Behavioral, biochemical, and endocrine stress responses in zebrafish exposed to azithromycin, nimesulide, and their combination

The widespread use of pharmaceuticals has led to their frequent detection in aquatic environments, raising concerns about adverse effects on non-target organisms. This study examined behavioral and endocrine responses to acute repetitive exposure (4 days, 15 min day −1 ) to environmentally relevant concentrations of azithromycin (AZT, 12.5 μg mL −1 ), nimesulide (NIME, 0.4 μg mL −1 ), and their combination in zebrafish ( Danio rerio ). Behavior was assessed using the Novel Tank Test, Social Preference Test, and Light/Dark Test. Whole-body cortisol, acetylcholinesterase (AChE) and catalase (CAT) activities, lipid peroxidation (TBARS), and non-protein thiols (NTP) were measured as stress biomarkers. NIME significantly reduced locomotion (∼30%) and time spent in upper zones ( p < 0.05), indicating hypolocomotion and anxiety-like behavior. AZT elevated cortisol (p < 0.05) but caused minimal behavioral changes. Co-exposure intensified behavioral impairments and further increased cortisol ( p < 0.01), suggesting synergistic effects. NIME increased TBARS, indicating oxidative damage. AChE activity was inhibited in the brain by NIME, while AZT and the combination increased systemic AChE. CAT activity rose in the combination group, possibly reflecting a compensatory response to reactive oxygen species. These findings demonstrate that short-term, repeated exposure to mixtures of commonly detected pharmaceuticals, even at environmentally relevant concentrations, can disrupt behavioral, biochemical, and endocrine homeostasis in aquatic organisms. This highlights the ecological risks posed by multi-compound contamination in aquatic environments.