The enormous investments in nanotechnology have led to an exponential increase of new manufactured nano-enabled materials whose impact in the aquatic systems is still largely unknown. Ecotoxicity and nanosafety studies mostly resulted in contradictory results and generally failed to clearly identify biological patterns that could be related specifically to nanotoxicity. Generation of reactive oxygen species (ROS) is one of the most discussed nanotoxicity mechanism in literature. ROS can induce oxidative stress (OS), resulting in cyto- and genotoxicity. The ROS overproduction can trigger the induction of anti-oxidant enzymes such as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidases (GPx), which are used as biomarkers of response. A critical overview of the biochemical responses induced by the presence of NPs on freshwater organisms is performed with a strong interest on indicators of ROS and general stress. A special focus will be given to the NPs transformations, including aggregation, and dissolution, in the exposure media and the produced biochemical endpoints.