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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.

Silicon nitride (Si3N4) is an industrial ceramic used in spinal fusion and maxillofacial reconstructionbecause of its excellent mechanical properties and good biocompatibility. This study compares ...

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.

The establishment of stormwater ponds for treatment of runoff water can be beneficial for amphibians due to creations of new habitats which often develop vegetative characteristics similar to that of natural wetlands. These ponds might help to promote stability in amphibian populations if the benefits of creation of new habitats exceed the risk for toxic impact. In the present study, Swedish stormwater ponds were searched for larval amphibians. Further, the impact of stormwater quality on embryo development was tested using Xenopus (Silurana) tropicalis. Data suggest that most of the studied stormwater ponds had water of proper quality for promoting amphibian embryo development. However there were sites in which the water caused premature hatching and/or reduced heart rate in X. tropicalis embryos. Pollutant toxicity might be one explanation for sublethal effects caused by some of the stormwater samples. In a majority of ponds, no amphibian larvae were detected and this may partly be explained by toxic compounds in the waters.

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.

Results are presented from a validation (with 5 laboratories) of the Fish Sexual Development Test (FSDT) developed to detect endocrine disrupters (EDs) and included in the OECD (Organisation for Economic Co-operation and Development) working program. The aromatase-inhibiting fungicide prochloraz was tested in zebrafish (Danio rerio) and fathead minnow (Pimephales promelas). The fish were exposed during sexual differentiation and development from 0 to 60 days post hatch (dph). After exposure, the vitellogenin (VTG) concentrations were quantified in head/tail homogenate and the sex ratio was determined (defined as female, male, intersex or undifferentiated). NOEC/LOEC and EC(x) designs were compared to optimize the test approach. Results show that both species are highly sensitive to prochloraz during sexual development. They respond by skewing of the sex ratio towards male phenotype and by a VTG decline in females. The NOEC/LOEC approach is preferred because sex ratio is difficult to analyze with a regression model. The mean NOEC/LOEC for prochloraz on the sex ratio was 43.3/134 μg/L and 101/293 μg/L for zebrafish and fathead minnow, respectively. The mean NOEC/LOEC on the decline in female VTG concentration was 65/110 μg/L and ~30/68 μg/L respectively. In conclusion, zebrafish and fathead minnow are suitable species in the FSDT and their sexual differentiation is equally labile to EDs.

The pharmaceutical estrogen 17alpha-ethinylestradiol (EE2) and the anabolic androgen 17beta-trenbolone (Tb) can interfere with the endocrine and reproductive systems of fish. The potency of these chemicals in zebrafish (Danio rerio) and Japanese medaka (Oryzias latipes) was assessed using the core end points vitellogenin (Vtg) concentration at 38 days post-hatch and sex ratio and gonad morphology at 60 days post-hatch. Vtg concentrations were measured in fish whole-body homogenate samples using enzyme-linked immunosorbent assay. Increased Vtg concentration and feminization of fish after exposure to 10 ng/L EE2, as well as masculinization after exposure to 50 ng/L of Tb, were observed in zebrafish. Intersex was observed in medaka exposed to EE2. A decrease in Vtg production after Tb exposure (50 ng/L) was measured in both zebrafish and medaka. Analyses of gonad morphology revealed increased testicular area and sperm percentage in Tb-exposed zebrafish, whereas increased sperm percentage was measured in Tb-exposed medaka. The higher sensitivity of zebrafish compared with medaka to both EE2 and Tb was revealed in the study.

Estrogenicity in Swedish wastewaters was surveyed. Estrogenicity was examined using a recombinant yeast cell test and analyses of the yolk protein precursor in the blood of caged juvenile rainbow trout at sites close to wastewater outlets or in continuous flow tanks of undiluted wastewater. Estrogenic effects corresponding to

Nitro musks are a group of nitrated benzenes mainly used by the fragrance industry as a substitute for natural musk. Two of the most common nitro musks, musk xylene and musk ketone, have been detected in water, fish, human adipose tissue, as well as in human breast milk. In this study, female zebrafish were dietary-exposed to musk ketone, prior to spawning with unexposed males. The fish were exposed for 8 weeks at two different dose levels. Accumulation of the compound in the fish as well as reproductive success were studied. Exposed females had reduced body weight and length, as well as reduced liver- and gonad somatic index. The results from the reproduction study showed a dose-dependent reduction in fecundity. Early life-stage mortality was increased and the median survival time was reduced. In addition, an embryo/larvae toxicity test was performed, using newly fertilized zebrafish eggs from unexposed parental fish. Eggs were exposed to a series of different concentrations of musk ketone via the surrounding water. A NOEC value of 10 micrograms/l and a LOEC value of 33 micrograms/l was determined. Conclusively, the present study clearly shows that musk ketone negatively affects reproduction and early life-stage survival in zebrafish.

Zebrafish (Danio rerio) were orally exposed to a mixture of 20 PCBs in three different dose levels (0.008, 0.08, and 0.4 microg of each congener per gram of freeze-dried chironomids). Generally, the PCBs accumulated in a dose-related manner. After 13 weeks of exposure body, liver, and ovary weights, as well as the liver and ovary somatic index, were significantly lower in exposed groups. In addition, the PCB mixture was an effective inducer of hepatic EROD activity. The reproduction study performed with exposed females and unexposed males after 9 weeks revealed that median survival time for larvae was only 7.7 days in the high-dose group as compared with 14 days in controls. Furthermore, egg production was reduced in all three groups exposed. No differences in hatching frequency or median hatching time were recorded. Histologically, females in both the intermediate and high-dose groups contained a reduced number of mature oocytes. The present study demonstrates that the potency of the mixture of selected PCBs induces hepatic EROD activity and has a clearly negative effect on zebrafish reproduction.

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.

Juvenile zebrafish (Danio rerio) were exposed to different dilutions (0, 0.67, 2.5, 10, and 50%) of effluent water from a Swedish pulp mill that previously has been reported to be androgenic to fish. Exposure was performed between days 10–38 days post-hatch. Fish were sampled for whole-body vitellogenin concentrations at day 38 post-hatch and for histological examination of gonads at day 60 post-hatch. In fish exposed to the highest concentration of pulp mill effluent, elevated concentrations of vitellogenin were measured. The androgenicity of the pulp mill water was confirmed by the increased number of males recorded at 60 days post-hatch. Image analysis of testes indicated stimulation of spennato genesis. Intersex fish were observed in all exposure groups. An androgenic activity equivalent to 5.6 ng/L dihydroxytestosterone was measured using the yeast androgen screen (YAS) assay. The present study demonstrates that both androgenic and estrogenic effects can be detected when exposing zebrafish during the juvenile period to complex mixtures of chemicals.

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.

The uptake and elimination of 20 structurally diverse tetra- to heptachlorinated biphenyls were studied in zebrafish (Danio rerio), three-spined stickleback (Gasterosteus aculeatus), and Arctic char (Salvelinus alpinus). The polychlorinated biphenyls (PCBs) were administered to the fish through food, intraperitoneal injection of peanut oil, or intraperitoneal implantation of silicone capsules. The retention of the PCBs in fish exposed through their diet was related with the substitution patterns of the compounds. Ortho-substituted congeners with no unsubstituted meta-para positions had high biomagnification potential. PCBs with low biomagnification all had adjacent vicinal hydrogens, indicating that congeners with this feature may have been metabolically eliminated. The retention characteristics of the PCBs in the diet-exposed and the injected zebrafish were similar. The pattern of congeners in Arctic char indicates that they have a lower capacity to metabolize PCBs compared to three-spined sticklebacks and zebrafish. The levels in the fish exposed to the PCBs through a silastic implant were negatively correlated with the hydrophobicity of the congeners. Most probably congener-specific release rates of the PCBs from the implants mask their retention characteristics. It is suggested that food, mimicking the natural intake route, should be used in PCB exposure studies to validate extrapolations to natural situations.

Zambia is a country with an extensive mining industry with the majority of mines located in the Copperbelt province. Through this region of the country, the Kafue River drains and receives effluent water from mining activities as well as from other industrial point sources. In addition, production of agricultural products and pest control requires use of different pesticides in the area. Information on industrial and agricultural pollution has not been clearly identified in Zambia, and little attention has been paid to pollution control and possible impact of metals, pesticides, and other persistent compounds in the environment. The objective of this study was to introduce and to evaluate a few methodologies based on in situ bioassays for environmental assessment to promote sustainable and environmentally sound water resource management of the Kafue River. The results show that caged threespot tilapia exposed downstream of industrial points sources rapidly bioaccumulate several trace elements, i.e., Cd, Co, Cu, Cr, Ni. These elements also occurred in much higher concentrations in water samples downstream of the industrial area compared with a locality upstream. Furthermore, the use of a semipermeable membrane device (SPMD) for passive absorption of lipophilic pollutants in the water showed relatively high concentration of several pesticides, i.e., DDT with major metabolites, PCB, and dieldrin. The present study shows that only 2 weeks of in situ studies in waters contaminated by pollutants affects in situ exposed fish and that the correlation between water and tissue concentrations was relatively good. Both trace elements and persistent organic pollutants occurred in such high concentrations that they must be considered from ecotoxicological aspects and may affect aquatic animal health.

Morphological studies of gonads from roach (Rutilus rutilus) in a small Swedish lake were performed. The lake is a potential recipient for drainage water from a refuse dumping area. All fishes were macroscopically identified as males, but histological examination of the gonads showed a high incidence of intersex. This high incidence might be caused by endocrine disrupting chemicals. The relationships between exposure to endocrine disrupters and intersex observations in wild fish is unknown and the effect of endocrine disrupters on zebrafish (Danio rerio) were evaluated in order to determine whether they might serve as a model laboratory species for effects of endocrine disrupting chemicals in wild fish. Zebrafish were exposed from pre-blastula stage until sexual maturation to either standardised water (controls), 17a-methyltestosterone (1, 10 μg l-1) or 17b-estradiol (1, 10 μg l-1). Exposure to both concentrations of 17bestradiol caused a significant feminisation, while a significant increase in the proportions of males was found after exposure to 1 mg 17a-methyltestosterone l-1. Furthermore, exposure to 1 and 10 mg 17a-methyltestosterone l-1 caused development of intersex individuals. Additionally, sexually mature male zebrafish were exposed to 1 μg 17b-estradiol l-1. After 10 weeks of exposure the fish were transferred to non-contaminated water for two weeks. The control group was kept in standardised water for 12 weeks. At termination of the experiment, measurement of whole-body vitellogenin concentrations were performed. Exposure to 17b-estradiol caused a significant increase in whole body vitellogenin concentrations. After 2 weeks in water without addition of 17b-estradiol, the vitellogenin concentration was reduced by approximately 50%. Roach and zebrafish may share properties which makes these species suitable for further comparisons to evaluate effects of endocrine disrupting chemicals as well as domestic and industrial effluents containing hormone-like substances.

The Fish Sexual Development Test (FSDT) has gone through two validations as an OECD test guideline for the detection of endocrine active chemicals with different modes of action. The validation has been finalized on four species: Zebrafish (Danio rerio), Japanese medaka (Oryzias latipes), three spined stickleback (Gasterosteus aculeatus) and fathead minnow (Pimephales promelas) and with four model compounds: the weak estrogens 4-tert Octylphenol and 4-tert pentylphenol, the aromatase inhibitor prochloraz and the androgen dihydrotestosterone. All species were tested in different laboratories to facilitate inter laboratory comparison. A NOEC-design with three exposure concentrations in four replicates was applied to all species. Flow through exposure to chemicals began with fertilised eggs and continued until 60 days post hatch. At sampling, the fish were either separated in three parts: A tail and a head part, which were pooled and homogenised for vitellogenin analysis and a body part that were fixed for histological evaluation or a blood sample were taken for vitellogenin analysis and the body were fixed for histology or the liver were sampled for vitellogenin analysis and the body were fixed for histology. For all three methods, the fish parts were numbered and histology could therefore be linked to the vitellogenin concentration in individual fish. The two core endocrine relevant endpoints were vitellogenin concentrations and phenotypic sex ratio. Change in the sex ratio is presented as a population relevant endpoint and the results of the two validation rounds will be discussed in relation to environmental risk assessment and species selection.