Pesticides are substances used for controlling, preventing, and repelling pests in agriculture. Among them, neonicotinoids have become the fastest-growing class of insecticides because of their efficiency in targeting pests. They work by strongly binding to nicotinic acetylcholine receptors (nAChRs) in the central nervous system of insects, leading to receptor blockage, paralysis, and death. Despite their selectivity for insects, these substances may be hazardous to non-target creatures, including earthworms. Although earthworms may be invasive in some regions like north America, they contribute to the development of soil structure, water management, nutrient cycling, pollution remediation, and cultural services, positively impacting the environment, particularly in the soil ecosystem. Thus, this study aimed to develop a novel earthworm behavior assay since behavior is a sensitive marker for toxicity assay, and demonstrated its application in evaluating the toxicity of various neonicotinoids. Here, we exposed to 1 and 10 ppb of eight neonicotinoids (acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram pestanal, thiacloprid, thiametoxam, and sulfoxaflor) for 3 days to observe their behavior toxicities. Overall, all of the neonicotinoids decreased their locomotion, showed by a reduction of average speed by 24.94–68.63% and increment in freezing time movement ratio by 1.51–4.25 times, and altered their movement orientation and complexity, indicated by the decrement in the fractal dimension value by 24–70%. Moreover, some of the neonicotinoids, which were acetamiprid, dinotefuran, imidacloprid, nitenpyram, and sulfoxaflor, could even alter their exploratory behaviors, which was shown by the increment in the time spent in the center area value by 6.94–12.99 times. Furthermore, based on the PCA and heatmap clustering results, thiametoxam was found as the neonicotinoid that possessed the least pronounced behavior toxicity effects among the tested pesticides since these neonicotinoid-treated groups in both concentrations were grouped in the same major cluster with the control group. Finally, molecular docking was also conducted to examine neonicotinoids' possible binding mechanism to Acetylcholine Binding Protein (AChBP), which is responsible for neurotransmission. The molecular docking result confirmed that each of the neonicotinoids has a relatively high binding energy with AChBP, with the lowest binding energy was possessed by thiametoxam, which consistent with its relatively low behavior toxicities. Thus, these molecular docking results might hint at the possible mechanism behind the observed behavior alterations. To sum up, the present study demonstrated that all of the neonicotinoids altered the earthworm behaviors which might be due to their ability to bind with some specific neurotransmitters and the current findings give insights into the toxicities of neonicotinoids to the environment, especially animals in a soil ecosystem.

中文翻译：

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通过新型运动跟踪测定评估新烟碱类杀虫剂对蚯蚓（Eisenia fetida）行为的毒性


农药是农业中用于控制、预防和驱除害虫的物质。其中，新烟碱类杀虫剂因其针对害虫的效率而成为增长最快的一类杀虫剂。它们的作用是与昆虫中枢神经系统中的烟碱乙酰胆碱受体 (nAChR) 强烈结合，导致受体阻断、麻痹和死亡。尽管它们对昆虫具有选择性，但这些物质可能对非目标生物（包括蚯蚓）有害。尽管蚯蚓在北美等一些地区可能具有入侵性，但它们有助于土壤结构的发展、水管理、养分循环、污染修复和文化服务，对环境，特别是土壤生态系统产生积极影响。因此，由于行为是毒性测定的敏感标记，本研究旨在开发一种新型的蚯蚓行为测定，并证明其在评估各种新烟碱类杀虫剂的毒性中的应用。在这里，我们暴露于 1 和 10 ppb 的八种新烟碱类杀虫剂（啶虫脒、噻虫胺、呋虫胺、吡虫啉、烯啶虫胺、噻虫啉、噻虫嗪和磺胺氟）3 天，观察其行为毒性。总体而言，所有新烟碱类杀虫剂都降低了它们的运动能力，表现为平均速度降低了 24.94-68.63%，冻结时间运动比率增加了 1.51-4.25 倍，并改变了它们的运动方向和复杂性，表现为分形的减少。尺寸值增加 24–70%。此外，一些新烟碱类药物，如啶虫脒、呋虫胺、吡虫啉、烯啶虫胺和氟啶虫胺，甚至可以改变它们的探索行为，这表现为在中心区域值花费的时间增加了6.94-12.99倍。 此外，根据主成分分析和热图聚类结果，发现噻虫嗪是受试农药中行为毒性效应最不显着的新烟碱类杀虫剂，因为这两个浓度的新烟碱类杀虫剂处理组与对照组属于同一主要聚类。最后，还进行了分子对接，以检查新烟碱类杀虫剂与负责神经传递的乙酰胆碱结合蛋白（AChBP）的可能结合机制。分子对接结果证实，各新烟碱类杀虫剂与AChBP具有较高的结合能，其中噻虫嗪的结合能最低，这与其较低的行为毒性相一致。因此，这些分子对接结果可能暗示了观察到的行为改变背后的可能机制。综上所述，本研究表明，所有新烟碱类杀虫剂都改变了蚯蚓的行为，这可能是由于它们与某些特定神经递质结合的能力，目前的研究结果使人们深入了解新烟碱类杀虫剂对环境的毒性，特别是土壤中的动物生态系统。