Neonicotinoid pesticides are a relatively new class of pesticides that have garnered significant attention owing to their potential ecological risks to nontarget organisms. A method combining solid phase extraction with liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) was developed for the rapid and accurate detection of eight neonicotinoid pesticides (dinotefuran, E-nitenpyram, thiamethoxam, clothianidin, imidacloprid, imidaclothiz, acetamiprid, and thiacloprid) in wastewater. The chromatographic mobile phase and MS parameters were selected, and a single-factor method was used to determine the optimal column type, extraction volume, sample loading speed, and pH for SPE. The optimal parameters were as follows: column type, HLB column (500 mg/6 mL); sample extraction volume, 500 mL; sample loading speed, 10 mL/min; and sample pH, 6-8. The matrix effects of the wastewater samples were reduced by optimizing the chromatographic gradient-elution program, examining the dilution factor of the samples, and using the isotope internal standard calibration method. Prior to analysis, the wastewater samples were diluted 5-fold with ultrapure water for pretreatment. Subsequently, 2 mmol/L ammonium acetate aqueous solution containing 0.1% (v/v) formic acid and methanol was used as mobile phases for gradient elution on a ZORBAX Eclipse Plus C18 column (100 mm×2.1 mm, 1.8 μm). The samples were quantified using positive-ion multiple reaction monitoring (MRM) mode for 10 min. Imidacloprid-d4 was used as the isotope internal standard. The SPE process was further optimized by applying response surface methodology to select the type and mass of rinsing and elution solvents. The optimal pretreatment of the SPE column included rinsing with 10% methanol aqueous solution and elution with methanol-acetonitrile (1∶1, v/v) mixture (7 mL). The eight neonicotinoid pesticides showed satisfactory linearity within the relevant range, with linear correlation coefficients (r) all greater than 0.9990. The method detection limits (MDLs) ranged from 0.2 to 1.2 ng/L, and the method quantification limits (MQLs) ranged from 0.8 to 4.8 ng/L. The average recoveries of the eight neonicotinoid pesticides were in the range of 82.6%-94.2% at three spiked levels, with relative standard deviations (RSDs) ranging from 3.9% to 9.4%. Finally, the optimized method was successfully applied to analyze wastewater samples collected from four sewage treatment plants. The results indicated that the eight neonicotinoid pesticides could be generally detected at concentrations ranging from not detected (ND) to 256 ng/L. The developed method has a low MDL and high accuracy, rendering it a suitable choice for the trace detection of the eight neonicotinoid pesticides in wastewater when compared with other similar methods. The proposed method can be utilized to monitor the environmental impact and assess the potential risks of neonicotinoid pesticides in wastewater, thus promoting the protection of nontarget organisms and the sustainable use of these pesticides in agriculture.

Cytochrome P450 monooxygenases (P450s) and UDP-glycosyltransferases (UGTs) are involved in the evolution of insecticide resistance. Leptinotarsa decemlineata (Say), the Colorado potato beetle (CPB), is a notorious insect that has developed resistance to various insecticides including neonicotinoids. This study investigated whether the differentially expressed P450 genes CYP9Z140 and CYP9AY1 and UGT gene UGT321AP1, found in our transcriptome results, conferred resistance to thiamethoxam in L. decemlineata. Resistance monitoring showed that the sampled field populations of L. decemlineata adults collected from Urumqi City and Qapqal, Jimsar, and Mulei Counties of Xinjiang in 2021-2023 developed low levels of resistance to thiamethoxam with resistance ratios ranging from 6.66- to 9.52-fold. Expression analyses indicated that CYP9Z140, CYP9AY1, and UGT321AP1 were significantly upregulated in thiamethoxam-resistant populations compared with susceptible populations. The expression of all three genes also increased significantly after thiamethoxam treatment compared with the control. Spatiotemporal expression patterns showed that the highest expression of CYP9Z140 and CYP9AY1 occurred in pupae and the midgut, whereas UGT321AP1 was highly expressed in adults and Malpighian tubules. Knocking down all three genes individually or simultaneously using RNA interference increased the sensitivity of adult L. decemlineata to thiamethoxam. These results suggest that overexpression of CYP9Z140, CYP9AY1, and UGT321AP1 contributes to the development of thiamethoxam resistance in L. decemlineata and provides a scientific basis for improving new resistance management of CPB.

Ecological risk assessments of agrochemicals have traditionally depended on in vivo guideline tests using northern bobwhite and mallard to provide relevant endpoints for avian species. However, these studies have limitations, including animal welfare concerns, the time and cost involved, limited potential for extrapolation to more realistic exposure conditions, and the lack of mechanistic understanding. The proof-of-concept work presented a case study for thiamethoxam in three avian species, demonstrating the potential of physiologically based kinetic (PBK) modeling to enable dosimetry extrapolations that inform hazard characterization in risk assessment, and reduce the use of avian testing. The model structure for northern bobwhite and mallard contained ten compartments, while an additional ovulation model was included for chicken in the physiological state of egg-laying. The model was first parameterized and evaluated for chicken and northern bobwhite using in vitro kinetic measurements and in vivo toxicokinetic (TK) data. The chicken model was then extrapolated to mallard based on allometric scaling. The models were then used to map the TK profiles across species by simulating internal dose metrics in different avian toxicology studies. These metrics, including peak blood concentrations (Cmax) and area under the curve (AUC) for blood concentration, were determined for acute, subacute, or chronic toxicity endpoints for mallard and northern bobwhite, enabling a quantitative cross-species and cross-route comparison of dosimetry. The results suggested that the chronic toxicological response of birds exposed to thiamethoxam is highly dependent on internal exposure, while mallard appeared to be more dynamically sensitive to thiamethoxam on an acute oral exposure basis. The case study increases the confidence in using new approach methodologies (NAMs) for interpreting avian toxicity studies and facilitating in vitro-in silico-based ecological risk assessments of agrochemicals.

Neonicotinoid insecticides move from targeted crops to wildflowers located in adjacent field margins, acting as a potential exposure source for wild pollinators and insect species of conservation concern, including monarch butterflies. Monarchs rely on milkweed over multiple life stages, including as a host plant for eggs and a food source for both larvae (leaves) and adults (flowers). Milkweeds, which are closely associated with field margins, can contain neonicotinoid residues, but previous assessments are constrained to a single plant tissue type. In 2017 and 2018, we sampled milkweeds from 95 field margins adjacent to crop fields (corn, soybean, hay, wheat, and barley) in agricultural landscapes of eastern Ontario, Canada. Milkweeds were sampled during the flower blooming period and leaves and flower tissues were analysed. The neonicotinoids acetamiprid, clothianidin, thiamethoxam, and thiacloprid were detected. Maximum concentrations in leaf samples included 10.30 ng/g of clothianidin in 2017, and 24.4 ng/g of thiamethoxam in 2018. Clothianidin and thiamethoxam percent detections in flowers (72 % and 61 %, respectively) were significantly higher than detections in leaves (24 % and 31 %, respectively). Thiamethoxam concentrations were significantly higher in paired flower samples than leaf samples (median 0.33 ng/g vs <0.07 ng/g) while clothianidin concentrations also trended higher in flowers (median 0.18-0.55 ng/g vs <0.18 ng/g). Only thiamethoxam showed significant differences between years, and we found no effect of crop type, with hay, soybean and corn fields all yielding 50-56 % detections in leaves. We found significantly higher concentrations in older milkweed flowers than young flowers or leaves (medians 0.87 ng/g vs <0.18 ng/g and 0.45 ng/g vs <0.07 ng/g for clothianidin and thiamethoxam, respectively). Our results highlight the importance of considering variation in milkweed tissue type and age of flowers in neonicotinoid exposure risk assessments. Efforts to increase milkweed availability in agricultural landscapes should consider how exposure to neonicotinoids can be mitigated.

Although the potential effects of neonicotinoids (NEOs) in early life have received considerable attention, data on the exposure of mothers and infants to NEOs are scarce. In this study, four parent NEOs and one metabolite were widely detected in paired maternal serum (MS), umbilical cord serum (UCS) and breast milk (BM) samples, with median total NEO concentrations (ΣNEOs) of 113, 160 and 69 ng/L, respectively. Decreasing trends were observed for N-desmethyl-acetamiprid (30 %/year), acetamiprid (22 %/year) and ΣNEOs (15 %/year) in breast milk between 2014 and 2022, whereas increasing trends were seen for clothianidin (17 %/year) and thiamethoxam (30 %/year). N-desmethyl-acetamiprid was the predominant compound in all matrices. However, the contributions of N-desmethyl-acetamiprid (35 %) and thiamethoxam (36 %) in breast milk were similar in 2022. Moreover, thiamethoxam has become the predominant contributor to the estimated daily intake of ΣNEOs since 2018, with the highest contribution of 71 % in 2022, suggesting the effects of NEOs continue to evolve and more attention should be paid to the new NEOs. Notably, the correlations and ratios of NEOs between paired UCS and MS were more significant and higher than those between paired BM and MS, respectively, indicating that NEO exposure was largely affected by the prenatal period.

Populations of various economic species of wireworms are increasing in the key cereal crop production areas of Canada and the United States. To address this problem, seed treatments are under development that both provide crop protection and significantly reduce populations equivalent in effectiveness to the formerly used but now deregistered organochlorine lindane. Herein, we evaluated isocycloseram (PLINAZOLIN technology), the first isoxazoline (GABA-gated Chloride Channel Allosteric Modulator) agricultural insecticide, as a seed treatment for the protection of cereal crops from the sugarbeet wireworm, Limonius californicus (Mannerheim). In wheat and barley field trials conducted over 4 years under extreme wireworm pressure, isocycloseram applied as a seed treatment at 5.0-7.5 g AI/100 kg seed was as effective as or more effective than the current industry standard thiamethoxam at 20.0 g AI/100 kg seed in protecting crop stand and yield. Isocycloseram also reduced neonate wireworms (produced from eggs during the growing season) and resident wireworms (in the field at the time of planting) to levels expected from the formerly used seed treatment lindane.

We investigated the effects of neonicotinoid pesticides (NEOs) on the spontaneous swimming and foraging behavior, as well as the morphological and physiological changes of goldfish. Most fish reared in thiamethoxam (THM)-sprayed rice fields showed the scales easily peeled off, and increased ascites. Some individuals showed decreased bio-defense activity and low plasma Ca2+. Similar changes were found in the exposure test to THM (1.0 and 20.0 μg/L) and dinotefuran (1.2 and 23.5 μg/L). Next, the effects of a low concentration of THM (1.0 μg/L) on the spontaneous swimming and foraging behavior of fish were examined. Fish exposed to THM for 1 week became restless and had increased the swimming performance, especially under natural light, white LED lighting and blue LED lighting. Goldfish exposed to THM had also increased intake of shiny white beads under green LED illumination. These results indicate that the exposure to NEO, even for a short period and at low levels, not only suppressed bio-defense activities and metabolic abnormalities, but also stress response, the swimming and foraging behavior of the fish are likely to be significantly suffered.

Precise, effective and green control plays an essential role in reducing environmental and ecosystem damage. Seed treatment has proven effective and long-lasting for target organisms, and exploring the reasons for long-term protection is important for sustainable agricultural development. This study examined the uptake and metabolism behaviour of thiamethoxam under seed treatment in wheat samples throughout the whole growth cycle, as well as the associated synergistic effects of thiamethoxam and its metabolites during the most severe period of aphid occurrence. Uptake and metabolism results showed that 41 % of thiamethoxam and its active metabolites (clothianidin and demethyl-clothianidin) accumulated mainly in flag leaves of wheat, severely harming aphids, which was significant in controlling leaf-feeding pests. Combined activity results showed that thiamethoxam, clothianidin and demethyl-clothianidin produced synergistic efficacy in controlling aphids, with cotoxicity coefficients ranging from 179.34 to 452.07. Compared with the control, thiamethoxam seed treatments at a rate of 1.5 a.i. g/kg seeds and 3.0 a.i. g/kg seeds can significantly enhance salicylic acid (55 % and 41 %) and jasmonic acid (168 % and 125 %) concentrations and invoke changes in the concentrations of plant secondary substances, which promoted wheat resistance to aphids. Future studies cannot ignore the synergistic effects of metabolites and plant secondary substances in pest control. These results provided data support for reducing pesticide use, increasing efficiency and making more rational use of neonicotinoid insecticides.

The stink bug, Riptortus pedestris (Fabricius) (Hemiptera: Alydidae), is a highly destructive pest that significantly damages legume crops in East and South Asia. Neonicotinoid insecticides containing thiamethoxam are widely used to control R. pedestris in soybean fields. However, the current knowledge on the impact of different thiamethoxam concentrations on R. pedestris growth and reproduction is lacking and insufficient. The present study investigated the effects of thiamethoxam on the biological traits of R. pedestris after treatment with LC10 (19.8 mg/L), LC20 (31.6 mg/L), LC30 (44.2 mg/L), LC40 (58.9 mg/L), and LC50 (77.0 mg/L) concentrations. These five thiamethoxam concentrations (LC10~LC50) reduced adult longevity and fecundity in the F1 generation females. Thiamethoxam treatment also significantly decreased the population trend index, intrinsic rate of increase, net reproductive rate, gross reproductive rate, and finite rate of increase and increased the mean generation time. These results show that thiamethoxam hinders and suppresses the development and growth of the F1 population of R. pedestris. Thiamethoxam is recommended for spray control during peak adult emergence, as it not only has a controlling effect on the parental generation but also a negative impact on the F1 generations.

Bees play a crucial role as pollinating insects in both natural and cultivated areas. However, the use of pesticides, such as thiamethoxam, has been identified as a contributing factor compromising bee health. The current risk assessment primarily relies on the model species Apis mellifera, raising concerns about the applicability of these assessments to other bee groups, including stingless bees. In this study, we investigated the acute toxicity of thiamethoxam on the stingless bee Frieseomelitta varia by determining the average lethal concentration (LC50) and mean lethal time (LT50). Additionally, we evaluated the enzymatic profile of Acetylcholinesterase (AChE), Carboxylesterase-3 (CaE-3), and Glutathione S-Transferase (GST), in the heads and abdomens of F. varia after exposure to thiamethoxam (LC50/10). The LC50 of thiamethoxam was determined to be 0.68 ng ai/μL, and the LT50 values were 37 days for the control group, 25 days at LC50/10, and 27 days at LC50/100. The thiamethoxam significantly decreased the survival time of F. varia. Furthermore, the enzymatic profile exhibited differences in CaE3 activity within one day in the heads and ten days in the abdomen. GST activity showed differences in the abdomen after one and five days of thiamethoxam exposure. These findings suggests that the abdomen is more affected than the head after oral exposure to thiamethoxam. Our study provides evidence of the toxicity of thiamethoxam at both the cellular and organismal levels, reinforcing the need to include non-Apis species in pollinator risk assessments. and provide solid arguments for bee protection.