Data Sheet 1_Effect of environmentally relevant concentrations of estrogens on the aggressive response of male zebrafish.docx

Environmental estrogens (EEs) are diverse and widespread in aquatic systems, influencing fish social behaviour. Prior studies have focused on individual EEs, but their combined effects, particularly at environmentally relevant concentrations, remain underexplored. In this study, adult male zebrafish were exposed to EE2-low (5.55 ng/L), EE2-high (11.1 ng/L), and Mix (4-NP, 62.2 ng/L; BPA, 250 ng/L; E1, 4.56 ng/L; E2, 5.53 ng/L; E3, 39.6 ng/L, with an estrogenic potency equal to EE2-low) for 60 days. Post-exposure assays (mirror test and dyadic interaction) revealed that Mix significantly reduced the frequency of approaching mirror, the attack mirror duration, and the frequency of chasing un-exposed fish, indicating a decrease in aggressive behaviour in Mix-fish. However, the ratio of 11-ketotestosterone (11-KT) to 17β-Estradiol (E2) was observed in EE2-exposed fish in addition to Mix-fish, indicating that, in addition to sex hormones, stress hormones and neurotransmitters may also be involved in Mix-altered aggression in zebrafish. In contrast, an elevation in plasma cortisol levels and a reduction in serotonin (5-HT) and dopamine (DA) levels in the brain were found only in Mix-fish, accompanied by altered expression of genes involved in the hypothalamic-pituitary-interrenal (HPI) axis and the 5-HT/DAergic system in the brain. The data suggest that a mixture of EEs may inhibit aggression in male zebrafish by disrupting the HPI/cortisol axis and the 5-HT/DAergic system, thus causing serious ecological consequences. These findings suggest that EEs mixtures may inhibit aggression by disrupting key physiological systems. This, in turn, could undermine the competitive and then survival abilities of zebrafish, and potentially affect their population number and structure.