A seafood risk tool for assessing and mitigating chemical and pathogen hazards in the aquaculture supply chain.

Intricate links between aquatic animals and their environment expose them to chemical and pathogenic hazards, which can disrupt seafood supply. Here we outline a risk schema for assessing potential impacts of chemical and microbial hazards on discrete subsectors of aquaculture-and control measures that may protect supply. As national governments develop strategies to achieve volumetric expansion in seafood production from aquaculture to meet increasing demand, we propose an urgent need for simultaneous focus on controlling those hazards that limit its production, harvesting, processing, trade and safe consumption. Policies aligning national and international water quality control measures for minimizing interaction with, and impact of, hazards on seafood supply will be critical as consumers increasingly rely on the aquaculture sector to supply safe, nutritious and healthy diets.

Applying a mechanistic model to predict interacting effects of chemical exposure and food availability on fish populations.

The potential environmental impacts of chemical exposures on wildlife are of growing concern. Freshwater ecosystems are vulnerable to chemical effects and wildlife populations, including fish, can be exposed to concentrations known to cause adverse effects at the individual level. Wild fish populations are also often subjected to numerous other stressors simultaneously which in temperate climates often include sustained periods of food limitation. The potential interactive effects of chemical exposures and food limitation on fish populations are however difficult to establish in the field. Mechanistic modelling approaches can be employed to help predict how the physiological effects of chemicals and food limitation on individuals may translate to population-level effects. Here an energy budget-individual-based model was developed and the control (no chemical) model was validated for the three-spined stickleback. Findings from two endocrine active chemical (EAC) case studies, (ethinyloestradiol and trenbolone) were then used to investigate how effects on individual fecundity translated into predicted population-level effects for environmentally relevant exposures. The cumulative effects of chemical exposure and food limitation were included in these analyses. Results show that effects of each EAC on the population were dependent on energy availability, and effects on population abundance were exacerbated by food limitation. Findings suggest that chemical effects and density dependent food competition interact to determine population responses to chemical exposures. Our study illustrates how mechanistic modelling approaches might usefully be applied to account for specific chemical effects, energy budgets and density-dependent competition, to provide a more integrated evaluation of population outcomes in chemical risk assessments.

Sustainable aquaculture through the One Health lens.

Aquaculture is predicted to supply the majority of aquatic dietary protein by 2050. For aquaculture to deliver significantly enhanced volumes of food in a sustainable manner, appropriate account needs to be taken of its impacts on environmental integrity, farmed organism health and welfare, and human health. Here, we explore increased aquaculture production through the One Health lens and define a set of success metrics - underpinned by evidence, policy and legislation - that must be embedded into aquaculture sustainability. We provide a framework for defining, monitoring and averting potential negative impacts of enhanced production - and consider interactions with land-based food systems. These metrics will inform national and international science and policy strategies to support improved aquatic food system design.

Exposure to an anti-androgenic herbicide negatively impacts reproductive physiology and fertility in Xenopus tropicalis.

Amphibians are threatened on a global scale and pollutants may be contributing to population declines, but how chemicals impact on their reproduction is poorly understood. We conducted a life cycle analysis to investigate the impacts of early life exposure to two anti-androgens (exposure until completion of metamorphosis;stage 66): flutamide, (50 µg/L)/linuron (9 and 45 µg/L)) on sexual development and breeding competence in Xenopus tropicalis. Our analyses included: mRNA levels of dmrt1, cyp17, amh, cyp19, foxl2 and ar (tadpoles/metamorphs), gonadal histomorphology (metamorphs/adults), mRNA levels of ar/gr (adult male brain/gonad/forelimb), testosterone/corticosterone levels (adult males), secondary sexual characteristics (forelimb width/nuptial pad: adult males) and breeding competence (amplexus/fertility: adult males). Compared to controls, feminised sex ratios and increased number of spermatogonia (adults) were observed after exposure to flutamide and the lower linuron concentration. Exposure to the lower linuron concentration also resulted in demasculinisation of secondary sexual characteristics and reduced male fertility. Flutamide exposure resulted in masculinisation of the nuptial pad and elevated mRNA levels of dmrt1, cyp17, amh and foxl2 in brains (metamorphs). Testosterone levels were higher in all treatment groups, however, overall few effects were observed in response to the higher linuron concentration. Our findings advance understanding of reproductive biology of X. tropicalis and illustrate negative effects of linuron on reproductive processes at a concentration measured in freshwater environments.

Can simple tank changes benefit the welfare of laboratory zebrafish Danio rerio?

This study examined the effects of simple changes in the tank environment on the wellbeing of laboratory-maintained zebrafish Danio rerio. Groups of D. rerio were either housed in stable environments (where they were maintained in the same tanks throughout the study) or in environments subject to change (where they were periodically moved to novel but identical tanks) and the effects of these treatments on morphometry, reproductive success and aggressive behaviour assessed. No effect of simple tank changes was found on body condition, reproductive output or aggression, for the periods of time studied, indicating that more complex scenarios in housing tank conditions are required for significant welfare benefits for captive D. rerio.

Stress and welfare in ornamental fishes: what can be learned from aquaculture?

The ornamental fish trade is estimated to handle up to 1·5 billion fishes. Transportation and handling of fishes imposes a range of stressors that can result in mortality at rates of up to 73%. These rates vary hugely, however, and can be as low as 2%, because they are generally estimated rather than based on experimental work. Given the numbers of ornamental fishes traded, any of the estimated mortality rates potentially incur significant financial losses and serious welfare issues. Industry bodies, such as the Ornamental Aquatic Trade Association (OATA), have established standards and codes of best practice for handling fishes, but little scientific research has been conducted to understand the links between stress, health and welfare in ornamental species. In aquaculture, many of the same stressors occur as those in the ornamental trade, including poor water quality, handling, transportation, confinement, poor social and physical environment and disease and in this sector directed research and some resulting interventions have resulted in improved welfare standards. This review considers the concept of welfare in fishes and evaluates reported rates of mortality in the ornamental trade. It assesses how the stress response can be quantified and used as a welfare indicator in fishes. It then analyses whether lessons from aquaculture can be usefully applied to the ornamental fish industry to improve welfare. Finally, this analysis is used to suggest how future research might be directed to help improve welfare in the ornamental trade.

Assessing variation in the potential susceptibility of fish to pharmaceuticals, considering evolutionary differences in their physiology and ecology.

Fish represent the planet's most diverse group of vertebrates and they can be exposed to a wide range of pharmaceuticals. For practical reasons, extrapolation of pharmaceutical effects from 'model' species to other fish species is adopted in risk assessment. Here, we critically assess this approach. First, we show that between 65% and 86% of human drug targets are evolutionarily conserved in 12 diverse fish species. Focusing on nuclear steroid hormone receptors, we further show that the sequence of the ligand binding domain that plays a key role in drug potency is highly conserved, but there is variation between species. This variation for the oestrogen receptor, however, does not obviously account for observed differences in receptor activation. Taking the synthetic oestrogen ethinyloestradiol as a test case, and using life-table-response experiments, we demonstrate significant reductions in population growth in fathead minnow and medaka, but not zebrafish, for environmentally relevant exposures. This finding contrasts with zebrafish being ranked as more ecologically susceptible, according to two independent life-history analyses. We conclude that while most drug targets are conserved in fish, evolutionary divergence in drug-target activation, physiology, behaviour and ecological life history make it difficult to predict population-level effects. This justifies the conventional use of at least a 10× assessment factor in pharmaceutical risk assessment, to account for differences in species susceptibility.

The role of relatedness in structuring the social network of a wild guppy population.

The role of relatedness in structuring animal societies has attracted considerable interest. Whilst a significant number of studies have documented kin recognition in shoaling fish under laboratory conditions, there is little evidence that relatedness plays a significant role in structuring social interactions in wild populations that are characterised by fission-fusion dynamics. Previous work has tended to compare relatedness within and among entire shoals. Such an approach however, does not have the ability to detect social sub-structuring within groups, which appears to be a major factor driving the social organisation of fission-fusion animal societies. Here, we use social network analysis combined with DNA microsatellite genotyping to examine the role of relatedness in structuring social relationships in a wild population of guppies (Poecilia reticulata). Consistent with previous findings, female-female dyads formed the strongest social relationships, which were stable over time. Interestingly, we also observed significant co-occurrence of male-male interactions, which is in contrast to previous work. Although we observed social sub-structuring in the population, we found no evidence for relatedness playing a significant role in underpinning this structure. Indeed, only seven first-degree relative dyads were identified among the 180 fish genotyped, indicating that the majority of individuals do not have a first-degree relative in the population. The high genetic diversity observed in this population is indicative of a large effective population size typical of lowland guppy populations. We discuss our findings in the context of the evolution of social organisation and the mechanisms and constraints that may drive the observed patterns in wild populations.

Review: Do engineered nanoparticles pose a significant threat to the aquatic environment?

Nanotechnology is a rapidly growing industry of global economic importance, exploiting the novel characteristics of materials manufactured at the nanoscale. The properties of engineered nanoparticles (ENPs) that make them useful in a wide range of industrial applications, however, have led to concerns regarding their potential impact on human and environmental health. The aquatic environment is particularly at risk of exposure to ENPs, as it acts as a sink for most environmental contaminants. This paper critically evaluates what is currently known about sources and discharge of ENPs to the aquatic environment and how the physicochemical characteristics of ENPs affect their fate and behaviour and thus availability for uptake into aquatic organisms, and assesses reported toxicological effects. Having reviewed the ecotoxicological information, the conclusion is that whilst there are data indicating some nanoparticles have the potential to induce harm in exposed aquatic organisms, there is insufficient evidence for harm, for known/modelled environmental concentrations for almost all ENPs considered. This conclusion, however, must be balanced by the fact that there are significant gaps in our understanding on the fate and behaviour of ENPs in the aquatic environment. Greater confidence in the assessments on ENP impacts in aquatic systems to enable effective comparisons across studies urgently requires more standardised approaches for ENP hazard identification, and critically, more thorough characterisations on the exposed particles. There is also an urgent need for the advancement of tools and techniques that can accurately quantify and visualise uptake of nanoparticles into biological tissues.

Effects of a weak oestrogenic active chemical (4-tert-pentylphenol) on pair-breeding and F1 development in the fathead minnow (Pimephales promelas).

A fish full life-cycle (FFLC) is the most comprehensive test to determine reproductive toxicity of chemicals to fish and this is likely to apply equally to endocrine active chemicals (EACs). However, FFLC tests use large numbers of animals, are expensive and time consuming. Alternative chronic tests, to the FFLC, potentially include sensitive life-stage windows of effect, such as sexual differentiation, early gonadal development and reproduction. In this paper, a fish pair-breeding study was applied to assess the biological effects of a weak environmental oestrogen, 4-tert-pentylphenol (4TPP), on reproduction and subsequent development of the F1 generation. The results of this study were then compared with the results for a published FFLC study, with this chemical. Fathead minnows (Pimephales promelas) were held in pairs and their reproductive performance assessed over two concurrent 21-day periods, the first without exposure to the test chemical, followed by the second with exposure to the test chemical, in a flow-through system at 25+/-1 degrees C. Embryos from two pairs, per treatment, were subsequently grown up in clean water until 90 days post-hatch to assess developmental effects of the parental exposure on the F1 generation. Nominal (measured geometric mean, time weighted) test concentrations of 4TPP were 56 (48), 180 (173) and 560 (570) microg l(-1). A significant decrease in fecundity was observed in all 4TPP exposed fish (mean number of eggs spawned per pair and number of spawns per pair) when compared to the solvent control. Vitellogenin (VTG) was significantly elevated in F0 males exposed to 560 microg 4TPPl(-1). Somatic endpoints, secondary sexual characteristics (SSC) and gonadosomatic index (GSI) were not affected by the 4TPP exposure. In the F1 generation, there were no treatment-related effects on hatching success, survival, growth, SSC or GSI. Histological examination of the gonads of the F1 fish revealed no treatment-related effects on sex ratio, sexual differentiation or sexual development. However, plasma VTG concentrations were significantly elevated in F1 male fish, derived from parents that had previously been exposed to 4TPP at concentrations of > or = 180 microg l(-1). These data show that the reproductive performance test is suitable for detecting weak environmental oestrogenic chemicals and that exposure of adult fish to oestrogens can result in altered biomarker expression (VTG) of the F1 generation. Our findings indicate that the reproductive performance test was as sensitive for detecting effects on reproduction when compared with a published FFLC test for 4TPP.

Evaluation of a carp primary hepatocyte culture system for screening chemicals for oestrogenic activity.

The presence of endocrine disrupting chemicals (EDCs) in the environment has driven the development of screening and testing assays to both identify chemicals with hormonal activity and evaluate their potential to cause adverse effects. As the number of animals used for research and regulatory purposes rises, and set against a desire to reduce animal testing, there is increased emphasis on the development and application of in vitro techniques to evaluate chemical risks to the environment. Induction of vitellogenin (VTG) in isolated fish liver cells has been used successfully to identify a wide range of EDCs, including both natural and synthetic oestrogens and a variety of other xenoestrogens. However, the vitellogenic response reported for hepatocytes in culture has been shown to vary widely, making comparisons between studies difficult. The work presented in this paper explored the variability of the vitellogenic response in primary cultures of common carp (Cyprinus carpio) hepatocytes following exposure to the model oestrogenic compound, 17beta-oestradiol (E2). As expected, variability in the vitellogenic response was observed, both in terms of the sensitivity and magnitude of VTG induction, for hepatocytes isolated from different fish. An apparent difference was observed in the response of isolated hepatocytes based on the sex of the donor fish; maximum levels of E2-stimulated VTG synthesis in hepatocytes derived from females appeared higher (1962 ng mL(-1)+/-487 [n=9] compared with 1194 ng mL(-1)+/-223 for hepatocytes from males [n=9]) and EC(50) values lower (1.61+/-0.4 microM E2 for females and 2.12+/-0.2 microM E2 for males). However, these differences were not statistically significant, likely in part due to the variation observed in the vitellogenic response. In particular, hepatocytes derived from female fish showed more variation than their male counterparts (the co-efficient of variation for females was 77% compared to 28% for males). Despite the variation observed in the vitellogenic response between different cultures, data from the different donor fish could be compared by standardising responses relative to the maximum VTG induction in each culture following exposure to E2. Adopting this approach in the future will allow for data from different hepatocyte cultures and from donor fish of different sexes, age and stage of maturity to be compared with greater consistency. Measurement of vtg mRNA expression was relatively more sensitive to the oestrogenic effects of E2 exposure than measurement of VTG protein (the LOEC at the transcriptome level was 10-fold lower [0.01 microM E2] than at the protein level [0.1 microM E2]) and changes in vtg mRNA expression showed less variation between individual hepatocyte isolations. Measurement of vtg mRNA in the hepatocyte culture system therefore may offer the most sensitive and consistent option for the screening of chemicals with oestrogenic activity in fish primary hepatocyte cultures.

Genetic variation in strains of zebrafish (Danio rerio) and the implications for ecotoxicology studies.

There is substantial evidence that genetic variation, at both the level of the individual and population, has a significant effect on behaviour, fitness and response to toxicants. Using DNA microsatellites, we examined the genetic variation in samples of several commonly used laboratory strains of zebrafish, Danio rerio, a model species in toxicological studies. We compared the genetic variation to that found in a sample of wild fish from Bangladesh. Our findings show that the wild fish were significantly more variable than the laboratory strains for several measures of genetic variability, including allelic richness and expected heterozygosity. This lack of variation should be given due consideration for any study which attempts to extrapolate the results of ecotoxicological laboratory tests to wild populations.

Ontogeny of sexual development in the roach (Rutilus rutilus) and its interrelationships with growth and age.

The roach (Rutilus rutilus) has become a sentinel species for the study of sexual disruption in wild fish populations as a consequence of exposure to endocrine disrupting chemicals (EDCs). Little is known, however, about the normal ontogeny of sexual development in this species. Here, we analyzed the ontogeny of sexual development in captive-bred roach and assessed how growth rate and fish size affected the timing of both sexual differentiation and sexual development over a 2-year period. Ovarian differentiation was first recorded at 68 days post-fertilization (dpf) and this preceded testicular differentiation (first recorded at 98 dpf). In contrast, sexual maturation occurred at an earlier age in males (300 dpf) compared with females (728 dpf). No differences in body size (length or weight) were recorded between male and female roach until the fish were 415 dpf. Studies on three populations of roach which grew at different rates showed that the timing of sexual differentiation was highly variable and more related to fish size than to fish age. Time to sexual maturation was also variable among populations but, subsequent to their first year of life, gonadal status was less well associated with fish size. Interestingly, the sex ratio of the population was biased towards females in populations that grew more rapidly during early life. The findings presented here provide a valuable foundation of work to support both field- and laboratory-based assessments on the effects of EDCs, and other stressors, on sexual differentiation and development in the roach.

Molecular characterisation of growth differentiation factor 9 (gdf9) and bone morphogenetic protein 15 (bmp15) and their patterns of gene expression during the ovarian reproductive cycle in the European sea bass.

Members of the transforming growth factor-beta superfamily, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), have crucial roles in primary follicle growth in mammals. To initiate investigations into their significance in teleost oogenesis, we set out to clone and characterise the cDNAs of gdf9 and bmp15 and analysed their patterns of gene expression during the ovarian reproductive cycle in the European sea bass (Dicentrachus labrax). Sea bass gdf9 and bmp15 cDNAs were 2200 and 2049 bp long, coding for 438 and 459 amino acids (aas), respectively, and were most similar to zebrafish gdf9 and bmp15 (64.4 and 56.1%, respectively). By Northern analysis, sea bass gdf9 and bmp15 mRNA transcripts were detected in the ovary only of the tissues analysed and their sizes were 2.2 and 2.1 kb, respectively. Dot-blot analysis revealed high levels of gdf9 and bmp15 expression in the ovary during primary oocyte growth and previtellogenesis (July to October), with a significant decline at the onset of vitellogenesis (November) and remaining low until the beginning of new oocyte growth (April/May). There was a highly significant positive correlation (r=0.939) between gdf9 and bmp15 gene expression in individual samples. The high levels of gdf9 and bmp15 mRNA transcripts in the ovary, especially during the previtellogenic growth period suggest an important role for these factors in early primary oocyte growth in the European sea bass.

PERMANENT GENETIC RESOURCES: Identification of microsatellite loci for parentage analysis in roach Rutilus rutilus and eight other cyprinid fish by cross-species amplification, and a novel test for detecting hybrids between roach and other cyprinids.

In order to identify microsatellite loci for parentage analysis in roach Rutilus rutilus, 59 published primer sets were tested on roach and eight other cyprinid fish. Twenty polymorphic loci were identified for roach, of which the polymerase chain reaction products of seven could be pooled for sequencer analysis. Together, these seven loci have an exclusion probability of 0.997 for parentage, when no parents are known. We also describe a novel test for hybrids between roach and four other cyprinids, based on intraspecies length differences of internal transcribed spacer region 1.

Evidence for the existence of a functional Kiss1/Kiss1 receptor pathway in fish.

In mammals, the Kiss1 receptor (Kiss1r) and its kisspeptin ligands are key factors regulating the onset of puberty. In fish, however, the mechanisms underlying the initiation of puberty are poorly understood and the role of the Kiss1r/kisspeptin pathway in this process has not been established. In this study, a bioinformatics approach was used to identify the genes for Kiss1 and Kiss1r in five teleost genomes and the information used to clone the corresponding transcripts from zebrafish. Zebrafish kiss1r was expressed predominantly in the brain, with a minor level of expression in the eye, and zebrafish kiss1 was expressed in brain, intestine, adipose tissue and testis. Analysis of the chromosome region containing the kiss1 locus showed high synteny across vertebrate genomes. In contrast to their mammalian homologues, teleost Kiss1 protein sequences were poorly conserved with the exception of the region representing kisspeptin-10. Signal peptide sequences and likely cleavage and amidation sites in the teleost Kiss1 sequences were determined and found to be similar to those in mammalian Kiss1. This is the first report of the existence and characterization of the Kiss1 gene outside the mammalian taxa, suggesting that a functional Kiss1/Kiss1 receptor pathway is conserved across vertebrate species.

Molecular basis of sex and reproductive status in breeding zebrafish.

The zebrafish (Danio rerio) is used extensively as a model species for studies on vertebrate development and for assessing chemical effects on reproduction. Despite this, the molecular mechanisms controlling zebrafish reproduction are poorly understood. We analyzed the transcriptomic profiles of the gonads of individual zebrafish, using a 17k oligonucleotide microarray, to define the molecular basis of sex and reproductive status in sexually mature fish. The gonadal transcriptome differed substantially between sexes. Among the genes overexpressed in females, 11 biological processes were overrepresented including mitochondrion organization and biogenesis, and cell growth and/or maintenance. Among the genes overexpressed in males, six biological processes were overrepresented including protein biosynthesis and protein metabolism. Analysis of the expression of gene families known to be involved in reproduction identified a number of genes differentially expressed between ovaries and testes including a number of sox genes and genes belonging to the insulin-like growth factor and the activin-inhibin pathways. Real-time quantitative PCR confirmed the expression profiles for nine of the most differentially expressed genes and indicated that many transcripts are likely to be switched off in one of the sexes in the gonads of adult fish. Significant differences were seen between the gonad transcriptomes of individual reproductively active females reflecting their stage of maturation, whereas the testis transcriptomes were remarkably similar between individuals. In summary, we have identified molecular processes associated with (gonadal) sex specificity in breeding zebrafish and established a strong relationship between individual ovarian transcriptomes and reproductive status in females.

Development of chronic tests for endocrine active chemicals. Part 1. An extended fish early-life stage test for oestrogenic active chemicals in the fathead minnow (Pimephales promelas).

An extended early-life stage test (based on OECD test guideline 210) was developed to allow the evaluation of a weak environmental oestrogen, 4-tert-pentyphenol (4TPP), on sexual differentiation and gonadal development. Fathead minnow (Pimephales promelas) embryos were exposed to three concentrations of 4TPP (56, 180 and 560 microg l(-1)) in a flow-through system, at 25+/-1 degrees C, for <107 days post-hatch (dph). In addition, some embryos were exposed to 180 microg 4TPPl(-1) until 30 or 60 dph, after which they were exposed to dilution water only until 107 dph. At 30, 60 and 107 dph fish were evaluated for growth and gonadal development (via histology), and at 107 dph fish were also evaluated for secondary sexual characteristics (SSC), gonadosomatic index (GSI) and plasma vitellogenin (VTG). There were no effects of 4TPP on hatching success or survival, however, there was a delay in the time taken for embryos to hatch (560 microg 4TPPl(-1)). No treatment-related effects were observed on fish growth, with the exception of at 107 dph when the condition factor in female fish was reduced in all 4TPP continuous exposure treatments. Plasma VTG was only elevated in female fish exposed to 180 microg 4TPPl(-1) and inhibition of gonadal growth (GSI) occurred only in females exposed to 560 microg 4TPPl(-1). Histological examination of the gonads revealed delays and disruption in male sexual differentiation and development (180 microg 4TPPl(-1)) and no testicular tissue was observed in any fish exposed to 560 microg 4TPPl(-1). Mixed gonads (predominately testes with a scattering of primary oocytes) were present in fish exposed to all doses of 180 microg 4TPPl(-1) at 107 dph. Feminisation of the reproductive ducts (formation of an ovarian like cavity) occurred in the testis of all males exposed to 180 microg l(-1), regardless of length of 4TPP exposure. Results indicate that the period of 30-60 dph appears to be the sensitive window for disruption of formation of the reproductive duct and this effect is not reversible when the fish are transferred to dilution water. The data also show that this integrative test is suitable for the detection of a weak environmental oestrogen and comparisons of these results with that of a fish full life-cycle, in medaka, indicate that this test could be a suitable surrogate for a fish full life-cycle.

Molecular characterization of estrogen receptors 1, 2a, and 2b and their tissue and ontogenic expression profiles in fathead minnow (Pimephales promelas).

There are two estrogen receptor (ER) subtypes in fish, Esr1 and Esr2 (formerly ERalpha and ERbeta), and in some species the Esr2 subtype has two forms, Esr2b (formerly ERbeta1) and Esr2a (formerly ERbeta2 or ERgamma). There is little information, however, on the different characteristics and functional significance of the two receptor subtypes in fish, and this is especially relevant for understanding the disruption of ER signaling by chemicals with estrogenic activity. In this study, the full-length cDNAs for esr1 (3167 base pairs [bp]) and esr2b (2318 bp), and a partial-length (267 bp) cDNA for esr2a, were cloned and characterized in fathead minnow (fhm; Pimephales promelas), and their patterns of expression established during development and in adults. Real-time polymerase chain reaction revealed some clear distinctions in the ontogenic and tissue expression of fhm esr1, esr2b, and esr2a, suggesting different functions for each ER subtype. Fhm ERs were expressed in brain, pituitary, liver, gonad, intestine, and gill of male and female fish, esr2b and esr2a were also expressed in muscle. Fhm esr1 and esr2b were expressed predominantly in the liver, whereas fhm esr2a was expressed predominantly in intestine and was lowest expressed in liver. Responses of the different hepatic ERs in male fathead minnow exposed to 100 ng estradiol/L differed, with a significant induction (5-fold) of fhm esr1 but no effect on esr2b or esr2a expression, suggesting different mechanisms of regulation for the different ERs. The detailed characterization of ERs in fathead minnow provides the foundation for understanding the molecular basis of estrogenic disruption in fish.

Mixtures of estrogenic contaminants in bile of fish exposed to wastewater treatment works effluents.

Most effluents from wastewater treatment works (WwTWs) contain estrogenic chemicals that include steroidal estrogens and xenoestrogens. We investigated the nature of mixtures of estrogenic contaminants taken up by two species of fish exposed to two WwTWs effluents. Sexually immature rainbow trout, Oncorhynchus mykiss, and sexually mature roach, Rutilus rutilus, were exposed to tap water, river water, or one of two estrogenic WwTWs effluents for up to 10 days, when the fish were sacrificed and tissues removed for chemical analysis. Estrogenic contaminants in the bile and gonads were hydrolyzed, concentrated by solid-phase extraction, and fractionated by RP-HPLC. Active fractions were detected and quantified using a yeast estrogen receptor transcription screen (YES assay) and the identities of estrogenic components in the fractions determined by GC-MS. Bile from rainbow trout exposed to either tap water or river water contained low amounts of 17beta-estradiol (E2) and estrone (E1) with a total estrogenic activity (mean+/-standard error) of 10+/-5 and 31+/-9 ng of E2 equivalents/mL (ng of E2eq/mL) for male and female fish, respectively. In effluent-exposed trout the total estrogen content of bile was considerably higher with the following composition and concentrations (ng of E2eq/mL) of individual estrogens: E2 (male, 591+/-125; female, 710+/-207), E1 (male, 338+/-75; female, 469+/-164), ethinylestradiol, EE2 (male, 32+/-2; female, 40+/-6), nonylphenol (NP) and short-chain NP polyethoxylates (male, 21+/-4; female, 22+/-3). An additional estrogenic compound, 17beta-dihydroequilenin (DHQ), was identified for the first time in effluent-exposed fish, and was present in trout bile at concentrations of (male) 40+/-9 and (female) 30+/-5 ng of E2 eq/mL. DHQ, E2, E1, and EE2, but not NP or NP polyethoxylates, were also detected in bile of effluent-exposed roach, and the concentrations of all these steroidal estrogens in ng of E2eq/mL were lower in male (E2, 62+/-2; E1, 35+/-11; EE2, 10+/-2; DHQ, 1+/-1) compared with female (E2, 740+/-197; E1, 197+/-37; EE2, 40+/-6; DHQ, 8+/-2) roach. The synthetic estrogen EE2 was also detected in the testes and ovaries of effluent-exposed roach. This study shows that a mixture of estrogenic contaminants present in WwTWs effluents bioconcentrate in fish tissues, resulting in the induction of vitellogenin, and are likely to contribute to feminizing effects in wild fish living in U.K. rivers. The composition of the mixture of estrogenic contaminants in the bile is species dependent and may determine the susceptibility of fish to the effects of exposure to estrogenic effluents.