Toxic effects of diazepam on crucian carp (Carassius auratus): insights into hepatic injury and underlying metabolic mechanisms

IntroductionDiazepam enters aquatic environments via wastewater and illegal use in live fish transport, yet its hepatic toxicity mechanisms in crucian carp (Carassius auratus)—a key species linking freshwater and estuarine food webs—remain unclear. This study established a multi‑level toxicity framework to clarify diazepam‑induced hepatotoxicity and ecological risks.MethodsAcute toxicity tests determined 96‑h LC50 and safe concentration (SC) in juveniles. A 10‑day chronic exposure (0.004, 0.04, 0.4 mg/L) assessed hepatic injury markers (ALT/AST/AKP/ACP), oxidative/antioxidant indices (SOD/CAT/GSH‑PX/GST/MDA), and 14‑day exposure (0.4 mg/L) hepatic proteomics (DIA‑MS) was used to identify molecular targets via functional enrichment.ResultsDiazepam showed moderate acute toxicity (96h LC50 = 9.06 mg/L; SC = 3.52 mg/L, far above typical environmental levels). Chronic exposure caused dose‑ and time‑dependent liver injury: aminotransferases (ALT/AST) responded earliest, followed by phosphatases (AKP/ACP). Antioxidant/detoxification enzymes showed biphasic activation‑then‑inhibition at ≥0.04 mg/L, with sustained MDA accumulation above this threshold. Proteomics identified 220 differentially expressed proteins enriched in endoplasmic reticulum protein processing and cytochrome P450‑mediated xenobiotic metabolism, plus tyrosine kinase domain overrepresentation.DiscussionDiazepam poses low acute lethality risk but induces chronic hepatotoxicity via a cascade: phase I metabolism‑triggered ROS burst overwhelms defenses, causing ER stress and protein homeostasis disruption. The 0.04 mg/L oxidative damage threshold and phased biomarker responses support ecological risk assessment of pharmaceuticals in aquaculture systems.