The Iguaçu River, located at the Southern part of Brazil, has a great socioeconomic and environmental importance due to its high endemic fish fauna and its potential to generate hydroelectric power. However, Iguaçu River suffers intense discharge of pollutants in the origin of the river. In a previous report, the local environmental agency described water quality to improve along the river course. However, no study with integrated evaluation of chemical analysis and biological responses has been reported so far for the Iguaçu River. In the current study, three different Brazilian fish species (Astyanax bifasciatus, Chrenicicla iguassuensis, and Geophagus brasiliensis) were captured in the five cascading reservoirs of Iguaçu River for a multi-biomarker study. Chemical analysis in water, sediment, and muscle indicated high levels of bioavailable metals in all reservoirs. Polycyclic aromatic hydrocarbons (PAHs) were detected in the bile of the three fish species. Integration of the data through a FA/PCA analysis demonstrated the poorest environmental quality of the reservoir farthest from river’s source, which is the opposite of what has been reported by the environmental agency. The presence of hazardous chemicals in the five reservoirs of Iguaçu River, their bioaccumulation in the muscle of fish, and the biological responses showed the impacts of human activities to this area and did not confirm a gradient of pollution between the five reservoirs, from the source toward Iguaçu River’s mouth. Therefore, diffuse source of pollutants present along the river course are increasing the risk of exposure to biota and human populations.
