In the context of increasing pollution pressure on aquatic ecosystems, it is essential to improve our knowledge of habitat quality and its suitability for organisms. It is particularly relevant to better integrate early life stages of fish into pollution biomonitoring programs, as they are reliable indicators of ecosystem integrity and because of their high sensitivity to pollutants. To avoid the influence of environmental parameters on their development, a lab-on-field approach, called the ex-situ exposure method, was developed. Aquatic organisms were exposed to a continuous flux of water under semi-controlled temperature, oxygen, and photoperiod conditions to avoid the influence of these confounding factors when interpreting the results. To investigate the potential role of water contamination, this active biomonitoring method was applied to the Garonne River (Southwest France), where migratory fish populations have declined. Two model species from different taxa were used: embryos of the Japanese medaka (Oryzias latipes) and adults of the crustacean Gammarus fossarum. The results showed a significant impact of water quality on embryo mortality and early hatching in two separate experiments on Japanese medaka. In addition, an induction of feeding rate was observed in exposed gammarids, but no impact on their embryo survival, suggesting differences in sensitivity between the two species selected. Chemical and biological analyses did not identify trace metals, pesticides, or microorganisms as potential sources of toxicity in medaka embryos or G. fossarum. These results raise concerns about the quality of the water in the Garonne River and its toxicity to aquatic organisms.