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Abstract
Background
The anti-diabetic pharmaceutical metformin is frequently analysed in the aquatic environment. Its impact on the fish microbiome is studied to get a deeper knowledge about the consequence of the metformin presence in river systems. Gut microbiome analyses were performed on larval brown trout with metformin including environmental concentrations. Therefore, the fish were exposed to metformin in concentrations of 0, 1, 10, 100, and 1000 µg/L. Especially, the lower metformin concentrations were measured in river waters containing percentages of conditioned wastewater from municipal wastewater treatment plants.

Results
Two complementary molecular biological methods for population analysis targeting the 16S rRNA gene regions V1–V3, i.e.: (1) 16S amplicon sequencing and (2) polymerase chain reaction (PCR) combined with denaturing gradient gel electrophoresis (DGGE). Both analyses demonstrated significant microbiome alterations even at low metformin concentrations being analysed in German rivers. The amplicon sequencing revealed the most distinct shifts in the Firmicutes phylum, or more specifically, within the Bacillales order, which were most affected by metformin exposure. Within the Bacillales order, the Planococcaceae family, which is described to provide essential amino acids for the fish, completely disappeared after metformin treatment. Conversely, the percentage of other bacteria, such as Staphylococcaceae, increased after exposure to metformin. Similarity profiles of the microbiomes could be generated using the Sørensen index calculation after PCR-DGGE analyses and confirmed shifts in the composition of the brown trout intestinal microbiome after metformin exposures. In vitro gene expression analyses of virulence factors from fish pathogens, previously identified in the fish microbiomes DNA extracts, were conducted in the presence or absence of environmentally relevant concentrations. Here, marker genes of Enterococcus faecium, Enterococcus faecalis, and Aeromonas hydrophila were detected and quantified via PCR approaches, firstly. An increased expression of the species-specific virulence genes was observed after normalisation with control data and ribosomal housekeeping genes.

Conclusion
Environmentally relevant concentrations of metformin can alter the composition in gut microbiome of brown trout in different ways. Both, the metformin-induced expression of virulence genes in fish pathogens in vitro and the impact of metformin on the microbiome composition in vivo in larval brown trout open the discussion about a possible long-term effect on the vitality, growth, and development in more mature brown trouts.

Abstract
Background
Glyphosate is a broad-spectrum, non-selective systemic herbicide with a commonly assumed low potential for accumulation in biota. Nevertheless, glyphosate has been shown to bioaccumulate in the tissues of several organisms. To understand the bioconcentration dynamics of glyphosate in fish, brown trout (Salmo trutta forma fario) of different age were exposed to different concentrations of glyphosate, the formulation Roundup® LB Plus, and the major transformation product aminomethylphosphonic acid (AMPA) for two, three, or four weeks at different temperatures in the laboratory. Mortality rates were determined, and tissue samples were collected at the end of the experiment to ascertain concentrations of glyphosate and AMPA residues by liquid chromatography coupled to mass spectrometry (LC–MS/MS).

Results
Brown trout mortality during exposure to glyphosate or AMPA was considerably higher at 15 °C than at 7 °C. Also, a significant increase in glyphosate concentrations in samples containing muscle, head, backbone, and caudal fin tissue with increasing exposure concentrations and temperatures was observed. Six-month-old fish contained more glyphosate per kg wet weight after exposure than ten-month-old fish. The bioconcentration factors (BCFs) for glyphosate and AMPA were much higher at 15 °C than at 7 °C, but in both cases decreased with higher glyphosate concentrations. The BCF for glyphosate formulated in Roundup® was higher than the one for the parent compound. Approximately 30–42% of the organ-absorbed glyphosate and AMPA remained in the tissues even when the fish were kept in clean water lacking the test substances for three weeks after termination of exposure.

Conclusion
Our study demonstrated that there is an interaction between glyphosate and ambient temperature in terms of toxicity. Further it was shown that increasing concentrations of glyphosate and AMPA in the surrounding media lead to significantly increased concentrations of these substances in brown trout tissues, although neither bioconcentration nor bioaccumulation of glyphosate in animal tissues is expected due to the high water solubility of this chemical. As a consequence, the uptake of glyphosate by humans through the consumption of contaminated edible fish is very likely.

AbstractBackgroundDuring the last decades, a worldwide increase in the number of cases of depression accompanied by rising prescription rates of antidepressants was recorded. In Germany, the two most prescribed antidepressants are the selective serotonin reuptake inhibitor (SSRI) citalopram and the serotonin and noradrenalin reuptake inhibitor (SNRI) venlafaxine, taking about 30% of the market share. Both antidepressants are found frequently in surface waters and have the potential of adversely affecting aquatic organisms. Most studies dealing with antidepressants address apical endpoints and behaviour; however, only few studies investigate biochemical biomarkers and histopathological alterations. We conducted citalopram and venlafaxine exposure experiments over 5 months, starting with brown trout eggs in the eyed-ova stage, as well as with juvenile brown trout for 4 weeks. Exposure concentrations ranged from environmentally relevant 1 µg/L up to 1 mg/L. In this study, we investigated the effects of the antidepressants on b-esterase activity (neurotoxicity), stress protein level (proteotoxicity) and superoxide dismutase activity (oxidative stress). Additionally, we assessed the health status of the liver by means of histopathological analyses.ResultsWe were able to show that both antidepressants did neither induce proteotoxic nor neurotoxic effects in brown trout. But for venlafaxine, the biochemical biomarker for oxidative stress (superoxide dismutase activity) was significantly increased in larvae exposed to at least 10-µg/L venlafaxine at 7 °C. With regard to liver histopathology, fish exposed to higher citalopram concentrations were in a worse health condition than control fish irrespective of their life stage. Also, the energy storage of fish exposed to 1 mg/L citalopram was reduced.ConclusionThus, we here report citalopram-dependent histopathological alterations in brown trout liver, and the induction of oxidative stress by venlafaxine.