Behavioral Effects And Reproductive Toxicity Of Thiamethoxam On Zebrafish

Neonicotinoid insecticides(NEOs)have become the most commonly used insecticides worldwide due to their high efficiency and selectivity,low toxicity to vertebrates,environmental stability,broad spectrum,and strong endosmosis,and are widely detected in various water bodies in several countries.Thiamethoxam(THM)is a newly structured second-generation NEO with highly efficient and low toxicity,but most of the ecotoxicity studies on it so far have been focused on terrestrial organisms and insects,and in-depth studies on its effects on aquatic organisms and aquatic ecosystems are seriously inadequate.The work on the toxic effects and mechanisms of THM on non-target aquatic organisms can provide an important basis for revealing the aquatic ecological risks of THM,rational planning for the safe use of insecticides,protecting the safety of aquatic ecosystems,and formulating relevant control policies.Therefore,this study selected the classical aquatic model organism zebrafish as the test object,and evaluated the behavioral effects,distribution and bioconcentration in vivo,reproductive toxicity and mechanism of THM on zebrafish through a series of toxicity tests.It provides an important reference for aquatic ecological risk assessment.The main findings are as follows:(1)Adult zebrafish were exposed to 0,0.1,10 and 1000 μg/L THM solutions for4,14 and 45 days,respectively.In the behavioral experiments of zebrafish,THM significantly modulated locomotor activity,anxiety,innate preference,courage,and memory accuracy of individual adult fish,while the shoal cohesion,aggression,and other social attributes(preference and recognition)were also significantly enhanced,and these patterns of behavioral change were modulated by both exposure time and exposure concentration.Males chronically exposed to THM in reproductive behavior chased females frequently and briefly,but their courtship behavior was significantly inhibited.In addition,the larvae exposed to THM for 6 days post-fertilization by different routes were stimulated by the light-dark cycle,colored-light cycle,and startle cycle,reflecting behavioral characteristics such as sluggishness,excitement,instability,and insensitivity.For the actual water environment polluted with THM(0.1 μg/L),the fish behavior was affected roughly as follows: larvae visual stimulus-response > adult individual behavior > adult social behavior > adult reproductive behavior ≈ larvae startle stimulus-response.(2)The concentrations of THM,its metabolite,and some endogenous metabolites were further quantified in various tissues of adult fish;their distribution patterns were affected by exposure time and exposure concentration,and there were significant tissue differences.The average concentration of THM basically followed the order of liver >heart > brain/intestine > gonads ≈ muscle,with the highest residual concentration in the liver(424.77 ng/g),which was the main target organ of THM in fish;while the metabolite clothianidin was extremely low.Furthermore,the bioconcentration factors of THM in various tissues ranged from 0.11-126.64 L/kg(4 days),0.11-1851.61 L/kg(14 days),and 0.11-169.86 L/kg(45 days),and the bioconcentration capacity of THM in zebrafish was higher at environmentally relevant concentrations(0.1 μg/L).The tissue distribution of some endogenous metabolites in exposed fish was complex and changeable,which indicated that THM may impair the normal physiological functions of fish tissues and organs and interfere with the neural signaling processes,which in turn regulate locomotor behavior.(3)Based on the abnormal reproductive behavior of male zebrafish,the specific reproductive toxicity endpoints in male zebrafish were determined after 45 days of THM exposure.The results showed stronger binding energy and stable binding patterns between THM small molecule and the estrogen receptor α agonist conformation and androgen receptor antagonist conformation in molecular docking.Damage to the gonadal tissue of exposed males was observed,with widened intercellular stroma,disturbed germinal tubules,and abnormal germ cell occupancy.Results of enzymelinked immunosorbent assay and real-time fluorescence quantitative polymerase chain reaction showed significant decreases in testosterone(T)and 17β-estradiol(17β-E2)in the brain and blood,and vitellogenin(VTG)in the liver compared to control(p < 0.05),while 11-ketotestosterone(11-KT)did not show significant changes(p > 0.05).In the hypothalamic-pituitary-gonadal-liver axis(HPGL)of male fish,significant upregulation(p < 0.05)of gnrh2,gnrh3,cyp11 a,hsd3b,hsd17 b,and cyp19 a expression in brain and gonads was the main process of positive feedback regulating the restoration of T and 17β-E2 to homeostasis in gonads.The significant down-regulation of cyp11b(p < 0.05)and up-regulation of hsd11b(p < 0.05)in gonads together maintained the stability of 11-KT in all tissues and organs;the expression of vtg1 was significantly down-regulated by 0.49±0.04-0.5±0.09 fold in the liver(p < 0.05),but VTG synthesized in other extrahepatic tissues maintained the homeostasis of the internal environment.Thus,THM has some estrogenic endocrine disrupting effects and impairs the reproductive system of fish.