Pharmaceuticals and drugs of abuse (DoA) are continuously released into aquatic environments, but their biological uptake and effects in wild fish remain poorly characterized. Neuroactive substances, including antidepressants, opioids, and stimulants, are particularly concerning due to their potential to alter fish behavior and physiology. The present study aimed to investigate the bioaccumulation of 26 neuroactive substances in wild fish associated with an outfall from a municipal wastewater treatment plant (WWTP). First, an extraction method was developed for small-bodied fish that combines ultrasonic solvent extraction with solid-phase extraction. Using LC-MS/MS analysis, the method achieved limits of detection ranging from 0.02 to 1.3 ng g -1 . Subsequently, the method was applied to male and female wild darters (rainbow, greenside, and fantail; Etheostoma spp.) collected upstream and downstream of a tertiary filtration WWTP in southern Ontario, Canada. Of the 26 analytes monitored, only fentanyl, methadone, venlafaxine, and O-desmethylvenlafaxine were consistently detected in all species, with higher concentrations at the downstream location. Sex-specific differences in accumulation were observed, with males accumulating higher concentrations than females. This study presents a novel analytical method for detecting DoA in fish tissue and demonstrates the potential for fish exposed to treated wastewater to bioaccumulate selected DoA and antidepressants. • An analytical method was developed for DoA and antidepressants in fish tissue. • Detection of DoA and antidepressants in wastewater, river water, and wild darters. • Site- and sex-specific accumulation differences were observed in darters.
Cárdenas-Soracá et al. (2026) studied this question.