A common analytical method for determining the active ingredients of insecticides and piperonyl butoxide in filter papers from laboratory impregnation and field trials: report of a study

A C O M M O N A N A LY T IC A L M E T H O D F O R D E T ER M IN IN G T H E  A C T IV E  IN G R ED IE N T S O F IN S EC T IC ID ES A N D P IP ER O N Y L B U T O X ID E IN F IL T ER P A P ER S FR O M L A B O R A T O R Y IM P R EG N A T IO N A N D F IE LD T R IA LS A common analytical method for determining the active ingredients of insecticides and piperonyl butoxide in filter papers from laboratory impregnation and field trials Report of a study

A common analytical method for determining the active ingredients of insecticides and piperonyl butoxide in filter papers from laboratory impregnation and field trials Report of a study A common analytical method for determining the active ingredients of insecticides and piperonyl butoxide in filter papers from laboratory impregnation and field trials: report of a study ISBN 978-92-4-003993-3 (electronic version) ISBN 978-92-4-003994-0 (print version) © World Health Organization 2021 Some rights reserved. This work is available under the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 IGO licence (CC BY-NC-SA 3.0 IGO; https://creativecommons.org/licenses/by-nc-sa/3.0/igo). Under the terms of this licence, you may copy, redistribute and adapt the work for non-commercial purposes, provided the work is appropriately cited, as indicated below. In any use of this work, there should be no suggestion that WHO endorses any specific organization, products or services. 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The responsibility for the interpretation and use of the material lies with the reader. In no event shall WHO be liable for damages arising from its use. Contents Acknowledgements ........................................................................................................... iv Abbreviations and acronyms .............................................................................................. v 1. Introduction ............................................................................................................... 1 2. Summary of results and conclusion ........................................................................... 5 3. Materials .................................................................................................................. 19 3.1 Filter papers .................................................................................................................20 3.2 Analytical standards ...................................................................................................22 3.3 Internal standard .........................................................................................................34 3.4 Insecticide formulations used to validate the method ............................................34 4. Analytical conditions ............................................................................................... 41 4.1 Outline of method ........................................................................................................42 4.2 Apparatus ....................................................................................................................42 4.3 Reagents and solvents ...............................................................................................42 4.4 Analytical procedure .................................................................................................. 43 5. Results of analysis ................................................................................................... 55 5.1 Specificity and non-analyte interference...................................................................56 5.2 Linearity of the detector response .............................................................................56 5.3 Efficacy of extraction (accuracy) ...............................................................................81 5.4 Recovery (accuracy, repeatability and intermediate precision) ..............................85 5.5 Limit of quantification .................................................................................................98 5.6 Pesticide stability in calibration solutions ..............................................................102 6. Evaluation and discussion of results ..................................................................... 103 Annex. Representative chromatograms of the pesticides and piperonyl butoxide content of filter papers ................................................................................ 107 iv Acknowledgements The World Health Organization (WHO) Department of Control of Neglected Tropical Diseases (WHO/ NTD) thanks the following personnel of the Protection, Control Products and Residues Unit of the Walloon Agricultural Research Centre (CRA-W), Gembloux, Belgium: Olivier Pigeon, Scientific Director; Marie Baes, Laboratory Manager; Natacha Garcia Albeniz, responsible for tests; and Marie Lambeau, Sophie Vandecandelaere, Ilian Goffin, Laurent Laduron and Laurette Salmon (technical staff) for developing and validating the common analytical method. The following people reviewed and commented on the draft document: Simon Baker (Switzerland), Jim Garvey (Ireland), Elen Karassali (Greece), Yumiko Kozuki (Japan) and Woramon Suriyachan (Thailand). The study was led by Rajpal S. Yadav and Raman Velayudhan (WHO/NTD). The draft of the document was revised on the basis of comments received after a peer review and finalized by Olivier Pigeon and Rajpal S. Yadav. The study was conducted as part of a WHO project supported financially by the Bill & Melinda Gates Foundation, Seattle (WA), USA. vAbbreviations and acronyms CIPAC Collaborative International Pesticides Analytical Council CRA-W Walloon Agricultural Research Centre DAD ultraviolet diode array spectrophotometry detection FID flame ionization detection GC gas chromatography HPLC high-performance liquid chromatography IRS indoor residual spraying IS internal standard LOQ limit of quantification MS mass spectrometry PBO piperonyl butoxide r2 determination coefficient RSD relative standard deviation RSDr repeatability relative standard deviation RSDR reproducibility relative standard deviation SANCO Directorate-General Health and Consumer Protection SD standard deviation

Introduction 1 2For quality control of filter paper impregnation or field applications, the content of various insecticide active ingredients and/or piperonyl butoxide (PBO; a synergist) must be determined for scientific studies, such as: y laboratory studies in which filter papers are treated with various concentrations of insecticides or PBO to test their bioefficacy; y studies of dose–response relations; y product evaluation trials, in which filter papers are placed on indoor surfaces of experimental huts for quality control of spraying with insecticides; y in large-scale field trials of indoor residual spraying (IRS), in which filter papers are placed on indoor surfaces of houses and sprayed with insecticides; and y laboratory studies in which filter papers are impregnated with well-defined target discriminating concentrations of insecticides to evaluate resistance (or susceptibility) in mosquito, sand fly and triatomine vectors. Currently, chemical testing laboratories use individual methods to determine the content of insecticide active ingredient or PBO in filter papers. These methods involve different extraction solvents, internal standards, laboratory glassware, extraction equipment and conditions, chromatographic columns and conditions as well as different calibration procedures. In view of the absence of a common standard operating procedure (SOP) for analysis of insecticide active ingredients and PBO on filter papers, WHO has developed and validated a common analytical method in collaboration with the Walloon Agricultural Research Centre (CRA-W), Gembloux, Belgium. CRA-W conducted a laboratory study with the following objectives: y to develop a common chromatographic analytical method – gas chromatography with flame ionization detection (GC-FID), gas chromatography with mass spectrometry detection (GC-MS) or high-performance liquid chromatography with ultraviolet diode array spectrophotometry detection (HPLC-DAD) – for determining (identifying and quantifying) insecticide active ingredients applied on filter papers; and y to validate the analytical method by assessing the parameters for validation recommended by the European (SANCO 3030/99) and Collaborative International Pesticides Analytical Council (CIPAC) guidelines, namely specificity, non-analyte interference, linearity of the chromatographic response, accuracy (efficacy of extraction, recovery, repeatability, intermediate precision) and limit of quantification (LOQ). This document describes the materials, analytical method and method validation. The analytical method will be useful to: y research institutions and laboratories that conduct testing and field trials of insecticides sprayed indoors to determine their efficacy in the laboratory and in the field; y research laboratories that determine the actual insecticide content of filter papers treated for determining lethal and discriminating concentrations as part of dose-response studies; y pesticide quality control laboratories; and y research and development laboratories, including those of the pesticide industry. Use of this common analytical method could be extended to other insecticides and pesticides after proper validation and performance verification. 3The advantages for chemical testing laboratories of using common analytical methods to determine insecticides and pesticides on filter papers are: y minimization of the number and quantity of solvents, reagents, internal standards, extraction equipment, glassware and chromatographic columns to be used; y minimization of the changes over time between one sample and the next; y applicability of the same extraction, chromatographic and calibration conditions to all insecticides and pesticides; y a significant reduction in the time required for chemical analysis; y minimization the analytical errors or mistakes in the laboratory; y more efficient use of human, equipment and consumable resources in laboratories; y less consumption of solvents and reagents and thus less laboratory waste; y a significant reduction in the cost of analysis; and y faster quality control of filter papers treated with insecticides. The study was conducted at CRA-W: report no. WHO /24845/2019; CRA-W Study N° 24845: Scientific director: Dr Olivier Pigeon Laboratory manager: Marie Baes Person responsible for testing: Natacha Garcia Albeniz Study starting date: 21 January 2019 Study completion date: 6 July 2021 The study was conducted according to the following guidelines: y Technical active substance and plant protection products: Guidance for generating and reporting methods of analysis in support of pre-registration and post-registration data requirements for Annex (Section 4) of Regulation (EU) No 283/2013 and Annex (Section 5) of Regulation (EU) No 284/2013. Document SANCO 3030/99, rev. 5, 22 March 2019 (https://ec.europa.eu/food/system/ files/2019-03/pesticides_ppp_app-proc_guide_phys-chem-ana_3030.pdf). y CIPAC guidelines on method validation to be performed in support of analytical methods for agrochemical formulations (document No. 380). Abingdon: Collaborative International Pesticides Analytical Council; 2003 (https://www.cipac.org/images/pdf/validat.pdf). Study sponsor WHO. Veterinary Public Health, Vector Control and Environment unit, Department of Control of Neglected Tropical Diseases, Avenue Appia, 20, CH-1211, Geneva 27, Switzerland Study monitor Dr Rajpal S. Yadav, WHO. Veterinary Public Health, Vector Control and Environment unit, Department of Control of Neglected Tropical Diseases, Avenue Appia, 20, CH-1211, Geneva 27, Switzerland Laboratory manager Marie Baes, CRA-W, Knowledge and Valorization of Products Department (D4), Protection, Control Products and Residues Unit (U10), Carson Building, Rue du Bordia, 11, B-5030 Gembloux, Belgium 4Person responsible for tests Natacha Garcia Albeniz, CRA-W, Knowledge and Valorization of Products Department (D4), Protection, Control Products and Residues Unit (U10), Carson Building, Rue du Bordia, 11, B-5030 Gembloux, Belgium Technical personnel Natacha Garcia Albeniz, Ilian Goffin, Laurent Laduron, Marie Lambeau, Laurette Salmon, Sophie Vandecandelaere Archivist Laurette Salmon Archives All the documentation related to this study will be kept under the study number in the archives of the test facility, presently located at 11 rue du Bordia, B-5030 Gembloux, Belgium, for ≥ 10 years from the study completion date, unless instructions to the contrary are received from the sponsor. The archived documents include, as a minimum, the following: y CRA-W study contract and WHO technical services agreement y relevant correspondence y protocols y raw data y final report The remaining samples after analysis will be kept by the test facility in a deep-freeze for ≥ 1 year from the study completion date. After this time and after receipt of study monitor agreement, they will be destroyed. 5Summary of results and conclusion 2 6Samples Filter papers treated with 11 insecticides (p,p΄ -DDT, malathion, pirimiphos-methyl, bendiocarb, propoxur, alpha-cypermethrin, cyfluthrin, deltamethrin, etofenprox, lambda-cyhalothrin, permethrin) or PBO were analysed. Analytical method The insecticides and PBO were extracted from filter papers by ultrasonication for 30 min with acetonitrile in the presence of dicyclohexyl phthalate as the internal standard, cleaned by dispersive solid-phase extraction and determined by capillary GC-MS or capillary GC-FID. Criteria for acceptance of results of method validation The criteria for accepting the validation results were based on the guidelines on method validation for pesticide formulations (document SANCO 3030/99, rev. 5, 22 March 2019 and CIPAC document No. 3807, 28 July 2003). To test for accuracy and repeatability, the guidelines were adapted for filter papers treated with insecticides, for which no published guidelines are available for validation. The criteria for accuracy and repeatability were based on measurement uncertainty linked to spiking of filter paper samples, in addition to measurement uncertainty linked to the analytical method. The results of the study are summarized in Tables 1–12. Conclusions The analytical method with GC-MS or GC-FID for determination of insecticides and PBO on filter papers was validated for each parameter according to the guidelines. 7Table 1. Results of validation of GC-MS analytical method for p,p΄ -DDT Parameter Results day 1 Results day 2 Criterion of acceptance Specificity and non-analyte interference Good separation of the peak of p,p -΄DDT from those of o,p -΄DDT and other pesticides No interference likely to affect the chromatographic peak of p,p -΄DDT Good separation of the peak of p,p -΄DDT from those of o,p -΄DDT and other pesticides No interference likely to affect the chromatographic peak of p,p -΄DDT Capable of determining each pesticide in the presence of other pesticides and each isomer from other isomers, if applicable < 3% interference Linearity Curve (five points): the response factor (peak area for p,p -΄DDT divided by the peak area for dicyclohexyl phthalate) is linear in the following ranges: r² = 0.9948 (244–2006 µg/mL) r² = 0.9985 (50–602 µg/mL) Curve (five points): the response factor (peak area for p,p -΄DDT divided by the peak area for dicyclohexyl phthalate) is linear in the following ranges: r² = 0.9984 (244–2006 µg/mL) r² = 0.9998 (50–602 µg/mL) r² > 0.98 Accuracy Efficacy of extraction Acetonitrile with a sonication time of about 30 min (≥ 20 min) is appropriate for correct extraction of p,p -΄DDT from filter papers Acetonitrile with a sonication time of about 30 min (≥ 20 min) is appropriate for correct extraction of p,p -΄DDT from filter papers – Accuracy and repeatability Recovery - 358–353 mg/m² - 1792–1767 mg/m² - 3571–3571 mg/m² Mean = 100% (n = 5) RSD = 0.9% Mean = 119% (n = 5) RSD = 1.9% Mean = 115% (n = 5) RSD = 1.5% Mean = 115% (n = 5) RSD = 1.5% Mean = 99% (n = 5) RSD = 4.1% Mean = 95% (n = 5) RSD = 6.7% Mean recovery 80–120% RSDr < 10% Intermediate precision Recovery - 358–353 mg/m² - 1792–1767 mg/m² - 3571–3571 mg/m² RSDR = 10.1% (n = 10) RSDR = 13.4% (n = 10) RSDR = 14.3% (n = 10) RSDR < 15% LOQ (practical) LOQ = 163 mg/m² LOQ = 220 mg/m² – LOQ = limit of quantification; r2 = determination coefficient; RSD = relative standard deviation; RSDr = repeatability RSD; RSDR = reproducibility RSD. 8Table 2. Results of validation of GC-MS analytical method for malathion Parameter Results day 1 Results day 2 Criterion of acceptance Specificity and non-analyte interference Good separation of the peak of malathion from those of other pesticides No interference likely to affect the chromatographic peak of malathion Good separation of the peak of malathion from those of other pesticides No interference likely to affect the chromatographic peak of malathion Capable of determining each pesticide in the presence of other pesticides and each isomer from other isomers, if applicable < 3% interference Linearity Curve (five points): the response factor (peak area for malathion divided by the peak area for dicyclohexyl phthalate) is linear in the following ranges: r² = 0.9932 (56–2222 µg/mL) r² = 0.9890 (56–1136 µg/mL) Curve (five points): the response factor (peak area for malathion divided by the peak area for dicyclohexyl phthalate) is linear in the following ranges: r² = 0.9994 (56–2222 µg/mL) r² = 1.0000 (56–667 µg/mL) r² > 0.98 Accuracy Efficacy of extraction Acetonitrile with a sonication time of about 30 min (≥ 20 min) is appropriate to extract malathion from filter papers correctly Acetonitrile with a sonication time of about 30 min (≥ 20 min) is appropriate to extract malathion from filter papers correctly – Accuracy and repeatability Recovery - Level 405–405 mg/m² - Level 2024–2024 mg/m² - Level 4048–4048 mg/m² Mean = 88% (n = 5) RSD = 0.4% Mean = 89% (n = 5) RSD = 5.0% Mean = 80% (n = 4) RSD = 3.2% Mean = 93% (n = 5) RSD = 3.3% Mean = 103% (n = 5) RSD = 1.5% Mean = 95% (n = 5) RSD = 4.1% Mean recovery 80–120% RSDr < 10% Intermediate precision Recovery - Level 405–405 mg/m² - Level 2024–2024 mg/m² - Level 4048–4048 mg/m² RSDR = 3.0% (n = 10) RSDR = 9.8% (n = 10) RSDR = 11.9% (n = 9) RSDR < 15% LOQ (practical) LOQ = 169 mg/m² LOQ = 222 mg/m² – r2 = determination coefficient; LOQ = limit of quantification; RSD = relative standard deviation; RSDr = repeatability RSD; RSDR = reproducibility RSD. 9Table 3. Results of validation of GC-MS analytical method for pirimiphos-methyl Parameter Results day 1 Results day 2 Criterion of acceptance Specificity and non-analyte interference Good separation of the peak of pirimiphos-methyl from those of other pesticides No interference likely to affect the chromatographic peak of pirimiphos-methyl Good separation of the peak of pirimiphos-methyl from those of other pesticides No interference likely to affect the chromatographic peak of pirimiphos-methyl Capable of determining each pesticide in the presence of other pesticides and each isomer from other isomers, if applicable < 3% interference Linearity Curve (six points): the response factor (peak area for pirimiphos-methyl divided by the peak area for dicyclohexyl phthalate) is linear in the following ranges: r² = 0.9963 (26–130 µg/mL) r² = 0.9850 (3–39 µg/mL) Curve (six points): the response factor (peak area for pirimiphos-methyl divided by the peak area for dicyclohexyl phthalate) is linear in the following ranges: r² = 0.9950 (26–130 µg/mL) r² = 0.9889 (3–39 µg/mL) r² > 0.98 Accuracy Efficacy of extraction Acetonitrile with a sonication time of about 30 min (≥ 20 min) is appropriate to extract pirimiphos-methyl from filter papers correctly Acetonitrile with a sonication time of about 30 min (≥ 20 min) is appropriate to extract pirimiphos-methyl from correctly filter papers correctly – Accuracy and repeatability Recovery - Level 21–20 mg/m² - Level 104–101 mg/m² - Level 190–203 mg/m² Mean = 100% (n = 5) RSD = 2.2% Mean = 98% (n = 5) RSD = 6.7% Mean = 87% (n = 5) RSD = 3.1% Mean = 95% (n = 5) RSD = 2.8% Mean = 93% (n = 5) RSD = 2.5% Mean = 90% (n = 5) RSD = 9.6% Mean recovery 80–120% RSDr < 10% Intermediate precision Recovery - Level 21–20 mg/m² - Level 104–101 mg/m² - Level 190–203 mg/m² RSDR = 4.1% (n = 10) RSDR = 5.6% (n = 10) RSDR = 6.8% (n = 10) RSDR < 15% LOQ (practical) LOQ = 11 mg/m² LOQ = 19 mg/m² – LOQ = limit of quantification; r2 = determination coefficient; RSD = relative standard deviation; RSDr = repeatability RSD; RSDR = reproducibility RSD. 10 Table 4. Results of validation of GC-FID analytical method for bendiocarb Parameter Results day 1 Results day 2 Criterion of acceptance Specificity and non-analyte interference Good separation of the peak of bendiocarb from those of other pesticides No interference likely to affect the chromatographic peak of bendiocarb Good separation of the peak of bendiocarb from those of other pesticides No interference likely to affect the chromatographic peak of bendiocarb Capable of determining each pesticide in the presence of other pesticides and each isomer from other isomers, if applicable < 3% interference Linearity Curve (six points): the response factor (peak area for bendiocarb divided by the peak area for dicyclohexyl phthalate) is linear in the following ranges: r² = 0.9966 (1.1–46 µg/mL) r² = 0.9971 (1.1–14 µg/mL) Curve (six points): the response factor (peak area for bendiocarb divided by the peak area for dicyclohexyl phthalate) is linear in the following ranges: r² = 0.9954 (1.1–46 µg/mL) r² = 0.9924 (1.1–14 µg/mL) r² > 0.98 Accuracy Efficacy of extraction Acetonitrile with a sonication time of about 30 min (≥ 20 min) is appropriate to extract correctly bendiocarb from the filter papers Acetonitrile with a sonication time of about 30 min (≥ 20 min) is appropriate to extract correctly bendiocarb from the filter papers – Accuracy and repeatability Recovery - Level 8.1–8.2 mg/m² - Level 41–41 mg/m² - Level 80–80 mg/m² Mean = 105% (n = 5) RSD = 7.1% Mean = 101% (n = 5) RSD = 1.0% Mean = 91% (n = 5) RSD = 0.8% Mean = 115% (n = 5) RSD = 2.2% Mean = 105% (n = 5) RSD = 0.4% Mean = 93% (n = 5) RSD = 0.9% Mean recovery, 80–120% RSDr < 10% Intermediate precision Recovery - Level 8.1–8.2 mg/m² - Level 41–41 mg/m² - Level 80–80 mg/m² RSDR = 8.1% (n = 10) RSDR = 2.5% (n = 10) RSDR = 1.4% (n = 10) RSDR < 15% LOQ (practical) LOQ = 3.5 mg/m² LOQ = 3.3 mg/m² – LOQ = limit of quantification; r2 = determination coefficient; RSD = relative standard deviation; RSDr = repeatability RSD; RSDR = reproducibility RSD. 11 Table 5. Results of validation of GC-FID analytical method for propoxur Parameter Results day 1 Results day 2 Criterion of acceptance Specificity and non-analyte interference Good separation of the peak of propoxur from those of other pesticides No interference likely to affect the chromatographic peak of propoxur Good separation of the peak of propoxur from those of other pesticides No interference likely to affect the chromatographic peak of propoxur Capable of determining each pesticide in the presence of other pesticides and each isomer from other isomers, if applicable < 3% interference Linearity Curve (six points): the response factor (peak area for propoxur divided by the peak area for dicyclohexyl phthalate) is linear in the following ranges: r² = 0.9997 (1.2–47 µg/mL) r² = 0.9992 (1.2–14 µg/mL) Curve (six points): the response factor (peak area for propoxur divided by the peak area for dicyclohexyl phthalate) is linear in the following ranges: r² = 0.9997 (1.2–47 µg/mL) r² = 0.9998 (1.2–14 µg/mL) r² > 0.98 Accuracy Efficacy of extraction Acetonitrile with a a sonication time of about 30 min (≥ 20 min) is appropriate to extract propoxur from filter papers correctly Acetonitrile with a sonication time of about 30 min (≥ 20 min) is appropriate to extract propoxur from filter papers correctly – Accuracy and repeatability Recovery - Level 8.2–8.2 mg/m² - Level 41–41 mg/m² - Level 82–82 mg/m² Mean = 114% (n = 5) RSD = 4.8% Mean = 109% (n = 5) RSD = 0.6% Mean = 88% (n = 5) RSD = 0.7% Mean = 107% (n = 5) RSD = 2.1% Mean = 107% (n = 5) RSD = 1.7% Mean = 89% (n = 5) RSD = 0.7% Mean recovery, 80–120% RSDr < 10% Intermediate precision Recovery - Level 8.2–8.2 mg/m² - Level 41–4.1 mg/m² - Level 82–82 mg/m² RSDR = 4.9% (n = 10) RSDR = 1.5% (n = 10) RSDR = 0.9% (n = 10) RSDR < 15% LOQ (practical) LOQ = 3.6 mg/m² LOQ = 3.7 mg/m² – LOQ = limit of quantification; r2 = determination coefficient; RSD = relative standard deviation; RSDr = repeatability RSD; RSDR = reproducibility RSD. 12 Table 6. Results of validation of GC-MS analytical method for alpha-cypermethrin Parameter Results day 1 Results day 2 Criterion of acceptance Specificity and non-analyte interference Good separation of the peak of alpha-cypermethrin (cis II) from those of alpha- cypermethrin isomer (cis I) and other pesticides No interference likely to affect the chromatographic peak of alpha-cypermethrin Good separation of the peak of alpha-cypermethrin (cis II) from those of alpha- cypermethrin isomer (cis I) and other pesticides No interference likely to affect the chromatographic peak of alpha-cypermethrin Capable of determining each pesticide in the presence of other pesticides and each isomer from other isomers, if applicable < 3% interference Linearity Curve (six points): the response factor (peak area for alpha-cypermethrin divided by the peak area for dicyclohexyl phthalate) is linear in the following ranges: r² = 0.9876 (0.6–18 µg/mL) r² = 0.9985 (0.6–12 µg/mL) Curve (six points): the response factor (peak area for alpha-cypermethrin divided by the peak area for dicyclohexyl phthalate) is linear in the following ranges: r² = 0.9921 (0.6–17 µg/mL) r² = 0.9902 (0.6–12 µg/mL) r² > 0.98 Accuracy Efficacy of extraction Acetonitrile with a sonication time of about 30 min (≥ 20 min) is appropriate to extract alpha-cypermethrin from filter papers correctly Acetonitrile with a sonication time of about 30 min (≥ 20 min) is appropriate to extract alpha-cypermethrin from filter papers correctly – Accuracy and repeatability Recovery - Level 3.9–3.8 mg/m² - Level 19–19 mg/m² - Level 37–38 mg/m² Mean = 94% (n = 5) RSD = 4.1% Mean = 86% (n = 5) RSD = 6.5% Mean = 89% (n = 5) RSD = 2.3% Mean = 113% (n = 5) RSD = 2.0% Mean = 91% (n = 5) RSD = 3.6% Mean = 99% (n = 5) RSD = 5.0% Mean recovery 80–120% RSDr < 10% Intermediate precision Recovery - Level 3.9–3.8 mg/m² - Level 19–19 mg/m² - Level 37–38 mg/m² RSDR = 13.5% (n = 10) RSDR = 6.2% (n = 10) RSDR = 8.7% (n = 10) RSDR < 15% LOQ (practical) LOQ = 1.9 mg/m² LOQ = 2.7 mg/m² – LOQ = limit of quantification; r2 = determination coefficient; RSD = relative standard deviation; RSDr = repeatability RSD; RSDR = reproducibility RSD. 13 Table 7. Results of validation of GC-MS analytical method for cyfluthrin Parameter Results day 1 Results day 2 Criterion of acceptance Specificity and non-analyte interference Good separation of the peaks of cyfluthrin from those of other pesticides No interference likely to affect the chromatographic peaks of cyfluthrin Good separation of the peaks of cyfluthrin from those of other pesticides No interference likely to affect the chromatographic peaks of cyfluthrin Capable of determining each pesticide in the presence of other pesticides and each isomer from other isomers, if applicable < 3% interference Linearity Curve (six points): the response factor (total peak area of the four isomers of cyfluthrin (divided by the peak area for dicyclohexyl phthalate) is linear in the following ranges: r² = 0.9927 (5.46–8 µg/mL) r² = 0.9995 (1.7–20 µg/mL) Curve (six points): the response factor (total peak area of the four isomers of cyfluthrin divided by the peak area for dicyclohexyl phthalate) is linear in the following ranges: r² = 0.9939 (5.0–31 µg/mL) r² = 0.9994 (0.9–10 µg/mL) r² > 0.98 Accuracy Efficacy of extraction Acetonitrile with a sonication time of about 30 min (≥ 20 min) is appropriate to extract correctly cyfluthrin from the filter papers Acetonitrile with a sonication time of about 30 min (≥ 20 min) is appropriate to extract correctly cyfluthrin from the filter papers – Accuracy and repeatability Recovery - Level 12–12 mg/m² - Level 58–58 mg/m² - Level 116–117 mg/m² Mean = 82% (n = 5) RSD = 4.0% Mean = 85% (n = 5) RSD = 5.1% Mean = 92% (n = 5) RSD = 2.0% Mean = 82% (n = 5) RSD = 5.0% Mean = 88% (n = 5) RSD = 2.1% Mean = 99% (n = 5) RSD = 0.7% Mean recovery 80–120% RSDr < 10% Intermediate precision Recovery - Level 12–12 mg/m² - Level 58–58 mg/m² - Level 116–117 mg/m² RSDR = 4.1% (n = 10) RSDR = 4.3% (n = 10) RSDR = 5.5% (n = 10) RSDR < 15% LOQ (practical) LOQ = 5.9 mg/m² LOQ = 3.9 mg/m² – LOQ = limit of quantification; r2 = determination coefficient; RSD = relative standard deviation; RSDr = repeatability RSD; RSDR = reproducibility RSD. 14 Table 8. Results of validation of GC-MS analytical method for deltamethrin Parameter Results day 1 Results day 2 Criterion of acceptance Specificity and non-analyte interference Good separation of the peak of deltamethrin from deltamethrin R-alpha isomer and other pesticides No interference likely to affect the chromatographic peak of deltamethrin Good separation of the peak of deltamethrin from deltamethrin R-alpha isomer and other pesticides No interference likely to affect the chromatographic peak of deltamethrin Capable of determining each pesticide in presence of other pesticides and each isomer from other isomers, if applicable < 3% of interference Linearity Curve (six points): the response factor (peak area for deltamethrin divided by the peak area for dicyclohexyl phthalate) is linear in the following ranges: r² = 0.9944 (3.6–24 µg/mL) r² = 0.9997 (0.6–12 µg/mL) Curve (six points): the response factor (peak area for deltamethrin divided by the peak area for dicyclohexyl phthalate) is linear in the following ranges: r² = 0.9894 (3.6–24 µg/mL) r² = 0.9893 (0.6–12 µg/mL) r² > 0.98 Accuracy Efficacy of extraction Acetonitrile with a sonication time of about 30 min (≥ 20 min) is appropriate to extract deltamethrin from filter papers correctly Acetonitrile with a sonication time of about 30 min (≥ 20 min) is appropriate to extract deltamethrin from filter papers correctly – Accuracy and repeatability Recovery - Level 3.8–3.8 mg/m² - Level 19–19 mg/m² - Level 38–38 mg/m² Mean = 83% (n = 5) RSD = 7.8% Mean = 86% (n = 5) RSD = 4.5% Mean = 86% (n = 5) RSD = 1.9% Mean = 101% (n = 5) RSD = 2.5% Mean = 80% (n = 5) RSD = 2.3% Mean = 82% (n = 5) RSD = 4.7% Mean recovery 80–120% RSDr < 10% Intermediate precision Recovery - Level 3.8–3.8 mg/m² - Level 19–19 mg/m² - Level 38–38 mg/m² RSDR = 14.5% (n = 10) RSDR = 3.2% (n = 10) RSDR = 4.3% (n = 10) RSDR < 15% LOQ (practical) LOQ = 2.0 mg/m² LOQ = 3.0 mg/m² – LOQ = limit of quantification; r2 = determination coefficient; RSD = relative standard deviation; RSDr = repeatability RSD; RSDR = reproducibility RSD. 15 Table 9. Results of validation of GC-MS analytical method for etofenprox Parameter Results day 1 Results day 2 Criterion of acceptance Specificity and non-analyte interference Good separation of the peak of etofenprox from those of other pesticides No interference likely to affect the chromatographic peak of etofenprox Good separation of the peak of etofenprox from those of other pesticides No interference likely to affect the chromatographic peak of etofenprox Capable of determining each pesticide in the presence of other pesticides and each isomer from other isomers, if applicable < 3% interference Linearity Curve (six points): the response factor (peak area for etofenprox divided by the peak area for dicyclohexyl phthalate) is linear in the following ranges: r² = 0.9890 (33–205 µg/mL) r² = 0.9999 (5–62 µg/mL) Curve (six points): the response factor (peak area for etofenprox divided by the peak area for dicyclohexyl phthalate) is linear in the following ranges: r² = 0.9958 (33–205 µg/mL) r² = 0.9795 (5–62 µg/mL) r² > 0.98 Accuracy Efficacy of extraction Acetonitrile with a sonication time of about 30 min (≥ 20 min) is appropriate to extract etofenprox from filter papers correctly Acetonitrile with a sonication time of about 30 min (≥ 20 min) is appropriate to extract etofenprox from filter papers correctly – Accuracy and repeatability Recovery - Level 37–37 mg/m² - Level 184–185 mg/m² - Level 367–368 mg/m² Mean = 91% (n = 5) RSD = 3.8% Mean = 93% (n = 5) RSD = 1.6% Mean = 97% (n = 5) RSD = 6.7% Mean = 105% (n = 5) RSD = 2.3% Mean = 100% (n = 5) RSD = 2.1% Mean = 100% (n = 5) RSD = 2.7% Mean recovery 80–120% RSDr < 10% Intermediate precision Recovery - Level 37–37 mg/m² - Level 184–185 mg/m² - Level 367–368 mg/m² RSDR = 10.9% (n = 10) RSDR = 5.5% (n = 10) RSDR = 5.1% (n = 10) RSDR < 15% LOQ (practical) LOQ = 15 mg/m² LOQ = 18 mg/m² – LOQ = limit of quantification; r2 = determination coefficient; RSD = relative standard deviation; RSDr = repeatability RSD; RSDR = reproducibility RSD. 16 Table 10. Results of validation of GC-MS analytical method for lambda-cyhalothrin Parameter Results day 1 Results day 2 Criterion of acceptance Specificity and non-analyte interference Good separation of the peak of lambda-cyhalothrin from those of cyhalothrin isomers and other pesticides No interference likely to affect the chromatographic peak of lambda-cyhalothrin Good separation of the peak of lambda-cyhalothrin from those of cyhalothrin isomers and other pesticides No interference likely to affect the chromatographic peak of lambda-cyhalothrin Capable of determining each pesticide in the presence of other pesticides and each isomer from other isomers, if applicable < 3% interference Linearity Curve (six points): the response factor (peak area for lambda-cyhalothrin divided by the peak area for dicyclohexyl phthalate) is linear in the following ranges: r² = 0.9936 (0.6–19 µg/mL) r² = 1.0000 (0.6–8 µg/mL) Curve (six points): the response factor (peak area for lambda-cyhalothrin divided by the peak area for dicyclohexyl phthalate) is linear in the following ranges: r² = 0.9950 (0.6–24 µg/mL) r² = 0.9895 (0.6–7 µg/mL) r² > 0.98 Accuracy Efficacy of extraction Acetonitrile with a sonication time of about 30 min (≥ 20 min) is appropriate to extract lambda-cyhalothrin from filter papers correctly Acetonitrile with a sonication time of about 30 min (≥ 20 min) is appropriate to extract lambda-cyhalothrin from filter papers correctly – Accuracy and repeatability Recovery - Level 3.7–3.8 mg/m² - Level 19–19 mg/m² - Level 38–38 mg/m² Mean = 104% (n = 5) RSD = 1.7% Mean = 90% (n = 5) RSD = 2.6% Mean = 92% (n = 5) RSD = 1.5% Mean = 106% (n = 5) RSD = 2.4% Mean = 91% (n = 5) RSD = 2.0% Mean = 94% (n = 5) RSD = 2.4% Mean recovery, 80–120% RSDr < 10% Intermediate precision Recovery - Level 3.7–3.8 mg/m² - Level 19–19 mg/m² - Level 38–38 mg/m² RSDR = 2.5% (n = 10) RSDR = 2.2% (n = 10) RSDR = 2.6% (n = 10) RSDR < 15% LOQ (practical) LOQ = 2.6 mg/m² LOQ = 2.5 mg/m² – LOQ = limit of quantification; r2 = determination coefficient; RSD = relative standard deviation; RSDr = repeatability RSD; RSDR = reproducibility RSD. 17 Table 11. Results of validation of GC-MS analytical method for permethrin Parameter Results day 1 Results day 2 Criterion of acceptance Specificity and non-analyte interference Good separation of the peaks of permethrin (cis and trans isomers) from those of other pesticides No interference likely to affect the chromatographic peaks of permethrin (cis and trans isomers) Good separation of the peaks of permethrin (cis and trans isomers) from those of other pesticides No interference likely to affect the chromatographic peaks of permethrin (cis and trans isomers) Capable of determining each pesticide in the presence of other pesticides and each isomer from other isomers, if applicable < 3% interference Linearity Curve (six points): the response factor (total peak area of cis and trans isomers of permethrin divided by the peak area for dicyclohexyl phthalate) is linear in the following ranges: r² = 0.9870 (51–430 µg/mL) r² = 0.9853 (11–129 µg/mL) Curve (six points): the response factor (total peak area of cis and trans isomers of permethrin divided by the peak area for dicyclohexyl phthalate) is linear in the following ranges: r² = 0.9939 (51–430 µg/mL) r² = 0.9891 (11–170 µg/mL) r² > 0.98 Accuracy Efficacy of extraction Acetonitrile with a sonication time of about 30 min (≥ 20 min) is appropriate to extract permethrin from filter papers correctly Acetonitrile with a sonication time of about 30 min (≥ 20 min) is appropriate to extract permethrin from filter papers correctly – Accuracy and repeatability Recovery - Level 56–55 mg/m² - Level 278–276 mg/m² - Level 550–552 mg/m² Mean = 105% (n = 5) RSD = 1.2% Mean = 91% (n = 5) RSD = 1.8% Mean = 83% (n = 5) RSD = 4.1% Mean = 108% (n = 5) RSD = 1.2% Mean = 93% (n = 5) RSD = 0.8% Mean = 88% (n = 5) RSD = 1.6% Mean recovery, 80–120% RSDr < 10% Intermediate precision Recovery - Level 56–55 mg/m² - Level 278–276 mg/m² - Level 550–552 mg/m² RSDR = 2.4% (n = 10) RSDR = 2.4% (n = 10) RSDR = 5.1% (n = 10) RSDR < 15% LOQ (practical) LOQ = 39 mg/m² LOQ = 37 mg/m² – LOQ = limit of quantification; r2 = determination coefficient; RSD = relative standard deviation; RSDr = repeatability RSD; RSDR = reproducibility RSD. 18 Table 12. Results of validation of GC-MS analytical method for piperonyl butoxide Parameter Results day 1 Results day 2 Criterion of acceptance Specificity and non-analyte interference Good separation of the peak of PBO from those of other pesticides No interference likely to affect the chromatographic peak of PBO Good separation of the peak of PBO from those of other pesticides No interference likely to affect the chromatographic peak of PBO Capable of determining each pesticide in the presence of other pesticides and each isomer from other isomers, if applicable < 3% interference Linearity Curve (six points): the response factor (peak area for PBO divided by the peak area for dicyclohexyl phthalate) is linear in the following ranges: r² = 0.9976 (39–1549 µg/mL) r² = 0.9875 (39–465 µg/mL) Curve (six points): the response factor (peak area for PBO divided by the peak area for dicyclohexyl phthalate) is linear in the following ranges: r² = 0.9994 (39–1549 µg/mL) r² = 0.9979 (39–465 µg/mL) r² > 0.98 Accuracy Efficacy of extraction Acetonitrile with a sonication time of about 30 min (≥ 20 min) is appropriate to extract PBO from filter papers correctly Acetonitrile with a sonication time of about 30 min (≥ 20 min) is appropriate to to extract PBO from filter papers correctly – Accuracy and repeatability Recovery - Level 294–294 mg/m² - Level 1472–1472 mg/m² - Level 2929–2929 mg/m² Mean = 91% (n = 5) RSD = 2.6% Mean = 109% (n = 5) RSD = 0.6% Mean = 93% (n = 5) RSD = 2.5% Mean = 92% (n = 5) RSD = 1.5% Mean = 98% (n = 5) RSD = 1.0% Mean = 112% (n = 5) RSD = 8.2% Mean recovery, 80–120% RSDr < 10% Intermediate precision Recovery - Level 294–294 mg/m² - Level 1472–1472 mg/m² - Level 2929–2929 mg/m² RSDR = 2.3% (n = 10) RSDR = 7.6% (n = 10) RSDR = 14.1% (n = 10) RSDR < 15% LOQ (practical) LOQ = 118 mg/m² LOQ = 129 mg/m² – LOQ = limit of quantification; r2 = determination coefficient; RSD = relative standard deviation; RSDr = repeatability RSD; RSDR = reproducibility RSD. 19 Materials 3 20 3.1 Filter papers The table below lists the filter papers untreated or treated freshly with different insecticides or PBO for the study, supplied by the Vector Control Research Unit, Universiti Sains Malaysia, Malaysia for method development. Product class Insecticide Number of control (untreated) papersa Number of treated papersb Organochlorine p,p -΄DDT 10 4 Organophosphates Malathion 10 4 Pirimiphos-methyl 4 Carbamates Bendiocarb 10 4 Propoxur 4 Pyrethroids Alpha-cypermethrin 10 4 Cyfluthrin 4 Deltamethrin 4 Etofenprox 4 Lambda-cyhalothrin 4 Permethrin 4 Synergist PBO 10 4 Total 50 48 a 10 control (untreated) papers per class group. b 4 treated papers per insecticide or PBO. 21 The following table lists the filter papers untreated or treated freshly with different insecticides or PBO for the study and supplied by the Vector Control Research Unit, Universiti Sains Malaysia, Malaysia for method validation. Product class Insecticide Number of control (untreated)a Number of treated papersb Organochlorine p,p -΄DDT 20 4 Organophosphates Malathion 20 4 Pirimiphos-methyl 4 Carbamates Bendiocarb 20 4 Propoxur 4 Pyrethroids Alpha-cypermethrin 20 4 Cyfluthrin 4 Deltamethrin 4 Etofenprox 4 Lambda-cyhalothrin 4 Permethrin 4 Synergist PBO 20 4 Total 100 48 a 20 control (untreated) papers per class group. b 4 treated papers per insecticide or PBO. Filter papers were received on 6 May 2019 and immediately stored in a deep-freeze at < –18 °C until analytical determination. Samples of carrier oils (silicone oil, olive oil and risella oil) that were used by the Vector Control Research Unit to treat the filter papers were received on 6 June 2019 and immediately stored in a refrigerator at 4 °C (± 3 °C). 22 3.2 Analytical standards The following analytical standards purchased by CRA-W were used. p,p΄ -DDT Common name p,p΄-DDT IUPAC name 1,1,1-Trichloro-2,2-bis(4-chlorophenyl)ethane Chemical Abstracts name 1,1’-(2,2,2-Trichloroethylidene)bis[4-chlorobenzene] Molecular formula C₁₄H₉Cl₅ Molecular weight 354.49 Chemical structure ClCl Cl Cl Cl CAS number [50-29-3] Supplier LGC Labor GmbH (Dr Ehrenstorfer), Germany Code number C12082000 DRE-C12082000 Batch number G133143 G832442 Purity 99.40% 99.85% Expiry date 13 March 2023 22 June 2024 Certificate of analysis Established by Dr Ehrenstorfer, LGC Labor GmbH on 13 March 2017 Established by Dr Ehrenstorfer, LGC Labor GmbH on 22 June 2018 Date of receipt at the test facility 1 December 2017 29 November2019 Registration number at the test facility SR/6525 SR/7078 - 7079 - 7080 Storage At 4 °C (± 3 °C) 23 Malathion Common name Malathion IUPAC name Diethyl (dimethoxyphosphinothioylthio)succinate Chemical Abstracts name Diethyl ((dimethoxyphosphinothioyl)thio)butanedioate Molecular formula C10H19O6PS2 Molecular weight 330.36 Chemical structure CH3 CH3 CH3 CH3 O O O O O O P S S CAS number [121-75-5] Supplier LGC Labor GmbH (Dr Ehrenstorfer), Germany Code number DRE-C14710000 Batch number G150822 Purity 99.46% Expiry date 27 February 2023 Certificate of analysis Established by Dr Ehrenstorfer, LGC Labor GmbH on 19 December 2017 Date of receipt at the test facility 29 November 2019 Registration number at the test facility SR/7090 - 7091 - 7092 - 7093 Storage At 4 °C (± 3 °C) 24 Pirimiphos-methyl Common name Pirimiphos-methyl IUPAC name O-2-Diethylamino-6-methylpyrimidin-4-yl O,O-dimethyl phosphorothioate Chemical Abstracts name O-(2-(Diethylamino)-6-methyl-4-pyrimidinyl) O,O-dimethyl phosphorothioate Molecular formula C11H20N3O3PS Molecular weight 305.33 Chemical structure CH3 CH3 CH3 CH3 CH3 N N N O OO P S CAS number [29232-93-7] Supplier Syngenta, Switzerland Code number PP511 Batch number ASJ10084-07 Purity 99.20% Expiry date 28 February 2022 Certificate of analysis Established by Syngenta Crop Protection AG on 19 March 2018 Date of receipt at the test facility 7 January 2020 Registration number at the test facility SR/7108 Storage At 4 °C (± 3 °C) 25 Bendiocarb Common name Bendiocarb IUPAC name 2,2-Dimethyl-1,3-benzodioxol-4-yl methylcarbamate Chemical Abstracts name 2,2-Dimethyl-1,3-benzodioxol-4-yl methylcarbamate Molecular formula C11H13NO4 Molecular weight 223.23 Chemical structure CH3 CH3 CH3N OHO O O CAS number [22781-23-3] Supplier Bayer CropScience, Germany Code number 1700478 Batch number 860402 Purity 98.50% Expiry date 24 January 2025 Certificate of analysis Established by Bayer Crop Science on 27 January 2017 Date of receipt at the test facility 27 February 2019 Registration number at the test facility SR/6848 Storage At 4 °C (± 3 °C) 26 Propoxur Common name Propoxur IUPAC name 2-Isopropoxyphenyl methylcarbamate Chemical Abstracts name 2-(1-Methylethoxy)phenyl methylcarbamate Molecular formula C11H15NO3 Molecular weight 209.24 Chemical structure CH3 CH3 CH3 N OH OO CAS number [114-26-1] Supplier HPC Standards GmbH, Germany Code number 672883 Batch number 783508 Purity 99.90% Expiry date 1 February 2023 Certificate of analysis Established by HPC Standards GmbH on 9 January 2018 Date of receipt at the test facility 30 September 2019 Registration number at the test facility SR/6981 Storage At 4 °C (± 3 °C) 27 Alpha-cypermethrin Common name Alpha-cypermethrin IUPAC name A racemate comprising (R)-α-cyano-3-phenoxybenzyl (1S)-cis- 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate and (S)-α-cyano-3-phenoxybenzyl (1R)-cis-3-(2,2-dichlorovinyl)- 2,2-dimethylcyclopropanecarboxylate Chemical Abstracts name (R)-Cyano(3-phenoxyphenyl)methyl (1S,3S)-rel-3- (2,2-dichloroethenyl)-2,2-dimethylcyclopropanecarboxylate and (S)-cyano(3-phenoxyphenyl)methyl (1R,3R)-rel-3- (2,2-dichloroethenyl)-2,2-dimethylcyclopropanecarboxylate Molecular formula C22H19Cl2NO3 Molecular weight 416.3 Chemical structure CH3 CH3 Cl Cl N O O O CH3 CH3 Cl Cl N O O O CAS number [67375-30-8] Supplier BASF, Germany HPC Standards GmbH, Germany Code number 4078193 673988 Batch number L80-24 778183 Purity 99.40% 99.60% Expiry date Expected 1 September 2019 1 October 2021 Certificate of analysis Established by BASF on 13 April 2015 Established by HPC Standards GmbH on 19 September 2016 Date of receipt at the test facility 21 April 2015 19 September 2016 Registration number at the test facility SR/5735 SR/6982 Storage At 4 °C (± 3 °C) 28 Cyfluthrin Common name Cyfluthrin IUPAC name (RS)-α-Cyano-4-fluoro-3-phenoxybenzyl (1RS,3RS;1RS,3SR)- 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate Chemical Abstracts name Cyano(4-fluoro-3-phenoxyphenyl)methyl 3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropanecarboxylate Molecular formula C22H18Cl2FNO3 Molecular weight 434.29 Chemical structure CH3CH3 Cl Cl F N O O O CAS number [68359-37-5] Supplier HPC Standards GmbH, Germany Code number 677696 Batch number 790889 Purity 98.00% Expiry date 1 September 2024 Certificate of analysis Established by HPC Standards GmbH on 20 August 2019 Date of receipt at the test facility 30 September 2019 7 November 2019 Registration number at the test facility SR/6984 SR/7056 Storage At 4 °C (± 3 °C) 29 Deltamethrin Common name Deltamethrin IUPAC name (S)-α-Cyano-3-phenoxybenzyl (1R,3R)-3-(2,2-dibromovinyl)- 2,2-dimethylcyclopropanecarboxylate Chemical Abstracts name (S)-Cyano(3-phenoxyphenyl)methyl (1R,3R)-3- (2,2-dibromoethenyl)-2,2-dimethylcyclopropanecarboxylate Molecular formula C22H19Br2NO3 Molecular weight 505.2 Chemical structure CH3 CH3 Br Br N O O O CAS number [52918-63-5] Supplier LGC Labor GmbH (Dr Ehrenstorfer), Germany Code number C12120000 Batch number G139484 Purity 99.73% Expiry date 1 February 2023 Certificate of analysis Established by Dr Ehrenstorfer, HPC Standards GmbH on 19 September 2017 Date of receipt at the test facility 30 September 2019 14 October 2019 Registration number at the test facility SR/6987 SR/7014 Storage At 4 °C (± 3 °C) 30 Etofenprox Common name Etofenprox IUPAC name 2-(4-Ethoxyphenyl)-2-methylpropyl 3-phenoxybenzyl ether Chemical Abstracts name 1-((2-(4-Ethoxyphenyl)-2-methylpropoxy) methyl)-3-phenoxybenzene Molecular formula C25H28O3 Molecular weight 376.49 Chemical structure CH3 CH3 CH3 O O O CAS number [80844-07-1] Supplier Mitsui Chemicals, Japan Code number AGPM-19-52 Batch number Y160802 Purity 99.98% Expiry date 31 May 2022 Certificate of analysis Established by Mitsui Chemicals Agro on 24 May 2019 Date of receipt at the test facility 17 June 2019 Registration number at the test facility SR/6917 Storage At 4 °C (± 3 °C) 31 Lambda-cyhalothrin Common name Lambda-cyhalothrin IUPAC name (R)-α-Cyano-3-phenoxybenzyl (1S)-cis-3-[(Z)-2-chloro- 3,3,3-trifluoropropenyl]-2,2-dimethylcyclopropanecarboxylate and (S)- α-cyano-3-phenoxybenzyl (1R)-cis-3-[(Z)-2-chloro- 3,3,3-trifluoropropenyl]-2,2-dimethylcyclopropane-carboxylate Chemical Abstracts name (R)-Cyano(3-phenoxyphenyl)methyl (1S,3S)-rel- 3-((1Z)-2-chloro-3,3,3-trifluoro-1-propenyl)- 2,2-dimethylcyclopropanecarboxylate and (S)-cyano (3-phenoxyphenyl)methyl (1R,3R)-rel-3-((1Z)-2-chloro- 3,3,3-trifluoro-1-propenyl)-2,2-dimethylcyclopropanecarboxylate Molecular formula C23H19ClF3NO3 Molecular weight 449.85 Chemical structure CH3CH3 Cl F F F N O O O CH3CH3 Cl F F F N O O O CAS number [91465-08-6] Supplier Chem Service, Germany LGC Labor GmbH (Dr Ehrenstorfer), Germany Code number N-12307 C11860000 Batch number 7613800 G130561 Purity 99.50% 99.10% Expiry date 31 January 2022 15 March 2021 Certificate of analysis Established by Chem Service INC on 19 September 2017 Established by Dr Erhenstorfer, LGC Labor GmbH on 15 March 2017 Date of receipt at the test facility 6 September 2018 8 August 2018 Registration number at the test facility SR/6729 SR/6711 Storage At 4 °C (± 3 °C) 32 Permethrin Common name Permethrin 40/60 IUPAC name 3-Phenoxybenzyl (1RS,3RS;1RS,3SR)-3-(2,2-dichlorovinyl)- 2,2-dimethylcyclopropanecarboxylate or 3-phenoxybenzyl (1RS)-cis-trans-3-(2,2-dichlorovinyl)- 2,2- dimethylcyclopropanecarboxylate Chemical Abstracts name (3-Phenoxyphenyl)methyl 3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropanecarboxylate Molecular formula C21H20Cl2O3 Molecular weight 391.3 Chemical structure CH3 CH3 Cl Cl O O O O CAS number [52645-53-1] Supplier Sumitomo Chemical Company, Japan Code number C11890100 Batch number PMPO000713 Purity 97.2% Expiry date 11 September 2020 Certificate of analysis Established by Sumitomo Chemical Co., Ltd on 12 September 2017 Date of receipt at the test facility 13 August 2018 Registration number at the test facility SR/6718 Storage At 4 °C (± 3 °C) 33 Piperonyl butoxide Common name Piperonyl butoxide IUPAC name 5-[2-(2-Butoxyethoxy)ethoxymethyl]-6-propyl-1,3-benzodioxole Chemical Abstracts name 5-[[2-(2-Butoxyethoxy)ethoxy]methyl]-6-propyl-1,3-benzodioxole Molecular formula C19H30O5 Molecular weight 338.44 Chemical structure CH3 CH3 O O O O O CAS number [51-03-6] Supplier LGC Labor GmbH (Dr Ehrenstorfer), Germany Code number DRE-C16240000 DRE-C16240000 Batch number G687415 G1006213 Purity 96.80% 95.80% Expiry date 10 April 2020 1 March 2021 Certificate of analysis Established by LGC Labor GmbH on 10 April 2018 Established by LGC Labor GmbH on 1 April 2019 Date of receipt at the test facility 8 April 2019 29 November 2019 Registration number at the test facility SR/6897 SR/7096 - 7097 Storage At 4 °C (± 3 °C) 34 3.3 Internal standard Common name Dicyclohexyl phthalate CAS number [84-61-7] Supplier Acros Organics Code number 37505 Batch number A0358546 Purity 99.9% Date of receipt at the test facility 1 December 2017 Registration number at the test facility INV 2616 Storage At 4 °C (± 3 °C) 3.4 Insecticide formulations used to validate the method Test item 1 Name DDT 75% WP Other name/code – Type of formulation Wettable powder Active substance(s) content - nominal concentration - analysed concentration p,p΄-DDT, 540 g/kg p,p΄-DDT, 538 g/kg Supplier Partnership for Supply Chain Management Container(s) 1 non-commercial aluminized bag containing about 300 g of product Manufacturer Hindustan Insecticides Limited, Rasayani, India Batch number HDTAI-19 Manufacture date March 2017 Expiry date February 2019a Date of receipt at the test facility 10 April 2017 Registration number at the test facility Ch.6810 Storage at the test facility (before the beginning of the experimental phase) At room temperature (10–30 °C) in an appropriate storage area, well ventilated and not exposed to direct sunlight. The temperature was monitored continuously. a The test item is expired, but this has no impact on the result, as it is used only for recovery. 35 Test item 2 Name Malathion 44% w/v EW Other name/code – Type of formulation Emulsion, oil in water (EW) Active substance(s) content - nominal concentration - analysed concentration Malathion 440 g/L Malathion 450 g/L Supplier Tagros Chemicals India Pvt Ltd, Chennai, India Container(s) One non-commercial high-density polyethylene/ethylene vinyl alcohol bottle containing about 1 L of product Manufacturer Tagros Chemicals India Pvt Ltd, Chennai, India Batch number 70E2019 Manufacture date May 2019 Expiry date May 2021 Date of receipt at the test facility 5 May 2019 Registration number at the test facility Ch.7214 Storage at the test facility (before the beginning of the experimental phase) At room temperature (10–30 °C) in an appropriate storage area, well ventilated and not exposed to direct sunlight. The temperature was monitored continuously. Test item 3 Name Actellic 300 CS Other name/code – Type of formulation Capsule suspension Active substance(s) content - nominal concentration - analysed concentration Pirimiphos-methyl 300 g/L Pirimiphos-methyl 295 g/L Supplier Partnership for Supply Chain Management Container(s) Two commercial high-density polyethylene bottles each containing 833 mL of product Manufacturer Syngenta Crop Protection AG, Basel, Switzerland Batch number BSN8K0580 Manufacture date November 2018 Expiry date November 2020 Date of receipt at the test facility 16 May 2019 Registration number at the test facility Ch.7210/8 Storage at the test facility (before the beginning of the experimental phase) At room temperature (10–30 °C) in an appropriate storage area, well ventilated and not exposed to direct sunlight. The temperature was monitored continuously. 36 Test item 4 Name Ficam W Other name/code – Type of formulation Wettable powder in water-soluble bag Active substance(s) content - nominal concentration - analysed concentration Bendiocarb 800 g/kg Bendiocarb 790 g/kg Supplier UNDP, Copenhagen, Denmark Container(s) 11 commercial sealed aluminized bags containing each five water soluble bags with about 125 g of product Manufacturer Bayer SAS, Environmental Science, Spain. Batch number EQ13001809 Manufacture date October 2018 Expiry date October 2020a Date of receipts at the test facility 20 December 2018 and 14 January 2019 Registration number at the test facility Ch.7101 Storage at the test facility (before the beginning of the experimental phase) At room temperature (10–30 °C) in an appropriate storage area, well ventilated and not exposed to direct sunlight. The temperature was monitored continuously. The temperature was monitored continuously. a The test item is expired, but this has no impact on the result, as it is used only for recovery. Test item 5 Name Propoxur 50% WP Other name/code – Type of formulation Wettable powder Active substance(s) content - nominal concentration - analysed concentration Propoxur 500 g/kg Propoxur 504 g/kg Supplier Tagros Chemicals India Pvt Ltd, Chennai, India Container(s) Two non-commercial aluminized bags each containing about 500 g of product Manufacturer Tagros Chemicals India Pvt Ltd, Chennai, India Batch number 508E2019 Manufacture date May 2019 Expiry date May 2021 Date of receipt at the test facility 20 May 2019 Registration number at the test facility Ch.7213 Storage at the test facility (before the beginning of the experimental phase) At room temperature (10–30°C) in an appropriate storage area, well ventilated and not exposed to direct sunlight. The temperature was monitored continuously. 37 Test item 6 Name alpha-cypermethrin 10% SC Other name/code – Type of formulation Suspension concentrate Active substance(s) content - nominal concentration - analysed concentration alpha-cypermethrin 100 g/L alpha-cypermethrin 103 g/L Supplier Tagros Chemicals India Pvt Ltd, Chennai, India Container(s) One non-commercial bottle containing about 200 mL of product Manufacturer Tagros Chemicals India Pvt Ltd, Chennai, India Batch number AASC18T023 Manufacture date March 2019 Expiry date February 2021 Date of receipt at the test facility 18 April 2019 Registration number at the test facility Ch.7187/9 Storage at the test facility (before the beginning of the experimental phase) At room temperature (10–30 °C) in an appropriate storage area, well ventilated and not exposed to direct sunlight. The temperature was monitored continuously. Test item 7 Name Cyfluthrin WP10 Other name/code – Type of formulation Wettable powder Active substance(s) content - nominal concentration - analysed concentration Cyfluthrin 100 g/kg Cyfluthrin 104 g/kg Supplier Bayer AG Crop Science, Leverkusen, Germany Container(s) Two non-commercial high-density polyethylene bottles, the first containing about 600 g of product and the second 400 g of product Manufacturer Bayer AG Crop Science, Leverkusen, Germany Batch number EQ15001448 Manufacture date N/A Expiry date N/Aa Date of receipt at the test facility 20 May 2019 Registration number at the test facility Ch.7212 Storage at the test facility (before the beginning of the experimental phase) At room temperature (10–30 °C) in an appropriate storage area, well ventilated and not exposed to direct sunlight. The temperature was monitored continuously. a The test item has no expiry date, but this has no impact on the result, as it is used only for recovery. 38 Test item 8 Name K-Othrine WG250 Other name/code – Type of formulation Water-dispersible granules Active substance(s) content - nominal concentration - analysed concentration Deltamethrin 250 g/kg Deltamethrin 252 g/kg Supplier Partnership for Supply Chain Management Container(s) 16 commercial paper bags each containing about 25 g of product Manufacturer Bayer SAS, Environmental Science, Lyon, France Batch number IL11001031 Manufacture date November 2018 Expiry date November 2021a Date of receipt at the test facility 15 February 2019 Registration number at the test facility Ch.7146 Storage at the test facility (before the beginning of the experimental phase) At room temperature (10–30 °C) in an appropriate storage area, well ventilated and not exposed to direct sunlight. The temperature was monitored continuously. a The test item has no expiry date, but this has no impact on the result, as it is used only for recovery. Test item 9 Name Vectron 20WP Other name/code – Type of formulation Wettable powder Active substance(s) content - nominal concentration Etofenprox 200 g/kg Supplier Mistui Chemicals Agro Inc., Tokyo, Japan Container(s) Five non-commercial aluminized bags each containing about 100 g of product Manufacturer Mistui Chemicals Agro Inc., Tokyo, Japan Batch number 78SE0008 Manufacture date N/A Expiry date N/Aa Date of receipt at the test facility 6 June 2019 Registration number at the test facility Ch.7215/4 Storage at the test facility (before the beginning of the experimental phase) At room temperature (10–30 °C) in an appropriate storage area, well ventilated and not exposed to direct sunlight. The temperature was monitored continuously. a The test item has no expiry date, but this has no impact on the result, as it is used only for recovery. 39 Test item 10 Name Icon 10 CS Other name/code – Type of formulation Capsule suspension Active substance(s) content - nominal concentration - analysed concentration lambda-cyhalothrin 100 g/L lambda-cyhalothrin 100 g/L Supplier Partnership for Supply Chain Management Container(s) 25 commercial aluminized sachets with cardboard packing, each containing 62.5 mL of product Manufacturer Syngenta Crop Protection AG, Basel, Switzerland Batch number SBR8B1680 Manufacture date February 2018 Expiry date February 2020a Date of receipt at the test facility 11 June 2018 Registration number at the test facility Ch.7017 Storage at the test facility (before the beginning of the experimental phase) At room temperature (10–30 °C) in an appropriate storage area, well ventilated and not exposed to direct sunlight. The temperature was monitored continuously. a The test item has no expiry date, but this has no impact on the result, as it is used only for recovery. Test item 11 Name Permas 100 EC Other name/code – Type of formulation Emulsifiable concentrate Active substance(s) content - nominal concentration - analysed concentration Permethrin (25–75) 100 g/L Permethrin (25–75) 95 g/L Supplier Denka International B.V., Barneveld, Netherlands Container(s) 1 polyethylene bottle containing about 250 mL of product Manufacturer Denka International B.V., Barneveld, Netherlands Batch number* 14122015 Manufacture date 14 December 2015 Expiry date 14 December 2017a Date of receipt at the test facility 17 December 2015 Registration number at the test facility Ch.6429 Storage at the test facility (before the beginning of the experimental phase) At room temperature (10–30 °C) in an appropriate storage area, well ventilated and not exposed to direct sunlight. The temperature was monitored continuously. a The test item has no expiry date, but this has no impact on the result, as it is used only for recovery. 40 Test item 12 Name Piperonyl butoxide TC Other name/code – Type of formulation Technical material Active substance(s) content - nominal concentration - analysed concentration PBO, minimum 920 g/kg PBO, minimum 941 g/kg Supplier Endura, Italy Container(s) 200-g glass bottle Manufacturer Endura, Italy Batch number R1912002 Manufacture date 4 December 2019 Expiry date 3 December 2022 Date of receipt at the test facility 27 January 2020 Registration number at the test facility SR/7127 Storage at the test facility (before the beginning of the experimental phase) At room temperature (10–30 °C) in an appropriate storage area, well ventilated and not exposed to direct sunlight. The temperature was monitored continuously. 41 Analytical conditions 4 42 4.1 Outline of method The insecticides and PBO are extracted from filter papers by ultrasonication for 30 min with acetonitrile in the presence of dicyclohexyl phthalate as the internal standard, cleaned by dispersive solid-phase extraction and determined by capillary GC-MS or capillary GC-FID. 4.2 Apparatus y analytical balance (to the nearest 0.1 mg) y ultrasonic bath y 50-mL Falcon centrifuge flask y centrifuge y oscillating shaker or vortex mixer y laboratory glassware y GC-MS with split injection y GC-FID and pulsed split injection (for bendiocarb and propoxur) y capillary column, fused silica, 30 m x 0.25 mm (internal diameter), 0.25 µm film thickness, coated with methyl siloxane (HP-1MS) (or equivalent material with the same selectivity), used for GC-MS and GC-FID. 4.3 Reagents and solvents y pesticide, certified analytical standard of known purity y deionized water y dicyclohexyl phthalate, internal standard, of known purity y acetonitrile, analytical reagent grade y sodium chloride, analytical reagent grade y dispersive solid-phase extraction 15-mL tube, fatty samples, European standard EN 15662, containing 150 mg primary secondary amine sorbent, 150 mg C18 and 900 mg magnesium sulfate (Agilent Technologies) y citric acid, 10% solution in acetone, analytical reagent grade (for alpha-cypermethrin, cyfluthrin, lambda-cyhalothrin and deltamethrin) y nylon filter with maximum 0.45-µm pore size. 43 4.4 Analytical procedure 4.4.1 Internal standard solutions Weigh, to the nearest 0.1 mg, about 100 mg dicyclohexyl phthalate into a 200-mL volumetric flask. Add acetonitrile (150 mL), and place the flask in an ultrasonic bath until complete dissolution. Allow the solution to cool to ambient temperature, and fill to the mark (at 20 °C) with acetonitrile. Mix thoroughly (= solution internal standard [IS]-1 at 500 µg/mL). Transfer 10 mL into a 100-mL volumetric flask, and fill to the mark (at 20 °C) with acetonitrile. Mix thoroughly (= solution IS-2 at 50 µg/mL). 4.4.2 Pesticide calibration solutions Calibration working solutions can be prepared individually or in a mixture. p,p΄ -DDT Weigh (to the nearest 0.1 mg) about 250 mg of p,p΄ -DDT analytical standard into a 10-mL volumetric flask. Add acetonitrile (8 mL), and place the flask in an ultrasonic bath until complete dissolution. Allow the solution to cool to room temperature, and fill to the mark with acetonitrile. Mix thoroughly (= solution CA at 25 000 µg/mL). Weigh (to the nearest 0.1 mg) about 75 mg of p,p΄ -DDT analytical standard into a 10-mL volumetric flask. Add acetonitrile (8 mL), and place the flask in an ultrasonic bath until complete dissolution. Allow the solution to cool to room temperature, and fill to the mark with acetonitrile. Mix thoroughly (= solution CB at 7500 µg/mL). Prepare the following p,p΄ -DDT working calibration solutions into 25-mL and 100-mL volumetric flasks (the internal standard solution shall be added at exactly 20 °C) (= calibration solutions C1, C2, C3, C4 and C5). Code IS-1 CA CB Acetonitrile Total volume p,p΄-DDT (µg/mL) C1 4 mL 0.2 mL – Up to volume 100 mL 50 C2 1 mL – 0.8 mL Up to volume 25 mL 240 C3 1 mL 0.6 mL – Up to volume 25 mL 600 C4 1 mL – 4.0 mL Up to volume 25 mL 1200 C5 1 mL 2.0 mL – Up to volume 25 mL 2000 Transfer an aliquot of each flask into separate GC vials. Malathion Weigh (to the nearest 0.1 mg) about 280 mg of malathion analytical standard into a 10-mL volumetric flask. Add acetonitrile (8 mL), and place the flask in an ultrasonic bath until complete dissolution. Allow the solution to cool to room temperature, and fill to the mark with acetonitrile. Mix thoroughly (= solution CA at 28 000 µg/mL). 44 Weigh (to the nearest 0.1 mg) about 70 mg of malathion analytical standard into a 10-mL volumetric flask. Add acetonitrile (8 mL), and place the flask in an ultrasonic bath until complete dissolution. Allow the solution to cool to room temperature, and fill to the mark with acetonitrile. Mix thoroughly (= solution CB at 7000 µg/mL). Prepare the following malathion working calibration solutions into 25-mL and 100-mL volumetric flasks (the internal standard solution shall be added at precisely 20 °C) (= calibration solutions C1, C2, C3, C4, and C5). Code IS-1 CA CB Acetonitrile Total volume Malathion (µg/mL) C1 4 mL 0.2 mL – Up to volume 100 mL 56 C2 1 mL – 0.8 mL Up to volume 25 mL 224 C3 1 mL 0.6 mL – Up to volume 25 mL 672 C4 1 mL – 4.0 mL Up to volume 25 mL 1120 C5 1 mL 2.0 mL – Up to volume 25 mL 2240 Transfer an aliquot of each flask into separate GC vials. Pirimiphos-methyl Weigh (to the nearest 0.1 mg) about 40 mg of pirimiphos-methyl analytical standard into a 25-mL volumetric flask. Add acetonitrile (20 mL), and place the flask in an ultrasonic bath until complete dissolution. Allow the solution to cool to room temperature, and fill to the mark with acetonitrile. Mix thoroughly (= solution CA at 1600 µg/mL). Weigh (to the nearest 0.1 mg) about 30 mg of pirimiphos-methyl analytical standard into a 25-mL volumetric flask. Add acetonitrile (20 mL), and place the flask in an ultrasonic bath until complete dissolution. Allow the solution to cool to room temperature, and fill to the mark with acetonitrile. Mix thoroughly (= solution CB at 1200 µg/mL). Prepare the following pirimiphos-methyl working calibration solutions into 25-mL and 100-mL volumetric flasks (the internal standard solution shall be added at precisely 20 °C) (= calibration solutions C1, C2, C3, C4, C5, and C6). Code IS-1 CA CB Acetonitrile Total volume Pirimiphos- methyl (µg/mL) C1 4 mL 0.2 mL – Up to volume 100 mL 3.2 C2 1 mL – 0.5 mL Up to volume 25 mL 24 C3 1 mL 0.6 mL – Up to volume 25 mL 38 C4 1 mL – 1.4 mL Up to volume 25 mL 67 C5 1 mL – 2.0 mL Up to volume 25 mL 96 C6 1 mL 2.0 mL – Up to volume 25 mL 128 Transfer an aliquot of each flask into separate GC vials. 45 Bendiocarb Weigh (to the nearest 0.1 mg) about 30 mg of bendiocarb analytical standard into a 50-mL volumetric flask. Add acetonitrile (45 mL), and place the flask in an ultrasonic bath until complete dissolution. Allow the solution to cool to room temperature, and fill to the mark with acetonitrile. Mix thoroughly (= solution CA at 600 µg/mL). Weigh (to the nearest 0.1 mg) about 30 mg of bendiocarb analytical standard into a 50-mL volumetric flask. Add acetonitrile (45 mL), and place the flask in an ultrasonic bath until complete dissolution. Allow the solution to cool to room temperature, and fill to the mark with acetonitrile. Mix thoroughly (= solution CB at 600 µg/mL). Prepare the following bendiocarb working calibration solutions into 25-mL and 100-mL volumetric flasks (the internal standard solution shall be added at precisely 20 °C) (= calibration solutions C1, C2, C3, C4, C5, and C6). Code IS-1 CA CB Acetonitrile Total volume Bendiocarb (µg/mL) C1 4 mL 0.2 mL – Up to volume 100 mL 1.2 C2 1 mL – 0.3 mL Up to volume 25 mL 7.2 C3 1 mL 0.6 mL – Up to volume 25 mL 14 C4 1 mL – 1.0 mL Up to volume 25 mL 24 C5 1 mL – 1.5 mL Up to volume 25 mL 36 C6 1 mL 2.0 mL – Up to volume 25 mL 48 Transfer an aliquot of each flask into separate GC vials. Propoxur Weigh (to the nearest 0.1 mg) about 30 mg of propoxur analytical standard into a 50-mL volumetric flask. Add acetonitrile (45 mL), and place the flask in an ultrasonic bath until complete dissolution. Allow the solution to cool to room temperature, and fill to the mark with acetonitrile. Mix thoroughly (= solution CA at 600 µg/mL). Weigh (to the nearest 0.1 mg) about 25 mg of propoxur analytical standard into a 50-mL volumetric flask. Add acetonitrile (45 mL), and place the flask in an ultrasonic bath until complete dissolution. Allow the solution to cool to room temperature, and fill to the mark with acetonitrile. Mix thoroughly (= solution CB at 500 µg/mL). Prepare the following propoxur working calibration solutions into 25-mL and 100-mL volumetric flasks (the internal standard solution shall be added at precisely 20 °C) (= calibration solutions C1, C2, C3, C4, C5, and C6). 46 Code IS-1 CA CB Acetonitrile Total volume Propoxur (µg/mL) C1 4 mL 0.2 mL – Up to volume 100 mL 1.2 C2 1 mL – 0.4 mL Up to volume 25 mL 8 C3 1 mL 0.6 mL – Up to volume 25 mL 14 C4 1 mL – 1.2 mL Up to volume 25 mL 24 C5 1 mL – 1.8 mL Up to volume 25 ml 36 C6 1 mL 2.0 mL – Up to volume 25 mL 48 Transfer an aliquot of each flask into separate GC vials. Alpha-cypermethrin Weigh (to the nearest 0.1 mg) about 30 mg of alpha-cypermethrin analytical standard into a 100-mL volumetric flask. Add acetonitrile (90 mL), and place the flask in an ultrasonic bath until complete dissolution. Allow the solution to cool to room temperature, and fill to the mark with acetonitrile. Mix thoroughly (= solution CA at 300 µg/mL). Weigh (to the nearest 0.1 mg) about 30 mg of alpha-cypermethrin analytical standard into a 100-mL volumetric flask. Add acetonitrile (90 mL), and place the flask in an ultrasonic bath until complete dissolution. Allow the solution to cool to room temperature, and fill to the mark with acetonitrile. Mix thoroughly (= solution CB at 300 µg/mL). Prepare the following alpha-cypermethrin working calibration solutions into 25-mL and 100-mL volumetric flasks (the internal standard solution shall be added at precisely 20 °C) (= calibration solutions C1, C2, C3, C4, C5, and C6). Code IS-2 CA CB Acetonitrile Total volume alpha-cypermethrin (µg/mL) C1 4 mL 0.2 mL – Up to volume 100 mL 0.6 C2 1 mL – 0.3 mL Up to volume 25 mL 3.6 C3 1 mL 0.6 mL – Up to volume 25 mL 7.2 C4 1 mL – 1.0 mL Up to volume 25 mL 12 C5 1 mL – 1.5 mL Up to volume 25 mL 18 C6 1 mL 2.0 mL – Up to volume 25 mL 24 Transfer an aliquot of each flask into separate GC vials. Cyfluthrin Weigh (to the nearest 0.1 mg) about 40 mg of cyfluthrin analytical standard into a 50-mL volumetric flask. Add acetonitrile (45 mL), and place the flask in an ultrasonic bath until complete dissolution. Allow the solution to cool to room temperature, and fill to the mark with acetonitrile. Mix thoroughly (= solution CA at 800 µg/mL). 47 Weigh (to the nearest 0.1 mg) about 30 mg of cyfluthrin analytical standard into a 50-mL volumetric flask. Add acetonitrile (45 mL), and place the flask in an ultrasonic bath until complete dissolution. Allow the solution to cool to room temperature, and fill to the mark with acetonitrile. Mix thoroughly (= solution CB at 600 µg/mL). Prepare the following cyfluthrin working calibration solutions into 25-mL and 100-mL volumetric flasks (the internal standard solution shall be added at precisely 20 °C) (= calibration solutions C1, C2, C3, C4, C5, and C6). Code IS-2 CA CB Acetonitrile Total volume Cyfluthrin (µg/mL) C1 4 mL 0.2 mL – Up to volume 100 mL 1.6 C2 1 mL – 0.3 mL Up to volume 25 mL 7.2 C3 1 mL 0.6 mL – Up to volume 25 mL 19 C4 1 mL – 1.5 mL Up to volume 25 mL 36 C5 1 mL – 2.0 mL Up to volume 25 mL 48 C6 1 mL 2.0 mL – Up to volume 25 mL 64 Transfer an aliquot of each flask into separate GC vials. Deltamethrin Weigh (to the nearest 0.1 mg) about 30 mg of deltamethrin analytical standard into a 100-mL volumetric flask. Add acetonitrile (90 mL), and place the flask in an ultrasonic bath until complete dissolution. Allow the solution to cool to room temperature, and fill to the mark with acetonitrile. Mix thoroughly (= solution CA at 300 µg/mL). Weigh (to the nearest 0.1 mg) about 30 mg of deltamethrin analytical standard into a 100-mL volumetric flask. Add acetonitrile (90 mL), and place the flask in an ultrasonic bath until complete dissolution. Allow the solution to cool to room temperature, and fill to the mark with acetonitrile. Mix thoroughly (= solution CB at 300 µg/mL). Prepare the following deltamethrin working calibration solutions into 25-mL and 100-mL volumetric flasks (the internal standard solution shall be added at precisely 20 °C) (= calibration solutions C1, C2, C3, C4, C5, and C6). Code IS-2 CA CB Acetonitrile Total volume Deltamethrin (µg/mL) C1 4 mL 0.2 mL – Up to volume 100 mL 0.6 C2 1 mL – 0.3 mL Up to volume 25 mL 3.6 C3 1 mL 0.6 mL – Up to volume 25 mL 7.2 C4 1 mL – 1.0 mL Up to volume 25 mL 12 C5 1 mL – 1.5 mL Up to volume 25 mL 18 C6 1 mL 2.0 mL – Up to volume 25 mL 24 Transfer an aliquot of each flask into separate GC vials. 48 Etofenprox Weigh (to the nearest 0.1 mg) about 65 mg of etofenprox analytical standard into a 25-mL volumetric flask. Add acetonitrile (20 mL), and place the flask in an ultrasonic bath until complete dissolution. Allow the solution to cool to room temperature, and fill to the mark with acetonitrile. Mix thoroughly (= solution CA at 2600 µg/mL). Weigh (to the nearest 0.1 mg) about 35 mg of etofenprox analytical standard into a 25-mL volumetric flask. Add acetonitrile (20 mL), and place the flask in an ultrasonic bath until complete dissolution. Allow the solution to cool to room temperature, and fill to the mark with acetonitrile. Mix thoroughly (= solution CB at 1400 µg/mL). Prepare the following etofenprox working calibration solutions into 25-mL and 100-mL volumetric flasks (the internal standard solution shall be added at precisely 20 °C) (= calibration solutions C1, C2, C3, C4, C5, and C6). Code IS-1 CA CB Acetonitrile Total volume Etofenprox (µg/mL) C1 4 mL 0.2 mL – Up to volume 100 mL 5.2 C2 1 mL – 0.6 mL Up to volume 25 mL 34 C3 1 mL 0.6 mL – Up to volume 25 mL 62 C4 1 mL – 1.8 mL Up to volume 25 mL 101 C5 1 mL – 2.5 mL Up to volume 25 mL 140 C6 1 mL 2.0 mL – Up to volume 25 mL 208 Transfer an aliquot of each flask into separate GC vials. Lambda-cyhalothrin Weigh (to the nearest 0.1 mg) about 30 mg of lambda-cyhalothrin analytical standard into a 100-mL volumetric flask. Add acetonitrile (90 mL), and place the flask in an ultrasonic bath until complete dissolution. Allow the solution to cool to room temperature, and fill to the mark with acetonitrile. Mix thoroughly (= solution CA at 300 µg/mL). Weigh (to the nearest 0.1 mg) about 30 mg of lambda-cyhalothrin analytical standard into a 100-mL volumetric flask. Add acetonitrile (90 mL), and place the flask in an ultrasonic bath until complete dissolution. Allow the solution to cool to room temperature, and fill to the mark with acetonitrile. Mix thoroughly (= solution CB at 300 µg/mL). Prepare the following lambda-cyhalothrin working calibration solutions into 25-mL and 100-mL volumetric flasks (the internal standard solution shall be added at precisely 20 °C) (= calibration solutions C1, C2, C3, C4, C5, and C6). Code IS-2 CA CB Acetonitrile Total volume lambda-cyhalothrin (µg/mL) C1 4 mL 0.2 mL – Up to volume 100 mL 0.6 C2 1 mL – 0.3 mL Up to volume 25 mL 3.6 C3 1 mL 0.6 mL – Up to volume 25 mL 7.2 C4 1 mL – 1.0 mL Up to volume 25 mL 12 C5 1 mL – 1.5 mL Up to volume 25 mL 18 C6 1 mL 2.0 mL – Up to volume 25 mL 24 Transfer an aliquot of each flask into separate GC vials. 49 Permethrin Weigh (to the nearest 0.1 mg) about 100 mg of permethrin analytical standard into a 25-mL volumetric flask. Add acetonitrile (20 mL), and place the flask in an ultrasonic bath until complete dissolution. Allow the solution to cool to room temperature, and fill to the mark with acetonitrile. Mix thoroughly (= solution CA at 4000 µg/mL). Weigh (to the nearest 0.1 mg) about 40 mg of permethrin analytical standard into a 10-mL volumetric flask. Add acetonitrile (8 mL), and place the flask in an ultrasonic bath until complete dissolution. Allow the solution to cool to room temperature, and fill to the mark with acetonitrile. Mix thoroughly (= solution CB at 4000 µg/mL). Prepare the following permethrin working calibration solutions into 25-mL and 100-mL volumetric flasks (the internal standard solution shall be added at precisely 20 °C) (= calibration solutions C1, C2, C3, C4, C5, and C6). Code IS-1 CA CB Acetonitrile Total volume Permethrin (µg/mL) C1 4 mL 0.2 mL – Up to volume 100 mL 8 C2 1 mL – 0.3 mL Up to volume 25 mL 48 C3 1 mL 0.6 mL – Up to volume 25 mL 96 C4 1 mL – 1.0 mL Up to volume 25 mL 160 C5 1 mL – 1.5 mL Up to volume 25 mL 240 C6 1 mL 2.0 mL – Up to volume 25 mL 320 Transfer an aliquot of each flask into separate GC vials. Piperonyl butoxide Weigh (to the nearest 0.1 mg) about 200 mg of PBO analytical standard into a 10-mL volumetric flask. Add acetonitrile (8 mL), and place the flask in an ultrasonic bath until complete dissolution. Allow the solution to cool to room temperature, and fill to the mark with acetonitrile. Mix thoroughly (= solution CA at 20 000 µg/mL). Weigh (to the nearest 0.1 mg) about 50 mg of PBO analytical standard into a 10-mL volumetric flask. Add acetonitrile (8 mL), and place the flask in an ultrasonic bath until complete dissolution. Allow the solution to cool to room temperature, and fill to the mark with acetonitrile. Mix thoroughly (= solution CB at 5000 µg/mL). Prepare the following PBO working calibration solutions into 25-mL and 100-mL volumetric flasks (the internal standard solution shall be added at precisely 20 °C) (= calibration solutions C1, C2, C3, C4, and C5). Code IS-1 CA CB Acetonitrile Total volume Piperonyl butoxide (µg/mL) C1 4 mL 0.2 mL – Up to volume 100 mL 40 C2 1 mL – 0.8 mL Up to volume 25 mL 160 C3 1 mL 0.6 mL – Up to volume 25 mL 480 C4 1 mL – 4 mL Up to volume 25 mL 800 C5 1 mL 2.0 mL – Up to volume 25 mL 1600 Transfer an aliquot of each flask into separate GC vials. 50 4.4.3 Preparation of samples As shown below, cut a filter paper measuring 12 × 15 cm into four quarters according to the diagonals. Cut two opposite quarters into pieces of approximately 1.5 cm × 1.5 cm. Pool the pieces in order to obtain a representative subsample of filter paper, and place in a 50-mL Falcon centrifuge flask. Replace the remaining filter paper immediately into the deep-freeze at < –18 °C. For recovery, spike the untreated sample with the appropriate formulation solution. 4.4.4 Extraction of pesticides from filter papers y Add 5 mL of deionized water. Put the flask into an ultrasonic bath at ambient temperature for 10 min, and let stand for about 10 min. y Add precisely 1 mL of the internal standard (IS) solution at 20 °C (IS-1 or IS-2, depending on the pesticide to be analysed and in the same way as the calibration solutions) and 24 mL of acetonitrile. Put the flask in an ultrasonic bath at ambient temperature for about 30 min (≥ 20 min). 4.4.5 Freezing out y Add 1 g of sodium chloride, and shake vigorously by hand. Put the flask into the deep-freeze at < –18 °C for a minimum of 1 h in order to freeze and separate the carrier oils used to treat the filter papers. y Allow the solution to warm to ambient temperature. y Centrifuge at 4500 rpm for 5 min. 4.4.6 Clean up by dispersive solid-phase extraction y Transfer about 6 mL of acetonitrile (upper layer) into a 15-mL dispersive solid-phase extraction tube containing 150 mg primary or secondary amine, 150 mg C18 and 900 mg magnesium sulfate. y Close the tube, and shake vigorously in an oscillating shaker or vortex mixer for about 1 min. y Centrifuge at 4500 rpm for 5 min. 4.4.7 Chromatographic determination by GC-MS or GC-FID y Filter the supernatant through a 0.45 µm nylon membrane filter into an injection vial. y Add 50 µL 10% citric acid solution in acetone into a GC injection vial (only for filter papers treated with alpha-cypermethrin, cyfluthrin, lambda-cyhalothrin or deltamethrin). y Analyse the final extract by capillary GC-MS or capillary GC-FID for determination of the pesticides with internal standard calibration and under the following chromatographic conditions. 51 4.4.8 Chromatographic conditions Apparatus Gas chromatograph Gas chromatograph Gas chromatograph Injector Automatic sampler (split mode) Automatic sampler (pulsed split mode) Detector Inert mass spectrometer with selective ion monitoring Flame ionization Software/integrator Software for integration Software for integration Chromatographic parameters Column Stationary phase (type) 100% dimethyl polysiloxane 100% dimethyl polysiloxane Length, internal diameter (ID), film thickness Length, 30 m; ID, 0.25 mm; film, 0.25 µm Length, 30 m; ID, 0.25 mm; film, 0.25 µm Injection system  Injection system Split injection Pulsed split injection Split ratio 50:1 or 20:1 5:1 Injection volume 1 µL 1 µL Temperature Injection port 280 °C 200 °C Oven 110 °C for 0.5 min; 25 °C/min to 200 °C; 10 °C/min to 250 °C; 250 °C for 8 min; 10 °C/min to 280 °C; 280 °C for 3 min 110 °C for 0.5 min; 25 °C/min to 200 °C; 10 °C/min to 250 °C; 250 °C for 8 min; 10 °C/min to 280; 280 °C for 3 min (option 1) 110 °C for 0.5 min; 25 °C/min to 200 °C; 200 °C for 8 min; 10 °C/min to 280 °C; 280 °C for 3 min (option 2)a Capillary direct interface 280 °C – Ionization source 230 °C – Quadrupole filter 150 °C – Gas  Carrier gas (type and flow rate) 1 mL/min (helium) 1 mL/min (helium) Retention times  p,p΄–DDT ± 9.900 – Malathion ± 7.000 – Pirimiphos-methyl ± 6.850 – Bendiocarb – ± 5.150 (option 1) – 6.900 (option 2) 52 Propoxur – ± 4.800 Alpha-cypermethrin ± 14.900 – Cyfluthrin ± 14.000 (average for the four isomers) – Deltamethrin ± 18.950 – Etofenprox ± 16.650 – Lambda-cyhalothrin ± 11.500 – Permethrin ± 13.600 (cis) – ± 13.850 (trans) – Piperonyl butoxide ± 10.200 – Dicyclohexyl phthalate ± 10.600 – 11.350 ± 11.000 (option 1) – ± 19.750 (option 2) Ions  p,p΄–DDT 165 – 235 – 237 – Malathion 152 – 158 – 173 – Pirimiphos-methyl 276 – 290 – 305 – Bendiocarb 126 – 151– 166 – Propoxur 110 – 111 – 152 – Alpha-cypermethrin 163 – 165 –180 – Cyfluthrin 163 – 165 – 206 – Deltamethrin 172 –174 – 181 – Etofenprox 163 – 164 – 183 – Lambda-cyhalothrin 141 – 181 – 208 – Permethrin 165 – 165 – 183 – Piperonyl butoxide 176 – 290 – 305 – Dicyclohexyl phthalate 149 – 150 – 167 – a Option 2 was used by mistake but had no impact on the results. Option 1 is recommended. The chromatographic conditions may be adapted to ensure good chromatographic separation. Representative chromatograms are shown in the Annex. Addition of 5 mL deionized water before extraction with acetonitrile, freezing out and clean-up by dispersive solid-phase extraction, as well as analysis by GC-MS, are necessary only for susceptibility filter papers containing carrier oils to avoid interference, particularly with silicone oils. These steps are also unnecessary for the analysis of filter papers used in the laboratory, experimental hut and large-scale field studies to evaluate the quality of IRS (not containing carrier oils), except for the addition of 5 mL of deionized water for pirimiphos-methyl. The analysis can be performed by GC-FID. 53 For bendiocarb and propoxur, GC-FID is recommended in all cases (with and without carrier oil) because it is more sensitive than GC-MS. The carrier oil (olive oil) used with these insecticides does not result in interference. 4.4.9 Calculation and expression of results The amount of pesticide in the sample solutions is determined qualitatively by comparing the retention time of the pesticide peak in the sample solutions with those of duplicate injections of the standard solutions. The concentration of pesticide in the sample solutions is determined quantitatively by comparing the detector response factor (peak area for pesticide divided by the peak area for dicyclohexyl phthalate) in the sample solutions with that of the standard solutions from a calibration curve calculated from the standard solutions bracketing the sample solutions. For sample solutions containing permethrin or cyfluthrin, the peak area is the sum of the peak areas of the isomers. The calibration curve for a pesticide is obtained by the internal standard calibration method from injection of pesticide standard solutions containing dicyclohexyl phthalate and plotting the detector response factor versus the pesticide concentration (in µg/mL). The curve is calculated by least squares linear regression. The amount of pesticide in filter paper is expressed as mg pesticide per m² filter paper, with account for the dilution factor and sample area. Pesticide (mg/m² filter paper) = Cc × D × 10 / W where Cc =concentration of pesticide in the sample solution in µg/mL (from the calibration curve), D = dilution factor in the sample solution (= 25) and W = area sample in cm².

55 Results of analysis 5 56 The analytical method by GC-FID was validated for bendiocarb and propoxur The analytical method by GC-MS was validated for all the other insecticides and PBO. 5.1 Specificity and non-analyte interference Analysis of blank solvent (solvent of extraction without IS or sample), blank method (solvent of extraction with IS but without sample) and blank sample (solvent of extraction with untreated sample and IS) and treated sample without IS and comparison with analysis of calibration solutions and treated filter paper samples showed that no interference is likely to affect the chromatographic peaks of the pesticides and the internal standard. The retention times of the pesticides did not deviate significantly from those of the sample solutions in comparison with the calibration solutions. The method is capable of determining each pesticide in the presence of each other and each isomer from each other, when applicable. Use of MS detection with selective ion monitoring allows separation of pesticides from interfering carrier oils used to treat susceptibility filter papers. Representative chromatograms for each of the pesticides are shown in the Annex. 5.2 Linearity of the detector response p,p΄-DDT Linearity was demonstrated over concentrations ranging from 50 µg/mL to 2000 µg/mL expressed as p,p΄ -DDT by measuring the detector response (peak area for p,p΄ -DDT divided by the peak area for dicyclohexyl phthalate) versus the p,p΄ -DDT concentration for a series of five calibration solutions of known concentration (50, 240, 600, 1200 and 2000 µg/mL). Linear regression (least squares method) was used to calculate the mean of a minimum of two injections of each calibration solution. 57 Calibration curve of p,p΄ -DDT by GC-MS (244–2006 µg/mL) – day 1 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Calibration curve of p,p΄ -DDT by GC-MS (50–602 µg/mL) – day 1 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. 58 Calibration curve of p,p΄ -DDT by GC-MS (244–2006 µg/mL) – day 2 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Calibration curve of p,p΄ -DDT by GC-MS (50–602 µg/mL) – day 2 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Malathion Linearity was demonstrated over concentrations ranging from 56 µg/mL to 2240 µg/mL expressed as malathion by measuring the detector response (peak area for malathion divided by the peak area for 59 dicyclohexyl phthalate) versus the malathion concentration for a series of five calibration solutions of known concentration (56, 224, 672, 1120 and 2240 µg/mL). Linear regression (least squares method) was used to calculate the mean of a minimum of two injections of each calibration solution. Calibration curve of malathion by GC-MS (56–2222 µg/mL) – day 1 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Calibration curve of malathion by GC-MS (56–1136 µg/mL) – day 1 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. 60 Calibration curve of malathion by GC-MS (56–2222 µg/mL) – day 2 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Calibration curve of malathion by GC-MS (56–667 µg/mL) – day 2 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Pirimiphos-methyl Linearity was demonstrated over concentrations ranging from 3 µg/mL to 128 µg/mL expressed as pirimiphos-methyl by measuring the detector response (peak area for pirimiphos-methyl divided by the peak 61 area for dicyclohexyl phthalate) versus the pirimiphos-methyl concentration for a series of six calibration solutions of known concentration (3, 24, 38, 67, 96 and 128 µg/mL). Linear regression (least squares method) was used to calculate the mean of a minimum of two injections of each calibration solution. Calibration curve of pirimiphos-methyl by GC-MS (26–130 µg/mL) – day 1 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Calibration curve of pirimiphos-methyl by GC-MS (3–39 µg/mL) – day 1 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. 62 Calibration curve of pirimiphos-methyl by GC-MS (26–130 µg/mL) – day 2 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Calibration curve of pirimiphos-methyl by GC-MS (3–39 µg/mL) – day 2 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Bendiocarb Linearity was demonstrated over concentrations ranging from 1.2 µg/mL to 48 µg/mL expressed as bendiocarb by measuring the detector response (peak area for bendiocarb divided by the peak area for 63 dicyclohexyl phthalate) versus the bendiocarb concentration for a series of six calibration solutions of known concentration (1.2, 7, 14, 24, 36 and 48 µg/mL). Linear regression (least squares method) was used to calculate the mean of a minimum of two injections of each calibration solution. Calibration curve of bendiocarb by GC-FID (1.1–46 µg/mL) – day 1 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Calibration curve of bendiocarb by GC-FID (1.1–14 µg/mL) – day 1 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. 64 Calibration curve of bendiocarb by GC-FID (1.1–46 µg/mL) – day 2 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Calibration curve of bendiocarb by GC-FID (1.1–14 µg/mL) – day 2 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. 65 Propoxur Linearity was demonstrated over concentrations ranging from 1.2 µg/mL to 48 µg/mL expressed as propoxur by measuring the detector response (peak area for propoxur divided by the peak area for dicyclohexyl phthalate) versus the propoxur concentration for a series of six calibration solutions of known concentration (1.2, 8, 14, 24, 36 and 48 µg/mL). Linear regression (least squares method) was used to calculate the mean of a minimum of two injections of each calibration solution. Calibration curve of propoxur by GC-FID (1.2–47 µg/mL) – day 1 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Calibration curve of propoxur by GC-FID (1.2–14 µg/mL) – day 1 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. 66 Calibration curve of propoxur by GC-FID (1.2–47 µg/mL) – day 2 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Calibration curve of propoxur by GC-FID (1.2–14 µg/mL) – day 2 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Alpha-cypermethrin Linearity was demonstrated over concentrations ranging from 0.6 µg/mL to 18 µg/mL expressed as alpha-cypermethrin by measuring the detector response (peak area for alpha-cypermethrin divided by 67 the peak area for dicyclohexyl phthalate) versus the alpha-cypermethrin concentration for a series of five calibration solutions of known concentration (0.6, 4, 7, 12 and 18 µg/mL). Linear regression (least squares method) was used to calculate the mean of a minimum of two injections of each calibration solution. Calibration curve of alpha-cypermethrin by GC-MS (0.6–18 µg/mL) – day 1 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Calibration curve of alpha-cypermethrin by GC-MS (0.6–12 µg/mL) – day 1 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. 68 Calibration curve of alpha-cypermethrin by GC-MS (0.6–17 µg/mL) – day 2 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Calibration curve of alpha-cypermethrin by GC-MS (0.6–12 µg/mL) – day 2 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Cyfluthrin Linearity was demonstrated over concentrations ranging from 2 µg/mL to 64 µg/mL expressed as cyfluthrin by measuring the detector response (peak area for cyfluthrin divided by the peak area for 69 dicyclohexyl phthalate) versus the cyfluthrin concentration for a series of six calibration solutions of known concentration (2, 7, 19, 36, 48 and 64 µg/mL for day 1 and 0.9, 5, 10, 15, 19 and 31 µg/mL for day 2). Linear regression (least squares method) was used to calculate the mean of a minimum of two injections of each calibration solution. Calibration curve of cyfluthrin by GC-MS (5.4–68 µg/mL) – day 1 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Calibration curve of cyfluthrin by GC-MS (1.7–20 µg/mL) – day 1 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. 70 Calibration curve of cyfluthrin by GC-MS (5.0–31 µg/mL) – day 2 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Calibration curve of cyfluthrin by GC-MS (0.9–10 µg/mL) – day 2 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Deltamethrin Linearity was demonstrated over concentrations ranging from 0.6 µg/mL to 24 µg/mL expressed as deltamethrin by measuring the detector response (peak area for deltamethrin divided by the peak area for 71 dicyclohexyl phthalate) versus the deltamethrin concentration for a series of six calibration solutions of known concentration (0.6, 4, 7, 12, 18 and 24 µg/mL). Linear regression (least squares method) was used to calculate the mean of a minimum of two injections of each calibration solution. Calibration curve of deltamethrin by GC-MS (3.6–24 µg/mL) – day 1 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Calibration curve of deltamethrin by GC-MS (0.6–12 µg/mL) – day 1 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. 72 Calibration curve of deltamethrin by GC-MS (3.6–24 µg/mL) – day 2 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Calibration curve of deltamethrin by GC-MS (0.6–12 µg/mL) – day 2 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Etofenprox Linearity was demonstrated over concentrations ranging from 5 µg/mL to 208 µg/mL expressed as etofenprox by measuring the detector response (peak area for etofenprox divided by the peak area for 73 dicyclohexyl phthalate) versus the etofenprox concentration for a series of six calibration solutions of known concentration (5, 34, 62, 101, 140 and 208 µg/mL). Linear regression (least squares method) was used to calculate the mean of a minimum of two injections of each calibration solution. Calibration curve of etofenprox by GC-MS (33–205 µg/mL) – day 1 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Calibration curve of etofenprox by GC-MS (5–62 µg/mL) – day 1 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. 74 Calibration curve of etofenprox by GC-MS (33–205 µg/mL) – day 2 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Calibration curve of etofenprox by GC-MS (5–62 µg/mL) – day 2 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is close to 0.98. Lambda-cyhalothrin Linearity was demonstrated over concentrations ranging from 0.6 µg/mL to 24 µg/mL expressed as lambda- cyhalothrin by measuring the detector response (peak area for lambda-cyhalothrin divided by the peak 75 area for dicyclohexyl phthalate) versus the lambda-cyhalothrin concentration for a series of six calibration solutions of known concentration (0.6, 4, 7, 12, 18 and 24 µg/mL). Linear regression (least squares method) was used to calculate the mean of a minimum of two injections of each calibration solution. Calibration curve of lambda-cyhalothrin by GC-MS (0.6–19 µg/mL) – day 1 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Calibration curve of lambda-cyhalothrin by GC-MS (0.6–8 µg/mL) – day 1 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. 76 Calibration curve of lambda-cyhalothrin by GC-MS (0.6–24 µg/mL) – day 2 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Calibration curve of lambda-cyhalothrin by GC-MS (0.6–7 µg/mL) – day 2 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Permethrin Linearity was demonstrated over concentrations ranging from 8 µg/mL to 320 µg/mL expressed as permethrin by measuring the detector response (peak area for permethrin divided by the peak area for 77 dicyclohexyl phthalate) versus the permethrin concentration for a series of six calibration solutions of known concentration (8, 48, 96, 160, 240 and 320 µg/mL). Linear regression (least squares method) was used to calculate the mean of a minimum of two injections of each calibration solution. Calibration curve of permethrin by GC-MS (51–430 µg/mL) – day 1 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Calibration curve of permethrin by GC-MS (11–129 µg/mL) – day 1 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. 78 Calibration curve of permethrin by GC-MS (51–430 µg/mL) – day 2 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Calibration curve of permethrin by GC-MS (11–170 µg/mL) – day 2 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Piperonyl butoxide Linearity was demonstrated over concentrations ranging from 40 µg/mL to 1600 µg/mL expressed as PBO by measuring the detector response (peak area for PBO divided by the peak area for dicyclohexyl 79 phthalate) versus the PBO concentration for a series of five calibration solutions of known concentration (40, 160, 480, 800 and 1600 µg/mL). Linear regression (least squares method) was used to calculate the mean of a minimum of two injections of each calibration solution. Calibration curve of piperonyl butoxide by GC-MS (39–1549 µg/mL) – day 1 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Calibration curve of piperonyl butoxide by GC-MS (39–465 µg/mL) – day 1 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. 80 Calibration curve of piperonyl butoxide by GC-MS (39–1549 µg/mL) – day 2 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. Calibration curve of piperonyl butoxide by GC-MS (39–465 µg/mL) – day 2 a.i. = active ingredient The determination coefficient (R²) of the calibration curve is ≥ 0.98. 81 5.3 Efficacy of extraction (accuracy) The efficacy of extraction of pesticides from the filter papers (extraction kinetics) was verified by analysis of treated filter papers extracted at four different times: one corresponding to the extraction time stated in the method (30 min), a shorter extraction time (20 min) and two longer extraction times (45 and 60 min). p,p΄ -DDT Freshly treated paper Extraction time (min) Extraction value (g/kg)a Extraction value (%) Extraction value (g/kg)a Extraction value (%) Day 1 Day 2 20 8.66 100 6.96 94 Packet 3–4 30 8.69 100 7.41 100 Mo 698 45 8.67 100 7.68 104 60 8.16 94 7.51 101 a Each result is the mean of two chromatographic injections. Malathion Freshly treated paper Extraction time (min) Extraction value (g/kg) a Extraction value (%) Extraction value (g/kg)a Extraction value (%) Day 1 Day 2 20 11.36 96 16.92 104 Packet 3–4 30 11.78 100 16.30 100 Mo 698 45 11.98 102 16.88 104 60 11.73 100 15.19 93 a Each result is the mean of two chromatographic injections. Pirimiphos-methyl Freshly treated paper Extraction time (min) Extraction value (g/kg)a Extraction value (%) Extraction value (g/kg)a Extraction value (%) Day 1 Day 2 20 0.94 100 0.97 94 Packet 3–4 30 0.94 100 1.03 100 Mo 698 45 0.93 99 1.01 98 60 0.95 101 1.02 99 a Each result is the mean of two chromatographic injections. 82 Bendiocarb Freshly treated paper Extraction time (min) Extraction value (g/kg)a Extraction value (%) Extraction value (g/kg)a Extraction value (%) Day 1 Day 2 20 0.13 115 0.30 101 Packet 3–4 30 0.12 100 0.30 100 Mo 698 45 0.12 101 - b - b 60 0.12 102 0.30 102 a Each result is the mean of two chromatographic injections. b Sample lost. Propoxur Freshly treated paper Extraction time (min) Extraction value (g/kg)a Extraction value (%) Extraction value (g/kg)a Extraction value (%) Day 1 Day 2 20 0.30 94 0.28 107 Packet 3–4 30 0.32 100 0.26 100 Mo 698 45 0.33 105 0.29 109 60 0.32 100 0.28 107 a Each result is the mean of two chromatographic injections. Alpha-cypermethrin Freshly treated paper Extraction time (min) Extraction value (g/kg)a Extraction value (%) Extraction value (g/kg)a Extraction value (%) Day 1 Day 2 20 0.12 95 0.13 113 Packet 3–4 30 0.13 100 0.11 100 Mo 698 45 0.12 93 0.11 100 60 0.12 95 0.12 103 a Each result is the mean of two chromatographic injections. 83 Cyfluthrin Freshly treated paper Extraction time (min) Extraction value (g/kg)a Extraction value (%) Extraction value (g/kg)a Extraction value (%) Day 1 Day 2 20 0.42 103 0.35 100 Packet 3–4 30 0.41 100 0.35 100 Mo 698 45 0.41 100 0.35 101 60 0.41 99 0.35 101 a Each result is the mean of two chromatographic injections. Deltamethrin Freshly treated paper Extraction time (min) Extraction value (g/kg)a Extraction value (%) Extraction value (g/kg)a Extraction value (%) Day 1 Day 2 20 0.14 104 0.10 88 Packet 3–4 30 0.13 100 0.12 100 Mo 698 45 0.12 91 0.11 94 60 0.12 93 0.12 98 a Each result is the mean of two chromatographic injections. Etofenprox Freshly treated paper Extraction time (min) Extraction value (g/kg)a Extraction value (%) Extraction value (g/kg)a Extraction value (%) Day 1 Day 2 20 1.44 104 1.43 98 Packet 3–4 30 1.38 100 1.45 100 Mo 698 45 1.47 106 1.45 100 60 1.49 107 1.44 99 a Each result is the mean of two chromatographic injections. 84 Lambda-cyhalothrin Freshly treated paper Extraction time (min) Extraction value (g/kg)a Extraction value (%) Extraction value (g/kg)a Extraction value (%) Day 1 Day 2 20 0.14 102 0.16 99 Packet 3–4 30 0.14 100 0.16 100 Mo 698 45 0.14 97 0.15 95 60 - b - b 0.16 98 a Each result is the mean of two chromatographic injections. b Sample lost Permethrin Freshly treated paper Extraction time (min) Extraction value (g/kg)a Extraction value (%) Extraction value (g/kg)a Extraction value (%) Day 1 Day 2 20 2.29 100 2.23 100 Packet 3–4 30 2.30 100 2.23 100 Mo 698 45 2.25 98 2.25 101 60 2.30 100 2.22 99 a Each result is the mean of two chromatographic injections. Piperonyl butoxide Freshly treated paper Extraction time (min) Extraction value (g/kg)a Extraction value (%) Extraction value (g/kg)a Extraction value (%) Day 1 Day 2 20 9.78 94 11.84 99 Packet 3–4 30 10.4 100 11.91 100 Mo 698 45 9.58 92 12.21 103 60 10.6 102 12.36 104 a Each result is the mean of two chromatographic injections. Determination of the extraction kinetics on treated filter papers showed that the concentrations of pesticides after 20, 45 and 60 min of ultrasonication remained the same as those after 30 min of extraction. Addition of 5 mL deionized water with a sonication time of 10 min, followed by 25 mL acetonitrile with a sonication time of about 30 min (≥ 20 min), is therefore appropriate for extracting all the pesticides from filter paper samples (90 cm ²) correctly. 85 5.4 Recovery (accuracy, repeatability and intermediate precision) Recovery values were calculated by fortifying untreated samples of filter paper with known amounts of pesticide. For each pesticide, five recoveries were performed at the target concentration, five at a concentration twice as high as the target concentration and five at a concentration five times lower than the target concentration. p,p΄ -DDT Fortification level (mg/m²) Recoverya (mg/m²) Recoverya (%) Fortification level (mg/m²) Recoverya (mg/m²) Recoverya (%) Day 1 Day 2 353 99 409 116 361 101 412 117 358 359 100 353 411 116 361 101 397 112 359 100 409 116 Mean (%) 100 Mean (%) 115 SD (%) 1 SD (%) 2 RSD 0.9% RSD 1.5% Confidence interval (%)b 1 Confidence interval (%)b 2 Intermediate precision standard deviation (%) = 11 Intermediate precision RSD (RSDR) = 10.1% 2127 119 1649 93 2131 119 1743 99 1792 2209 123 1767 1810 102 2121 118 1826 103 2108 118 1727 98 Mean (%) 119 Mean (%) 99 SD (%) 2 SD (%) 4 RSD 1.9% RSD 4.1% Confidence interval (%)b 3 Confidence interval (%)b 5 Intermediate precision standard deviation (%) = 15 Intermediate precision RSD (RSDR) = 13.4% 86 Fortification level (mg/m²) Recoverya (mg/m²) Recoverya (%) Fortification level (mg/m²) Recoverya (mg/m²) Recoverya (%) Day 1 Day 2 4147 116 3256 91 4178 117 3240 91 3571 4155 116 3571 3762 105 4062 114 3447 97 4035 113 3237 91 Mean (%) 115 Mean (%) 95 SD (%) 2 SD (%) 6 RSD 1.5% RSD 6.7% Confidence interval (%)b 2 Confidence interval (%)b 8 Intermediate precision standard deviation (%) = 15 Intermediate precision RSD (RSDR) = 14.3% a Each result is the mean of two chromatographic injections. b Student t test with a probability of 95%. 87 Malathion Fortification level (mg/m²) Recoverya (mg/m²) Recoverya (%) Fortification level (mg/m²) Recoverya (mg/m²) Recoverya (%) Day 1 Day 2 356 88 355 88 356 88 371 92 405 358 88 405 384 95 355 88 356 88 359 89 370 91 Mean (%) 88 Mean (%) 91 SD (%) 0 SD (%) 3 RSD 0.4% RSD 3.3% Confidence interval (%)b 0 Confidence interval (%)b 4 Intermediate precision standard deviation (%) = 3 Intermediate precision RSD (RSDR) = 3.0% 1856 92 2082 103 1812 90 2001 99 2024 1638 81 2024 2037 101 1853 92 2041 101 1804 89 2064 102 Mean (%) 89 Mean (%) 101 SD (%) 4 SD (%) 2 RSD 5.0% RSD 1.5% Confidence interval (%)b 6 Confidence interval (%)b 2 Intermediate precision standard deviation (%) = 9 Intermediate precision RSD (RSDR) = 9.8% 3280 81 3944 97 3189 79 3554 88 4048 3349 83 4048 3894 96 3117 77 3712 92 - c - c 3797 94 Mean (%) 80 Mean (%) 93 SD (%) 3 SD (%) 4 RSD 3.1% RSD 4.1% Confidence interval (%)b 4 Confidence interval (%)b 5 Intermediate precision standard deviation (%) = 10 Intermediate precision RSD (RSDR) = 11.9% a Each result is the mean of two chromatographic injections. b Student t test with a probability of 95%. c Sample lost. 88 Pirimiphos-methyl Fortification level (mg/m²) Recoverya (mg/m²) Recoverya (%) Fortification level (mg/m²) Recoverya (mg/m²) Recoverya (%) Day 1 Day 2 21 100 19 96 20 96 19 94 21 21 102 20 19 93 21 101 19 94 21 100 20 99 Mean (%) 100 Mean (%) 95 SD (%) 2 SD (%) 3 RSD 2.2% RSD 2.8% Confidence interval (%)b 3 Confidence interval (%)b 3 Intermediate precision standard deviation (%) = 4 Intermediate precision RSD (RSDR) = 4.1% 99 95 92 90 99 95 94 93 104 114 109 101 98 97 98 94 94 92 98 94 96 95 Mean (%) 98 Mean (%) 93 SD (%) 7 SD (%) 2 RSD 6.7% RSD 2.5% Confidence interval (%)b 8 Confidence interval (%)b 3 Intermediate precision standard deviation (%) = 5 Intermediate precision RSD (RSDR) = 5.6% 166 87 203 100 174 91 162 80 190 160 84 203 174 86 162 85 198 98 166 87 173 86 Mean (%) 87 Mean (%) 90 SD (%) 3 SD (%) 9 RSD 3.1% RSD 9.6% Confidence interval (%)b 3 Confidence interval (%)b 11 Intermediate precision standard deviation (%) = 6 Intermediate precision RSD (RSDR) = 6.8% a Each result is the mean of two chromatographic injections. b Student t test with a probability of 95%. 89 Bendiocarb Fortification level (mg/m²) Recoverya (mg/m²) Recoverya (%) Fortification level (mg/m²) Recoverya (mg/m²) Recoverya (%) Day 1 Day 2 7.9 98 9.6 118 8.2 101 9.4 115 8.1 8.6 106 8.2 9.1 112 8.3 103 9.5 116 9.6 117 9.6 118 Mean (%) 105 Mean (%) 115 SD (%) 7 SD (%) 3 RSD 7.1% RSD 2.2% Confidence interval (%)b 9 Confidence interval (%)b 3 Intermediate precision standard deviation (%) = 9 Intermediate precision RSD (RSDR) = 8.1% 41 101 43 104 41 101 43 105 41 40 100 41 43 105 41 102 43 104 41 102 43 105 Mean (%) 101 Mean (%) 105 SD (%) 1 SD (%) 0 RSD 1.0% RSD 0.4% Confidence interval (%)b 1 Confidence interval (%)b 1 Intermediate precision standard deviation (%) = 3 Intermediate precision RSD (RSDR) = 2.5% 72 90 74 92 73 92 75 93 80 73 91 80 75 94 73 91 74 92 74 92 74 93 Mean (%) 91 Mean (%) 93 SD (%) 1 SD (%) 1 RSD 0.8% RSD 0.9% Confidence interval (%)b 1 Confidence interval (%)b 1 Intermediate precision standard deviation (%) = 1 Intermediate precision RSD (RSDR) = 1.4% a Each result is the mean of two chromatographic injections. b Student t test with a probability of 95%. 90 Propoxur Fortification level (mg/m²) Recoverya (mg/m²) Recoverya (%) Fortification level (mg/m²) Recoverya (mg/m²) Recoverya (%) Day 1 Day 2 9.8 119 8.8 107 9.6 116 9.0 109 8.2 9.4 114 8.1 8.9 109 9.0 110 8.9 108 8.6 105 8.5 104 Mean (%) 114 Mean (%) 107 SD (%) 5 SD (%) 2 RSD 4.8% RSD 2.1% Confidence interval (%)b 7 Confidence interval (%)b 3 Intermediate precision standard deviation (%) = 5 Intermediate precision RSD (RSDR) = 4.9% 44 108 45 110 44 108 43 106 41 45 109 41 44 107 44 108 43 106 45 109 44 108 Mean (%) 109 Mean (%) 107 SD (%) 1 SD (%) 2 RSD 0.6% RSD 1.7% Confidence interval (%)b 1 Confidence interval (%)b 2 Intermediate precision standard deviation (%) = 2 Intermediate precision RSD (RSDR) = 1.5% 73 89 72 88 72 87 74 90 81 72 88 82 73 89 72 87 73 89 73 89 73 89 Mean (%) 88 Mean (%) 89 SD (*) 1 SD (%) 1 RSD 0.7% RSD 0.7% Confidence interval (%)b 1 Confidence interval (%)b 1 Intermediate precision standard deviation (%) = 1 Intermediate precision RSD (RSDR) = 0.9% a Each result is the mean of two chromatographic injections. b Student t test with a probability of 95%. 91 Alpha-cypermethrin Fortification level (mg/m²) Recoverya (mg/m²) Recoverya (%) Fortification level (mg/m²) Recoverya (mg/m²) Recoverya (%) Day 1 Day 2 3.7 95 4.2 111 3.8 98 4.2 111 3.9 3.6 93 3.8 4.4 116 3.4 88 4.2 111 3.6 93 4.4 115 Mean (%) 94 Mean (%) 113 SD (%) 4 SD (%) 2 RSD 4.1% RSD 2.0% Confidence interval (%)b 5 Confidence interval (%)b 3 Intermediate precision standard deviation (%) = 14 Intermediate precision RSD (RSDR) = 13.5% 16 84 16 86 19 96 18 94 19 16 81 19 18 92 17 86 18 93 16 84 17 91 Mean (%) 86 Mean (%) 91 SD (%) 6 SD (%) 3 RSD 6.5% RSD 3.6% Confidence interval (%)b 7 Confidence interval (%)b 4 Intermediate precision standard deviation (%) = 5 Intermediate precision RSD (RSDR) = 6.2% 34 91 41 107 33 89 36 95 37 32 86 38 36 96 33 90 37 98 33 88 38 101 Mean (%) 89 Mean (%) 99 SD (%) 2 SD (%) 5 RSD 2.3% RSD 5.0% Confidence interval (%)b 3 Confidence interval (%)b 6 Intermediate precision standard deviation (%) = 8 Intermediate precision RSD (RSDR) = 8.7% a Each result is the mean of two chromatographic injections. b Student t test with a probability of 95%. 92 Cyfluthrin Fortification level (mg/m²) Recoverya (mg/m²) Recoverya (%) Fortification level (mg/m²) Recoverya (mg/m²) Recoverya (%) Day 1 Day 2 9.4 81 10.3 88 10.0 85 9.5 82 12 9.6 82 11 9.7 83 9.0 78 9.0 77 10.0 86 9.3 79 Mean (%) 82 Mean (%) 82 SD (%) 3 SD (%) 4 RSD 4.0% RSD 5.0% Confidence interval (%)b 4 Confidence interval (%)b 5 Intermediate precision standard deviation (%) = 3 Intermediate precision RSD (RSDR) = 4.1% 49 84 49 85 53 91 52 88 58 46 80 58 51 88 49 84 52 89 50 85 52 90 Mean (%) 85 Mean (%) 88 SD (%) 4 SD (%) 2 RSD 5.1% RSD 2.1% Confidence interval (%)b 5 Confidence interval (%)b 2 Intermediate precision standard deviation (%) = 4 Intermediate precision RSD (RSDR) = 4.3% 109 94 116 100 110 94 114 98 116 105 90 117 116 99 106 91 116 99 106 91 117 100 Mean (%) 92 Mean (%) 99 SD (%) 2 SD (%) 1 RSD 2.0% RSD 0.7% Confidence interval (%)b 2 Confidence interval (%)b 1 Intermediate precision standard deviation (%) = 5 Intermediate precision RSD (RSDR) = 5.5% a Each result is the mean of two chromatographic injections. b Student t test with a probability of 95%. 93 Deltamethrin Fortification level (mg/m²) Recoverya (mg/m²) Recoverya (%) Fortification level (mg/m²) Recoverya (mg/m²) Recoverya (%) Day 1 Day 2 3.6 94 3.7 99 3.1 81 3.7 100 3.8 3.1 82 3.8 3.9 105 2.9 76 3.7 99 3.2 82 3.8 102 Mean (%) 83 Mean (%) 101 SD (%) 6 SD (%) 2 RSD 7.8% RSD 2.5% Confidence interval (%)b 8 Confidence interval (%)b 3 Intermediate precision standard deviation (%) = 13 Intermediate precision RSD (RSDR) = 14.5% 16 83 15 80 18 92 16 83 19 16 85 19 15 79 17 88 15 80 16 82 15 80 Mean (%) 86 Mean (%) 80 SD (%) 4 SD (%) 2 RSD 4.5% RSD 2.3% Confidence interval (%)b 5 Confidence interval (%)b 2 Intermediate precision standard deviation (%) = 3 Intermediate precision RSD (RSDR) = 3.2% 34 87 32 85 32 83 29 78 38 33 86 38 30 79 33 85 31 82 33 86 33 87 Mean (%) 86 Mean (%) 82 SD (%) 2 SD (%) 4 RSD 1.9% RSD 4.7% Confidence interval (%)b 2 Confidence interval (%)b 5 Intermediate precision standard deviation (%) = 4 Intermediate precision RSD (RSDR) = 4.3% a Each result is the mean of two chromatographic injections. b Student t test with a probability of 95%. 94 Etofenprox Fortification level (mg/m²) Recoverya (mg/m²) Recoverya (%) Fortification level (mg/m²) Recoverya (mg/m²) Recoverya (%) Day 1 Day 2 35 96 40 108 32 88 40 108 37 33 88 37 38 104 33 89 39 105 34 92 38 102 Mean (%) 91 Mean (%) 105 SD (%) 3 SD (%) 2 RSD 3.8% RSD 2.3% Confidence interval (%)b 4 Confidence interval (%)b 3 Intermediate precision standard deviation (%) = 11 Intermediate precision RSD (RSDR) = 10.9% 168 91 183 99 172 93 179 97 184 169 92 185 187 102 175 95 183 99 169 92 189 102 Mean (%) 93 Mean (%) 100 SD (%) 2 SD (%) 2 RSD 1.6% RSD 2.1% Confidence interval (%)b 2 Confidence interval (%)b 3 Intermediate precision standard deviation (%) = 5 Intermediate precision RSD (RSDR) = 5.5% 319 87 375 102 343 94 368 100 367 366 100 368 359 97 377 103 383 104 369 101 361 98 Mean (%) 97 Mean (%) 100 SD (%) 6 SD (%) 3 RSD 6.7% RSD 2.7% Confidence interval (%)b 8 Confidence interval (%)b 3 Intermediate precision standard deviation (%) = 5 Intermediate precision RSD (RSDR) = 5.1% a Each result is the mean of two chromatographic injections. b Student t test with a probability of 95%. 95 Lambda-cyhalothrin Fortification level (mg/m²) Recoverya (mg/m²) Recoverya (%) Fortification level (mg/m²) Recoverya (mg/m²) Recoverya (%) Day 1 Day 2 4.0 106 4.1 108 3.9 105 4.1 108 3.7 3.9 104 3.8 4.1 108 3.8 101 4.0 104 3.8 103 3.9 103 Mean (%) 104 Mean (%) 106 SD (%) 2 SD (%) 3 RSD 1.7% RSD 2.4% Confidence interval (%)b 2 Confidence interval (%)b 3 Intermediate precision standard deviation (%) = 3 Intermediate precision RSD (RSDR) = 2.5% 16 88 17 89 17 94 17 90 19 17 89 19 17 90 17 90 18 92 16 88 18 93 Mean (%) 90 Mean (%) 91 SD (%) 2 SD (%) 2 RSD 2.6% RSD 2.0% Confidence interval (%)b 3 Confidence interval (%)b 2 Intermediate precision standard deviation (%) = 2 Intermediate precision RSD (RSDR) = 2.2% 35 94 37 98 35 92 35 93 38 35 93 38 36 95 34 91 36 94 34 91 35 92 Mean (%) 92 Mean (%) 94 SD (%) 1 SD (%) 2 RSD 1.5% RSD 2.4% Confidence interval (%)b 2 Confidence interval (%)b 3 Intermediate precision standard deviation (%) = 2 Intermediate precision RSD (RSDR) = 2.6% a Each result is the mean of two chromatographic injections. b Student t test with a probability of 95%. 96 Permethrin Fortification level (mg/m²) Recoverya (mg/m²) Recoverya (%) Fortification level (mg/m²) Recoverya (mg/m²) Recoverya (%) Day 1 Day 2 59 107 60 109 58 104 60 108 55 58 105 55 59 107 58 104 60 109 59 106 60 109 Mean (%) 105 Mean (%) 108 SD (%) 1 SD (%) 1 RSD 1.2% RSD 1.2% Confidence interval (%)b 2 Confidence interval (%)b 2 Intermediate precision standard deviation (%) = 3 Intermediate precision RSD (RSDR) = 2.4% 251 90 257 93 253 91 254 92 278 247 89 276 257 93 258 93 259 94 248 89 259 94 Mean (%) 91 Mean (%) 93 SD (%) 2 SD (%) 1 RSD 1.8% RSD 0.8% Confidence interval (%)b 2 Confidence interval (%)b 1 Intermediate precision standard deviation (%) = 2 Intermediate precision RSD (RSDR) = 2.4% 426 77 496 90 461 84 486 88 550 459 83 552 474 86 476 87 484 88 446 81 482 87 Mean (%) 83 Mean (%) 88 SD (%) 3 SD (%) 1 RSD 4.1% RSD 1.6% Confidence interval (%)b 4 Confidence interval (%)b 2 Intermediate precision standard deviation (%) = 4 Intermediate precision RSD (RSDR) = 5.1% a Each result is the mean of two chromatographic injections. b Student t test with a probability of 95%. 97 Piperonyl butoxide Fortification level (mg/m²) Recoverya (mg/m²) Recovera (%) Fortification level (mg/m²) Recoverya (mg/m²) Recoverya (%) Day 1 Day 2 258 88 270 92 263 89 271 92 294 268 91 294 275 93 274 93 267 91 273 93 278 94 Mean (%) 91 Mean (%) 92 SD (%) 2 SD (%) 1 RSD 2.6% RSD 1.5% Confidence interval (%)b 3 Confidence interval (%)b 2 Intermediate precision standard deviation (%) = 2 Intermediate precision RSD (RSDR) = 2.3% 1605 109 1446 98 1588 108 1422 97 1472 1610 109 1472 1442 98 1609 109 1436 98 1606 109 1460 99 Mean (%) 109 Mean (%) 98 SD (%) 1 SD (%) 1 RSD 0.6% RSD 1.0% Confidence interval (%)b 1 Confidence interval (%)b 1 Intermediate precision standard deviation (%) = 8 Intermediate precision RSD (RSDR) = 7.6% 2677 91 3233 110 2814 96 3066 105 2929 2822 96 2929 3025 103 2707 92 3646 124 2693 92 3484 119 Mean (%) 93 Mean (%) 112 SD (%) 2 SD (%) 9 RSD 2.5% RSD 8.2% Confidence interval (%)b 3 Confidence interval (%)b 11 Intermediate precision standard deviation (%) = 15 Intermediate precision RSD (RSDR) = 14.1% a Each result is the mean of two chromatographic injections. b Student t test with a probability of 95%. 98 5.5 Limit of quantification The limit of quantification (LOQ) for each pesticide on filter paper was appraised in two ways. y A practical LOQ, expressed as mg/m², was set at the pesticide concentration corresponding to the lowest concentration on the calibration curve. This corresponds to a concentration 10 times lower than the concentration of the target pesticide on the filter paper. y A theoretical LOQ (when possible), expressed as mg/m², was set at the pesticide concentration corresponding to the lowest chromatographic peak that can still be easily integrated and quantified. This corresponds to a concentration of pesticide that yields a peak signal ≥ 5 times the baseline noise. Representative chromatograms are shown in the Annex. p,p΄ -DDT Sample Calculation LOQa (mg/m² as p,p΄-DDT) LOQa (mg/m² as p,p΄-DDT) Day 1 Day 2 Filter paper Practical 163 corresponding to a standard solution of 58.63 µg/mL 220 corresponding to a standard solution of 79.32 µg/mL Theoretical 1.0 corresponding to a standard solution of 0.35 µg/mL 4.3 corresponding to a standard solution of 1.53 µg/mL a Each result is the mean of four chromatographic injections. Malathion Sample Calculation type LOQa (mg/m² as malathion) LOQa (mg/m² as malathion) Day 1 Day 2 Filter paper Practical 169 corresponding to a standard solution of 60.74 µg/mL 222 corresponding to a standard solution of 80.10 µg/mL Theoretical 7.5 corresponding to a standard solution of 2.70 µg/mL 3.8 corresponding to a standard solution of 1.36 µg/mL a Each result is the mean of four chromatographic injections. 99 Pirimiphos-methyl Sample Calculation type LOQa (mg/m² as pirimiphos-methyl) LOQa (mg/m² as pirimiphos-methyl) Day 1 Day 2 Filter paper Practical 11 corresponding to a standard solution of 4.00 µg/mL 19 corresponding to a standard solution of 6.77 µg/mL Theoretical 3.3 corresponding to a standard solution of 1.17 µg/mL – a Each result is the mean of four chromatographic injections. Bendiocarb Sample Calculation type LOQa (mg/m² as bendiocarb) LOQa (mg/m² as bendiocarb) Day 1 Day 2 Filter paper Practical 3.5 corresponding to a standard solution of 1.25 µg/mL 3.3 corresponding to a standard solution of 1.20 µg/mL Theoretical – – a Each result is the mean of four chromatographic injections. Propoxur Sample Calculation type LOQa (mg/m² as propoxur) LOQa (mg/m² as propoxur) Day 1 Day 2 Filter paper Practical 3.6 corresponding to a standard solution of 1.31 µg/mL 3.7 corresponding to a standard solution of 1.34 µg/mL Theoretical – – a Each result is the mean of four chromatographic injections. 100 Alpha-cypermethrin Sample Calculation type LOQa (mg/m² as alpha-cypermethrin) LOQa (mg/m² as alpha-cypermethrin) Day 1 Day 2 Filter paper Practical 1.9 corresponding to a standard solution of 0.68 µg/mL 2.7 corresponding to a standard solution of 0.99 µg/mL Theoretical – – a Each result is the mean of four chromatographic injections. Cyfluthrin Sample Calculation type LOQa (mg/m² as cyfluthrin) LOQa (mg/m² as cyfluthrin) Day 1 Day 2 Filter paper Practical 5.9 corresponding to a standard solution of 2.13 µg/mL 3.9 corresponding to a standard solution of 1.42 µg/mL Theoretical – – a Each result is the mean of four chromatographic injections. Deltamethrin Sample Calculation type LOQa (mg/m² as deltamethrin) LOQa (mg/m² as deltamethrin) Day 1 Day 2 Filter paper Practical 2.0 corresponding to a standard solution of 0.72 µg/mL 3.0 corresponding to a standard solution of 1.08 µg/mL Theoretical – – a Each result is the mean of four chromatographic injections. 101 Etofenprox Sample Calculation type LOQa (mg/m² as etofenprox) LOQa (mg/m² as etofenprox) Day 1 Day 2 Filter paper Practical 15 corresponding to a standard solution of 5.53 µg/mL 18 corresponding to a standard solution of 6.66 µg/mL Theoretical 8.4 corresponding to a standard solution of 3.03 µg/mL 6.8 corresponding to a standard solution of 2.45 µg/mL a Each result is the mean of four chromatographic injections. Lambda-cyhalothrin Sample Calculation type LOQa (mg/m² as lambda-cyhalothrin) LOQa (mg/m² as lambda-cyhalothrin) Day 1 Day 2 Filter paper Practical 2.6 corresponding to a standard solution of 0.93 µg/mL 2.5 corresponding to a standard solution of 0.90 µg/mL Theoretical – 0.2 corresponding to a standard solution of 0.09 µg/mL a Each result is the mean of four chromatographic injections. Permethrin Sample Calculation type LOQa (mg/m² as permethrin) LOQa (mg/m² as permethrin) Day 1 Day 2 Filter paper Practical 39 corresponding to a standard solution of 14.02 µg/mL 37 corresponding to a standard solution of 13.23 µg/mL Theoretical 11 corresponding to a standard solution of 3.96 µg/mL 6.9 corresponding to a standard solution of 2.49 µg/mL a Each result is the mean of four chromatographic injections. 102 Piperonyl butoxide Sample Calculation type LOQa (mg/m² as PBO) LOQa (mg/m² as PBO) Day 1 Day 2 Filter paper Practical 118 corresponding to a standard solution of 42.57 µg/mL 129 corresponding to a standard solution of 46.39 µg/mL Theoretical 5.2 corresponding to a standard solution of 1.87 µg/mL 3.7 corresponding to a standard solution of 1.35 µg/mL a Each result is the mean of four chromatographic injections. 5.6 Pesticide stability in calibration solutions The following table shows the stability of the pesticides in acetonitrile at 4 °C. Pesticide Preparation date (dd/mm/yyyy) First injection date (dd/mm/yyyy) Last injection date (dd/mm/yyyy) Stable for ≥ p,p΄-DDT 21/02/2020 06/03/2020 8/06/2020 108 days Malathion 8/01/2020 19/05/2020 25/11/2020 322 days Pirimiphos-methyl 13/01/2020 8/05/2020 24/06/2020 163 days Bendiocarb 3/12/2019 23/10/2020 12/12/2020 375 days Propoxur 8/01/2020 26/10/2020 1/11/2020 298 days Alpha-cypermethrin 3/12/2019 3/04/2020 7/05/2020 156 days Cyfluthrin 21/10/2019 3/04/2020 7/05/2020 156 days Deltamethrin 3/12/2019 3/04/2020 7/05/2020 156 days Etofenprox 8/01/2020 26/05/2020 18/07/2020 192 days Lambda-cyhalothrin 3/12/2019 3/04/2020 7/05/2020 156 days Permethrin 21/02/2020 26/05/2020 27/08/2020 188 days Piperonyl butoxide 8/01/2020 26/05/2020 18/07/2020 192 days 103 Evaluation and discussion of results 6 104 The initial objective of this study was to develop a common GC-FID and HPLC-DAD analytical method for determining (identifying and quantifying) insecticide active ingredients on filter papers. During method development, it was found that GC-FID works very well for most pesticides in standard solutions, and HPLC-DAD works well for some of them. Nevertheless, we faced the problem of chromatographic interference with the pesticides when analysing susceptibility papers from the Vector Control Research Unit, Universiti Sains Malaysia. The interference is due to the carrier oils (silicone oil, olive oil and risella oil) and is particularly problematic with silicone oil. Several attempts were made to eliminate these oils during sample preparation, before chromatographic injection, but this was unsuccessful for filter papers treated with silicone oil. Thus, GC-FID and HLPC-DAD work for the analysis of pesticides in filter papers from IRS studies but not for the analysis of susceptibility filter papers treated with oils. GC-MS detection was therefore used to eliminate the interference from oils. This method works well for all pesticides, is more specific than GC-FID and HPLC-DAD and is also very sensitive. Preliminary tests with GC-MS were highly promising, and the method was then optimized for sample preparation (extraction and clean-up) for all the pesticides. A GC-FID method for filter papers collected from IRS studies without mineral or vegetal carrier oils was also developed. The following active substances of different product classes were included in the analysis. The target active ingredient content, as provided by the Vector Control Research Unit, Universiti Sains Malaysia, is shown in the following table. Product class Insecticide Active ingredient content (mg/m²) Target discriminating concentration (%)a Organochlorine p,p΄-DDT 1786.60 4 Organophosphates Malathion 1978.02 5 Pirimiphos-methyl 98.90 Not applicable Carbamates Bendiocarb 39.56 0.1 Propoxur 39.56 0.1 Pyrethroids Alpha-cypermethrin 18.37 0.05 Cyfluthrin 55.10 0.15 Deltamethrin 18.37 0.05 Etofenprox 183.67 0.5 Lambda-cyhalothrin 18.37 0.05 Permethrin 275.51 0.75 Synergist PBO 1469.39 4 a Source: World Health Organization, 30 June 2021. The following parameters were assessed during development of the GC-MS and GC-FID analytical methods: y preparation of standard and calibration solutions (e.g. solvent, calibration curve with various active ingredients, concentrations, range); y definition of a unique internal standard (solvent, concentration); y subsample preparation (e.g. cutting of filter papers before analysis, subsample of a representative aliquot); 105 y extraction solvent for samples (e.g. acetonitrile, acetone, methanol); y extraction glassware or disposable tubes, volume for samples, dilution; y ultrasonication extraction conditions (e.g. time, temperature); and y GC-MS and GC-FID parameters (e.g. injection mode, liner, temperature, injection volume, column type, dimension and film thickness, oven temperature programme, carrier gas and flow rate, detection parameters). A common GC-MS analytical method was finally developed and validated for p,p΄ -DDT, malathion, pirimiphos-methyl, alpha-cypermethrin, cyfluthrin, deltamethrin, etofenprox, lambda-cyhalothrin, permethrin and PBO to overcome interfering carrier mineral or vegetable oils used to treat susceptibility filter papers. For filter papers collected from IRS studies without mineral or vegetal oils, a GC-FID method was also developed and validated for bendiocarb and propoxur. The following validation parameters were determined over 2 days: y specificity and non-analyte interference: y two extraction solvents (blank solvent) y two internal standard solutions (blank method) y two control (untreated) filter paper samples (blank sample) y two treated filter paper samples without internal standard y linearity of the chromatographic response: measurement of the detector response after injection of standard solutions of known concentrations (≥ five concentrations); y accuracy (efficacy of extraction): four extraction kinetics samples (assay after extraction of samples after four different times): y one corresponding to the extraction time recommended for the method, y one shorter extraction time and y two longer extraction times; y accuracy, repeatability and intermediate precision (recoveries): y five recoveries at the target concentration, y five recoveries at a concentration twice as high as the target concentration and y five recoveries at a concentration five times lower than the target concentration; and y limit of quantification (LOQ). The GC-MS or GC-FID analytical method for determination of insecticides and PBO on filter papers was successfully validated for each validation parameter according to the guidelines of the European Union (SANCO 3030/99, rev. 5, March 22, 2019) and CIPAC (CIPAC document No. 3807, 28 July 2003). The results of method validation are summarized in sections 2 and 5 of this report.

107 Annex. Representative chromatograms of the pesticides and piperonyl butoxide content of filter papers 108 p,p’-DDT content in filter paper determined by GC-MS Blank solvent solution Blank method solution 109 Blank sample solution Sample without internal standard solution 110 p,p’-DDT calibration solution (C1) – Practical LOQ p,p’-DDT calibration solution (C3) 111 Untreated sample of filter paper spiked with p,p’-DDT – 358 mg/m² fortification Untreated sample of filter paper spiked with p,p’-DDT – 1792 mg/m² fortification 112 Untreated sample of filter paper spiked with p,p’-DDT – 3571 mg/m² fortification 113 Malathion content in filter paper determined by GC-MS Blank solvent solution Blank method solution 114 Blank sample solution Sample without internal standard solution 115 Malathion calibration solution (C1) – Practical LOQ Malathion calibration solution (C3) 116 Untreated sample of filter paper spiked with malathion – 405 mg/m² fortification Untreated sample of filter paper spiked with malathion – 2024 mg/m² fortification 117 Untreated sample of filter paper spiked with malathion – 4048 mg/m² fortification 118 Pirimiphos-methyl content in filter paper determined by GC-MS Blank solvent solution Blank method solution 119 Blank sample solution Sample without internal standard solution 120 Pirimiphos-methyl calibration solution (C1) – Practical LOQ Pirimiphos-methyl calibration solution (C3) 121 Untreated sample of filter paper spiked with pirimiphos-methyl – 21 mg/m² fortification Untreated sample of filter paper spiked with pirimiphos-methyl – 104 mg/m² fortification 122 Untreated sample of filter paper spiked with pirimiphos-methyl – 190 mg/m² fortification 123 Bendiocarb content in filter paper determined by GC-FID Blank solvent solution Blank method solution 124 Blank sample solution – oven temperature conditions 1 Sample without internal standard solution – oven temperature conditions 1 125 Bendiocarb calibration solution (C1) – Practical LOQ – oven temperature conditions 1 Bendiocarb calibration solution (C1) – Practical LOQ – oven temperature conditions 2 126 Bendiocarb calibration solution (C3) – oven temperature conditions 1 Bendiocarb calibration solution (C3) – oven temperature conditions 2 127 Untreated sample of filter paper spiked with bendiocarb – 8.1 mg/m² fortification Untreated sample of filter paper spiked with bendiocarb – 41 mg/m² fortification 128 Untreated sample of filter paper spiked with bendiocarb – 80 mg/m² fortification 129 Propoxur content in filter paper determined by GC-FID Blank solvent solution Blank method solution 130 Blank sample solution Sample without internal standard solution 131 Propoxur calibration solution (C1) – Practical LOQ Propoxur calibration solution (C3) 132 Untreated sample of filter paper spiked with propoxur – 8.2 mg/m² fortification Untreated sample of filter paper spiked with propoxur – 41 mg/m² fortification 133 Untreated sample of filter paper spiked with propoxur – 82 mg/m² fortification 134 Alpha-cypermethrin content in filter paper determined by GC-MS Blank solvent solution Blank method solution Chromatogram Bl. solvant 3 C:\GCMSsolution\RESIDUS\24845\VALID5021.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC Chromatogram Bl. méthode 3 C:\GCMSsolution\RESIDUS\24845\VALID5025.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC D ic yc lo he xy lp ht ha la te /1 0. 58 8 135 Blank sample solution Sample without internal standard solution Chromatogram Bl. éch 3 C:\GCMSsolution\RESIDUS\24845\VALID5029.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC D ic yc lo he xy lp ht ha la te /1 0. 59 1 /1 1. 06 4 /1 1. 29 2 L am bd a- cy ha lo th ri ne /1 1. 49 0 /1 1. 56 0 /1 9. 26 4 Chromatogram Alpha ss IS 3 C:\GCMSsolution\RESIDUS\24845\VALID5033.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC /1 1. 06 6 /1 1. 29 2 L am bd a- cy ha lo th ri ne /1 1. 48 7 A lp ha -c yp er m et hr in e/ 14 .9 17 /1 9. 26 4 136 Alpha-cypermethrin calibration solution (C1) – Practical LOQ Alpha-cypermethrin calibration solution (C3) Chromatogram C1 C:\GCMSsolution\RESIDUS\24845\VALID5017.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC D ic yc lo he xy lp ht ha la te /1 0. 58 9 L am bd a- cy ha lo th ri ne /1 1. 53 1 C yf lu th ri ne 1/ 13 .7 93 C yf lu th ri ne 2/ 13 .9 96 C yf lu th ri ne 3/ 14 .1 81 C yf lu th ri ne 4/ 14 .2 89 A lp ha -c yp er m et hr in e/ 14 .9 14 D el ta m ét hr in e/ 18 .9 54 Chromatogram C3 C:\GCMSsolution\RESIDUS\24845\VALID5014.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC D ic yc lo he xy lp ht ha la te /1 0. 59 3 /1 0. 87 2 /1 1. 25 9 L am bd a- cy ha lo th ri ne /1 1. 53 6 C yf lu th ri ne 1/ 13 .7 97 C yf lu th ri ne 2/ 13 .9 96 C yf lu th ri ne 3/ 14 .1 88 C yf lu th ri ne 4/ 14 .2 93 A lp ha -c yp er m et hr in e/ 14 .9 13 D el ta m ét hr in e/ 18 .9 51 137 Untreated sample of filter paper spiked with alpha-cypermethrin – 4 mg/m² fortification Untreated sample of filter paper spiked with alpha-cypermethrin – 19 mg/m² fortification Chromatogram TR1 F C:\GCMSsolution\RESIDUS\24845\VALID5052.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC D ic yc lo he xy lp ht ha la te /1 0. 59 1 /1 1. 06 3 /1 1. 29 1 L am bd a- cy ha lo th ri ne /1 1. 53 6 C yf lu th ri ne 1/ 13 .7 95 C yf lu th ri ne 2/ 14 .0 02 C yf lu th ri ne 3/ 14 .1 88 C yf lu th ri ne 4/ 14 .2 92 A lp ha -c yp er m et hr in e/ 14 .9 13 D el ta m ét hr in e/ 18 .9 53 /1 9. 25 8 Chromatogram TR2 G C:\GCMSsolution\RESIDUS\24845\VALID5061.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC D ic yc lo he xy lp ht ha la te /1 0. 58 9 /1 1. 06 0 /1 1. 29 2 L am bd a- cy ha lo th ri ne /1 1. 53 6 C yf lu th ri ne 1/ 13 .7 89 C yf lu th ri ne 2/ 13 .9 91 C yf lu th ri ne 3/ 14 .1 79 C yf lu th ri ne 4/ 14 .2 83 A lp ha -c yp er m et hr in e/ 14 .9 06 D el ta m ét hr in e/ 18 .9 46 /1 9. 25 1 138 Untreated sample of filter paper spiked with alpha-cypermethrin – 38 mg/m² fortification Chromatogram TR3 F C:\GCMSsolution\RESIDUS\24845\VALID5073.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC D ic yc lo he xy lp ht ha la te /1 0. 58 1 /1 1. 05 1 /1 1. 28 0 L am bd a- cy ha lo th ri ne /1 1. 52 6 C yf lu th ri ne 1/ 13 .7 79 C yf lu th ri ne 2/ 13 .9 80 C yf lu th ri ne 3/ 14 .1 70 C yf lu th ri ne 4/ 14 .2 73 A lp ha -c yp er m et hr in e/ 14 .8 93 D el ta m ét hr in e/ 18 .9 34 139 Cyfluthrin content in filter paper determined by GC-MS Blank solvent solution Blank method solution Chromatogram Bl. solvant 3 C:\GCMSsolution\RESIDUS\24845\VALID5021.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC Chromatogram Bl. méthode 3 C:\GCMSsolution\RESIDUS\24845\VALID5025.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC D ic yc lo he xy lp ht ha la te /1 0. 58 8 140 Blank sample solution Sample without internal standard solution Chromatogram Bl. éch 3 C:\GCMSsolution\RESIDUS\24845\VALID5029.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC D ic yc lo he xy lp ht ha la te /1 0. 59 1 /1 1. 06 4 /1 1. 29 2 L am bd a- cy ha lo th ri ne /1 1. 49 0 /1 1. 56 0 /1 9. 26 4 Chromatogram Cyflu ss IS 3 C:\GCMSsolution\RESIDUS\24845\VALID5041.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC /1 1. 06 7 /1 1. 29 4 L am bd a- cy ha lo th ri ne /1 1. 49 1 C yf lu th ri ne 1/ 13 .7 96 C yf lu th ri ne 2/ 13 .9 99 C yf lu th ri ne 3/ 14 .1 87 C yf lu th ri ne 4/ 14 .2 91 /1 9. 26 2 141 Cyfluthrin calibration solution (C1) – Practical LOQ Cyfluthrin calibration solution (C3) Chromatogram C1 C:\GCMSsolution\RESIDUS\24845\VALID5017.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC D ic yc lo he xy lp ht ha la te /1 0. 58 9 L am bd a- cy ha lo th ri ne /1 1. 53 1 C yf lu th ri ne 1/ 13 .7 93 C yf lu th ri ne 2/ 13 .9 96 C yf lu th ri ne 3/ 14 .1 81 C yf lu th ri ne 4/ 14 .2 89 A lp ha -c yp er m et hr in e/ 14 .9 14 D el ta m ét hr in e/ 18 .9 54 Chromatogram C3 C:\GCMSsolution\RESIDUS\24845\VALID5014.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC D ic yc lo he xy lp ht ha la te /1 0. 59 3 /1 0. 87 2 /1 1. 25 9 L am bd a- cy ha lo th ri ne /1 1. 53 6 C yf lu th ri ne 1/ 13 .7 97 C yf lu th ri ne 2/ 13 .9 96 C yf lu th ri ne 3/ 14 .1 88 C yf lu th ri ne 4/ 14 .2 93 A lp ha -c yp er m et hr in e/ 14 .9 13 D el ta m ét hr in e/ 18 .9 51 142 Untreated sample of filter paper spiked with cyfluthrin – 12 mg/m² fortification Untreated sample of filter paper spiked with cyfluthrin – 58 mg/m² fortification Chromatogram TR1 F C:\GCMSsolution\RESIDUS\24845\VALID5052.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC D ic yc lo he xy lp ht ha la te /1 0. 59 1 /1 1. 06 3 /1 1. 29 1 L am bd a- cy ha lo th ri ne /1 1. 53 6 C yf lu th ri ne 1/ 13 .7 95 C yf lu th ri ne 2/ 14 .0 02 C yf lu th ri ne 3/ 14 .1 88 C yf lu th ri ne 4/ 14 .2 92 A lp ha -c yp er m et hr in e/ 14 .9 13 D el ta m ét hr in e/ 18 .9 53 /1 9. 25 8 Chromatogram TR2 G C:\GCMSsolution\RESIDUS\24845\VALID5061.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC D ic yc lo he xy lp ht ha la te /1 0. 58 9 /1 1. 06 0 /1 1. 29 2 L am bd a- cy ha lo th ri ne /1 1. 53 6 C yf lu th ri ne 1/ 13 .7 89 C yf lu th ri ne 2/ 13 .9 91 C yf lu th ri ne 3/ 14 .1 79 C yf lu th ri ne 4/ 14 .2 83 A lp ha -c yp er m et hr in e/ 14 .9 06 D el ta m ét hr in e/ 18 .9 46 /1 9. 25 1 143 Untreated sample of filter paper spiked with cyfluthrin – 117 mg/m² fortification Chromatogram TR3 F C:\GCMSsolution\RESIDUS\24845\VALID5073.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC D ic yc lo he xy lp ht ha la te /1 0. 58 1 /1 1. 05 1 /1 1. 28 0 L am bd a- cy ha lo th ri ne /1 1. 52 6 C yf lu th ri ne 1/ 13 .7 79 C yf lu th ri ne 2/ 13 .9 80 C yf lu th ri ne 3/ 14 .1 70 C yf lu th ri ne 4/ 14 .2 73 A lp ha -c yp er m et hr in e/ 14 .8 93 D el ta m ét hr in e/ 18 .9 34 144 Deltamethrin content in filter paper determined by GC-MS Blank solvent solution Blank method solution Chromatogram Bl. solvant 3 C:\GCMSsolution\RESIDUS\24845\VALID5021.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC Chromatogram Bl. méthode 3 C:\GCMSsolution\RESIDUS\24845\VALID5025.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC D ic yc lo he xy lp ht ha la te /1 0. 58 8 145 Blank sample solution Sample without internal standard solution Chromatogram Bl. éch 3 C:\GCMSsolution\RESIDUS\24845\VALID5029.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC D ic yc lo he xy lp ht ha la te /1 0. 59 1 /1 1. 06 4 /1 1. 29 2 L am bd a- cy ha lo th ri ne /1 1. 49 0 /1 1. 56 0 /1 9. 26 4 Chromatogram Delta ss IS 3 C:\GCMSsolution\RESIDUS\24845\VALID5037.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC D ic yc lo he xy lp ht ha la te /1 0. 59 2 /1 1. 06 7 /1 1. 29 4 L am bd a- cy ha lo th ri ne /1 1. 49 2 D el ta m ét hr in e/ 18 .9 57 /1 9. 26 8 146 Deltamethrin calibration solution (C1) – Practical LOQ Deltamethrin calibration solution (C3) Chromatogram C1 C:\GCMSsolution\RESIDUS\24845\VALID5017.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC D ic yc lo he xy lp ht ha la te /1 0. 58 9 L am bd a- cy ha lo th ri ne /1 1. 53 1 C yf lu th ri ne 1/ 13 .7 93 C yf lu th ri ne 2/ 13 .9 96 C yf lu th ri ne 3/ 14 .1 81 C yf lu th ri ne 4/ 14 .2 89 A lp ha -c yp er m et hr in e/ 14 .9 14 D el ta m ét hr in e/ 18 .9 54 Chromatogram C3 C:\GCMSsolution\RESIDUS\24845\VALID5014.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC D ic yc lo he xy lp ht ha la te /1 0. 59 3 /1 0. 87 2 /1 1. 25 9 L am bd a- cy ha lo th ri ne /1 1. 53 6 C yf lu th ri ne 1/ 13 .7 97 C yf lu th ri ne 2/ 13 .9 96 C yf lu th ri ne 3/ 14 .1 88 C yf lu th ri ne 4/ 14 .2 93 A lp ha -c yp er m et hr in e/ 14 .9 13 D el ta m ét hr in e/ 18 .9 51 147 Untreated sample of filter paper spiked with deltamethrin – 4 mg/m² fortification Untreated sample of filter paper spiked with deltamethrin – 19 mg/m² fortification Chromatogram TR1 F C:\GCMSsolution\RESIDUS\24845\VALID5052.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC D ic yc lo he xy lp ht ha la te /1 0. 59 1 /1 1. 06 3 /1 1. 29 1 L am bd a- cy ha lo th ri ne /1 1. 53 6 C yf lu th ri ne 1/ 13 .7 95 C yf lu th ri ne 2/ 14 .0 02 C yf lu th ri ne 3/ 14 .1 88 C yf lu th ri ne 4/ 14 .2 92 A lp ha -c yp er m et hr in e/ 14 .9 13 D el ta m ét hr in e/ 18 .9 53 /1 9. 25 8 Chromatogram TR2 G C:\GCMSsolution\RESIDUS\24845\VALID5061.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC D ic yc lo he xy lp ht ha la te /1 0. 58 9 /1 1. 06 0 /1 1. 29 2 L am bd a- cy ha lo th ri ne /1 1. 53 6 C yf lu th ri ne 1/ 13 .7 89 C yf lu th ri ne 2/ 13 .9 91 C yf lu th ri ne 3/ 14 .1 79 C yf lu th ri ne 4/ 14 .2 83 A lp ha -c yp er m et hr in e/ 14 .9 06 D el ta m ét hr in e/ 18 .9 46 /1 9. 25 1 148 Untreated sample of filter paper spiked with deltamethrin – 38 mg/m² fortification Chromatogram TR3 F C:\GCMSsolution\RESIDUS\24845\VALID5073.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC D ic yc lo he xy lp ht ha la te /1 0. 58 1 /1 1. 05 1 /1 1. 28 0 L am bd a- cy ha lo th ri ne /1 1. 52 6 C yf lu th ri ne 1/ 13 .7 79 C yf lu th ri ne 2/ 13 .9 80 C yf lu th ri ne 3/ 14 .1 70 C yf lu th ri ne 4/ 14 .2 73 A lp ha -c yp er m et hr in e/ 14 .8 93 D el ta m ét hr in e/ 18 .9 34 149 Etofenprox content in filter paper determined by GC-MS Blank solvent solution Blank method solution 150 Blank sample solution Sample without internal standard solution 151 Etofenprox calibration solution (C1) – Practical LOQ Etofenprox calibration solution (C3) 152 Untreated sample of filter paper spiked with etofenprox – 37 mg/m² fortification Untreated sample of filter paper spiked with etofenprox – 184 mg/m² fortification 153 Untreated sample of filter paper spiked with etofenprox – 367 mg/m² fortification 154 Lambda-cyhalothrin content in filter paper determined by GC-MS Blank solvent solution Blank method solution Chromatogram Bl. solvant 3 C:\GCMSsolution\RESIDUS\24845\VALID5021.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC Chromatogram Bl. méthode 3 C:\GCMSsolution\RESIDUS\24845\VALID5025.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC D ic yc lo he xy lp ht ha la te /1 0. 58 8 155 Blank sample solution Sample without internal standard solution Chromatogram Bl. éch 3 C:\GCMSsolution\RESIDUS\24845\VALID5029.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC D ic yc lo he xy lp ht ha la te /1 0. 59 1 /1 1. 06 4 /1 1. 29 2 L am bd a- cy ha lo th ri ne /1 1. 49 0 /1 1. 56 0 /1 9. 26 4 Chromatogram Lam ss IS 3 C:\GCMSsolution\RESIDUS\24845\VALID5045.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC /1 1. 06 7 /1 1. 29 5 L am bd a- cy ha lo th ri ne /1 1. 54 2 /1 9. 25 7 156 Lambda-cyhalothrin calibration solution (C1) – Practical LOQ Lambda-cyhalothrin calibration solution (C3) Chromatogram C1 C:\GCMSsolution\RESIDUS\24845\VALID5017.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC D ic yc lo he xy lp ht ha la te /1 0. 58 9 L am bd a- cy ha lo th ri ne /1 1. 53 1 C yf lu th ri ne 1/ 13 .7 93 C yf lu th ri ne 2/ 13 .9 96 C yf lu th ri ne 3/ 14 .1 81 C yf lu th ri ne 4/ 14 .2 89 A lp ha -c yp er m et hr in e/ 14 .9 14 D el ta m ét hr in e/ 18 .9 54 Chromatogram C3 C:\GCMSsolution\RESIDUS\24845\VALID5014.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC D ic yc lo he xy lp ht ha la te /1 0. 59 3 /1 0. 87 2 /1 1. 25 9 L am bd a- cy ha lo th ri ne /1 1. 53 6 C yf lu th ri ne 1/ 13 .7 97 C yf lu th ri ne 2/ 13 .9 96 C yf lu th ri ne 3/ 14 .1 88 C yf lu th ri ne 4/ 14 .2 93 A lp ha -c yp er m et hr in e/ 14 .9 13 D el ta m ét hr in e/ 18 .9 51 157 Untreated sample of filter paper spiked with lambda-cyhalothrin – 4 mg/m² fortification Untreated sample of filter paper spiked with lambda-cyhalothrin – 19 mg/m² fortification Chromatogram TR1 F C:\GCMSsolution\RESIDUS\24845\VALID5052.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC D ic yc lo he xy lp ht ha la te /1 0. 59 1 /1 1. 06 3 /1 1. 29 1 L am bd a- cy ha lo th ri ne /1 1. 53 6 C yf lu th ri ne 1/ 13 .7 95 C yf lu th ri ne 2/ 14 .0 02 C yf lu th ri ne 3/ 14 .1 88 C yf lu th ri ne 4/ 14 .2 92 A lp ha -c yp er m et hr in e/ 14 .9 13 D el ta m ét hr in e/ 18 .9 53 /1 9. 25 8 Chromatogram TR2 G C:\GCMSsolution\RESIDUS\24845\VALID5061.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC D ic y cl o h ex y lp h th al at e/ 1 0 .5 8 9 /1 1 .0 6 0 /1 1 .2 9 2 L am b d a- cy h al o th ri n e/ 1 1 .5 3 6 C y fl u th ri n e1 /1 3 .7 8 9 C y fl u th ri n e2 /1 3 .9 9 1 C y fl u th ri n e3 /1 4 .1 7 9 C y fl u th ri n e4 /1 4 .2 8 3 A lp h a- cy p er m et h ri n e/ 1 4 .9 0 6 D el ta m ét h ri n e/ 1 8 .9 4 6 /1 9 .2 5 1 158 Untreated sample of filter paper spiked with lambda-cyhalothrin – 38 mg/m² fortification Chromatogram TR3 F C:\GCMSsolution\RESIDUS\24845\VALID5073.qgd min intensity 200,000 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 0 50000 100000 150000 200000 TIC D ic y cl o h ex y lp h th al at e/ 1 0 .5 8 1 /1 1 .0 5 1 /1 1 .2 8 0 L am b d a- cy h al o th ri n e/ 1 1 .5 2 6 C y fl u th ri n e1 /1 3 .7 7 9 C y fl u th ri n e2 /1 3 .9 8 0 C y fl u th ri n e3 /1 4 .1 7 0 C y fl u th ri n e4 /1 4 .2 7 3 A lp h a- cy p er m et h ri n e/ 1 4 .8 9 3 D el ta m ét h ri n e/ 1 8 .9 3 4 159 Permethrin content in filter paper determined by GC-MS Blank solvent solution Blank method solution 160 Blank sample solution Sample without internal standard solution 161 Permethrin calibration solution (C1) – Practical LOQ Permethrin calibration solution (C3) 162 Untreated sample of filter paper spiked with permethrin – 56 mg/m² fortification Untreated sample of filter paper spiked with permethrin – 278 mg/m² fortification 163 Untreated sample of filter paper spiked with permethrin – 550 mg/m² fortification 164 Piperonyl butoxide content in filter paper determined by GC-MS Blank solvent solution Blank method solution 165 Blank sample solution Sample without internal standard solution 166 Piperonyl butoxide calibration solution (C1) – Practical LOQ Piperonyl butoxide calibration solution (C3) 167 Untreated sample of filter paper spiked with piperonyl butoxide – 294 mg/m² fortification Untreated sample of filter paper spiked with piperonyl butoxide – 1472 mg/m² fortification 168 Untreated sample of filter paper spiked with piperonyl butoxide – 2929 mg/m² fortification