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Bisphenol A (BPA) is an industrial chemical, which has raised human health and environmental concerns due to its endocrine-disrupting properties. BPA analogues are less well-studied despite their wide use in consumer products. These analogues have been detected in water and aquatic organisms around the world, with some analogues showing toxic effects in various species including fish. Here, we present novel organ-specific time-course distribution data of bisphenol Z (BPZ) in female zebrafish (Danio rerio), including concentrations in the ovaries, liver, and brain, a rarely sampled organ with high toxicological relevance. Furthermore, fish-specific in vitro biotransformation rates were determined for 11 selected bisphenols. A physiologically based toxicokinetic (PBTK) model was adapted for four of these bisphenols, which was able to predict levels in the gonads, liver, and brain as well as the whole body within a 2-5-fold error with respect to experimental data, covering several important target organs of toxicity. In particular, predicted liver concentrations improved compared to currently available PBTK models. Predicted data indicate that studied bisphenols mainly distribute to the carcass and gonads and less to the brain. Our model provides a tool to increase our understanding on the distribution and kinetics of a group of emerging pollutants.

To understand the dynamics of antimicrobial resistance (AMR), in a One-Health perspective, surveillance play an important role. Monitoring systems already exist in the human health and livestock sectors, but there are no environmental monitoring programs. Therefore there is an urgent need to initiate environmental AMR monitoring programs nationally and globally, which will complement existing systems in different sectors. However, environmental programs should not only identify anthropogenic influences and levels of AMR, but they should also allow for identification of transmissions to and from human and animal populations. In the current review we therefore propose using antimicrobial resistant Escherichia coli as indicators for monitoring occurrence and levels of AMR in the environment, including wildlife.

Carbamazepine (CBZ) is an anticonvulsant medication with highly persistent properties in the aquatic environment, where it has the potential to affect nontarget biota. Because CBZ and many other pharmaceuticals are not readily removed in conventional sewage treatment plants (STP), additional STP effluent treatment technologies are being evaluated and implemented. Whole effluent ozonation is a prospective method to remove pharmaceuticals such as CBZ, yet knowledge on the toxicity of CBZ ozonation byproducts (OBPs) is lacking. This study presents, for the first time, in vivo individual and mixture toxicity of four putative OBPs, that is, carbamazepine 10,11-epoxide, 10,11-Dihydrocarbamazepine, 1-(2-benzaldehyde)-4-hydro-(1H,3H)-quinazoline-2-one (BQM), and 1-(2-benzaldehyde)-(1H,3H)-quinazoline-2,4-dione (BQD) in developing zebrafish (Danio rerio) embryos. BQM and BQD were isolated from the ozonated solution as they were not commercially available. The study confirmed that the OBP mixture caused embryotoxic responses comparable to that of ozonated CBZ. Individual compound embryotoxicity assessment further revealed that BQM and BQD were the drivers of embryotoxicity. OBP chemical stability in ozonated CBZ water solution during 2 week dark storage at 22 °C was also assessed. The OBP concentrations remained over time, except for BQD which decreased by 94%. Meanwhile, ozonated CBZ persistently induced embryotoxicity over 2 week storage, potentially illustrating environmental concern.

Perfluorononanoic acid (PFNA) is one of the perfluoroalkyl acids present in human tissues. In this study, effects on early embryo development after PFNA exposure were investigated using the bovine in vitro production system. Oocytes were exposed to PFNA during maturation in vitro (10 μg mL−1 and 0.1 μg mL−1), and then fertilized and cultured in parallel with control groups. Developmental parameters (cleavage, blastocyst formation) were followed and embryo quality evaluated (stage, grade). Embryos developed after exposure to 0.1 μg mL−1 were stained to distinguish nuclei, active mitochondria and neutral lipids. 10 μg mL−1 of PFNA had a severe negative effect on blastocyst formation (OR: 0.27 p

Sewage effluent ozonation can reduce concentrations of chemical pollutants including pharmaceutical residues. However, the formation of potentially toxic ozonation byproducts (OBPs) is a matter of concern. This study sought to elucidate toxicity mechanisms of ozonated carbamazepine (CBZ), an anti-epileptic drug frequently detected in sewage effluents and surface water, in zebrafish embryos (Danio rerio). Embryos were exposed to ozonated and non-ozonated CBZ from 3 h post-fertilization (hpf) until 144 hpf. Embryotoxicity endpoints (proportion of dead and malformed embryos) were assessed at 24, 48, and 144 hpf. Heart rate was recorded at 48 hpf. Exposure to ozonated CBZ gave rise to cardiovascular-related malformations and reduced heart rate. Moreover, embryo-larvae exposed to ozonated CBZ displayed a lack of swim bladder inflation. Hence, the expression patterns of CBZ target genes involved in cardiovascular and embryonal development were investigated through a stepwise gene co-expression analysis approach. Two co-expression networks and their upstream transcription regulators were identified, offering mechanistic explanations for the observed toxicity phenotypes. The study presents a novel application of gene co-expression analysis elucidating potential toxicity mechanisms of an ozonated pharmaceutical with environmental relevance. The resulting data was used to establish a putative adverse outcome pathway (AOP). (C) 2021 The Author(s). Published by Elsevier Ltd.

Carbamazepine (CBZ) is an anticonvulsant medication with highly persistent properties in the aquatic environment, where it has the potential to affect nontarget biota. Because CBZ and many other pharmaceuticals are not readily removed in conventional sewage treatment plants (STP), additional STP effluent treatment technologies are being evaluated and implemented. Whole effluent ozonation is a prospective method to remove pharmaceuticals such as CBZ, yet knowledge on the toxicity of CBZ ozonation byproducts (OBPs) is lacking. This study presents, for the first time, in vivo individual and mixture toxicity of four putative OBPs, that is, carbamazepine 10,11-epoxide, 10,11-Dihydrocarbamazepine, 1-(2-benzaldehyde)-4-hydro-(1H,3H)-quinazoline-2-one (BQM), and 1-(2-benzaldehyde)-(1H,3H)-quinazoline-2,4-dione (BQD) in developing zebrafish (Danio rerio) embryos. BQM and BQD were isolated from the ozonated solution as they were not commercially available. The study confirmed that the OBP mixture caused embryotoxic responses comparable to that of ozonated CBZ. Individual compound embryotoxicity assessment further revealed that BQM and BQD were the drivers of embryotoxicity. OBP chemical stability in ozonated CBZ water solution during 2 week dark storage at 22 °C was also assessed. The OBP concentrations remained over time, except for BQD which decreased by 94%. Meanwhile, ozonated CBZ persistently induced embryotoxicity over 2 week storage, potentially illustrating environmental concern.

Pharmaceutical residues and other micro-contaminants may enter aquatic environments through effluent from sewage treatment plants (STPs) and could cause adverse effects in wild fish. One strategy to alleviate this situation is to improve wastewater treatment by ozonation. To test the effectiveness of full-scale wastewater effluent ozonation at a Swedish municipal STP, the added removal efficiency was measured for 105 pharmaceuticals. In addition, gene expression, reproductive and behavioral endpoints were analyzed in zebrafish (Danio rerio) exposed on-site over 21 days to ozonated or non-ozonated effluents as well as to tap water. Ozone treatment (7 g O

The use of in vitro bioassays for studies of toxic activity in environmental water samples is a rapidly expanding field of research. Cell-based bioassays can assess the total toxicity exerted by a water sample, regardless whether the toxicity is caused by a known or unknown agent or by a complex mixture of different agents. When using bioassays for environmental water samples, it is often necessary to concentrate the water samples before applying the sample. Commonly, water samples are concentrated 10–50 times. However, there is always a risk of losing compounds in the sample in such sample preparation. We have developed an alternative experimental design by preparing a concentrated cell culture medium which was then diluted in the environmental water sample to compose the final cell culture media for the in vitro assays. Water samples from five Swedish waste water treatment plants were analyzed for oxidative stress response, estrogen receptor (ER), and aryl hydrocarbon receptor (AhR) activity using this experimental design. We were able to detect responses equivalent to 8.8–11.3 ng/L TCCD for AhR activity and 0.4–0.9 ng/L 17β-estradiol for ER activity. We were unable to detect oxidative stress response in any of the studied water samples. In conclusion, we have developed an experimental design allowing us to examine environmental water samples in toxicity in vitro assays at a concentration factor close to 1, without the risk of losing known or unknown compounds during an extraction procedure. Electronic supplementary material The online version of this article (10.1007/s11356-018-1656-4) contains supplementary material, which is available to authorized users.

Drugs are excreted from the human body as both original substances and as metabolites and enter aquatic environment through waste water. The aim of this study was to widen the current knowledge considering the effects of waterborne antidepressants with different modes of action—amitriptyline, venlafaxine, sertraline—on embryos of non-target aquatic biota—fish (represented by Danio rerio) and amphibians (represented by Xenopus tropicalis). The tested concentrations were 0.3; 3; 30; 300 and 3000 μg/L in case of amitriptyline and venlafaxine and 0.1; 1; 10; 100 and 1000 μg/L for sertraline. Test on zebrafish embryos was carried out until 144 h post fertilization, while test on Xenopus embryos was terminated after 48 h. Lethal and sublethal effects as well as swimming alterations were observed at higher tested concentrations that are not present in the environment. In contrast, mRNA expression of genes related to heart, eye, brain and bone development (nkx2.5, otx 2, bmp4 and pax 6) seems to be impacted also at environmentally relevant concentrations. In a wider context, this study reveals several indications on the ability of antidepressants to affect non target animals occupying environments which may be contaminated by such compounds.

Pharmaceutical residues are polluting the surface water environments worldwide. Sewage and wastewater treatment, therefore, needs to be improved in order to remove pharmaceutical residues from the effluent. One such treatment improvement is effluent ozonation. Even though ozonation has proven to be very efficient in reducing pharmaceutical parent compound concentrations in wastewater effluents, much remains unclear regarding potentially toxic ozonation by-product (OBP) formation. In this study, we sought to elucidate the aquatic toxicity of ozonated pharmaceuticals in zebrafish (Danio rerio) embryos in a static 144 h post fertilization (hpf) fish embryotoxicity (ZFET) assay. Three pharmaceuticals commonly detected in wastewater effluents, i.e. carbamazepine, diclofenac, and oxazepam, were selected for testing. Toxicity was assessed before and after 1 min ozonation (0.053 mg L

Wastewater treatment plants (WWTPs) are considered the main sources of chemicals of emerging concern (CECs) in aquatic environments, and can negatively impact aquatic ecosystems. In this study, WWTP influent, effluent, and sludge, and upstream and downstream waters from the WWTP recipient were investigated at 15 locations for a total of 164 CECs, including pharmaceuticals, personal care products, industrial chemicals, per- and polyfluoroalkyl substances (PFASs), and pesticides. In addition, zebrafish (Dania rerio) embryo toxicity tests (ZFET) were applied to WWTP influent and effluent, and upstream and downstream waters from WWTP recipients. A total of 119 CECs were detected in at least one sample, mean concentrations ranging from 0.11 ng/L (propylparaben) to 64,000 ng/L (caffeine), in wastewater samples and from 0.44 ng/L (ciprofloxacin) to 19,000 ng/L (metformin) in surface water samples. Large variations of CEC concentrations were found between the selected WWTPs, which can be explained by differences in CEC composition in influent water and WWTP treatment process. The sludge-water partitioning coefficient (K-d) of CECs showed a significant linear con-elation to octanol/warer partition coefficient (K-ow) (p < 0.001), and thus could be used for predicting their fare in the aqueous and solid phase. The Sigma CEC concentrations in WWTPs declined by on average 60%, based on comparisons of WWTP influent and effluent concentrations. The high concentrations of CECs in WWTP effluent resulted in, on average, 50% higher concentrations of CECs in water downstream of WWTPs compared with upstream. Some WWTP samples showed toxicity in ZFET compared with the respective control group, but no individual CECs or groups of CECs could explain this toxicity. These results could provide a theoretical basis for optimization of existing treatment systems of different designs, and could significantly contribute to protecting recipient waters. (C) 2020 The Authors. Published by Elsevier B.V.

Environmental estrogens and androgens can be present simultaneously in aquatic environments and thereby interact to disturb multiple physiological systems in organisms. Studies on interaction effects in fish of androgenic and estrogenic chemicals are limited. Therefore, the aim of the present study was to evaluate feminization and masculinization effects in zebrafish (Danio rerio) exposed to combinations of two synthetic steroid hormones detected in environmental waters: the androgen 17β-trenbolone (Tb) and the oestrogen 17α-ethinylestradiol (EE2). Juvenile zebrafish were exposed between days 20 and 60 post-hatch to different binary mixtures of Tb (1, 10, and 50 ng/L) and EE2 (2 and 5 ng/L). The endpoints studied were whole-body homogenate vitellogenin concentration at 40 days post-hatch, and sex ratio including gonad maturation at 60 days post-hatch. The feminizing potency of 5 ng/L of EE2, alone as well as in combination with Tb, was clear in the present study, with exposures resulting in almost all-female populations and females being sexually immature. Masculinization effects with male-biased sex ratios were observed when fish were exposed to 2 ng/L of EE2 in combination with Tb concentrations. Intersex fish were observed after exposure to mixtures of 2 ng/L EE2 with 50 ng/L Tb. Sexual maturity generally increased among males at increasing concentrations of Tb. The results of the present study show that exposure to environmentally relevant mixtures of an oestrogen and androgen affects the process of gonad differentiation in zebrafish and lead to sexual disruption.

Per- and polyfluoroalkyl substances (PFASs) are contaminants of emerging concern (CECs) that cause concern regarding their environmental impact and risk to human health. In this study, zebrafish (Danio rerio) embryos were exposed to PFASs for six days, to investigate behaviour toxicity and bioconcentration factor (BCF). Nine individual PFASs (five C-4-C-8 perfluoroalkyl carboxylates (PFCAs) (PFPeA, PFHxA, PFHpA, PFOA, PFNA), three C-4, C-6 and C-8 perfluoroalkyl sulfonates (PFSAs) (PFBS, PFHxS, PFOS) and 6:2 fluorotelomersulfonate (6:2 FTSA)) and a mixture of these were investigated at seven concentrations ranging from environmentally relevant to acutely toxic levels. In exposed embryos, significant differences were found in total swimming distance (PFHpA, PFOA, PFNA, PFHxS, PFOS, 6:2 FTSA, PFAS mixture), burst activity (PFOA, PFNA, PFHxS, PFOS, PFAS mixture) and startle response (PFNA, PFHxS, PFOS, PFAS mixture). Toxicity was only observed at concentrations well above environmental levels. The toxicity of the PFAS mixture generally followed that of the individual substances, but the mixture reduced the potencies of individual PFASs. BCF was determined for all nine PFASs and ranged between 0.9 (PFPeA) and 2700 (PFOS). Long-chain PFASs (C-8) and PFASs with sulfonate as an active group showed the greatest toxic potential, while short-chain PFASs (C-6 and C-7) also caused significant behaviour alterations and accumulated in the embryos. To our knowledge, this is the first study to compare the behaviour toxicity of a PFAS mixture with that of the individual PFASs. Follow-up studies are needed to identify the mechanistic responses to PFAS mixtures. (C) 2019 Elsevier Ltd. All rights reserved.

Veterinary antiparasitic pharmaceuticals have been detected in surface waters and several of these pharmaceuticals act on the nervous system on the target organisms implying that neurological effects also might be of concern in non-target animals such as fish. We tested if exposure to antiparasitic pharmaceuticals affect swimming activity in 6 days old zebrafish larvae. The results revealed that most pharmaceuticals did not cause any effects in swimming activity. However, larvae exposed to 0.58 mg/L doramectin displayed reduced swimming activity even though they were classified as normal, having no morphological abnormalities.

Perfluorooctanoic acid (PFOA) is a long-chain perfluorinated chemical that has been shown to be non-degradable and persistent in the environment. Laboratory studies on bioconcentration and compound-specific tissue distribution in fish can be valuable for prediction of the persistence and environmental effects of the chemicals. In the present study male and female zebrafish (Danio rerio) were continuously exposed to 10μg/L of radiolabeled perfluorooctanoic acid ((14)C-PFOA) for 40 days, after which the exposed fish were transferred to fresh clean water for another 80 days wash-out period. At defined periodic intervals during the uptake and wash-out, fish were sampled for liquid scintillation counting and whole body autoradiography to profile the bioconcentration and tissue distribution of PFOA. The steady-state concentration of (14)C-PFOA in the zebrafish was reached within 20-30 days of exposure. The concentration-time course of (14)C-PFOA displayed a bi-exponential decline during washout, with a terminal half-life of approximately 13-14 days. At steady-state the bioconcentration of (14)C-PFOA into whole-body fish was approximately 20-30 times greater than that of the exposure concentration, with no differences between females and males. The bioconcentration factors for liver and intestine were approximately 100-fold of the exposure medium, while in brain, ovary and gall bladder the accumulation factors were in the range 15-20. Whole-body autoradiograms confirmed the highest labeling of PFOA in bile and intestines, which implies enterohepatic circulation of PFOA. The (14)C-PFOA was also observed in maturing vitellogenic oocytes, suggesting chemical accumulation via yolk proteins into oocytes with plausible risk for adverse effects on early embryonic development and offspring health. The bioconcentration at several (14)C-PFOA exposure concentrations were also investigated (0.3-30μg/L). This showed that bioconcentration increased linearly with tank exposure in the present in vivo model under steady-state conditions. From this model tissue concentrations of PFOA can be predicted when the external exposure level is known. The present study has generated experimental data on PFOA kinetics in zebrafish that can be valuable for aquatic environmental risk assessment.

The toxicity of individual perfluoroalkyl acids (PFAAs) has been suggested to be determined by the carbon chain length as well as the functional group attached. We tested seven different PFAAs including both sulfonic and carboxylic PFAAs with different chain length to evaluate the developmental toxicity in zebrafish embryos. Generally, the acute toxicity of PFAAs is relatively low to zebrafish embryos. The EC50 values ranged from 1.5 to 2200mg/L. We observed a relationship between higher toxicity with longer carbon chain. In addition, we also observed a higher toxicity for sulfonic PFAAs than for carboxylic PFAAs.

Perfluoroalkyl acids (PFAAs) are persistent organic contaminants that have been detected in wildlife, humans and the environment. Studies have shown that the toxicity of PFAAs is determined by the carbon chain length as well as the attached functional group. The locomotor activity of zebrafish larvae has become widely used for evaluation of chemicals with neurotoxic properties. In the present study the behavioral effects of seven structurally different PFAAs (i.e. TFAA, PFBA, PFOA, PFNA, PFDA, PFBS and PFOS) were evaluated in zebrafish larvae. Exposure to high concentrations of TFAA, PFNA, PFBS and PFOS resulted in distinct changes in behavioral patterns. Based on redundancy analysis, our results demonstrate three main factors affecting zebrafish larval locomotor behavior. The strongest effect on behavior was determined by the carbon chain length and the attached functional group. PFAAs with longer carbon chain length as well as PFAAs with attached sulfonic groups showed larger potential to affect locomotor behavior in zebrafish larvae. Also the concentration of the PFAAs determined the behavior responses. The results of the present study are in agreement with previous studies showing correlations between the chemical structure of PFAAs and the toxicological effects.

Very high levels of a range of pharmaceuticals have been reported recently in the effluent from a wastewater treatment plant near Hyderabad, India. The plant serves approximately 90 manufacturers of bulk drugs that primarily are exported to the world market. Fluoroquinolone antibiotics were found at levels that are highly toxic to various microorganisms. Even though milligram-per-liter levels of drugs targeting human proteins also have been found, it is difficult to conclude whether these levels are sufficiently high to adversely affect fish or amphibians due to the lack of relevant chronic toxicity data for most human pharmaceuticals. To assess potential effects on aquatic vertebrates, tadpoles of Xenopus tropicalis were exposed to three dilutions of effluent (0.2, 0.6, and 2%) over 14 d, starting at developmental stage 51. Additionally, newly fertilized zebrafish (Danio rerio) were exposed to diluted effluent in 96-well plates for up to 144 h postfertilization (hpf). The tadpoles' body lengths, weights, and developmental stages were recorded, whereas a larger number of sublethal and lethal endpoints were studied in the zebrafish. A 40% reduced growth of the exposed tadpoles was demonstrated at the lowest tested effluent concentration (0.2%), indicating potent constituents in the effluent that can adversely affect aquatic vertebrates. The median lethal concentration (LC50) for zebrafish at 144 hpf was between 2.7 and 8.1% in different experiments. Reduced spontaneous movements, pigmentation, and heart rate were recorded within 48 hpf at 8 and 16% effluent concentrations. Treated effluent from a plant that serves as an important link in the global supply chain for bulk drugs is thus shown to cause adverse effects to aquatic vertebrates even at very high dilutions.

In a partial life-cycle test, the impact of 17alpha-ethinylestradiol (EE2) and 17alpha-methyltestosterone (MT) on juvenile zebrafish was evaluated by use of vitellogenin measurements and gonadal development. Exposure to EE2 (1-25 ng/l) resulted in a dose-dependent increase in vitellogenin production starting at 2 ng/l. Significant changes in sex ratios in female direction were detected at 1 ng/l, with complete sex reversal taking place after exposure to 2 ng/l. No intersex fish were observed after exposure to EE2. Exposure to MT resulted in decreased vitellogenin concentrations. Complete sex reversal was detected in all MT concentrations used (26-1000 ng/l). A large proportion of intersex fish was observed after exposure to 1000 ng MT/l. The period of gonadal sex reversal in non-exposed zebrafish was also studied. The main morphological features of the transformation of ovaries into testis were observed 4-5 weeks after hatching.