The Mechanism And Ecological Effect Of Antimony Pollution On Soil Enzymes

Antimony（Sb）is a kind of metalloid element.Recently,with the rapid increase in the production and use of antimony products,the activities of antimony mining and smelting are frequent,which makes a large amount of antimony enter the soil environment and threaten human health.Therefore,it is particularly urgent to timely and accurately assesses soil Sb pollution and provide early warning information for its pollution supervision and control.Soil enzymes participate in the processes of soil geochemical elements cycling,energy metabolism,pollutants degradation,etc.,which are good biological indicators for soil pollution monitoring.Soil enzymes have been widely used in monitoring and evaluating soil heavy metal and metalloid pollution such as mercury,cadmium,copper,zinc,and arsenic.At present,increasing investigations have reported that plants,animals,and microorganisms were used to monitor and evaluate soil antimony pollution,and there are few reports on the ecological effects of antimony on soil enzymes.Studying the relationships between antimony pollution and soil enzymes and the action mechanism of antimony on soil enzymes are very important for the scientific and accurate risk assessment of soil Sb pollution,taking timely and effective prevention and control measures.In this study,the soils with different properties and typical Sb mining area soils were selected as the research objects.The simulation studies in the laboratory were used to systematically investigate the ecotoxicity of Sb to soil enzymes under acute Sb stress,Sb aging,and Sb mining area pollution.Based on soil enzymatic reaction kinetics and thermodynamics,these studies explored the action mechanism of Sb to soil enzymes,and analysed the main factors causing ecotoxicity differences of Sb in various soils.Antimony ecological thresholds were obtained under Sb acute,aging,and mining area pollution.All the above results will have laid a solid foundation for constructing reasonable and accurate assessment indicators of Sb pollution.The main conclusions are as follows:It was found that there were slight changes in the activities of alkaline phosphatase,dehydrogenase,and fluorescein diacetate hydrolase under acute Sb stress.The activities ofβ-glucosidase and urease were decreased under acute Sb stress,and the maximum inhibition rates of Sb to these enzyme activities were less than 11%.While Sb significantly inhibited arylsulfatase activity and the inhibition rate reached 29.63%,suggesting that soil arylsulfatase was sensitive to acute Sb stress.The 17 soils with different properties from China were collected to a conduct simulation study of acute antimony pollution for exploring the toxicity differences of Sb to arylsulfatase.The study showed that the inhibition effects of Sb on arylsulfatase activities increased with the increase of Sb concentration.When the concentration of Sb in soils was 7000 mg kg^-1,the inhibition rates of Sb on arylsulfatase activity was 27.38 to 51.20%in acidic soils（S1-S9）and 2.83 to 26.7%in alkaline soils（S10-S17）.Obviously,the toxicity of Sb to arylsulfatase activity in acidic soils was stronger than those in alkaline soils.The dose-response relationship between Sb concentrations and enzyme activities significantly fitted the partial inhibition model.The ecological dose values(ED₁₀)of total Sb and available Sb concentrations to arylsulfatase activities were 78 to 865 mg kg^-1 and27 to 603 mg kg^-1,respectively.Multiple stepwise regression analysis showed that the available phosphorus content in soils was the main factor affecting the toxicity of Sb to arylsulfatase in various soils.The kinetic characteristics of enzymatic reaction(V_max,K_m)revealed that the inhibition of Sb on arylsulfatase in acidic soils（S1-S9）was noncompetitive and linear mixed inhibition,while the inhibition mechanism existed competitive inhibition and uncompetitive types in the alkaline soils（S10-S17）.The competitive(K_ic)and non-competitive(K_iu)inhibition constants of Sb on arylsulfatase were 0.058 to 0.142 mM and 0.075 to 0.503 mM,respectively.Besides,the values of inhibition constants were smaller than K_m（1.3 to 4.35 mM）indicating that the binding strength of Sb to arylsulfatase and the arylsulfatase-substrate complex were stronger than the affinity between arylsulfatase and the substrate,thus leading to decrease arylsulfatase activity.In addition,the inhibition constants(K_ic:0.058 to 0.085 mM and K_iu:0.075 to 0.153mM)of acidic soils（S1-S9）were lower than those of alkaline soils(K_ic:0.140 to 142 mM and K_iu:0.124 to 0.503 mM)suggesting that Sb has a greater affinity for enzyme and enzyme-substrate complex in acidic soils,so the inhibition rates of Sb to arylsulfatase activities were higher in acidic soils.The thermodynamic studies of arylsulfatase found that these parameters,such as activation energy（E_a）,activation enthalpy（ΔH^*）,and activation entropy（ΔS^*）,were increased under acute Sb stress,indicating that the inhibition of Sb to arylsulfatase enzymatic reaction were controlled through enthalpy process.The study on the aging of exogenous antimony in soils and its soil enzyme effects showed that the available Sb（the sum of water-soluble,ion exchangeable,and carbonate bound Sb）content in red（pH 4.90）and gray desert soils（pH 8.12）decreased along with the aging time.The aging process of Sb in soils fitted the Elovich equation significantly,suggesting that the aging process of Sb in soils was a heterogeneous dispersion process.The aging rate constant（|b|）of Sb in the red soil was 1.2 to 1.9 times that of the gray desert soil.This result illustrated that the Sb aging in the red soil first reached an approximate equilibrium state.Antimony presented different effects on soil enzyme activities under various aging times.To obtain the soil enzymology indexes that can evaluate soil Sb pollution during the whole aging period,the soil enzyme activities at different aging times were measured.The enzyme activities were divided into the extracellular,intracellular,and total enzyme by chloroform fumigation.For extracellular enzymes,the ED₁₀values of Sb to arylsulfatase were 42.3 to 228.3 mg kg^-1,which were only used to evaluate Sb contamination within 60 days.The ED₁₀values of Sb to urease ranged from 29.6 to 1791.2 mg kg^-1,which were used to access Sb pollution during aging 120 days.For the total enzyme,the ED₁₀ values of Sb to arylsulfatase ranged from167.86 to 520.88 mg kg^-1for two soils;the ED₁₀ of Sb to urease was just presented in the gray desert soil.Although the arylsulfatase existed in intracellular enzyme activity,it was not sensitive to Sb pollution.These results illustrated that extracellular enzyme activities are more suitable to assess antimony pollution in soils.The comprehensive indexes of extracellular enzyme activities included the total enzyme activity index（TEI）and the geometric mean（GM）.The ED₁₀ values of Sb to TEI and GM were 24.9 to 184.7 and 25.7 to 199.4 mg kg^-1,respectively.Therefore,the comprehensive indexes of extracellular enzyme activity can also evaluate soil Sb pollution.The distribution of Sb fractions and microbial community diversity based on enzyme activity of typical Sb contaminated soil in the mining area were studied.The Sb content in these soils reached 110.59 to 8398.19 mg kg^-1.Except for L15 soil,the Sb content in other soils was higher than 360 mg kg^-1（GB36600-2018）,which was the Sb pollution control value in the secondary soil standard of construction land in China.The residual Sb in all soils was the main fraction accounting for 76.51 to 95.29%of the total Sb content.Available Sb contents in all soils were 3.95 to 526.79 mg kg^-1.This suggested that soils in the Sb mining area were heavy pollution and serious ecological environmental risks.The variation coefficients of geometric mean（GM）,weighted mean（WM）,and total enzyme activity index（TEI）of L1-L15 soils are greater than the Shannon index and Simpson index of enzyme activity,indicating that the functional diversity of microbial communities in Sb contaminated soils in the mining area is higher than the structural diversity.The Sb contents in soils were closely related to the indexes of soil enzyme activity.Both urease activity and total enzyme activity indexes were significantly negatively correlated with total Sb concentration.The ED₁₀ value of Sb to urease activity was 242.40 mg kg^-1.Total organic carbon and available phosphorus concentration in soils were significantly positive with urease activity.In conclusion,there was the most toxic effect of Sb on soil arylsulfatase under acute pollution compared to other soil hydrolases.The action mechanism of Sb to arylsulfatase was mainly non-competitive and linear mixed inhibition.Meanwhile,the enzymatic reaction rates were reduced by increasing the enzymatic reaction barrier under acute pollution.The aging of Sb in soils is a heterogeneous dispersion process,and the aging rate of Sb in red soil is higher than that in gray desert soil.Compared with total enzyme and intracellular enzyme,the extracellular enzyme activity was more suitable to access Sb pollution at different aging times.In general,the arylsulfatase extracellular enzyme could evaluate the Sb pollution within 60days,while the urease extracellular enzyme,total enzyme activity index,and geometric mean value of enzyme activity could evaluate the Sb pollution within 120 days.Urease activity can be used to evaluate the Sb pollution in typical Sb contaminated soil of mining areas.This study expanded the theoretical information on the response and mechanism of soil enzymes to Sb pollution and provided evidence for evaluating the degree of Sb pollution and soil environmental risk.