The Effects of Ditch Management in Agroecosystems on Embryonic and Tadpole Survival, Growth, and Development of Northern Leopard Frogs (Lithobates pipiens).

Agricultural drainage ditches help remove excess water from fields and provide habitat for wildlife. Drainage ditch management, which includes various forms of vegetation clearing and sediment dredging, can variably affect the ecological function of these systems. To determine whether ditch conditions following dredging/vegetation clearing management affected the survival, growth, and development of embryos and tadpoles of northern leopard frogs (Lithobates pipiens), we conducted three field studies using in situ cages over 2 years. We measured nutrients, pesticides, and other water quality properties in vegetated/unmanaged (i.e., no clearing or dredging) and newly cleared/dredged (i.e., treeless, then dredged), clay-bottomed drainage ditches in a river basin in Eastern Ontario, Canada. Nutrients, atrazine, and total neonicotinoid concentrations were generally lower at the cleared/dredged sites, whereas glyphosate was at higher concentrations. In contrast, water-quality variables measured in situ, particularly temperature, dissolved oxygen, and turbidity, tended to be higher in the cleared/dredged sites. Total phosphorous and total organic carbon concentrations at all sites were above the recommended limits for amphibian assays. No significant differences were detected in the survival, hatching success, or development of embryos among the ditch management treatments, but premature hatching was observed at one vegetated/unmanaged site where high specific conductivity may have been formative. We found the cleared/dredged sites supported earlier tadpole growth and development, likely as a result of the higher water temperatures. Increased temperature may have offset other growth/development stressors, such as those related to water chemistry. However, the long-term consequences of these differences on amphibian populations requires further study.

Clothianidin alters leukocyte profiles and elevates measures of oxidative stress in tadpoles of the amphibian, Rana pipiens.

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.

Effects of two pesticides on northern leopard frog (Lithobates pipiens) stress metrics: Blood cell profiles and corticosterone concentrations.

Amphibians are declining globally. Exposure to pesticides has been implicated in decreasing amphibian immune function, thus increasing their susceptibility to parasites and disease and thereby negatively affecting individuals and populations. Amphibians are likely exposed to neonicotinoids because these widely used insecticides are highly soluble in water and because amphibian freshwater habitats are often embedded in agroecosystems. Herein, we investigate the effects of long-term exposure to two individual neonicotinoids (clothianidin or thiamethoxam) at either low or high concentrations (2.5 or 250 µg/L) on northern leopard frog (Lithobates pipiens) blood cell profiles and concentrations of corticosterone, an energy-mediating hormone associated with stress. Larval frogs from Gosner stage 25 to 46 were exposed to pesticide and control treatments in outdoor mesocosms. Corticosterone concentrations were measured after 6 d of exposure, and blood cell profiles were assessed once frogs reached Gosner stage 46 (following 8 w of exposure). No significant changes were found in erythrocyte counts, leukocyte counts, monocyte to leukocyte ratios or corticosterone concentrations between treatments. However, exposure to either 2.5 or 250 µg/L of clothianidin, or 250 µg/L of thiamethoxam decreased neutrophil to lymphocyte ratios and neutrophil to leukocyte ratios, and exposure to 2.5 µg/L of clothianidin or 250 µg/L of thiamethoxam decreased eosinophil to leukocyte ratios. Our results indicate that long-term exposure to neonicotinoids can alter leukocyte profiles, indicative of a stress response. Future studies should investigate whether chronic exposure to neonicotinoids affect multiple measures of stress differently or influences the susceptibility of amphibians to parasites and pathogens. Our work underscores the importance of continued use of multiple measures of stress for different amphibian species when undertaking ecotoxicological assessments.

Ecotoxicity assessment and bioconcentration of a highly brominated organophosphate ester flame retardant in two amphibian species.

Restrictions on the production and use of some highly toxic and persistent flame retardants has resulted in the increased use of alternative phosphate flame retardants that are less-well characterized. The brominated organophosphate ester flame retardant, tris(tribromoneopentyl) phosphate (CAS 19186-97-1, molecular formula C15H24Br9O4P, molecular weight 1018.47 g/mol, acronym TTBrNP) is a compound with potential to bioaccumulate and disrupt endocrine functions. To determine the toxicity of TTBrNP, two Canadian native amphibian species, Lithobates sylvaticus and L. pipiens, were acutely (embryos and Gosner stage 25 (GS25) tadpoles) or sub-chronically (GS25-41 tadpoles) exposed to the following nominal concentrations of TTBrNP: 0 (water and solvent controls), 30.6, 61.3, 122.5 and 245.0 µg/L. Note, measured concentrations declined with time (i.e., 118%-30% of nominal). There was high survival for both species after acute and sub-chronic exposures, where 75%-100% survived the exposures, respectively. There were no differences in the occurrence of abnormalities or hatchling size between controls and TTBrNP treatments for either species exposed acutely as embryos or tadpoles. Furthermore, after 30 d of sub-chronic exposure of L. pipiens tadpoles to TTBrNP there were no effects on size, developmental stage, liver somatic index or sex ratio. Bioconcentration factors were low at 26 ± 3.1 L/kg ww in tadpoles from all treatments, suggesting biotransformation or limited bioavailability via aquatic exposures. Thus, using two species of anurans at different early larval stages, we found TTBrNP up to 245 µg/L to have no overt detrimental effects on survival or morphological responses that would suggest fitness-relevant consequences.

Assessment of sublethal ecotoxicity of solvents on larvae of a model native amphibian (Lithobates pipiens).

Carrier solvents are used frequently in toxicity testing to assist hydrophobic chemicals into solution, but such solvents may have toxic effects on test subjects. Amphibians are model organisms in toxicity studies; however, little is known about the direct effects of solvents on native amphibians. Following modifications to standardized guidelines for native species, we used acute 96-hour exposures to assess the direct effects of three common solvents on survival, differences in morphology and occurrence of abnormalities of northern leopard frog larvae (Lithobates pipiens). The solvents, dimethyl sulfoxide (DMSO), ethanol (ETOH) and acetone (ACE) were used at nominal concentrations ranging from 1 to 100 µL/L. We also conducted a 30-day exposure to assess the direct chronic effects of DMSO at 1 and 5 µL/L, on larval growth, development and sex differentiation, but found no effects. Acute exposure to solvents also had no effect on the survival of larvae, but we found significant abnormalities in tadpoles acutely exposed to 100 µL/L ACE. Acute exposure to DMSO and ETOH had further concentration-dependent effects on larval morphological traits. Our study suggests that DMSO and ETOH at ≤20 µL/L may be used as solvents in amphibian ecotoxicological studies, but ACE should be limited to ≤50 µL/L in ecotoxicity studies and perhaps much less (≤10 µL/L) in studies with other amphibians, based on a review of existing literature. We emphasize pilot studies when using solvents on acute and chronic ecotoxicity tests, using native amphibians.