It is well know that some aquatic environments are contaminated by heavy metals from diverse kind of wastes. At present there are not many biomarkers of metal pollution and many of the biotic indices which are in use are mostly based on structural changes occurring at community level which are expected to integrate biotic responses over relatively long periods of time (from weeks to months depending on the life time of the organisms investigated). Thus, it is of great interest to develop new metal toxicity bioindicators focusing on both early responses and chronic effects in order to complete the information provided by the available ones. This will improve our understanding of the causes of ecosystem damage as required by the water framework directive (WFD, 2000/60/EC). In rivers and streams, the biofilm (also known as phytobenthos or periphyton) has been widely used as bioindicator of pollution due its capacity to detect early effects produced by toxic substances, as are heavy metals, providing an ecotoxicology approach at community level with high ecological relevance. Metals interfere with the metabolism of organisms at different stages. The pulse amplitude modulation (PAM) fluorometry has been widely used to assess the direct and indirect functional effects of toxicants on photosynthetic organisms. Antioxidant enzyme activities (AEA) have been studied as well, giving to the main conclusions that AEA can be used as early warning systems because they have a functional response detectable before than structural changes (i.e. algal biomass or species composition). Moreover, these enzyme activities might also be biomarkers of adaptation, since their activation is expected to contribute to metal detoxification under chronic exposure. The aim of this study was to investigate metal toxicity on fluvial biofilms using functional (PAM), metabolic (AEA) as well as structural (diatom species composition) endpoints. To reach this goal, a biofilm translocation experiment was performed in the Osor River, a tributary of the Ter river located in a former mining area. This river presents high levels of dissolved zinc (Zn) and iron (Fe) reaching up to 600 µg Zn/L and 750 µg Fe/L after the entrance of a mining source. Biofilms were translocated from non-polluted to polluted sites in order to evaluate the different responses obtained over a metal concentration gradient at different temporal scales (from hours to several weeks of exposure). In spite of the high temporal variability observed, we identified several functional and metabolic parameters related with the metal gradient. Metal concentrations found in Osor River affected the fluvial biofilm causing transitory physiological responses (short exposure) and structural and functional alterations (chronic exposure). Moreover, at the end of the experiment (after 5 weeks of exposure), dominant diatom species were also related with the metal gradient, showing more teratological forms and a smaller biovolume at the metalpolluted sites. Our results suggest that PAM and AEA of fluvial biofilms can be used as early-warning tools as well as biomarkers of adaptation in metal polluted rivers complementing the information provided by diatom studies (including taxonomical and morphological attributes).