the Hindu symbol of sauvastika (right hand)

Fig. 1.2 Hieroglyphs carved out of stone on a stele outside the Pergamon museum, Berlin, Germany (courtesy Erika and Katja Hühnerfuss)

1.3 Chirality in Living Organisms

3

hieroglyphs carved out of stone such that they are mirrored on both sides of the so-called ankh cross. This ankh cross plays an important role in the Egyptian mythology representing the symbol for live. The pharaohs firmly believed in life after death, and accordingly, they wanted to link the symbol for life with their coffins as well as the mural paintings and stelae of the burial chamber. In addition, the hieroglyphs closely related to the ankh cross are appearing twice, that is, in the form of their two mirror images. This ancient interpretation of chirality/life/death is well in line with our modern convention to assume life processes, for example, enzymatic transformation processes, to be highly enantioselective, but also dead organic matter may be transformed and in part mineralised by very enantioselective processes.

1.3

Chirality in Living Organisms

Detailed evidence for the influence of symmetry and asymmetry on life processes and the obvious consequences for our daily life can be found in the realm of plants and animals, where numerous examples for symmetric and asymmetric macroscopic structures can be observed (Schilthuizen and Davison 2005; Makela and Annila 2010; Barron 2012a, b; Wensink and Morales-Anda 2015; Jedrzejewska and Szumna 2017). For example, the occurrence of a horizontal radial symmetric shape for plants may be attributed to the influence of the sun light, which circles around the stem in the course of a day. Branches and leaves tend to grow towards the light, whereas roots are not at all attracted by light but rather follow the gravitational force. Wherever the symmetric lateral plane is being placed, it will divide the object into (more or less) equal parts. On the other hand, a special case of radial symmetry, the so-called bilateral symmetry, is also often encountered in the realm of plants and animals. These kinds of objects only possess one vertical or horizontal internal plane of symmetry, sometimes referred to as “mirror plane”. A symmetry of this kind can be found for the human body. The symmetric plane divides the human body vertically into two more or less even parts with one ear, one eye, one nostril, one arm and one leg at each side. This type of symmetry is closely connected to the evolutionary development of the organism. In general, animals that are sessile and do not move are assumed to show a classical bilateral symmetry, while organisms that have developed locomotive properties are preferentially characterised by radial symmetry. However, many exceptions from this rule can be found in natural systems, and, in addition, spherical, conical, cylindrical symmetries are also encountered. On the other hand, macroscopic asymmetric structures possessing no plane of symmetry are also quite general in nature. Classic examples for asymmetric objects are helical structures, which are found for climbing and twining plants. For example, the “honeysuckle (Linnaea borealis L.)” always twines in a left-handed helix, whereas the “morning glory (Convolvulus sabatius L.)” twines in a right-handed helix. Helical structures are also known for animals. Most prominent examples can be found for marine and freshwater molluscs (snails and shells). Normally, only one

4

1 Introduction

preferential type of “handedness” is common for one species, but both right- and left-handed helical structures are known and found for all these asymmetric species. Several fossil molluscs demonstrate the evolutionary potential of asymmetric structures, for example, ammonites and the Nautilus pompilius (L.) shell are the most prominent examples for asymmetric natural shapes in pre-historical time, where links have been revealed between ammonites and the present cephalopodae, while Nautilus represents one of those rare examples that survived and almost exactly exhibit their former shape (living fossils). Certain asymmetric helical structures can also be found for humans, for example, the umbilical cord of the newborn child shows a clear helical structure. Cords were found to be winded left- and righthanded. The reason why a certain direction is preferred is still under discussion (Fletcher 1993). Another type of non-helical asymmetry is being represented by a passerine bird species which can be found in both North America and Middle Europe, a bird species called cross bill (Loxia curvirostra L.). The bird’s upper and lower beaks crossover each other, where two ways are possible, to the left- or the right-hand side of each other. These two ways represent two mirror image-like procedures. Interestingly enough, the upper bill of the American population crosses to the left-hand side of the bird, whereas the opposite can be observed for the European birds. An unusual type of asymmetric shape occurs in the marine flatfish family (Pleuronectiformes), for example, halibut (Hippoglossus hippoglossus L.), sole (Glyptocephalus cynoglossus L.) and flounder (Pleuronectes platessa L.) belong to this group of marine fish species (Suzuki and Kurokawa 2000; Falk-Petersen 2005). During embryo development, one eye slowly migrates to the other side of the organism. As an adult animal, the flatfish lies on the ground with one side and both eyes upwards, hunting small crustaceans and fish. The side which is preferentially directed upwards differs from one species to the other; however, this side is characteristic of one species. “Left- and right-sided” individuals are known for several species (e.g. soles ¼ Limanda limanda). In general, it can be assumed for plants and animals that a radial or bilateral symmetry is coupled with small asymmetric structures (Gardner 1990). During the evolution of the present plants and animals, particularly bilateral structures have proven to be important for survival strategies. Nevertheless, there cannot be any doubt that for certain purposes, asymmetric structures have shown to be better adapted than bilateral symmetric shapes.

1.4

Chirality in Chemistry

The first to discover the principle of asymmetry from a chemical point of view was Louis Pasteur who succeeded in separating two types of sodium ammonium tartrate crystals in 1848 (Pasteur 1848). He showed that separate solutions of those two types of crystals are able to rotate the plane of linearly polarised light in different directions, to the right- and left-hand side, respectively. Depending on this

1.5 Chirality as Indicator Feature for Living Processes

5

characteristic direction, the two isomers were called “left-handed” and “righthanded”. The principal properties of chirality were found. In a subsequent experiment, Pasteur discovered that mould degrades only one type of molecule (left- or right-handed), whereas the other remains intact. This was without any doubt the first step in the direction of modern biochemistry (Burke and Henderson 2002). A few years later, this structural phenomenon was named CHIRALITY by Lord Kelvin (after the Greek word χειρ which means hand). At that time, the majority of the scientists did not consider these results to be very important for biological processes. However, in the late nineteenth century, a vivid discussion was initiated, where chemists and biologists discussed the implication of chirality for natural processes. These discussions lasted relatively long (until the early years of the twentieth century) and were one of those typical examples in the history of science, where unconventional researchers, leaving the traditional ways of scientific thinking, had to fight against ignorance raised by the established scientific community and had to struggle, in order to break through the wall of traditions.

1.5

Chirality as Indicator Feature for Living Processes

Today, the crucial role of chirality in life-defining processes is undisputed anymore. According to our current scientific understanding, a homochiral environment, created by enantiomeric pure forms of amino acids (L-forms) and here from deriving peptides and proteins as well as nucleic acids (D-forms), is the basic foundations for the complex physiological processes creating life in the biosphere.

1.5.1

Chirality as Tools for the Search of Extraterrestrial Life

According to today’s understanding, molecular chirality is acknowledged as a basic feature for living processes (Fuss 2009; Hein and Blackmond 2012; Woolf 2015). For the past decades, the origin of life is still vividly debated in science (Brack et al. 1998; Burke and Henderson 2002; Cintas 2008; Fuss 2009; Barron 2012a, b). On the one hand, evolutionary scientists explain the continuous evolutionary principles of living processes as exclusive adoptions to terrestrial environments. This theory is supported by many renowned scientists acknowledging that the evolutionary development of living processes and organisms is exclusively terrestrial (Soffen 1986; Bonner 1998; Breslow and Cheng 2009; Brack et al. 2010; Pizzarello and Shock 2010; Spitzer 2013). Other experts, however, discuss and investigate the introduction of important building blocks, for example, complex organic chemicals like amino acids and peptides, for the evolution of life on earth by the action of extraterrestrial sources like meteorites and comets. This hypothesis is also supported by the presence of amino acid-like organic molecules found in archived meteorites and other extra-terrestrial materials collected from the earth’s surface (Donaldson et al.

6

1 Introduction

2004; Bernstein 2006; Pavlov et al. 2006; Pizzarello 2006; Sandford 2008; Bada 2009; Glavin and Dworkin 2009; de Duve 2011; Sojo 2015). Chirality plays a crucial role in the current struggle to unravel this important principal scientific question. Thus, refined enantiomer-selective separation methods were actively applied earlier for the identification and characterisation of chiral organic molecules in extra-terrestrial materials (Engel et al. 1990; Cronin and Pizzarello 1997; Pizzarello and Cronin 2000). As a further step, enantiomer-selective chromatography coupled to a mass-selective detector was installed on the Rosetta Mission of the European Space Agency (ESA) in the early 2000s aiming at in situ identification and enantiomer-selective analysis of amino acids and other chiral organic molecules in surface samples from the Churyumov-Gerasimenko comet (Szopa et al. 2002a, b; Evans et al. 2012). However, due to challenges during the landing procedures of the Philae lander, the representative measurements could not be completed and the final proof for the presence of enantiomeric amino acids on extra-terrestrial objects still remains to be confirmed.

1.6

General Principles of Chirality in Chemistry

Nowadays, the general principles of chirality and the associated nomenclature for enantiomeric compounds are included in basic lectures of organic chemistry (Prelog 1976; Helmchen 2016). Therefore, we do not wish to duplicate this general information in the present monograph but rather refer to the general literature in chemistry. On the other hand, scientists of other disciplines may not be that familiar with the terminology and basic rules, which are assumed to be crucial for a comprehensive understanding of molecular structures determining chiral molecules and enantioselective processes, as discussed in the present monograph. Therefore, we decided to form a uniform “stage” for readers of all disciplines by giving a brief survey on some crucial aspects ruling chiral molecules and enantioselective processes. In order to meet the requirements of the present monograph as closely as possible, examples for the different types of chirality will be represented by chiral environmental pollutants discussed herein. Chirality in chemistry is a very important field of Stereoisomerism according to the following definitions: stereoisomers are chemical substances made up of the same atoms, bonded by the same sequence of bonds, but possessing different threedimensional structures which are not interchangeable. These three-dimensional structures are called configurations. In the same way as many things around us, such as our hands and pairs of shoes, are not identical, but the mirror images of one another, non-identical stereoisomers exist, in which the only distinction between them is that one is the mirror image of the other. However, these mirror images are not superimposable. A simple example of this type of stereoisomerism is represented by the herbicide dichlorprop (or DCPP), that is, 2-(2,4-dichlorophenoxy)propionic acid (Fig. 1.3), which can exist in two spatial configurations that correspond to reflections of each other. These stereoisomers are specifically called enantiomers. A

1.6 General Principles of Chirality in Chemistry

7 Cl

Cl

Cl

O

H3C

CH3

H

H C

C

Cl

HOOC

COOH

(R)-(+)-Dichlorprop

O

mirror

(S)-(-)-Dichlorprop

Fig. 1.3 Not superimposable mirror images, so-called (R)- and (S)-enantiomers, of the herbicide dichlorprop [or DCPP, i.e. 2-(2,4-dichlorophenoxy)propionic acid)

1:1 mixture of both enantiomers forms a racemate or racemic mixture. In addition to these general definitions, some rules have to be summarised that allow a discrimination between chiral and achiral molecules.

1.6.1

Chiral Environmental Pollutants with a Stereogenic Centre

The most common origin of chirality in molecules, and the one originally recognised by van’t Hoff and Le Bel, is the presence of one or more atoms, mainly carbon atoms, each of which forms non-coplanar bonds to four different atoms or groups (tetrahedral carbon atom). The atom that carries the four different substitutes is called the asymmetric or stereogenic centre. This is the case for the example given in Fig. 1.3, where the stereogenic centre bonds to a hydrogen atom, a methyl, a carboxyl and a 2,4-dichlorophenoxy group. In evaluating a chemical structure for chirality, a carbon carrying four different attached groups may be one indication for the presence of a chiral compound. Many important natural compounds such as amino acids and carbohydrates possess one or more stereogenic centres linked with four different atoms or groups. But also several environmental pollutants discussed in the present monograph exhibit this stereochemical characteristic, which can be easily recognised also by inexperienced readers. A list of these environmental pollutants can be found in Table 1.1. Furthermore, it is important to note that one of the four different atoms and groups, respectively, carried by the stereogenic centre may be substituted by a free pair of electrons. This type of chiral compounds can be found in the homological series of tertiary amines or sulfoxides. A parameter that limits the possibility of isolating and analysing enantiomers by enantioselective chromatographic approaches is the inversion barrier. Fast inversion between the two enantiomers would render it impossible to separate them chromatographically. Examples for this type of chirality will be discussed in Chap. 3 (naloxone; one stereogenic nitrogen centre). Waxman et al. (1982) studied the enantioselective sulfoxidation of 4-tolyl

8

1 Introduction

Table 1.1 Chiral environmental pollutants, which are discussed in the present monograph, with one or more stereogenic centres linked with four different substituents; examples for central asymmetry Chiral environmental pollutant Dichlorprop (DCPP; i.e. (R,S)-2-(2,4-dichlorphenoxy)propionic acid) Methyl dichlorprop (i.e. methyl-(R,S)-2-(2,4-dichlorphenoxy) propionate) MCPP (i.e. 2-(4-chloro-2-methyl-phenoxy)propionic acid) Ruelene, i.e. (R,S)-4-tertbutyl-2-chlorophenylmethyl-N-methyl phosphoramidate (stereogenic phosphorus-Centre) o,p0 -DDT o,p0 -DDD Chlordanes (cis-, trans-, other congeners) Oxychlordane Chlordene and metabolites Photochlordene Heptachlor Heptachlor exoepoxide Bromocyclen Toxaphene® HHCB (1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethyl-cyclopenta [g]-2-benzopyrane; Galaxolide®; two stereogenic centres) Galaxolidone (1,3,4,6,7,8-hexahydro-4,6,6,7,8,8hexamethylcyclopenta[g]-2-benzopyrane-1-one metabolite of Galaxolide®; two stereogenic centres) AHTN (1-(5,6,7,8-tetrahydro-3,5,5,6,8,8-hexamethyl-2naphthalenyl)-ethanone; Tonalide®) ATII (1-[2,3-dihydro-1,1,2,6-tetramethyl-3-(1-methyl-ethyl)-1Hinden-5-yl]-ethanone; Traseolide®; two stereogenic centres) AHDI (1-(2,3-dihydro-1,1,2,3,3,6-hexamethyl-1H-inden-5-yl)ethanone; Phantolide®) Thalidomide (Contergan®) Naloxone (three stereogenic carbon centres, one nitrogen Centre) Tetrodotoxin Saxitoxin Anatoxin-a and homoanatoxin-a

Chapters of the present monograph 8.1; 8.2; 10.5 8.1; 8.2 8.1 8.2 5.1; 5.4; 8.1; 8.2; 8.3; 9.3; 10.2 5.4; 8.1; 8.2; 9.3; 9.4; 10.1; 5.4; 8.1; 8.2; 9.3; 10.2 5.4; 8.1; 8.2; 9.3; 10.2 8.2 8.2 5.1; 8.1; 8.2; 9.3, 10.2 5.1; 8.1; 8.2; 9.3, 10.2 8.1; 8.2 8.2; 10.2 1.6; 8.2 8.2

8.2 8.2 8.2 9.1 1.6 10.2 10.2 10.2

ethylsulfide applying two cytochrome P-450 isoenzymes purified from phenobarbital-induced rat liver, both of which generated 4-tolyl ethyl sulfoxide (Fig. 1.4), exhibiting predominantly the (S)-()-configuration. An example for a stereogenic phosphorus centre is represented by ruelene, that is, (R,S)-4-tertbutyl-2chlorophenylmethyl-N-methyl phosphoramidate.

1.6 General Principles of Chirality in Chemistry

O

9

H5C2

C2H5

O

S

S

(R)-4-tolyl ethyl sulfoxide

mirror

(S)-4-tolyl ethyl sulfoxide

Fig. 1.4 The sulfoxidation products of 4-tolyl ethylsulphide, (R)-4-tolyl ethyl sulfoxide (left-hand side) and (S)-4-tolyl ethyl sulfoxide (right-hand side)

Cl

ring B

Cl

Cl

ring A Cl

Cl

PCB 96

Cl

ring B

Cl

Cl

Cl

Cl

ring A

Cl

PCB 136

Fig. 1.5 The PCB congener on the left-hand side possesses a plane of symmetry perpendicular to ring B, which is assumed to be oriented perpendicular to ring A, and hence PCB 96 is achiral. By contrast, PCB 136 possesses no plane of symmetry and thus is a chiral PCB congener

1.6.2

Environmental Pollutants with Axial Chirality

Certain compounds that do not contain asymmetric atoms may nevertheless be chiral, if they contain a structure represented, for example, by a sub-group of polychlorinated biphenyls (PCBs) and their metabolites (Fig. 1.5). Biphenyls containing four large substituents in ortho positions cannot freely rotate about the central single bond because of steric hindrance. In such compounds, the two-ring systems are oriented in perpendicular planes or at least in planes between which an angle of >0–90 degrees exists. If either or both rings are symmetrical and perpendicular to each other, the molecule has a so-called plane of symmetry. For example, in PCB 96 (Fig. 1.5), ring B is symmetrical. A plane drawn perpendicular to ring B such that the part on one side of the plane is the exact reflection of the part on the other side contains all the atoms and groups in ring A, that is, this plane would cut the molecule into two equal parts. A mirror image of PCB 96 reflected parallel to this plane of symmetry would not meet the requirement of being non-superimposable with the original molecule, and hence, this compound is achiral. On the other hand, if an asymmetric substitution of both ring systems in a PCB is encountered, as represented by PCB 136 in Fig. 1.5, no plane of symmetry exists and, as a consequence, this PCB congener is chiral. It is not always necessary for four large

10

1 Introduction

Table 1.2 IUPAC nomenclature and chlorine substitution pattern of atropisomeric PCB congeners that are stable against racemisation at room temperature PCB congener PCB 45 PCB 84 PCB 88 PCB 91 PCB 95 PCB 131 PCB 132 PCB 135 PCB 136 PCB 139

Chlorine substitution 2,20 ,3,6 2,20 ,3,30 ,6 2,20 ,3,4,6 2,20 ,3,40 ,6 2,20 ,3,50 ,6 2,20 ,2,30 ,4,6 2,20 ,3,30 ,6 2,20 ,3,30 ,5,60 2,20 ,3,30 ,6,60 2,20 ,3,4,40 ,6

PCB congener PCB 144 PCB 149 PCB 171 PCB 174 PCB175 PCB 176 PCB 183 PCB 196 PCB 197

Chlorine substitution 2,20 ,3,4,50 ,6 2,20 ,3,40 ,50 ,6 2,20 ,3,30 ,4,40 ,6 2,20 ,3,30 ,4,5,60 2,20 ,3,30 ,4,6,60 2,20 ,3,30 ,4,6,60 2,20 ,3,4,40 ,50 ,6 2,20 ,3,30 ,4,40 ,5 2,20 ,3,30 ,4,40 ,6

ortho groups to be present, in order to inhibit free rotation. Compounds with three and even two groups, if large enough, can exhibit hindered rotation and, if suitably substituted, can be resolved into the two enantiomeric forms. Basically 78 PCB congeners out of 209 are chiral, however, only 19 of which turned out to be stable against racemisation at room temperature (Table 1.2). The rotational barrier of the latter group of PCBs attains values of at least 105–250 kJ/mol (Haglund and Harju 1999; Harju and Haglund 1999). PCB 136 shown in Fig. 1.5 belongs to this group. PCB stereoisomers that can be separated because rotation about single bonds is prevented or greatly slowed are called atropisomers. Very detailed investigations on the rotation barriers in dependence on the substituents and the substitution pattern of biphenyl congeners were carried out in the 1990s by the research groups of Schurig et al. (Glausch et al. 1995), König et al. (Weseloh et al. 1996; Wolf et al. 1996) and Haglund (Harju et al. 2003). For details on this aspect, the reader is referred to the above-cited publications. The relevance of atropisomeric structures in environmental transformation processes has been studied in recent follow-up studies (Ross et al. 2011; Dai et al. 2014; Megson et al. 2015; Wu et al. 2015; Ma et al. 2016; Feng et al. 2017; Lei et al. 2017). Furthermore, it should be noted that if either ring is symmetrical with respect to ortho- and meta-positions, the molecule also possesses a plane of symmetry, if any additional group in para-position is present, because such groups cannot cause lack of symmetry.

1.6.3

Asymmetry of Cyclic Environmental Pollutants

Sometimes, it is difficult to recognise the asymmetry of certain cyclic environmental pollutants such as hexachlorocyclohexane (HCH), because some additional rules must be applied, in order to discriminate reliably between achiral and chiral stereoisomers. Although the ultimate criterion is, of course, non-superimposability on the

1.6 General Principles of Chirality in Chemistry

4

H

4

11

Cl Cl

Cl

4

H

Cl

4

H Cl

Cl

H

Cl

H

H

1

H

H H

Cl

Cl

1

1

1

H

b -HCH

COOH

Cl

H H

Cl

Cl H

g -HCH

Ph

Cl

H

H

*

H

H

Cl

Cl

H H

Cl COOH a -Truxillic acid

Ph

H H

Cl H

Cl a -HCH

Fig. 1.6 Left-hand side top: γ-HCH possessing a plane of symmetry (H1, Cl1, C1, H4, Cl4, C4); right-hand side top: β-HCH exhibiting a plane of symmetry (H1, Cl1, C1, H4, Cl4, C4); left-hand side below: α-truxillic acid possessing a centre of symmetry (asterisk *); right-hand side below: (α)1,2,3,4,6,-hexachlorocyclohexane (α-HCH) exhibiting neither a plane nor a centre nor an alternating axis of symmetry

mirror image, simple tests like the presence of a plane of symmetry (see the previous chapter) may be often successfully applied. For instance, the plane of symmetry can be easily found in molecules of two most prominent HCH isomers, γ-HCH and β-HCH, presumably also by scientists who are not so familiar with chiral compounds: if we adopt the nomenclature of carbohydrates, where the two possible chair conformations are denominated 4C1 (conformation, in which the C4 is drawn upwards and the C1 downwards as shown in Fig. 1.6) and 1C4, we can define exactly the plane of symmetry, which in both cases can be laid through the H1, Cl1, C1, H4, Cl4, C4 atoms. Hence, these two HCH isomers as well as five other HCH isomers, where similar tests may be applied, are achiral. On the other hand, this simple test may be misleading. Compounds possessing such a plane are always achiral, but there are known a few cases in which compounds lack a plane of symmetry and are nevertheless achiral. Such substances may possess a centre of symmetry, for instance, α-truxillic acid (asterisk in Fig. 1.6), a component of the cocaine alkaloids. A centre of symmetry is a point within an object such that a straight line is drawn from any part or element of the object to the centre and extended an equal distance on the other side encounters an equal part or element. Furthermore, it has to be checked as to whether or not an alternating axis of symmetry can be found. An alternating axis of symmetry of the order n (also called an improper axis of rotation) is an axis such that when an object containing such an axis is rotated by 360 /n about the axis and then the reflection is effected across a

12

1 Introduction

plane at right angles to the axis, a new object is obtained that is indistinguishable from the original one. In general, a one-fold alternating axis is equivalent to a plane of symmetry and a two-fold alternative axis is identical with a centre of inversion. Therefore, the fundamental symmetry condition for optical activity is the absence of an improper axis. In the case of α-HCH, neither a plane nor a centre nor an alternating axis of symmetry is encountered, and, as a consequence, α-HCH is chiral, the only chiral of eight conceivable HCH isomers. In lectures and monographs of basic organic chemistry (Dalton 2011; Koskinen 2012; Boikess 2015; Tro 2018; Timberlake and Orgill 2019), additional types of chirality are usually discussed. For completeness, we briefly mention these types without going into detail, because we do not necessarily need these aspects for the present monograph. These types of chirality include allene-type chirality (Webster et al. 2016), in which the central carbon atom is sp-bonded with even numbers of double bonds, both sides being asymmetrically substituted, helical asymmetry (represented by hexahelicene) and planar asymmetry (represented by substituted paracyclophanes) such as described earlier (Zhao et al. 2014). Basically, the same tests described above (plane or centre or alternating axis of symmetry) can be applied, in order to find out as to whether or not such molecules are chiral.

1.6.4

Chiral Environmental Pollutants with Two or More Stereogenic Centres

As described in comprehensive review publication and repeated here (Hühnerfuss and Shah 2009), when a molecule possesses two asymmetric centres, each centre has to be attributed its own configuration. The first centre may exhibit R- or S-configuration, and so may the second. Accordingly, a systematic variation of all possibilities at each stereogenic centre shows that two times two, that is, four stereoisomers can be formulated. Generalising, the maximum number of stereoisomers existing for a molecule with n stereogenic centres thus may be 2n stereoisomers. In order to distinguish between these different possibilities, the R,S-nomenclature that is based on the so-called CIP rules is strongly recommended, because these rules can be universally used also in those cases, where the old D,L-nomenclature is ambiguous. It would be beyond the scope of the present monograph to develop the exact CIP rules herein. For descriptions of the system and sets of sequence rules, the reader should refer to basic organic monographs (Boikess 2015; Tro 2018; Timberlake and Orgill 2019), or to the original publications by Cahn, Ingold and Prelog (¼ CIP) (Cahn et al. 1956, 1966; Prelog and Helmchen 1982; Helmchen 2016). In the latter case, the more complicated assignments such as those for (+)-α-1S,2R,3R,4S,5S,6S-HCH will become accessible, though some time will be required to infer the exact nomenclature for such cyclic compounds. Although four is the maximum number of isomers when the compound exhibits two chiral centres, this number may be reduced when the three groups on one

1.6 General Principles of Chirality in Chemistry

COOH

COOH HO H

13

H

H HO

OH

OH

H

OH

HO

H

H

H

OH

HO

H

COOH

COOH

COOH

COOH

COOH

(2S,3S)-(-)-TA mirror (2R,3R)-(+)TA

COOH

meso-Tartaric acid

Fig. 1.7 Left-hand side: the non-superimposable mirror images (¼ enantiomers) (2S,3S)-()- and (2R,3R)-(+)- tartaric acid; right-hand side: the two meso-forms of tartaric acid which exhibit a plane of symmetry (-------)

enantiomers

A d i a s t

d

C

s ia

t.

di

as

enantiomers

B

t.

d i a s t

D

mirror Fig. 1.8 Relationship between the four stereoisomers A, B, C and D that can be formulated for chiral compounds with two stereogenic centres and thus 2n ¼ 4 stereoisomers. A/B and C/D are mirror images of each other and thus enantiomers, while all other relationships are designated by the term diastereomers (diast)

stereogenic centre are the same as those on the other. In this case, one of the isomers has a plane of symmetry and hence is achiral, even though it exhibits two asymmetric centres. A typical example, tartaric acid, is shown in Fig. 1.7, for which only three isomers exist: a pair of enantiomers and an achiral so-called meso-form. Therefore, it is strongly recommended that one should exclude or verify the existence of a mesoform, if chiral compounds with two stereogenic centres are being investigated, where the four groups on one asymmetric centre are the same as those on the other chiral atom. This confirmation is necessary, because this will have consequences for the enantioselective chromatography, an aspect which will be discussed in more detail below (Hühnerfuss and Shah 2009). In the case of a molecule with two stereogenic centres, four stereoisomers A, B, C and D can be formulated, as outlined above. Since a molecule can only possess one mirror image, each of the four stereoisomers can only be the reflection of one of the three remaining stereoisomers. As a consequence, two enantiomeric pairs exist, A/B and C/D. As schematically shown in Fig. 1.8, the pairs A/C, A/D, B/C and B/D cannot be mirror images of each other. These relationships are designated by the

14

1 Introduction

term diastereomers. It is important to note that diastereomers in general possess different physical properties. By contrast, enantiomers exhibit identical physical and chemical properties except in two important respects: • They rotate the plane of polarised light in opposite directions, though in equal amounts. The isomer that rotates the plane to the left (counterclockwise) is called the levo isomer which is indicated with (), while the one that rotates the plane to the right (clockwise) is called the dextro isomer and is designated (+). • They react at different rates with other chiral compounds. These rates may be so close together that the distinction is practically useless, or they may be so far apart that one enantiomer undergoes the reaction at a convenient rate, while the other does not react at all. This is the reason that many compounds are biologically active while their enantiomers are not. However, enantiomers react at the same rate as achiral compounds. Furthermore, enantiomers may react at different rates with achiral molecules, if a chiral catalyst is present, they may show different solubilities in a chiral solvent, and they may exhibit different indexes of refraction or absorption spectra when examined with circularly polarised light. In most cases, these differences are too small to be useful and are often too small to be measured. In general, it can be concluded that enantiomers possess identical properties in a symmetrical environment, but their properties may differ in an unsymmetrical environment. This basic principle forms the background for enantioselective chromatography, where chiral stationary phases are being used which form the asymmetric environment, as required. Thus, diastereomeric complexes between the respective enantiomers and the chiral stationary phase emerge that give rise to different retention times, hence allowing an enantioselective separation. By contrast, diastereomers, which possess different physical properties, can also be separated on achiral stationary phases. The basic principles of chirality summarised above set the stage for an understanding of the different chromatographic characteristics of chiral and achiral environmental pollutants with one or more stereogenic centres. As the separation of diastereomers can be separated on achiral stationary phases, the maximum number of peaks to be expected will be identical with the number of diastereomers. The separation of enantiomers requires an asymmetric environment and thus chiral stationary phases. Then, the maximum number of peaks to be expected will be 2n, provided that no meso-forms will be present and accordingly reduce this number. An example for a chiral environmental pollutant that exhibits two asymmetric centres and which could be successfully separated in its two diastereomeric pairs of enantiomers (i.e. four peaks) by enantioselective capillary gas chromatography is represented by the musk compound HHCB (Fig. 1.9; for further details, see Chap. 3.3).

References Fig. 1.9 The molecular structure of the musk compound HHCB (Galaxolide®), possessing two diastereomeric centres (asterisks *)

15 CH 3

H 3C

CH 3 CH 3

O H 3C

CH 3

HHCB )Galaxolide )

References Bada JL (2009) Enantiomeric excesses in the Murchison meteorite and the origin of homochirality in terrestrial biology. Proc Natl Acad Sci U S A 106(32):E85. author reply E86 Bakhtiar R, Ramos L, Tse FL (2001) Use of atmospheric pressure ionization mass spectrometry in enantioselective liquid chromatography. Chirality 13(2):63–74 Barron LD (2012a) Cosmic chirality both true and false. Chirality 24(12):957–958 Barron LD (2012b) From cosmic chirality to protein structure: Lord Kelvin's legacy. Chirality 24 (11):879–893 Basheer AA (2017) Chemical chiral pollution: impact on the society and science and need of the regulations in the 21(st) century. Chirality Bernstein M (2006) Prebiotic materials from on and off the early earth. Philos Trans R Soc Lond Ser B Biol Sci 361(1474):1689–1700. discussion 1700-1682 Boikess RS (2015) Chemical principles for organic chemistry. Cengage Learning, Stamford, CT Bonner WA (1998) Homochirality and life. EXS 85:159–188 Brack A, Clancy P, Fitton B, Hoffmann B, Horneck G, Kurat G, Maxwell J, Ori G, Pillinger C, Raulin F, Thomas N, Westall F (1998) Search for life on mars. Biol Sci Space 12(2):119–123 Brack A, Horneck G, Cockell CS, Berces A, Belisheva NK, Eiroa C, Henning T, Herbst T, Kaltenegger L, Leger A, Liseau R, Lammer H, Selsis F, Beichman C, Danchi W, Fridlund M, Lunine J, Paresce F, Penny A, Quirrenbach A, Rottgering H, Schneider J, Stam D, Tinetti G, White GJ (2010) Origin and evolution of life on terrestrial planets. Astrobiology 10(1):69–76 Breslow R, Cheng ZL (2009) On the origin of terrestrial homochirality for nucleosides and amino acids. Proc Natl Acad Sci U S A 106(23):9144–9146 Burke D, Henderson DJ (2002) Chirality: a blueprint for the future. Br J Anaesth 88(4):563–576 Cahn RN, Ingold CK, Prelog V (1956) The specification of asymmetric configuration in organic chemistry. Experientia 12:43 Cahn RN, Ingold CK, Prelog V (1966) Spezifikation der molekularen Chiralität. Angewandte Chemie-International Edition in English 78:33 Cintas P (2008) Chirality and chemical processes: a few afterthoughts. Chirality 20(1):2–4 Cronin JR, Pizzarello S (1997) Enantiomeric excesses in meteoritic amino acids. Science 275 (5302):951–955 Dai S, Wong CS, Qiu J, Wang M, Chai T, Fan L, Yang S (2014) Enantioselective accumulation of chiral polychlorinated biphenyls in lotus plant (Nelumbonucifera spp.). J Hazard Mater 280:612–618 Dalton DR (2011) Foundations of organic chemistry: unity and diversity of structures, pathways, and reactions. Wiley, Hoboken, NJ de Duve C (2011) Life as a cosmic imperative? Philos Trans A Math Phys Eng Sci 369 (1936):620–623 Donaldson DJ, Tervahattu H, Tuck AF, Vaida V (2004) Organic aerosols and the origin of life: an hypothesis. Orig Life Evol Biosph 34(1–2):57–67

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Engel MH, Macko SA, Silfer JA (1990) Carbon isotope composition of individual amino acids in the Murchison meteorite. Nature 348(6296):47–49 Evans AC, Meinert C, Giri C, Goesmann F, Meierhenrich UJ (2012) Chirality, photochemistry and the detection of amino acids in interstellar ice analogues and comets. Chem Soc Rev 41 (16):5447–5458 Falk-Petersen IB (2005) Comparative organ differentiation during early life stages of marine fish. Fish Shellfish Immunol 19(5):397–412 Feng W, Zheng J, Robin G, Dong Y, Ichikawa M, Inoue Y, Mori T, Nakano T, Pessah IN (2017) Enantioselectivity of 2,20 ,3,50 ,6-Pentachlorobiphenyl (PCB 95) Atropisomers toward ryanodine receptors (RyRs) and their influences on hippocampal neuronal networks. Environ Sci Technol 51(24):14406–14416 Fletcher S (1993) Chirality in the umbilical cord. Br J Obstet Gynaecol 100(3):234–236 Fuss W (2009) Does life originate from a single molecule? Chirality 21(2):299–304 Gardner M (1990) The ambidextrous universe: mirror asymmetry and time-reversed worlds, Scribne Glausch A, Hahn J, Schurig V (1995) Enantioselective determination of chiral 2,20 ,3,30 ,4,60 -hexachlorobiphenyl (PCB 132) in human milk samples by multidimensional gas chromatography/electron capture detection and by mass spectrometry. Chemosphere 30(11):2079–2085 Glavin DP, Dworkin JP (2009) Enrichment of the amino acid L-isovaline by aqueous alteration on CI and CM meteorite parent bodies. Proc Natl Acad Sci U S A 106(14):5487–5492 Haglund P, Harju M (1999) Chromatographic separation of atropisomeric environmental pollutants. Abstr Pap Am Chem Soc 218:U569–U569 Harju MT, Haglund P (1999) Determination of the rotational energy barriers of atropisomeric polychlorinated biphenyls. Fresen J Anal Chem 364(3):219–223 Harju M, Bergman A, Olsson M, Roos A, Haglund P (2003) Determination of atropisomeric and planar polychlorinated biphenyls, their enantiomeric fractions and tissue distribution in grey seals using comprehensive 2D gas chromatography. J Chromatogr A 1019(1–2):127–142 Hein JE, Blackmond DG (2012) On the origin of single chirality of amino acids and sugars in biogenesis. Acc Chem Res 45(12):2045–2054 Helmchen G (2016) The 50th anniversary of the Cahn-Ingold-Prelog specification of molecular chirality. Angew Chem Int Ed Engl 55(24):6798–6799 Hühnerfuss H, Shah MR (2009) Enantioselective chromatography – a powerful tool for discrimination of biotic and abiotic transformation processes of chiral environmental pollutants. J Chromatogr A 1216/3:481–502 Hühnerfuss H, Faller J, Kallenborn R, König WA, Ludwig P, Pfaffenberger B, Oehme M, Rimkus G (1993) Enantioselective and nonenantioselective degradation of organic pollutants in the marine ecosystem. Chirality 5(5):393–399 Jedrzejewska H, Szumna A (2017) Making a right or left choice: chiral self-sorting as a tool for the formation of discrete complex structures. Chem Rev 117(6):4863–4899 Koskinen A (2012) Asymmetric synthesis of natural products. Wiley, Hoboken, NJ Kraft P, Frater G (2001) Enantioselectivity of the musk odor sensation. Chirality 13(8):388–394 Lei H, Lin X, Han L, Ma J, Ma Q, Zhong J, Liu Y, Sun T, Wang J, Huang X (2017) New metabolites and bioactive chlorinated benzophenone derivatives produced by a marine-derived fungus Pestalotiopsis heterocornis. Mar Drugs 15(3) Liu W, Ye J, Jin M (2009) Enantioselective phytoeffects of chiral pesticides. J Agric Food Chem 57 (6):2087–2095 Ma C, Zhai G, Wu H, Kania-Korwel I, Lehmler HJ, Schnoor JL (2016) Identification of a novel hydroxylated metabolite of 2,20 ,3,50 ,6-pentachlorobiphenyl formed in whole poplar plants. Environ Sci Pollut Res Int 23(3):2089–2098 Makela T, Annila A (2010) Natural patterns of energy dispersal. Phys Life Rev 7(4):477–498 Megson D, Focant JF, Patterson DG, Robson M, Lohan MC, Worsfold PJ, Comber S, Kalin R, Reiner E, O'Sullivan G (2015) Can polychlorinated biphenyl (PCB) signatures and enantiomer

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fractions be used for source identification and to age date occupational exposure? Environ Int 81:56–63 Pasteur L (1848) Mémoire sur la relation qui peut exister entre la forme crystalline et la composition chimique, et sur la cause de la polarisation rotatoire. C R Acad Sci 26:4 Pavlov AK, Kalinin VL, Konstantinov AN, Shelegedin VN, Pavlov AA (2006) Was earth ever infected by Martian biota? Clues from radioresistant bacteria. Astrobiology 6(6):911–918 Pizzarello S (2006) The chemistry of life's origin: a carbonaceous meteorite perspective. Acc Chem Res 39(4):231–237 Pizzarello S, Cronin JR (2000) Non-racemic amino acids in the Murray and Murchison meteorites. Geochim Cosmochim Acta 64(2):329–338 Pizzarello S, Shock E (2010) The organic composition of carbonaceous meteorites: the evolutionary story ahead of biochemistry. Cold Spring Harb Perspect Biol 2(3):a002105 Prelog V (1976) Chirality in chemistry. Science 193(4247):17–24 Prelog V, Helmchen G (1982) Grundlagen des CIP-Systems und Vorschläge für eine Revision. Angew Chem Int Ed Engl 94:27 Queneau Y, Dumoulin F, Cheaib R, Chambert S, Andraud C, Bretonniere Y, Blum LJ, Boullanger P, Girard-Egrot A (2011) Two-dimensional supramolecular assemblies involving neoglycoplipids: self-organization and insertion properties into Langmuir monolayers. Biochimie 93(1):101–112 Ross MS, Pulster EL, Ejsmont MB, Chow EA, Hessel CM, Maruya KA, Wong CS (2011) Enantioselectivity of polychlorinated biphenyl atropisomers in sediment and biota from the turtle/Brunswick River estuary, Georgia, USA. Mar Pollut Bull 63(5–12):548–555 Sandford SA (2008) Terrestrial analysis of the organic component of comet dust. Annu Rev Anal Chem (Palo Alto, Calif) 1:549–578 Schilthuizen M, Davison A (2005) The convoluted evolution of snail chirality. Naturwissenschaften 92(11):504–515 Soffen GA (1986) Is there a single origin of life? Adv Space Res 6(11):57–60 Sojo V (2015) On the biogenic origins of homochirality. Orig Life Evol Biosph 45(1–2):219–224 Speranza M, Rondino F, Satta M, Paladini A, Giardini A, Catone D, Piccirillo S (2009) Molecular and supramolecular chirality: R2PI spectroscopy as a tool for the gas-phase recognition of chiral systems of biological interest. Chirality 21(1):119–144 Spitzer J (2013) Emergence of life from multicomponent mixtures of chemicals: the case for experiments with cycling physicochemical gradients. Astrobiology 13(4):404–413 Suzuki T, Kurokawa T (2000) Organogenesis in flounder. Tanpakushitsu Kakusan Koso 45 (17 Suppl):2752–2758 Szopa C, Meierhenrich UJ, Coscia D, Janin L, Goesmann F, Sternberg R, Brun JF, Israel G, Cabane M, Roll R, Raulin F, Thiemann W, Vidal-Madjar C, Rosenbauer H (2002a) Gas chromatography for in situ analysis of a cometary nucleus. IV. Study of capillary column robustness for space application. J Chromatogr A 982(2):303–312 Szopa C, Sternberg R, Coscia D, Raulin F, Vidal-Madjar C, Rosenbauer H (2002b) Gas chromatography for in situ analysis of a cometary nucleus III. Multi-capillary column system for the cometary sampling and composition experiment of the Rosetta lander probe. J Chromatogr A 953(1–2):165–173 Timberlake KC, Orgill M (2019) General, organic, and biological chemistry: structures of life. Pearson Education, San Francisco, CA Tro NJ (2018) Chemistry: structure and properties. Hoboken, NJ, Pearson Ulrich EM, Morrison CN, Goldsmith MR, Foreman WT (2012) Chiral pesticides: identification, description, and environmental implications. Rev Environ Contam Toxicol 217:1–74 Waxman DJ, Light DR, Walsh C (1982) Chiral sulfoxidations catalyzed by rat liver cytochromes P-450. Biochemistry 21(10):2499–2507 Webster S, Sutherland DR, Lee AL (2016) Chirality transfer in gold(I)-catalysed Hydroalkoxylation of 1,3-Disubstituted Allenes. Chemistry 22(51):18593–18600

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Wensink HH, Morales-Anda L (2015) Chiral assembly of weakly curled hard rods: effect of steric chirality and polarity. J Chem Phys 143(14):144907 Weseloh G, Wolf C, König W (1996) New technique for the determination of interconversion processes based on capillary zone electrophoresis: studies with axially chiral biphenyls. Chirality 8:5 Wolf C, Hochmuth DH, König WA, Roussel C (1996) Influence of substituents on the rotational energy barrier of axially chiral biphenyls. Part 2. Liebigs Ann 1996:357–363 Woolf NJ (2015) A hypothesis about the origin of biology. Orig Life Evol Biosph 45(1–2):257–274 Wu X, Barnhart C, Lein PJ, Lehmler HJ (2015) Hepatic metabolism affects the atropselective disposition of 2,20 ,3,30 ,6,60 -hexachlorobiphenyl (PCB 136) in mice. Environ Sci Technol 49 (1):616–625 Zhang Y, Ye J, Liu M (2017) Enantioselective biotransformation of chiral persistent organic pollutants. Curr Protein Pept Sci 18(1):48–56 Zhao T, Zhang Q, Long H, Xu L (2014) Graph theoretical representation of atomic asymmetry and molecular chirality of benzenoids in two-dimensional space. PLoS One 9(7):e102043

Chapter 2

Criteria for the Selection of a Proper Enantiomer-Selective Analytical Method

During the past two decades and after the first edition of this monograph was published in 2001, tremendous progress has been made with respect to sensitivity and selectivity in scientific technology for ultra-trace analysis. Today, chromatographic separation and modern mass-selective detection have been significantly improved as compared with the earlier methods introduced during the first edition. High-resolution liquid chromatography today is the separation method of choice for enantiomer-selective separation of many chiral environmental toxicants (Hühnerfuss and Shah 2009; Kasprzyk-Hordern 2010; Lehmler et al. 2010; Li et al. 2010; Nillos et al. 2010; Ali et al. 2012; Brooks et al. 2012; Ulrich et al. 2012; Kania-Korwel and Lehmler 2016; Basheer 2017; Guo et al. 2017; Peluso et al. 2017). Many updated monographs, books and reviews are today illustrating the versatility of enantiomerselective analytical methods, not just for environmental pollutant research (Inoue and Ramamurthy 2004; Reddy and Miḥvar 2004; Francotte and Lindner 2006; Subramanian 2007; Eeckhaut and Michotte 2009; Berthod 2010; Cutillas and Timms 2010; Dasgupta 2010; Busch and Busch 2011; Garrison et al. 2011; Lin et al. 2011). However, today and in contrast to the majority of other application areas, ultratrace analytical methods used for the determination of environmental pollutants are highly sensitive, but unfortunately, also still quite selective towards the compounds to be analysed. Depending on physical parameters like boiling point, polarity, water solubility, etc., each method is only reliable for a limited number of analytes, and therefore, in addition, an adjustment and optimisation for each compound group analysed are required. In general, highly sophisticated separation and detection methods are available for the multi-component monitoring of the majority of the known environmental pollutants. Today, the challenge for the modern environmental chemist is to identify and characterise new potential pollutants and develop effective multi-compound or even non-target trace analytical methods for their detection (Farre et al. 2012). Chromatographic separations driven by the solvent strength of the mobile phases, temperature or pressure differences during the separation as well as electric forces (e.g. electrophoresis, electro kinetic chromatography) © Springer Nature Switzerland AG 2021 R. Kallenborn et al., Chiral Environmental Pollutants, https://doi.org/10.1007/978-3-030-62456-9_2

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allow an effective starting point for the selectivity challenges of the new tasks of modern environmental chemistry (Fischer et al. 2012; Kalachova et al. 2013). However, also today, methods that are specially optimised for the needs of chiral trace analysis have to be developed, keeping in mind that the separation of two enantiomers by chromatography leads to a considerable reduction in sensitivity (Trivedi et al. 2007; Samuelsson et al. 2009; Camacho-Munoz and KasprzykHordern 2015; Adhikari et al. 2016; Kazsoki et al. 2016; Naile et al. 2016; Sanganyado et al. 2017). Therefore, only highly sensitive, but very selective detection methods can be used for the ultra-trace analysis of chiral environmental pollutants. In addition, today all commercially available stationary phases used for separation purposes in high-resolution liquid and gas and liquid chromatography are highly selective (Bidleman et al. 2002; Qi et al. 2016). Thus, these methods are only able to separate a very limited number of compounds simultaneously. In order to increase in the effectivity of these stationary phases, different technical approaches have been investigated for all types of chromatographic separation methods. A detailed and updated discussion of these different methods will be given in the next sections of this chapter. For new emerging environmental contaminants as well as for conventional environmental pollutants, two important physical parameters have to be considered when choosing a suitable chromatographic separation and a subsequent detection method.

2.1

Polarity Expressed as Octanol–Water Partitioning Coefficient (KOW)

One of the most comprehensively applied parameters for the characterisation of chromatographic separation as well as for the assessment of environmental behaviour of chemicals during the past four decades is the log KOW (¼ the logarithm of the octanol–water partition coefficient). According to the general partition theory, the distribution of a substance, S, between two immiscible solvents (such as aqueous and organic phases) is based on the substance-specific equilibrium condition that is described by the following equation: Saq , Sorg where Saq is the amount of substance “S” in the aqueous phase and Sorg is the amount of substance “S” in the organic phase. Thus, the equilibrium constant “K” for this equilibrium condition represents the quotient of the respective concentrations:

2.2 Dipole Moment

21



 K ¼ Sorg = Saq “K” is also called the partition or distribution coefficient. Thus, for the distribution of solute, S, in our specific solvent system, octanol/water can be assumed:

 K OW ¼ ½Soctanol = Saq and log K OW ¼ log ½Soctanol = log Saq The log KOW can, for example, be used to estimate whether gas chromatographic or liquid chromatographic methods should be employed for the analysis of a specific analyte “S”. In general, an analytical chemist in charge of developing a new analytical method can, thus, assume that compounds with a log KOW value in the range between 1 and 4 should be analysed by liquid chromatographic methods, whereas a log KOW value larger than 4 indicates the application of gas chromatographic methods for the analysis (Liu et al. 2014; Roman et al. 2014; Golubovic et al. 2016). More details about the methods for the determination of partitioning coefficients can be inferred from the literature (Hayward et al. 2006; Kah and Brown 2008; Levitt 2010; Rayne and Forest 2010; Kim et al. 2014; Roman et al. 2014).

2.2

Dipole Moment

A further important parameter is the dipole moment of a molecule analysed. The dipole moment (μ) is defined as the first moment of the charge distribution. In simple terms, this means that it is the difference between the average location of the positive charge (the nuclei) and the average location of the negative charge (the electrons). If the average locations are the same, no dipole moment exists and, hence, the molecule is non-polar (Bonincontro and Risuleo 2003; Salieres et al. 2012; Buckingham 2015). If the locations of the charges are different, the molecule will exhibit a dipole moment and, hence, be polar. The magnitude of the dipole moment is a measure of the polarity and is typically measured in CGS units of Debye (Bonincontro and Risuleo 2003), which in the SI system have to be converted as follows: 1 Debye ¼ 333564  1030 Cm. Mathematically, the dipole moment is given by the following formula Z μ¼

X i  hX qi Ri þ dr Nx dRNo P r Nx , RNo er j

where P is the probability of finding the electrons and nuclei at specific positions in space, qi is the charge on nucleus i, Ri is the location of nucleus i, e is the charge of an electron and rj is the location of electron j. A further parameter which may be important for the choice of an analytical method is solubility. However, the extent to which an ionic compound is soluble in a solvent is dependent on a parameter explained before, the polarity. As a general

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rule, it can be assumed that the solubility of an ionic compound in an organic solvent decreases as the charge on the ions increases.

2.3

Analyte–Stationary Phase Interactions in Chromatography Separation

In general, small differences in the interactions between the stationary phase and every single compound lead to the desired separation effect of a compound mixture by chromatographic methods. A variety of weak interactions is known and actively applied to substance-specific retention in chromatographic separation. For a more in-depth insight into these comprehensive interactions in modern chromatographic systems, we refer to recent general reviews (Cecchi 2011; Guo and Gaiki 2011; Jian et al. 2011; Buszewski and Noga 2012; Sherma 2013; Shi et al. 2015; Ciura et al. 2017; Jandera and Janas 2017; McCalley 2017; Jandera and Hajek 2018; Taraji et al. 2018) and to modern standard text book on advanced organic chromatographic separation. For chiral compounds, however, due to the special stereoselective structural character of these groups of compounds, William Pirkle established a general rule for an optimised chromatographic separation (Hu and Ziffer 1991; Caccamese 1993; Forjan et al. 2007; Badaloni et al. 2010), the so-called three-point rule: Chiral recognition during chromatographic separation processes requires a minimum of three simultaneous but independent compound-stationary phase (SP) interactions for enantiomer selective separation and at least one of the enantiomers with at least one of the interactions being enantiomer selective.

The following interactions are known to be important for a chromatographic separation: • • • • •

Hydrogen bonds π–π interactions. Dipole–dipole interactions Van der Waals interactions Acid–base–ionic interactions.

This simple principle of chromatographic interaction rules the general development of chromatographic methods, the selectivity and sensitivity as important prerequisites for the optimisation of new chiral stationary phases.

References

2.4

23

Rotation Energy

One type of chirality demands additional considerations concerning chromatographic separations. As already described in the introduction, atropisomers gain their chirality by a rotation hindrance between two cyclic molecule substructures with different substitution patterns (Perez-Fernandez et al. 2012; Dai et al. 2017; Li et al. 2018). Only if the substituents are of a certain size, the rotation barrier can be preserved (LaPlante et al. 2011; Glunz 2018). Today, it is possible to calculate the energy of the rotation barrier and the temperature limit below with chromatographic separation (gas or liquid chromatographic methods) (Ghosn and Wolf 2011; Yan et al. 2015). Furthermore, theoretical calculations may also be applied to determine the temperature-dependent stability of atropisomers (Hesek et al. 2001; Casarini et al. 2005; Callear et al. 2015; Buevich 2016). One of the predominant environmental pollution groups featuring atropisomers in environmental pollution research is the group of polychlorinated biphenyls (PCBs). PCBs will be introduced later in the group-specific chapters (Warner et al. 2009; Lu et al. 2013). Based on the careful application of the above-described interactions to chromatography in combination with sensitive detectors, analytical chemists have developed a multitude of selective and sensitive methods for the chromatographic separation of environmentally relevant chiral trace compounds into their respective enantiomers. However, based on modern ultra-high-resolution mass spectrometry, new methods based on enantiomer-selective mass-selective separation focussing on enantiomer selectivity in ion mobility and MALDI are currently under development. These methods are considered as a new promising enantiomer-selective detection method also for low-level environmental applications (Awad and El-Aneed 2013; Jurcek et al. 2015; Nachtigall et al. 2018; Li et al. 2019).

References Adhikari S, Kang JS, Lee W (2016) A convenient and validated enantiomer separation of chiral aliphatic amines as nitrobenzoxadiazole derivatives on polysaccharide-derived chiral stationary phases under simultaneous ultraviolet and fluorescence detection. Chirality 28(12):789–794 Ali I, Aboul-Enein HY, Sanagi MM, Wan-ibrahim WA (2012) Chirality and its role in environmental toxicology. In: Luch A (ed) Molecular, clinical and environmental toxicology, vol 3. Springer, Heidelberg, pp 413–436 Awad H, El-Aneed A (2013) Enantioselectivity of mass spectrometry: challenges and promises. Mass Spectrom Rev 32(6):466–483 Badaloni E, Cabri W, Ciogli A, D'Acquarica I, Deias R, Gasparrini F, Giorgi F, Kotoni D, Villani C (2010) Extending the use of “inverted chirality columns approach” for enantiomeric excess determination in absence of reference samples: application to a water-soluble camptothecin derivative. J Chromatogr A 1217(7):1024–1032 Basheer AA (2017) Chemical chiral pollution: impact on the society and science and need of the regulations in the 21(st) century. Chirality 30(4):402–406 Berthod A (2010) Chiral recognition in separation methods: mechanisms and applications. Springer, Heidelberg; New York

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Bidleman TF, Leone AD, Falconer RL, Harner T, Jantunen LM, Wiberg K, Helm PA, Diamond ML, Loo B (2002) Chiral pesticides in soil and water and exchange with the atmosphere. Sci World J 2:357–373 Bonincontro A, Risuleo G (2003) Dielectric spectroscopy as a probe for the investigation of conformational properties of proteins. Spectrochim Acta A Mol Biomol Spectrosc 59 (12):2677–2684 Brooks BW, Berninger JP, Kristofco LA, Ramirez AJ, Stanley JK, Valenti TW (2012) Pharmaceuticals in the environment: lessons learned for reducing uncertainties in environmental risk assessment. Prog Mol Biol Transl Sci 112:231–258 Buckingham AD (2015) Chiral discrimination in NMR spectroscopy. Q Rev Biophys 48 (4):421–423 Buevich AV (2016) Atropisomerization of 8-membered Dibenzolactam: experimental NMR and theoretical DFT study. J Org Chem 81(2):485–501 Busch KW, Busch MA (2011) Chiral analysis. Elsevier Science, Amsterdam Buszewski B, Noga S (2012) Hydrophilic interaction liquid chromatography (HILIC)--a powerful separation technique. Anal Bioanal Chem 402(1):231–247 Caccamese S (1993) Direct high-performance liquid chromatography (HPLC) separation of etodolac enantiomers using chiral stationary phases. Chirality 5(3):164–167 Callear SK, Johnston A, McLain SE, Imberti S (2015) Conformation and interactions of dopamine hydrochloride in solution. J Chem Phys 142(1):014502 Camacho-Munoz D, Kasprzyk-Hordern B (2015) Multi-residue enantiomeric analysis of human and veterinary pharmaceuticals and their metabolites in environmental samples by chiral liquid chromatography coupled with tandem mass spectrometry detection. Anal Bioanal Chem 407 (30):9085–9104 Casarini D, Coluccini C, Lunazzi L, Mazzanti A (2005) Stereolabile and configurationally stable atropisomers of hindered aryl carbinols. J Org Chem 70(13):5098–5102 Cecchi T (2011) Retention mechanism for ion-pair chromatography with Chaotropic reagents. From ion-pair chromatography toward a unified salt chromatography. Adv Chromatogr 49:1–35 Ciura K, Dziomba S, Nowakowska J, Markuszewski MJ (2017) Thin layer chromatography in drug discovery process. J Chromatogr A 1520:9–22 Cutillas PR, Timms JF (2010) LC-MS/MS in proteomics: methods and applications. Humana Press, New York, NY Dai J, Wang C, Traeger SC, Discenza L, Obermeier MT, Tymiak AA, Zhang Y (2017) The role of chromatographic and chiroptical spectroscopic techniques and methodologies in support of drug discovery for atropisomeric drug inhibitors of Bruton's tyrosine kinase. J Chromatogr A 1487:116–128 Dasgupta A (2010) Advances in chromatographic techniques for therapeutic drug monitoring. CRC Press/Taylor & Francis, Boca Raton Eeckhaut A v, Michotte Y (2009) Chiral separations by capillary electrophoresis. Taylor & Francis, Boca Raton Farre M, Kantiani L, Petrovic M, Perez S, Barcelo D (2012) Achievements and future trends in the analysis of emerging organic contaminants in environmental samples by mass spectrometry and bioanalytical techniques. J Chromatogr A 1259:86–99 Fischer K, Fries E, Korner W, Schmalz C, Zwiener C (2012) New developments in the trace analysis of organic water pollutants. Appl Microbiol Biotechnol 94(1):11–28 Forjan DM, Gazic I, Vinkovic V (2007) Role of the weak interactions in enantiorecognition of racemic dihydropyrimidinones by novel brush-type chiral stationary phases. Chirality 19 (6):446–452 Francotte E, Lindner W (2006) Chirality in drug research. Wiley-VCH, Weinheim Garrison AW, Gan JJ, Liu W, American Chemical Society. Division of Agrochemicals (2011) Chiral pesticides: stereoselectivity and its consequences. American Chemical Society, Washington, DC

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LaPlante SR, Edwards PJ, Fader LD, Jakalian A, Hucke O (2011) Revealing atropisomer axial chirality in drug discovery. ChemMedChem 6(3):505–513 Lehmler HJ, Harrad SJ, Hühnerfuss H, Kania-Korwel I, Lee CM, Lu Z, Wong CS (2010) Chiral polychlorinated biphenyl transport, metabolism, and distribution: a review. Environ Sci Technol 44(8):2757–2766 Levitt DG (2010) Quantitative relationship between the octanol/water partition coefficient and the diffusion limitation of the exchange between adipose and blood. BMC Clin Pharmacol 10:1 Li L, Zhou S, Jin L, Zhang C, Liu W (2010) Enantiomeric separation of organophosphorus pesticides by high-performance liquid chromatography, gas chromatography and capillary electrophoresis and their applications to environmental fate and toxicity assays. J Chromatogr B Analyt Technol Biomed Life Sci 878(17–18):1264–1276 Li X, Parkin SR, Lehmler HJ (2018) Absolute configuration of 2,20 ,3,30 ,6-pentachlorinatedbiphenyl (PCB 84) atropisomers. Environ Sci Pollut Res Int 25(17):16402–16410 Li G, DeLaney K, Li L (2019) Molecular basis for chirality-regulated Abeta self-assembly and receptor recognition revealed by ion mobility-mass spectrometry. Nat Commun 10(1):5038 Lin G-Q, You Q-D, Cheng J-F (2011) Chiral drugs: chemistry and biological action. Wiley, Hoboken, NJ Liu D, Zou X, Gao M, Gu M, Xiao H (2014) Hydrophilic organic/salt-containing aqueous two-phase solvent system for counter-current chromatography: a novel technique for separation of polar compounds. J Chromatogr A 1356:157–162 Lu Z, Kania-Korwel I, Lehmler HJ, Wong CS (2013) Stereoselective formation of mono- and dihydroxylated polychlorinated biphenyls by rat cytochrome P450 2B1. Environ Sci Technol 47 (21):12184–12192 McCalley DV (2017) Understanding and manipulating the separation in hydrophilic interaction liquid chromatography. J Chromatogr A 1523:49–71 Nachtigall FM, Rojas M, Santos LS (2018) MALDI coupled to modified traveling-wave ion-mobility mass spectrometry for fast enantiomeric determination. J Mass Spectrom 53 (8):693–699 Naile JE, Garrison AW, Avants JK, Washington JW (2016) Isomers/enantiomers of perfluorocarboxylic acids: method development and detection in environmental samples. Chemosphere 144:1722–1728 Nillos MG, Gan J, Schlenk D (2010) Chirality of organophosphorus pesticides: analysis and toxicity. J Chromatogr B Analyt Technol Biomed Life Sci 878(17–18):1277–1284 Peluso P, Mamane V, Aubert E, Cossu S (2017) Recent trends and applications in liquid-phase chromatography enantioseparation of atropisomers. Electrophoresis 38(15):1830–1850 Perez-Fernandez V, Castro-Puyana M, Gonzalez MJ, Marina ML, Garcia MA, Gomara B (2012) Simultaneous enantioselective separation of polychlorinated biphenyls and their methyl sulfone metabolites by heart-cut MDGC: determination of enantiomeric fractions in fish oils and cow liver samples. Chirality 24(7):577–583 Qi P, Yuan Y, Wang Z, Wang X, Xu H, Zhang H, Wang Q, Wang X (2016) Use of liquid chromatography- quadrupole time-of-flight mass spectrometry for enantioselective separation and determination of pyrisoxazole in vegetables, strawberry and soil. J Chromatogr A 1449:62–70 Rayne S, Forest K (2010) Dow and Kaw, eff vs. Kow and Kaw degrees: acid/base ionization effects on partitioning properties and screening commercial chemicals for long-range transport and bioaccumulation potential. J Environ Sci Health A Tox Hazard Subst Environ Eng 45 (12):1550–1594 Reddy IK, Miḥvar RA (2004) Chirality in drug design and development. Marcel Dekker, New York Roman IP, Mastromichali A, Tyrovola K, Canals A, Psillakis E (2014) Rapid determination of octanol-water partition coefficient using vortex-assisted liquid-liquid microextraction. J Chromatogr A 1330:1–5 Salieres P, Maquet A, Haessler S, Caillat J, Taieb R (2012) Imaging orbitals with attosecond and angstrom resolutions: toward attochemistry? Rep Prog Phys 75(6):062401

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Samuelsson J, Arnell R, Fornstedt T (2009) Potential of adsorption isotherm measurements for closer elucidating of binding in chiral liquid chromatographic phase systems. J Sep Sci 32 (10):1491–1506 Sanganyado E, Lu Z, Fu Q, Schlenk D, Gan J (2017) Chiral pharmaceuticals: a review on their environmental occurrence and fate processes. Water Res 124:527–542 Sherma J (2013) Review of advances in the thin layer chromatography of pesticides: 2010–2012. J Environ Sci Health B 48(6):417–430 Shi X, Qiao L, Xu G (2015) Recent development of ionic liquid stationary phases for liquid chromatography. J Chromatogr A 1420:1–15 Subramanian G (2007) Chiral separation techniques: a practical approach. Wiley-VCH, Weinheim Taraji M, Haddad PR, Amos RIJ, Talebi M, Szucs R, Dolan JW, Pohl CA (2018) Chemometricassisted method development in hydrophilic interaction liquid chromatography: a review. Anal Chim Acta 1000:20–40 Trivedi RK, Dubey PK, Mullangi R, Srinivas NR (2007) Development and validation of an enantioselective HPLC-UV method using Chiralpak AD-H to quantify (+)- and ()-torcetrapib enantiomers in hamster plasma--application to a pharmacokinetic study. J Chromatogr B Analyt Technol Biomed Life Sci 857(2):224–230 Ulrich EM, Morrison CN, Goldsmith MR, Foreman WT (2012) Chiral pesticides: identification, description, and environmental implications. Rev Environ Contam Toxicol 217:1–74 Warner NA, Martin JW, Wong CS (2009) Chiral polychlorinated biphenyls are biotransformed enantioselectively by mammalian cytochrome P-450 isozymes to form hydroxylated metabolites. Environ Sci Technol 43(1):114–121 Yan TQ, Riley F, Philippe L, Davoren J, Cox L, Orozco C, Rai B, Hardink M (2015) Chromatographic resolution of atropisomers for toxicity and biotransformation studies in pharmaceutical research. J Chromatogr A 1398:108–120

Chapter 3

Enantiomer-Selective High- and UltraHigh-Performance Liquid Chromatography

The analytical separation technologies with most applications to enantiomerselective chromatographic separations are high-performance liquid chromatography (HPLC) and the more advanced ultrahigh-performance liquid chromatography (UHPLC) based on stationary phases with particles 1), while in the latter case, a preferential transformation of the (+)-α-HCH was determined (ER < 1.00). Furthermore, Jantunen and Bidleman analysed the marine water samples collected during the AOS-94 cruise also for the enantiomeric ratios ((+)-/()-

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Table 8.4 Enantiomeric ratios of α-HCH, β-PCCH and γ-PCCH as determined for seawater samples obtained at the North Sea and Baltic Sea stations shown in Fig. 8.7

Station no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Average value Baltic Sea 15 16 17 18 19 20 21 Average value North Sea

Enantiomeric ratios (+)-α-HCH/()-αHCH 0.89 0.79 0.87 0.85 0.83 0.85 0.83 0.84 0.85 0.83 0.85 0.92 0.84 0.82 0.85  0.03 0.81 0.84 0.87 0.83 0.88 0.94 0.94 0.87  0.05

β1-PCCH/β2PCCH b b b b b b b b b b b b b b 0.97b

γ 1-PCCH/γ 2PCCH 1.16 1.16 1.17 1.12

c c 1.13c c

1.15  0.02

b ¼ value of pooled station 1–10 c ¼ value of pooled stations 7–10 (Hühnerfuss and Kallenborn 1992)

enantiomers) of trans-chlordane, cis-chlordane and heptachlor exoepoxide, a major metabolite of heptachlor (Table 8.5). The ER values determined for heptachlor exoepoxide ranged between 1.47 and 1.76, which implies a preferential formation of the (+)-enantiomer. The range of ERs was tight considering the wide expanse covered by the stations. The two chlordane isomers trans- and cis-chlordane were close to racemic in the dissolved phase (ER ¼ 0.94-1.06). Although HCHs were > 99% in the dissolved phase at most stations, levels of HCHs in water were high enough to allow ER values to be measured on the filters of the large volume samples. The particulate α-HCH showed the same, or more enantioselective transformation than the dissolved fraction (Table 8.5). Both fractions were depleted in the same enantiomer at most stations, but there appeared to be no relationship between the two and reversal in the depleted enantiomer was found at stations 16 and 29. Some particulate samples showed the enhanced transformation of α-HCH compared to the dissolved phase. For example, the ER of dissolved α-HCH

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8 Enantiomer-Specific Fate and Behaviour of Chiral Contaminants

Fig. 8.8 Cruise track of AOS-94. Dots running from the Chukchi Sea to the Greenland Sea correspond to the station numbers in Table 8.5 (Map provided by AMAP)

at station 20 was 0.82, whereas the ER on the filter was 0.18. The ERs for dissolved α-HCH generally decreased with depth. At stations 37 and 38 north of Spitsbergen, surface ERs were 0.82, decreased to 0.64 at 109 m, 0.45 at 235 m and 0.14 at 762 m. Jantunen and Bidleman offer two explanations for the greater enantioselectivity with depth: As particles settle from the surface, the sorbed αHCH is metabolised and released back into the dissolved phase. Alternatively, the ERs may be typical of older, Atlantic-layer water, which lies below the pycnoline. The study by Franke et al. (1998) extended the spectrum of chiral target pollutant analysis with enantiomer-selective cGC methods for the first time towards the group of halogenated ethers. The authors investigated the chlorinated bis (propyl) ethers (ClxBPE; x ¼ 2–4), used as solvent or in metallurgic applications, as an important new class of environmental contaminants in the river Elbe from the border to the Czech Republic to the river mouth in the German Bight (Franke et al. 1998). Quantitative analysis during the period 1992–1995 revealed total concentrations of up to 30 μg/L close to the Czech border and a gradual decrease to 4–2 μg/L towards the central part of the river. The authors conjectured that this change in concentration cannot solely be attributed to the diluting effect caused by uncontaminated tributaries, because their water supply is insufficient to account for this. The source of Cl4BPE (Fig. 8.9) is identified as a production site for epichlorohydrin close to the Czech border emitting considerable amounts of these compounds as side product wastes.

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125

Table 8.5 Enantiomeric ratios of α-HCH, heptachlor exo-epoxide (HEPX), trans-chlordane (TC) and cis-chlordane (CC) in surface water samples collected at the stations shown in Fig. 8.8; two different chiral phases were applied: BGB-172 and Beta-DEX, respectively Station no. Standard 1 2 11 13 16 20 24 25 28 29 31 35 37 39

α-HCH/BGB-172 Dissolved Particle 1.00 1.11 1.27 1.09 1.05 1.06 na 0.95 0.98 na 1.10 na 0.21 0.92 0.88 0.96 0.93 0.92 0.91 0.90 1.16 0.96 0.42 0.91 0.84 0.85 na 0.71 0.80

α-HCH/Beta-DEX Dissolved Particle 0.99 1.08 1.26 1.09 1.03 1.05 0.98 0.89 0.93 0.83 1.07 0.82 0.18 0.87 0.95 0.89 0.95 0.87 0.89 0.88 1.23 0.90 0.42 0.88 0.86 0.78 na 0.68 0.79

HEPX Dissolved 1.01 1.52 1.47 1.64 1.58 na na 1.76 1.59 1.59 1.65 1.67 1.63 1.56 1.67

TC Dissolved 0.99 1.00 1.00 1.00 1.06 0.98 0.99 0.97 0.98 1.01 0.97 1.01 0.99 0.97 1.03

CC Dissolved 0.99 1.00 1.04 1.06 1.02 na na 0.99 1.01 0.94 na na 1.01 0.98 1.01

na not analysed

Fig. 8.9 Structures of three Cl4BPE: 1 bis(2,3-dichloro1-propy)ether (2,3,20 ,30 -Cl4BPE), 2 1,3-dichloro-2-propyl-2,3dichloro1-propyl ether (1,3,20 ,30 - Cl4BPE), 3 bis (1,3-dichloro-2-propyl)ether (1,3,10 ,30 - Cl4BPE)

During the investigation period, three Cl4BPE isomers were present in an almost constant pattern over the upper and central part of the river, which corresponded to the pattern of the emitted derivatives (Fig. 8.9): 1,3,10 ,30 -Cl4BPE approx. 10–15%, the 2,3,20 ,3-Cl4-isomer approx. 28–35% and 1,3,20 ,30 -Cl4BPE approx. 55–57%. A shift in the isomeric pattern was, however, verified for the lower reaches of the river:

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8 Enantiomer-Specific Fate and Behaviour of Chiral Contaminants

1,3,10 ,30 -Cl4BPE increased in concentration, while the 2,3,20 ,30 -Cl4BPE decreased. Basically, the shift in the isomeric proportions towards the lower reaches of the river may result from a discrimination of the 2,3,20 ,30 -Cl4BPE by physical or biochemical processes. A decision about this alternative was expected by an investigation of the enantiomeric composition over the entire length of the river, since 2,3,20 ,30 -Cl4BPE is a chiral compound. A change of the enantiomeric ratios would be indicative of enzymatic microbial degradation, while retention of a racemic composition would point to a non-enzymatic process. As earlier noted already, appropriate chiral selectors in cGC for solving such problems often are modified cyclodextrin phases. However, the separation potential of a cyclodextrin-type chiral stationary phase is still difficult to predict and, therefore, needs to be tested empirically. In this case, several cyclodextrin derivatives had to be evaluated with respect to their selectivity towards the chiral bis(chloropropyl) ethers with mixtures of standard compounds of different degrees of chlorination. Experienced analytical chemists will confirm that similar time-consuming tests need to precede the determination of the enantiomer signature as a part of the quality control procedure, when analytical methods for emerging chiral environmental pollutants are developed. As an example for the evaluation of the CSP selectivity and determination of the enantiomer-specific elution order in the optimised cGC, separation of a potential of some cyclodextrin phase is illustrated for chloro-bis(propyl) ethers in the herediscussed report (Franke et al. 1998). A satisfactory enantiomeric separation of 1,20 -Cl2BPE and 1,3,20 -Cl3BPE (the digits indicate the position of the chlorine atoms; this simplified nomenclature is unambiguous) is achieved with octakis(3-O-butyryl-2,6-di-O-n-pentyl)-γ-cyclodextrin (Lipodex E). The stereoisomers of the tetrachloro compounds, however, are not resolved on this phase. Heptakis(6-O-tertbutyldimethylsilyl-2,3-di-O-methyl)-βcyclodextrin resolves 1,3,20 -Cl3BPE and the tetrachloro compounds 1,3,20 ,30 -Cl4BPE and 2,3,20 ,3-Cl4BPE with the (S,S)-enantiomer of 2,3,20 ,30 -Cl4BPE and the (+)-enantiomer of 1,3,20 ,30 -Cl4BPE co-eluting. Similarly, separations can be achieved with the heptakis(6-O-tertbutyldimethylsilyl-2-O-methyl-3-O-n-pentyl)β-cyclodextrin column as long as a slower temperature program is applied. In addition, 1,20 ,30 -Cl3BPE is completely separated into four stereoisomers. No separations of haloethers were observed on the column with the mixed phase of heptakis(2,6-di-O-methyl-3-O-n-pentyl)-β-cyclodextrin and heptakis(6-O-methyl2,3-di-O-n-pentyl)-β-cyclodextrin. None of the investigated cyclodextrin phases was able to resolve all compounds of interest. But since Franke et al. had known from earlier investigations that the contamination of the Elbe river with tetrachloro compounds was more severe than with tri- and dichloro homologues, they focused on the investigation of the tetrachloro bis(propyl) ethers, and as a consequence, they chose the heptakis(6-O-tertbutyldimethylsilyl-2,3-di-O-methyl)-β-cyclodextrin column which separated these homologues well. The assignment of the order of elution of the enantiomers of 2,3,20 ,30 -Cl4BPE was achieved by enantioselective synthesis of the 2(R,20 R/S)-stereoisomer and the subsequent cGC analysis of the achieved enantiomer. The determination of the absolute configuration of one of the two stereogenic centres is sufficient, since the mesoform (note: 2R,20 S ¼ 2S, 20 R) can

8.1 Microbial Transformation of Chiral Environmental Pollutants

127

be easily detected in the gas chromatogram of the racemate by its peak height which is approximately twice that of the enantiomers of the chiral stereoisomers. The enantiomers of 1,3,20 ,30 -Cl4BPE could be correlated by co-injection with an enantiomerically enriched sample (40% ee), which was obtained by preceding preparative gas chromatography of the corresponding racemate. Although co-elution of the (S,S)-isomer of 2,3,20 ,3-Cl4BPE and the (+)enantiomer of 1,3,20 ,30 -Cl4BPE was encountered, a quantitative determination of the enantiomeric proportion was possible by virtue of the specific mass spectrometric fragmentation of the isomers, using compound-specific ion traces (m/z) for the respective selection ion monitoring (SIM). For a detailed discussion on this aspect, the reader should refer to the report of here-discussed study (Franke et al. 1998). The results thus obtained are summarised in Table 8.6. The enantiomeric ratios of 1,3,20 ,30 -Cl4BPE showed values between 0.72 and 1.20. In the samples P1, P5 and P7 (see map in Fig. 8.9), a slight shift in the ER towards the (+)-isomer was observed. This trend was even more obvious in the samples of September 1995. A shift of the ER of the ()-enantiomer to 0.72 (P5) was far beyond the medium deviation of the experimental method. Significantly different were the proportions in sample P9 with an increase in the ER of the ()-enantiomer to 1.20. The enantiomeric ratios of the 2,3,20 ,30 -Cl4BPE show a significant discrimination of the (R,R)-enantiomer in the samples “Schmilka”, “Schnackenburg” and “Seemannshöft” (February 1995), as well as in the samples of September 1995 with ER values as low as 0.27. This shift of the enantiomeric ratios in the middle part of the Elbe River must be attributed to a decrease in the concentration of (R,R)2,3,20 ,30 -Cl4BPE. Due to the relatively low levels of these compounds in the series taken in September 1995, the enantioselective discrimination and the concomitant shift in the enantiomeric ratios can be clearly observed, since it is not superimposed Table 8.6 Enantiomeric ratios of chiral tetrachloro bis(propyl) ethers in the River Elbe at the stations shown in Fig. 8.10 Sample Location No February 1995 Bilina P1 Schmilka P3 Magdeburg P5 Schnackenburg P6 Zollenspieker P7 Seemannshöft P8 Grauerort P9 Cuxhaven September 1995 P1 Schmilka P2 Zehren P3 Magdeburg P4 Tangermünde P5 Schnackenburg

1,3,20 ,30 -Cl4BPE (-)/(+)

2,3,20 ,30 -Cl4BPE (R/R0 )/(S,S0 )

M/(S/S0 )

1.02 0.85 0.92 0.89 1.01 0.87 1.05 1.20

0.94 0.63 1.05 0.78 0.94 0.41 1.02 1.59

1.91 1.66 2.06 1.87 1.92 1.16 2.01 2.88

0.86 1.00 0.84 0.82 0.72

0.54 0.83 0.53 0.44 0.27

0.74 0.83 1.75 1.42 0.92

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8 Enantiomer-Specific Fate and Behaviour of Chiral Contaminants

Fig. 8.10 Sampling sites P1–P9 along the Elbe River (Northern Germany) during the campaign carried out by Franke et al. (1998)

by the continuous inflow of racemic material. In addition, the biological activity may have been larger during the summer months, which may also have given rise to a more intense enantioselective discrimination. Similar to the values for 1,3,20 ,30 -Cl4BPE, the ER values for the samples “Magdeburg”, “Zollenspieker” and “Grauerort” were in the range of the racemate, considering the variability of the method. Again, the sample “Cuxhaven” with an ER value far above 1 deviates significantly from the other samples. The corresponding concentration was relatively high, but Franke et al. could not offer a scientifically convincing explanation for this observation (Fig. 8.10). In the following years, in line with the continuous progress of trace analytical methods, technology and research focus in the field of enantiomer-selective trace analysis of chiral organic pollutants; the list of target substances continuously increased. Due to conscious environmental screening, a new environmental contaminant, the insecticide bromocyclen (CAS 1715-40-8), was identified in biota (Pfaffenberger et al. 1994a, b; Bethan et al. 1997). This brominated and chlorinated bicycloheptene contact insecticide (tradename Bromodan®, Alugan®) is the DielsAlder adduct of hexachlorocyclopentadiene and allyl bromide. It possesses neither a plane nor a centre of symmetry, and therefore, it exists in two enantiomeric forms (Fig. 8.11).

8.1 Microbial Transformation of Chiral Environmental Pollutants

Cl

Cl

Cl

Cl

Cl Cl

Cl

Cl

129

Br Cl

Cl

Br Cl

Cl

Fig. 8.11 The enantiomers of bromocyclen

Due to its very low mammalian toxicity in Europe, bromocyclen is being used against ectoparasites for the treatment of domestic animals in lifestocks. In this study (Bethan et al. 1997), ten water samples were collected from the river Stör, a tributary of the Elbe River in Northern Germany, and one water sample from the Elbe River close to the mouth of the Stör River. Moreover, samples from the influent, as well as from the effluent of various sewage plants along the river, were collected. Particular emphasis was placed upon the question how bromocyclen enters the river Stör and if the enantiomeric ratios determined in the fish samples correlate with the concentrations. Close to the spring of the Stör River, concentrations of 37 pg/L were measured, followed by strong increases to 213 pg/L (near Neumünster) and 261 pg/L (near Itzehoe) presumably due to effluents of communal wastewater treatment plants, and a decrease to 51 pg/L in the course of its downstream movements towards the estuary, obviously caused by dilution effects. As the “hot spots” in the Stör River pointed to two local wastewater treatment plants as potential sources for bromocyclen, samples from their influents as well as from their effluents were analysed. The concentrations in 1995, which varied between 3.3 and 11.5 ng/L, were up to 100-fold higher than the increases in concentration that were found in the river. For example, an increase of 104 pg/L in the bromocyclen contamination of the Stör water, from the station upstream of the sewage plant of Itzehoe to the next station downstream of the sewage plant, reflects a concentration of 11.5 ng/L in the effluent of the sewage treatment plant. With regard to the analysis of the enantiomeric excesses of bromocyclen in the Stör and Elbe water samples, no significant differences in the concentrations of the two enantiomers were found (Bethan et al. 1997). The enantiomeric ratios of all nine water sample extracts ranged between 1.01 and 1.05. These nearly racemic distribution indicates that bromocyclen is not or only insignificantly transformed by microbial processes in the river water. Also, the polycyclic musk compounds were identified as relevant chiral environmental pollutants. Polycyclic musks are a group of artificial fragrances applied in a personal care product. As such, these substances are directly applied to human skin during daily washing and cosmetic procedures. High levels and corresponding enantiomeric patterns are reported in a variety of environmental compartments.

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This includes product samples (Martinez-Giron et al. 2010), water samples (Wang et al. 2013a, b; Lee et al. 2016), aquatic organisms (Franke et al. 1999; Gatermann et al. 2002a, b) and others. A first review on the environmental implications of polycyclic musks was already published in 1999 (Kallenborn et al. 1999a, b, c) and comprehensively described in a dedicated book (Rimkus 2004). In addition to potential toxicological aspects, the chirality of polycyclic musks and their transformation products is currently also discussed in the context of pheromone-like effects and potential behavioural consequences for higher organisms (Kallenborn et al. 1999a, b, c; Rimkus 2004; van der Burg et al. 2008). Detailed information and relevant examples on this interesting compound group will be provided when uptake and distribution profile in higher organisms, as well as human exposure is discussed. In recent years, scientific information on enantiomer-selective microbial transformation has been extended into several emerging contaminant groups containing chiral member substances. This includes currently used pesticides (Monkiedje et al. 2003; Monkiedje and Spiteller 2005; Tan et al. 2008; Garrison et al. 2011a, b; Liu et al. 2015a, b, 2016a, b; Frkova et al. 2016), polychlorinated biphenyl atropisomers (Pakdeesusuk et al. 2003; Ross et al. 2011a, b), cyclodiene pesticides (Huang et al. 2017; Sanchez-Osorio et al. 2017) and pharmaceuticals (Ribeiro et al. 2014; Sanganyado et al. 2017).

8.2

Transformation/Accumulation of Chiral Xenobiotics in Higher Organisms

8.2.1

Marine and Freshwater Organisms

8.2.1.1

α-Hexachlorocyclohexane

In 1991, Kallenborn et al. were the first to report on the successful application of enantioselective cGC in vertebrate tissue samples. The here-reported study aimed at investigating the enantiomer-selective transformation of α-HCH in seabird tissue samples (Kallenborn et al. 1991). The authors collected carcasses from common Eiders (Somateria mollissima (L.)) drown in local fishing nets from the Oehe/ Schleimünde wildlife refuge on the German Baltic coast. Organ samples were taken, thus, only from healthy animals. The common Eider duck was chosen, because it largely favours molluscs, but in that region particularly blue mussels (Mytilus edulis L.) in its diet (Kallenborn et al. 1994a, b). Blue mussels, in turn, are capable of strongly enriching pollutants and thus serve as “indicator organisms” in many national monitoring programs in order to provide insight into the state of an aquatic environment (Beyer et al. 2017). Thus, due to the highly specialist food habits of common eiders in this region, a simple and almost complete “food chain” can be assumed (water ! mussel ! common Eider duck) for this study. The tissue samples (mussel, liver, kidney and pectoral muscle) were homogenised with a threeto five-fold amount of anhydrous sodium sulphate (Na2SO4), followed by the

8.2 Transformation/Accumulation of Chiral Xenobiotics in Higher Organisms Fig. 8.12 Enantiomer separation of α-HCH extracted from a Baltic Sea water sample, from a blue mussel (Mytilus edulis L.) and from the liver of a common Eider duck (Somateria mollissima (L.)) using a fused-silica capillary column coated with 50% heptakis(2,3,6-tri-O-npentyl)-β-cyclodextrin and 50% OV-1701. Column temperature programme: initial 323 K, increased at 10 K/min to 388 K; carrier gas, helium (45 kPa); on-column injection; ee ¼ enantiomeric excess

Water (ER = 0.83) ‐

Blue mussel (Mylus edulis) (ER = 0.73)

131

Common Eider liver (Sommateria mollissima) (ER = 18.7) +

+ ‐

+

‐

30

40

50

30

40

50

30

40

50 [min]

addition of the internal standard ε-HCH and 8–12 h extraction in a Soxhlet apparatus with n-hexane. The detailed procedure including the clean-up and fractionation steps can be found in Kallenborn et al. (1991). In Fig. 8.12, the gas chromatograms of the α-HCH enantiomers are shown for the complete food chain, that is, surface sea water ! blue mussel ! common Eider (liver), using a modified cyclodextrin phase as chiral selector (Hühnerfuss and Kallenborn 1992). Whereas the blue mussel largely reflects the characteristics of the adjacent water area, that is, an excess of the ()-α-HCH, in the liver of the common Eider duck, the (+)-α-HCH is dominant. The detailed analyses of the extracts of all three common Eider duck tissues revealed that (+)-α-HCH was clearly enriched; almost enantiomerically pure (+)-αHCH was present in the liver extracts. The enantiomeric purity of (+)-α-HCH isolated from liver extracts was so high that after purification by HPLC, it can be used directly in model experiments. By contrast, the enantiomeric ratio (+)-α-HCH/ ()-α-HCH was about 7 in muscle extracts and about 1.6 in extracts, whereby the values for these organs were slightly larger or smaller for different common Eider ducks. In this first study, the organs from a total of six common Eider ducks were investigated, so that the results can be considered sufficiently reliable. An exact explanation for the appearance of different enantiomeric ratios of (+)-αHCH in the organs of common Eider ducks was not presented. However, it may be assumed that the reason lies in the different physiological functions of the organs. For muscle and kidney, whose main functions are “locomotion” and “excretion”,

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8 Enantiomer-Specific Fate and Behaviour of Chiral Contaminants

respectively, the content of extractable lipids is about 2%; these organs can, therefore, store lipophilic pollutants. Liver, which contains about 2.5% of extractable organic matrix (EOM) which mainly consists of lipids, peptides and larger molecules, serves as a “detoxification organ” and, therefore, is not only capable of storing toxic compounds but can also metabolise them to substances that the body can tolerate or excrete. Since the (+)-α-HCH found in the liver of common Eider ducks is almost enantiomerically pure, ()-α-HCH is presumably more readily transformed enzymatically than the (+)-enantiomer in the common eider liver. Although such a nearly enantioselective transformation had already been observed previously for biogenic organic compounds, the study by Kallenborn et al. (1991) represents the first evidence for the enantiomer-selective degradation of chiral synthetic pollutants in vertebrates. Additional and, at first glance, even more surprising, results were reported by Möller et al. (1994) who analysed brain tissue of the same Eider duck animals that had already been investigated by Kallenborn et al. with regard to liver, kidney and muscle tissues. It turned out that an additional enantioselective process that thus far escaped the attention of environmental toxicologists has to be considered when assessing the potential risk of environmental pollutants in vertebrates and higher organisms. The permeation through the blood–brain barrier seems to be of highly enantiomer-selective character for biogenic substances and anthropogenic chiral pollutants. After these early investigation by Kallenborn et al. who demonstrated the enantioselective metabolisation of α-HCH in common Eiders (Somateria mollissima (L.)) (Kallenborn et al. 1991), increased attention has been paid to the chromatographic enantiomer separation of chiral xenobiotics and their metabolites in environmental biota samples. Today, the enantiomer distribution profiles in higher organisms are considered an indicator parameter for bioaccumulation and bioactivity of chiral pollutants in the respective food web and the investigated organisms (Müller and Kohler 2004; Wong 2006; Smith 2009). In Table 8.7, a selection of relevant chiral environmental pollutants is listed, as well as the references, in which a successful enantiomer separation of the respective standard compounds is reported. Table 8.8 summarises environmental pollutants together with the respective biota samples and the references, in which results about these compounds with regard to enantioselective analyses and processes can be found. As it is already clarified in the previously discussed reports, the chiral pollutant that has been investigated earliest and most intensively during the past decades and throughout all environmental compartments is still, without any doubt, α-HCH. Its chemical structure and its main microbial metabolism were already described earlier. In this chapter, the emphasis is placed on enzymatic transformation and enantiomerselective uptake of α-HCH in higher organisms. Already at lower trophic levels (microbial transformation in water, mussel), preferential depletion of one α-HCH enantiomer, be it the (+)- or the ()-enantiomer, has been verified. These first results encouraged to perform a more systematic investigation that addressed the problem as to whether or not different enzymatic

8.2 Transformation/Accumulation of Chiral Xenobiotics in Higher Organisms

133

Table 8.7 Standard compounds of selected environmental chiral xenobiotics, the successful enantiomer separation of which was thus far reported in the literature Substance α-HCH, β-PCCH,γ-PCCH

PCCH II, III, V-VIII Heptachlor cis-chlordane

trans-chlordane

Chlordene cis-heptachlorepoxide

trans-heptachlorepoxide

oxychlordane

Octachlordanes MC4, MC5, MC7, Bromocyclen

o,p'-DDT o,p'-DDD Hepta-, octa-, nonachlorobornanes toxaphene, chloropinene, Melipax

PCB 45/139

Standards [References] Hardt et al. (1994), Möller et al. (1994), Pfaffenberger et al. (1994a, b), Mossner and Ballschmiter (1997), Moisey et al. (2001), Hoekstra et al. (2003a, b, c), Suar et al. (2005), Geueke et al. (2013a, b, c), Zhang et al. (2014a, b, c), Liu et al. (2019) Mossner and Ballschmiter (1997) König et al. (1991), Champion et al. (2004), Kania-Korwel and Lehmler (2013) König et al. (1991), Oehme et al. (1994), Mattina et al. (2002), White et al. (2002), Hoekstra et al. (2003a, b, c), Champion et al. (2004), Bondy et al. (2005), Kania-Korwel and Lehmler (2013) König et al. (1991), Oehme et al. (1994), Mattina et al. (2002), White et al. (2002), Hoekstra et al. (2003a, b, c), Champion et al. (2004), Bondy et al. (2005), Kania-Korwel and Lehmler (2013) König et al. (1991) Buser and Müller (1993), Jantunen and Bidleman (1998), Wong et al. (2002), Champion et al. (2004), Venier and Hites (2007) Buser and Müller (1993), Jantunen and Bidleman (1998), Wong et al. (2002), Champion et al. (2004), Venier and Hites (2007) Buser and Müller (1993), Jantunen and Bidleman (1998), Wong et al. (2002), Champion et al. (2004), Venier and Hites (2007) Hühnerfuss (2000), Vetter (2001), Hühnerfuss and Shah (2009), Ulrich and Falconer (2011) Pfaffenberger et al. (1994a, b), Bethan et al. (1997), Fromme et al. (1999), Fidalgo-Used et al. (2008) (Ali and Aboul-Enein (2002), Muñoz-Arnanz and Jiménez (2011), Bosch et al. (2015) Ali and Aboul-Enein (2002) Venier and Hites (2007) Buser and Müller (1994a, b), Vetter and Schurig (1997), Jantunen and Bidleman (1998), Vetter and Luckas (2000), Vetter and Kirchberg (2001), Vetter et al. (2001a, b), Bidleman and Leone (2004), Hamed et al. (2005), Maruya et al. (2005), Bordajandi et al. (2006), Wong et al. (2009), Bidleman et al. (2013a, b), Zhang et al. (2017a, b) König et al. (1993), Hardt et al. (1994) (continued)

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8 Enantiomer-Specific Fate and Behaviour of Chiral Contaminants

Table 8.7 (continued) Substance PCB 84 PCB 88 PCB 91/136 PCB 95 PCB 131/135/175 PCB 132

PCB 144 PCB 149 PCB 171 PCB 174 PCB 176 PCB 183 PCB 196/197

PCB methyl sulfones: 3-91, 4-91, 3-95, 4-95, 3-149, 4-149, 3-132, 4-132, 3-174, 4-174 Hydroxy-PCBs Atropisomeric PCBs (in general)

2-(2,4-dichlorophenoxy)propionic acid

2-(4-chloro-2-methylphenoxy)propionic acid 2-(2,4,5-trichlorophenoxy)propionic acid (S)-bio allethrin Methamidophos

Acephate

Standards [References] Hardt et al. (1994), Vetter et al. (1997a, b, c, d, e, f) Hardt et al. (1994) Schurig and Glausch (1993), Glausch et al. (1994), Hardt et al. (1994) König et al. (1991, 1993), Schurig and Glausch (1993), Glausch et al. (1994), Hardt et al. (1994) Hardt et al. (1994) Schurig and Glausch (1993), Glausch et al. (1994), Hardt et al. (1994), Vetter et al. (1997a, b, c, d, e, f) Haglund and Wiberg (1996) Schurig and Glausch (1993), Hardt et al. (1994), Vetter et al. (1997a, b, c, d, e, f) Vetter et al. (1997a, b, c, d, e, f) Hardt et al. (1994), Haglund and Wiberg (1996), Vetter et al. (1997a, b, c, d, e, f) Hardt et al. (1994), Haglund and Wiberg (1996) Hardt et al. (1994), Vetter et al. (1997a, b, c, d, e, f), Toda et al. (2012) Haglund (1996a, b), Marina et al. (1996), Magnusson et al. (2000), Holoubek and Robertson (2003) Kania-Korwel et al. (2008), Cooper et al. (2012), Perez-Fernandez et al. (2012), Kania-Korwel and Lehmler (2016) Kania-Korwel et al. (2011), Zhai et al. (2013) Harju and Haglund (2001), Edwards and Shamsi (2002), Pham-Tuan et al. (2005), Bucheli and Brandli (2006), Lehmler et al. (2010), Xu et al. (2011), Vetter (2016) König et al. (1991), Miura et al. (1999), Messina and Sinibaldi (2007), Caballo et al. (2013), Menestrina et al. (2018) König et al. (1991), Miura et al. (1999), Caballo et al. (2013), Menestrina et al. (2018) König et al. (1991), Miura et al. (1999), Caballo et al. (2013), Menestrina et al. (2018) Hardt et al. (1994), Shea et al. (1999), Zhou et al. (2002) König et al. (1991), Hardt et al. (1994), Emerick et al. (2010), Wang et al. (2013a, b), JimenezJimenez et al. (2019) Chae et al. (1994), Hardt et al. (1994), Wang et al. (2013a, b) (continued)

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135

Table 8.7 (continued) Substance Trichlorfon Malaoxon Bromoacil Fonofos HHCB (galaxolide) AHTN (tonalide) (2R,20 R/S)-bis(2,3-dichloro-1-propyl) ether Selected chiral pharmaceuticals

Standards [References] König et al. (1991), Hardt et al. (1994), FidalgoUsed et al. (2006) Jing et al. (2016) Hua et al. (2020) Hardt et al. (1994), Polec et al. (2007), Zhang et al. (2013a, b) Hardt et al. (1994), Gritti et al. (2017) König et al. (1991), Ellington et al. (2001), Nillos et al. (2010) Franke et al. (1999), Gatermann et al. (2002a, b), Wang et al. (2013a, b), Wang and Khan (2014) Franke et al. (1999), Gatermann et al. (2002a, b), Wang et al. (2013a, b), Wang and Khan (2014) (Franke et al. (1998) Colombo et al. (2020), Hancu et al. (2020), Hühnerfuss and Shah (2009), Lin et al. (2020), Liu et al. (2020)

processes may also be revealed by the analysis of enantiomeric ratios of α-HCH in marine biota of different trophic levels. Therefore, during a research cruise in 1991, Pfaffenberger et al. collected water samples, blue mussels as well as flounders (Platychthys flesus L.) and common Eider from three areas typical of the German Bight, that is, the mouth of the river, two coastal sites and in the central/northwestern part of the German Bight (Pfaffenberger et al. 1992). All samples investigated were taken in marine areas that had been studied and characterised thoroughly since 1987 during several research cruises of two national research projects (i.e.: ZISCH ¼ “Zirkulation und Schadstoffumsatz in der Nordsee” and PRISMA ¼ “Prozesse im Schadstoffkreislauf Meer-Atmosphäre: Ökosystem Deutsche Bucht”) coordinated by the University of Hamburg. Thus, complementary background information about nutrients, heavy metals and various non-chiral organic pollutants was also available for the here-performed assessment. Thus, the sampling strategy was developed on already available information on “more polluted” and “less polluted” sampling sites for the here-conducted study. On the basis of this valuable background information, as “more polluted sampling site” the estuary of the river Elbe (F1, E1, M1, W1, W2; see Table 8.9) was chosen, while “less polluted sampling sites” include the estuary of the river Eider (F2), the isle of Amrum (E2, M2) and the German Bight area of the river Elbe plume (W3, W4). After field work and analysis, the results were interpreted focussing on the question of whether or not higher levels of pollutants may induce stronger enzymatic activities and thus larger shifts of the enantiomeric ratios of α-HCH. The mean enantiomeric ratio of α-HCH determined in the sea water samples of the German Bight was determined with an average value of 0.84  0.03. This result compared well within the error limits with earlier values for the eastern part of the North Sea and the Skagerrak (Faller et al. 1991a, b). Furthermore, this value, which is assumed

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8 Enantiomer-Specific Fate and Behaviour of Chiral Contaminants

Table 8.8 Selected environmental chiral or prochiral xenobiotics reported in different matrices Substance α-HCH

γ-HCH β-PCCH d-PCCH γ-PCCH

trans- and cis-chlordane

cis-heptachlorepoxide

Water/Tissue/Air [References] Water (Hühnerfuss et al. 1993; Falconer et al. 1995a, b; Jantunen and Bidleman 1998; Bidleman et al. 2002; Law et al. 2004; Pucko et al. 2012; Zhang et al. 2012a, b, c) Sea bird tissues (Kallenborn et al. 1991; Hühnerfuss et al. 1993; Moisey et al. 2001; Corsolini et al. 2006) Seal: blubber, brain, liver, lung (Hühnerfuss et al. 1993; Hummert et al. 1995; Klobes et al. 1998a, b, c; Moisey et al. 2001; Hoekstra et al. 2003a, b, c; Carlsson et al. 2014a, b) Fish oil (Hühnerfuss et al. 1993; Koske et al. 1999; Wiberg et al. 2006); fish liver [(Hühnerfuss et al. 1993; Koske et al. 1999; Hoekstra et al. 2003a, b, c; Konwick et al. 2006; Wiberg et al. 2006)]; mollusks (Pfaffenberger et al. 1992; Hühnerfuss et al. 1993; Zhou et al. 2014a, b) Whale blubber (Hühnerfuss et al. 1993; Hummert et al. 1995; Hoekstra et al. 2003a, b, c; Carlsson et al. 2014a, b; Zhou et al. 2018) Polar bear: liver, fat (Wiberg et al. 2000; Vetter 2001; Hoekstra et al. 2003a, b, c); roe deer: liver (Pfaffenberger et al. 1994a, b); sheep: fat, liver, brain (Möller et al. 1993); atmospheric deposition (Bidleman 1999; Wiberg et al. 2001a, b; Gouin et al. 2007; Jantunen et al. 2008; Covaci et al. 2010; Cabrerizo et al. 2011; Wang et al. 2016) Water (Shao et al. 2016; Cui et al. 2017; Chang 2018; Li et al. 2018; Ogbeide et al. 2019; Lorenzo et al. 2020) Microbial (Suar et al. 2005) water (Hühnerfuss et al. 1992a, b; Ludwig et al. 1992a) [1,2,21,39]; air [2] Microbial (Geueke et al. 2013a, b, c) Microbial (Trantirek et al. 2001) water (Hühnerfuss et al. 1992a, b; Ludwig et al. 1992a) [1,2,21]; air (Hühnerfuss et al. 1992a, b) Water (Jantunen and Bidleman 1998; Bidleman et al. 2002; Genualdi et al. 2011; Huang et al. 2013; Jin et al. 2017); soil/ sediment (Eitzer et al. 2001; Wiberg et al. 2001a, b; Bidleman and Leone 2004; Kurt-Karakus et al. 2005); amphipods (Carlsson et al. 2014a, b) Marine biota and mammals (Borga and Bidleman 2005; Carlsson et al. 2014a, b); cod liver oil (Hühnerfuss et al. 1993; Koske et al. 1999; Wong et al. 2002; Hoekstra et al. 2003a, b, c) Water (Jantunen and Bidleman 1998; Bidleman et al. 2002; Vetter et al. 2006; Zhang et al. 2012a, b, c); bird eggs (Hühnerfuss et al. 1993; Buser and Müller 1994a, b); marine biota (Hühnerfuss et al. 1993; Vetter and Schurig 1997; Fisk et al. 2002; Hegeman and Laane 2002; Konwick et al. 2006) ; cod liver oil (Borga and Bidleman 2005; Carlsson et al. 2014a, b); cod liver oil (Hühnerfuss et al. 1993; Koske et al. 1999; Wong et al. 2002; Hoekstra et al. 2003a, b, c) (continued)

8.2 Transformation/Accumulation of Chiral Xenobiotics in Higher Organisms

137

Table 8.8 (continued) Substance

Oxychlordane

Octachlordanes MC4, MC5, MC7

Photo-cis-chlordanes [2,5and 1,5] Photoheptachlor Bromocyclen Chlorobornanes

Atropisomeric PCBs PCB 88 PCB 95 PCB 132

PCB 136 PCB 144 PCB 149 PCB 171 PCB 174 PCB 183 PCB methyl sulfones: 3-132, 3-149

Water/Tissue/Air [References] Roe deer liver (Pfaffenberger et al. 1994a, b); rat liver (KaniaKorwel and Lehmler 2013); human adipose (Buser and Müller 1992) Bird eggs (Hühnerfuss et al. 1993; Herzke et al. 2002); marine biota (Hühnerfuss et al. 1993; Vetter and Schurig 1997; Fisk et al. 2002; Hegeman and Laane 2002; Konwick et al. 2006); cod liver oil (Borga and Bidleman 2005; Carlsson et al. 2014a, b); cod liver oil (Hühnerfuss et al. 1993; Koske et al. 1999; Wong et al. 2002; Hoekstra et al. 2003a, b, c) Roe deer liver (Pfaffenberger et al. 1994a, b) Marine biota (Buser et al. 1992; Buser and Müller 1993; Müller et al. 1997) nonachlor III MC6 marine biota (Buser et al. 1992; Buser and Müller 1993; Müller et al. 1997) Marine biota (Buser and Müller 1993) Marine biota (Buser and Müller 1993; Zhu et al. 1995) polar bear, human plasma (Zhu et al. 1995) Water (Bethan et al. 1997); fish (Pfaffenberger et al. 1994a, b; Fidalgo-Used et al. 2008) Sea water (Jantunen and Bidleman 1998); soil (Bidleman and Leone 2004); bird eggs (Hamed et al. 2005); marine biota (Buser and Müller 1994a, b; Vetter and Luckas 2000; Herzke et al. 2002; Wong et al. 2009; Zhang et al. 2017a, b) Bacteria (Singer et al. 2002; Harju et al. 2003; Pakdeesusuk et al. 2003) Blue mussel (Hühnerfuss et al. 1995) Human milk, doe liver, eel (Glausch et al. 1994) Doe liver, eel (Vetter et al. (1997a, b, c, d, e, f); human milk (Glausch et al. 1994; Glausch et al. 1995), transformation (Uwimana et al. 2019) E-waste location (Chen et al. 2014), human tissue (Wu et al. 2013) Biota (Vetter et al. (1997a, b, c, d, e, f) Blue mussel (Hühnerfuss et al. 1995); human milk, doe liver, eel (Glausch et al. 1994); Blue mussel (Hühnerfuss et al. 1995) Blue mussel (Hühnerfuss et al. 1995) Blue mussel (Hühnerfuss et al. 1995), human tissue (Toda et al. 2012) Human adipose tissue (Karásek et al. 2007); human liver (Ellerichmann, Bergman et al. 1998); rat (Larsson et al. 2002; Norström et al. 2006); soil (Robson and Harrad 2004); marine biota (Chu et al. 2003a, b; Larsson et al. 2004a, b; Karásek et al. 2007) (continued)

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Table 8.8 (continued) Substance 2-(2,4-dichlorophenoxy) propionic acid 2-(4-chloro-2methylphenoxy)propionic acid HHCB (galaxolide) AHTN (tonalide) ATII (traseolide)

Water/Tissue/Air [References] Water (Ludwig et al. 1992a; Hühnerfuss et al. 1993; Buser and Müller 1998; Caballo et al. 2013); soil (Romero et al. 2001) Water (Buser and Müller 1998; Caballo et al. 2013); Sediment Microbial (Zipper et al. 1996) Water (Lee et al. 2016; Gao et al. 2019); Sediment (Song et al. 2015); fish (Gatermann et al. 2002a, b) Water (Wang et al. 2013a, b); sediment (Song et al. 2015); fish (Gatermann et al. 2002a, b) Water (Wang and Khan 2014; Gao et al. 2019); sediment (Song et al. 2015); fish (Gatermann et al. 2002a, b)

to represent the enantioselective transformation of α-HCH by marine microorganisms, seems to be independent of the seasonal variability of the biological activity. The enantiomeric ratios determined for the blue mussel samples exhibit a larger variability; however, the values (mean ER value 0.89  0.14) largely reflect the fact that the mussels accumulate persistent organic pollutants like α-HCH, but cannot significantly degrade such persistent halogenated compounds. As a consequence, a similar enantiomeric ratio of α-HCH is observed as in the water, which implies that at least no enantioselective transformation of this compound takes place in the blue mussel. A dramatic shift of the enantiomeric ratio of α-HCH was, however, observed in the liver of the Common eiders whose diet, as outlined above, almost completely consists of molluscs (Kallenborn et al. 1994a, b). In all instances, the ratios (+)-αHCH/()-α-HCH turned out to be significantly larger than 1, thus confirming the earlier results by Kallenborn et al. (1991, Hühnerfuss and Kallenborn 1992). A more detailed analysis in the study by Pfaffenberger included aspects of the physiological fitness of the investigated birds, as well as the contamination status of the sampling area (Pfaffenberger et al. 1992). Animals suffering on chronic illnesses, that is, two specimen (samples #5 and #8; Table 8.9) suffered from a severe parasitic disease, showed enantiomeric α-HCH ratios in their organ tissues between 1.4 (E2, sample #6) and 2.8 (E1, sample # 1), while individuals of better physiological conditions (collected after drowning in the local fishing nets) exhibited enantiomeric α-HCH ratios up to nearly 1 (E1, samples # 2 and #3), which implies that the ()-α-HCH enantiomer had been nearly completely decomposed, and only the (+)-α-HCH enantiomer was enriched in the liver. A comparison between samples of the more polluted site E1 and the less polluted site E2 shows no clear tendency, and also the absolute concentrations of αHCH in the liver samples (Table 8.9) give no clear indication as to whether or not higher pollution of the area gives rise to stronger enzymatic activity. However, common Eiders must be considered migrating marine birds, during spring and autumn moving long distances along the North Sea coast. Thus, a direct

8.2 Transformation/Accumulation of Chiral Xenobiotics in Higher Organisms

139

Table 8.9 Concentrations and enantiomeric ratios of α-HCH for North Sea water, blue mussels and for liver samples of common Eider ducks and of flounders, respectively

Sample North Sea water sample

Blue mussel

Sampling site W1 W2 W3 W4 M1 M2

Common eider duck (liver)

E1

E2

Flounder (liver)

F1 (6/1991)

F1 (1/1991)

F2 (1/1991)

Sample # 1 2 3 4 1 2 3 4 5 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 9 10 11 12 13

Concentration (ng/g wet weight) – – – – 0.93 0.78 0.57 1.35 0.35 3.12 0.21 0.11 1.67 0.16 0.53 2.17 2.0 3.1 1.1 0.7 1.9 2.4 1.1 1.0 2.1 2.0 1.6 0.6 1.3 1.8

(+)-α-HCH ()-αHCH 0.81 0.84 0.87 0.83 0.70 0.97 0.83 0.93 1.04 2.8 1 1 1.8 9.5 1.4 25 5.5 0.92 0.91 0.91 0.98 0.97 0.83 0.84 0.76 0.76 0.88 0.86 0.89 0.91

Mean enantiomeric ratio

0.84  0.03

0.89  0.14

0.94  0.04

0.80  0.05

0.89  0.03

correlation between local pollutant levels and the individual concentrations in the common Eider duck tissues has not to be expected. Moreover, in the case of the common Eiders, the ability to transform α-HCH in the liver appears to be rather dependent on the physical conditions of the animals, which in turn may be influenced by the sum of physiological stress induced by marine pollutants. However, regardless of the absolute values of the enantiomeric ratios, in all instances the enzymatic processes in the liver of common Eider ducks gave rise to a faster (or nearly exclusive) degradation of ()-α-HCH.

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8 Enantiomer-Specific Fate and Behaviour of Chiral Contaminants

A different result was obtained from enantiomer-selective analysis of liver samples derived from the corresponding flounders (Platichthys flesus). The mean values of the different test sites vary from 0.80 to 0.94 (Table 8.9), which reflect an enzymatic transformation process that prefers common structural elements represented by (+)-α-HCH. Since the enantiomeric ratios of α-HCH determined in the liver samples of the flounders in the two test sites F1 and F2 show relatively low variability for the respective areas, a comparison between these two data sets appears to be feasible: In the Test site F1, a mean enantiomeric ratio of 0.8  0.05 was determined for the flounders caught in January 1991, while the flounders caught during the same period in the less polluted area F2 showed a mean value of 0.89  0.03. The result seems to support the assumption that a stronger enzymatic activity is induced in the liver of the flounders caught in the highly polluted Elbe estuary than in the liver of flounders living in the Eider estuary. However, it should be noted that the effects observed by Pfaffenberger et al. is based on a consistent but small data set, and, furthermore, the effect is only slightly beyond the error limits. In addition, seasonal effects, for example, different input of pollutants into the sea during winter and summer periods may modify the results. This is demonstrated by a comparison of the enantiomeric ratios of α-HCH determined in flounders caught in the Elbe estuary in January 1991 (mean value of 0.80  0.05; F1/samples # 6–9, Table 8.9) and in June 1991 (mean value 0.94  0.04; F1/samples # 1–5). Therefore, caution has to be applied when interpreting the flounder data summarised in Table 8.9 and follow-up study should be encouraged in order to verify the here-presented indications. Meanwhile, several authors have addressed the scientific challenge posed by the topic enantioselective transformation of α-HCH at low trophic levels (Hoekstra et al. 2003a, b, c; Borga and Bidleman 2005), organisms associated with higher trophic levels like other domestic and wild birds (Corsolini et al. 2006; Yang et al. 2010a, b; Liu et al. 2016a, b), Canadian wolverine (Hoekstra et al. 2003a, b, c), seals (Hühnerfuss et al. 1993; Hummert et al. 1995; Klobes et al. 1998a, b, c; Moisey et al. 2001; Fisk et al. 2002; Hoekstra et al. 2003a, b, c; Carlsson et al. 2014a, b), whales (Hummert et al. 1995; Hoekstra et al. 2003a, b, c; Carlsson et al. 2014a, b) and polar bears (Wiberg et al. 1998a, b, c; Ross et al. 2011a, b) applying chiral selectors on the basis of modified cyclodextrin CSPs. The results are summarised in Table 8.10. In detail, different scientific aspects have been covered by the earlier studies summarised above. For example, Hühnerfuss et al. (1993) compared the results of tissue sample extracts of dead found harbour seals (Phoca vitulina L.) from the German Bight and from hunted animals from Iceland, respectively, in order to study the impact of different contamination levels on the enantioselective processes. In the seal tissues obtained from animals of the German Bight, enantiomeric ratios of 1.5–4.5 were determined for blubber tissue, while brain tissue from the same animals yielded enantiomer ratios of 7.9–19.9. These values have to be compared with those from Iceland seals, that is, enantiomeric ratios in blubber 1.2–1.4 and in brain tissues 55.6, 66.2 and six values of nearly 1 (¼ infinite). Though the general tendency of the data from both regions is comparable, the enantiomeric ratios in the blubber of

8.2 Transformation/Accumulation of Chiral Xenobiotics in Higher Organisms

141

Table 8.10 Enantiomeric ratios of α-HCH in extracts of marine biota tissues of higher trophic levels; for comparison selected cod samples (oil and whole body, respectively) are included

Sample Neonatal fur seal (healthy) (Callorhinus ursinus) Stillborn fur seal (Callorhinus ursinus) Neonatal fur seal (disease) Female fur seal Cod liver oil North Atlantic Harbour seal (German Bight) (Phoca vitulina) Harbour seal (German Bight) (Phoca vitulina) Harbour seal (German Bight) (Phoca vitulina) Harbour seal (Iceland) (Phoca vitulina)

Harbour seal (Iceland) (Phoca vitulina)

Harbour seal (Iceland) (Phoca vitulina)

Harbour seal (Iceland)

Tissue Blubber Liver Lung

Enantiomeric ratios Column Column A B 1.88 1.67 1.66 1.45 1.55 1.47

Column C – 1.64 –

Brain Blubber Liver

30.0 1.85 1.83

26.2 1.60 1.64

26.2 – –

Brain Blubber Liver Milk

32.9 1.20 1.30 1.58 0.98

31.4 1.18 1.27 1.47 0.99

– – – – –

Blubber

4.47

–

–

Spinal marrow Blubber

19.9

–

–

1.54

–

–

Brain Blubber

16.3 2.83

– –

– –

Brain Blubber

7.9 1.36

– –

– –

Brain Spinal marrow Blubber

1 1

– –

– –

1.26

–

–

Brain Spinal marrow Blubber

1 99.5

– –

– –

1.21

–

–

Brain Spinal marrow Brain

1 1

– –

– –

1

–

–

References Mössner et al. (1992)

Mössner et al. (1992)

Mössner et al. (1992) Mössner et al. (1992) Mössner et al. (1992) Hühnerfuss et al. (1992a, b), Möller et al. (1994)

Hühnerfuss et al. (1992a, b), Möller et al. (1994) Hühnerfuss et al. (1992a, b), Möller et al. (1994) Hühnerfuss et al. (1992a, b), Möller et al. (1994)

Hühnerfuss et al. (1992a, b), Möller et al. (1994)

Hühnerfuss et al. (1992a, b), Möller et al. (1994)

Hühnerfuss et al. (1992a, b), Möller et al. (1994) (continued)

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8 Enantiomer-Specific Fate and Behaviour of Chiral Contaminants

Table 8.10 (continued)

Sample (Phoca vitulina) Harbour seal (Iceland) (Phoca vitulina) Harbour seal (Iceland) (Phoca vitulina) Harbour seal (Iceland) (Phoca vitulina) Harbour seal (Iceland) (Phoca vitulina) Harbour seal (Phoca vitulina) Harbour seal (Phoca vitulina) Whale blubber (Ph. Phoceona) Whale blubber (Ph. Phoceona) Cod (Boreogadus saida) Ringed seal (Phoca hispida) Polar bear (Ursus maritimus) Harbour seal F (Phoca vitulina) F F F F M M M

Tissue Spinal marrow Brain

Enantiomeric ratios Column Column A B 1 –

Column C –

References

66.2

–

–

Spinal marrow Brain

30.2

–

–

55.6

–

–

Spinal marrow Brain

1

–

–

1

–

–

Spinal marrow Brain

1

–

–

1

–

–

Spinal marrow Blubber

22.8

–

–

2.33

2.04

–

Müller (1992)

Blubber

2.17

2.17

–

Müller (1992)

Blubber

2.78

2.51

–

Müller (1992)

Blubber

2.86

2.63

–

Müller (1992)

Whole body Blubber Liver Fat Liver Blubber Blubber Blubber Blubber Blubber Blubber Blubber Blubber Mean value

1

–

–

Wiberg et al. (1998a, b, c)

1  1.4  1.7  2.5 1.0 1.3 1.3 1.3 1.8 1.2 1.3 1.5 1.4

– – – –

– – – –

Hühnerfuss et al. (1992a, b), Möller et al. (1994)

Hühnerfuss et al. (1992a, b), Möller et al. (1994)

Hühnerfuss et al. (1992a, b), Möller et al. (1994)

Hühnerfuss et al. (1992a, b), Möller et al. (1994)

Wiberg et al. (1998a, b, c) Wiberg et al. (1998a, b, c)

(continued)

8.2 Transformation/Accumulation of Chiral Xenobiotics in Higher Organisms

143

Table 8.10 (continued)

Sample (Halichoerus grypus) F F F F M M M

Cod liver oil Fish oil

Tissue Blubber Blubber Blubber Blubber Blubber Blubber Blubber Mean value

Enantiomeric ratios Column Column A B 1.3

Column C

References

1.2 1.2 1.5 1.5 1.5 1.2 1.3

Wiberg et al. (1998a, b, c)

1.00– 1.23 1.0

Koske et al. (1999)

Iceland animals, that is, from the less polluted area, appear to be lower, and in the brain samples from the eight Iceland seals almost exclusively (+)-α-HCH was found. As the α-HCH concentrations in blubber of the German Bight seals were about five times higher than those of the Iceland seals, it is tentatively assumed that the higher concentrations may have induced higher enzymatic activity, which in turn is expected to give rise to higher enantiomeric shifts in blubber. However, this postulate requires further in-depth investigations. The aspect of enantioselective permeation of α-HCH enantiomers through the blood–brain barrier, which can be also inferred from the data set thus far available, will be discussed separately later in this chapter. Mössner et al. (1992; Mossner and Ballschmiter 1997) focused on the enantioselective transformation of α-HCH in ill and healthy neonatal, as well as adult fur seals (Callorhinus ursinus (L.)). Tissues of three dead neonatal fur seals with different health status were collected on St. Paul Island (Pribilof Islands, Alaska, USA) in July 1990 in co-operation with the Alaska Marine Mammal Tissue Archival Project and the National Marine Fisheries Service. The animals were characterised as follows: first, a neonatal fur seal (healthy): death due to massive head trauma; body condition good; age 1–2 weeks; male. Second, a stillborn fur seal; female; third, a neonatal fur seal (disease); white muscle syndrome; body condition good; age 7–10 days; male. Furthermore, a milk sample, which was taken from the stomach of a fur seal pup that died of pneumonia, was included in this study (Mössner et al. 1992). A cod liver oil originating from the North Atlantic, reflecting in part the sum of abiotic and biotic transformation processes of the diet, was analysed as a reference (SRM 1588, NIST, Gaithersburg, Maryland, USA). For blubber, liver, lung and milk tissues of the fur seals, Mössner et al. obtained enantiomeric ratios of α-HCH in the range 1.2–1.9 (Table 8.10), that is, values that

144

8 Enantiomer-Specific Fate and Behaviour of Chiral Contaminants

are in accordance with those reported by Hühnerfuss et al., for the Iceland seal blubber tissues and for the two brain tissues; also high ratios of 28 and 32 were determined. The ratio found in the milk sample reflects enantioselective processes in the mother animal. The slightly enhanced ratios in the tissues of the normal and stillborn fur seals are assumed to be indicative of additional metabolic discrimination of the later eluting enantiomer. However, the neonatal fur seal, which died of the white muscle syndrome, shows the smallest ratios. It cannot be excluded that the disease may have influenced the enzyme induction potential and thus reduced the potential to metabolise xenobiotics like α-HCH. Müller et al. (1996a, b) extended the data sets thus far available for the enantiomeric composition of α-HCH in seal tissues by a systematic investigation including two different seal species, eight harbour seals (Phoca vitulina L.) and eight grey seals (Halichoerus grypus (FABR.)), which were shot on western Iceland. For both seal species, ER values of ER+/ > 1 were determined in blubber extracts, which is fully in accordance with the results of other authors (Table 8.10). With mean values of ER ¼ 1.4 (harbour seals) and ER ¼ 1.3 (grey seals), no species-dependent effects were observed for these two seal species. Up to now, hooded seals are the only marine mammals for which ER < 1 for α-HCH were reported (Hummert et al. 1995). Müller et al. did not verify any correlation between α-HCH levels or enantiomeric ratios with age or sex for the two seal species. Wiberg et al. (1998a, b, c) investigated enantioselective processes of organochlorines including α-HCH in the Arctic marine food chain, placing special emphasis on the polar bear (Ursus maritimus PH.) food chain, which is simply due to the limited biodiversity in the Arctic marine environment. The main food for polar bears is the blubber of ringed seals (Phoca hispida, ERX.), which in turn largely consume fish and pelagic crustaceans, in particular Arctic cod (Boregadus saida LEP.) and amphipods. The Arctic cod tissue extracts showed near-racemic ERs, indicating that bioaccumulation takes place without or with minor selective metabolism. This conclusion is fully in accordance with results of other authors who reported enantiomeric ratios of α-HCH in Atlantic cod liver (Gadus morhua) or fish oil extracts from other species between 0.98 and 1.23 (Mössner et al. 1992; Koske et al. 1999; Wong et al. 2002; Wiberg et al. 2006). The tissue extracts of ringed seal and polar bear exhibited large ER changes. Obviously, (+)-α-HCH becomes more abundant relative to ()-α-HCH in top predators (Fig. 8.13). Therefore, Wiberg et al. calculated separate biomagnification factors (BMFs) for the (+)- and the ()-enantiomers. For the first step, from cod to seal, the BMFs are comparable ((+)-α-HCH: BMF ¼ 2.0; ()-α-HCH: BMF ¼ 1.7); however, during the next step, from seal to polar bear, only the (+)-enantiomer biomagnifies (BMF ¼ 1.4), in contrast to the ()enantiomer (BMF ¼ 0.7). For the complete chain, from cod to polar bear, the values are for the (+)-enantiomer BMF ¼ 2.8 and for the ()-enantiomer BMF ¼ 1.2, respectively. The data set used for the calculations by Wiberg et al. comprised 40 samples. At least qualitatively, the results are fully in line with the observation that the (+)-enantiomer is increasingly dominant up the polar bear food chain. The above selection of research studies illustrates the importance of α-HCH as the first and most comprehensively investigated chiral persistent environmental

8.2 Transformation/Accumulation of Chiral Xenobiotics in Higher Organisms

145

Fig. 8.13 Average enantiomeric ratios (ER)  standard deviation of αHCH in the polar bear (PB) food chain; RS ¼ ringed seal (Wiberg et al. 2000)

pollutant. Additional updated information about levels and distribution in higher organisms can be found in several recently published studies, reviews and books (Ali and Aboul-Enein 2004; Busch and Busch 2011; Garrison et al. 2011a, b; Genualdi et al. 2011; Ma et al. 2013; Carlsson et al. 2014a, b; Wang et al. 2015a, b; Zhang et al. 2017a, b).

8.2.1.2

Other Chlorinated Pesticides

Beside α-HCH, several other persistent polychlorinated insecticides are chiral and many have been applied as racemates in agricultural application. Some of these substances were banned or are severely restricted in use for about three decades already. However, all of the conventional POPs including polychlorinated insecticides like HCHs, Toxaphene® and chlordanes®, after the ban of their application in the United States, Asia and most European countries, are still found in the environment, even in remote ecosystems at all trophic levels. An important member group are cyclodiene pesticides, for example, cis and trans-chlordane, heptachlor, aldrin and dieldrin, as well as some of their oxygenated metabolites like oxychlordane and heptachlor exoepoxide. These compounds were extensively used in many countries (e.g. U.S.A.), but reportedly not in others, for example, not in Scandinavia. The fact that they are still being detected in biota from remote areas, for example, the Arctic and Antarctic, points to global distribution mechanisms such as long-range atmospheric transport. This latter aspect will be presented in a separate section of this chapter. In 1991, König et al. were the first to report the successful enantiomer separation of cis- and trans-chlordane, oxychlordane, heptachlor, heptachlor exoepoxide (see Fig. 8.14) using selectively derivatised cyclodextrin phases (König et al. 1991). Although the authors were able to resolve the individual enantiomeric pairs of cis-

146

8 Enantiomer-Specific Fate and Behaviour of Chiral Contaminants

Enantiomers Cl

Cl Cl

cis-chlordane

Cl Cl Cl

Cl

Cl Cl

Cl

Cl

Cl

Cl O

Cl

Cl

Cl

Cl Cl Cl H

H

Cl

Cl

Cl

O Cl

H

Cl Cl

Cl Cl H H O

Cl

Cl

H

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl O

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl Cl

Cl

Cl O

Cl

Cl

Cl

trans-heptachlorepoxid

Cl Cl

Cl Cl

cis-heptachlorepoxid

Cl

Cl

Cl

Heptachlor

Cl Cl

Cl

Oxy-chlordane

Cl

Cl Cl

Cl

Cl Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl Cl

Cl Cl

trans-chlordane

Cl

Cl

Cl H

H

Cl

Cl

O

Fig. 8.14 The molecular structures of cis- and trans-chlordane, oxychlordane, heptachlor, heptachlor exoepoxide, the chiral MC compounds MC 4, MC 5, MC 6, MC 7 (Miyazaki et al. 1985) and the achiral trans- and cis-nonachlor

and trans-chlordane on several cyclodextrin derivatives (e.g. heptakis(3-O-methyl2,6-di-O-n-pentyl)-β-cyclodextrin or per-O-methyl-β-cyclodextrin), the simultaneous separation of all four stereoisomers was possible only on heptakis(2-Omethyl-3,6-di-O-n-pentyl)-β-cyclodextrin. Meanwhile, several authors applied this new experimental approach to the investigation of enzymatic transformation of cyclodiene pesticides and their metabolites in marine and freshwater biota; see Tables 8.11 and 8.12 (Buser et al. 1992; Oehme

8.2 Transformation/Accumulation of Chiral Xenobiotics in Higher Organisms

147

Table 8.11 Enantiomeric ratios of cis-chlordane (cis-CD) and trans-chlordane (trans-CD), oxychlordane (OXY), heptachlor (HEPTA), heptachlor exo-epoxide (exoHEP) determined in tissue extracts of marine and limnic biota; F ¼ female (age); M ¼ male (age)

Biota/tissue Baltic herring

Enantiomeric ratios cistransCD CD OXY 1.35 0.42 –

HEPTA –

exoHEP –

References Buser et al. (1992), Buser and Müller (1993)

Baltic salmon

0.38

 0.02 1.19

Baltic seal

 0.03 –

 0.02 0.60

–

–

–

Antarctic penguin Harbour seal: liver Blubber

–

 0.04 –

Buser et al. (1992), Buser and Müller (1993)

–

–

–

Buser et al. (1992), Buser and Müller (1993)

–

–

0.57

–

0.06

–

–

0.66

–

0.14

Harbour seal: liver Blubber Seagull egg

–

–

0.45

–

0.05

– –

– –

0.54 2.3

–

0.18 –

Seagull egg

–

–

2.1

–

Seagull egg

–

–

1.8

–

Seagull egg

–

–

1.5

2.7

Cod liver oil NIST Cod: muscle F6 Gonad Liver Cod: muscle F12 Gonad Liver Cod: muscle F11 Gonad Liver Cod: muscle M9 Gonad Liver

0.95

1.13

1.33

–

1.60

0.71 0.77 0.71 0.83 1.0 0.83 0.91 1.1 0.91 1.14 1.1 1.2

0.54 0.68 0.54 0.73 0.77 0.62 0.46 0.62 0.41 1.9 2.0 2.2

– – – – – – – – – – – –

– – – – – – – – – – – –

– – – – – – – – – – – –

 0.1

–

–

–

Buser et al. (1992), Buser and Müller (1993)

König et al. (1994a, b, c), Hühnerfuss et al. (1996a, b) König et al. (1994a, b, c), Hühnerfuss et al. (1996a, b) König et al. (1994a, b, c), Hühnerfuss et al. (1996a, b) König et al. (1994a, b, c), Hühnerfuss et al. (1996a, b) König et al. (1994a, b, c), Hühnerfuss et al. (1996a, b) König et al. (1994a, b, c), Hühnerfuss et al. (1996a, b) Müller et al. (1997) Karlsson et al. (1997a, b)

Karlsson et al. (1997a, b)

Karlsson et al. (1997a, b)

Karlsson et al. (1997a, b)

(continued)

148

8 Enantiomer-Specific Fate and Behaviour of Chiral Contaminants

Table 8.11 (continued)

Biota/tissue Cod: muscle M6 Gonad Liver Herring F3 M3 M3 M3 M3 F3 F3 F3 F3 Cod liver oil Fish oil Grey and harbour. seal blubber F F F F F M M Mean value Grey seal blubber F F F F F M M M Mean value

Enantiomeric ratios cistransCD CD OXY 1.0 1.1 – 1.0 1.1 – 1.06 1.2 – 1.1 0.48 – 1.2 0.20 – 1.2 0.27 – 1.3 0.58 – 1.5 0.47 – 1.2 0.36 – 1.2 0.35 – 1.6 0.27 – 1.6 0.32 – 0.50– 0.80– – 1.02 3.49 1.00– 1.00– – 1.18 1.06 – – 0.7

HEPTA – – – – – – – – – – – – –

exoHEP – – – – – – – – – – – – –

References Karlsson et al. (1997a, b)

–

–

Koske et al. (1999)

–

–

Müller et al. (1996a, b)

Müller et al. (1996a, b)

– – – – – – – –

– – – – – – – –

0.7 0.8 0.6 0.7 0.7 0.9 0.7 1.6

– – – – – – – –

– – – – – – – –

– – – – – – – –

– – – – – – – –

1.5 1.4 1.2 1.1 1.1 1.4 1.5 1.3

– – – – – – – –

– – – – – – – –

Karlsson et al. (1997a, b)

Koske et al. (1999)

et al. 1994; Borga and Bidleman 2005; Ross et al. 2008; Genualdi et al. 2011; Zhang et al. 2012a, b, c; Bidleman et al. 2013a, b; Carlsson et al. 2014a, b; Zhou et al. 2018). Buser et al. (1992) analysed herring oil and tissues of a salmon, a seal and a penguin for the determination of the enantiomeric signature of chiral chlordanes. The

8.2 Transformation/Accumulation of Chiral Xenobiotics in Higher Organisms

149

Table 8.12 Enantiomeric ratios of additional cyclodiene pesticides, in particular, chiral chlordane components observed in technical chlordane mixtures and bromocyclen (BC), determined in tissue extracts of marine and limnic biota; F ¼ female (age); M ¼ male (age) Biota/ tissue Baltic herring Baltic salmon Baltic seal Antarctic penguin Cod liver oil NIST Cod: muscle F6 Gonad Liver Cod: muscle F12 Gonad Liver Cod: muscle F11 Gonad Liver Cod: muscle M9 Gonad Cod: muscle M6 Gonad Liver Herring F3 M3 M3 M3 M3 F3 F3 F3

Enantiomeric ratios MC4 MC5 MC6 0.7 0.81 –

MC7 0.83

K –

U81 –

U82 –

BC –

0.7

0.75

–

0.92

–

–

–

–

2.7

0.24

–

0.86

–

–

–

–

2.2

0.91

–

1.35

–

–

–

–

–

0.87

–

–

–

–

–

–

–

0.29

–

–

0.85

not res. 0.73

References Buser and Müller (1992), Buser et al. (1992) Buser and Müller (1992), Buser et al. (1992) Buser and Müller (1992), Buser et al. (1992) Buser and Müller (1992), Buser et al. (1992) Müller et al. (1997)

–

Karlsson et al. (1997a, b)

– – –

– – –

0.23 0.11 0.32

– – –

– – –

1.0 0.93 0.77

0.76 0.76 0.80

– – –

Karlsson et al. (1997a, b)

– – –

– – –

0.32 0.26 0.54

– – –

– – –

0.76 0.88 0.83

0.75 0.76 0.82

– – –

Karlsson et al. (1997a, b)

– – –

– – –

0.35 0.24 1.6

– – –

– – –

0.85 0.78 0.93

0.74 0.76 0.97

– – –

Karlsson et al. (1997a, b)

– –

– –

1.0 1.8

– –

– –

0.92 –

0.76 0.92

– –

Karlsson et al. (1997a, b)

– – –

– – –

1.1 1.0 –

– – –

– – –

1.1 0.86 –

0.89 0.89 1.1

– – –

Karlsson et al. (1997a, b)

– – – – – – –

– – – – – – –

– – – – – – –

– – – – – – –

– – – – – – –

– – – – – – –

1.2 1.2 1.1 1.2 1.0 1.0 1.1

– – – – – – – (continued)

150

8 Enantiomer-Specific Fate and Behaviour of Chiral Contaminants

Table 8.12 (continued) Biota/ tissue F3 Trout

Enantiomeric ratios MC4 MC5 MC6 – – – – – –

MC7 – –

K – –

U81 – –

U82 1.0 –

BC – 0.84

Orfe

– – – – –

– – – – –

– – – – –

– – – – –

– – – – –

– – – – –

– – – – –

0.93 1.00 0.94 0.84 0.80

Bream

–

–

–

–

–

–

–

0.89

Pike

–

–

–

–

–

–

–

0.85

Trout: muscle

–

–

–

–

–

–

–

0.66

– –

– –

– –

– –

– –

– –

– –

0.71 1.19

– – – – – – –

– – – – – – –

– – – – – – –

– – – – – – –

– – – – – – –

– – – – – – –

– – – – – – –

1.22 1.08 1.34 1.56 2.13 1.93 2.05

Bream: muscle

References Pfaffenberger et al. (1994a, b)

Pfaffenberger et al. (1994a, b) Pfaffenberger et al. (1994a, b) Pfaffenberger et al. (1994a, b) Bethan et al. (1997), Karlsson et al. (1997a, b) Bethan et al. (1997), Karlsson et al. (1997a, b)

herring oil was prepared from fresh herring (Clupea harengus L.) collected from the Gulf of Bothnia (June 1988). Preparation of the oil was performed at the Norwegian Herring Oil and Meal Industry Research Institute, Bergen, Norway. The salmon muscle tissue stemmed from a female salmon (Salmo salar L.) caught in the Ume River at Stornorrfors, Sweden, in 1989. The seal sample was a composite of liver tissue of adult grey seal (Halichoerus grypus (FABR.)) collected from the Baltic Sea along the Swedish south-eastern coastline during the 1980s. Finally, the penguin tissue was a sample from a juvenile Adelie penguin (Pygoscelis adelis (L.)) found dead at Shackleton’s Hut, Ross Island, Antarctica, in March 1988. The frozen tissue was shipped to the University of Umeå, Sweden, where extraction and clean-up were carried out (Buser et al. 1992). The authors stress that no individual chlordane congeners were available as standards for their investigations at that time (today commercially available). Instead, they used the technical chlordane mixture, in which all compounds of interest could be identified and then assigned in the environmental samples using published retention data. Technical chlordane consists of a complex mixture of primarily hepta-, octa- and nona-chlorinated tricyclic

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compounds. It is prepared by the Diels–Alder reaction of hexachlorocyclopentadiene and cyclopentadiene to chlordene, an unsaturated hexachlorinated, tricyclic compound. Subsequent chlorination leads to products containing two to three additional chlorines in the cyclopentane ring, that is, octaand nona-chlordanes. The main constituents in technical chlordane, cis- and transchlordane and trans-nonachlor, belong to these groups. Furthermore, there is a number of more complex structures present in the technical mixture, resulting from rearrangement reactions, in particular, Wagner-Meerwein rearrangements (Dearth and Hites 1991). In Fig. 8.14, the molecular structures of cis- and transchlordane, oxychlordane, heptachlor, heptachlor exoepoxide, U82, MC4, MC5, MC6, MC7 and trans- and cis-nona-chlor are shown. All derivatives possess an endo configuration, which can be easily explained on the basis of the frontier orbital theory valid for pericyclic reactions like the Diels–Alder reaction (Fleming 2009). Furthermore, all compounds shown in Fig. 8.14 are chiral, apart from cis- and transnona-chlor. The chlordane components detectable in all four biota samples investigated by Buser et al. (1992) were the chiral congeners cis- and trans-chlordane, MC4, MC5, MC6, MC7 and U82, the prochiral cis- and trans-nona-chlor, as well as various minor components. Component K was not found to be present in these extracts. For some congeners, a complete enantiomer separation was achieved by Buser et al., and for other components enantiomer separation remained incomplete (see Tables 8.11 and 8.12). The largest enantiomeric excess (ER value of 0.24) was observed for MC5 in Baltic seal. The major octa-chlordane component in both fish samples was cis-chlordane, wherein herring the earlier and in salmon the later eluting enantiomer predominated., that is, the enantiomeric ratios of cis-chlordane between the two fish species are thus reversed. For trans-chlordane, the authors also observed a reversal in the enantiomeric ratios between the two species. In this case, the later eluting enantiomer predominated in herring and the earlier eluting in salmon. In seal and penguin, the concentrations of cis- and trans-chlordane were much lower and, therefore, enantiomeric ratios were much more difficult to determine. In these two warm-blooded species, the components U82 and MC5 were dominating. U82 is a 5+3 type chiral octa-chlordane of, at that time, unknown configuration (Buser et al. 1992). At first, Buser and co-workers were not able to resolve the enantiomers of U82, but their endeavour was successful in a subsequent investigation, when they applied a different chiral selector, heptakis(6-O-tertbutyldimethylsilyl-2,3-di-O-methyl)-βcyclodextrin (TBDMS-CD, to a technical chlordane mixture) (Buser et al. 1992). Furthermore, Karlsson et al. meanwhile successfully elucidated the structure of U82 (see Fig. 8.14) (Karlsson et al. 1998, 1999). By contrast, MC5 was clearly separated into enantiomers (Table 8.12). The later eluting enantiomer predominated in seal tissue, and some preference for this enantiomer was still observed in tissues of the other aquatic species included in the study. Some differences in the enantiomeric composition of components MC4 and MC7 were also inferred from the data set. For component MC4, the chromatograms showed a clear predominance of the earlier eluting enantiomer in the warmblooded species, whereas this can hardly be observed in the two fish. For component

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8 Enantiomer-Specific Fate and Behaviour of Chiral Contaminants

MC7, the chromatograms indicated a small preference for the later eluting enantiomer in herring, salmon and seal, whereas in the penguin a small prevalence of the earlier eluting enantiomer can be recognised. Some of the separation problems encountered by Buser et al. with regard to components of the technical chlordane mixture were overcome by Karlsson and co-workers who used heptakis(2,3,6-O-tertbutyl-dimethylsilyl)-β-cyclodextrin as chiral selector. The latter authors analysed Atlantic cod (Gadus morhua L.) samples carrying out a very comprehensive study. Atlantic cod, on the winter migration from the Barents Sea to the Lofoten Islands for spawning, was caught outside Kvaløya, Tromsø (70  N, 17  E). Age, sex, size, as well as the maturation stage of the gonads, were determined. Liver and gonads were weighted, and samples were taken including a portion of the filet. The extraction and clean-up procedure can be found in a corresponding publication (Karlsson et al. 1997a, b). As can be inferred from Tables 8.11 and 8.12, the enantiomeric ratios (ER; area of (+)-/()-enantiomer or first eluting enantiomer divided by the second one] in Atlantic cod deviate substantially from the racemic ratio. With a few exceptions, the ER found in all three cod tissues are similar within the uncertainty of the method (3–5%). This means that any tissue can be used for an ER determination. It is worth noting that ER of the octa-chloro congener U82 was below one in cod and above one in herring. No differences in the ERs between male and female herring were observed. However, in all cod samples analysed by Karlsson et al., the ERs for trans-chlordane and MC6 were very different between males and females. Enantiomer transformation was opposite in male and female cod leading to changes of the ERs by a factor of 3–5. A similar but not so unequivocal trend was also obtained for cis-chlordane. No gender difference was observed for U81 and U82. The differences in the influence of sex on the ERs of the congeners cannot be explained yet. Karlsson et al. (1997a, b) concluded that U81 and U82 have a 5+3 chlorine distribution between ring 2 and 1, while all other congeners are of the 6+2 (octa-chloro congeners) or 6+3 type (nona-chloro congeners). The technical pesticide chlordane has shown a significant synergism concerning artificial estrogenic effects together with other pesticides such as dieldrin (Arnold et al. 1996; Arnold and McLachlan 1996; Ribeiro et al. 2009; Chighizola and Meroni 2012). This might be an explanation for the different ERs in male and female cod. However, this does not explain the missing gender influence at herring. Other factors such as position in the food chain and unlike enzyme systems can also be of importance. The enantioselective analysis of organochlorines in the Arctic food chain “cod/ seal/polar bear” carried out by Wiberg et al. (1998a, b, c) also included the chlordane congeners MC4, MC5 and MC6. The results are summarised in Fig. 8.15. Similar to the situation encountered in the case of α-HCH, near-racemic ERs were found in the cod tissue extracts. With regard to the octachlordane MC5, the second eluting enantiomer became more abundant up the food chain. Different enantiomeric prevalence was observed for MC4: while the first eluting enantiomer dominated in blubber and liver of the ringed seal, the second eluting enantiomer was preferentially found in the liver tissue extract of the polar bear. In the case of MC6, both in the ringed seal and polar bear tissues the second eluting enantiomer was dominating. A

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Fig. 8.15 Average enantiomeric ratios (ER)  standard deviation of the chlordane congeners MC4, MC5 and MC6 in the polar bear food chain; RA ¼ ringed seal; PB ¼ polar bear; Cod ¼ Atlantic cod

strange reversal of the enantiomers up the food chain was determined for the oxygenated metabolites. The (+)-enantiomer of heptachlor exoepoxide was in excess in cod and polar bear, whereas the ()-enantiomer dominated in ringed seals. The mean ER for oxychlordane was similar for all species in the food chain. Complementary aspects on the enantiomer-selective distribution patterns along a typical Polar food web were conducted by consecutive studies mainly by international research groups (Borga and Bidleman 2005; Corsolini et al. 2006; Bidleman et al. 2013a, b). Corsolini and co-workers (2006) investigated the enantiomeric signature of organochlorine pesticides in an Antarctic food web; from krill to Adelie penguins (Pygoscelis adelis). Beside α-HCH the enantiomeric distribution of oxychlordane was included. The study confirmed that enantioselective biotransformation increased proportionately with trophic level also in Antarctic marine habitats. Borga and Bidleman, however, focused on enantiomer-selective uptake and transfer along the low trophic levels of an Arctic food web (Borga and Bidleman 2005). The enantiomeric signature of α-HCH, trans- and cis-chlordane, MC5, o,p'-DDT were determined in Arctic marine invertebrates (ice-associated amphipods ¼ Gammarus wilkitzkii, pelagic copepods ¼ Calanus hyperboreus, krill ¼ Thysanoessa inermis and amphipods ¼ Themisto libellula, incl. benthic amphipods ¼ Paramphithoe hystrix). The corresponding enantiomer fractions (EFs) were determined in order to investigate the influence of habitat, geographic area and diet on selective bioaccumulation of the ()- or (+)-enantiomer of the target residues. Depletion of the (+)-α-HCH enantiomer increased with increasing trophic levels from ice fauna to zooplankton to benthos. These results correspond to previous reports on enantiomeric signatures. Chlordanes and o,p'-DDT also showed the strongest enantioselective bioaccumulation in benthic amphipods and less so in zooplankton and ice fauna, which had closer to racemic EFs (¼0.5). Neither diet nor geographic area explained EF differences among samples. Non-racemic EFs in benthos may be related to enantiomer-selective biotransformation, but is most likely reflecting the vertical distribution of EFs in the water column and sediments, as demonstrated earlier for α-HCH in the Canadian and European Arctic (Jantunen and Bidleman 1998). Bidleman et al. (2013a, b) investigated the enantiomeric distribution of chiral

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8 Enantiomer-Specific Fate and Behaviour of Chiral Contaminants

organochlorine pesticides in deep-sea Arctic amphipods (Bidleman et al. 2013a, b). For this purpose, archived amphipod samples from Eurythenes gryllus were analysed, collected between 1983 and 1998 in water depths between 2075 to 4250 from locations in the western and central Arctic Ocean. The total persistent organic pollutants (POPs) ranged from 9750 to 156,000 ng/g (lipid weight). Enantiomerselective accumulation was found for o,p'-DDT, cis- and trans-chlordane, nonachlor MC6 and oxychlordane. As already reported for α-HCH before, Wiberg et al. also calculated separate biomagnification factors (BMFs) for the (+)- and the ()-enantiomers of the oxygenated compounds. For the first step, from Atlantic cod to seal, the BMFs are comparable for oxychlordane ((+)-OXY: BMF ¼ 152; ()-OXY: BMF ¼ 124), and also for the second step from seal to polar bear similar values were calculated ((+)OXY: 7.1; ()-OXY: 6.7) As a consequence, the overall values from cod to polar bear are also comparable ((+)-OXY: 1075; ()-OXY: 834). The strange reversal of the enantiomeric ratios observed for heptachlor exoepoxide up the food chain is reflected by the following BMFs: from cod to seal for (+)-HEPX BMF ¼ 4.8 and for ()-HEPX BMF ¼ 9.3; from seal to bear for (+)-HEPX BMF ¼ 7.4 and for ()HEPX BMF ¼ 2.3; from cod to bear for (+)-HEPX BMF ¼ 35 and for ()-HEPX BMF ¼ 21. After the first successful separation of the enantiomers of oxychlordane and heptachlor exoepoxide (König et al. 1991), the main metabolites of cis-/transchlordane and heptachlor, respectively, Hühnerfuss et al. performed systematic investigations on the enantiomeric distribution of these metabolites in various marine biota samples (König et al. 1994a, b, c; Hühnerfuss et al. 1996a, b). Oxychlordane was found in all five sea-gull eggs investigated, while the concentrations of heptachlor exoepoxide were below the detection limit in three eggs. The presence of heptachlor exoepoxide shows that the transformation of both exoheptachlor and endoheptachlor to heptachlor exoepoxide is not confined to mammalian biota like seals, but it is also the exclusive transformation pathway in sea birds. The ER values of oxychlordane and heptachlor exoepoxide in seagull eggs are of particular interest: in both cases, the values are larger than one, indicating a preferential accumulation of the respective (+)-enantiomer. In contrast, the corresponding values determined in different tissues of seals were smaller than one. This may possibly suggest that seagulls, representing in this case a coastal environmental food web, are reflecting rather terrestrial characteristics than marine profiles. This holds at least for the enzymatic processes that give rise to enantioselective accumulation of oxychlordane and/or heptachlor exoepoxide. Oxychlordane enantiomers were also analysed by Müller et al. (1996a, b) in harbour seals (Phoca vitulina L.) and grey seals (Halichoerus grypus (FABR.)). They reported the notable result that higher ()-oxychlordane levels were present in harbour seals, while in grey seals (+)-oxychlordane was dominating (Table 8.11). A more detailed analysis of the data set revealed that neither levels nor enantiomeric ratios of α-HCH and oxychlordane did correlate in harbour seal or grey seals. Also in this case (as reported from previous studies already), no trend in the enantiomeric profiles of oxychlordane with regard to age or sex could be inferred from the data obtained in the here-reported study.

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Fig. 8.16 Enantiomer separation of bromocyclen extracted from fish samples: (a) Standard solution; (b) rainbow trout from a Danish fish farm

In the follow-up studies on environmental fate of cyclodiene pesticides, the chiral insecticide bromocyclen has also been investigated with respect to its enantiomeric distribution patterns (Pfaffenberger et al. 1994a, b; Bethan et al. 1996, 1997; Fidalgo-Used et al. 2008). Since this substance possesses neither a plane nor a centre of symmetry and, therefore, it exists in two enantiomeric forms (Fig. 8.16). cGC conditions: 30-m fused silica capillary column coated with 50% (w/w) heptakis(6-O-tert-butyldimethylsilyl-2,3-O-methyl)-β-cyclodextrin and 50% OV-1701. Column temperature program: initial 333K, increased at 10K/min to 413 K, 40 min isothermal; carrier gas: H2 (60 kPA); on-column injection, ECD-detection; from (Pfaffenberger et al. 1994a, b). Due to its very low mammalian toxicity, in Europe bromocyclen is still used against ectoparasites for the treatment of domestic animals. In 1992, Rimkus and Wolf were the first to report about the contamination of both German and imported

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8 Enantiomer-Specific Fate and Behaviour of Chiral Contaminants

samples of rainbow trout with this new environmental pollutant (Rimkus and Wolf 1992). This was at that time a surprising result, as there is no official registration of this substance for fish farming. The appearance of this new environmental contaminant triggered two follow-up studies by Pfaffenberger et al. (1994a, b) and by Bethan et al. (1996, 1997). Pfaffenberger et al. analysed fish samples from various fish farms in Denmark and from the river Stör, a tributary of the Elberiver in the northern German state Schleswig-Holstein. They focused on the problem as to whether a significant enzymatic transformation of bromocyclen is possible at that trophic level. Particular emphasis was placed upon the question of whether a correlation between the concentration of bromocyclen and its enantiomeric ratio can be inferred from enantioselective cGC using modified cyclodextrins as chiral selector. The fish samples were collected during the spring of 1990 and the autumn of 1992. The rainbow trouts (Oncorhynchus mykiss (WAL.)) were imported from various fish farms in Denmark. The other fish species orfe (Leuciscus idus L.), bream (Abramisbrama orientalis BERG) and pike (Esox lucius (L.)) were caught in the river Stör. Sample preparation and clean-up can be found in the here-discussed report (Pfaffenberger et al. 1994a, b). In all eight fish samples, in part, remarkably high concentrations of bromocyclen were determined. They varied between 0.093 and 1.200 mg/kg, regardless of whether the fishes lived in the artificial environment of a fish farm or in a natural riverine environment like the river Stör. Furthermore, no correlation between the fish species and the observed concentrations were found. With regard to the determination of enantiomeric excesses, Pfaffenberger et al. were the first to separate the enantiomers of bromocyclen in fish tissue extracts. An example of these first successful enantiomer separations with the help of a 25-m fused-silica capillary column, coated with 50% (w/w) heptakis(6-Otertbutyldimethylsilyl-2,3-di-O-methyl)-β-cyclodextrin and 50% OV-1701, is shown in Fig. 8.16. The enantiomeric ratios as determined in the fish samples are summarised in Table 8.11. The ERs vary between 0.84 and 1.00. In all samples of the different fish species, the first eluting ()-enantiomer was preferentially transformed, with the exception of one sample which showed an ER of 1.00 (the assignment of the order of elution of enantiomers is based on optical rotation measurements after enantiomeric resolution by preparative packed-column GC (König et al. 1994a, b, c)). This effect was observed regardless of whether the sample originated from fish farms or from the river Stör. Therefore, it can be safely assumed that an enzymatic process, which is common to all fish species investigated by Pfaffenberger et al., gave rise to the enantioselective transformation of bromocyclen. As for the samples from a fish farm, a correlation between the enantiomeric ratios and the concentration can be inferred (Fig. 8.17). However, caution has to be applied when interpreting this result from a statistical point of view. The observed correlation is based on a consistent but small data set, and it is, therefore, not statistically significant. However, the observed enantiomeric ratios in the fish samples imply that at least the ()-enantiomer can be metabolised to some degree. The source of contamination which led to the high concentration in this matrix was unclear. It could not be excluded that effluents of municipal sewage treatment

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Fig. 8.17 Enantiomeric ratios (ER) and concentrations of bromocylen in five fish samples from Danish fish farms

plants into the river Stör could have been one source of contamination for the three fishes caught in this river. However, the severe impact of bromocyclen on fish stemming from fish farms remained unclear thus far. In order to gain deepened insight into potential sources, Bethan et al. (1997) carried out a comprehensive study that included the analysis of bromocyclen in water and fish samples from different stations of the river Stör. Moreover, samples from the influent, as well as from the effluent of various sewage plants along the river, were collected. Particular emphasis was placed upon the question of on how bromocyclen gets into the river Stör and if the enantiomeric ratios determined in the fish samples correlate with the concentrations. The latter aspect was intended to verify the hypothesis by Pfaffenberger et al. outlined above. The water samples, which were already discussed earlier, were collected from May to August in 1995 in the river Stör. The fish samples, that is, two brook trouts (Salmo trutta fario (L.)) and eight breams (Abramis brama orientalis BERG) were also caught in the river Stör from July to August 1995. In addition, three samples of the effluents of sewage plants of the cities of Hamburg, Itzehoe and Neumünster (Germany) were taken during this period. Furthermore, two samples of the effluents and from the influents of the sewage treatment plants in Neumünster and in Itzehoe were collected in the autumn of 1996. The sample preparation and the clean-up procedure is described in Bethan et al. (1996, 1997). The concentrations in the ten fish samples varied between 0.01 and 0.24 mg/kg (extractable organic material) and showed the same general characteristics in the decreasing trend towards the estuary as those received from the water samples. High concentrations were found in the trout that had been caught in Neumünster downstream of the influx of the sewage plant’s wastewater, while the fish samples collected at the estuary of the river showed low concentrations. But it has to be

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8 Enantiomer-Specific Fate and Behaviour of Chiral Contaminants

noted that the bromocyclen concentration in fish reflects a long-term accumulation, while the water samples represent the state during the sample collection. The enantiomeric ratios of bromocyclen for the extracts from two trout and eight bream samples from the river Stör are summarised in Table 8.12. In the muscle tissue of bream, the ER values turned out to be higher than 1 and varied between 1.08 and 2.13. The values imply that the (+)-enantiomer was preferentially degraded, that is, at least the (+)-enantiomer can be metabolised, or, alternatively, the ()-enantiomer was preferentially accumulated. This result appears to be at variance with the results from a previous investigation of rainbow trouts from a Danish fish farm that led to the contrary assumption (Pfaffenberger et al. 1994a, b). However, the two trout filet samples included in the study by Bethan et al. also showed enantiomeric ratios smaller than one. Therefore, it can be tentatively assumed that bream and trout are activating different metabolic pathways with regard to bromocyclen transformation. Moreover, a correlation between the enantiomeric ratios and the concentrations can be inferred from the data set of Bethan et al. (Fig. 8.18). Though their result is based on two farmed rainbow trout and eight bream samples only, it is evident that higher concentrations correlate with a higher relative concentration of the (+)-enantiomer. It is worth noting that nothing is known about the toxicity of the single enantiomers of bromocyclen even until today (20 years after the studies were conducted). After the above reported studies (in Germany), no further studies on the enantiomer-selective behaviour of bromocyclen is reported except a study on spiked trout samples (Fidalgo-Used et al. 2008). In this study, a new online solid-phase micro-extraction method (SPME) was developed for the determination of the enantiomer distribution

Fig. 8.18 Concentrations and enantiomeric ratios of bromocyclen in two trout (F1, F2) and eight bream sample (F3–F10) extracts

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159

with cGC on a Chirasil-DEX column. Beside this investigation, bromocyclen was included in various surveys and monitoring programs applying non-stereoselective quantitative analytical methods (Li et al. 2014; Hildmann et al. 2015; Perugini et al. 2018). Toxaphene® is a broad-range pesticide produced in the US, which was used primarily on cotton and soya bean in the south-eastern United States. Similar products were produced under the brandname Melipax® (Germany) and Polychloropinene® (former Soviet Union). For simplification reasons, in the following section, single compounds associated with these pesticide mixtures will only be called Toxaphene® congeners (de Geus et al. 1999, 2000). Toxaphene® use was banned by the US Environmental Protection Agency in 1986 because of its persistence and environmental toxicity. However, residues of technical toxaphene are still found in biota far removed from sites of its former application. Technical toxaphene is synthesised by the exhaustive chlorination of camphene, whereas the former Sowjet product Polychloropinene® used natural α-pinene as starting material (Shinova 1974; Trukhin et al. 2007), followed by a Wagner-Meerwein rearrangement taking place under the usual experimental conditions, thus giving rise to the formation of polychlorinated 1,7,7-trimethyl-bicyclo[2,2,1]heptanes (bornanes). Theoretically, 32767 chlorinated bornanes are conceivable, 16128 of which exist as enantiomeric pairs, while only 511 are achiral (Vetter and Schurig 1997). As a consequence, Toxaphene® (and other brands) is an extremely complex mixture of hexa- to decachlorinated bornanes, bornenes, camphenes and camphedienes. In technical mixtures, only selected congeners have been detected thus far (Fig. 8.19). The nomenclature of this class of compounds is however unsatisfactory, because the IUPAC names are very long and can hardly be used in a manuscript for scientific publications. Therefore, several suggestions for acronyms and alternative nomenclatures were made for more convenient applications, two of which seem to dominate, that is, the Palar numbers (Coelhan and Parlar 1996) and the systematic code names proposed by Andrews and Vetter (1995). These will also be used in the present monograph (Table 8.13). In addition, suggestions by Oehme and Kallenborn (1995), and by Nikiforov et al. (1995) are worth mentioning. As few of its numerous congeners have been synthesised thus far, toxaphene congeners can only be separated and analysed effectively with a highly selective analytical technique. In addition to the large number of congeners, the following problems have to be overcome: First, gas chromatographic enantiomer separation of toxaphene congeners requires relatively high temperatures, which turned out to be unfavourable to a good chromatographic performance (Coelhan and Parlar 1996; Vetter and Schurig 1997). Second, Buser and Müller (1994a, b) presented experimental evidence that non-racemic congeners may be present in technical mixtures of toxaphenes. At first, these reports caused some scepticism among the scientists. However, recently Vetter et al. were able to isolate the heptachlorobornane B7-1453 from the technical product Melipax (produced in the former German Democratic Republic), which showed different peak abundance of the enantiomers (ER ¼ 1.26  0.03). Furthermore, the authors excluded interferences and co-elution with another compound. Therefore, they conclude that B7-1453 and possibly other chlorobornanes are

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8 Enantiomer-Specific Fate and Behaviour of Chiral Contaminants

Fig. 8.19 Chemical structure of the chlorobornanes Parlar 62 and Parlar 50

present in a non-racemic composition in technical mixtures. An explanation is provided by the use of natural precursors (α-pinene, terpene and camphene) in the toxaphene synthesis, which may give rise to chiral induction in the course of the preparation pathway. Similar conclusions were drawn by a study on the Soviet pesticide mixture Polychloropinene® (Trukhin et al. 2007). As a consequence, caution has to be applied when interpreting enantiomeric excesses of toxaphene congeners determined in biota, because deviations from a racemic composition in biological samples may not only arise from enantioselective transformation processes. The first to report enantiomeric excesses of two major abundant chlorobornanes in biological samples were Kallenborn et al. (1994a, b), as well as Buser and Müller (1994a, b). In both studies, the same sample was used, that is, blubber of a harbour seal (Phoca vitulina L.), from which the major chlorobornanes were isolated. B9-1679 (Parlar50, Fig. 8.19) showed good agreement with ERs of 1.06 vs. 1.06 (Table 8.13), but B8-1413 (Parlar26) deviated a little with an ER of 1.02 vs. 1,12. In

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Table 8.13 Systematic code names and Parlar numbers of toxaphenes (chlorbornanes), as well as their enantiomeric ratios determined in extracts of biota tissues

Biota/tissue Harbour seal Techn. toxaphene mixture Harbour seal

Enantiomeric ratios B8B91413 1679 (Parlar (Parlar 26) 50) 1.02 1.06

1.12 0.04

Baltic herring Harp seal Antarctic penguin Herring (Baltic Sea)

0.95

Redfish (North Atlantic

1.10

Herring (North Sea) Mackerel (North Atlantic)

1.06

0.03 0.10

0.98 0.03

B82229 (Parlar 44) –

B71453

B81412

() –

–

1.13  0.02 1.08  0.04

–

–

–

–

–

–

0.98  0.08 1.19  0.03 1.34  0.02 1.08  0.03

–

–

–

–

–

–

–

–

–

–

–

–

Buser and Müller (1994a, b) Buser and Müller (1994a, b) Buser and Müller (1994a, b) Alder et al. (1996a, b)

1.08  0.07

–

–

–

Alder et al. (1996a, b)

1.08  0.03 1.08  0.03

–

–

–

Alder et al. (1996a, b)

–

–

–

Alder et al. (1996a, b)

1.11  0.03

–

–

–

Alder et al. (1996a, b)

Vetter et al. (1997a, b, c, d, e, f) Vetter et al. (1997a, b, c, d, e, f) Vetter et al. (1997a, b, c, d, e, f) Vetter et al. (1997a, b, c, d, e, f) Vetter et al. (1997a, b, c, d, e, f)

Mackerel (North Sea)

1.13

Weddell seal, male, adult Weddell seal, male, adult Leopard seal, unknown, ad. Baltic cod liver

1.2

1.3

2.6

–

–

1.1

1.3

2.8

–

–

1.3

1.6

4.3

–

–

1.0

–

1.26

–

Techn. toxaphene mixture Baltic cod liver

0.12

0.03 0.4 0.7 0.3 0.6

References Kallenborn et al. (1994a, b) Buser and Müller (1994a, b) Buser and Müller (1994a, b)

Klobes et al. (1995)

(continued)

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8 Enantiomer-Specific Fate and Behaviour of Chiral Contaminants

Table 8.13 (continued)

Biota/tissue

Enantiomeric ratios B8B91413 1679 (Parlar (Parlar 26) 50)

B82229 (Parlar 44)

B71453

B81412

()

References

Grey seal

0.5 0.4

Weddell seal

0.4

Technical mixture TC6 Antarctic penguin TC6 Technical mixture TC9 Baltic salmon TC9

1.04  0.03 0.91  0.02 1.00  0.03 1.4

Technical mixture TC1

1.02

Antarctic penguin TC1

0.74  0.03

0.02

Vetter et al. (1997a, b, c, d, e, f) Vetter et al. (1997a, b, c, d, e, f) Buser and Müller (1994a, b) Buser and Müller (1994a, b) Buser and Müller (1994a, b) Buser and Müller (1994a, b) Buser and Müller (1994a, b) Buser and Müller (1994a, b)

the latter case, baseline separation of the enantiomers was not achieved in both studies, which might explain the larger discrepancy of the results. Furthermore, Buser et al. investigated Baltic herring (Clupea harengus L.), harp seal (Phoca groenlandica ERX.) from Greenland and Adelie penguin (Pygoscelis adelis (L.)) from Antarctica. These samples were prepared at the Institute of Environmental Chemistry, University of Umeå, Sweden. It should be noted that the enantiomeric ratios for these biota sample extracts summarised in Table 8.13. have to be compared with the value for the technical toxaphene mixture. For example, for the congener B9-1679 (Parlar50) an ER of 1.13  0.02 was reported, that is, a value that slightly differs from a racemic mixture. Therefore, the ERs determined for the warm-blooded animals are less significantly larger than the value in the technical mixture, as usually to be expected from technical compounds. It is worth noting that the congener B9-1679 (Parlar50, see Fig. 8.19) is a major component in all aquatic biota samples, but only a minor component of the technical product. A very comprehensive survey was reported by Alder et al. (1996a, b) who analysed eight fish samples including six different species (Table 8.13). The enantiomeric ratios ranged between 0.91 and 1.13, that is, when taking into account the experimental error of the method, only small differences from racemic distributions were encountered, and no species-dependent transformation can be inferred from the data set. Vetter et al. (1997a, b, c, d, e, f, 1998a, b) analysed three Weddel seals (Leptonychotes weddellii LES.) and one Leopard seal (Hydrurga leptonyx BLA.)

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with regard to the enantiomeric distribution profile (ER) of B8-1413 (Parlar26), B9-1679 (Parlar50) and B8-2229 (Parlar44). For all animals, the second eluting enantiomer of B8-2229 was below 40% of the first eluting one (Table 8.13). Vetter et al. also isolated B7-1453 from a Baltic cod (Gadus morhua L.) liver extract and determined the ER with 1.0. This value has to be compared with the isolate from the technical Melipax® mixture, which exhibited an ER value of 1.26  0.03, as mentioned above. In this special case, that is, application of an enantiomer-enriched toxaphene congener, the alteration of the ER was owing to a faster transformation of the first eluting enantiomer, which finally led to an ER of 1.0. According to the authors’ conclusions, this spectacular result, enantioselective transformation leading to equal amounts of both enantiomers, is the result of biotransformation of enantiomers with different reaction speeds, which accidentally led to an ER of 1.0. The interpretation of this result would be erroneous, if synthetic (racemic) standards had been used as a reference instead of the B7-1453 isolate from Melipax. It cannot be excluded that a similar situation may be encountered in connection with other toxaphene congeners. Therefore, caution has to be applied, when interpreting enantiomeric ratios of toxaphene congeners determined from environmental samples. In a subsequent investigation, Vetter and co-workers confirmed the enantiomeric ratios published for the four toxaphene congeners B7-1453 (Vetter et al. 1998a, b), B8-1413 (Vetter et al. 1997a, b, c, d, e, f, B8-2229 (Vetter et al. 1997a, b, c, d, e, f) and B9-1679 (Vetter et al. 1997a, b, c, d, e, f) by carefully studying and excluding co-elution effects and other artefacts that may have been caused, for example, at least in the case of cGC/ECNI-MS application, as already discussed in Chap. 7 (Vetter and Luckas 1998). Klobes et al. succeeded in separating the enantiomers of toxaphene congener B8-1412, a major component in biota, using a 30-m long column coated with 35% heptakis(6-O-tertbutyldimethylsilyl-2,3-di-O-methyl)-β-cyclodextrin diluted in OV1701 (Klobes et al. 1998a, b, c). In all Atlantic cod liver samples from different areas of the Baltic Sea, enantiomeric ratios of B8-1412 were significantly < 1 (Table 8.13). Both in blubber extracts from a grey seal grypus (FABR.) from Iceland and in blubber extracts from a Weddell seal (Leptonychotes Weddeli LES.), an ER of 0.4 was found. Although the number of samples investigated thus far is low, the results obtained by Klobes et al. clearly indicate an enantioenriched B8-1412 in all samples. A Norwegian-Russian study reported the successful isolation and synthesis of a major enantiomerically pure Toxaphene®-related compound from the former soviet pesticide Polychloropinene® (Hansen et al. 2004). This enantiomerically pure potential congener (C10H9Cl9) has been isolated from a reaction mixture obtained by the free radical chlorination of (1S)-2-endo-chlorobornane and its absolute configuration determined after crystallisation with X-ray crystallography. It crystallises in the monoclinic space group C2 with two molecules in the asymmetric unit. This was the first report of the preparation of a single enantiomer of a synthetic polychloroterpene on a multi-milligram scale (Hansen et al. 2004). As earlier reported, Garrison and co-workers were not able to verify any enantioselectivity for the transformation of o,p-DDT to o,p0 -DDD using the Elodea-water reaction medium (Garrison et al. 1997). By way of contrast,

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enantioselectivity was proven by these authors for the occurrence of o,p0 -DDD in fish tissue (Garrison et al. 1997). Extracts from 21 fishes captured from river waters where the sediment has a history of severe DDT contamination from a long-defunct pesticide manufacturing plant were analysed by enantioselective cGC and CE, respectively, for o,p0 -DDD. The fish species were channel catfish (Ictalurus punctatus (RAF.)), buffalo (Ictiobus cyprinellus (VAL.)) and largemouth bass (Micropterus salmoides (NM)). As Garrison et al. used a modified γ-phase as CSP, they assumed that the ()-enantiomer eluted first, in line with suggestions made by earlier investigations (Buser and Müller 1995) who separated these enantiomers on a γ-cyclodextrin-based HPLC column. Among the 21 samples, only two showed positive ER values (both values were 1.10). The range of ratios of the 19 samples with a negative ER was 0.25 to 0.98, but 14 of these fell between 0.40 and 0.79. Apparently, the biological uptake mechanism or some membrane transport process, or both, favoured the ()-enantiomer, or else the (+)-enantiomer was metabolised faster by the fish. However, there was no correlation between fish species and direction or degree of enantioselectivity. Basically, it cannot be excluded that the o,p-DDD was partially degraded by an enantioselective process in the environment, probably in the sediment. Garrison and co-workers later verified this hypothesis by additional analyses of all DDT-related derivatives including all major transformation products in fish, water and sediment samples. The here-derived quantitative results were comprehensively discussed with respect to transformation and distribution pathways for all target DDT-derivatives at the here-investigated contaminated location (Garrison et al. 2014). All major transformation products including DDAs were still found in milligram levels in sediment and water samples. For the chiral o,p0 -DDD, enantiomeric fractions (EF) < 0,5 (determined in fish, sediment and water) were determined confirming similar transformation mainly of the (+)- enantiomer in all trophic levels of the local food web. Obviously, this EF did not change significantly over the 15 years since the last survey was conducted (Garrison et al. 1997).

8.2.1.3

Non-Chlorinated Pesticides

All the organochlorine pesticides were banned in 1970s and 1980s from their agricultural application due to their considerable hazardous effect (Carson 1964). New products need to be developed in order to satisfy the growing demand on agricultural products on a global scale. Many new non-chlorinated pesticides and other agricultural aids were developed and marketed by the industry for agricultural use (Phipps et al. 1986; Becker 1997; Wheeler 2002; Lee and Aizawa 2003; Sideris and Moore 2008; Hester and Harrison 2012). In 2011, more than 30% of the worldwide used pesticides were chiral organic chemicals (Garrison et al. 2011a, b). However, only 7% of them are currently marketed enantiomerically enriched or even in their pure enantiomeric form. In a comprehensive review, Ulrich et al. reviewed 1693 chiral pesticides currently on the marked for agricultural applications published as global assessment of the US Environmental Protection Agency (Ulrich et al. 2012). This number has only

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marginally changed since this report was published. According to Jeschke (Jeschke 2018), however, only few enantiomerically pure products are produced on an industrial scale (>100 t annual production). Since 2007 ca. 43% of the 44 products launched were chiral and ca. 47% of those were produced in the racemic form. Nevertheless, this quite overwhelming number of chiral agrochemicals potentially applied in unknown quantities on agricultural soils today presents the most important environmental platform (in addition to the growing concern on chiral pharmaceutic residues) for method development and risk assessments in modern enantiomer-selective environmental chemistry. Also here, as already demonstrated for other contaminant classes, the classical single compound approach for environmental assessments is expected to meet its limits when it comes to comprehensive environmental risk and exposure assessments (Meyer 2003; Albuquerque et al. 2016; Kim Tiam et al. 2016; Aamir et al. 2017; Panizzi et al. 2017; de Albuquerque et al. 2018). Toxicological aspects and exposure as well as the role of enantiomer-selective analysis associated with new evaluation approaches will be discussed in detail in Chap. 10. However, the importance of chirality and enantiomer-selective analytical methods for the determination of enantiomeric profiles is stressed as an essential feature for the assessment of bioavailability and transformation pathways in target suspect and even-non-target strategies today (Meng et al. 2009; Ye et al. 2010; Zhou et al. 2014a, b). Based upon similar considerations, Garrison (2006) argued that the sole application of enantiomeric pure pesticides products would consequently lead to more effective targetfocussed strategies in the fight against pests in modern agriculture. Nevertheless, taking into account the above reported overwhelming number of chiral modern pesticides released into the environment via agricultural applications, it is not possible to give a comprehensive report and appreciation within the limits of this book. For details on the product characterisation and potential environmental toxicological consequences, we wish to direct the interested reader to already available scientific literature on this topic (Nillos et al. 2010; Garrison et al. 2011a, b; Ulrich et al. 2012; Ye et al. 2015; de Albuquerque et al. 2018; Jeschke 2018; Zhao et al. 2018a, b, c). Here, however, we will present selected relevant examples from the current literature in order to demonstrate the levels and consequences of unintended pesticide release into the aqueous environment. Already in 2006, Wong reported on the presence of several contaminants of emerging concern in a general review (Wong 2006). Beside the classical chlorinated substances, the emphasis was placed on modern pesticides including pyrethroid- and phosphorus-containing pesticides. A list of 20 modern pesticides was presented for which the enantiomeric profile was determined mainly in soil and aquatic samples. The already earlier discussed phenoxyalkanoic acids (e.g. dichlorprop and mecoprop) were identified as the most prevailing current used pesticides in this survey. Other groups like acetamides, organophosphorus pesticides and pyrethroids were also found to be relevant environmental pollutants. A new multi-compound method for the enantiomer-selective determination of modern pesticides was developed by Ye et al. (2009). This research group developed an optimised method for the enantiomer separation of synthetic pyrethroids,

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organophosphate pesticides, acylanilides, imidazolinones, phenoxypropanoic acid herbicides and triazoles with HPLC separation techniques. In recent years, the list of available methods for the trace level determination of pesticide enantiomers continued to grow in line with the continuous development of the available analytical methods. The enantiomer-selective transformation and up-take of the triazole fungicide hexaconazole was studied in vegetables and agricultural soil. These results were presented in a recent report (Li et al. 2013). A new enantiomer-selective HPLC separation method with tandem mass spectrometry (LC-MS/MS) was developed and validated for measuring hexaconazole enantiomers in tomato, cucumber and soil. The enantiomer separation was performed on an RP-phase Chiralcel OD-RH column (CSP see above), under isocratic conditions (mobile phase: acetonitrile-2 mM ammonium acetate in water (60/40, v/v), flow rate of 0.4 mL/min.). This analytical method was used for the investigation of the enantiomeric signature of rachexaconazole in vegetables and soil. The enantiomer-selective transformation of the two hexaconazole enantiomers was confirmed but the velocity and kinetics seem matrix dependent: The (+)-enantiomer showed a faster transformation in plants, while the ()-enantiomer dissipated faster in field soil, resulting in relative enrichment of the opposite enantiomer. Today, the enantiomer specific multi-compound analysis is the favourable attempt to cope with ever increasing potential contaminant numbers. A Chinese study recently reported the simultaneous enantiomer-selective determination of 18 currently used chiral pesticides (CUPs) both in solid and liquid environmental samples (Zhao et al. 2018a, b, c). In this study modified carbon nanotube amended enantioselective liquid chromatography was applied to reach the required chromatographic resolution. For the environmental measurements, samples were collected in Shenyang (Liaoning province, PRC). River water and sediment from the Hunhe River were taken for enantiomer-selective analysis. The analysis was performed on UHPLC/QqQ in positive and negative ESI, simultaneously. The commercially available ChiralPak IG CSP (separation condition: acetonitrile/water in 5-mM ammonium ethanoate and 0.05% methanoic acid) was used for the simultaneous enantiomer-selective separation (producer Chiral Technology, West Chester, PA). Most of the target compounds were identified in the (waste-) water samples analysed with trans-2R,4R-(+)-defenoconazole in the highest levels (46 ng/L) in influent wastewater. In river water, however, paclobutrazol E1 (stereo-specific structure unknown yet) was found in the highest concentrations (9 ng/L). Ulrich et al. examined the enantiomer distribution of five CUPs in aquatic systems (Ulrich et al. 2018). The here-performed survey was a part of an ongoing US-EPA monitoring. Previously optimised enantiomer-selective methods were applied to the enantiomer determination of five chiral CUPs (fipronil, cis-bifenthrin, cis- and trans-permethrin, cypermethrin and cyfluthrin) in 90 aqueous samples. Enantiomer fractions were found for fipronil, bifenthrin and cis-permethrin target compounds. However, 98% of fipronil, 82% of bifenthrin and 43% of cis-permethrin EF measured (n ¼ 75) were racemic indicating minor biotransformation potential.

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8.2.1.4

167

Chiral Industrial Chemicals

Polychlorinated biphenyls (PCBs), highly persistent lipophilic industrial chemical compounds, have been in focus as a major and ubiquitous environmental contaminant for more than five decades already. Basically, 209 structurally and chemically related congeners are conceivable, representing a wide range of physicochemical properties. PCBs have been used as industrial fluids, flame retardants, diluents, hydraulic fluids and dielectric fluids for capacitors and transformers (Lang 1992; Swanson et al. 1995; Carpenter 1998; Zabik and Zabik 1999). An extensive contamination with PCBs has occurred during the period of their industrial use, from the early 1930s until the 1980s (Wania and Su 2004; Shi et al. 2016). Although PCBs were banned from industrial applications since the early 1980s, they are still entering the environment due to emissions from decommissioned installations and waste sites. Primary sources are leakages from old so-called closed systems, such as capacitors and transformers, and the disposal of materials contaminated with PCBs, such as old paints, painted construction materials, lubricant oils, sealing material and fire retardants in old fire extinguishers. Today, scientific estimations conclude that around 40% of the totally used PCBs is still in use in old electrical devices, paints and etc. Furthermore, a number of secondary sources of PCBs can also be identified including resuspended river sediments, leakages from dump sites, dumping of sewage sludges and long-range atmospheric transport (Wania and Su 2004; Shi et al. 2016). Jensen and co-workers were the first to report PCBs extracts of Swedish whitetailed sea eagles (Haliaeetus albicilla) in the late 1960s (Jensen 1966), and subsequent analytical studies have demonstrated the presence of PCBs in almost every compartment of the global ecosystem including the air, water, sediments, fish, wildlife and humans. In particular, in the marine environment PCBs were not only encountered in seawater, but also in species of different trophic levels of the marine ecosystem (Safe 1984; Hühnerfuss and Kallenborn 1992; Lang 1992; Kamrin and Ringer 1994; Ritter et al. 2002; Beyer and Biziuk 2009; Su et al. 2013; Gioia et al. 2014; Fang et al. 2015; Kaw and Kannan 2017; Carlsson et al. 2018). Analytical methods for the determination of PCBs have improved in recent years, thus permitting the determination of all 209 congeners of technical PCB mixtures in environmental samples (Matsumoto et al. 2014; Kraft et al. 2017). In the last few years, increasing attention has been paid to the analysis of coplanar and atropisomeric congeners. The toxicological implications related to the structures of these two groups will be discussed further in Chap. 10. Herein, emphasis will be placed upon the analysis of chiral atropisomeric PCBs in environmental samples. Basically, 78 out of 209 PCB congeners display axial chirality in their nonplanar conformations, the so-called atropisomerism. In a pioneering account, Kaiser predicted that 19 PCBs, nearly all of which are present in commercially available technical PCB formulations, exist as stable atropisomers at ambient temperatures due to restricted rotation around the C-C bond of the biphenyl system (G* ¼

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104–242 kJ/mol; 25–58 kcal/mol) with “a bearing on the toxicity and metabolic interactions of these chemicals” (Kaiser 1974, Harju and Haglund 1999). The first cGC separations of atropisomeric PCBs were published in 1993 and 1994 (König et al. 1993; Schurig and Glausch 1993; Glausch et al. 1994; Hardt et al. 1994; Wong and Garrison 2000), and in the years 1994 and 1995 the first reports on the enantioselective determination of an atropisomeric PCB in biological samples appeared (Hühnerfuss et al. 1994, 1995; Wong et al. 2001a, b; Ross et al. 2011a, b; Megson et al. 2015). Meanwhile, several enantiopure or enantio-enriched atropisomeric PCBs have been isolated by application of enantioselective HPLC and can thus be used as standard compounds (Haglund 1995, 1996a, b; Schurig et al. 1995), for example, for the determination of the elution order of PCB atropisomers in enantioselective cGC (Wong et al. 2001a, b, 2007). With regard to enantioselective analysis of atropisomeric PCBs in marine and limnic biota tissues, only few studies were reported in the literature during the past decades. In 1994, Hühnerfuss et al. (Hardt et al. 1994; Hühnerfuss et al. 1994) reported for the first time about the identification of five atropisomeric PCBs in blue mussels (Mytilus edulis L.) on an achiral CP-Sil 5/C 18 CB fused-silica capillary column (Chrompack; length 100 m). Higher levels of PCB88, PCB149, PCB174 and PCB183 were determined in spring as compared to autumn at six sampling sites in the Weser, Jade and Elbe river estuaries (German Bight). Furthermore, the enantiomers of PCB149 in all mussels collected during the spring and autumn period were separated. The enantiomeric ratios ranged between 1.0 and 1.2, which implies a weak enantio-enrichment of the first eluting enantiomer. Blanch et al. (1996) also used enantioselective cGC for the determination of three atropisomeric PCBs in liver samples of shark (Centroscymnus coelolepis, B. & C.). While PCB95 and PCB149 were present in racemic compositions, the second eluting enantiomer of PCB132, that is, (+)-PCB132, predominated in most of the samples (ER ¼ 0.75–0.89). Ramos et al. (1996) analysed nine atropisomeric PCBs in two other samples. The ERs were in part extremely high, although the report is lacking a detailed experimental description. Enantioenrichment of PCB149 in blubber of an adult female harbour seal (Phoca vitulina L.) sample from Iceland was determined by Vetter et al. (1997a, b, c, d, e, f). They report that the first eluted peak was significantly higher than the second one. In a later study, enantioenriched PCB149 was also found in blubber of further harbour seals, as well as grey seals (Halichoerus grypus (FABR.)), and a Caspian seal (Phoca caspica L.) (Vetter et al. 1997a, b, c, d, e, f). Reich et al. (1998) determined enantiomeric ratios of atropisomeric PCBs in Mediterranean striped dolphins (Stenella coeruleoalba (ME.)). The six dolphins were found dead along the Italian coast (Lygurian and Tirrhenian seas) in the period 1989–1990. The enantiomeric ratios (defined as relation of the first eluting enantiomer to the second one) of the nine atropisomeric PCBs (84, 91, 95, 132, 135, 136, 149, 174, 176) obtained in the samples studied revealed that PCB95 (ER ¼ 0.71–1.07) PCB136 (ER ¼ 0.84–1.07), PCB174 (ER ¼ 0.58–1.16) and PCB176 (ER ¼ 0.72–0.97) were racemic or nearly racemic in almost all samples. PCB95 exhibited an enantiomeric excess (ee) of 17% of the second eluting atropisomer in two liver samples, and PCB174 showed an ee of 26.6% in one liver sample. PCB132

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(ER ¼ 0.55–0.92), PCB135 (ER ¼ 0.63–0.76) and PCB149 (ER ¼ 0.58–0.91) revealed an ee of the second eluting enantiomer in almost all sample extracts. No ee was found for PCB132 and PCB149 in one sample each. The differences observed in the enantiomeric ratios of the atropisomeric PCBs could not be explained by the relationship between structure and metabolism. PCB95, PCB132, PCB135, PCB136, PCB149, PCB174 and PCB176 belong to the readily metabolised PCBs. They possess vicinal hydrogen atoms in both ortho-/meta- and meta-/para-positions (PCB132), in two meta-/para-positions (PCB95, PCB136) or in one meta-/paraposition (PCB135, PCB149, PCB174, PCB176). It is, therefore, not possible, on the basis of its structure, to explain why PCB95 (with two vicinal H atoms in meta-/ para-positions) only shows slight enantiomeric enrichment, while PCB149 (with only one free meta-/para-position) exhibited higher enantiomeric enrichment. Thus, Reich et al. conclude that the differences found in the metabolic transformation pathway between the two atropisomers of these PCBs could be better explained by the enantioselective character of the enzymatic biotransformation process. Wong et al. (2002) provided a first overview of enantiomer signatures of chiralchlorinated organic pollutants in certified reference materials. In their survey, they covered the most important chiral organochlorine pesticides, as well as atropisomeric polychlorinated biphenyls. In their study, the authors investigated the enantiomeric profiles of cis- and trans-chlordane, heptachlor exo-epoxide, oxychlordane, U82, MC5, MC6, MC7, o,p'-DDT, as well as the atropisomeric PCB congeners 91, 95, 136, 149, 174, 176 and 183. The enantiomeric fractions (EF) of the respective compounds were determined in SRM 1588a (organics in cod liver oil), SRM 1945 (organics in whale blubber), Marine Mammal Quality Assurance Exercise Control Material IV (NIST IV, organics in whale blubber), CRM trout and CRM EC-5 (sediment). For EC-5 (sediment), mainly racemic EFs for OCP and atropisomeric PCBs were determined. In contrast, SRM1588 (cod liver oil) was characterised by non-racemic EFs, especially for PCBs. “CRM trout” also showed mainly non-racemic EFs. The enantiomer residues in the two pilot whale reference materials (SRM 1945 and NIST IV) were racemic for many target analytes. This feature is mainly explained by a combination of metabolisation and non-chiral accumulation in cetaceans. This first comprehensive survey extended the value of the here-investigated certified materials and should be followed up by a proper robin round laboratory intercomparison. The enantiomeric profile was determined in a study on 11 harbour porpoise livers (Phocoena phocoena) found dead in the southern North Sea (Chu et al. 2003a, b). The concentration levels and enantiomeric ratios (ER) for polychlorinated biphenyl (PCB) atropisomers PCB 95, PCB 149 and PCB 132, were measured. Non-racemic enantiomeric ratios (ERs) were found in several individuals. The value of ERs in three of the four juvenile porpoises was close to racemic. However, the ERs in all adults differed from racemic and ranged from 1.31 to 2.54 for PCB 95; from 1.19 to 1.81 for PCB 149 and from 0.45 to 0.94 for PCB 132. There were no relationships between the total concentration of PCBs and ERs found in this investigation. A

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kinetic model was developed for the elimination and uptake processes of PCBs in the porpoises. In the model, a clear trend between the enantiomeric ratios and the ratio between PCB 153 and PCB 101 was confirmed. Due to the fact that PCB 153 is one of the most persistent PCB congeners in marine mammals and PCB 101 considered to be easily metabolised, this tendency confirms that the enantiomeric ratio most likely reflects the proportion of the metabolised congener. The authors concluded (Chu et al. 2003a, b) that the determination of enantiomeric profiles in wildlife, combined with information on their anthropometric data, health status, diet and habitat conditions, might be good indicators of pollution status in pristine marine environments. A study on a marine Arctic ecosystem, the biosphere of the North Water Polynya (NOW), was reported by a Canadian research group (Warner et al. 2005). The enantiomer distribution of atropisomeric PCBs was examined in a typical Arctic marine food web. Typical members, representing different trophic levels were pelagic zooplankton, Arctic cod (Boreogadus saida), seabirds and ringed seals (Pusa hispida). Along with previously reported studies on similar species, this investigation confirmed biomagnification in the NOW food web. The heredetermined, highly non-racemic enantiomeric fractions (EFs) in l seabird species and ringed seals indicated biotransformation and selective bioaccumulation of atropisomeric PCBs. However, racemic EFs were found in their prey (zooplankton and fish), which, in turn, indicates, that biotransformation in the top predators are the main reason for the non-racemic EFs determined. The here-presented results are consistent with previously reported biotransformation activity for chiral organochlorine pesticides in these species and demonstrate the versatility of chiral analysis of PCBs for the assessment of biotransformation within Arctic food webs.

8.2.2

Terrestrial Ecosystems

In the initial phase of research on the behaviour of chiral pollutants in the environment, enantioselective analyses in terrestrial ecosystems largely focused on the questions: Can the conclusions drawn from marine biota analyses with regard to enantioselective transformation of xenobiotics be transferred to terrestrial animals? Can species-dependent and/or concentration-dependent effects be observed? Early investigations addressed these scientific questions were Möller et al. (1993) determined the enrichment of α-HCH enantiomers as determined in fat, liver and brain tissue samples of sheep (Ovis ammon L.) bred in the northern German state of Schleswig-Holstein (Table 8.14; Fig. 8.20). In fat and liver, a depletion of the (+)enantiomer was observed, while in brain vice versa the (+)-enantiomer is dominating. These values were compared with those from marine biota like blue mussels (Mytilus edulis L.; ER between 0.67 and 0.89), flounder (Platychthys flesus L.; ER 0.80–0.94), Common Eider duck (Somateria mollissima (L.); liver: ER ¼ 1.4–1;

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Table 8.14 Comparison of the residue contents and enantiomeric ratios of α-HCH in sheep liver, fat and brain samples from Schleswig Holstein (Möller 1993; Möller et al. 1993) Sample no. 1 2 3 4 5 6 7

Liver Conc. (μg/ g EOM) 0.010 0.006 0.007 0.011 0.015 0.013 0.012

ER (+)/(–) 0.96 0.69 0.89 0.75 0.85 0.75 0.79

Fat Conc. (μg/g EOM) 0.008 0.004 0.005 0.013 0.012 0.013 0.009

ER (+)/(–) 0.85 0.64 0.70 0.56 0.96 0.64 0.82

Brain Conc. (μg/ g EOM)