Assessment of Stability of Pesticides During Sample Processing

1. Assessment of Stability of Pesticides During Sample Processing Richard Fussell CSL York, UK e-mail: r.fussell@csl.gov.uk

2. Analytical Strategies for Pesticide Residues Analysis Derivatisation Oxidation SAMPLING RECEIPT OF SAMPLES SAMPLE PROCESSING ambient / cryogenic / acid EXTRACTION ethyl acetate / acetone / acetonitrile / SFE CLEAN-UP GPC / HPGPC / SPE / SPME DETECTION / QUANTITATION / CONFIRMATION Gas Liquid Chromatography Liquid Chromatography AED / ECD / NPD / FPD / MS / MS-MS FL / UV / DAD / MS / MS-MS REPORTS

3. Ambient Temperature Processing ‘Tomato purée

4. Sample Processing - Losses at Ambient Temperature • 1994 CSL reported rapid losses of chlorothalonil in lettuce • Subsequent investigations have indicated losses of a number of pesticides during sample processing at ambient temperatures: bitertanol dichlofluanid isofenphos captan dicofol tebuconazole captafol etridiazole tolylfluanid chlorothalonil folpet

5. Mechanisms for Losses during Sample Processing No single mechanism involved • nature, temperature, duration of processing • volatility e.g. fumigants, biphenyl, dichlorvos • hydrolysis (exposure to water) • Interaction with chemicals/enzymes released from plant cells • pH

6. Sample Processing - Implications of the Losses • Unlikely to cause an underestimate of consumer risk • MRL exceedances and misuse may not be detected ‘Cryogenic’ processing may help to minimise some of the losses and is used in the generation of registration data

7. ‘Cryogenic’ Sample Processing - Temperature Conditions • Freeze Sample (-20°C) • Add ‘dry ice’ (-69°C) • Homogenise to produce a flowable powder (-25 to -30°C) samples must not defrost

8. Sample Processing - Before ‘Cryogenic’ Processing

9. ‘Cryogenic’ Processing

10. After ‘Cryogenic’ Sample Processing

11. Sample Processing of Tomatoes - Ambient Vs ‘Cryogenic’

12. Development of a ‘ Cryogenic’ Processing Protocol for Apples Protocol designed to reflect intended practice • whole fruit spiked prior to freezing • spiking at realistic levels (0.05 mg/kg) • 106 different pesticides assessed for stability • parallel method recoveries • extraction using ethyl acetate • measurement using GC-MS, GC-FPD, LC-FL

13. Spiking Procedure 250 µl of pesticides standard in ethyl acetate

14. Development of a ‘ Cryogenic’ Processing Protocol for Apples Protocol designed to reflect intended practice • whole fruit spiked prior to freezing • spiking at realistic levels (0.05 mg/kg) • 106 different pesticides assessed for stability • parallel method recoveries • extraction using ethyl acetate • measurement using GC-MS, GC-FPD, LC-FL

15. Sample Processing Protocol - Other Considerations • calculation of mass balance - sum of pesticides on filters, in mill washes and sample • chlorpyrifos-methyl employed as an internal deposition standard - assumed to be stable • duplicate analysis on 7 different days, duplicate injection (n=28)

16. Processing Individual fruit

17. ‘Cryogenic’ Processing - Summary of Results • Uncorrected Data AQC - % mean recoveries 85-90%, %CVs 5-10% mass balance - 75 - 85%, %CVs 5 -10% (equates to loss of 0.5 - 1 µg of each pesticide) • CPM Corrected Data AQC & ‘Survival’ (processing recovery minus AQC recovery) very similar - % mean recoveries 97 - 102%, %CVs 2 - 5% • >90% survival for >100 pesticides • malaoxon only 80% survival

18. ‘Cryogenic’ Processing - Notable Successes Survival (%) Pesticide Ambient ‘Cryogenic’ bitertanol 5 100 captan* 41 81 captafol* 62 95 dichlofluanid 46 90 folpet* 50 93 heptenophos 50 93 isofenphos 60 100 tolylfluanid 52 96

19. ‘Cryogenic’ Processing - Future Work • Re-assessment of ‘problem’ pesticides in apples • Assessment of dodine, dithianon, dinocap and dithiocarbamates in apples • Assessment of stability of a wide range of pesticides in other commodities (carrots, lettuce, onions, oranges and a brassica)

20. ‘Cryogenic’ Processing -CPM Corrected Data Pesticides with Poor AQC Pesticide Survival Recovery %CV acephate 62 7321 methamidophos 61 7317 omethoate 71 8116 chlozolinate 87 104 24 biphenyl 46 74 17 dichlorvos 54 8021 carbendazim* 80 93 13 thiabendazole* 82 93 13 (*not corrected) phosalone, pyrimethanil, chlorothalonil - AQC failures ethoxyquin, tebuconazole, n-methyl carbamates - not included

21. ‘Cryogenic’ Processing - Future Work • Re-assessment of ‘problem’ pesticides in apples • Assessment of dodine, dithianon, dinocap and dithiocarbamates in apples • Assessment of stability of a wide range of pesticides in other commodities (carrots, lettuce, onions, oranges and a brassica)

22. ‘Cryogenic’ Processing - Pros and Cons • Advantages • more accurate results • improved homogeneity of sample • lower costs if samples can be milled for dithiocarbamates • Disadvantages • more stress on the equipment • more stress on the operator (noise) Cryogenic Processing is being used for more commodities in the UK monitoring programme - But cryogenic Processing does not solve all problems

23. Sample Processing - Chlorothalonil in Onions • Complete loss during processing at ambient temperatures and 80-90% loss during cryogenic Processing - possible reaction with sulphur compounds • Add 2.2 M phosphoric acid prior to processing - at 1 mg/kg recovery = 105%, %CV = 6.8

24. Sample Processing - Further Information • ARC Hill et al - Effects of Sample Processing on Pesticide Residues in Fruit and Vegetables Principles and Practices of Method Validation 2000 Ed A Fajelj and A Ambrus, RSC, ISBN 0-85404-783-2 • R Fussell et al - Assessment of the Stability of Pesticides During Cryogenic sample Processing Part 1: Apples J. Agricultural & Food Chemistry, 2002, 50, 5, 441-448 • www.iaea.org/programmes/ri/uc.html

25. Acknowledgments • Alan Hill, CSL, UK • Pesticides Safety Directorate, UK • Pesticides Team, CSL, UK