Copper (Cu) isa major contaminant of freshwater ecosystems, however, the sublethal effects of metals on early life stages of fish and the changes to their biomolecular makeup are not well understood. This study assessed the changes in amino acid composition and tissue composition of purple spotted gudgeons (PSG) in response to exposure to environmentally realistic Cu concentrations representing the EC10 and EC50 (ECx: effect concentration that affected X% of test organisms) values of 12 (9 - 15) µg Cu L-1 and 22 (19 - 24) µg Cu L-1, respectively. Copper exposure caused a decrease in total amino acid content and changed the AA profile of PSG compared to the controls. Proton-induced X-ray emission (PIXE) mapping techniques showed accumulation of Cu in the retinal tissues within the eyes of the sacfry, and the redistribution of other elements such as zinc. Synchrotron Fourier-transform infrared (FTIR) microspectroscopy of control and Cu treated eye tissues revealed a change in protein secondary structure in retinal tissues in response to Cu accumulation, as well as decreased levels of retinal, consistent with the degradation of rhodopsin, a key protein in the visual sensory system. This is the first study to demonstrate the multi-level responses of PSG arising from exposure to environmentally realistic Cu concentrations and suggests that AA profiling can serve as a useful tool to assess the impacts of metals on freshwater organisms.