Effect of environmentally relevant concentrations of estrogens on the fear-like response of male zebrafish

Environmental estrogens (EEs), diversely and widely distributed, are established to affect fish behaviour. However, information on the effects of EEs mixtures at environmentally relevant concentrations on fish fear-like responses is currently limited. In this study, adult male zebrafish were exposed to MIX (EEs mixtures at environmental concentrations: BPA, 250 ng/L; 4-NP, 62.2 ng/L; E1, 4.56 ng/L; E2, 5.53 ng/L; E3, 39.6 ng/L), as well as to EE2-low (5.55 ng/L, with oestrogenic potency equivalent to that of MIX) and EE2-high (11.1 ng/L) for 60 days. After exposure, fear-like response was evaluated using a novel tank diving test with alarm substance (AS) stimulation. Our data revealed that only MIX significantly delayed the onset time to upper layer, increased the duration in lower layer and reduced the frequency of entry to upper layer, indicating exacerbated fear-like responses in MIX-exposed fish. Additionally, for the response tests, plasma cortisol levels and brain 5-HIAA (5-hydroxyindoleacetic acid) and DOPAC (3,4-Dihydroxyphenylacetic acid) levels were elevated while brain serotonin (5-HT), dopamine (DA), and norepinephrine (NE) levels were reduced in MIX-fish. Furthermore, significant alterations were also observed in the gene mRNA expression involved in hypothalamus-pituitary-interrenal (HPI) axis and 5-HT/DA/NEergic systems in brain. These findings suggest that MIX may exacerbate fear-like responses in male zebrafish by disrupting the HPI/cortisol axis and the 5-HT/DA/NEergic pathways, potentially affecting fish population structure and leading to severe ecological consequences.