Does salinity mediate the toxicity of perfluorooctanesulfonic acid (PFOS) in an estuarine fish?

Perfluorooctanesulfonic acid (PFOS) is detected in estuarine environments, where salinity levels fluctuate regularly. We investigated the effects of salinity on the toxicity of PFOS in embryos and larvae of Cyprinodon variegatus. We crossed six PFOS treatments (0, 1-10,000 µg/L) with two salinities (10, 30 ppt). Larvae exposed to the highest concentration of PFOS under high salinity accumulated over twice the amount of PFOS compared to larvae maintained under low salinity. Embryonic survival was unaffected by PFOS, salinity, or their interaction. PFOS delayed time to hatch and increased salinity reduced time to hatch regardless of PFOS treatment; however, no salinity by PFOS interactions were observed. Conversely, PFOS and salinity interacted in the larval stage, with decreased survival at 30 ppt salinity. This is one of the first studies evaluating interactive effects of PFOS and high salinity and highlights the importance of assessing PFAS toxicity across life stages.

Toxicities of Legacy and Current-Use PFAS in an Anuran: Do Larval Exposures Influence Responses to a Terrestrial Pathogen Challenge?

Legacy polyfluoroalkyl substances (PFAS) [perfluorooctanesulfonate (PFOS) and perfluorooctanoic acid (PFOA)] are being replaced by various other fluorinated compounds, such as hexafluoropropylene oxide dimer acid (GenX). These alternatives are thought to be less bioaccumulative and, therefore, less toxic than legacy PFAS. Contaminant exposures occur concurrently with exposure to natural stressors, including the fungal pathogen Batrachocytrium dendrobatidis (Bd). Despite evidence that other pollutants can increase the adverse effects of Bd on anurans, no studies have examined the interactive effects of Bd and PFAS. This study tested the growth and developmental effects of PFOS, PFOA, and GenX on gray treefrog (Hyla versicolor) tadpoles, followed by a Bd challenge after metamorphosis. Despite PFAS exposure only occurring during the larval stage, carry-over effects on growth were observed post metamorphosis. Further, PFAS interacted with Bd exposure to influence growth; Bd-exposed animals had significantly shorter SVL [snout-vent length (mm)] with significantly increased body condition, among other time-dependent effects. Our data suggest that larval exposure to PFAS can continue to impact growth in the juvenile stage after exposure has ended. Contrary to predictions, GenX affected terrestrial performance more consistently than its legacy congener, PFOA. Given the role of Bd in amphibian declines, further investigation of interactions of PFAS with Bd and other environmentally relevant pathogens is warranted.

A Critical Review of Amphibian Per- and Polyfluoroalkyl Substance Ecotoxicity Research Studies: Identification of Screening Levels in Water and Other Useful Resources for Site-Specific Ecological Risk Assessments.

With the goal of aiding risk assessors conducting site-specific risk assessments at per- and polyfluoroalkyl substance (PFAS)-contaminated sites, this critical review synthesizes information on the ecotoxicity of PFAS to amphibians in 10 amphibian species and 16 peer-reviewed publications. The studies in this review consisted of spiked-PFAS chronic toxicity experiments with perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorohexane sulfonate (PFHxS), and 6:2 fluorotelomer sulfonate (6:2 FTS) that evaluated apical endpoints typical of ecological risk-based decision making (survival, growth, and development). Body mass was the most sensitive endpoint, showing clear and biologically meaningful population level adverse effect sizes (≥20% adverse effects). From these results, we recommend chronic no observed effect concentration (NOEC) screening levels of 590 µg/L for PFOS and 130 µg/L for PFOA. At or above recommended chronic lowest observed effect concentration screening levels of 1100 µg/L PFOS and 1400 µg/L PFOA, there is an increased chance of adverse biologically relevant chronic effects. Biologically relevant adverse effects were not observed for PFHxS and 6:2 FTS, so unbounded NOECs of 1300 µg/L PFHxS and 1800 µg/L 6:2 FTS are recommended. Screening levels are also provided for the concentration of PFAS in an amphibian diet, amphibian tissue, and moss substrate. In addition, we recommend bioconcentration factors that can be useful to predict concentrations of PFAS in amphibians using concentrations in water; these values are useful for food web modeling to understand risks to vertebrate wildlife that prey on amphibians. Overall, the present study provides a guide to the wealth of ecotoxicological research on PFAS conducted by our research group and highlights the need for additional work that would improve the understanding of chemical risks to amphibians. Environ Toxicol Chem 2023;42:2078-2090. © 2023 SETAC.

An Environmentally Relevant Mixture of Perfluorooctanesulfonic Acid and Perfluorohexanesulfonic Acid Does Not Conform to Additivity in Northern Leopard Frogs Exposed Through Metamorphosis.

Per- and polyfluoroalkyl substances (PFAS) are chemicals associated with adverse health effects. At aqueous film-forming foam sites, they occur as mixtures, with perfluorooctanesulfonic acid (PFOS) and perfluorohexanesulfonic acid (PFHxS) commonly co-occurring in the highest concentrations. Although PFOS and PFHxS toxicities have been studied, few studies have tested their potential interaction. Using Rana pipiens, the present study compared toxicities of a 1:1 PFOS:PFHxS mixture to PFOS and PFHxS individually with the prediction that responses would be additive. Gosner stage 25 (GS 25) tadpoles were exposed through metamorphosis (GS 46) to 0.5 and 1 ppb PFOS or PFHxS alone or to a mixture of 0.5 ppb PFOS and 0.5 ppb PFHxS. Tadpoles were weighed and measured (snout-vent length [SVL]) at day 31, metamorphic climax (GS 42), and GS 46. These values were used to calculate the scaled mass index (SMI), a measure of body condition. Body burdens were quantified on day 31 and at GS 46. The PFOS and PFHxS body burdens were elevated relative to controls at GS 46. No effects were observed on survival, SVL, or mass. Single PFAS effects included a 17% reduction in SMI at day 31 (0.5 ppb PFHxS) and a 1.1-day longer metamorphic period (1 ppb PFHxS) relative to controls. Mixture results deviated from additivity-SMIs were higher than expected on day 31 and lower than expected at GS 42. In addition, time to GS 42 in the PFAS mixture exceeded expected additivity by 12 days. Results from a chronic exposure to a 1:1 PFOS:PFHxS mixture resulted in changes in body condition and length of metamorphosis that deviated from additivity. More PFAS mixture toxicity studies conducted at relevant ratios and concentrations are needed. Environ Toxicol Chem 2022;41:3007-3016. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Oil induced cardiac effects in embryonic sheepshead minnows, Cyprinodon variegatus.

Following the Deepwater Horizon oil spill in April 2010, much research has been conducted on the cardiotoxic effects of oil on fish. Sensitive life history stages, such as the embryonic period, have been targeted to elucidate the effects of polycyclic aromatic hydrocarbons (PAHs) on the developing cardiovascular systems of fish. However, much of this research has focused on rapidly developing pelagic species, with little emphasis on estuarine species with longer embryological periods. Moreover, previous studies have used heart rate as the primary endpoint to measure cardiac performance in embryos and larvae; an endpoint that on its own may overlook impairment in cardiac performance. This study aims to fill these knowledge gaps and provide a more holistic approach for assessing the effects of PAHs on cardiac function by exposing sheepshead minnow (Cyprinodon variegatus) embryos to two oil doses (150 and 300 µg/L tPAH nominally) throughout embryonic development and measuring cardiac responses through the identification of cardiotoxic phenotypes (pericardial edema) as well as calculation of cardiac output at 4 days post fertilization. Results of this study show significant increases in pericardial edema at both oil doses relative to controls as well as significantly reduced cardiac output - driven by reductions in ventricular stroke volume. This study is one of the first to assess cardiac output in embryonic fish exposed to oil and methods described here allow for more physiologically relevant measures of cardiac performance in early life stages through established and non-invasive measures.

Haemodynamic dependence of mechano-genetic evolution of the cardiovascular system in Japanese medaka.

The progression of cardiac gene expression-wall shear stress (WSS) interplay is critical to identifying developmental defects during cardiovascular morphogenesis. However, mechano-genetics from the embryonic to larval stages are poorly understood in vertebrates. We quantified peak WSS in the heart and tail vessels of Japanese medaka from 3 days post fertilization (dpf) to 14 dpf using in vivo micro-particle image velocimetry flow measurements, and in parallel analysed the expression of five cardiac genes (fgf8, hoxb6b, bmp4, nkx2.5, smyd1). Here, we report that WSS in the atrioventricular canal (AVC), ventricular outflow tract (OFT), and the caudal vessels in medaka peak with inflection points at 6 dpf and 10-11 dpf instead of a monotonic trend. Retrograde flows are captured at the AVC and OFT of the medaka heart for the first time. In addition, all genes were upregulated at 3 dpf and 7 dpf, indicating a possible correlation between the two, with the cardiac gene upregulation preceding WSS increase in order to facilitate cardiac wall remodelling.

The influence of hypoxia on the cardiac transcriptomes of two estuarine species - C. variegatus and F. grandis.

Increased nutrient loading has led to eutrophication of coastal shelf waters which has resulted in increased prevalence of persistent hypoxic zones - areas in which the dissolved oxygen content of the water drops below 2 mg/L. The northern Gulf of Mexico, fed primarily by the Mississippi River watershed, undergoes annual establishment of one of the largest hypoxic zones in the world. Exposure to hypoxia can induce physiological impacts in fish cardiac systems that include bradycardia, changes in stroke volume, and altered cardiovascular vessel development. While these impacts have been addressed at the functional level, there is little information regarding the molecular basis for these changes. This study used transcriptomic analysis techniques to interrogate the effects of hypoxia exposure on the developing cardiovascular system in newly hatched larvae of two estuarine species that occupy the same ecological niche - the sheepshead minnow (Cyprinodon variegatus) and the Gulf killifish (Fundulus grandis). Results suggest that while differential gene expression is largely distinct between the two species, downstream impacts on pathways and functional responses such as reduced cardiac hypertrophy, modulation of blood pressure, and increased incidence of apoptosis appear to be conserved. Further, differences in the magnitude of these conserved responses may suggest that the length of embryonic development could impart a level of resiliency to hypoxic perturbation in early life stage fish.

Effects of polycyclic aromatic hydrocarbons and abiotic stressors on Fundulus grandis cardiac transcriptomics.

Following the 2010 Deepwater Horizon oil spill, extensive research has been conducted on the toxicity of oil and polycyclic aromatic hydrocarbons (PAHs) in the aquatic environment. Many studies have identified the toxicological effects of PAHs in estuarine and marine fishes, however, only recently has work begun to identify the combinatorial effect of PAHs and abiotic environmental factors such as hypoxia, salinity, and temperature. This study aims to characterize the combined effects of abiotic stressors and PAH exposure on the cardiac transcriptomes of developing Fundulus grandis larvae. In this study, F. grandis larvae were exposed to varying environmental conditions (dissolved oxygen (DO) 2, 6 ppm; temperature 20, 30 °C; and salinity 3, 30 ppt) as well as to a single concentration of high energy water accommodated fraction (HEWAF) (∑PAHs 15 ppb). Whole larvae were sampled for RNA and transcriptional changes were quantified using RNA-Seq followed by qPCR for a set of target genes. Analysis revealed that exposure to oil and abiotic stressors impacts signaling pathways associated with cardiovascular function. Specifically, combined exposures appear to reduce development of the systemic vasculature as well as strongly impact the cardiac musculature through cardiomyocyte proliferation resulting in inhibited cardiac function and modulated blood pressure maintenance. Results of this study provide a holistic view of impacts of PAHs and common environmental stressors on the cardiac system in early life stage estuarine species. To our knowledge, this study is one of the first to simultaneously manipulate oil exposure with abiotic factors (DO, salinity, temperature) and the first to analyze cardiac transcriptional responses under these co-exposures.

Effects of Deepwater Horizon crude oil on ocular development in two estuarine fish species, red drum (Sciaenops ocellatus) and sheepshead minnow (Cyprinodon variegatus).

Polycyclic aromatic hydrocarbons (PAHs) present in crude oil have been shown to cause the dysregulation of genes important in eye development and function, as well as morphological abnormalities of the eye. However, it is not currently understood how these changes in gene expression are manifested as deficits in visual function. Embryonic red drum (Sciaenops ocellatus) and sheepshead minnow (Cyprinodon variegatus) were exposed to water accommodated fractions (WAFs) of weathered crude oil and assessed for visual function using an optomotor response assay in early life-stage larvae, with subsequent samples taken for histological analysis of the eyes. Larvae of both species exposed to increasing concentrations of oil exhibited a reduced optomotor response. The mean diameters of retinal layers, which play an important role in visual function and image processing, were significantly reduced in oil-exposed sheepshead larvae, though not in red drum larvae. The present study provides evidence that weathered crude oil has a significant effect on visual function in early life-stage fishes.

Carbon dioxide induced plasticity of branchial acid-base pathways in an estuarine teleost.

Anthropogenic CO2 is expected to drive ocean pCO2 above 1,000 µatm by 2100 - inducing respiratory acidosis in fish that must be corrected through branchial ion transport. This study examined the time course and plasticity of branchial metabolic compensation in response to varying levels of CO2 in an estuarine fish, the red drum, which regularly encounters elevated CO2 and may therefore have intrinsic resilience. Under control conditions fish exhibited net base excretion; however, CO2 exposure resulted in a dose dependent increase in acid excretion during the initial 2 h. This returned to baseline levels during the second 2 h interval for exposures up to 5,000 µatm, but remained elevated for exposures above 15,000 µatm. Plasticity was assessed via gene expression in three CO2 treatments: environmentally realistic 1,000 and 6,000 µatm exposures, and a proof-of-principle 30,000 µatm exposure. Few differences were observed at 1,000 or 6,000 µatm; however, 30,000 µatm stimulated widespread up-regulation. Translocation of V-type ATPase after 1 h of exposure to 30,000 µatm was also assessed; however, no evidence of translocation was found. These results indicate that red drum can quickly compensate to environmentally relevant acid-base disturbances using baseline cellular machinery, yet are capable of plasticity in response to extreme acid-base challenges.