土壤汞污染的物理化学行为及其微生物学特征
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摘要
本研究是国家重点基础研究发展规划(973)项目(编号：2002CB410804)“长江、珠江三角洲地区土壤和大气环境质量变化规律与调控原理”子课题“重金属在土壤—植物系统中迁移、转化、积累规律与农产品安全”的组成部分。根据国家项目的特定研究内容、目标的要求，本研究选择了长江三角洲的小粉土、黄红壤和青紫泥三种代表性人为耕作土壤作为研究对象，通过实验室模拟分析及网室盆栽试验研究了土壤中主要污染物汞的吸附解吸行为，生物有效性，土壤—水稻(蔬菜)系统中土壤微生物的汞生态效应、群落结构和酶活性等。取得了以下有一定特色和意义的研究结果：
1．土壤对汞离子的吸附-解吸行为与土壤性质密切相关。有机质和粘粒含量较高的黄红壤对汞离子的吸附容量最大，其次是青紫泥，最小的是小粉土。小粉土、黄红壤和青紫泥对汞离子的吸附可以用Langmuir和Freundlich吸附模型很好的拟合，Langmuir方程的相关系数更高一些，说明它更适用于描述汞的吸附。小粉土、黄红壤与青紫泥对汞的最大吸附量(K_L)分别为111.11 mg kg~(-1)、212.77 mg kg~(-1)和188.68 mg kg~(-1)。三种供试土壤在吸附汞离子后平衡液的pH随添加汞浓度的增加而显著降低。随汞浓度增加，三种土壤中汞的吸附分配系数呈指数下降。在汞加入浓度1000 mg kg~(-1)时，用0.1 M KCl连续解吸5次，小粉土吸附汞中有24.40％被解吸，而黄红壤则有14.37％吸附汞被解吸，青紫泥有18.35％吸附汞被解吸。
pH对土壤吸附汞离子的解吸影响较大。小粉土、青紫泥和黄红壤吸附Hg~(2+)的解吸率均随pH值升高先降低，后变平缓，又升高，在pH为3.00-5.00范围内下降，pH为5.00-7.00曲线趋于平缓，pH为7.00-9.00曲线趋于升高，这说明酸性的环境(pH＜5.4)有利于Hg~(2+)解吸，并且对Hg~(2+)解吸率随pH增加而减少，因而pH也是控制土壤中汞有效性一个重要因素。
在加入Cu~(2+)和Zn~(2+)后，三种供试土壤吸附汞的解吸量大大增加，在相同Cu~(2+)和Zn~(2+)离子浓度下，Cu~(2+)对Hg~(2+)解吸量增加的幅度较Zn~(2+)大。铜加入量从0 mM到15 mM，小粉土、青紫泥和黄红壤吸附汞的解吸量分别从1.50 mg L~(-1)、1.64 mg L~(-1)和2.06 mg L~(-1)增加到5.45 mg L~(-1)、4.48 mg L~(-1)和2.68 mg L~(-1)；锌加入量从0 mM到20 mM，小粉土、青紫泥和黄红壤吸附汞的解吸量分别从1.50 mg L~(-1)、1.64 mg L~(-1)和2.06 mg L~(-1)增加到4.53mg L~(-1)、3.96 mg L~(-1)和2.56 mg L~(-1)。
研究表明有机酸对三种土壤中汞的解吸行为影响极大，随加入的有机酸浓度增大，三种土壤吸附汞的解吸量增大。对小粉土而言，低浓度(＜10~(-4) M)的有机酸可抑制对Hg~(2+)的解吸，而高浓度(＞10~(-4) M)有机酸可促进Hg~(2+)的解吸。不同有机酸对土壤吸附汞的解吸行为的影响也不同，本实验研究的四种有机酸对三种土壤吸附汞的解吸行为的影响中，按对土壤吸附汞离子解吸的促进作用强弱排序为：柠檬酸＞酒石酸＞苹果酸＞草酸。
2．本文从土壤化学角度出发，对HCl、NH_4OAC、DTPA和CaCl_2四种提取剂对小粉土、黄红壤和青紫泥中汞的化学提取条件及提取性作了初步研究。结果表明，随着平衡时间延长增加，汞提取量有逐渐增加的趋势。在三种土壤中各提取剂的提取率达到稳定的提取时间分别为：小粉土(HCl 10分钟；CaCl_2 10分钟；DTPA 10分钟；NH_4OAc 10分钟)；黄红壤(HCl 10分钟；CaCl_2 30分钟；DTPA 30分钟；NH_4OAc 30分钟)；青紫泥(HCl 10分钟；CaCl_2 30分钟；DTPA 30分钟；NH_4OAc 30分钟)。可见，提取时间30分钟时，有效汞含量基本达到平衡，因此，对四种提取剂来说，30分钟可作为提取三种供试土壤汞有效态的最佳提取时间。
研究表明，随着土液比的不断减小，三种土壤四种提取剂Hg的提取量明显提高。为减少试验误差，确定1：5为较适宜的土液比。
四种提取剂在三种供试土壤中的提取能力有显著的差异。对于小粉土，其顺序为：NH_4OAc＜CaCl_2＜DTPA＜HCl；对于黄红壤，其顺序为：NH_4OAc＜CaCl_2≈DTPA＜HCl；对于青紫泥，其顺序为：DTPA＜NH_4OAc＜HCl＜CaCl_2。这表明HCl和DTPA，特别是HCl提取效率较高。
用水稻籽粒、小白菜叶、萝卜根含汞量与提取剂提取汞量进行相关统计分析。结果表明：对土壤—水稻系统来说，小粉土和黄红壤用DTPA作提取剂，青紫泥用CaCl_2作提取剂，在振荡平衡时间30min，土液比1：5条件下，测定土壤中有效态Hg是较为适宜的方法；
对土壤—小白菜系统来说，在振荡平衡时间30min，土液比1：5条件下，由于三种供试土壤上小白菜叶Hg含量与4种提取剂提取Hg量相关不显著，因此，需要选择其它的提取剂来提取；
对土壤—萝卜系统来说，在振荡平衡时间30min，土液比1：5条件下，小粉土和黄红壤上萝卜根Hg含量与提取剂HCl提取Hg量相关系数最大，而青紫泥上萝卜根Hg含量与提取剂CaCl_2提取Hg量相关系数最大。因此，小粉土和黄红壤用HCl作提取剂，青紫泥用CaCl_2作提取剂，在振荡平衡时间30min，土液比1：5条件下，测定土壤中有效态Hg是较为适宜的方法。
通过综合分析4种提取剂的提取能力、提取结果与植物汞含量的关系，确定HCl为评价本研究三种供试土壤中汞元素有效性的最佳化学提取剂。
3．土壤—水稻作物系统中，本实验条件下，汞污染对土壤环境质量微生物学和酶学指标有较大的影响。水稻收获后，除部分土壤的呼吸强度和代谢商随汞处理浓度的增加而持续增加外，不同Hg处理后土壤微生物量碳、呼吸强度，土壤脲酶、酸性磷酸酶和脱氢酶均表现为在低浓度Hg处理时有促进作用，在高浓度时则表现出抑制作用。不同指标出现峰值时的Hg浓度分别是0.5、1、1.5、2mg kg~(-1)不等。综合来看，土壤微生物商是Hg处理后比较敏感的微生物学指标。Hg不同处理的土壤酶活性强弱依次为：黄红壤＞青紫泥＞小粉土。通过对重金属生态剂量ED50的分析，表明Hg对小粉土脲酶活性的生态毒性比相应的青紫泥和黄红壤强；对黄红壤磷酸酶活性的生态毒性比相应的青紫泥和小粉土强；对青紫泥脱氢酶活性的生态毒性比相应的小粉土和黄红壤强。
本文也证实了BIOLOG系统是反映土壤微生物生理轮廓和微生物群落结构的有效手段。微生物群落代谢剖面AWCD值和McIntosh指数可以较好的反应本实验三种土壤微生物群落结构和汞污染之间的剂量关系。汞污染严重的土壤AWCD值、McIntosh指数均显著低于无污染和轻度污染的土壤，表明汞污染引起了红砂土和黄筋泥微生物群落结构功能多样性的下降，减少了能利用有关碳源底物的微生物的数量，最终导致土壤微生物群落功能结构多样性发生变化。
4．土壤—(小白菜)作物系统中分析测定的大部分土壤微生物学和酶学等生态指标，均与土壤—水稻系统表现出相似的规律。本试验中，就平均而言，青紫泥中的土壤微生物量碳最高，其次是黄红壤，最小的是小粉土。同时，小粉土中对汞污染的敏感性最强，其次是青紫泥，最弱的是黄红壤。不同浓度汞处理后，土壤微生物活性的变化与土壤微生物量的变化规律相似。即，随汞处理浓度增大土壤微生物活性而增强，到一定浓度后转为减弱。三种供试土壤的微生物商差异显著，黄红壤的微生物商最大，其次是青紫泥，最小的是小粉土。比较而言，微生物商是对土壤微生物比较敏感的指标。
平均而言，黄红壤和青紫泥中的土壤酶活性高于小粉土。除磷酸酶外，Hg多数处理浓度对土壤酶活性表现出抑制作用，与种植水稻时多数处理浓度起促进作用正好相反。Hg污染条件下小粉土脲酶活性受抑制的幅度最大，黄红壤的受抑制最小。种植小白菜后，除磷酸酶和小粉土中的脱氢酶外，其它土壤中脲酶和脱氢酶活性与重金属处理间均达负相关水平，但未达到显著水平。研究表明Hg对小粉土脲酶活性的生态毒性比相应的青紫泥和黄红壤强；Hg对黄红壤磷酸酶活性的生态毒性比相应的青紫泥和小粉土强；Hg对小粉土脱氢酶活性的生态毒性比相应的青紫泥和黄红壤强。
此外，在小白菜收获后，采用碳素利用法(BIOLOG测试系统)对土壤微生物功能和群落结构多样性分析，结果表明：随Hg各处理浓度增加，BIOLOG ECO盘平均吸光值(AWCD)降低，群落代谢剖面值与培养时间间呈非线性关系，其变化过程符合微生物种群生长动态模型，呈“S”形增长模式(logistic模型)。
5．土壤—(萝卜)作物系统中Hg的生态效应与土壤—水稻或蔬菜系统有相似之处。就平均而言，青紫泥和黄红壤中平均Cmic高于小粉土。从土壤看，青紫泥和黄红壤中土壤呼吸强度要高于小粉土。黄红壤和青紫泥的土壤代谢商除了峰值外差异不大，但小粉土的最高，其次是黄红壤，最低的是青紫泥。与栽培小白菜后的变化不同，青紫泥的微生物商最大，其次是小粉土，最小的是黄红壤。
不同浓度汞处理后，土壤酶活性在供试的三种土壤中都表现出一定的差异性。起初，随着汞处理浓度的增高，三种土壤中酶的活性增强，小粉土到1 mg kg~(-1)处理时出现峰值，黄红壤和青紫泥到1.5 mg kg~(-1)处理时出现峰值，然后随汞浓度增高而下降。
种植萝卜后，Biolog生态盘的AWCD随培养时间的增加而增强，呈“S”型指数增长模式。三种土壤的平均吸光值依次为：青紫泥＞黄红壤＞小粉土，表明青紫泥和黄红壤中微生物活性超过小粉土。
本研究阐明了在长江、珠江三角洲地区主要土壤类型中汞的物理化学行为、生物有效性及生物学特征；揭示汞在土壤—作物系统中迁移、积累、平衡规律及其主控因子；明确土壤—作物系统中汞污染的生态毒理、预测方法及其调控机制。在土壤-植物系统中汞的迁移、转化和积累规律及其健康效应方面的基础理论研究方面取得较大突破，为国家制定该地区土壤汞污染控制、削减、修复行动计划提供科学依据。
This study was a part of the research project "the mechanisms of movement,transform, and accumulation of typical heavy metals in agricultural soil and theirrelationships to plant ecosystem and farm produce safety", a sub-project of the NationalKey Basic Research Program of China (973)"the temporal and spatial variation andadjusting and controlling principles of the environmental quality of soil and atmosphere inthe areas of Yangtse Rive and Pearl Rive Deltas"(No. 2002CB410804)". According to thedemands of the specific research content and aims of the national program, Three paddysoils with contrasting properties were studied. A silty loam soil (Paddy soil 1) wascollected from Huajiachi campus, Zhejiang University, Hangzhou, a yellowish red soil(Paddy soil 2) from Deqing County, and a purplish clayey soil (Paddy soil 3) from JiaxingCounty in Zhejiang Province, China. All of them belong to Gleyi-Stagnic Anthrosols inFAO/UNESCO nomenclature.
Both laboratory and greenhouse pot experiments using rice, Chinese cabbage andradish as indicator plants were conducted to investigate the adsorption-desorptioncharacteristics and availability of Hg, and the ecological effects of increasing Hg loadingson microbial biomass, microbial activity, microbial community structure, and soil enzymeactivities. The major objectives of this study were to understand biogeochemical behaviorof Hg in three tested soils and to compare the potential of chemical, biochemical, andmicrobiological parameters for indicating and predicting Hg contamination. The resultswere summarized as follows:
1. Adsorption-desorption of Hg at contaminated levels in three tested soils wereinvestigated. In all cases, Hg~(2+) adsorption decreased in the order: YRS soil＞PCS soil＞SLSsoil, with YRS having the highest sorption capacity for Hg. The maximum adsorption (K_L)value was 111.11 mg kg~(-1) for SLS soil, 212.77 mg kg~(-1) for YRS soil and 188.68 mg kg~(-1)for PCS soil. The isotherms of Hg~(2+) adsorption in the soils can be well described by thesimple Langmuir and Freudlich model. The simple Langrnuir model gave a relativelybetter representation of the experimental data based on the fitting correlation coefficients(r~2).
Adsorption of Hg~(2+) decreased soil pH by 0.75 unit for the SLS soil, 0.91 unit for theYRS soil and 0.55 unit for the PCS at the highest loading. The distribution coefficient (k_d)of Hg in the soils decreased exponentially with increasing Hg~(2+) loading. The adsorptionequilibrium pH of YRS was higher than that of SLS at the same Hg~(2+) concentration.Most of the adsorbed Hg~(2+) in the soils was not desorbed in the 0.01 M KCl solution. Afterfive successive desorptions, the accumulative amounts of Hg~(2+) desorbed accounted foronly 24.40％of the adsorbed Hg~(2+) for SLS soil, 14.37％for YRS soil and 18.35％for PCS soil, indicating that the YRS soil had the greatest affinity for Hg~(2+) at the same Hg~(2+)loading. Different mechanisms may be involved in Hg~(2+) adsorption/desorption atdifferent levels of Hg~(2+) loading and among three testedsoils.
Desorption characteristics as affected by desorption solution pH, organic acids, andcompetitive ions were also studied, pH was the most important factor controlling Hg~(2+)desorption. Mercury desorption with increasing solution pH was characteristic of a "U"pattern and consisted of a desorption decreasing stage at pH 3.0-5.0, a precipitation andminimal desorption stage at pH 5.0-7.0; and a desorption increasing stage at pH 7.0-9.0.
Organic acids remarkably influenced the desorption behavior of Hg~(2+) in the paddysoils. The presence of organic acids at low concentrations (＜10~(-4) M) tended to inhibit Hg~(2+)desorption, but enhanced Hg~(2+) desorption at higher concentrations (＞10~(-4) M). Citric acid athigh concentrations (＞10~(-3) M) was the most effective in increasing Hg~(2+) desorption,followed by tartaric acid and malic acid, and oxalic acid was the least effective. Thedesorption of Hg~(2+) was also affected by the presence of competitive cations (Cu~(2+) or Zn~(2+)).The desorption of adsorbed Hg~(2+) increased with increasing concentrations of added Cu~(2+)or Zn~(2+), and Cu~(2+) was more effective than Zn~(2+) in competing with Hg~(2+) for adsorbing sitesat the same concentration levels.
2. Four extractants, including 0.1 M HCl, 1 M NH_4OAc(pH7.0), 0.005 M DTPA and 0.1M CaCl_2 (pH5.0) were evaluated for their extraction of available Hg~(2+) in the three testedsoils at different equilibrium times and different soil: solution ration.
The amount of Hg~(2+) extracted increased with the increasing of equilibrium time. Theequilibrium time required for maximum extraction of the added Hg~(2+) varied among thefour extractants: 10 min for the four extractants in the SLS soil; 10 min for the HCl, 30min for the other extractants for the YRS and the PCS soil. Overall, 30 min is the bestequilibrium time. The amount of Hg~(2+) extracted decreased with increasing soil: solutionratio. Our study shows that the soil:solution ratio of 1:5 is adequate. The amount of Hg~(2+)extracted increased in the order of NH_4OAc＜CaCl_2＜DTPA＜HCl for the SLS soil,NH_4OAc＜CaCl_2≈DTPA＜HCl for the YRS soil, and DTPA＜NH_4OAc＜HCl＜CaCl_2 for thePCS soil.
There were significant correlations between concentrations of Hg in edible tissue andthe amounts of Hg extracted with the four extractants for soil-rice system and soil-radishsystem, but not for soil-Chinese cabbage system. According to the capability of differentextractants and the correlation of the extractable Hg~(2+) and Hg contents in edible planttissues, the 0.1M HCl was the best extractant among the four extractants and couldindicate plant availability of Hg in soil-plant systems.
3. The changes of soil microbial activities and the functional diversity of microbialcommunity can be used to indicate the dose effect of Hg~(2+) on soil ecological system. Somesoil microbial parameters such as microbial biomass carbon, microbial quotient, AWCD, and urease activity were sensitive to Hg pollution.
Mercury ecological dose responses of basal respiration, microbial biomass carbon,metabolism quotient, microbial quotient, and microbial community diversity wereinvestigated in the SLS, YRS, and PCS soils at Hg~(2+) loadings of 0, 0.25, 0.5, 1, 1.5, 2, 3,6mg kg~(-1). The results showed that mercury (Hg~(2+)) pollution had significant effects on themicrobial and enzymatic indices. There were several ecological effect characteristics ofHg in the soil-rice system. After harvesting rice plant, except for basal respiration andmetabolism quotient, which increased with increasing level of Hg treatments, all measuredmicrobial and enzymatic indices including microbial biomass carbon, microbial quotientincreased with the increasing level of Hg treatments at low concentrations (＜2mg kg~(-1)) anddecreased at the 0.5, 1, 1.5, 2mg kg~(-1) Hg treatments. The results also indicate that soilmicrobial quotient is more sensitive to Hg contamination than the other microbial indices.
Some important soil enzymes including urease, dehydrogenase, and acid phosphatase,which are related to cycling of soil C, N and P, were investigated. Mercury input had verydifferent effects on the enzyme activities. Enzymatic activity of the YRS soil was highest,followed by the PCS soil, and the SLS soil was the least.. The analysis of ED50 indicatedthat ecological toxicity of urease was the strongest for the SLS soil; ecological toxicity ofacid phosphatase was the strongest for the YRS soil; and ecological toxicity ofdehydrogenase was the strongest for the PCS soil.
After harvesting rice plants, soil microbial function and community structurediversity were analyzed using BIOLOG, soil microbial functional diversities of microbialcommunity were changed to varying extent under the stress of Hg~(2+) pollution. The averagewell color development (AWCD) on the ECO plate reduced with increasing level of Hg.There was a nonlinearly relationship between AWCD and incubation time, and therelationship followed a growth dynamic model of microbial population (Logistic curve).The microbial community richness and McIntosh index decreased in polluted soil than thecontrol soil. The results suggest that the structure of microbial community has beenchanged under Hg~(2+) stress. Mercury pollution decreased the functional diversity ofmicrobial community, and reduced the microbial utilization of different carbon resources.
4. The ecological effect of Hg in soil-vegetable (Chinese cabbage and radish) systemwas similar to those in soil—rice system. Namely, small addition of Hg (＜2mg kg~(-1)) to soilcould promot above soil microbial and enzymatic indices, but excessive addition of Hg(＞2mg kg~(-1)) to soil declined the soil microbial and enzymatic indices.
In the soil-Chinese cabbage system, the results indicate that soil microbial quotient ismore sensitive to Hg contamination than the other microbial indices. In addition,enzymatic activities were higher in the YRS and PCS soil than the SLS soil. Afterharvesting Chinese cabbage, except for acid phosphatase whose activity increased withincreasing level of Hg treatments, most of Hg treatments had inhibitory effects on enzymatic activity in contrast with soil-rice system. Mercury treatments had the mostinhibitory effects on urease activity in SLS soil, less in PCS soil, and the least in YRS soil.There were no significant relationships between dehydrogenase and soil Hg loadings, norbetween urease activity and soil Hg~(2+) loadings except for acid phosphatase anddehydrogenase in SLS soil. The analysis of ED50 indicated that ecological toxicity ofurease and dehydrogenase was the strongest in the SLS soil; ecological toxicity of acidphosphatase was the strongest in the YRS soil after harvesting Chinese cabbage.
After harvesting radish, microbial biomass carbon in SLS soil was the highest, butbasal respiration of SLS soil was the smallest. In soil-radish system, the peak values ofsoil enzymatic activity varied with soils, 1 mg kg~(-1) for SLS soil, 1.5 mg kg~(-1) for YRS andPCS soils. The soil-radish system differed from the soil-Chinese cabbage system in thatthe microbial quotient of three tested soils in the radish system decreased in the order ofPCS soil＞SLS soil＞YRS soil.
The analysis of BIOLOG after harvesting radish also indicated that the average wellcolor development (AWCD) on the ECO plate was reduced with increasing level of Hg.There was a nonlinear relationship between AWCD and incubation time, and therelationship followed a growth dynamic model of microbial population (Logistic curve),similar to other cropping system.
The study illuminated physicochemical and biological characterization of mercurycontamination in representative paddy soils in the areas of Yangtse Rive and Pearl RiveDeltas, revealed transplant, accumulation,and equilibium law of mercury and someimportant factors controlling mercury contamination in soil-crop system, and highlightedecological toxicity, prediction method, and regulation and control mechanism of mercurycontamination in soil—crop system. It also made major breakthrough in fundamentalresearch aspect consisting of transplant, transformation, and accumulation law of mercuryand healthy effect, which would supply scientific theoretics gist of control, restore projectof soil mercury pollution for our country.
1．土壤对汞离子的吸附-解吸行为与土壤性质密切相关。有机质和粘粒含量较高的黄红壤对汞离子的吸附容量最大，其次是青紫泥，最小的是小粉土。小粉土、黄红壤和青紫泥对汞离子的吸附可以用Langmuir和Freundlich吸附模型很好的拟合，Langmuir方程的相关系数更高一些，说明它更适用于描述汞的吸附。小粉土、黄红壤与青紫泥对汞的最大吸附量(K_L)分别为111.11 mg kg~(-1)、212.77 mg kg~(-1)和188.68 mg kg~(-1)。三种供试土壤在吸附汞离子后平衡液的pH随添加汞浓度的增加而显著降低。随汞浓度增加，三种土壤中汞的吸附分配系数呈指数下降。在汞加入浓度1000 mg kg~(-1)时，用0.1 M KCl连续解吸5次，小粉土吸附汞中有24.40％被解吸，而黄红壤则有14.37％吸附汞被解吸，青紫泥有18.35％吸附汞被解吸。
pH对土壤吸附汞离子的解吸影响较大。小粉土、青紫泥和黄红壤吸附Hg~(2+)的解吸率均随pH值升高先降低，后变平缓，又升高，在pH为3.00-5.00范围内下降，pH为5.00-7.00曲线趋于平缓，pH为7.00-9.00曲线趋于升高，这说明酸性的环境(pH＜5.4)有利于Hg~(2+)解吸，并且对Hg~(2+)解吸率随pH增加而减少，因而pH也是控制土壤中汞有效性一个重要因素。
在加入Cu~(2+)和Zn~(2+)后，三种供试土壤吸附汞的解吸量大大增加，在相同Cu~(2+)和Zn~(2+)离子浓度下，Cu~(2+)对Hg~(2+)解吸量增加的幅度较Zn~(2+)大。铜加入量从0 mM到15 mM，小粉土、青紫泥和黄红壤吸附汞的解吸量分别从1.50 mg L~(-1)、1.64 mg L~(-1)和2.06 mg L~(-1)增加到5.45 mg L~(-1)、4.48 mg L~(-1)和2.68 mg L~(-1)；锌加入量从0 mM到20 mM，小粉土、青紫泥和黄红壤吸附汞的解吸量分别从1.50 mg L~(-1)、1.64 mg L~(-1)和2.06 mg L~(-1)增加到4.53mg L~(-1)、3.96 mg L~(-1)和2.56 mg L~(-1)。
研究表明有机酸对三种土壤中汞的解吸行为影响极大，随加入的有机酸浓度增大，三种土壤吸附汞的解吸量增大。对小粉土而言，低浓度(＜10~(-4) M)的有机酸可抑制对Hg~(2+)的解吸，而高浓度(＞10~(-4) M)有机酸可促进Hg~(2+)的解吸。不同有机酸对土壤吸附汞的解吸行为的影响也不同，本实验研究的四种有机酸对三种土壤吸附汞的解吸行为的影响中，按对土壤吸附汞离子解吸的促进作用强弱排序为：柠檬酸＞酒石酸＞苹果酸＞草酸。
2．本文从土壤化学角度出发，对HCl、NH_4OAC、DTPA和CaCl_2四种提取剂对小粉土、黄红壤和青紫泥中汞的化学提取条件及提取性作了初步研究。结果表明，随着平衡时间延长增加，汞提取量有逐渐增加的趋势。在三种土壤中各提取剂的提取率达到稳定的提取时间分别为：小粉土(HCl 10分钟；CaCl_2 10分钟；DTPA 10分钟；NH_4OAc 10分钟)；黄红壤(HCl 10分钟；CaCl_2 30分钟；DTPA 30分钟；NH_4OAc 30分钟)；青紫泥(HCl 10分钟；CaCl_2 30分钟；DTPA 30分钟；NH_4OAc 30分钟)。可见，提取时间30分钟时，有效汞含量基本达到平衡，因此，对四种提取剂来说，30分钟可作为提取三种供试土壤汞有效态的最佳提取时间。
研究表明，随着土液比的不断减小，三种土壤四种提取剂Hg的提取量明显提高。为减少试验误差，确定1：5为较适宜的土液比。
四种提取剂在三种供试土壤中的提取能力有显著的差异。对于小粉土，其顺序为：NH_4OAc＜CaCl_2＜DTPA＜HCl；对于黄红壤，其顺序为：NH_4OAc＜CaCl_2≈DTPA＜HCl；对于青紫泥，其顺序为：DTPA＜NH_4OAc＜HCl＜CaCl_2。这表明HCl和DTPA，特别是HCl提取效率较高。
用水稻籽粒、小白菜叶、萝卜根含汞量与提取剂提取汞量进行相关统计分析。结果表明：对土壤—水稻系统来说，小粉土和黄红壤用DTPA作提取剂，青紫泥用CaCl_2作提取剂，在振荡平衡时间30min，土液比1：5条件下，测定土壤中有效态Hg是较为适宜的方法；
对土壤—小白菜系统来说，在振荡平衡时间30min，土液比1：5条件下，由于三种供试土壤上小白菜叶Hg含量与4种提取剂提取Hg量相关不显著，因此，需要选择其它的提取剂来提取；
对土壤—萝卜系统来说，在振荡平衡时间30min，土液比1：5条件下，小粉土和黄红壤上萝卜根Hg含量与提取剂HCl提取Hg量相关系数最大，而青紫泥上萝卜根Hg含量与提取剂CaCl_2提取Hg量相关系数最大。因此，小粉土和黄红壤用HCl作提取剂，青紫泥用CaCl_2作提取剂，在振荡平衡时间30min，土液比1：5条件下，测定土壤中有效态Hg是较为适宜的方法。
通过综合分析4种提取剂的提取能力、提取结果与植物汞含量的关系，确定HCl为评价本研究三种供试土壤中汞元素有效性的最佳化学提取剂。
3．土壤—水稻作物系统中，本实验条件下，汞污染对土壤环境质量微生物学和酶学指标有较大的影响。水稻收获后，除部分土壤的呼吸强度和代谢商随汞处理浓度的增加而持续增加外，不同Hg处理后土壤微生物量碳、呼吸强度，土壤脲酶、酸性磷酸酶和脱氢酶均表现为在低浓度Hg处理时有促进作用，在高浓度时则表现出抑制作用。不同指标出现峰值时的Hg浓度分别是0.5、1、1.5、2mg kg~(-1)不等。综合来看，土壤微生物商是Hg处理后比较敏感的微生物学指标。Hg不同处理的土壤酶活性强弱依次为：黄红壤＞青紫泥＞小粉土。通过对重金属生态剂量ED50的分析，表明Hg对小粉土脲酶活性的生态毒性比相应的青紫泥和黄红壤强；对黄红壤磷酸酶活性的生态毒性比相应的青紫泥和小粉土强；对青紫泥脱氢酶活性的生态毒性比相应的小粉土和黄红壤强。
本文也证实了BIOLOG系统是反映土壤微生物生理轮廓和微生物群落结构的有效手段。微生物群落代谢剖面AWCD值和McIntosh指数可以较好的反应本实验三种土壤微生物群落结构和汞污染之间的剂量关系。汞污染严重的土壤AWCD值、McIntosh指数均显著低于无污染和轻度污染的土壤，表明汞污染引起了红砂土和黄筋泥微生物群落结构功能多样性的下降，减少了能利用有关碳源底物的微生物的数量，最终导致土壤微生物群落功能结构多样性发生变化。
4．土壤—(小白菜)作物系统中分析测定的大部分土壤微生物学和酶学等生态指标，均与土壤—水稻系统表现出相似的规律。本试验中，就平均而言，青紫泥中的土壤微生物量碳最高，其次是黄红壤，最小的是小粉土。同时，小粉土中对汞污染的敏感性最强，其次是青紫泥，最弱的是黄红壤。不同浓度汞处理后，土壤微生物活性的变化与土壤微生物量的变化规律相似。即，随汞处理浓度增大土壤微生物活性而增强，到一定浓度后转为减弱。三种供试土壤的微生物商差异显著，黄红壤的微生物商最大，其次是青紫泥，最小的是小粉土。比较而言，微生物商是对土壤微生物比较敏感的指标。
平均而言，黄红壤和青紫泥中的土壤酶活性高于小粉土。除磷酸酶外，Hg多数处理浓度对土壤酶活性表现出抑制作用，与种植水稻时多数处理浓度起促进作用正好相反。Hg污染条件下小粉土脲酶活性受抑制的幅度最大，黄红壤的受抑制最小。种植小白菜后，除磷酸酶和小粉土中的脱氢酶外，其它土壤中脲酶和脱氢酶活性与重金属处理间均达负相关水平，但未达到显著水平。研究表明Hg对小粉土脲酶活性的生态毒性比相应的青紫泥和黄红壤强；Hg对黄红壤磷酸酶活性的生态毒性比相应的青紫泥和小粉土强；Hg对小粉土脱氢酶活性的生态毒性比相应的青紫泥和黄红壤强。
此外，在小白菜收获后，采用碳素利用法(BIOLOG测试系统)对土壤微生物功能和群落结构多样性分析，结果表明：随Hg各处理浓度增加，BIOLOG ECO盘平均吸光值(AWCD)降低，群落代谢剖面值与培养时间间呈非线性关系，其变化过程符合微生物种群生长动态模型，呈“S”形增长模式(logistic模型)。
5．土壤—(萝卜)作物系统中Hg的生态效应与土壤—水稻或蔬菜系统有相似之处。就平均而言，青紫泥和黄红壤中平均Cmic高于小粉土。从土壤看，青紫泥和黄红壤中土壤呼吸强度要高于小粉土。黄红壤和青紫泥的土壤代谢商除了峰值外差异不大，但小粉土的最高，其次是黄红壤，最低的是青紫泥。与栽培小白菜后的变化不同，青紫泥的微生物商最大，其次是小粉土，最小的是黄红壤。
不同浓度汞处理后，土壤酶活性在供试的三种土壤中都表现出一定的差异性。起初，随着汞处理浓度的增高，三种土壤中酶的活性增强，小粉土到1 mg kg~(-1)处理时出现峰值，黄红壤和青紫泥到1.5 mg kg~(-1)处理时出现峰值，然后随汞浓度增高而下降。
种植萝卜后，Biolog生态盘的AWCD随培养时间的增加而增强，呈“S”型指数增长模式。三种土壤的平均吸光值依次为：青紫泥＞黄红壤＞小粉土，表明青紫泥和黄红壤中微生物活性超过小粉土。
本研究阐明了在长江、珠江三角洲地区主要土壤类型中汞的物理化学行为、生物有效性及生物学特征；揭示汞在土壤—作物系统中迁移、积累、平衡规律及其主控因子；明确土壤—作物系统中汞污染的生态毒理、预测方法及其调控机制。在土壤-植物系统中汞的迁移、转化和积累规律及其健康效应方面的基础理论研究方面取得较大突破，为国家制定该地区土壤汞污染控制、削减、修复行动计划提供科学依据。
This study was a part of the research project "the mechanisms of movement,transform, and accumulation of typical heavy metals in agricultural soil and theirrelationships to plant ecosystem and farm produce safety", a sub-project of the NationalKey Basic Research Program of China (973)"the temporal and spatial variation andadjusting and controlling principles of the environmental quality of soil and atmosphere inthe areas of Yangtse Rive and Pearl Rive Deltas"(No. 2002CB410804)". According to thedemands of the specific research content and aims of the national program, Three paddysoils with contrasting properties were studied. A silty loam soil (Paddy soil 1) wascollected from Huajiachi campus, Zhejiang University, Hangzhou, a yellowish red soil(Paddy soil 2) from Deqing County, and a purplish clayey soil (Paddy soil 3) from JiaxingCounty in Zhejiang Province, China. All of them belong to Gleyi-Stagnic Anthrosols inFAO/UNESCO nomenclature.
Both laboratory and greenhouse pot experiments using rice, Chinese cabbage andradish as indicator plants were conducted to investigate the adsorption-desorptioncharacteristics and availability of Hg, and the ecological effects of increasing Hg loadingson microbial biomass, microbial activity, microbial community structure, and soil enzymeactivities. The major objectives of this study were to understand biogeochemical behaviorof Hg in three tested soils and to compare the potential of chemical, biochemical, andmicrobiological parameters for indicating and predicting Hg contamination. The resultswere summarized as follows:
1. Adsorption-desorption of Hg at contaminated levels in three tested soils wereinvestigated. In all cases, Hg~(2+) adsorption decreased in the order: YRS soil＞PCS soil＞SLSsoil, with YRS having the highest sorption capacity for Hg. The maximum adsorption (K_L)value was 111.11 mg kg~(-1) for SLS soil, 212.77 mg kg~(-1) for YRS soil and 188.68 mg kg~(-1)for PCS soil. The isotherms of Hg~(2+) adsorption in the soils can be well described by thesimple Langmuir and Freudlich model. The simple Langrnuir model gave a relativelybetter representation of the experimental data based on the fitting correlation coefficients(r~2).
Adsorption of Hg~(2+) decreased soil pH by 0.75 unit for the SLS soil, 0.91 unit for theYRS soil and 0.55 unit for the PCS at the highest loading. The distribution coefficient (k_d)of Hg in the soils decreased exponentially with increasing Hg~(2+) loading. The adsorptionequilibrium pH of YRS was higher than that of SLS at the same Hg~(2+) concentration.Most of the adsorbed Hg~(2+) in the soils was not desorbed in the 0.01 M KCl solution. Afterfive successive desorptions, the accumulative amounts of Hg~(2+) desorbed accounted foronly 24.40％of the adsorbed Hg~(2+) for SLS soil, 14.37％for YRS soil and 18.35％for PCS soil, indicating that the YRS soil had the greatest affinity for Hg~(2+) at the same Hg~(2+)loading. Different mechanisms may be involved in Hg~(2+) adsorption/desorption atdifferent levels of Hg~(2+) loading and among three testedsoils.
Desorption characteristics as affected by desorption solution pH, organic acids, andcompetitive ions were also studied, pH was the most important factor controlling Hg~(2+)desorption. Mercury desorption with increasing solution pH was characteristic of a "U"pattern and consisted of a desorption decreasing stage at pH 3.0-5.0, a precipitation andminimal desorption stage at pH 5.0-7.0; and a desorption increasing stage at pH 7.0-9.0.
Organic acids remarkably influenced the desorption behavior of Hg~(2+) in the paddysoils. The presence of organic acids at low concentrations (＜10~(-4) M) tended to inhibit Hg~(2+)desorption, but enhanced Hg~(2+) desorption at higher concentrations (＞10~(-4) M). Citric acid athigh concentrations (＞10~(-3) M) was the most effective in increasing Hg~(2+) desorption,followed by tartaric acid and malic acid, and oxalic acid was the least effective. Thedesorption of Hg~(2+) was also affected by the presence of competitive cations (Cu~(2+) or Zn~(2+)).The desorption of adsorbed Hg~(2+) increased with increasing concentrations of added Cu~(2+)or Zn~(2+), and Cu~(2+) was more effective than Zn~(2+) in competing with Hg~(2+) for adsorbing sitesat the same concentration levels.
2. Four extractants, including 0.1 M HCl, 1 M NH_4OAc(pH7.0), 0.005 M DTPA and 0.1M CaCl_2 (pH5.0) were evaluated for their extraction of available Hg~(2+) in the three testedsoils at different equilibrium times and different soil: solution ration.
The amount of Hg~(2+) extracted increased with the increasing of equilibrium time. Theequilibrium time required for maximum extraction of the added Hg~(2+) varied among thefour extractants: 10 min for the four extractants in the SLS soil; 10 min for the HCl, 30min for the other extractants for the YRS and the PCS soil. Overall, 30 min is the bestequilibrium time. The amount of Hg~(2+) extracted decreased with increasing soil: solutionratio. Our study shows that the soil:solution ratio of 1:5 is adequate. The amount of Hg~(2+)extracted increased in the order of NH_4OAc＜CaCl_2＜DTPA＜HCl for the SLS soil,NH_4OAc＜CaCl_2≈DTPA＜HCl for the YRS soil, and DTPA＜NH_4OAc＜HCl＜CaCl_2 for thePCS soil.
There were significant correlations between concentrations of Hg in edible tissue andthe amounts of Hg extracted with the four extractants for soil-rice system and soil-radishsystem, but not for soil-Chinese cabbage system. According to the capability of differentextractants and the correlation of the extractable Hg~(2+) and Hg contents in edible planttissues, the 0.1M HCl was the best extractant among the four extractants and couldindicate plant availability of Hg in soil-plant systems.
3. The changes of soil microbial activities and the functional diversity of microbialcommunity can be used to indicate the dose effect of Hg~(2+) on soil ecological system. Somesoil microbial parameters such as microbial biomass carbon, microbial quotient, AWCD, and urease activity were sensitive to Hg pollution.
Mercury ecological dose responses of basal respiration, microbial biomass carbon,metabolism quotient, microbial quotient, and microbial community diversity wereinvestigated in the SLS, YRS, and PCS soils at Hg~(2+) loadings of 0, 0.25, 0.5, 1, 1.5, 2, 3,6mg kg~(-1). The results showed that mercury (Hg~(2+)) pollution had significant effects on themicrobial and enzymatic indices. There were several ecological effect characteristics ofHg in the soil-rice system. After harvesting rice plant, except for basal respiration andmetabolism quotient, which increased with increasing level of Hg treatments, all measuredmicrobial and enzymatic indices including microbial biomass carbon, microbial quotientincreased with the increasing level of Hg treatments at low concentrations (＜2mg kg~(-1)) anddecreased at the 0.5, 1, 1.5, 2mg kg~(-1) Hg treatments. The results also indicate that soilmicrobial quotient is more sensitive to Hg contamination than the other microbial indices.
Some important soil enzymes including urease, dehydrogenase, and acid phosphatase,which are related to cycling of soil C, N and P, were investigated. Mercury input had verydifferent effects on the enzyme activities. Enzymatic activity of the YRS soil was highest,followed by the PCS soil, and the SLS soil was the least.. The analysis of ED50 indicatedthat ecological toxicity of urease was the strongest for the SLS soil; ecological toxicity ofacid phosphatase was the strongest for the YRS soil; and ecological toxicity ofdehydrogenase was the strongest for the PCS soil.
After harvesting rice plants, soil microbial function and community structurediversity were analyzed using BIOLOG, soil microbial functional diversities of microbialcommunity were changed to varying extent under the stress of Hg~(2+) pollution. The averagewell color development (AWCD) on the ECO plate reduced with increasing level of Hg.There was a nonlinearly relationship between AWCD and incubation time, and therelationship followed a growth dynamic model of microbial population (Logistic curve).The microbial community richness and McIntosh index decreased in polluted soil than thecontrol soil. The results suggest that the structure of microbial community has beenchanged under Hg~(2+) stress. Mercury pollution decreased the functional diversity ofmicrobial community, and reduced the microbial utilization of different carbon resources.
4. The ecological effect of Hg in soil-vegetable (Chinese cabbage and radish) systemwas similar to those in soil—rice system. Namely, small addition of Hg (＜2mg kg~(-1)) to soilcould promot above soil microbial and enzymatic indices, but excessive addition of Hg(＞2mg kg~(-1)) to soil declined the soil microbial and enzymatic indices.
In the soil-Chinese cabbage system, the results indicate that soil microbial quotient ismore sensitive to Hg contamination than the other microbial indices. In addition,enzymatic activities were higher in the YRS and PCS soil than the SLS soil. Afterharvesting Chinese cabbage, except for acid phosphatase whose activity increased withincreasing level of Hg treatments, most of Hg treatments had inhibitory effects on enzymatic activity in contrast with soil-rice system. Mercury treatments had the mostinhibitory effects on urease activity in SLS soil, less in PCS soil, and the least in YRS soil.There were no significant relationships between dehydrogenase and soil Hg loadings, norbetween urease activity and soil Hg~(2+) loadings except for acid phosphatase anddehydrogenase in SLS soil. The analysis of ED50 indicated that ecological toxicity ofurease and dehydrogenase was the strongest in the SLS soil; ecological toxicity of acidphosphatase was the strongest in the YRS soil after harvesting Chinese cabbage.
After harvesting radish, microbial biomass carbon in SLS soil was the highest, butbasal respiration of SLS soil was the smallest. In soil-radish system, the peak values ofsoil enzymatic activity varied with soils, 1 mg kg~(-1) for SLS soil, 1.5 mg kg~(-1) for YRS andPCS soils. The soil-radish system differed from the soil-Chinese cabbage system in thatthe microbial quotient of three tested soils in the radish system decreased in the order ofPCS soil＞SLS soil＞YRS soil.
The analysis of BIOLOG after harvesting radish also indicated that the average wellcolor development (AWCD) on the ECO plate was reduced with increasing level of Hg.There was a nonlinear relationship between AWCD and incubation time, and therelationship followed a growth dynamic model of microbial population (Logistic curve),similar to other cropping system.
The study illuminated physicochemical and biological characterization of mercurycontamination in representative paddy soils in the areas of Yangtse Rive and Pearl RiveDeltas, revealed transplant, accumulation,and equilibium law of mercury and someimportant factors controlling mercury contamination in soil-crop system, and highlightedecological toxicity, prediction method, and regulation and control mechanism of mercurycontamination in soil—crop system. It also made major breakthrough in fundamentalresearch aspect consisting of transplant, transformation, and accumulation law of mercuryand healthy effect, which would supply scientific theoretics gist of control, restore projectof soil mercury pollution for our country.
引文
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