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.

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.

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 2 Neonicotinoid Insecticides on Blood Cell Profiles and Corticosterone Concentrations of Wood Frogs (Lithobates sylvaticus).

Neonicotinoids are widely used insecticides that are detectable in agricultural waterways. These insecticides are of concern due to their potential impacts on nontarget organisms. Pesticides can affect development of amphibians and suppress the immune system, which could impact disease susceptibility and tolerance. No previous studies on amphibians have examined the effects of these insecticides on differential blood cell proportions or concentrations of corticosterone (a general stress hormone). We investigated the effects of chronic exposure to 2 neonicotinoids, thiamethoxam and clothianidin, on immunometrics of wood frogs (Lithobates sylvaticus). Frogs were exposed to single, chronic treatments of 2.5 or 250 µg/L of clothianidin or thiamethoxam for 7 wk from Gosner stages 25 to 46. The juvenile frogs were then maintained for 3 wk post metamorphosis without exposure to neonicotinoids. We measured water-borne corticosterone twice: at 6 d and 8 wk after exposure in larval and juvenile frogs, respectively. We assessed differential blood cell profiles from juvenile frogs. Corticosterone was significantly lower in tadpoles exposed to 250 µg/L of thiamethoxam compared with other tadpole treatments, but no significant differences in corticosterone concentrations were found in treatments using juvenile frogs. Anemia was detected in all treatments compared with controls with the exception of tadpoles exposed to 2.5 µg/L of clothianidin. Neutrophil-to-leukocyte and neutrophil-to-lymphocyte ratios were elevated in frogs exposed to 250 µg/L of thiamethoxam. Collectively, these results indicate that chronic exposure to neonicotinoids has varied impacts on blood cell profiles and corticosterone concentrations of developing wood frogs, which are indicative of stress. Future studies should investigate whether exposure to neonicotinoids increases susceptibility to infection by parasites in both larval and adult wood frogs. Environ Toxicol Chem 2019;38:1273-1284. © 2019 Crown in the right of Canada. Published by Wiley Periodicals Inc. on behalf of SETAC.

What killed Frame Lake? A precautionary tale for urban planners.

Frame Lake, located within the city of Yellowknife, Northwest Territories, Canada, has been identified as requiring significant remediation due to its steadily declining water quality and inability to support fish by the 1970s. Former gold mining operations and urbanization around the lake have been suspected as probable causes for the decline in water quality. While these land-use activities are well documented, little information is available regarding their impact on the lake itself. For this reason, Arcellinida, a group of shelled protozoans known to be reliable bioindicators of land-use change, were used to develop a hydroecological history of the lake. The purpose of this study was to use Arcellinida to: (1) document the contamination history of the lake, particularly related to arsenic (As) associated with aerial deposition from mine roaster stacks; (2) track the progress of water quality deterioration in Frame Lake related to mining, urbanization and other activities; and (3) identify any evidence of natural remediation within the lake. Arcellinida assemblages were assessed at 1-cm intervals through the upper 30 cm of a freeze core obtained from Frame Lake. The assemblages were statistically compared to geochemical and loss-on-ignition results from the core to document the contamination and degradation of conditions in the lake. The chronology of limnological changes recorded in the lake sediments were derived from 210Pb, 14C dating and known stratigraphic events. The progress of urbanization near the lake was tracked using aerial photography. Using Spearman correlations, the five most significant environmental variables impacting Arcellinida distribution were identified as minerogenics, organics, As, iron and mercury (p < 0.05; n = 30). Based on CONISS and ANOSIM analysis, three Arcellinida assemblages are identified. These include the Baseline Limnological Conditions Assemblage (BLCA), ranging from 17-30 cm and deposited in the early Holocene >7,000 years before present; the As Contamination Assemblage (ACA), ranging from 7-16 cm, deposited after ∼1962 when sedimentation began in the lake again following a long hiatus that spanned to the early Holocene; and the Eutrophication Assemblage (EA), ranging from 1-6 cm, comprised of sediments deposited after 1990 following the cessation of As and other metal contaminations. The EA developed in response to nutrient-rich waters entering the lake derived from the urbanization of the lake catchment and a reduction in lake circulation associated with the development at the lake outlet of a major road, later replaced by a causeway with rarely open sluiceways. The eutrophic condition currently charactering the lake-as evidenced by a population explosion of eutrophication indicator taxa Cucurbitella tricuspis-likely led to a massive increase in macrophyte growth and winter fish-kills. This ecological shift ultimately led to a system dominated by Hirudinea (leeches) and cessation of the lake as a recreational area.