1. Assessing effects of pesticides on the breakdown of organic matter Geoff Frampton Susan Jones University of Southampton, UK Thomas Knacker Joerg Roembke Bernhard Foerster ECT Oekotoxikologie, Germany Juliane Filser Holger Mebes University of Bremen, Germany Funded by

2. Review of available and potential test methods Limitations of the current trigger pathway Alternatives ? Assessing effects of pesticides on the breakdown of organic matter

3. “ An assessment of effects on breakdown of organic matter is required …” Authorisations Directive 91 / 414 / EEC (as amended by 96 / 12 / EC) Annex III (Formulated products) Point 10.6.2 (Effects on other soil non-target organisms) based on chemical persistence data (DT 90 field )

4. Test for pesticide impact on OM breakdown: exposure in soil is likely, and risk to soil microflora, earthworms, or other soil fauna (e.g. Collembola) DT 90 100 to 365 days Test required IF… DT 90 < 100 daysTest not required DT 90 > 365 daysTest required

5. Tests relevant to OM breakdown ? Litter bag Minicontainer Cotton-strip assay Isotopes Bait lamina

6. Litter bags OM (e.g. straw) enclosed in gauze bagsPrinciple: Duration: Factors measured: Exposure of OM: On soil surface or buried Comments: Typically 6-12 months Mesh size and exposure crucial for realism Litter mass loss (rate)

8. Minicontainer Similar to litter bag, but smaller PVC containers Principle: Duration:2 - 6 months

9. Cotton strip assay Measurement of cellulose decompositionPrinciple: Factors measured: Comments:Fungi may increase tensile strength Loss of tensile strength of cotton strips buried in soil

10. 15 N, 14 C, 13 C Isotopes Detection of isotopes from labelled OM which is mixed directly into soil Principle: Comments:Mainly applied in laboratory studies

11. Bait-lamina method Detects feeding activity of soil organismsPrinciple: Factors measured: Duration: OM bait removed from small (1mm diam.) holes in PVC strips inserted into soil 1-4 weeks

12. 1. Relevance to RA scheme 2. Ecological relevance 3. Experience 4. Flexibility 5. Robustness 6. Practicability 7. Sensitivity 8. Data assessment 9. Reproducibility, repeatability 10. Standardisation, validation Assessment criteria

13. Litter bag Mini- container Cotton strip Isotopes Bait- lamina 1. Relevance to ERA 2. Ecological relevance 3. Experience 4. Flexibility 5. Robustness 6. Practicability 7. Sensitivity 8. Data assessment 9. Reproducibility, repeatability 10. Standardisation, validation ? ? ? ? ? ? ? ? ? ? ? ? ? ?

14. Litter bag is the most appropriate method at present for assessing effects of pesticides on OM breakdown Conclusion… But…

15. Relevant only to early (microbially-mediated) stages of OM breakdown Limitations of the litter-bag method Acceptability of effects on mass loss are unclear Realistic simulation of exposure for repeat- application chemicals is difficult

16. Relevant only to early mass loss phase of OM breakdown; does not assess later mineralisation Limitations of the litter-bag method Acceptability of effects on mass loss are unclear Realistic simulation of exposure for repeat- application chemicals is difficult

17. Relevant only to early (microbially-mediated) stages of OM breakdown Limitations of the litter-bag method Acceptability of effects on mass loss are unclear Realistic simulation of exposure for repeat- application chemicals is difficult

18. Relevant only to early (microbially-mediated) stages of OM breakdown Limitations of the litter-bag method Acceptability of effects on mass loss are unclear Realistic simulation of exposure for repeat- application chemicals is difficult

19. Limitations of the overall RA scheme relevance to OM breakdown unclear representativeness of test species unproven functional redundancy and ecological complexity Are soil fauna tests appropriate as a trigger for the OM breakdown test ?

20. relevance to OM breakdown unclear representativeness of test species unproven Limitations of the overall RA scheme Are soil fauna tests appropriate as a trigger for the OM breakdown test ? functional redundancy and ecological complexity

21. relevance to OM breakdown unclear representativeness of test species unproven Limitations of the overall RA scheme Are soil fauna tests appropriate as a trigger for the OM breakdown test ? functional redundancy and ecological complexity

22. Are soil fauna tests appropriate as a trigger for the OM breakdown test ? relevance to OM breakdown unclear representativeness of test species unproven functional redundancy and ecological complexity Limitations of the overall RA scheme

23. Could functional lower or middle tier tests be used to predict OM breakdown ?

24. Existing tests related to system function FURTHER TESTS : Nitrification Soil respiration Soil enzymes TIER I TESTS : N transformation C mineralization ?

25. Enzymes as predictors of OM breakdown ? YES … Because some enzymes have clear roles in OM turnover YES … Because some enzymes (e.g. lignocellulases) are specific to the decomposer fauna BUT … Single enzyme tests are poor predictors of pesticide effects on soil processes

26. Simultaneous multi-substrate assays for predicting OM breakdown ?

27. Microbial community profiling using Biolog analyses Catabolic Response Profile - CRP Possible approaches

28. BiologCRP Test and control soils added to range of enzyme substrates in microtiter plate Range of substrates added to test and control soils After 1 week incubation, enzyme activities in test and control soils determined colorimetrically Over 12h, substrate-induced respiration measured for all substrates

29. BiologCRP Enzyme activity profiles for test and control soils Catabolic activity profiles for test and control soils Risk assessment scheme ?

30. Standard methods available, homogeneous substrates Substrate profile could be customised for OM processing Rapid results Biolog / CRP - pros cons Biolog is restricted to culturable microorganisms Predictive capability for OM processing requires clarification Refined method would require validation …these limitations are no worse than for the existing single-species tests !

31. The litter-bag method is the most appropriate… Concluding remarks But it still has key limitations and uncertainties The OM breakdown trigger pathway for medium-persistence substances does not make ecological sense Could Biolog and / or CRP be more appropriate ‘middle-tier’ (functional) methods than the existing (structural) tests ? Thanks for listening !