Poly and per-fluoroalkyl substances (PFAS) are currently of high concern to environmental regulators and the public due to their widespread occurrence, resistance to degradation and reported toxicity. However, there is a lack of research concerning the ecological effects of PFAS at environmentally relevant exposure levels. Environmental toxicological studies can be greatly enhanced when coupled with biochemical methods such as metabolomics, which can indicate early responses to low concentrations of contaminants. Determining the dose where PFAS begins to interrupt the biochemistry of organisms will improve our overall understanding of the mode of action, effects, and toxicity of PFAS in ecosystems. In this study we assessed if such markers could be developed using metabolomics. We exposed the freshwater amphipod, Austrochiltonia subtenuis to a range of environmentally relevant concentrations of Perfluoro-octane sulfonic acid (PFOS), Hexafluoropropylene oxide dimer acid (GenX) and Perfluorohexanesulphonic acid (PFHxS). Here, we investigated the biochemical response in the amphipods over seven days and three separate treatments, using Gas Chromatography-Mass Spectrometry. A metabolic response was detected following PFAS exposure in all concentrations and treatments even though survival rates between treatments and controls did not differ significantly except at the highest exposure levels. Interestingly only PFOS was found to bioaccumulate. PFHxS and GenX were quickly eliminated from the amphipods but still induced metabolic changes. All three PFAS was found to induce disruptions to metabolites contributing to fatty acid metabolism and the TCA cycle. This study has improved our understanding of the of the sub-lethal effects of PFAS and their environmental fate and behavior. This may lead to better regulation of these compounds in future.