Plant uptake and leaching of copper during EDTA-enhanced phytoremediation of repacked and undisturbed soil重填土与原状土EDTA强化植物修复过程中铜的植物吸收与淋溶

T. Thayalakumaran1,2, B.H. Robinson1, I. Vogeler1,4, D.R. Scotter1,2, B.E. Clothier1 & H.J. Percival3

¹Environment and Risk Management Group, Hortresearch, Private Bag 11030, Palmerston North, New Zealand. ²Institute of Natural Resources, Massey University, Private Bag 11222, Palmerston North, New Zealand. ³Landcare Research, Private Bag 11052, Palmerston North, New Zealand. 4Corresponding author^∗

¹Hortresearch环境与风险管理组，Private Bag 11030，Palmerston North，新西兰。 ²梅西大学自然资源研究所，Private Bag 11222，Palmerston North，新西兰。 ³Landcare Research，Private Bag 11052，Palmerston North，新西兰。 4通讯作者^∗

Received 2 July 2002; in revised form 28 February 2003

Key words: copper, EDTA, phytoremediation, plant uptake, leaching

关键词：铜，EDTA，植物修复，植物吸收，淋溶

1Abstract摘要

EDTA-enhanced phytoremediation of copper contaminated soil was evaluated. Up to 740 µg g⁻¹ of Na2H2 EDTA in solution was added to repacked soil columns, and intact cores of a sandy loam of volcanic origin, that was growing Agrostis tenuis. The soil contained up to 400 µg g⁻¹ of copper due to a history of fungicide spraying. EDTA application increased the herbage copper concentration of the grass growing in repacked soil from 30 to 300 µg g⁻¹, but the same application to an intact core only brought about an increase from 10 to 60 µg g⁻¹. More copper accumulated in the herbage when the EDTA was applied in numerous small doses than in just one or two larger amounts. Calculation of the concentration of copper in the water taken up by the grass revealed this to be two orders of magnitude lower than that in the soil solution. As a result of the EDTA applications, about 100 times more copper was leached than was taken up by the herbage. This means that a strategy for managing leaching losses needs to be part of any plan for EDTA-enhanced phytoremediation.

本研究评价了EDTA强化植物修复铜污染土壤的效果。向种植Agrostis tenuis的火山源砂壤土重填土柱和原状土芯中施加溶液态Na2H2 EDTA，用量最高达740 µg g⁻¹。由于长期喷施杀菌剂，该土壤铜含量高达400 µg g⁻¹。施加EDTA使重填土中生长的牧草地上部铜浓度由30升至300 µg g⁻¹，但对原状土芯施加相同剂量仅使其由10升至60 µg g⁻¹。与一次或两次大剂量施加相比，多次小剂量施加EDTA时地上部积累的铜更多。计算表明，牧草所吸收水分中的铜浓度比土壤溶液中的铜浓度低两个数量级。施加EDTA后，淋溶损失的铜约为牧草地上部吸收量的100倍。这意味着任何EDTA强化植物修复方案都必须包含控制淋溶损失的策略。

2Introduction引言

Contamination of land with heavy metals has become a problem in some industrial and agricultural areas. Copper is one such contaminant in many soils around the world, as it is used as an active ingredient in fungicides in orchards. Also, along with chromium and arsenic, it is used as a timber preservative. Typically the concentration of copper in the soil at contaminated sites in New Zealand ranges from 100 to 8000 µg g⁻¹ (Roberts et al., 1996). Copper is usually strongly bound to soil organic matter and thus relatively unavailable to living organisms. However if the soil adsorption sites in the soil become saturated with copper, it then becomes toxic for microbes and plants, resulting in decreased soil fertility (Merry et al., 1983). This threshold concentration depends on the organism, soil pH, and the organic matter and clay content. The guideline for the maximum acceptable concentration of copper in horticultural soils in Australia by the NSW Environmental Protection Agency is 100 µg g⁻¹. This is well below the levels of contaminated soils in New Zealand. Using plants to remediate such sites has been proposed as a relatively inexpensive process, as compared to conventional engineeringbased solutions. This so-called phytoremediation has been suggested as an economic and environmentally sound method for extracting heavy metals from moderately polluted soils (Brooks, 1998; Cunningham et al., 1996).

土地重金属污染已成为部分工业区和农业区面临的问题。铜是世界上许多土壤中的一种此类污染物，因为它被用作果园杀菌剂的有效成分。此外，铜还与铬和砷一起用作木材防腐剂。新西兰污染场地土壤中的铜浓度通常在100～8000 µg g⁻¹之间（Roberts et al., 1996）。铜通常与土壤有机质强烈结合，因而对生物的有效性相对较低。然而，一旦土壤吸附位点被铜饱和，铜便会对微生物和植物产生毒性，导致土壤肥力下降（Merry et al., 1983）。该阈值浓度取决于生物种类、土壤pH以及有机质和黏粒含量。澳大利亚新南威尔士州环境保护局规定园艺土壤中铜的最大可接受浓度为100 µg g⁻¹，远低于新西兰污染土壤中的含量水平。与传统的工程修复方法相比，利用植物修复此类场地被认为是一种成本相对低廉的方法。这种所谓的植物修复被认为是从中度污染土壤中提取重金属的一种经济且环境友好的方法（Brooks, 1998; Cunningham et al., 1996）。

Although several conditions must be met in order for phytoremediation to be effective, the bioavailability of the target metal in the soil is a critical requirement for plant uptake to occur. Usually little copper is taken up by plants, mainly due to its low concentration in the soil solution. This lack of bioavailability is a result of copper- forming complexes with organic matter, as well as being sorbed onto oxides and clays, and being occluded by oxides and clay lattices (McLaren and Crawford, 1973). This low solution concentration also means that the mobility of copper is limited. Therefore copper is usually concentrated in the surface soil at contaminated sites.

植物修复要取得成效须满足若干条件，其中土壤中目标金属的生物有效性是植物吸收得以发生的关键前提。植物通常很少吸收铜，这主要是由于土壤溶液中铜浓度较低。铜生物有效性低的原因在于其与有机质形成络合物，被氧化物和黏土矿物吸附，并被氧化物和黏土晶格包蔽（McLaren and Crawford, 1973）。土壤溶液中铜浓度低也意味着铜的迁移性有限。因此，污染场地中的铜通常富集于表层土壤。

∗ E-mail: ivogeler@hortresearch.co.nz

Chelates have long been used in nutrient solutions to increase the solubility and plant availability of trace elements. They have also been used to remediate contaminated soils using soil-washing techniques (Tuin and Tels, 1990; Yu and Klarup, 1994). More recently, many glass-house studies and a few field studies (Kayser et al., 2000) have demonstrated the potential for chelates to enhance the plant uptake of metals that exist in soil predominantly in a sorbed state. Different metals have been targeted, either individually or in a multi-metal contaminated environment. Amongst the metals that have been targeted individually are Pb (Blaylock et al., 1997; Cooper et al., 1999; Epstein et al., 1999), Zn (Ebbs and Kochian, 1998), Cs (Lasat et al., 1998), U (Huang et al., 1998) and Au (Anderson et al., 1998). Furthermore Lombi et al. (2001), Grcman et al. (2001) and Kayser et al. (2000)ˇ have evaluated chelate-assisted phytoremediation in a multi-metal contaminated environment.

螯合剂长期以来被用于营养液中，以提高微量元素的溶解度和植物有效性。螯合剂也被用于土壤淋洗技术修复污染土壤（Tuin and Tels, 1990; Yu and Klarup, 1994）。近年来，许多温室研究和少数田间研究（Kayser et al., 2000）表明，对于土壤中主要以吸附态存在的金属，螯合剂具有促进植物吸收的潜力。已有研究针对不同金属开展，既有单一金属污染，也有多金属复合污染。单独研究的金属包括Pb（Blaylock et al., 1997; Cooper et al., 1999; Epstein et al., 1999）、Zn（Ebbs and Kochian, 1998）、Cs（Lasat et al., 1998）、U（Huang et al., 1998）和Au（Anderson et al., 1998）。此外，Lombi et al. (2001)、Grcman et al. (2001)和Kayser et al. (2000)ˇ评价了多金属复合污染环境中螯合剂辅助的植物修复。

Enhanced-phytoremediation makes use of highbiomass crops that are induced to take up large amounts of metals when their mobility in soil is enhanced by the chelates. Brassica juncea is one plant that produces high rates of biomass under field conditions, and also has the capacity to accumulate substantial metal concentration in its shoots (Kumar et al., 1995; Blaylock et al., 1997). Of the chelates tested for this enhanced technology, ethylene diamine tetraacetic acid (EDTA) has been found to be the most effective for increasing the uptake of various metals (Blaylock et al., 1997; Brooks, 1998; Ebbs and Kochian, 1998; Epstein et al., 1999; Grcman et al., 2001; Huang et al.,ˇ 1997; Lombi et al., 2001), including copper (Deram et al., 2000).

强化植物修复利用高生物量作物，在螯合剂提高土壤中金属迁移性的条件下诱导其大量吸收金属。Brassica juncea是一种在田间条件下生物量高、且地上部能积累大量金属的植物（Kumar et al., 1995; Blaylock et al., 1997）。在用于这一强化技术的各种螯合剂中，乙二胺四乙酸（EDTA）被发现对提高多种金属的吸收最为有效（Blaylock et al., 1997; Brooks, 1998; Ebbs and Kochian, 1998; Epstein et al., 1999; Grcman et al., 2001; Huang et al.,ˇ 1997; Lombi et al., 2001），其中包括铜（Deram et al., 2000）。

In situ application of such chelates can increase the risk of water pollution by promoting uncontrolled metal solubilisation and leaching. Although some of the studies referred to above mention the risk of target metal being leached below the root zone to the ground water, little attention has been given to measuring or predicting such leaching. The exception is the paper of Grcman et al. (2001). While they found that EDTAˇ did enhance heavy metal uptake by Brassica rapa, it also resulted in the loss, by leaching of 38, 10 and 56% of the total Pb, Zn and Cd, respectively, present in their repacked soil columns.

原位施加此类螯合剂会促使金属不受控制地溶解和淋溶，从而增加水体污染风险。尽管上述部分研究提到目标金属可能被淋溶至根区以下并进入地下水，但对此类淋溶的测定或预测关注甚少。例外的是Grcman et al. (2001)的研究。他们发现EDTAˇ确实提高了Brassica rapa对重金属的吸收，但同时也导致其重填土柱中Pb、Zn和Cd总量的38%、10%和56%经淋溶损失。

Clearly the potential mobility of chelated metals needs to be evaluated before there is widespread acceptance of this technology. The main objective of our study was to investigate the relative importance of plant uptake and leaching of copper following the addition of EDTA.

显然，在该技术被广泛接受之前，需要评价螯合态金属的潜在迁移性。本研究的主要目的是探讨添加EDTA后铜的植物吸收与淋溶的相对重要性。

The key to chelate-induced phytoremediation is to maintain an increased bioavailability of the target metal long enough for plants to take it up. Hence the soluble complex which the target metal forms with EDTA needs to be relatively stable. Although EDTA itself is quite stable in soil (Hong et al., 1999), many metal–EDTA complexes are not (Jardine et al., 1993; Szecscody et al., 1994). In particular, Cu-EDTA can react with soil Fe over time, leaving the more stable Fe-EDTA in solution whereupon the Cu is released and re-adsorbed by the soil (Thayalakumaran et al., 2003a,b). Therefore a second objective of this study was to compare the effect on the plant copper uptake of numerous small EDTA applications over time, as compared to just two widely-spaced applications.

螯合剂诱导植物修复的关键在于使目标金属的生物有效性维持在较高水平，且持续时间足以供植物吸收。因此，目标金属与EDTA形成的可溶性络合物需要相对稳定。尽管EDTA本身在土壤中相当稳定（Hong et al., 1999），但许多金属–EDTA络合物并不稳定（Jardine et al., 1993; Szecscody et al., 1994）。特别是，Cu-EDTA会随时间推移与土壤中的Fe反应，使更稳定的Fe-EDTA留在溶液中，而Cu则被释放并重新被土壤吸附（Thayalakumaran et al., 2003a,b）。因此，本研究的第二个目的是比较随时间多次少量施加EDTA与仅间隔较长时间施加两次EDTA对植物铜吸收的影响。

Except for the study of Kayser et al. (2000), the chelate-induced phytoremediation studies conducted so far have all used repacked soil columns. Kayser et al. (2000) conducted a field study to investigate nitrilotriacetic acid (NTA)-enhanced phytoremediation of soil contaminated with Cu, Zn and Cd by the emissions from a nearby brass smelter. They reported that the NTA-amended soil increased the solubility of Zn, Cd and Cu by factors of 21, 58 and 9, respectively, but plant accumulation of these metals was only increased by a factor of 2–3. They did not investigate any leaching of the NTA-complexed metals. Water movement and root growth in repacked soil may not be the same as in intact soil with its quite different pore geometry. Hence, the third objective was to compare EDTAinduced copper uptake by grass grown in repacked soil columns, with that of grass in intact soil cores.

除Kayser et al. (2000)的研究外，迄今开展的螯合剂诱导植物修复研究均采用重填土柱。Kayser et al. (2000)开展了一项田间研究，考察次氮基三乙酸（NTA）强化植物修复受附近黄铜冶炼厂排放物污染的Cu、Zn和Cd污染土壤。他们报道，施加NTA的土壤中Zn、Cd和Cu的溶解度分别提高了21、58和9倍，但植物对这些金属的积累仅提高了2～3倍。他们未研究NTA络合态金属的淋溶。重填土中的水分运移和根系生长可能与孔隙结构截然不同的原状土不同。因此，本研究的第三个目的是比较重填土柱与原状土芯中生长的牧草在EDTA诱导下的铜吸收。

3Materials and methods材料与方法

A copper-tolerant grass from Parys Mountain, Amlwch, Anglesey, UK, Agrostis tenuis was used in this study. A copper-tolerant grass was used, rather than a copper hyperaccumulator, such as the Haumanistrum spp. These identified hyperaccumulators have a small biomass, and are not tolerant of temperate climatic conditions, as they are native of the Democratic Republic of Congo (Deram et al., 2000). A preliminary test showed Brassica juncea, a commonly used plant for enhanced phytoremediation, can suffer from severe phytotoxicity when EDTA mobilizes the copper.

本研究采用一种来自英国Anglesey岛Amlwch的Parys Mountain的耐铜草本植物Agrostis tenuis。之所以选用耐铜草本植物而非铜超富集植物（如Haumanistrum spp.），是因为已鉴定的这些超富集植物生物量小，且原产于刚果民主共和国，不耐温带气候条件（Deram et al., 2000）。预备试验表明，强化植物修复中常用的植物Brassica juncea,在EDTA活化铜时会遭受严重的植物毒害。

The soil used was collected from a site near Opotiki, New Zealand, that had been used to grow passion fruit for the preceding 10 years. The top 20 mm had become contaminated with copper due to the legacy of a heavy use of copper-based fungicide sprays. The soil was Opotiki sandy loam, a Typic Udivitrand Ashy Thermic in Soil Taxonomy. The top 100 mm had a bulk density of 0.9 Mg m⁻³, a relatively high cation exchange capacity of 22 cmolc kg⁻¹, and an organic matter content of 12–16%. The pH of the soil in a 1:2.5 water suspension was 5.6.

供试土壤采自新西兰Opotiki附近一处场地，该场地此前10年一直种植百香果。由于长期大量喷施铜基杀菌剂，表层20 mm土壤已受到铜污染。该土壤为Opotiki砂壤土，按美国土壤系统分类为Typic Udivitrand Ashy Thermic。表层100 mm土壤容重为0.9 Mg m⁻³，阳离子交换量（CEC）相对较高，为22 cmolc kg⁻¹，有机质含量为12%～16%。土水比1:2.5悬液的pH为5.6。

Two experiments were conducted, which we will refer to as I and II. Experiment I used repacked soil columns, while Experiment II used intact soil cores. The soil for Experiment I was collected from the top 100 mm of the A horizon in the copper-contaminated orchard and passed through a 2-mm nylon sieve while still moist. A depth of 150 mm of soil was packed at a bulk density of 0.75 Mg m⁻³ into plastic tubes with an internal diameter of 100 mm and a nylon mesh base. The average acid-extractable (conc. HNO3) copper content in the sieved soil was 276 µg g⁻¹. The intact cores for Experiment II were collected carefully from the same orchard site. After the removal of the above ground vegetation, plastic cylinders 150 mm long and 100 mm in diameter were gently pressed into the soil with minimal disturbance to the soil, and the cores retrieved by excavation. The bulk density ranged from 0.91 to 0.96 Mg m⁻³. Several smaller 35-mm diameter soil cores were taken adjacent to the larger cores to measure the copper distribution in the soil profile. The cores and repacked columns were placed in a glasshouse and seeds were sown into the surface soil.

本研究开展了两项试验，以下分别称为试验I和试验II。试验I采用重填土柱，试验II采用原状土芯。试验I的土壤采自铜污染果园A层表层100 mm，在湿润状态下过2 mm尼龙筛。将土壤以0.75 Mg m⁻³的容重装填至内径100 mm、底部为尼龙网的塑料管中，装填深度为150 mm。过筛土壤中酸提取态（浓HNO3）铜的平均含量为276 µg g⁻¹。试验II的原状土芯在同一果园场地谨慎采集。去除地上植被后，将长150 mm、直径100 mm的塑料圆筒轻轻压入土壤，尽量减少对土壤的扰动，然后挖出土芯。土芯容重为0.91～0.96 Mg m⁻³。在大土芯旁另采集若干直径35 mm的小土芯，用于测定土壤剖面中铜的分布。将土芯和重填土柱置于温室中，并将种子播于表层土壤。

In all cases, EDTA application commenced 4 weeks after seedling emergence. The EDTA solution applied was 0.01 M Na2H2EDTA, which had been raised to pH 6.0 with KOH to ensure optimal plant growth as well as mobility of the Cu-EDTA complex. To ameliorate the soil dispersion effect likely to be caused by the sodium, 0.01 M CaCl2 was also added to the EDTA solution. Where there were nine or 18 applications of this solution, the EDTA solution was mixed with the required amount of irrigation water and applied to the column or core. However, when there were only one or two EDTA applications, the EDTA solution was applied without mixing with water.

所有处理均在幼苗出土4周后开始施加EDTA。所施EDTA溶液为0.01 M Na2H2EDTA，并用KOH将pH调至6.0，以保证植物最佳生长以及Cu-EDTA络合物的迁移性。为缓解钠可能引起的土壤分散效应，还在EDTA溶液中加入0.01 M CaCl2。当该溶液分9次或18次施加时，将EDTA溶液与所需量的灌溉水混合后施加于土柱或土芯；而当仅施加1次或2次时，EDTA溶液不与水混合直接施加。

In Experiment I, three repacked columns were used. The first column was kept as a control with no EDTA addition. The second column had a total of 1800 µmol of EDTA sprinkled on to it in two applications of 900 µmol, the second application was 16 days after the first. The third column also received a total of 1800 µmol, but in 18 applications, each of 100 µmol, and applied on alternate days. As the columns contained soil with a dry mass of 880 g, the 1800 µmol of EDTA added corresponded to 0.74 mg of Na2H2EDTA per gram of dry soil.

试验I使用3个重填土柱。第1个土柱作为对照，不添加EDTA。第2个土柱分两次喷洒共1800 µmol EDTA，每次900 µmol，第2次施加在第1次施加后16天。第3个土柱同样共接受1800 µmol，但分18次施加，每次100 µmol，隔天施加一次。由于土柱中土壤干重为880 g，所添加的1800 µmol EDTA相当于每克干土0.74 mg Na2H2EDTA。

Experiment II involved five intact soil cores. The experimental treatments used in Experiment I were imposed on three of the cores. The other two cores both had 900 µmol of EDTA added to them. This was added as a single application to one core, and as nine 100 µmol applications applied at 4-day intervals to the other core.

试验II包括5个原状土芯。其中3个土芯施加与试验I相同的处理。其余2个土芯均添加900 µmol EDTA：一个土芯一次性施加，另一个土芯分9次、每次100 µmol、每隔4天施加一次。

Every day the water loss from each of the columns and cores was measured, and water applied to correct for the weight loss. No leachate emerged from the base of the columns or cores, as the soil water content was just brought back to ‘pot capacity’ each day. Forty-four days after EDTA was first applied, the grass was harvested from all columns and cores and placed in a drying oven at 80 ^◦C until a constant weight was achieved. Samples were analysed for copper as described below.

每天测定各土柱和土芯的失水量，并补充水分以弥补质量损失。由于每天仅将土壤含水量恢复至“盆栽持水量”，土柱或土芯底部没有渗滤液流出。首次施加EDTA后第44天，收获所有土柱和土芯上的牧草，置于80 ^◦C烘箱中烘至恒重。样品按下文所述方法测定铜含量。

After harvesting, the columns and cores were leached with about seven liquid-filled pore volumes (PV) of water under unsaturated conditions. The apparatus used to do this is described by Magesan et al. (1995). In brief, a disk permeameter sat atop each soil column to allow solution entry at a pre-set pressure potential. Unsaturated control was maintained by a bubbling tower and a vacuum system. A similar apparatus underneath maintained the same pressure potential at the base. A pressure head of −100 mm was maintained at the top and bottom of each core or column. Thin layers of sand ensured good contact between the soil and the porous plates. The leachate was collected and analysed for copper and iron as described below. Then the soil in each column or core was cut into six segments. The soil in each segment was homogenised, sub-sampled, and analysed as described below.

收获后，在非饱和条件下用约7个充液孔隙体积（PV）的水淋洗土柱和土芯。所用装置见Magesan et al. (1995)的描述。简言之，在每个土柱顶部放置一个圆盘渗透仪，使溶液在预设压力势下进入。通过鼓泡塔和真空系统维持非饱和状态。底部的类似装置维持相同的压力势。每个土芯或土柱的顶部和底部均维持−100 mm的压力水头。薄砂层确保土壤与多孔板良好接触。收集淋出液，按下文所述方法测定铜和铁。随后将各土柱或土芯中的土壤切分为6段，每段土壤经均匀混合、取亚样后按下文所述方法分析。

The plant material analysed for copper was prepared in the following manner. Sub-samples (1 g) of grass were weighed into 10-mL borosilicate test tubes. The tubes were heated in a muffle furnace overnight at 500 ^◦C. The next day 5 mL of warm 2 M HCl was added to each tube, mixed in with a vortex mixer, and then the solution was stored for analyses. The acid-extractable copper concentrations in the soil were determined by digesting the soil. To do this about 0.2 g of sieved soil was weighed into boiling tubes. Ten mL of concentrated HNO3 were then added and the mixture boiled until a final volume of 3 mL was reached. A further 10 mL of concentrated HCl were then added and the mixture again evaporated to 3 mL. The solution was made up to 10 mL, filtered and stored for analyses (Deram et al., 2000).

用于测定铜的植物样品按以下方法制备。称取牧草亚样（1 g）置于10 mL硼硅酸盐试管中，在马弗炉中于500 ^◦C加热过夜。次日向每支试管加入5 mL温热的2 M HCl，用涡旋混合器混匀后保存溶液待测。土壤酸提取态铜浓度通过土壤消解测定：称取约0.2 g过筛土壤置于消煮管中，加入10 mL浓HNO3，煮沸至最终体积为3 mL；再加入10 mL浓HCl，再次蒸发至3 mL。溶液定容至10 mL，过滤后保存待测（Deram et al., 2000）。

All samples were analysed for copper using flame atomic adsorption spectroscopy (FAAS).

所有样品均采用火焰原子吸收光谱法（FAAS）测定铜。

4Results and discussion结果与讨论

The dry matter yields at harvest are shown in Table 1, and these ranged from an equivalent of 331–433 g m⁻². There was no observable decrease in yield attributable to EDTA application, probably because the grass had been allowed to establish and grow for about 45 days before the EDTA was applied. During the 44- day period between the first application of EDTA and harvest, the evapotranspiration from the columns and cores ranged from 10 to 13 mm day⁻¹ on a pot crosssectional basis. Because of the overhang of the plants, the effective leaf area was large accounting for such high rates of water use on an area basis. There was no observable decrease in transpiration after the addition of EDTA.

收获时的干物质产量见表1，折合为331～433 g m⁻²。未观察到施加EDTA导致产量下降，这可能是由于在施加EDTA之前牧草已定植并生长了约45天。在首次施加EDTA至收获的44天内，以盆横截面积计，土柱和土芯的蒸散量为10～13 mm day⁻¹。由于植株冠层伸出盆缘，有效叶面积较大，因此按面积计的耗水速率如此之高。添加EDTA后未观察到蒸腾量下降。

In the repacked soil columns, pre-mixing of the soil meant that the initial soil copper concentration was reasonably uniform, although some variation was observed in the amount of total acid-extractable copper (Table 1). In contrast the copper in the intact cores was concentrated near the surface, as copper binds strongly to organic matter and therefore has limited mobility. As we had no way of non-destructively measuring the initial copper distribution in the cores, we used the data from the smaller cores taken close to each large core to infer the copper distribution in the larger cores. However due to soil variability, this did not necessarily give an exact measure of the total amount of initial copper in each large core. We found the total amount of copper by adding the amount of copper leached and the amount left in the soil after leaching. To get the correct mass balance for each core we used these total values, in conjunction with the shape of the distribution found in the smaller cores, to infer the initial copper distribution in each large core. Again some variation was observed in the total amount of copper in the cores, as expected in the field (Table 1). On average the repacked columns contained about 50% more copper than in the intact cores.

在重填土柱中，由于土壤经预先混合，初始土壤铜浓度较为均匀，但酸提取态总铜量仍存在一定差异（表1）。相比之下，原状土芯中的铜集中于表层附近，因为铜与有机质强烈结合，迁移性有限。由于无法以非破坏方式测定土芯中的初始铜分布，我们利用在每个大土芯附近采集的小土芯数据推断大土芯中的铜分布。然而，由于土壤的空间变异性，这种方法并不一定能准确给出每个大土芯中的初始铜总量。我们将淋溶的铜量与淋溶后残留于土壤中的铜量相加得到铜总量。为使每个土芯的质量平衡准确，我们利用这些总量值并结合小土芯中铜分布的形态，推断每个大土芯中的初始铜分布。正如田间条件下所预期的，各土芯的铜总量同样存在一定差异（表1）。平均而言，重填土柱的铜含量比原状土芯约高50%。

The 1800 µmol of EDTA applied to the contaminated soil corresponded to 740 µg g⁻¹ in the repacked columns and 670 µg g⁻¹ in the intact cores. This EDTA was sufficient to complex about half of the acid-extractable copper present, assuming a 1:1 molar reaction between copper and EDTA. However, EDTA forms a soluble complex with not only copper but with most other heavy metals, and has a strong affinity for iron when applied to soil. Therefore we expected less than half of the copper to be brought into solution by the EDTA.

向污染土壤施加的1800 µmol EDTA在重填土柱中相当于740 µg g⁻¹，在原状土芯中相当于670 µg g⁻¹。假设铜与EDTA按1:1摩尔比反应，该EDTA用量足以络合约一半的酸提取态铜。然而，EDTA不仅与铜形成可溶性络合物，还与大多数其他重金属形成络合物，且施入土壤后对铁具有很强的亲和力。因此，我们预计被EDTA带入溶液的铜不到一半。

Figure 1 shows that the addition of EDTA to the copper contaminated soil resulted in a substantial increase in the copper concentration in the leaves of A. tenuis, in both the repacked columns and intact cores. In the absence of EDTA, copper uptake was minimal. The highest herbage copper-concentration measured was 300 µg g⁻¹ and this is comparable to the 472 µg g⁻¹ measured by Aseni et al. (1999) in Erica andevalensis, a metal-tolerator, grown in crushed copper ore containing 1926 µg g⁻¹ of copper and with 2000 µg g⁻¹ of EDTA added. But our value was well below the 2500 µg g⁻¹ found in Arrhenatherum elatius, also a metal-tolerator, by Deram et al. (2000) after the addition of EDTA at a rate of 500 µg g⁻¹ to a copper ore with a total concentration of just 188 µg g⁻¹. These variations in leaf copper concentration could be due to differences in the form of copper present in the substrate, the age of the copper in the soil, or to differences between plant species. Copper ores mostly contain copper in inorganic form, whereas the copper in orchard topsoil is most likely to be bound to organic matter.

图1表明，向铜污染土壤中添加EDTA使重填土柱和原状土芯中A. tenuis叶片铜浓度均大幅增加。未施EDTA时，铜吸收极少。测得的地上部最高铜浓度为300 µg g⁻¹，与Aseni et al. (1999)在金属耐性植物Erica andevalensis中测得的472 µg g⁻¹相当，后者生长于含铜1926 µg g⁻¹的破碎铜矿石中，并添加了2000 µg g⁻¹ EDTA。但本研究的数值远低于Deram et al. (2000)在同为金属耐性植物的Arrhenatherum elatius中测得的2500 µg g⁻¹，后者是在总铜浓度仅为188 µg g⁻¹的铜矿石中按500 µg g⁻¹添加EDTA后测得的。叶片铜浓度的这些差异可能源于基质中铜的存在形态、铜在土壤中的老化时间或植物种间差异。铜矿石中的铜多为无机形态，而果园表土中的铜很可能与有机质结合。

The herbage copper concentration was much higher for the repacked columns than for the intact cores (Figure 1). In part this can be attributed to more copper being present in the repacked soil than intact cores (Table 1). But we suggest it was mainly due to a greater spatial separation between the copper adsorption sites and the roots in the intact cores, as compared to the repacked soil columns. In intact cores, the humus responsible for the copper adsorption sites tends to be more associated with the clay in the soil’s matrix inside the aggregates, while roots tend to grow in the interstitial larger cracks and bio-pores. In contrast, in the repacked soil columns, the roots would have a more uniform distribution. Additionally, the physical disturbance caused by sieving of the soil for repacked columns would make some of the copper adsorption sites more accessible.

重填土柱的地上部铜浓度远高于原状土芯（图1）。这在一定程度上可归因于重填土中的铜含量高于原状土芯（表1）。但我们认为，其主要原因是与重填土柱相比，原状土芯中铜吸附位点与根系之间的空间隔离更大。在原状土芯中，构成铜吸附位点的腐殖质往往更多地与团聚体内部土壤基质中的黏粒结合，而根系则倾向于在团聚体间较大的裂隙和生物孔隙中生长。相反，在重填土柱中，根系分布较为均匀。此外，重填土柱在过筛过程中造成的物理扰动会使部分铜吸附位点更易接近。

Figure 1. Copper accumulation in shoots of A. tenuis grown on copper contaminated repacked soil columns and intact soil cores in response to the addition of EDTA (1800 µmol per column, or core).图1. 在铜污染重填土柱和原状土芯中生长的A. tenuis地上部铜积累对添加EDTA（每个土柱或土芯1800 µmol）的响应。

The amount of copper uptake by the plants also varied with the amount and frequency of EDTA application (Table 1). As might be expected, the addition of 1800 µmol of EDTA induced more uptake from repacked soil columns than did the addition of 900 µmol. But also, when 1800 µmol of EDTA was applied in 18 small aliquots rather than two large doses, the copper concentration in the herbage (Figure 1) and total amount of copper uptake (Table 1) doubled, both in the repacked column and intact core.

植物的铜吸收量还随EDTA施加量和施加频率而变化（表1）。正如预期，添加1800 µmol EDTA比添加900 µmol诱导重填土柱中产生更多吸收。此外，当1800 µmol EDTA分18次少量施加而非两次大剂量施加时，无论是重填土柱还是原状土芯，地上部铜浓度（图1）和铜吸收总量（表1）均增加了一倍。

The reasons for this are not clear. However it is possible that the application of 900 µmol of EDTA on one day produced such high local concentrations of copper in the soil solution near the surface that the roots became inactive there. Thus they may have taken up most of their water from lower down where there was little EDTA, and so available copper, in the soil solution. This explanation is consistent with the finding that the uptake difference due to this treatment was less pronounced when only 900 µmol of EDTA was applied either as one application or in nine aliquots (Table 1).

其原因尚不清楚。但可能是一天内施加900 µmol EDTA使表层附近土壤溶液中局部铜浓度过高，导致该处根系失去活性。因此，根系可能主要从下层吸收水分，而下层土壤溶液中EDTA很少，有效铜也因而很少。这一解释与以下结果一致：当仅施加900 µmol EDTA时，无论一次施加还是分9次施加，该处理引起的吸收差异均不明显（表1）。

Thus far, the discussion has mainly centered on plant uptake. Enhanced-uptake occurred because EDTA brought copper into the soil solution. But if this copper is not all taken up by the plants, there is the risk that it could leach into the groundwater. To assess this risk, following plant harvest we measured the amount of copper leached from each of the columns and cores when 7 PV, about 500 mm, of water were applied. Table 1 shows that between 10 and 20% of the copper in the soil was leached from the columns and cores that received EDTA. A lot of iron was also leached. About 23–58% of the EDTA applied apparently was complexed with iron when it was leached from soil. Figures 2 and 3 show the copper distributions in the columns and cores after leaching. Most of the copper from the top 30 mm has been brought into solution by the EDTA, and then it was leached downward. The final copper concentration was about 84 µg g⁻¹ on the surface soil in the two repacked columns to which EDTA had been applied. This concentration is similar to the 90 µg g⁻¹ of copper that Thayalakumaran et al. (2003a, b) found remaining in contaminated Opotiki umaran et al., 2003a, b). The negligible amount of copper measured in the leachate at the end of the 7 PV of leaching confirmed that the increased copper concentration below 80 mm depth was not due to incomplete leaching of the copper present in the soil solution. Between 58 and 70% of the applied EDTA was leached in association with either copper or iron. The rest of the EDTA probably was complexed with calcium or the small amount of zinc in the soil. Alternatively it could have been adsorbed or broken down in the soil.

至此，讨论主要集中于植物吸收。吸收之所以增强，是因为EDTA将铜带入了土壤溶液。但若这些铜未被植物全部吸收，便存在淋溶进入地下水的风险。为评估这一风险，我们在植物收获后施加7 PV（约500 mm）的水，测定了各土柱和土芯的铜淋溶量。表1显示，施加EDTA的土柱和土芯中有10%～20%的土壤铜被淋溶。同时也有大量铁被淋溶。所施EDTA中约23%～58%在从土壤中淋出时显然是与铁络合的。图2和图3显示了淋溶后土柱和土芯中的铜分布。表层30 mm中的大部分铜已被EDTA带入溶液，随后向下淋溶。在两个施加EDTA的重填土柱中，表层土壤的最终铜浓度约为84 µg g⁻¹。该浓度与Thayalakumaran et al. (2003a, b)在受污染的Opotiki umaran et al., 2003a, b)中发现的残留铜浓度90 µg g⁻¹相近。7 PV淋溶结束时淋出液中铜含量可忽略不计，证实80 mm深度以下铜浓度的增加并非由于土壤溶液中的铜淋溶不完全所致。所施EDTA中有58%～70%与铜或铁结合而被淋出。其余EDTA可能与钙或土壤中少量的锌络合，也可能被土壤吸附或在土壤中降解。

Comparing the amount of copper uptake by the grass, with the amount leached, provides a risk assessment of how effective an EDTA-enhanced phytoremediation might be in terms of the added benefit of removing copper by plant uptake, relative to the increased chance of polluting the groundwater. For the two repacked columns with EDTA added, 25 and 62 times more copper was leached than was taken up by plants. This uptake-leaching ratio was even higher for the intact cores, ranging from 99 to 169. EDTA-enhanced phytoremediation is unlikely to be risk-free in humid environments, unless a drainage system can be installed and the leachate collected and recycled, or unless drainage can be otherwise minimized. If the leachate were collected, then it rather than plant uptake would provide the major Cu removal pathway. Alternatively there may be scope for using deep-rooted, higher water-use trees to reduce drainage.

比较牧草的铜吸收量与淋溶量，可以对EDTA强化植物修复的有效性进行风险评估，即权衡通过植物吸收去除铜的额外收益与污染地下水可能性的增加。对于两个添加EDTA的重填土柱，铜淋溶量分别为植物吸收量的25倍和62倍。原状土芯的淋溶与吸收之比更高，为99～169。在湿润环境中，除非能够设置排水系统并收集和循环利用淋出液，或能以其他方式将排水降至最低，否则EDTA强化植物修复不太可能是无风险的。若收集淋出液，则去除Cu的主要途径将是淋出液而非植物吸收。另外，也可考虑利用根系较深、耗水量较大的树木来减少排水。

Figure 2. The initial copper concentration (—) and the final measured copper concentrations (�) in the repacked soil columns in Experiment I. (a) Control column; (b) two EDTA applications; and (c) 18 EDTA applications.图2. 试验I重填土柱中的初始铜浓度（—）和最终实测铜浓度（�）。(a) 对照土柱；(b) 施加2次EDTA；(c) 施加18次EDTA。

sandy loam after leaching with an excess of EDTA solution. The higher copper concentration measured in the top 30 mm of the intact cores could be due to the amount of EDTA applied not being enough to solubilise the higher initial copper concentration at the surface soil.

砂壤土中残留的铜浓度，该结果是在用过量EDTA溶液淋洗后得到的。原状土芯表层30 mm测得的铜浓度较高，可能是由于所施EDTA量不足以溶解表层土壤中较高的初始铜。

Beyond a depth of 80 mm, the final copper concentration in the soil was greater than the initial concentration in all columns and cores apart from the controls (Figures 2 and 3). We attribute this increase to the resorption of EDTA-solubilised copper. Another study on the same soil has shown that with EDTA residence times of a week or more, CuEDTA slowly reacts with iron in the soil to form FeEDTA. The once-mobilised copper ions are then adsorbed by the soil (Thayalak- A simple calculation allows us to address the interesting question of to what extent the grass roots might exclude copper in the soil solution. As an example, we consider the repacked soil column with the highest copper concentration in the leaves. Over the 50 days between the first EDTA application and harvest, 450 mm or 3.5 kg of water was lost. We can reasonably assume that this was mostly lost via transpiration. The A. tenuis leaves took up 14.8 µmol, or 941 µg of copper. Therefore the average copper concentration in the water entering the roots must have been 0.27 µg g⁻¹. A total of 363 µmol, or 23 mg of copper was leached from that column after harvest. We consider this an approximate estimate of the Cu in the soil solution over the 50 days. The volume of soil in the column was 1.2 L, and the water content at ‘pot capacity’ was 0.5 m³ m⁻³. So the column would have typically contained 600 g of water. Thus we can estimate the average copper concentration in the soil solution as being 23 mg/600 g, or 38 µg g⁻¹. Looked at this way, the copper concentration in the water entering the roots was, 0.27/38, or 0.007, some two orders of magnitude less than the concentration in the soil solution. Hence the grass was quite effective at screening out the copper in the soil solution. This high filter efficiency poses a limitation on the phytoremediation of copper-contaminated soils using A. tenuis, unless leachate recycling can be used to allow the plant repeated opportunities to take up the copper.

除对照外，所有土柱和土芯在80 mm深度以下的最终土壤铜浓度均高于初始浓度（图2和图3）。我们将这一增加归因于EDTA溶解态铜的再吸附。对同一土壤的另一项研究表明，当EDTA停留时间达到一周或更长时，CuEDTA会与土壤中的铁缓慢反应形成FeEDTA，随后曾被活化的铜离子又被土壤吸附（Thayalak- 通过简单计算，可以探讨一个有趣的问题：牧草根系在多大程度上排斥土壤溶液中的铜。以叶片铜浓度最高的重填土柱为例。在首次施加EDTA至收获的50天内，共损失水分450 mm，即3.5 kg。可以合理假设这些水分主要经蒸腾损失。A. tenuis叶片吸收了14.8 µmol即941 µg铜。因此，进入根系的水中铜的平均浓度应为0.27 µg g⁻¹。收获后该土柱共淋出363 µmol即23 mg铜。我们将其视为50天内土壤溶液中Cu量的近似估计值。土柱中土壤体积为1.2 L，“盆栽持水量”下的含水量为0.5 m³ m⁻³，因此土柱中通常含水约600 g。由此可估算土壤溶液中铜的平均浓度为23 mg/600 g，即38 µg g⁻¹。由此看来，进入根系的水中铜浓度为土壤溶液的0.27/38，即0.007，约比土壤溶液中的浓度低两个数量级。因此，牧草能相当有效地滤除土壤溶液中的铜。这种高过滤效率限制了利用A. tenuis对铜污染土壤进行植物修复，除非采用淋出液循环方式，使植物有多次机会吸收铜。

Figure 3. The initial copper concentration (—) and the final measured copper concentrations (�) in the soil cores in Experiment II. (a) Control core; (b) one EDTA application of 900 µmol; (c) nine EDTA applications totalling 900 µmol; (d) two applications totalling 1800 µmol; and (e) 18 EDTA applications totalling 1800 µmol.图3. 试验II土芯中的初始铜浓度（—）和最终实测铜浓度（�）。(a) 对照土芯；(b) 一次施加900 µmol EDTA；(c) 分9次施加EDTA，共900 µmol；(d) 分2次施加，共1800 µmol；(e) 分18次施加EDTA，共1800 µmol。

The current study was set up as a pilot study, to look at both, the uptake of copper following various application rates of EDTA, and the risk of leaching, treatments were not replicated. As most of the differences found were large and consistent across treatments, critical inferences can be made.

本研究作为一项预备研究，旨在同时考察不同EDTA施加量下的铜吸收和淋溶风险，各处理未设重复。由于所发现的差异大多较大且在各处理间一致，仍可得出关键推论。

5Conclusions结论

EDTA application of 1800 µmol to 0.9 kg of copper-contaminated soil in a repacked column growing A. tenuis, increased the shoot copper concentration from 30 µg g⁻¹ to 300 µg g⁻¹. However the EDTA-enhanced phytoremediation was less effective in the intact cores, in which the same EDTA application to 1.0 kg of soil only increased the herbage copper concentration from 10 to 60 µg g⁻¹. More copper accumulated in the herbage when the EDTA was applied in numerous small doses, rather than in just one or two larger amounts. The concentration of copper in the water taken up by the grass was two orders of magnitude lower than that in the soil solution.

向种植A. tenuis的重填土柱中0.9 kg铜污染土壤施加1800 µmol EDTA，使地上部铜浓度由30 µg g⁻¹增至300 µg g⁻¹。然而，EDTA强化植物修复在原状土芯中效果较差，对1.0 kg土壤施加相同量的EDTA仅使地上部铜浓度由10增至60 µg g⁻¹。与一次或两次大剂量施加相比，多次小剂量施加EDTA时地上部积累的铜更多。牧草所吸收水分中的铜浓度比土壤溶液中的铜浓度低两个数量级。

After the EDTA application 25 to 169 times more copper was leached than was taken up by the herbage. This means that a strategy for managing leaching losses needs to be part of any plan for EDTA-enhanced phytoremediation.

施加EDTA后，铜的淋溶量为牧草地上部吸收量的25～169倍。这意味着任何EDTA强化植物修复方案都必须包含控制淋溶损失的策略。

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