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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.

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 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.

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.

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.