The present work describes a field survey aiming at assessing the impact of a sewage treatment plant (STP) effluent on fish health by means of biomarkers. Indigenous fish were absent downstream of the STP. To elucidate the reason behind this, brown trout (Salmo trutta f. fario) were exposed in floating steel cages up- and downstream of a STP located at the Neckar River near Tübingen (Southern Germany), for 10 and 30 days. A combination of biomarker methods (histopathological investigations, analysis of the stress protein Hsp70, micronucleus test, B-esterase assays) offered the possibility to investigate endocrine, geno-, proteo- and neurotoxic effects in fish organs. Biological results were complemented with chemical analyses on 20 accumulative substances in fish tissue. Even after short-term exposure, biomarkers revealed clear evidence of water contamination at both Neckar River sites; however, physiological responses of caged brown trout were more severe downstream of the STP. According to this, similar bioaccumulation levels (low μg/kg range) of DDE and 12 polycyclic aromatic hydrocarbons (PAHs) were detected at both sampling sites, while up to fourfold higher concentrations of four PAHs, methyl-triclosan and two synthetic musks occurred in the tissues of downstream-exposed fish. The results obtained in this study suggest a constitutive background pollution at both sites investigated at the Neckar River and provided evidence for the additional negative impact of the STP Tübingen on water quality and the health condition of fish.

The Neckar River (Southern Germany) represents an aquatic system strongly affected by numerous anthropogenic activities. Thus, it is an excellent model for ecotoxicological investigations. The present study aims to assess time and spatial variations of embryo- and proteotoxic effects in surface water and sediment. For this end, embryos of zebrafish (Danio rerio) were exposed to Neckar River samples collected in the Tübingen region in different seasons over 2 years. Additionally, quantification of the heat shock (stress) protein Hsp70 was carried out in newly hatched larvae; furthermore, physico-chemical water parameters were measured in order to gain baseline information about the limnologic conditions.

Theba pisana (Müller, 1774), a Mediterranean snail, is known for its heat tolerance and its remarkable shell colour polymorphism ranging from pale white to darkly striped, although darker morphs are considered less vital in hot habitats due to presumably stronger absorption of radiation. Melanin, the black pigment of these snails, is known as an effective antioxidant, and elevated temperatures can increase oxidative stress. By analysing oxidative waste products via the ferrous oxidation xylenol orange (FOX) assay as a marker for the lipid peroxidation level, we aimed at investigating possible links between heat stress, colouration and antioxidant defence capacity in T. pisana. Although we found increasing levels of peroxidation products with increasing heat exposure duration, there was no difference in antioxidant defence capacity observable between different morphs of T. pisana. Hence the avoidance of lipid peroxidation as an environmentally relevant factor for the maintenance of melanistic morphs in strongly illuminated habitats can be considered improbable.

Ecotoxicological effect studies often expose test organisms under optimal environmental conditions. However, organisms in their natural settings rarely experience optimal conditions. On the contrary, during most of their lifetime they are forced to cope with sub-optimal conditions and occasionally with severe environmental stress. Interactions between the effects of a natural stressor and a toxicant can sometimes result in greater effects than expected from either of the stress types alone. The aim of the present review is to provide a synthesis of existing knowledge on the interactions between effects of “natural” and chemical (anthropogenic) stressors. More than 150 studies were evaluated covering stressors including heat, cold, desiccation, oxygen depletion, pathogens and immunomodulatory factors combined with a variety of environmental pollutants. This evaluation revealed that synergistic interactions between the effects of various natural stressors and toxicants are not uncommon phenomena. Thus, synergistic interactions were reported in more than 50% of the available studies on these interactions. Antagonistic interactions were also detected, but in fewer cases. Interestingly, about 70% of the tested chemicals were found to compromise the immune system of humans as judged from studies on human cell lines. The challenge for future studies will therefore be to include aspects of combined stressors in effect and risk assessment of chemicals in the environment.

The paper addresses problems arising from effects of natural environmental factors on toxicity of pollutants to organisms. Most studies on interactions between toxicants and natural factors, including those completed in the EU project NoMiracle (Novel Methods for Integrated Risk Assessment of Cumulative Stressors in Europe) described herein, showed that effects of toxic chemicals on organisms can differ vastly depending purely on external conditions. We compiled data from 61 studies on effects of temperature, moisture and dissolved oxygen on toxicity of a range of chemicals representing pesticides, polycyclic aromatic hydrocarbons, plant protection products of bacterial origin and trace metals. In 62.3% cases significant interactions (p ≤ 0.05 or less) between natural factors and chemicals were found, reaching 100% for the effect of dissolved oxygen on toxicity of waterborne chemicals. The meta-analysis of the 61 studies showed that the null hypothesis assuming no interactions between toxic chemicals and natural environmental factors should be rejected at p = 2.7 × 10− 82 (truncated product method probability). In a few cases of more complex experimental designs, also second-order interactions were found, indicating that natural factors can modify interactions among chemicals. Such data emphasize the necessity of including information on natural factors and their variation in time and across geographic regions in ecological risk assessment. This can be done only if appropriate ecotoxicological test designs are used, in which test organisms are exposed to toxicants at a range of environmental conditions. We advocate designing such tests for the second-tier ecological risk assessment procedures.

Two independent types of stressors, chemicals and high temperatures, which frequently act together in the environment, are addressed in this study. Pesticides (imidacloprid and chlorpyrifos) as well as a metal salt (nickel chloride) were investigated for their toxic effect at different temperatures. Tests focused on the early development of zebrafish (Danio rerio) embryos and larvae (from fertilization up to 168 h postfertilization) when exposed to the three respective chemicals at an optimum temperature (26 degrees C) and three higher temperatures (up to 33.5 degrees C). At all temperatures tested, the two pesticides did not have a significant impact on the early development of the zebrafish at the highest test concentrations (imidacloprid, 50 mg/l; chlorpyrifos, 1 mg/l). Nickel led to a significant decrease of hatching success at all temperatures; the combination of elevated temperature and nickel exposure revealed a synergistic effect of both stressors.

Two different classes of chemicals were tested in a multilevel approach in this study: NiCl2 as a representative for heavy metals and chlorpyrifos, a pesticide. Both, the single substances and mixtures of them were investigated for their effects on embryonic development, histological alterations, and the stress protein (Hsp70) response in the zebrafish Danio rerio. Fishes were exposed from fertilization of eggs up to a maximum of 168 h post fertilization, depending on the investigated endpoint. NiCl2 led to effects in all tests which, however, were less severe at the histopathological level than in developmental (hatching success) and stress protein studies. Chlorpyrifos did not lead to developmental alterations but it was found to induce the Hsp70 response as well as histopathological damages. Mixtures of both substances resulted in similar results as the single substances; the results suggest an independent mode of action of these two substances and additivity of their effects. © 2009 Wiley Periodicals, Inc. Environ Toxicol, 2010.

In this study the effects of 3,4-dichloroaniline (3,4-DCA), a decomposition product of the herbicides propanil and diuron (and other pesticides), and diazinon, a neurotoxic insecticide, on early life stages of zebrafish Danio rerio were assessed. The toxicity of these substances with different modes of action (acetylcholine esterase inhibitor vs. polar narcosis) was tested for single substances as well as in binary mixtures. To study effects on different biological organisation levels (from the molecular up to the whole organism level) the molecular stress response regarding Hsp70, the embryonic and larval development and the locomotor activity were investigated as integrative biomarkers. In single substance tests 3,4-dichloroaniline elicited deformations at ≥0.25 mg/l during the 11 days subchronic test, whereas locomotor activity and mortality were impaired at ≥0.5 mg/l. Diazinon effects on those parameters were obvious at ≥2 mg/l, except for the deformation rate (11 days:1 mg/l). In equitoxic mixtures of both substances concentration additivity was observed for deformation rate and mortality (11 days). An increase in the Hsp70 content occurred in zebrafish exposed to 0.25 mg 3,4-DCA/l as well as to 0.05 mg diazinon/l; in mixtures concentration additivity could be shown. The investigated endpoints varied in respect to their sensitivity, with deformations and Hsp70 levels as most sensitive parameters concerning 3,4-DCA and Hsp70 as most sensitive parameter concerning diazinon. Accordingly, for an integrated understanding of the effects of chemicals and their mixtures on fish, a battery of different test methods should be applied.

Chemical ultraviolet (UV) filters are used in various products and could be released into the environment, for example, via sunscreens used at swimming lakes. UV filters have been found in surface waters in the past but only a few investigations have concentrated on the effects of these substances in the environment. This study investigates the effects of a UV filter in the amphipod Gammarus fossarum at the cellular and molecular level. Stress protein (Hsp70) responses and reactions of hepatopancreatic cells and cells of gut appendices were investigated in the freshwater amphipod Gammarus fossarum after short-term exposure (4 days) to five different concentrations of the UV filter 3-benzylidene camphor (3-BC; 33 ng/L, 330 ng/L, 3.3 μg/L, 33 μg/L, 330 μg/L) and two control conditions (water and solvent ethanol). Male as well as female gammarids showed increased Hsp70 levels after exposure to low concentrations of 3-BC, with a maximum response at 3.3 μg/L, while the higher concentrations resulted in lower Hsp70 levels. This effect was most likely due to a cessation of Hsp70 synthesis following pathological impact as indicated by strong cellular responses and cellular damage obtained in epithelia of the hepatopancreas and the gut appendices after treatment with 330 μg/L 3-BC. Although environmentally relevant concentrations of 3-BC did not seem to have an adverse effect in this short-term study, higher concentrations of 3-BC are surely detrimental. It is known that chronic exposure generally requires much lower concentrations to cause harm than acute exposure. Additionally, juvenile stages may be even more sensitive than the adults tested. Therefore, on the basis of this study, 3-BC-effects in the field cannot be excluded and should be regarded possible.

In order to assess potential effects of human pharmaceuticals in aquatic wildlife, laboratory experiments were conducted with carbamazepine, clofibric acid, metoprolol, and diclofenac using fish as test organisms. For each substance, at least one environmentally relevant concentration was tested. In liver, kidney, and gills of trout and carp exposed to carbamazepine, clofibric acid, and metoprolol, ultrastructural effects were qualitatively described and semi-quantitatively assessed. The obtained assessment values were compared with previously published data for diclofenac-induced effects in rainbow trout tissues. Quantitative analyses of protein accumulated in kidneys of diclofenac-exposed trout corroborated previously published data which indicated that diclofenac induced a severe glomerulonephritis resulting in a hyaline droplet degeneration of proximal kidney tubules. The investigations provided information on the general health status of the pharmaceutical-exposed fish, and allowed a differential diagnosis of harmful effects caused by these human pharmaceuticals in non-target species. For the different cytological effects observed, lowest observed effect concentration (LOECs) for at least three of the test substances (diclofenac, carbamazepine, metoprolol) were in the range of environmentally relevant concentrations (1 μg/L).

Along a downstream stretch of River Mureş, Romania, adult males of two feral fish species, European chub ( Leuciscus cephalus ) and sneep ( Chondrostoma nasus ) were sampled at four sites with different levels of contamination. Fish were analysed for the biochemical markers hsp70 (in liver and gills) and hepatic EROD activity, as well as several biometrical parameters (age, length, wet weight, condition factor). None of the biochemical markers correlated with any biometrical parameter, thus biomarker reactions were related to site-specific criteria. While the hepatic hsp70 level did not differ among the sites, significant elevation of the hsp70 level in the gills revealed proteotoxic damage in chub at the most upstream site, where we recorded the highest heavy metal contamination of the investigated stretch, and in both chub and sneep at the site right downstream of the city of Arad. In both species, significantly elevated hepatic EROD activity downstream of Arad indicated that fish from these sites are also exposed to organic chemicals. The results were indicative of impaired fish health at least at three of the four investigated sites. The approach to relate biomarker responses to analytical data on pollution was shown to fit well the recent EU demands on further enhanced efforts in the monitoring of Romanian water quality.