Per and polyfluoroalkyl substances (PFAS) are of concern to environmental regulators due to their widespread occurrence, persistence and reported toxicity. However, little data exist on the effects of PFAS at environmentally relevant concentrations. The development of molecular markers for PFAS exposure would therefore be useful to better understand the environmental risks of these compounds. In this study, we assessed if such markers could be developed using Gas Chromatography–Mass Spectrometry-based 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) for 7 days at five concentrations. A metabolic response was detected in all concentrations and treatments even though the survival rates only differed significantly at the highest exposure levels. The metabolic response differed between compounds but all three PFAS induced changes in the levels of amino acids, fatty acids, and cholesterol, in line with the literature. PFOS was found to bioaccumulate. Both GenX and PFHxS were eliminated from the amphipods, but PFHxS was eliminated at a slower rate than GenX. This information improves our understanding of the sublethal effects of PFAS as well as their environmental fate and behaviour.

Environmental pollutants such as heavy metals and fungicides pose a serious threat to waterways worldwide. Toxicological assessment of such contaminants is usually conducted using single compound exposures, as it is challenging to understand the effect of mixtures on biota using standard ecotoxicological methods; whereas complex chemical mixtures are more probable in ecosystems. This study exposed Simplisetia aequisetis (an estuarine annelid) to sublethal concentrations of a metal (zinc) and a fungicide (boscalid), both singly and as a mixture, for two weeks. Metabolomic analysis via gas and liquid chromatography-mass spectrometry was used to measure the stress response(s) of the organism following exposure. A total of 75 metabolites, including compounds contributing to the tricarboxylic acid cycle, the urea cycle, and a number of other pathways, were identified and quantified. The multiplatform approach identified distinct metabolomic responses to each compound that differed depending on whether the substance was presented singly or as a mixture, indicating a possible antagonistic effect. The study demonstrates that metabolomics is able to elucidate the effects and mode of action of contaminants and can identify possible outcomes faster than standard ecotoxicological endpoints, such as growth and reproduction. Metabolomics therefore has a possible future role in biomonitoring and ecosystem health assessments.

It is crucial to understand the effects caused by experimental parameters such as temperature, light, and food type on lab and field-based ecotoxicology experiments, as these variables, and combinations thereof, can affect results. The type of substrate used in exposure experiments, however, is generally assumed to have no effect. This may not always be correct. The metabolic changes in the freshwater crustacean, Austrochiltonia subtenuis exposed to copper, using three common substrates, gauze; toilet paper; and cellulose were investigated. Substrate alone did not affect survival, but each substrate elicited a different metabolic response and adult and juvenile amphipods had different substrate preferences. Several classes of metabolites were shown to change in response to different substrates and toxicant. These included disaccharides, monosaccharides, fatty acids, and tricarboxylic acid cycle intermediates. The results illustrate that metabolomic responses can differ in response to experimental factors that were previously thought not to be significant. In fact, our data indicate that substrate should be viewed as an experimental factor as important to control for as more well-known confounders such as temperature or food, thus challenging the current paradigm. Assuming substrate type has no effect on the experiment could potentially lead to errors in contaminant toxicity assessments. We propose that ideal good practise would be that all experimental factors should be evaluated for their potential influence on metabolomic profiles prior to contaminant response experiments being undertaken.