Toxicological Effects Of Acetochlor And Metribuzin Combination On Escherichia Coli K12 And Bacillus Subtilis B7

Acetochlor is widely used as an amide herbicide with a wide range of weeding and low cost.Due to its strong mobility,non-degradability,and high water solubility,it will remain in the soil for a long time and cause environmental pollution problems.Metribuzin is widely used as a triazine herbicide with high control effect and small residue.Because it has been recognized as an endocrine disruptor,it has attracted widespread attention.In recent years,in order to improve the weeding effect and reduce the cost,most of the combined application of acetochlor and metribuzin to expand the broad spectrum of weeding.However,the current toxicology research mainly focuses on the impact of single pollution on environmental organisms,and there are relatively few toxicological studies on compound pollution.Therefore,in this study,E.coli K12 and B.subtilis B7 were used as the test strains,and the toxic effects and toxic mechanisms of the above two strains under the combined conditions of acetochlor and metribuzin were studied respectively.The main research results are as follows:（1）Effects of acetochlor and metribuzin on the growth of E.coli K12 and B.subtilis B7 under single and combined stresses showed that:Under a single stress,acetochlor and metribuzin can inhibit the growth of E.coli K12 and B.subtilis B7,and the inhibitory effect increases with the increase of the concentration.Fitting shows that under compound conditions,the toxicity of acetochlor and metribuzin to E.coli K12 is first synergistic and then antagonistic,and the effect on B.subtilis B7 is antagonistic.The cell viability of the strain was investigated by flow cytometry c FDA/PI double staining method.The results showed that compared with single stress,the apoptosis rate of E.coli K12 cells under the combined stress of acetochlor and metribuzin at low concentrations increased significantly to 32.3%,while the proportion of live cells decreased to12.6%.However,the apoptosis rates of B.subtilis B7 under low-concentration complex stress and E.coli K12 and B.subtilis B7 under high-concentration complex stress were significantly reduced,which were 13.9%,22.6%,and 37.7%,respectively,while living cells accounted for the proportion However,it rose to 19.4%,16.0%and 13.6%respectively.（2）Effects of acetochlor and metribuzin on the morphology of E.coli K12 and B.subtilis B7cells under single and combined stress were investigated by scanning electron microscopy.The results showed that:Acetochlor and metribuzin can damage the cell membrane structure of E.coli K12 and B.subtilis B7.The degree of cell membrane damage of E.coli K12 and B.subtilis B7strains increased with the increase of the concentration of acetochlor or metribuzin.Compared with single stress,the cell membrane damage of E.coli K12 strain under low-concentration compound stress is aggravated,while the cell membrane damage degree of B.subtilis B7 under low-concentration compound stress and E.coli K12 and B.subtilis B7 strain under high-concentration compound stress All weakened.On this basis,the effect on the cell membrane was further investigated by measuring the extracellular LDH enzyme activity of the strain and the Zeta potential on the surface of the strain.The results showed that:The extracellular LDH enzyme activity of E.coli K12 and B.subtilis B7 strains increased with the increase of the concentration of acetochlor and metribuzin.Compared with single stress,E.coli K12 extracellular LDH enzyme activity was significantly increased under low-concentration complex stress,while the extracellular LDH enzyme activity of B.subtilis B7 under low concentration compound stress and E.coli K12 and B.subtilis B7 strains significantly decreased under high concentration compound stress.Compared with single stress,the surface potential of E.coli K12 under low-concentration complex stress showed an increasing trend,while the surface potential of B.subtilis B7 under low-concentration complex stress and E.coli K12 and B.subtilis B7 strain under high-concentration complex stress had both shows a downward trend.（3）By detecting the effects of acetochlor and metribuzin on the oxidative stress indicators of E.coli K12 and B.subtilis B7 strains under single and combined stress,the results showed that:Compared with single stress,low-concentration combined stress of acetochlor and metribuzin increased the SOD,CAT enzyme activity,MDA content and ROS content of E.coli K12.The ROS content was 3.06 times that of the blank control group,and the GST enzyme activity was suppressed.However,the SOD enzyme activity,ROS content,and MDA content of E.coli K12under high-concentration complex stress decreased,and the CAT and GST enzyme activities increased significantly.The combined stress of acetochlor and metribuzin significantly decreased the SOD,CAT,GST enzyme activities,ROS content and MDA content of B.subtilis B7.（4）Effects of acetochlor and metribuzin on E.coli K12 and B.subtilis B7 strains were investigated by determining the content of 8-OHd G in strain DNA,yield and components of extracellular polymers and accumulation concentration of surface and intracellular pollutants.The results showed that compared with single stress,the intracellular concentration of acetochlor in E.coli K12 strain increased under the combined stress of low concentration of acetochlor and metribuzin,resulting in a significant increase in ROS content,which resulted in an increase in8-OHd G content in DNA to 31.32 ng·m L^-1,DNA oxidative damage is aggravated.However,the intracellular concentrations of acetochlor in E.coli K12 and B.subtilis B7 strains under low-concentration complex stress and high-concentration complex stress both decreased,reducing the 8-OHd G content in DNA to 28.47,23.63,and 37.08 ng·m L^-1,respectively,DNA oxidative damage is weakened.The stress of acetochlor and metribuzin can increase the EPS production of E.coli K12 and B.subtilis B7 strains.The scanning results of three-dimensional fluorescence spectroscopy showed that under the combined stress of low concentration of acetochlor and metribuzin,the protein substances tryptophan and humic acid substances in EPS of E.coli K12strain could be fluorescently quenched and combined with acetochlor.However,the fluorescence quenching of tryptophan in the EPS of B.subtilis B7 and E.coli K12 and B.subtilis B7 strains under complex stress of low concentration and complex stress of high concentration combine d with acetochlor occurred.（5）The results of the comprehensive biomarker response index analysis showed that the combined stress conditions of acetochlor and metribuzin significantly increased the cell membrane,oxidative damage and genomic response of E.coli K12,and the total biomarker IBR score was12.37,more than 38.21%of acetochlor single stress treatment,which led to increased toxicity.However,the cell membrane,oxidative stress and genome response levels of B.subtilis B7 and both bacteria under high concentration compound stress,and the IBR scores were 4.74,10.79 and14.90 respectively,which were lower than glylamine single stress,indicating diminished toxicity.