Multiple stressors are linked to declines of insects and important pollinators, such as bees. Recently, interactive effects of multiple agrochemicals on bees have been highlighted, including fungicides, which increase toxicity of neonicotinoid insecticides. Here, we use a semi-field study across two seasons in controlled foraging tunnels to test the effects of a field application of a commercial fungicide product with two active ingredients (pyraclostrobin and metconazole) applied at label rates. We also examine its interactive effects with the neonicotinoid insecticide clothianidin, at a conservative field-realistic dose of 2.23 ppb, on 48 honey bee colonies. We found combined effects of pesticide exposure, including additive 2.93-fold increases in mortality, and an additional effect of increased infestation levels of the ectoparasitic mite, Varroa destructor. Pesticide treatments also reduced colony activity, reduced colony weight, and increased sugar consumption of whole colonies. These findings indicate that typical sublethal exposure levels to common, co-occurring agrochemicals in the field significantly affect the health of whole honey bee colonies, highlighting an unintended consequence of increasing pesticide applications.

Pesticides, especially the newly developed neonicotinoids, are increasingly used in many countries around the world, including Cameroon, to control pests involved in crop destruction or disease transmission. Unfortunately, the pesticides also pose tremendous environmental problems because a predominant amount of their residues enter environmental matrices to affect other nontargeted species including humans. This therefore calls for continuous biomonitoring of these insecticides in human populations. The present study sought to assess the neonicotinoid insecticide exposures in two agrarian regions of Cameroon, the South-West region and Littoral region. The study involved 188 men, including 125 farmers and 63 nonfarmers. Spot urine samples were obtained from these subjects and subjected to liquid chromatographic-tandem mass spectrometric analysis for concentrations of neonicotinoid compounds, including acetamiprid, clothianidin, dinotefuran, imidacloprid, thiacloprid, nitenpyram, thiamethoxam, and N-dm-acetamiprid. Neonicotinoid compounds were detected in all study participants, and residues of all the screened pesticides were detected among participants. N-dm-Acetamiprid and imidacloprid were the most prevalent among the subjects (100.0% and 93.1%, respectively), whereas nitenpyram was less common (3.2%). The median values of imidacloprid and total urinary neonicotinoid concentrations were elevated among farmers (0.258 vs. 0.126 µg/L and 0.829 vs. 0.312 µg/L, respectively). Altogether the findings showed that both the farmer and nonfarmer study populations of Cameroon were exposed to multiple residues of neonicotinoids, with relatively higher levels of pesticides generally recorded among farmers. Although exposure levels of the neonicotinoids were generally lower than their respective reference doses, these results warrant further research on the health risk evaluation of multiple residues of the pesticides and reinforcement of control measures to minimize the exposure risks, especially among farmers. Environ Toxicol Chem 2024;43:952-964. © 2024 SETAC.

BACKGROUND: Recent studies have reported the effect of neonicotinoid (NEO) exposures on development in human. However, information about the risk of childhood development delays due to NEO exposures is limited.
OBJECTIVE: The study aimed to examine the association between NEO exposure and child development up to 4 years of age using data of the Japan Environment and Children\'s Study.
METHODS: The study employed urinary NEO and metabolite concentrations in the first and second or third trimesters; the Japanese translation of the Ages and Stages Questionnaire (third edition; J-ASQ-3) scores on developmental delay in five domains, namely communication, gross motor skills, fine motor skills, problem solving, and personal-social characteristics from 6 months to 4 years of age; and self-reported questionnaire data. The associations between urinary NEO concentrations and J-ASQ-3 results were analysed using the treed distributed lag mixture model. A total of 8538 participants were included in statistical analyses.
RESULTS: The determination rates of urinary acetamiprid-N-desmethyl (dm-ACE), clothianidin (CLO), dinotefuran (DIN) and thiamethoxam (THX) were greater than 50%. Median urinary dm-ACE, CLO, DIN and THX concentrations were 0.34, 0.14, 0.22 and 0.05 ng/ml, respectively, in samples collected during gestational weeks < 23, and 0.28, 0.12, 0.18 and 0.04 ng/ml, respectively, in those collected during gestational weeks ≥ 23. The binomial scores divided by the cut-off values of the J-ASQ were used in the treed distributed lag mixture model. The highest percentage for a domain with a value less than the cut-off value was \'problem solving\' at 6 months of age among all the J-ASQ-3 scores (10.5%). There was no statistically significant association between maternal urinary dm-ACE, CLO, DIN and THX concentrations during pregnancy and the J-ASQ-3 results up to 4 years of age. Objective assessment of child development in different populations may be warranted to confirm our findings.

Thiamethoxam is a neonicotinoid that has been used to control insect pests. The literature reports a few behavioral studies evaluating the toxic effect of thiamethoxam in ants; however, there are scarce studies at the cellular level. The present research evaluated the effects of thiamethoxam in labial (LG) and mandibular glands (MG), fat bodies (FB), and Malpighian tubules (MT) of workers of Atta sexdens, using transmission electron microscopy. The duct and secretory cells of LG were profoundly affected, then the production of saliva can be compromised, as well as its quality and subsequent use. In MG, reservoir and canaliculi cells presented slight alterations; however, MG secretory cells presented vacuoles containing lamellar structures, increased lipid production, and a large amount of mitochondria, which may lead to organ\'s malfunctioning. The FB cell alterations do not seem enough to cause significant changes that lead to cell death. Prominent changes in MT, such as loss of the electron-dense concentric ring, increased smooth endoplasmic reticulum, loss of basal infolds, vacuoles containing mineralized granules, and lamellar structures associated with mitochondria, suggest that their excretory function is compromised. In conclusion, thiamethoxam acts not only in the nervous system but also contributes to systemic toxicity on the target organism.

Exposure to insecticides containing organophosphate (OP) and neonicotinoid (NEO) compounds has been associated with adverse reproductive health outcomes. This study characterized and identified predictors of exposure to OP and NEO among 100 reproductive-age farmworkers from two intensive farming areas in Chiang Mai Province, Thailand, including 50 each from the Fang (FA) and Chom Thong (CT) districts. OP exposure was determined by measuring the urinary concentrations of six dialkylphosphates (DAPs), whereas NEO exposure was determined by measuring the urinary concentrations of NEO compounds and their metabolites (NEO/m). The most frequently detected OPs were diethylphosphate (DEP) and diethylthiophosphate (DETP), with DETP having the highest geometric mean (GM) concentration, 8.9 μg/g-creatinine. The most frequently detected NEO/m were N-desmethyl-acetamiprid (N-dm-ACE), imidacloprid (IMI), and thiamethoxam (THX), with IMI having the highest GM concentration, 8.7 μg/g-creatinine. Consumption of well water was the predominant determinant of OP and NEO exposure in this population. In addition to encouraging workers to use personal protective equipment, exposure of farmworkers to these compounds may be reduced by nation-wide monitoring agricultural insecticides and other pesticides in community drinking water resources.

The dispersion of clothianidin from treated seeds was studied in a commercial winter canola field. During planting, using a John Deere 1890 single disk air seeder, a proportion, an estimated 14.2 ± 2.9% (mean ± standard error), of the clothianidin that was applied to the seed escaped into the atmosphere from the seeder\'s exhaust. We suggest that this source of environmental contamination may be the main contributor that explains the off-target detection of neonicotinoid residues in soils and water near canola plantings better than movement from seed after it is placed in the soil, which is often proposed in the literature. Environ Toxicol Chem 2020;39:2420-2423. © 2020 SETAC.

Despite the restriction of the use of neonicotinoids in the EU, including thiamethoxam and clothianidin, the debate over their risk on honey bees has not been fully settled. This study presents results of a three-year study working with 180 honey bee colonies in ten replicates. Colonies were sorted into three treatments (60 colonies per treatment) exposed to sunflower blooms grown from seeds treated with thiamethoxam, clothianidin and a non-treated control. Each colony was assessed at six moments: one before to exposition to sunflower, two during the exposition (short-time risk), two after exposition (medium-time risk) and one after wintering (long-time risk). The health and development of the colonies were assessed by monitoring adult bee population, brood development, status of the queen, food reserves and survival. No significant difference among treatments when raw data was considered. However, when evolution from initial status of the colony was evaluated, a significant difference was observed from the first week of exposure to sunflower blooms. In this period, the number of adult bees and the amount of brood were slightly lower in the bee hives exposed to neonicotinoids, although such differences disappeared in subsequent evaluations. The concentration of residues in samples of beebread and adult bees was at the level of ng·g-1. Magnitude of the effect of the treatment factor on the variability of colony health and development related parameters was low. The most important factor was the hive, followed by the replicate and year, and to a lesser extent the initial strength of the colonies.

Thiamethoxam is a neonicotinoid insecticide used widely in agriculture to control a broad spectrum of insect pests. To assess potential risks from this compound to non-target aquatic organisms, an outdoor mesocosm study was performed. Mesocosms (1300 L) were treated once with a formulated product with the active substance (a.s.) thiamethoxam at nominal concentrations of 1 (n = 3), 3 (n = 3), 10 (n = 4), 30 (n = 4), and 100 (n = 2) μg a.s./L, plus untreated controls (n = 4). Primary producers (phytoplankton), zooplankton, and macroinvertebrates were monitored for up to 93 days following treatment. Thiamethoxam was observed to have a water column dissipation half-life (DT50) of ≤1.6-5.2 days in the mesocosms. Community-based principal response curve analysis detected no treatment effects for phytoplankton, zooplankton, emergent insects, and macroinvertebrates, indicating a lack of direct and indirect effects. A number of statistically significant differences from controls were detected for individual phytoplankton and zooplankton species abundances, but these were not considered to be treatment-related due to their transient nature and lack of concentration-response. After application of 30 μg a.s./L, slight temporary effects on Asellus aquaticus could not be excluded. At 100 μg a.s./L, there was an effect with no clear recovery of Asellus observed, likely due to their inability to recolonize these isolated test systems. A statistically significant but transient reduction in the emergence of chironomids by day 23 at the 100 μg a.s./L treatment was observed and possibly related to direct toxicity from thiamethoxam on larval stages. Therefore, a conservative study specific No Observed Ecological Adverse Effect Concentration (NOEAEC) is proposed to be 30 μg a.s./L. Overall, based on current concentrations of thiamethoxam detected in North American surface waters (typically <0.4 μg/L), there is low likelihood of direct or indirect effects from a pulsed exposure on primary producers, zooplankton, and macroinvertebrates, including insects, as monitored in this study.

Acetylcholine binding proteins (AChBPs) are homologs of extracellular domains of nicotinic acetylcholine receptors (nAChRs) and serve as models for studies on nAChRs. Particularly, studies on invertebrate nAChRs that are limited due to difficulties in their heterologous expression have benefitted from the discovery of AChBPs. Thus far, AChBPs have been characterized only in aquatic mollusks, which have shown low sensitivity to neonicotinoids, the insecticides targeting insect nAChRs. However, AChBPs were also found in spiders based on the sequence and tissue expression analysis. Here, we report five AChBP subunits in Pardosa pseudoannulata, a predator enemy against rice insect pests. Spider AChBP subunits shared higher sequence similarities with nAChR subunits of both insects and mammals compared with mollusk AChBP subunits. The AChBP1 subunit of P. pseudoannulata (Pp-AChBP) was then expressed in Sf9 cells. The Ls-AChBP from Lymnaea stagnalis was also expressed for comparison. In both AChBPs, one ligand site per subunit was present at each interface between two adjacent subunits. Neonicotinoids had higher affinities (7.9-18.4 times based on Kd or Ki values) for Pp-AChBP than for Ls-AChBP, although epibatidine and α-bungarotoxin showed higher affinities for Ls-AChBP. These results indicate that spider AChBP could be used as an alternative model to study the interaction between insect nAChRs and neonicotinoids.

Non-target organisms, including human and wildlife, are susceptible to deleterious effects of pesticide mixtures in their environment. Present study demonstrated the disruption of the hypothalamic-pituitary-thyroid (HPT) axis in a seasonally breeding wildlife bird Amandava amandava on co-exposure to dithiocarbamate mancozeb/MCZ and neonicotinoid imidacloprid/IMI, at concentrations even lower than respective environmentally realistic exposure level of each of the pesticide. Adult male birds (n=8/group) were exposed individually to 0.25% LD50 of each of MCZ (0.14mg) and IMI (2.75μl) followed by co-exposure to their equimixture as MIX-I (0.25% LD50 of each) and MIX-II (0.5% LD50 of each) through food for 30d in preparatory phase of reproductive cycle. Disruptions of thyroid gland and pituitary-thyroid axis were evident. Altered thyroid weight and volume, follicles with inactive colloids and lesions, decrease of height and nucleus-to-cytoplasm ratio of follicular epithelial cells were noted. Plasma levels of T4 and T3 were decreased, more significant in mixture groups than in individually exposed groups. Within co-exposed groups, comparatively high plasma T4 and T3 levels in MIX-II than MIX-I indicated dose non-responsiveness of the pesticides in mixtures; a characteristic displayed by endocrine disrupters. Plasma TSH level was increased in MCZ- and IMI- but decreased in MIX-I and MIX-II suggesting the disruption of the negative feedback and impairment of the HPT axis in co-exposed groups. Effects were more prominent in co-exposed groups due to combinatorial action and cumulative toxicity of pesticides. Considering the role of thyroid hormones in reproductive development, pesticides even in low dose could affect the thyroid homeostasis and reproductive axis.