Perfluorooctane sulfonate (PFOS), a member of the perfluoroalkyl substances (PFAS) family, is known for its widespread use in various industrial and consumer applications. It has raised concerns due to its persistence, bioaccumulation, and potential toxicity to organisms. When assessing the ecological impact of PFOS, it is important to consider its varying effects on different species. We conducted chronic bioassays using a water flea, Daphnia carinata and a midge, Chironomus tepperi. Apical endpoints, such as growth and reproduction success, were assessed. PFOS exhibited greater toxicity to midges compared to daphnids. This difference in sensitivity can be attributed to variations in their physiological and biochemical characteristics, including feeding and respiration modes. To delve deeper into PFOS toxicity, we employed a targeted GC-MS-based metabolomics approach, which revealed the concentration-dependent effect of PFOS on midge larvae. However, daphnia exposed to PFOS did not exhibit the same trend. The impaired amino acid metabolisms could lead to many other metabolic disturbances that affect health and survival of midge larvae. This study highlights the need for further metabolomics and omics research to unravel the underlying mechanisms of PFOS toxicity and inform the development of effective mitigation strategies to safeguard vulnerable ecosystems.