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Neonicotinoids are neurotoxic insecticides that are commonly applied to combat agricultural pests. Due to widespread application and select physicochemical characteristics, mixtures of different neonicotinoids are frequently detected in freshwater environments. This is of potential concern because these freshwater habitats are populated with ecologically important benthic macroinvertebrates (e.g. Chironomidae), which are markedly sensitive to neonicotinoid compounds. Despite the likelihood of continuous and/or repeated exposure, previous studies have primarily evaluated the individual toxicities of these neurotoxic compounds. Yet, little is known about how mixtures affect sensitive aquatic insects under real world exposure scenarios. Thus, the objectives of this research were to (1) evaluate acute and chronic toxicities of three commonly used neonicotinoids (imidacloprid (IMI), clothianidin (CLO), and thiamethoxam (TMX)) and their mixtures to Chironomidae using Chironomus dilutus as a representative test species, (2) validate single compound and neonicotinoid mixture toxicity predictions to Chironomidae populations under field settings, and (3) identify mechanisms behind species-, life stage-, and compound-specific differences in neonicotinoid toxicity for these sensitive aquatic insects. To address the first objective of this research, acute (96 h, endpoint = lethality) and chronic (28 d, endpoint = cessation of emergence) laboratory-based toxicity tests were carried out, characterizing the toxicities of IMI, CLO, TMX and their binary and ternary mixtures to larval C. dilutus. Using the MIXTOX approach (a statistical technique based on fitting mixture toxicity data to pre-defined mixture models), the nature and magnitude of cumulative toxicity was classified for each neonicotinoid mixture. Several mixtures were found to display cumulative toxicity that significantly deviated from direct, concentration-based additivity. Under acute exposure settings, all IMI-containing mixtures (IMI-CLO, IMI-TMX, and IMI-CLO-TMX) exhibited synergism when the concentrations of IMI in the solution were dominant (up to 7 %, 28 %, and 6 % decreases in survival, respectively), and some mixtures (IMI-CLO and IMI-TMX) displayed antagonism when the other mixture constituent was dominant (up to 19 % and 30 % increases in survival, respectively). Under chronic exposure settings all binary mixtures demonstrated dose-ratio dependent deviation from direct additivity (concentration addition), displaying synergism at high concentrations of CLO (IMI-CLO: 13 % decrease in emergence) or TMX (CLO-TMX and IMI-TMX: 2 % and 4 % decreases in emergence, respectively) and antagonism at high concentrations of IMI (IMI-CLO and IMI-TMX: 5 %, and 2 % increases in emergence, respectively). Under chronic exposures, the ternary mixture (IMI-CLO-TMX) elicited an overall antagonistic effect (2 % increase in emergence). Thus, laboratory-derived bioassays indicated that under both acute and chronic exposure settings, neonicotinoid mixtures had the potential to display cumulative toxicities that deviated from direct additivity. Furthermore, although toxicities of neonicotinoid mixtures were not exactly parallel across different exposure settings, acute tests generally predicted which mixtures were likely to display significant synergism under chronic exposure settings. To determine if laboratory-derived predictions could be used to estimate the toxicities of neonicotinoids and their mixtures under more environmentally realistic exposure settings (Objective 2), chronic (56 day), semi-controlled field studies were carried out in a natural wetland in Saskatchewan’s Prairie Pothole Region. Using in situ limnocorrals fitted with emergence traps, the effects of predicted equitoxic concentrations of IMI, CLO, TMX, and their binary mixtures (concentrations equivalent to 28 d EC50 values; mixtures at 1:1 ratio) were characterized for all emerged aquatic insects (endpoint: abundance) and Chironomidae (endpoint: abundance, biomass, and sex ratios) at the community level. In all treated limnocorrals, there were subtle shifts in insect community composition. Furthermore, at concentrations tested, neonicotinoids and their mixtures significantly impacted Chironomidae abundance and biomass. However, contrary to laboratory predictions, IMI-CLO and IMI-TMX mixtures did not elicit greater-than-additive effects. Furthermore, exposure to IMI, CLO, TMX, and CLO-TMX elicited greater-than-expected declines in Chironomidae abundance and biomass. In addition, CLO significantly shifted sex-ratios of emerged Chironomidae towards female-dominated populations. Thus, although laboratory-derived toxicity estimates could adequately predict relative effects of IMI, CLO, and TMX on Chironomidae populations (e.g. toxicity: IMI ≥ CLO >> TMX), they frequently underestimated the magnitudes of single-compound and neonicotinoid mixture effects under semi-controlled field settings. To better characterize patterns of observed toxicity (e.g. differences among compounds, species, and life-stages), the binding properties of IMI, CLO, and TMX to their molecular target (nicotinic acetylcholine receptors (nAChRs)) were investigated in Chironomidae (Objective 3). Using radioligand binding studies with tritium-labeled IMI ([3H]-IMI) and unlabeled competitors (IMI, CLO, and TMX), nAChR density and neonicotinoid binding affinity were characterized for and compared across two species (C. dilutus and Chironomus riparius) at two different life stages (larval and adult). Despite marked differences in neonicotinoid toxicity, there were no significant species-specific differences in neonicotinoid binding or nAChR density. However, there were life stage-specific differences in nAChR density and binding, and compound-specific differences in binding affinity that reflected previously described patterns in neonicotinoid toxicity (e.g. higher larval sensitivity and relative toxicity of IMI ≥ CLO >> TMX). Furthermore, compared to other insects, Chironomidae displayed relatively high densities of nAChRs with high neonicotinoid affinity, which reflected their sensitivity to these insecticides. Ultimately this work provides a comprehensive characterization of the toxicity of three commonly used neonicotinoid insecticides (IMI, CLO, and TMX) and their mixtures to the sensitive aquatic insect group, Chironomidae. This can help inform regulators and risk assessors focused on assessing risks of neonicotinoids in freshwater environments. Furthermore, by characterizing effects at three levels of biological organization (molecular, individual, and communities), this work provides a basis through which a relative toxicity pathway could be formed, highlighting techniques that could be potentially used to predict large-scale effects for Chironomidae inhabiting neonicotinoid-contaminated aquatic environments. Finally, this work highlights areas worthy of further investigation and provides methodology through which these studies can be carried out, including the characterization of the binding properties and/or expression profiles of nAChRs for other neonicotinoid-sensitive aquatic insects, evaluation of the nAChR binding profiles for other nAChR agonists (e.g. other neonicotinoids, sulfoximines, and butenolides), and further characterization of nAChR binding profiles in Chironomidae (e.g. with α-bungarotoxin or epibatidine) to allow for a more comprehensive, mechanistic understanding of neonicotinoid mixture toxicity.



Neonicotinoid, mixture toxicity



Doctor of Philosophy (Ph.D.)


Toxicology Centre




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