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Agriculture and Environment Division, IACR-Rothamsted, Harpenden, Hertfordshire AL5 2JQ, UK
* Corresponding author (steve.mcgrath{at}bbsrc.ac.uk)
Received for publication August 1, 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Abbreviations: EDTA, ethylenediaminetetraacetic acid • ICP–AES, inductively coupled plasma atomic emission spectroscopy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Phytoremediation of heavy metal–contaminated soil is an emerging technology that aims to extract or inactivate metals in soils (McGrath, 1998; Salt et al., 1998). It has attracted attention in recent years for the low cost of implementation and environmental benefits. Moreover, the technology is likely to be more acceptable to the public than other traditional methods. It has been estimated that the market for phytoextraction of metals from soils in the USA alone was approximately $1–2 million in 1997, with a potential to increase to $15–25 million by 2000 and $70–100 million by 2005 (Glass, 2000).
Two approaches have been proposed for phytoextraction of heavy metals, namely continuous or natural phytoextraction and chemically enhanced phytoextraction (Salt et al., 1998). The first is based on the use of natural hyperaccumulator plants with exceptional metal-accumulating capacity. These plants have several beneficial characteristics such as the ability to accumulate metals in their shoots and an exceptionally high tolerance to heavy metals (Baker et al., 2000). On the other hand, many hyperaccumulator plants tend to be slow-growing and produce low biomass, with the exception of some Ni hyperaccumulator species. With the plant materials currently available, years or decades are needed to clean up a contaminated site. For instance, McGrath et al. (1993), using field data, calculated that nine croppings of T. caerulescens would be required to decrease Zn concentration in the soil from 440 to 300 mg Zn kg-1. Similarly, Brown et al. (1994) estimated that 28 yr of T. caerulescens cultivation would be necessary to remove all the Zn from a soil containing 2100 mg Zn kg-1. Another problem with the continuous phytoextraction of metals from soils is related to the fact that some metals such as Pb are largely immobile in soil and their extraction rate is limited by solubility and diffusion to root surface.
Chemically enhanced phytoextraction has been developed to overcome these problems (Huang and Cunningham, 1996; Blaylock et al., 1997; Huang et al., 1997; Blaylock, 2000). This approach makes use of high-biomass crops that are induced to take up large amounts of metals when their mobility in soil is enhanced by chemical treatments. Several chelating agents, such as citric acid, EDTA, CDTA, DTPA, EGTA, EDDHA, and NTA, have been studied for their ability to mobilize metals and increase metal accumulation in different plant species (Huang et al., 1997; Cooper et al., 1999). Different metals have been targeted, such as Pb (Blaylock et al., 1997; Huang et al., 1997), U (Huang et al., 1998), 137Cs (Lasat et al., 1998), and Au (Anderson et al., 1998). However, at the moment, the most promising application of this technology is for the remediation of Pb-contaminated soils using Indian mustard [Brassica juncea (L.) Czern.] in combination with EDTA (e.g., Blaylock, 2000). Despite the success of this technology, some concerns have been expressed regarding the enhanced mobility of metals in soil and their potential risk of leaching to ground water (Cooper et al., 1999). However, no detailed studies regarding the persistence of metal–EDTA complexes in contaminated soils have been conducted.
Soils contaminated with multiple heavy metals can present a difficult challenge for both approaches to phytoextraction. Although some hyperaccumulators appear to be capable of accumulating elevated concentrations of several heavy metals simultaneously, there is still considerable specificity in metal hyperaccumulation (Baker et al., 2000). Also, Pb and Cu hyperaccumulators have been noted, but largely unproven (Baker et al., 2000). For chemically enhanced phytoextraction, establishment of a high-biomass crop is required before chelate application. This may be difficult to achieve if the soil is heavily contaminated with metals such as Zn, Cd, and Cu, which are usually much more bioavailable, and thus more phytotoxic, than Pb.
In the present study, we compared natural phytoextraction using the well-known Zn and Cd hyperaccumulator T. caerulescens, and chemically enhanced phytoextraction using maize and EDTA treatment. Two soils contaminated with multiple heavy metals were used. The primary aim was to evaluate which approach is likely to be more successful to remediate soils contaminated with multiple heavy metals. Furthermore, we investigated the potential environmental risk associated with metal mobilization by EDTA.
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MATERIALS AND METHODS |
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We grew each plant species for three consecutive growth cycles over a 391-d period, allowing about 1 mo between croppings. Both were resown each time. One plant of maize, or four plants of T. caerulescens, grew in each pot containing 1 kg soil (oven-dry basis). There were four replicates for each treatment. At the beginning of each cropping cycle, 240, 120, and 150 mg kg-1 of N, P, and K, respectively, were added to the pots as fertilizer. Deionized water was used throughout.
In each cycle of maize, when it reached the early flowering stage, EDTA (as disodium salt) was applied to the soil at a rate of 1 g per kg (2.7 mmol kg-1 of soil). Maize was harvested 4 wk after the EDTA treatment. To assess the effect of EDTA application on the metal uptake by maize, we also included a control of maize without EDTA, only at the same time as the first cropping + EDTA. The control was replicated four times for each soil. All pots were arranged randomly.
At the end of each growth cycle (approximately 4 mo), the shoots of T. caerulescens and the shoots and roots of maize were harvested. Plant materials were washed thoroughly with deionized water and dried at 80°C for 16 h, and their dry weight was recorded. Dried samples were ground to <0.5 mm before analysis.
Nylon-coated soil moisture samplers (Rhizosphere Research Products, Wageningen, the Netherlands) were placed inside the soil in each pot to collect soil pore water, following the method described by Knight et al. (1998). In the pots with T. caerulescens, soil pore water was collected at the beginning of the experiment and immediately before each harvest. In the pots with maize, soil pore water samples were collected at the beginning of the experiment, before each EDTA application and at five different times after the application up to 4 wk.
Chemical and Statistical Analyses
Total concentrations of heavy metals in the soils were determined using inductively coupled plasma atomic emission spectroscopy (ICP–AES; Fisons Accuris, Ecublens, Switzerland), following aqua regia digestion (McGrath and Cunliffe, 1985). Soil pH was measured in the 1:2.5 suspension of soil and water. Total C and N were determined using a LECO (St. Joseph, MI) CNS-2000. The sand, silt, and clay contents, and soil cation exchange capacity (CEC) were determined using standard methods (Avery and Bascomb, 1982). Soils before and after phytoextraction were determined for extractable metals using 1 M NH4NO3 (Deutsch Institut für Normung, 1995).
Soil pore water samples were analyzed for total soluble metal concentrations using ICP–AES. In addition, we quantified metal–EDTA complexes by ion chromatography (Dionex [Sunnyvale, CA] DX500), using a hydrophilic anion column (Dionex IonPac AS5) and a guard column (Dionex IonPac AG5). The eluant consisted of 2.24 mM Na2CO3 and 1.76 mM NaHCO3, which was pumped through the columns isocratically at a flow rate of 1 mL min-1 (Sun et al., 2001).
Subsamples of plant materials (0.2 g) were digested with a mixture of HNO3 and HClO4. Metal concentrations of the digests were determined using ICP–AES.
Statistical analysis was performed using Genstat 5 (Genstat 5 Committee, 1993). Means and standard errors are presented for all data. Comparisons between croppings were made using t tests.
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RESULTS |
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The concentrations of Cd, Cu, Ni, Pb, and Zn in the shoots of T. caerulescens varied considerably across the three harvests (Table 2). On average, the concentrations of Zn and Cd in the shoots of T. caerulescens were 2139 and 116 mg kg-1 in the UK soil, and 8700 and 263 mg kg-1 in the French soil, respectively. The concentrations of Cd obtained in most harvests on both soils were greater than the value used to define Cd hyperaccumulation (100 mg kg-1; Baker et al., 2000), whereas the concentrations of Zn obtained on the French soil were close to the threshold value for hyperaccumulation (10000 mg kg-1). The concentrations of both metals did not decrease with cropping (Table 2). Thlaspi caerulescens grown on the French soil had higher concentrations of Pb than plants on the UK soil. In contrast, the concentrations of Cu were considerably higher on the UK soil than those on the French soil, reflecting a much higher level of Cu contamination in the former. For Cu, Pb, and Ni, the concentrations in the shoots of T. caerulescens were far below the values used to define hyperaccumulation (1000 mg kg-1).
In maize, the concentrations of all heavy metals determined were much higher in the roots than in the shoots (Table 2). In the first cycle, application of EDTA to both soils increased the concentrations of Cd, Cu, and Zn in the roots by one- to threefold (P < 0.05). In the French soil, which had a high concentration of Pb (Table 1), application of EDTA increased the concentration of Pb in the roots by 16-fold (P < 0.001). In contrast, application of EDTA had no significant effect on the concentrations of metals in the shoots, except for Zn in the UK soil (50% increase, P < 0.01) and Pb in the French soil (2.5-fold increase, P < 0.05). Compared with the first crop cycle, the metal concentrations in both roots and shoots increased successively in the two following croppings. In the French soil, a much-reduced biomass in the third cropping may partly explain the large increases in the metal concentrations in maize.
Phytoextraction of Heavy Metals
The amounts of Cd and Zn removed by maize (shoots) and T. caerulescens are shown in Fig. 1
. The amounts of other metals extracted by both plants were small, and are not presented. In the French and UK soils, T. caerulescens extracted, respectively, 63 and 8 times more Cd than maize (P < 0.001). In particular, T. caerulescens removed a total of more than 8 mg Cd per kg of soil in the three croppings in the French soil. Thlaspi caerulescens also extracted approximately 4 times more Zn (197 mg kg-1 soil) than the EDTA-treated maize (25 mg kg-1 soil) from the French soil. The removal of Zn by the two plant species from the UK soil was similar.
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DISCUSSION |
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The French soil had high concentrations of Zn, Cd, and Pb, but low Cu, and the biomass of T. caerulescens was comparable with that of maize. Three successive croppings of T. caerulescens removed approximately 8 and 200 mg kg-1 of Cd and Zn, respectively, representing 43 and 6.8% of the total Cd and Zn contents in the French soil. It is interesting to note that the phytoextraction efficiency for Zn and Cd did not decrease with cropping. In the third cropping, which produced the largest biomass, the concentrations of Cd and Zn in the shoots were also the highest or among the highest. This suggests that optimization of the growing conditions may result in an increased yield without decreasing metal concentrations in this species. Similarly, Bennett et al. (1998) showed that optimizing N fertilization of T. caerulescens increased yield without decreasing the Zn concentration in the plant. Assuming a constant removal rate, approximately six croppings of T. caerulescens would be required to decrease the total Cd in the French soil from 19 to 3 mg kg-1. This very encouraging result was obtained with an ecotype of T. caerulescens that is particularly efficient in hyperaccumulating Cd (Lombi et al., 2000). In the case of Zn, 40 croppings would be required to reduce its total concentration from 2920 to 300 mg kg-1. This estimate is very similar to that of Brown et al. (1994).
Brassica species are often used in chemically enhanced phytoextraction (Blaylock, 2000), although other plant species such as maize and pea (Pisum sativum L.) have also been used (Huang and Cunningham, 1996; Huang et al., 1997). However, Indian mustard suffered from severe phytotoxicity when grown on both soils used in this study, even before EDTA was applied. Elevated tolerance to heavy metals in soils is a prerequisite for a successful phytoextraction. Unlike Pb, which is usually low in bioavailability in soil, metals like Zn, Cd, and Cu tend to have much higher bioavailability, and thus are more phytotoxic. This means that phytoextraction using nontolerant cultivars of Brassica species is unlikely to succeed in soils contaminated with large concentrations of Zn, Cd, or Cu.
Monocotyledon species are usually more tolerant to metals than dicotyledon species (Marschner, 1995). Maize was able to grow on both soils, showing only limited signs of phytotoxicity. However, the growth of maize on the French soil was severely stunted in the second and third cropping, following the EDTA treatments in the first and second cropping. It is likely that Zn toxicity was responsible for this inhibition of growth. The concentrations of soluble Zn in the soil pore waters collected from the French soil at the beginning of the second and third cropping were >100 mg L-1, compared with <50 mg L-1 before the EDTA treatment (Fig. 2). In contrast, the EDTA treatments did not decrease the biomass of maize in the second and third cropping in the UK soil, probably because this soil had a lower concentration of total Zn and a lower concentration of soluble Zn in the pore water.
Although EDTA increased the concentrations of soluble metals dramatically (Fig. 2), metal uptake by maize was minimal (Table 2). Also, the EDTA treatment increased the metal concentrations in the roots far more than in the shoots, suggesting that EDTA was far more efficient in overcoming the diffusion limitation of metals to root surface than the barrier of root to shoot translocation. The results are consistent with those of Ebbs and Kochian (1998), who showed that EDTA increased the concentration of Zn in the shoots of Indian mustard by less than twofold, and did not enhance Zn uptake by oat (Avena sativa L.) and barley. Similarly, Kayser et al. (2000) showed that NTA and elemental S increased the solubility of Cd, Cu, and Zn in the soil by a factor of 58, 9, and 21 respectively, but accumulation of these metals in maize, Indian mustard, and other plants was only increased by a factor of 2 to 3. The enhancing effect of EDTA observed here, and by Ebbs and Kochian (1998), was much smaller than that reported for Pb by Blaylock et al. (1997) and Huang et al. (1997), and for Zn by Blaylock et al. (1997). The latter spiked a low-Zn soil with zinc carbonate, which may be more readily solubilized than the Zn in the aged contaminated soils used in this study.
Our results indicate that EDTA alone increases metal mobility in soil and accumulation in roots, but does not substantially increase the transfer of metals to shoots. In their patent on induced hyperaccumulation of metals in plant shoots, Ensley et al. (1999) described chemically induced phytoextraction as a two-step process in which plants first accumulate metals in their roots and then, by application of an inducing agent, enhanced transfer of the metals to the shoots occurs. This transfer is due to disrupting the plant metabolism that regulates the transport of metal to the shoots. Ensley et al. (1999) maintain that chelating agents (such as EDTA) and acids increase both solubility of metals in the soil and root to shoot transfer of metals. However, our results indicated that transfer of metals to the shoots was not greatly enhanced by EDTA. It is not clear whether application of an herbicide, as proposed by Ensley et al. (1999), enhances metal accumulation in shoots through disruption of plant metabolism.
Compared with natural phytoextraction using T. caerulescens, EDTA-enhanced phytoextraction of Cd and Zn by maize was much less efficient from the French soil. The amounts of Cd and Zn removed by maize + EDTA over the three croppings were only 1.6 and 11.5%, respectively, of those removed by T. caerulescens. Natural phytoextraction with T. caerulescens performed poorly because of the high Cu concentrations in the UK soil. Even so, its removal of Cd was still eight times greater than that by maize + EDTA. The amounts of Zn removed by T. caerulescens and by maize + EDTA from the UK soil were similar.
The marked increases in soluble metals in soil pore water following EDTA applications may pose a serious concern in terms of potential leaching of heavy metals to ground water. Furthermore, the concentrations of soluble metals remained very high several weeks after the application of the chelating agent. In fact, 5 mo after EDTA application (which was the time interval between EDTA applications), metal–EDTA complexes were still found in the soil pore water, indicating the persistence of metal–EDTA complexes. This is in agreement with the work of Hong et al. (1999), who reported that EDTA is relatively biologically stable even under conditions favoring biodegradation. This may be because metal–EDTA complexes with high stability constants (i.e., chelates of Cu, Fe, Pb, and Zn) are degraded more slowly (Satroutdinov et al., 2000) than complexes with low stability constants (i.e., chelates of Ca, Mg, and Mn). In the contaminated soils used in our study, virtually all the EDTA added formed complexes with metals of high stability constants, and this may contribute to the slow degradation and the persistent mobility of the metals.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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In chemically enhanced phytoextraction, we used maize instead of Indian mustard, because the latter was not tolerant to the heavy metals presented in the two soils, even before EDTA applications. The EDTA treatment greatly increased the concentrations of soluble metals, but increased metal extraction by maize shoots to a much smaller degree. Maize also suffered severe phytotoxicity in the second and third cropping in the industrial soil. Thlaspi caerulescens was far more efficient than maize + EDTA treatments in phytoextracting Cd from both soil types. Thlaspi caerulescens also extracted much more Zn than maize + EDTA treatments from the low-Cu soil, whereas both approaches extracted similar amounts of Zn from the high-Cu soil.
In chemically enhanced phytoextraction, the dramatically increased metal solubility and the persistence of metal–EDTA complexes in soil pore water may pose an environmental risk of leaching to ground water.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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