Neurodevelopmental and behavioral effects of early-life esketamine hydrochloride exposure in zebrafish (Danio rerio)

Esketamine hydrochloride is increasingly used as a rapid-acting antidepressant, and its expanding clinical and non-medical use has raised concerns regarding its release into aquatic systems via wastewater treatment plant effluents as an emerging psychoactive contaminant. However, its potential neurodevelopmental toxicity in aquatic organisms remains insufficiently characterized. In this study, zebrafish embryos were exposed to esketamine hydrochloride during early development, and its toxic effects were evaluated using an integrated framework combining developmental, behavioral, histological, transcriptomic, oxidative stress-related, and apoptosis-related endpoints. Early-life esketamine exposure altered multiple developmental indicators, including head length, eye depth, interocular distance, and body length, and disrupted locomotor regulation at later stages, particularly light-dark responsiveness and spatial preference. Histological examination further revealed exposure-related alterations in brain tissue organization. Transcriptomic profiling identified coordinated changes in pathways associated with redox homeostasis, protein synthesis, and phototransduction-related signaling. Targeted validation demonstrated significant upregulation of oxidative stress-related genes, including sod1 and sod2, while ELISA-based assays showed exposure-dependent alterations in SOD, CAT, GSH, and MDA levels. Acridine orange (AO) staining showed increased apoptosis-related fluorescence signals in the head region, with AO-positive puncta density differing significantly among groups. Integrative correlation analysis further linked developmental, behavioral, oxidative stress-related, and apoptosis-related endpoints. Notably, these effects occurred in the absence of overt lethality. Collectively, these findings demonstrate that esketamine interferes with neurodevelopmental and behavioral processes in zebrafish larvae and support the incorporation of early-life neurobehavioral endpoints into risk assessment frameworks for neuroactive pharmaceuticals.