Sandra Stepić

Effect of temephos on cholinesterase activity in the earthworm Eisenia fetida (Oligochaeta, Lumbricidae)

In this study, adult Eisenia fetida earthworms were exposed to the sub-lethal concentrations of temephos using the contact filter paper test procedure. Since temephos is an organophosphate pesticide, its effects on earthworms were determined by measuring ChE inhibition--a known biomarker of exposure. The ChE activity was measured after a short time of exposure--1 and 2 h. As expected, the lowest ChE activity (72.70% and 38.03% inhibition) was measured at the highest concentration of temephos (120 ng cm(-2)) applied. More interestingly, at the 0.12 ng cm(-2) concentration the ChE activity increased up to 36.28% of activity in the control in all three conducted experiments. Dose-response curves showed an inverted U-shape characteristic for hormesis. This hormetic-like effect could be important for health status of an earthworm.

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.

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.

Differences in enzymes (biomarkers) activities in fish after cage exposure in Drava and Danube Rivers (Croatia)

Biomonitoring using molecular biomarkers in fish isvery important in environmental risk assessment. Theneed for sensitive and reliable methods to assess theimpact of chemical pollution on the aquatic environ-ment has generated considerable interest in biochem-ical and physiological indices of contaminant expo-sure and effects in fish (Srrn et al. 1992). Two mo-lecular biomarkers were used in this study: EROD(etoxyresorufin-O-deethylase) and AChE (acetyl-cholinesterase). Induction of EROD activity is acommonly used biomarker of exposure to xenobi-otics such as polycyclic aromatic hydrocarbons(PAHs), biphenyls, dibenzodioxines and dibenzofu-rans. The intensiveness of induction is proportionalto their concentration in sediment (NennoNNe et al.l99l) and in water. Furthermore, inhibition ofAChEactivity is a biomarker of effect to organophosphateand carbamate pesticides, detergents and cadmium inwater, although some organic compounds simultane-ously are EROD inducers and AChE inhibitors (Gnl-cANr et al. 1992, PavNe et al. 1996, GurLueRurNo etal. 1998). We used the XAD-7 column adsorptiontechnique to exmct and concentrate lipophilic xeno-biotics (EROD inducers) from water samples andtook advantage of its ability to adsorb aromatic com-pounds such as PAHs and heterocyclic amines(HCA) with two or more fused rings. The extracts ob-tained from XAD-7 column were used in the labora-tory induct-test followed by EROD and AChE meas-urements in order to determine effects on their activ-ities.Key words: EROD; AChE; fish; Carassius auratusgibelio; Cyprinus carpio

Effects of formalin on some biomarker activities of earthworms pre-exposed to temephos

Despite its negative effects, formalin has been often used for the expulsion of earthworms due to its high efficiency; however it is not known whether it will affect any significant measurable molecular processes in sampled earthworms. The aim of this research was to investigate effects of formalin on the activities of chosen molecular biomarkers in Eisenia andrei earthworms previously exposed to temephos. Additionally, the inhibitory effect of temephos, hitherto evaluated only on laboratory-bred earthworm species, was confirmed on two earthworm species obtained from their natural environment -Dendrobaena octaedra and Lumbricus rubellus. Earthworms were first exposed to the sub-lethal concentration of temephos for 2h and then to formalin 15 min in order to simulate the sampling procedure. Besides acetylcholinesterase (AChE) inhibition - a known biomarker of exposure to organophosphate insecticides - the concentration of oximes and the activities of catalase (CAT) and efflux pump were measured. Results showed that in all species temephos caused inhibition of AChE and CAT activity. Exposure of E. andrei to formalin caused inhibition of AChE, however after post-exposure to formalin for 15 min significant increase in AChE activity was recorded. Similar results were obtained with the measurement of oximes concentrations. Exposure to only formalin and combination of temephos (2h) and formalin (15 min) led to an increase in the CAT activity. The obtained results showed that exposure to formalin during the sampling could affect measured molecular biomarkers and also may change effects caused by exposure to temephos.

Detection of mutagens and BaPMO inducers in river water using the Blue Cotton adsorption technique.

Induction of Mixed Function Oxidase (MFO) activity and bioactivation potential were measured in experimental carp and in native fish species from two rivers with different pollution level (Sava and Mrežnica). The experimental carp were intraperitoneally exposed to various water volume equivalents of Blue Cotton extracts from both rivers. The induction of MFO measured as a Benzo(a)pyrene monooxygenase (BaPMO) activity was increased up to 9.3‐fold in experimental carp and up to 11.3‐fold in native fish from Sava River, whereas the values from Mrežnica River showed only a slight increase when compared with the control (highest increase of 1.8‐fold in nose carp). Accordingly, bioactivation potential using modified Ames test was higher in both experiments with Sava River. Both measured parameters in experimental carp increased in a dose‐dependent manner in accordance to river volume equivalents. Different induction potential of native fish species comparable between two rivers confirmed the known possibility of their usage in biomonitoring studies. These results give qualitatively a new support to the idea of using Blue Cotton extraction technique combined with induction of MFO activity and Ames test in fish as a good biomarker for assessing risk of exposure to mutagens/carcinogens such as Polycyclic Aromatic Hydrocarbons (PAHs), especially due to the simplicity and cost‐effectiveness of these methods.