Site-specific spatiotemporal occurrence and molecular congener distributions of naphthenic acids in Athabasca oil sands wetlands of Alberta, Canada.

The Athabasca oil sands region (AOSR) of Alberta, Canada is notable for its considerable unconventional petroleum extraction projects, where bitumen is extracted from naturally-occurring oil sands ore. The large scale of these heavy crude oil developments raises concerns because of their potential to distribute and/or otherwise influence the occurrence, behaviour, and fate of environmental contaminants. Naphthenic acids (NAs) are one such contaminant class of concern in the AOSR, so studies have examined the occurrence and molecular profiles of NAs in the region. We catalogued the spatiotemporal occurrence and characteristics of NAs in boreal wetlands in the AOSR over a 7-year period, using derivatized liquid chromatography-tandem mass spectrometry (LC-MS/MS). Comparing median concentrations of NAs across these wetlands revealed a pattern of NAs suggesting NAs in surface waters derived from oil sands deposits. Opportunistic wetlands that formed adjacent to reclaimed overburden and other reclamation activities had the highest concentrations of NAs and consistent patterns suggestive of bitumen-derived inputs. However, similar patterns in the occurrence of NAs were also observed in undeveloped natural wetlands located above the known surface-mineable oil sands deposit that underlies the region. Intra-annual sampling results along with inter-annual comparisons across wetlands demonstrated that differences in the spatial and temporal NA concentrations were dependent on local factors, particularly when naturally occurring oil sands ores were observed in the wetland or drainage catchment.

Effects of Herbicides and the Chytrid Fungus Batrachochytrium dendrobatidis on the growth, development and survival of Larval American Toads (Anaxyrus americanus).

Pesticides and pathogens adversely affect amphibian health, but their interactive effects are not well known. We assessed independent and combined effects of two agricultural herbicides and the fungal pathogen Batrachochytrium dendrobatidis (Bd) on the growth, development and survival of larval American toads (Anaxyrus americanus). Wild-caught tadpoles were exposed to four concentrations of atrazine (0.18, 1.8, 18.0, 180 µg/L) or glyphosate (7, 70, 700, 7000 µg a.e./L), respectively contained in Aatrex® Liquid 480 (Syngenta) or Vision® Silviculture Herbicide (Monsanto) for 14 days, followed by two doses of Bd. At day 14, atrazine had not affected survival, but it non-monotonically affected growth. Exposure to the highest concentration of glyphosate caused 100% mortality within 4 days, while lower doses had an increasing monotonic effect on growth. At day 65, tadpole survival was unaffected by atrazine and the lower doses of glyphosate. Neither herbicide demonstrated an interaction effect with Bd on survival, but exposure to Bd increased survival among both herbicide-exposed and herbicide-control tadpoles. At day 60, tadpoles exposed to the highest concentration of atrazine remained smaller than controls, indicating longer-term effects of atrazine on growth, but effects of glyphosate on growth disappeared. Growth was unaffected by any herbicide-fungal interaction but was positively affected by exposure to Bd following exposure to atrazine. Atrazine exhibited a slowing and non-monotonic effect on Gosner developmental stage, while exposure to Bd tended to speed up development and act antagonistically toward the observed effect of atrazine. Overall, atrazine, glyphosate and Bd all showed a potential to modulate larval toad growth and development.

Transformation of bitumen-derived naphthenic acid fraction compounds across surface waters of wetlands in the Athabasca Oil Sands region.

Bitumen is extracted from oil sands in the Athabasca Oil Sands region (AOSR) of Alberta, Canada. Much of the bitumen-derived toxicity in mine waste is attributable to naphthenic acid fraction compounds (NAFCs). Mines in the AOSR are required to be returned to a natural state after closure; thus, cost-effective strategies are needed to reduce toxicity from NAFCs. Previous studies have demonstrated the capability of constructed wetlands to attenuate NAFCs. However, the capacity of wetlands in the natural environment to degrade and transform NAFCs to less toxic components is poorly understood. To better understand the spatial distribution and fate of NAFCs in natural wetlands, samples were collected across the surfaces of two mature opportunistic wetlands near active oil sands mines. The first wetland has a well-defined surface flow pathway and inflows affected by overburden containing lean bitumen ore. The second wetland, in contrast, is a stagnant water body with raw bitumen visible along its edges. For the wetland with a well defined flow path, NAFCs decreased in concentration down gradient, while oxidized NAFCs constituted a greater proportion of NAFCs with increase in flow path. Likewise there was a decrease in the molecular weights of NAFCs, similar to trends observed in constructed wetland treatment systems. In comparison, NAFCs were more uniformly distributed across the relatively stagnant wetland. Overall, these data provide new evidence that mature opportunistic wetlands in the AOSR can promote the degradation and oxidation of bitumen-derived naphthenic acids into less toxic compounds.

Characterization of naphthenic acid fraction compounds in water from Athabasca oil sands wetlands by Orbitrap high-resolution mass spectrometry.

Classical naphthenic acids (NAs) are known to be primary aquatic toxicants of concern in the Athabasca oil sands region (AOSR), and are a component of naphthenic acid fraction compounds (NAFCs). Recent studies conducted in the AOSR have examined metals and polycyclic aromatic hydrocarbons in regional wetlands. However, few studies have described NAs and/or NAFCs in AOSR wetlands. To address this gap, we examined NAFC profiles in the water of different wetlands in the AOSR, including naturalized borrow pits (i.e., abandoned pits created by excavation of road-building materials), and opportunistically-formed wetlands associated with reclamation activities. For comparison, NAFC profiles from these wetlands were compared to an opportunistic wetland formed from tailings pond dyke seepage. Samples were prepared using solid-phase extraction and analyzed using negative-ion high-resolution Orbitrap mass spectrometry. Principal component analyses (PCA) revealed patterns to the NAFC profiles in the wetlands. The first distinct grouping of wetlands included water bodies associated with reclamation activities that are located on and/or adjacent to mining overburden. One other wetland, HATS5w, separated from all other wetlands during PCA, and had a unique NAFC profile; detailed examination of NAFCs revealed HATS5w contained the heaviest (i.e., high m/z components) and most unsaturated NAFCs among study locations, demonstrating the usefulness of high-resolution mass spectrometry for characterizing individual wetlands. The NAFCs of HATS5w are also substantially different from bitumen-derived inputs in overburden-adjacent opportunistic wetlands. Collectively, the NAFC profiles presented provide new information on background levels of polar bitumen-derived organics in AOSR wetlands.

The Use of Geographic Information Systems for Spatial Ecological Risk Assessments: An Example from the Athabasca Oil Sands Area in Canada.

There is an acknowledged need in ecotoxicology for methods that integrate spatial analyses in risk assessment. This has resulted in the emergence of landscape ecotoxicology, a subdiscipline of ecotoxicology. However, landscape ecotoxicology has yet to become common practice in risk assessment due to the underdevelopment of techniques and a lack of standardized methods. In the present study, we demonstrate how geographic information systems (GISs) can serve as a standardized platform to integrate data, assess spatial patterns of ecotoxicological data for multiple species, and assess relationships between chemical mixture exposures and effects on biota for landscape ecotoxicological risks assessment. We use data collected under the Joint Oil Sands Monitoring Program in the Athabasca Oil Sands Region in Alberta, Canada. This dataset is composed of concentrations of contaminants including metals and polycyclic aromatic compounds, and health endpoints measured in 1100 biological samples, including tree swallows, amphibians, gull and tern eggs, plants, and mammals. We present 3 examples using a GIS as a platform and geospatial analysis to: 1) integrate data and assess spatial patterns of contaminant exposure in the region, 2) assess spatial patterns of exposures to complex mixtures, and 3) examine patterns of exposures and responses across the landscape. We summarize the methods used in the present study into a workflow for ease of use. The GIS methods allow researchers to identify hot spots of contamination, use georeferenced monitoring data to derive quantitative exposure-response relationships, and assess complex exposures with more realism. Environ Toxicol Chem 2019;38:2797-2810. © 2019 SETAC.

Extracts of Passive Samplers Deployed in Variably Contaminated Wetlands in the Athabasca Oil Sands Region Elicit Biochemical and Transcriptomic Effects in Avian Hepatocytes.

Recent contaminant monitoring in boreal wetlands situated in Alberta's Athabasca oil sands region revealed increased concentrations of polycyclic aromatic compounds (PACs) in passive sampling devices deployed in wetlands close to bitumen surface mining operations. In this study, graded concentrations of semipermeable membrane device (SPMD) extracts, collected from 4 wetlands with variable burdens of PACs, were administered to chicken and double-crested cormorant (DCCO) embryonic hepatocytes to determine effects on 7-ethoxyresorufin-O-deethylase (EROD) activity and mRNA expression. Concentrations and composition of PACs detected in SPMDs varied among sites, and the proportion of alkyl PACs was greater than parent compounds at all sites. ΣPACs was the highest in SPMDs deployed within 10 km of mining activity (near-site wetland [5930 ng SPMD-1]) compared to those ∼50 km south (far-site wetland [689 ng SPMD-1]). Measures of EROD activity and Cyp1a4 mRNA expression allowed the ranking of wetland sites based on aryl hydrocarbon receptor-mediated end points; EROD activity and Cyp1a4 mRNA induction were the highest at the near-site wetland. ToxChip PCR arrays (one chicken and one DCCO) provided a more exhaustive transcriptomic evaluation across multiple toxicological pathways following exposure to the SPMD extracts. Study sites with the greatest PAC concentrations had the most genes altered on the chicken ToxChip (12-15/43 genes). Exposure of avian hepatocytes to SPMD extracts from variably contaminated wetlands highlighted traditional PAC-related toxicity pathways as well as other novel mechanisms of action. A novel combination of passive sampling techniques and high-throughput toxicity evaluation techniques shows promise in terms of identifying hotspots of chemical concern in the natural environment.

Naphthenic Acid Mixtures and Acid-Extractable Organics from Oil Sands Process-Affected Water Impair Embryonic Development of Silurana (Xenopus) tropicalis.

Naphthenic acids (NAs) are carboxylic acids naturally occurring in crude oils and bitumen and are suspected to be the primary toxic substances in wastewaters associated with oil refineries and mining of oil sands. Oil sands process-affected water (OSPW) generated by the extraction of bitumen from oil sands are a major source of NAs and are currently stored in tailings ponds. We report on the acute lethality and teratogenic effects of aquatic exposure of Silurana (Xenopus) tropicalis embryos to commercial NA extracts and from the acid extractable organics (AEOs) fraction of a Canadian OSPW. Using electrospray ionization-high resolution mass spectrometry, we determined that the O2 species proportion were 98.8, 98.9 and 58.6% for commercial mixtures Sigma 1 (S1M) and Sigma 2 (S2M), and AEOs, respectively. The 96h LC50 estimates were 10.4, 11.7, and 52.3 mg/L for S1M, S2M, and the AEOs, respectively. The 96h EC50 estimates based on frequencies of developmental abnormalities were 2.1, 2.6, and 14.2 mg/L for S1M, S2M, and the AEOs, respectively. The main effects observed were reduced body size, edema, and cranial, heart, gut and ocular abnormalities. Increasing concentrations of the mixtures resulted in increased severity and frequency of abnormalities ( p < 0.05). The rank-order potency was S1M > S2M > AEO based on LC50 and EC50 estimates. These data provide insight into the effects NAs in amphibian embryos and can contribute to the development of environmental guidelines for the management of OSPW.

Using wood frog (Lithobates sylvaticus) tadpoles and semipermeable membrane devices to monitor polycyclic aromatic compounds in boreal wetlands in the oil sands region of northern Alberta, Canada.

Several recent studies have reported evidence that surface mining operations of bitumen in northern Alberta's oil sands (OS) region contribute significantly to the atmospheric deposition of metals and polycyclic aromatic compounds (PACs) within the vicinity of OS development. The present study examines the accumulation of PACs in boreal wetlands at varying distance from OS industrial activities with the use of semipermeable membrane devices (SPMDs) and wood frog (Lithobates sylvaticus) tadpoles. SPMDs were deployed in shallow lentic waterbodies adjacent to wood frog egg masses and were retrieved, along with tadpoles, approximately 35-45 days later. The highest concentrations of PACs were detected in SPMDs deployed within a 25 km radius of surface mining activity, consistent with snow deposition studies of PACs in the region. In wetlands located within the vicinity of surface mining activity, PAC profiles of SPMDs and wood frog tadpoles were dominated by C1-C4 alkylated PACs, including alkylated dibenzothiophenes, which are strongly indicative of petrogenic sources. Contrary to differences seen in the SPMD PAC concentrations, there were no obvious differences in the ∑PACs in wood frog tissue between wetland study sites, although alkylated fluorenes were found to be higher in tadpoles collected from a wetland located within 10 km of two bitumen upgrading facilities. The use of SPMDs in tandem with wood frog tadpoles can help assess the potential exposure of aquatic organisms to PACs in boreal wetlands.

Sublethal effects on wood frogs chronically exposed to environmentally relevant concentrations of two neonicotinoid insecticides.

Neonicotinoids are prophylactically used globally on a variety of crops, and there is concern for the potential impacts of neonicotinoids on aquatic ecosystems. The intensive use of pesticides on crops has been identified as a contributor to population declines of amphibians, but currently little is known regarding the sublethal effects of chronic neonicotinoid exposure on amphibians. The objective of the present study was to characterize the sublethal effect(s) of exposure to 3 environmentally relevant concentrations (1 µg/L, 10 µg/L, and 100 µg/L) of 2 neonicotinoids on larval wood frogs (Lithobates sylvaticus) using outdoor mesocosms. We exposed tadpoles to solutions of 2 commercial formulations containing imidacloprid and thiamethoxam, and assessed survival, growth, and development. Exposure to imidacloprid at 10 µg/L and 100 µg/L increased survival and delayed completion of metamorphosis compared with controls. Exposure to thiamethoxam did not influence amphibian responses. There was no significant effect of any treatment on body mass or size of the metamorphs. The results suggest that current usage of imidacloprid and thiamethoxam does not pose a threat to wood frogs. However, further assessment of both direct and indirect effects on subtle sublethal endpoints, and the influence of multiple interacting stressors at various life stages, is needed to fully understand the effects of neonicotinoids on amphibians. Environ Toxicol Chem 2017;36:1101-1109. © 2017 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Characterization and Developmental Expression Profile of the Steroidogenic Acute Regulatory Protein (StAR) in the Gonad-Mesonephros Complex of Lithobates sylvaticus.

The steroidogenic acute regulatory (StAR) protein is responsible for the movement of cholesterol across mitochondrial membranes and is therefore a key factor in regulating the timing and rate of steroidogenesis. In this study, we characterized the coding region of the star gene in the ranid Lithobates sylvaticus and studied star mRNA levels in steroidogenic tissues during development and under natural conditions. Our results support previous research showing that the StAR sequence is well conserved. We determined that star is expressed in both the interrenal and gonadal tissues of adults and in the tadpole gonad-mesonephros complex (GMC). The mRNA levels of star in the GMC were found to increase during tadpole development, reaching a maximum between Gosner stages (Gs) 32 and 38. We observed a significant drop in star mRNA levels at the end of prometamorphosis (Gs40-41), just before the start of the metamorphic climax. Significant differences in star levels between females and males, with males presenting higher levels than females, were detected at Gs36-38. To our knowledge, this is the first study that reports transitory star sex differences in tadpoles' developing GMC. Our results suggest an involvement of StAR in anuran late male GMC formation and development that requires further investigation.

Mortality of American bullfrog tadpoles Lithobates catesbeianus infected by Gyrodactylus jennyae and experimentally exposed to Batrachochytrium dendrobatidis.

The fungal pathogen Batrachochytrium dendrobatidis (Bd), which causes the disease chytridiomycosis in postmetamorphic amphibians, has been linked to amphibian population declines. Different amphibian species, however, exhibit different susceptibility to Bd pathogenicity. At the same time, agricultural pesticides commonly found contaminating aquatic habitats have been reported to increase the susceptibility of amphibians to pathogens. To investigate whether certain pesticides are able to alter the pathogenicity of Bd to larval amphibians, we exposed larval American bullfrogs Lithobates catesbeianus to end-use formulations of the herbicides atrazine or glyphosate, and then exposed them to Bd. Following the experimental exposures, a preexisting infection of the tadpoles by the monogenean ectoparasite Gyrodactylus jennyae was detected in all experimental and control tadpoles. Gyrodactylus jennyae infection intensity varied, and individuals with heavy G. jennyae infections suffered more skin erosion due to grazing by the parasite. Tadpoles experimentally exposed to Bd, or to Bd and either herbicide, had significantly reduced survival rates compared with untreated tadpoles that were only infected by G. jennyae. Increased mortality was also correlated with degree of skin erosion; survival of tadpoles with severe skin erosion was significantly reduced compared with that of tadpoles with no, or mild, skin erosion. While infected with G. jennyae, the group of tadpoles with the lowest survival rate (exposed only to Bd) included significantly more individuals exhibiting severe skin erosion and significantly fewer individuals without skin erosion, compared with the control group. These results emphasize the potential pathogenicity of gyrodactylid infections in larval amphibian hosts and suggest that concomitant exposures to Bd may enhance infections and effects of G. jennyae in bullfrog tadpoles.

Effects of herbicides and the chytrid fungus Batrachochytrium dendrobatidis on the health of post-metamorphic northern leopard frogs (Lithobates pipiens).

Effects of exposure to contaminants such as pesticides along with exposure to pathogens have been listed as two major contributors to the global crisis of declining amphibian populations. These two factors have also been linked in explanations of the causes of these population declines. We conducted a combined exposure experiment to test the hypothesis that exposure to two agricultural herbicides would increase the susceptibility of post-metamorphic northern leopard frogs (Lithobates pipiens) to the amphibian fungal pathogen Batrachochytrium dendrobatidis (Bd). We assessed the independent and interactive effects of these exposures on the health and survival of the frogs. Wild-caught frogs underwent a 21-day exposure to a nominal concentration of either 2.1 µg/L atrazine (Aatrex(®) Liquid 480) or 100 µg a.e./L glyphosate (Roundup(®) Original), followed by Bd, and then were observed until 94 days post-initial exposure to the herbicides. Actual levels of atrazine were between 4.28 ± 0.04 µg/L and 1.70 ± 0.26 µg/L while glyphosate degraded from 100 µg a.e./L to approximately 7 µg a.e./L within 6 days of initial exposure to the herbicides. Compared to controls, the glyphosate formulation reduced the snout-vent length of frogs during the pesticide exposure (at Day 21), and the atrazine formulation reduced gain in mass up to Day 94. No treatment affected survival, splenosomatic or hepatosomatic indices, the densities and sizes of hepatic and splenic melanomacrophage aggregates, the density and size of hepatic granulomas, proportions of circulating leucocytes, the ratio of neutrophils to lymphocytes, or the ratio of leucocytes to erythrocytes. Histological assessment of samples collected at Day 94 revealed no evidence of Bd infection in any Bd-exposed frogs, while real-time PCR detected only one case of light infection in a single atrazine- and Bd-exposed frog. Frogs exposed to Bd shed their skin significantly more frequently than Bd-unexposed frogs, which may have helped them resist or clear infection, and could explain why no interaction between the herbicides and Bd was detected. The results suggest that these frogs were resistant to Bd infection and that pre-exposure to the herbicides did not alter this resistance. The effects seen on the growth following herbicide exposure is a concern, as reduced growth can lower the reproductive success and survival of the amphibians.

Effects of chytrid fungus and a glyphosate-based herbicide on survival and growth of wood frogs (Lithobates sylvaticus).

Anthropogenic-derived stressors in the environment, such as contaminants, are increasingly considered important cofactors that may decrease the immune response of amphibians to pathogens. Few studies, however, have integrated amphibian disease and contaminants to test this multiple-stressor hypothesis for amphibian declines. We examined whether exposure to sublethal concentrations of a glyphosate-based herbicide and two strains of the pathogenic chytrid fungus, Batrachochrytrium dendrobatidis (Bd) could: (1) sublethally affect wood frogs (Lithobates sylvaticus) by altering the time to and size at metamorphosis, and (2) directly affect survivability of wood frogs after metamorphosis. Neither Bd strain nor herbicide exposure alone significantly altered growth or time to metamorphosis. The two Bd strains did not differ in their pathogenicity, and both caused mortality in post-metamorphic wood frogs. There was no evidence of an interaction between treatments, indicating a lack of herbicide-induced susceptibility to Bd. However, the trends in our data suggest that exposure of wood frogs to a high concentration of glyphosate-based herbicide may reduce Bd-caused mortality compared to animals exposed to Bd alone. These results exemplify the complexities inherent when populations are coping with multiple stressors. In this case, the perceived stressor, glyphosate-based herbicide, appeared to affect the pathogen more than the host's immune system, relieving the host from disease-caused effects. This suggests caution when invoking multiple stressors as a cause for increased disease susceptibility and indicates that the effects of multiple stressors on disease outcome depend on the interrelationships of stressors to both the pathogen and the host.

Exposure of juvenile green frogs (Lithobates clamitans) in littoral enclosures to a glyphosate-based herbicide.

The majority of studies on the toxicity of glyphosate-based herbicides to amphibians have focused on larval life stages exposed in aqueous media. However, adult and juvenile amphibians may also be exposed directly or indirectly to herbicides. The potential for such exposures is of particular interest in the littoral zone surrounding wetlands as this is preferred habitat for many amphibian species. Moreover, it may be argued that potential herbicide effects on juvenile or adult amphibians could have comparatively greater influence on overall recruitment, reproductive potential and thus stability of local populations than effects on larvae. In this experiment, juvenile green frogs (Lithobates clamitans) were exposed to two concentrations (2.16 and 4.27 kg a.e./ha) of a glyphosate-based herbicide formulation (VisionMax®), which were based on typical application scenarios in Canadian forestry. The experimental design employed frogs inhabiting in situ enclosures established at the edge of small naturalized wetlands that were split in half using an impermeable plastic barrier. When analyzed using nominal target application rates, exposure to the glyphosate-based herbicide had no significant effect on survival, body condition, liver somatic index or the observed rate of Batrachochytrium dendrobatidis infection. However, there were marginal trends in both ANOVA analysis and post-hoc regressions regarding B. dendrobatidis infection rates and liver somatic index in relation to measured exposure estimates. Results from this study highlight the importance of field research and the need to include multiple endpoints when examining potential effects of a contaminant on non-target organisms.

Low levels of the herbicide atrazine alter sex ratios and reduce metamorphic success in Rana pipiens tadpoles raised in outdoor mesocosms.

BACKGROUND: There are conflicting reports regarding the effects of atrazine (ATZ) on amphibian development. Therefore, further studies are needed to examine the potential mechanisms of action of ATZ in amphibians. OBJECTIVES: Our aim in this study was to determine whether low concentrations of ATZ affect gonadal development and metamorphosis in the Northern leopard frog, Rana pipiens. METHODS: Tadpoles were exposed in outdoor mesocosms to nominal concentrations of 0.1 and 1.8 microg/L of formulated ATZ from Gosner stage 27 (G27) to metamorphic climax (G42). Exposure to 17alpha-ethinylestradiol (EE2; 1.5 microg/L) provided a positive control for induction of testicular oocytes in males. Endocrine-related gene expression and gonadal histopathology were examined at G42 and in a subset of premetamorphic G34 tadpoles that failed to metamorphose. RESULTS: Gonadal gross morphology revealed that the 1.8-microg/L ATZ treatment produced 20% more females compared with the control. Histologic analysis revealed that 22% of EE2-treated males had testicular oocytes, whereas none were observed in any animals from the control or either ATZ groups. ATZ increased brain estrogen receptor alpha mRNA to 2.5 times that of the control at premetamorphosis and altered liver levels of 5beta-reductase activity at metamorphosis. In contrast, brain aromatase mRNA level and activity did not change. ATZ treatments significantly reduced metamorphic success (number of animals reaching metamorphosis) without affecting body weight, snout-vent length, or age at metamorphosis. Gene expression analysis indicated that ATZ decreased the expression of deiodinase type 3 in the tail at premetamorphosis. CONCLUSIONS: Our study indicates that exposure to low concentrations of ATZ in experimental mesocosms alters gonadal differentiation and metamorphosis in developing R. pipiens.

Fadrozole and finasteride exposures modulate sex steroid- and thyroid hormone-related gene expression in Silurana (Xenopus) tropicalis early larval development.

Steroidogenic enzymes and their steroid products play critical roles during gonadal differentiation in amphibians; however their roles during embryogenesis remain unclear. The objective of this study was to investigate the expression and activity of aromatase (cyp19; estrogen synthase) and 5 beta-reductase (srd5 beta; 5 beta-dihydrotestosterone synthase) during amphibian embryogenesis. Expression and activity profiles of cyp19 and srd5 beta were first established during Silurana (Xenopus) tropicalis embryogenesis from Nieuwkoop-Faber (NF) stage 2 (2-cell stage; 1h post-fertilization) to NF stage 46 (beginning of feeding; 72 h post-fertilization). Exposures to fadrozole (an aromatase inhibitor; 0.5, 1.0 and 2.0 microM) and finasteride (a putative 5-reductase inhibitor; 25, 50 and 100 microM) were designed to assess the consequences of inhibiting these enzymes on gene expression in early amphibian larval development. Exposed embryos showed changes in both enzyme activities and sex steroid- and thyroid hormone-related gene expression. Fadrozole treatment inhibited cyp19 activity and increased androgen receptor and thyroid hormone receptor (alpha and beta) mRNAs. Finasteride treatment inhibited srd5 beta (activity and mRNA), decreased cyp19 mRNA and activity levels and increased estrogen receptor alpha mRNA. Both treatments altered the expression of deiodinases (thyroid hormone metabolizing enzymes). We conclude that cyp19 and srd5 beta are active in early embryogenesis and larval development in Silurana tropicalis and their inhibition affected transcription of genes associated with the thyroid and reproductive axes.

Expression and T3 regulation of thyroid hormone- and sex steroid-related genes during Silurana (Xenopus) tropicalis early development.

In amphibians, thyroid hormones (THs) are the primary regulators of metamorphosis; however, their physiological role during embryogenesis remains unclear. First, we established complete developmental profiles for TH receptors (tr alpha and tr beta), deiodinases (dio; types 1, 2, 3), estrogen receptors (er alpha and er beta) and androgen receptor (ar) mRNA levels during embryogenesis and early larval stages in Silurana (Xenopus) tropicalis (from Nieuwkoop and Faber (NF) stage 2 until NF 46). Real-time RT-PCR analyses in whole embryos and larvae revealed that all transcripts except tr alpha were detected throughout development; tr alpha only appears after gastrulation. The first significant increase in the expression of tralpha and tr beta was observed before hatching, between NF 21 and NF 27 (2.5- and 11-fold, respectively). In order to test if these genes could be regulated by THs during early larval development, embryos were exposed to triiodothyronine (T3; 0.5, 5.0, 50 nM) from NF 27 to NF 46. T3 exposure caused a dose-dependent increase relative to control in the expression of tr alpha, tr beta, dio (types 2 and 3), ar, and 5 alpha-reductase type 1 in whole larvae. These results indicate that in S. tropicalis, tr and dio can be induced by T3 as early as NF 46, a response that had only been characterized later during frog metamorphosis. In addition, T3 also affected androgen-related gene expression, supporting our hypothesis that THs are involved in male development in frogs.

Hepatic porphyria induced by the herbicide tralkoxydim in small mammals is species-specific.

Tralkoxydim is the active ingredient in a postemergent herbicide used in cereal crops. During preregistration trials, tralkoxydim was observed to cause hepatic porphyria and cholestasis in laboratory mice. Porphyria was not seen in similarly exposed rats or hamsters, but data were not collected regarding the susceptibility of any wild small mammal species to the tralkoxydim-induced porphyria. To address this data gap, we exposed small mammals to tralkoxydim, to 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC; a known porphyrinogenic chemical), or to sunflower oil alone. We studied small mammal species that might be exposed following agricultural applications of the herbicide, including the white-footed mouse (Peromyscus leucopus), the deer mouse (P. maniculatus), and the meadow vole (Microtus pennsylvanicus). Because of their known susceptibility to both tralkoxydim- and DDC-induced porphyria, commercially supplied Mus musculus (CD-1 Swiss mice) were exposed as positive-control animals. We also exposed offspring of wild-caught M. musculus to compare their responses to those of the commercially supplied animals. Potential hepatotoxicity was determined by assessing the accumulation of liver protoporphyrin. Of the species tested, only M. musculus was susceptible to the porphyrinogenic action of tralkoxydim, and no significant accumulation of protoporphyrin was observed in any of the other species exposed to the herbicide.

Toxicity of glyphosate-based pesticides to four North American frog species.

Glyphosate-based herbicides are among the most widely used pesticides in the world. We compared the acute toxicity of the glyphosate end-use formulation Roundup Original to four North American amphibian species (Rana clamitans, R. pipiens, R. sylvatica, and Bufo americanus) and the toxicity of glyphosate technical, the polyethoxylated tallowamine surfactant (POEA) commonly used in glyphosate-based herbicides, and five newer glyphosate formulations to R. clamitans. For R. clamitans, acute toxicity values in order of decreasing toxicity were POEA > Roundup Original > Roundup Transorb > Glyfos AU; no significant acute toxicity was observed with glyphosate technical material or the glyphosate formulations Roundup Biactive, Touchdown, or Glyfos BIO. Comparisons between the four amphibian species showed that the toxicity of Roundup Original varied with species and developmental stage. Rana pipiens tadpoles chronically exposed to environmentally relevant concentrations of POEA or glyphosate formulations containing POEA showed decreased snout-vent length at metamorphosis and increased time to metamorphosis, tail damage, and gonadal abnormalities. These effects may be caused, in some part, by disruption of hormone signaling, because thyroid hormone receptor beta mRNA transcript levels were elevated by exposure to formulations containing glyphosate and POEA. Taken together, the data suggest that surfactant composition must be considered in the evaluation of toxicity of glyphosate-based herbicides.

Gonadal differentiation in frogs exposed to estrogenic and antiestrogenic compounds.

Exposure of amphibians to endocrine disrupting compounds (EDCs) may alter differentiation of gonads, especially when exposures begin during early life stages. Gonadal differentiation was observed in leopard frogs (Rana pipiens) and wood frogs (Rana sylvatica) exposed as tadpoles to estrogenic (estradiol, ethinylestradiol, nonylphenol) and antiestrogenic compounds (an aromatase inhibitor, flavone, and an antiestrogen, ICI 182780). Exposure to all compounds at micrograms/L concentrations altered gonadal differentiation in some animals by inducing either complete feminization or an intersex condition, and altered testicular tubule morphology, increased germ cell maturation (vitellogenesis), and oocyte atresia. Comparisons between the two species indicate that R. pipiens are more susceptible to sex reversal and development of intersex gonads. However, R. sylvatica also showed alterations to testicular morphology, germ cell maturation, and ooctye atresia. These laboratory results indicate that amphibians could be susceptible to altered gonadal differentiation and development when exposed to estrogenic and antiestrogenic compounds in aquatic environments, such as those impacted by agricultural, industrial, and municipal runoff.