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Co- authors : Manier Nicolas 1 , Chabot Laure 1 , Bauda Pascale 2 and Pandard Pascal 1

Caenorhabditis elegans ( Nematoda ) as « new » ecotoxicological tool in test batteries for the assessment of organic materials used in agriculture. Presented by Pierre Huguier 1 In PhD position.

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Co- authors : Manier Nicolas 1 , Chabot Laure 1 , Bauda Pascale 2 and Pandard Pascal 1

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  1. Caenorhabditiselegans(Nematoda) as « new » ecotoxicologicaltool in test batteries for the assessment of organicmaterialsused in agriculture Presented by Pierre Huguier1 In PhD position Co-authors: Manier Nicolas1, Chabot Laure1, Bauda Pascale2 and Pandard Pascal1 INERIS, Parc technologique ALATA, 60550, Verneuil-en-Halatte (France) LIEC, UMR 7569, Université de Lorraine, rue du Général Delestraint, 57070, Metz (France)

  2. CONTENT • Context • Materials and Methods • Results • Conclusion

  3. CONTENT • Context • Materials and Methods • Results • Conclusion

  4. I. Context Water treatment plants Industries (paper mills, composting plants,…) Animal feces Other use than for feeding (for fibers, for oil, …) Livestock Human beings Organic materials Soil

  5. I. Context Water treatment plants Industries (paper mills, composting plants,…) Animal feces Other use than for feeding (for fibers, for oil, …) Livestock Human beings Organic materials Soil

  6. I. Context Water treatment plants Industries (paper mills, composting plants,…) Animal feces Fertilizing compounds, water retention characteristics, liming properties,… Potential source of pollutants, i.e. heavy metals, PAHs, … Other use than for feeding (for fibers, for oil, …) Livestock Human beings Organic materials Soil

  7. I. Context Water treatment plants Industries (paper mills, composting plants,…) Animal feces Fertilizing compounds, water retention characteristics, liming properties,… Potential source of pollutants, i.e. heavy metals, PAHs, … Other use than for feeding (for fibers, for oil, …) Livestock Human beings Soil fauna and ecosystem functioning Necessity to assess potential toxicity before field application, using ecotoxicological tests Organic materials Soil

  8. I. Context Interest of the nematodeCaenorhabditiselegansas test organism • Biologically and ecologicallyspeaking: • Key functions of nematodes on decomposition, nutrientscyclingprocesses and on microbialregulation • Bacterial feeder, complementary in term of trophic chain • Microfaunaorganism, onlymesofaunaorganisms in ourterrestrial test battery

  9. I. Context Interest of the nematodeCaenorhabditiselegansas test organism • Biologically and ecologicallyspeaking: • Key functions of nematodes on decomposition, nutrientscyclingprocesses and on microbialregulation • Bacterial feeder, complementary in term of trophic chain • Microfaunaorganism, onlymesofaunaorganisms in ourterrestrial test battery • Toxicologicallyspeaking: • Well-studiedorganism in ecotoxicology (mainly pure substances and naturalsamples) but neverused for the assessment of complex matrices • Standard protocolsavailable (ASTM, ISO) • Evaluation of chronic (ISO) toxicity • Quick test (4d) • Possibility of testingliquid, soil and sedimentsamples

  10. CONTENT • Context • Materials and Methods • AMIOM studied • Test strategy • Results • Conclusion

  11. II. Materials & Methods 1. AMIOM studied Agricultural, Municipal and IndustrialOrganicMaterials (AMIOM)

  12. II. Materials & Methods 1. AMIOM studied Agricultural, Municipal and IndustrialOrganicMaterials (AMIOM) Intrisiccharacteristics Fertilizing compounds Heavymetals

  13. II. Materials & Methods 2. Test strategy AMIOM Doses tested: 1, 5, 10, 50 and 100 times the respective application rate a. Diagram Mixture leachate Terrestrial bio-assays Aquatic bio-assays

  14. II. Materials & Methods 2. Test strategy AMIOM Doses tested: 1, 5, 10, 50 and 100 times the respective application rate a. Diagram Mixture leachate Terrestrial bio-assays Aquatic bio-assays Higher plants (Avenasativa rootsgrowth and biomass, Brassicarapabiomass) ISO 11269-1 and 2, 2012. Bacteria (Vibriofischeriluminescence) ISO 11348-1, 2007. Crustacean (Daphnia magna mobility) ISO 6341, 2012. Nematode (Caenorhabditiselegansgrowth and reproduction) Huguier et al., 2013. Algae (Pseudokirchneriellasubcapitatagrowth) ISO 8692, 2012. Nematode (Caenorhabditiselegansgrowth and reproduction) ISO 10872, 2010. Earthworms (Eiseniafetida avoidancebehavior and reproduction) ISO 17512-1, 2008 and ISO 11268-2, 2012. Predatory mites (Hypoaspisaculeiferreproduction) OECD 226, 2008. Rotifers (Brachionuscalyciflorusreproduction) ISO 20666, 2008.

  15. II. Materials & Methods 2. Test strategy AMIOM Doses tested: 1, 5, 10, 50 and 100 times the respective application rate a. Diagram Mixture leachate Terrestrial bio-assays Aquatic bio-assays Higher plants (Avenasativa rootsgrowth and biomass, Brassicarapabiomass) ISO 11269-1 and 2, 2012. Bacteria (Vibriofischeriluminescence) ISO 11348-1, 2007. Crustacean (Daphnia magna mobility) ISO 6341, 2012. Nematode (Caenorhabditiselegansgrowth and reproduction) Huguier et al., 2013. Algae (Pseudokirchneriellasubcapitatagrowth) ISO 8692, 2012. Nematode (Caenorhabditiselegansgrowth and reproduction) ISO 10872, 2010. Earthworms (Eiseniafetida avoidancebehavior and reproduction) ISO 17512-1, 2008 and ISO 11268-2, 2012. Predatory mites (Hypoaspisaculeiferreproduction) OECD 226, 2008. Rotifers (Brachionuscalyciflorusreproduction) ISO 20666, 2008.

  16. II. Materials & Methods 2. Test strategy b. Mixtures preparation and leaching Mixtures of soilwith AMIOM Artificial ISO soil Mixed witheachAgricultural, Municipal and IndustrialOrganicMaterialsat the desired application rate Moisture: 20% d.w.; Equilibration time

  17. II. Materials & Methods 2. Test strategy b. Mixtures preparation and leaching Mixtures of soilwith AMIOM Leaching of mixtures Artificial ISO soil Mixtures Distilled water (ratio 1:10 soil:water) Eluate Mixed witheachAgricultural, Municipal and IndustrialOrganicMaterialsat the desired application rate Horizontal rotating agitation, 24h Centrifugation, 30min 2500g Supernatantrecovery Not filtered Filtered Moisture: 20% d.w.; Equilibration time

  18. CONTENT • Context • Materials and Methods • Results • Overview of AMIOM ecotoxicity • Comparison of bio-assaysresponses • Conclusion

  19. III. Results 1. Overview of AMIOM ecotoxicity Resultsoverview: • Significanttoxic (coding: red) as well as significanthormesis (coding: green) effects

  20. III. Results 1. Overview of AMIOM ecotoxicity Resultsoverview: • Significanttoxic (coding: red) as well as significanthormesis (coding: green) effects • For bothterrestrial and aquatic bio-assays, AMIOM studied not ecotoxicattheir respective application rates

  21. III. Results 1. Overview of AMIOM ecotoxicity Resultsoverview: • Significanttoxic (coding: red) as well as significanthormesis (coding: green) effects • For bothterrestrial and aquatic bio-assays, AMIOM studied not ecotoxicattheir respective application rates • AMIOM ecotoxicity assessmentathigher application rates

  22. III. Results 1. Overview of AMIOM ecotoxicity Resultsoverview: • Significanttoxic (coding: red) as well as significanthormesis (coding: green) effects • For bothterrestrial and aquatic bio-assays, AMIOM studied not ecotoxicattheir respective application rates • AMIOM ecotoxicity assessmentathigher application rates • Terrestrial bio-assaysshowedhighersensitivitythanaquaticones

  23. III. Results 2. Comparison of bio-assaysresponses Principal component analyses of terrestrial bio-assays Correlationcircle axes 1-2 PCA plot of the data • Selected axes: • 1: 60.51% • 2: 15.99% • 3: 10.92%

  24. III. Results 2. Comparison of bio-assaysresponses Principal component analyses of terrestrial bio-assays Correlationcircle axes 1-2 PCA plot of the data • Selected axes: • 1: 60.51% • 2: 15.99% • 3: 10.92% • Results: • First axis: AMIOM toxicity, mainly due to high application rates of ashes of compostedmaterials and limedsewagesludge • Second axis: sensitivity of bio-assays, related to AMIOM • Plants biomassgroupedtogether, highersensitivity of Brassicarapa • Specificity of Avenasativarootselongation • Earthwormsendpointsgroupedtogether, highersensitivity of avoidancebehavior • Nematodeendpointsgroupedwith plants, highersensitivity of reproduction

  25. III. Results 2. Comparison of bio-assaysresponses Principal component analyses of aquatic bio-assays Correlationcircle axes 1-2 PCA plot of the data • Selected axes: • 1: 70.3% • 2: 17.6%

  26. III. Results 2. Comparison of bio-assaysresponses Principal component analyses of aquatic bio-assays Correlationcircle axes 1-2 PCA plot of the data • Selected axes: • 1: 70.3% • 2: 17.6% • Results: • First axe: sensitivity of bio-assays, mainly due to low application rates of organicmaterials • Second axis: alsolinked to bio-assayssensitivity • Bacteria, algae and crustaceanendpointsresponsesgroupedtogether, highestsensitivity of the algaePseudokirchneriellasubcapitata • Nematodeendpointsgroupedtogetheragainstotherorganisms (hormesis?), highersensitivity of reproduction

  27. CONTENT • Context • Materials and Methods • Results • Conclusion

  28. IV. Conclusion • AMIOM ecotoxicity • - AMIOM studiednot ecotoxicattheir respective application rates for all tests • - Toxicityassessmentonlyat high application rates • - Possibility of hormesiseffects, especially in aquatic tests for rotifer and nematode • Test strategy • - Terrestrial bio-assays more sensitive thanaquaticones • - For terrestrial tests: redundancy of plants biomassendpoints • - For aquatic tests: redundancy of bacteria and algaeendpoints • Nematoda • Organismhaving the lagestscale of responses (toxicity and hormesis) • No redundancywithotherorganisms • Highersensitivity in terrestrialthan in aquatic phase • Growth and reproduction endpointsshowedsame • tendencies, but reproduction more sensitive

  29. Work in progress… • Increase the number and diversity of AMIOM studied • Data acquisition for predaceous mite Hypoaspisaculeifer(Acaria) in terrestrial test battery • Get more data with the nematodeCaenorhabditiselegans to conclude on its relevance in the test batteries • Select optimal aquatic and terrestrial test strategy, to beincluded in furtherregulatoryassessment of AMIOM

  30. Thanks for your attention! ACKNOWLEDGEMENTS • The financial support from the French Ministry of Ecology, Sustainable Development, Transport and Housing and from the French Environment and Energy Management Agency (contract 0975C0061) is gratefully acknowledged. The Caenorhabditiselegans strain was provided by the CGC, which is funded by NIH Office Reasearch Infrastructure Programs (P40 OD010440).

  31. I. Context

  32. III. Results 1. Ecotoxicity of AMIOM Results of terrestrial tests:

  33. III. Results 1. Ecotoxicity of AMIOM Results of terrestrial tests: • Most sensitive endpoint: • Amonghigher plants: Avenasativarootelongation • Amongearthworm: Eiseniafetidaavoidancebehavior • Amongnematode: Caenorhabditiselegans reproduction

  34. III. Results 1. Ecotoxicity of AMIOM Results of terrestrial tests: Results of aquatic tests: • Most sensitive endpoint: • Amonghigher plants: Avenasativarootelongation • Amongearthworm: Eiseniafetidaavoidancebehavior • Amongnematode: Caenorhabditiselegans reproduction

  35. III. Results 1. Ecotoxicity of AMIOM Results of terrestrial tests: Results of aquatic tests: • Most sensitive endpoint: • Amonghigher plants: Avenasativarootelongation • Amongearthworm: Eiseniafetidaavoidancebehavior • Amongnematode: Caenorhabditiselegans reproduction • Most sensitive endpoint: • Amongnematodes: Caenorhabditiselegans reproduction • Among bio-assays: • Pseudokirchneriellasubcapitatagrowth

  36. III. Results 1. Ecotoxicity of AMIOM Results of terrestrial tests: Results of aquatic tests: Resultsoverview: • Most sensitive endpoint: • Amonghigher plants: Avenasativarootelongation • Amongearthworm: Eiseniafetidaavoidancebehavior • Amongnematode: Caenorhabditiselegans reproduction • Most sensitive endpoint: • Amongnematodes: Caenorhabditiselegans reproduction • Among bio-assays: • Pseudokirchneriellasubcapitatagrowth • For bothterrestrial and aquatic bio-assays, AMIOM studied not ecotoxicattheir respective application rates

  37. III. Results 1. Ecotoxicity of AMIOM Results of terrestrial tests: Results of aquatic tests: Resultsoverview: • Most sensitive endpoint: • Amonghigher plants: Avenasativarootelongation • Amongearthworm: Eiseniafetidaavoidancebehavior • Amongnematode: Caenorhabditiselegans reproduction • Most sensitive endpoint: • Amongnematodes: Caenorhabditiselegans reproduction • Among bio-assays: • Pseudokirchneriellasubcapitatagrowth • For bothterrestrial and aquatic bio-assays, AMIOM studied not ecotoxicattheir respective application rates • Terrestrial bio-assaysshowedhighersensitivitythanaquaticones

  38. II. Materials & Methods 3. Statistical analyses Objectives Statistical analyses • Ecotoxicity of the AMIOM studied • Hierarchy of the AMIOM studied • Interest of Caenorhabditiselegansin the test batteries

  39. II. Materials & Methods 3. Statistical analyses Objectives Statistical analyses • Ecotoxicity of the AMIOM studied • Hierarchy of the AMIOM studied • Interest of Caenorhabditiselegansin the test batteries • Significantdiferenceswith control, colorcodinghormesis as well as toxicity

  40. II. Materials & Methods 3. Statistical analyses Objectives Statistical analyses • Ecotoxicity of the AMIOM studied • Hierarchy of the AMIOM studied • Interest of Caenorhabditiselegansin the test batteries • Significantdiferenceswith control, colorcodinghormesis as well as toxicity • Hierarchical cluster analysis of AMIOM, properties Vs bio-assays

  41. II. Materials & Methods 3. Statistical analyses Objectives Statistical analyses • Ecotoxicity of the AMIOM studied • Hierarchy of the AMIOM studied • Interest of Caenorhabditiselegansin the test batteries • Significantdiferenceswith control, colorcodinghormesis as well as toxicity • Hierarchical cluster analysis of AMIOM, properties Vs bio-assays • Principle component analyses of bio-assays + Hierarchical cluster analysis of bio-assays

  42. III. Results 2. Hierarchy of the AMIOM studied Hierarchical cluster analyses of AMIOM using physico-chemicalproperties

  43. III. Results 2. Hierarchy of the AMIOM studied Hierarchical cluster analyses of AMIOM using physico-chemicalproperties Physico-chemicalhierarchy: - Deinkingsludge (DS) rankedwithcowmanure (CM) - Limedsewagesludge (LS) rankedwithcompostedwastes (CW) - LS, CW, DS and CM rankedaltogetheragainstashes of compostedmaterials (AC)

  44. III. Results 2. Hierarchy of the AMIOM studied Hierarchical cluster analyses of AMIOM using bio-assaysresponses Hierarchical cluster analyses of AMIOM using physico-chemicalproperties Physico-chemicalhierarchy: - Deinkingsludge (DS) rankedwithcowmanure (CM) - Limedsewagesludge (LS) rankedwithcompostedwastes (CW) - LS, CW, DS and CM rankedaltogetheragainstashes of compostedmaterials (AC)

  45. III. Results 2. Hierarchy of the AMIOM studied Hierarchical cluster analyses of AMIOM using bio-assaysresponses Hierarchical cluster analyses of AMIOM using physico-chemicalproperties Physico-chemicalhierarchy: - Deinkingsludge (DS) rankedwithcowmanure (CM) - Limedsewagesludge (LS) rankedwithcompostedwastes (CW) - LS, CW, DS and CM rankedaltogetheragainstashes of compostedmaterials (AC) • Bio-assayshierarchy: • Deinkingsludge (DS) rankedwithcowmanure (CM), altogetherwithcompostedwastes (CW) • Limedsewagesludge (LS) rankedwithashes of compostedmaterials (AC)

  46. III. Results 3. Interest of Caenorhabditiselegansin the test batteries Principal component analyses of terrestrial and aquatic bio-assays • Results: • Plants endpointsgroupedtogether (SBrasB, SAveR, SAveB) • Earthwormsendpointsgroupedtogether (SEisR, SEisA) • Nematodeendpoints in liquid phase for LUFA eluatesgroupedtogether (LCaeGL, LCaeRL) • Nematodeendpoints in solid phase groupedwithaquaticorganisms (SCaeG, SCaeR)

  47. III. Results 3. Interest of Caenorhabditiselegansin the test batteries Hierarchical cluster analyses of terrestrial bio-assays

  48. III. Results 3. Interest of Caenorhabditiselegansin the test batteries Hierarchical cluster analyses of terrestrial bio-assays • Hierarchy of bio-assays: • Earthwormendpointsresponses (EisAv and EisRep) rankedtogetherwithAvenasativarootselongation (AveRoo) • Nematodeendpointsresponses (CaeGro and CaeRep) rankedtogether • Plants endpointsresponsesrankedapartfromterrestrialorganisms

  49. III. Results 3. Interest of Caenorhabditiselegansin the test batteries Hierarchical cluster analyses of aquatic bio-assays

  50. III. Results 3. Interest of Caenorhabditiselegansin the test batteries Hierarchical cluster analyses of aquatic bio-assays • Hierarchy of bio-assays: • Algae (PseGro), bacteria (VibLum) and crustacean (DapMob) endpointsresponsesrankedtogether • Nematodegrowthendpointsresponses (CaeGroISO and CaeGroLUF) rankedtogether • Nematode reproduction endpointsresponses (CaeRepISO and CaeRepLUF) rankedtogether • Rotiferendpointresponses (BracRep) not rankedwithother test

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