The Impacts Mechanism Of Pesticide Chlorothalonil On Soil Microbial Denitrification And N₂O Emission

Pesticide chlorothalonil?2,4,5,6-tetrachloroisophthalonitrile?is a high efficient and broad-spectrum fungicide,and has been widely used in modern agricultural production.Soil is the hot zone for pesticides.Fungicide chlorothalonil frequently and repeatedly applied to protect plants against pathogens will eventually accumulated in soil environemts,potentially influencing soil health and microbial activity.During nitrogen cycle,microbial denitrification catalyzed by bacteria and fungi is a special step,because it can cause nitrogen loss from soils and also lead to the potent greenhouse gas N₂O emission.Hence,soil denitrification process has long been regarded as the research point.Fungicide chlorothalonil and its metabolites potentially impact soil heath,deteriorate microbial activity,and thus disturb soil denitrification.However,fewer studies are found about the effects of pesticides on soil denitrification and N₂O emission,and the underlying mechanism is still unknown.Therefore,this study chose chlorothalonil as a typical pesticide,and investigated the impacts of chlorothalonil and its metabolite 4OH-chlorothalonil on soil denitrification and N₂O emission.In detail,our study revealed the microbial mechanism of chlorothalonil impacting on soil denitrification;distinguish bacterial and fungal denitrification and N₂O emission pathways;and establish the relationships between denitrification rate and microbial metabolism process.Conclusions were listed below:?1?In the short-term experiment,chlorothalonil dissipation rate of 5 mg kg^-1?T5?treatment was 24.8%,which was significantly higher than the dissipation rate of 10 and25 mg kg^-1?T10 and T25?treatment?10.3%and 4.6%?.This indicated that there was a significant chlorothalonil degradation behavior in T5 treatment,and the lower dissipation of T10 and T25 treatments could be due to the toxicity of high concentration of chlorothalonil on soil microorganisms.In addition,the metabolite of chlorothalonil?4-hydroxy chlorothalonil?was only detected in T5 treatment(0.41 mg kg^-1)but not in the T10 and T25 treatments.During the long-term experiment,chlorothalonil dissipation rates of T5 and T10 treatments were 51.1%⁵9.7%and 22.3%⁴0.6%,respectively,which were significantly higher than the T25 treatment?11.8%to 15.6%?.At the end of the experiment,the chlorothalonil residues of T5,T10 and T25treatments were 11.23±0.27 mg kg^-1,36.07±0.28 mg kg^-1 and 110.25±1.62 mg kg^-1,respectively.The 4-OH chlorothalonil was detected in all three treatments,and concentrations were maintained at 0.07 mg kg^-12.18 mg kg^-1.?2?Soil respiration rate?basal and substrate-induced respiaration?was used to reveal the effects of chlorothalonil on soil health and microbial activity.Results showed that chlorothalonil,rather than 4OH-chlorothalonil significantly impacted soil health and microbial activity.Regardless of short-term or long-term experiments,soil basal respiration rates?16.74%⁵7.66%?and substrate-induced respiration rates?27.68%⁵8.58%?were significantly decreased,which indicated that chlorothalonil lowerd the healthy status of soil and inhibited soil microbial activity.During the short-term experiment,chlorothalonil didn't influence the soil microbial community structure;however,in long-term experiment,chlorothalonil significantly changed the soil microbial community.Additionally,abundances of Firmicutes,Proteobacteria and Actinobacteria were most sensitive,and were decreased by 27.68%,27.68%and58.58%,respectively.?3?In this study,we used ¹⁵N isotopic tracing to explore the effects of chlorothalonil on soil denitrificatrion process and N₂O emission.Results showed that chlorothalonil significantly inhibited soil nitrate and nitrite reductions,and decreased denitrification rates but remarkably promoted N₂O accumulations.After the experiment,the final nitrate and nitrite concentrations were only 0.7 mg kg^-1 and 8.7l mg kg^-1 in the control,3.9 mg kg^-11 and 16.0 mg kg^-1 in the T5 treatment,7.6 mg kg^-1 and 19.0 mg kg^-1in the T10 treatment,and 17.2 mg kg^-11 and 31.7 mg kg^-11 in the T25 treatment.Denitrification rate in the control was 14.44?mol ¹⁵N kg^-1 dry soil h^-1,which was decreased by 27.08%in the T5 treatment,53.12%in the T10 treatment and 72.16%in the T25 treatment.The release of N₂O in control was 0.21 mg kg^-1,which was increased by 85.71%in the T5 treatment,185.71%in the T10 treatment,and 323.81%in the T25 treatment.Results from the long-term experiment showed the similar trends with the short-term experiment,but the strength was stronger than the short-term test.?4?Respiration inhibition experiment showed that N₂O emission from bacterial and fungal denitrification was closely related to chlorothalonil concentrations.In low chlorothalonil treatments?control and T5?,fungal denitrification dominated in N₂O emission,contributing 55.24%⁷2.74%of N₂O emission.In high chlorothalonil treatments?T10 and T25?,bacterial denitrification dominated in N₂O emission,contributing 55.86%⁶8.13%of N₂O emission.Our study also indicated that chlorothalonil did not showed the significant inhibitions on fungal denitrification,but significantly stimulated bacterial denitrification.?5?Chlorothalonil significantly inhibited the activity of four key denitrifying enzymes:nitrate reductase?NAR?,nitrite reductase?NIR?,nitric oxide reductase?NOR?and nitrous oxide reductase?NOS?.In control,the activities of NAR?NIR?NOR and NOS respectively were 0.153,0.767,2.135 and 4.309?mol g^-1 h^-1.However,chlorothalonil did not alter the abundances of functional gene:narG,nirK,nirS,norB,and nosZ,which indicated that chlorothalonil inhibited denitrification process and promoted N₂O release in the short-term by affecting denitrifying enzyme activities rather than directly altering gene abundances.During the long-term experiment,however,chlorothalonil also significantly inhibited the activity of four denitrifying enzymes,reduced the abundances of narG,nirS and norB,but did not significantly change the abundance of nosZ.This indicated that the effects of chlorothalonil on the release of N₂O was controlled by NOS activity not the nosZ gene in the long-term experiment.?6?During the microbial metabolism,chlorothalonil did not affect the initial use process of carbon source glucose?glucose?glucose phosphate 6?,nor did it inhibit the activity of hexokinase to catalyze this step(0.075 nm mg^-1 protein min^-10.089 nm mg^-1 protein min^-1).However,chlorothalonil significantly inhibited the most critical step in the glycolysis process?glyceraldehyde phosphate?phosphoglycerate?,the activity of glyceraldehyde phosphate dehydrogenase which catalyzes this step was0.124 nm mg^-1 protein min^-1 in the control treatment,0.101 nm mg^-1 protein min^-1 in the T5 treatment,0.061 nm mg^-1 protein min^-1 in the T10 treatment,and 0.026 nm mg^-1protein min^-1 in the T25 treatment.In addition,chlorothalonil significantly reduced the production of electron donor?NADH?,energy substance?ATP?and pyruvate in denitrification.The contents of NADH,ATP and pyruvate were 14.02?mol g^-1,7.83?mol kg^-1 and 21.06?mol kg^-1 in the control treatment,and 3.12?mol g^-1,1.11?mol kg^-1 and 12.71?mol kg^-1 in the T25 treatment.This study also found that chlorothalonil had a significant impact on microbial electron transfer system activity?ETSA?.Compared with the control treatment,ETSA of T5,T10 and T25 treatment decreased by 21.93%,36.84%and 44.74%,respectively.Based on the above results,although chlorothalonil does not affect the initial degradation of carbon source,it significantly reduces the generation and transmission ability of electron during the microbial denitrification process,thereby deteriorating the efficiency of soil denitrification.?7?Path analysis showed that the electron donor?NADH?and electron transfer system activity?ETSA?of microbial metabolism indirectly affect soil denitrification by interfering with denitrifying enzyme activity.In addition,the structural equation model shows that ETSA?indirect influence coefficient is 0.831?and NOS activity?direct influence coefficient is 0.628?are the key factors to predict soil denitrification under chlorothalonil accumulation.