Occurrence and Dietary Health Risk Assessment of Diazepam Residues in Freshwater Fish from Suining, China

Diazepam, an emerging contaminant frequently detected in farmed fish, has unclear residue distribution patterns and associated health risks in the aquaculture supply chain. This study systematically investigated diazepam residues in freshwater fish from Suining, a typical aquaculture region in southwestern China. We collected 241 fish, 122 water, and 69 bait samples across breeding, transportation, and sales stages, analyzed residues via high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), evaluated diazepam's binding affinity to crucian carp GABAA Receptor-Associated Protein through molecular docking, and assessed human health risks using the hazard quotient (HQ) method. Results showed diazepam accumulated along the supply chain, with the highest detection rate (38.78%) at the sales stage; crucian carp had significantly higher residues (2.65 μg/kg) than other carp species (p < 0.05), with 86.36% detection rate in aquaculture water and 46.38% in bait. The binding energy of diazepam to the target protein was -6.884 kcal/mol, and HQ values for all age groups were far below the safety threshold of 1. This study revealed the bioaccumulation of diazepam in the aquaculture supply chain, identified bait as a potential contamination source, and provided a scientific basis for targeted aquaculture regulation and food safety standard refinement.