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NATO SfP 982590 project workshop, Dubrovnik, Croatia – Sep 23-26, 2010 Characterisation of hazardous chemical contamination – from environmental chemistry and toxicology to risk assessment Effect of organophosphates malathion and temephos on cholinesterase activity in the earthworm Eisenia fetida (Oligochaeta, Lumbricidae) Mirna Velki, Sandra Stepić, Davorka Jarić, Branimir K. Hackenberger Department of Biology, Josip Juraj Strossmayer University in Osijek, Ljudevita Gaja 6, HR-31000 Osijek, Croatia Earthworms common test organisms and good indicator species for ecotoxicological research they have highly differentiated organs and tissues and an immune system that is comparable with that of vertebrates they can be exposed to pesticides either through dermal contact or by ingestion Biomarkers many biomarkers have been identified in environmental pollution dealing with pesticides and organochlorine contaminants acetylcholinesterase (AChE) activity is routinely used as a biomarker of the exposure to organophosphates and carbamates compounds many unsolved questions in biomarker applicability in real time environmental pollution monitoring effects of small so called sublethal doses Experiment adult earthworms (Eisenia fetida) were exposed to the sub-lethal concentrations of organophosphates malathion and temephos contact filter paper test procedure ChE activity was determined according to the method of Ellman et al. (1961) Experiment Malathion one of the most often used organophospahte insecticide in the world insecticide and acaricide with contact, gut and respiratory action in insects and mammals is metabolically converted to its structurally-similar metabolite, malaoxon malaoxon inhibits AChE resulting in respiratory, myocardial and neuromuscular transmission impairment structure of malathion Experiment Temephos nonsystemic organophosphorus insecticide used to control mosquito, midge, and black fly larvae it is used in lakes, ponds, and wetlands only organophosphate with larvicidal use temephos affects the central nervous system through inhibition of cholinesterase in larvae, this results in death before reaching the adult stage structure of temephos Experiment PESTICIDE CLASS OF PESTICIDE TYPE OF PESTICIDE MODE OF ACTION MALATHION organophosphate acaricide, insecticide indirect inhibitor of cholinesterase TEMEPHOS organophosphate insecticide (larvicidae) cholinesterase inhibitor Experiment PRELIMINARY EXPERIMENT Determination of EC50 and LC50 values Choosing the concentrations for final experiments FINAL EXPERIMENT Determination of ChE activity MALATHION TEMEPHOS 0.05 ng/cm2 0.1 ng/cm2 0.2 ng/cm2 0.4 ng/cm2 0.6 ng/cm2 1 ng/cm2 4 ng/cm2 8 ng/cm2 0.025 ng/cm2 0.01 ng/cm2 0.05 ng/cm2 0.12 ng/cm2 0.25 ng/cm2 0.05 ng/cm2 1.24 ng/cm2 2.5 ng/cm2 Results Concentration-response curves for 24, 48, and 72 h of exposure to different temephos concentrations with mortality as an endpoint, calculated from the first-preliminary experiment. Curves representing: (white) 24h, (grey ) 48 h, and (black) 72 h of exposure. Results Activity of ChE measured in E. fetida earthworms after exposure to different concentrations of temephos for 2 h. *Significantly different vs. control activity (p<0.05). Results Activity of ChE (nmol min-1 mg-1) 50 * 45 * 40 35 30 * 25 20 15 10 5 0 0 0.05 0.1 0.2 0.4 0.6 1 4 8 Concentration of malathion (ng cm -2) Activity of ChE measured in E. fetida earthworms after exposure to different concentrations of malathion for 2 h. *Significantly different vs. control activity (p<0.05). low dose stimulation, high dose inhibition opposite effect in small doses (sub-lethal, sub-effect) compared to large doses Role of hormetic effect? Importance of the hormetic effect? INTERPRETATION OF RESULTS, ESPECIALLY IN INTERPRETATION OF ENVIRONMENTAL MONITORING ↓ ENVIRONMENTAL RISK ASSESSMENT Response to: Low-level concentrations Time! Objective of biomonitoring early detection of the biologically relevant concentrations of pollutants early detection of the effect of pollutants on biological systems Important to detect any variation from BENCHMARK VALUES for test organisms Mathematical models which describe response on presence of pollutant often show high sensitivity on so called starting conditions ↓ Mixtures! Is hormesis doubtful? Why is hormetic effect difficult to prove? Small concentrations! (researchers usually work with concentrations in range of expected response) Biomarkers often show relatively large variance (especially measured in situ organisms; in vivo) ↓ using cell cultures could result in more accurate results and more precise determination of hormetic effect Hormetic effect is NOT doubtful! 1. 2. 3. 4. 5. 6. Hose, J.E, Puffer, H.W., 1984. Oxygen consumption rates of grunion (Leuresthes tenuis) embryos exposed to the petroleum hydrocarbon, benzo[a]pyrene. Environ. Res. 35, 413-420. 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Conclusions Some dose-response curves can show an inverted Ushape characteristic for hormesis as it was seen by temephos and malathion in earthworms. This hormetic-like effect could be important for health status of an earthworm as well for correct interpretation of biomonitoring results. Conclusions The significance of hormetic effect detection: 1) in environmental biomonitoring, where very low concentrations are usually expected, it is obvious that hormetic effect can theoretically be anticipated as a marker of exposure to such concentrations 2) when earthworms are used for toxicological parameters research, models that include hormetic effect should be taken into account THANK YOU FOR YOUR ATTENTION! Questions... ?