The lack of knowledge about the sensitivity of the endangered freshwater pearl mussel (FWPM) Margaritifera margaritifera to environmental pollution and the rapid decline of its populations in Europe, have led to the need of developing non-destructive experimental protocols in order to assess the impact of such pollution. This species has a complex life cycle and the early life stages are considered the most sensitive. This study deals with the development of a methodology for the assessment of juvenile mussels' locomotor behavior using an automated video tracking system. Different parameters were determined such as the duration of the video recording and light exposure as a stimulus during the experiment. Locomotion behavior pattern of juveniles was assessed in control condition and also following exposure to sodium chloride as a positive control in order to validate the experimental protocol developed in this study. Results showed that juveniles locomotion behavior was stimulated under light exposure. Moreover, exposure to sublethal concentrations of sodium chloride (0.8 and 1.2 g/L) for 24 h was found to decrease juveniles' locomotion by almost three-times, thus validating our experimental methodology. This study allowed to provide a new tool for the assessment of stress condition impacts on the juveniles of the endangered FWPM, highlighting the interest of such non-destructive biomarker of health for protected species. Consequently, this will help in the improvement of our knowledge on M. margaritifera sensitivity to environmental pollution.Read less <