Short-term exposure to diazepam during zebrafish embryogenesis induces intergenerational neurobehavioral disruption later in life.

Diazepam (DZP) is a widely prescribed benzodiazepine that is increasingly detected in aquatic systems and known to impair neural function in aquatic organisms. However, its potential to elicit intergenerational neurobehavioral disruption, along with the underpinning mechanism in fish, remains uncharacterized. In this study, zebrafish (Danio rerio) embryos were exposed to DZP (0.1-1000 μg/L) during early development, and behavioral endpoints were assessed from larval to adult stages in the parental (F0) generation and in F1 offspring not directly exposed via water. Embryonic DZP exposure reduced larval locomotor activity and altered anxiety-related thigmotaxis and social interaction in F0 adults; similar behavioral alterations were also observed in F1 larvae not directly exposed via water. Transcriptomic profiling identified that the top enriched pathways altered in DZP-exposed fish were GABA receptor signaling. Targeted neurochemistry and fluorescence imaging revealed sustained activation of the GABAergic system in the F0 and F1 fish brain, as evidenced by elevated GABA levels and increased gad1b-associated signals. Collectively, these results suggest that exposure to DZP can cause long-lasting and intergenerational behavioral changes in zebrafish. This highlights the importance of considering the developmental stage at which exposure occurs when assessing the ecological risk of benzodiazepines.