| Arsenic(As)pollution has become one of the most widespread and toxic metal pollution at present.It not only influence soil normal ecological functions,but also do harmful to human beings through food chains to enter the body.Thus,it is very necessary to assess and monitor the environmental risk of soil As pollution.Soil enzyme plays an important role in soil substance cycling and biochemical process,and is a good bioindicator to monitor soil pollution.Studying the relationships of soil enzyme and As pollution and the action mechanism is meaningful for revealing the effect of As on soil ecological function and pollution monitoring and restoration.In this paper,the effects of simulated As pollution and long-term field As pollution on soil enzyme activity,kinetics,thermodynamic,functional diversity and stability were studied..We calculated the toxicity threshold of As pollution by fitting the relationship between As concentration and enzyme response.This study enriched the theory about soil enzyme as an index to assess heavy metal pollution.The main conclusions are as follows:(1)The effects of simulated acute As pollution on soil enzyme activity were studied.As had no inhibitory effect on soil invertase activity;arsenate showed no significant inhibition on soil urease,but high concentration of arsenite inhibited urease activity;arsenate strongly decreased soil alkaline phosphatase activity,but arsenite had little effect on it;while soil dehydrogenase was very sensitive to both of arsenate and arsenite.Soil fertility affected As inhibition on soil enzyme.For the same type of soils,the enzyme in soil with higher fertility showed less sensitivity to As pollution.(2)The kinetics and thermodynamics approaches were used to study the inhibition mechanism of As on alkaline phosphatase and acid phosphatase in soils with varied properties.The inhibition of As on activities of alkaline phosphatase and acid phosphatase was influenced by substrate concentration.In the same As concentration,the rate of inhibition of activity declined with the increase in substrate concentration.Under arsenic pollution,the Vmax remained unchanged or declined at high As concentration;Km,however,increased linearly with increasing As concentration.Thus the type of inhibition was competitive or mixed inhibition(including competitive and noncompetitive)type,with the former one being the dominated type.The kinetics of acid phosphatase showed that Vmax decreased and Km remained unchanged or increased linearly.The inhibition of As on acid phosphatase belonged to noncompetitive or mixed inhibition,the noncompetitive type was the dominant one.The competitive inhibition constant for alkaline phosphatase(Kic)ranged from 0.17~0.70 m M,which was smaller than the constant of enzyme for its substrate(Km).The noncompetitive inhibition constant Kiu ranged from 2.51-5.92 m M.The Kic of acid phosphatase ranged from 0.38~3.65 m M and Kiu was from 0.84 to 7.43 m M.The Kic and Kiu of the phosphatase determined the affinity and binding site of As with phosphatase,resulting in the difference in the inhibitory mechanism and ecological toxicity.Soil properties affected the inhibitory mechanism of As on phosphatase and its ecological toxicity by influencing the affinity and binding site of As with phosphatae.Soil PH,organic matter,amorphous Fe and CEC were the major affecting factors.The thermodynamics of alkaline phosphatase indicated that the inhibition by As was an enthalpy controlled process and the values of Ea,?H*,?S* and ?G* were increased with a different extent under As pollution.(3)The response of clay immobilized alkaline phosphatase to As pollution was investigated.Results suggested that the kinetic and thermodynamic responses in free and immobilized alkaline phosphatase to As were similar with those in soil alkaline phosphatase.The inhibition belonged to competitive inhibition and it was an enthalpy controlled process.The clay of montmorillonite and goethite decreased the rate of inhibition of alkaline phosphatase by As.The changes in kinetic parameters(Km?Vmax/Km)of alkaline phosphatase caused by As were alleviated by clay immobilization.Montmorillonite alleviated the effects of As on alkaline phospahtase thermodynamic properties(Ea,?H* and ?S*),while goethite had the opposite function,which indicated different effects of different clay on As toxicity to alkaline phosphatase.In general,enzyme immobilization on clay minerals alleviated As toxicity and showed protection effect from As pollution.(4)The contamination status of As and its impact on soil enzyme ecological functions in realgar mine area in Shimen county has been studied.Soil As concentrations in mine area were generally exceed the national limit and the maximum one reached 4008.5 mg kg-1.The main fraction of As in soil was the residual one;the ratio of water soluble,exchangeable and carbonate bound fractions to total As content were small and the proportion was below 3%;the coefficient of As bioavailablity was inferior,however,because of the high content of total As leading the high content of 3 easily migrate As fractions,the risk of toxicity is high.Soil enzyme activities were closely related with the concentration of As.?-glucosidase,invertase,urease,acid phosphatase,arylsulfatase,FDA hydrolase and dehydrogenase showed significant negative correlation with different fractionations of As.Soil ?-glucosidase,acid phosphatase and dehydrogenase(Vmax?Vmax/Km)showed significant negative correlation with total As content,alkaline phosphatase has insignificant relation with As.The functional stability properties of soil enzyme suggested that the heat stability for 4 soil enzyme ranked: urease > ?-glucosidase > acid phosphatase > FDA hydrolase.The contamination of As decreased the heat stability of soil acid phosphatase and FDA hydrolase;as for ?-glucosidase,the mid-level polluted soils showed the highest stability.Soil enzyme activities,kinetics and stability were not only influenced by As content but also affect by soil properties.The CEC,total nitrogen and p H were main factors impact on enzyme activities;enzyme kinetics was related with total nitrogen,alkali-hydrolyzale nitrogen,p H,CEC,available phosphorus,with the total nitrogen and alkali-hydrolyzale being the most significant factors;the stability of soil enzyme showed a significant correlation with total nitrogen,alkali-hydrolyzale nitrogen and available phosphorus.(5)The ecological doses ED10 and ED50(the concentration of As caused 10% and 50% decrease in enzyme parameters)were calculated.In laboratory simulation contamination test,the ED10 and ED50 for Vmax/Km of alkaline phosphatase were 7.4~37.3 and 66.9~335.6 mg kg-1,respectively,which was the lowest ones among all indices of soil enzyme.In long term contaminated soils,dehydrogenase was the most sensitive enzyme to long term As pollution;the ED10 for activity,Vmax and Vmax/Km of dehydrogenase were 71.9,55.8,34.9 mg kg-1,respectively;Vmax/Km was more sensitive than enzyme activity and Vmax to As pollution,the similar result has been found in ?-glucosidase and acid phosphatase.The diversity indices,total enzyme activity,geometric means and weighted means was weaker than single sensitive enzyme indices to assess As pollution.To sum up,kinetic parameter of sensitive enzyme to As contamination,namely Vmax/Km,was the most sensitive index to assess As contamination,compared with traditional enzyme activity index.The critical value of slight As pollution(ED10)from laboratory simulated contamination test was 7.4 mg kg-1,which could be the threshold for acute contamination.Under long term field pollution condition,the threshold of slight pollution was 35 mg kg-1,which was identified with the second contamination level for As in soils(for agriculture use)in national soil environmental quality standard.This confirmed that soil enzyme was a sensitive and accurate index for soil contamination assessment. |
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