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.

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.

Adaptive Management to Reduce Nest Inundation of a Critically Endangered Freshwater Turtle: Confirming the Win-win.

Inundation of Australian freshwater turtle nests has been identified as a threat to recruitment and long-term viability of species such as the critically endangered white-throated snapping turtle (Elseya albagula). Water level fluctuations within water storage infrastructure can inundate significant proportions of E. albagula nests in any year. Using an ecological risk assessment framework, operating rules for a water storage in the Burnett River (South East Queensland, Australia) were implemented to support nesting of E. albagula. Turtles were encouraged to nest at higher elevations on riverbanks by maintaining higher water levels in the impoundment during the nesting season, followed by lowering of water levels during the incubation period to minimise rates of nest inundation from riverine inflows. To verify the success of the new rules, a three-year confirmation monitoring program of nest heights and water levels was undertaken. Results of confirmation monitoring showed that 3% (2018), 11% (2019) and 0% (2020) of E. albagula nests were inundated under the new operating rules, compared to previously estimated nest inundation rates of >20% in ~24% of years of a 118-year simulation period (1890-2008) under previous storage operating rules. Emergency releases from an upstream storage in 2019 and 2020 for dam safety did not affect the success of the rule, demonstrating its resilience to natural and artificial flow regimes. This study demonstrates the importance of confirmation monitoring in verifying the efficacy of targeted changes to water management, and highlights potential application across other water storage infrastructure with threatened freshwater turtle populations requiring adaptive management.

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.

Bioaccumulation and metabolic response of PFAS mixtures in wild-caught freshwater turtles (Emydura macquariimacquarii) using omics-based ecosurveillance techniques.

PFAS mixtures in the environment are common and identifying PFAS constituents, bioaccumulation, and biological impacts of mixtures remains a challenge. Here, an omics-based ecosurveillance approach was taken to investigate the impacts of PFAS pollution in freshwater turtles (Emydura macquariimacquarii). Four turtles were collected from an impacted waterway downstream from an industrial source of PFAS contamination in Queensland, Australia and analysed for 49 different PFAS. One turtle was collected from a suitable control site. PFAS concentrations were quantified in turtle serum using an established targeted methodology. The serum PFAS concentration was ten-fold greater at the impacted site (Σ49 PFAS 1933 ± 481 ng/mL) relative to the control sample (Σ49 PFAS 140 ng/mL). Perfluorooctane sulfonate (PFOS; 889 ± 56 ng/mL) was 235 times higher in turtle serum than in the water that they were collected from (ΣPFAS 32.0 µg/L). Perfluorobutane sulfonamide (FBSA; 403 ± 83 ng/mL) and perfluorohexane sulfonamide (FHxSA; 550 ± 330 ng/mL) were also reported at substantial concentrations in the serum of impacted turtles. Biochemical profiles were analysed using a mixture of liquid chromatography triple quadrupole (QqQ) and quadrupole time-of-flight (QToF) mass spectrometry methodologies. These profiles demonstrated a positive correlation in the impacted turtles exposed to elevated PFAS with an enhanced purine metabolism, glycerophosphocholines and an innate immune response, which suggest an inflammation response, metabolic preservation and re-routing of central carbon metabolites. Conversely, lipid transport and binding activity were negatively correlated. Using these preliminary data, we were able to demonstrate the negative metabolic impact from PFAS mixtures on turtle metabolic health. With further research on a larger turtle cohort, omics-based data will contribute towards linking adverse outcome pathways for turtle populations exposed to PFAS mixtures. Moreover, expanding the use of ecosurveillance tools will inform mechanistic toxicological data for risk assessment and regulatory applications.

Biochemical and hematological reference intervals for Krefft's turtles Emydura macquarii krefftii from the Burnett River Catchment, Australia.

Biochemical and hematological reference intervals have not previously been reported for Emydura macquarii krefftii. In 2009, 56 E. m. krefftii were captured by hand from the Burnett Catchment, clinically assessed to determine health status and blood sampled. Reference intervals were calculated from the 35 clinically healthy turtles using techniques established in other chelonid species. Aberrant blood results were identified from the 21 clinically unhealthy turtles. Low numbers of observed cases of creatine kinase, glucose, magnesium, phosphorus and uric acid outside of the blood biochemistry reference interval were recorded, as were high numbers of observed cases of estimated eosinophils, thrombocytes and total leukocyte counts outside of the hematological reference interval. Lesions of the shell and plastron (shell rot) were observed in 38% (21/56) of the examined healthy and unhealthy turtles. Microbiological assessment of a subsample (n=7) of these lesions grew Aeromonas veronii 100% (7/7), Aeromonas hydrophila 29% (2/7) and Acinetobacter baumannii 14% (1/7). Of the examined turtles, 13% (7/56) had evidence of opacity of the lens or anterior chamber of the eye and 70% (39/56) had erythema of the neck, axillary and inguinal soft tissues. Not all observed cases of erythema were associated with clinical ill-health. The anomalous blood results and clinical findings identified in this study suggest disease processes which may have resulted from causative agents in the surrounding environment.