Naproxen affects multiple organs in fish but is still an environmentally better alternative to diclofenac.

The presence of diclofenac in the aquatic environment and the risks for aquatic wildlife, especially fish, have been raised in several studies. One way to manage risks without enforcing improved wastewater treatment would be to substitute diclofenac (when suitable from a clinical perspective) with another non-steroidal anti-inflammatory drug (NSAID) associated with less environmental risk. While there are many ecotoxicity-studies of different NSAIDs, they vary extensively in set-up, species studied, endpoints and reporting format, making direct comparisons difficult. We previously published a comprehensive study on the effects of diclofenac in the three-spined stickleback (Gasterosteus aculeatus). Our present aim was to generate relevant effect data for another NSAID (naproxen) using a very similar setup, which also allowed direct comparisons with diclofenac regarding hazards and risks. Sticklebacks were therefore exposed to naproxen in flow-through systems for 27 days. Triplicate aquaria with 20 fish per aquarium were used for each concentration (0, 18, 70, 299 or 1232 µg/L). We investigated bioconcentration, hepatic gene expression, jaw lesions, kidney and liver histology. On day 21, mortalities in the highest exposure concentration group unexpectedly reached ≥ 25 % in all three replicate aquaria, leading us to terminate and sample that group the same day. On the last day (day 27), the mortality was also significantly increased in the second highest exposure concentration group. Increased renal hematopoietic hyperplasia was observed in fish exposed to 299 and 1232 µg/L. This represents considerably higher concentrations than those expected in surface waters as a result of naproxen use. Such effects were observed already at 4.6 µg/L in the experiment with diclofenac (lowest tested concentration). Similar to the responses to diclofenac, a concentration-dependent increase in both relative hepatic gene expression of c7 (complement component 7) and jaw lesions were observed, again at concentrations considerably higher than expected in surface waters. Naproxen bioconcentrated less than diclofenac, in line with the observed effect data. An analysis of recent sales data and reported concentrations in treated sewage effluent in Sweden suggest that despite higher dosages used for naproxen, a complete substitution would only be expected to double naproxen emissions. In summary, naproxen and diclofenac produce highly similar effects in fish but the environmental hazards and risks are clearly lower for naproxen. Hence, if there are concerns for environmental risks to fish with diclofenac, a substitution would be advisable when naproxen presents an adequate alternative from a clinical point-of-view.

Thyroid disruption properties of three indoor dust chemicals tested in Silurana tropicalis tadpoles.

Indoor dust contains a multitude of industrial chemicals, and ingestion of dust is considered an important exposure route to organic contaminants. Some of these contaminants have been shown to interfere with the thyroid system, which may result in significant consequences on public health. The amphibian metamorphosis is a thyroid hormone-dependent process, which can be used as an in vivo model for studies on thyroid hormone-disrupting potency. Three contaminants of indoor dust were tested on metamorphosing Silurana (Xenopus) tropicalis tadpoles. The tested chemicals were Tris (1,3-dichloroisopropyl) phosphate (TDCiPP), tetrabromobisphenol-A (TBBPA) and propylparaben (PrP). Measurements reflecting general growth, development progress and thyroid epithelial cell height were performed on the exposed tadpoles as well as chemical analyses of the exposure water. It was shown that TDCiPP acts as a thyroid hormone-disrupting chemical in metamorphosing tadpoles by causing increased epithelial cell height in thyroid glands after exposure to a nominal concentration of 0.010 mg/L and in higher concentrations. TBBPA caused reductions in general growth of tadpoles at the nominal concentration 0.125 mg/L, and PrP caused acute toxicity at the nominal concentration 12.5 mg/L. However, no evident indications of specific thyroid-disrupting effects caused by TBBPA or PrP were observed.

Embryotoxicity of ozonated diclofenac, carbamazepine, and oxazepam in zebrafish (Danio rerio).

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-1 peak O3 concentration) and 10 min ozonation (0.147 mg L-1 peak O3 concentration). Chemical analysis showed that carbamazepine and diclofenac were largely removed by ozone (90 ±â€¯11% and 97 ±â€¯3.8%), whereas oxazepam was removed to a lesser extent (19 ±â€¯5.7%). The ZFET assay revealed diverging toxicities. Diclofenac embryotoxicity decreased with increasing ozonation. Oxazepam did not cause embryotoxicity in the ZFET assay either pre- or post ozonation, but larvae swimming activity was affected at 144 hpf. Carbamazepine embryotoxicity, on the other hand, increased with increasing ozonation. Chemical analysis showed the formation of two OBPs (carbamazepine-10,11-epoxide and 10,11-dihydrocarbamazepine), possibly explaining the increased embryotoxicity. The results of this study highlight the importance of new chemical and toxicological knowledge regarding the formation of OBPs in post-ozonated effluents.

Effects of ozonated sewage effluent on reproduction and behavioral endpoints in zebrafish (Danio rerio).

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 O3/m3) removed pharmaceuticals by an average efficiency of 77% in addition to the conventional treatment, leaving 11 screened pharmaceuticals above detection limits. Differences in biological responses of the exposure treatments were recorded in gene expression, reproduction and behavior. Hepatic vitellogenin gene expression was higher in male zebrafish exposed to the ozonated effluent compared to the non-ozonated effluent and tap water treatments. The reproductive success was higher in fish exposed to ozonated effluent compared to non-ozonated effluent and to tap water. The behavioral measurements showed that fish exposed to the ozonated STP effluent were less active in swimming the first minute after placed in a novel vessel. Ozonation is a capable method for removing pharmaceuticals in effluents. However, its implementation should be thoroughly evaluated for any potential biological impact. Future research is needed for uncovering the factors which produced the in vivo responses in fish.

Diclofenac affects kidney histology in the three-spined stickleback (Gasterosteus aculeatus) at low µg/L concentrations.

Diclofenac, a commonly used non-steroidal anti-inflammatory drug, is considered for regulation under the European water framework directive. This is because effects on fish have been reported at concentrations around those regularly found in treated sewage effluents (∼1µg/L). However, a recent publication reports no effects on fish at 320µg/L. In this study, three-spined sticklebacks (Gasterosteus aculeatus) were exposed to 0, 4.6, 22, 82 and 271µg/L diclofenac in flow-through systems for 28days using triplicate aquaria per concentration. At the highest concentration, significant mortalities were observed already after 21days (no mortalities found up to 22µg/L). Histological analysis revealed a significant increase in the proportion of renal hematopoietic tissue (renal hematopoietic hyperplasia) after 28days at the lowest concentration and at all higher concentrations, following a clear dose-response pattern. Skin ulcerations of the jaw were noted by macroscopic observations, primarily at the two highest concentrations. No histological changes were observed in the liver. There was an increase in the relative hepatic mRNA levels of c7 (complement component 7), a gene involved in the innate immune system, at 22µg/L and at all higher concentrations, again following a clear dose-response. The bioconcentration factor was stable across concentrations, but lower than reported for rainbow trout, suggesting lower internal exposure to the drug in the stickleback. In conclusion, this study demonstrates that diclofenac causes histological changes in the three-spined stickleback at low µg/L concentrations, which cause concern for fish populations exposed to treated sewage effluents.

Optimizing the design of a reproduction toxicity test with the pond snail Lymnaea stagnalis.

This paper presents the results from two ring-tests addressing the feasibility, robustness and reproducibility of a reproduction toxicity test with the freshwater gastropod Lymnaea stagnalis (RENILYS strain). Sixteen laboratories (from inexperienced to expert laboratories in mollusc testing) from nine countries participated in these ring-tests. Survival and reproduction were evaluated in L. stagnalis exposed to cadmium, tributyltin, prochloraz and trenbolone according to an OECD draft Test Guideline. In total, 49 datasets were analysed to assess the practicability of the proposed experimental protocol, and to estimate the between-laboratory reproducibility of toxicity endpoint values. The statistical analysis of count data (number of clutches or eggs per individual-day) leading to ECx estimation was specifically developed and automated through a free web-interface. Based on a complementary statistical analysis, the optimal test duration was established and the most sensitive and cost-effective reproduction toxicity endpoint was identified, to be used as the core endpoint. This validation process and the resulting optimized protocol were used to consolidate the OECD Test Guideline for the evaluation of reproductive effects of chemicals in L. stagnalis.

Sexual disruption in zebrafish (Danio rerio) exposed to mixtures of 17α-ethinylestradiol and 17ß-trenbolone.

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 17ß-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.

Tissue uptake, distribution and elimination of (14)C-PFOA in zebrafish (Danio rerio).

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.

PFOS induces behavioral alterations, including spontaneous hyperactivity that is corrected by dexamfetamine in zebrafish larvae.

Perfluorooctane sulfonate (PFOS) is a widely spread environmental contaminant. It accumulates in the brain and has potential neurotoxic effects. The exposure to PFOS has been associated with higher impulsivity and increased ADHD prevalence. We investigated the effects of developmental exposure to PFOS in zebrafish larvae, focusing on the modulation of activity by the dopaminergic system. We exposed zebrafish embryos to 0.1 or 1 mg/L PFOS (0.186 or 1.858 µM, respectively) and assessed swimming activity at 6 dpf. We analyzed the structure of spontaneous activity, the hyperactivity and the habituation during a brief dark period (visual motor response), and the vibrational startle response. The findings in zebrafish larvae were compared with historical data from 3 months old male mice exposed to 0.3 or 3 mg/kg/day PFOS throughout gestation. Finally, we investigated the effects of dexamfetamine on the alterations in spontaneous activity and startle response in zebrafish larvae. We found that zebrafish larvae exposed to 0.1 mg/L PFOS habituate faster than controls during a dark pulse, while the larvae exposed to 1 mg/L PFOS display a disorganized pattern of spontaneous activity and persistent hyperactivity. Similarly, mice exposed to 0.3 mg/kg/day PFOS habituated faster than controls to a new environment, while mice exposed to 3 mg/kg/day PFOS displayed more intense and disorganized spontaneous activity. Dexamfetamine partly corrected the hyperactive phenotype in zebrafish larvae. In conclusion, developmental exposure to PFOS in zebrafish induces spontaneous hyperactivity mediated by a dopaminergic deficit, which can be partially reversed by dexamfetamine in zebrafish larvae.

Comparison of embryo toxicity using two classes of aquatic vertebrates.

Toxicity tests of musk ketone (MK) and tetrabromobisphenol-A (TBBPA) on embryos were conducted in two amphibian species, Xenopus (Silurana) tropicalis and the Swedish native species Rana arvalis. TBBPA was also tested on fish embryos of Danio rerio. All species were tested in similar experimental setup. Musk ketone caused decreased heart rates at concentrations from 10 and 100 µg/L in R. arvalis and X. tropicalis, respectively. TBBPA caused effects at 1000 µg/L in all three species. The responses were comparable between all three species which supports the relevance for using data from non-native species in national risk assessment.

Locomotor behavior in zebrafish (Danio rerio) larvae exposed to perfluoroalkyl acids.

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.

Science and policy on endocrine disrupters must not be mixed: a reply to a "common sense" intervention by toxicology journal editors.

The "common sense" intervention by toxicology journal editors regarding proposed European Union endocrine disrupter regulations ignores scientific evidence and well-established principles of chemical risk assessment. In this commentary, endocrine disrupter experts express their concerns about a recently published, and is in our considered opinion inaccurate and factually incorrect, editorial that has appeared in several journals in toxicology. Some of the shortcomings of the editorial are discussed in detail. We call for a better founded scientific debate which may help to overcome a polarisation of views detrimental to reaching a consensus about scientific foundations for endocrine disrupter regulation in the EU.

Comparison of developmental toxicity of seven perfluoroalkyl acids to zebrafish embryos.

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.

Toxicity of 15 veterinary pharmaceuticals in zebrafish (Danio rerio) embryos.

Extensive use of veterinary pharmaceuticals may result in contamination of water bodies adjacent to pasture land or areas where animal manure has been applied. In order to evaluate the potential risk to fish embryos 15 veterinary pharmaceuticals were investigated by use of an extended zebrafish embryo toxicity test. Chemical analysis of the exposure medium was performed by solid phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) for 11 of the compounds and potential metabolism by the embryos was studied for albendazole, febantel, fenbendazole and oxfendazole. Newly fertilized zebrafish eggs were exposed under static conditions in 96-well plates for 6 days to the pharmaceuticals: 5 antibacterials and 10 antiparasitics. Endpoints including mortality, malformations and other sublethal responses were recorded at 24, 48 and 144 h post fertilization (hpf). The pharmaceuticals causing the highest toxicity were antiparasitics whereas the tested antibacterials, danofloxacin, enrofloxacin, tylosine, trimethoprim and oxytetracyclin had a much lower toxic potency in zebrafish embryos. Most toxic were fenbendazole, albendazole and flumethrin with no observed effect concentrations (NOECs) around 0.02 mg/L. The overall NOEC was determined by lethality for the following pharmaceuticals: albendazole, fenbendazole and oxfendazole. Sublethal endpoints, including malformations, side-laying embryos, tremors, reduced movements and altered heart rate increased the sensitivity of the tests and determined the overall NOECs for febantel, doramectin, ivermectin, flumethrin and toltrazuril. Exposure to doramectin and ivermectin caused a decrease in movements at 24 hpf and a decrease in heart rate at 48 hpf. Flumethrin exposure resulted in decreased time to hatching, except at the highest concentrations, and caused an increase in heart rate at 48 hpf. In contrast, toltrazuril caused an increased time to hatching and a decrease in heart rate. Chemical analysis of the exposure medium after the tests revealed great differences between nominal and measured concentrations, emphasizing the need of including analysis of the actual exposure concentrations. The results indicated that metabolism of albendazole into its sulfoxide protected the embryos from toxicity. Albendazole was metabolized efficiently into albendazole sulfoxide at lower exposure concentrations, resulting in reduced toxicity. At higher concentrations, an increasing proportion of albendazole remained unmetabolized and embryo mortality occurred. Metabolism by the embryos of febantel into fenbendazole and oxfendazole and of fenbendazole into oxfendazole was demonstrated. It is suggested that the toxic effect of febantel in zebrafish embryos is due to metabolism into fenbendazole.

Sows exposed to octylphenol in early gestation: no estrogenic effects in male piglets, but increased rate of stillbirth.

Octylphenol is an industrial chemical with estrogenic effects both in vitro and in vivo. In this study the effects of short-term intramuscular exposure to 0.1 mg/kg of body weight and 1.0 mg/kg of body weight in early gestation were evaluated in pregnant sows with respect to reproductive parameters in the newborn male piglets, as compared with male piglets from unexposed control sows. The male piglets were examined immediately after birth with respect to the macroscopic appearance of the reproductive organs and testosterone concentration in serum. It was not possible to identify any estrogenic effects in the newborn male piglets. However, in the sows exposed at the highest level of octylphenol, there was an increased number of stillborn piglets and an increased proportion of sows with stillborn piglets in the litter (P < 0.05). This was an unexpected finding which has not been reported previously.

Retention and maternal transfer of environmentally relevant polybrominated dibenzo-p-dioxins and dibenzofurans, polychlorinated dibenzo-p-dioxins and dibenzofurans, and polychlorinated biphenyls in zebrafish (Danio rerio) after dietary exposure.

High levels of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), mono- and non-ortho-polychlorinated biphenyls (PCBs), and polybrominated dibenzo-p-dioxins (PBDDs) are found in fish from coastal areas in the Baltic Sea, which may cause ecotoxicological effects. To increase our understanding of the persistency of the emerging pollutants polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/Fs), fish feed was spiked with 21 PBDD/Fs, 17 PCDD/Fs, and 30 PCBs and fed to zebrafish (Danio rerio). Concentrations in fish and eggs were examined during a 6- or 12-week uptake period, and a 6-week elimination period. Steady-state was reached for 2-, 3-, 7-, and/or 8-substituted tri- and tetra-BDD/Fs; 2,3,7,8-tetra-BDD (2,3,7,8-TeBDD) was the most strongly retained. Steady-state was not reached for tetra- to hexa-CDDs. Non-2,3,7,8 congeners showed little or no retention. Most PCBs had high retention and did not reach steady state. Half-lives decreased in the order PCBs > PCDD/Fs > PBDD/Fs. Concentrations of 2,3,7,8-substituted penta- to octa-CDD/Fs decreased with their degree of chlorination, suggesting that the rate-limiting factor for uptake is low bioavailability. Maternal transfer was observed for all retained compounds, with most transfer factors <1, indicating that transfer rates are affected by the poor water solubility of the compounds. The limited retention of the major PBDD congeners found in Baltic Sea fish suggests that they are exposed to high or very high concentrations via either food or water.

Comparison of zebrafish (Danio rerio) and fathead minnow (Pimephales promelas) as test species in the Fish Sexual Development Test (FSDT).

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.

Oral exposure of adult zebrafish (Danio rerio) to 2,4,6-tribromophenol affects reproduction.

The bromophenol 2,4,6-tribromophenol (TBP) is widely used as an industrial chemical, formed by degradation of tetrabromobisphenol-A, and it occurs naturally in marine organisms. Concentrations of TBP in fish have been related to intake via feed, but little is known about effects on fish health after oral exposure. In this study, we exposed adult male and female zebrafish (Danio rerio) to TBP via feed in nominal concentrations of 33, 330, and 3300 µg/g feed (or control feed) for 6 weeks to assess the effects of TBP on reproductive output, gonad morphology, circulatory vitellogenin levels, and early embryo development. The aim was also to investigate the extent to which TBP was metabolised to 2,4,6-tribromoanisole (TBA) in dietary exposed zebrafish, and the amounts of TBP and TBA found in offspring. After 6 weeks of exposure, we found about 3% of the daily dose of TBP in adult fish and the mean concentration of TBA was 25-30% of the TBP concentration. TBP and TBA were detected in offspring with wet weight-based egg/fish concentration ratios well below one. Exposure to TBP significantly reduced the fertilization success and disturbed the gonad morphology, i.e. fewer spermatid cysts in males and increased presence of atretic follicles and oocytes with decreased vitellogenesis in females. In females, the disturbed gonad morphology was accompanied by increased levels of circulating vitellogenin. Significant effects were observed at 3300 µg/g feed. Offspring early development was not significantly affected, but yolk-sac oedema tended to increase in frequency in exposed groups with time. Our results show that dietary exposure to TBP, at concentrations found in marine organisms that are part of the natural diet of wild fish, can interfere with reproduction in zebrafish. We also observed low accumulation from feed of TBP in zebrafish and biotransformation of TBP to TBA. This is the first paper showing gonadal histopathological changes and effects on fertility in TBP exposed fish.

Uptake and biotransformation of structurally diverse brominated flame retardants in zebrafish (Danio rerio) after dietary exposure.

Zebrafish (Danio rerio) were fed a diet containing a mixture of 11 structurally diverse brominated flame retardants (BFRs) at nominal concentrations of either 1 or 100 nmol/g for up to 42 d, followed by an elimination period of 14 d. Uptake rates and elimination constants for five of the BFRs were calculated from measurements of their concentrations in the male fish during the exposure and elimination phases. Observed uptake efficiencies were highest for 2,4,4'-tribromodiphenyl ether (BDE 28) and 1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane (TBECH) and were lowest for decabromodiphenyl ether (BDE 209). Estimated half-lives for TBECH and 2,4,6-tribromophenol were short (<2 d). Four BFR metabolites were identified in the fish: 2,2',3,4',5',6-Hexabromodiphenyl ether (BDE 149), 2,2',4,4',5,6'-hexabromodiphenyl ether (BDE 154), 2,4,6-tribromoanisole, and 1,2,4,5-hexabromobenzene. These metabolites were still present in the zebrafish after the 14-d elimination period. No relationship between the BFR concentrations in the zebrafish and their log octanol-water partition coefficient (Kow) values was found. Generally, low tendencies to bioaccumulate were observed for perbrominated and hydroxylated compounds. The observed accumulation of BFR metabolites in fish, however, shows that low concentration of a BFR does not provide, in isolation, a sound indication that the BFR poses low risks.

Maternal transfer of brominated flame retardants in zebrafish (Danio rerio).

In many species reproduction and embryonic development have been shown to be sensitive to environmental contaminants. Understanding embryonic exposure to environmental contaminants is thus highly important. In this study concentrations of brominated flame retardants (BFRs) were measured in zebrafish eggs after parental exposure for 42 days via the diet. Zebrafish were exposed to two doses of eleven structurally-diverse BFRs. Eight BFRs were detected in the female zebrafish and maternal transfer to eggs was evident for all eight compounds. The highest concentrations in eggs were observed for hexabromocyclododecane (HBCD) and 2,4,4'-tribromodiphenyl ether (BDE 28), followed by 2,2',3,4,4',5',6-heptabromodiphenyl ether (BDE 183) and tetrabromobisphenol A 2,3-dibromopropyl ether (TBBPA DBPE). Five potential BFR metabolites were tentatively identified in female fish and maternal transfer was observed also for these compounds. The lipid adjusted concentrations in eggs were significantly higher than the concentrations in female fish for several of the BFRs. Further, the results showed a generally higher transfer in the lower exposure level and also indicated a dependency on the physico-chemical properties of the compounds.