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.

Microbiota of Four Tissue Types in American Alligators (Alligator mississippiensis) Following Extended Dietary Selenomethionine Exposure.

Selenium represents an essential trace nutrient that is necessary for biological functions. Deficiencies can induce disease, but excess can induce toxicity. Selenium deficiency is a major concern in underdeveloped countries, while also posing as a toxic pollutant in waterways surrounding landfills, agricultural areas, and fossil fuel production sites. We examined the microbiome of selenomethionine (SeMet) fed American alligators (Alligator mississippiensis) at the beginning and end of a 7-week exposure experiment. Alligators were randomly divided into three groups: control and 1000 or 2000 ppm SeMet. DNA from before exposure (oral and cloaca swabs) and post-exposure (oral, cloaca, small & large intestines) sampling were extracted and amplified for bacterial 16 s rRNA. While treatment did not seem to have much effect, we observed a predominance of Fusobacteriaceae and Porpyromonodaceae across all tissue types. Cetobacterium and Clostridium are the most abundant genera as potential indicators of the aquatic and carrion feeding lifestyle of alligators.

Examining the Effects of Chronic Selenium Exposure on Traditionally Used Stress Parameters in Juvenile American Alligators (Alligator mississippiensis).

Environmental contaminants, such as the trace element selenium (Se), are a continuing concern to species worldwide due to their potential pathophysiological effects, including their influence on the stress response mediated through glucocorticoids (GCs; stress hormones). Environmental concentrations of Se are increasing due to anthropogenic activities, including the incomplete combustion of coal and subsequent disposal of coal combustion wastes. However, most studies examining how Se affects GCs have been focused on lower trophic organisms. The objectives of this study were to investigate the effects of long-term Se exposure on traditionally used stress parameters and to identify which of these parameters best indicate Se accumulation in liver and kidney of the American alligator (Alligator mississippiensis), a top trophic carnivore found in the southeastern United States and known to inhabit Se-containing areas. Alligators were divided into three dietary treatments and fed prey spiked with 1000 or 2000 ppm of selenomethionine (SeMet) or deionized water (control treatment) for 7 weeks. Following the 7-week treatment protocol, blood and tissue samples were obtained to measure plasma corticosterone (CORT; the main crocodilian GC), tail scute CORT, the ratio of peripheral blood heterophils (H) to lymphocytes (L) as H/L ratio, and body condition. To evaluate which parameter best indicated Se accumulation in the liver and kidney, principal component and discriminant analyses were performed. The only parameter significantly correlated with liver and kidney Se concentrations was scute CORT. Our results suggest that measurement of CORT in tail scutes compared with plasma CORT, H/L ratios, and body condition is the best indicator of Se-exposure and accumulation in crocodilians.

Corticosterone in American alligator (Alligator mississippiensis) tail scutes: Evaluating the feasibility of using unconventional samples for investigating environmental stressors.

Baseline plasma corticosterone (CORT) concentrations have been widely used to investigate the effects of stressors in wild and captive crocodilians. However, collecting baseline plasma CORT samples from wild crocodilians may be particularly difficult due to the capture and handling protocols used for large individuals. Thus, it may prove beneficial to use recently modified techniques for extracting CORT deposited in keratinized and non-keratinized tissues to better quantify the effects of long-term stress in crocodilians. In this study, we investigated the feasibility of using American alligator (Alligator mississippiensis) tail scute tissues to quantify CORT by collecting blood and tail scutes from 40 alligators before and after a short-term handling stressor. The objective of the current study was to better understand CORT deposition in crocodilian scutes and whether short-term increases in CORT could be detected. We found that CORT can be reliably extracted from alligator scute tissue and quantified using a commercially available enzyme immunoassay. However, there was a significant increase in scute CORT concentrations following an alligator being exposed to a short-term stressor (p = 0.017), although the magnitude of change was less than observed in plasma samples from the same individuals (p = 0.002). Furthermore, our results indicate that there was a significant effect of body condition on an alligator's post-stressor CORT concentration (p = 0.02). While our study is among the first to experimentally examine the usefulness of tissue CORT in crocodilians, a combination of field and laboratory experiments are needed to better understand deposition rates of CORT in scute tissues and to further validate the usefulness of tissue glucocorticoids for evaluating the effects of stress.

Dietary Selenomethionine Administration and Its Effects on the American Alligator (Alligator mississippiensis): Oxidative Status and Corticosterone Levels.

Selenium (Se) is an essential nutrient which in excess causes toxicity. The disposal of incompletely combusted coal, which often is rich in Se, into aquatic settling basins is increasing the risk of Se exposure worldwide. However, very few studies have looked at the physiological effects of Se exposure on long-lived, top trophic vertebrates, such as the American alligator (Alligator mississippiensis). During a 7-week period, alligators were fed one of three dietary treatments: mice injected with deionized water or mice injected with water containing 1000 or 2000 ppm selenomethionine (SeMet). One week after the last feeding alligators were bled within 3 min of capture for plasma corticosterone (CORT). A few days later, all alligators were euthanized and whole blood and tail tissue were harvested to measure oxidative damage, an antioxidant-associated transcription factor, and antioxidant enzymes [glutathione peroxidase-1 (GPX1), superoxide dismutase-1 (SOD1), and SOD2] by Western blotting. There was a dose-dependent increase in baseline CORT levels in alligators administered SeMet. Except for blood SOD2 levels, SeMet treatment had no effect (p > 0.05 for all) on oxidative status: oxidative damage, GPX1, SOD1, and muscle SOD2 levels were similar among treatments. Our results illustrate that high levels of Se may act as a stressor to crocodilians. Future studies should investigate further the physiological effects of Se accumulation in long-lived, top-trophic vertebrates.

Effects of selenium exposure on the hematology, innate immunity, and metabolic rate of yellow-bellied sliders (Trachemys scripta scripta).

Selenium (Se) is a naturally occurring essential element that can be toxic to vertebrates at high concentrations. Despite studies that have documented that wild reptile species can accumulate copious amounts of Se, little is known regarding specific toxicologic effects of Se. In this study, 70 juvenile yellow-bellied sliders (Trachemys scripta scripta) were exposed to one of three seleno-L-methionine (SetMet) treatments (control, n = 24; 15 mg/kg, n = 23; and 30 mg/kg, n = 23) via weekly oral gavage for 5 weeks. At the conclusion of the experiment, kidney, liver, muscle, and blood samples were collected for quantitative Se analysis. Turtles in the SeMet treatment groups accumulated significantly higher amounts of Se in all tissue types relative to controls (all p < 0.001). Turtles in the 30 mg/kg SeMet group also accumulated significantly higher amounts of Se compared to the 15 mg/kg group (all p < 0.001). Although toxicity thresholds for reptiles have not been established, Se concentrations in liver tissue from both SeMet treatment groups exceeded reported avian toxicity thresholds for liver tissue. Neither oxygen consumption nor innate bactericidal capacity were impacted by SeMet exposure. However, turtles in the 30 mg/kg SeMet group exhibited anemia, which has been reported in other vertebrates exposed to Se. Furthermore, juvenile T. s. scripta in the 30 mg/kg SeMet group experienced 17% mortality compared to 0% in the 15 mg/kg treatment and control groups. To our knowledge, this study is the first to report dose-dependent Se-associated anemia and mortality in a chelonian species.

Dietary Selenomethionine Administration in the American Alligator (Alligator mississippiensis): Hepatic and Renal Se Accumulation and Its Effects on Growth and Body Condition.

Selenium (Se) is an essential trace nutrient, but in excess, it can induce toxicity. Incomplete combustion of coal produces coal combustion wastes, which are enriched in Se and often disposed of in aquatic basins. While a multitude of studies have investigated Se accumulation in vertebrates, few studies have examined its effects on longer-lived top trophic carnivores, such as the American alligator (Alligator mississippiensis). In this study, alligators were fed one of three Dietary Treatments: mice injected with water (controls) or water supplemented with 1000 or 2000 ppm selenomethionine (SeMet). Dietary Treatment significantly affected Se levels in both the liver (p < 0.0001; raw mean ± SE: 1000 ppm group, 35.20 ± 6.32 ppm; 2000 ppm group, 49.97 ± 4.00 ppm) and kidney (p < 0.0001; raw mean ± SE: 1000 ppm group, 101.60 ± 8.64 ppm; 2000 ppm, 96.38 ± 5.81 ppm), which were significantly higher in alligators fed SeMet than in controls. Post-treatment head length, used to control for size variation, was negatively related to both kidney (p = 0.0142) and liver (p = 0.0010) Se concentrations. Dietary treatment with SeMet significantly reduced body condition (1000 ppm, p < 0.0029; 2000 ppm, p = 0.0075), but it significantly increased growth (1000 ppm, p < 0.0001; 2000 ppm, p = 0.0316). Body condition and growth remained unchanged in control alligators (p > 0.05). Our results demonstrate alligators are capable of accumulating high levels of Se through trophic transfer. The positive effects of accumulation on growth may demonstrate Se essentiality, whereas the negative effects on condition may demonstrate toxicity. Accumulation also was associated with mortality, further demonstrating toxicity. Future studies should further investigate the physiological effects of Se accumulation in long-lived, top-trophic carnivores.

Chronic Ingestion of Coal Fly-Ash Contaminated Prey and Its Effects on Health and Immune Parameters in Juvenile American Alligators (Alligator mississippiensis).

Coal-burning power plants supply approximately 37 % of the electricity in the United States. However, incomplete combustion produces ash wastes enriched with toxic trace elements that have historically been disposed of in aquatic basins. Organisms inhabiting such habitats may accumulate these trace elements; however, studies investigating the effects on biota have been primarily restricted to shorter-lived, lower-trophic organisms. The American alligator (Alligator mississippiensis), a long-lived, top-trophic carnivore, has been observed inhabiting these basins, yet the health or immune effects of chronic exposure and possible accumulation remains unknown. In this study, we investigated how chronic dietary ingestion of prey contaminated with coal combustion wastes (CCWs) for 25 months, and subsequent accumulation of trace elements present in CCWs, affected juvenile alligator immune function and health. Alligators were assigned to one of four dietary-treatment groups including controls and those fed prey contaminated with CCWs for one, two, or three times a week. However, no effect of Dietary Treatment (p > 0.05) was observed on any immune parameter or hematological or plasma analyte we tested. Our results suggest that neither exposure to nor accumulation of low doses of CCWs had a negative effect on certain aspects of the immune and hematological system. However, future studies are required to elucidate this further.

First case of ranavirus and associated morbidity and mortality in an eastern mud turtle Kinosternon subrubrum in South Carolina.

Ranaviruses are double-stranded DNA viruses that infect amphibians, fish, and reptiles, causing global epidemics in some amphibian populations. It is important to identify new species that may be susceptible to the disease, particularly if they reside in the same habitat as other at-risk species. On the Savannah River Site (SRS) in Aiken, South Carolina, USA, ranaviruses are present in several amphibian populations, but information is lacking on the presence, prevalence, and morbidity of the virus in reptile species. An eastern mud turtle Kinosternon subrubrum captured on the SRS in April 2014 exhibited clinical signs of a ranaviral infection, including oral plaque and conjunctivitis. Quantitative PCR analyses of DNA from liver tissue, ocular, oral, nasal, and cloacal swabs were all positive for ranavirus, and sequencing of the template confirmed infection with a FV3-like ranavirus. Histopathologic examination of postmortem tissue samples revealed ulceration of the oral and tracheal mucosa, intracytoplasmic epithelial inclusions in the oral mucosa and tongue sections, individualized and clusters of melanomacrophages in the liver, and bacterial rods located in the liver, kidney, heart, stomach, and small intestine. This is the first report of morbidity and mortality of a mud turtle with a systemic ranaviral infection.

Influence of collection time on hematologic and immune markers in the American alligator (Alligator mississippiensis).

Crocodilians are important keystone species and indicators of environmental health. Much remains unknown, however regarding utility of field-collected crocodilian blood samples for ecologic assessments. Field sampling sites are also often distant to analysis centers, necessitating development of new techniques and panels of assays that will yield environmentally relevant data. Stability and viability of hematological and immunological indices have been of particular interest for linking ecosystem health to biomarkers in resident species. In this study, we investigated the effect of time at analysis post-blood sampling at 4 and 24 hr on a panel of potential biomarkers in alligator blood. Our results suggest alligator blood samples can be reliably evaluated for both hematologic and immunologic profile 24 hr after sampling.

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.

Coal combustion residues and their effects on trace element accumulation and health indices of eastern mud turtles (Kinosternon subrubrum).

Coal combustion is a major energy source in the US. The solid waste product of coal combustion, coal combustion residue (CCR), contains potentially toxic trace elements. Before 1980, the US primarily disposed of CCR in aquatic settling basins. Animals use these basins as habitat and can be exposed to CCR, potentially affecting their physiology. To investigate the effects of CCR on eastern mud turtles (Kinosternon subrubrum), we sampled 30 turtles exposed to CCRs and 17 unexposed turtles captured in 2015-2016 from the Savannah River Site (Aiken, SC, USA). For captured turtles, we (1) quantified accumulation of CCR in claw and blood samples, (2) used bacterial killing assays to assess influences of CCR on immune responses, (3) compared hemogregarine parasite loads, and (4) compared metabolic rates via flow-through respirometry between CCR-exposed and unexposed turtles when increased temperature was introduced as an added stressor. Turtles exposed to CCR accumulated CCR-associated trace elements, corroborating previous studies. Blood Se and Sr levels and claw As, Se, and Sr levels were significantly higher in turtles from contaminated sites. Average bacterial killing efficiency was not significantly different between groups. Neither prevalence nor average parasite load significantly differed between CCR-exposed and reference turtles, although parasite load increased with turtle size. Regardless of site, temperature had a significant impact on turtle metabolic rates; as temperature increased, turtle metabolic rates increased. The effect of temperature on turtle metabolic rates was less pronounced for CCR-exposed turtles, which resulted in CCR-exposed turtles having lower metabolic rates than reference turtles at 30 and 35 °C. Our results demonstrate that turtles accumulate CCR from their environment and that accumulation of CCR is associated with changes in turtle physiological functions when additional stressors are present.

Accumulation of coal combustion residues and their immunological effects in the yellow-bellied slider (Trachemys scripta scripta).

Anthropogenic activities such as industrial processes often produce copious amounts of contaminants that have the potential to negatively impact growth, survival, and reproduction of exposed wildlife. Coal combustion residues (CCRs) represent a major source of pollutants globally, resulting in the release of potentially harmful trace elements such as arsenic (As), cadmium (Cd), and selenium (Se) into the environment. In the United States, CCRs are typically stored in aquatic settling basins that may become attractive nuisances to wildlife. Trace element contaminants, such as CCRs, may pose a threat to biota yet little is known about their sublethal effects on reptiles. To assess the effects of CCR exposure in turtles, we sampled 81 yellow-bellied sliders (Trachemys scripta scripta) in 2014-2015 from CCR-contaminated and uncontaminated reference wetlands located on the Savannah River Site (Aiken, SC, USA). Specific aims were to (1) compare the accumulation of trace elements in T. s. scripta claw and blood samples between reference and CCR-contaminated site types, (2) evaluate potential immunological effects of CCRs via bacterial killing assays and phytohaemagglutinin (PHA) assays, and (3) quantify differences in hemogregarine parasite loads between site types. Claw As, Cd, copper (Cu), and Se (all p ≤ 0.001) and blood As, Cu, Se, and strontium (Sr; p ≤ 0.015) were significantly elevated in turtles from CCR-contaminated wetlands compared to turtles from reference wetlands. Turtles from reference wetlands exhibited lower bacterial killing (p = 0.015) abilities than individuals from contaminated sites but neither PHA responses (p = 0.566) nor parasite loads (p = 0.980) differed by site type. Despite relatively high CCR body burdens, sliders did not exhibit apparent impairment of immunological response or parasite load. In addition, the high correlation between claw and blood concentrations within individuals suggests that nonlethal tissue sampling may be useful for monitoring CCR exposure in turtles.

Evaluating the effect of sample type on American alligator (Alligator mississippiensis) analyte values in a point-of-care blood analyser.

The assessment of wildlife health has been enhanced by the ability of point-of-care (POC) blood analysers to provide biochemical analyses of non-domesticated animals in the field. However, environmental limitations (e.g. temperature, atmospheric humidity and rain) and lack of reference values may inhibit researchers from using such a device with certain wildlife species. Evaluating the use of alternative sample types, such as plasma, in a POC device may afford researchers the opportunity to delay sample analysis and the ability to use banked samples. In this study, we examined fresh whole blood, fresh plasma and frozen plasma (sample type) pH, partial pressure of carbon dioxide (PCO2), bicarbonate (HCO3 (-)), total carbon dioxide (TCO2), base excess (BE), partial pressure of oxygen (PO2), oxygen saturation (sO2) and lactate concentrations in 23 juvenile American alligators (Alligator mississippiensis) using an i-STAT CG4+ cartridge. Our results indicate that sample type had no effect on lactate concentration values (F 2,65 = 0.37, P = 0.963), suggesting that the i-STAT analyser can be used reliably to quantify lactate concentrations in fresh and frozen plasma samples. In contrast, the other seven blood parameters measured by the CG4+ cartridge were significantly affected by sample type. Lastly, we were able to collect blood samples from all alligators within 2 min of capture to establish preliminary reference ranges for juvenile alligators based on values obtained using fresh whole blood.

Hepatic and renal trace element concentrations in American alligators (Alligator mississippiensis) following chronic dietary exposure to coal fly ash contaminated prey.

Little is known about the propensity of crocodilians to bioaccumulate trace elements as a result of chronic dietary exposure. We exposed 36 juvenile alligators (Alligator mississippiensis) to one of four dietary treatments that varied in the relative frequency of meals containing prey from coal combustion waste (CCW)-contaminated habitats vs. prey from uncontaminated sites, and evaluated tissue residues and growth rates after 12 mo and 25 mo of exposure. Hepatic and renal concentrations of arsenic (As), cadmium (Cd) and selenium (Se) varied significantly among dietary treatment groups in a dose-dependent manner and were higher in kidneys than in livers. Exposure period did not affect Se or As levels but Cd levels were significantly higher after 25 mo than 12 mo of exposure. Kidney As and Se levels were negatively correlated with body size but neither growth rates nor body condition varied significantly among dietary treatment groups. Our study is among the first to experimentally examine bioaccumulation of trace element contaminants in crocodilians as a result of chronic dietary exposure. A combination of field surveys and laboratory experiments will be required to understand the effects of different exposure scenarios on tissue residues, and ultimately link these concentrations with effects on individual health.

Blood and Plasma Biochemistry Reference Intervals for Wild Juvenile American Alligators ( Alligator mississippiensis ).

: American alligators ( Alligator mississippiensis ) are one of the most studied crocodilian species in the world, yet blood and plasma biochemistry information is limited for juvenile alligators in their northern range, where individuals may be exposed to extreme abiotic and biotic stressors. We collected blood samples over a 2-yr period from 37 juvenile alligators in May, June, and July to establish reference intervals for 22 blood and plasma analytes. We observed no effect of either sex or blood collection time on any analyte investigated. However, our results indicate a significant correlation between a calculated body condition index and aspartate aminotransferase and creatine kinase. Glucose, total protein, and potassium varied significantly between sampling sessions. In addition, glucose and potassium were highly correlated between the two point-of-care devices used, although they were significantly lower with the i-STAT 1 CG8+ cartridge than with the Vetscan VS2 Avian/Reptile Rotor. The reference intervals presented herein should provide baseline data for evaluating wild juvenile alligators in the northern portion of their range.