Mirna Velki

Oxidative stress elicited by insecticides: A role for the adipokinetic hormone

Adipokinetic hormones (AKHs) are insect neuropeptides responding to stress situations including oxidative stress. Two insecticides - endosulfan and malathion - were used to elicit oxidative stress conditions in the firebug Pyrrhocoris apterus, and the physiological functions of AKHs and their ability to activate protective antioxidative reactions were studied. The insecticide treatments elicited only a slight increase of the AKH level in CNS, but more intensive increase in haemolymph, which indicates an immediate involvement of AKH in the stress response. The treatment also resulted in a significant increase of catalase activity in the bugs body and depletion of the reduced glutathione pool in the haemolymph, however, co-application of the insecticides with the AKH (80 pmol) reduced the effect. It has also been found that co-application of the insecticides with AKH increased significantly the bug mortality compared to that induced by the insecticides alone. This enhanced effect of the insecticides probably resulted from the stimulatory role of AKH on bug metabolism: the carbon dioxide production was increased significantly after the co-treatment by AKH with insecticides compared to insecticide treatment alone. It was hypothesized that the increased metabolic rate could intensify the insecticide action by an accelerated rate of exchange of metabolites accompanied by faster penetration of insecticides into tissues.

Species-specific differences in biomarker responses in two ecologically different earthworms exposed to the insecticide dimethoate

Earthworms ingest large amounts of soil and therefore are continuously exposed to contaminants through their alimentary surfaces. Additionally, several studies have shown that earthworm skin is a significant route of contaminant uptake as well. In order to determine effects of dimethoate, a broad-spectrum organophosphorous insecticide, two ecologically different earthworm species were used - Eisenia andrei and Octolasion lacteum. Although several studies used soil organisms to investigate the effects of dimethoate, none of these studies included investigations of dimethoate effects on biochemical biomarkers in earthworms. Earthworms were exposed to 0.001, 0.005, 0.01, 0.5 and 1 μg/cm(2) of dimethoate for 24 h, and the activities of acetylcholinesterase, carboxylesterase, catalase and efflux pump were measured. In both earthworm species dimethoate caused significant inhibition of acetylcholinesterase and carboxylesterase activities, however in E. andrei an hormetic effect was evident. Efflux pump activity was inhibited only in E. andrei, and catalase activity was significantly inhibited in both earthworm species. Additionally, responses of earthworm acetylcholinesterase, carboxylesterase and catalase activity to dimethoate were examined through in vitro experiments. Comparison of responses between E. andrei and O. lacteum has shown significant differences, and E. andrei has proved to be less susceptible to dimethoate exposure.

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.

Changes in exposure temperature lead to changes in pesticide toxicity to earthworms: A preliminary study

The occurring climate changes will have direct consequences to all ecosystems, including the soil ecosystems. The effects of climate change include, among other, the changes in temperature and greater frequency and intensity of extreme weather conditions. Temperature is an important factor in ecotoxicological investigations since it can act as a stressor and influence the physiological status of organisms, as well as affect the fate and transport of pollutants present in the environment. However, most of so far conducted (eco)toxicological investigations neglected the possible effects of temperature and focused solely on the effects of toxicants on organisms. Considering that temperature can contribute to the toxicity of pollutants, it is of immense importance to investigate whether the change in the exposure temperature will impact the strength of the toxic effects of pollutants present in soil ecosystems. Therefore, in the present study the toxicity of several commonly used pesticides to earthworms was assessed under different exposure temperatures (15, 20 and 25°C). The results showed that changes in exposure temperature lead to changes in susceptibility of earthworms to particular pesticides. Namely, exposures to the same pesticide concentration at different temperatures lead to different toxicity responses. Increase in exposure temperature in most cases caused increase in toxicity, whereas decrease in temperature mostly caused decrease in toxicity. This preliminary study points to need for an in-depth investigation of mechanisms by which temperature affects the toxicity of pesticides and also provides important data for future research on the effects of temperature change on the soil ecosystems.

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.

Investigation of the soluble metals in tissue as biological response pattern to environmental pollutants (Gammarus fossarum example).

In the present study, Gammarus fossarum was used to investigate the bioaccumulation and toxic effects of aquatic pollutants in the real environmental conditions. The novelty of the study is the evaluation of soluble tissue metal concentrations in gammarids as indicators in early assessment of metal exposure. In the Sutla River, industrially/rurally/agriculturally influenced catchment in North-Western Croatia, physico-chemical water properties pointed to disturbed ecological status, which was reflected on population scale as more than 50 times lower gammarid density compared to the reference location, Črnomerec Stream. Significantly higher levels of soluble toxic metals (Al, As, Cd, Pb, Sb, Sn, Sr) were observed in gammarids from the Sutla River compared to the reference site and reflected the data on higher total dissolved metal levels in the river water at that site. The soluble metal estimates were supplemented with the common multibiomarker approach, which showed significant biological responses for decreased acetylcholinesterase activity and increased total soluble protein concentrations, confirming stressed environmental conditions for biota in the Sutla River. Biomarker of metal exposure, metallothionein, was not induced and therefore, toxic effect of metals was not confirmed on molecular level. Comparable between-site pattern of soluble toxic metals in gammarids and total dissolved metal levels in water suggests that prior to biomarker response and observed toxic impact, soluble metals in tissue might be used as early warning signs of metal impact in the aquatic environment and improve the assessment of water quality.

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.

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.