Effects of environmentally relevant ibuprofen and valproic acid exposure on zebrafish behavior

Active pharmaceutical ingredients (APIs) are increasingly entering aquatic environments due to human and veterinary use, wastewater discharges, and inadequate waste containment, raising significant concerns for both ecosystems and human health. Ibuprofen and valproic acid are among the pharmaceuticals detected in surface waters, primarily due to incomplete metabolism and the limited removal efficiency of conventional wastewater treatment systems. Ibuprofen, readily available over the counter, is frequently found in high concentrations, while valproic acid, which is available only by prescription, is detected less often, likely reflecting its more restricted use. This study employed the established behavioral ecotoxicology model, Danio rerio, to investigate the effects of environmentally relevant concentrations of ibuprofen (20 µg L-1) and valproic acid (3 µg L-1) on zebrafish (8 months old) after 96 hours of exposure. Both compounds influenced locomotor and anxiety-related endpoints, with changes in social preference primarily associated with valproic acid exposure. Single compound exposures resulted in reduced total distance traveled and average velocity, while combined exposure did not differ from the control group, indicating no additive locomotor impairment. Inactivity duration decreased in both individual treatments, most significantly with valproic acid, whereas the mixture produced no significant effect. Only ibuprofen reduced counterclockwise rotations, suggesting a mild anxiolytic-like response. Given the ecological importance of social cohesion and locomotor performance in predator avoidance, foraging, and reproduction, such behavioral disruptions may compromise population stability. These findings highlight the necessity of integrating behavioral endpoints and considerations of mixture toxicity into ecological risk assessments of pharmaceutical contaminants in aquatic systems.