不同结合相的梯度薄膜扩散技术（DGT）评估土壤重金属生物有效性
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摘要
土壤重金属污染对农产品的质量安全造成了严重威胁，探寻土壤重金属生物有效态的评价方法一直是农业环境科研领域研究的热点。由于重金属元素有效态的提取与元素种类、土壤类型、pH值、温度、提取剂的性质等有着密切的关系，因此国际上土壤重金属生物有效态提取方法缺乏统一的标准。梯度薄膜扩散技术（DGT）是一种新型原位采集并测量重金属有效态或生物可给性的方法，大量研究表明，DGT技术比传统的形态分析方法提取的重金属生物有效态浓度更接近生物可利用态的浓度，能更好地反映值物体所吸收的重金属。目前DGT技术的知识产权为英国，材料一般为英国及美国产，需要进口，导致成本较高，难以在我国大面积推广。开发具有自主产权的DGT技术，实现DGT装置的国产化，并在我国土壤上进行推广应用，以此来指导农业生产，是亟待解决的问题之一。
本研究在引进国外DGT技术的基础上，吸收并创新了DGT装置的扩散相和结合相，选择市售的9种膜为扩散相，以聚丙烯酸钠（PAAS）作为结合相，考察Cd在不同扩散层中的扩散系数及离子强度对Cd在不同扩散层中扩散系数的影响；在16个不同理化性质实际Cd污染土壤上进行种植黑麦草盆栽试验，研究了两种结合相的DGT技术预测黑麦草吸收Cd的效果，并与传统提取方法进行了比较；采用两种结合相的DGT技术对广西桑田土壤中有效态Pb进行了累积和测定，融合土壤理化各项指标，运用多元统计分析建立了逐步回归预测模型；以两种结合相的DGT技术为基础，研究了CO_2浓度升高及真菌诱导条件下黑麦草及美洲商陆根际土中生物有效态Cd含量的变化。得出以下几点结论：
（1）选择截留分子量为14000，7000，5000，3500，1000的透析膜、氟膜、聚醚砜膜、醋酸纤维素膜和混合纤维素膜为扩散相，以PAAS作为结合相测定Cd在9种不同扩散层的扩散系数。总体来说，在低离子强度条件下（尤其是离子强度小于5mM），扩散系数有一定的波动；当离子强度大于25mM时，扩散系数变化不大。从成本、扩散通量、精密度等方面综合考虑，截留分子量为14000的透析膜是液体结合相DGT技术中扩散层的首选。
（2）综合考虑土壤理化指标（pH、有机质、机械组成、CEC）的影响，建立DGT方法和传统方法提取的有效Cd含量对黑麦草吸收Cd的多元回归预测模型，结果表明基于两种不同结合相的DGT技术（chelex100-DGT和PAAS-DGT）提取的土壤有效态Cd含量所构建的预测模型要显著优于4种传统方法（土壤溶液、CaCl2提取法、 EDTANa2提取法和HAc提取法）。4种传统提取方法构建的多元回归预测模型受到了pH和CEC的显著影响，而DGT技术综合了两种主成分对土壤有效态Cd含量的影响，所构建的模型几乎不受土壤基本理化性质的影响，且模型回归关系极显著，说明DGT技术可以预测重金属污染土壤中Cd的生物有效性而不受土壤理化性质的影响，是一种预测黑麦草吸收Cd的较好方法。以PAAS为液体结合相，截留分子量为14000的透析膜为扩散相的PAAS-DGT在本研究中较好地预测了黑麦草对土壤中Cd的吸收，大大扩展了DGT技术的应用范围。
（3）采用两种不同结合相的DGT技术（chelex100-DGT和PAAS-DGT）对广西桑田土壤中有效态Pb进行了累积和测定，融合土壤pH、阳离子交换量(CEC)、有机质(OM%)和土壤颗粒组成等理化指标影响，运用多元统计分析，建立了逐步回归模型。多元统计分析表明，两种结合相的DGT技术所构建的回归模型是可靠的,其调整判定系数R_(adj)~2分别为0.87，0.89，0.96和0.95，且预测结果融合了影响土壤有效态Pb含量的pH、CEC、有机质和土壤质地等主要因素，研究结果表明两种结合相的DGT装置均能较好预测桑田土壤中Pb的生物有效性。
（4）CO_2浓度升高或接种真菌的单独或联合作用均能显著增加黑麦草和商陆的Cd的生物量及对Cd的吸收总量，两种不同结合相的DGT技术的测定结果均表明CO_2浓度升高和接种真菌可以显著增加植物根际土中生物有效态重金属的含量，揭示了CO_2浓度升高以及真菌诱导能够增强植物吸收重金属的直接原因。与正常CO_2浓度相比，CO_2浓度升高使黑麦草地上部和地下部干重增加了42.08-89.63%和17.93-51.43%；相比之下， CO_2浓度升高条件下， DGT测得黑麦草根际土中有效态Cd含量的增加只有2.01-25.17%。CO_2浓度升高条件下，根际土生物有效性重金属的增加远远低于植物生物量的增加的幅度，从而影响了对植物对重金属的吸收，因此产生了“稀释效应”。CO_2浓度升高条件下，商陆也存在相同的现象。
综上，chelex100-DGT和PAAS-DGT是一种快速、准确的土壤重金属有效态表征和预测方法。DGT技术具有传统化学提取方法无法比拟的优势，它可以很好的模拟预测植物对重金属的吸收而不受土壤理化性质的影响。以PAAS为结合相，截留分子量14000的透析膜为扩散相的DGT装置也成功的预测了植物对重金属Pb、Cd的吸收，研究扩大了DGT技术的应用范围，同时为DGT技术在我国大范围推广提供了理论依据和技术支撑。
Heavy metal pollution in soils poses a serious threat to the quality and safety of agriculturalproducts. Thus developing effective methods for evaluate heavy metal bioavailability has been a hottopic in the field of agricultural environment research. The extraction of heavy metals depended onelement types, soil texture, pH, temperature, the nature of the solvent, and so on. There was still a lackof effective uniform standards of extraction bioavailable heavy metals from soil. A new technique,diffusive gradients in thin films （DGT）, measures labile species and bioavailability of heavy metals insitu. Many studies have shown the heavy metals measured by DGT were closer to the bioavailabilityconcentrations, and DGT technique could better predict the potential runoff of heavy metals in plantsthan conventional methods. The intellectual property right of DGT technique was belongs to U.K., andthe masteries were generally product by U.K. and U.S.A., which needed to be imported and resulting inhigher cost, so it was hard to promote it in a large area in China. Developing DGT technique with ourown intellectual property right, producing DGT device localization, and promoting the application ofDGT technique in China in order to guide agricultural production, is one of the problems to be solved.
The diffusive phase and binding phase of DGT device were absorbed and improved based on theDGT technique introduced form foreign country. Nine kinds of commercially available membraneswere selected as diffusive layers to study the diffusion coefficients of Cd in these diffusive layers andthe impact of ionic strength on the diffusion coefficients of Cd, with sodium polyacrylate （PAAS） asbinding phase.16natural Cd contaminated soils with diverse properties were collected, and ryegrasswas grown on these soils by pot experiment. Prediction models for evaluating cadmium accumulation inryegrass were built based on Cd bioavailable concentrations measured by DGT technique with twodifferent binding phases, which was also compared with prediction models built by traditional chemicalmethods. The bioavaliability of lead in the rhizosphere soils of Moraceae was accumulated andmeasured by different binding phases DGT devices. Multivariate analyses were performed andregression models were established, which also consider the impact of the physical and chemicalindicators of soils. The bioavailability concentrations of Cd in rhizosphere soils of ryegrass andpokeweed grown under fungal inoculation and elevated CO_2were measured by DGT withtwo different binding phases.The main conclusions were as follows:
（1）The dialysis membranes with14000，7000，5000，3500and1000interception molecularweight, fluoride membrane, polyethersulfone membrane, cellulose acetate membrane and mixedcellulose membrane were chosen as diffusive phases. The diffusion coefficients of Cd in these9kindsof diffusive phases were determined with sodium polyacrylate （PAAS） as binding phase. In general,the diffusion coefficients had a flux under low ionic strength （ionic strength greater than5mM）, andthe diffusion coefficients had no change nearly when the ionic strength higher than25mM. Considerfrom all aspects, such as cost, the diffusive flux, scientific and so on, it was the best choose to usedialysis membranes with14000interception molecular weight as diffusive layer in liquid binding phaseDGT technique.
（2）Multiple regression prediction models for evaluating cadmium accumulation in ryegrasswere built based on Cd bioavailable concentrations extracted by DGT technique and traditional methods,which also the impact of pH, cation exchanged capacity （CEC）, organic materials （OM） and textureof the soils. Results showed that prediction models based on chelex100-DGT and PAAS-DGTtechniques were significantly better than those built by traditional techniques （soil solution, CaCl2,EDTANa2and HAc extractions）. Results showed that pH and CEC had significant effects on predictionmodels built by4traditional methods, but there was little effect on the prediction model based on DGTtechnique. DGT measurement incorporated the main factors affected bioavailability, and could betterpredict the potential runoff of Cd from soil and the accumulation of Cd in ryegrass than traditionalchemical methods. Results also showed that DGT technique with dialysis membranes with14000interception molecular weight as diffusive layer and PAAS as binding phase could predict the potentialrunoff of Cd from soil and the accumulation of Cd in s ryegrass, which expanded the applicable range ofDGT technique.
（3）The bioavaliability of lead in the rhizosphere soils of Moraceae was accumulated and measuredby different binding phases DGT devices (chelex100-DGT and PAAS-DGT). Multivariate analysisshowed that the regression models established by the two binding phase DGT were reliable, and theadjusted coefficients were0.87,0.89,0.96and0.95, respectively. Predicted results from these modelsincorporated the main factors affecting available Pb in soils, such as pH, cation exchanged capacity(CEC), soil organic matter (OM) and texture. Results showed that both DGT devices could predict thepotential runoff of Pb from soil and the accumulation of Pb in leaves of Moraceae, which expanded theapplicable range of DGT technique.
（4）Elevated CO_2and fungal inoculation, either singly or in combination, could significantlyincreased plant biomass and total Cd uptake per pot ryegrass and pokeweed. The bioavailable Cdconcentrations in the rhizosphere soils measured by DGT were increased, which revealed the directcause that elevated CO_2and fungal inoculation could enhance the heavy metals accumulated in plants.Our present study showed that the shoot and root dry weight biomass of ryegrass grown at elevated CO_2increased by42.08–89.63and17.93–51.43%, respectively, compared to the ambient CO_2control. Bycontrast, the increase in DGT-measured Cd concentrations in the rhizosphere soil of ryegrass at elevatedCO_2was only2.01–25.17and15.79–38.31%, respectively, compared to the ambient CO_2control. It isclear that there was a substantial difference between the increased rate of the shoot and root dry weightbiomass of ryegrass grown at elevated CO_2, and the increased magnitude of DGT-measured Cdconcentrations in the rhizosphere soil of ryegrass elevated CO_2. This implies that the apparent increasein size of the bioavailable metal pools in the rhizosphere of ryegrass due to elevated CO_2might not meetthe potential for plant uptake of metals in association with the plant biomass increase and, as a result, a‘dilution effect’ took place under elevated CO_2. The situation was similar for pokeweed.
In conclusion, chelex100-DGT and PAAS-DGT techniques could be used as an effective andaccurate method to characterize and predict heavy metal bioavailable concentrations in soils.PAAS-DGT technique with14000interception molecular weight dialysis membranes as diffusive layer could predict the potential runoff of Cd and Pb from soil and the accumulation of Cd and Pb in plants,which expanded the applicable range of DGT technique, and provided theoretical basis and technicalsupport for promoting the application of DGT technique in China.
本研究在引进国外DGT技术的基础上，吸收并创新了DGT装置的扩散相和结合相，选择市售的9种膜为扩散相，以聚丙烯酸钠（PAAS）作为结合相，考察Cd在不同扩散层中的扩散系数及离子强度对Cd在不同扩散层中扩散系数的影响；在16个不同理化性质实际Cd污染土壤上进行种植黑麦草盆栽试验，研究了两种结合相的DGT技术预测黑麦草吸收Cd的效果，并与传统提取方法进行了比较；采用两种结合相的DGT技术对广西桑田土壤中有效态Pb进行了累积和测定，融合土壤理化各项指标，运用多元统计分析建立了逐步回归预测模型；以两种结合相的DGT技术为基础，研究了CO_2浓度升高及真菌诱导条件下黑麦草及美洲商陆根际土中生物有效态Cd含量的变化。得出以下几点结论：
（1）选择截留分子量为14000，7000，5000，3500，1000的透析膜、氟膜、聚醚砜膜、醋酸纤维素膜和混合纤维素膜为扩散相，以PAAS作为结合相测定Cd在9种不同扩散层的扩散系数。总体来说，在低离子强度条件下（尤其是离子强度小于5mM），扩散系数有一定的波动；当离子强度大于25mM时，扩散系数变化不大。从成本、扩散通量、精密度等方面综合考虑，截留分子量为14000的透析膜是液体结合相DGT技术中扩散层的首选。
（2）综合考虑土壤理化指标（pH、有机质、机械组成、CEC）的影响，建立DGT方法和传统方法提取的有效Cd含量对黑麦草吸收Cd的多元回归预测模型，结果表明基于两种不同结合相的DGT技术（chelex100-DGT和PAAS-DGT）提取的土壤有效态Cd含量所构建的预测模型要显著优于4种传统方法（土壤溶液、CaCl2提取法、 EDTANa2提取法和HAc提取法）。4种传统提取方法构建的多元回归预测模型受到了pH和CEC的显著影响，而DGT技术综合了两种主成分对土壤有效态Cd含量的影响，所构建的模型几乎不受土壤基本理化性质的影响，且模型回归关系极显著，说明DGT技术可以预测重金属污染土壤中Cd的生物有效性而不受土壤理化性质的影响，是一种预测黑麦草吸收Cd的较好方法。以PAAS为液体结合相，截留分子量为14000的透析膜为扩散相的PAAS-DGT在本研究中较好地预测了黑麦草对土壤中Cd的吸收，大大扩展了DGT技术的应用范围。
（3）采用两种不同结合相的DGT技术（chelex100-DGT和PAAS-DGT）对广西桑田土壤中有效态Pb进行了累积和测定，融合土壤pH、阳离子交换量(CEC)、有机质(OM%)和土壤颗粒组成等理化指标影响，运用多元统计分析，建立了逐步回归模型。多元统计分析表明，两种结合相的DGT技术所构建的回归模型是可靠的,其调整判定系数R_(adj)~2分别为0.87，0.89，0.96和0.95，且预测结果融合了影响土壤有效态Pb含量的pH、CEC、有机质和土壤质地等主要因素，研究结果表明两种结合相的DGT装置均能较好预测桑田土壤中Pb的生物有效性。
（4）CO_2浓度升高或接种真菌的单独或联合作用均能显著增加黑麦草和商陆的Cd的生物量及对Cd的吸收总量，两种不同结合相的DGT技术的测定结果均表明CO_2浓度升高和接种真菌可以显著增加植物根际土中生物有效态重金属的含量，揭示了CO_2浓度升高以及真菌诱导能够增强植物吸收重金属的直接原因。与正常CO_2浓度相比，CO_2浓度升高使黑麦草地上部和地下部干重增加了42.08-89.63%和17.93-51.43%；相比之下， CO_2浓度升高条件下， DGT测得黑麦草根际土中有效态Cd含量的增加只有2.01-25.17%。CO_2浓度升高条件下，根际土生物有效性重金属的增加远远低于植物生物量的增加的幅度，从而影响了对植物对重金属的吸收，因此产生了“稀释效应”。CO_2浓度升高条件下，商陆也存在相同的现象。
综上，chelex100-DGT和PAAS-DGT是一种快速、准确的土壤重金属有效态表征和预测方法。DGT技术具有传统化学提取方法无法比拟的优势，它可以很好的模拟预测植物对重金属的吸收而不受土壤理化性质的影响。以PAAS为结合相，截留分子量14000的透析膜为扩散相的DGT装置也成功的预测了植物对重金属Pb、Cd的吸收，研究扩大了DGT技术的应用范围，同时为DGT技术在我国大范围推广提供了理论依据和技术支撑。
Heavy metal pollution in soils poses a serious threat to the quality and safety of agriculturalproducts. Thus developing effective methods for evaluate heavy metal bioavailability has been a hottopic in the field of agricultural environment research. The extraction of heavy metals depended onelement types, soil texture, pH, temperature, the nature of the solvent, and so on. There was still a lackof effective uniform standards of extraction bioavailable heavy metals from soil. A new technique,diffusive gradients in thin films （DGT）, measures labile species and bioavailability of heavy metals insitu. Many studies have shown the heavy metals measured by DGT were closer to the bioavailabilityconcentrations, and DGT technique could better predict the potential runoff of heavy metals in plantsthan conventional methods. The intellectual property right of DGT technique was belongs to U.K., andthe masteries were generally product by U.K. and U.S.A., which needed to be imported and resulting inhigher cost, so it was hard to promote it in a large area in China. Developing DGT technique with ourown intellectual property right, producing DGT device localization, and promoting the application ofDGT technique in China in order to guide agricultural production, is one of the problems to be solved.
The diffusive phase and binding phase of DGT device were absorbed and improved based on theDGT technique introduced form foreign country. Nine kinds of commercially available membraneswere selected as diffusive layers to study the diffusion coefficients of Cd in these diffusive layers andthe impact of ionic strength on the diffusion coefficients of Cd, with sodium polyacrylate （PAAS） asbinding phase.16natural Cd contaminated soils with diverse properties were collected, and ryegrasswas grown on these soils by pot experiment. Prediction models for evaluating cadmium accumulation inryegrass were built based on Cd bioavailable concentrations measured by DGT technique with twodifferent binding phases, which was also compared with prediction models built by traditional chemicalmethods. The bioavaliability of lead in the rhizosphere soils of Moraceae was accumulated andmeasured by different binding phases DGT devices. Multivariate analyses were performed andregression models were established, which also consider the impact of the physical and chemicalindicators of soils. The bioavailability concentrations of Cd in rhizosphere soils of ryegrass andpokeweed grown under fungal inoculation and elevated CO_2were measured by DGT withtwo different binding phases.The main conclusions were as follows:
（1）The dialysis membranes with14000，7000，5000，3500and1000interception molecularweight, fluoride membrane, polyethersulfone membrane, cellulose acetate membrane and mixedcellulose membrane were chosen as diffusive phases. The diffusion coefficients of Cd in these9kindsof diffusive phases were determined with sodium polyacrylate （PAAS） as binding phase. In general,the diffusion coefficients had a flux under low ionic strength （ionic strength greater than5mM）, andthe diffusion coefficients had no change nearly when the ionic strength higher than25mM. Considerfrom all aspects, such as cost, the diffusive flux, scientific and so on, it was the best choose to usedialysis membranes with14000interception molecular weight as diffusive layer in liquid binding phaseDGT technique.
（2）Multiple regression prediction models for evaluating cadmium accumulation in ryegrasswere built based on Cd bioavailable concentrations extracted by DGT technique and traditional methods,which also the impact of pH, cation exchanged capacity （CEC）, organic materials （OM） and textureof the soils. Results showed that prediction models based on chelex100-DGT and PAAS-DGTtechniques were significantly better than those built by traditional techniques （soil solution, CaCl2,EDTANa2and HAc extractions）. Results showed that pH and CEC had significant effects on predictionmodels built by4traditional methods, but there was little effect on the prediction model based on DGTtechnique. DGT measurement incorporated the main factors affected bioavailability, and could betterpredict the potential runoff of Cd from soil and the accumulation of Cd in ryegrass than traditionalchemical methods. Results also showed that DGT technique with dialysis membranes with14000interception molecular weight as diffusive layer and PAAS as binding phase could predict the potentialrunoff of Cd from soil and the accumulation of Cd in s ryegrass, which expanded the applicable range ofDGT technique.
（3）The bioavaliability of lead in the rhizosphere soils of Moraceae was accumulated and measuredby different binding phases DGT devices (chelex100-DGT and PAAS-DGT). Multivariate analysisshowed that the regression models established by the two binding phase DGT were reliable, and theadjusted coefficients were0.87,0.89,0.96and0.95, respectively. Predicted results from these modelsincorporated the main factors affecting available Pb in soils, such as pH, cation exchanged capacity(CEC), soil organic matter (OM) and texture. Results showed that both DGT devices could predict thepotential runoff of Pb from soil and the accumulation of Pb in leaves of Moraceae, which expanded theapplicable range of DGT technique.
（4）Elevated CO_2and fungal inoculation, either singly or in combination, could significantlyincreased plant biomass and total Cd uptake per pot ryegrass and pokeweed. The bioavailable Cdconcentrations in the rhizosphere soils measured by DGT were increased, which revealed the directcause that elevated CO_2and fungal inoculation could enhance the heavy metals accumulated in plants.Our present study showed that the shoot and root dry weight biomass of ryegrass grown at elevated CO_2increased by42.08–89.63and17.93–51.43%, respectively, compared to the ambient CO_2control. Bycontrast, the increase in DGT-measured Cd concentrations in the rhizosphere soil of ryegrass at elevatedCO_2was only2.01–25.17and15.79–38.31%, respectively, compared to the ambient CO_2control. It isclear that there was a substantial difference between the increased rate of the shoot and root dry weightbiomass of ryegrass grown at elevated CO_2, and the increased magnitude of DGT-measured Cdconcentrations in the rhizosphere soil of ryegrass elevated CO_2. This implies that the apparent increasein size of the bioavailable metal pools in the rhizosphere of ryegrass due to elevated CO_2might not meetthe potential for plant uptake of metals in association with the plant biomass increase and, as a result, a‘dilution effect’ took place under elevated CO_2. The situation was similar for pokeweed.
In conclusion, chelex100-DGT and PAAS-DGT techniques could be used as an effective andaccurate method to characterize and predict heavy metal bioavailable concentrations in soils.PAAS-DGT technique with14000interception molecular weight dialysis membranes as diffusive layer could predict the potential runoff of Cd and Pb from soil and the accumulation of Cd and Pb in plants,which expanded the applicable range of DGT technique, and provided theoretical basis and technicalsupport for promoting the application of DGT technique in China.
引文
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