Behavior And Metabolic Transformation Of Vanisulfane In Rats And Hens,and Biological Effects Of Its Contaminated Chicken Manure On Vegetables

Pesticides can enter human habitats through the environment,such as water and soil,and then transfer and accumulate in biological chains in the form of parent and metabolites,which will cause human exposure risk of pesticide residues.For example,pesticides can be used on fodder crops,and their residues might influence human health through the cascade effect of livestock and poultry.Meanwhile,pesticides might also be rereleased to the environment with the application of animal excrements in the field,leading to secondary pollution hazards of pesticides in soil and plants.In addition,human beings might have an exposure risk caused by the direct ingestion of plants polluted by pesticides.Therefore,it undoubtedly has important scientific significance and practical value to clarify the behavior and fate of pesticides in the whole process of“pesticides→plants→mammal/livestock and poultry→plant?soil environment”.Vanisulfane is a new pesticide against plant virus independently developed in China,and it has relatively good application prospects.However,there have been few studies expounding on its behavior and fate and human health risk from the whole process of“vanisulfane→mammal/livestock and poultry→livestock and poultry excrements→plant?soil environment”.Therefore,in this study,mammalian rats and livestock laying hens were adopted as model organisms,with[benzyl-¹⁴C]-vanisulfane and[benzene-¹⁴C]-vanisulfane,which can objectively reflect the molecular metabolism characteristics of vanisulfane as isotope tracers,to clarify the fate and metabolic transformation mechanism of vanisulfane in mammals and livestock via radioactive isotope tracing and frontier instrument analysis methods.On this basis,a discussion was made on the biological effect of excrement return on vegetables and soil residual transformation characteristics after livestock were exposed to this pesticide.This study aimed to conduct an in-depth analysis of the residual and metabolic transformation process of vanisulfane in the ecological environment,to systematically and scientifically explain the environmental safety and health risks of vanisulfane and to provide referable research technology systems and methods for related research on pesticides developed by China in complex systems.The main results are as follows:（1）The residual distribution law of vanisulfane in mammalian rats and livestock laying hens was analyzed.Twenty-four hours after intragastric administration of ¹⁴C-vanisulfane,the radioactivity excreted in the form of urine and feces occupied 21.32～31.06%and52.24～65.35%of the initial radioactivity,respectively.The ranking of male rat tissues in residual concentration from high to low was kidney>liver>lung/spleen/heart>testis>brain,and kidney>liver>ovary>lung>heart>spleen>brain in female rat tissues.The residual concentrations in the liver and kidney were 0.669～0.903 mg kg^-1and 0.853～0.992 mg kg^-1,respectively,indicating that vanisulfane and its metabolites mainly accumulated in the metabolic organs of mammals.Seven days after the laying hens were orally administered ¹⁴C-vanisulfane continuously,radioactivity exceeding 80%of the initial was excreted from the laying hens in the form of excrement.Within the first 12 h after oral administration each day,the radioactivity content in the excrement was higher than that within 12-24 h after oral administration.Vanisulfane mainly remained in liver(0.265～0.512 mg kg^-1)and kidney tissues(0.276～0.480 mg kg^-1)and showed biological accumulation in the egg samples.In addition,pollutant accumulation occurred more easily in the egg yolk than in the egg white.It was demostrated in this study that after oral administration,vanisulfane and its metabolites have a high excretion rate in mammalian rats and livestock laying hens,and are widely distributed in tissues,mainly in liver and kidney.（2）The behavioral differences in vanisulfane between the two sexes in the mammalian rats were clarified.Twenty-four hours after oral administration,the clearance rate of pollutants in male rats was lower than that in female rats,and male rats and female rats tended to eliminate vanisulfane and its metabolites through bile excretion and kidney excretion,respectively.During metabolism,male rats formed downstream products earlier than females and were more inclined to form sulfonated metabolite,suggesting that vanisulfane was metabolized more rapidly and completely in males.In addition,through7-day consecutive exposure,it was found that rats exposed to vanisulfane exhibited hepatic steatosis in both sexes,along with gonadal effects in males（the seminiferous tubule presented minor atrophy,and the spermatogenic cells decreased in the tube）,indicating that continuous exposure to vanisulfane would cause potential hepatotoxicity and male reproductive toxicity.Based on the sex differences in mammals,it is suggested that the transport and metabolic characteristics,and toxicological evaluation of vanisulfane in mammals should be considered comprehensively from the sex dimension.（3）The exposure evaluation indicated that vanisulfane would not cause dietary risks in laying hens.According to the evaluation guidelines of the World Health Organization and International Food and Agriculture Organization on dietary exposure of compounds in food,the estimated dietary intake（EDI）and margin of exposure（MOS）of vanisulfane and its metabolites were estimated.After one-week successive administration,the EDI value of the large intestine was the highest in the edible tissues of laying hens and reached 0.6636～0.8152μg（kg d）^-1.The EDI value of other tissues was all lower than0.2μg（kg d）^-1.In general,the MOS of vanisulfane in various edible tissues of laying hens was lower than 1.0,that is,the dietary exposure risk of vanisulfane was relatively low.（4）The metabolic pathways and metabolic transformation mechanisms of vanisulfane in rats and laying hens were expounded.By applying high-specific activity ¹⁴C-vanisulfane of two labeling positions,the quantitative and qualitative analysis of vanisulfane and its metabolites in animals was established via the combined analytical technologies of LC-LSC and LC-QTOF-MS.The results showed that vanisulfane mostly existed in the form of metabolites in rats and laying hens.A total of twelve metabolites（M1–M12）were identified.Among them,metabolites M1-M8 were observed in rats and metabolites M1,M3,M5 and M9-M12were discovered in laying hens.Metabolite M7（2-（（（4-（（4-chlorobenzyl）oxy）-3-methoxyphenyl）（（2-hydroxyethyl）thio）methyl）thio）acetic acid）was the major metabolite in rat tissues,and the concentrations of various metabolites in laying hen edible tissues were all lower than 0.05 mg kg^-1.In this study,the metabolic pathways of vanisulfane in mammals and livestock were deduced,and it was discovered that vanisulfane underwent phase I and phase II metabolism in rats and laying hens.Phase I metabolism was mainly oxidation,hydroxylation,dechlorination and demethylation reactions to form metabolites M4–M11;in terms of phase II metabolism,conjugation reactions mainly occurred with endogenous small molecules to form glucuronic acid（M1）and glycine（M2）conjugates.In addition,the sulfonation reaction occurred easily with the chlorobenzene of vanisulfane to form metabolite M3.Among them,glycine-binding reaction occurred only in rats,while demethylation and dechlorination reactions occurred only during laying hens’metabolism.（5）The biological effect of applying chicken manure contaminated with vanisulfane to vegetables and the transformation characteristics of its residues were revealed.To elucidate the differences in the behavior and fate of vanisulfane contaminated with chicken manure in the environment when it is present alone and when it coexists in the form of parent and metabolites,two types of chicken manure were used for vegetable（radish and cabbage）-soil incubation experiments.Chicken manure I（CM-I）was manure artificially spiked with vanisulfane and chicken manure II（CM-II）was manure excreted by chickens orally exposed to vanisulfane（containing a mixture of vanisulfane and its metabolites）.Unspiked chicken manure was set as the control group.It was found that the accumulation of vanisulfane and its metabolites in plants after the introduction of both chicken manures into the vegetable-soil system was very limited,and the radioactivity detected in both vegetables was less than 2%of introduced amount.Pollutants were more likely to remain enriched in vegetabales when chicken manure II was applied compared to chicken manure I.The bioconcentration factor of pollutant under the treatment with CM-I was 0.42～1.14,and the factor under the CM-II treatment was 1.05～1.95.Vanisulfane and its metabolites were mainly present in the form of bound residue（BR）in soil,and BR bonded with soil was rereleased at 35 d.Compared to the degradation half-life（5.50～5.99 d）of the parent in soil when chicken manure was contaminated with vanisulfane only（CM-I）,the half-life increased when the metabolites coexisted with the parent in chicken manure（CM-II）,indicating that the existence of metabolites would influence the degradation rate of vanisulfane in soil.In the soil where the chicken manure II was applied,a new metabolite,M13(2-(4-（2-（4-（（4-chlorobenzyl）oxy）-3-methoxyphenyl）-1,3-oxathiolane）,was discovered in addition to the original metabolites（M8,M11 and M12）in the excrement.After the field application of chicken manure in which only vanisulfane was added,vanisulfane in the soil was transformed into metabolites M8,M11,M12 and M13.The research showed that the degradation and transformation of vanisulfane in the soil system mainly included oxidation,cyclization,ring opening and demethylation processes,in which the formation process of oxythiacyclopentane occurred only in soil metabolism.Despite the low residue accumulation in vegetables,significant growth toxicity（reduction in plant height,root length and plant biomass）was observed in vegetables after application of chicken manure II compared to the blank control group.To further explain this phenomenon,an experiment on seed germination and seedling culture of vegetables was conducted via the reference substances of vanisulfane and its metabolites in this study.The research showed that metabolite M12（4-（（4-chlorobenzyl）oxy）-3-methoxybenzaldehyde）,with an aldehyde structure in vanisulfane,was the major compound causing growth toxicity in plants.Compared with the control group,the seeds exposed to M12 presented an obviously lower seed germination rate,and the follow-up growth of radish was significantly restrained.It is demostrated in this study that the application of chicken manure polluted by vanisulfane will cause secondary environmental pollution risk and plant toxicity,of which the metabolite M12is an important factor causing plant toxicity and needs to be monitored in the actual application.