We investigated the suitability of an active biomonitoring approach, using the ecologically relevant species Gammarus fossarum, to assess trends of bioavailable contamination in continental waters. Gammarids were translocated into cages at 27 sites, in the Rhône-Alpes region (France) during early autumn 2009. Study sites were chosen to represent different physico-chemical characteristics and various anthropic pressures. Biotic factors such as sex, weight and food availability were controlled in order to provide robust and comparable results. After one week of exposure, concentrations of 11 metals/metalloids (Cd, Pb, Hg, Ni, Zn, Cr, Co, Cu, As, Se and Ag) and 38 hydrophobic organic substances including polycyclic aromatic hydrocarbons (PAHs), polychlorobiphenyles (PCBs), pentabromodiphenylethers (PBDEs) and organochlorine pesticides, were measured in gammarids. All metals except Ag, and 33 organic substances among 38 were quantified in G. fossarum, showing that this species is relevant for chemical biomonitoring. The control of biotic factors allowed a robust and direct inter-site comparison of the bioavailable contamination levels. Overall, our results show the interest and robustness of the proposed methodological approach for assessing trends of bioavailable contamination, notably for metals and hydrophobic organic contaminants, in continental waters. Furthermore, we built threshold values of bioavailable contamination in gammarids, above which measured concentrations are expected to reveal a bioavailable contamination at the sampling site. Two ways to define such values were investigated, a statistical approach and a model fit. Threshold values were determined for almost all the substances investigated in this study and similar values were generally derived from the two approaches. Then, levels of contaminants measured in G. fossarum at the 27 study were compared to the threshold values obtained using the model fit. These threshold values could serve as a basis for further implementation of quality grids to rank sites according to the extent of the bioavailable contamination, with regard to the applied methodology.