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The aim of this research was to determine the effect of nine essential oils from the Croatian native flora (Mentha x piperita, Salvia officinalis, Rosmarinus officinalis, Lavandula hybrida, Origanum compactum, Thymus vulgaris, Hiperici oleum, Achillea millefolium and Helichrysum italicum) on the Botrytis cinerea mycelial growth suppression. The research results demonstrated that the oils of Thymus vulgaris and Mentha x pipereta in both investigated methods (the volatile and the macrodillution one) have exerted a positive influence on the suppression of mycelial growth. Also, the aforementioned oils had a fungistatic effect in all investigated concentrations long after 96 hours. The essential oils of Achillea millefolium and Hiperici oleum in the volatile method have not manifested a suppression effect, while the effect of suppression of a mycelial growth was recorded in the macrodillution method. The essential oil of Helichrysum italicum stimulated the growth of B. cinerea mycelium in both investigated methods. Considering a reduction of the mycelium growth, the essential oils, as a biocontrol agent, could be a positive substitution for the traditional pesticides in grapevine gray mold control. Additional investigation with regard to the essential oils in the Botrytis control are needed both the in vitro and the in vivo conditions.

White stork nestlings can provide quantitative data on the quality of the environment, as they are dependent on their parents that provide locally foraged food. Blood was sampled from the brachial vein (n = 109) and the sampling was performed in parallel with ringing during breeding season 2020 from five areas in eastern Croatia: Lonjsko polje, Jelas polje, Slavonski Brod-east, Podunavlje, and Donje Podravlje. In the present study, for the first time in Croatia, the following enzymatic biomarkers were assessed in white stork nestlings: activities of acetylcholinesterase (AChE), carboxylesterase (CES), glutathione S-transferase (GST), and glutathione reductase (GR), as well as nonenzymatic biomarkers: levels of glutathione (GSH) and reactive oxygen species (ROS). All endpoints were measured in two blood fractions: plasma and a postmitochondrial fraction (S9). Nestlings from Podunavlje and Donje Podravlje, areas known for intensive agriculture, showed lower AChE and CES activity when compared to the other investigated areas, indicating the presence of inhibitory xenobiotics. Higher oxidative stress was observed in Slavonski Brod-east, an area surrounded by metal and engineering industry, and Podunavlje compared to the other sampling areas. Hence, this study shows the impact of pollutants from the surrounding metal, petroleum, and agricultural industry might have on the biomarkers in white stork nestlings, which are often seen as early-warning signals.

Microplastics are small plastic fragments that are widely distributed in marine and terrestrial environments. While the soil ecosystem represents a large reservoir for plastic, research so far has focused mainly on the impact on aquatic ecosystems and there is a lack of information on the potentially adverse effects of microplastics on soil biota. Earthworms are key organisms of the soil ecosystem and are due to their crucial role in soil quality and fertility a suitable and popular model organism in soil ecotoxicology. Therefore, the aim of this study was to gain insight into the effects of environmentally relevant concentrations of microplastics on the earthworm Eisenia andrei on multiple levels of biological organization after different exposure periods. Earthworms were exposed to two types of microplastics: (1) polystyrene-HBCD and (2) car tire abrasion in natural soil for 2, 7, 14 and 28d. Acute and chronic toxicity and all subcellular investigations were conducted for all exposure times, avoidance behavior assessed after 48 h and reproduction after 28d. Subcellular endpoints included enzymatic biomarker responses, namely, carboxylesterase, glutathione peroxidase, acetylcholinesterase, glutathione reductase, glutathione S-transferase and catalase activities, as well as fluorescence-based measurements of oxidative stress-related markers and multixenobiotic resistance activity. Multiple biomarkers showed significant changes in activity, but a recovery of most enzymatic activities could be observed after 28d. Overall, only minor effects could be observed on a subcellular level, showing that in this exposure scenario with environmentally relevant concentrations based on German pollution levels the threat to soil biota is minimal. However, in areas with higher concentrations of microplastics in the environment, these results can be interpreted as an early warning signal for more adverse effects. In conclusion, these findings provide new insights regarding the ecotoxicological effects of environmentally relevant concentrations of microplastics on soil organisms.

The functioning of soil ecosystems greatly depends on the interactions occurring between soil biota communities. It is well known that earthworms are an important soil component that substantially affects its function, including their meaningful impact on the development of different phytopathogenic soil fungi. Phytopathogenic fungi are responsible for crop disease and cause great economic damage. It has previously been established that earthworms’ coelomic fluid can suppress the growth of phytopathogenic fungi, but the exact molecular mechanism is unknown. The present study aimed at broadening the proof of this observed phenomenon by investigating the effects of the coelomic fluid extract of three different earthworm species (Eisenia andrei, Dendrobaena veneta and Allolobophora chlorotica) on the growth of six different phytopathogenic fungi species (Berkeleyomyces basicola, Fusarium culmorum, Globisporangium irregulare, Rhizoctonia solani, Macrophomina phaseolina, and Sclerotinia sclerotiorum). Coelomic fluid extract was obtained by electrostimulation or usage of extraction buffer (only in case of A. chlorotica) and prepared in three different concentrations by diluting the obtained coelomic fluid with physiological saline. The coelomic fluid extract of the three investigated earthworm species had an inhibitory effect on the growth of all six phytopathogenic fungi species. The greatest inhibitory effect was achieved with the E. andrei coelomic fluid extract reducing the growth of R. solani fungi. The findings of this research confirm the antifungal activity of coelomic fluid obtained from earthworm species belonging to different ecological categories and may be of potential use in crop protection against phytopathogenic fungi.

Industrialization and urbanization have led to an increased accumulation of waste materials that are transformed into a nutrient-rich and high-quality product called vermicompost by the vermicomposting process. Vermicomposting is an ecofriendly and economically favorable biotechnological process that involves the interaction of earthworms and microorganisms. Due to the importance of this process and its great potential in dealing with the consequences of waste accumulation, this review aims to provide key insights as well as highlight knowledge gaps. It is emphasized that there is a great challenge in understanding and clarifying the mechanisms involved in the vermicomposting process. The optimization of the factors affecting the possible application of vermicompost is crucial for obtaining the final product. Information on the composition of bacterial communities, amount of vermicompost, effect on heavy metal content, plant pathogens, diseases and organic waste selection is here recognized as currently the most important issues to be addressed. By answering these knowledge gaps, it is possible to enable wider utilization of vermicompost products.

Animals 11(8), 2341 (2021). doi:10.3390/ani11082341 special issue: "Special Issue "Biomarkers of Oxidative Stress in Animals : Advances in Their Measurement and Practical Applications" / Special Issue Editors: Dr. Camila Peres Rubio, Guest Editor; Dr. Breno Fernando Martins de Almeida, Guest Editor", Published by MDPI, Basel

Hydroxylation of polyaromatic compounds through cytochromes P450 (CYPs) is known to result in potentially estrogenic transformation products. Recently, there has been an increasing awareness of the importance of alternative pathways such as aldehyde oxidases (AOX) or N-methyltransferases (NMT) in bioactivation of small molecules, particularly N-heterocycles. Therefore, this study investigated the biotransformation and activity of methylated quinolines, a class of environmentally relevant N-heterocycles that are no native ligands of the estrogen receptor (ER), in the estrogen-responsive cell line ERα CALUX. We found that this widely used cell line overexpresses AOXs and NMTs while having low expression of CYP enzymes. Exposure of ERα CALUX cells to quinolines resulted in estrogenic effects, which could be mitigated using an inhibitor of AOX/NMTs. No such mitigation occurred after coexposure to a CYP1A inhibitor. A number of N-methylated but no hydroxylated transformation products were detected using liquid chromatography-mass spectrometry, which indicated that biotransformations to estrogenic metabolites were likely catalyzed by NMTs. Compared to the natural ER ligand 17β-estradiol, the products formed during the metabolization of quinolines were weak to moderate agonists of the human ERα. Our findings have potential implications for the risk assessment of these compounds and indicate that care must be taken when using in vitro estrogenicity assays, for example, ERα CALUX, for the characterization of N-heterocycles or environmental samples that may contain them.

Investigations of deleterious effects on non-target species, including earthworms, have been conducted for a number of pesticides, but there is a need for additional assessments of potential adverse effects. In the present study, the acute toxicity of eight pesticides to the earthworm Eisenia andrei was assessed and compared. The exposures were conducted using the filter paper contact toxicity method. Based on the 48-h LC50 values, one pesticide was classified as supertoxic (combined fungicide containing difenoconazole and fludioxonil), four as extremely toxic (combined herbicide containing pethoxamide and terbuthylazine, combined fungicide containing fluopyram and tebuconazole, fungicide containing pyrimethanil, and combined fungicide containing thiram and carboxin), two as very toxic (combined fungicide containing flutriafol and thiabendazole, and herbicide containing fluroxypyr-meptyl), and one as moderately toxic (insecticide containing thiamethoxam). Additionally, effects of pesticides on the multixenobiotic resistance (MXR) activity were measured. Results showed that four pesticides caused significant effects with a recorded inhibition of the activity, which can consequently lead to a higher toxicity due to longer retention of the pesticides in the cells. Finally, for three chosen pesticides, gene expression of cat, sod, and gst was measured, and significant changes were observed. The obtained results show that earthworms could be significantly affected by pesticides commonly used in agriculture.

Grapevine plants can form mutualistic associations with endophytic fungi. However, to date, there is not much information on the influence of endophytic Fusarium isolates on grapevine growth, oenological characteristics or their capabilities to suppress disease. In the present study, it was demonstrated on two grapevine varieties tested in a field experiment in vineyards under natural conditions that three isolates of Fusarium do indeed enter into mutualistic interactions with their host. Results indicate a complementary relationship between grape yield and Fusarium colonization. Plants treated with Fusarium isolates not only showed increases in yield, but also had fewer losses due to shriveling and also had more vigorous development under unfavorable weather conditions. Furthermore, treatment with isolates of Fusarium solani and Fusarium subglutinans resulted in significantly lower gray mold incidence at rates of 80 %.

This study represents the first analysis of heavy metals: lead and cadmium, as well as two metalloids: arsenic and selenium, in the feathers of Grey Herons breeding in Croatia. The Grey Heron was chosen as a model apex predator of the wetland ecosystem due to high trophic level, feeding habits, long lifespan and abundance. Sampling was conducted during the 2019 breeding season in April and May from six Grey Herons colonies in Croatia (Mrsunjski lug, Kopački rit, Piljenice, Kravarsko, Čepin, Slovinci), whose environment is heavily influenced by rivers Danube, Drava and Sava tributaries. The primary and secondary flight feathers were collected under the colonies as shed feathers or from dead birds. The feathers are a suitable non-invasive method for metal and metalloid analysis, and can reflect internal concentrations, depending on the metal. In the Slovinci colony, we measured the highest lead (Pb) and cadmium (Cd) concentrations (2194.53 ± 416.20 μg kg-1 dw and 79.30 ± 9.37 μg kg-1 dw, respectively). The highest arsenic (As) and selenium (Se) concentrations were found in the Čepin colony (359.70 ± 301.46 μg kg-1 dw and 3375.61 ± 2502.80 μg kg-1 dw, respectively). All the measured levels of heavy metals and metalloids are below concentrations that can cause adverse health effects in birds., Antropogenim aktivnostima povećava se razina metala i polumetala u okolišu rezultirajući njihovim zagađenjem. Mjerenje teških metala i polumetala neinvazivnim metodama dobar je pokazatelj stanja okoliša. U radu su predstavljeni rezultati prve analize teških metala: olova i kadmija te dva metaloida: arsena i selena u perju sivih čaplji Ardea cinerea koje se gnijezde u Hrvatskoj. Uzorkovanje pera sivih čaplji provedeno je tijekom sezone gniježđenja, u travnju i svibnju 2019. iz šest hrvatskih kolonija (Mrsunjski lug, Kopački rit, Piljenice, Kravarsko, Čepin i Slovinci). Primarna i sekundarna letna pera odraslih ptica prikupljena su ispod kolonija. Najveće koncentracije olova (Pb) zabilježene su u perju iz Slovinaca (2194,53 ± 416,20 μg kg-1 suhe tvari) i Kopačkog rita (1955,85 ± 1373,07 μg kg-1 suhe tvari). U Čepinu su izmjerene najviše koncentracije arsena (As; 359,70 ± 301,46 μg kg-1 suhe tvari) i selena (Se; 3375,61 ± 2502,80 μg kg-1 suhe tvari), a najviša razina kadmija (Cd) izmjerena je u koloniji Slovinci (79,30 ± 9,37 μg kg-1 suhe tvari). Sve izmjerene razine metala i polumetala su ispod koncentracija koje se smatraju toksičnima za ptice. Preliminarni rezultati ovog istraživanja ukazuju na relativno nisku zagađenost okoliša kontinentalne Hrvatske, no potrebna su dodatna istraživanja i uspostava kontinuiranog praćenja metala i polumetala u okolišu te njihov utjecaj na vršne predatore

The assessment of the exposure of aquatic wildlife to complex environmental mixtures of chemicals originating from both point and diffuse sources and evaluating the potential impact thereof constitutes a significant step towards mitigating toxic pressure and the improvement of ecological status. In the current proof-of-concept study, we demonstrate the potential of a novel Aggregated Biomarker Response (ABR) approach involving a comprehensive set of biomarkers to identify complex exposure and impacts on wild brown trout (Salmo trutta fario). Our scenario used a small lowland river in Germany (Holtemme river in the Elbe river catchment) impacted by two wastewater treatment plants (WWTP) and diffuse agricultural runoff as a case study. The trout were collected along a pollution gradient (characterised in a parallel study) in the river. Compared to fish from the reference site upstream of the first WWTP, the trout collected downstream of the WWTPs showed a significant increase in micronucleus formation, phase I and II enzyme activities, and oxidative stress parameters in agreement with increasing exposure to various chemicals. By integrating single biomarker responses into an aggregated biomarker response, the two WWTPs' contribution to the observed toxicity could be clearly differentiated. The ABR results were supported by chemical analyses and whole transcriptome data, which revealed alterations of steroid biosynthesis and associated pathways, including an anti-androgenic effect, as some of the key drivers of the observed toxicity. Overall, this combined approach of in situ biomarker responses complemented with molecular pathway analysis allowed for a comprehensive ecotoxicological assessment of fish along the river. This study provides evidence for specific hazard potentials caused by mixtures of agricultural and WWTP derived chemicals at sublethal concentrations. Using aggregated biomarker responses combined with chemical analyses enabled an evidence-based ranking of sites with different degrees of pollution according to toxic stress and observed effects.

The usage of pesticides has been steadily increasing over the last decades, and among them herbicides are the most commonly used ones. Despite their main mode of action targeting plant organisms, they can also have adverse effects on non-target animal organisms. In soil ecosystems, earthworms play an important role due to their positive impacts on the soil functioning and they represent good model organisms in soil ecotoxicology. The aim of the present study was to assess the effects of two herbicides on several endpoints at different levels of biological organization in the earthworm Eisenia andrei. Diuron and fluazifop-p-butyl were selected for the investigation and their lethal concentrations were determined: LC50 48 h: 89.087 μg/cm2 for diuron and 6.167 μg/cm2 for fluazifop-p-butyl. Furthermore, measurements of enzymatic biomarkers (catalase (CAT), acetylcholinesterase (AChE), carboxylesterase (CES) and glutathione S-transferase (GST)), multixenobiotic resistance (MXR) activity and gene expression of antioxidative enzymes (only for fluazifop-p-butyl) were conducted. Enzymatic biomarker responses showed no significant differences compared to the control after the exposure to the investigated herbicides, whereas the MXR activity was significantly inhibited. The gene expression level of superoxide dismutase (sod) and glutathione S-transferase (gst) after fluazifop-p-butyl exposure showed a significant increase. Finally, avoidance behavior in soil was assessed and it was determined that both herbicides caused significant avoidance response. The obtained results show that both investigated herbicides significantly affect earthworms on different levels of biological organization. This emphasizes the importance of comprehensive ecotoxicological assessment of herbicide effects on non-target organisms at all organizational levels.

Interactions between soil biota communities play an important role as intrinsic factors of suppression and degradation of different phytopathogenic soil fungi. There is significant evidence that earthworms can directly affect fungal populations by feeding or by dispersing fungal propagules. Possible effects of indirect contact by secretion of mucus and coelomic fluid were not assessed previously. In the present laboratory study the effects of earthworms' coelomic fluid extract on the growth of Fusarium oxysporum , a phytopathogenic fungi species important in agricultural production, were assessed. The results showed that extracts of coelomic fluid of both tested earthworm species ( Dendrobaena veneta and Eisenia fetida ) have negative effects on fungal growth. After 48 h, a significant growth reduction was observed in groups treated with extracts containing 2250, 4000 and 4500 coelomocytes/mL. Growth reduction was even more pronounced 72 h after the treatment. The obtained results indicate the possibility that earthworms can affect soil fungi not only by ingestion, but also by contact interaction. This study proved that earthworm coelomic fluid extract shows antifungal activity in in vitro testing. For better understanding of the exact mechanism, studies with soil as a substrate are required, as well as in depth investigation of contact interactions between earthworms and fungi.

Massive toxic blooms of cyanobacteria represent a major threat to water supplies worldwide. Here, the biological activities of lipopolysaccharide (LPS) isolated from Microcystis aeruginosa, the most prominent cyanobacteria in water bloom, were studied. LPS was isolated from complex environmental water bloom samples dominated by M. aeruginosa, and from laboratory cultures of non-axenic as well as axenic M. aeruginosa strains PCC7806 and HAMBI/UHCC130. Employing human blood-based in vitro tests, the LPS isolated from complex water bloom revealed the priming of both major blood phagocyte population monocytes and polymorphonuclear leukocytes documented by the increased surface expression of CD11b and CD66b. This was accompanied by a water bloom LPS-mediated dose-dependent induction of tumor necrosis factor α, interleukin-1β, and interleukin-6 production. In accordance with its priming effects, water bloom LPS induced significant activation of p38 and ERK1/2 kinases, as well as NF-κB phosphorylation, in isolated polymorphonuclear leukocytes. Interestingly, the pro-inflammatory potential of LPS from the axenic strain of M. aeruginosa was not lower compared to that of LPS isolated from non-axenic strains. In contrast to the biological activity, water bloom LPS revealed almost twice higher pyrogenicity levels compared to Escherichia coli LPS, as analyzed by the PyroGene test. Moreover, LPS from the non-axenic culture exhibited higher endotoxin activity in comparison to LPS from axenic strains. Taking the above findings together, M. aeruginosa LPS can contribute to the health risks associated with contamination by complex water bloom mass.

Background Zebrafish have been increasingly used for monitoring and assessing the effects of different contaminants in the aquatic environments. In the present study, zebrafish embryos and larvae were used to study the effects of the insecticide diazinon and the herbicide diuron in regard to occurrence of oxidative stress-related cellular responses, multixenobiotic resistance (MXR)-related efflux transporter activity—which represents the first line of defense against xenobiotics in many aquatic organisms—and responses of different molecular and biochemical biomarkers. Results The recently established non-invasive quantitative plate assay, which uses the fluorescent probes CM-H2DCFDA and CellTracker™ Green CMFDA and measures the fluorescence in whole zebrafish larvae, was applied to assess changes in reactive oxidative species (ROS) and glutathione (GSH) after exposure to the investigated pesticides. The results showed a significant increase of GSH after 1h exposure of zebrafish larvae to both diazinon and diuron. Regarding the ROS induction, no significant increases in fluorescence could be detected after 2h exposure to the investigated pesticides. Applying a newly adapted assay for MXR activity, it was determined that diuron caused no change after a 24-h exposure, but caused a significant induction of MXR activity after a 48-h exposure (indicated by a decreased amount of accumulated rhodamine B). On the other hand, diazinon caused an inhibition of MXR activity after both 24h and 48h exposure (indicated by an increased amount of accumulated rhodamine B). Regarding the biomarkers, different set-ups and exposure periods were applied and both molecular (gene expression) and biochemical (enzymatic activities) responses were assessed. Diazinon caused an inhibition of carboxylesterase (CES) and acetylcholinesterase (AChE) activity in zebrafish larvae, diuron inhibited AChE activity in in vitro testing, and both pesticides significantly affected gene expression and activities of some of the cytochrome P450 (CYP) family enzymes. Conclusions The obtained results show various effects of the investigated pesticides and will help to elucidate how aquatic animals cope with pesticides present in their environment. Additionally, the recently developed fluorescence-based assay and the newly adapted MXR activity assay proved to be useful tools for ecotoxicological risk assessment to further investigate pesticide effects.

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.

Metabolism has to be considered during the toxicological assessment of chemical and environmental samples because it is an important process in the mammalian liver. It can be assessed in vitro via liver homogenates called S9-fractions, an external metabolic activation system. However, the external metabolic activation systems can vary greatly in their composition due to biological variations among individual animals and animal strains that the S9-fraction are derived as well as the differences in the production treatment. To gain more insight into these variances, three different but commonly used rat-derived S9-fractions were compared in the present study for their variance and performance with a reference compound in the Ames fluctuation assay with Salmonella typhimurium strains TA 98 and TA 100 according to ISO 11350. Severe shortcomings of conventional rat-derived S9-fractions were observed in the present study, such that S9-fractions differed significantly within the same rat strain and for different types of induction procedures in regards to the metabolic capability. An intrinsic mutagenic potential of the three rat-derived S9-fractions were identified in the Ames fluctuation assay with varying S9-fraction concentrations. To address some of the shortcomings of the animal-derived S9-fraction, the present study investigated the use and performance of a biotechnological, animal-free alternative, ewoS9R, in comparison to one of the rat-derived S9-fraction as the others showed a mutagenic potential themselves. Specifically, 12 different chemicals were used as a reference to determine if ewoS9R could serve as an adequate and more consistent replacement of traditional rat-derived metabolic activation systems: 8 pro-mutagenic compounds (i.e., require metabolic activation to show a mutagenic potential), one pro-mutagenic compound but not in the tested strains, one mutagenic compound without metabolic activation and two compounds that are equivocal in the literature. EwoS9R was evaluated as a promising approach in the Ames fluctuation assay with 5 compounds observed to have similar results with both rat-derived S9-fraction and ewoS9R (41%), for 3 compounds ewoS9R was a better metabolization system than the rat-derived S9-fraction (16%). Further research is necessary to determine the full potential of ewoS9R in comparison to rat-derived S9-fractions.

This study presents a high-throughput (HTP) micronucleus assay in multi-well plates with an automated evaluation for risk assessment applications. The evaluation of genotoxicity via the micronucleus assays according to international guidelines ISO 21427-2 with Chinese hamster (Cricetulus griseus) V79 cells was the starting point to develop our methodology. A drawback of this assay is that it is very time consuming and cost intensive. Our HTP micronucleus assay in a 48-well plate format allows for the simultaneous assessment of five different sample-concentrations with additional positive, negative and solvent controls with six technical replicates each within a quarter of the time required for the equivalent evaluation using the traditional slide method. In accordance with the 3R principle, animal compounds should be replaced with animal-free alternatives. However, traditional cell culture-based methods still require animal derived compounds like rat-liver derived S9-fraction, which is used to simulate the mammalian metabolism in in vitro assays that do show intrinsic metabolization capabilities. In the present study, a recently developed animal-free biotechnological alternative (ewoS9R) was investigated in the new high-throughput micronucleus assay. In total, 12 different mutagenic or genotoxic chemicals were investigated to assess the potential use of the animal-free metabolization system (ewoS9R) in comparison to a common rat-derived product. Out of the 12 compounds, one compound did not induce micronuclei in any treatment and 2 substances showed a genotoxic potential without the need for a metabolization system. EwoS9R demonstrated promising potential for future applications as it shows comparable results to the rat-derived S9 for 6 of the 9 pro-genotoxic substances tested. The remaining 3 substances (2-Acetamidofluorene, Benzo[a]pyrene, Cyclophosphamide) were only metabolized by rat-derived S9. A potential explanation is that ewoS9R was investigated with an approx. 10-fold lower enzyme concentration and was only optimized for CYP1A metabolization that may be improved with a modified production procedure. Future applications of ewoS9R go beyond the micronucleus assay, but further research is necessary.

The modes of action of pollutants are diverse, and a common consequences to pollutant exposure is oxidative stress. This phenomenon is caused by an imbalance or disurption in the control of Reactive Oxygen Species (ROS) resulting in an accumulation of free radicals. Oxidative stress may cause damages to the DNA, phospholipids and proteins, and lead to cell death. Due to the possible contribution of oxidative stress to pollutant toxicity , it is valuable to assess its occurrence, role and mechanism. Detection of oxidative stress at low concentrations soon after the onset of exposure can be a sensitive, general marker for contamination. This study aimed at developing and benchmarking a set of novel fluorescence-based procedures to assess the occurrence of oxidative stress in zebrafish larvae (96 hpf) by measuring the antioxidant glutathione (GSH) and general ROS. Zebrafish larvae were exposed to tert-butyl hydroperoxide (t-BHP). ROS and GSH were made visible by means of specific fluorescent molecular probes in different experimental scenarios. The induction was qualified using microscopy and quantified through photometric measurement. For quantitative assessment, an approach based on homogenized larvae and a non-invasive plate assay were developed. The novel procedures proved suitable for oxidative stress detection. Comparisons of qualitative to quantitative data showed that the orientation of the larvae in the well can influence fluorescence data evaluation. The non-invasive quantitative assay proved robust against any influence of the orientation of the larvae. The developed protocols promise to be useful tools for the detection of oxidative stress in zebrafish larvae.

Selenium (Se) is an essential element for humans, animals, and certain lower plants, but can be toxic at high concentration. Even though Se is potentially toxic, little information is available about the effects of Se on soil animals. The aim of this study was to assess the impact of different concentrations of two Se forms, selenate and selenite, on earthworm Eisenia andrei. In order to obtain comprehensive overview on the Se effects, different parameters were measured. Namely, acute toxicity, apoptosis, efflux pump activity, different enzymatic and non-enzymatic biomarkers (acetylcholinesterase, carboxylesterase, glutathione S-transferase, catalase, glutathione reductase and superoxide dismutase activities, lipid peroxidation level and GSH/GSSG ratio) and expression of genes involved in oxidative and immune response have been investigated. Additionally, measurement of metallothioneins concentration and concentration of Se in exposed earthworms has been also performed. The assessment of acute toxicity showed a greater sensitivity of E. andrei to selenite exposure, whereas Se concentration measurements in earthworms showed higher accumulation of selenate form. Both Se forms caused inhibition of the efflux pump activity. Decrease in superoxide dismutase activity and increase in lipid peroxidation and glutathione reductase activity indicate that Se has a significant impact on the oxidative status of earthworms. Selenate exposure caused an apoptotic-like cell death in the coelomocytes of exposed earthworms, whereas decreased mRNA levels of stress-related genes and antimicrobial factors were observed upon the exposure to selenite. The obtained data give insight into the effects of two most common forms of Se in soil on the earthworm E. andrei.

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.

The use of zebrafish for aquatic vertebrate (eco)toxicity testing allows the assessment of effects on a wide range of biological levels – from enzymes to sensory organs and behavioral endpoints. The present study investigated the effects of the insecticide diazinon and the herbicide diuron regarding the acute toxicity and behavior of zebrafish embryos and larvae. After conducting the fish embryo toxicity test, three concentrations (1, 2 and 3.5 mg L −1 for diazinon and 1, 2 and 3.8 mg L −1 for diuron) were evaluated for effects on embryonic spontaneous movement and heartbeat, larval light-dark transition response, and thigmotaxis. Although the modes-of-action are different, both pesticides proved to be moderately toxic to early life stages of zebrafish with 96 h LC 50 of approximately 6.5 mg L −1 and similar EC 50 values of approximately 4 mg L −1 . Changes in behavioral endpoints were detected 24 h of exposure, suggesting that behavioral measurements can serve as sensitive and early indicators of pesticide exposure. Changes in behavior, such as decrease in spontaneous coiling movements of embryos and reduction of thigmotaxis in larvae, were pronounced for diuron, indicating the usefulness of the application of behavioral endpoints to assess the effects of other herbicides. In the case of diazinon, the effects were less prominent, but the detected changes in ratios between activity in light and darkness also point to the possibility of using behavioral changes for evaluation of insecticide effects. The obtained results support the usage of behavioral endpoints in zebrafish embryos and larvae for the detection of early effects of pesticides.

The zebrafish as a test organism enables the investigation of effects on a wide range of biological levels from molecular level to the whole-organism level. The use of fish embryos represents an attractive model for studies aimed at understanding toxic mechanisms and the environmental risk assessment of chemicals. In the present study, a zebrafish (Danio rerio) in vivo model was employed in order to assess the effects of two commonly used pesticides, the insecticide diazinon and the herbicide diuron, on zebrafish early life stages. Since it was previously established that diazinon and diuron cause effects at the whole-organism level, this study assessed the suborganismic responses to exposure to these pesticides and the enzymatic responses (biochemical level) and the gene expression changes (molecular level) were analyzed. Different exposure scenarios were employed and the following endpoints measured: acetylcholinesterase (AChE), carboxylesterase (CES), ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST), catalase (CAT) and glutathione peroxidase (GPx) activities ; and gene expressions of the corresponding genes: acetylcholinesterase (ache), carboxylesterase (ces2), cytochrome P450 (cyp1a), glutathione-S- transferase (gstp1), catalase (cat), glutathione peroxidase (gpx1a) and additionally glutathione reductase (gsr). Significant changes at both the biochemical and the molecular level were detected. In addition, different sensitivities of different developmental stages of zebrafish were determined and partial recovery of the enzyme activity 48 h after the end of the exposure was observed. The observed disparity between gene expression changes and alterations in enzyme activities points to the necessity of monitoring changes at different levels of biological organization. Different exposure scenarios, together with a comparison of the responses at the biochemical and molecular level, provide valuable data on the effects of diazinon and diuron on low organizational levels in zebrafish embryos and larvae.

Selenium (Se) is an essential micronutrient for humans, animals and certain lower plants, but at higher concentrations Se becomes toxic to organisms. The boundary between the Se beneficial effect and its toxicity is narrow and depends on its chemical form, applied concentration, and other environmentally regulating factors. Due to the potential risk of toxicity in higher concentration, the aim of this study was to estimate the impact of increased concentrations of different forms of Se on the response of wheat-soil-earthworm system. Soil, earthworms and wheat grains were exposed to the Se in form of selenite and selenate in concentrations of 0.01, 0.1 and 1 mg/kg. As an indicator of oxidative stress in wheat, lipid peroxidation levels (LPO) and total H2O2 content were determined, while antioxidative response was determined by catalase (CAT), glutathione peroxidase (GPX) and glutathione reductase (GR) activities. The biomarker responses in earthworms were determined by acetylcholinesterase (AChE), carboxylesterase (CES) and antioxidative enzymes (CAT and glutathione S-transferase) activities. Selenite and selenate increased Se content in the wheat and earthworms, while selenate application was more efficient, indicating higher bioaccumulation of this Se form. Both Se forms did not cause significant changes in the LPO level and H2O2 content, while GPX activities were elevated in all treatments, suggesting that oxidative stress was not induced in wheat. In earthworms, Se significantly reduced activities of AChE and CAT at some concentrations, while CES activity was increased at all concentrations applied. This study showed significant impact of Se on measured biochemical responses in wheat and earthworms, indicating the disruption of homeostasis. Obtained results can serve as basis for further studies on Se effects and will help in including different aspects necessary for understanding of Se impact on different components of soil ecosystems.

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.

This study investigated the direct and indirect toxic effects of two 19 forms of microplastics and nanoplastics toward zebrafish (Danio rerio) larvae locomotor activity. Results showed that microplastics alone 23 exhibited no significant effects, except for the upregulated zfrho visual gene expression ; whereas nanoplastics inhibited the larval locomotion by 22% during the last darkness period, and significantly reduced larvae body length by 6%, inhibited the acetylcholinesterase activity by 40%, and upregulated gfap, α1-tubulin, zfrho and zfblue gene expression significantly. When co-exposed with 2 μg/L 17 α-ethynylestradiol (EE2), microplastics led to alleviation on EE2’s inhibition effect on locomotion, which was probably due to the decreased freely dissolved EE2 concentration. However, though nanoplastics showed stronger adsorption ability for EE2, the hypoactivity phenomenon still existed in the nanoplastics co-exposure group. Moreover, when co-exposed with a higher concentration of EE2 (20 μg/L), both plastics showed an enhanced effect on the hypoactivity. Principal component analysis was performed to reduce data dimensions and four principal components were reconstituted in terms of oxidative stress, body length, nervous and visual system related genes explaining 84% of total variance. Furthermore, oxidative damage and body length reduction were evaluated to be main reasons for the hypoactivity. Therefore, nanoplastics alone supressed zebrafish larvae locomotor activity and both plastic particles can change the larvae swimming behavior when co-exposed with EE2. This study provides new insights into plastic particles’ effects on zebrafish larvae, improving the understanding of their environmental risks to the aquatic environment.

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.

The importance and beneficial effects of earthworms on soil structure and quality is well-established. In addition, earthworms have proved to be important model organisms for investigation of pollutant effects on soil ecosystems. In ecotoxicological investigations effects of various pollutants on earthworms were assessed. But some important issues regarding the effects of pollutants on earthworms still need to be comprehensively addressed. In this review several issues relevant to soil ecotoxicological investigations using earthworms are emphasized and guidelines that should be adopted in ecotoxicological investigations using earthworms are given. The inclusion of these guidelines in ecotoxicological studies will contribute to the better quantification of impacts of pollutants and will allow more accurate prediction of the real field effects of pollutants to earthworms.

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.

The importance and beneficial effects of earthworms on soil structure and quality is well-established. In addition, earthworms have proved to be important model organisms for investigation of pollutant effects on soil ecosystems. In ecotoxicological investigations effects of various pollutants on earthworms were assessed. But some important issues regarding the effects of pollutants on earthworms still need to be comprehensively addressed. In this review several issues relevant to soil ecotoxicological investigations using earthworms are emphasized and guidelines that should be adopted in ecotoxicological investigations using earthworms are given. The inclusion of these guidelines in ecotoxicological studies will contribute to the better quantification of impacts of pollutants and will allow more accurate prediction of the real field effects of pollutants to earthworms.

Earthworms contribute to the functioning and stability of soil processes which are equally fundamental to both forestry and agriculture. Earthworms are generally exposed to chemicals present in their terrestrial environment and have been used for assessing the chemical environmental pollution. Reliable and efficient extraction methods are required for studies of earthworms. However, when ecotoxicological and biomonitoring studies are conducted, besides the efficacy, it is also important that the extraction method does not influence the physiological status of the earthworms. Allyl isothiocyanate (AITC) application and the electrical octet method are non-destructive alternative extraction methods. The focus of this research was to investigate effects of electroshocking and AITC on molecular biomarkers in earthworms, since there is no data about possible side effects of these methods on physiological status of earthworms. Therefore, an experimental study was conducted using the earthworm species Eisenia andrei earthworms and the activities of acetylcholinesterase (AChE), catalase (CAT) and efflux pump were measured. Measurements showed that exposure to AITC on filter paper and direct application of electricity significantly affected measured biomarkers. Electroshocking caused decrease in AChE activity, whereas AITC caused decrease of AChE activity, as well as increase of CAT activity and increase in efflux pump activity. The obtained results demonstrate that these extraction methods could influence the physiological status of earthworms and therefore are unsuitable for ecotoxicological research and biomonitoring studies when sampled earthworms are used for biomarker measurements.

Formalin, the aqueous solution of formaldehyde, is used as a standard earthworm expellant. Since the possible biochemical effects of formalin to earthworms were not investigated, in the present study adult individuals of the earthworm species Eisenia andrei were exposed to sub-lethal concentrations of formalin in order to determine whether its usage as an expellant will influence the physiological status of the earthworms. In all experiments filter paper contact test was used and experiments were conducted under controlled laboratory conditions. Earthworms were exposed to 0.005, 0.01, 0.05, 0.1 and 0.2 mg ml −1 of formalin for 5 min, 15 min, 30 min and 2 h, and the acetylcholinesterase (AChE) activity, catalase (CAT) activity and concentration of oximes were measured. As expected, the lowest AChE activity was measured at the highest concentration of formalin applied (0.2 mg ml −1 ). However, following a 2 h exposure to concentration of 0.01 mg ml −1 , the AChE activity increased up to 1.12 times the activity in the control. Similar results were obtained when concentration of oximes was measured: the lowest concentration of oximes occurred following 2 h exposure to the highest concentration (0.2 mg ml −1 ); and the highest concentration of oximes—equating to 1.18 times increase relative to the control—occurred after a 2 h exposure at 0.01 mg ml −1 . Dose–response curves for AChE activity showed an inverted U-shape characteristic for hormesis and concentration of oximes indicates a role in maintaining the normal AChE activity in the organism. Measurement of CAT activity measurement showed dose and time dependent induction, indicating the occurrence of oxidative stress. The obtained results showed formalin causes measurable effects on the metabolism of E. andrei , therefore the usage of formalin as an earthworm expellant is unsuitable for ecotoxicological research or biomonitoring.

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.

Tribolium castaneum, the red flour beetle, is one of the major pests of cereal grains and their products. Infestation of raw food products by T. castaneum can be tackled by use of insecticides which offer a rapid and effective control method for different insect pests. In the present study, toxicity and repellency of three widely used insecticides was assessed on adult red flour beetles to investigate how a flour substrate affects the results of the standard laboratory toxicity and repellency test methods. The red flour beetles were exposed to the organophosphates dimethoate and pirimiphos-methyl and the pyrethroid deltamethrin, following the original and two modified residual film methods (toxicity assessment) and by the area preference method to assess repellency. Since the conditions of exposure (glass and filter paper surfaces as exposure substrates) in the original methods are quite different compared to the exposure carried out in stored-product protection, methods were modified to include flour as an exposure substrate. The results showed that according to the LC50 values, toxicity of the investigated insecticides could be arranged in the following order: pirimiphos-methyl > dimethoate > deltamethrin. Also, both mortality and repellency were dependent on the exposure methods, i.e. presence and treatment of flour substrate. Mortality was significantly reduced in comparison to the original method. Repellency was recorded when beetles were exposed to deltamethrin using flour as a substrate, whereas using the filter paper surface (original method) repellency was not obtained. The results of the present study clearly indicate that there is an influence of substrate on the susceptibility of T. castaneum to insecticides and that the efficacy of different insecticides is affected by exposure substrate.

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.

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 2 h 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 (2 h) 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.

This study examined the impact of oxidative stress indicated by thiobarbituric acid reactive substances (TBARS) and protein carbonyl (PC), induced by intensive exercise and cadmium chloride (CdCl2) on ethoxyresorufin-O-deethylase (EROD) activity in juvenile carp (Cyprinus carpio). In the first experiment, fish were divided into three groups: (1) control, (2) carp exposed to intensive exercise, and (3) carp that was not exercised but previously, as well as carp in group 2, received single dose of 3-methylcholantrene (3-MC). The third and sixth day fish were sacrificed and the measurements were conducted. In the second experiment, fish were divided into (1) control, (2) carp in water containing CdCl2, and (3) carp in dechlorinated tap water (2 and 3 received single dose of 3-MC on the seventh day after exposure to CdCl2). The carp were killed 6 days later and livers were excised for biochemical analyses. In the first experiment, on the sixth day after treatment with 3-MC, results show statistically significant increase in EROD activity in non-exercised carp, while that increase in carp exposed to intensive exercise was significantly lower. Three days after exposure to 3-MC, statistically significant increase in TBARS was observed in both exercised and non-exercised carp. Six days after exposure to 3-MC, PC levels were significantly higher in exercised carp. Pretreatment with CdCl2, in the second experiment, caused oxidative stress and reduction of EROD activity. Results show linkage between expression of EROD activity and oxidative stress biomarkers and possible influence of oxidative stress on the cell membrane structures and consequently on EROD activity.

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.