Per- and polyfluoralkyl substances (PFAS) accumulate from the environment in freshwater fish. A plethora of individual PFAS compounds occur in the environment, but little is known about the accumulation of these into the freshwater fish food chain. In this study, the accumulation of these compounds in fish is studied from a low trophic level via fish to otters. The accumulation will be linked to the molecular characteristics of every compound. Moreover, it will be explored if analytical approaches can be developed for determination of PFAS as a group (total-PFAS), to allow a comprehensive understanding of all PFAS that accumulate in the food chain. Lastly, in-vitro effect assays will be applied to relate potential effects to the in the organisms to the bioaccumulating PFAS. This work will allow a comprehensive understanding of the hazards and potential risks of PFAS in freshwater fish.