Response Of Soil Intracellular And Extracellular β-glucosidase And Evaluation Of Ecotoxicity In Cr(Ⅵ) Contaminated Soils

Soil chromium（Cr（Ⅵ））pollution seriously affects the ecological environment and human health.How to accurately evaluate the ecology of soil Cr（Ⅵ）pollution and its remediation treatment is an important research content of current environmental science.Soil enzymes are important indicators in soil biology,which can reflect soil biological activity and soil health.Soil enzymes can be used as biomarkers to assess soil heavy metal ecotoxicity.However,due to the morphological transformation of Cr（Ⅵ）in soil and the sensitivity of different soil enzyme libraries（intracellular enzyme/extracellular enzyme）to pollutants,the response of soil enzyme activity to Cr（Ⅵ）pollution is inconsistent,and the response mechanism of soil intracellular and extracellular enzymes to Cr（Ⅵ）toxicity is unclear.Therefore,this study took soilβ-glucosidase associated with the carbon cycle as the object,distinguished soil intracellular and extracellularβ-glucosidase by chloroform fumigation,studied the effects of simulated acute and aging pollution of Cr（Ⅵ）on soil intracellular and extracellularβ-glucosidase,explored the kinetic mechanism of Cr（Ⅵ）on soil intracellular and extracellularβ-glucosidase,and calculated the toxicity threshold of Cr（Ⅵ）acute pollution and aging pollution by dose-effect model.The characteristics of soil enzyme activity and microbial biomass carbon under Cr（Ⅵ）stress with time were elucidated,which provided accurate and reliable soil enzymatic theoretical support for soil Cr（Ⅵ）pollution toxicity assessment and environmental risk assessment.The following results and conclusions were obtained in this study:（1）After chloroform fumigation,soil intracellularβ-glucosidase accounted for69.39%of the total enzymes,and soil extracellularβ-glucosidase accounted for 30.61%,indicating that soil intracellular enzymes occupied an important position in soil enzyme banks.Acute contamination of Cr（Ⅵ）significantly inhibited the activities of soil totalβ-glucosidase and intracellularβ-glucosidase,and inhibited the activity of extracellularβ-glucosidase in some of the tested soils,indicating that the activity of totalβ-glucosidase and intracellularβ-glucosidase in soil can characterize the degree of Cr（Ⅵ）pollution.Under the condition of acute Cr（Ⅵ）pollution,the ecological dose ED₅₀（Cr（Ⅵ）concentration corresponding to a 50%decrease in enzyme activity）of totalβ-glucosidase and intracellularβ-glucosidase calculated by dose-effect model was2.41～16.43 mg kg^-1,1.72～6.48 mg kg^-1.It was found that soil microbial biomass carbon,organic matter,total nitrogen and cation exchange were the factors affecting the toxicity of Cr（Ⅵ）to totalβ-glucosidase and intracellularβ-glucosidase by Pearson correlation analysis.（2）The mechanism of acute contamination of Cr（Ⅵ）on the inhibition of soil totalβ-glucosidase,intracellular and extracellularβ-glucosidase was investigated by enzyme kinetic analysis.Under acute contamination of Cr（Ⅵ）,the Michaelian constant ofβ-glucosidase kinetics（characterizing the affinity of enzyme-substrate binding）remained unchanged,and the maximum reaction rate V_max（characterizing the ability of enzyme-substrate complexes to break down into enzymes and products）decreased,from which it was inferred that the inhibition of soil totalβ-glucosidase,intracellular and extracellularβ-glucosidase by acute Cr（Ⅵ）pollution was mainly non-competitive inhibition.The non-competitive inhibitory constants K_iu of Cr（Ⅵ）for totalβ-glucosidase,intracellular enzyme and extracellularβ-glucosidase were between0.005～0.032,0.002～0.088 and 0.002～0.200 m M,respectively,and their K_iu were much smaller than the Michael’s constant K_m,indicating that the affinity of Cr（Ⅵ）with soil totalβ-glucosidase total,intracellular and extracellularβ-glucosidase were much greater than that of substrate and enzyme activity.The ecological doses of Cr（Ⅵ）to soil totalβ-glucosidase V_max and V_max/K_m were 3.10～18.00 and 1.01～13.29 mg kg^-1,respectively,and the ecological doses of intracellularβ-glucosidase V_max and V_max/K_mwere 1.22～7.38 and 1.19～9.13 mg kg^-1.It can be revealed to a certain extent to characterize the acute pollution degree of soil Cr（Ⅵ）.（3）Through the aging pollution test of Cr（Ⅵ）in gray desert soil and aquic soil,it was found that Cr（Ⅵ）significantly inhibited the activities of soil microbial biomass char（MBC）,totalβ-glucosidase and intracellularβ-glucosidase,and still showed a strong inhibitory effect on the extracellularβ-glucosidase of aquic soil,and still showed strong inhibitory effect on extracellularβ-glucosidase in aquic soil,but had an activating effect on extracellularβ-glucosidase in gray desert soil,indicating that different types of soil extracellularβ-glucosidase had different responses to the toxicity of Cr（Ⅵ）,and with the extension of the culture time,the adverse effects of Cr（Ⅵ）on soilβ-glucosidase activity and MBC existed for a long time.Two-way ANOVA showed that the incubation time and Cr（Ⅵ）had significant effects on soilβ-glucosidase activity and MBC.The ecological dose of totalβ-glucosidase in aquic soil was 0.81～3.75 mg kg^-1,and the ED₅₀ of intracellularβ-glucosidase was 0.46～2.84 mg kg^-1.The ecological dose ED₅₀ of totalβ-glucosidase in gray desert soil was 7.24～31.23 mg kg^-1,and the ED₅₀ of intracellularβ-glucosidase was 2.70～7.10 mg kg^-1.The ecological dose values of aquic soil were much smaller than those of gray desert soil,indicating that Cr（Ⅵ）was more toxic to aquic soil during aging.Through correlation analysis,it was found that totalβ-glucosidase and intracellularβ-glucosidase were positively correlated with soil MBC during the aging process of Cr（Ⅵ）,revealing that soil enzyme activity was regulated by microbial biomass.（4）Compared with acute Cr（Ⅵ）pollution,the inhibition type of soilβ-glucosidase by Cr（Ⅵ）aging is different,mainly due to the different ways and pollution times of soil pollution.The inhibition type of Cr（Ⅵ）aging pollution on totalβ-glucosidase total enzyme and intracellularβ-glucosidase components in gray desert soil was anticompetitive inhibition,but there was no inhibitory effect on extracellularβ-glucosidase in gray desert soil.The types of inhibition of Cr（Ⅵ）onβ-glucosidase total enzyme,intracellular and extracellularβ-glucosidase components in aquic soil were anticompetitive inhibition or linear mixed inhibition.The ecological doses of totalβ-glucosidase and intracellularβ-glucosidase component V_max in aquic soil were1.06～3.24 and 0.72～2.21 mg kg^-1,respectively.The ecological doses of tatalβ-glucosidase and intracellularβ-glucosidase V_max in gray desert soil were 4.52～10.34and 2.98～8.69 mg kg^-1,respectively.The ecological dose value ofβ-glucosidase V_maxin aquic soil was much smaller than that in gray desert soil,indicating that Cr（Ⅵ）was more toxic toβ-glucosidase in aquic soil,revealing that the resistance of different types of soil to Cr（Ⅵ）toxicity was different.In summary,soil intracellular enzymes play an important role in evaluating the toxicity of soil Cr（Ⅵ）,and Cr（Ⅵ）contamination significantly inhibits the activities of soil totalβ-glucosidase and intracellularβ-glucosidase.In the acute pollution and aging pollution test of Cr（Ⅵ）,the ecological dose values calculated by intracellularβ-glucosidase were smaller than the total enzyme,indicating that intracellularβ-glucosidase was more sensitive to Cr（Ⅵ）pollution,so it was more accurate to use soil intracellularβ-glucosidase as the ecotoxicity evaluation index of soil Cr（Ⅵ）pollution.The results of this study will provide a basis for the toxicity evaluation of soil Cr（Ⅵ）pollution.