Chronic exposure to environmentally relevant diazepam alters behaviour, gut microbiome, and offspring phenotype in zebrafish (Danio rerio).

The widespread consumption and incomplete wastewater removal of the psychoactive drug diazepam (DZP) have led to its ubiquitous presence in aquatic environments, posing profound ecological risks. While the direct toxicity of DZP is well-documented, its chronic and mechanistic impacts, particularly those concerning the gut-brain axis and intergenerational effects on offspring, remain largely unexplored. To investigate the multigenerational toxicity of DZP, adult female zebrafish were chronically exposed to environmentally relevant concentrations (1 and 10 μg/L) for 28 days, and their offspring were subsequently collected. Our findings revealed that DZP exposure induced severe gut damage and disrupted the gut microbiota and metabolite profiles in female zebrafish. This microecological dysbiosis was linked to impaired oocyte development and reduced brain γ-aminobutyric acid (GABA) levels, culminating in reproductive dysfunction and abnormal neurobehavior. Crucially, these toxicological effects were maternally transferred to the offspring, which exhibited corresponding developmental abnormalities and neurobehavioral deficits. Overall, this study demonstrates that environmentally relevant concentrations of DZP exert covert but profound intergenerational toxicity, potentially mediated by disruptions in the gut-brain axis, making it highly necessary to establish stricter regulatory guidelines regarding the discharge of psychoactive drugs into water environments.