1. PHAGOCYTIC ACTIVITY OF EARTHWORM COELOMYCYTES USED AS A SUBLETHAL BIOMARKER OF METAL TOXICITY Sauvé S, Hendawi M, Brousseau P, Fournier M Département de chimie – Université de Montréal INRS-Institut Armand Frappier-Santé Humaine

2. © Sauvé 2001 Earthworms Earthworms lead an essential ecological role for maintaining soil health, fertility, structure, nutrient cycling, … Standard worm assays evaluate lethality Sublethal markers such as reproduction and immunocompetence can be quantified

3. © Sauvé 2001 Worm species Terrestrial earthworms  Lumbricus terrestris  Eisenia fetida  Aporrectodea turgida Aquatic worm  Tubifex tubifex

4. © Sauvé 2001 Cell Extrusion By puncture with pasteur pipette or syringe Ethanol extrusion Electrical stimulation Ultrasound Extruded cells are suspended in a Lumbricus Basal Salt Solution (LBSS)

5. © Sauvé 2001 Species-specific parameters E. fetidaL. terrestrisA. turgidaT. tubifex Fresh weight (g) 0.374.240.540.005 Total cells / animals (10 6 ) 2.6513.43.280.14 Viability (%) Trypan Blue 81.987.477.776.2 Viability (%) PI66.374.890.974.1

6. © Sauvé 2001 Phagocytosis Earthworm coelomocytes (Eisenia fetida)

7. © Sauvé 2001 Flow Cytometry

8. © Sauvé 2001 Phagocytosis SpeciesControl Toxic (10 -4 M HgCl 2 ) L. terrestris E. fetida

9. © Sauvé 2001 Silver and Arsenic

10. © Sauvé 2001 Cadmium and Copper

11. © Sauvé 2001 Mercury

12. Lead and Zinc

13. © Sauvé 2001 In vitro Toxicity thresholds Compound EC 50 (mg L -1 ) AgNO 3 6.6 Na 2 HAsO 4 ND CdCl 2 2.4 CoCl 2 ND KCr 2 O 7 ND CuCl 2 1.5 HgCl 2 1.0 Compound EC 50 (mg L -1 ) CH 3 HgCl0.4 MoCl 4 47.5* NiCl 2 6.4 PbCl 2 134* NaSeO 3 51447* ZnCl 2 24853* ND = not detected * = extrapolated

14. © Sauvé 2001 Transfer in vitro to in vivo In vitro cellular exposure Petri dish, solution exposure Soil incubation (using the OECD reproduction protocol) Field, in situ exposure in mesocosm « worm bags » Some recent field applications to the evaluation of cement factory emissions