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.

Decadal differences in polycyclic aromatic compound (PAC) concentrations in two seabird species in Arctic Canada.

Seabirds are exposed to a variety of environmental contaminants in the Arctic. While the persistence, bioaccumulation, and toxicity of some groups of contaminants have been well-studied in seabirds since the 1970s, there is less known about polycyclic aromatic compounds (PACs). With increased vessel traffic, and potential oil and gas development in the Arctic region, there is a need to understand existing PAC exposure in biota against which to compare potential effects of anticipated increases of PACs in the marine region. Thick-billed murres (Uria lomvia) and northern fulmars (Fulmarus glacialis) collected in the Baffin Bay - Davis Strait region during the International Polar Year (IPY; 2007-08), and during a recent Strategic Environmental Assessment (2018; SEA) were examined for hepatic PAC concentrations. We found that fulmars generally had higher concentrations of PACs than the murres, but murres and fulmars sampled in 2007/08 had higher concentrations of most groups of PACs compared to birds from 2018. The one exception to this pattern was that the sum of the alkylated congeners of the heterocyclic aromatic compounds containing a sulfur atom (dibenzothiophene; ΣAHET) was significantly higher in murres in the more recent sampling period (2018) as compared to 2007/08. ΣAHETs likely reflect recent exposure to more refined petroleum products associated with small boats, such as diesel, gasoline and motor oil. This work highlights the need for longitudinal studies on PAC concentrations in biota for us to gain a better understanding of how Arctic biota are exposed to this group of contaminants, and the potential deleterious effects associated with PACs.

Metabolomic profiles in relation to benchmark polycyclic aromatic compounds (PACs) and trace elements in two seabird species from Arctic Canada.

While exposure of birds to oil-related contaminants has been documented, the related adverse effects this exposure has on Arctic marine birds remain unexplored. Metabolomics can play an important role to explore biologically relevant metabolite biomarkers in relation to different stressors, even at benchmark levels of contamination. The aim of this study was to characterize the metabolomics profiles in relation to polycyclic aromatic compounds (PACs) and trace elements in the liver of two seabird species in the Canadian Arctic. In July 2018, black guillemots (Cepphus grylle) and thick-billed murres (Uria lomvia) were collected by hunters from a region where natural oil seeps occur in the seabed near Qikiqtarjuaq, Nunavut, Canada. A total of 121 metabolites were identified in liver tissue samples using reversed phase and hydrophilic interaction liquid chromatography coupled to high resolution mass spectrometry platforms to detect non-polar and polar metabolites, respectively. Sixty-nine metabolites showed excellent repeatability and linearity and were used to examine possible effects of oil-related contaminants exposure (PACs and trace elements). Metabolites including 3-hydroxy anthranilic acid, adenine, adenosine, adenosine mono-phosphate, ascorbic acid, butyrylcarnitine, cholic acid, guanosine, guanosine mono-phosphate, inosine, norepinephrine and threonine showed significant differences (more than two fold) between the two species. Elevated adenine and adenosine, along with decreased reduced/oxidized glutathione ratio, highlighted the potential for oxidative stress in murres. Lipid peroxidation and superoxide dismutase activity assays also confirmed these metabolomic findings. These results will help to characterize the baseline metabolomic profiles of Arctic seabird species with different foraging behaviour and trace element burden.

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.

ToxChip PCR Arrays for Two Arctic-Breeding Seabirds: Applications for Regional Environmental Assessments.

Increasing pollution in the Arctic poses challenges in terms of geographical and ecological monitoring. The Baffin Bay-Davis Strait (BBDS) region in the Canadian Arctic Archipelago is of particular concern due to the potential for increased shipping traffic and oil exploration. However, data on background contaminants associated with oil exploration/spills/natural seeps (e.g., polycyclic aromatic compounds [PAC]) and measures of potential effects for Arctic birds are limited. We developed a toxicogenomics approach to investigate the background gene expression profiles for two Arctic-breeding seabirds, the thick-billed murre (Uria lomvia) and the black guillemot (Cepphus grylle), which will aid effects-based monitoring efforts. Chemical burdens (53 PACs and 5 trace elements) and transcriptomic profiles (31 genes using a ToxChip PCR array) were examined in liver tissues (n = 30) of each species collected from the Qaqulluit and Akpait National Wildlife Areas in the BBDS region. While chemical and transcriptomic profiles demonstrated low variability across individuals for each species, gene expression signatures were able to distinguish guillemots collected from two distinct colonies. This toxicogenomics approach provides benchmark data for two Arctic seabirds and is promising for future monitoring efforts and strategic environmental assessments in this sensitive ecosystem and areas elsewhere in the circumpolar Arctic that are undergoing change.

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.

Geospatial analysis of the patterns of chemical exposures among biota in the Canadian Oil Sands Region.

Understanding the patterns of chemical exposure among biota across a landscape is challenging due to the spatial heterogeneity and complexity of the sources, pathways, and fate of the different chemicals. While spatially-driven relationships between contaminant sources and biota body burdens of a single chemical are commonly modelled, there has been little effort on modelling chemical mixtures across multiple wildlife species in the Canadian Oil Sands region. In this study, we used spatial principal components analysis (sPCA) to assess spatial patterns of the body burdens of 22 metals and Potentially Toxic Elements (PTEs) in 492 individual wildlife, including fur-bearing mammals, colonial waterbirds, and amphibians collected from the Canadian Oil Sands region in Canada. Spatial analysis and mapping both indicate that some of the complex exposures in the studied biota are distributed randomly across a landscape, which suggests background or non-point source exposures. In contrast, the pattern of exposure for seven metals and PTEs, including mercury, vanadium, lead, rubidium, lithium, strontium, and barium, exhibited a clustered pattern to the east of the open-pit mining area and in regions downstream of oil sands development which indicates point-source input. This analysis demonstrated useful methods for integrating monitoring datasets and identifying sources and potential drivers of exposure to chemical mixtures in biota across a landscape. These results can be used to support an adaptive monitoring program by identifying regions needing additional monitoring, health impact assessments, and possible intervention strategies.

Polycyclic aromatic compounds (PACs) and trace elements in four marine bird species from northern Canada in a region of natural marine oil and gas seeps.

There is a growing understanding of how oil pollution can affect aquatic ecosystems, including physical and chemical effects. One of the biggest challenges with detecting the effects of oil-related contaminants on biota from resource development is understanding the background levels and potential effects of the exposure of biota to contaminants from various natural and anthropogenic sources prior to large scale oil and gas operations. Seabirds are effective indicators of pollution, and can be useful for tracking oil-related contaminants in the marine environment. We sampled four seabird species (black guillemot, Cepphus grylle; thick-billed murre, Uria lomvia; black-legged kittiwake, Rissa tridactyla; and northern fulmar, Fulmarus glacialis) in the Baffin Bay-Davis Strait region of the Northwest Atlantic and Arctic oceans, an area where natural oil and gas seeps are present but lacking any large-scale oil and gas projects. We found detectable levels of PACs and several trace elements in all species examined. Alkylated PAC levels were higher than parent compounds in all four seabird species examined, with fulmars and murres having the highest levels detected; mean hepatic concentrations of ∑16PAC were 99.05, 46.42, 12.78 and 9.57 ng/g lw, respectively, for guillemots, murres, fulmars and kittiwakes. Overall, PAC concentrations in the seabird species examined were similar to PAC concentrations measured in other bird species in regions with more industrialization. These findings provide data which can be used to assess the current oil-related contaminant exposure of biota in the region. As well, they provide background levels for the region at a time when shipping activity is relatively low, which can used for future comparisons following expected increases in shipping and oil and gas activities in the region.

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.

Environmental dependency of amphibian-ranavirus genotypic interactions: evolutionary perspectives on infectious diseases.

The context-dependent investigations of host-pathogen genotypic interactions, where environmental factors are explicitly incorporated, allow the assessment of both coevolutionary history and contemporary ecological influences. Such a functional explanatory framework is particularly valuable for describing mortality trends and identifying drivers of disease risk more accurately. Using two common North American frog species (Lithobates pipiens and Lithobates sylvaticus) and three strains of frog virus 3 (FV3) at different temperatures, we conducted a laboratory experiment to investigate the influence of host species/genotype, ranavirus strains, temperature, and their interactions, in determining mortality and infection patterns. Our results revealed variability in host susceptibility and strain infectivity along with significant host-strain interactions, indicating that the outcome of an infection is dependent on the specific combination of host and virus genotypes. Moreover, we observed a strong influence of temperature on infection and mortality probabilities, revealing the potential for genotype-genotype-environment interactions to be responsible for unexpected mortality in this system. Our study thus suggests that amphibian hosts and ranavirus strains genetic characteristics should be considered in order to understand infection outcomes and that the investigation of coevolutionary mechanisms within a context-dependent framework provides a tool for the comprehensive understanding of disease dynamics.

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.