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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Analytical methodology for the identification of estrogenic contaminants in fish bile.

Effluents from wastewater treatment works (WwTWs) contain estrogenic contaminants that can cause feminised responses in fish. In order to assess the identity of estrogenic contaminants taken up by fish exposed to effluents, an analytical method was developed to detect estrogenic substances in fish bile, where many xenobiotics are excreted and concentrated. Estrogenic metabolites in bile were deconjugated using enzymatic hydrolysis and the estrogenic activity was determined using a yeast estrogen receptor transcription screen (YES). Hydrolysed samples were concentrated by solid-phase extraction (SPE) prior to fractionation by reversed-phase high-performance liquid chromatography (HPLC). Active HPLC fractions were detected by YES assay and analysed by gas chromatography-mass spectrometry (GC-MS) after trimethylsilylation. The method was validated using bile samples from immature female rainbow trout, which had been exposed to either tap water or an undiluted estrogenic effluent for 10 days. Hydrolysis of bile from effluent-exposed fish was complete within 16 h add most of the estrogenic activity in the bile was released by 3-glucuronidase rather than sulfatase or 3-glucosidase treatment. The estrogenic activity of hydrolysed bile from effluent-exposed fish ranged between 530 and 1440 ng E2eq/mL and was 17-48-fold greater than the activity of bile from reference fish exposed to tap water. The estrogenic activity of bile samples decreased with time in storage (at-70 degrees C by 7% per month). The recovery of estrogenic activity from SPE was 96 +/- 7% (mean +/- SD), from HPLC fractionation 87 +/- 7% and for the whole method 81 +/- 7% (n = 7). 17beta-Estradiol, estrone, 17alpha-ethinylestradiol, nonylphenol and short-chain nonylphenol polyethoxylates were all identified from GC-MS analysis of active HPLC fractions of bile from effluent-exposed trout, whereas only 17beta-estradiol was detected in bile from fish exposed to tap water. There were also several other minor estrogenic components, at present unidentified, in bile of effluent-exposed fish. The work shows that fractionation of fish bile is a useful approach to identifying mixtures of estrogenic contaminants taken up by fish from WwTW effluents and has the potential for application in the detection of other endocrine disrupting chemicals in fish tissues.

Molecular characterisation of ovarian cathepsin D in the rainbow trout, Oncorhynchus mykiss.

In fish, cathepsin D, an aspartyl protease, is believed to mediate the processing of yolk proteins in the oocyte. Cathepsin D, therefore, is vital for the production of a viable egg. This study set out to isolate and sequence the cDNA encoding cathepsin D, and to determine the developmental expression of the message in the ovary and subsequently during embryogenesis in the rainbow trout, Oncorhynchus mykiss. The full-length trout cathepsin D cDNA is 1847 base pairs (bp) long, encoding a protein of 400 amino acids (aa). The sequence consists of a putative signal peptide of 18 aa, a prosequence extending 46 aa and a mature peptide of 336 aa. The deduced sequence of rainbow trout ovarian cathepsin D shows significant homology with cathepsin D in mammals (human; 81% aa similarity), in the chicken (80% aa similarity) and in Xenopus (74% aa similarity). Our data support the contention that the primary structure of cathepsin D is highly conserved across the vertebrate phyla, from mammals to fish. Unlike cathepsin Ds in other species, however, rainbow trout cathepsin D appears to have only one putative N-glycosylation site, rather than two. The mRNA for 'ovarian' cathepsin D was expressed in both ovarian and non-ovarian tissues (liver, muscle, spleen and testis). During the development of the ovary, the highest expression levels of cathepsin D mRNA were seen at around the onset of vitellogenesis, a time when the oocytes are starting to sequester large quantities of yolk proteins. Northern hybridisation did not detect cathepsin D mRNA in either unfertilised eggs, or in fertilised eggs until after gastrulation, indicating that there is little, if any, de novo synthesis of this message at these stages of development. However, the mRNA for cathepsin D was detectable at the eyed embryo stage, and the expression of the gene increased towards the end of embryonic development.

Fish p53 as a possible biomarker for genotoxins in the aquatic environment.

The p53 gene is a tumour suppressor gene which has a fundamental role in cell cycle control and division, and in mammals certain genotoxic agents induce specific mutations in p53, leading to tumourigenesis. Fish have been investigated as models for studying carcinogens, but as yet very little data exists that links exposure to specific chemicals with the aetiology of tumours found in wild populations. In this study, p53 was sequenced from five species of fish with a view to the possible use of mutations in the highly conserved domains of p53 to identify genotoxins in the aquatic environment. A 0.8 kb fragment of the cDNA encompassing the conserved DNA-binding domain of p53 was sequenced in three Oncorhynchus salmonid fish: coho (O. kisutch), chum (O. keta), and chinook (O. tshawytscha) and full-length p53 cDNAs were sequenced in the puffer fish (Tetraodon miurus) and the barbel (Barbus barbus). The full-length puffer fish and barbel p53 cDNAs were 1834 bp and 1790 bp in length, encoding a 367 aa protein and a 369 aa protein, respectively. The deduced aa sequences of the p53 cDNA in the Oncorhynchus salmon shared a 100% identity in the five conserved regions (I-V). Comparisons of the deduced aa sequences for puffer fish and barbel p53 with other fish p53s revealed a high homology within the conserved DNA binding domain (68-86% for puffer fish and between 66-88% for barbel). "Conserved" domain I was not highly conserved in fish, as it is in mammals, and, therefore, conserved domains II-V are most likely to provide the valuable sequences in fish p53 for use in mutational studies to fingerprint genotoxins in the aquatic environment.

Molecular characterization of the first non-mammalian p73 cDNA.

The p53 gene is believed to be mutated or deficient in over 50% of human tumours, and is therefore considered to be instrumental in the process of carcinogenesis. Recently in humans, homologues of p53 (such as p73 and p63) have been isolated. In our studies in fish, we have been isolating tumour suppressor genes with a view to their potential use to study genotoxins in the aquatic environment. In this paper, we report the characterisation of the first non-mammalian p73 cDNA, isolated from barbel (Barbus barbus), a freshwater cyprinid fish indigenous to UK rivers. The deduced barbel p73 amino acid sequence has a high homology with human p73 alpha: the proteins are 641 and 636 aa in length, respectively, and there is a 72% identity over the entire sequence length of the protein (over 90% in the putative DNA binding domain). The level of conservancy for p73 is considerably higher across class (from man to fish), than for p53 and it may therefore have particular value in studies on environmental mutagenesis. Northern analysis showed expression of three p73 mRNA transcripts/homologues. The patterns of p73 tissue expression in the barbel differed from the expression of p53 mRNA, suggesting specific functional roles for the two genes.

Gonadotropins, their receptors, and the regulation of testicular functions in fish.

The pituitary gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) regulate steroidogenesis and spermatogenesis by activating receptors expressed by Leydig cells (LH receptor) and Sertoli cells (FSH receptor), respectively. This concept is also valid in fish, although the piscine receptors may be less discriminatory than their mammalian counterparts. The main biological activity of LH is to regulate Leydig-cell steroid production. Steroidogenesis is moreover modulated in an autoregulatory manner by androgens. The male sex steroids (testosterone in higher vertebrates, 11-ketotestosterone in fish) are required for spermatogenesis, but their mode of action has remained obscure. While piscine FSH also appears to have steroidogenic activity, specific roles have not been described yet in the testis. The feedback of androgens on gonadotrophs presents a complex pattern. Aromatizable androgens/estrogens stimulate LH synthesis in juvenile fish; this effect fades out during maturation. This positive feedback on LH synthesis is balanced by a negative feedback on LH release, which may involve GnRH neurones. While the role of GnRH as LH secretagogue is evident, we have found no indication in adult male African catfish for a direct, GnRH-mediated stimulation of LH synthesis. The limited available information at present precludes a generalized view on the testicular feedback on FSH.

Impacts of 17beta-estradiol, including environmentally relevant concentrations, on reproduction after exposure during embryo-larval-, juvenile- and adult-life stages in zebrafish (Danio rerio).

Zebrafish (Danio rerio) were exposed for 3 weeks to low concentrations of estradiol including environmentally relevant concentrations (5, 25 and 100 ng/l), encompassing either their embryo-larvae (from fertilization to 21 day post-fertilization (dpf)), juvenile (from 21 to 42 dpf) or adult life stages (>200 dpf) with a view to investigating the most sensitive life stage of the zebrafish to 17beta-estradiol (E2). At all sampling points, whole-body vitellogenin concentrations and gonadal development were analyzed in order to investigate the effects of estrogen exposure on these endpoint in the zebrafish. In the adult stage, additional endpoints were measured including secondary sexual characteristics (manifestation of the uro-genital papillae (UGP) in males), gonadal growth (the gonado-somatic index (GSI)) and sex ratio. For all the different life stage exposures, reproductive performance of the F0 generation was assessed (egg production) and survival and development of the F1 embryo-larvae. Exposure to low concentrations of E2 resulted in vitellogenin induction whatever the life stage exposed but these effects were reversible after depuration. The effective concentration for vitellogenin induction in zebrafish early life stages was 100 ng E2/l, and in adult male zebrafish the effective concentration for vitellogenin induction (between 5 and 25 ng/l) was lower than for the early life stage fish. Exposure to E2 prior to (from fertilization to 21 dpf) and during the time of sex differentiation (from 21 to 42 dpf) also caused disruptions in the process of sexual differentiation (resulting in formation of a retrogonadal cavity in presumptive male, germ cell development and leading to a significant change of the sex ratio towards the female sex at the dose of 100 ng E2/l for the fish exposure as embryo-larvae) and altered patterns of egg production in the subsequent adults. Exposure of adult fish to E2 resulted in a modification of the secondary sexual characteristic in males at 25 and 100 ng E2/l as well as a dose-dependent inhibition of egg production. The findings from this study show that the nature and intensity of the reproductive effects of E2 are dependent of the time and concentration of exposures of zebrafish to E2, some of these effects being permanent (effect on the sexual differentiation) while others being reversible (effect on the Vtg induction). This study demonstrated that early life stages of zebrafish are sensitive to low concentrations of E2 and provides relevant data that could be used for the adaptation of existing fish early life stage test for the in vivo testing of estrogenic compounds. The data presented raise further concerns about the effects of steroid estrogens in the environment on fish reproductive health.

Androgenic and estrogenic effects of the synthetic androgen 17alpha-methyltestosterone on sexual development and reproductive performance in the fathead minnow (Pimephales promelas) determined using the gonadal recrudescence assay.

The effects of the androgen, 17alpha-methyltestosterone were assessed on sexual development and reproductive performance in the fathead minnow (Pimephales promelas) using a gonadal recrudescence assay. In this assay, mature male and female fathead minnow, previously kept under simulated winter conditions (15 degrees C; 8:16 h light:dark regime) were transferred to simulated summer conditions (25 degrees C water temperature; 16:8 h light:dark regime) to induce gonadal recrudescence. To assess sexual development fish were exposed to nominal concentrations of 0, 0.1, 1, 5 and 50 microg/L 17alpha-methyltestosterone. After 3 weeks of chemical exposure, effects on condition (condition factor, CF), plasma vitellogenin (VTG), secondary sex characteristics, gonad growth (gonadosomatic index; GSI) and gonad histology were investigated. Reproductive performance, including reproductive output (egg production), spawning behaviour, and fertilisation rate were measured over a subsequent 3-week-period in breeding adults maintained in clean water. 17alpha-Methyltestosterone had no effects on the condition of fish at any of the doses tested. 17alpha-Methyltestosterone induced both androgenic and estrogenic effects with females generally more affected by 17alpha-methyltestosterone than males: atretic follicles and male-specific sex characteristics (androgenic effect) were induced in females at > or = 0.1 and > or = 1 microg/L 17alpha-methyltestosterone, respectively. An inhibitory effect on ovary growth occurred at an exposure concentration of 50 microg/L 17alpha-methyltestosterone. In males 1 microg/L 17alpha-methyltestosterone induced a concentration-response induction of plasma vitellogenin (estrogenic effect) likely due to its conversion into 17alpha-methylestradiol, rather to the competition with endogenous steroids and their cross reactivity with the estrogen receptor. In the fish breeding studies, concentration-dependent reductions in egg number, fertilisation rate and increases in abnormal sexual behaviour in females were observed. All of these effects occurred at exposure concentrations of > or = 5 microg/L 17alpha-methyltestosterone. Thus, it could be assumed that the observed estrogenic effects in male fathead minnow were likely to the conversion of 17alpha-methyltestosterone into the estrogen 17alpha-methylestradiol, rather to the acting of 17alpha-methyltestosterone itself. In conclusion to this, showing hormonally activity of 17alpha-methyltestosterone in fish down to 100 ng/L, indicates that its potency was close to the range of several naturally occurring estrogens.