Exposure to PFAS contaminated urban wetland water causes similar metabolic alterations to laboratory-based exposures in the freshwater amphipod Austrochiltonia subtenuis.

Assessing the harm caused by pollutants in urban ecosystems remains a significant challenge. Traditional ecotoxicological endpoints are often not sensitive enough to detect the effects of toxicants at environmentally relevant concentrations (≤ng/L). A potential solution is using molecular biology methods to look at small biochemical changes caused by exposure to ng/L concentrations of contaminants. This has been tested in the lab but not conclusively demonstrated in the field. We exposed the freshwater amphipod (Austrochiltonia subtenuis) to water from an urban wetland containing known concentrations of per-and polyfluoroalkyl substances (as well as very low concentrations of pesticides) for 14 days and analyzed their metabolite profiles. Mannose, Myo-inositol, and Isopropyl propionate were found to change in PFAS exposed amphipods, a similar response to that previously observed in laboratory exposures to the same PFAS, but not pesticides. The results give a better understanding of PFAS toxicity at environmentally relevant concentrations and conditions.

Changes in metabolic profiles of amphipods Allorchestes compressa after acute exposures to copper, pyrene, and their mixtures.

Amphipods are ideal indicators for biomonitoring and ecotoxicological studies of environmental contaminants because they are extensively distributed in aquatic environments, are easy to collect and are important in nutrient cycling. Marine amphipods (Allorchestes compressa) were exposed to two concentrations of copper and pyrene, and their mixtures, for 24 and 48 h. Changes in polar metabolites were assessed using Gas Chromatography Mass Spectrometry (GC-MS)-based untargeted metabolomics. Generally, limited metabolite changes were observed for copper and pyrene single exposures (eight and two significant metabolites, respectively), while 28 metabolites had changed following exposures to mixtures. Furthermore, changes were mainly observed after 24 h but had seemingly returned to control levels after 48 h. Multiple types of metabolites were affected including amino acids, Tricarboxylic acid (TCA) cycle intermediates, sugars, fatty acids, and hormones. This study highlights the sensitivity of metabolomics in assessing the impacts of low concentrations of chemicals compared to traditional ecotoxicological endpoints.

Exploring the potential application of alternative nuclei in NMR based metabolomics.

INTRODUCTION: Nuclear magnetic resonance (NMR) is widely used in metabolomics but it focusses on 1H over other NMR-active nuclei. OBJECTIVES: To evaluate the potential of alternative NMR-sensitive nuclei to generate useful metabolomic data. METHOD: Proton, carbon, phosphorus, and nitrogen-based NMR metabolomics was undertaken on extracts from mint and European honey bee tissue. RESULTS: Carbon NMR provided useful information but required larger sample sizes. Phosphorus produced overlapping peaks in one dimensional (1D) analysis but showed potential in 2D experiments. 15N NMR was found to not be sensitive enough for general metabolomic work. CONCLUSIONS: Alternative NMR active nuclei are useful for metabolomics.

Is substrate choice an overlooked variable in ecotoxicology experiments?

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.

Exposure to Environmentally Relevant Levels of PFAS Causes Metabolic Changes in the Freshwater Amphipod Austrochiltonia subtenuis.

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.

A review of omics-based PFAS exposure studies reveals common biochemical response pathways.

Per and Polyfluoroalkyl Substances (PFAS) are a diverse group of man-made chemicals with a range of industrial applications and which are widespread in the environment. They are structurally diverse but comprise a common chemical feature of at least one (though usually more) perfluorocarbon moiety (-CnF2n-) attached to a functional group such as a carboxylic or sulphonic acid. The strength of the Carbon-Fluorine bond means the compounds do not break down easily and can thus bioaccumulate. PFAS are of high concern to regulators and the public due to their potential toxicity and high persistence. At high exposure levels, PFAS have been implicated in a range of harmful effects on human and environmental health, particularly problems in/with development, cholesterol and endocrine disruption, immune system function, and oncogenesis. However, most environmental toxicology studies use far higher levels of PFAS than are generally found in the environment. Additionally, since the type of exposure, the PFAS used, and the organisms tested all vary between studies, so do the results. Traditional ecotoxicology studies may thus not identify PFAS effects at environmentally relevant exposures. Here we conduct a review of omics-based PFAS exposure studies using laboratory ecotoxicological methodologies and environmentally relevant exposure levels and show that common biochemical response pathways are identified in multiple studies. A major pathway identified was the pentose phosphate shunt pathway. Such molecular markers of sublethal PFAS exposure will greatly benefit accurate and effective risk assessments to ensure that new PFAS regulations can consider the full effects of PFAS exposure on environmental and human health receptors.

What are the effects of PFAS exposure at environmentally relevant concentrations?

The group of synthetic chemicals known as 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, little data exists on the effects of exposure to PFAS at environmentally relevant concentrations and this hampers the effective management of these compounds. This paper reviews current research on the occurrence and ecotoxicology of PFAS at environmentally relevant doses to assess their potential biological impacts. Hazard Quotient (HQ) analysis was undertaken as part of this assessment. Most PFAS detected in the environment were found to have a HQ risk value of <1 meaning their reported concentrations are below their predicted no effect concentration. This indicates many reported toxic effects of PFAS are, theoretically, unlikely to occur outside the laboratory. However, lack of information on new PFAS as well as their precursors and degradation products, coupled with lack of knowledge of their mixture toxicity means our understanding of the risks of PFAS is incomplete, especially in regard to sub-lethal and/or chronic effects. It is proposed that the development of molecular markers for PFAS exposure are needed to aid in the development of environmental PFAS regulations that are effective in fully protecting the environment.

Metabolite Changes in an Estuarine Annelid Following Sublethal Exposure to a Mixture of Zinc and Boscalid.

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.

Using metabolomics to assess the sub-lethal effects of zinc and boscalid on an estuarine polychaete worm over time.

INTRODUCTION: Zinc is a heavy metal commonly detected in urban estuaries around Australia. Boscalid is a fungicide found in estuaries, both in water and sediment, it enters the system predominantly through agricultural run-off. Zinc is persistent while boscalid breaks down, with a half-life of 108 days. Both contaminants are widely distributed and their effects on ecosystems are not well understood. OBJECTIVES: The aim of this study was to determine the metabolite changes in Simplisetia aequisetis (an estuarine polychaete) following laboratory exposure to a sub-lethal concentration of zinc or boscalid over a 2-week period. METHODS: Individuals were collected at six time points over a 2-week period. Whole polychaete metabolites were extracted and quantified using a multi-platform approach. Polar metabolites were detected using a semi-targeted GC-MS analysis and amine containing compounds were analysed using a targeted LC-MS analysis. Total lipid energy content was also analysed for Simplisetia aequisetis. RESULTS: The pathways that responded to zinc and boscalid exposure were alanine, aspartate and glutamate metabolism (AAG); glycine, serine and threonine metabolism (GST) and metabolites associated with the tricarboxylic acid cycle (TCA). Results showed that changes in total abundance of some metabolites could be detected as early as 24-h exposure. Changes were detected in the metabolites before commonly used total lipid energy assays identified effects. CONCLUSION: A multi-platform approach provided a holistic overview of the metabolomic response to contaminants in polychaetes. This approach shows promise to be used in biomonitoring programs to provide early diagnostic indicators of contamination and exposure.

Bifenthrin Causes Toxicity in Urban Stormwater Wetlands: Field and Laboratory Assessment Using Austrochiltonia (Amphipoda).

Stormwater wetlands are engineered to accumulate sediment and pollutants from stormwater and provide environmental value to urban environments. Therefore, contaminated sediment risks causing toxicity to aquatic fauna. This research identifies contaminants of concern in urban wetland sediments by assessing sediment toxicity using the amphipod Austrochiltonia subtenuis. Sediments from 98 wetlands were analyzed for contaminants, and laboratory bioassays were performed with A. subtenuis. Wild Austrochiltonia spp. were also collected from wetlands to assess field populations. Random forest modeling was used to identify the most important variables predicting survival, growth, and field absence of Austrochiltonia spp. Bifenthrin was the most frequently detected pesticide and also the most important predictor of Austrochiltonia spp. responses. Copper, permethrin, chromium, triclosan, and lead were also important. The median lethal effect concentration (LC50) of bifenthrin to laboratory-based A. subtenuis (1.09 (±0.08) µg/gOC) exposed to wetland sediments was supported by a bifenthrin-spiked sediment experiment, indicating A. subtenuis is a suitable test species. Furthermore, Austrochiltonia spp. were absent from all sites that exceeded the calculated bifenthrin LC50, demonstrating the impact of this contaminant on wild populations. This research demonstrates the sensitivity of Austrochiltonia spp. to urban sediment contamination and identifies bifenthrin as a contaminant of concern in urban wetlands.