Adaptation strategies of soil biodiversity (earthworms) to pesticides: physiological mechanisms and soil ecological implications

Transcription

1 Adaptation strategies of soil biodiversity (earthworms) to pesticides: physiological mechanisms and soil ecological implications Nicolas Givaudan, Claudia Wiegand, Barbara Le Bot, David Renault, Frédérique Pallois, Stéphanie Llopis, Françoise Binet

2 CONTEXT AND OBJECTIVES Adaptation strategies of soil biodiversity (earthworms) to pesticides: physiological mechanisms and soil ecological implications

3 Context Earthworms: ecosystem «engineers» Jones et al, 1997 Ecology Formation of soil structure (porosity) Organic matter and nutrient turnover Stimulation of microbial activity Decrease of lumbricids populations in intensively cultivated plots Conventional wheat/corn crop 107 ind/m 2 Organic leguminous crop 344 ind/m 2 Smith et al, 2008 Soil & Tillage Research They face chronic pesticide exposition(fungicides, insecticides, herbicides, growth regulators)

4 Context Soil residues (ng.g -1 ) of an herbicide and a fungicide in five agricultural fields Bretagne, France Active molecule Conventional 1 Conventional 2 Conventional 3 Organic Pasture Alachlore 2.9 [± 0.6] 4.2 [± 0.4] 8.8 [± 3.1] nd nd Epoxiconazole 11.1 [± 2.7] 4.4 [± 1.1] 4.2 [± 0.5] nd nd (nd = not detected) Givaudan et al, 2012 Regular applications of pesticides on crops wheat: 6 pesticide applications / year 2-3 fungicides, 2 herbicides, 1 growth regulator Pesticide losses (not reaching target) + persistency = accumulation as residues in the soil

5 Objectives Organic cropped field Conventional cropped field Is there physiological adaptation to pesticides in earthworms from conventional cropped field? Does it have consequences for the soil ecosystem? Adaptation to heavy metals Lumbricus rubellus Simonsen and Scott-Fordsmand, 2003 Fisker et al, 2011 Dendrobaena octaedra Bengtsson, 1992 Genetic variation of esterase enzymes Higher Cd-binding capability Higher growth rate

6 MATERIALS AND METHODS Adaptation strategies of soil biodiversity (earthworms) to pesticides: physiological mechanisms and soil ecological implications

7 Materials and methods Two A. caliginosa populations Organic field (reference) Epoxiconazole Not detected Fungicide-exposed groups (7, 28 days) Experimental exposure to epoxiconazole (OPUS ) Epoxiconazole 11.1 ± 2.7 ng.g -1 ) Control groups (0, 7, 28 days) Conventional field (pre-exposed) Aporrectodea caliginosa An endogeic species Adults and sub-adults sampled from the fields Individual worm exposure in soil microcosm predicted field concentration = 100 ng.g -1 dry soil 25% humidity, T 12 C Time exposure 0, 7, 28 days

8 Biological parameters Methodology At each 0,7,28 sampling days and for the control and exposed groups Soil parameters Pesticide fate 1. Live worm out of the soil Respiration (CO 2 production) micro gas chromatograph 2. Freeze-drying and grinding (Liquid-liquid extraction-lc-ms) Cast production = burrowing activity Capowiez et al, 2010 Energy resources Glycogen Total proteins Total lipids Metabolomics by GC-MS 22 metabolites

9 RESULTS Adaptation strategies of soil biodiversity (earthworms) to pesticides: physiological mechanisms and soil ecological implications

10 mg glycogen g -1 worm (DW) Results : glycogen and respiration CO 2 production Glycogen levels # 70 µg CO2 g -1 worm (FW) hour * * # days days Pre-exposed earthworms (conventional field) Fungicide-exposed Control (clean soil) Naïve earthworms (organic field) Fungicide-exposed Control (clean soil) Respiration is increased in fungicide-exposed groups after 7 and 28 days (*) Glycogen is decreased in both populations but with a temporal delay

11 Results : metabolomics Molecules detected and classification Hydrophilic Amino-Acids Asparagine Aspartate Lipophilic Amino-acids Alanine Isoleucine Leucine Methionine Phenylalanine Proline Valine Sugars Ribose Mannose Glucose Organic acids (citric acid circle, energy production) Aminobutyrate Citrate Fumarate Lactate Succinate 22 molecules detected Data analysis: a) PCA multivariate analysis b) Functional group responses Neutral Amino-Acids Glycine Serine Threonine Urea cycle metabolites Ornithine Putrescine

12 (11% var) (23% var) Results : metabolomics Axis 2 vs 3 Axis 1 vs 2 Clear pesticide effect on metabolome (21% var) (38% var) Naïve metabolome changes along with time but not with pesticide Scores plots (metabolic profiles) of principal component analyses (3 axes, 70% variability explained) realised on the whole metabolomic dataset for each population (22 variables). Crosses= mean scores ± Std Error of the Mean (SEM)

13 Functional group responses to epoxiconazole = % percentage of mean control value Results : metabolomics Increase in most amino acids and one organic acid in the conventional population when exposed Increase in putrescine in both populations Amino-acids: neutral hydrophilic lipophilic Sugars: Glucose Mannose Ribose Organic acids: Aminobutyrate Citrate Fumarate Lactate Succinate Urea cycle compounds: Ornithine Putrescine

14 Pesticide levels in the soil Results : soil Cast production * Soil containing naïve worms Soil containing no worms (natural dissipation) Soil containing pre-exposed worms Increased pesticide disappearance with pre-exposed earthworms * Cast Production in mg (dry cast) g -1 worm (fresh weight) day Pre-exposed population # ** Day 7 Day 28 Naïve population Control Epoxiconazole Changes in burrowing activity only under fungicide exposure: rapid increase after seven days of pre-exposed earthworms

15 SUMMARY AND CONCLUSION Adaptation strategies of soil biodiversity (earthworms) to pesticides: physiological mechanisms and soil ecological implications

16 Summary: Responses of Earthworms to Epoxiconazole Conventional crooped soil 11.1 ng g -1 Organic cropped soil Not detected Respiration rate (28 days) Glycogen reserve (7 days) Epoxi Ctrl (28 days) Metabolic profiles Epoxi Ctrl (7/12) (Putrescine) Amino-acids Urea cycle compounds (Putrescine) (7 days) (28 days) Cast production (28 days) Pesticide disappearance

17 Conclusion Chemical stress response in both naïve and conventional populations (i.e, respiration rate or putrescine concentrations) But differences in handling of energetic processes (delay in glycogen use or different metabolic patterns) evidence of physiological adaptation Changes in burrowing behaviour of the pre-exposed earthworms With significant enhanced pesticide disappearance All together our findings evidence that earthworms have developped physiological adaptation to cope with chronic chemical exposures that are installed in agricultural landscapes, and this mechanism lead to a difference in burrowing behaviour. Questions that need to be adressed : What is the Link between physiological adaptation and increased burrowing? Compensatory mechanism? What are the ecological consequences for the soil?

18 THANK YOU FOR YOUR ATTENTION

19 THANK YOU FOR YOUR ATTENTION

20 Supplementary results Initial population parameters (weight, glycogen, proteins, lipids, CO 2 ) Weight change in % initial weight