Abstract

Understanding how chemical pollution translates into biological effects under environmentally realistic conditions remains a central challenge for freshwater ecotoxicology. The aim of this study was to evaluate whether an integrated in situ approach combining exposure and behavioral biomarkers, with chemical indicators can detect sublethal impairment associated with chemical pollution in river ecosystems. We performed a 72-h in situ caging experiment using the native fish Galaxias maculatus in Cruces River (Chile), comparing a reference site with a site impacted by urban, agricultural, and industrial discharges. Neurotoxicity and detoxification responses were assessed though brain acetylcholinesterase (AChE) activity and hepatic ethoxyresorufin-O-deethylase (EROD) activity, respectively, while behavior (activity, social proximity, immobility) was quantified through video tracking. To characterize chemical exposure, the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was quantified in sediments and fish tissues. Fish from the impacted site exhibited marked AChE inhibition (∼60%) and a five-fold induction of EROD activity relative to reference fish. Tissue analyses revealed a two-fold higher accumulation of 2,4-D at the impacted site, despite comparable sediment concentrations, indicating increased internal exposure. These biochemical responses were accompanied by reduced activity, decreased social proximity, and increased immobility. Cluster analysis revealed a shift toward hypoactive and isolated behavioral phenotypes at the impacted site. By integrating targeted chemical analyses with multi-level biological endpoints in a native sentinel species, this study highlights the value of in situ approaches for supporting the interpretation that pollutant exposure is associated with functional impairment and supports their application in freshwater pollution monitoring and risk assessment frameworks.