Broad host susceptibility of North American amphibian species to Batrachochytrium salamandrivorans suggests high invasion potential and biodiversity risk.

Batrachochytrium salamandrivorans (Bsal) is a fungal pathogen of amphibians that is emerging in Europe and could be introduced to North America through international trade or other pathways. To evaluate the risk of Bsal invasion to amphibian biodiversity, we performed dose-response experiments on 35 North American species from 10 families, including larvae from five species. We discovered that Bsal caused infection in 74% and mortality in 35% of species tested. Both salamanders and frogs became infected and developed Bsal chytridiomycosis. Based on our host susceptibility results, environmental suitability conditions for Bsal, and geographic ranges of salamanders in the United States, predicted biodiversity loss is expected to be greatest in the Appalachian Region and along the West Coast. Indices of infection and disease susceptibility suggest that North American amphibian species span a spectrum of vulnerability to Bsal chytridiomycosis and most amphibian communities will include an assemblage of resistant, carrier, and amplification species. Predicted salamander losses could exceed 80 species in the United States and 140 species in North America.

Emerging Pathogens and a Current-Use Pesticide: Potential Impacts on Eastern Hellbenders.

Populations of the eastern hellbender Cryptobranchus alleganiensis alleganiensis have been declining for decades, and emerging pathogens and pesticides are hypothesized to be contributing factors. However, few empirical studies have attempted to test the potential effects of these factors on hellbenders. We simultaneously exposed subadult hellbenders to environmentally relevant concentrations of either Batrachochytrium dendrobatidis (Bd) or a frog virus 3-like ranavirus (RV), a combination of the pathogens, or each pathogen following exposure to a glyphosate herbicide (Roundup). Additionally, we measured the ability of the skin mucosome to inactivate Bd and RV in growth assays. We found that mucosome significantly inactivated RV by an average of 40% but had no negative effects on Bd growth. All treatments that included RV exposure experienced reduced survival compared to controls, and the combination of RV and herbicide resulted in 100% mortality. Histopathology verified RV as the cause of mortality in all RV-exposed treatments. No animals were infected with Bd or died in the Bd-only treatment. Our results suggest that RV exposure may be a significant threat to the survival of subadult hellbenders and that Roundup exposure may potentially exacerbate this threat.

Factors influencing the toxicity of Headline® fungicides to terrestrial stage toads.

Amphibians are susceptible to exposure from contaminants via multiple pathways. Pyraclostrobin fungicides have been shown to be toxic to terrestrial amphibians at environmentally relevant concentrations; however, these studies did not account for factors that may influence exposure and effects, such as fungicide formulation, age of the individual, exposure route, and physiological state of the individual. We examined Headline® and Headline AMP® fungicide toxicity to adult Anaxyrus cognatus and Anaxyrus woodhousii by direct overspray, as well as acute toxicity of Headline AMP to juvenile A. cognatus through direct overspray, previously exposed soils, and diet. We also assessed effects of hydration state on fungicide toxicity in juvenile A. cognatus and sublethal effects of fungicide exposure on prey-orientation ability of juvenile A. cognatus. Neither formulation of Headline caused mortality of adult A. cognatus and A. woodhousii at up to 5 times the maximum label rate in North American corn (1052 and 879 mL formulation/ha for Headline AMP and Headline fungicides, respectively, corresponding to 1.52 and 2.2 µg pyraclostrobin/cm2 , respectively). Exposures of juvenile A. cognatus to Headline AMP via direct overspray and previously exposed soils (2 types) resulted in median lethal rate (LR50) values of 2.4, 3.34, and 3.61 µg pyraclostrobin/cm2 , respectively. Dietary Headline AMP exposure was not toxic, prey-orientations were not influenced by exposure, and effects were similar between dehydrated and hydrated treatments (LR50 = 2.4 and 2.3 µg pyraclostrobin/cm2 , respectively). These results, combined with exposure concentrations reported in previous studies, suggest that risk of acute mortality for amphibians in terrestrial environments is low and is dictated by body size and site-specific factors influencing exposure. Environ Toxicol Chem 2017;36:2679-2688. © 2017 SETAC.

Occurrence of current-use fungicides and bifenthrin in Rainwater Basin wetlands.

Wetlands in the Rainwater Basin (RWB) of Nebraska are commonly in close proximity to or embedded within row-crop agriculture. Several fungicides and bifenthrin are applied aerially to corn throughout the RWB during tassel stage. Thus, aerial spray drift and runoff may result in pesticide contamination of wetlands. The primary objective of this study was to determine water concentrations of five fungicides and bifenthrin in wetlands located in and near fields during a heavy application period (July 16th-26th 2014) to evaluate the influence of distance from crop field on wetland contamination. In addition, the sampling sites were grouped based upon the type of water body sampled and environmental concentrations were compared to relevant "levels of concern" (LOCs) from invertebrates, fish, and amphibians selected from the literature based upon relevant toxicity data. Bifenthrin was the most frequently detected analyte, appearing in 83% of the samples. The 95th percentile concentrations across all wetlands were found to be 0.07 µg/L for bifenthrin, 0.28 µg/L for pyraclostrobin, 0.28 µg/L for azoxystrobin, and <0.14 µg/L for all other analytes. Analyte concentrations did not differ by wetland type and were not correlated with distance from closest crop. Environmental concentrations of fungicides were lower than LOCs, indicating limited acute toxicity risk. However, bifenthrin concentrations were frequently higher than LOCs for aquatic arthropods. Because the water samples included particulate bound pesticides, further work is needed to determine if this bifenthrin is bioavailable and contamination levels within the sediment.

Effects of Maternally-Transferred Methylmercury on Stress Physiology in Northern Water Snake (Nerodia sipedon) Neonates.

Biomagnification of methylmercury in aquatic systems can cause elevated tissue mercury (Hg) and physiological stress in top predators. Mercury is known to affect stress hormone levels in mammals, birds and fish. In this study, the effects of maternally-transferred methylmercury on the stress physiology of Northern Water Snake (Nerodia sipedon) neonates were tested. Gravid females were dosed via force-fed capsules during late gestation with 0, 0.01, or 10 µg methylmercury per gram of body mass. Plasma corticosterone levels and leukocyte differentials were analyzed in baseline and confinement-stressed neonates from all dose levels. Neither Hg nor confinement stress had a significant effect on leukocyte differentials nor was Hg related to corticosterone levels. However, stress group neonates showed lower heterophil/lymphocyte ratios and this study was the first to show that neonate N. sipedon can upregulate CORT in response to stress. These results indicate that N. sipedon may be somewhat tolerant to Hg contamination.

Acute toxicity of Headline® fungicide to Blanchard's cricket frogs (Acris blanchardi).

Previous laboratory studies have suggested that pyraclostrobin-containing fungicide formulations are toxic to amphibians at environmentally relevant concentrations. However, it is unknown if all pyraclostrobin formulations have similar toxicity and if toxicity occurs in different amphibian species. We investigated the acute toxicity of two formulations, Headline(®) fungicide and Headline AMP(®) fungicide, to Blanchard's cricket frogs (Acris blanchardi) based on a direct overspray scenario. In addition, we examined body residues of fungicide active ingredients in A. blanchardi following direct exposure to Headline AMP fungicide. Headline fungicide and Headline AMP fungicide had similar toxicity to A. blanchardi with calculated median lethal doses of 2.1 and 1.7 µg pyraclostrobin/cm(2), respectively, which are similar to the suggested maximum label rate in North American corn (2.2 and 1.52 µg pyraclostrobin/cm(2), respectively). Tissue concentrations of pyraclostrobin were lower than predicted based on full uptake of a direct dose, and did not drop during the first 24 h after exposure. Headline fungicides at corn application rates are acutely toxic to cricket frogs, but acute toxicity in the field will depend on worst-case exposure.

Terrestrial exposure and effects of Headline AMP(®) Fungicide on amphibians.

Recent studies have demonstrated that a pyraclostrobin-containing fungicide (Headline(®) Fungicide--Headline(®) Fungicide and Headline AMP(®) Fungicide are registered trademarks of BASF) is toxic to amphibians at environmentally relevant concentrations. However, these studies were performed in a laboratory setting of a worst-case direct exposure in clean media. Interception of spray by the crop canopy and ground cover used by animals for security cover will influence exposure. Thus, risk to amphibians is unclear in an environmentally realistic field environment. We tested exposure and toxicity of Headline AMP(®) Fungicide to amphibians in multiple agricultural habitat scenarios (e.g., within treated crop vs. grassy areas adjacent to crop) and at two rates during routine aerial application. Specifically, we placed Woodhouse's toads (Bufo woodhousii) and Blanchard's cricket frogs (Acris blanchardi) in enclosures located within treated and untreated corn (VT stage, approximate height = 3 m), and in the potential drift area (adjacent to treated corn) during aerial application of Headline AMP Fungicide at either 731 or 1052 ml/ha (70 and 100 % the maximum application rate in corn, respectively). Mean concentrations of pyraclostrobin measured at ground level were ≤19 % of nominal application rate in all areas. Overall, mean mortality of recovered individuals of both species was ≤15 %, and mortality within Headline AMP Fungicide-treated corn (where risk was anticipated to be highest) was <10 %. It is important to understand that application timing, interception by the crop canopy (which varies both within and between crop systems), and timing of amphibian presence in the crop field influences risk of exposure and effects; however, our results demonstrate that amphibians inhabiting VT stage corn during routine aerial application of Headline AMP Fungicide are at low risk for acute mortality, matching existing laboratory results from acute toxicity studies of Headline Fungicide.