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Reactive oxygen species (ROS) in biological tissues of elected biosentinels represent an optimal biomarker for eco-monitoring of polluted areas. Electron spin resonance (ESR) is the most definitive method for detecting, quantifying and possibly identifying radicals in complex systems.A non-invasive method for monitoring polluted areas by the quantitative determination of ROS in frog skin biopsy is presented.We assessed by ESR spectroscopy the ROS level in adult male of Pelophylax bergeri, specie not a risk of extinction, collected from the polluted Sarno River (SA, Italy) basin. The spin-trap ESR method was validated by immunohistochemical analysis of the well-assessed pollution biomarkers cytochrome P450 aromatase 1A (CYP1A) and glutathione S-transferase (GST), and by determining the poly(ADPribose) polymerase (PARP) and GST enzymatic activity.ROS concentration in skin samples from frogs collected in the polluted area is significantly higher than that determined for the unpolluted reference area. Immunohistochemical analysis of CYP1A and GST supported the reliability of our approach, even in the absence of evident morphological and ultrastructural differences. PARP activity assay, connected to possible oxidative DNA damage, and the detoxification index by GST enzymatic assay give statistically significant evidence that higher levels of ROS are associated to alterations of the different biomarkers.ROS concentration, measured by ESR on isolated frog skin, through the presented non-lethal method, is a reliable biomarker for toxicity screening and represents a useful basic datum for future modelling studies on environmental monitoring and biodiversity loss prevention.ROS levels in frog skin biopsy were determined by spin-trapping ESR spectroscopy.Skin of frogs collected in polluted areas contains higher ROS concentration.ROS levels determined in skin biopsy follow the same trend of those from liver samples.Immunohistochemical analyses and enzymatic activity tests validate the method.ROS concentration determined by ESR in biosentinel biopsy is an effective biomarker.