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Analytical methods for agricultural contaminants
 9780128159408, 0128159405

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ANALYTICAL METHODS FOR AGRICULTURAL CONTAMINANTS

ANALYTICAL METHODS FOR AGRICULTURAL CONTAMINANTS Edited by

BRITT MAESTRONI Food and Environmental Protection Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria

VICTORIA OCHOA Food and Environmental Protection Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria

ANDREW CANNAVAN Food and Environmental Protection Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright r 2019 International Atomic Energy Agency. Published by Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-815940-8 For Information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Charlotte Cockle Senior Acquisition Editor: Patricia Osborn Editorial Project Manager: Karen R. Miller Production Project Manager: Bharatwaj Varatharajan Cover Designer: Mark Rogers Typeset by MPS Limited, Chennai, India

DEDICATION To the memory of David H. Byron

DISCLAIMER This book is distributed without warranties of any kind. Mention of trade names or commercial products does not constitute endorsement or recommendation by the Food and Agriculture Organization of the United Nations, the International Atomic Energy Agency, or the RALACA network of participating laboratories.

LIST OF CONTRIBUTORS Mara M. de Andre´a Laboratorio de Ecologia de Agroquı´micos, Instituto Biolo´gico, Sa˜o Paulo, Brazil Edwin Samir Barbosa Angel Laboratorio nacional de Insumos Agricolas Lania (LANIA), Mosquera, Colombia Fabiano Barreto Laborato´rio Nacional Agropecua´rio (LANAGRO), Porto Alegre, Brazil Beatriz Brena Evaluation and Environmental Quality Control Service, Montevideo, Uruguay Andrew Cannavan Food and Environmental Protection Laboratory (FEPL), Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria Elizabeth Carazo Centro de Investigacio´n en Contaminacio´n Ambiental (CICA), Universidad de Costa Rica, San Jose´, Costa Rica ˇ Eduardo Egan˜a Cerni Laboratorio de Bromatologia, Montevideo, Uruguay Pedro Enriquez Alfaro Servicio Agrı´cola y Ganadero de Chile (SAG), Laboratorio Quı´mica Ambiental y Alimentaria, Pudahuel, Santiago, Chile Marı´a Vero´nica Cesio Facultad de Quı´mica, Universidad de la Repu´blica (UdelaR), Montevideo, Uruguay Jairo Arturo Guerrero Dallos Laboratory of Pesticide Residue Analysis (LARP), Ciudad Universitaria, Bogota, Colombia Heitor Daguer Ministe´rio da Agricultura, Pecua´ria e Abastecimento (LANAGRO), Sa˜o Jose´, Brazil Susana Franchi Departamento Laboratorio Quı´mico, Seccio´n Residuos de Plaguicidas. Ministerio de Ganaderı´a Agricultura y Pesca (MGAP), Direccio´n General de Servicios Agrı´colas, Montevideo, Uruguay Patricia Gatti Chromatography Laboratory, Technological Research Centre of the Dairy Industry, National Institute of Industrial Technology (INTI), San Martı´n, Argentina Horacio Heinzen Facultad de Quı´mica, Universidad de la Repu´blica (UdelaR), Montevideo, Uruguay

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List of Contributors

Julio Ernesto Payes Herna´ndez Centro de Investigaciones y Aplicaciones Nucleares (CIAN), Engineering and Architecture Faculty, Ciudad Universitaria, Mexico, Mexico Bert Kohlmann Universidad EARTH, San Jose´, Costa Rica Ruth Miriam Loewy Laboratory of Chromatography, UNCo- IDEPA- CONICET, Neuque´n, Argentina Luiz Carlos Luchini Laboratorio de Ecologia de Agroquı´micos, Instituto Biolo´gico, Sa˜o Paulo, Brazil Pablo Macchi Universidad Nacional de Rı´o Negro, Rı´o Negro, Argentina Britt Maestroni Food and Environmental Protection Laboratory (FEPL), Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria Mario Alberto Ması´s Mora Laboratorio de Ana´lisis de Residuos de Plaguicidas, Centro de Investigacio´n en Contaminacio´n Ambiental (CICA), Universidad de Costa Rica, San Jose´, Costa Rica Paula Aguilar Mora Laboratorio de Metabolismo y Degradacio´n de Contaminantes, Centro de Investigacio´n en Contaminacio´n Ambiental (CICA), Universidad de Costa Ric, San Jose´, Costa Rica Adriana Nario Laboratory of Soil Analysis, Agriculture Section, Chilean Nuclear Energy Commission (CCHEN), Las Condes, Santiago, Chile Victoria Ochoa Food and Environmental Protection Laboratory (FEPL), Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria Patricia Ohaco Instituto Nacional de Tecnologı´a Industrial (INTI), San Martin-Buenos Aires, Argentina Rodrigo Palma Servicio Agrı´cola y Ganadero (SAG), Temuco, Chile Juan Salvador Chin Pampillo Laboratorio de Metabolismo y Degradacio´n de Contaminantes, Centro de Investigacio´n en Contaminacio´n Ambiental (CICA), Universidad de Costa Rica, San Jose´, Costa Rica Patricia Pappolla Chromatography Laboratory, Technological Research Centre of the Dairy Industry, National Institute of Industrial Technology (INTI), San Martı´n, Argentina

List of Contributors

Ana Maria Parada Laboratory of Soil Analysis, Agriculture Section, Chilean Nuclear Energy Commission (CCHEN), Las Condes, Santiago, Chile Lucia Pareja Grupo de Ana´lisis de Contaminantes Trazas (GACT), Polo Agroalimentario y Agroindustrial de Paysandu´, Centro Universitario de Paysandu´, Paysandu´, Uruguay Andres Perez Evaluation and Environmental Quality Control Service, Montevideo, Uruguay M. Alejandra Rodrı´guez Chromatography Laboratory, Technological Research Centre of the Dairy Industry, National Institute of Industrial Technology (INTI), San Martı´n, Argentina Mauricio Rodriguez Evaluation and Environmental Quality Control Service, Montevideo, Uruguay G. Jacqueline Rojas Servicio Agrı´cola y Ganadero de Chile (SAG), Laboratorio Quı´mica Ambiental y Alimentaria, Pudahuel, Santiago, Chile Pablo Sa´nchez Chromatography Laboratory, Technological Research Centre of the Dairy Industry, National Institute of Industrial Technology (INTI), San Martı´n, Argentina Ximena Videla Laboratory of Soil Analysis, Agriculture Section, Chilean Nuclear Energy Commission (CCHEN), Las Condes, Santiago, Chile Eliane Vieira Laboratorio de Ecologia de Agroquı´micos, Instituto Biolo´gico, Sa˜o Paulo, Brazil

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GLOSSARY OF TERMS Analyte The component of interest in a sample. Analytical portion A well-homogenized subsample of the test sample which has to be analyzed. Confirmatory method A method that provides complimentary information in agreement with a previous result. Internal standard A chemical compound added to the sample extract in a known quantity just prior to analysis to compensate for matrix effects during instrumental analysis. The internal standard needs to be similar to the analyte of interest. Limit of detection The lowest concentration of analyte in a sample that can be detected but not necessarily quantified. Limit of determination The validated lowest residue concentration that can be quantified with a validated method. Limit of quantitation/quantification (LOQ) The minimum injected amount that produces quantitative measurements in the target matrix with acceptable accuracy and precision. Matrix The material or component sampled. Matrix blank Sample material containing no detectable concentration of the analyte(s) of interest. Matrix-matched standards Standard solutions prepared in a matrix extract similar to that of the sample to be analyzed which compensate for matrix effects and acceptable interference, if present. Multiresidue method (MRM) Analytical method that measures a number of analytes simultaneously. Quantitative method A method capable of producing analyte concentration results that comply with established criteria (precision, accuracy, etc.). Range The interval between the upper and lower levels that have been demonstrated to be determined with precision, accuracy, and linearity using the method as written. Reference material A matrix characterized with respect to its analyte content. Repeatability The closeness of agreement between results of measurements on identical test material subject to the following conditions: same analyst, same instrumentation, same location, same conditions of use, repetition over a short period of time. Reproducibility The closeness of agreement between results of measurements on identical test material where individual measurements are carried under changing conditions such as: analyst, instrumentation, location, conditions of use, time. Screening method A method that meets predetermined criteria to detect the presence of an analyte or class of analyte at or above the minimum concentration of interest. Selectivity The extent to which a method can be used to determine particular analytes in mixtures or matrices without interferences from other components of similar behavior. Sensitivity The capability of a method to discriminate small differences in concentration or mass of the test analyte. In practical terms, sensitivity is the slope of the calibration curve that is obtained by plotting the response against the analyte concentration or mass.

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Specificity The ability of the detector to provide signals that effectively identify the analyte. Surrogate standard A chemical compound added in a known quantity to the sample analytical portion or to another stage of the analysis to check the correct execution of the analytical procedure (or part of it). The surrogate standard needs to be similar to the analyte of interest.

CHAPTER 1

Introduction to the Book of Methods Britt Maestroni, Victoria Ochoa and Andrew Cannavan Food and Environmental Protection Laboratory (FEPL), Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria

The book of analytical methods for the control of agricultural residues and contaminants is an initiative of the “Red Analitica de Latino America y el Caribe” (RALACA) network operated under the technical coordination of the Food and Environmental Protection Laboratory of the Joint Food and Agriculture Organization and International Atomic Energy Agency (FAO/ IAEA) Division on Nuclear Techniques in Food and Agriculture. To protect the health of consumers, facilitate trade, and ensure a sustainable environment, it is essential to strengthen the capabilities of analytical laboratories to generate reliable, accurate, and reproducible scientific data for monitoring food contaminants and residues. The IAEA, through the Joint FAO/IAEA Division on Nuclear Techniques in Food and Agriculture, assists Member States in applying nuclear, nuclearrelated, and complimentary techniques to produce more, better, and safer food and agricultural products, while sustaining natural resources. Ensuring the safety and quality of food and agricultural commodities has the twin impacts of protecting consumers and helping to facilitate international trade. In the field of analytical techniques and food control mechanisms, the Joint FAO/IAEA Division supports countries in using nuclear-related and complementary technologies to detect and monitor chemical residues and contaminants in food and the environment, thereby assisting in the adoption and implementation of Codex Alimentarius Standards in support of international trade. Nuclear techniques can be used to advantage in a number of ways in food safety and environmental protection applications. Radiolabeled compounds can be used as radiotracers to optimize sample preparation, extraction, clean-up, and other steps during the development of analytical methods that are used in regulatory programmes for the analysis of Analytical Methods for Agricultural Contaminants. Copyright © 2019 International Atomic Energy Agency. DOI: https://doi.org/10.1016/B978-0-12-815940-8.00001-6 Published by Elsevier Inc. All rights reserved.

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residues of pesticides and other contaminants in food and environmental samples. Radiolabeled compounds can assist in improving method performance characteristics and the estimation of uncertainty associated with the method. Radiotracers are also invaluable tools for studies on pesticide transfer in the environment and translocation in plants, and for characterizing the behavior of pesticides in various environmental compartments, such as their binding characteristics in soil. Stable isotope-labeled compounds can be used as internal standards to improve the precision and accuracy of analytical methods using mass spectrometry, to meet stringent international or national standards and trading requirements. The real power of nuclear and related techniques in this field is in their application in a package along with complementary, nonnuclear techniques, to provide solutions to complex analytical problems and reliable data to inform decision making in a policy or regulatory context. The Joint Division has been supporting institutions in Latin America and the Caribbean for many years through training workshops, technical cooperation projects, and coordinated research projects. All of these activities had, as a main objective, the goal of strengthening the quality assurance systems of the participating laboratories and of providing exemplar methods and standard operating procedures that could be applied in the laboratory. As a result, several multiresidue analytical methods were developed, validated, and shared among laboratories and related institutions. In most cases, general methods were modified and adapted to the needs of each individual laboratory to meet local challenges and their requirements in terms of analytical scope and the availability of reagents and equipment/instrumentation. For example, the original QuEChERS (quick, easy, cheap, effective, rugged, and safe) method developed for the analysis of multiple pesticide residues in fruits, vegetables, cereals. and processed products, makes use of acetonitrile as the extraction solvent. To enable the application of the method to a simple gas chromatograph coupled to electron capture detector or nitrogen phosphorus detector (GC with ECD/NPD) the original method was modified to use ethylacetate for the extraction of samples. Through these activities, staff and laboratory capabilities were enhanced in several areas, including analytical work, radioisotope techniques, mass spectrometry, data interpretation, sampling procedures, biomonitoring, investigating and understanding pesticide transport processes, catchment modeling, and in feeding back laboratory results to stakeholders while supporting farming communities. Each RALACA laboratory has validated

Introduction to the Book of Methods

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analytical methods and contributed to the joint work between the FAO/ IAEA and RALACA. This manual provides laboratory personnel with general guidelines on specifications and requirements for analytical methods for residues of pesticides, veterinary drugs, and other chemicals in food and the environment. The methods are intended to provide a harmonized baseline analytical capability, upon which more sophisticated methods can be developed to address the ever-growing list of analytes of concern to meet national priorities. The manual comprises methods varying greatly in terms of their objective, scope, sample treatment, and detection system. For example, guidelines cover the requirements for methods to detect residues and contaminants in food, soil, and water. Also covered are general methods for testing the uncertainty of analytical steps, standard procedures for calibration, for analysis of method validation data for testing water quality, and for measuring bioaccumulation of pollutants and the effect of pesticides, drugs, and chemical compounds on the soil respiration using biological indicators. Contaminants range from pesticide and veterinary drug residues, to heavy metals. Other analytes include marker compounds for irradiated food and radiolabeled compounds The last part of this manual highlights current literature and provides some useful links to FAO, Codex Alimentarius, and other sources where the reader can access information on sampling procedures, quality assurance and quality control measures, and guidelines for validation of methods and estimation of measurement uncertainty. It is the hope of the RALACA network that this manual will be helpful in building, enhancing, or consolidating analytical and food safety control capabilities in interested laboratories and institutes. Contact details are included for each method, so that interested readers can contact the responsible institution and obtain more detailed information. The FAO/IAEA acknowledges the contribution of all RALACA participants to this publication and the numerous reviewers of this first edition. The FAO/IAEA officers responsible for this publication were Britt Maestroni, Victoria Ochoa, and Andrew Cannavan of the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture.

CHAPTER 2

General Requirements for Food Safety Analysis Britt Maestroni Food and Environmental Protection Laboratory (FEPL), Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria

SETTING UP A RESIDUES LABORATORY Setting up an analytical (residue) laboratory is a very challenging task that requires a multiteam/multidisciplinary approach in addition to finances, staff, and time. The ideal situation when planning an analytical laboratory is having an infinite budget. However, that is very rarely the case. Therefore careful planning should start from the outset, focusing on what needs to be implemented and what can realistically be done.

Define the mandate of the laboratory The management should define the mandate of the laboratory and the areas of intervention. Typical questions to be answered are: • What are the tasks and aims of the laboratory? • Is the laboratory operating in the official, mandatory, or voluntary area? • What quality systems are planned for/to be put in place at the laboratory?

Establish the scope The scope of the laboratory activities needs to be defined. Typical questions to answer are: • What analyses are to be performed? • What is envisaged in terms of the types of residues, and the matrices? • What is the expected laboratory turnover in terms of number of samples per day/month/year? • What are the desired turnaround time-frames for the analyses/clients? Analytical Methods for Agricultural Contaminants. Copyright © 2019 International Atomic Energy Agency. DOI: https://doi.org/10.1016/B978-0-12-815940-8.00002-8 Published by Elsevier Inc. All rights reserved.

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Define the types of methods and limitations desired Expert advice usually is needed to address the following points: • Scope of the analytical methods • Screening/routine/confirmatory methods • Required limits of quantification (LOQ)/limits of detection (LOD) desired • Applicability

Determine the analytical equipment requirements to perform the laboratory’s tasks The definition of the methodologies helps defining the type of instruments and equipment needed at the analytical laboratory. Categories to be considered are: • Major and minor equipment • Consumables and other materials (chemicals, solvents, reagents, corrosives, etc.) • Standards, reference materials • Books, literature, journals, periodicals Laboratory management information system, and IT requirements • Extra facilities (i.e., uninterrupted power supply, gas installation, and waste disposal)

Define requirements for sustainability of the laboratory Defining all needs to meet the demand is essential to ensure the sustainability of the laboratory operations. A checklist includes: • Formal accreditation of the quality systems • Staff training and educational support • Local infrastructure, i.e., procurement and supply • External support, i.e., finance • Instrument replacement and turnover • Instrument maintenance and contracts

Specify the conditions of the analytical laboratory Planning the space requirements for the laboratory should be done by the staff working in the laboratory together with an architect. Checklists shall include: • rooms for sample reception, sample preparation, weighing, analytical equipment, cleaning, washing, storage, library, meetings, etc.

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supply of electricity, water, temperature control, gases (inside/outside), fume hoods • restricted access areas, special provisions such as for negative pressure rooms • safety precautions, fire extinguisher, showers, eye washes, blankets, medical and emergency kits • furnishing of the laboratory with work benches, tables, fume hoods, cupboards, coolers, freezers • Facilities for storage of chemicals such as solvents, reagents, and corrosives. • special requirements for floor quality, wall and ceiling surface and painting, supply lines, IT wiring and networking, etc. • HVAC systems (heating, venting, air conditioning/cooling) needed for different areas such as wet chemistry labs (hoods) and instrument rooms • special requirements for sound reduction systems, i.e., for pumps • waste disposal The points above are indicative and not exhaustive. A feasibility study conducted by a team of architects, laboratory staff, and managers can identify options for a construction that complies with country regulations with regard to safety, waste disposal, etc., while at the same time optimizing the available budget. The following information provides examples of manufacturers and companies that can be contacted to obtain information for laboratory furniture, supplies, chemicals, instruments, and standards.

Examples of resources • •



Laboratory furniture: AdvanceLab, Flores Valdes, Koettermann, Waldner, VWR, local providers, designers General laboratory supply and consumables: Alltech-Grace, Beckmann, C3, Coulter, Bibby, Buechi, Carl Roth, Cole Parmer, Eppendorf, Fisher Bioblock, IKA, J & K, Hach, Hettich, Kern, LAT, Macherey & Nagel, Metrohm, Millipore, Sartorius, Schott, Supelco/ Sigma-Aldrich, Upchurch, Varian, VWR, Winkworth, Whatman Major analytical instrumentation and accessories: GC and GC/MS: Agilent, Finnigan, Dani, GL Sciences, Hamilton, Koni, LATEK, Perkin-Elmer, Restek, Schambeck, SGE, Shimadzu, Varian; LC and LC/MS: Agilent, Bio-Rad, Bischoff, Dionex, ERC, GBC, GL

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Sciences, Gilson, Hamilton, Hitachi, Jasco, Knauer, LI-COR, Phenomenex, Pickering, Rheodyne, Schambeck, Shimadzu, Shodex, Sykam, Thermo, Tosoh, Valco, Varian, Waters; TLC: CAMAG Chemicals, standards, reference material: FAPAS, Fisher Bioblock, Fluka, Merck, Rohm & Haas, Scharlau, Sigma-Aldrich, Varian, VWR, Ehrenstorfer, International Isotopes Inc., LGC Standards/ Promochem, Progetto Trieste

SAMPLING IN THE CONTEXT OF FOOD SAFETY Food control systems aim to protect the health of the population and to promote trade. An appropriate and properly designed sampling plan for the purpose of monitoring food safety and quality is required for this purpose. The objective of sampling is to provide the laboratory with samples for analysis (and results) that represent the entire population being monitored (e.g., batch, lot, orchard, farm, and shipment) In this context, effective monitoring/testing schemes depend on the coexistence of sound sampling plans, valid analytical methods, and regulatory limits at a minimum. Sampling should be seen as an extremely useful tool for gathering important information that might otherwise be unattainable or unaffordable. In general, separate sampling plans must be prepared for monitoring, surveillance, and targeted sampling. Monitoring is both preliminary and performed routinely to ensure that residues of concern fall below acceptable maximum limits (MLs). Surveillance is undertaken whenever data from monitoring reveal that country specific (legal limits) or internationally acceptable ML values have been exceeded, and it aims at providing a basis for centralized and qualified feedback. Targeted sampling is undertaken when there is evidence or a history (e.g., from previous analysis) of excessive residues or past trade rejection. It focuses on samplingspecific populations suspected to be noncompliant (e.g., goods produced or stored under bad conditions and food derived from animals showing clinical signs of intoxication) or that are intended for more sensitive consumers (e.g., babies and immunocompromised patients). It is important to understand that sampling plans may have different objectives. For example, an acceptable sampling plan for quality control (QC) purposes may be very different from a sampling plan for testing commodities at harvest for contaminants.

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To be able to design a valid sampling plan it is important to answer the following questions: Why? • What is the sampling objective[s]? • What decisions will be made based upon the results obtained? What? • What samples are to be taken? Where? • What is the population or domain to be sampled? When? • When should samples to be taken to meet the sampling objective[s]? • When will they be processed? • When will a report on the sampling be available? How? • How will the samples be located? • How will the samples be taken, processed and documented? All the above are simple questions to help the scientist collect enough information to be able to start planning a sampling protocol. Statistical considerations must be factored in when developing a sampling plan. Several options may exist and expert advice is strongly recommended to ensure that the resulting sample is representative, economical, practical, and statistically valid. Care should also be taken to ensure that the integrity of the batch/lot being sampled is not compromised during the sampling exercise. The analytical laboratory can play an active role in relation to the following aspects concerning sampling: 1. The design of the sampling plan (mainly for research purposes) 2. The sampling itself—e.g., by ensuring that the samples are collected according to an established protocol and transported to the laboratory under conditions that prevent their integrity being compromised (mainly for compliance monitoring) 3. Ensuring that adequate information about the sampling is recorded and conveyed to enable the correct interpretation of the analytical data (this is mostly the case for risk assessment studies) In the case of compliance monitoring, sampling protocols are available describing recommended procedures for the sampling of many types of materials and chemical components. These protocols are sometimes specified in national regulations or international agreements. Examples are: • Codex (2004) General Guidelines on Sampling; CAC/GL-2004 (http:// www.codexalimentarius.net/download/standards/10141/CXG_050e.pdf, 2016)

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Commission Regulation (EU) No. 589/2014 methods of sampling and analysis for the control of levels of dioxins, dioxin-like polychlorinated biphenyls (PCBs) and nondioxin-like PCBs in certain foodstuffs (http://eur-lex.europa.eu/legal-content/EN/TXT/?uri 5 CELEX% 3A32014R0589, 2016) Commission Regulation 519/2014, sampling of large lots, spices and food supplements, performance criteria for T-2, HT-2 toxin and citrinin and screening methods of analysis (http://eur-lex.europa.eu/legalcontent/EN/TXT/?uri 5 uriserv%3AOJ.L_.2014.147.01.0029.01.ENG, 2016) Commission Directive 2002/27/EC sampling methods and methods of analysis for aflatoxins in foodstuffs (http://eur-lex.europa.eu/legalcontent/EN/TXT/?uri 5 celex%3A32002L0027, 2016) Commission Directive 2002/26/EC sampling methods and methods of analysis for ochratoxin A in foodstuffs (http://faolex.fao.org/cgibin/ faolex.exe?rec_id 5 030851&database 5 faolex&search_type 5 link& table 5 result&lang 5 eng&format_name 5 @ERALL, 2016) Commission Directive 2002/63/EC of July 11, 2002 establishing Community methods of sampling for the official control of pesticide residues in and on products of plant and animal origin

SAMPLE PREPARATION AND PROCESSING In analytical chemistry, sample preparation is the general term used to describe the ways in which a sample is treated prior to its analysis. It may involve solvent extraction, reaction with some chemical species, pulverizing, treatment with a chelating agent, filtering, dilution, subsampling, and/or many other techniques. However, other definitions may be encountered especially when working in the area of food contaminant analysis. According to the definitions introduced by Hill and Reynolds (1999) in the area of pesticide residue analysis the preparation of the analytical sample from which the analytical portion for analysis is withdrawn may consist of two distinct procedures: sample preparation and sample processing. Sample preparation is the procedure used, if required, to convert the laboratory sample into the analytical sample by removal of parts (soil, stones, bones, stems, etc.—parts not normally consumed and therefore not included in the analytical sample) not to be analyzed. Sample

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processing is the procedure (e.g., cutting, grinding, and mixing) used to make the analytical sample homogeneous with respect to the analyte distribution and ready for extraction, prior to removal of the analytical portion. The analytical portion is the test portion that is solvent-extracted and analyzed using an approved and validated analytical procedure to quantify/qualify the analyte(s) of interest. The ultimate goal of sample preparation and sample processing is to ensure that the analytical portion (aliquot/subsample taken for extraction, clean-up, and analysis) is representative of the entire population of the batch/lot sampled. Sample preparation and processing are likely to be the greatest sources of contamination and errors (both random and systematic) between sampling and analysis. Hence great care must be taken to minimize sources of errors and contamination during preparation and processing. To ensure an uncontaminated and representative sample, each laboratory must create and strictly follow scientifically sound and management-approved documented procedures—Standard Operating Procedures—for sample preparation and processing. As a requirement for compliance with ISO/IEC 17025 guidelines, the uncertainty resulting from the sample processing step must be evaluated and factored into the overall uncertainty in the measurement resulting from the entire analytical procedure. Useful guidelines are given by Fajgelj and Ambrus (2000).

QUALITY ASSURANCE AND QUALITY CONTROL PROCEDURES Laboratories can improve their performance and ensure reliability of their test results by implementing quality assurance (QA) and QC procedures. QA is defined by ISO as that component of quality management, focused on providing confidence that quality requirements are fulfilled. The CITAC/Eurachem Guide to Quality in Analytical Chemistry (https://eurachem.org/index.php/publications/guides/qa, 2016) refers to QA as the overall measures that a laboratory uses to ensure the quality of its operations. QC refers to the operational techniques and activities that are used to fulfill requirements for quality. For example, to ensure the quality of a specific batch of samples one can make use of reference materials to check for recoveries of spiked samples and monitor those in control charts. The purpose of QA is to provide confidence in the results produced and that all data generated in the laboratory are fit for their

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intended purpose. This demonstrates that the laboratory has adequate facilities/equipment and competent staff; the work is carried out in a controlled manner; the methods are validated and the process is well documented. The CITAC/Eurachem guide also mentions that QA should focus on the key issues which determine quality results, costs and timeliness and avoid diversion of energies into less important issues. In general laboratories may opt to design their own QA system; however, it is recommended to follow an established QA system to be able to claim compliance according to that system and to obtain an independent assessment or endorsement by a qualified body (accreditation or certification). Formal and recognized QA standards are: 1. ISO/IEC 17025 (http://www.iso.org/iso/catalogue_detail.htm? csnumber 5 39883, 2016) (2005)—“Technical competence of laboratories to carry out tests and calibrations.” This standard combines laboratory-specific requirements with the quality management principles of ISO 9001. ISO/IEC 17025 contains all the requirements that testing and calibration laboratories have to meet if they wish to demonstrate that they are technically competent, operate a quality management system, and are able to generate technically valid results. The acceptance of testing and calibration results between countries should be facilitated if laboratories comply with this International Standard and if they obtain accreditation from bodies which have entered into mutual recognition agreements with equivalent bodies in other countries using the International Standard ISO/IEC 17025. The standard sets a number of requirements: a. management driven: these relate to the legal entity, the structure and the responsibilities, the policies and the procedures, and the resources of the organization and the formal documentation of the quality management system. b. support systems driven: these relate to document control, review of requests, tenders and contracts, subcontracting of tests and calibrations, purchasing services and supplies, services to clients, complaints, control of nonconformities, continuous improvement, corrective actions, preventative actions, control of records, internal audit, and management review c. technically driven: these requirements relate to personnel, test site and environment, calibration and test methods/procedures, laboratory developed methods, nonstandard methods, method validation,

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measurement of uncertainty, data control, equipment, measurement traceability, transportation/storage, sampling, handling of test items, QC procedures, test reports, proficiency testing. 2. ISO 9001:2015 (http://www.iso.org/iso/home/store/catalogue_ics/ catalogue_detail_ics.htm?csnumber 5 62085, 2016)—“Quality management systems Requirements.” This standard provides guidance and tools to ensure that the products and services offered by an organization consistently meet customer requirements, and that quality is consistently improved. 3. OECD Series on Principles of Good Laboratory Practice (GLP) (http://www.oecd.org/chemicalsafety/testing/oecdseriesonprinciplesofgoodlaboratorypracticeglpandcompliancemonitoring.htm, 2016). GLP is the basis for a quality system concerned with the organizational process and conditions under which nonclinical health and environmental safety studies are planned, performed, recorded, reported, archived, and monitored. GLP is, in essence, a managerial concept for the organization of studies. The terms “accreditation” and “certification” are sometimes used interchangeably. However, they are not synonymous. Accreditation is the procedure carried out by an authoritative body (usually a national accreditation body) which gives formal recognition that the laboratory is competent to carry out specific tests or calibrations against a recognized standard (usually ISO/IEC 17025). Accreditation is usually given for specific combinations of analyte, matrix, and method. Certification is the procedure carried out by a third party against a recognized standard (usually ISO 9001) giving formal assurance that a product, process, or service conforms to specified requirements. Technical competence is not specifically addressed through certification but is normally addressed via accreditation. In general the requirements for a laboratory quality system can be summarized by the following: • Ensures management commitment and resources • Ensures the availability of staff with necessary skills and training • Ensures infrastructure and conditions suitable to perform analyses • Provides training on QA to managers and analysts • Develops the right “mind set” Work in an analytical laboratory should be carried out in a systematic way, where the processes are stepwise. It is important to define the sequence of actions necessary to ensure that sample identity and integrity are maintained throughout the process. Critical parameters are those that refer to

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sample registration, when a unique identification number is assigned, and details relating to the sample are logged and analytical tests scheduled. Samples have to be securely labeled, and stored, if necessary, under proper conditions. High-quality analyses are based on three essential elements: • Trained staff with records of on-job training • Adequate technical infrastructure with properly maintained and calibrated equipment • Good analytical methods which are “fit for purpose” and validated. A good reference document for analytical laboratories is the European Commission Directorate General for Health and Food Safety’s (SANTE) document SANTE/11945/2015 on “Method Validation and Quality Control Procedures for Pesticide Residues Analysis in Food and Feed” (http://ec.europa.eu/food/plant/docs/plant_pesticides_mrl_guidelines_ wrkdoc_11945_en.pdf, 2016). Although the document targets the field of pesticide residues analysis, it constitutes an excellent resource for information on QA/QC measures that should be implemented in the laboratory. Since performance in analytical testing varies from batch to batch and from day to day, the guide suggests two types of QC procedures: 1. On-going Analytical QC to verify and supplement method validation on a regular basis. 2. Additional QC/Assurance procedures for monitoring the validity of tests (e.g., proficiency tests). The SANTE Document SANTE/11945/2015 provides detailed recommendations for sampling, transport, processing and storage of samples, standards, calibration solutions, extraction and concentration, contamination and interference, analytical calibration, representative analytes, matrix effects and chromatographic integration, analytical method validation and performance criteria, routine recovery determination, proficiency testing and analysis of reference materials, confirmation and reporting of results. Reading is highly recommended.

METHOD VALIDATION The purpose of Method Validation is to show that the method of analysis chosen is capable of producing accurate, precise, and reproducible results for the analytes of interest in the specified matrices. The data collected (and retained) to prove the validity of the method provide the basic

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evidence to support the validity of the results subsequently generated using the method for sample analysis. In other words, a method must be fit for the purpose of analysis and should provide reliable results. Method validation is a requirement for seeking accreditation under ISO/IEC 17025. Essential information for the characterization of a method may be gathered during the development or adaptation of an analytical procedure, the establishment of acceptable performance (in house validation), the regular performance verification of methods applied in the laboratory, the demonstration of acceptable performance in a second or third laboratory (AOAC Peer-Verified Method), and participation in interlaboratory collaborative studies. Validation must be carried out following completion of the method development and before introducing the method for routine analysis. Additional validation is essential when the method is transferred to another laboratory or whenever the conditions or method parameters for which the method was initially validated have changed. Important parameters to be studied during method validation are: • Stability of analytes during sample storage, sample processing, extract storage and in analytical standards. It is important to note that stability of analyte(s) in solutions and sample extracts may vary greatly according to the chemical or physicochemical properties of the analyte(s), the solvent used, storage conditions/temperatures and the nature of the extract, and must be investigated. • Efficiency of extraction. Rigorous validation of extraction efficiency of organic analytes can only be performed with samples containing analyte(s) incurred by the route through which the trace levels would normally be expected to arise. To this end the use of radiotracer studies can be of great advantage. In practice, it is often not possible to obtain incurred samples and extraction efficiency must be estimated using blank samples spiked or fortified with a known amount of the target analytes. However, this method does not give a true reflection of the efficiency of extraction of incurred samples. • Homogeneity of distribution of the analyte in the processed sample. This is very specific for the laboratory equipment and the sample matrix processed and therefore needs to be investigated. • Selectivity. The ability of an analytical procedure to assess the analyte in the presence of interferences such as matrix components, impurities, and degradation products needs to be investigated.

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Specificity of analyte detection. The detection system response used for calibration must be demonstrated as being completely attributable to the analyte, preferably by mass spectrometry in full-scan or multiple reaction monitoring or exact-mass/high resolution mass spectrometry • Calibration function. The relationship between the observed signal from the target analyte in the sample extract and known quantities of the analyte prepared as calibrant must be defined for its linearity, analytical range, limit of detection and limit of determination, and response range. • Matrix effects. These are described as the possible impacts of sample matrix components on the measurement of analyte concentration (analyte signal). If present, matrix effects may need to be compensated for, e.g., through the use of matrix-matched calibration. • Precision and reproducibility. The precision of a method is the extent to which the individual test results of multiple injections of a series of standards agree. The measured standard deviation can be expressed as repeatability, intermediate precision, and reproducibility. • Accuracy. The accuracy of an analytical method is the extent to which test results generated by the method agree with the known value (either as a true or accepted reference value). The validity of a specific method should be demonstrated in laboratory experiments using samples or standards that are similar to unknown samples to be analyzed routinely. The preparation and execution of a method validation study should follow a validation protocol, preferably written in a step-by-step instruction format. The objective of analytical method validation is to ensure that valid analytical data are generated both during initial use of the method and also during its entire lifetime of application. However, appropriate QC checks should be included during routine sample analysis to verify that the performance of the method and the system has not changed from the initial method validation. Several references available to describe the requirements of method validation are: • CAC/GL 71-2009: Guidelines for the design and implementation of national regulatory food safety assurance program associated with the use of veterinary drugs in food producing animals (http://www.agricultura.gov.br/arq_editor/file/CRC/CAC-GL%2071-2009.pdf, 2016)

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CAC/GL 40-1993 Guidelines on Good Laboratory Practice in Pesticide Residue Analysis (CAC/GL 40-1993) (http://www.fao.org/ faowhocodexalimentarius/shproxy/en/?lnk 5 1&url 5 https%253A% 252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FStandards %252FCAC%2BGL%2B40-1993%252Fcxg_040e.pdf, 2016) SANTE/11495/2015 Guidance document on analytical QC and method validation procedures for pesticide residues analysis in food and feed (http://ec.europa.eu/food/plant/docs/plant_pesticides_mrl_ guidelines_wrkdoc_11945_en.pdf, 2016) FDA Regulations and specific guidelines for the validation of analytical methods and procedures (http://www.fda.gov/downloads/Drugs/ GuidanceComplianceRegulatoryInformation/Guidances/UCM386366. pdf, 2016) In essence, the steps involved in a method validation study include: Preparation of a validation plan by specifying the method requirements (accuracy, precision, selectivity, specificity, sensitivity -LOD, LOQ-, linearity, dynamic range, and ruggedness) selecting matrices and analytes and fortification levels Establishment of experimental design Running of experiments Statistical analysis of data Comparison of results with requirements, guidelines/standards Setting the parameters for verification of method performance in routine application

UNCERTAINTY ESTIMATION The main purpose of making measurements is to use the results to help in making decisions. The reliability of the decisions made depends, therefore, on the uncertainty of the results, or their fitness for purpose. It is therefore essential that effective procedures are established for estimating the uncertainties arising from all parts of the process that lead to a result. These include sampling, sample preparation and processing, extraction, clean-up, and analysis. While many texts and guidelines exist for measuring the uncertainty component of an analytical measurement, only one document exists for estimating the sampling uncertainty. Eurachem has

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published a guideline entitled, Measurement Uncertainty Arising from Sampling A Guide to Methods and Approaches (https://eurachem.org/ images/stories/Guides/pdf/UfS_2007.pdf, 2016). When the analytical laboratory is responsible for conducting the sampling, it is recommended that a validated sampling protocol is established and that this includes a calculation of uncertainty resulting from the sampling procedure, as part of the method validation process. Analytical laboratories, however, are mostly concerned with estimating the uncertainty of measurements for the results they produce for a given sample. Uncertainty of measurement has been defined as the range around the reported result within which the true value is expected to lie with a specified probability. It is important to note that estimation and reporting of the uncertainty of measurement is a requirement under ISO/IEC 17025. To this end, food testing laboratories operating under the Guidelines on Good Laboratory Practice in Pesticide Residue Analysis (CAC/GL 401993) should have sufficient data available, derived from method validation/verification, interlaboratory studies, and in-house QC activities to enable estimation of the uncertainty of a measurement, particularly for the routine methods undertaken in the laboratory. There are several approaches to estimating the measurement uncertainty. One approach is described in the Guide to the Expression of Uncertainty in Measurement (GUM) published by ISO (http://www.bipm.org/utils/common/ documents/jcgm/JCGM_100_2008_E.pdf, 2016). Also referred to as the “bottom-up” approach, and shared by Eurachem, this consists of identifying and quantifying the contribution of each step of the analysis separately and combining all contributions into an overall uncertainty estimate. An alternative approach, referred as the “top-down approach,” attempts to estimate the all-inclusive variability of the measurement using information from proficiency testing, collaborative studies (Analytical Methods Committee, 1995) or from in-house validation data. Both approaches are acceptable within the ISO/IEC 17025 standard. The “top-down” approach is probably simpler but provides little information as to how uncertainty could be improved. In contrast, the investigation and estimation of the contributions made in the “bottom-up” approach is more complicated and time consuming, but is the more useful approach for any laboratory aiming to improve its methodology. Once the information about uncertainty has been generated, the next step is to use this for compliance assessment. Assuming that the objective is to assess whether or not a sample contains a compound above a set (or

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Figure 2.1 Interpretation of results for compliance to a certain upper limit. Error bars and distribution curves indicate the uncertainty of the measured result.

permissible) upper limit, four possible scenarios are possible when comparing the result with the upper limit (see Fig. 2.1): 1. The result of the measurement exceeds the upper limit by more than the measurement uncertainty. 2. The result of measurement exceeds the upper limit but the upper limit is within the measurement uncertainty. 3. The result of the measurement is below the limit but the upper limit is within the measurement uncertainty. 4. The result of measurement and the measurement uncertainty are below the upper limit. It is easy to conclude that case (1) is truly a “noncompliant sample” and case (4) is truly a compliant sample. However, in cases (2) and (3), rules are needed to decide whether or not a sample is compliant or noncompliant. The Eurachem CITAC guide describes the need for decision rules and the setting of acceptability levels with acceptance zones (for the product to be declared compliant) and rejection zones (for the product to be declared noncompliant). Those decision rules are in general set by national legislation. Sources of information on Uncertainty: • CAC/GL 59-2006 Guidelines on estimation of uncertainty of results • ISO Guide to the Expression of Uncertainty in Measurement (GUM) is a key document used as the basis of evaluating the uncertainty in the output of a measurement system (http://www.bipm.org/utils/ common/documents/jcgm/JCGM_100_2008_E.pdf, 2016) • Eurachem/CITAC Guide: Quantifying Uncertainty in Analytical Measurement, third Edition (2012) is an interpretation for chemical measurement including a number of worked examples (http://www.eurachem.org/index.php/publications/guides/quam (accessed 17.08.16.))

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SANTE/11495/2015 Guidance document on analytical QC and method validation procedures for pesticide residues analysis in food and feed (http://ec.europa.eu/food/plant/docs/plant_pesticides_mrl_ guidelines_wrkdoc_11945_en.pdf, 2016) Eurachem/CITAC Guide, Use of Uncertainty Information in Compliance Assessment, first Edition (2007). (https://www.eurachem.org/images/ stories/Guides/pdf/Interpretation_with_expanded_uncertainty_2007_v1. pdf (accessed 17.08.16.))

CHOOSING THE RIGHT METHOD FOR YOUR PURPOSE Murphy (1955) wrote an editorial on the selection of analytical methods. He stated that “selecting the right analytical method . . . this now presents a very real problem in many laboratories.” This challenge for the analytical scientist is still relevant after so many years and so many analytical revolutions. The concept expressed by Murphy about the need for a multidisciplinary approach to problem solving is still valid and real. To be able to choose the right analytical method it is important that the scientist/chemist is aware of the following issues at a minimum: Why? • What is the objective[s] of the analysis? • Is the method needed for routine testing/quantification? • Is there a need for a screening or a confirmatory method? What? • What is the sample matrix to be analyzed? • What is the target analyte(s)? • Is there a need for a single analyte or a multianalyte method? • What instrumentation/equipment is available? • What reference materials or analytical standards are available? • What current validated methods are available from standards/ literature? • What criteria have to be met for accuracy, precision, sensitivity, selectivity, robustness, and ruggedness? Where? • What are the current national/regional/international legislative requirement(s) in terms of method performance criteria?

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• Where and when will the analytical results be used? • How quickly do the results need to be reported?” How? • How much can be spent for each sample? • How much analytical uncertainty can be tolerated? With the above information at hand the analyst should conduct a proper bibliographical review and identify options for suitable methods. These criteria are not mutually independent, and it is often necessary to find an acceptable balance between them. In cases where a method does not exist, the analytical scientist should add an additional component for method development to the total investment plan. It is important to note that any method to be adapted for use in the laboratory should undergo a complete method validation meeting internationally acceptable standards (as suggested by regulatory authorities or reputable bodies such as the Codex Alimentarius Commission) prior to routine use of the method for sample analysis. The European approach, also more recently adopted by Codex Alimentarius, requires methods to comply with certain performance criteria, and in general is more accommodating in terms of the methods used, provided these deliver results according to the criteria established by the European Commission. The US approach is more defined in terms of the methods that can be used, e.g., the tolerance (maximum limit of residue) enforcement program imposes certain requirements and restrictions on the method to be used. Only collaboratively studied methods and stringent guidelines specifying the analytical procedure are permissible.

REFERENCES Analytical Methods Committee, 1995. Uncertainty of measurement: implications of its use in analytical science. Analyst 120, 2303 2308. Fajgelj, A., Ambrus, A., 2000. Principles and Practice of Method Validation. The Royal Society of Chemistry, Special Publication NR. 257, Cambridge. Hill, A.R.C., Reynolds, S.L., 1999. Guidelines for in-house validation of analytical methods for pesticide residues in food and animal feeds. Analyst 124, 953 958. ,http://ec.europa.eu/food/plant/docs/plant_pesticides_mrl_guidelines_wrkdoc_11945_en. pdf. (accessed 17.08.16.). ,http://ec.europa.eu/food/plant/docs/plant_pesticides_mrl_guidelines_wrkdoc_11945_en. pdf. (accessed 20.08.16.). ,http://eur-lex.europa.eu/legal-content/EN/TXT/?uri 5 celex%3A32002L0027. (accessed 29.08.16.). ,http://eur-lex.europa.eu/legal-content/EN/TXT/?uri 5 CELEX%3A32014R0589. (accessed 29.08.16.).

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,http://eur-lex.europa.eu/legal-content/EN/TXT/?uri 5 uriserv%3AOJ.L_.2014.147.01. 0029.01.ENG. (accessed 29.08.16.). ,http://faolex.fao.org/cgi-bin/faolex.exe?rec_id 5 030851&database 5 faolex&search_type 5 link&table 5 result&lang 5 eng&format_name 5 @ERALL. (accessed 29.08.16.). ,http://www.agricultura.gov.br/arq_editor/file/CRC/CAC-GL%2071-2009.pdf. (accessed 20.08.16.). ,http://www.bipm.org/utils/common/documents/jcgm/JCGM_100_2008_E.pdf. (accessed 17.08.16.). ,http://www.codexalimentarius.net/download/standards/10141/CXG_050e.pdf. (accessed 29.08.16.) ,http://www.fao.org/fao-who-codexalimentarius/sh-proxy/en/?lnk 5 1&url 5 https%253A %252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FStandards%252FCAC% 2BGL%2B40-1993%252Fcxg_040e.pdf. (accessed 20.08.16.). ,http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/ Guidances/UCM386366.pdf. (accessed 20.08.16.). ,http://www.iso.org/iso/catalogue_detail.htm?csnumber 5 39883. (accessed 17.08.16.). ,http://www.iso.org/iso/home/store/catalogue_ics/catalogue_detail_ics.htm?csnumber 5 62085. (accessed 17.08.16.). ,http://www.oecd.org/chemicalsafety/testing/oecdseriesonprinciplesofgoodlaboratorypracticeglpandcompliancemonitoring.htm. (accessed 17.08.16.). ,https://eurachem.org/images/stories/Guides/pdf/UfS_2007.pdf. (accessed 25.08.16.). ,https://eurachem.org/index.php/publications/guides/qa. (accessed 17.08.16.) Murphy, W., 1955. Editorial. Selecting the right analytical method. Anal. Chem. 27 (10), 1509. http://pubs.acs.org/doi/abs/10.1021/ac60106a602 (accessed 17.08.16.).

FURTHER READING Food Contaminant and Residue Information System (FCRIS), accessible through http:// nucleus.iaea.org/fcris/. The FCRIS resource contains information on analytical techniques for the detection of food contaminants such as pesticide and veterinary drug residues. Joint Research Centre https://ec.europa.eu/jrc/en/science-area/health-and-consumerprotection. FDA laboratory methods http://www.fda.gov/food/foodscienceresearch/laboratorymethods/ default.htm. Official Methods of Analysis of AOAC INTERNATIONAL http://www.aoac.org/ imis15_prod/AOAC/AOAC_Member/PUBSCF/OMACF/OMAP_M.aspx. Search engines: Science Direct http://www.sciencedirect.com/. Pubmed http://www.ncbi.nlm.nih.gov/pubmed. Web of science https://apps.webofknowledge.com/UA_GeneralSearch_input.do? product 5 UA&search_mode 5 GeneralSearch&SID 5 S2hEKqLqAvOIeeJHAZJ& preferencesSaved 5 . Journals (among many options): Analytical Chemistry http://pubs.acs.org/journal/ancham. Food Chemistry http://www.journals.elsevier.com/food-chemistry/. Journal of Food Science http://onlinelibrary.wiley.com/journal/10.1111/(ISSN) 1750-3841. Analytical Methods http://pubs.rsc.org/en/journals/journalissues/ay#!recentarticles&all. Journal of Chromatography A http://www.journals.elsevier.com/journal-ofchromatography-a.

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Journal of Chromatography B http://www.journals.elsevier.com/journal-ofchromatography-b/. Journal of AOAC INTERNATIONAL http://www.aoac.org/imis15_prod/AOAC/ AOAC_Member/PUBSCF/JAOACCF/JN_M.aspx. Journal of Environmental Science and Health, Part B http://www.tandfonline.com/ toc/lesb20/current.

CHAPTER 3

Integrated Analytical Approaches for Food Safety and Environmental Sustainability EXAMPLES OF METHODS FOR FOOD CONTAMINANT ANALYSIS METHOD 1: Pesticide residue determination in fresh fruits and vegetables using ethyl acetate extraction and QuEChERS clean-up 1. Laboratory name and address: Food and Environmental Protection Laboratory (FEPL), Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna International Centre, PO Box 100, 1400 Vienna, Austria 2. Contact person: Andrew Cannavan email: [email protected] 3. Title of the analytical method: Pesticide residue determination in fresh fruits and vegetables using ethyl acetate extraction and GPC clean-up 4. Principle: The original QuEChERS method (Anastassiades et al., 2003) was modified to use ethyl acetate for extraction to enable the application of gas chromatography (GC) using selective detectors such as electron capture detector (ECD) or nitrogen phosphorus detector (NPD). According to the modified procedure, a portion of 30 g homogenized sample was extracted with 60 mL ethylacetate by using an Ultra Turrax blender and drying with anhydrous sodium sulfate. The clean-up step was applied without any deviation from the original QuEChERS method. Cleaned ethyl acetate extracts were injected into the GC-ECD and NPD without solvent change. Analytical Methods for Agricultural Contaminants. Copyright © 2019 International Atomic Energy Agency. DOI: https://doi.org/10.1016/B978-0-12-815940-8.00003-X Published by Elsevier Inc. All rights reserved.

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5. Scope: The method is validated for 22 representative pesticides in tomatoes, apple, and frozen green bean samples in the range 0.055 mg/kg. The analytes are as follows: EPTC, mevinphos, heptenophos, propachlor, dimethoate, diazinon, pirimicarb, vinclozolin, fenitrothion, chlorfenvinfos, folpet, methidation, triazophos, propyconazole, fenpropathrin, azinphosmethyl, fenarimol, coumaphos, cyfluthrin, fenvalerate, lindane, and α-endosulfan. 6. Equipment and instruments: • 250 mL centrifuge bottles, HDPE • Top loading and/or analytical balance (capable of weighing 100 g within 0.1 g precision and weighing 1 g within 0.01 g precision) • Spoon/spatulas, weighing boats • Centrifuge capable of at least 4000 rpm and head(s) able to hold 250 mL centrifuge • Bottles and 15 mL centrifuge tubes • Vortex mixer and optional automatic shaker capable of shaking 250 mL bottles • 1015 mL conical glass centrifuge tubes • 1.82 mL auto sampler vials • Pasteur pipettes and/or syringes • Volumetric flasks • Sealable 40 mL glass vials or bottles • Ultraturrax • Chopper 7. Reagents and materials: • Sodium sulfate anhydrous (Na2SO4), residue grade • Sodium hydrogen carbonate (NaHCO3), residue grade • Magnesium sulfate anhydrous (MgSO4), residue grade; note: this should be heated for at least 5 h at 500 C to remove phthalates— this additionally requires a high-temperature oven. The phthalates are not a problem except potentially in mass spectrometric detection. • Internal standards: chlorpyrifos methyl, bromophos methyl • Ethyl acetate (pesticide or HPLC grade) • Primary secondary amine (PSA) sorbent of 40 μm particle size • Toluene • Certified analytical standards with known purity

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8. Standard solutions: • Pesticide stock solutions of 1 mg/mL were prepared in 85:15 (v:v) acetone:isooctane, and working solutions were prepared in EtOAc. Stored at 220 C. The stock solutions were stable for 6 months. • Internal standard IS1: chlorpyrifos methyl (0.5 mg/kg sample equivalent): 150 ng/μL in toluene, IS2: bromophos methyl (0.5 mg/kg sample equivalent): 150 ng/μL in EtAc. • Prepare six calibration levels and inject in duplicate. The standard solutions are prepared at 2, 12.5, 25, 50, 100, and 125 pg/μL concentrations for ECD and 125, 250, 500, 1000, 1250, and 2500 pg/μL for NPD. 9. Detailed procedure (protocol): Comminution and extraction of the sample • Chop/grind the sample and store the homogenous sample in plastic bags or glass jars in a deep-freezer at , 220 C, or take subsamples right away for extraction and analysis. • Allow sample to partially thaw prior to starting extraction procedure. • Label all containers to be used in the experiment while the sample is thawing and standards are reaching room temperature. • Prepare a number of 40 mL vials that contain 30 g anhydrous Na2SO4 and 5 g NaHCO3 separately. The vials are stored capped at room temperature. • Tare the 250 mL centrifuge bottles (high-density polyethylene) on the top-loading balance, and weigh 30 6 0.1 g previously homogenized sample into each centrifuge bottle. • Add 5 g NaHCO3 and mix it by gently shaking. • Dispense 60 mL ethyl acetate (EA) into all bottles, add 100 μL of IS1 solution (0.5 mg/kg 3 sample equivalent), cap well and shake briefly. • Add 30 6 0.1 g anhydrous Na2SO4 one by one and immediately start extraction. • Centrifuge for 3 min at 2500 rpm, ensuring that the centrifuge is balanced. Dispersive-SPE clean-up • Weigh 0.25 6 0.01 g PSA sorbent and 1.5 6 0.01 g anhydrous MgSO4, all mixed into 15 mL conical glass centrifuge tubes. Cap the tubes.

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Transfer 10 mL extract to the tubes, cap the tubes well, and Vortex for 30 s. • Centrifuge the tubes for 1 min at 2500 rpm. Preparation of the extracts for GC analysis: • For GC/ECD analysis take 500 μL of the final extract, add 10 μL of IS2, bring to 1 mL with EA and inject 1 μL into the GC. • For GC/NPD analysis take 500 μL of the extract, add 10 μL of IS2, bring to 1 mL with EA and inject 1 μL into the GC. Gas chromatographic conditions: GC-NPD conditions: • Injector: EPC PTV inlet, splitless mode • PTV temperature program: 0.1 min at 73 C; raise the temperature to 250 C at a rate of 600 C/min, 2 min; cool to 73 C at a rate of 600 C/min • Column: HP-5, constant flow, 2 mL/min • Oven: 1 min at 70 C; raise at a rate of 20 C/min to 160 C, then with a rate of 4 C/min until the final temperature of 270 C, hold for 5 min • Detector: temperature 300 C, H2 flow 2 mL/min, air flow 60 mL/min, make-up gas flow 36 mL/min GC-ECD conditions: • On column injection • Oven: 1 min at 70 C; raise at a rate of 20 C/min to 160 C, thenwith a rate of 4 C/min until final temperature of 270 C, hold for 5 min • Detector temperature: 300 C • Auxiliary gas flow rate: 50 mL/min 10. Calculations: A five-point calibration curve is constructed with matrix-matched calibrators and an internal standard is applied to correct for volumetric losses and recovery variation. A weighted linear calibration model is applied. Goodness of the calibration curves was checked for standard deviation of relative residuals which should be less than 0.1. 11. Quality control: The GCs were optimized according to daily maintenance routine and system suitability was checked with test mixtures before analysis of the samples. Performance/noise reports, chromatograms, and reports related to peak asymmetry for chosen peaks were stored in related log books of the GCs.

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As a quality control (QC) check of the performance of the GCs, a 15 ng/μL bromophos-methyl solution (QC-std) was also prepared in EA and added to all final extracts prior to injection. 12. Interferences, troubleshooting, and safety: Label all glassware to be used before starting the procedure. Strictly follow safety regulations related to general laboratory operations. 13. References: EN 15662, 2007. J. Environ. Sci. Health Part B 42, 481490. 14. Minimum method validation data: Range of matrices: tomatoes, apples, and frozen green beans Range of validation: 0.055 mg/kg LOD: 0.0050.01 mg/kg Recoveries: 70%110% CV: 10%

METHOD 2: Extraction of pesticide residues present in fresh fruits and vegetables using the QuECHERS method 1. Laboratory name and address: Laboratorio Nacional de Insumios Agricolas  LANIA, Km 14. Via Bogota Mosquera, Mosquera, Colombia 2. Contact person: Edwin Samir Barbosa Angel email: [email protected] 3. Title of the analytical method: Extraction of pesticides residues present in fresh fruits and vegetables using the QuECHERS method 4. Principle: A portion of a processed and homogenized sample is extracted with acetonitrile (ACN). Subsequently, a mixture of anhydrous magnesium sulfate and sodium chloride is added to promote the separation of phases and a mixture of sodium citrate dihydrate and sodium hydrogen citrate sesquihydrate is added to adjust the pH. After vigorous shaking and centrifugation, an aliquot of the organic phase is cleaned up by adding primary secondary amine (PSA) and anhydrous magnesium sulfate is added to remove remaining water. The extract is shaken vigorously and centrifuged. Finally, an aliquot of the extract is filtered for the analysis by HPLC-MS/MS.

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5. Scope: The current method is recommended for the determination of nonionic pesticide residues in fresh fruits and vegetables with high water content. 6. Equipment and instruments: • Dispenser bottle with variable volume 1525 mL • Porcelain capsule • Spatulas • 15 mL Amber bottles with cap • Disposable syringes 2 mL • 10. 5 mL volumetric flask • Microspatulas • Balance to weight substances • 10 mL volumetric pipette • 15 mL centrifuge tube with cap • 50 mL centrifuge tubes with cap • 0.45 or 0.22 μM filtration units for solvent • Liquid chromatograph vials • Freezer at 220 C • Cooler • Food processor • Industrial blender • Balance 60.01 g • Analytical balance 60.0001 g • Fix volume micropipettes, 10, 20, 50, 100, 200, 500, and 1000 μL • Centrifuge with adapters required for 50 and 15 mL tubes • HPLC solvents filtration system • Ultrasound bath • Type I water production system • Oven to operate at 500 C • Automatic shakers for tubes • Vortex mixer 7. Reagents and materials: • Standards of pesticides .96% purity with certified and uncertainty associated • ACN, chromatographic grade 99.9% • Water, HPLC grade • Sodium citrate dihydrate residue grade

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• Sodium chloride A.R. • Sodium hydrogencitrate sesquihydrate A.R. • PSA, from Varian (part nr. 12213024) • Magnesium sulfate anhydrous residue grade 8. Standard solutions: 8.1. Preliminaries NOTE: For each series of samples from the same matrix (extraction/analysis batch) the following must be performed: one (1) reagent blank, two (2) replicates of each sample and matrix blank fortified for calibration curve levels and the detection limit. The results obtained from a low- and a highlevel spike should be considered as controls of the method performance. The sample must be stored frozen between sampling and analysis. Prior to analysis the sample should be quartered (if needed), blended, and homogenized taking the amount needed for the analysis. The remaining sample should be stored frozen again. Phthalates and MgSO4 humidity elimination: Take 100 g of anhydrous magnesium sulfate and place it into a porcelain capsule. Place the capsule into a flask and heat to 550 C for 5 h. Turn off the heating and allow to cool. Transfer the porcelain capsule to a desiccator previously stabilized. 8.2. Preparation of solutions Preparation of stock solution of 1000 mg/L for each pesticide. Weigh 10 6 0.5 mg standard pesticide, predilute with ACN and dilute to 10 mL. Shake and label the solution. Register the preparation. Solution of 10 mg/L: take 100 μL of the stock solution of each pesticide and bring to 10 mL in a volumetric flask with ACN. Shake and label the solution as mix solution. Register the preparation. If the number of pesticides to analyze is greater than 30, it is possible to prepare different mixed solutions. Solution of 1 mg/L: measure 500 μL of each mixed solution of 10 mg/L and bring to 5 mL in a volumetric flask with ACN. Shake and label solution. Register the preparation. 8.3. Preparation of calibration curve in matrix blank The calibration curve is prepared in the matrix. Homogenize the sample matrix blank in the processor. Weigh 10 6 0.1 g homogenized sample in seven 50 mL centrifuge

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32

Table 3.1 Calibration levels and volumes used for fortification of matrix blank Level Volume of working solution Final concentration at the at 1 mg/L (µL) 10 g sample (mg/kg)

0 1 2 3 4 5 6 7

0 10 20 50 100 200 500 1000

Method Blank 0.001 0.002 0.005 0.010 0.020 0.050 0.100

tubes. Add the following quantities of the 1 mg/L pesticide mixture solution and continue the process of sample preparation as indicated by the method. 9. Detailed procedure (protocol): Sample extraction • Chop process and homogenize the sample. • Weigh 10 6 0.1 g into a 50 mL centrifuge tube. • Add 10 mL ACN. • Shake the mixture vigorously in a Vortex for 1 min. • Add the following salt mixture: • 4.0 6 0.2 g of anhydrous magnesium sulfate • 1.00 6 0.05 g sodium chloride • 1.00 6 0.05 g sodium citrate dihydrate • 0.50 6 0.03 g disodium hydrogen citrate sesquihydrate • Shake the mixture vigorously in a Vortex mixer for 1 min. • Centrifuge the mixture at 4000 rpm for 5 min. • Weigh 150 mg PSA in a 15 mL centrifuge tube and add 900 mg anhydrous magnesium sulfate. • Measure 6 mL aliquots from the upper ACN phase and transfer to 15 mL centrifuge tubes. • Shake the mixture vigorously in a Vortex mixer. • Centrifuge at 4000 rpm for 6 min. • Filter the final extract through 0.45 μm filtration units with disposable syringes directly into liquid chromatography vials. Instrumental analysis The extracts obtained are ready for instrumental analysis using the method described in “Analysis of pesticide residues by HPLC MS/MS in agricultural products” analytical method number 3 in this manual.

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10. Quality control: The analytical batch includes • one reagent blank • two (2) replicates from each sample to be analyzed • matrix blank fortified for calibration curve levels and the detection limit The results of recovery obtained from a low- and a high-level spike should be considered as controls of the method performance. 11. Remarks: Processing or milling of the sample should be done in the cold to prevent possible degradation of the pesticide by heating. 12. Interferences, troubleshooting, and safety: Products with a high content of pigments, fats, acids, and sugars require addition of activated carbon, C18 or greater amount of PSA in the cleaning step. 13. References: AOAC Official Method 2007.01. Pesticide Residues in Foods by Acetonitrile Extraction and Partitioning with Magnesium Sulfate. Gas Chromatography/Mass Spectrometry and Liquid Chromatography/Tandem Mass Spectrometry First Edition 2007. ,http://quechers.cvua-stuttgart.de/index.php?nav1o 5 2&nav2o 5 1&nav3o 5 0.. EURL-FV. Multiresidue Method using QuEChERS followed by GC-QqQ/MS/MS and LC-QqQ/MS/MS for fruits and vegetables. ,http://www.crl-pesticides.eu/library/docs/fv/CRLFV_Multiresidue_ methods.pdf.. SANTE/11495/2015 Guidance document on analytical quality control and method validation procedures for pesticide residues analysis in food and feed. ,http://ec.europa.eu/food/plant/docs/ plant_pesticides_mrl_guidelines_wrkdoc_11945_en.pdf.. 14. Minimum method validation data: Range of matrices: The current method is recommended for nonionic pesticide determination in fresh fruits and vegetables with high water content. Range of validation: LOD: LOQ: Recoveries: CV %

0.010.1 mg/kg 0.0010.01 mg/kg 0.010.02 mg/kg 70%120% ,20

34

Analytical Methods for Agricultural Contaminants

METHOD 3: Analysis of pesticide residues by HPLC MS/MS in fresh fruits and vegetables 1. Laboratory name and address: Laboratorio Nacional de Insumos Agricolas-(Lania), Km 14 Via Bogota Mosquera, Mosquera, Colombia 2. Contact person: Edwin Samir Barbosa Angel email: [email protected] 3. Title of the analytical method: Analysis of pesticide residues by HPLC MS/MS in fresh fruits and vegetables 4. Principle: Extracts of fresh fruits and vegetables are analyzed by injecting 10 μL into a liquid chromatograph coupled to a mass spectrometer (HPLC-MS /MS) with electrospray ionization in positive mode. Initially the chromatographic system separates the compounds and subsequently the mass spectrometer quantifies and confirms the identity of the analytes. A precursor ion for each analyte is selected in the first quadrupole and the ion is refragmented in the second quadrupole to produce daughter ions. A specific or set of daughter ions for each precursor is analyzed in the third quadrupole. The identified pesticides are quantified by external standard calibration. The system has been established and standardized for each analyte including precursor ions, daughter ions, and the corresponding collision energies. 5. Scope: The method is recommended for nonionic pesticide determination in fresh fruits and vegetables. 6. Equipment and instruments: • HPLC MS MS triple quadrupole chromatograph • Analytical balance • Solvent filtration equipment • Freezer at 220 C • Freezer 210 C to 240 C • Nitrogen generator • HPLC-MS/MS chromatograph • Micropipettes fixed volume of 10, 20, 50, 100, 200, and 5001000 μL • Refrigerator • Vortex

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• Chromatographic column Zorbax C18 150 mm 3 3 mm 3 3.5 μm • 2 mL disposable syringes • 2, 5, 10, and 50 mL volumetric flasks • 2, 5, 10, and 50 mL volumetric flasks • 0.45 or 0.22 μm PVDF membrane filtration • Microspatulas • Balance • Precolumn • Analytical column C18 5 μm 3 30 mm 3 3.2 mm • Tips for 5200 and 2001000 μL micropipettes • 0.45 or 0.22 μm PVDF filtration units • Liquid chromatography equipment vials with septa and caps 7. Reagents and materials: • ACN, HPLC grade • Formic acid, residue analysis grade, purity .95% • Water, HPLC grade • Ammonium formate, residue analysis grade • Nitrogen (nitrogen generator) • Tuning solution • Triphenyl phosphate (TPP), .95% purity • Pesticide standards with certified analysis and associated uncertainty 8. Standard solutions: Preparation of the mobile phases Mobile Phase A: 5 mM aqueous solution HCOONH4 0.01% HCOOH; weigh 0.324 g ammonium formate and dissolve slowly with 900 mL type I water. Add 100 mL formic acid, shake and bring up to 1 L. Filter the solution with 0.45 μm filter. Register and label the solution as Phase A. Mobile Phase B: acetonitrile/Phase A (95:5, v-v). Take 50 mL of Phase A and bring up to 1 L with ACN. Filter the solution with 0.45 μm filter. Register and label the solution as Phase B. Preparation of TPP control solution TPP solution at 1000 mg/L: weigh 10 6 0.5 mg of TPP, bring to 10 mL with ACN and shake, register, and label. TPP solution at 20 mg/L: take 200 mL of the solution at 1000 mg/L and transfer to a 10 mL flask, bring to 10 mL with ACN and shake, register, and label. Control solution (TPP solution of 0.5 mg/L): take 50 mL of a 20 mg/L solution and transfer to a 2 mL flask, bring up to 2 mL with ACN and shake, register, and label.

36

Analytical Methods for Agricultural Contaminants

Control of the equipment: Control of the system is done by injecting regularly TPP at 0.5 mg/L and checking that no change is observed during time for the chromatographic working conditions for TPP. Register the initial pressure in a control chart, the retention time of the TPP and the transition ions response ratio. 9. Detailed procedure (protocol): Preinstrumental • Prepare the mobile phases and TPP control solution. • Turn on the HPLC-MS/MS. • Perform test equipment calibration mass (tuning) following instructions. • Record and save the calibration test. • Load the instrumental method. • Run the TPP and evaluate the condition of the equipment (see Annexes 3.1 and 3.2). • Check that the retention time and the area ratio of the transitions are between the tolerances allowed. Instrumental • Load the working pesticide method. • Check the chromatographic (Annex 3.1) and spectrometric conditions or the method (Annex 3.2) • Create the sequence (work list) of the samples. • Inject the vials in the following sequence: • Reagent blank • Matrix blank extract • Sample replicates extract • Matrix calibration curve • Analyze the reagent blank and matrix blank chromatograms to rule out possible interferences. Record if there is any possible interference. • Review the chromatograms of the injected extracts to determine the possible presence of pesticides. • Compare the results of the replicate samples verifying the retention time (Rt), the two transition ions, and the transition responses ratios. 10. Calculations: Pesticide concentrations are determined by interpolation using a linear weighted regression line obtained from the calibration curve of the standards in the linear range.

Integrated Analytical Approaches for Food Safety and Environmental Sustainability

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  C 3ðInterpolated concentration in the curve ðmg=LÞ  ACN volume ðLÞ C mg=kg 5 Sample weight ðkgÞ % Recovery 5

Experimental concentration ðmg=kgÞ 3 100 Theoretical concentration ðmg=kgÞ

11. Quality control: The analytical batch includes • One reagent blank • Two (2) replicates of each sample • Matrix blank fortified for calibration curve levels and the detection limit. The results of recovery obtained from a low and a high level should be considered as controls of the method performance and recorded in a control chart. 12. Remarks: It is important to set up the system and to standardize for each analyte the precursor ions, the transition ions, and the corresponding collision energies and other voltages. If the samples are giving results outside the linear working calibration range, they should be diluted in order to have the residues within the linear range. In this case, the dilution factor should be taken into account in determining the final residue value. The reported pesticide concentration (mg/kg) in the sample is the average of the concentration results obtained for the analytes in replicate samples. 13. References: AOAC Official Method 2007.01. Pesticide Residues in Foods by Acetonitrile Extraction and Partitioning with Magnesium Sulfate. Gas Chromatography/Mass Spectrometry and Liquid Chromatography/Tandem Mass Spectrometry First Action 2007. ,http://quechers.cvua-stuttgart.de/index.php?nav1o 5 2&nav2o 5 1&nav3o 5 0.. EURL-FV. Multiresidue Method using QuEChERS followed by GC-QqQ/MS/MS and LC-QqQ/MS/MS for fruits and vegetables. ,http://www.crl-pesticides.eu/library/docs/fv/CRLFV_Mμltiresidue_ methods.pdf.. SANTE/11495/2015 Guidance document on analytical quality control and method validation procedures for pesticide residues

Analytical Methods for Agricultural Contaminants

38

analysis in food and feed. ,http://ec.europa.eu/food/plant/docs/ plant_pesticides_mrl_guidelines_wrkdoc_11945_en.pdf.. 14. Minimum method validation data: Range of matrices: The current method is recommended for nonionic pesticide determination in fresh fruits and vegetables with high water content. Range of validation: LOD: LOQ: Recoveries: CV

0.010.1 mg/kg 0.0010.01 mg/kg 0.010.02 mg/kg 70%120% ,20

15. Annexes: Annex 3.1 Chromatographic conditions Equipment HPLC 1200 Agilent QQQ 6400

Precolumn

Column

Stationary phase Length Internal diameter Particle diameter Stationary phase Length Internal diameter Particle diameter

C18 30 mm 3.2 mm 5 μm C18 150 mm 3 mm 3,5 μm

Injection volume

10 μL

Column flow

0.5 mL/min

Mobile phase A

5 mM HCOONH4 0.01% HCOOH aqueous solution

Mobile phase B

ACN/Phase A (95:5; v:v)

Gradient

Run time

Time (min)

%B

Flow (mL/min)

0.00 1.00 16.00 22.00 22.01 30.00 30 min

5 5 100 100 5 5

0.5 0.5 0.5 0.5 0.5 0.5

Annex 3.2 Spectrometric conditions for triphenyl phosphate (TPP) and other pesticides Pesticide CAS number MW Ionization Precursor Fragmentor Product mode ion voltage (V) ion 1

TPP Methamidophos Aldicarb sulfoxide Aldicarb sulfone Methomyl Carbendazim 3 OH Carbofuran Dimethoate Aldicarb Carbofuran Carbaryl Metalaxyl Clomazone Methiocarb Azoxystrobin Diflubenzuron Iprodione Spinosad Fipronil Difenoconazole Trifloxystrobin Profenofos Chlorpyrifos Carbosulfan

Collision energy (V)

Product ion 2

Collision energy (V)

Rt (min)

115-86-6 10265-92-6 1646-87-3

326.3 141.1 [M 1 1] 206.26 [M 1 1]

327 142.0 207.1

80 80 80

275 125 132

20 10 5

152 94 89

15 15 5

1.95 6.78

1646-88-4 16752-77-5 10605-21-7 16655-82-6

222.26 162.2 191.2 237.2

[M 1 1] [M 1 1] [M 1 1] [M 1 1]

223.1 163.1 192.1 238.0

80 80 90 80

148 106.0 160.1 220.0

5 5 20 10

76 88.0 132.1 163.0

5 5 25 15

7.28 8.00 9.04 9.27

60-51-5 116-06-3 1563-66-2 25-Feb 57837-19-1 81777-89-1 2032-65-7 1318660-33-8 35367-38-5 36734-19-7 131929-63-0 120068-37-3 119446-68-3 141517-21-7 41198-08-7 2921-88-2 55285-14-8

229.3 190.26 221.3 201.2 279.3 239.7 225.3 403.4 310.68 330.2 745.99 437.2 406.3 408.37 373.6 350.6 380.5

[M 1 1] [M 1 1] [M 1 1] [M 1 1] [M 1 1] [M 1 1] [M 1 1] [M 1 1] [M 1 1] [M 1 1] [M 1 1] [M 1 NH4] [M 1 1] [M 1 1] [M 1 1] [M 1 1] [M 1 1]

230.0 116.1 222.1 202.1 280.2 240.1 226.1 404.1 311 330.0 746.5 454.0 406.1 409.1 372.9 351.7 381.2

80 80 120 80 120 120 80 120 99 120 140 70 160 120 120 80 80

199.0 89 165.1 145.1 220.1 125 169.1 372.1 158 288 142.1 436.9 337.0 206.1 344.9 323.8 160.1

5.0 5 10 4 10 20 5 10 8 10 35 4 15 10 10.0 10 15

171.0 70.1 123.0 127.1 192 89 121.1 344.1 141 245 98 368 251.0 186.1 302.9 200.0 118.1

10.0 5 15 28 15 30 10 15 32 10 55 20 20 15 15.0 10 15

9.72 11.00 12.27 12.64 12.99 13.95 14.24 14.61 15.20 15.39 15.64 15.87 16.53 17.23 17.57 18.40 20.858

Analytical Methods for Agricultural Contaminants

40

METHOD 4: Pesticide residue determination in potato using the dispersive QuEChERS template with ammonium acetate 1. Laboratory name and address: Grupo de Ana´lisis de Contaminantes Trazas (GACT), Ca´tedra de Farmacognosia, Facultad de Quı´mica, General Flores 2124 and Polo Agroalimentario y Agroindustrial de Paysandu´ (PAAP), Ruta 3 Km 393, Centro Universitario de Paysandu´, Universidad de la Repu´blica (UdelaR) 2. Contact persons: Horacio Heinzen Marı´a Vero´nica Cesio Lucia Pareja

email: [email protected] email: [email protected] email: [email protected]

3. Title of the analytical method: Pesticide residue determination in potato using the dispersive QuEChERS template with ammonium acetate 4. Principle: Dispersive extraction of pesticide residues based on a QuEChERS template with acetonitrile and ammonium acetate (ACN/(NH4)Ac) as extractive media. 5. Scope: This method is validated for 50 LC amenable pesticides as described in Table 3.2. 6. Responsibilities: The analyst is responsible for following the protocol and report any deviations. 7. Equipment and instruments: • Analytical balance • Vortex mixer • Centrifuge and centrifuge tubes • High-precision pipette • Vials: amber glass, 2 mL capacity with TFE-fluorocarbon-lined screw-caps • HPLC-MS/MS: ESI (6) • LC conditions: Column eclipse XDB-C18, Agilent Zorbax C18, 5 μm particle size, 4.6 3 150 mm • Gradient, see Table 3.2 • Flow: 0.6 mL/min • Volume injection: 5 μL

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Table 3.2 Scope of the method, selected transitions, and operational parameters Pesticide Precursor Fragment DP CE CXP ESI ion (m/z) ion (m/z) (eV) (eV) mode

2,4-Dimethyl-phenylformamidine 2,4-Dimethyl-phenylformamidine Acetamiprid Acetamiprid Amitraz Amitraz Azoxystrobin Azoxystrobin Boscalid Boscalid Boscalid Buprofezin Buprofezin Carbaryl Carbaryl Carbendazim Carbendazim Carbofuran Carbofuran Chlorpyrifos ethyl Chlorpyrifos ethyl Clorfenvinfos Clorfenvinfos Clothianidin Clothianidin Coumaphos Coumaphos Diazinon Diazinon Difenoconazole Difenoconazole Dimethoate Dimethoate Epoxiconazole Epoxiconazole Epoxiconazole Epoxiconazole

163.1

105.1

135

20

10

ESI pos

163.1

127.1

135

19

10

ESI pos

223.2 223.2 294 294 404.1 404.1 343.1 343.1 345 306.1 306.1 202.2 202.2 192.1 192.1 222.1 222.1 349.9 349.9 360.3 360.3 250 250 363.1 363.1 304.2 304.2 406 406 230.1 230.1 330.1 330.1 330.1 330.1

126.1 99.2 163.1 122.1 372.1 344 139.8 112.2 271.3 201 116 145 127.1 160 132.1 165.1 123.1 198.1 97 99 155 169 132 307 227.1 199.2 111.2 337 251.1 198.9 125.1 121.3 70.2 141.1 101.2

55 55 100 100 72 72 89 89 89 100 100 68 68 80 80 102 102 80 80 80 80 56 56 100 100 80 80 90 90 50 50 75 100 100 100

25 47 19 37 19 31 24 57 39 17 25 12 35 22.8 41.9 12 31 23 38 31 20.8 17 19 22 33 15.2 20.9 21.4 37.2 13 28 27 48 23 65

10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10

ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI

pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos

(Continued)

Analytical Methods for Agricultural Contaminants

42 Table 3.2 (Continued) Pesticide

Ethion Ethion Fenhexamide Fenhexamide Fipronil Fipronil Flusilazole Flusilazole Flutriafol Flutriafol Hexythiazox Hexythiazox Imazalil Imazalil Imidacloprid Imidacloprid Iprodione Iprodione Kresoxim-methyl Kresoxim-methyl Mepanipyrim Mepanipyrim Malaoxon Malaoxon Malathion Malathion Malathion Methamidophos Methamidophos Methamidophos Methidathion Methidathion Methiocarb Methiocarb Methomyl Methomyl Omethoate Omethoate Oxadixyl

Precursor ion (m/z)

Fragment ion (m/z)

DP (eV)

CE (eV)

CXP

ESI mode

406.9 406.9 302 304 434.9 436.9 316.1 316.1 302 302 353.1 353.1 297 298.9 256.1 256.1 330.1 330.1 315 315 224 224 315.1 315.1 331.2 331.2 331.2 142 142 142 303 303 226.2 226.2 163.1 163.1 214 214 279.1

171.1 199 97 97 329.8 331.8 247.2 165.3 70.1 123.3 168.1 228.1 159 161.1 209.1 175.1 245.1 288 135.2 256.9 77.1 106 127 99.1 127 284.9 99 112.1 93.9 124.8 144.9 85 169.1 121.1 88 105.9 183 155 219.1

30 30 120 120 247 247 100 100 100 100 70 70 130 130 86 86 64 64 100 100 55 55 97 97 200 56 56 51 51 51 49 49 78 78 44 44 70 70 90

38.3 22.9 34 34 224 225 26 40 35 37 34 23 32 30 22 23 21 16 17 8 55 34.7 17 29 60 11 35 19 19 21 13 29 13 26 12 14 17 22 12

10 10 10 10 210 210 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10

ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI

pos pos pos pos neg neg pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos

(Continued)

Integrated Analytical Approaches for Food Safety and Environmental Sustainability

Table 3.2 (Continued) Pesticide

Oxadixyl Parathion methyl Parathion methyl Parathion methyl Pirimiphos-methyl Pirimiphos-methyl Procloraz Procloraz Propiconazole Propiconazole Pyraclostrobin Pyraclostrobin Pyrimetanil Pyrimetanil Pyriproxifen Pyriproxifen Spiroxamine Spiroxamine Tebuconazole Tebuconazole Thiacloprid Thiacloprid Thiamethoxam Thiamethoxam Tiabendazole Tiabendazole Trifloxystrobin Trifloxystrobin Triflumuron Triflumuron Triflumuron Triflumuron

43

Precursor ion (m/z)

Fragment ion (m/z)

DP (eV)

CE (eV)

CXP

ESI mode

279.1 264 264 264 306.1 306.1 376 376 338.9 338.9 388.1 388.1 200 200 322.2 322.2 298.1 298.1 308.1 308.1 253.1 255 292 292 202.1 202.1 409 409 357.3 357.3 356.9 356.9

102.1 124.9 231.9 79 108.1 164.1 308 266 118.9 182.9 194.2 163.1 107.2 168.2 185.1 227.1 144.2 100.2 125 70.3 126 128 211.1 181.2 175.1 131.1 186 206 239.1 121.1 175.9 153.9

90 71 71 71 130 130 78 78 130 130 67 67 40 40 50 50 90 90 85 85 98 98 88 88 100 100 70 70 77 77 275 275

12 27 23 23 42 31 15 24 79 47 17 39 31.1 36.9 30.2 19.5 28 40 45 40 28 25 15 29 34 42.9 20.8 18.1 11 14 234 218

10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 210 210

ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI ESI

pos pos pos pos pos pos pos pos neg neg pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos neg neg

CE, collision energy; CXP, collision cell exit; DP, declustering potential; ESI, electrospray ionization.

Mobile phase A: Water 0.1% formic acid B: ACN The gradient is described in Table 3.3. Injection volume (μL): 5

Analytical Methods for Agricultural Contaminants

44

Table 3.3 HPLC solvent gradient and flow rate Total time (min) Flow rate (µL/min)

A (%)

0 1 15 25 25 35

90 90 0 0 90 90

600 600 600 600 600 600

Source parameters: Interface turbo pump Analyzer turbo pump Sample introduction status Source/ion path electronics Source type Source temperature (at setpoint) Source exhaust pump Interface heater

Normal Normal Ready On Turbo spray 425 C Ok Ready

Acquisitions parameters: Scan type Scheduled MRM Polarity Scan mode Ion source MRM detection window Target scan time: Resolution Q1 Resolution Q3

MRM (MRM) Yes Positive and negative separately N/A Turbo spray 70 s 10,000 s Unit Unit

8. Reagents and materials: • ACN, HPLC quality • Pesticide standards prepared in ACN at a concentration of 100 mg/mL. • Ammonium acetate (CH3COONH4) • Magnesium sulfate anhydrous 9. Standard solutions: Stock solutions of individual analytes at 1000 mg/L were prepared in ACN; three mixed standard stock solutions were prepared and serially diluted with ACN to produce a series of working standard solutions of 0.00520 mg/L. The latter solutions were used to

Integrated Analytical Approaches for Food Safety and Environmental Sustainability

45

construct the calibration curves and to prepare the fortified samples. Stock solutions and the working standard solutions were stored in a deep freeze (223 C), and renewed at weekly intervals. Matrixmatched calibration solutions (0.0051 μg/mL) were prepared by drying 1.0 mL potato extracts under a N2 stream and fortified with 1.0 mL working standard solutions of pesticides at various concentrations. These matrix-matched solutions were used to prepare calibration curves, to evaluate the linear range, and to calculate recoveries. 10. Detailed procedure (protocol): • In a 50 mL capped Teflon tube suspend 10 g of the homogenized sample in 10 mL of 1% HAc in ACN. • Shake the suspension manually and vigorously for 2 min and add 4 g MgSO4 plus 1 g CH3COONH4. Shake manually the extract for 4 min and centrifuge at 5000 rpm for 5 min. • Take a 1 mL aliquot from the supernatant and transfer to a 2 mL autosampler vial for LC-MS/MS analysis. • Prepare calibration standards at five concentration levels by adding accurately measured volumes of each pesticide stock standard solution to volumetric flasks and diluting to volume with an appropriate solvent. One of the external standards should be representative of a concentration near, but above, the method detection limit. • The other concentrations should correspond to the range of concentrations expected in the sample or should define the working range of the detector. • Using injections of 5 μL of each calibration standard, tabulate peak height or area responses against the concentration injected. The results can be used to prepare a calibration curve for each pesticide. 11. Calculations: Determine the pesticide residue concentration in the sample. Calculate the concentration of material injected from the peak response using the calibration curve. Area i 5 ½Slope 3 ðP Þi 1 Intercept ðP Þi 5

Area i 2 Intercept Slope

where (P)i 5 pesticide concentration in the vial extract in ng/mL. The concentration of pesticide residues in the sample (Cs) is expressed in mg/kg. It is calculated using the equation below

46

Analytical Methods for Agricultural Contaminants

considering the amount of pesticide residues in 10 mL extract, and the amount of sample weighted (Mi) in g. Csðng=gÞ 5 ðPiðng=mLÞ  10ðmLÞÞ=MiðgÞ 12. Quality control: Each laboratory using this method is required to operate within a formal quality control program. The minimum requirements for this program consist of an initial demonstration of laboratory capability and the analysis of spiked samples as a continuing check on performance. The laboratory is required to maintain performance records to define the quality of data generated. The developed methodology was fully validated at three concentration levels: 10, 50, and 250 μg/kg, with 5 replicates and the evaluated parameters were those defined by the SANCO document for determination of pesticide residues in food and feed. The HPLC operating parameters were established as those indicated in Table 3.1. The working calibration curve was verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varied from the predicted response by more than 6 10%, the test was repeated using a fresh calibration standard. Alternatively, a new calibration curve was prepared. The laboratory must spike and analyze a number of control samples corresponding to a minimum of 10% of all samples to monitor continuously the performance. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as chromatography with a dissimilar column, another detection system or a different sample preparation procedure should be employed. Whenever possible, the laboratory should participate in relevant performance evaluations, such as proficiency tests. 13. Remarks: Most of the studied pesticides presented detection and quantification limits (LOD and LOQ) below 10 μg/kg except for some compounds such as amitraz, chlorpyrifos, flutriafol and parathion-methyl which showed higher LOQs. 14. Interferences, troubleshooting, and safety: From the evaluated pesticides only chlorpyrifos, coumaphos, hexythiazox, omethoate, and thiabendazole presented a matrix effect greater than 25%.

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47

Some compounds such as parathion methyl, propiconazole, and spiroxamine could not be recovered with the methodology described. 15. References: ˇ Anastassiades, M., Lehotay, S.J., Stajnbaher, D., Schenck, F.J., 2003. J. AOAC Int. 86, 412431. Document N SANTE/11495/2015. Method Validation and Quality Control Procedures for Pesticide Residues Analysis in Food and Feed 2011. EURL-DataPool. European Reference Laboratories for Pesticide Residues. ,http://www.eurl-pesticides-datapool.eu.. Garrido Frenich, A., Martı´n Ferna´ndez, M.M., Dı´az Moreno, L., Martı´nez Vidal, J.L., Lo´pez-Gutie´rrez, N., 2012. J. AOAC Int. 95, 13191330. 16. Minimum method validation data: Range of matrices: potato Range of validation: 0.0051.00 mg/kg LCL: 0.005 mg/kg Recovery range: 80%119% CV: ,15%

METHOD 5: Multiresidue method for pesticides in potato using by QuECHERS and GC-MSD detection 1. Laboratory name and address: Food and Environmental Protection Laboratory (FEPL), Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna International Centre, PO Box 100, 1400 Vienna, Austria 2. Contact persons: Britt Maestroni email: [email protected] Victoria Ochoa email: [email protected] 3. Title of the analytical method: Multiresidue method for pesticides in potato using by QuECHERS and GC-MSD detection 4. Principle: Processed and homogenized samples are extracted with acidified ACN. A mixture of anhydrous magnesium sulfate, sodium chloride and sodium citrate salts is added for pH adjustment and phase separation. After shaking and centrifugation, an aliquot of the organic phase is

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Analytical Methods for Agricultural Contaminants

cleaned up adding PSA and anhydrous magnesium sulfate. The cleanedup extract is concentrated over nitrogen and redissolved in ethyl acetate. The final extract is filtered and injected for analysis by GC-MSD. 5. Scope: The current method is applicable to the determination of GC amenable pesticides in potato: azoxystrobyn, benalaxyl, alfa endosulfan, beta endosulfan, endosulfan sulfate, bromopropylate, chlorothalonil, chlorpyrifos methyl, chlorpyrifos, deltamethryn, diazinon dimethoate, ethoprophos, fipronil, fludioxinil, flutolanil, kresoximmethyl, metalaxyl, methidathion, parathion methyl, pirimiphos ethyl, pirimiphos methyl, propyzamide, pyraclostrobyn, pyrimethanil, tebuconazole, tolclofos methyl, and vinclozolin. 6. Equipment and instruments: • Dispenser bottle with variable volume 525 mL • Spatulas • 50 mL tubes • 15 mL Amber bottles with cap • Volumetric flasks • Microspatulas • Volumetric pipettes • Centrifuge tubes with cap • Evaporation tubes • 0.22 μM filtration discs • Gas chromatography vials • Freezer at 220 C • Food processor • Analytical balance 6 0.0001 g • High-speed centrifuge with adapters required for 50 and 15 mL tubes • Ultrasonic bath • Oven to operate at 500 C • Automatic shakers for tubes • Vortex mixer • Nitrogen evaporation station • Gas chromatograph coupled to mass selective detector (GC-MSD) 7. Reagents and materials: • Standards of pesticides .96% purity with certified and uncertainty associated • ACN, chromatographic grade 99.9%

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• Ethyl acetate, chromatographic grade 99.9% • Sodium citrate dihydrate A.R. • Sodium chloride A.R. • Sodium hydrogencitrate sesquihydrate A.R. • PSA, Varian # 12213024 • Magnesium sulfate anhydrous A.R. 8. Standard solutions: • Pesticide mixture stock solution: prepared in acetone: isooctane (15:85, v-v) at concentration 2 mg/mL and stored at 220 C. The stock solutions were stable for 6 months. • Internal standard stock solution: PCB 18 prepared in ethyl acetate at concentration 1 mg/mL and stored at 220 C. The stock solutions were stable for 6 months. The working solution was prepared freshly. • Surrogate standard: triphenyl phosphate (TPP) prepared in ACN at concentration 1 mg/mL and stored at 220 C. The stock solutions were stable for 6 months. The working solution was prepared freshly. • Pesticide mixture solution prepared at 25 ng/μL, stored in freezer and stable for 3 months. Dilutions of the pesticide mixture at 5 and 0.5 ng/μL are prepared on the day of analysis and used for preparation of matrix matched calibrators. • Prepare 510 calibration levels and inject in duplicate. Suggested calibration range is from 0.003 to 1.5 ng/μL. 9. Detailed procedure (protocol): • Chop, process, and homogenize the sample. • Weigh 10 6 0.1 g in a 50 mL centrifuge tube. • Add 10 mL of acidified ACN containing the surrogate internal standard TPP at 0.2 ng/μL (prepare the acidified ACN mixture as follows: add 0.5 g ascorbic acid dissolved in 10 mL water, add 10 mL glacial acetic acid, and bring to 1 L with ACN). • Shake vigorously in a Vortex for 1 min. • Add to following mixture of salts: • 4.0 6 0.2 g anhydrous magnesium sulfate • 1.00 6 0.05 g sodium chloride • 1.00 6 0.05 g sodium citrate dihydrate • 0.50 6 0.03 g disodium hydrogen citrate sesquihydrate • Shake vigorously in a Vortex mixer for 1 min and then place on a horizontal shaker and shake at high speed for 30 min.

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• • • • • • •





• •

Centrifuge the mixture at 10,000 rpm for 5 min at 18 C. Weigh 150 mg PSA in a 15 mL centrifuge tube and add 900 mg anhydrous magnesium sulfate. Measure a 6 mL aliquot of the upper solvent phase and transfer to the 15 mL centrifuge tube. Shake the mixture vigorously in a Vortex mixer. Centrifuge at 11,000 rpm for 5 min at 18 C. Take 5 mL of the clean-up extract and evaporate to dryness using a nitrogen evaporator. Reconstitute all extracts with acidified ethyl acetate to a defined final volume (to prepare acidified ethyl acetate add 0.5 g ascorbic acid dissolved in 10 mL water, add 10 mL glacial acetic acid, and bring to 1 L volume with ethyl acetate). Prepare the matrix-matched calibration standards by adding the proper pesticide mix standard amount and the internal standard and bring to the same final volume as in the sample extracts. Transfer the final extract through a 0.22 μm filtration unit with a disposable syringe directly into gas chromatography vial with inserts. Transfer to GC/MS vial. MSD analysis condition: • Inlet mode splitless, 1.0 μL injected • Inlet temperature 250 C • Pressure 2025 psi (chlorpyrifos-methyl RT relocked to 18.51 min) • Purge flow 40.0 mL/min • Purge time 1 min • Total flow 54.7 mL/min • Gas saver 20 mL/min • Gas type—helium • Inlet liner splitless, single-taper, deactivated • Oven temperature program: Initial temperature 70 C for 1.5 min Ramp 40 C/min to 110 C not hold Ramp 6 C/min to 260 C not hold Ramp 8 C/min to 280 C hold for 2.75 min Total run time 40.5 min (last standard elutes around 35 min) Equilibration time 0.5 min • Column HP 5 MS, 30 m 3 0.25 mm, 0.25 μm

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• Acquisition mode: concurrent SIM and SCAN mode • Electron energy—70 eV • EMV mode: gain, gain factor: 5 • Quad temperature 150 C • Source temperature 230 C • Target and qualifier ions are listed in Table 3.4. 10. Calculations: A minimum of a five-point calibration curve is constructed with matrix-matched calibration standards and an internal standard is applied to correct for volumetric losses and recovery variation. A weighted linear calibration model is applied. Table 3.4 Target and qualifier ions Compound TG

Q1

Q2

Azoxystrobyn Benalaxyl beta endosulfan Bromopropylate Chlorothalonil Chlorpyrifos methyl Chlorpyrifos Deltamethryn Diazinon Dimethoate Ethoprophos Fipronil Fludioxinil Flutolanil Kresoxim-methyl Metalaxyl Methidathion Parathion methyl PCB 18 Pirimiphos ethyl Pirimiphos methyl Propyzamide Pyraclostrobyn Pyrimethanil Tebuconazole Tolclofos methyl TPP Vinclozolin

388 91 207 185 264 288 97 253 137 93 43 213 127 145 131 206 367 125 256 333 276 175 164 199 250 125 325 198

345

344 148 195 341 266 286 197 181 179 87 158 367 248 173 116 45 145 109 186 318 290 173 132 198 125 265 326 212

Q3

159 183 268 314 77 199 125 126 154 281 206 132 213 263

198 304 97

233

304 145 44 70 267 187

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52

11. Quality control: In each batch of analysis one recovery sample fortified at 0.1 mg/kg and run through the extraction procedure is injected at the beginning and at the end of each run. Blank sample, reagent blank, and solvents are also prepared and introduced in the batch in a randomized way. 12. Remarks: Processing or milling of the sample should be done under cryogenic (e.g., using liquid nitrogen or dry ice) conditions to prevent possible degradation of the pesticides. The processed sample should be kept frozen until analysis. To eliminate interference from phthalates and humidity, condition the MgSO4 in a furnace to 550600 C for 5 h before use. 13. Interferences, troubleshooting, and safety: Label all glassware to be used before starting the procedure. Strictly follow safety regulations related to general laboratory operations. 14. References: ,http://quechers.cvua-stuttgart.de/index.php?nav1o 5 2&nav2o 5 1&nav3o 5 0.. 15. Minimum method validation data: Range of matrices: The current method is recommended for potato samples Range of validation: Recoveries: LOD: CVR

0.010.5 mg/kg 70%103% 0.0040.01 mg/kg ,20%

METHOD 6: Processing and extraction of fruits and vegetables with possible pesticide residue content 1. Laboratory name and address: Instituto Nacional de Tecnologı´a Industrial (INTI), Centro Neuque´n, Av Gral Paz 5445-San Martin-Buenos Aires, Argentina 2. Contact person: Patricia Ohaco email: [email protected] 3. Title of the analytical method: Processing and extraction of fruits and vegetables with possible pesticide residue content 4. Principle: Mini Luke liquidliquid phase extraction method.

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5. Scope: The method was validated for carrots. Analytes covered by the method are: acephate, a-HCH, dimethoate, B-HCH, chlorpyrifos, phosphamidon, endosulfan I (alpha), dieldrin, endosulfan II (beta), ethion, and endosulfan sulfate. Applies to fruits and vegetables with fat content lower than 2% and moisture content greater than 30%. 6. Equipment and instruments: • Balance • Vacuum pump • Extraction hood • Food blender • High-speed blender • Rotavapor • Timer • Micropipette 1000 μL • 250 mL volumetric balloon • Spatula • Knife • Glass funnel • Buchner funnel • 50 mL amber bottle • Stainless steel tray • Vacuum flask • Absorbent paper • Filter paper • 25 mL pipette • Pasteur pipettes • Squeeze bottle containing acetone • Squeeze bottle containing distilled water • 100 mL test tube • 20 mL graduated cylinders • Pro-pipette for 25 mL pipette • Pro-pipette for Pasteur pipette • Two separatory funnels • Caps for separatory funnels • 2 mL vial 7. Reagents and materials: • Pesticide residue analysis grade acetone • Pesticide residue analysis grade dichloromethane

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• Pesticide residue analysis grade petroleum ether • Pesticide residue analysis grade normal hexane • Sodium sulfate, anhydrous, powder, granular • Sodium chloride 8. Detailed procedure (protocol): • The sample must be prepared conditioned before being processed, following the Codex Alimentarius guidelines. Avoid washing samples. • Cut the sample into small pieces with a knife on a stainless steel vessel. • Process the sample in a food chopper. • Weigh 50 g of the homogenized sample • Take 100 mL acetone in a graduated cylinder and transfer into the blender with the sample. Cover and blend vigorously for 5 min. • Place a filter paper, previously rinsed with acetone, on the Buchner funnel and apply the vacuum. The necessary vacuum for filtration is about 28 cm Hg. Filter the sample. • Transfer 16 mL acetone sample extract from the vacuum flask to a separatory funnel with a pipette, identified as Nr. 1, having previously checked that the stopcock is closed. • Transfer the remaining sample to a 50 mL amber bottle. Label and store the extract solution in the freezer. • Add 40 mL petroleum etherdichlorometane (1:1) (v:v) to the separatory funnel 1. • Shake for 1 min, taking care to open occasionally because the high volatility of the reagents can eject the cap causing damage. • Place the separatory funnel 1 in a clamp and allow to stand until two distinct layers are visible. • If an emulsion is generated, add 1 g sodium chloride to the funnel, shake again and let stand. • Transfer only the lower layer (aqueous phase) to a separating funnel, defined as Nr. 2, having previously checked that the stopcock is closed. • Transfer the remaining layer (organic phase) of separating funnel 1 to a 250 mL balloon by drying over 20 g sodium sulfate (placed over a filter paper into a glass funnel placed over the balloon). • Add 20 mL methylene chloride to the separating funnel 2. • Shake for 1 min, taking care to open occasionally because the volatility of the reagents can eject the tip causing damage.

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9.

10.

11.

12.

55

Place separatory funnel 2 in a clamp and allow to stand until two distinct layers are visible. • Transfer only the lower layer (now organic phase) to the 250 mL balloon as indicated above. • Add 20 mL methylene chloride to the remaining liquid in separator funnel 2 and shake for 1 min. Repeat as above. • Place the balloon with all the collected organic phase extracts in a rotary evaporator in a water bath at 35 C (acceptable variation of 6 1 C) with a rotation speed of around 75 rpm and the maximum possible flow rate of cooling water (keep all parameters constant). Caution: Do not allow to evaporate to dryness. • Concentrate to obtain a volume of 1 mL. • Extract the content of the balloon with a Pasteur pipette and transfer to vial. Rinse the balloon twice with n-hexane and transfer to vial. Dry the vial with a nitrogen flow. • Add 1000 μL of n-hexane with a micropipette. Cover and store in the freezer until analysis. The shelf life of the final extracts should not exceed 1 week. Calculations: Matrix concentration (mg/kg) 5 (Cv*1 mL*100 mL)/(16 mL*50 g) Cv 5 final concentration in the 1000 μL extract (mg/L) Quality control: To assure the performance of this method it is necessary to: • Perform this procedure in duplicate. • Analyze a fortified sample at a level of 1 mg/L simultaneously with the batch of samples. Interferences, troubleshooting, and safety: Analysts should be aware that dangerous substances are being used and therefore should know the safety rules for these substances References: Codex Alimentarius Volume 2, FAO, 1996. IRAM 301 - ISO/IEC 17025, Edition 2005. Laboratory training manual for pesticide residue laboratorios, AOAC International, 1998. Luke, et al., 1981. Improved multiresidue gas chromatographic determination of organophosphorus, organonitrogen, and organohalogen pesticides in produce, using flame photometric and electrolytic conductivity detectors. J. Assoc. Off. Anal. Chem. 64 (5), 11871195.

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Pesticide Analytical Manual, FDA, 1994. ,http://www.fda. gov/Food/FoodScienceResearch/LaboratoryMethods/ucm2006955. htm.. 13. Minimum method validation data (range of matrices, range of validation, LOQ or lowest calibrated level (LCL), recoveries, and CV%): Matrix: carrot LOQ: 1 mg/L Recovery: 82%108% CV: 3%5%

METHOD 7: Determination of selected pesticides in tomato samples by column extraction and gel permeation chromatography clean-up 1. Laboratory name and address: Food and Environmental Protection Laboratory (FEPL), Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna International Centre, PO Box 100, 1400 Vienna, Austria 2. Contact person: Ms Britt Maestroni email: [email protected] 3. Title of the analytical method: Determination of selected pesticides by a column extraction multiresidue method 4. Principle: A multiresidue method for determination of selected pesticide residues in tomato using florisil as dispersive matrix agent, gel permeation chromatography (GPC) and gas chromatographic determination. 5. Scope: Determination of dichlorvos, atrazine, chlorpyrifos methyl, chlorpyrifos ethyl, and chlorfenvinphos in tomato using a matrix solidphase dispersion agent such as florisil. 1. Equipment and instruments: • Food homogenizer • Warring blender • Top load balance, capable of weighing 100 g within 0.1 g precision

Integrated Analytical Approaches for Food Safety and Environmental Sustainability

• • • • • • • •

57

Glass columns for extraction Mortar and pestle Hamilton syringe 0.25 mL Automatic pipette 0.5 mL Rotary evaporator Nitrogen evaporator Fraction collector through GPC Gas chromatograph equipped with selective detectors (NPD/ MSD/ECD) 2. Reagents and materials: • Florisil, 60100 mesh. Florisil activated at 675 C. Florisil should be stored in closed glass containers. • Sodium sulfate (Na2SO4) anhydrous, pesticide residue grade • Ethyl acetate (EA), pesticide residue grade • Cyclo-hexane (C-hex), pesticide residue grade • Milli-Q water • Internal standard: triphenyl phosphate • GC column: HP 5, 30 m 3 0.25 mm, 0.25 μm 3. Standard solutions: a. Pesticide mixture stock solution: prepared in 15:85 (v:v) acetone: isooctane at concentration 1000 mg/L and stored at 220 C. The stock solutions were stable for 6 months. b. Internal standard triphenyl phosphate stock solution: prepared in isooctane at concentration 1000 mg/L and stored at 220 C. The stock solution was stable for 6 months. c. Pesticide mixture working solution: prepared after dilution of stock solution in isooctane. 4. Detailed procedures Sample preparation • For tomato remove stems and homogenize the whole commodity Sample processing • Process 2 kg matrix using a food homogenizer. • Transfer 200 g of ground/homogenized matrix into a Waring blender. • Thoroughly homogenize. • Sufficient blank matrix should be set aside for use in preparation of matrix matched standards.

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Extraction/clean-up procedure • Add 10 g florisil to each 5 g aliquot of homogenized material. • Thoroughly mix the florisil into the matrix by use of a pestle. Continue mixing until a uniform mix is obtained. • Column loading: place anhydrous sodium sulfate (corresponding to one full spoon) in an empty extraction column. Transfer powdery sample mixture onto the column in a uniform manner. Tap the glass column gently. • Rinse the mortar and pestle four times with 5 mL ethyl acetate (total 20 mL), pour the rinsing solvent onto column. • Adjust the flow rate of the eluate to 5 mL/min. • Complete the extraction by adding additional 50 mL ethyl acetate. • Collect all the eluates in a round bottom flask. • Quickly concentrate the extract to approximately 1 mL using a rotary evaporator. • Transfer to turbovap test tube by washing five times with 1 mL EA. Evaporate on turbovap down to 1 mL. Add 1 mL cyclohexane, mix the solvents, and evaporate to 0.81.0 mL. • Adjust the volume to exactly 1 mL with a mixture of cyclohexane:ethyl acetate (1:1) using a Hamilton syringe Gel permeation chromatography clean-up: • Ensure the GPC has been calibrated before use. Establish the volume to be discarded. Before injection of sample extract turn elution switch to “ON” position (ensure that the elution solvent is running). Run the eluting solvent through the column for at least 12 min before making an injection. Ensure syringe is carefully washed before and after injection to the GPC. Wash 10 times with acetone and 10 times with GPC solvent (EA:C-hex;1:1). • Adjust the flow rate to 1.52 mL/min making use of a stop watch. • Inject a 500 μL aliquot of the extract onto the top middle section of the column with a 500 μL Hamilton syringe. (Be careful: the injection should result in a well-defined plug in the column and the injected extract should not remain in the injector port.) • Attach a collector vessel for the first fractions. According to the established GPC calibration, collect the first fraction and discard. • Change the collector flask to a turbovap test tube and collect 22 mL eluent (subject to elution pattern, check if late eluting

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compounds are present in the 22 mL collected, otherwise use round-bottomed flask, collect 30 mL and proceed with concentration on rotary evaporator). • Evaporate the collected eluent to 0.5 mL on turbovap. Add 1.0 mL isooctane and evaporate again to 1 mL. Observe the test tubes closely to avoid evaporation to dryness. • Add 100 μL internal standard in the final fraction after GPC. Adjust the final volume with Isooctane to 2 mL. • Dilute the extract with 1:1 in isooctane/blank matrix (1.25 g/ mL) for GC-determination. Detection by gas chromatography a. GC-ECD: • splitless injection, 1 μL • Injector port T 5 250 C • Detector T 5 300 C • Oven: T col 5 70 C, hold 1 min, increase to 160 C at 20 C/ min, increase to 270 C at 4 C/min hold 1 min. b. GC-NPD: • splitless injection, 1 μL • Injector port T 5 250 C • Detector T 5 280 C • Oven: T col 5 70 C, hold 1 min, increase to 160 C at 20 C/ min, increase to 270 C at 4 C/min hold 1 min. 4. Calculations: A five-point calibration curve is constructed with matrixmatched calibrators and an internal standard is applied to correct for volumetric losses and recovery variation. A weighted linear calibration model is applied. 5. Quality control: In each batch of analysis one recovery sample fortified at 0.25 mg/kg should be injected at the beginning of the run and at the end of the run. Blank sample, reagent blank, and solvents are also introduced in the batch in a randomized way. 6. Interferences, troubleshooting, and safety: The laboratory is responsible for maintaining a safe work environment and a current awareness of material safety data sheet (MSDS) for the safe handling of the chemicals listed in this method. The MSDSs should be available to all personnel involved in these analyses.

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7. References: Column extraction multi-residue method from Kadenski, et al., 1992. J. AOAC Int. 75 (1). 8. Minimum method validation data: Range of validated matrices: tomato Validation range: 0.250.5 mg/kg Lowest calibrated level: 50 ng/mL Recovery range: 60%113% CV: ,20%

METHOD 8: Determination of carbamate pesticides residues in honey by HPLC with postcolumn derivatization and fluorescence detector (FLD) 1. Laboratory name and address: Chromatography Laboratory, Technological Research Centre of the Dairy Industry, National Institute of Industrial Technology (INTI), Avenida General Paz 5445—San Martı´n, Argentina 2. Contact persons: M. Alejandra Rodrı´guez email:[email protected] Pablo Sa´nchez Patricia Pappolla Patricia Gatti 3. Title of the analytical method: Determination of carbamate pesticide residues in honey by HPLC with fluorescence detection 4. Principle: This describes a method for analysis of pesticide residues in products with low water content, like honey. Water has to be added to enhance the solubility of all components. The extraction process uses MgSO4 and NaCl to promote the salting out phenomenon in which the carbamate pesticides in the sample are transferred to the acetonitrile phase. The clean-up stage uses PSA (primary/secondary amine) to remove the organic acid pigments and MgSO4 to reduce the water content. Finally, the extract is concentrated and filtered for subsequent analysis by HPLC-FLD detector and postcolumn derivatization. 5. Scope: This methodology is used to determine the following carbamate pesticides in honey: aldicarb, aldicarb sulfoxide, aldicarb sulfone, carbofuran, 3-hydroxy-carbofuran, and carbaryl.

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6. Equipment and instruments: • High-performance liquid chromatograph (HPLC) • Postcolumn derivatization equipment • Analytical balance • Evaporator • Vacuum pump • Sonicator • Water purifier • Vortex • Centrifuge • HPLC analytical column, Pickering, Carbamate Analysis—C18, 250 mm 3 4.6 mm 3 5 μm 7. Reagents and materials: Reagents • Magnesium sulfate heptahydrate, granular, p.r. (CAS N 1003499-8) • Purified water Mill-Q • Acetone P.A.R.P. (CAS N 67-64-1) • ACN, HPLC grade (CAS N 75-05-8) • Methanol, HPLC grade (CAS N 67-56-1) • Sodium chloride, P.A. (CAS N 7647-14-5) • PSA (primary/secondary amine) • OPA, chromatographic grade (CAS N 643-79-8) • Thiofluor, chromatography grade (CAS N 13242-44-9) • Standards (SIGMA) • Hydrolyzing solution 0.2% NaOH preparation: • Weigh 20 g NaOH and add water in a 100 mL flask. • Take 10 mL and dilute in a 1000 mL flask. • Filter the solution before use. • Hydrolyzing solution OPA preparation: • Weigh 4 g anhydrous sodium borate and dissolve with hot water in a 1000 mL flask. Cool and filter the solution before use. • Weigh 2 g thiofluor and dilute in 5 mL of the previous solution. • Weigh 100 mg solid OPA and dilute in 10 mL MeOH. Mix solutions with OPA diluent solution (945 mL). Materials: • Falcon tubes 15 and 50 mL • 50 mL beakers • 50 mL round bottomed flask

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Analytical Methods for Agricultural Contaminants

• 2 mL vials for HPLC • 10 mL pipette • Pasteur pipette • Volumetric pipettes (1, 5, and 10 mL) • Membrane filter PVDF 0.45 μm pore • Flasks (10, 25, 50, 100, and 1000 mL) • Graduate syringes for preparation of gravimetric solutions 8. Standard solutions: • Aldicarb—CAS No% : 116-06-3 • Aldicarb sulfone—CAS No% : 1646-88-4 • Aldicarb sulfoxide—CAS No% : 1646-87-3 • 3-Hydroxi-carbofuran—CAS No% : 16655-82-6 • Carbofuran—CAS No% : 1563-33-2 • Carbaryl—CAS No% : 63-25-2 9. Detailed procedure (protocol): • Weigh 5.0 6 0.1 g of honey sample (50 mL Falcon tubes). • Add 10 mL Milli-Q water and homogenize with a rod. • Add 10 mL ACN with volumetric pipette, wash the rod and remove. • Shake vigorously for 1 min. • Add 6.0 6 0.1 g MgSO4  7H2O and 2.0 6 0.1 g NaCl. • Shake vigorously for 1 min. • Centrifuge at 3000 rpm for 5 min. • Take an aliquot of 5 mL of the supernatant using a volumetric pipette, add 700 mg of MgSO4  7H2O and 150 mg PSA, and mix. • Shake vigorously for 30 s. • Centrifuge at 3000 rpm for 5 min. • Transfer supernatant to a 50 mL round bottomed flask. • Wash the precipitate with 5 mL ACN and shake vigorously for 15 s. • Centrifuge at 3000 rpm for 5 min. • Take supernatant and transfer to the 50 mL round bottomed flask. • Concentrate on a rotary evaporator in a water bath at 38 C, until one drop is present. • Evaporate until dryness under nitrogen flow. • Add 0.5 mL H2O: ACN (90:10) (v:v) mixture to the round bottomed flask, sonicate for 1 min and transfer the extract to a HPLC-vial. Rinse the flask twice with the same solvent mixture to a final volume of 1 mL in a HPLC-vial. • Filter the extract through a 0.45 μm pore. • Inject 100 μL into the HPLC system.

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Table 3.5 Elution gradient H2O: ACN for separation of carbamates Time (min) Water (%) ACN (%)

0 35 45 65

90 34 90 90

10 66 10 10

Chromatographic conditions • HPLC equipped with fluorescencedetector (FLD) with excitation wavelength (λ): 330 nm and emission wavelength (λ): 465 nm • Mobile phase: H2O: ACN gradient, see Table 3.1 for gradient elution. • Sample temperature thermostat control: 4 C • Flow: 1 mL/min. • Injection volume: 100 μL • Postcolumn derivatization: reaction solution 1: hydrolyzing solution NaOH 0.2%, Temperature: 100 C, Flow: 0.1 mL/min. Reaction solution 2: hydrolyzing solution OPA, Temperature: 30 C, Flow: 0.1 mL/min. • The gradient is given in Table 3.5. 10. Calculations: Extracts were analyzed in the chromatograph using the following sequence: a. Calibration curve b. Reagent blank c. Matrix blank d. Samples e. Calibration curve Identification of the compounds was carried out qualitatively by comparing the retention times (Rt) 6 0.05 min of the compounds in the samples and the standards. Confirmation of the identity of the compounds was carried out by comparing the fluorescence spectra obtained for the samples and standards. Quantification is performed by constructing a calibration curve using a linear calibration model. Calibration levels were: 12.5, 25, 50, 75, and 100 ng/g. 11. Quality control: A matrix blank and a recovery control spiked sample are processed together with each batch of samples. The percentage recovery of the control sample should be between 60% and 110%, to consider

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the batch acceptable; if not, the causes should be studied and eliminated and the batch reprocessed. Stabilize the equipment to obtain a stable baseline. Perform 10 successive carbaryl injections of 100 μL of working concentration. Calculate the mean, standard deviation, and percentage coefficient of variation for areas and retention times. Conditions of acceptance: CV% areas # 5%; CV% retention time # 2%. If the coefficients of variation obtained are not acceptable, the causes should be studied and the source of malfunction eliminated solved before the next run to assure the quality of the determination. 12. Remarks: The use of nonporous gloves during preparation of reagents is advised because OPA and thiofluor can cause skin irritation. The OPA reagent is sensitive to air oxidation, degrades over time, and should be prepared fresh for optimum sensitivity. OPA reagent maintains its activity for 1 week when stored under an inert gas. The water in the solvent reservoir should be changed every 3 or 4 days to prevent possible bacterial growth. Avoid purging the system with 100% acetonitrile because precipitation of borate salt in the reactor might occur. If acetonitrile is used as the mobile phase, do not exceed 70%. Do not store the column in water. 13. References: Anastassiades, M., Lechotay, S.J., Tajnbaher, D., Schenck, F.J., 2003. Fast and easy multiresidue method employing acetonitrile extraction/ partitioning and dispersive solid-phase extraction for determination of pesticide residue in produce. J. AOAC Int. 86 (22), 412431. Document SANTE N 11495, 2015. Method validation and quality control procedures for pesticide residues analysis in food and feed. Pickering Laboratories, Pinnacle PCX, 2010. 14. Minimum method validation data (range of matrices, range of validation, LOQ or lowest calibrated level (LCL), recoveries, and CV%): Matrix: honey Validation range: 0.01250.100 mg/kg LCL: 12.5 ng/g honey Recovery range: 60%107% CV ,20%

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METHOD 9: Determination and confirmation of chloramphenicol in honey, fish, and prawns by liquid chromatographytandem mass spectrometry 1. Laboratory name and address: Laborato´rio Nacional Agropecua´rio/RS—LANAGRO/RS, Estrada da Ponta Grossa, 3036, 91780-580, Porto Alegre/RS, Brazil 2. Contact person: Fabiano Barreto email: [email protected] 3. Title of the analytical method: Determination and confirmation of chloramphenicol (CAP) in honey, fish, and prawns by liquid chromatographytandem mass spectrometry 4. Principle: Although a banned compound for animal production, CAP remains under control due a risk of use and recurrent identification in different countries. The procedure is based on organic solvent extraction without solid phase extraction (SPE) steps. It provides a reliable high-throughput method for CAP residue control under the National Residues Control Plan. 5. Scope: Determination of chloramphenicol residues in honey, fish, and prawns 6. Equipment and instruments: • Ultra-Turrax • Centrifuge shaker • Thermostat water bath with nitrogen stream • LC-MS/MS 7. Reagents and materials: • Chloramphenicol (99.0%) • Deuterated chloramphenicol-d5 (98.0%)—internal standard • Methanol HPLC grade • CN, HPLC grade • Ethyl acetate HPLC grade • Chloroform HPLC grade • Ultrapure water • Column C18 XTerra 2.1 3 100 (3.5 μm) 8. Standard solutions: • CAP stock solution: prepared in methanol at a concentration of 1 mg/mL and stored at 220 C. The stock solutions were stable for 6 months.

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CAP-d5 stock solution: prepared in methanol at a concentration of 10 μg/mL after ampoule dilution and stored at 220 C. The stock solutions were stable for 6 months. • CAP intermediate solution: prepared after dilution of stock solution at a concentration of 10 μg/mL in methanol and stable for 2 months. • CAP-d5 intermediate solution: prepared after dilution of stock solution at a concentration of 10 μg/mL in methanol and stable for 2 months. • CAP working solution: prepared after dilution of intermediate solution at a concentration of 20 ng/mL in methanol and stable for 1 month. • CAP-d5 working solution: prepared after dilution of the intermediate solution at a concentration of 6 ng/mL in methanol and stable for 1 month. 9. Detailed procedure (protocol): Fish and prawns samples • Weigh 2.0 g chopped muscle in 50 mL tube. • Add 100 μL of internal standard working solution. • Extract samples with 10 mL ACN and apply ultra Turrax to complete tissue disruption. • Keep in agitation for 20 min • Centrifuge at 3000 g • Transfer the supernatant to 50 mL tube and add 5 mL chloroform. • The chloroform addition promotes a water separation and water content is removed by aspiration. • Dry the supernatant under nitrogen stream at 45 C. • Reconstitute the sample with a solution of water:ACN (1:1, v/v) • LC-MS/MS Honey samples • Weigh 2.0 g honey in 50 mL tube. • Add 1 mL hot water (40 C) to promote matrix dissolution. • Add 4 mL of ethyl acetate and Vortex for 1 min. • Remove 1 mL organic layer and dry. • Reconstitute with 1 mL water:ACN (1:1, v/v). The calibration details, elution gradient, and MS parameters are given in Tables 3.6, 3.7,and 3.8, respectively.

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Table 3.6 Calibration curve and quality control sample concentrations applied to honey, fish, and prawns samples Sample Sample size (g) IS (µL) Standard (µL)

0 0.2 0.3 0.45 0.6 1 Blank R1 R2 R3 TS1 TS2 TS3

2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0

100 100 100 100 100 100 0 100 100 100 100 100 100

0 20 30 45 60 100 0 30 30 30 30 30 30

Table 3.7 Elution gradient conditions. LC-MS/MS: Mobile phase: elution gradient of 10 mM ammonium acetate 0.1% glacial acetic acid (A) and methanol (B) Time (min) Flow (µL/min) A (%) B (%)

4 (Equilibrate) 1 3 5 7 8

300 300 300 300 300 300

95 95 5 5 95 95

Table 3.8 MS/MS parameters employed Precursor ion

Q1/Q3

Dwell time (ms)

Compounds Declustering potential

321.1 321.1 326 326

152 257 156.9 262

50 50 150 150

CAP1 CAP2 CAP-D5 1 CAP-D5 2

IS voltage Curtain gas CAD GS1 GS2 TEM

24500 15 10 55 55 700

285 285 285 285

5 5 95 95 5 5

Entrance Collision potential energy

Exit cell potential

210 210 210 210

29 213 211 211

224 216 224 216

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10. Calculations: The calibration curves are constructed in a matrix-matched mode and internal standard applied for correction. A nonweighted linear calibration model is applied. 11. Quality control: For routine control, three samples fortified at regulatory level, reagent blank sample, and extraction solvent are included in each batch analyzed. 12. Interferences, troubleshooting, and safety: Different honey samples and within-year differences can produce different chemical profiles and interference patterns in MRM transitions. A wide selective evaluation is therefore necessary for honey samples. 13. References: Barreto, F., Ribeiro, C., Hoff, R.B., Costa, T.D., 2012. Determination and confirmation of chloramphenicol in honey, fish and prawns by liquid chromatography-tandem mass spectrometry with minimum sample preparation: validation according to 2002/ 657/EC Directive. Food Addit. Contam. Part A Chem. Anal. Control Expo Risk Assess. 29 (4), 550558. Martins Ju´nior, H.A., Bustillos, O.V., Pires, M.A.F., Lebre, D.T., Wang, A.Y., 2006. Determinac¸a˜o de resı´duos de cloranfenicol em amostras de leite e mel industrializados utilizando a te´cnica de espectrometria de massas em “tandem” (CLAE-EM/EM). Quı´mica Nova. 29, 586592. 14. Minimum method validation data: LOQ: 0.2 μg/kg Working range: 0.21.0 μg/kg Recovery: 80%120% LMDR: 0.3 μg/kg

METHOD 10: Determination of avermectins and milbemycin residues in food of animal origin by LC-MS/MS 1. Laboratory name and address: Laborato´rio Nacional Agropecua´rio/RS—LANAGRO/RS, Estrada da Ponta Grossa, 3036, 91780-580, Porto Alegre/RS, Brazil 2. Contact person: Fabiano Barreto email: [email protected]

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3. Title of the analytical method: Determination of avermectins and milbemycin residues in food of animal origin by LC-MS/MS 4. Principle: Residues of avermectins and milbemycins can be present in matrixes such as milk and meat as the result of widespread usage of antiparasitic drugs for the treatment and prevention of parasitic diseases in food producing animals. These residues are extracted from the matrix using ACN. The extract is cleaned up by freezing at 220 C overnight, and concentrated by solvent evaporation. The dry residue is dissolved in ACN, transferred to a polypropylene vial, and then injected into the LC-MS/MS system. Analyses are performed in MRM mode, monitoring two transitions for each analyte; the most intense transition is dedicated for analyte quantification, and the remaining transition for analyte confirmation. Method calibration is performed by matrix-matched calibration curves using internal standardization. 5. Scope: The present methodology was validated according to 2002/657/ EC (EC, 2002) for analyses of avermectins and milbemycins residues in bovine milk (dairy farm milk and commercial UHT milk), but is not restricted to such products. Whole and defatted powdered milk, whole or defatted pasteurized milk, milk of nonbovine animals, etc. may be considered for analysis after method validation or scope extension. This is also true for analyses of edible tissues other than bovine muscle, in which the method was also validated. The method is applicable to determination of avermectins abamectin (ABA), doramectin (DOR), eprinomectin (EPR), ivermectin (IVR), milbemycin and moxidectin (MOX) in the matrixes listed above, at concentrations higher than 2.5 μg/mL (milk) or 2.5 μg/kg (muscle). 6. Equipment and instruments: • Micropipettes (10100, 1001000, and 10005000 μL) • Mechanical shaker • Food processorblender • Centrifuge • Nitrogen evaporator with thermostatted water bath • pH meter • Water purification system, model Milli-Q

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Mass spectrometer triple quadrupole, equipped with electrospray ionization interface (ESI) • Liquid chromatograph with a quaternary pump, a vacuum degasser, and an autosampler. • Tanalytical column Luna C18, 150 mm 3 2.1 mm, i.d., 5 μm (Phenomenex, Torrence, CA, USA) • C18 guard column, 4 mm 3 2.1 mm, i.d., 5 μm (Phenomenex, Torrence, CA, USA). • A diverter valve is used between the chromatograph and the mass spectrometer, to minimize contamination of the ion source. 7. Reagents and materials: • Polypropylene centrifuge tubes (15 and 50 mL) • Polypropylene LC vials (1 mL) • Plastic rods • Can, spectrometric grade and acetic acid HPLC grade • Ammonium acetate and sodium chloride P.A. 8. Standard solutions: Abamectin 89% B1a, doramectin 81.1% B1, emamectin benzoate 99.1% B1a (internal standard), eprinomectin 93.9% B1a, moxidectin 92.3%, and ivermectin 82.9% B1a 9. Detailed procedure (protocol): Solutions • Mobile phases: ultrapure water (A); ACN (B); 100 mM ammonium acetate buffer pH 5 (C) obtained by dissolving 770 mg acetate salt in approximately 50 mL ultrapure water. Adjust pH using pH meter, by adding a few drops of acetic acid into the solution until pH 5. Adjust the volume to 100 mL with ultrapure water. • Standard solutions: individual stock solutions of 1.0 mg/mL were prepared by dissolving 10 mg of solid standard in 10 mL ACN. Working solution was prepared by combining aliquots of each stock solution in 10 mL ACN to obtain a final concentration of 1 μg/mL for all analytes. EMA was used as internal standard (IS) and its working solution was prepared and stored in a separate flask at 1.0 μg/mL in ACN. All standard solutions were stored at 220 C in polypropylene tubes. Note: stock solutions are stable for 6 months, and working solutions for 1 month when stored in polypropylene flasks, at 220 C.

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Sample preparation Frozen milk samples are thawed until they reach room temperature (c. 22 C). Homogenize the sample and aliquot 5 mL into a 50 mL polypropylene centrifuge tube. Frozen muscle samples are separated from fat tissue and the muscle fraction finely chopped and homogenized. Five gram aliquots of the homogenized muscle sample are weighed into 50 mL polypropylene centrifuge tubes. All samples are stored frozen until analysis. Samples free of avermectin and milbemycin residues (blank samples) are used to prepare matrix-matched calibration curves and quality control samples (QC). Calibration curves are prepared by adding 0, 2.5, 12.5, 25, 50, 75, and 100 μL working solution, and 25 μL of IS, in each tube of blank samples. “Recovery” samples (R) are prepared by adding 50 μL working solution and 25 μL of IS in three tubes of blank samples. “Tissue” samples (TS) are prepared by only adding 25 μL of IS. Standards are added (50 μL working solution and 25 μL of IS) into TS only after the extraction procedure. Extraction of milk samples Milk sample are extracted by successive additions of two aliquots of 2.5 mL and one of 5 mL ACN, with manual homogenization between each ACN addition. After the last addition, the samples are shaken thoroughly on a shaking table, at 180 rpm for 20 min. Approximately 2 g sodium chloride are then added to each sample, followed by homogenization for 5 min, and subsequent centrifugation for 10 min at 2200 g. The top layer is transferred to a 15 mL polypropylene centrifuge tube and stored in the freezer at 220 C for 12 h, in order to perform the clean-up. After this time, the liquid phase is transferred to a 50 mL centrifuge tube and evaporated to dryness at 4045 C, under a nitrogen flow. The dry residue is reconstituted with 1 mL of ACN and transferred to a polypropylene vial for further LC-MS/MS analysis. Muscle samples Add 2.5 mL ultrapure water into sample tubes and homogenize the mixture manually with a plastic rod. Sample extraction is performed by successive additions of two aliquots of 2.5 mL and one of 5 mL ACN, with manual homogenization between each ACN addition. After the last addition, samples are shaken thoroughly on a shaking table, at 180 rpm for 20 min. Approximately 2 g sodium chloride are then added to each sample, followed by homogenization for

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5 min, and subsequent centrifugation for 10 min at 2200 g. The top layer is transferred to a 15 mL polypropylene centrifuge tube and stored in the freezer at 220 C for 12 h in order to perform extract clean-up. After this time, the liquid phase is transferred to a 50 mL centrifuge tube and evaporated to dryness at 4045 C, under a nitrogen flow. The dry residue is reconstituted with 1 mL of ACN and transferred to a polypropylene vial for further LC-MS/MS Instrument settings • Liquid chromatography Chromatography is performed in solvent gradient mode, at a mobile phase flow of 0.2 μL/min. The gradient was programmed to start with a mobile phase composition of 50% A, 45% B, and 5% C, and then programmed to 95% B and 5% C after 2 min. This composition was maintained for 15 min before returning to the start condition. The injection volume was 10 μL. A divert valve was used to direct the eluent flow to waste for the first 3.5 min to help remove any matrix impurities from entering the MS/MS. • Mass spectrometer The mass spectrometer conditions optimized for the present method are shown in Table 3.9, using scanning mode in MRM and a dwell time of 200 ms for each transition.

Table 3.9 Mass spectrometry setting for MS/MS analysis for avermectins and milbemycin in positive mode Analytes RT Molecular Precursor Product Declustering Collision (min) ion or ion (m/z) ions (m/z) potential (V) energy (eV)

ABA

9.6

[M 1 NH4]1 890.4

DOR

10.5

[M 1 NH4]1 916.5

EPR

8.7

[M 1 H]1

EMA IVR

8.4 12

886.1 [M 1 H]1 [M 1 NH4]1 892.5

MOX

10.9

[M 1 H]1

914.5

640.4

*, Most abundant fragment ion; RT, retention time.

305.6* 567.3 331.3* 593.3 468.2 186.1* 158.0* 307.3* 569.4 528.2* 498.3

96 96 66 66 106 76 76 71 71 30 28

33 35 35 19 17 29 57 33 21 9 17

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The electrospray ionization source (ESI) is operated in positive mode at 500 C, using curtain gas (CUR) 25, collision gas (CAD) 6, ion source gas 1 and 2 (GS1 and GS2) 50 each, and ion spray 5500 V. Analyte confirmation is obtained based on the ion ratio criteria established in the 2002/657/EC Guide for LC-MS/MS, in which the relative intensity of two transitions for each analyte should correspond to those of the calibration standard, in addition to retention time criteria. Additionally, LOD can be calculated using qualifier transitions, and LOQ can be calculated using quantifier transitions (most intense transitions). 10. Calculations: Positive samples are quantified using linear regression equations obtained from matrix-matched calibration curves and internal standardization, which already consider effects from matrix and recovery, respectively. Recovery for QC samples is calculated by the equation QCR 5 100 3 R/TS, in which QCR 5 recovery of QC samples R 5 concentration of analytes added before sample extraction TS 5 concentration of analytes found 11. Quality control: Quality controls are performed at two distinct concentrations, one at MRL concentrations (in triplicate) and named “recovery samples,” and the other at an arbitrary concentration (in single replicate), within the working range. 12. Remarks: • The present methodology was validated according to 2002/657/ EC Guide, which requires validation around the maximum limit of residue (MRL) for authorized drugs. Although, ivermectin is not authorized for use in lactating dairy cows whose milk is destined for human consumption, a MRL of 10 μg/mL was adopted for regulatory actions. • Samples are analyzed using a single replicate. Samples with residues above MRL concentrations or above the calibration working range must be submitted to a new extraction, in triplicate. In this case, results are reported as mean values. • Muscle and milk samples are generally stable for 1 year when stored at 220 C.

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13. Interferences, troubleshooting, and safety: Samples intended for analysis should be preserved for transport or stored at 220 C. Special attention should be paid to frozen edible tissues, which can lose water during and after thawing, causing variations of residue concentration. To overcome this problem, tissue samples should be kept frozen (or partially frozen) until processing (chopping, homogenization, and weighting). For milk samples, it is imperative that all samples are homogenized at room temperature before sampling. All analytes are lipophilic compounds and tend to accumulate in the fat. 14. References: EC, 2002 (Commission Directive/2002/26 EC). Commission of the European Communities, implementing Council Directive 96/ 23/EC, concerning the performance of analytical methods and the interpretation of results. Off. J. Eur. Commun. L209, 514. Ru¨bensam, G., Barreto, F., Hoff, R.B., Kist, T.B.L., Pizzolato, T.M., 2011. A liquid-liquid extraction procedure followed by a low temperature purification step for the analysis of macrocyclic lactones in milk by liquid chromatography-tandem mass spectrometry and fluorescence detection. Anal. Chim. Acta, 705, 2429. Ru¨bensam, G., Barreto, F., Hoff, R.B., Pizzolato, T.M., 2013. Determination of avermectin and milbemycin residues in bovine muscle by liquid chromatography-tandem mass spectrometry and fluorescence detection using solvent extraction and low temperature clean-up. Food Control 29, 5560. 15. Minimum method validation data Recovery: 70%120% Working Range: 2.520 μg/kg LOD: 1 μg/kg LOQ: 2.5 μg/L CCa: 10.614.6 CCb: 11.319.3

METHOD 11: Determination of sulfonamides in eggs by high-pressure liquid chromatography detection and diode arrangement (HPLC/DAD) 1. Laboratory name and address: Servicio Agrı´cola y Ganadero de Chile, Laboratorio Quı´mica Ambiental y Alimentaria, Ruta 68 Km. 12 Complejo Lo Aguirre, Pudahuel, Santiago, Chile

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2. Contact persons: Pedro Enriquez Alfaro email: [email protected] Jacqueline Rojas email: [email protected] 3. Title of the analytical method: Determination of sulfonamides in eggs by high-pressure liquid chromatography detection and diode arrangement (HPLC/DAD) 4. Principle: This method enables determination of 10 sulfonamides in egg samples using high-resolution liquid chromatography. Five gram homogenized egg (yolk and albumin) are extracted twice with ACN, stirred and centrifuged. The ACN extract is concentrated under a nitrogen stream and cleaned up using solid phase extraction cartridge columns. The dried extract is reconstituted and injected into the HPLC/DAD. 5. Scope: Determination of sulfonamides: sulfadiazine (SDZ), sulfatiazole (STZ), sulfapyridine (SPD), sulfamerazine (SME), sulfamethoxypyridazine (SPZ), sulfachlorpyridazine (SCP), sulfadoxine (SDX), sulfamethoxazole (SMZ), sulfadimethoxine (SDA), and sulfaquinoxaline (SQX) in fresh eggs intended for human consumption. 6. Equipment and instruments: • Refrigerator for storing standards at 28 C • Refrigerator for storing samples at 210 C to 220 C • Fume hood • Ultrasonic sonicator • Digital Vortex shaker • High resolution liquid chromatograph coupled to diode array detector (DAD) • Analytical balance • Blender • Solid phase extraction manifold 7. Reagents and materials: Materials • 10 mL flask • 50 mL flask • 1000 mL flask • 1000 mL graduated cylinder • 1001000 μL micropipette • 5 mL glass vials

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• 50 mL Falcon centrifuge tubes • Pasteur pipettes • 10 mL volumetric pipettes • 50 and 100 mL beakers • Membrane filters (acrodiscs) • HLB OASIS solid phase extraction column, 200 mg sorbent, 6 mL • 1 mL disposable syringe • Nitrogen evaporator Reagents • Methanol HPLC grade • CN, HPLC grade • MilliQ or HPLC grade water • Sodium phosphate (NaH2PO4) • Sulfonamides standards 8. Standard solutions: • Prepare a sodium phosphate (NaH2PO4) solution 50 mM: Weigh 6 g anhydrous NaH2PO4, dissolve and transfer to a 1000 mL flask. Bring to volume with MilliQ water. This solution can be stored at 28 C for 1 week. Standards, calibration curve • 1000 ppm stock solution: weigh 25 mg of each sulphonamide standard, dissolve with a small amount of solvent and dilute to 25 mL. The respective solvents are: • Stock solutions prepared in ACN: sulfatiazole (STZ), sulfamerazine (SME), sulfamethoxypyridazine (SPZ), sulfamethoxazole (SMZ), sulfachlorpyridazine (SCP), and sulfadimethoxine (SDA) • Stock solutions prepared in methanol: sulphapiridina (SPD), sulphadoxina (SDX), and sulfaquinoxaline (SQX) • Stock solutions prepared in 0.2 N NaOH: sulfadiazine (SDZ) These solutions should be frozen at 220 C and are stable for 1 year. Correct the concentration according to the percentage purity of the standard used. • 10 ppm intermediate multistandard solution: take 250 μL of each stock solution and transfer to a 25 mL flask, bringing to 25 mL with ACN. These solutions should be frozen at under 220 C and are stable up to 6 months. • Calibration curve (2501500 ppb): prepare a calibration curve in 50 mM sodium phosphate with increasing addition of aliquots taken from the 10 ppm intermediate multistandard solution.

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• This solution must be prepared daily or each time a test is performed. 9. Detailed procedure (protocol): Sample preparation a. Homogenize whole eggs (yolk and albumin) without shells in a blender. Samples and fortifications follow the same treatment. b. Weigh approximately 5 g homogenized egg sample in a 50 mL Falcon tube. c. Fortified samples: add the corresponding aliquot of the fortification and let the sample stand for 10 min in a cold bath prior to extraction. d. Add 15 mL ACN. e. Shake for 5 min in a Vortex. f. Centrifuge at 4000 rpm for 10 min at 02 C. g. Transfer the supernatant to another centrifuge tube and repeat the extraction steps (d), (e), and (f). Combine both supernatants. h. Add 3 mL deionized water to each tube containing ACN. i. Concentrate to 1 mL under nitrogen at 4045 C. (Critical step) j. Condition a HLB SPE cartridge (6 mL, 200 mg) sequentially with 6 mL ACN and 6 mL water, and leave 1 mL water in the cartridge to prevent drying. k. Shake the extract in a Vortex and load the sample with a flow of 12 drops/s. l. Once the entire sample is loaded, dry the cartridge under vacuum. m. Label and weigh 5 mL glass tubes previously cleaned and dried. Record the weight of each tube. n. Elute with 3 mL ACN and collect in previously weighed 5 mL tube. o. Add 1 mL water to each tube and Vortex. p. Evaporate under nitrogen stream at 40 C to 0.5 mL. q. Dilute to 1 mL with 50 mM NaH2PO4, shake in a Vortex for 2 min and sonicate for 5 min. r. Filter the sample in 0.2 μm membrane and collect the sample into a vial for chromatographic analysis. Instrumental analysis: Determination of sulfonamides is through HPLC, using the diode array detector (DAD) at 270 nm wavelength. Chromatographic conditions are summarized in Table 3.10.

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Table 3.10 Summary chromatographic conditions (HPLC-DAD)

Column chromatography Precolumn Flow Mobile phase

C18, 250 3 4. 6 mm, 5 μm particle diameter C18, 4 3 3 mm, 5 μm particle diameter 0.8 mL/min A: NaH2PO4 50 mM B: CAN C: Methanol Gradient:

Injection volume Temperature Detector

Time (min)

%A

(%) B

%C

Flow (mL/min)

0 30 31 45 46 50

83 83 60 60 83 83

17 17 0 0 17 17

0 0 40 40 0 0

0.8 0.8 0.8 0.8 0.8 0.8

50 μL 30 C Diode’s, quantification at 270 nm

Once chromatographic conditions are optimized, the sequence consisting of calibration curve, blanks, samples and fortified samples is injected. Construct the calibration curve with the integrated areas and determine the slope, intercept, and correlation coefficient. Interpolate the samples in the calibration curve and calculate the concentration of the sample. 10. Quality control: Each analysis process should include at least: • Blank • Solvent control • Standards-curve (four or more levels), standard control • Fortified at detection limit and at the reporting limit Acceptability criteria: Blank and solvent without signals at the retention times of the analyses (selectivity) Standard curve, check: stability standards Fortified controls: reproducibility method CV ,30%.

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11. Remarks: N/A. 12. Interferences, troubleshooting, and safety: The method has no interference in the retention time of the analysis. 13. References: Heller, D.N., Ngoh, M.A., Donoghue, D., Podhorniak, L., Righter, H., Thomas, M.H., 2002. Identification of incurred sulfonamide residues in eggs: methods for confirmation by liquid chromatography-tandem mass spectrometry and quantitation by chromatography with ultraviolet detection. J. Chromatogr. B 774, 3952. Internal procedure QAA/I-40: Determinacio´n de sulfonamidas en tejido animal mediante Cromatografı´a Liquida de Alta Resolucio´n. Laboratorio Quı´mica Ambiental y Alimentaria—SAG. ,[email protected].. Manual Merck de Veterinaria fourth Edition, 1993. Editorial Merck Co INC. Publishedby MerckCo INC, Rahway, N.J. EUA y Oce´ano-Centrum, Barcelona, Espan˜a 1993. 14. Minimum method validation data (range of matrices, range of validation, LOQ or lowest calibrated level (LCL), recoveries, and CV %): Matrix: egg samples Recovery: 70%94% Validated range: 50150 ppb LOD: 10 ppb CCa: 105136 ppb

METHOD 12: Determination of ractopamine in swine feed by LC-MS/MS. 1. Laboratory name and address: Ministe´rio da Agricultura, Pecua´ria e Abastecimento, SLAV/SC/ LANAGRO/RS, Rua Joa˜o Grumiche´, 117/bloco T, Sa˜o Jose´, SC 88102-600, Brazil 2. Contact person: Heitor Daguer email: [email protected] 3. Title of the analytical method: Determination of ractopamine in swine feed by LC-MS/MS 4. Principle: Ractopamine (Fig. 3.1) is a β-adrenergic agonist used as feed supplement in animal production, mainly during the late growth stage of swine.

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HO

OH

N H

OH

Figure 3.1 Chemical structure of ractopamine.

Ractopamine (RAC) is extracted from feed by solid:liquid extraction with acidified water and ACN, and partially purified by centrifugation and n-hexane extraction. Ractopamine is determined by LC-MS/MS, using clenbuterol-D9 as internal standard. 5. Scope: This method aims to determine the concentration of ractopamine in swine feed by LC-MS/MS. 6. Equipment and instruments: Equipment Analytical balance ( 6 0.0001 g) Centrifuge equipped with a rotor with 50 mL centrifuge tube holders and capable of attaining 3000 g (c. 4000 rpm). High-speed microcentrifuge equipped with a fixed-angle rotor with 2 mL microcentrifuge tube holders and capable of attaining 16,000 g (c. 12,000 rpm). Micropipettes: 220, 20200, and 10005000 μL Ultra-Turrax@homogenizer wand Orbital shaker Vortex mixer Water bath Nitrogen-evaporator Instruments HPLC Triple quadrupole mass spectrometer Mass spectrometer recording device—HPLC Column-C18, Venusil XBP (Agela), 3 μm 100 A˚ 100 3 2.1 mm HPLC guard column-C18, Phenomenex, 5 μm, 4.0 mm 3 3.0 mm 7. Reagents and materials: Reagents ACN, HPLC grade Ammonium acetate, reagent grade Formic acid 95%, reagent grade Nitrogen gas

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Methanol, HPLC grade N-Hexane, HPLC grade Distilled deionized water (ultrapure water) generated in-house with an electrical resistance of at least 18.2 mΩ. Materials Volumetric flasks: glass, 10, 100, and 500 mL 50 mL polypropylene centrifuge tubes (Falcon) 1.5 mL polypropylene centrifuge tubes (Eppendorf) 2.0 mL autosampler vials 0.2 mL vial inserts 8. Standard solutions: Ractopamine HCl (RAC) Clenbuterol-D9 (CBT) 9. Detailed procedure (protocol): Solutions Sample extraction solutions 0.1% formic acid Add 50 mL ultrapure water and 100 μL formic acid in a 100 mL volumetric flask. Complete the volume with ultrapure water. 0.1% formic acid in acetonitrile Add 50 mL of acetonitrile and 500 μL formic acid in a 500 mL volumetric flask. Complete the volume with acetonitrile. 100 mM ammonium acetate Dissolve 0.77 g ammonium acetate in 100 mL volumetric flask using ultrapure water as solvent. HPLC mobile phase Phase A Add 25 mL of 100 mM ammonium acetate and 500 μL formic acid in a 500 mL volumetric flask. Complete the volume with ultrapure water. Phase B Add 25 mL of 100 mM ammonium acetate and 500 μL formic acid in a 500 mL volumetric flask. Complete the volume with acetonitrile. Extract diluent Prepare a mixture of HPLC mobile phase A and B (75:25, v-v). Add 75 mL of HPLC mobile phase A and 25 mL of HPLC mobile phase B into a 100 mL volumetric flask.

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Standard solutions Stock solutions (1000 ppm) Dissolve 0.01 g of each standard (ractopamine and clenbuterolD9) in separate 10 mL volumetric flasks using methanol as solvent. Fortification solutions (5 ppm) Add 50 μL of each standard stock solution (ractopamine and clenbuterol-D9) in separate 10 mL volumetric flasks using methanol as solvent. Sample preparation Weigh 2.0 6 0.1 g homogenized feed into a 50 mL centrifuge tube. Weigh 2.0 6 0.1 g of homogenized blank material Feed samples previously confirmed for the absence of ractopamine should be selected as blank samples. Calibration curve in matrix The calibration curve is prepared by adding fortification standard solutions on blank samples as described in Table 3.11. Quality control samples Negative sample Reserve one blank sample and proceed with the extraction without addition of any standard solution. Recovery samples Prepare three blank samples by addition of 20 μL of both fortification standard solutions (RAC and CBT) and proceed with sample extraction.

Table 3.11 Preparation of calibration curve Calibration standard Calibration Volume of concentration (ng/mL) level ractopamine 5 (ppb) ppm (µL)

Volume of Clenbuterol-D9 5 ppm (µL)

1 2 3 4 5 6

20 20 20 20 20 20

0 4 10 20 30 40

0 10 25 50 75 100

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Tissue standard (TS) samples Reserve three blank samples and proceed with the extraction without addition of any standard solution. For extract dilution add 20 μL of both fortification standard solutions (RAC and CBT) and 0.66 mL of extract diluent solution. Extraction Weigh 2.0 6 0.1 g homogenized feed in 50 mL centrifuge tube. Add ractopamine fortification standard solution to calibration curve samples. Add 20 μL clenbuterol-D9 fortification standard solution, except to blank and tissue samples. Add 1 mL of 0.1% formic acid Add 10 mL 0.1% formic acid in ACN Process extract on Ultra Turrax, adjusted to level 4, for 5 s Shake for 10 min on an orbital shaker Centrifuge at 4000 rpm for 10 min at 4 C Transfer the supernatant into a 50 mL centrifuge tube Evaporate the extract to dryness under a stream of nitrogen on a 50 C water bath Add 0.7 mL extract diluent solution and Vortex for 10 s Add 0.7 mL hexane and Vortex for 10 s Shake for 10 min on an orbital shaker Transfer solution into a 1.5 mL centrifuge tube. Centrifuge at 12,000 rpm for 10 min at 4 C. Transfer 0.2 mL of the aqueous phase into a vial insert. Instrument conditions The instrument conditions are listed in Tables 3.123.14. Table 3.12 HPLC conditions

Column Guard column Column temperature Flow rate Injection volume Gradient

˚ 100 3 2.1 mm C18, Venusil XBP (Agela), 3 μm 100 A C18, Phenomenex, 5 μm, 4.0 mm 3 3.0 mm 40 C 0.3 mL/min 10 μL Time (min) % Mobile phase A % Mobile phase B 0 95 5 3 10 90 6 10 90 7 95 5 12 95 5

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84 Table 3.13 MS conditions RF lens (V)

Positive

Capillary (kV) Source temperature ( C) Desolvation temperature ( C) Cone gas flow (L/h) Desolvation gas flow (L/h)

3.5 120 400 50 400

Table 3.14 Analyte dependant parameters Analyte Precursor ion Product ions (m/z) (m/z)

Dwell time (s)

Cone (V)

Collision (V)

Ractopamine

0.2

25

0.2

23

16 16 15 15

302.2

Clenbuterol-D9 286.1

164.2 121.3 204.3 268.4

10. Calculations: The analyte concentration in the sample is calculated according to the following equation, obtained from the equation of the calibration curve: y 5 ax 1 b where: y 5 analyte concentration in ppb; x 5 area ratio of ractopamine/clenbuterol-D9; a 5 slope; b 5 linear coefficient. The accuracy on recovery quality control samples is calculated using the following equation: Accuracy ð%Þ 5

Calculated analyte concentration 3 100 Nominal concentration

The recovery is calculated from: Average calculated analyte concentration on recovery quality control samples Recovery ð%Þ 5 3 100 Average calculated analyte concentration on TS quality control samples The ion-abundance ratio from the two monitored ions of ractopamine is calculated from:

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Ratio 5

85

Area of the product ion 164:2 Area of the product ion 121:3

11. Quality control: The negative quality control sample must not contain ractopamine or clenbuterol-D9. The results are accepted when the correlation coefficient (r) of the calibration curve is $ 0.95. The accuracy is acceptable within the range of 220% to 110%. Samples that present more than 50 ppb of ractopamine should be reexamined in duplicate and the result calculated by the arithmetic mean of the calculated concentrations. The coefficient of variation within the recovery quality control samples is accepted if under 20%. The ion-abundance ratio of the analyte in the samples is accepted if arithmetically within 25% of the average abundance ratio from calibration curve samples. The calculated recovery is evaluated to identify failures in the extraction process. 12. Interferences, troubleshooting, and safety: This method employs harmful chemicals and procedures that may represent a risk to the operator. All procedures should be performed with use of protective equipment and appropriate clothing. Preparation of reagent solutions should be done in fume hoods. Food samples should be considered biohazards, avoiding direct contact with skin and mucous membranes. 13. References: Commission Decision 2002/657/EC, 2002. J. Eur. Commun. L221, 8. 14. Minimum method validation data Minimum validation data are given in Table 3.15.

METHOD 13: Determination of histamine in fish and fish products by capillary zone electrophoresis 1. Laboratory name and address: Ministe´rio da Agricultura, Pecua´ria e Abastecimento, SLAV/SC/ LANAGRO/RS, Rua Joa˜o Grumiche´, 117/bloco T, Sa˜o Jose´, SC 88102-600, Brazil

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Table 3.15 Range of matrices, range of validation, LOQ or lowest calibrated level (LCL), recoveries, and CV %

Range of matrices Action level LOD LOQ Recovery Repeatability (CV) Reproducibility (CV) CCα CCβ Measurement uncertainty

Swine feed 50 μg/kg 2.5 μg/kg 10.0 μg/kg 105% ,20% ,20% 62.30 μg/kg 74.61 μg/kg 3.93 μg/kg

2. Contact person: Heitor Daguer email: [email protected] 3. Title of the analytical method: Determination of histamine in fish and fish products by capillary zone electrophoresis 4. Principle: Histamine is a biogenic amine present at various levels in many foods. It forms in food by decarboxylation of the amino acid histidine by L-histidine decarboxylase in the presence of decarboxylase-positive microorganisms, and by conditions that allow bacterial growth and decarboxylase activity. Free histidine can be found naturally in foods or may be liberated by proteolysis during processing or storage. Therefore high concentrations of histamine in foods are related to microbial fermentation and histamine can be used as an indicator of hygienic food quality. Furthermore, foods containing high levels of histamine can bring about food-borne illnesses. Capillary electrophoresis is an analytical technique that separates ions based on their migration within a capillary using an electric current. The present method relies on the extraction and determination of histamine by capillary zone electrophoresis (CZE) using appropriate conditions that allow high sampling rate (run time shorter than 1 min), simple sample preparation, minimal waste generation, and reagent consumption. 5. Scope: This method aims to determine the concentration of histamine in fish samples (Scombridae, Scombresocidae, Coryphaenidae, and Clupeidae) by CZE.

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6. Equipment and instruments: Equipment • Analytical balance ( 6 0.0001 g) • Centrifuge • Micropipettes: 105000 μL • Polypropylene tubes: 50 mL • Food processor • Ultra-Turrax • Orbital shaker • Vials Instruments • CE equipment: CE detector wavelength diode array, deuterium lamp • CE capillary: fused-silica capillary from Polymicro Technologies, measuring 32 cm (23.5 cm effective length) 3 75 μm internal diameter 3 375 μm outside diameter. 7. Reagents: • Methanol (MeOH), HPLC grade • Sodium hydroxide (NaOH) 8. Standards: • Histamine (Hist), Sigma-Aldrich • Methylimidazole (PI), Across • α-Hydroxyisobutyric acid (HIBA), Sigma-Aldrich Preparation of solutions • Sodium hydroxide solution 1.0 M Weigh 4.0 g NaOH in a volumetric flask (100 mL) and dissolve in ultrapure water in sufficient quantity to prepare 100 mL. Identify and store in plastic flask at ambient temperature. • Sodium hydroxide solution 100 mM Weigh 0.515 g of NaOH in a volumetric flask (100 mL) and dissolve in ultrapure water in sufficient quantity to prepare 100 mL. Identify and store at ambient temperature. • Acid α-hydroxy-isobutyric 100 mM Weigh 1.041 g HIBA in a volumetric flask (100 mL) and dissolve in ultrapure water in sufficient quantity to prepare 100 mL. Identify and store at ambient temperature. • Background electrolyte buffer solution To be prepared before each use, take an aliquot (1500 μL) of a HIBA stock solution 100 mM and mix with 250 μL of a NaOH stock solution 100 mM, 500 μL of MeOH, and 250 μL of ultrapure water.

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Methylimidazole standard solution 10,000 ppm Weigh 100 mg methylimidazole standard in a volumetric flask (10 mL) and dissolve in ultrapure water in sufficient quantity to prepare 10 mL. Identify and store refrigerated. • Histamine standard solution 10,000 ppm Weigh 100 mg histamine standard in a volumetric flask (10 mL) and dissolve in MeOH in sufficient quantity to prepare 10 mL. Identify and store refrigerated. 9. Detailed procedure (protocol): Fish sample preparation The test requires a nine units packed separately (fresh fish units must weigh at least 500 g and canned fish units must be from the same lot). Each unit should be analyzed individually. Allow the samples to cool to a temperature of 10 C. Unpack the samples and remove the aponeurosis, fat, and bones. Remove muscle portions from various regions of the fish, cut into smaller samples and process with a food processor. Weigh 2.0 6 0.1 g samples in 50 mL polypropylene tubes. • Canned fish Grind and mix the entire content of the can (liquid and solid) in a food processor. Weigh 2.0 6 0.1 g samples in 50 mL polypropylene tubes. Submit to extraction. Calibration curve preparation Weigh 2.0 6 0.1 g blank samples in 50 mL polypropylene tubes and add the volumes of stock solution of histamine and methylimidazole (internal standard) as shown in Table 3.16.

Table 3.16 Preparation of the calibration curve for determination of histamine in fish by CZE Volume of stock Point Concentration in the Volume of stock matrix (mg/kg) solution of histamine solution of methylimidazole 10,000 ppm (µL) 10,000 ppm (µL)

0 1 2 3 4 5

0 25 50 100 150 200

0 5 10 20 30 40

40 40 40 40 40 40

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Extraction • Add 10 mL MeOH into each tube, mix the samples and the solvent using Ultra Turrax. Make sure you rinse the shaft between a sample and the next with water and methanol. • Mix on an orbital shaker for 10 min. • Centrifuge at 4000 rpm for 10 min at 4 C. Transfer 200 μL supernatant (fresh fish) or 50 μL (canned fish) to the vial; prevent aspiration of films and waste adhering to the tube. • Add 400 μL ultrapure water to vial and mix. Electrophoresis Samples are analyzed by CE system under the conditions specified in Table 3.17 10. Calculations: The analyte concentration in samples is calculated according to the following equation, obtained from the equation of the calibration curve: y 5 ax 1 b where: y 5 analyte concentration in mg/kg x 5 area ratio of histamine/methylimidazole a 5 slope b 5 linear coefficient 11. Quality control: The results are accepted when the correlation coefficient (r) of the calibration curve is $ 0.95. Otherwise, the curve must be repeated. Table 3.17 Instrumental conditions for determination of histamine in fish by CE

CE system Capillary Wavelength (λ) Sample injection Voltage separation BGO

Temperature Capillary conditioning Preconditioning

32 cm (Ltot) 3 23.5 cm (Ldet) 3 75 μm 210 nm 50 mbar/5 s 30 kV 60 mmol/L HIBA 10 mmol/L NaOH 20% MeOH 25 C 15 min NaOH/15 min H2O/15 min BGO 30 s H2O/40 s BGO

90

12. 13.

14.

15.

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The recovery/accuracy is acceptable within the range of 220% to 110%. Remarks: Salted fish may need proper dilution. Interferences, troubleshooting, and safety: A greater dilution is required to minimize the effects of the high ionic strength in samples of canned fish. This method employs harmful chemicals and procedures that may represent risk to the operator. All procedures should therefore be performed with use of individual protective equipment. Reagent solutions should be prepared under laminar flow. Food samples should be considered biohazards, avoiding direct contact with skin and mucous membranes. References: Vitali, L., et al., 2012. Development of a fast and selective separation method to determine histamine in tuna fish samples using capillary zone electrophoresis. Talanta 106, 181185. Minimum method validation data Range of matrices: fresh fish, canned fish, salted fish (Scombridae, Scombresocidae, Coryphaenidae, and Clupeidae) LOD: 15 mg/kg LOQ: 25 mg/kg Recovery: 102% CV: ,10% CCa: 118.8 mg/kg CCb: 137.7 mg/kg

EXAMPLES OF METHODS FOR SOIL AND WATER ANALYSIS METHOD 14: Determination of carbamates and triazines in soil and sediment samples by high-pressure liquid chromatography coupled to mass spectrometry 1. Laboratory names and addresses: Laboratorio de Ana´lisis de Residuos de Plaguicidas, Centro de Investigacio´n en Contaminacio´n Ambiental, Universidad de Costa Rica

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2. 3.

4.

5.

6.

91

Sede Rodrigo Facio, Universidad de Costa Rica, San Pedro de Montes de Oca, San Jose´, Costa Rica Contact persons: Mario Alberto Ması´s Mora email: [email protected] Elizabeth Carazo email: [email protected] Title of the analytical method: Determination of carbamates and triazines in soil and sediment samples by high-pressure liquid chromatography coupled to mass spectrometry Principle: Extracts of soil and sediment samples are analyzed in a liquid chromatographic system coupled to mass spectrometry using electrospray ionization in positive mode. While the chromatographic system separates the compounds, the mass spectrometric system detects and confirms the identity of the pesticides. Scope: Determination of carbamates and triazines in soil and sediment samples using a high pressure liquid chromatograph coupled to a single quadrupole mass spectrometer (LC-MS) and an ultra-high pressure liquid chromatograph coupled to tandem mass spectrometry (LC-MS/ MS) as a detection system. Equipment and instruments: Equipment • Refrigerated centrifuge to be used with 15 and 50 mL centrifuge tubes. • Analytical balance with four digits • Nitrogen evaporation system • Mechanical Vortex mixer • Volumetric micropipettes with adjustable volume: 10100, 1001000, and 0.55 mL • Fume hood • Liquid dispenser, 025 mL • Thermometer, 0100 C Instruments • High-pressure liquid chromatograph coupled to a single quadrupole mass spectrometer • High-pressure liquid chromatograph coupled to a triple quadrupole mass spectrometer

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7. Reagents and materials: Reagents • Acetonitrile, HPLC grade • Methanol, HPLC grade • Sodium chloride, residue analysis grade • Sodium acetatetrihydrate or sodium acetate monohydrate • Sorbent C18, 50 μm • Magnesium sulfate, anhydrous 97% purity • Deionized water • Nitrogen • Formic acid 98%100% volume fraction, analysis grade • Glacial acetic acid, 98% purity, analysis grade • Pesticide analytical standards with purity above 95% for the following compounds: aldicarb, carbaril, carbofuran, methomil, methcarb, 3-cetocarbofuran, 3-hidroxicarbofuran, oxamil, atrazine, cianazine, terbutrine, amethrine, simetrine, simazine, and promethrine. • Deuterated analytical standards with analytical purity above 95% for carbendazim-D4 and carbofuran-D3. Materials • Polypropylene tubes, 15 and 50 mL • Centrifuge glass tubes, 15 mL • Pasteur pipettes • 25 mL graduated cylinder • Spatulas • Glass stirring rod • Amber glass vials, 50 mL • 0.45 μm PTFE filters 47 mm diameter • PTFE filters 0.45 μm diameter • 2 mL HPLC vials with glass inserts 8. Standard solutions: Formic acid (0.1% volume fraction) in deionized water • Add deionized water in a 1 L volumetric flask and bring to half capacity. • Add 1 mL formic acid at 98% with a micropipette and fill the flask to the 1 L mark with deionized water. • Stopper and shake. • Transfer the contents to an amber glass vial which is then used in the liquid chromatography system. • This solution can be stored in a refrigerator at 210 C.

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Formic acid (0.1% volume fraction) in methanol • Add methanol in a 1 L volumetric flask and bring to half capacity. • Add 1 mL formic acid at 98% with a micropipette and fill the flask to the 1 L mark with methanol. • Stopper and shake. • Transfer the contents to a glass vial which is then used in the liquid chromatography system. • This solution can be stored in a refrigerator at 210 C. Solution of acetic acid (1% volume fraction) in ACN • Add ACN in a 1 L volumetric flask and bring to half capacity. • Add 1 mL concentrated acetic acid and fill the flask to the 1 L mark with ACN. • Stopper and shake. • Transfer the contents into a glass vial. • Store the solution in a refrigerator at 210 C. Primary solutions of analytical standards Prepare 10 mL of individual stock standards solutions of each pesticide in a volumetric flask according to Table 3.18. Table 3.18 Recommended solvents for the preparation of stock standard solutions of pesticides Pesticide Solvent Weight (mg) Concentration (mg/L)

Aldicarb Carbofuran Carbaryl 3-Hydroxycarbofuran 3-Cetocarbofuran Methomil Methiocarb Oxamyl Atrazine Cianazine Ametrine Terbutrine Simetrine Simazine Prometrine Carbofuran-D3 Carbendazim-D4

ACN 10 ACN 10 ACN 10 ACN 10 ACN 10 ACN 10 ACN 10 ACN 10 Methanol 10 Methanol 10 Methanol or acetone 10 Methanol or acetone 10 Acetonitrile 10 can 10 Acetone 10 ACN 10 Methanol 10

1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 200a

a Carbendazim-D4, is very soluble in methanol up to a concentration of 200 mg/L. In this case it is advisable to use a volumetric 50 mL flask.

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Table 3.19 Intermediate solutions used for preparation of calibration curves Level Intermediate concentrations Equipment of the pesticide mixes (mg/L)

1 2 3 4 5 6 7 8

0.05 0.10 0.20 0.30 0.50 0.90 1.30 2.80

LC-MS, LC-MS/MSa LC-MS, LC-MS/MS LC-MS, LC-MS/MS LC-MS, LC-MS/MS LC-MS, LC-MS/MS LC-MS, LC-MS/MS LC-MS, LC-MS/MS LC-MS, LC-MS/MS

a In the case of LC-MS, the calibration curve can be prepared starting with an intermediate dilution of 0.005 mg/L.

Solutions for preparing the calibration curve The calibration curve is prepared in matrix. Therefore intermediate solutions of between 8 and 10 levels must be prepared. For LC-MS equipment only six levels are required (Table 3.19). The intermediate solutions are prepared as a mixture (not individually). Other solutions Solution 1: carbendazim-D4, 5 mg/L. This solution must be prepared in acidified water with formic acid. It is be used as internal standard. Solution 2: carbofuran-D3, 5 mg/L. This solution must be prepared in ACN. It is used as standard recovery. 9. Detailed procedure (protocol): Weighing of samples • Weigh 5 g soil sample in a 50 mL polypropylene centrifuge tube. • In a glass beaker weigh 5 g sodium acetate trihydrate or 1.5 g sodium monohydrate and 1 g sodium chloride (extraction salts). • In a 15 mL polypropylene tube weigh 75 mg C18 sorbent and 900 mg sodium sulfate anhydrous (clean-up salts). Sample extraction • Add 50 μL solution 25 g soil. Let rest for 30 min. • Add 10 mL deionized water and shake for 1 min using a Vortex mechanical mixing system. • Let rest for a further 30 min. • Add 15 mL of 1% acetic acid solution in ACN using a 25 mL liquid dispenser Hand shake for 1 min. • Continue adding the extraction salts.

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• •

Carefully close the tubes and agitate vigorously for 2 min. Centrifuge the samples for 7 min at 4500 rpm and at 10 C to separate the organic and the aqueous phases. Sample clean-up • Carefully remove the centrifuges tubes from the centrifuge. • Withdraw 8 mL of the organic phase (upper phase) using a micropipette and place this into a 15 mL centrifuge tube which already contains the clean-up salts. Close the tubes hermetically and vigorously agitate for 1 min on a mechanical Vortex mixer. • Centrifuge the tube for 7 min at 4500 rpm and at 10 C to precipitate the clean-up salts. • Solvent exchange. • Take 6 mL of supernatant and place in a conical glass tube. • Concentrate extracts to dryness under a nitrogen stream and in a water bath at 2030 C. Take care not to lose analytes of interest when the extract reaches dryness. • Add 50 μL solution 1 and 1.5 mL acidified water to the dry extracts. Carefully Vortex each glass tube • Filter sample extracts through 0.45 μm PTFE filters and collect the extracts in 2 mL HPLC vials. Preparation of calibration curve • Label eight 50 mL propylene tubes. • In each tube weigh 5 g blank soil • Add 50 μL Solution 2 • Add 1 mL of each intermediate solution prepared according to Table 3.2. The final concentration levels are shown in Table 3.20. • Shake and let rest for 30 min. Table 3.20 Final concentration levels of calibration curve Level Intermediate conc. from Table 3.2 (µg/mL)

Concentration in each calibrator level (ng/g)

1 2 3 4 5 6 7 8

10 20 40 60 100 180 260 560

0.05 0.10 0.20 0.30 0.50 0.90 1.30 2.80

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Add 14 mL of 1% acetic acid solution in ACN using the liquid dispenser. Shake vigorously for 1 min. • Continue with the procedure as described above from the addition of extraction salts. • Chromatographic analysis using an LC-MS • Inject the sample into the liquid chromatograph coupled to a mass spectrometry detector. The chromatographic conditions are described in Table 3.21. Chromatographic analysis using a tandem mass spectrometer (LC-MS/MS) Inject the sample into the liquid chromatograph ultra-high resolution tandem mass spectrometry detector. The chromatographic conditions are shown in Table 3.22. 10. Calculations: The results obtained by this method are expressed in mg/kg or μg/kg. Eq. (3.1) is used to calculate the estimated concentration in mg/kg and the value obtained is divided by 1000 to express the results in μg/kg. The quantification is done by interpolation on the calibration curve. No adjustment is made because samples are treated in the same way at each concentration level (Eq. 3.1). Areacompound in sample 5 m  Cncompound in sample 1 b

(3.1)

11. Quality control: The internal and recovery standards are used as quality control. Recovery for each sample must be calculated and compared with calibration curve results. The sample is quantified and experimental concentration is determined which then compared with the theoretical concentration to obtain the recovery rate.   Experimental concentration ng=g    100 %Rec 5 (3.2) Theoretical concentration ng=g %RecRovery Standard 5

ARSS =AISS  100 ARSC =AISC

(3.3)

where: ARSS and AISS are, respectively, the recovery and internal standard areas of the samples.

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Table 3.21 Chromatographic conditions for the mass spectrometric detection (LC-MS)

Column Flow of mobile phase Column temperature Injection volume Mobile phase Time between injections Run time Ionization source

Mobile phase composition: Gradient Time (min) Flow (mL/min) 0 0.4 3.0 0.4 25.0 0.4 30.0 0.4 30.1 0.4 36.00 0.4 Positive ion mode and SIM Pesticide Oxamyl Methomyl 3-Hydroxycarbofuran Aldicarb Carbofuran Carbaril Methiocarb 3-Cetocarbofuran Carbendazim-D4 Carbofuran-D3 Ametrin Terbutrin Atrazine Cianazine Simetrine Simazine Promethrin

C18 5 μm, (4.6 3 150) mm 0.4 mL/min 30 C 5 μL Gradient of acidified Milli-Q water/acidified methanol 36 min 30 min Electrospray Capillary voltage 2000 V Drying gas 350 C temperature Flow of drying gas 5 L/min Nebulizer pressure 60 psi (0.414 MPa) Vaporization 200 C temperature Phase A: 0.1% formic acid acidified water Phase B: 0.1% formic acid acidified methanol % Phase A % Phase B Fragmentor voltage (V) 40 60 90 40 60 90 0 100 90 0 100 90 40 60 90 40 60 90 Ion (m/z) 242 185 163 and 260 213 222 and 244 145 and 224 226 and 248 179 and 236 196 247 228 and 229 242 216 and 218 241 and 243 214 202 242

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Table 3.22 Chromatographic conditions for LC-MS/MS analysis

Column Mobile phase flow Column temperature Injection volume Autosampler Binary pump Software Mobile phase Time between injection Ionization source Detector Ionization source

Mobile phase composition

Poroshell 120 EC-C18 2.7 μm, (2.1 3 100) mm 0.3 mL/min 30 C 2 μL Agilent Technologies 1290 Infinity series Agilent Technologies 1290 Infinity series Mass Hunter Milli-Q acidified water/methanol acidified/ (gradient) 27 min 22.1 min Mass tandem Agilent Technologies spectrometer 6460 Electrospray Capillary voltage 2000 V Drying gas temperature 350 C Drying gas flow 11 L/min Nebulizer pressure 60 psi (0.414 MPa) Vaporizer temperature 250 C Phase A: Acidified deionized water at 0.1% (fraction volume) with formic acid Phase B: Acidified methanol at 0.1 % (fraction volume) with formic acid

Gradient Time (min) % Phase A 0 70 3.0 70 18.0 0 22.0 0 22.1 70 27.00 70 Method parameters for each compound of interest Pesticide Precursor Product Retention ion ion time (min) 3-Hydroxicarbofuran 236 163 6.15 107 3-Cetocarbofuran 238 179 3.66 161 Aldicarb 213 116 5.97 89 Ametrine 228 186 8.64 96 Atrazine 216 174 9.58 96 Carbaryl 202 145 8.74 127

% Phase B 30 30 100 100 30 30 Fragmentor Collision voltage (V) energy 72 9 33 82 9 17 106 9 13 106 17 25 106 17 25 60 9 29

(Continued)

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Table 3.22 (Continued)

Carbofuran

222

Carbofuran-D3

225

Carbendazim-D4

196

Cianazine

241

Methiocarb

226

Methomyl

185

Oxamyl

242

Simazine

202

Simetryn

214

Terbutryn

242

Prometryne

242

165 123 165 123 164 136 214 104 169 121 128 64 121 72 124 104 124 96 186 91 200 158

7.98

82

7.98

86

1.5

102

7.29

100

11.15

72

1.68

116

1.36

106

7.64

106

6.24

106

9.72

96

8.50

126

9 21 9 21 17 34 15 30 5 17 5 9 9 17 17 25 17 25 17 29 17 21

ARSC and AISC are respectively the average areas of the recovery internal standards areas relative to the calibration curve. The results obtained are plotted on a control chart for trueness having fixed limits of 70% and 120%. 12. Interferences, troubleshooting, and safety: This method provides a listing of transitions; however, it is recommended that each laboratory identifies ions and transitions for each of the pesticides using the actual conditions (solvents and adduct formation) of the target instrument. 13. References: The method was developed at CICA. 14. Minimum method validation data: Recovery range 70%115% CVR 5 0.8%10.1% Detection limit (LOD) for LC-MS: range 1088 μg/kg; for LCMS/MS: range is 2 μg/kg. Quantification limit (LOQ) for LC-MS: range 21164 μg/kg; for LC-MS/MS: range is 5 μg/kg.

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Linearity range for LC-MS is 0.121.2 mg/L; for LC-MS/MS, range is 0.010.3 mg/L; R2 . 0.99. Calibration range for LC-MS is 0.125 mg/L; for LC-MS/MS range is 0.015.

METHOD 15: Determination of organophosphorus and other pesticides in soil through ultrasound treatment 1. Laboratory name and address: Laboratory of Pesticide Residue Analysis (LARP), Ciudad Universitaria, Bogota, Colombia 2. Contact person: Jairo Arturo Guerrero Dallos email: [email protected] 3. Title of the analytical method: Analysis of pesticides in soil through ultrasound treatment 4. Principle: This method describes the determination of organophosphorus pesticides in soil by gas chromatography (GC) with microelectron capture detection (μ-ECD) and selective nitrogenphosphorus detection (NPD). The method involves extracting compounds with different physicochemical properties using a first extraction with ethyl acetate, which removes the less polar compounds, and a subsequent extraction with methanol which removes the more polar compounds. 5. Scope: Determination of organophosphorus and other pesticides such as cymoxanil, dimethoate, chlorothalonil, m-parathion, malathion, folpet, profenofos, oxadicil, cyproconazole, L-cyhalothrina, difenoconazole, propiconazole, deltamethrin, methamidophos, monocrotophos. metalaxyl, dimethomorph, azoxystrobin, and tebuconazole in soil through ultrasound treatment. 6. Equipment and instruments: Gas chromatography system with electronic pressure control and split/splitless inlet connected to a borosilicate flow splitter (Y) attached to a HP-5 capillary column (30 m 3 0.32 mm, 0.25 μm particle size) coupled to a microelectronic capture detector ECD63Ni and HP-50 (30 m, 0.32 mm d.i, 0.25 μm) capillary column in parallel with a nitrogenphosphorus detector (NPD) in parallel. 7. Reagents and materials: Ethyl acetate, trace analysis grade Methanol, HPLC grade Anhydrous sodium sulfate

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8. Standard solutions: Internal standard for u-ECD, PCB52 Internal standard for NPD, sulfotep Surrogate for μ-ECD, PCB 103 Surrogate for NPD, triphenyl phosphate (TPP) 9. Detailed procedure (protocol): • Air dry the sample. • Use a wooden roller on the samples to undo agglomerates that could be formed during drying. • Sift the sample through a No. 8 sieve which corresponds to a particle size of 2380 μm. • Weigh 5 g dry sifted soil in 50 mL centrifuge tubes. • Add 320 μL surrogate compounds mixture. • Add 30 mL ethyl acetate, trace analysis grade. • Place it into the ultrasound bath for 30 min. • Centrifuge • Filter the ethyl acetate solvent through sodium sulfate and collect 15 mL solvent in a graduated cylinder. • Transfer quantitatively the 15 mL collected fraction into a round bottomed flask. • Dry the soil fraction into the centrifuge tubes under nitrogen flow. • Add 30 mL methanol to the dried soil fraction. • Place it into an ultrasound bath for 30 min. • Centrifuge. • Filter the solvent through sodium sulfate and collect 15 mL methanol solvent in a graduated cylinder. • Transfer quantitatively to the round bottom flask that already contains the ethyl acetate extract. • Concentrate to a volume of about 0.5 mL in a rotaevaporator in a water bath at 35 C and transfer quantitatively to a 2 mL volumetric flask and fill to 2 mL volume. • Take 125 μL of extract and transfer to a 1 mL volumetric flask, in which a 20 μL internal standard solution mixture had been previously added and dilute to 1 mL with trace analysis grade ethyl acetate. • Inject the extract obtained into the gas chromatographic system. Chromatographic conditions • 2 μL injection volume • Injection mode: pulsed splitless with pulse pressure of 65 psi for 0.8 min

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• • • • •

Purge time 0.6 min Purge flow 40 mL/min Injector temperature 256 C Carrier gas: nitrogen Oven temperature program: 52 C (0 min) to 100 C at a rate of 4 C/min, subsequently the temperature is increased to 110 C at 2 C/min, then to 130 C at a rate of 20 C/min, then brought to 195 C at 4 C/min and finally a temperature of 280 C is reached through a rate increase of 5 C/min. The total analysis time is 64.25 min. Calibration and fortification Calibration and fortification levels are given in Tables 3.23 and 3.24.

Table 3.23 Concentration of the mixtures solution for matrix fortification and concentration ranges for the calibration curve Compound Mixture concentration Concentration range (µg/mL) (µg/mL)

Cimoxanyl Dimethoate Chlorothalonil m-Parathion Malathion Folpet Profenofos Oxadixyl L-Cyhalothrin Difenoconazole Cyproconazole Propiconazole Deltamethrin Methamidophos Monocrotophos Metalaxyl Dimetomorf Azoxystrobin Tebuconazole

29.31 12.34 4.3 6.31 5.36 6.02 3.7 9.74 0.91 9.3 10.91 2.04 1.81 28.75 25 16.88 26.46 9.47 4.94

0.24 0.78 0.07 0.13 0.09 0.05 0.06 0.19 0.01 0.15 0.2 0.03 0.03 0.46 0.2 0.27 0.35 0.15 0.08

1.18 3.88 0.34 0.66 0.43 0.24 0.3 0.95 0.14 0.74 1 0.16 0.14 2.28 1.02 1.38 1.76 0.76 0.4

Table 3.24 Surrogate and intern standard mixture Surrogate compound Internal standard compound

PCB 103 TPP

Mixture conc. (μg/mL) 15 25

PCB 52 Sulfotep

Mixture conc. (μg/mL) 7.1 22.6

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10. Calculations: The calibration curve is made with a blank matrix and five calibration levels. Internal standards are added to each level and the surrogate compounds are also added to the second level of the calibration curve. The calibration curve is performed according to the following equation: Acompounds 5 m  Concentration 1 b Ais where m and b are, respectively, the slope and the intercept of the calibration curve. Acompounds is the area of the compound to be analyzed and Ais is the internal standard area. Table 3.6 shows the concentration of each compound in the mixture and the concentration range of the calibration curve. Table 3.7 shows the concentrations of the surrogate and internal standards. The response of the surrogate and internal standards is near the second level of calibration for the individual pesticides. The value of each sample is interpolated on the calibration curve and multiplied by 6.4 to be expressed in mg/kg. This value can be calculated by considering 1.25 g/mL of soil sample equivalent for each extract and a total injected sample equivalent of 0.15625 g/mL. The ratio between what is present in the soil in mg/kg and the concentration of the injected amount in μg/mL gives 6.4, which is the conversion factor to be used. 11. Quality control: Recoveries are performed by fortifying 5 g soil sample with 1 mL of the mixtures as shown in Table 3.6, respectively, following the described procedure. The recoveries must fall within the range of the recovery rates calculated in the validation of the method. 12. Interferences, troubleshooting, and safety: Soil blank extractions are necessary to compare with the fortified blanks and determine possible interferences of the compounds to be analyzed. 13. References: Fenoll, J., Hellı´n, P., Marı´n, C., Martı´nez, C.M., Flores, P., 2005. J. Agric. Food Chem. 53, 76617666.

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Mojica, A.P., 2010.Determinacio´n de residuos de plaguicidas en aguas y suelos de la regio´n del lago de tota  Municipio de Aquitania, Universidad Nacional de Colombia. Mojica, A.P., Guerrero, J.A., 2010. Extraccio´n de residuos de plaguicidas en suelos asistida por ultrasonido. Rev. Colomb. Quı´m. 39 (3), 371387. Sa´nchez-Brunete, C., Miguel, E., Tadeo, J.L., 1998. J. Chromatogr. A 823, 1724. Sa´nchez Brunete, C., Tadeo, J.L., 2001. J. Chromatogr. A 918, 371380. Sa´nchez-Brunete, C., Miguel, E., Tadeo, J.L., 2002. J. Chromatogr. A 976, 319327. 14. Minimum method validation data: Matrix: soil Validation range: 0.0110 mg/kg Detection limit: 0.020.68 mg/kg Quantification limit: 0.075.30 Recovery range: 64%107% CV ,14%

METHOD 16: Determination of selected pesticides in soil by GC-MSD 1. Laboratory name and address: Food and Environmental Protection Laboratory (FEPL), Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna International Centre, PO Box 100, 1400 Vienna, Austria 2. Contact person: Britt Maestroni email: [email protected] 3. Title of the analytical method: Determination of selected pesticides in soil samples by GC-MSD 4. Principle: A multiresidue method for determination of selected pesticide residues in soil. The analytes are extracted from humidified soil with ACN and matrix interferences are cleaned up by primary secondary ammine.

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5. Scope: The current method is validated for representative pesticides in soil in the range 100500 μg/kg. The analytes are as follows: atrazine, azinphos-methyl, azoxystrobin, chlorothalonil, chlorpyrifos, cypermethrin, o,p,-DDT, p,p-DDT, diazinon, dieldrin, lindane, kresoxim-methyl, parathion-methyl, pirimicarb, and pyrimethanil. Methyl parathion represents the low persistence group (less than 30 days persistence of the parent compound in soil), atrazine and diazinon represent the moderate persistence group (half-life 30100 days), and lindane and DDT represent the high persistence group (half-life .100 days). 6. Equipment and instruments: • Centrifuge • Gas chromatograph equipped with mass selective detector • GC column: HP 5 MS, 30 m 3 0.25 mm, 0.25 μm 7. Reagents and materials: • Magnesium sulfate anhydrous, residue grade • Sodium chloride (NaCl), residue grade • Di-sodium hydrogen citrate sesquihydrate, residue grade • Sodium citrate dehydrate, residue grade • Internal standard: triphenyl phosphate and sulfotep • Certified analytical standards with declared purity • Chlorpyrifos methyl used for retention time locking (RTL) • Water, HPLC grade • ACN, HPLC grade • Isooctane, HPLC grade 8. Standard solutions: Pesticide mixture stock solution: prepared in acetone: isooctane (15:85, v-v) at concentration 1 mg/ mL and stored at 220 C. The stock solutions were stable for 6 months. Internal standard stock solution: prepared in isooctane at concentration 1 mg/mL and stored at 220 C. The stock solutions were stable for 6 months. Prepare five calibration levels and inject in duplicate. Calibration range is from 0.025 to 3 ng/μL. 9. Detailed procedure (protocol): • Weigh 10 g soil in centrifuge tubes. • Add 4 mL of HPLC grade water.

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• • • • • • • • • • • •

Shake and leave for 30 min. Extract with 20 mL ACN. Shake. Add 8 g MgSO4 1 2 g NaCl 1 1 g disodium hydrogen citrate sesquihydrate 1 2 g sodium citrate dehydrate. Shake and centrifuge at 2500 rpm for 3 min. Prepare 1.5 g MgSO4 and 250 mg PSA in a 20 mL centrifuge tube. Add 10 mL aliquot of the extract. Add 100 μL surrogate standard sulfotep at 5 ng/μL. Vortex and centrifuge at 1500 rpm for 1 min. Carefully evaporate to dryness, add 100 μL of a 5 ng/μL internal standard solution and bring to 1 mL with isooctane. Transfer to GC/MS vial. MSD analysis condition: • Inlet mode splitless, 1.0 μL injected • Inlet temperature 250 C • Pressure 2025 psi (chlorpyrifos-methyl RT relocked to 17.41 min) • Purge flow 50.0 mL/min • Purge time 1 min • Total flow 54.7 mL/min • Gas saver 20 mL/min • Gas type helium • Inlet liner splitless, single-taper, glass wool, deactivated • Oven temperature program:  Initial temperature 60 C for 1.5 min  Ramp 1 40 C/min to 110 C not hold  Ramp 6 C/min to 260 C not hold  Ramp 8 C/min to 280 C hold for 2.75 min  Ramp 10 C/min to 280 C hold for 8 min  Total run time 40.5 min (last standard elutes around 35 min)  Equilibration time 0.5 min • Column HP 5 MS, 30 m 3 0.25 mm, 0.25 μm • Acquisition mode: SIM mode • Electron energy: 70 eV • EMV mode: gain, gain factor: 5 • Quad temperature: 150 C • Source temperature: 230 C • Target and qualifier ions are given in Table 3.25.

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Table 3.25 Target and qualifier ions TG

Q1

Q2

Q3

Sulfotep Atrazine Lindane Pyrimethanil Diazinon Chlorothalonil Pirimicarb Parathion-methyl Chlorpyrifos Kresoxim-methyl Azinphos-methyl Cypermethrin Azoxystrobin

202 202 183 199 179 264 238 125 199 131 132 165 388

215 217

217 219

199 268 72 263 314 206 105 181 345

304

322 200 181 198 137 266 166 109 197 116 160 163 344

233 132

10. Calculations: A five-point calibration curve is constructed with matrixmatched calibrators and an internal standard applied to correct for volumetric losses and recovery variation. A weighted linear calibration model is applied. 11. Quality control: In each batch of analysis one recovery sample fortified at 0.5 mg/ kg is present at the beginning and again at the end of the run. Blank sample, reagent blank, and solvents are also introduced in the batch in a randomized way. 12. Remarks: The amount of water added to soil needs to be evaluated, e.g., using an empirical method. Based on the soils available for method validation it was found that the proper amount of water to be added was 40%. 13. Interferences, troubleshooting, and safety: Label all glassware to be used before starting the procedure. Strictly follow safety regulations related to general laboratory operations. 14. References: Foods of plant origin  Determination of pesticide residues using GC-MS and/or LC-MS/MS following acetonitrile extraction/ partitioning and clean-up by dispersive SPE  QuEChERS-method; EN 15662:2008.

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15. Minimum method validation data: Matrix: dried soil LOD: 0.410.95 μg/kg Average recoveries: 45%120% RSD range: 3%24%

METHOD 17: Procedure for extraction of organophosphate pesticides in water using solid phase extraction 1. Laboratory name and address: Laboratory of Chromatography, UNCo, IDEPA, CONICET, 1400 Buenos Aires, Neuque´n, Argentina 2. Contact person: Ms. Ruth Miriam Loewy email: [email protected] 3. Title of the analytical method: Procedure for extraction and measurement of organophosphate pesticides in water using solid phase extraction 4. Principle: This method uses a solid phase extraction (SPE) to selectively determine organophosphorus pesticides in water. 5. Scope: Organophosphorus pesticides residues in water: chlorpyrifos, pirimiphos ethyl, pirimiphos ethyl, parathion, malathion, methyl parathion, diazinon, dichlorvos, phosmet, fenitrothion, tetrachlorvinphos, azamethiphos, and azinphos-methyl. 6. Responsibilities: The laboratory head is responsible for overseeing that the method is performed according to the procedure. The technical manager is responsible for performing the procedure and complete the associated data records. 7. Equipment and instruments: • Analytical balance • Peristaltic pump • TurboVap evaporator • GC equipped with PTV injector and MSD, NPD and FID detector 8. Reagents and materials: • Methanol • Water, HPLC quality • Methylene chloride, pesticide quality

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n-Hexane, pesticide quality 510 mL pipettes Cartridges (equivalent to Strata-X 33 μm polymetric reversedphase) • 10 mL graduated tubes • 250 mL glass vials 9. Detailed procedure (protocol): Conditioning the SPE cartridges Load 6 mL methanol and then 6 mL water into SPE cartridges. Prevent the surface of the cartridge from drying between solvents. Elute both solvents with the desired flow rate for the sample (ideally between 5 and 6 mL/min). Sample extraction After conditioning the SPE cartridges, load the sample of water to be extracted (1 L) at a controlled flow rate (less than 6 mL/min). Choose the best option to load the total volume of sample to respect this maximum flow rate. Drying cartridge Air drying: once the sample is transferred to the cartridge, place the cartridge into the vacuum manifold and apply approx. 220 Mg Hg for 10 min, verifying that the selected channel is open and the water contained in the cartridge is removed by vacuum and discharged into the glass cuvette. Nitrogen drying: prepare the water bath at 50 C under a fume hood and after 10 min vacuum, place the nitrogen line over the cartridge, open the nitrogen key and increase the pressure gradually. Keep drying operation for 15 min. Observe the appearance of the cartridge filling which must turn into “dry powder.” Elution of the cartridge filling Once dry, place the cartridge into the vacuum manifold and place a 10 mL graduated tube just below the cartridge. Load 3 mL n-hexane and when the first drop is falling, stop the elution, wait 5 min, then allow gravity elution. Proceed by loading 6 mL methylene chloride, preventing the surface from drying, and allow slow gravity elution for the time necessary to obtain a volume of 2 mL in the tube below. Then stop elution and wait 5 min. Open and continue the elution. Collect both solvents in the same tube. • • •

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When the elution is finished, remove the tube from the manifold, and concentrate the elution to less than 0.25 mL under a nitrogen stream in a water bath (30 C) and under the fume hood. Addition of internal standard After concentrating the eluate, add the internal standard (sulfotep, 25 μL at 1 ng/μL). Make up to 0.25 mL and shake in a Vortex. Transfer to vials Transfer the samples with an automatic pipette to 400 μL vials. Measurement: Organophosphate pesticides are measured by Gas Chromatography equipped with NPD detector and the positive samples are confirmed by GC-MS (conditions listed in Tables 3.26 and 3.27). 10. Calculations: The quantification is performed using a calibration curve with internal standard. After calculating the concentration in the vial with the regression equation, the concentration in the sample is calculated using the following: ½Sample ðppbÞ5½Vialconcentration ðppmÞ0:25mL1000=Vsample ðmLÞ

Table 3.26 Chromatographic conditions (NPD)

Column Gas carrier Injector temperature Mode Oven temperature Detector temperature

Capillary column HP-5 30 m; 250 μm; 0.25 μm Nitrogen 250 C Splitless T0: 70 C, R1: 20 C/min, T1: 160 C, R2: 4 C/min, T2: 240 C (9 min) 350 C

Table 3.27 Chromatographic conditions (MS)

Column Gas carrier Injector Oven temperature Acquisition mode

Capillary column HP-5MS 30 m; 250 μm; 0,25 μm Helium PTV T0: 35 C, R1: 15 C/min, T1: 95 C, R2: 6 C/min, T2: 300 C (18 min) SIM

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11. Quality control: A blank and a recovery for each extraction should be performed. 12. Interferences, troubleshooting, and safety: Laboratory coat, nitrile gloves, protector glasses and mask. 13. References: EPA Method 3535A ISO Standard 17025 OECD GLP Principles 14. Minimum method validation data:: Matrix: river water Validation range: 0.0250.10 μg/L LOD/LOQ: 0.030.06 μg/L Recoveries: 80%120% CV , 20%

METHOD 18: Procedure for extraction of organochlorinated pesticides in water 1. Laboratory name and address: Laboratory of Chromatography, UNCo, IDEPA, CONICET, Buenos Aires 1400, Neuque´n, Argentina 2. Contact person: Ruth Miriam Loewy email: [email protected] 3. Title of the analytical method: Procedure for extraction and measurement of organochlorinated pesticides in water 4. Principle: Liquidliquid microextraction of organochlorinated pesticides in water. 5. Scope: Extraction and measurement of organochlorinated pesticide residues in river water. α-HCH, β-HCH, γ-HCH, δ-HCH, aldrin, endrin, dieldrin, heptacloro, heptacloro epoxido, op0 -DDT, pp0 -DDT, op0 -DDD, pp0 -DDD, op0 -DDE, pp0 -DDE, endosulfan I, endosulfan II, endrin aldehyde, metoxicloro, and endosulfan sulfate. 6. Responsibilities: The analyst is responsible for performing the procedure and complete the associated data records.

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7. Equipment and instruments: • Microextraction glass equipment • TurboVap evaporator • Beam balance • Gas Chromatograph equipped with MicroECD Detector 8. Reagents and materials: • n-Hexane, pesticide quality • Anhydrous sodium sulfate • Glass wool • 10 mL graduated pipette • Pro-pipette • Glass funnel • 25 mL graduated tubes • Microextractor • Variable speed magnetic stirrer • Magnetic bar • 500 μL glass vials 9. Detailed procedure (protocol): Place the sample (at room temperature) in a microextractor flask (1000 mL) Weigh 12 g anhydrous sodium sulfate and transfer to the extraction flask (see drawing at point 12) with the sample. Close the flask with the cap and shake until complete dissolution of the salt. Add 10 mL of solvent (n-hexane) shaking vigorously for 10 min. Rest for 5 min. Remove the cap of the microextraction flask and place the microextractor tube on it. Add distilled water, raising the hexane level until its complete displacement through the lateral tube and collecting it into a 25 mL tube through a funnel with glass wool and sodium sulfate. If an emulsion is produced in the system, place glass wool in the extractor tube. Concentrate the sample (in the extraction tube) to 0.5 mL under a nitrogen stream in a water bath (40 C) kept in a fume hood. Transfer to a 10 mL graduated tube with a Pasteur pipette, rinse 23 times with hexane, and concentrate to 1 mL in the same way as before. Finally transfer 0.5 mL to a labeled vial.

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Table 3.28 Chromatographic conditions

Column Gas carrier Injector temperature Mode Oven temperature Detector temperature

Capillary column HP-5 30 m; 250 μm; 0,25 μm Nitrogen 250 C Splitless T0: 70 C (1 min), R1: 20 C/min, T1:160 C, R2: 4 C/min, T2: 270 C (17 min) 300 C

10. Measurement: Organochlorine pesticides are measured by gas chromatography equipped with μ-ECD detector. Chromatographic conditions are given in Table 3.28. 11. Calculations: Quantification is performed using a calibration curve and an external standard. After calculating the concentration in the vial with the regression equation, the concentration in the sample is calculated by: ½SampleðppbÞ 5 ½Vial concentrationðppmÞ  1 mL=V sampleðmLÞ 12. Quality control: A blank and a recovery should be performed for each extraction. 13. Remarks: The glass microextractor is placed on a 1 L amber glass bottle. The shape and dimensions of the extractor device are shown in Fig. 3.2. 14. Interferences, troubleshooting, and safety: Laboratory coat, nitrile gloves, protector glasses, and mask. 15. References: Organochlorine compounds determination: DIN 38 407 F2 16. Minimum method validation data (range of matrices, range of validation, LOQ or lowest calibrated level (LCL), recoveries, and CV%): Matrix: river water Validation range: 0.0010.01 μg/L LOD/LOQ: 0.00030.001 μg/L Recoveries: 70%100% CV , 20%

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Figure 3.2 Microextractor scheme (dimensions in mm).

METHOD 19: Determination of organochlorine insecticides in water by GC/MSD 1. Laboratory name and address: Laboratorio de Bromatologia, Intendencia de Montevideo, Isla de Flores, 1323 Montevideo, Uruguay 2. Contact person: ˇ Eduardo Egan˜a Cerni email: [email protected] 3. Title of the analytical method: Determination of organochlorine insecticides and trifluralin by GC/MSD 4. Principle: Organochlorine insecticides are extracted from water using solid phase extraction and detected by using gas chromatography and mass selective detection 5. Scope: Determination of organochlorine insecticides in water: lindane, heptachlor, heptachlor epoxide, endosulfan I, dieldrin, p,p-DDE, endrin, endosulfan II, o,p-DDT, and p,pDDT. 6. Equipment and instruments: • Gas chromatograph coupled to mass selective detector • Manifold

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7. Reagents and materials: • Sodium hydrogen carbonate (NaHCO3), pesticide residue grade • Ethyl acetate, HPLC grade • Methanol, HPLC grade • Double distilled water • Column SPE C18, 6 mL, 500 mg • 2 mL volumetric flask • Adapter for columns • 50 mL disposable syringe (without plunger or needle) 8. Detailed procedure (protocol): • Filter 500 mL water sample. Condition the column with two column volumes of ethyl acetate, one volume of methanol, and finally a volume of distilled water. • Apply the 500 mL water sample to the column slowly (40 drops/min) by applying a slight vacuum if necessary. • Wash the column with a volume of distilled water. • Vacuum dry the column, ensuring it is dry by determining its mass every 5 min until constant to one-hundredth of a gram. • Elute in a 2 mL volumetric flask with two volumes each of 500 μL ethyl acetate, dilute with ethyl acetate, mix and transfer to a 2 mL vial for injection into GC-MSD. • The GC-MSD instrumental conditions are as follows: Oven Program • 50 C for 1 min. • then 25 C/min to 150 C for 10 min. • then 3 C/min to 230 C for 1 min. • Postrun: 320 C hold 1 min. Injection Volume: 2 μL Front Inlet: 250 C Mode: Pulsed Splitless Injection Pulse: 15 psi until 0.5 min. Detector: MSD Transfer Line: 280 C Column: HP5 5% Phenyl Methyl Siloxan 30 m 3 250 μm 3 0.5 μm Tune File: HighSense Tune Acquistion Mode: SIM Solvent Delay: 15 min

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Table 3.29 Target and qualifiers ions for the SIM method Target Qualifiers

Group 1 alfa BHC Group 2 17.9 min Lindane Group 3 23.0 min Heptacloro Group 4 28.0 min Heptacloro epoxido Group 5 30.0 min Endosulfan I Group 6 33.0 min Dieldrin Group 7 33.7 min p,p-DDE Group 8 34.4 min Endrin/endosulfan II Group 9 36.0 min p,p-DDT/o,p-DDT Group 10 38.0 min p,p-DDT

9.

10.

11.

12.

224

185

219

109, 183

272

274, 337

351

353, 355

339

170, 195

277

263, 345

318

248, 316

241, 275

261, 263, 279

235

165, 237, 246

235

165, 237

Relative Voltage: 1400 V SIM Parameters: see Table 3.29 Resolution: Low Calculations: The sample is concentrated 250 times during the analytical procedure. This must be taken into account when reporting results. Quality control: Testing of blank samples and recovery tests should be performed in parallel. Quantification is carried out using alphaBHC-d6 internal standard. Interferences, troubleshooting, and safety: Drying the solid phase of the columns is critical in order to avoid analyte losses in the water phase. References: Pesticides: organochlorine insecticides from water, MN Appl. No. 301700, pp 178. Solid Phase Extraction Application Guide, Macherey-Nagel

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13. Minimum method validation data Range of matrices: drinking water, groundwater, and river water LOQ: 0.010 μg/L Recovery range: 39%172% CV: , 31%

METHOD 20: Determination of selected pesticides in water by GC-MSD 1. Laboratory name and address: Food and Environmental Protection Laboratory (FEPL), Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna International Centre, PO Box 100, 1400 Vienna, Austria 2. Contact person: Britt Maestroni email: [email protected] 3. Title of the analytical method: Determination of selected pesticides in water samples by GC-MSD. 4. Principle: A multiresidue method for determination of selected pesticide residues in water. The nonpolar analytes are extracted from water with hexane. The polar pesticides are concentrated on to solid phase extraction cartridges and eluted with methanol and ACN, the extracts are concentrated and the solvent exchanged and injected on to the GC-MSD. 5. Scope: The current method is validated for representative pesticides in water in the range 0.050.5 μg/L. The analytes are as follows: alachlor, atrazine, azinphos-ethyl, azoxystrobin, chlorfenvinphos, chlorpyrifos ethyl, chlorpyrifos methyl, DDT, diazinon, dieldrin, endosulfan, kresoxim-methyl, lindane, parathion-methyl, pendimethalin, pirimicarb, profenofos, promethryn, trifluralin, and vinclozolin. 6. Equipment and instruments: • Microseparation device (see Fig. 3.3) • Rotary evaporator • Gas chromatograph equipped with mass selective detector • Magnetic stirrer and stir bar

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Figure 3.3 The microseparator device on a flask containing the water sample. A is the place where HPLC grade water is added. B represents the exit, where pesticideenriched hexane is collected.

7. Reagents and materials: • Sodium sulfate anhydrous, residue grade • Po´dium colorido (Mal), residuo grade • Internal standard: triphenyl phosphate, sulfotep or other • Certified analytical standards with declared purity • Methanol, HPLC grade • CN, HPLC grade • Isooctane, HPLC grade • SPE Phenomenex strata X-33, 200 mg/6 mL • N-Hexane, trace analysis grade 8. Standard solutions: a. Pesticide mixture stock solution: prepared in acetone at 1 mg/mL and stored at 220 C. The stock solutions were stable for 6 months. b. Internal standard stock solution: prepared in n-hexane at 1 mg/ mL and stored at 220 C. The stock solutions were stable for 6 months. c. Prepare five calibration levels and inject in duplicate. Calibration range is from 0.0250.6 μg/L. 9. Detailed procedure (protocol): Determination of nonpolar compounds • In a brown 1 L bottle flask add 20 g NaCl and 20 mL hexane to 1 L water. Add an internal standard solution. Add a magnetic stirrer and place on a magnetic stirring device.

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• • •

119

Stir for 10 min using magnetic rotation. Stop the stirring action. Allow to rest for 5 min for phase separation. Attach a microseparator device (Fig. 3.3), to the brown flask. Make sure no leakage occurs at the connection between the device and the neck of the bottle. • Slowly add 130 mL HPLC grade water to the inlet (A) of the microseparation device. The n-hexane layer will be displaced through outlet (B). Collect the displaced n-hexane (20 mL) into a beaker. This is emptied into a round bottomed flask (100 mL) through a layer of 2.5 g NaSO4 using a funnel and a filter. • Wash the collecting beaker with an additional 5 mL n-hexane and add to the round bottomed flask. • Carefully evaporate to less than 0.5 mL on a rotary evaporator. Quantitatively transfer the solution to a 1 mL volumetric flask, add 100 μL of a 5 ng/μL internal standard and bring to 1 mL with n-hexane. • Transfer to GC vial for analysis. Determination of polar compounds • Precondition SPE cartridges by adding three times 1 mL of methanol:ACN (1:1) mixture followed by adding 2 mL of HPLC grade water three times. • Slowly add 1 L of the water sample to preconditioned SPE cartridge. The flow rate should be at least 6 mL/min. • After sample loading, dry the SPE cartridges with pure nitrogen gas. • Elute three times with 1 mL methanol:ACN (1:1, v-v). • Add 100 μL of a 5 ng/μL internal standard solution prior to evaporation. • Concentrate to less than 1 mL on a rotary evaporator and exchange to n-hexane paying attention not to dry. Add 5 mL of n-hexane and repeat the evaporation step. • Evaporate to less than 0.5 mL on rotary evaporator, add 100 μL of an internal standard solution and bring to 1 mL with n-hexane. • Transfer to a GC vial for analysis. • MSD analysis condition: • Inlet mode: splitless, 1.0 μL injected • Inlet temperature: 250 C • Gas flow: 1.2 mL/min • Chlorpyrifos-methyl used for retention time locking at 17.41 min

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• • • • • •

• • • • • •

Purge flow: 50.0 mL/min Purge time: 1 min Gas saver: 20 mL/min Gas type: helium Inlet liner splitless, single-taper, deactivated Oven temperature program: Initial temperature 60 C for 1.5 min Ramp 40 C/min to 110 C Ramp 6 C/min to 260 C Ramp 8 C/min to 280 C hold for 2.75 min Total run time: 40.5 min (last standard elutes around 35 min) Equilibration time: 0.5 min Column HP 5 MS, 30 m 3 0.25 mm, 0.25 μm Electron energy: 70 eV EMV mode: gain, gain factor: 5 Quad temperature: 150 C Source temperature: 230 C Acquisition mode SIM mode: see Table 3.30 for target and qualifier ions

Table 3.30 Target and qualifier ions RT

TG

Q1

Sulfotep (ISTD) Atrazine Lindane Diazinon Pirimicarb Parathion-methyl Chlorpyrifos methyl Alachlor Prometryn Metolachlor Chlorpyrifos Pendimethalin Endosulfan, alphaProfenofos Kresoxim-methyl Endosulfan, betaTPP (ISTD) Azinphos-ethyl Azoxystrobin

322 200 181 137 166 109 286 160 241 162 197 252 195 208 116 195 326 160 344

202 202 183 179 238 125 289 146 184 238 199 253 237 139 131 237 325 132 388

13.821 14.894 15.094 15.905 16.792 17.383 17.41 17.684 17.879 18.899 19.066 20.135 21.206 21.714 22.676 23.063 25.291 27.331 35.798

Q2

Q3

215 217 199 72 263

217 219 304

188 226 240 314 162 241 337 206 241 327 105 345

Q4

233

211

239 339 132 239

206

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10. Calculations: A five-point calibration curve is constructed in solvent, and internal standards are applied to correct for volumetric losses and recovery variation. A weighted linear calibration model is applied. 11. Quality control: In each batch of analysis one recovery sample fortified at 0.5 μg/L is present at the beginning and at the end of the run. Blank samples, reagent blanks, and solvents are also introduced in the batch in a randomized way. 12. Remarks: The reference DIN 38 407 F2 provides the drawing of the microseparator device with the exact measurements. 13. Interferences, troubleshooting, and safety: Label all glassware to be used before starting the procedure. Strictly follow safety regulations related to general laboratory operations. 14. References: German standard methods for the determination of low volatile halogenated hydrocarbons by gas chromatography. DIN 38 407 F2/1993-02 15. Minimum method validation data: Range of matrices: fresh river waters Range of validation: 0.050.5 mg/L LCL: 0.025 mg/L Recoveries: 64%105% CV: 8%20%

METHOD 21: Analysis of residues of glyphosate and aminomethylphosphonic acid (AMPA) in water 1. Laboratory name and address: Departamento Laboratorio Quı´mico, Seccio´n Residuos de Plaguicidas, Ministerio de Ganaderı´a Agricultura y Pesca, Direccio´n General de Servicios Agrı´colas, Milla´n 4703, Montevideo, Uruguay. C. P. 12900 2. Contact person: Susana Franchi email: [email protected] [email protected] 3. Title of analytical method: Analysis of residues of glyphosate and AMPA in water

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4. Principle: An aliquot of the water sample is evaporated to dryness. The glyphosate and the principal metabolite (AMPA) are derivatized by the addition of a sodium borate solution, which generates adequate pH conditions for the reaction with the FMOC-Chloride derivatizing agent. Two complex fluorescents for glyphosate and AMPA, respectively, which are stable for at least 30 days at 4 C and in darkness, are formed from this reaction. The detection is performed by HPLC-FLD under the conditions detailed in the procedure. 5. Scope: Analysis of glyphosate and AMPA in water from different sources, except water from industrial effluents. 6. Responsibilities: The analyst is responsible for ensuring compliance to the standard operating procedure 7. Equipment and instruments: • Glass material: 100 mL concentration flask with cap, 200.0, 100.0, and 20.0 mL volumetric flasks, 25 and 1000 mL graduated cylinders • Rotary evaporator • Precision balance (sensitivity balance (sensitivity 0.01 g) • High-pressure (performance) liquid chromatograph with FLD detector • Column TSK Gel TOSH Bioscience LLC QAE 25 W 250 mm 3 4.6 mm 5 μm • Automatic pipettes (1.05.0 mL, 1001000 μL, and PL 10100 μL) • pH meter 8. Reagents and materials: • 0.05 M sodium tetraborate decahydrate solution (Na2B4O7  10H2O): dissolve 19.07 g Na2B4O7.10H2O in 100 mL distilled water. • 0.1 M sodium tetraborate decahydrated solution: dissolve 38.14 g of Na2B4O7  10H2O in 100 mL distilled water. • Fluoroenilmethyloxycarbonyl chloride solution 0.1% w/v in acetone: dissolve 1.00 g f FOMC-Cl for derivatization in 1000 mL acetone. • Acetone (analytical grade) • Ethyl acetate (HPLC grade) • ACN (HPLC grade) • Methanol (HPLC grade)

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• Acetic acid (analytical grade). • Phosphoric acid (analytical grade) • MilliQ water • Standard certificate of glyphosate • Standard certificate of AMPA 9. Standard solutions: • Standard solution mixture of glyphosate and AMPA at 100 mg/L: dissolve 10 mg glyphosate and AMPA certificated standard in MilliQ quality water in a 100 mL volumetric flask. • Standard solution mixture of glyphosate and AMPA at 20 mg/L: prepare from the 100 mg/L solution in MilliQ water. • Standard solution mixture of glyphosate and AMPA at 5 mg/L: prepare from the 100 mg/L solution in MilliQ water. 10. Detailed procedure (protocol): Conditioning the sample Take the sample from the freezer. The sample can be stored in a freezer at 224 C for a period of 3 months if needed. Place the sample in a water bath (30 6 5 C) for thawing. Stabilize at room temperature. Measure and record the pH of the sample, according to the instructions for the pH meter. Method of analysis: • Include in each batch of analyses a negative and a positive control prepared as indicated below. • Shake the sample. Allow to settle for 5 min. Take 10 mL and transfer into a 100 mL concentration flask. Rotoevaporate at 50 C to dryness. • Add 4.0 mL Na2B4O7  10H2O 0.05 M solution and shake slowly. • Add 4.0 mL of the FMOC-Cl 0.1% solution in acetone and shake slowly. • Rest for 20 min. • Add 20 mL ethyl acetate. Shake vigorously by hand for 1 min with a vertical movement. • Rest overnight. • Take around 1 mL of the bottom layer with a Pasteur pipette and transfer into an HPLC vial. This extract can be stored in a freezer for up to 1 month, protected from light. • To prepare the positive control, use 10.0 mL distilled water enriched with 30 mL of the glyphosate and AMPA standard solution at 5 mg/L. For the negative control use 10.0 mL distilled water prepared as indicated below.

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Shake the sample. Allow to settle for 5 min. Take 10 mL and transfer into a 100 mL concentration flask. Rotoevaporate at 50 C to dryness. • To prepare glyphosate and AMPA derivatized standards, transfer 2.0 mL of the glyphosate and AMPA standard solution at 20 mg/L to a 100 mL concentration flask. Add 2.0 mL Na2B4O7  10 H2O 0.1 M solution. Add 4.0 mL of the FMOC-Cl 0.1% solution in acetone and shake slowly. • Rest for 20 min. • Add 20 mL ethyl acetate. Shake vigorously by hand for 1 min with a vertical movement. • Rest overnight. Transfer approximately 2 mL of the bottom layer to a vial with a Pasteur pipette. Transfer 1000 μL from the vial to a 200 mL flask. The standard solution concentration is 50.0 μg/ L and it can be stored for a month in the refrigerator, protected from light. Chromatographic detection Chromatographic conditions: HPLC, FLD wavelengths: λexc 255 nm and λem 315 nm, Column, TOSOH Bioscence LLC TSK Gel QAE 25 W 250 mm 3 4.6 mm 5 μm Flow rate: 1.0 mL/min Column temperature: 40 C ACN mobile phase: methanol:water:acetic acid:phosphoric acid 600:50:350:16:2 Glyphosate retention time: 16.973 min and AMPA: 13.134 min. Run time: 25 min. • Prepare a series of standard solution for a calibration curve (0.5, 5.0 μg/L, 12.5, 25.0, 37.5, and 50 μg/L) and an adequacy test solution, following the instructions above for preparation of glyphosate and AMPA standard solutions. 11. Calculations: The calibration curve is calculated using HPLC software, according to the expression: H 5C  a1b

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This is the expression of the linear regression obtained by the least squares method. The concentration is determined according to the following formula: Vial C 5

ðH 2 bÞ a

C sample 5 mL C vialT4 mL/10 mL H 5 height of the peak corresponding to glyphosate or AMPA A and b 5 slope and intercept, respectively C vial 5 concentration of glyphosate and AMPA in the vial expressed in μg/L C sample 5 concentration of sample or AMPA glyphosate in the sample expressed in μg/L. 12. Quality control: Each batch of 10 samples to be analyzed includes a blank and a control sample. For the blank sample, use distilled water; for the control sample distilled water with a known amount of standard added. For samples of unknown origin, a known amount of standard is added to the water sample of the batch to study the recovery in the matrix. Perform traceability of preparation of standards, stock solutions of glyphosate and AMPA, and derivatized standards. Prepare stock solutions once per year, derivatized standards once a month. Check the new standards against the previous. The tolerances for these parameters are: Sample blank: no response at the retention times of glyphosate and AMPA Control sample: 70%120% of the amount added Variation allowed for traceability standards: 90%110% 13. Interferences, troubleshooting, and safety: Documents to consult • Safety data sheets • “Seguridad en el laboratorio: 15 recomendaciones ba´sicas” UNASEG guide • Procedure for use of HPLC • Rotavapor instructions

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Personal protective equipment • Nitrile gloves • Safety glasses • Lab coat Infrastructure • Extraction system in sample preparation room 14. References: Informe final del Proyecto con la Agencia Japonesa de Cooperacio´n Internacional: Asistencia para la construccio´n de un modelo de evaluacio´n ambiental para el registro de productos fitosanitarios’ 20092011, DGSA-INIA-JICA. Me´todo Oficial de Ana´lisis de Glifosato y AMPA en agua, Publicacio´n del Ministerio de Salud Pu´blica y Trabajo, Departamento de Seguridad Alimentaria fecha: 24/01/105. Seguridad en el Laboratorio: 15 recomendaciones ba´sicas, UNASEG. 15. Minimum method validation data: Matrices: from surface water courses, rivers and streams, tap water, drinking water Lowest calibration level: 0.5 μg/L Range of validation: 0.550.0 μg/L Recoveries: glyphosate: 84.6% CV: 14.5% (N 5 45); AMPA: 84.7%; CV: 12.5% (N 5 45)

METHOD 22: Determination of nitrate and nitrite in water using unsuppressed ion chromatography with UV detection 1. Laboratory name and address: Evaluation and Environmental Quality Control Service, Montevideo, Uruguay 2. Contact persons: Beatriz Brena email: [email protected] Alejandro Caaman˜o email: alejandro.caaman˜[email protected] Cristina Cacho email: [email protected] 3. Title of the analytical method: Determination of nitrate and nitrite in water using unsuppressed ion chromatography with UV detection 4. Principle: The separation method is based on an ion exchange between the mobile phase and the exchangeable ionic groups attached to a

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support material. The stationary phases are generally based on polystyrene, ethylvinylbenzene, or copolymerized resins of methacrylate groups with divinylbenzene modified by ionic exchange groups. Ion chromatography is used for separation of anions and cations. Nitrate and nitrite ions in the sample are separated on an anionic silica column of modified with quaternary ammonia groups and subsequently detected by ultraviolet light at 210 nm. Treatment of samples with high salinity is performed by solid phase extraction on sulfonic acid anion exchanger cartridges leading to chloride selective retention 5. Scope: The method is useful for the determination of nitrate (NO3-N 0.050.010 mg/L) and nitrite NO2-N 0.0310 ng/L) in drinking water, surface water, sewage, estuary, and sea waters with a salinity lower than 30 μS/cm. 6. Equipment and instruments: • Liquid chromatograph system (HPLC) equipped with an ultraviolet (UV) detector with a fixed (210 nm) or variable wavelength • Analytical conditions: • Oven temperature: 30 C • Deuterium lamp mode • λ measured 5 210 nm • Pump flow 1.0 mL/min in isocratic mode • Cell temperature: 40 C • Anion exchange chromatography column (particle size 9 μm, 250 mm long 3 4.00 mm i.d.) of quaternary ammonium group (columns suggested: Shim-pack IC-SA2 or Hamilton PRPX100) including a guard column • Conductivity meter with temperature compensation • Cation exchange chromatographic column with sulfonic groups (9 μm particle size, 250 mm long 3 4 mm i.d.) (Suggested column Shim-pack IC-SC1 or equivalent) 7. Reagents and materials: • Glass volumetric material, A class • Water, ASTM Type 1 or MilliQ quality) • Automatic pipettes, 20200 μL • Cellulose acetate filters, HDPV 13 mm i.d. with 0.45 μm size pore • Exchange cartridge, IC-Ag (Alltech, EE.UU), benzene estyrendiviny support, sulfonic acid functional group (0.5 mL of capacity) • 20 mL plastic hypodermic syringe

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HPLC auto sampler vials NIST certified and traceable standard solutions (1000 mg/L) • NaHCO3 and NaCO3, ACS quality ( . 99%) 8. Standard solutions: Prepare the following standard solutions from the NO3-N and NO2-N concentrated standard stock solution at 50 mg N/L with an automatic pipette: 0.05, 0.10, 0.25, 0.50, 1.00, 2.00, and 5.00 mg containing both analytes. These solutions can be stored and used for a month at 4 6 2 C. Note: units are mg nitrogen per liter (mg N/L) 9. Detailed procedure (protocol): Take 5 mL of sample into a 50 mL volumetric flask with a pipette and dilute (1/10) in water. Two strategies are used depending of the salinity content of the samples: • Samples with salinity content below 8 (B900 μS/cm): filter the diluted solution through a 0.45 μm filter coupled to a syringe. Discard the first mL and collect it into a vial. Then proceed to samples analysis. • Samples with salinity content above 8 (B900 μS/cm): condition the SPE IC-Ag cartridge with water, coupling a syringe to the cartridge and injecting 10 mL water. Attach a holder with a 0.45 μm filter to the bottom side. Perform the interference extraction and the filtration with a flow of 5 mL/min by manual pressure. Collect the sample in into a vial after discarding the first mL. Clean the system with 5 mL water between samples to prevent cross-contamination. Chromatographic process Mobile phase preparation: weigh 1.008 g NaHCO3 (1.7 mM) and 0.0636 g Na2Co3 (1.8 mM). Dissolve the salts in 1 L water HPLC quality. Filter the mobile phase with a 0.45 μm filter before using. Stabilize the instrument for at least an hour before passing mobile phase through the system. Inject a set of samples and a concentration standard in the following order: blank, standard, and samples. Inject a control solution between each 10 samples. 10. Calculations: Make the calibration curve by plotting area versus concentration (N-mg/L) taking the blank into consideration. Calculate the linear adjustment on the calibration curve and then obtain the intercept (b) and the slope (a)

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For the calibration curves: y1 5 a1x 1 b1 y2 5 a2x 1 b2

for N-NO3 for N-NO2

ðArea obtained  b1Þ 3 10 a1   ðArea obtained  b2Þ Concentration N-NO2 mg=L 5 3 10 a2

Concentration ðN-NO3 mg=LÞ 5

11. Quality control: The blank area must be smaller than the area of the first point in the curve. Otherwise repeat the process for preparing the calibration curve. Measure the response factor of the calibration curve daily in each sequence. This factor should be maintained between 80% and 12% of the historic values. 12. Remarks: Particular care should be taken with filtering the solutions before injection into the chromatographic system to avoid problems with column obstruction. Also, special care should be taken in cleaning the system when the run is finished. Cleaning with water ASTM Type 1 or Milli-Q quality is recommended. 13. Interferences, troubleshooting, and safety: The high salinity in estuarine water arising from high sodium chloride content can inhibit analyte separation due to column saturation. For this reason, a cut-off point of 8 has been established and waters with greater salinity should be treated by solid phase extraction to remove the interference. Cartridge reuse is recommended only when the quality control demonstrates that there is no cross-contamination. The number of times that a cartridge can be reused depends on the salinity content of the samples. Sample filtration is necessary to prevent obstruction problems in the liquid chromatographic system and to enhance the life of the chromatographic column. Fig. 3.4 shows a typical chromatographic run with standard solutions and the effect of using the extraction cartridge to eliminate chloride interference.

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Estándar

Muestra con IC-Ag

Muestra sin IC-Ag

0.0

2.5

5.0

7.5

N-NO2

10.0

12.5

15.0 min

N-NO3

Figure 3.4 Chromatogram showing the effect of the extraction cartridge.

14. References: ASTM 2001 Document D4327-03. Standard Test Method for Anions in Water by Chemically Suppressed Ion Chromatography, p. 1. Dahllo¨f, I., Svensson, O., Tortensson, C., 1997. Optimizing the determination of nitrate and phosphate in sea water with ion chromatography using experimental design. J. Chromatogr. A 771, 163168. Miller, J.N., Miller, J.C., 2001 Estadistica y Quimiometria para Quimica Analitica, fourth ed., Prentice Hall, Madrid Espana. ORganismo Uruguayo de Acreditacion (OUA). 20010 Documento OUA-DOC033, directriz para validacion de metodos de ensayo, p. 1. US. Environmental Protection Agency (USEPA), 1993. Method 300.1 Determination of Inorganic Anions by Ion Chromatography, p. 1. Williams, R.J., 1983. Determination of inorganica anions by ion chromatography with ultraviolet absorbance detection. Anal. Chem. 55, 851854. 15. Minimum method validation data: The method retention times, range, LOD and LOQ are given in Table 3.31. Table 3.31 Calibration parameters obtained Analyte TR (min) Dynamic range (mg/L) 2

N-NO2 N-NO32

6.1 8.3

0.035.00 0.065.00

LOD (mg N/L)

LOQ (mg N/L)

0.01 0.02

0.03 0.06

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The reproducibility measured by participation in an interlaboratory test showed excellent results with a Z-score of 0.223.

METHOD 23: Determination of ammonium in wastewater using unsuppressed ion chromatography with conductivity detection 1. Laboratory name and address: Evaluation and Environmental Quality Control Service, Montevideo, Uruguay 2. Contact persons: Beatriz Brena email: [email protected] Alejandro Caaman˜o email: alejandro.caaman˜[email protected] Cristina Cacho email : [email protected] 3. Title of the analytical method: Determination of ammonium in wastewater using unsuppressed ion chromatography with conductivity detection 4. Principle: Separation is based on ion exchange between the mobile phase and the exchangeable ionic groups attached to a support material. The stationary phases are generally based on polystyrene, ethylvinylbenzene, or copolymerized resins of methacrylate groups with divinylbenzene modified by ionic exchange groups. Ion chromatography is used for separation of anions and cations. In the case of cations, the groups used are sulfonates, carboxylic, or phosphate. Generally, diluted acid solutions are used as the mobile phase, requiring thereby a system with inert materials such as PEEK. Solid-phase extraction is used for the retention of organic compounds that can damage the system. 5. Scope: This method is useful for the determination of NH4-N 0.0630 mg/L in surface water, sewage, and estuarine waters with low salinity 6. Equipment and instruments: • Ion chromatographic system equipped with a conductivity detector (CD). • Conditions of analysis: oven temperature 40 C, pump flow 1.2 mL/min (isocratic), cell conductivity temperature 43 C. Response: 1.0 s and polarity 1, Intensity units: S/cm. • Cation exchange chromatographic column with sulfonic groups (9 μm particle size, 250 mm long 3 4 mm i.d.). Suggested column Shim-pack IC-SC1 or equivalent.

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7. Reagents and materials: • Glass volumetric material, A class • Water, ASTM Type 1 or Milli-Q quality • Automatic pipettes, range 20200 μL • 13 mm i.d. cellulose acetate filters, HDPV with 0.22 μm pore and 13 mm filter holders • Solid phase extraction cartridges, Sep-Pack Vac, Waters (200 mg) C18 support (3 mL capacity • 20 mL plastic hypodermic syringe • NH4-N standards traceable to NIST • Citric acid, ACS ( . 99%) grade • Extraction manifold and vacuum pump • Methanol, HPLC grade 8. Standard solutions: Prepare the following standard solution from the concentrated standard solution with automatic pipette: 0.05, 0.2, 1.0, 3.0, 5.0, and 30.0 mg N/L These solutions can be stored and used for a month at 4 6 2 C 9. Detailed procedure (protocol): Sample preparation • Turn on the vacuum extraction system. • Condition the cartridge with 5 mL of Milli-Q water, 5 mL methanol and finally with 10 mL distilled water. Wait 10 min after conditioning. • Take 5 mL sample with a pipette and dilute (1/10) in Milli-Q water in a 50 mL volumetric flask. • Charge 5 mL of sample on the cartridge and discard the first 2 mL of the eluate. Complete the elution and collect the eluate (sample) in a clean flask. • Arm the filtration system with 0.22 μm filters, and filter the extracted solution. Transfer the filtered solution into a labeled HPLC vial. • Wash the Sep-Pak cartridge with 5 mL distilled water. • Wash with distilled water between filtrations to prevent crosscontamination. Chromatographic process • Mobile phase preparation: weigh 0.576 g of citric acid and dissolve in 1 L of HPLC grade water. Filter the mobile phase with a 0.45 μm filter before using. Stabilize the instrument for at least 1 h by passing the mobile phase through the system.

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• Inject a set of samples and a standard in the following order: blank, standard, samples. Inject a control solution between each 10 samples. 10. Calculations: Make a calibration curve by plotting area versus concentration (N-mg/L) taking the blank into consideration. Calculate the linear adjustment on the calibration curve and then obtain the intercept (b) and the slope (a) Area 5 Conc: ðmg=LÞ  a 1 b Calculate final concentration as follows Conc: ðN-NH4 mg=LÞ 5

F dil  ðsignal area  bÞ a

where F dil 5 sample dilution factor 11. Quality control: The blank area must be smaller than the area of the first point on the curve. Otherwise repeat the process for preparing the calibration curve. Inject a control standard solution each 10 samples (intermediate concentration between two points on the curve) and check the system status. Determine the recovery as follows: R (%) 5 (Determined concentration (N-NH4 mg/L) Theoretical concentration (N-NH4 mg/L))/Theoretical concentration (N-NH4 mg/L) 12. Remarks: Special care should be taken with filtering the solutions before injection into the chromatographic system to avoid problems arising from obstruction of the column. Care also needs to be taken with cleaning the system when the run is finished. Cleaning with water ASTM Type1 or Milli-Q quality is recommended. 13. Interferences, troubleshooting, and safety: Depending on the column used, sodium can act as interference (the elution order generally used is: Li, Na, NH4, K, Mg, and Ca). Verification of this phenomenon is recommended with Nalco and NH4 solutions of known concentration. The sodium effect in determination of a 0.1 mg/L N-NH41 solution is exemplified in Fig. 3.5.

Analytical Methods for Agricultural Contaminants

134 µV 200 150 100 50 0 –50 –100 10.0

12.5

15.0

min

Figure 3.5 Effect of Na1 on the determination of NH41. A solution of 20 mg Na1/L is shown in black, a solution of 40 mg Na1/L is shown in pink, a solution of 60 mg Na1/L is shown in blue, and the NH41 standard solution is shown in brown.

14. References: Duran, M.C., 2011. Informe de practicando: Validacion de cormatografia ionica en muestras ambientales, Intendencia de Montevideo, Montevideo, Uruguay. Fritz, J.S., Gjerde, D.T., 2009. Ion Chromatography, WILEY  vch Verlag GmbH & Co. KGaA, Weinheim (ISBN: 978-3-52732052-3). Jackson, P.E., 2000. Ion chromatography in environmental analysis. In: Meyers, R.A. (Ed.), Encyclopedia of Analytical Chemistry. John Wiley & Sons Ltd, Chischester, pp. 27792801. Shimadzu Ion Chromatograph System Application Data Book, 3295-06406-10a-ik. Weiss, J., 2004. Handbook of Ion Chromatography, WILEYVHC Verlag GmbH & Co. KgaA Weinheim, (3-527-28701-9). 15. Minimum method validation data: The method range, LOD anfd LOQ are given in Table 3.32. Table 3.32 Calibration parameters obtained (matrix: sewage) Analyte TR (min) Dynamic range (mg/L) LOD (mg N/L)

LOQ (mg N/L)

N-NH4

0.07

10.5

0.0530.0

0.05

LOD, limit over detection; LOQ, limit over quantification; TR, retention time.

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Recovery: 106%, RSD: 5% (Standard: Merck, lot HC085895) The reproducibility measured by participation in an interlaboratory test showed excellent results with a Z-score 0.223.

EXAMPLES OF METHODS USING NUCLEAR AND BIOLOGICAL APPROACHES METHOD 24: Detection of irradiated fat containing foods through the analysis of 2-alkylcyclobutanones using gas chromatography coupled to mass spectrometry. 1. Laboratory name and address: Centro de Aplicaciones Tecnolo´gicas y Desarrollo Nuclear (CEADEN), Dpto. Radiobiologı´a., Calle 30 No. 502, esq. 5ta Ave., Miramar Playa, Cuba 2. Contact person: Damaris L. Moreno Alvarez email: [email protected] 3. Title of the analytical method: Detection of irradiated fat containing foods through the analysis of 2-alkylcyclobutanones using gas chromatography coupled to mass spectrometry 4. Principle: This method is applicable to food containing fat which are subject to irradiation treatment. Radiation-induced 2-alkylcyclobutanones are formed from the fatty acids of the irradiated fats. The method is based on the detection of 2-alkylcyclobutanones by gas chromatography coupled to mass spectrometry. During irradiation, the acyl-oxygen bond in triglycerides is cleaved and this reaction results in the formation of 2-alkylcyclobutanones (2-ACBs). 2-ACBs comprise a four-membered ring with a keto group at position 1 and a side chain at position 2 containing the same number of carbon atoms as the parent fatty acid. Thus if the fatty acid composition is known, the 2-alkylcyclobutanones formed can be predicted. The 2-alkyilcyclobutanones are extracted together with the fatty phase using n-hexane or n-pentane, and the extract is fractionated using column chromatography and analyzed by gas chromatography coupled to mass spectrometry.

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5. Scope: This method is applicable for the identification of irradiated fat containing. The 2-alkylcyclobutanones analyzed by this method are the 2-dodecylcyclobutanone (2-DCB) and the 2-tetradecylcyclobutanone (2-TCB) which are formed from palmitic acid and stearic acid, respectively, during the irradiation process. 6. Equipment and instruments: • Food homogenizer • Soxhlet extractor • Water bath • Glass chromatography column (300 mm length, 20 mm internal diameter) • Rotoevaporator • Glass vials • Gas chromatograph coupled to mass spectrometry 7. Reagents and materials: • Milli-Q water • n-Hexane trace analysis grade • Anhydrous sodium sulfate reagent grade • Ethyl alcohol • Florisil 150250 μm 8. Standard solutions: Preparation of 2-cyclohexylcyclohexanone 0.5 μg/mL, 2-dodecylcyclohexanon and 2-tetradecylcyclobutanone 10 μg/mL 9. Detailed procedure (protocol): 9.1 Soxhlet fat extraction method: Sampling of the fat containing tissue or parts shall be conducted according to standardized operating procedures of the laboratory. Weigh 20 g of anhydrous sodium sulfate and 20 g of wellhomogenized sample. Place in an extraction vessel and plug with cotton wool. If the simple has high water amount increase the amount of sodium sulfate needed for drying the sample. Freeze-drying can be used for papaya and mango, always checking that the 2-alkyl cyclobutanone recovery is constant. Add 100 mL of n-hexane in a 250 mL glass balloon, cover with an extraction unit (see Fig. 3.6 parts 3,6, and 7), place the extraction thimble (4) containing the sample (5) and add 40 mL of n-hexane.

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Figure 3.6 A schematic representation of a Soxhlet extractor. 1, Stirrer bar; 2, still pot (the still pot should not be overfilled and the volume of solvent in the still pot should be 3 to 4 times the volume of the Soxhlet chamber); 3, distillation path; 4: thimble; 5, solid; 6, Siphon top; 7, Siphon exit; 8, expansion adapter; 9, condenser; 10, cooling water in; 11, cooling water out (Wikipedia, 2014).

Place the glass balloon in a waterbath and attach a condensation device above the extractor unit. Heat the solvent until reflux and slowly extract during 6 h. The solvent shall exceed the siphon level four times per hour. Remove the flask from the heat and discard the thimble and the remaining n-hexane present in the extractor chamber (around the thimble). 9.2 Lipid content determination: Dry the glass vials, at least 3 h, in oven at 100 C and allow cooling in a desiccator, weight and add an aliquot of the extracted sample, evaporate the solvent under nitrogen flow.

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Weigh again. This procedure is repeated until the weight is constant. The required volume of the extract should contain about 200 mg of lipids for its calculation. 9.3 Florisil column chromatography: A 2021 cm Florisil column is prepared. Florisil is activated by heating at 550 C for 5 h, allow cooling in a desiccator. Activate the column adding 20:100 (wt/wt) of water: florisil (around 30gr of activated florisil is required for each column). Place a known volume of extract containing approximately 200 mg of lipids in the column. Elute at a flow rate of 2 mL/min and collect the eluent in a flask using 1% ethyl alcohol in 99% n-hexane, collect the eluate and concentrate, if necessary, in a rotoevaporator. Transfer to test tube and concentrate under nitrogen flow. Suspend in 200 μL of a 1-cyclohexyl cyclohexanone solution (internal standard). 9.4 Separation and detection: The 2-alkyl cyclobutanone are separated by capillary column and identified by the mass spectrum working in selected ion monitoring mode for ions of mass/charge (m/z) 98 and 112. 9.5 Analysis: The 2-alkylcyclobutanones analyzed by this method are the 2-dodecylcyclobutanone (2-DCB) and the 2-tetradecylcyclobutanone (2-TCB). The ions monitored in the gas chromatograph coupled to mass spectrometry are 98 and 112 m/z. 10. Calculations: A calibration curve for 2-DCB and 2-TCB is constructed in the range 0.5, 0.2, 1, 2, and 5 ppm. The quantification is done through the ratio of the area of the compound to the area of the internal standard. 11. Quality Control: A nonirradiated control sample and a control sample traced with 200 and 100 μL of 2-DCB and 2-TCB at 10 μg/mL in n-hexane, in duplicate, are used as quality control. These samples are processed in the same way that the sample to study. The recovery percent is calculated using the traced samples. 12. References: Crews, C., Driffield, M., Thomas, C., 2002. Analysis of 2-alkylcyclobutanones for detection of food irradiation: Curre status, needs and propects. J. Food Compos. Anal. 26, 111.

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EN 1785 Foodstuffs  Detection of irradiated food containing fat  Gas chromatographic/Mass spectrometric analysis of 2-Alkylcyclobutanones http://ec.europa.eu/food/fs/sfp/fi07_met1785_en.pdf Hijaz, F., Kumar A., Smith, S., 2010. A rapid direct solvent. Extraction method for the extraition of the 2-dodecylcyclobutanone from irradiated ground beef patties using acetonitrile. J. Food Sci. 75 (6). Horvatovich, P., Werner, D., Jung, S., Miesch, M., Delincee, H., Hasselmann, C., et al., 2006. Determination of 2alkylcyclobutanones with electronic impact and chemical ionization gas chromatography/mass spectrometry (GC/MS) in irradiated foods. J. Agric. Food Chem. 54, 19901996, 2006 Obana, H., Furuta, M., Tanka, Y., 2005. Analysis of 2alkylcyclobutanonas with accelerated solvent extraction to detect irradiated meat and fish. J. Agric. Food Chem. 53 (17), 66036608. Stewart, E.M., Moore, S., Graham, W.D., Mc Roberts, W.C., Hamilton, J.T.G., 2000. 2-Alkylcyclobutanones as markers for the detection of irradiated mango, papaya, Camembert cheese and salmon meat. J. Sci. Food Agric. 80, 121130. 13. Minimum method validation data: Range of matrices: mango and papaya Recovery %: in mango: 83% for 2-DCB and 92% for 2-TCB in papaya: 89% for2-DCB and 96% for 2-TCB

METHOD 25: Determination of radon in water 1. Laboratory name and address: Centro de Investigaciones y Aplicaciones Nucleares (CIAN), Engineering and Architecture Faculty. Final 25 Avenida Norte, Ciudad Universitaria, San Salvador, El Salvador, C.A. 2. Contact person: Mr. Julio Ernesto Payes email: [email protected] Herna´ndez [email protected] 3. Title of the analytical method: Determination of radon in water 4. Principle: This method is based on the scintillation counting of 222Rn and its progeny. 222Rn is the seventh element in a series created as a decay product of 226Ra.

140

5. 6.

7.

8.

9.

Analytical Methods for Agricultural Contaminants

Radon dissolved in water is extracted with an organic solvent (toluene), taking advantage of the major solubility of radon in such kinds of solvents. The extract is mixed with a suitable scintillation cocktail and analyzed in a liquid scintillation counter. Scope: Potable water and spring water Equipment and instruments: • Sampling funnel • Plastic vials with caps, 20 mL capacity • Liquid scintillation counter Reagents and materials: • Distilled water • Toluene scintillation degree • 1,4-bis(5-phenyloxazol-2-yl) benzene, known as POPOP (scintillation compound) C24H16N2O • 2,5-Diphenyloxazole, known as PPO (scintillation compound) C15H11NO • Liquid scintillation counter accessories Standard solutions: • Toluene-based scintillation cocktail, prepared at the laboratory • Preparation: • Slowly dissolve 4.0 g of PPO and 0.01 g of POPOP in 40 mL of toluene. • 226Radium-standard solution, NIST traceable Detailed procedure (protocol): • Collect 1 L of water in a plastic container, remove air bubbles. • Filter the water and collect it in a separatory funnel. • Add 40 mL of scintillation cocktail • Stir the mixture vigorously for 1 min in the separatory funnel. • Place the separatory funnel in a clamp and let stand until two distinct layers are visible. • Transfer 20 mL of the organic phase to a polyethylene terephthalate (PET) vial. • Seal the vial, clean it with a cloth dampened with alcohol, allow to stand for 3 h in darkness. • Place the vial in the liquid scintillation counter and measure for 60 min at open window of 02000 kevs.

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10. Calculations: Calculation of Radon activity (Bq/L) is done by: AC 5

Rn ðCF ÞðDÞðVa Þ

where AC, concentration of radon activity (Bq/L) Rn, net count rate (s21), CF, calibration factor, D, decay correction factor, Va, sample volume Net count rate: Rn 5 Ra 2 Rb where Ra, sample count rate (s21) Rb, blank count rate (s21) Calibration factor (CF): CF 5

ðCCS 2 CB Þ ACS

where CCS, count rate from the calibration standard (s21), CB, count rate from the blank sample (s21), ACS, calibration standard activity (Bq). Decay correction factor:  0:693ðT Þ 2 D 5 e t1=2 T, elapsed time (days) from sample collection till sample analysis t1/2, half life for radon (3.82 days). Limit of detection or minimum detection activity: qffiffiffiffi ð2:71ÞðJÞ 1 4:65 Rt b t MDC 5 ðCFÞðDÞðVa Þ where MDC, minimum detectable radon activity concentration (Bq/L) t, counting time of the sample and background

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J value 5 index of dispersion for the net counts produced by radon and its progeny: c51 P3 c e2λi t i50 i J5 1 2 e2λ0 t where λ values are tabulated decay constants and c are tabulated coefficients. Uncertainty calculation of the radon activity concentration (AC). Standard uncertainty (µ): u 2 ð Rn Þ 5

J 3 Rn 1 2Rb t

Combined standard uncertainty: pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi u2 ðRn Þ 1 Rn2 3 ðu2r ðCF Þ 1 u2r ðVa Þ 1 u2r ðDÞÞ uc ðAC Þ 5 CF 3 D 3 Va 11. Quality control: Quality control measures and acceptability criteria are described in Table 3.33. 12. Interferences, troubleshooting, and safety: Interferences: other radionuclides soluble in the scintillation cocktail may interfere. Troubleshooting: a reduced or increased counting efficiency may result if sample quenching is significantly different than that of the Table 3.33 Quality control measures and criteria applied to the method Parameter Procedure Frequency Accepting criteria

Liquid scintillation counter efficiency

Background

Use a sealed standard solution of known activity Ra-226 to calculate Rn-222 concentration at secular equilibrium Use a blank solution

Daily

Measurements should be within 2 standard deviations from the average

Twice a day

Measurements should be within 2 standard deviations from the average

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calibration standard. To overcome this, compare the quenching indicator parameter (QIP) (usually provided by the counter during the measurements). Safety: observe specific procedures for handling liquid radioactive materials (Radium-226 standard solutions). 13. References: ASTM, 2009. Standard test method for radon in drinking water. 5 pp., D5072-09. Kappel, R.J., Keller, G., Kreienbrock, L., Nickels, R., 1992. An epidemiological study using passive radon measurement by liquid scintillation counting. From: Noakes, J., Scho¨nhofer, F., Polach, H., (Eds.), 1993. International Conference on Advances in Liquid Scintillation Spectrometry. 522 pp. Radiocarbon, Vienna. 14. Minimum method validation data: Minimum detectable activity for Rn concentration 5 0.068 Bq/L Limit of quantification 5 0.13 Bq/L. Recovery 5 101% Reproducibility: 10% Efficiency of detection 5 91% Calibration curve shown in Fig. 3.7.

12.0

Conteos (s –1)

10.0

R2 = 0.9991

8.0 6.0 4.0 2.0 0.0 0.0

0.2

0.4

0.6

0.8

1.0

1.2

Activity Rn(Bq)

Figure 3.7 Calibration curve.

1.4

1.6

1.8

2.0

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METHOD 26: Procedure for the generation of adsorption isotherms in soils using radiotracers 1. Laboratory name and address: Laboratory of Soil Analysis, Agriculture Section, Chilean Nuclear Energy Commission, Avda. Nueva Bilbao 12501, Las Condes, Santiago, Chile 2. Contact person: Adriana Nario email: [email protected] Ana Maria Parada email: [email protected] Ximena Videla email: [email protected] 3. Title of the analytical method: Procedure for the generation of adsorption isotherms in soils using radiotracers 4. Principle: Determination of adsorption curves at different 14C-pesticides concentrations in soil samples in order to understand the behavior of pesticides molecules in soil and the potential mobility in it. 5. Scope: Samples from agricultural soil with agrochemical application management 6. Equipment and instruments: • Horizontal shaker • Vortex mixer • Centrifuge • Liquid scintillation counter • Analytical balance 7. Reagents and materials: • 20 mL centrifuge glass or plastic tubes • 20 mL scintillation counting glass vials with hermetic caps • Aqueous solution of 0.01 M CaCl2 • Liquid scintillation cocktail • 14C-labeled pesticide • Pesticide active ingredient 8. Standard solutions: a. Fortified Solution 1: For the determination of the kinetics of adsorption (equilibration time, ET) (1) prepare a 14C-Pesticide solution with approximate activity of 5000 dpm/mL in 0.01 M CaCl2 aqueous solution.

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b. Fortified Solution 2: For the determination of adsorption isotherms (2) prepare a 14C-Pesticide solution with approximate activity of 5000 dpm/mL in 0.01 M CaCl2 aqueous solution having a pesticide active ingredient concentration of 1.0, 0.8, 0.6, 0.4, and 0.2 μg/mL. 9. Detailed procedure (protocol): a. Determination of the adsorption kinetics (Equilibrium Time, ET) • Weigh three replicate 2 g samples of soil in a glass tube • Add 10 mL of the fortified solution (1) • Shake the tubes in a Vortex mixer for 2 min • Place the tubes in a shaker and shake for 1, 10, 20, 30, 60, 90 min, and 24 h (target time spans), at 170 rpm and 20 6 1 C • After each target time span, remove the tubes from the shaker and centrifuge for 10 min at 3500 rpm • Withdraw a 1 mL aliquot of the supernatant solution into a 20 mL glass bottle • Add 10 mL of liquid scintillation cocktail • Determine the activity expressed in dpm in a liquid scintillation counter b. Determination of the adsorption isotherm (at the Equilibrium Time as obtained in a) • Weigh 2 g of soil in a glass tube, three replicates, for each concentration prepared in 8b. • Add 10 mL of the fortified solution (8b) to each • Shake the tubes in a Vortex mixer for 2 min • Place the tubes in a shaker and shake for 1, 10, 20, 30, 60, and 90 min (target time spans), at 170 rpm and 20 6 1 C • After each target time span, remove the tubes from the shaker and centrifuge for 10 min at 3500 rpm • Withdraw a 1 mL aliquot of the supernatant solution into a 20 mL glass bottle • Add 10 mL of liquid scintillation cocktail • Determine the activity expressed in dpm in a liquid scintillation counter

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10. Calculations: The concentration of pesticide present in the aqueous solution at equilibrium (Ceq) is calculated using the following formula: Ceq 5

Ci  Aeq Ai

where Ceq 5 Concentration of pesticide present in the aqueous solution at equilibrium (μg/mL) Ci 5 Concentration of pesticide in each standard (μg/mL) Aeq 5 Activity at equilibrium in the aqueous phase (dpm/mL) corrected for the blank response Ai 5 Activity of the added standard (dpm/mL) To estimate the equilibrium time, plot the different obtained Ceq in a graph. The equilibrium time corresponds to the pesticide adsorption in different evaluated time. Obtaining a graph of the percentage of pesticide adsorbed versus time, the plateau of the graph will provide the equilibrium time. The concentration of pesticide present in the soil at equilibrium (Cs) is calculated using the formula: Cs 5

ðCi 2 Ceq Þ  VT ms

where Cs 5 Concentration of pesticide present in the soil at equilibrium (μg/g) Ci 5 Concentration of the pesticide in each standard (μg/mL) Ceq 5 Concentration of pesticide present in the aqueous phase at equilibrium (μg/mL) ms 5 soil mass (g) VT 5 total volume (mL) The values obtained for Ceq and Cs can be plotted in a graph to adjust to the Freundlich equation which represents the amount of adsorbed pesticide in soil (Cs) in relation to the amount of pesticide in equilibrium in the solution (Ceq) according to the following formula: Cs 5 Kf  Ceq 1=n

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where Cs 5 amount of adsorbed pesticide in the soil (mg/g) Kf 5 adsorption Freundlich coefficient Ceq 5 amount of pesticide equilibrium in the aqueous phase (mg/mL) 1/n 5 adsorption variation and the curve linearity To build the Freundlich Isotherm calculate the logarithm values of Cs (log Cs) and Ceq (log Ceq). Plot the values in a graph, where y-axis 5 log (Cs) and x-axis 5 log (Ceq). Construct a linear regression of the data and estimate the following parameters according to the following formula: Log Cs 5 1=n Log Ceq 1 Log Kf 1 n5 m Kf 5 10b Kof 5 Kf U

100 %co

where m 5 slope of the regression curve and is equal to 1/n b 5 intercept of the regression curve and is equal to log Kf The Freundlich Adsorption Coefficient Normalized to the organic matter content of the soil (Kof) can be estimated using the following formulas: n5

1 m

Kf 5 10b Kof 5 Kf U

100 %co

where Kf 5 Freundlich Adsorption Coefficient (similar to Kd (L/kg)) %co 5 amount of organic matter (g/g) Kof 5 Freundlich Adsorption Coefficient Normalized to the organic matter content of the soil (similar to Koc (L/kg)) 11. Quality control: A blank set of samples is prepared by weighing 2 g of soil, adding 10 mL of 0.01 M CaCl2 aqueous solution (without pesticide), and

148

12.

13.

14.

15.

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proceeding with the shaking times and conditions as described above for samples. Remarks: The laboratory shall have an authorization for working with radioisotopes. The soil: CaCl2 aqueous solution proportion could range from 1:1, 1:5, 1:25, depending on the matrix (soil and sediments) characteristics (percentage of clay, organic carbon content). Interferences, troubleshooting, and safety: Label all glassware to be used before starting the procedure. Strictly follow safety regulations related to general laboratory operations. References: ISO 9001:2015. Quality Management Systems  Requirements. OECD (Organization for Cooperation of Economic and Development), 2000. Guidelines for the Testing of Chemicals: Adsorption-Desorption Using a Batch Equilibrium Method (Guideline 106). SA-CCHEN, 1992. Guia Practica de Proteccion Radiologica. Seccion Agricultura.  DAN. Vers. 2. Skoog A.D., Holler, J.F., Nieman, A.T., 2001. Principios de Ana´lisis instrumental. 5a% Edicio´n. Mc Graw Hill, Madrid, 1028 pp. Minimum method validation data: The reading in disintegration per minute (dpm) is considered acceptable when the value is between 4 and 6 times of the value of the blank. Coefficient of variation of the samples should be , 5%.

METHOD 27: Effect of pesticides, drugs, and chemical compounds on the soil respiration using 14C-glucose as a radiotracer 1. Laboratory name and address: Laboratorio de Ecologia de Agroquı´micos  Instituto Biolo´gico  Sa˜o Paulo  SP, Brazil www.biologico.sp.gov.br 2. Contact persons: Mara M. de Andre´a email: [email protected] 3. Title of the analytical method: Effect of pesticides, drugs, and chemical compounds on the soil respiration using 14C-glucose as a radiotracer

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4. Principle: The soil microorganisms are very important in the nutrient cycle because they act directly on the breakdown and transformation of organic matter, which are crucial to soil fertility. The influence of xenobiotics on the soil bioactivity was already observed (Papini and Andre´a, 2000; Andre´a et al., 2003; Steffani et al., 2012) and thus some interference on the transformation processes may also occur. The method is based on the addition of known amounts of pollutants to batches of soil. Soil microorganisms are given 14C-labeled glucose as a nutritional substrate and the amount of labeled CO2 produced is measured in a liquid scintillation counter. The amount of labeled CO2 produced by treated soil versus control soils is compared. 5. Scope: Laboratory bioassay to evaluate the effect of pollutants (agrochemicals and other drugs) that are applied or discarded in the soil on its bioactivity. 6. Equipment and instruments: • Liquid scintillation counter (LSC) • Dark room with controlled temperature • Biometric flasks (an example is shown in Fig. 3.8) 7. Reagents and materials: • 14C-labeled glucose • Glucose • NaOH or KOH, technical grade

Figure 3.8 Biometric flask used for the procedure.

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• Deionized water • Ethanol residue grade • Liquid scintillation cocktail according to Mesquita & Ruegg (1984) 8. Standard Solutions: • Prepare using deionized water an aqueous 14C-labeled glucose solution containing 0.5 μCi of 14C-glucose and 200 mg of glucose/mL • Prepare 0.1 M of NaOH or 0.1 M of KOH 9. Detailed procedure (refer to Fig. 3.8): • Dry the soil sample from the field (preferably with no history of contamination, or with a well-known history of the field site) under room temperature • Sieve the soil with a 2.0 mm mesh • Determine the soil moisture content • One week before the start of the treatments adjust the soil moisture content to 60% of the Maximum Water Holding Capacity (MWHC) less 1.0 mL. This is done to activate the soil microbial activity (Stefani et al., 2012) • Ensure a constant temperature of storage of the soil samples (e.g., 22 C or 25 C) • Three batches of three replicates each of 50 g (dry weight equivalent) soil samples are placed into biometric flasks • For studying the behavior of agrochemicals: • One batch represents the controls. These soil samples will be treated only with the same volume of solvent as used in the other batches • A second batch will be treated with a concentration of pollutant that reflects the maximum amount of the pollutant that may reach the soil after agrochemical application, i.e., the full recommended dose • A third batch will receive a multiple of the second batch treatment (e.g., five times higher dose) • The concentration of the agrochemicals added to soil are calculated assuming the uniform incorporation of the recommended dose per hectare of the active ingredient to a depth of 5 cm and a soil bulk density of 1.5 (OECD, 2000). • For agrochemicals that are applied directly to soil, or for chemicals for which the quantity reaching the soil can be predicted, the test concentration recommended is the Predicatable Environmental Concentration (PEC) and five times that concentration.

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If nonagrochemicals are tested, a geometric series of at least five concentrations is used. The concentrations tested should cover the range needed to determine the ECx values. • Carefully and quickly incorporate into all biometric flasks, at the soil surface, 1.0 mL of an aqueous 14C-labeled glucose solution containing 0.5 μCi of 14C-glucose and 200 mg of glucose/mL. • Immediately close the soil container. • Add 10 mL of NaOH (0.1 M) or KOH (0.1 M) solution in the side tube (C) of the biometric flask. • Close the alkali container with an inert rubber stopper (A). • Store the biometric flasks under controlled temperature in the dark. • Change the alkali solution, at a minimum after 7, 14, and 28 days of incubation period. It is however recommended to change the alkaline solution more frequently, every 2 h during the first 10 h of the study as well at more frequent intervals such as, 1, 3, 5, 7, 14, and 28 days after the treatment. • To change the alkaline solution, remove the rubber stopper on the side arm (C) and connect a syringe to the needle (B); remove the rubber stopper (A) placed on top of the ascarite tower (F) and open the glass tap; aspirate the alkaline solution through the syringe. • Place the alkaline solution into a test tube and seals it with a stopper or a glass lid. • Put freshly prepared alkaline solution back in the side tube. • Close the needle (B), the glass tap (G), and cover again the ascarite tower with the rubber stopper. • To measure the amount of CO2 produced by soil microorganism take 2.0 mL of each of the collected alkaline solution and mix with 10 mL of scintillation cocktail that is compatible with aqueous solutions and measure the activity in a LSC counter. 10. Calculations: The 14CO2 produced and trapped by the alkaline solution is calculated from the 14C amount detected by LSC, taking into account the 10 mL of alkali collected each sampling time, and compared with the added amount (200 mg) to each 50 g soil (dry weight equivalent). Results are expressed as mg CO2/kg dry soil per hour and the mean of the respiration rate in the pollutant-treated samples is

152

11.

12.

13.

14.

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compared with the mean in the control samples (OECD, 2000). If the mean between the treated and control samples is equal or greater than 25%, the measurements should continue for additional 14 days or until a maximum of 100 days, with fortnight sampling of the alkaline solution (OECD, 2000). Quality control: • The actual 14C-activity (radiochemical activity) in dpm in the labeled glucose is checked by counting using an LSC at least, two replicate 50 μL aliquots of the 14C-glucose solution. • Three replicates for each batch are recommended for statistical significance. • Daily checks of the standard carbon-14, and the standard of background radiation shall be checked to ensure accurate measurements and to ensure the efficiency of the liquid scintillation counting. Remarks: The 14C-glucose aqueous solution should be prepared in 10 mL volumetric flask to reach 0.5 μCi of 14C-glucose and 200 mg of glucose/mL. 0.1% (10 μL) of ethanol may be added to avoid contamination and degradation of the glucose. If KOH is used, it is necessary to keep the scintillation vials during 24 h in the dark before counting, for decay of the naturally occurring 40K. The moisture content of the soil samples should be kept between 40% and 60% of the MWHC, which may be assured by weighting each complete biometric flask in the beginning of the study and the weekly checkings. Deionized water may be added when necessary, at the time of the exchange of the alkaline solution. Interferences, troubleshooting, and safety: All the laboratory personnel must be warned and trained on the safety and correct handling of the pollutants and radioactive materials. Attention and correction shall be paid if unmiscible phases may occur in the scintillation vials during LSC counting. Minimum method validation data (range of matrices, range of validation, LOQ or lowest calibrated level (LCL): The method validation was conducted using four classes of soils with variation ranges of the following attributes:

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Organic Matter: 1354 g/dm3; Microbial Biomass: 0.171.21 mgC/g and pH: 5.46.4 The Limit of Detection (LOD) was calculated as the mean 1 3 times of the activity (dpm) standard deviation of the blanks (2.0 mL of 0.1 M NaOH or KOH solution mixed with 10 mL of scintillation cocktail), estimated as 285.9 dpm (4.8 Bq). The Limit of Quantification (LOQ) was defined as the mean 1 10 times of the activity (dpm) standard deviation of the blanks, estimated as 484.0 dpm (8.1 Bq). The accuracy obtained for 14C-Glucose standard detection (dpm) was # 2%. 15. References: Andre´a, M.M., Peres, T.B., Luchini, L.C., Bazarin, S., Papini, S., Matallo, M.B., et al., 2003. Influence of repeated applications of glyphosate on its persistence and soil bioactivity. Pesq. Agropec. Bras. 38 (11), 13291335. Geisseler, D., Horwath, W.R., Scow, K.M., 2011. Soil moisture and plant residue addition interact in their effect on extracellular enzyme activity. Pedobiologia 54, 7178. Mesquita, T.B., Ruegg, E.F., 1984. Influeˆncia de agents tensoativos na detecc¸a˜o da radiac¸a˜o beta. Cieˆnc. Cultura, 36, 446450. OECD  (Organization for Economic Co-operation and Development), 2000. OECD guideline No 217. Soil Microorganisms: Carbon Transformation Test. 10 pp.% Papini, S., Andre´a, M.M., 2000. Enhanced degradation of metalaxyl in Gley Humic and Dark Red Latosol. Rev. Bras. Cieˆnc. Solo 4, 469474. Stefani Jr., A., Felı´cio, J.’A., Andre´a, M.M., 2012. Comparative assessment of the effect of synthetic and natural fungicides on soil respiration. Sensors 12, 32433252.

METHOD 28: Determination of the mineralization curve of pesticides in soil using a radiometric technique 1. Laboratory name and address: Laboratorio de Metabolismo y Degradacio´n de Contaminantes, Centro de investigacio´n en Contaminacio´n Ambiental, Universidad de Costa Rica 2. Contact persons: Juan Salvador Chin Pampillo email: [email protected]

154

3. 4.

5.

6.

7.

8.

9.

Analytical Methods for Agricultural Contaminants

Elizabeth Carazo Rojas email: [email protected] Paula Aguilar Mora email: [email protected] Title of the analytical method: Determination of the mineralization curve of pesticides in soil using a radiometric technique Principle: A pesticide applied to the soil is used as a carbon source by soil microorganisms and is converted to CO2 and H2O as major transformation products. When a 14C labeled pesticide is applied to a soil, 14CO2 will be produced and it can be fixed in an alkaline solution. By measuring the activity of the alkaline solution and monitoring the accumulated activity during time it is possible to calculate the percentage of the pesticide applied to the soil and which is mineralized by soil microorganisms. Scope: Determination of the mineralization rate of any low volatile or nonvolatile pesticide in any kind of soil, under controlled humidity and temperature conditions, using carbon-14 radiolabeled pesticides as a radiotracer. Equipment and instruments: • Incubator • Liquid scintillation counter (LSC) • Balance. Reagents and materials: • Radiolabeled pesticide with a 14 carbon. • Pesticide standard (nonlabeled) • Potassium hydroxide solution at 0.1 M • CO2 adsorbent solid medium, as ascarite. • Liquid scintillation cocktail • Biometric flasks • 15 mL centrifuge plastic tubes, falcon type • 20 mL Liquid scintillation vials Standard solutions: • Prepare an alkaline solution at 0.1 mol/L of Potassium hydroxide • Prepare 14C-pesticide solution, so that the total activity is between 6000 and 10,000 dpm/g of soil Detailed procedure (protocol): • Dry the soil at ambient temperature. • Sift the soil (,2 mm) and homogenize. • Weigh 50 g of dry soil and place it into a biometric flask.

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Add the required volume of water for adjusting the humidity to 75% of field capacity. • Add an aliquot of the pesticide solution which approximates the field dose (see Eq. 3.1). • Add an aliquot of the 14C-pesticide solution, so that the total activity is between 6000 and 10,000 dpm/g soil. Carefully agitate the biometric flask to homogenize the soil. • Close the flask with a cap having a CO2 absorbing medium trap (i.e., ascaria). • Close the side tube of the biometric flask with a cap having a closed needle installed. • Add 10 mL of the alkaline solution (solution of KOH 0.1 mol/L). Place the flask in an incubator at a constant temperature and let stand. • Renew the alkaline solution at 1, 2, 4, 6, 8, 12, 16, 24, 32, 40, 48, 56, and 64 days. • To renew the alkaline solution remove the cap containing the CO2 trap, open the stopcock and remove the cap from the needle. This will allow the air entry into the system. Place a syringe on the needle and remove the alkaline solution. Transfer it to graduated tubes. • Add 10 mL of fresh solution of KOH 0.1 mol/L to the biometric flasks. Remove the syringe, place the needle cap on the side tube, close the stopcock, and place the trap cap. • Place the flask back into the incubator. • Take 2 mL of the removed solution and transfer it to a liquid scintillation vial. • Add 10 mL of scintillation cocktail and shake to homogenize the mixture. • Let stand samples in dark for 24 h. • Determine the activity in the liquid scintillation counter. • Schedule the reading of the activity in the LSC for 510 min. • Express the result as the percentage of the initially added compound (accumulated), and plot it versus time. 10. Calculations: Calculation of the concentration of pesticide in soil:     D kg=ha  106 mg=kg     Csoil ðmg=kgsoil Þ 5 l ðmÞ  104 m2 =ha  d kgsuelo =m3 D 5 Dose of pesticide in kg/ha.

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l 5 Penetration depth of the pesticide in the soil, in m. d 5 Soil apparent density Calculation of the collected instantaneous activity: At 5

Am ðdpmÞ  10 mL Vm ðmLÞ

Am 5 activity measured in the aliquot at each time of study in dpm. Vm 5 volume of the aliquot of KOH solution at 0.1 mol/L, measured in mL. Calculation of percentage of 14C-applied pesticide converted to 14 C-CO2: Pn At %P 5 t50 3 100 A0

11.

12.

13.

14.

At 5 instant activity at each time of study in dpm. A0 5 initial total activity added to the soil in dpm Quality control: A control sample must be performed, consisting of the whole process without adding the pesticide or 14C-pesticide. This is used to correct the background activity. The assay shall be performed at least in duplicate, or triplicate as optimal. LOD and LOQ: depend on liquid scintillation counter parameters. Remarks: Prepare and label the tubes to be used. The humidity of the soil shall be kept constant during the method. The total mass of the system without KOH shall be measured at the beginning and at regular intervals. Deionized water can be added when changing the alkaline solution to keep the humidity to 75% of field capacity Interferences, troubleshooting, and safety: The CO2 trapping method is not specific. A blank must be prepared to be able to carry out a background activity subtraction as indicated in point 12. References: International Atomic Energy Agency, 1991. Laboratory Training Manual on the Use of Nuclear Techniques in Pesticide and Associated Research. Vienna.

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OECD, 1981. OECD Guideline for Testing of Chemicals 304A: Inherent Biodegradability in Soil. OECD, 2002. Guideline for the Testing of Chemicals 307: Aerobic and Anaerobic Transformation in Soil.

METHOD 29: Biomonitoring of water quality using macroinvertebrate as bioindicators 1. Laboratory names and addresses: Universidad EARTH, AP 4442-1000, San Jose´, Costa Rica; Servicio Agrı´cola y Ganadero, Regio´n de la Araucanı´a, Bilbao 931, Temuco, Chile; Universidad Nacional de Rı´o Negro, Isidro Lobo 516 General Roca, Rı´o Negro, Argentina 2. Contact persons: Bert Kohlmann email: [email protected] Rodrigo Palma email: [email protected] Pablo Macchi email: [email protected] 3. Title of the analytical method: Biomonitoring of water quality using macroinvertebrates as bioindicators 4. Principle: Biomonitoring is the use of biological responses to assess changes in the environment, generally caused by human activities. Macroinvertebrate community structural changes are studied and evaluated as indicators of pollution (organic and inorganic) processes. One of the most used biomonitoring indices in Latin America is the BMWP (Biological Monitoring Working Party) (National Water Council, 1981) adapted to country or regional/local conditions (Prat et al., 2009; Springer, 2012). 5. Scope: This method covers the macroinvertebrate community biomonitoring and applies to freshwater bodies following the Costa Rican water law (MINAE, 2007). 6. Equipment and instruments: D-net, kick-net, tweezers, plastic bottles, vials, plastic gloves and goggles, rubber wading pants, safety-line, white trays, brush, cooler, strainer, squeeze bottle, sprayer, plastic bags, pencils, paper, and laboratory microscope. 7. Reagents and materials: 70% alcohol, distilled water

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8. Detailed procedure (protocol): • Use a plastic strainer with a diameter of 2025 cm and 1 mm mesh size and tweezers to directly collect macroinvertebrates (Fig. 3.9). • The strainer is to be used in the following ways: • to collect organisms living in the water column or close to the river bank, as well as those living on submerged vegetation, by pulling the strainer through the water; • the strainer is to be held behind or underneath rocks or other substrates, while they are lifted; organisms can also be removed by hand from lifted substrates and caught with the strainer while drifting; • ground substrates can be kicked with the heels and the strainer used for sifting through the current with the separated material; and • leaf packs are to be put inside the strainer and searched with tweezers for macroinvertebrates. • Tweezers are to be used mainly when macroinvertebrates live tightly attached to the substrate. • The average strainer “emptying rate” is after each pass, up to three passes in the case of rock lifting. Total sampling time is divided

Figure 3.9 Using a strainer for collecting water in Brazil. IAEA Latin American technicians training course (2010).

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• •



159

into approximately 50% of the time for using the strainer and 50% for using the tweezers. To carry out sampling of the macroinvertebrates in a river, start the collection at one side of the river and continue until you crossed the river to the other bank. Note: for rivers deeper than 1 m or broader than 15 m, a boat and a strainer attached to a pole are usually needed. The river is usually recrossed some meters further upstream in a zigzag fashion towards the original starting site. The main criterion for this semiquantitative collecting method is time; there are no defined sampling areas. To make the sampling representative, all types of microhabitats present at a particular site in a river have to be equally examined for macroinvertebrates. Collected organisms are to be fixed immediately in 70% ethanol at the time of sampling. Similarly a D-net can be used to sample a river (Fig. 3.10) or a kick-net (Fig. 3.11). With the D-net, rocks, the bottom of the river, as well as vegetation can be sifted. Note: very efficient sampling can be carried by a minimum of two persons. One holds the D-net or the kick-net against the bottom of the river; a second person stands in front of the net upriver. The second person starts kicking the bottom of the river with his/her heels. The river current will then carry the macroinvertebrates into the net.

Figure 3.10 Using a D-net for sampling. A second person stands up-current in front of the net and kicks the river bottom with their heels. The flow carries the macroinvertebrates to the net. IAEA Latin American technicians training course in Uruguay (2012).

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Figure 3.11 Sampling with a kick-net in the Tijeral River, Chile (2012).

Figure 3.12 Accumulated number of aquatic macroinvertebrate taxa in a presampling campaign (December 2004) at the Dos Novillos River, Guácimo, Limón, Costa Rica (Stein et al., 2008).

Presampling Before proper sampling, a presampling has to be carried out in the area under study in order to establish a representative sampling time. This can be done by sampling the various microhabitats in a river every 30 min (Stein et al., 2008). An accumulated taxa curve then needs to be elaborated, such as the one shown in Fig. 3.12. When a plateau of the response has been reached this corresponds to the minimum sampling time.

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Table 3.34 Categories of water quality as defined by the numerical values of the BMWP-CR index adapted to Costa Rica and their color equivalents for mapping purposes Water quality Color BMWP0 -CR ChBMWP BMPS

Excellent quality Good quality, no pollution Regular quality, eutrophic Bad quality, polluted Bad quality, very polluted Very bad quality, extremely polluted

.120 101120 61100 3660 1635 ,15

.100 61100 3660 1635 ,15

$ 150 101149 61100 3660 1635 , 15

The same index adapted to Chile (ChBMWP, Figueroa et al., 2007) and Patagonia, Argentina (BMPS, Miserendino and Pizzolo´n, 1999).

9. Calculations: The BMWP index is used to define water quality (Table 3.34), based on identification of organisms to family level, and is calculated in the following way. Each macroinvertebrate gathered during biomonitoring is identified to family level using key tools and identification guides (Fig. 3.13). Each family is assigned a sensitivity value ranging from 1 to 10, reflecting tolerance to pollution based on knowledge of its distribution and abundance. The values for each family are then summed (independently from any value of abundance or genera or species diversity). Sensitivity scores higher than 120 points indicate undisturbed aquatic ecosystems, while low values indicate serious contamination (mostly organic) of the environment. Table 3.35 shows the family-assigned BMWP values modified for Costa Rica, Chile, and Argentina. Fig. 3.14 shows the typical way of reporting the BMWP index values by superimposing bar graphs on a color scale. If no index is used (or cannot be used) for whatever reason, another way of representing and analyzing water quality is to depict the macroinvertebrate order composition as a percentage of the number of individuals (Fig. 3.14). For example, an increase in the number of snails (Gastropoda), as shown in site 6, is an indication of a heavier organic load in that specific place, whereas high beetle numbers (Coleoptera), as shown in site 1, are an indication of good ecosystem condition in tropical areas (Fig. 3.15). The biomonitoring approach method can also be used for analyzing the environmental condition of a particular river or watershed

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Figure 3.13 Part of a field guide for Costa Rica with some macroinvertebrate families as bioindicators of excellent to good water quality and their associated BMWP-CR sensitivity numbers. Modified for Costa Rica (Vásquez, D., Springer, M., Castro, A., Kohlmann, B., 2010. Bioindicadores de la Calidad del Agua: Cuenca del Río Tempisque. Universidad EARTH, Costa Rica, 8 pp.).

along its length (Fig. 3.16). The calculated BMWP index can be indicated along different parts of the same river system, and it can be expanded to indicate the ecological condition at different times of the year by using a pie chart (Fig. 3.7). 10. Quality control: Establishment of an international group of experts in taxonomy and bioindication, providing also advisory services to the different countries that employ the biomonitoring methodology in the region.

Table 3.35 Macroinvertebrate families and their associated BMWP value adapted for Costa Rica, Chile, and Argentina a b c Family BMWP-CR Family ChBMWP Family BMPS

Heptageniidae, Perlidae, Hydrobiosidae Leptophlebiidae, Corduliidae, Blaberidae, Calamoceratidae, Glossosomatidae, Leptoceridae Platystictidae, Ptilodactylidae, Lutrochidae, Gomphidae Philopotamidae, Psephenidae Euthyplociidae, Polycentropodidae, Hydroptilidae Libellulidae, Corydalidae, Xiphocentronidae

Austroperlidae, Diaphipnoidae, Eustheniidae, Notonemouridae, Perlidae, Nesameletidae, Ameletopsidae, Oligoneuriidae, Coloburiscidae, Anomalopsychidae, Calamoceratidae, Helicophidae, Kokriidae, Philopotamidae, Sericostomatidae, Stenopsychidae, Blephariceridae, Limnichidae, Psephenidae Leptophlebidae, Glossosomatidae, Limnephilidae, Athericidae, Dixidae Oniscigastridae, Phylorheytidae, Polycentropodidae, Tasiimidae, Calopterygidae, Libellulidae, Parastacidae Gripopterygiidae, Ecnomidae, Hydrobiosidae, Leptoceridae, Lastidae, Gomphidae, Corduliidae, Coenagrionidae Hydroptilidae, Ceratopogonidae, Petaluridae, Aeshnidae, Elmidae, Aeglidae, Hyallelidae, Ancylidae, Chilinidae, Hyriidae

Eustheniidae, Diamphipnoidae, Perlidae, Notonemouridae Gripopterygidae, Austroperlidae, Ameletopsidae, Leptophlebiidae, Siphlonuridae, Leptoceridae Sericostomatidae, Philorheithridae Helicophidae, Anomalopsychidae Tasimiidae, Kokiriidae, Athericidae Blephariceridae

Value

10

9 Coloboruscidae, Glossossomatidae Philopotamidae, Calamoceratidae Odontoceridae, Helicopsychidae

8

Hidrobiosidae, Polycentropodidae, Limnephilidae, Gomphidae

7

Baetidae, Hydroptilidae, Gelastocoridae, 6 Lestidae Austropetaliidae, Aeshnidae, Petaluridae, Gomphomacromiidae Neopetaliidae, Coenagrioniidae Libellulidae, Hyalelilidae, Chilinidae (Continued)

Table 3.35 (Continued) a Family BMWP-CR

b

Family ChBMWP

c

Family BMPS

Baetidae, Elmidae, Leptohyphidae, Baetidae, Hydropsychidae, Corydalidae, Ecnomidae, Hydraenidae, Eubriidae, Pyralidae, Helicopsychidae, Tipulidae, Simuliidae, Dryopidae, Scirtidae, Elmidae, Corydalidae, Hydropsychidae, Hidracarina, Gyrinidae, Turbellaria, Amnicolidae Hydropsychidae, Tipulidae, Simuliidae, Pseudothelphusidae, Hyalellidae, Parastacidae, Aeglidae, Dugesiidae, Isopoda, Palaemonidae Ancylidae, Phreodrilidae, Osnylidae Coenagrionidae, Stratiomydae, Tipulidae, Caenidae, Sialidae, Tabanidae, Caenidae, Haliplidae, Chrysomelidae, Ceratopogonidae, Tabanidae, Simuliidae, Stratiomydae, Empididae, Limoniidae, Curculionidae, Tabanidae, Neritidae, Caenidae, Calopterygidae, Notonectidae, Psychodidae, Haliplidae, Curculionidae, Stratiomydae, Empididae, Naucoridae, Pleidae, Belostomatidae, Sphephenidae, Belostomatidae, Acari Ceratopogonidae, Psychodidae, Corixidae, Veliidae, Mesoveliidae, Tanyderidae, Thaumaleidae, Sialidae, Dytiscidae, Noteridae, Staphylinidae, Hyriidae, Hydracarina Scirtidae Psychodidae, Hydrophilidae, Unionidae, Hydrophilidae, Dytiscidae, Gerridae, Notonectidae, Corixidae, Mesoveliidae, Sphaeriidae, Thiaridae, Hydrobiidae, Notonectidae, Corixidae, Lymnaeidae, Hydrometridae, Belostomatidae, Ampullariidae, Planorbidae, Physidae, Physidae, Planorbidae, Sphaeriidae, Dytiscidae, Gyrinidae, Hydrophilidae, Glossiphonidae Janiiridae, Hirudinea Mycetopodidae, Sphaeriidae, Hydrobiidae, Lymnaeidae, Physidae, Planorbidae, Glossiphoniidae, Semiscoleidae, Macrobdellidae Chironomidae, Culicidae Chironomidae, Culicidae, Ephydridae Chironomidae, Culicidae, Muscidae, Ephydridae, Syrphidae Tubificidae, Oligochaeta Syrphidae, Oligochaeta Lumbriculidae, Tubificidae, Naididae a

Adapted for Costa Rica (MINAE, 2007). Adapted for Chile (Figueroa et al. 2007). Adapted for Argentina (Miserendino and Pizzolo´n, 1999).

b c

Value

5

4

3

2 1

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Water quality

Mercedes

Desembocadura

P. Hamaca

Chiquitín (B)

Chiquitín (A)

La Argentina

Don Eladio

BMWP

140 130 120 110 100 90 80 70 60 50 40 30 20 10 0

Figure 3.14 An example of reporting biomonitoring data using the international color system (see Table 3.1), indicating BMWP0 values for different sampling locations. In this example Don Eladio represents an undisturbed tropical rain forest, La Argentina is cattle pasture (3 ha), Chiquitín is a small village (5660 inhabitants), Puente Hamaca is a gallery forest, Desembocadura is a banana plantation (100 ha), and Mercedes represents the effluent discharge of a banana packing plant (750,000 boxes/year).

Figure 3.15 Percentage abundances of the seven most common taxonomic groups along six different collecting sites of the Dos Novillos River, Costa Rica. Groups marked with an asterisk (*) have statistically significant frequency differences (Chisquare; P , .0001). Site 1 represents an undisturbed tropical rain forest, site 2 is cattle pasture (3 ha), site 3 is a small village (5660 inhabitants), site 4 is a gallery forest, site 5 is a banana plantation (100 ha), and site 6 represents the effluent discharge of a banana packing plant (750,000 boxes/year) (Kohlmann et al., in press).

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Analytical Methods for Agricultural Contaminants

Figure 3.16 Water quality evaluation of different river localities in Guanacaste, Costa Rica’s agricultural area in the dry tropics (taken from Vásquez, D., Springer, M., Castro, A., Kohlmann, B., 2010. Bioindicadores de la Calidad del Agua: Cuenca del Río Tempisque. Universidad EARTH, Costa Rica, 8 pp.). Note how water quality decreases along the way from river springs to the agricultural landscape (Liberia-Filadelfia axis).

11. Remarks: Under ideal conditions weekly biomonitoring is recommended; if time and money are limiting factors, then samples should be collected monthly. It is important to standardize the time and day of sampling on a routine basis. Sampling is recommended to take place

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167

during early morning and under normal water current situations. During high water flows or flooding conditions macroinvertebrates will hide to avoid being carried away by strong water currents. 12. References: Black, R., Munn, M., 2004. Using macroinvertebrates to identify biotaland cover optima at multiple scales in the Pacific Northwest, USA. J. North Am. Benthol. Soc. 23 (2), 340362. Bonada, N., Prat, N., Resh, V.H., Statzner, B., 2006. Developments in aquatic insect biomonitoring: a comparative analysis of recent approaches. Annu. Rev. Entomol. 51, 495523. Castillo, L.E., Martı´nez, E., Ruepert, C., Savage, C., Gilek, M., Pinnock, M., et al., 2006. Water quality and macroinvertebrate community response following pesticide applications in a banana plantation, Limo´n, Costa Rica. Sci. Total Environ. 367, 418432. Collier, K., Quinn, J., 2003. Land-use influences macroinvertebrate community response following a pulse disturbance. Freshwater Biol. 4, 14621481. Domı´nguez, E., Ferna´ndez, H.R. (Eds.), 2009. Macroinvertebrados Bento´nicos Sudamericanos: Sistema´tica y Biologı´a. Fundacio´n Miguel Lillo, Tucuma´n, 656 pp. Figueroa, R., Palma, A., Ruiz, V., Niell, X., 2007. Ana´lisis comparativo de ´ındices bio´ticos utilizados en la evaluacio´n de la calidad de la aguas en un rı´o mediterra´neo de Chile: rı´o Chilla´n, VIII Regio´n Rev. Chil. Hist. Nat. 80, 225242. Hauer, F., Lambert, G., 1996. Methods in Stream Ecology, second ed. Academic Press, New York, NY, 877 pp. Heino, J., 2014. Taxonomic surrogacy, numerical resolution and responses of stream macroinvertebrate communities to ecological gradients: are the inferences transferable among regions? Ecol. Indic. 36, 186194. Kohlmann, B., Arroyo, A., Springer, M., Va´squez, D., in press. Agrorural ecosystem effects on the macroinvertebrate assemblage of a tropical river. In: Blanco, J. (Ed.), Biodiversity in Ecosystems  Linking Structure and Function. InTech Publishers, Rijeka. Macchi, P.A., 2007. Calidad del agua en ecosistemas fluviales utilizando macroinvertebrados bento´nicos. Cuenca del Arroyo Pocahullo, San Martı´n de los Andes. Tesis de Licenciatura. Universidad Nacional del Comuahue, Neuque´n, p. 79.

168

Analytical Methods for Agricultural Contaminants

Martin, S., Bertaux, A., Le Ber, F., Maillard, E., Imfeld, G., 2012. Seasonal changes of macroinvertebrate communities in a stormwater wetland collecting pesticide runoff from a vineyard catchment (Alsace, France). Arch. Environ. Contam. Toxicol. 62, 2941. MINAE (Ministerio de Ambiente y Energı´a, CR), 2007. Reglamento para la Evaluacio´n y Clasificacio´n de la Calidad de Cuerpos de Agua Superficiales (en lı´nea). Decreto Ejecutivo No 33903-MINAE-S. Alcance No. 8 a La Gaceta No. 78, lunes 17 de% septiembre del 2007. San Jose´, CR, 7 pp. Disponible en: ,http://historico.gaceta.go.cr/pub/2007/09/17/COMP_17_09_2007.html.. Miserendino, M.L., Pizzolo´n, L.A., 1999. Rapid assessment of river water quality using macroinvertebrates: a family level biotic index for the Patagonic Andean zone. Acta Limnol. Bras. 11, 137148. National Water Council, 1981. River Quality: The 1980 Survey and Future Outlook. NWC, London, 39 pp. Prat, N., Rı´os, B., Acosta, R., Rieradevall, M., 2009. Los macroinvertebrados como indicadores de la calidad de las aguas. In: Domı´nguez, E., Ferna´ndez, H.R. (Eds.), Macroinvertebrados Bento´nicos Sudamericanos: Sistema´tica y Biologı´a. Fundacio´n Miguel Lillo, Tucuma´n, pp. 631654. Rizo-Patro´n, F., Kumar, A., McCoy Colton, M.B., Springer, M., Trama, F.A., 2013. Macroinvertebrate communities as bioindicators of water quality in conventional and organic irrigated rice fields in Guanacaste, Costa Rica. Ecol. Indic. 29, 6878. Springer, M., 2012. Biomonitoreo acua´tico. In: Springer, M., Ramı´rez, A., Hanson, P. (Eds.), Macroinvertebrados de Agua Dulce de Costa Rica I. Rev. Biol. Trop. 58 (4), 5359. Stein, H., Springer, M., Kohlmann, B., 2008. Comparison of two sampling methods for biomonitoring using aquatic macroinvertebrates in the Dos Novillos River, Costa Rica. In: Kohlmann, B., Mitsch, M. J. (Eds.), Ecological Management and Sustainable Development in the Humid Tropics of Costa Rica. Ecol. Eng. 34 (4), 267275. Va´squez, D., Springer, M., Castro, A., Kohlmann, B., 2010. Bioindicadores de la Calidad del Agua: Cuenca del Rı´o Tempisque. Universidad EARTH, Costa Rica, 8 pp. Weigel, B., 2003. Development of stream macroinvertebrate models that predict watershed and local stressors in Wisconsin. J. North Am. Benthol. Soc. 22 (1), 123142.

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Woodward, G., Gessner, M., Giller, P., Gulis, V., Hladyz, S., Lecerf, A., et al., 2012. Continental-Scale effects of nutrient pollution on stream ecosystem functioning. Science 336, 1438. 13. Method validation: It is recognized that, due to evolutionary and adaptive processes, macroinvertebrate communities respond to climatic and geomorphological conditions, as well as to water quality at the regional (tropical or neotropical) and local levels (altitude or latitude); thus they are subject to similar geoclimatic patterns, but with specific response differences (Heino, 2014). Similarly, there are local effects of anthropogenic activities on aquatic communities, specifically on the biodiversity and water quality (Woodward et al., 2012). These and other factors justify the need to adapt biotic indices when establishing biomonitoring programmes (Bonada et al., 2006). To date, biomonitoring methods have been validated for some cultivars, e.g., Rizo-Patro´n et al. (2013) for conventional and organic rice, Castillo et al. (2006) and Kohlmann et al. (in press) for bananas, and Martin et al. (2012) for vineyards. Similarly, they have been used for watersheds having a mixture of forestry and agricultural landscapes (Black and Munn, 2004; Collier and Quinn, 2003; Weigel, 2003). Similarly, books on the subject of rivers devote a chapter to biomonitoring, where macroinvertebrate sensitivity versus response are matched to different water quality levels and help define standardized measurement methodologies (see Hauer and Lamberti, 2007; Dominguez and Ferna´ndez, 2009).

METHOD 30: Estimation of the bioaccumulation factor of radiolabeled pollutants in compost worms Eisenia andrei/ fetida 1. Laboratory name and address: Laboratorio de Ecologia de Agroquı´micos, Instituto Biolo´gico, Sa˜o Paulo SP, Brazil 2. Contact persons: Luiz Carlos Luchini email: [email protected] Mara M. de Andre´a email: [email protected] Elaine Vieira email: [email protected] 3. Title of the analytical method: Estimation of the bioaccumulation factor of radiolabeled pollutants in compost worms Eisenia andrei/fetida

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Analytical Methods for Agricultural Contaminants

4. Principle: As earthworms are an integral part of many soil food webs, they may serve as bioindicators for the potential hazards posed by pollutants (pesticides, drugs, and metals) to soil biota. The method presented is a laboratory bioassay to evaluate the bioaccumulation of radiolabeled (using a 14C-label) pollutants in compost worms that grow in soil and incorporate a certain amount of radiolabeled pollutant. Liquid scintillation counting is used to estimate the amount of radioactivity present in organic extracts prepared from compost worms and soil. 5. Scope: The current method is applicable to the determination of radiolabeled pollutants in soil using earthworms as soil bioindicator organisms. 6. Equipment and instruments: • Liquid scintillation counter (LSC) • Laboratory microwave • Rotary evaporator • Freezer • Homogenizer 7. Reagents and materials: • Organic solvents, residue analysis grade • 14C-labeled target pollutant • Pollutant (nonlabeled) • Adult Eisenia andrei earthworms 8. Detailed procedure (protocol): • One week before the beginning of the study, weigh 200 g samples of different soils in 500 mL glass beakers and moisten to 60% of the maximum water holding capacity (MWHC) with deionized water, as recommended by OECD guide No% 317. • Weigh enough sample replicates to have at least three soil replicates for the treatment with the pollutant and three replicates for the control. • For acclimation to laboratory conditions at least 1 day before the beginning of the study, place five adult earthworms per replicate each weighing more than 300 mg with clitellum in untreated soil with at least 50 g soil per earthworm. • On day 1 of the study, slowly treat and mix the soils with a solution of 14C-target pollutant. The samples remain in a fume hood until the solvent has evaporated.

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Only after the solvent has evaporated from the soil the acclimatized adult earthworms are placed in the treated or control soil. • Cover the surface of the glass beakers with perforated plastic film to allow air exchange. • Weigh each complete glass beaker containing the moistened soil and the compost worms to evaluate the dynamics of water evaporation during the study period (Vampre´ et al., 2010). • Keep the glass beakers at a constant controlled temperature (e.g., 25 C) and standardized light periods (e.g., 12 h). • Every 3 days weigh the beakers and adjust the water content to keep the soil moisture at 60% MWHC. • After 14 days, disassemble the beakers and separate the worms; wash in tap water, dry in filter paper and weigh to compare with the initial weight and determine the effect of the pollutant on growth of the organisms. • Maintain the earthworms at 4 C for 24 h on wet filter paper, which is changed after two periods of 12 h to allow emptying of soil or substrate particles from their guts. Freeze for 24 h. Follow the extraction method by Papini et al. (2006) to recover the pollutants from the organic extracts prepared from compost worms and soil. All organisms of each replicate are extracted together by this method. 9. Calculations: The bioaccumulation factor is determined by dividing the amount of pollutant present in the earthworms (Co, in kBq/g or μCi/g) by the amount in the soil (Cs, in kBq/g of soil dry weight): BAF 5

Co Cs

10. Quality control: Preliminary experiments should be conducted to determine the extraction recovery of the target pollutant from the soil and earthworm tissue. The recovery should be at least 85% for the added 14 C-labeled target pollutant. To estimate the extraction recovery from earthworms, wash five adult worms (with clitellum and more than 300 mg each) in tap water in a plastic sieve, dry carefully in filter paper and freeze at 218oC for 24 h. Spike the frozen animals with a solution containing a mixture of the 14C-pollutant and a reasonable—but not lethal—

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amount of the pollutant. Cut the animals into small pieces of about 1 cm and homogenize for about 15 min under a fume hood to allow evaporation of the spiking solvent. Material from the treated earthworms is extracted by addition of a solvent or a solvent mixture by microwave-assisted extraction (MASE) using frequent short (a few seconds) low energy cycles as the earthworms contain a significant amount of water. The heating into microwave is intercalated by temperature decrease through immersion in ice-bath (method by Andre´a et al., 2001). Depending on the solvent used, few mL aliquots of the extract obtained are directly counted by LSC using a scintillation cocktail. If the solvent is a quencher, it can be rotary-evaporated and resuspended in another suitable solvent compatible with liquid scintillation counting. To estimate the recovery following extraction from soil, an adequate soil extraction method should be established. For example, the samples may be extracted by MASE in various cycles of few seconds and low level of microwave energy, also intercalated by ice-bath. The extraction recovery should be established in triplicates using 3.0 g wet samples spiked with a known amount of the pollutant. Extraction recovery is calculated as the ratio between the amount of 14C-pollutant recovered in soil and earthworm extracts, and the amount of 14C-pollutant added into the samples, taking into account the dilution factors. 11. Remarks: For a test to be valid, the mortality of the bioindicator organism should not exceed 10% of the total number of earthworms in each replicate of control or treated samples. Ensure that the measurement conditions of the LSC are checked to enable valid measurements. These include daily checks of the carbon-14 calibration standard and the standard of background radiation to verify the efficiency of counting. 12. Interferences, troubleshooting, and safety: All laboratory personnel must be trained on the safety and correct handling of the pollutants, radioactive materials and the quenchers for LSC. 13. References: Andre´a, M.M., Papini, S., Nakagawa, L.E., 2001. Optimizing microwave-assisted solvent extraction (MASE) of pesticides from soil.

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J. Environ. Sci. Health. Part B, Pesticides, Food Cont. Agric. Wastes, B 36, 8793. OECD  (Organization for Economic Co-operation and Development), 2010. OECD guideline No 317. Bioaccumulation in % Terrestrial Oligochaetes. 30 pp. Papini, S., Langenbach, T., Luchini, L.C., Andre´a, M.M., 2006. 14 C-Paraquat in compost worms Eisenia foetida. J. Environ. Sci. Health. Part B, Pesticides, Food Cont. Agric. Wastes, B 41, 523530. Vampre´, T.M., Fuccillo, R., Andre´a, M.M., 2010. Oligoqueta Eisenia andrei como bioindicador de contaminac¸a˜o de solo por hexaclorobenzeno. R. Bras. Ci. Solo 34, 5966. 14. Minimum method validation data: The method validation was conducted using 14C-Paraquat and 14 C-Simazine herbicides (Tables 3.36 and 3.37). The percentage total radiocarbon recovery as 14C-extractable 1 14 C-bound in soil and earthworms at the end of the experiment was: 93.9 6 9%. The range of variation on the efficiency of the biological combustion method to determine the 14C-bound in soil and earthworms was 91%95%. The Limit of Detection (LOD) was calculated as the mean 1 3 times of the activity (dpm) standard deviation of the blanks (6:4 of monoethanolamine and scintillation cocktail), estimated as 285 dpm (4.8 Bq).

Table 3.36 Radiocarbon recoveries from 5 (five) earthworms spiked with 14 C-herbicides using different methods Method Solvent Volume Extraction Conditions (mL)

Recovery (% 6 SD)

1

Ethyl acetate

100

100.2 6 2

2

Methanol

150

3 4

Methanol 150 Methanol: 15 dichloromethane (10:5)

Shaker

4 cycles, 6 h each Shaker 5 cycles, 6 h each Soxhlet 1 cycle, 8 h Microwave 15 cycles, 30 s each and 1120W

88.9 6 2 116.8 6 5 99.2 6 8

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174

Table 3.37 Radiocarbon recoveries from soil spiked with 14C-herbicides using different methods Method Solvent Volume Extraction Conditions Recovery (mL) (% 6 SD)

1

Methanol

2 3

Methanol 100 Water:methanol: 15 dichloromethane (1:8:6) Hexane:Acetone 15 (75:25)

4

100

3 cycles, 8 h 97.1 6 2 each Soxhlet 1 cycle, 12 h 105.1 6 2 Microwave 10 cycles, 20 s 89.4 6 1 each and 800W Microwave 12 cycles. 2 min 81. 1 6 1 each and 480W Shaker

The Limit of Quantification (LOQ) was defined as the mean 1 10 times of the activity (dpm) standard deviation of the blanks, estimated as 593.0 dpm (9.9 Bq). The accuracy obtained for 14C-pesticides standard detection (dpm) was # 10%.

USEFUL LINKS AND RESOURCES

Analytical Methods and Other Related Food Safety and Security Information (Many Useful Links) http://nucleus.iaea.org/fcris/ Codex Standards and Guidelines: http://www.codexalimentarius.org/standards/list-of-standards/en/? no_cache 5 1 Codex Pesticide MRLs: http://www.codexalimentarius.net/pestres/data/index.html?lang 5 en Codex Veterinary Drug MRLs: http://www.codexalimentarius.net/vetdrugs/data/vetdrugs/index. html?lang 5 en Codex Thematic Compilations: http://www.codexalimentarius.org/standards/thematic-compilations/ en/ Use of Nuclear Technology IAEA, 1991. Technical report series No.329. Laboratory Training Manual on the Use of Nuclear and Associated Techniques in Pesticide Research. Vienna, Austria. ISBN 92-0-115091-1 IAEA Division of Food and Environmental Protection: http://www-naweb.iaea.org/nafa/fep/index.html RALACA Web Page: http://www.red-ralaca.net/

175

INDEX Note: Page numbers followed by “f ” and “t” refer to figures and tables, respectively.

A Abamectin (ABA), 69 2-ACBs. See 2-Alkylcyclobutanones (2-ACBs) Accreditation, 12 13, 15 Accuracy, 1 2, 16 Acetonitrile (ACN), 29 Adsorption isotherms in soils using radiotracers, 144 148 Agrochemicals, 150 Air drying, 109 110 2-Alkylcyclobutanones analysis, 135 139 2-Alkylcyclobutanones (2-ACBs), 135 α-Hydroxyisobutyric acid (HIBA), 87 Aminomethylphosphonic acid (AMPA), 121 126 Ammonium acetate ((NH4)Ac), 40, 44 pesticide residue determination in potato, 40 47 Ammonium determination in wastewater, 131 135 AMPA. See Aminomethylphosphonic acid (AMPA) Analytical laboratories, 1, 9 conditions, 6 7 Analytical methods, 8 multiresidue, 2 AOAC Peer-Verified Method, 15 Avermectins determination in food, 68 74

B Bioaccumulation factor estimation radiocarbon recoveries, 173t, 174t of radiolabeled pollutants, 169 174 Bioindicators, macroinvertebrate as, 157 169 Biological approach, 135 174 adsorption isotherm generation in soils, 144 148

bioaccumulation factor estimation, 169 174 biomonitoring of water quality using macroinvertebrate, 157 169 irradiated fat containing food detection, 135 139 mineralization curve of pesticides in soil, 153 157 effect of pesticides, drugs, and chemical compounds, 148 153 radon determination in water, 139 143 Biological Monitoring Working Party (BMWP), 157 Biometric flasks, 149, 149f Biomonitoring of water quality accumulated number of aquatic macroinvertebrate taxa, 160f categories of water quality, 161t example of reporting biomonitoring data, 165f using macroinvertebrate as bioindicators, 157 169 macroinvertebrate families and associated BMWP value, 163t part of field guide for Costa Rica, 162f percentage abundances, 165f water quality evaluation of different river localities, 166f 1,4-Bis(5-phenyloxazol-2-yl) benzene (POPOP), 140 BMWP. See Biological Monitoring Working Party (BMWP) “Bottom-up” approach, 18

C Calibration curve, 103 function, 16 Calibration factor (CF), 141 142

177

Index

178 CAP. See Chloramphenicol (CAP) Capillary zone electrophoresis (CZE), 86 histamine determination, 85 90 Carbamate determination in soil and sediment samples, 90 100 Carbamate pesticides residues determination in honey, 60 64 CBT. See Clenbuterol-D9 (CBT) CD. See Conductivity detector (CD) Centro de Aplicaciones Tecnolo´gicas y Desarrollo Nuclear (CEADEN), 135 Centro de Investigaciones y Aplicaciones Nucleares (CIAN), 139 Certification, 12 13 CF. See Calibration factor (CF) 14 C-glucose as radiotracer, 148 153 CH3COONH4. See Ammonium acetate ((NH4)Ac) Chelating agent, 10 Chemical compounds, 148 153 C-hex. See Cyclo-hexane (C-hex) Chloramphenicol (CAP), 65 determination and confirmation in honey, fish, and prawns, 65 68 CIAN. See Centro de Investigaciones y Aplicaciones Nucleares (CIAN) CITAC/Eurachem Guide to Quality in Analytical Chemistry, 11 12 Clenbuterol-D9 (CBT), 81 Codex Alimentarius, 3, 21 guidelines, 54 Codex Alimentarius Commission, 21 Column extraction technique, 56 60 Compost worms (Eisenia andrei/fetida), 169 174 Concentration of pesticide, 146 147 of pesticide in soil, 155 156 Conductivity detection, 131 135 Conductivity detector (CD), 131 Contamination, 11 14 C-pesticides concentrations, 144 Cyclo-hexane (C-hex), 57 CZE. See Capillary zone electrophoresis (CZE)

D 2-DCB. See 2-Dodecylcyclobutanone (2DCB) Decay correction factor, 141 142 Detection of irradiated fat containing foods, 135 139 or minimum detection activity limit, 141 142 Dilution, 10 2,5-Diphenyloxazole (PPO), 140 Disintegration per minute (dpm), 148 D-net sample, 159, 159f 2-Dodecylcyclobutanone (2-DCB), 136, 138 Doramectin (DOR), 69 Drugs, 148 153

E EA. See Ethyl acetate (EA) Earthworms, 170 ECD. See Electron capture detector (ECD) μ-ECD. See Microelectron capture detection (μ-ECD) Eggs, sulfonamides determination in, 74 79 Eisenia andrei/fetida. See Compost worms (Eisenia andrei/fetida) Electron capture detector (ECD), 25 Electrospray ionization interface (ESI), 70 Environmental sustainability, integrated analytical approaches for food contaminant analysis, methods for, 25 90 nuclear and biological approaches, methods using, 135 174 soil and water analysis, methods for, 90 135 Eprinomectin (EPR), 69 Equilibration time (ET), 144 145 ESI. See Electrospray ionization interface (ESI) ET. See Equilibration time (ET) Ethyl acetate (EA), 57 extraction, 25 29 Eurachem CITAC guide, 19

Index

European Commission Directorate General for Health and Food Safety (SANTE), 14 Exact-mass/high resolution mass spectrometry, 16 Extraction. See also Solid phase extraction (SPE) column extraction technique, 56 60 EA, 25 29 efficiency, 15 of fresh fruits and vegetables, 52 56 MASE, 171 172 organochlorinated pesticide extraction procedure, 111 113 organophosphate pesticide extraction procedure, 108 111 of pesticide residues present in fresh fruits and vegetables, 29 33

F FAO/IAEA. See Food and Environmental Protection Laboratory of Joint Food and Agriculture Organization and International Atomic Energy Agency (FAO/IAEA) Fatty phase, 135 FEPL. See Food and Environmental Protection Laboratory (FEPL) Filtering, 10 Fish determination and confirmation of chloramphenicol in, 65 68 histamine determination in, 85 90 FLD. See Fluorescence detector (FLD) Florisil, 56 column chromatography, 138 Fluorescence detector (FLD), 63 carbamate pesticides residues determination in honey, 60 64 Food and Environmental Protection Laboratory (FEPL), 25, 47, 56, 104, 117 Food and Environmental Protection Laboratory of Joint Food and Agriculture Organization and International Atomic Energy Agency (FAO/IAEA), 1 3

179 Food contaminant analysis, 10 carbamate pesticide residues in honey, 60 64 determination and confirmation of CAP, 65 68 determination of avermectins and milbemycin residues, 68 74 histamine determination in fish and fish products, 85 90 pesticide residue analysis by HPLC MS/MS, 34 39 determination in fresh fruits and vegetables, 25 29 extraction present in fresh fruits and vegetables, 29 33 pesticides in tomato samples, 56 60 potato multiresidue method for pesticides in, 47 52 pesticide residue determination in, 40 47 processing and extraction of fruits and vegetables, 52 56 RAC determination in swine feed, 79 85 sulfonamide determination in eggs, 74 79 Food contaminants, 1 Food control, 1, 8 Food safety analysis method validation, 14 17 quality assurance and quality control procedures, 11 14 right method for purpose, 20 21 sample preparation and processing, 10 11 sampling in context of food safety, 8 10 setting up residues laboratory, 5 8 uncertainty estimation, 17 20 integrated analytical approaches methods for food contaminant analysis, 25 90 methods for soil and water analysis, 90 135 methods using nuclear and biological approaches, 135 174

Index

180 Fresh fruits and vegetables pesticide residue analysis by HPLC MS/MS, 34 39 determination, 25 29 extraction, 29 33 processing and extraction, 52 56

G GACT. See Grupo de Ana´lisis de Contaminantes Trazas (GACT) Gas chromatograph coupled to electron capture detector or nitrogen phosphorus detector (GC with ECD/NPD), 2 Gas chromatograph coupled to mass selective detector (GC-MSD), 48 detection, 47 52 determination of selected pesticides in soil, 104 108 target and qualifier ions, 107t determination of selected pesticides in water, 117 121 target and qualifier ions, 120t organochlorine insecticide determination in water, 114 117 Gas chromatography (GC), 25, 100 Gas chromatography coupled to mass spectrometry (GC-MS), 107, 135 139 GC. See Gas chromatography (GC) GC-MS. See Gas chromatography coupled to mass spectrometry (GC-MS) GC-MSD. See Gas chromatograph coupled to mass selective detector (GC-MSD) Gel permeation chromatography (GPC), 56 pesticide determination in tomato, 56 60 GLP. See Good Laboratory Practice (GLP) Glyphosate, residues analysis of AMPA and, 121 126 Good Laboratory Practice (GLP), 13 GPC. See Gel permeation chromatography (GPC) Grupo de Ana´lisis de Contaminantes Trazas (GACT), 40 Guide to the Expression of Uncertainty in Measurement (GUM), 18 19

H Heating, venting, air conditioning/cooling systems (HVAC systems), 7 HIBA. See α-Hydroxyisobutyric acid (HIBA) High pressure liquid chromatograph coupled to single quadrupole mass spectrometer (High pressure LCMS), 91 High-performance liquid chromatograph (HPLC), 61 with postcolumn derivatization, 60 64 High-pressure liquid chromatography detection and diode arrangement (HPLC/DAD), 74 79 Histamine (Hist), 87 Histamine determination in fish and fish products, 85 90 Honey carbamate pesticides residues determination in, 60 64 determination and confirmation of chloramphenicol in, 65 68 HPLC. See High-performance liquid chromatograph (HPLC) HPLC MS/MS, pesticide residue analysis by, 34 39 HPLC/DAD. See High-pressure liquid chromatography detection and diode arrangement (HPLC/DAD) HVAC systems. See Heating, venting, air conditioning/cooling systems (HVAC systems)

I Instituto Nacional de Tecnologı´a Industrial (INTI), 52, 60 Integrated analytical approaches examples of methods for soil and water analysis, 90 135 food contaminant analysis, methods for, 25 90 nuclear and biological approaches, methods using, 135 174 INTI. See Instituto Nacional de Tecnologı´a Industrial (INTI)

Index

Ion-abundance ratio, 84 Irradiated fat containing foods, 135 139 Ivermectin (IVR), 69

J Joint FAO/IAEA Division on Nuclear Techniques in Food and Agriculture, 1 3

K Kick-net, 159, 160f

L Laboratory mandate, 5 Laboratory quality system, 13 LC. See Liquid chromatography (LC) LC-MS/MS. See Liquid chromatographytandem mass spectrometry (LCMS/MS) LCL. See Lowest calibrated level (LCL) Limits of detection (LOD), 6, 152 153, 173 174 Limits of quantification (LOQ), 6, 56, 152 153, 173 174 Lipid content determination, 137 138 Liquid chromatography (LC), 72 Liquid chromatography-tandem mass spectrometry (LC-MS/MS), 91 determination of avermectins and milbemycin residues in food, 68 74 ractopamine determination in swine feed, 79 85 Liquid scintillation counter (LSC), 149, 154, 170 LOD. See Limits of detection (LOD) LOQ. See Limits of quantification (LOQ) Lowest calibrated level (LCL), 56, 64, 113, 152 153

M Macroinvertebrate as bioindicators, 157 169 Magnesium sulfate anhydrous (MgSO4), 26

181 MASE. See Microwave-assisted extraction (MASE) Mass spectrometer, 72 73 Material safety data sheet (MSDS), 59 Matrix effects, 16 Maximum limit of residue (MRL), 73 Maximum limits (MLs), 8 Maximum water holding capacity (MWHC), 150, 170 Methyl parathion, 105 Microelectron capture detection (μ-ECD), 100 Microextractor scheme, 113, 114f Microseparation device, 117, 118f Microwave-assisted extraction (MASE), 171 172 Milbemycin, 69 mass spectrometry setting for MS/MS analysis, 72t residues determination in food, 68 74 samples free of, 70 Mineralization curve of pesticides in soil, 153 157 MLs. See Maximum limits (MLs) Monitoring/testing schemes, 8 Moxidectin (MOX), 69 MRL. See Maximum limit of residue (MRL) MSDS. See Material safety data sheet (MSDS) Multiple pesticide residue analysis, 2 Multiresidue method analytical methods, 2 for pesticides in potato, 47 52 target and qualifier ions, 51t MWHC. See Maximum water holding capacity (MWHC)

N National Institute of Industrial Technology. See Instituto Nacional de Tecnologı´a Industrial (INTI) Net count rate, 141 142 Nitrate and nitrite determination in water calibration parameters, 130t extraction cartridge effect, 130f

Index

182 Nitrate and nitrite determination in water (Continued) using unsuppressed ion chromatography with UV detection, 126 131 Nitrogen drying, 109 110 Nitrogen phosphorus detector/detection (NPD), 25, 100 NPD. See Nitrogen phosphorus detector/ detection (NPD) Nuclear approach, 1 2, 135 174 bioaccumulation factor estimation, 169 174 biomonitoring of water quality using macroinvertebrate, 157 169 detection of irradiated fat containing foods, 135 139 determination of radon in water, 139 143 generation of adsorption isotherms in soils, 144 148 mineralization curve of pesticides in soil, 153 157 effect of pesticides, drugs, and chemical compounds, 148 153

O Organization for Cooperation of Economic and Development (OECD), 148 Organochlorinated pesticide extraction procedure in water, 111 113 chromatographic conditions, 113t Organochlorine insecticide determination in water by GC/MSD, 114 117 target and qualifiers ions for SIM method, 116t Organophosphate pesticide extraction procedure, 108 111 Organophosphorus determination and pesticides, 100 104 concentration of mixtures solution, 102t surrogate and intern standard mixture, 102t

P PAAP. See Polo Agroalimentario y Agroindustrial de Paysandu´ (PAAP)

PEC. See Predicatable Environmental Concentration (PEC) Pesticides effect, 148 153 residue analysis by HPLC MS/MS, 34 39 chromatographic conditions, 38t spectrometric conditions for triphenyl phosphate, 39t residue determination in fresh fruits and vegetables, 25 29 in potato using dispersive QuEChERS template, 40 47 residue extraction in fresh fruits and vegetables, 29 33 in soil using radiometric technique, 153 157 PET. See Polyethylene terephthalate (PET) Polo Agroalimentario y Agroindustrial de Paysandu´ (PAAP), 40 Polyethylene terephthalate (PET), 140 POPOP. See 1,4-Bis(5-phenyloxazol-2-yl) benzene (POPOP) Potato multiresidue method for pesticides, 47 52 pesticide residue determination in, 40 47 PPO. See 2,5-Diphenyloxazole (PPO) Prawns, determination and confirmation of chloramphenicol in, 65 68 Precision and reproducibility, 16 Predicatable Environmental Concentration (PEC), 150 Primary secondary amine (PSA), 26, 29, 60

Q Quality assurance (QA), 11 14 Quality control (QC), 8, 11 12, 28 29, 70 measures, 142, 142t procedures, 11 14 Quenching indicator parameter (QIP), 142 143 Quick, easy, cheap, effective, rugged, and safe method (QuEChERS method), 2

Index

extraction of pesticide residues present in fresh fruits and vegetables, 29 33 multiresidue method for pesticides in potato, 47 52 pesticide residue determination in fresh fruits and vegetables, 25 29 in potato with ammonium acetate, 40 47

R Ractopamine (RAC), 80 determination in swine feed, 79 85 Radiation-induced 2-alkylcyclobutanones, 135 Radiochemical activity, 152 Radiolabeled compounds, 1 2 Radiolabeled pollutants, bioaccumulation factor estimation of, 169 174 Radiometric technique, 153 157 Radiotracers adsorption isotherms in soils using, 144 148 soil respiration using 14C-glucose as, 148 153 Radon determination in water, 139 143 calibration curve, 143f “Recovery” samples (R), 70 Red Analitica de Latino America y el Caribe” network (RALACA network), 1 3 Residues, 1 Residues laboratory, setting up, 5 8 analytical equipment requirements to perform laboratory’s tasks, 6 conditions of analytical laboratory, 6 7 establish scope, 5 examples of resources, 7 8 mandate of laboratory, 5 requirements for sustainability of laboratory, 6 types of methods and limitations, 6 Retention time locking (RTL), 105 222 Rn element, 139 140 RTL. See Retention time locking (RTL)

183

S Sample preparation and processing, 10 11 Sampling in context of food safety, 8 10 SANTE. See European Commission Directorate General for Health and Food Safety (SANTE) SCP. See Sulfachlorpyridazine (SCP) SDA. See Sulfadimethoxine (SDA) SDX. See Sulfadoxine (SDX) SDZ. See Sulfadiazine (SDZ) Selectivity, 15 SME. See Sulfamerazine (SME) SMZ. See Sulfamethoxazole (SMZ) Sodium chloride (NaCl), 105 Sodium hydrogen carbonate (NaHCO3), 26, 115 Sodium hydroxide (NaOH), 87 Sodium phosphate (NaH2PO4), 76 Sodium sulfate (Na2SO4), 26, 57 Soil and water analysis, 90 135 ammonium determination in wastewater, 131 135 determination of carbamates and triazines in soil and sediment samples, 90 100 chromatographic conditions for LCMS/MS analysis, 98t chromatographic conditions for mass spectrometric detection, 97t final concentration levels of calibration curve, 95t intermediate solutions, 94t recommended solvents for preparation of stock standard solutions, 93t determination of selected pesticides in soil by GC-MSD, 104 108 in water by GC-MSD, 117 121 nitrate and nitrite determination in water, 126 131 organochlorinated pesticide extraction procedure in water, 111 113 organochlorine insecticide determination in water by GC/ MSD, 114 117 organophosphate pesticide extraction procedure in water, 108 111

Index

184 Soil and water analysis (Continued) organophosphorus determination and pesticides in soil, 100 104 residues analysis of glyphosate and AMPA in water, 121 126 Soil microorganisms, 149 pesticides in, 153 157 respiration using 14C-glucose as radiotracer, 148 153 Solid phase extraction (SPE), 65, 108. See also Extraction organophosphate pesticide extraction procedure in water using, 108 111 chromatographic conditions, 110t Soxhlet fat extraction method, 136 137, 137f SPD. See Sulfapyridine (SPD) SPE. See Solid phase extraction (SPE) SPZ. See Sulfamethoxypyridazine (SPZ) SQX. See Sulfaquinoxaline (SQX) Stable isotope-labeled compounds, 1 2 Staff and laboratory capabilities, 2 3 Standard uncertainty, 141 142 Strainer, 158 159, 158f STZ. See Sulfatiazole (STZ) Sulfachlorpyridazine (SCP), 75 76 Sulfadiazine (SDZ), 75 76 Sulfadimethoxine (SDA), 75 76 Sulfadoxine (SDX), 75 76 Sulfamerazine (SME), 75 76 Sulfamethoxazole (SMZ), 75 76 Sulfamethoxypyridazine (SPZ), 75 Sulfapyridine (SPD), 75 76 Sulfaquinoxaline (SQX), 75 76 Sulfatiazole (STZ), 75 76 Sulfonamides determination in eggs, 74 79 Support systems, 12 Sustainability of laboratory, 6 Swine feed, ractopamine determination in, 79 85

T 2-TCB. See 2-Tetradecylcyclobutanone (2-TCB) Technically driven, 12 13 2-Tetradecylcyclobutanone (2-TCB), 136, 138 Tissue samples (TS), 70 Tomato, pesticide determination in, 56 60 “Top-down” approach, 18 TPP. See Triphenyl phosphate (TPP) Triazine determination in soil and sediment samples, 90 100 Triphenyl phosphate (TPP), 49, 101 TS. See Tissue samples (TS)

U UdelaR. See Universidad de la Repu´blica (UdelaR) Ultra Turrax blender, 25 Ultrasound treatment, 100 104 Uncertainty estimation, 17 20 Universidad de la Repu´blica (UdelaR), 40 Unsuppressed ion chromatography ammonium determination in wastewater using, 131 135 nitrate and nitrite determination in water using, 126 131

W Water determination of selected pesticides in water by GC-MSD, 117 121 organochlorinated pesticide extraction procedure in, 111 113 organochlorine insecticide determination in water by GC/ MSD, 114 117 quality using macroinvertebrate as bioindicators, 157 169 radon determination in, 139 143 residues analysis of glyphosate and AMPA in, 121 126 Weighted linear calibration model, 28, 51