Exposure to estrogenic endocrine disruptors (EDCs) during of development affects fertility, reproductive and non-reproductive behavior in mammals and fish. These effects can also be transferred to coming generations. In fish, the effects of developmental EDC exposure on non-reproductive behavior is less well studied. Here, we analyze the effects of 17α-Ethinylestradiol (EE2) on anxiety, shoaling behavior and fertility in zebrafish after developmental treatment and remediation in clean water until adulthood. Zebrafish embryos were exposed from day 1 to day 80 post fertilization to actual concentrations of 1.2 and 1.6ng/L EE2. After remediation for 82days non-reproductive behavior and fertilization success were analyzed in both sexes. Males and females from the 1.2ng/L group, as well as control males and females, were bred, and behavior of the untreated F1 offspring was tested as adults. Developmental treatment with 1.2 and 1.6ng/L EE2 significantly increased anxiety in the Novel Tank test and increased shoaling intensity in both sexes. Fertilization success was significantly reduced by EE2 in both sexes when mated with untreated fish of opposite sex. Progeny of fish treated with 1.2ng/L EE2 showed increased anxiety in the Novel tank test and increased light avoidance in the Scototaxis test compared to control offspring. In conclusion, developmental exposure of zebrafish to low doses of EE2 resulted in persistent changes in behavior and fertility. The behavior of unexposed progeny were affected by their parents' exposure, which might suggest transgenerational effects.

Psychoactive drugs, such as selective serotonin reuptake inhibitors (SSRI) have been identified in high levels in effluents from Swedish sewage treatment plants (STP) at concentrations high enough to give pharmacological effects in fish. In humans SSRIs are used in the treatment of depression and they have anxiolytic effects. In the present study we exposed Endler guppy (Poecilia wingei) of both sexes to citalopram that showed the highest concentrations of SSRIs in STP effluents and studied reproductive and non-reproductive behaviour. Male courting behaviours were not affected compared to control fish after 14-28 days exposure to 1μgL^-1. In two experiments exposing both sexes to 0.2, 2.3 or 15μgL^-1 for 21 days, fish exposed to the two highest doses showed anxiolytic effects when placed in a novel environment (novel tank diving test, NT). Males were only affected by exposure to 15μgL^-1. They had significantly longer latency to explore the upper half of the aquarium, more visits and longer time spent in the upper half, and showed less bottom freezing behaviour, all markers of anxiolytic behaviour. In females exposure to 2.3 or 15μgL^-1 significantly increased freezing behaviour, while no effects on other behaviour variables were observed. No effects on shoaling behaviour could be discerned. These results show that citalopram have anxiolytic effects on guppy fish and thus affect ecologically relevant behaviours of importance to survival of fish.

The synthetic estrogen 17α-ethinyl estradiol (EE2) disturbs reproduction and causes gonadal malformation in fish. Effects on the transcription of genes involved in gonad development and function that could serve as sensitive biomarkers of reproductive effects in the field is, however, not well known. We have studied mRNA expression in testes and liver of adult zebrafish (Danio rerio) males treated with 0, 5 or 25ng/L EE2for 14days. qPCR analysis showed that the mRNA expression of four genes linked to zebrafish male sex determination and differentiation, Anti-Mullerian Hormone, Double sex and mab-related protein, Sry-related HMG box-9a and Nuclear receptor subfamily 5 group number 1b were significantly decreased by 25ng/L, but not 5ng/L EE2 compared with the levels in untreated fish. The decreased transcription was correlated with a previously shown spawning failure in these males (Reyhanian et al., 2011. Aquat Toxicol 105, 41-48), suggesting that decreased mRNA expression of genes regulating male sexual function could be involved in the functional sterility. The mRNA level of Cytochrome P-45019a, involved in female reproductive development, was unaffected by hormone treatment. The transcription of the female-specific Vitellogenin was significantly induced in testes. While testicular Androgen Receptor and the Estrogen Receptor-alpha mRNA levels were unchanged, Estrogen receptor-beta was significantly decreased by 25ng/L EE2. Hepatic Estrogen Receptor-alpha mRNA was significantly increased by both exposure concentrations, while Estrogen Receptor-beta transcription was unaltered. The decreased transcription of male-predominant genes supports a demasculinization of testes by EE2 and might reflect reproductive disturbances in the environment.

The effects of endocrine disruptors may vary with the timing of exposure. The physiological implications of adult exposure are present during and shortly after exposure while embryonic exposure can imprint changes manifested in adulthood. In this study, guppy (Poecilia reticulata) embryos were exposed to 2 ng/L and 20 ng/L of 17α-ethinylestradiol during development via the mother and reared in clean water from gestation until 6 months of age. As adults, fish exposed to 20ng/L during development showed significantly altered behaviour in the Novel Tank test, where anxiety is determined as the tendency to remain at the bottom upon introduction into an unfamiliar tank. 17α-ethinylestradiol treatment increased the latency time before swimming to the upper half of the tank and decreased the number of transitions to the upper half. In control females the basal stress behaviour responses were significantly higher than in males, as indicated by longer latency period and fewer and shorter visits to the upper half, supporting the importance of gonadal hormones for the behaviour. The anxiety increased, however, with treatment in both sexes, suggesting that the observed response is not entirely due to feminization of the males. Shoaling behaviour, analyzed as tendency to leave a shoal of littermates, was neither sex-differentiated nor changed by treatment. Also male reproductive behaviour, brain aromatase activity and testes histology, previously shown to respond to oestrogen exposure in adult guppy, were unaffected by the developmental treatment. This suggests that the stress system in the guppy is very sensitive to 17α-ethinylestradiol, which possibly causes an early organisational imprint on the brain circuit that regulates stress reactions.

The liver-X-receptors (LXRs) act as cholesterol sensors and participate in the regulation of lipid and cholesterol metabolism. The objective of this study was to determine the role of LXR during development using the zebrafish model. By in situ hybridization we showed distinct expression of lxr in the brain and the retina in the developing and adult zebrafish. Lxr ligand activation affected the expression of genes involved in lipid metabolism in zebrafish adult brain and eye as well as in zebrafish embryos. Morpholino knock down of lxr resulted in an overall impaired lipid deposition as determined by oil red O staining particularly in the head and around the eyes, and to significantly elevated levels of both total and free cholesterol in the yolk of lxr morphant embryos. The expression of genes involved in lipid and cholesterol metabolism was also changed in the lxr morphants. Furthermore, alcian blue staining revealed malformation of the pharyngeal skeleton in the lxr morphant. Our data show that Lxr is an important component of the regulatory network governing the lipid homeostasis during zebrafish development, which in turn may support a role of Lxr for normal development of the central nervous sytem, including the retina.

Behaviour studies are used in toxicology research as they are excellent tools to measure physiological end-points caused by exogenous chemicals. In mammals both reproductive and non-reproductive behaviours have been used for a long period of time, whereas in teleost fishes non-reproductive behaviours have received little attention compared to reproductive behaviours. Recent advances in measuring stress related behaviours in zebrafish have provided additional tools to understand behaviour toxicology in fish. One species with well documented reproductive behaviour disturbed by different toxicants is the guppy, which is better suited than zebrafish for reproductive behaviour studies and therefore might be a better model organism for comparative behaviour studies in fish toxicology. Here we report new applications for non-reproductive behaviours in guppy and test these behaviours on males treated with the endocrine disruptor 17α-ethynylestradiol at environmentally relevant concentrations. 17α-ethynylestradiol increased freezing and bottom-dwelling when fish were placed in a non-familiar aquarium, but did not significantly affect shoaling behaviour. These results are similar to the anxiogenic behaviours seen in rats treated perinatally with 17α-ethynylestradiol and add more concern to the impacts of endocrine disruptors on aquatic wildlife.

Brain estrogen production, performed by the enzyme aromatase, can be disrupted/affected in teleost fish exposed to endocrine disruptors found in polluted aquatic environments. The guppy (Poecilia reticulata) was previously studied and confirmed to suffer negative effects on reproductive behaviors following inhibition of the brain aromatase reaction. Here adult guppies (Poecilia reticulata) of both genders were subjected to known endocrine disruptors: the androgen androstenedione (A), the synthetic estrogen 17 alpha-ethinylestradiol (EE2), and the estrogenic surfactant 4-nonylphenol (NP), at high (50 mu g/L) and at environmentally relevant concentrations (10 ng/L EE2, 5 mu g/L NP, and 0.7 mu g/L A) for 2 weeks followed by measurements of brain aromatase activity (bAA). In the adult males, bAA was stimulated by A and EE2 at 50 mu g/L. Female activity was also stimulated by the higher estrogenic treatment. At environmentally relevant concentrations only the EE2 treatment affected bAA, and only in males. The alkylphenolic substance NP produced no effect in either of the experiments, not on males nor females. The results indicate that short-term steroid treatments have stimulatory effects on guppy brain aromatase even at concentrations that can be found in the environment. We thus suggest bAA of adult guppies to be a suitable bioindicator of endocrine disruptors.