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.

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.

Determining in situ periphyton community responses to nutrient and atrazine gradients via pigment analysis.

Agrochemicals, including fertilizers and herbicides, are significant contributors of non-point source pollution to surface waters and have the potential to negatively affect periphyton. We characterized periphyton communities using pigment markers to assess the effects of nutrient enrichment and the herbicide atrazine with in situ experimental manipulations and by examining changes in community structure along existing agrochemical gradients. In 2008, the addition of nutrients (20 mg/L nitrate and 1.25 mg/L reactive phosphate), atrazine (20 µg/L) and a combination of both nutrients and atrazine had no significant effect on periphyton biomass or community structure in a stream periphytometer experiment. In 2009, similar experiments with higher concentrations of atrazine (200 µg/L) at two stream sites led to some minor effects. In contrast, at the watershed scale (2010) periphyton biomass (mg/m(2) chlorophyll a) increased significantly along correlated gradients of nitrate and atrazine but no direct effects of reactive phosphate were observed. Across the watershed, the average periphyton community was composed of Bacillariophyceae (60.9%), Chlorophyceae (28.1%), Cryptophyceae (6.9%) and Euglenophyceae (4.1%), with the Bacillariophyceae associated with high turbidity and the Chlorophyceae with nitrate enrichment. Overall, effects of nitrate on periphyton biomass and community structure superseded effects of reactive phosphate and atrazine.

Atrazine contamination at the watershed scale and environmental factors affecting sampling rates of the polar organic chemical integrative sampler (POCIS).

Polar organic chemical integrative samplers (POCIS) were used to estimate atrazine contamination at 24 stream/river sites located across a watershed with land use ranging from 6.7 to 97.4% annual crops and surface water nitrate concentrations ranging from 3 to 5404 µg/L. A gradient of atrazine contamination spanning two orders of magnitude was observed over two POCIS deployments of 28 d and was positively correlated with measures of agricultural intensity. The metabolite desisopropyl atrazine was used as a performance reference compound in field calibration studies. Sampling rates were similar between field sites but differed seasonally. Temperature had a significant effect on sampling rates while other environmental variables, including water velocity, appeared to have no effect on sampling rates. A performance reference compound approach showed potential in evaluating spatial and temporal differences in field sampling rates and as a tool for further understanding processes governing uptake of polar compounds by POCIS.

Comparing the sensitivity of geographically distinct Lemna minor populations to atrazine.

The objectives of this study were to compare the sensitivities of field populations and a laboratory culture of a duckweed species (Lemna minor) to the herbicide atrazine using three different endpoints and to determine whether sensitivity to atrazine was affected by past exposure to the herbicide. L. minor cultures were purchased commercially or collected from field sites within an agricultural watershed and exposed to atrazine for 7 days under greenhouse conditions. Populations differed significantly in their sensitivity to atrazine. Biomass was more sensitive than frond number, while chlorophyll fluorescence was not a sensitive endpoint. Overall, the sensitivity of the various populations to atrazine was not strongly related to measures of past exposure to agriculture stressors. Positive correlations between biomass twenty-five percent inhibition concentrations (IC25s), biomass estimated marginal means and in-stream atrazine concentrations were observed, providing evidence that atrazine exposure is linked to a decrease in sensitivity to atrazine. However, IC25s generated for each population were similar, ranging from 19 to 40 and 57 to 92 µg/L atrazine for biomass and frond data respectively, and likely do not represent biologically significant differences in atrazine sensitivity. Given the small range in sensitivity observed between populations, commercial laboratory cultures appear to provide a good estimate of the sensitivity of field populations of L. minor to atrazine and should continue to be used in regulatory phytotoxicity testing.

Comparison of the effects of glyphosate and atrazine herbicides on nontarget plants grown singly and in microcosms.

Intensification of agriculture and the corresponding increase in herbicide use has led to concern regarding the effects these chemicals may have on nontarget plants of agroecosystems. Current pesticide registration guidelines are focused on testing crop species grown singly in pots under greenhouse conditions and may not provide adequate measures of protection to noncrop species. The objective of the present study was to compare the response of terrestrial and wetland plants to the herbicides glyphosate and atrazine when grown singly in pots versus under different microcosm conditions. Greenhouse microcosms were generally more sensitive than single-species tests. Plants grown for an extended test period or in seminatural field conditions were generally less sensitive to herbicides. Sensitivity was found to be dependent on interactions between species and test conditions. Changes in community structure were observed in herbicide-treated microcosms that would not be predicted from single-species testing. Single-species tests are useful because they are inexpensive, can demonstrate clear dose-response patterns uncomplicated by other factors influencing growth, and are able to provide a measure of the sensitivity of a given species to glyphosate and atrazine. However, they are unable to predict subtle changes in community structure that may have important long-term consequences.

Germination requirements for 29 terrestrial and wetland wild plant species appropriate for phytotoxicity testing.

BACKGROUND: Species selected for phytotoxicity testing have been limited to a few standard crop species owing to restrictive recommendations at the regulatory level. However, guidelines by the Organisation for Economic Development and Cooperation (OECD) were recently amended in 2006 to include a list of herbaceous non-crop plant species suitable for testing. The objective of this study was to outline the optimum germination requirements for a selection of wild species for which seeds were readily available from commercial suppliers. RESULTS: Of the 29 herbaceous terrestrial and wetland species included in this study, all achieved 50% germination and 23 reached > 70% germination to meet the criterion outlined in the OECD guidelines. Most species attained their maximum germination within 14 days or less. Cold stratification of imbibed seeds improved germination for 14 species. Increasing sowing soil depth did not improve seed germination. The variance attained in this experiment between replicates was low, especially for species with > 70% germination (standard error approximately 5%). CONCLUSION: The present study showed that 23 of the 29 species tested required minimal pretreatments and produced consistent, reliable and uniform germination reaching at least 70%. The inclusion of wild plant species in regulatory testing should be given real consideration.