Addressing chemical pollution in biodiversity research.

Climate change, biodiversity loss, and chemical pollution are planetary-scale emergencies requiring urgent mitigation actions. As these "triple crises" are deeply interlinked, they need to be tackled in an integrative manner. However, while climate change and biodiversity are often studied together, chemical pollution as a global change factor contributing to worldwide biodiversity loss has received much less attention in biodiversity research so far. Here, we review evidence showing that the multifaceted effects of anthropogenic chemicals in the environment are posing a growing threat to biodiversity and ecosystems. Therefore, failure to account for pollution effects may significantly undermine the success of biodiversity protection efforts. We argue that progress in understanding and counteracting the negative impact of chemical pollution on biodiversity requires collective efforts of scientists from different disciplines, including but not limited to ecology, ecotoxicology, and environmental chemistry. Importantly, recent developments in these fields have now enabled comprehensive studies that could efficiently address the manifold interactions between chemicals and ecosystems. Based on their experience with intricate studies of biodiversity, ecologists are well equipped to embrace the additional challenge of chemical complexity through interdisciplinary collaborations. This offers a unique opportunity to jointly advance a seminal frontier in pollution ecology and facilitate the development of innovative solutions for environmental protection.

Development of a capillary electrophoresis-mass spectrometry method for the analysis of metformin and its transformation product guanylurea in biota.

A method with capillary electrophoresis coupled to mass spectrometry was optimized to determine the uptake of metformin and its metabolite guanylurea by zebrafish (Danio rerio) embryos and brown trout (Salmo trutta f. fario) exposed under laboratory conditions. Metformin was extracted from fish tissues by sonication in methanol, resulting in an absolute recovery of almost 90%. For the extraction of guanylurea from brown trout, solid-phase extraction was implemented with a recovery of 84%. The use of a mixture of methanol and glacial acetic acid as a non-aqueous background electrolyte was vital to achieve robust analysis using a bare fused-silica capillary with an applied voltage of +30 kV. Problems with adsorption associated with an aqueous background electrolyte were eliminated using a non-aqueous background electrolyte made of methanol/acetic acid (97:3) with 25 mM ammonium acetate (for zebrafish embryos) or 100 mM ammonium acetate (for brown trouts), depending on the sample complexity and matrix influences. High resolution and high separation selectivity from matrix components were achieved by optimization of the ammonium acetate concentration in the background electrolyte. An extensive evaluation of matrix effects was conducted with regard to the complex matrices present in the fish samples. They required adapting the background electrolyte to higher concentrations. Applying this method to extracts of zebrafish embryos and brown trout tissue samples, limits of detection for both metformin and guanylurea in zebrafish embryos (12.2 µg/l and 15 µg/l) and brown trout tissues (15 ng/g and 34 ng/g) were in the low µg/l or ng/g range. Finally, metformin and guanylurea could be both quantified for the first time in biota samples from exposure experiments.

Does Bti (Bacillus thuringiensis var. israelensis) affect Rana temporaria tadpoles?

Biocides based on toxins of Bacillus thuringiensis var. israelensis (Bti) are established as alternatives to conventional chemical insecticides for mosquito control all across the globe since they are regarded ecologically compatible and harmless to non-target species. Since recent studies on amphibian larvae have called this opinion into question, we exposed Rana temporaria tadpoles to single (1 mg/L), tenfold (10 mg/L) and hundredfold (100 mg/L) field concentrations of VectoBac® WG (a water dispersible granule Bti formulation) in the laboratory for eleven days to investigate whether larvae were adversely affected by Bti and its endotoxin proteins. In addition to a negative (water) control, a positive control based on organic rice protein (50 mg/L) was run to check for the nutritional relevance of Bti proteins. There was no Bti-related mortality and a histopathological analysis of tadpole intestines revealed no adverse effects. Analyses of biomarkers for proteotoxicity (stress protein family, Hsp70) and neurotoxicity or metabolic action (b-esterases acetylcholine esterase (AChE) and carboxylesterases) revealed no significant differences between Bti treatments and the negative control. The responses of tadpoles in the protein-supplemented positive control differed from those of the negative control and the Bti treatments. Tadpoles in the positive control had reduced body mass and elevated AChE activity.

Drifting Away of a FET Endpoint: The Heart Rate in Danio rerio Embryos is Extremely Sensitive to Variation in Ambient Temperature.

Beyond the mere detection of presence or absence of heart beat in zebrafish (Danio rerio) embryos in a fish embryo test conducted referring to the OECD TG 236 at 48 hpf (hours post fertilization) onwards, embryo heart rate may serve as an additional and very sensitive endpoint in ecotoxicological studies. But by including heart rate as a sublethal endpoint, care has to be taken of separating effects exerted by a tested compound from those exerted by temperature. Therefore, profound knowledge on the natural variation of zebrafish heart rates at defined temperatures as a basis for the assessment of gained results is mandatorily needed. As such continuous information in D. rerio is lacking from the literature, we designed a study covering a span of 12°C (from 18 to 30°C in steps of 2°C) to quantify the relationship between heart rate and temperature in D. rerio embryos 48 hpf. Conducting a multiple regression analysis, we found a considerably strong relationship between treatment temperature and the log10 of the heart rate, ranging from 82.8 beats per minute at 18°C to 218.0 beats per minute at 30°C. Our results therefore may serve as a reference for heart rates measured under normal conditions to be able to detect potential effects of contaminants in other studies when working under certain temperatures.

Reduction of dioxin-like toxicity in effluents by additional wastewater treatment and related effects in fish.

Efficiency of advanced wastewater treatment technologies to reduce micropollutants which mediate dioxin-like toxicity was investigated. Technologies compared included ozonation, powdered activated carbon and granular activated carbon. In addition to chemical analyses in samples of effluents, surface waters, sediments, and fish, (1) dioxin-like potentials were measured in paired samples of effluents, surface waters, and sediments by use of an in vitro biotest (reporter gene assay) and (2) dioxin-like effects were investigated in exposed fish by use of in vivo activity of the mixed-function, monooxygenase enzyme, ethoxyresorufin O-deethylase (EROD) in liver. All advanced technologies studied, based on degradation or adsorption, significantly reduced dioxin-like potentials in samples and resulted in lesser EROD activity in livers of fish. Results of in vitro and in vivo biological responses were not clearly related to quantification of targeted analytes by use of instrumental analyses.

PIXE-electrophoresis shows starving collembolan reallocates protein-bound metals.

One of multiple functions of metalloproteins is to provide detoxification to excess metal levels in organisms. Here we address the induction and persistence of a range of low to high molecular weight copper- and zinc binding proteins in the collembolan species Tetrodontophora bielanensis exposed to copper- and zinc-enriched food, followed by a period of recovery from metal exposure, in absence and presence of food. After 10 days of feeding copper and zinc contaminated yeast, specimens were either moved to ample of leaf litter material from their woodland stand of origin or starved (no food offered). The molecular weight distribution of metal binding proteins was determined by native polyacryl gel electrophoresis. One gel was stained with Comassie brilliant blue and a duplicate gel dried and scanned for the amount of copper and zinc by particle-induced X-ray emission. Specimens exposed to copper and recovered from it with ample of food had copper bound to two groups of rather low molecular weight proteins (40-50 kDa) and two of intermediate size (70-80 kDa). Most zinc in specimens from the woodland stand was bound to two large proteins of about 104 and 106 kDa. The same proteins were holding some zinc in metal-exposed specimens, but most zinc was found in proteins <40 kDa in size. Specimens recovered from metal exposure in presence of ample of food had the same distribution pattern of zinc binding proteins, whereas starved specimens had zinc as well as copper mainly bound to two proteins of 8 and 10 kDa in size. Thus, the induction and distribution of copper- and zinc-binding proteins depend on exposure conditions, and the presence of low molecular weight binding proteins, characteristic of metallothioneins, was mainly limited to starving conditions.

Antioxidant defence and stress protein induction following heat stress in the Mediterranean snail Xeropicta derbentina.

The Mediterranean snail Xeropicta derbentina (Pulmonata, Hygromiidae), being highly abundant in Southern France, has the need for efficient physiological adaptations to desiccation and over-heating posed by dry and hot environmental conditions. As a consequence of heat, oxidative stress manifests in these organisms, which, in turn, leads to the formation of reactive oxygen species (ROS). In this study, we focused on adaptations at the biochemical level by investigation of antioxidant defences and heat shock protein 70 (Hsp70) induction, both essential mechanisms of the heat stress response. We exposed snails to elevated temperature (25, 38, 40, 43 and 45°C) in the laboratory and measured the activity of the antioxidant enzymes catalase (CAT) and glutathione peroxidase (GPx), determined the Hsp70 level and quantified lipid peroxidation. In general, we found a high constitutive level of CAT activity in all treatments, which may be interpreted as a permanent protection against ROS, i.e. hydrogen peroxide. CAT and GPx showed temperature-dependent activity: CAT activity was significantly increased in response to high temperatures (43 and 45°C), whereas GPx exhibited a significantly increased activity at 40°C, probably in response to high levels of lipid peroxides that occurred in the 38°C treatment. Hsp70 showed a maximum induction at 40°C, followed by a decrease at higher temperatures. Our results reveal that X. derbentina possesses a set of efficient mechanisms to cope with the damaging effects of heat. Furthermore, we demonstrated that, besides the well-documented Hsp70 stress response, antioxidant defence plays a crucial role in the snails' competence to survive extreme temperatures.

Shell colouration and parasite tolerance in two helicoid snail species.

The polymorphism of shell colouration in helicoid snails is a well-known phenomenon attributed to different factors such as predation and climatic effects. Another aspect contributing to this polymorphism could be the interplay of melanin production and phenoloxidase-related immunity. Therefore, in this study we aimed at answering the questions whether there is a differential sensitivity of different snail shell colour morphs to nematode infection, and whether this can be related to differences in phenoloxidase (PO) activity levels using the two helicoid, polymorphic snail species Cepaea hortensis and Cernuella virgata. Snails of both species were artificially infected with the parasitic nematode Phasmarhabditis hermaphrodita, and analysed for mortality and PO activity levels. We found C. virgata to be more severely affected by P. hermaphrodita infection than C. hortensis, and the dark C. virgata morphs to be more resistant to lethal effects of this infection than pale morphs. However, these differences in sensitivity to the parasite could not clearly be related to different PO activity levels.

Invertebrates as indicators for chemical stress in sewage-influenced stream systems: toxic and endocrine effects in gammarids and reactions at the community level in two tributaries of Lake Constance, Schussen and Argen.

The present study investigates the impact of releases from waste water treatment plants and storm water overflow basins on gammarids and other macrozoobenthos. The study relates to a recent upgrading of a waste water treatment plant (Langwiese) at the Schussen river, an important tributary to Lake Constance. Samples were taken at different sites at the Schussen river upstream and downstream of a storm water overflow basin and the waste water treatment plant Langwiese and, in parallel, at the Argen river, a less polluted reference stream. We assessed the influence of water quality on the distribution of macrozoobenthos and on the health of gammarid populations by a variety of ecotoxicological methods including biomarkers prior to the expansion of the waste water treatment plant. Through histopathological studies, the impact of parasites on host tissue health was evaluated. Analyses of heat shock protein (hsp70) levels allowed us to draw conclusions about the proteotoxicity-related stress status of the organisms. Furthermore, gammarid populations from all sites were investigated in respect to sex ratio, parasitism rate, and fecundity. Macrozoobenthos community integrity was determined by means of the saprobic index and the abundance as well as by the number of taxa. In gammarids, the sex ratio was significantly shifted towards females, fecundity was significantly decreased, and the hsp70 level was significantly increased downstream of the waste water treatment plant Langwiese, compared to the upstream sampling site. Similarly, these effects could be detected downstream of three small storm water overflow basins. In the macrozoobenthos communities, the abundance of taxa, the number of taxa, the number of ephemeroptera, plecoptera, and trichoptera taxa (EPT-taxa), and the number of sensitive taxa decreased downstream of the storm water overflow basin Mariatal as well as downstream of the waste water treatment plant Langwiese. Our study showed, that waste water treatment plants and storm water overflow basins affected macroinvertebrate communities and the health of gammarids.

External and internal shell formation in the ramshorn snail Marisa cornuarietis are extremes in a continuum of gradual variation in development.

BACKGROUND: Toxic substances like heavy metals can inhibit and disrupt the normal embryonic development of organisms. Exposure to platinum during embryogenesis has been shown to lead to a "one fell swoop" internalization of the shell in the ramshorn snail Marisa cornuarietis, an event which has been discussed to be possibly indicative of processes in evolution which may result in dramatic changes in body plans. RESULTS: Whereas at usual cultivation temperature, 26°C, platinum inhibits the growth of both shell gland and mantle edge during embryogenesis leading to an internalization of the mantle and, thus, also of the shell, higher temperatures induce a re-start of the differential growth of the mantle edge and the shell gland after a period of inactivity. Here, developing embryos exhibit a broad spectrum of shell forms: in some individuals only the ventral part of the visceral sac is covered while others develop almost "normal" shells. Histological studies and scanning electron microscopy images revealed platinum to inhibit the differential growth of the shell gland and the mantle edge, and elevated temperature (28 - 30°C) to mitigate this platinum effect with varying efficiency. CONCLUSION: We could show that the formation of internal, external, and intermediate shells is realized within the continuum of a developmental gradient defined by the degree of differential growth of the embryonic mantle edge and shell gland. The artificially induced internal and intermediate shells are first external and then partly internalized, similar to internal shells found in other molluscan groups.

LogD-based modelling and ΔlogD as a proxy for pH-dependent action of ionizable chemicals reveal the relevance of both neutral and ionic species for fish embryotoxicity and possess great potential for practical application in the regulation of chemicals.

Depending on the ambient pH, ionizable substances are present in varying proportions in their neutral or charged form. The extent to which these two chemical species contribute to the pH-dependant toxicity of ionizable chemicals and whether intracellular ion trapping has a decisive influence in this context is controversially discussed. Against this background, we determined the acute toxicity of 24 ionizable substances at up to 4 different pH values on the embryonic development of the zebrafish, Danio rerio, and supplemented this dataset with additional data from the literature. The LC50 for some substances (diclofenac, propranolol, fluoxetine) differed by a factor of even >103 between pH5 and pH9. To simulate the toxicity of 12 acids and 12 bases, six models to calculate a pH-dependant logD value as a proxy for the uptake of potentially toxic molecules were created based on different premises for the trans-membrane passage and toxic action of neutral and ionic species, and their abilities to explain the real LC50 data set were assessed. Using this approach, we were able to show that both neutral and charged species are almost certainly taken up into cells according to their logD-based distribution, and that both species exert toxicity. Since two of the models that assume all intracellular molecules to be neutral overestimated the real toxicity, it must be concluded, that the toxic effect of a single charged intracellularly present molecule is, on the average, lower than that of a single neutral molecule. Furthermore, it was possible to attribute differences in toxicity at different pH values for these 24 ionizable substances to the respective deltas in logD at these pH levels with high accuracy, enabling particularly a full logD-based model on the basis of logPow as a membrane passage descriptor to be used for predicting potential toxicities in worst-case scenarios from existing experimental studies, as stipulated in the process of registration of chemicals and the definition of Environmental Quality Standards (EQS).

Effects of the Antidepressant Amitriptyline on Juvenile Brown Trout and Their Modulation by Microplastics.

Pharmaceuticals such as antidepressants are designed to be bioactive at low concentrations. According to their mode of action, they can also influence non-target organisms due to the phylogenetic conservation of molecular targets. In addition to the pollution by environmental chemicals, the topic of microplastics (MP) in the aquatic environment came into the focus of scientific and public interest. The aim of the present study was to investigate the influence of the antidepressant amitriptyline in the presence and absence of irregularly shaped polystyrene MP as well as the effects of MP alone on juvenile brown trout (Salmo trutta f. fario). Fish were exposed to different concentrations of amitriptyline (nominal concentrations between 1 and 1000 µg/L) and two concentrations of MP (104 and 105 particles/L; <50 µm) for three weeks. Tissue cortisol concentration, oxidative stress, and the activity of two carboxylesterases and of acetylcholinesterase were assessed. Furthermore, the swimming behavior was analyzed in situations with different stress levels. Exposure to amitriptyline altered the behavior and increased the activity of acetylcholinesterase. Moreover, nominal amitriptyline concentrations above 300 µg/L caused severe acute adverse effects in fish. MP alone did not affect any of the investigated endpoints. Co-exposure caused largely similar effects such as the exposure to solely amitriptyline. However, the effect of amitriptyline on the swimming behavior during the experiment was alleviated by the higher MP concentration.

Heart rate as an early warning parameter and proxy for subsequent mortality in Danio rerio embryos exposed to ionisable substances.

Environmental risk assessments of organic chemicals usually do not consider pH as a key factor. Hence, most substances are tested at a single pH only, which may underestimate the toxicity of ionisable substances with a pKa in the range of 4-10. Thus, the ability to consider the pH-dependent toxicity would be crucial for a more realistic assessment. Moreover, there is a tendency in acute toxicity tests to focus on mortality only, while little attention is paid to sublethal endpoints. We used Danio rerio embryos exposed to ten ionisable substances (the acids diclofenac, ibuprofen, naproxen and triclosan and the bases citalopram, fluoxetine, metoprolol, propranolol, tramadol and tetracaine) at four external pH levels, investigating the endpoints mortality (LC50) and heart rate (EC20). Dose-response curves were fitted with an ensemble-model to determine the true uncertainty and variation around the mean endpoints. The ensemble considers eight (heart rate) or twelve (mortality) individual models for binominal and Poisson distributed data, respectively, selected based on the Akaike Information Criterion (AIC). In case of equally good models, the mean endpoint of all models in the ensemble was calculated, resulting in more robust ECx estimates with lower 'standard errors' as compared to randomly selected individual models. We detected a high correlation between mortality (LC50) at 96 hpf and reduced heart rate (EC20) at 48 hpf for all compounds and all external pH levels (r = 0.98). Moreover, the observed pH-dependent effects were strongly associated with log D and thus, likely driven by differences in uptake (toxicokinetic) rather than internal (toxicodynamic) processes. Prospectively, the a priori consideration of pH-dependent effects of ionisable substances might make testing at different pH levels redundant, while the endpoint of mortality might even be replaced by a reliable sublethal proxy that would reduce the exposure, accelerating the evaluation process.

Experimental simulation of environmental warming selects against pigmented morphs of land snails.

In terrestrial snails, thermal selection acts on shell coloration. However, the biological relevance of small differences in the intensity of shell pigmentation and the associated thermodynamic, physiological, and evolutionary consequences for snail diversity within the course of environmental warming are still insufficiently understood. To relate temperature-driven internal heating, protein and membrane integrity impairment, escape behavior, place of residence selection, water loss, and mortality, we used experimentally warmed open-top chambers and field observations with a total of >11,000 naturally or experimentally colored individuals of the highly polymorphic species Theba pisana (O.F. MÜller, 1774). We show that solar radiation in their natural Mediterranean habitat in Southern France poses intensifying thermal stress on increasingly pigmented snails that cannot be compensated for by behavioral responses. Individuals of all morphs acted neither jointly nor actively competed in climbing behavior, but acted similarly regardless of neighbor pigmentation intensity. Consequently, dark morphs progressively suffered from high internal temperatures, oxidative stress, and a breakdown of the chaperone system. Concomitant with increasing water loss, mortality increased with more intense pigmentation under simulated global warming conditions. In parallel with an increase in mean ambient temperature of 1.34°C over the past 30 years, the mortality rate of pigmented individuals in the field is, currently, about 50% higher than that of white morphs. A further increase of 1.12°C, as experimentally simulated in our study, would elevate this rate by another 26%. For 34 T. pisana populations from locations that are up to 2.7°C warmer than our experimental site, we show that both the frequency of pigmented morphs and overall pigmentation intensity decrease with an increase in average summer temperatures. We therefore predict a continuing strong decline in the frequency of pigmented morphs and a decrease in overall pigmentation intensity with ongoing global change in areas with strong solar radiation.

Turning snails into slugs: induced body plan changes and formation of an internal shell.

The archetypal body plan of conchiferan molluscs is characterized by an external calcareous shell, though internalization of shells has evolved independently in a number of molluscan clades, including gastropod families. In gastropods, the developmental process of torsion is regarded as a hallmark that is associated with a new anatomical configuration. This configuration is present in extant prosobranch gastropod species, which predominantly bear external shells. Here, we show that short-term exposure to platinum during development uncouples at least two of the processes associated with torsion of the freshwater snail Marisa cornuarietis. That is, the anus of the treated snails is located anteriorly, but the gill and the designated mantle tissue remains in a posterior location, thus preventing the formation of an external shell. In contrast to the prosobranchian archetype, platinum treatment results in the formation of a posterior gill and a cone-shaped internal shell, which persists across the lifetime. This first finding of artificially induced snail-slug conversion was also seen in the pulmonate snail Planorbarius corneus and demonstrates that selective alteration of embryonic key processes can result in fundamental changes of an existing body plan and-if altered regulation is inherited-may give rise to a new one.

Physiological and behavioural effects of imidacloprid on two ecologically relevant earthworm species (Lumbricus terrestris and Aporrectodea caliginosa).

Earthworms play key roles in soils and sub-lethal effects of environmental toxicants on these organisms should be taken seriously, since they might have detrimental effects on higher ecological levels. In laboratory experiments we have assessed sub-lethal effects (body mass change and cast production) of imidacloprid on two earthworm species commonly found in different agricultural soils (Lumbricus terrestris and Aporrectodea caliginosa). After 7 days of exposure in contaminated soil, a significant loss of body mass was found in both species exposed to imidacloprid concentrations as low as 0.66 mg kg(-1) dry soil. These losses ranged from 18.3 to 39% for A. caliginosa and from 7.4 to 32.4% for L. terrestris, respectively. Changes in cast production, a new biomarker previously validated using L. terrestris, was assessed by soil sieving using the recommended mesh size (5.6 mm) for L. terrestris and three different mesh sizes for A. caliginosa (5.6, 4 and 3.15 mm). The 4 mm mesh size proved to be the most suitable sieve size for A. caliginosa. Cast production increased by 26.2% in A. caliginosa and by 28.1% in L. terrestris at the lowest imidacloprid concentration tested (0.2 mg kg(-1) dry soil), but significantly decreased at higher concentrations (equal to and above 0.66 mg kg(-1) dry soil) in both earthworm species after the 7 days exposure experiment. These decreases in cast production ranged from 44.5 to 96.9% in A. caliginosa and from 42.4 to 95.7% in L. terrestris. The EC(50) for cast production were 0.84 (L. terrestris) and 0.76 mg kg(-1) dry soil (A. caliginosa), respectively. The detected sub-lethal effects were found close to the predicted environmental concentration (PEC) of imidacloprid, which is in the range of 0.33-0.66 mg kg(-1) dry soil. The biomarkers used in the present study, body mass change and changes in cast production, may be of ecological relevance and have shown high sensitivity for imidacloprid exposure of earthworms. The measurement of changes in cast production should be considered for inclusion in current standard tests.

Embryo development, stress protein (Hsp70) responses, and histopathology in zebrafish (Danio rerio) following exposure to nickel chloride, chlorpyrifos, and binary mixtures of them.

Two different classes of chemicals were tested in a multilevel approach in this study: NiCl(2) 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. NiCl(2) 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.

Impact of the antidepressant citalopram on the behaviour of two different life stages of brown trout.

BACKGROUND: Over the last two decades, there has been a constant increase in prescription rates of antidepressants. In parallel, neuroactive pharmaceuticals are making their way into aquatic environments at increasing concentrations. Among the antidepressants detected in the environment citalopram, a selective serotonin reuptake inhibitor, is one of the most commonly found. Given citalopram is specifically designed to alter mood and behaviour in humans, there is growing concern it can adversely affect the behaviour on non-target wildlife. METHODS: In our study, brown trout were exposed to citalopram (nominal concentrations: 1, 10, 100, 1000 µg/L) in two different life stages. Larvae were exposed at 7 and 11 °C from the eyed ova stage until 8 weeks post yolk sac consumption, and juvenile brown trout were exposed for 4 weeks at 7 °C. At both stages we measured mortality, weight, length, tissue citalopram concentration, behaviour during exposure and behaviour in a stressfull environment. For brown trout larvae additionally hatching rate and heart rate, and for juvenile brown trout the tissue cortisol concentration were assessed. RESULTS: During the exposure, both larvae and juvenile fish exposed to the highest test concentration of citalopram (1 mg/L) had higher swimming activity and spent longer in the upper part of the aquaria compared to control fish, which is an indicator for decreased anxiety. Most probably due to the higher swimming activity during the exposure, the juveniles and larvae exposed to 1 mg/L citalopram showed decreased weight and length. Additionally, in a stressful artificial swimming measurement device, brown trout larvae displayed the anxiolytic effect of the antidepressant by reduced swimming activity during this stress situation, already at concentrations of 100 µg/L citalopram. Chemical analysis of the tissue revealed rising citalopram tissue concentrations with rising exposure concentrations. Tissue concentrations were 10 times higher in juvenile fish compared to brown trout larvae. Fish plasma concentrations were calculated, which exceeded human therapeutic levels for the highest exposure concentration, matching the behavioural results. Developmental parameters like hatching rate and heart rate, as well as mortality and tissue cortisol content were unaffected by the antidepressant. Overall, we could trace the pharmacological mode of action of the antidepressant citalopram in the non-target organism brown trout in two different life stages.