Accumulation of heavy metals from single and combined olive mill wastewater and pomace in soil and bioaccumulation in tissues of two earthworm species: Endogeic (Aporrectodea trapezoides) and Epigeic (Eisenia fetida).

Soil and earthworms are threatened by anthropogenic contamination resulting from olive mill waste dumping on the soil due to their pollutant properties. While several studies have explored the effects of olive mill waste on soil properties and the accumulation of heavy metals in soil, there is currently a gap in the literature regarding the potential bioaccumulation of heavy metals from olive mill waste in earthworms. In this study, soil with earthworms from two ecological categories (endogeic: Aporrectodea trapezoides and epigeic: Eisenia fetida) was treated with increasing doses of olive mill wastewater (OMWW) and olive mill pomace (OMP), applied individually or combined, in an indoor experiment in plastic containers, under laboratory conditions. The results revealed the presence of significant concentrations of heavy metals in the two types of wastes ranging as follows: Fe˃ Zn˃ Cu˃ Cd˃ Cr for OMWW, and Fe˃ Zn˃ Cu˃ Cr for OMP (with Cd below the detection limit). The study demonstrated distinct effects of OMWW and OMP, both individually and in combination, on soil heavy metal content, ranging as follows: soil OMWW > soil Combination > soil OMP for Cd; soil Combination > soil OMWW > soil OMP for Cr and Fe; and soil Combination > soil OMP > soil OMWW for Cu and Zn. Additionally, our investigation showed that both earthworm species exhibited significant uptake of these metals into their tissues, particularly the endogeic species. Interestingly, the most significant difference between species was in the accumulation of Cu, with the epigeic species accumulating significantly lower amounts.

Comprehensive study of the effects of strobilurin-based fungicide formulations on Enchytraeus albidus.

Excessive application of fungicides in crop fields can cause adverse effects on soil organisms and consequently affect soil properties. Existing knowledge on the effects of strobilurin fungicides has been primarily based on toxicity tests with active ingredients, while the effects of fungicide formulations remain unclear. Therefore, this work aims to provide new data on the effects of three commercial formulations of strobilurin fungicides on the soil organism Enchytraeus albidus. The tested fungicide formulations were Retengo® (pyraclostrobin-PYR), Zato WG 50® (trifloxystrobin-TRI) and Stroby WG® (kresoxim-methyl-KM). In laboratory experiments, multiple endpoints were considered at different time points. The results showed that PYR had the greatest impact on survival and reproduction (LC50 = 7.57 mga.i.kgsoil-1, EC50 = 0.98 mga.i.kgsoil-1), followed by TRI (LC50 = 72.98 mga.i.kgsoil-1, EC50 = 16.93 mga.i.kgsoil-1) and KM (LC50 = 73.12 mga.i.kgsoil-1, EC50 ≥ 30 mga.i.kgsoil-1). After 7 days of exposure, MXR activity was inhibited at the highest concentration of all fungicides tested (6 mgPYRkgsoil-1, 15 mgTRIkgsoil-1 and 30 mgKMkgsoil-1). Furthermore, oxidative stress (induction of SOD, CAT and GST) and lipid peroxidation (increase in MDA) were also observed. In addition, there was a decrease in total available energy after exposure to PYR and KM. Exposure to fungicides resulted in a shift in the proportions of carbohydrates, lipids, and proteins affecting the amount of available energy. In addition to the initial findings on the effects of strobilurin formulations on enchytraeids, the observed results suggest that multiple and long-term exposure to strobilurin formulations in the field could have negative consequences on enchytraeid populations.

Toxicity of fungicide azoxystrobin to Enchytraeus albidus: Differences between the active ingredient and formulated product.

Due to the often-excessive usage of fungicides, increasing attention is being paid to their impact on soil and non-target organisms. Risk assessments are usually based on the pure active ingredient and not on the formulated products applied in the environment. The aim of this study was therefore to investigate how azoxystrobin, the best-selling strobilurin fungicide, affects non-target soil organisms Enchytraeus albidus. To investigate the effects of the different types of azoxystrobin, E. albidus was exposed to the pure active ingredient, AZO_AI, and the formulated product, AZO_FP. Survival, reproduction, and molecular biomarkers of E. albidus were determined for different exposure durations (seven and 21 days). AZO_FP (LC50 = 15.3 mga.i./kg) showed a slightly stronger effect on survival than AZO_AI (LC50 = 16.8 mga.i./kg), yet the impact on reproduction was much stronger. Namely, while the tested concentrations of AZO_AI (EC50≥ 8 mga.i./kg) had almost no effect on reproduction, AZO_FP (EC50 = 2.9 mga.i./kg) significantly inhibited reproduction in a dose-dependent manner. Changes in enzyme activities (superoxide dismutase, catalase, glutathione-s-transferase) and malondialdehyde levels in both treatments indicated oxidative stress. Although AZO_FP had a stronger negative effect, the impact depended on the exposure time and the tested concentration. The higher toxicity of AZO_FP was a consequence of increased bioavailability and activity of the active ingredient due to the presence of adjuvants. Overall stronger adverse effects of AZO_FP suggest that the toxicity of azoxystrobin in the agricultural environment on the enchytraeid population may be underestimated. Furthermore, the results of this study highlighted the importance of comparing the toxicity of the active ingredient and the formulated product.

Multidisciplinary Approach to Agricultural Biomass Ash Usage for Earthworks in Road Construction.

Agricultural biomass has great bioenergy potential due to its availability, and it is a carbon-free energy source. During biomass incineration, biomass ash is formed, which is still considered as a waste without proper disposal and management solutions. Various biomass ash utilization options were investigated, mainly concerning engineering issues (the mechanical characterization of newly produced building materials or products), and there is a lack of knowledge of environmental issues arising from this "waste" material utilization in civil engineering practice. The main aim of this research is discussion of a different agricultural biomass characteristics as a fuel, the impact of agricultural biomass ashes (ABA) on the mechanical properties of stabilized soil with a particular emphasis on the environmental impacts within this kind of waste management. The results of this study indicate improved geotechnical characteristics of low-plasticity clay stabilized by lime/ABA binder. In addition to mechanical characterization for materials embedded in road embankments and subgrades, appropriate environmental risk assessment needs to be performed, and the results of this study indicate that the amount of ABAs added to the soil for roadworks should not have adverse effects on the soil fauna in the surrounding environment.

Mitigation of OMW toxicity toward Enchytraeus albidus with application of additives.

The olive oil industry generates considerable amounts of olive mill wastewater (OMW) which is treated and used in agriculture, energy production, or discharged into evaporating ponds where OMW contaminated soil (OMWS) is formed. Due to the extremely high phenol content, untreated OMWS is not suitable for plants and soil organisms. This study aimed to determine the adverse effects of OMWS on Enchytraeus albidus and whether the addition of clay and diatomaceous earth can reduce these effects. Without additives, reproduction was reduced up to 35%, with high number of unhatched cocoons, indicated hatching impairment. Furthermore, acetylcholinesterase (AChE) activity decreased significantly at the highest OMWS ratio (40%), as did glutathione-S-transferase (GST) activity at two ratios (40%), indicating neurotoxic effects and oxidative stress. The application of additives (clay and diatomaceous earth) decreased phenol concentration up to 35 and 45%, respectively. Consequently, the number of juveniles increased significantly compared to the control and AChE and GST activities were not affected. However, an increased number of unhatched cocoons and lipid peroxidation were observed after diatomaceous earth treatment, indicating the occurrence of stress. Although additives can bind phenols, neutralize harmful effects and allow the use of OMWS as a fertilizer in agriculture, they should be carefully selected taking into account soil organisms as well. The use of additives to reduce the adverse effects of OMWS can be a turning point in the remediation of olive contaminated soil.

Assessment of adverse effects of olive mill waste water and olive mill waste contaminated soil on springtail Folsomia candida.

In recent years significant attention has been given to the problem of olive mill waste towards the environment. Still, there is a considerable gap in the knowledge of the impact of the olive mill wastewater (OMWW) and the olive mill waste contaminated soil (OMW CS) on non-target soil organisms. Springtails, as an important group of non-target soil organisms, are frequently used in ecotoxicological research. However, information on olive mill waste impact on the model species Folsomia candida is scarce. Therefore, in this study, we determined the effects of OMWW and OMW CS on survival, reproduction, neurotoxicity, oxidative stress, and available energy in springtail F. candida. The exposure to different ratios of OMWW and OMW CS showed higher toxicity of OMW CS in terms of survival (LC50 = 32.34% of OMWW; LC50 = 45.36% of OMW CS) and reproduction (EC50 = 10.10% of OMWW; EC50 = 19.44% of OMW CS). Furthermore, neurotoxicity (AChE induction), oxidative stress (SOD, GST, and MDA induction), and changes in available energy (decrease in lipid and carbohydrate content) have been observed. Those negative effects are likely consequences of the high phenol content specific to OMWW and OMW CS. Obtained results indicate that for the ecotoxicological assessment of various wastes it is essential to consider different tier level biomarkers to have a clear insight into the mechanism of action.

Effects of olive mill waste (OMW) contaminated soil on biochemical biomarkers and reproduction of Dendrobaena veneta.

Olive oil industry is economically important in Mediterranean countries. Disposal of olive mill waste (OMW) presents an environmental concern in those countries due to its high salinity and its high level of polyphenols. In order to reuse OMW, those properties have to change either through the filtration process and addition of adsorbents or by composting. One of the most important organisms in composting of organic wastes is earthworms. However, data on the effects of OMW on earthworms are scarce. The main aim of our study was to investigate whether OMW contaminated soil (OMW CS) causes adverse effects on molecular and organism level in epigeic earthworm Dendrobaena veneta and on microbiological activity. Changes of measured biochemical biomarkers (AChE, CAT, GST, lipids, MDA) varied depending on the quantity of added OMW CS and the exposure duration. Oxidative stress occurred after 7 days of exposure, while in most cases enzyme activity recovered after 28 days. At the highest ratio of contaminated soil (50%), reproduction was completely inhibited. The second aim was to investigate the impact of earthworms on phenol degradation and microbial activity, indicating an important role in the bioremediation of contaminated soils. Our results show that above a certain quantity an OMW CS has an adverse effect on earthworms, while the impact of earthworms on soil microbial activity was positive but transient. Yet, as the results also imply that earthworms have an impact on phenol degradation, they can be used to help remediation of OMW CS and its subsequent usage in agriculture. However, the quantity of OMW CS that can be safely added should be determined first.

Effects of strobilurin fungicides (azoxystrobin, pyraclostrobin, and trifloxystrobin) on survival, reproduction and hatching success of Enchytraeus crypticus.

Large quantities of strobilurin fungicides (SFs) are used worldwide, resulting in adverse effects on non-target organisms. SFs affect the reproduction and embryonic development of aquatic organisms, while the impact on soil organisms has been insufficiently researched. Therefore, we investigated the effects of three SFs (azoxystrobin (AZO), pyraclostrobin (PYR), and trifloxystrobin (TRI)) on the survival, reproduction, and hatching success of the non-target soil oligochaete Enchytraeus crypticus. The standard enchytraeid reproduction test (ERT) showed that, regarding survival, TRI (LC50 = 2.34 mg/kg) was the most toxic, followed by PYR (LC50 = 4.26 mg/kg) and AZO (LC50 ≥150 mg/kg). Reproduction was affected in the same order (TRI EC50 = 0.045 mg/kg, PYR EC50 = 1.85 mg/kg, and AZO EC50 = 93.10 mg/kg). Exposure to AZO and PYR showed a negative impact on hatching success with a significant increase in the number of unhatched cocoons. Prolonged hatching test was consequently carried out. As a result, a hatching delay was observed at lower AZO and PYR concentrations, while at higher concentrations hatching was completely stopped as the cocoons were no longer viable. Hence, hatching test enabled a discrimination between hatching delay and hatching impairment. Besides demonstrating the adverse effects of AZO, PYR, and TRI on the survival, reproduction, and hatching success of E. crypticus, the obtained results indicate the convenience of using several endpoints in reproduction tests. The usage of prolonged hatching tests and monitoring of hatching dynamics could fill the gap between standard reproduction tests and multigeneration tests and allow a better understanding of the adverse effects on reproduction.

Measurement of multixenobiotic resistance activity in enchytraeids as a tool in soil ecotoxicology.

The multixenobiotic resistance (MXR) mechanism is the first defense line against xenobiotics. Enchytraeids, a model organism in soil ecotoxicology, are often exposed to various xenobiotics, some of which may influence MXR activity. Since MXR activity has not been studied in these organisms, the aim of this paper was to establish a methodology for the implementation of the dye assay in enchytraeids. Enchytraeus albidus and Enchytraeus crypticus were exposed to model chemosensitizers: cyclosporine A (CA), dexamethasone (DEX), ivermectin (IVM), rifampicin (RIF), verapamil (VER), and fungicide propiconazole (PCZ). Thereafter, a dye assay with specific fluorescent dyes rhodamine B and rhodamine 123 was performed. Changes in MXR activity caused by variations in dye accumulation were measured fluorometrically. CA, IVM, and VER were found to inhibit the MXR system and increase the fluorescence 2.2-fold, while DEX and RIF induced the MXR system and decreased the fluorescence. CA was the strongest inhibitor in both E. albidus (IC50 5.48 ± 1.25 µM) and E. crypticus (IC50 5.20 ± 3.10 µM). In the validation experiment, PCZ was found to inhibit the MXR system. The IC50 varied between species and exposure substrates: water (E. albidus - IC50 0.74 ± 0.24 mg/L; E. crypticus - 1.31 ± 0.24 mg/L) or soil (E. albidus - 1.79 ± 0.42 mg/kg; E. crypticus - 1.79 ± 0.17 mg/kg). In conclusion, the tested compounds changed the MXR activity, which confirms the applicability of this method as a valuable complementary biomarker in soil ecotoxicology.

ErIK-a software-based identification key for earthworm species of Croatia.

The need for reliable taxonomic species identification is always present. Correct identification of specimens is based on taxonomic research, scattered in the scientific literature and accessible only for a restricted group of specialists or research institutions with extensive library facilities (Zuquim et al. 2017). Therefore, identification keys that summarize the knowledge of a group of biota are very important; they provide the link between producers and users of taxonomy. Recently, printed identification keys are increasingly being replaced or supplemented by computer-aided keys.

Effects of single and combined exposure to nano and bulk zinc-oxide and propiconazole on Enchytraeus albidus.

Organisms in soil are often exposed to different mixtures of contaminants. These contaminants may interact with each other and, consequently, may have a different effect on organisms than each of them alone. We wanted to investigate possible effects of ZnO mixtures in bulk and nano form and fungicide propiconazole (PCZ) on biochemical parameters and reproduction in Enchytreus albidus. These compounds were applied separately and in binary mixture. In the single exposure experiment the EC50 values for the number of juveniles were calculated: nano ZnO (641.21 ±â€¯88.82 mg kg-1), bulk ZnO (445.78 ±â€¯148.4 mg kg-1) and PCZ (3.63 ±â€¯1.68 mg kg-1), respectively. These concentrations were subsequently used in the binary exposure experiment. Calculated combination indices (CI), that allow quantitative determination of chemical interactions at different concentration and effect level, indicated additive or antagonistic interactions (CI ≥ 1) of applied mixture ratios. The only ratio that showed synergistic interaction (CI < 1) was 75% EC50 nZnO/25% EC50 PCZ. Both ZnO forms caused acetylcholinesterase (AChE) activity increase of up to 40% of control level, as well as increased catalase (CAT) and glutathione S-transferase (GST) activities and malondialdehyde (MDA) level. PCZ did not affect AChE and CAT activities, yet it increased GST activity and MDA level. Induced levels of measured biomarkers indicate an oxidative stress after binary exposure, as well. These effects were not enhanced after binary exposure but reflected the effects on biomarkers that corresponding concentrations of these compounds generated in a single exposure experiment.

Biochemical and reproductive effects of red mud to earthworm Eisenia fetida.

Red mud (RM) is the main waste of alumina production whose disposal poses a problem. The research of various possible effects of red mud on soil organisms has been scarce. We have exposed earthworms (Eisenia fetida) to red mud: artificial soil mixtures. The tested samples of red mud were of different origin: Croatian (CRRM) and Hungarian (HURM). The effects of exposure on the metabolic and oxidative status of earthworms were measured using several biochemical biomarkers (acetylcholinesterase, catalase and glutathione S-transferase activity and metallothionenin content) and reproductive success was assessed upon counting the number of hatched juveniles. The LC50 value for CRRM was 40% and for HURM 62% of red mud in the growth medium on weight basis, respectively. A significant effect (p < 0.001) of the RM concentration and origin, as well as significant interactions between the origin of RM and the applied concentrations on all measured biomarkers were observed. CRRM had a higher content of different metals as well as a higher conductivity in comparison to HURM. The reproduction was inhibited after exposure to both RMs. Namely, 25% CRRM caused a 53.26% reduction in the number of juveniles, whereas 18% HURM caused a 68.84% reduction, and 50% HURM caused 97.9% reduction, respectively. Both RMs caused changes in the measured biomarkers related to an oxidative stress. Consequently, the possible adverse effects on soil organisms before the environmental application of red mud should be assessed to avoid further environmental damage.

Acute and subchronic effects of three herbicides on biomarkers and reproduction in earthworm Dendrobaena veneta.

Earthworms are exposed to herbicides both through their skin and digestive system. Herbicides can influence earthworms' survival, physiology and reproduction. However, there is a lack of data on herbicide effects on earthworms as they are often regarded as low or non-toxic. The aim of our study was to investigate whether widely used commercial formulations of glyphosate (GLF), tembotrione (TBT) and nicosulfuron (NCS) each applied at three environmentally relevant concentrations have adverse effects on various biomarkers and reproduction in epigeic earthworm Dendrobaena veneta. The activities of measured biomarkers varied depending on the herbicide used and the exposure duration and suggest that oxidative stress plays an important role in the toxicity of tested herbicides. Namely, GLF caused an acetylcholinesterase (AChE) activity induction after seven days, and NCS after 28 days, while TBT caused an inhibition up to 47% (6.6 µg kgdw soil-1) after seven days. Only TBT caused a significant change (H2 = 13.96, p = 0.002) to catalase (CAT) after seven days of exposure. Malondialdehyde concentrations (MDA) were increased all the time after NCS exposure, but only after seven days in GLF and 28 days in TBT treatments, respectively. The tested herbicides did not have a significant effect on reproduction success, expect of NCS which increased the number of juveniles (p < 0.05).

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 organism's 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.

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.

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.

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.

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.