Investigating The Biochemical Effects of Exposure to Environmentally Relevant Concentrations of Per and Polyfluoroalkyl Substances (PFAS) In Aquatic Invertebrates
Creators: name: Sinclair, Georgia
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 340105
subject: Instrumental methods (excl. immunological and bioassay methods)
classificationCode: 310103
subject: Cell metabolism
classificationCode: 370104
subject: Atmospheric composition, chemistry and processes

description: Per- and polyfluoroalkyl substances (PFAS) encompass over seven million potential synthetic compounds that are currently listed on the PubChem database, following the updated definition of PFAS in 2021 by the Organisation for Economic Co-operation and Development (OECD) (Wang et al. 2021; Schymanski et al. 2023). The updated definition now describes PFAS as fluorinated substances that contain at least one fully fluorinated methyl or methylene carbon atom (without any H/Cl/Br/I atom attached to it, with a few noted exceptions, for example a carbon atom with a H/Cl/Br/I atom attached to it). PFAS is any chemical with at least a perfluorinated methyl group (−CF3−) or a perfluorinated methylene group (−CF2−) (Wang et al. 2021). There are thousands of these compounds that have been in use since the 1940s in a diverse range of mixtures and volumes for a wide variety of industrial/commercial applications. This includes their original use in Teflon® and Scotchgard™ as well as more modern uses in food packaging, cosmetics, waterproof and stain-proof textiles and carpets, aqueous film-forming foams (AFFF) and metal plating processes (Pelch et al. 2019). There are global concerns about the exposure to PFAS at high levels (mg/L), such as developmental toxicity (Lau et al. 2004; 2007); neurotoxicity (Mariussen 2012), carcinogenicity, cell membrane disruption, and genetic damage (Gong et al. 2019) that have all been linked to PFAS exposure in humans. This leads to concerns about their presence in the environment and organisms worldwide, however, their effects at environmentally relevant concentrations, in ng/L and µg/L on organisms remains poorly understood. The effect of low-level exposure is the knowledge gap I am endeavouring to understand in this thesis. 

The primary aim of this project is to address this significant gap in the published literature on the mechanisms of PFAS toxicity to aquatic organisms at a biochemical level and the specific concentration thresholds at which this occurs. Such insights are crucial for the effective management of PFAS in the environment. The objectives are to establish a robust scientific foundation to tackle this issue, employing the powerful analytical approach of metabolomics (the study of small biological metabolites). To begin understanding the extent of the knowledge gap, I first reviewed the existing ecotoxicology data on PFAS at environmentally relevant levels and conducted a Hazard Quotient Analysis, which is used to determine the risk of pollutants on ecosystems, using an environmental measurement and a concentration that causes an observed effect (Sardiña et al. 2019). The HQA showed a low-level risk at ng/L concentrations. I went on to test the analytical instruments, Nuclear Magnetic Resonance (NMR) and Gas Chromatography – Mass Spectrometry (GC-MS) to determine the best application for this project and develop my experimental design. Once the experimental design and analytical instrument had been determined, the effects of PFAS at environmentally relevant concentrations on aquatic organisms using metabolomics as a sublethal endpoint could be addressed.

The thesis conducted ecotoxicity experiments involving method development of metabolomic laboratory-based exposures, extraction, and analysis for Austrochiltonia subtenuis (a freshwater amphipod commonly found in Victorian waterways and wetlands). Before the amphipods were exposed to three PFAS compounds at environmentally relevant concentrations as well as concentrations above what you would usually find. While PFAS encompasses millions of compounds, the focus of my research zeroes in on three key compounds: perfluoro-octane sulfonic acid (PFOS), renowned as the most frequently detected PFAS compound globally; Hexafluoropropylene oxide dimer acid (GenX), a short chain replacement for PFOS with increased use and detection, and Perfluorohexanesulphonic acid (PFHxS), as this compound is of growing concern particularly in environments around Australia. Both shorter-chained PFAS, GenX and PFHxS, are relatively new and were thought to be safer than PFOA and PFOS as they had shorter chain lengths therefore were assumed to degrade faster than longer chained PFAS. However, concerns over the widespread detection of these shorter-chained compounds are increasing. In 2022, UN countries agreed to a global ban of PFHxS with no exceptions and it was added to Annex A of the Stockholm Convention on persistent organic pollutants (POPs) (Hogue 2022). GenX has recently been detected in ecosystems in Europe and North America, indicating that it may be just as persistent as other PFAS compounds (Xiao 2017; Hassell et al. 2019). Subsequently, samples were extracted and analysed using Gas Chromatography - Mass Spectrometry (GC-MS) and metabolites were identified from a polar metabolite reference library. Metabolic effects were observed at lower concentrations of PFAS than were seen with more traditional ecotoxicological endpoints such as survival, growth, or reproduction, indicating that our understanding of the effects of these compounds is not complete. Metabolomic pathways detected to be affected by PFAS included fatty acid, arginine, proline, glycolipid, galactose, and aspartate metabolism, which have been reported in other organisms to respond to PFAS exposure. A key finding was metabolomic responses were detected without accumulation of a toxicant, rather than the common perception that increasing accumulation can lead to a level of toxicity where it overwhelms the mechanisms the organism has in place.

Moving beyond controlled laboratory conditions, I exposed laboratory-cultured amphipods and chironomids (Chironomus tepperi) to water collected from a wetland with known concentrations of PFAS in Melbourne, Victoria, Australia. Amphipod metabolite responses were compared between PFAS standards (from the lab exposures) and PFAS from real-world environmental samples. Field-collected invertebrates (Chironomus species) from the PFAS-contaminated site were analysed using GC-MS, allowing for insightful comparisons in uptake and metabolite response between laboratory-cultured chironomids and resident organisms collected from their natural environment. These studies illustrate the challenges in understanding the biochemical effects of PFAS exposure within ecosystems, especially at concentrations assumed to not be of concern. The findings from these studies confirm previous studies that reported similar metabolomic pathway alterations in response to PFAS in other organisms to both environmental samples and controlled laboratory exposures to PFAS. A recommendation is to shift towards investigations involving 'real' environmentally relevant exposures of PFAS, rather than the effects of individual compounds in isolation at high exposure levels in a laboratory culture. This approach will hold the potential to unveil metabolomic markers indicative of PFAS exposure. 

The outcomes of this research have contributed to the understanding of how PFAS impact the biochemistry of aquatic organisms, particularly at environmentally relevant concentrations. In terms of the current guideline levels, the results show that levels deemed reasonable in an ecosystem can cause metabolomic alterations and PFAS accumulation to occur despite other endpoints not indicating an impact. These insights are instrumental in improving the effectiveness of environmental regulations governing these compounds, thereby advancing environmental protection and management.