Short-term, high-end environmentally -relevant diazepam exposure induces sex-specific behavioral and molecular alterations in adult zebrafish

Pharmaceuticals with psychoactive properties are increasingly recognized as emerging pollutants, raising concern due to their persistent release and long-term stability in aquatic environments. We investigated the behavioral and molecular effects of continuous 24-h exposure to diazepam (DZP) at worst-case environmentally relevant (12 μg/L) and one order of magnitude higher concentration (120 μg/L) in adult zebrafish (Danio rerio). Behavioral investigations, performed 24-h post exposure, revealed sex-specific responses: females exposed to 12 μg/L showed reduced dark preference and freezing time, indicative of anxiolytic effect, while males at 120 μg/L exhibited increased light avoidance, suggesting anxiogenic behavior, while locomotor parameters remained unchanged, and anxiety-related indices in the novel tank test (NTT) further confirmed that DZP, at these concentrations, did not exert sedative effects. At the molecular level, gabra1 gene expression was upregulated in females exposed to the lower concentration and downregulated in males exposed to the higher dose. In line with this finding, histological analyses revealed increased expression of GABA-A receptor subunit α in the telencephalon of females. These findings revealed that short-term, low-concentration DZP exposure induces pronounced sex-specific behavioral and neurochemical alterations in adult zebrafish, highlighting the ecological risk of benzodiazepine contamination and the relevance of sex differences research to environmental risk assessment.