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.

Sewage treatment plant effluents contain a complex mixture of pharmaceuticals, personal care products and industrial chemicals, thus exposing aquatic organisms. Still, the consequences of exposure to combinations of different classes of drugs is largely unknown. In this study, we expose adult zebrafish (Danio rerio) males and females to low, environmentally relevant concentrations of the endocrine disrupting chemical 17α-ethinyl estradiol (EE₂) and the selective serotonin re-uptake inhibitor (SSRI) citalopram, alone and in combination, and analyse three non-reproductive behaviours of importance for population fitness.

Two weeks exposure to 0.1 and 0.5 ng/LEE₂ resulted in increased anxiety in males in the scototaxis (light/dark preference) test. Significantly longer latency periods before entering the white zone and fewer visits in the white zone were observed in males exposed to both 0.1 and 0.5 ng/LEE₂ compared to unexposed males. No significant effects of citalopram alone (0.1 and 0.5 µg/L) were observed in the scototaxis test. The combined exposures (0.1 ng/L EE₂ + 0.1 µg/L citalopram and 0.5 ng/L EE₂ + 0.5 µg/L citalopram) resulted in abolishment of the anxiogenic effects of EE₂, with significantly shorter latency period (low dose) and more transitions to white (high and low dose) than in fish exposed to EE₂ alone. No significant effects of either EE₂, citalopramor the combination of the two were observed in females. In the novel tank test, significantly more transitions to the upper half of the tank were observed in males exposed to 0.1 µg/L citalopram alone compared to unexposed males while males exposed to 0.1 ng/lEE₂ had significantly shorter latency period to enter the upper half. Exposure to the combination of the two low concentrations did, however, result in a significantly longer latency and fewer transitions to upper half compared to both control, EE₂- and citalopram-exposed males. These males also spent significantly less time in the upper half than the fish exposed to 0.1 ng/l EE₂ or 0.1 µg/l citalopram alone. No significant effects on novel tank behaviour were observed in females or males exposed to the higher concentrations. In the shoaling test, males exposed to 0.1 µg/L citalopram and females exposed to 0.5 ng/l EE₂ made significantly fewer transitions away from peers while males exposed to 0.1 µg/L citalopram + 0.1 ng/l EE₂ performed significantly more transitions than the fish exposed to 0.1 µg/L citalopram alone.

In conclusion, this study shows that very low concentrations ofEE_2, at or slightly above the predicted noeffect concentration (NOEC), affects anxiety in zebrafish males. Furthermore, citalopram, in spite of marginal effect of its own at such low levels, counteracts the response to EE₂. This study represents an initial effort to understand the effects on water-living organisms of the cocktails of anthropogenic substances contaminating aquatic environments.

17α-Ethinylestradiol (EE2) is a ubiquitous aquatic contaminant shown to decrease fish fertility at low concentrations, especially in fish exposed during development. The mechanisms of the decreased fertility are not fully understood. In this study, we perform transcriptome analysis by RNA sequencing of testes from zebrafish with previously reported lowered fertility due to exposure to low concentrations of EE2during development. Fish were exposed to 1.2 and 1.6 ng/L (measured concentration; nominal concentrations 3 and 10 ng/L) of EE2 from fertilization to 80 days of age, followed by 82 days of remediation in clean water. RNA sequencing analysis revealed 249 and 16 genes to be differentially expressed after exposure to 1.2 and 1.6 ng/L, respectively; a larger inter-sample variation was noted in the latter. Expression of 11 genes were altered by both exposures and in the same direction. The coding sequences most affected could be categorized to the putative functions cell signalling, proteolysis, protein metabolic transport and lipid metabolic process. Several homeobox transcription factors involved in development and differentiation showed increased expression in response to EE2 and differential expression of genes related to cell death, differentiation and proliferation was observed. In addition, several genes related to steroid synthesis, testis development and function were differentially expressed. A number of genes associated with spermatogenesis in zebrafish and/or mouse were also found to be differentially expressed. Further, differences in non-coding sequences were observed, among them several differentially expressed miRNA that might contribute to testis gene regulation at post-transcriptional level. This study has generated insights of changes in gene expression that accompany fertility alterations in zebrafish males that persist after developmental exposure to environmental relevant concentrations of EE2 that persist followed by clean water to adulthood. Hopefully, this will generate hypotheses to test in search for mechanistic explanations.

The synthetic estrogen 17α-ethinylestradiol (EE2) is an endocrine disrupting compound of concern due to its persistence and widespread presence in the aquatic environment. Effects of developmental exposure to low concentrations of EE2 in fish on reproduction and behavior not only persisted to adulthood, but have also been observed to be transmitted to several generations of unexposed progeny. To investigate the possible biological mechanisms of the persistent anxiogenic phenotype, we exposed zebrafish embryos for 80 days post fertilization to 0, 3 and 10 ng/L EE2 (measured concentrations 2.14 and 7.34 ng/L). After discontinued exposure, the animals were allowed to recover for 120 days in clean water. Adult males and females were later tested for changes in stress response and shoal cohesion, and whole-brain gene expression was analyzed with RNA sequencing. The results show increased anxiety in the novel tank and scototaxis tests, and increased shoal cohesion in fish exposed during development to EE2. RNA sequencing revealed 34 coding genes differentially expressed in male brains and 62 in female brains as a result of EE2 exposure. Several differences were observed between males and females in differential gene expression, with only one gene, sv2b, coding for a synaptic vesicle protein, that was affected by EE2 in both sexes. Functional analyses showed that in female brains, EE2 had significant effects on pathways connected to the circadian rhythm, cytoskeleton and motor proteins and synaptic proteins. A large number of non-coding sequences including 19 novel miRNAs were also differentially expressed in the female brain. The largest treatment effect in male brains was observed in pathways related to cholesterol biosynthesis and synaptic proteins. Circadian rhythm and cholesterol biosynthesis, previously implicated in anxiety behavior, might represent possible candidate pathways connecting the transcriptome changes to the alterations to behavior. Further the observed alteration in expression of genes involved in synaptogenesis and synaptic function may be important for the developmental modulations resulting in an anxiety phenotype. This study represents an initial survey of the fish brain transcriptome by RNA sequencing after long-term recovery from developmental exposure to an estrogenic compound.

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 synthetic estrogen 17α-ethinylestradiol (EE2) is an endocrine disrupting chemical (EDC) of concern due to its persistent nature and widespread presence in the aquatic environment. In mammals, effects of developmental EDC exposure on reproduction and behavior not only persist to adulthood after discontinued exposure, but are also inherited by several consecutive unexposed generations. The results presented in this thesis demonstrate that non-reproductive behavior in fish is highly sensitive to the influence of EE2 during development and the effects do not appear to be restored after a long recovery period in clean water. We have shown that exposure to low doses of EE2 during development results in increased anxiety in two fish species (zebrafish and guppy) and their offspring. We have also demonstrated that the effects of EE2 on anxiety are apparent in both sexes and are transgenerationally transmitted to two consecutive generations of unexposed offspring in the guppy. In order to investigate the possible biological mechanisms of the observed persistent effects on non-reproductive behavior, we also performed an RNA sequencing analysis of the whole-brain transcriptome in developmentally exposed zebrafish after remediation in clean water until adulthood. Differential expression of 33 genes in males and 62 genes in females were observed as a result of EE2 exposure, with only one gene affected in both sexes. Functional analysis revealed cholesterol biosynthesis and circadian rhythm to be the top two affected pathways in males and females, respectively. Both pathways have previously been implicated in anxiety behavior and represent possible candidates connecting the transcriptome alterations to the observed behavioral phenotype. The study represents an initial survey of the fish brain transcriptome by means of RNA sequencing after long-term recovery from developmental exposure to an estrogenic compound.

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.

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.