Novel stressors introduced by human activities increasingly threaten freshwater ecosystems. The annual application of more than 2.3 billion kg of pesticide active ingredient and 22 billion kg of road salt has led to the contamination of temperate waterways. While pesticides and road salt are known to cause direct and indirect effects in aquatic communities, their possible interactive effects remain widely unknown. Using outdoor mesocosms, we created wetland communities consisting of zooplankton, phytoplankton, periphyton, and leopard frog (Rana pipiens) tadpoles. We evaluated the toxic effects of six broad-spectrum insecticides from three families (neonicotinoids: thiamethoxam, imidacloprid; organophosphates: chlorpyrifos, malathion; pyrethroids: cypermethrin, permethrin), as well as the potentially interactive effects of four of these insecticides with three concentrations of road salt (NaCl; 44, 160, 1600 Cl⁻ mg/L). Organophosphate exposure decreased zooplankton abundance, elevated phytoplankton biomass, and reduced tadpole mass whereas exposure to neonicotinoids and pyrethroids decreased zooplankton abundance but had no significant effect on phytoplankton abundance or tadpole mass. While organophosphates decreased zooplankton abundance at all salt concentrations, effects on phytoplankton abundance and tadpole mass were dependent upon salt concentration. In contrast, while pyrethroids had no effects in the absence of salt, they decreased zooplankton and phytoplankton density under increased salt concentrations. Our results highlight the importance of multiple-stressor research under natural conditions. As human activities continue to imperil freshwater systems, it is vital to move beyond single-stressor experiments that exclude potentially interactive effects of chemical contaminants.

Amphibian larvae are commonly used as indicators of aquatic ecosystem health because they are susceptible to contaminants. However, there is limited information on how species characteristics and trophic position influence contaminant loads in larval amphibians. Importantly, there remains a need to understand whether grazers (frogs and toads [anurans]) and predators (salamanders) provide comparable information on contaminant accumulation or if they are each indicative of unique environmental processes and risks. To better understand the role of trophic position in contaminant accumulation, we analyzed composite tissues for 10 metals from larvae of multiple co-occurring anuran and salamander species from 20 wetlands across the United States. We examined how metal concentrations varied with body size (anurans and salamanders) and developmental stage (anurans) and how the digestive tract (gut) influenced observed metal concentrations. Across all wetlands, metal concentrations were greater in anurans than salamanders for all metals tested except mercury (Hg), selenium (Se), and zinc (Zn). Concentrations of individual metals in anurans decreased with increasing weight and developmental stage. In salamanders, metal concentrations were less correlated with weight, indicating diet played a role in contaminant accumulation. Based on batches of similarly sized whole-body larvae compared to larvae with their digestive tracts removed, our results indicated that tissue type strongly affected perceived concentrations, especially for anurans (gut represented an estimated 46–97% of all metals except Se and Zn). This suggests the reliability of results based on whole-body sampling could be biased by metal, larval size, and development. Overall, our data shows that metal concentrations differs between anurans and salamanders, which suggests that metal accumulation is unique to feeding behavior and potentially trophic position. To truly characterize exposure risk in wetlands, species of different life histories, sizes and developmental stages should be included in biomonitoring efforts.

In order for Alberta's thick bitumen to be transported through pipelines, condensates are added creating a diluted bitumen (dilbit) mixture. Recent pipeline expansion projects have generated concern about potential dilbit spills on aquatic wildlife health. Studies have suggested that polycyclic aromatic compounds (PACs) are toxic to aquatic vertebrates and could potentially also interfere with their endocrine system. The research objectives of this study were to investigate the toxicity of dilbit to developing frog embryos and to identify the molecular mechanisms of action involved. Fertilized embryos of Western clawed frog (Silurana tropicalis) were exposed for 72 h to water accommodated fractions (WAF; 0.7–8.9 μg/L TPACs) and chemically-enhanced WAFs (CEWAF; 0.09–56.7 μg/L TPACs) of Access Western Blend (AWB) and Cold Lake Blend (CLB) dilbits. Both dilbit's CEWAFs significantly increased embryonic mortality and malformation incidence in the highest treatments tested, while WAF treatments led to no visible toxic effects. Increases of the cytochrome P450 1A (cyp1a) mRNA levels were observed for all WAF and CEWAF dilbit treatments suggesting that phase I detoxification is activated in the dilbit-exposed larvae. When exposed to PAC concentrations ranging from 0.09 to 8.9 μg/L, the frogs displayed no observable malformations, but expressed significant increases of cyp1a mRNA levels (2- to 25-fold; indicating a suitable biomarker of exposure); however, when concentrations were of 46.6 μg/L or higher, both malformed frog phenotype and induction of cyp1a mRNA level (>250-fold) were measured (indicating a suitable biomarker of response). The expression of several genes related to cellular detoxification and endocrine disruption were also measured, but were not significantly altered by the treatments. In sum, cyp1a mRNA level is a highly sensitive endpoint to measure subtle molecular changes induced by PAC exposure in the frog embryos and larvae, and data suggest that PAC concentration higher than 46 μg/L would be toxic to the developing S. tropicalis.

Tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA) are persistent toxic environmental pollutants that cause severe reproductive toxicity in animals. The goal of this study was to compare the reproductive toxic effects of TBBPA and TCBPA on male Rana nigromaculata and to expound on the mechanisms leading to these effects. Healthy adult frogs were exposed to 0, 0.001, 0.01, 0.1, and 1 mg/L of TBBPA and TCBPA for 14 days. Sperm numbers were counted by erythrometry. Sperm mobility and deformities were observed under a light microscope (400 ×). We used commercial ELISA kits to determine the serum content of testosterone (T), estradiol (E2), luteinizing hormone (LH) and follicle stimulating hormone (FSH). Expression of androgen receptor (AR) mRNA was detected using real-time qPCR. Sperm numbers and sperm mobility were significantly decreased and sperm deformity was significantly increased in a concentration dependent manner following exposure to TBBPA and TCBPA. Sperm deformity was significantly greater in the 1 mg/L TCBPA (0.549) treatment group than in the 1 mg/L TBBPA (0.397) treatment group. Serum T content was significantly greater in the 0.01, 0.1 and 1 mg/L TBBPA and TCBPA experimental groups compared with controls, while E2 content was significantly greater in only the 1 mg/L TBBPA and TCBPA experimental groups. Expression levels of LH and FSH significantly decreased in the 1 mg/L TBBPA and TCBPA treatment groups. AR mRNA expression decreased markedly in all the treated groups. Our results indicated that TBBPA and TCBPA induced reproductive toxicity in a dose-dependent manner, with TCBPA having greater toxicity than TBBPA. Furthermore, changes in T, E2, LH, and FSH levels induced by TBBPA and TCBPA exposure, which led to endocrine disorders, also caused disturbance of spermatogenesis through abnormal gene expressions of AR in the testes.

Previous studies reported that some species of the family Ceratophryidae are able to produce sounds during premetamorphic tadpole stages. We have now determined the effects of the cholinesterase-inhibiting insecticide chlorpyrifos (CPF) on sounds emitted by tadpoles of Ceratophrys ornata. Tadpoles were exposed individually in order to evaluate the progression of effects. Effects on sound production were complemented with common ecotoxicological endpoints (mortality, behavior, abnormalities and growth inhibition). C. ornata was found to be more sensitive than other native (= 67%, 50%) and non-native species (= 75%, 100%) considering lethal and sublethal endpoints, respectively. Effects on sounds appear along with alterations in swimming, followed by the presence of mild, then severe abnormalities and finally death. Therefore, sound production may be a good biomarker since it anticipates other endpoints that are also affected by CPF. Ceratophrys ornata is a promising new model species in ecotoxicology.

Advances in drilling techniques have facilitated a rapid increase in hydrocarbon extraction from energy shales, including the Williston Basin in central North America. This area overlaps with the Prairie Pothole Region, a region densely populated with wetlands that provide numerous ecosystem services. Historical (legacy) disposal practices often released saline co-produced waters (brines) with high chloride concentrations, affecting wetland water quality directly or persisting in sediments. Despite the potential threat of brine contamination to aquatic habitats, there has been little research into its ecological effects. We capitalized on a gradient of legacy brine-contaminated wetlands in northeast Montana to conduct laboratory experiments to assess variation in survival of larval Boreal Chorus Frogs (Pseudacris maculata) reared on sediments from 3 local wetlands and a control source. To help provide environmental context for the experiment, we also measured chloride concentrations in 6 brine-contaminated wetlands in our study area, including the 2 contaminated sites used for sediment exposures. Survival of frog larvae during 46- and 55-day experiments differed by up to 88% among sediment sources (Site Model) and was negatively correlated with potential chloride exposure (Chloride Model). Five of the 6 contaminated wetlands exceeded the U.S. EPA acute benchmark for chloride in freshwater (860 mg/L) and all exceeded the chronic benchmark (230 mg/L). However, the Wetland Site model explained more variation in survival than the Chloride Model, suggesting that chloride concentration alone does not fully reflect the threat of contamination to aquatic species. Because the profiles of brine-contaminated sediments are complex, further surveys and experiments are needed across a broad range of conditions, especially where restoration or remediation actions have reduced brine-contamination. Information provided by this study can help quantify potential ecological threats and help land managers prioritize conservation strategies as part of responsible and sustainable energy development.

Understanding the mechanisms of ¹³⁷Cs movement across different ecosystems is crucial for projecting the environmental impact and management of nuclear contamination events. Here, we report differential movement of ¹³⁷Cs in adjacent forest and stream ecosystems. The food webs of the forest and stream ecosystems in our study were similar, in that they were both dominated by detrital-based food webs and the basal energy source was terrestrial litter. However, the concentration of ¹³⁷Cs in stream litter was significantly lower than in forest litter, the result of ¹³⁷Cs leaching from litter in stream water. The difference in ¹³⁷Cs concentrations between the two types of litter was reflected in the ¹³⁷Cs concentrations in the animal community. While the importance of ¹³⁷Cs fallout and the associated transfer to food webs has been well studied, research has been primarily limited to cases in a single ecosystem. Our results indicate that there are differences in the flow of ¹³⁷Cs through terrestrial and aquatic ecosystems, and that ¹³⁷Cs concentrations are reduced in both basal food resources and higher trophic animals in aquatic systems, where primary production is subsidized by a neighboring terrestrial ecosystem.

Climate projections over the next century include disproportionately warmer nighttime temperatures (“asymmetrical warming”). Cool nighttime temperatures lower metabolic rates of aquatic ectotherms. In contaminated waters, areas with cool nights may provide thermal refugia from high rates of daytime contaminant uptake. We exposed Cope's gray tree frogs (Hyla chrysoscelis), southern leopard frogs (Lithobates sphenocephalus), and spotted salamanders (Ambystoma maculatum) to five concentrations of a mixture of cadmium, copper, and lead under three to four temperature regimes, representing asymmetrical warming. At concentrations with intermediate toxicosis at test termination (96 h), temperature effects on acute toxicity or escape distance were evident in all study species. Asymmetrical warming (day:night, 22:20 °C; 22:22 °C) doubled or tripled mortality relative to overall cooler temperatures (20:20 °C) or cool nights (22:18 °C). Escape distances were 40–70% shorter under asymmetrical warming. Results suggest potentially grave ecological impacts from unexpected toxicosis under climate change.

Microplastics (MPs) have been reported to threaten a wide variety of terrestrial, marine, and freshwater organisms. However, knowledge about the effects of MPs on anuran amphibians, one of the most threatened taxa worldwide, is still limited. To assess the effects of MPs on the growth and survival of the Italian agile frog (Rana latastei) and green toad (Bufotes balearicus), we exposed tadpoles to three different concentrations (1, 7, and 50 mg L⁻¹) of an environmental relevant mixture of microplastics (HPDE, PVC, PS and PES), recording data on their activity level, weight and mortality rates. While the effects of MPs on green toad tadpoles were negligible, Italian agile frog tadpoles were severely affected both in terms of growth and activity level, with high mortality rates even at the lowest MP density (1 mg L⁻¹). Our results suggest that MP contamination of freshwater habitats may contribute to the ongoing decline of anuran amphibians.

Neonicotinoid pesticide use is widespread and highly debated, as evidenced by recent attention received from the public, academics and pesticide regulatory agencies. However, relatively little is known about the physiological effects of neonicotinoid insecticides on aquatic vertebrates. Amphibians (larval stages in particular) are excellent vertebrate bioindicators in aquatic systems due to their risk of exposure and sensitivity to environmental stressors. Previous work with wood frog (Rana sylvatica) tadpoles exposed to formulated products containing thiamethoxam or clothianidin in outdoor mesocosms found significant shifts in leukocyte profiles, suggesting the tadpoles were physiologically stressed. The main objective of the present study was to characterize this stress response further using complementary measures of stress after exposure to clothianidin on northern leopard frogs (Rana pipiens) during their aquatic larval stages. Laboratory static-renewal exposures were conducted over eight weeks with the technical product clothianidin at 0, 0.23, 1, 10 and 100 μg/L, and diquat dibromide at 532 μg/L was used as a positive control. We assessed tadpole leukocyte profiles and measures of oxidative stress as these sub-lethal alterations could affect amphibian fitness. We found changes in several types of leukocytes at 1 and 10 μg/L, suggesting that these tadpoles exhibited signs of mild physiological stress. Clothianidin also induced an oxidative stress response at 0.23, 1 and 100 μg/L. However, we found no differences in survival, growth, development time or hepatosomatic index in frogs exposed to clothianidin. Our study indicates that tadpoles chronically exposed to clothianidin have increased stress responses, but in the absence of concentration-response relationships and effects on whole-organism endpoints, the implications on the overall health and fitness of these changes are unclear.