Biomarker responses in earthworm coelomocyte extract - Noninvasively collected sample for pesticide effect assessment.

Earthworms are often used as model organisms in ecotoxicological research because of their natural habitat where they can be exposed to many different pollutants, including pesticides. Since a number of them has to be sacrificed for sample collection, it would be useful to develop non-invasive methods and techniques suitable for the analysis of target parameters. The aim of this study is to determine whether the coelomocyte extract, obtained by the non-invasive method, can be used to measure responses of biochemical biomarkers and to establish if it can be used in assessing the effects of pesticides already known to have a negative impact on the earthworms. In the present study Eisenia andrei earthworms were exposed for 48 h to organophosphates dimethoate and pirimiphos-methyl using the filter paper contact test. Following exposure, coelomocyte extracts were prepared and acetylcholinesterase (AChE) and carboxylesterase (CES) activities were measured. The percentage of inhibition of the measured enzymes in the coelomocyte extract was compared with the inhibition of the same enzyme activities in the samples obtained from the whole body homogenate. AChE and CES inhibition was observed at all concentrations for both pesticides in different types of samples. Compared to the coelomocyte extract, the level of AChE inhibition was slightly stronger in the whole body homogenate. Inhibition of CES at the same concentrations in different types of samples did not always coincide, especially in the case of dimethoate, however significant inhibition of CES in coelomocyte extract was recorded. This study indicates the possibility of using the coelomocyte extract for measurement of biochemical biomarkers and assessment of pesticide effects.

Integrated assessment of biochemical markers in premetamorphic tadpoles of three amphibian species exposed to glyphosate- and methidathion-based pesticides in single and combination forms.

In this study, we evaluated the toxic effects of a glyphosate-based herbicide (GBH) and a methidathion-based insecticide (MBI), individually and in combination, on premetamorphic tadpoles of three anuran species: Pelophylax ridibundus, Xenopus laevis, and Bufotes viridis. Based on the determined 96-h LC50 values of each species, the effects of a series of sublethal concentrations of single pesticides and their mixtures after 96-h exposure and also the time-related effects of a high sublethal concentration of each pesticide were evaluated, with determination of changes in selected biomarkers: glutathione S-transferase (GST), glutathione reductase (GR), acetylcholinesterase (AChE), carboxylesterase (CaE), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH). Also, the integrated biomarker response (IBR) was used to assess biomarker responses and quantitatively evaluate toxicological effects. Isozyme differences in CaE inhibition were assessed using native page electrophoresis; results showed that GBH to cause structural changes in the enzyme but not CaE inhibition in P. ridibundus. In general, single MBI and pesticide mixture exposures increased GST activity, while single GBH exposures decreased GST activity in exposed tadpoles. The AChE and CaE activities were inhibited after exposure to all single MBI and pesticide mixtures. Also, higher IBR values and GST, GR, AST, and LDH activities were determined for pesticide mixtures compared with single-pesticide exposure. This situation may be indicative of a synergistic interaction between pesticides and a sign of a more stressful condition.

Cholin- and carboxylesterase activities in developing zebrafish embryos (Danio rerio) and their potential use for insecticide hazard assessment.

Insecticides are a potential hazard for non-target organisms like fish particularly at run off events. The study of effects to embryos of the zebra fish Danio rerio is already an accepted tool in waste water monitoring, but effects of various groups of substances (like most pesticides) to the zebrafish embryo remain to be studied. Enzymes are often taken as biomarkers of exposure and effect. Therefore cholinesterase isozymes and carboxylesterase were examined for their suitability as biomarkers of insecticide exposure. The activities of cholinesterase and of carboxylesterase were monitored in the first 48 h post-fertilization (hpf) of zebrafish development. Significant specific activities in the range of 0.5-25 U could be measured from the sixth somite stage (12 h) up to the Long Pec stage (48 h) for different cholinesterases using acetyl-, acetyl-beta-methyl-, butyryl- and propionylthiocholin as substrates. The specific activity of carboxylesterase ranged from 4 to 16 Umg(-1) protein in the respective developmental stages. Substrate specificity was analysed using specific inhibitors (eserine sulphate, DPDA, BW284c51). The results showed that the observed cholinesterase activities in the whole embryo may be attributed mainly to acetylcholinesterase with a partial capability to use propionylthiocholine as a second substrate. The potential use of cholin- and carboxylesterase as biomarkers was investigated using the organophosphate paraoxon-methyl. A 40% inhibition of enzyme activities was reached by 0.4 microM paraoxon-methyl indicating the possible use of these enzymes as biomarkers of exposure.