Davorka K. Hackenberger

Effects of individual and binary-combined commercial insecticides endosulfan, temephos, malathion and pirimiphos-methyl on biomarker responses in earthworm Eisenia andrei.

Laboratory tests were conducted in order to investigate the effects of individual and binary-combined commercial insecticides endosulfan, temephos, malathion and pirimiphos-methyl on the earthworm Eisenia andrei. The effects of individual insecticides were determined by measuring the activities of acetylcholinesterase (AChE), catalase (CAT) and glutathione-S-transferase (GST). After exposure to studied insecticides, dose-dependent decrease in AChE activity and dose-dependent increase in CAT activity was recorded. The activity of GST was without consistent dose-response reaction, but generally the investigated insecticides caused the increase in GST activity. In order to determine the effects of binary-combined mixtures, and interactions between the components in the mixture, the relationship between effective concentration of AChE inhibition for mixture and effective concentration of AChE inhibition for each component in the mixture was investigated. The obtained results showed additive effect for mixtures endosulfan+malathion; endosulfan+pirimiphos-methyl; temephos+malathion and temephos+pirimiphos-methyl, synergistic effect for mixture endosulfan+temephos and in the case of mixture malathion+pirimiphos-methyl the antagonistic effect was indicated.

Application of microcosmic system for assessment of insecticide effects on biomarker responses in ecologically different earthworm species

Earthworms from different ecological categories--epigeic Eisenia andrei and Lumbricus rubellus, endogeic Octolasion lacteum and anecic Lumbricus terrestris--were exposed in a microcosmic system to three commonly used insecticides. The effects of the insecticides were evaluated by measuring the following molecular biomarkers-the activities of AChE, CES, CAT, GST and the concentration of GSH. The results showed that environmentally relevant doses of organophosphates dimethoate and pirimiphos-methyl significantly affected the measured biomarkers, whereas pyrethroid deltamethrin did not affect the earthworms at the recommended agricultural dose. Considering the ecological category of earthworms, the results were inhomogeneous and species-specific differences in the biomarker responses were recorded. Since the biomarker responses of the investigated earthworm species were different after exposure to organophosphates in a microcosm compared to the exposure via standardized toxicity tests, two types of species sensitivity should be distinguished-physiological and environmental sensitivity. In addition, the hormetic effect of organophosphates on AChE and CES activities was recorded. The detection of hormesis in a microcosm is of great importance for future environmental research and soil biomonitoring, since in a realistic environment pollutants usually occur at low concentrations that could cause a hormetic effect. The results demonstrate the importance of the application of microcosmic systems in the assessment of the effects of environmental pollutants and the necessity of taking into account the possible differences between physiological and environmental species sensitivity.

First evidence for the presence of efflux pump in the earthworm Eisenia andrei.

Efflux pumps are transport proteins involved in the extrusion of toxic substrates from cells to the external environment. Activities of efflux pumps have been found in many organisms, however such activity has not been evidenced in earthworms. Adult Eisenia andrei earthworms were exposed to efflux modulators - verapamil (a known inhibitor of efflux pump protein) and dexamethasone (a known inducer of efflux activity) - and the amount of absorbed fluorescent dye rhodamine B was measured. The results showed that verapamil inhibited efflux activity and decreased removal of rhodamine B, whereas dexamethasone induced efflux activity and increased removal of rhodamine B. This is the first evidence of the presence of efflux pump in earthworm Eisenia andrei. Since earthworms are often used as test organisms due to their sensitive reactions towards environmental influences, the discovery of efflux pump activity can contribute to the better understanding of toxicity of certain pollutants.

Potentiation effect of metolachlor on toxicity of organochlorine and organophosphate insecticides in earthworm Eisenia andrei.

Acetylcholinesterase, glutathione-S-transferase and catalase activities were determined in earthworms Eisenia andrei exposed to insecticides (endosulfan, temephos, malathion, pirimiphos-methyl) alone and in a binary combination with the herbicide metolachlor. Metolachlor individually was not acutely toxic, even at high concentrations applied; however, in the treated earthworms metolachlor enhanced the toxicity of endosulfan and temephos by significantly reducing the acetylcholinesterase activity. In binary combination with malathion and pirimiphos-methyl, metolachlor did not increase toxicity. The potentiation character of metolachlor is specific rather than general, and probably depends on the chemical structure of pesticides in the mixture.

Influence of soil temperature and moisture on biochemical biomarkers in earthworm and microbial activity after exposure to propiconazole and chlorantraniliprole

Predicted climate change could impact the effects that various chemicals have on organisms. Increased temperature or change in precipitation regime could either enhance or lower toxicity of pesticides. The aim of this study is to assess how change in temperature and soil moisture affect biochemical biomarkers in Eisenia fetida earthworm and microbial activity in their excrements after exposure to a fungicide - propiconazole (PCZ) and an insecticide - chlorantraniliprole (CAP). For seven days, earthworms were exposed to the pesticides under four environmental conditions comprising combinations of two different temperatures (20°C and 25°C) and two different soil water holding capacities (30% and 50%). After exposure, in the collected earthworm casts the microbial activity was measured through dehydrogenase activity (DHA) and biofilm forming ability (BFA), and in the postmitochondrial fraction of earthworms the activities of acetylcholinesterase (AChE), catalase (CAT) and glutathione-S-transferase (GST) respectively. The temperature and the soil moisture affected enzyme activities and organisms response to pesticides. It was determined that a three-way interaction (pesticide concentration, temperature and moisture) is statistically significant for the CAT and GST after the CAP exposure, and for the AChE and CAT after the PCZ exposure. Interestingly, the AChE activity was induced by both pesticides at a higher temperature tested. The most important two-way interaction that was determined occurred between the concentration and temperature applied. DHA and BFA, as markers of microbial activity, were unevenly affected by PCZ, CAP and environmental conditions. The results of this experiment demonstrate that experiments with at least two different environmental conditions can give a very good insight into some possible effects that the climate change could have on the toxicity of pesticides. The interaction of environmental factors should play a more important role in the risk assessments for pesticides.

Effect of different river flow rates on biomarker responses in common carp (Cyprinus carpio).

The present study investigated effects of different river flow rates on basal activities of selected biomarkers and the occurrence of oxidative stress in the common carp (Cyprinus carpio). Juvenile carp were exposed to different river flow rates (5-120 cm/s) by caging for 3 weeks. After this period, one half of the fish were sacrificed and used for analysis. The other half received a single intraperitoneal injection of 3-methylcholanthrene (3-MC) and after 6 days were sacrificed and used for analysis. In order to investigate whether the physical activity of carp in the environment will influence the condition status of carp, following biomarkers were measured - activities of glutathione S-transferase (GST), catalase (CAT) and ethoxyresorufin-O-deethylase (EROD) and concentration of protein carbonyls (PC). The results showed that different flow rates significantly influenced biochemical biomarkers. The basal activity of GST did not change significantly after exposure to different river flow rates, whereas the activity of CAT increased with increasing river flow rates. The application of 3-MC caused significant increases in GST and CAT activities, but there were no difference between 3-MC control and 3-MC different flow rates. The occurrence of oxidative stress as a result of exposure to increased physical activity, i.e. increased river flow rates, was confirmed by measurement of PC levels - the level of PC increased with increasing river flow rates. Measurement of EROD basal activity showed that at lower river flow rates the EROD activity increased and at higher river flow rates decreased towards control levels demonstrating a close relationship between oxidative stress, PC levels and EROD activity. Obviously, biomarker responses in carp of different condition status can differ substantially. It can be concluded that flow rate may be an important factor in biomonitoring of rivers using biomarkers and since at different locations river water flow rate can vary significantly, the site selection is extremely important for proper design of river biomonitoring studies involving caging.