Evaluation Of The Toxicity Of Cr³⁺ To Soil Extracellular/intracellular Arylsulfatase

Chromium（Cr）pollution has attracted lot of attentions world-widely due to its toxicity to eco-environment and human health.Cr exists mainly in two states in the environment:Cr³⁺and Cr⁶⁺.Although Cr³⁺is less migratory than Cr⁶⁺in the environment,it can combine with organic molecules in the environment to form complexes which is difficult to monitor.It can cause extremely serious pollution to the environment when it is largely accumulated in the soil.Therefore,it is vital to monitor and assess the Cr³⁺contamination risk in the environment.Soil enzymes participate in the conversion of nutrients such as C,N,P,S.They are widely used for the evaluation of soil heavy metal contamination.Soil enzymes mainly exist in soil in two states:intracellular enzymes and extracellular enzymes.The difference of their response to heavy metals may affect the evaluation of heavy metal pollution.Thus,laboratory simulation tests were conducted in this study to explore the differences in the responses of total,extracellular and intracellular arylsulfatase enzyme to Cr³⁺pollution by the means of soil enzymatic kinetics and dose-response models.The object was to find suitable enzymatic indicators for the evaluation of Cr³⁺pollution.Furthermore,we also explored the influence of Cr³⁺concentration,pollution time,and soil physical and chemical properties on the Cr³⁺ecotoxicity to soil arylsulfatase.The main results and conclusions are as follows:（1）The proportion of extracellular arylsulfatase and intracellular arylsulfatase varied in different uncontaminated soils.The proportion of extracellular enzyme reached 80.25%in red soil,50.53%in paddy soil,while only 30.56%in black soil.The correlation analysis found that the total and intracellular arylsulfatase activities exhibited highly significant positive correlation with the SOM and TN contents in uncontaminated soils.Cr³⁺significantly inhibited the arylsulfatase activities with different existence states under acute contamination conditions,with the highest inhibition rates being 70%（total arylsulfatase）,72%（extracellular arylsulfatase）and 70%（intracellular arylsulfatase）,respectively.The ecological dose values(ED₁₀,the contaminant concentration that results in a 10%decrease in enzyme activities)of the inhibition of Cr³⁺on total,extracellular and intracellular enzyme ranged from 28.4 to 309.82 mg·kg^-1,28.27 to 551.56 mg·kg^-1and 20.61 to 173.77 mg·kg^-1,respectively.The intracellular enzyme,with the smallest ED₁₀value,was the most sensitive indicator to evaluate Cr³⁺acute contamination.Principal component analysis showed that SOM,TN and NH₄⁺-N were the main factors affecting the relationship between arylsulfatase activity and Cr³⁺under acute contamination conditions.（2）Under acute Cr³⁺contamination,the enzymatic kinetics parameters Michaelis constant（K_m）of arylsulfatase increased in different degree,while maximum reaction velocity(V_max)of arylsulfatase was constant or reduced,which were related to the soil properties and enzyme states.The inhibition types of Cr³⁺on total,extracellular and intracellular enzymes under acute contamination were mainly competitive inhibition.With the increase of Cr³⁺concentration,the catalytic efficiency(V_max/K_m)was significantly inhibited,and the ecological dose values(ED₁₀)of the inhibition of Cr³⁺on the V_max/K_mof total,extracellular and intracellular arylsulfatase in different soils were 51.92～140.63mg·kg^-1,45.72～257.26 mg·kg^-1and 65.36～274.64 mg·kg^-1,respectively.The paddy soil,with the highest organic matter content and stronger buffering effect on Cr³⁺,had the largest ecological dose values.Except for the extracellular enzyme in red soil,the competitive inhibition constant(K_ic)of the remaining soils were greater than the K_mvalues when uncontaminated,revealing that Cr³⁺might inhibit arylsulfatase activities by binding to arylsulfatase active sites.Correlation and principal component analysis showed that the inhibition effect of Cr³⁺on different state arylsulfatase was related to soil properties.（3）The proportion of extracellular and intracellular arylsulfatase in soil changed under Cr³⁺aging process.The percentage of extracellular enzymes in black soil increased under the Cr³⁺concentration of 1000 and 1500 mg·kg^-1.The total,extracellular and intracellular arylsulfatase activities and microbial biomass carbon were inhibited by Cr³⁺to different degrees during Cr³⁺aging process,and the higher the Cr³⁺concentration,the stronger the inhibition effect.Two-factor ANOVA revealed that Cr³⁺concentration,aging time and the interaction between them were all factors that affected the inhibition effect of Cr³⁺on different state arylsulfatase activities and microbial biomass carbon.The correlation analysis showed that total and intracellular arylsulfatase were significantly positive correlated with microbial biomass carbon.（4）The kinetic characteristics of arylsulfatase enzymes changed at the beginning and end of Cr³⁺aging process.Apparently,the inhibition types of Cr³⁺on total and intracellular arylsulfatase enzymes in black soil changed from linear mixed inhibition dominated by non-competitive inhibition to non-competitive inhibition.In cumulated irrigated soil,the inhibition type of Cr³⁺on total enzyme changed from non-competitive to linear mixed inhibition dominated by non-competitive inhibition in the silt soils,and the inhibition type of Cr³⁺on intracellular enzyme changed from competitive to linear mixed inhibition dominated by competitive inhibition.The ecological dose values of different state arylsulfatase varied in different soils after 90 days of aging,but all the ecological dose values showed in the order:intracellular enzyme<total enzyme<extracellular enzyme,indicating that the intracellular enzyme was more sensitive to Cr³⁺stress.The inhibition constants(K_icand K_iu)of the intracellular enzymes in different soils decreased and were both smaller than K_m（the affinity between the intracellular enzymes and the substrate）after 90 days of aging,resulting in a decrease in the catalytic efficiency of the intracellular enzymatic reactions.The changes in the inhibition constants of the extracellular enzymes after 90 days of aging were not consistent in different soils,which decreased in black soil,increased in cumulated irrigated soil.The results indicated that the inhibition effect of Cr³⁺on different states arylsulfatase is affected by the soil properties and aging time as well as the enzyme existence state.（5）This study found that intracellular arylsulfatase enzymes showed higher sensitivity in the evaluation of both acute and aging Cr³⁺contamination,and are more suitable as indicators for the enzymatic evaluation of Cr³⁺contamination.By comparing the ecological dose values of intracellular enzyme activity and catalytic efficiency,the overall ED value of intracellular enzyme activity was smaller,which was closer to the risk screening value and risk control value of farmland soil,and could provide reliable biological basis for determining the risk threshold of soil Cr pollution.