Partitioning of PFAS to serum, tissues, eggs, and hatchlings of an Australian freshwater turtle.

Turtles are a potential sentinel species of aquatic ecosystem health as they inhabit aquatic ecosystems, are long lived, and potentially have high exposure to anthropogenic chemicals via food and water. This study investigated per- and polyfluoroalkyl substances (PFAS) tissue partitioning in female Emydura macquarii macquarii turtle, and the maternal offloading of (PFAS) into eggs and then hatchlings as well as the accumulation of PFAS in male and female Emydura macquarii macquarii serum. Significantly higher levels of perfluorosulfonic acids (PFSAs) and perfluorocarboxylic acids (PFCAs) were measured in the male serum compared to the female turtle serum, whereas perfluoroalkane sulfonamides (FASAs) were significantly higher in the female turtle serum. Perfluorooctane sulfonate (PFOS) was the predominant PFAS in the turtles whereas PFHxA was the predominant PFAS found in the surrounding water. PFHxA was not reported in any turtle tissue or the serum. The short-chain PFSAs and FASAs appeared to be highly associated with blood; long-chain PFSAs and PFCAs were more likely to be associated with tissue. Half of the PFHxS and all the long-chain PFSAs and PFCAs reported in the yolks were transferred into the hatchlings (by mass), suggesting a potential intergenerational effect.

Metabolic disruptions and impaired reproductive fitness in wild-caught freshwater turtles (Emydura macquarii macquarii) exposed to elevated per- and polyfluoroalkyl substances (PFAS).

Per- and poly-fluoroalkyl substances (PFAS) pose a threat to organisms and ecosystems due to their persistent nature. Ecotoxicology endpoints used in regulatory guidelines may not reflect multiple, low-level but persistent stressors. This study examines the biological effects of PFAS on Eastern short-necked turtles in Queensland, Australia. In this study, blood samples were collected and analysed for PFAS, hormone levels, and functional omics endpoints. High levels of PFAS were found in turtles at the impacted site, with PFOS being the dominant constituent. The PFAS profiles of males and females differed, with males having higher PFAS concentrations. Hormone concentrations differed between impacted and reference sites in male turtles, with elevated testosterone and corticosterone indicative of stress. Further, energy utilisation, nucleotide synthesis, nitrogen metabolism, and amino acid synthesis were altered in both male and female turtles from PFAS-impacted sites. Both sexes show similar metabolic responses to environmental stressors from the PFAS-contaminated site, which may adversely affect their reproductive fitness. Purine metabolism, caffeine metabolism, and ferroptosis pathway changes in turtles can cause gout, cell death, and overall health problems. Further, the study showed that prolonged exposure to elevated PFAS levels in the wild could compromise turtle reproductive fitness by disrupting reproductive steroids and metabolic pathways.

Perturbation of the gut microbiome in wild-caught freshwater turtles (Emydura macquarii macquarii) exposed to elevated PFAS levels.

Per- and polyfluoroalkyl substances (PFAS) are environmentally persistent and pervasive. Understanding the toxicity of PFAS to wildlife is difficult, both due to the complexity of biotic and abiotic perturbations in the taxa under study and the practical and ethical problems associated with studying the impacts of environmental pollutants on free living wildlife. One avenue of inquiry into the effects of environmental pollutants, such as PFAS, is assessing the impact on the host gut microbiome. Here we show the microbial composition and biochemical functional outputs from the gut microbiome of sampled faeces from euthanised and necropsied wild-caught freshwater turtles (Emydura macquarii macquarii) exposed to elevated PFAS levels. The microbial community composition was profiled by 16S rRNA gene sequencing using a Nanopore MinION and the biochemical functional outputs of the gut microbiome were profiled using a combination of targeted central carbon metabolism metabolomics using liquid chromatography coupled to a triple quadrupole mass spectrometer (LC-QqQ-MS) and untargeted metabolomics using liquid chromatography coupled to a quadrupole time of flight mass spectrometer (LC-QToF-MS). Total PFAS was measured in the turtle serum using standard methods. These preliminary data demonstrated a 60-fold PFAS increase in impacted turtles compared to the sampled aquatic environment. The microbiome community was also impacted in the PFAS exposed turtles, with the ratio of Firmicutes-to-Bacteroidetes rising from 1.4 at the reference site to 5.5 at the PFAS impacted site. This ratio increase is indicative of host stress and dysfunction of the gut microbiome that was correlated with the biochemical metabolic function data, metabolites observed that are indications of stress and inflammation in the gut microbiome. Utilising the gut microbiome of sampled faeces collected from freshwater turtles provides a non-destructive avenue for investigating the impacts of PFAS in native wildlife, and provides an avenue to explore other contaminants in higher-order taxa within the environment.

Bioaccumulation and impact of maternal PFAS offloading on egg biochemistry from wild-caught freshwater turtles (Emydura macquarii macquarii).

Per- and polyfluoroalkyl substances (PFAS) are persistent synthetic contaminants that are pervasive in the environment. Toxicity resulting from elevated PFAS concentrations in wildlife has been studied, yet evidence of their accumulation, developmental toxicity and maternal offloading in egg-laying species is limited. Here we show the maternal offloading of PFAS in freshwater short-necked turtles (Emydura macquarii macquarii) exposed to elevated PFAS and the resulting biological impact on oviducal eggs. Total PFAS concentrations were determined in serum from adult females and harvested oviducal eggs collected from euthanised turtles exposed to low and high levels of PFAS and compared against turtle serum and eggs collected from a suitable reference site. Multi-omics assays were utilised to explore the biochemical impact of elevated PFAS on egg albumen, yolk and eggshell using a range of metabolomics, lipidomics, and proteomics techniques. Eggshells were also screened for metals by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Analysis of the serum collected from adult female turtles and their oviducal eggs demonstrated PFAS offloading and transference that is 1.6 and 5.3 times higher in the low and high PFAS impacted eggs, respectively, compared to maternal serum concentrations. Oviducal egg yolk comprised >90% of the bioaccumulated PFAS load. Multi-omic analysis of the dissected egg fractions illustrated PFAS impacted eggs are significantly elevated in purine metabolism metabolites, which are tied to potential biological dysfunctional processes. The yolks were significantly depleted in lipids and lipid quality tied to growth and development. The high PFAS impacted oviducal eggshells were lower in calcium, important developmental and immune response proteins, and higher in glycerophosphoethanolamines (PE) lipids and histidine metabolism metabolites that are tied to a weakened physical structure. Further investigation is needed to establish the rate of PFAS offloading and quantify the developmental impact on hatchling and hatchling success to fully demonstrate PFAS-developmental toxicity linkages.

Host-Gut Microbiome Metabolic Interactions in PFAS-Impacted Freshwater Turtles (Emydura macquarii macquarii).

Per-and polyfluoroalkyl substances (PFAS) are a growing concern for humans, wildlife, and more broadly, ecosystem health. Previously, we characterised the microbial and biochemical impact of elevated PFAS on the gut microbiome of freshwater turtles (Emydura macquarii macquarii) within a contaminated catchment in Queensland, Australia. However, the understanding of PFAS impacts on this species and other aquatic organisms is still very limited, especially at the host-gut microbiome molecular interaction level. To this end, the present study aimed to apply these leading-edge omics technologies within an integrated framework that provides biological insight into the host turtle-turtle gut microbiome interactions of PFAS-impacted wild-caught freshwater turtles. For this purpose, faecal samples from PFAS-impacted turtles (n = 5) and suitable PFAS-free reference turtles (n = 5) were collected and analysed. Data from 16S rRNA gene amplicon sequencing and metabolomic profiling of the turtle faeces were integrated using MetOrigin to assign host, microbiome, and co-metabolism activities. Significant variation in microbial composition was observed between the two turtle groups. The PFAS-impacted turtles showed a higher relative abundance of Firmicutes and a lower relative abundance of Bacteroidota than the reference turtles. The faecal metabolome showed several metabolites and pathways significantly affected by PFAS exposure. Turtles exposed to PFAS displayed altered amino acid and butanoate metabolisms, as well as altered purine and pyrimidine metabolism. It is predicted from this study that PFAS-impacted both the metabolism of the host turtle and its gut microbiota which in turn has the potential to influence the host's physiology and health.