Quantitative Nontarget Analysis of CECs in Environmental Samples Can Be Improved by Considering All Mass Adducts.

Quantitative nontarget analysis (qNTA) for liquid chromatography coupled to high-resolution mass spectrometry enables a more comprehensive assessment of environmental samples. Previous studies have shown that correlations between a compound's ionization efficiency and a range of molecular descriptors can predict the compound's concentration within a factor of 5. In this study, the qNTA approach was further improved by considering all mass adducts instead of only the protonated ion. The model was based on a quantitative structure-property relationship (QSPR), including 216 contaminants of emerging concern (CECs), of which 80 exhibited adduct formation that accounted for >10% of the total peak intensity. When all mass adducts were included, the test set coefficient of determination improved to Q2 = 0.855 compared to Q2 = 0.670 when only the protonated ions were considered (test set median RF error factor 1.6). The inclusion of all adducts was also important to transfer the RF QSPR model reliably. It was assumed that RF variations are sequence-dependent; therefore, a second QSPR model for the prediction of the transferability factor was built for each sequence. For validation, samples were analyzed up to two years apart. The median prediction fold change was 1.74 for analytical standards (63 compounds) and 2.4 for enriched wastewater effluent samples (41 compounds), with 80% of the compounds predicted within a fold change of 2.4 and 3.3, respectively. The model was also validated on a second instrument, where 80% of the 26 compounds in wastewater effluent were predicted within a factor of 3.8.

Catchment area, fate, and environmental risks investigation of micropollutants in Danish wastewater.

This study aimed to investigate the spatial distribution of micropollutants in wastewater related to catchment area, and their environmental risks and fate. About 24-h flow proportional effluent (n = 26) wastewater samples were collected from eight WWTPs across Denmark. From five of these WWTPs corresponding influent samples (n = 20) were collected. Samples were enriched by multi-layer solid phase and analysed by liquid chromatography-high-resolution mass spectrometry and comprehensive two-dimensional gas chromatography with high-resolution mass spectrometry detection. We detected and quantified 79 micropollutants from a list of 291 micropollutants in at least one influent or effluent wastewater sample. From this we found that 54 micropollutants decreased in concentrations during wastewater treatment, while O-desmethylvenlafaxine, carbamazepine, amitriptyline, benzothiazole, terbutryn, and citalopram increased in concentrations through the WWTP.The toxicity of effluent wastewater samples was assessed by EC50 using Raphidocelis subcapitata (R. subcapitata) and LC50 using the crustacean Daphnia magna (D. Magna), for which six micropollutants were detected above the predicted no-effect concentration. Our study demonstrates that catchment area influences the micropollutant composition of wastewater. Out of 19 pharmaceuticals, the measured concentration in influent wastewater was predicted within a factor of 10 from sale numbers and human excretion, which demonstrates the strong influence of catchment area on micropollutant composition.

Supercritical Fluid Chromatography Coupled to High-Resolution Mass Spectrometry Reveals Persistent Mobile Organic Compounds with Unknown Toxicity in Wastewater Effluents.

Broad screening approaches for monitoring wastewater are normally based on reversed-phase liquid chromatography (LC) coupled to high-resolution mass spectrometry (HRMS). This method is not sufficient for the very polar micropollutants, neglected in the past due to a lack of suitable analytical methods. In this study, we used supercritical fluid chromatography (SFC) to detect very polar and yet-undetected micropollutants in wastewater effluents. We tentatively identified 85 compounds, whereas 18 have only rarely been detected and 11 have not previously been detected in wastewater effluents such as 17α-hydroxypregnenolone, a likely transformation product (TP) of steroids, and 1H-indole-3-carboxamide, a likely TP from new synthetic cannabinoids. Suspect screening of 25 effluent wastewater samples from 8 wastewater treatment plants revealed several distinct potential pollution sources such as a pharmaceutical company and a golf court. The analysis of the same samples with LC-HRMS showed clearly how SFC increases the ionization efficiency for low-molecular-weight micropollutants (m/z < 300 Da) by a factor 2 to 87 times, which significantly improved the mass spectra for identifying very polar compounds. In order to assess which micropollutants might be of environmental concern, literature and toxicological databases were screened. There was a lack of available hazard and bio-activity data for regulatory-relevant in vitro and in vivo assays for >50% of the micropollutants. Especially, 70% of the data were lacking for the whole organism (in vivo) tests.

A non-target screening study of high-density polyethylene pipes revealed rubber compounds as main contaminant in a drinking water distribution system.

Polyethylene (PE) pipes are often the material of choice for water supply systems, thanks to their favorable properties, such as high strength-density ratio and corrosion resistance. However, previous studies have shown that organic compounds can migrate from PE pipes to the water. This study aimed to identify potential organic compounds migrating from high-density PE (HDPE) pipes used to distribute drinking water in Denmark, based on laboratory experiments and sampling in the distribution system using a two-tiered study design. In the first tier, migration of volatile and semi-volatile organic compounds (VOCs and semi-VOCs) from HDPE pipes were investigated over one, three, and nine days in laboratory experiments, performed according to modified standards for migration testing (EN 12,873-1). The analytical workflow consisted of solid-phase extraction (SPE) for 10,000 times enrichment and gas chromatography - mass spectrometry (GC-MS) analysis from the water phase after migration. A total of 133 compounds originating from the PE pipes were detected. Thirty-one compounds were detected by suspect screening (SS), while the remaining 102 compounds were detected by non-target screening (NTS) analysis. Among the detected compounds were also hindered amine stabilizers (HALS), flame retardant, and plasticizer tris(2-chloroethyl) phosphate. In the second tier, drinking water from a water distribution system in Copenhagen, Denmark, with a newly installed HDPE pipe was sampled and analyzed with GC-MS and liquid chromatography high-resolution mass spectrometry (LCHRMS). A total of 51 compounds were detected in the water, 12 of which were assigned to migration from HDPE. Surprisingly, HDPE antioxidants and their degradation products contributed only a relatively small percentage of the total measured compound intensities in the drinking water distribution system. Instead, a larger proportion of the compounds detected were assigned to rubber seals, used upstream in the water system from the abstraction site to delivery at the consumer tap. Seals are considered trifle in the larger picture of materials in contact with drinking water, however these results may cause a reconsideration of this position.

Non-target screening of micropollutants and transformation products for assessing AOP-BAC treatment in groundwater.

Standard monitoring programs give limited insight into groundwater status, especially transformation products (TPs) formed by natural processes or advanced oxidation processes (AOP), are normally underrepresented. In this study, using suspect and non-target screening, we performed a comprehensive analysis of groundwater before and after AOP by UV/H2O2 and consecutively installed biological activated carbon filters (BAC). By non-target screening, up to 413 compounds were detected in the groundwater, with an average 70% removal by AOP. However, a similar number of compounds were formed during the process, shown in groundwater from three waterworks. The most polar compounds were typically the most stable during the AOP. A subsequent BAC filter showed removal of 95% of the TPs, but only 46% removal of the AOP remaining precursors. The BAC removal for polar compounds was highly dependent on the acidic and basic functional groups of the molecules. 49 compounds of a wide polarity range could be identified by supercritical fluid chromatography (SFC) and liquid chromatography (LC) with high resolution mass spectrometry (HRMS); of these, 29 compounds were already present in the groundwater. To the best of our knowledge, five compounds have never been reported before in groundwater (4-chlorobenzenesulfonic acid, dibutylamine, N-phenlybenzenesulfonamide, 2-(methylthio)benzothiazole and benzothiazole-2-sulfonate). A further five rarely reported compounds are reported for the first time in Danish groundwater (2,4,6-trichlorophenol, 2,5-dichlorobenzenesulfonic acid, trifluormethansulfonic acid, pyrimidinol and benzymethylamine). Twenty of the identified compounds were formed by AOP, of which 10 have never been reported before in groundwater. All detected compounds could be related to agricultural and industrial products as well as artificial sweeteners. Whereas dechlorination was a common AOP degradation pathway for chlorophenols, the (ultra-) short chain PFAs showed no removal in our study. We prioritized 11 compounds as of concern, however, the toxicity for many compounds remains unknown, especially for the TPs.

From data to reliable conclusions: Identification and comparison of persistent micropollutants and transformation products in 37 wastewater samples by non-target screening prioritization.

In this study, micropollutants in wastewater effluents were prioritized by monitoring the composition of influent and effluent wastewater by liquid chromatography - high-resolution mass spectrometry (LCHRMS) non-target screening (NTS) analysis. The study shows how important data pre-processing and filtering of raw data is to produce reliable NTS data for comparison of compounds between many samples (37 wastewater samples) analyzed at different times. Triplicate injections were critical for reducing the number of false-positive detections. Intensity drift corrections within and between batches analyzed months apart made peak intensities comparable across samples. Adjustment of the feature detection threshold was shown to be important, due to large intensity variations for low abundance compounds across batches. When the threshold correction cut-offs, or the filtering of relevant compounds by the occurrence frequency, were too stringent, a high number of false positive transformation products (TPs) were reported. We also showed that matrix effect correction by internal standards can over- or under-correct the intensity for unknown compounds, thus the TIC matrix effect correction was shown as an additional tool for a retention time dependent matrix effect correction. After these preprocessing and filtering steps, we identified 78 prioritized compounds, of which 36 were persistent compounds, defined as compounds with a reduction in peak intensity between influent and effluent wastewater <50%, and 13 compounds were defined as TPs because they occurred solely in the effluent samples. Some examples of persistent compounds are 1,3-diphenylguanidine, amisulpride and the human metabolites from losartan, verapamil and methadone. To our knowledge, nine of the identified TPs have not been previously described in effluent wastewater. The TPs were derived from metoprolol, fexofenadine, DEET and losartan. The screening of all identified compounds in effluent samples from eight wastewater treatment plants (WWTPs) showed that potential drugs of abuse, anti-psychotic and anti-depressant drugs were predominant in the capital city region, whereas the anti-epileptic agents and agricultural pesticides were dominant in more rural areas.

Non-target screening for the identification of migrating compounds from reusable plastic bottles into drinking water.

Reusable plastic sports bottles are used extensively worldwide, and little is known about the migration of chemicals from the bottles into drinking water. In this study, we investigated the chemical migration into drinking water stored for 24 h in new bottles, used bottles and bottles washed in the dishwasher. Non-target screening (NTS) by liquid-chromatography - high-resolution mass spectrometry (LC-HRMS) was performed to identify these compounds. We detected > 3500 dishwasher related compounds, with 430 showing migration even after subsequent flushing of the bottles. In addition, more than 400 plastic related compounds were detected, with high peaks for oligomers suspected to originate from the biodegradable polyester polycaprolactone, and aromatic amines, which may have been introduced as slip agents or antioxidants. These compounds have never been reported before in bottled water. Most of the identified compounds migrating out of the used bottles were plasticizers, antioxidants or photoinitiators. The presence of photoinitiators are of particular concern, due to possible endocrine disrupting effects. Furthermore, diethyltoluamide (DEET) was detected, which may have been formed from the plasticizer laurolactam. Typically, the dishwashing process enhanced the leaching of plastic related compounds, and even after additional water flushing, the average peak intensity of these compounds was only reduced by half.

Correction of Matrix Effects for Reliable Non-target Screening LC-ESI-MS Analysis of Wastewater.

Matrix effects are well-known challenges for accurate and comparable measurements with liquid chromatography (LC) electrospray ionization mass spectrometry (ESI-MS). This study describes a three-step method to evaluate and compensate for matrix effects in enriched wastewater extracts using LC ESI-high-resolution MS (HRMS). As a first step, the "dilute and shoot" approach was used to determine the optimal relative enrichment factor (REF) for a direct comparison between wastewater influent (REF 10) and effluent (REF 50) extracts. However, the rapid decrease in the number of non-target compounds detected with increasing dilution leads to the need for a correction of the matrix effect for analyzing samples with higher REFs. As a second step, the observed matrix effect at higher REFs was corrected by the retention time-dependent matrix effect. A new scaling (TiChri scale) of the matrix effect was introduced, which demonstrates that the total ion chromatogram (TIC) predicts the matrix effect as effectively as post-column infusion (PCI) approaches; thus, the average median matrix effect was improved from -65 to 1% for influent (REF 100) and from -46 to -2% for effluent extracts (REF 250). The TIC traces for concentrated (REF 250) influent and effluent samples were successfully used to correct the matrix effects and allowed the extent of micropollutant degradation in three WWTPs to be quantified. As a final step, the residual structure-specific matrix effect was predicted and corrected by quantitative structure-property relationships (QSPR), which led to a further correction of the matrix effect to 0 ± 7% for 65 compounds.

Identification of more than 100 new compounds in the wastewater: Fate of polyethylene/polypropylene oxide copolymers and their metabolites in the aquatic environment.

100 ethylene oxide (EO)/propylene oxide (PO) copolymer precursor and metabolites were detected in wastewater effluents. The homopolymers of EO and PO as well as the EO/PO copolymers are widely used as surfactants, e.g., for the production of cosmetics, pharmaceuticals and lubricants. Concomitantly, these compounds are discharged into the wastewater and the environmental fate of the PO homopolymers, also called polypropylene glycols (PPGs), and EO/PO copolymers is mostly unknown. In the present study, we identified hitherto unknown copolymer EO/PO homologous series and their metabolites in wastewater effluent. The identified compounds occur in homologous series and consist of PPGs and EO/PO copolymers, and their carbonylated, carboxylated and dicarboxylated metabolites. MBBR lab incubations of PPGs and EO/PO copolymers showed the successive degradation by cleavage of individual PO and EO groups, with high removal (>90%) in the initial 8 h for most of the copolymers. Carbonylated and carboxylated metabolites were degraded within 40 h. EO/PO copolymers with a higher number of EO and PO units showed a higher removal in MBBR and conventional activated sludge wastewater treatment plants. Polymers with lower molecular weight were initially formed by degradation of the EO/PO polymers. The mono-carboxylated metabolites were also detected in surface waters. Overall, our results provide new knowledge about degradation pathways of PO containing compounds and show the hitherto unnoticed occurrence of EO/PO copolymers and metabolites in the water cycle.

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.

Norfluoxetine Is the Only Metabolite of Fluoxetine in Zebrafish (Danio rerio) Embryos That Accumulates at Environmentally Relevant Exposure Scenarios.

Fluoxetine has been recognized as one of the most toxic pharmaceuticals in the aquatic environment. Since there is growing evidence that the toxic potential of fluoxetine in surface waters is markedly influenced by its own metabolism in aquatic species, this study investigated the biotransformation of fluoxetine in the zebrafish embryo - an aquatic model organism of intermediate complexity. Zebrafish embryos were exposed to 0.1, 1.0, 10, 50, and 5000 µg/L of fluoxetine from 48 to 120 h post-fertilization (hpf), and the accumulation of fluoxetine and its metabolites was analyzed over time. Additionally, depuration of fluoxetine and its metabolites from 96 to 120 hpf was investigated, and autoinhibitory effects of fluoxetine on phase I biotransformation were analyzed. Exposure to 5000 µg/L fluoxetine resulted in elevated 7-ethoxyresorufin-O-deethylase (EROD) activity of cytochrome P450 enzymes and continuous accumulation of fluoxetine and 11 fluoxetine metabolites. Embryos exposed to 10 and 50 µg/L fluoxetine were able to reduce fluoxetine accumulation from 94 to 120 hpf. During depuration, accumulation of fluoxetine and most metabolites was clearly reduced, and biotransformation shifted in favor of norfluoxetine, the primary fluoxetine metabolite in humans. Findings demonstrated that norfluoxetine is the only metabolite of fluoxetine that accumulates in zebrafish embryos at environmentally relevant exposure scenarios.

Effects of guanylurea, the transformation product of the antidiabetic drug metformin, on the health of brown trout (Salmo trutta f. fario).

BACKGROUND: Guanylurea is the main transformation product of the antidiabetic drug metformin, which is one of the most prescribed pharmaceuticals worldwide. Due to the high rate of microbial degradation of metformin in sewage treatment plants, guanylurea occurs in higher concentrations in surface waters than its parent compound and could therefore affect aquatic wildlife. In this context, data for fish are scarce up to now which made us investigate the health of brown trout (Salmo trutta f. fario) in response to guanylurea. METHODS: In two experiments, eggs plus developing larvae and juvenile brown trout were exposed to three different concentrations of guanylurea (10, 100 and 1,000 µg/L) and, as a negative control, filtered tap water without this compound. Low internal concentrations were determined. The investigated parameters were mortality, length, weight, condition factor, tissue integrity of the liver and kidney, levels of stress proteins and lipid peroxides, as well as behavioural and developmental endpoints. It was found that guanylurea did not significantly change any of these parameters in the tested concentration range. RESULTS: In conclusion, these results do not give rise to concern that guanylurea could negatively affect the health or the development of brown trout under field conditions. Nevertheless, more studies focusing on further parameters and other species are highly needed for a more profound environmental risk assessment of guanylurea.

Transformation Products of Fluoxetine Formed by Photodegradation in Water and Biodegradation in Zebrafish Embryos ( Danio rerio).

The present study investigates the transformation of the antidepressant fluoxetine (FLX) by photo- and biodegradation and shows similarities and differences in transformation products (TPs). TPs were identified using LC-high-resolution mass spectrometry with positive and negative electrospray ionization. In a sunlight simulator, photodegradation was carried out using ultrapure water (pH 6, 8, and 10) and surface water (pH 8) to study the effect of direct and indirect photolysis, respectively. The well-known metabolite norfluoxetine (NFLX) proved to be a minor TP in photolysis (≤2% of degraded FLX). In addition, 26 TPs were detected, which were formed by cleavage of the phenolether bond ( O-dealkylation) which primarily formed 3-(methylamino)-1-phenyl-1-propanol (TP 166) and 4-(trifluoromethyl)phenol, by hydroxylation of the benzyl moiety, by CF3 substitution to benzoic aldehyde/acid, and by adduct formation at the amine group ( N-acylation with aldehydes and carboxylic acids). Higher pH favors the neutral species of FLX and the neutral/anionic species of primary TPs and, therefore, photodegradation. In zebrafish embryos, the bioconcentration factor of FLX was found to be 110, and about 1% of FLX taken up by the embryos was transformed to NFLX. Seven metabolites known from photodegradation and formed by hydrolysis, hydroxylation, and N-acylation as well as three new metabolites formed by N-hydroxylation, N-methylation, and attachment of an amine group were identified in zebrafish embryos. The study highlights the importance of considering a broad range of TPs of FLX in fresh water systems and in ecotoxicity tests and to include TP formation in both environmental processes and metabolism in organisms.

Aerobic and anaerobic formation and biodegradation of guanyl urea and other transformation products of metformin.

The aim of the study was to investigate the biodegradability of guanyl urea (GU) and the behavior of other transformation products (TPs) of Metformin (MF). Most biodegradation studies of MF with activated sludge of waste water treatment plants (WWTPs) showed GU as the only bacterial dead-end metabolite without further degradation. In this study, batch experiments with activated sludge revealed biodegradability of GU. GU degradation was much faster under anaerobic than under aerobic conditions. Degradation kinetics for MF was much slower under anaerobic conditions. Adsorptive removal of up to 20% was an additional elimination process of MF and GU. The batch experiments were conducted with sludge of 2 WWTPs, WWTP 1 showed decreasing concentrations of GU from influent to effluent and the other increasing concentrations. This indicates a different adaption of the sludge to GU and may explain the better GU degradation capability of the sludge from WWTP 1. Furthermore, the biodegradation potential of MF was confirmed and in addition, occurrence of the TPs methylbiguanide (MBG), 2-amino-4-methylamino-1,3,5-triazine (2,4-AMT) and the secondary TP 2,4-diamino-1,3,5-triazine (2,4-DAT) was observed in batch experiments with activated sludge of WWTP 1. After fast formation, degradation in turn was slower, especially for 2,4-AMT. In general, TPs played a minor role in MF and GU degradation.

Formation and occurrence of transformation products of metformin in wastewater and surface water.

The aim of this work was to investigate the occurrence and fate of the antidiabetic metformin (MF) and its transformation products (TPs) in wastewater and surface water samples. New TPs of MF were approached by electrochemical degradation with a boron-doped-diamond electrode (at 1.5V for 10min). 2,4-Diamino-1,3,5-triazine (2,4-DAT), methylbiguanide (MBG), 2-amino-4-methylamino-1,3,5-triazine (2,4-AMT) and 4-amino-2-imino-1-methyl-1,2-dihydro-1,3,5-triazine (4,2,1-AIMT) were identified by hydrophilic interaction chromatography (HILIC) with quadrupole time-of-flight mass spectrometry (QTOF-MS) and accurate mass fragmentation. However, the well-known transformation product guanyl urea (GU) could not be formed electrochemically. In samples from wastewater treatment plants (WWTP), 2,4-AMT and 2,4-DAT showed an increasing trend from influents to effluents, which implies formation of the TPs during WWT. MBG is also formed by hydrolysis of MF and therefore didn't show this trend in WWTPs. Compared to GU, the concentrations of other TPs are generally three orders of magnitude lower. MBG and 2,4-DAT were also detected in surface water which was impacted by waste water, while 4,2,1-AIMT could not be detected in any sample. The concentrations of MF were in an expected range for influent (14 to 95µg/l), effluent (0.7 to 6.5µg/l), surface water (up to 234ng/l) and tap water (34ng/l). GU concentrations, however, were in one of the two investigated WWTP much higher in the influent (between 158µg/l and 2100µg/l) than in the effluent (between 26 and 810µg/l). This is a rather unexpected result which has not been reported yet. Obviously, GU has been already formed in parts of the sewer system from MF or from other biguanide compounds like antidiabetics or disinfection chemicals. Furthermore, lower concentrations of GU in the effluents than in the influents indicate degradation processes of guanyl urea in the waste water treatment.

Does the antidiabetic drug metformin affect embryo development and the health of brown trout (Salmo trutta f. fario)?

BACKGROUND: Due to the rising number of type 2 diabetes patients, the antidiabetic drug, metformin is currently among those pharmaceuticals with the highest consumption rates worldwide. Via sewage-treatment plants, metformin enters surface waters where it is frequently detected in low concentrations (µg/L). Since possible adverse effects of this substance in aquatic organisms have been insufficiently explored to date, the aim of this study was to investigate the impact of metformin on health and development in brown trout (Salmo trutta f. fario) and its microbiome. RESULTS: Brown trout embryos were exposed to 0, 1, 10, 100 and 1000 µg/L metformin over a period from 48 days post fertilisation (dpf) until 8 weeks post-yolk sac consumption at 7 °C (156 dpf) and 11 °C (143 dpf). Chemical analyses in tissues of exposed fish showed the concentration-dependent presence of metformin in the larvae. Mortality, embryonic development, body length, liver tissue integrity, stress protein levels and swimming behaviour were not influenced. However, compared to the controls, the amount of hepatic glycogen was higher in larvae exposed to metformin, especially in fish exposed to the lowest metformin concentration of 1 µg/L, which is environmentally relevant. At higher metformin concentrations, the glycogen content in the liver showed a high variability, especially for larvae exposed to 1000 µg/L metformin. Furthermore, the body weight of fish exposed to 10 and 100 µg/L metformin at 7 °C and to 1 µg/L metformin at 11 °C was decreased compared with the respective controls. The results of the microbiome analyses indicated a shift in the bacteria distribution in fish exposed to 1 and 10 µg/L metformin at 7 °C and to 100 µg/L metformin at 11 °C, leading to an increase of Proteobacteria and a reduction of Firmicutes and Actinobacteria. CONCLUSIONS: Overall, weight reduction and the increased glycogen content belong to the described pharmaceutical effects of the drug in humans, but this study showed that they also occur in brown trout larvae. The impact of a shift in the intestinal microbiome caused by metformin on the immune system and vitality of the host organism should be the subject of further research before assessing the environmental relevance of the pharmaceutical.