Chemosphere 74 (2009) 1279–1291

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Chemosphere

journal homepage: www.elsevier .com/locate /chemosphere

Review

EDTA-assisted Pb phytoextraction

Saifullah a, E. Meers b,*, M. Qadir c, P. de Caritat d, F.M.G. Tack b, G. Du Laing b, M.H. Zia a,e

a Institute of Soil and Environmental Sciences, University of Agriculture, Fasialabad-38040, Pakistanb Laboratory of Analytical Chemistry and Applied Ecochemistry, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, T.a.v. Prof. Verloo, 9000 Ghent,Oost-Vlaanderen, Belgiumc International Center for Agricultural Research in the Dry Areas (ICARDA), P.O. Box 5466, Aleppo, Syriad Cooperative Research Centre for Landscape Environments and Mineral Exploration (CRC LEME), GPO Box 378, Canberra, ACT 2601, Australiae Technical Services Department, Fauji Fertilizer Company Ltd., 11 Shahrah-e-Aiwan-i-Tijarat, Lahore, Pakistan

a r t i c l e i n f o

Article history:Received 21 June 2008Received in revised form 3 November 2008Accepted 4 November 2008Available online 1 January 2009

Keywords:PhytoremediationPbEDTAPhytoextractionSoil remediationHeavy metal

0045-6535/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.chemosphere.2008.11.007

* Corresponding author. Tel.: +32 9 2645947; fax: +E-mail address: erik.meers@ugent.be (E. Meers).

a b s t r a c t

Pb is one of the most widespread and metal pollutants in soil. It is generally concentrated in surface lay-ers with only a minor portion of the total metal found in soil solution. Phytoextraction has been proposedas an inexpensive, sustainable, in situ plant-based technology that makes use of natural hyperaccumula-tors as well as high biomass producing crops to help rehabilitate soils contaminated with heavy metalswithout destructive effects on soil properties. The success of phytoextraction is determined by theamount of biomass, concentration of heavy metals in plant, and bioavailable fraction of heavy metalsin the rooting medium. In general, metal hyperaccumulators are low biomass, slow growing plant speciesthat are highly metal specific. For some metals such as Pb, there are no hyperaccumulator plant speciesknown to date. Although high biomass-yielding non-hyperaccumulator plants lack an inherent ability toaccumulate unusual concentrations of Pb, soil application of chelating agents such as EDTA has been pro-posed to enhance the metal concentration in above-ground harvestable plant parts through enhancingthe metal solubility and translocation from roots to shoots. Leaching of metals due to enhanced mobilityduring EDTA-assisted phytoextraction has been demonstrated as one of the potential hazards associatedwith this technology. Due to environmental persistence of EDTA in combination with its strong chelatingabilities, the scientific community is moving away from the use of EDTA in phytoextraction and is turningto less aggressive alternative strategies such as the use of organic acids or more degradable APCAs (ami-nopolycarboxylic acids). We have therefore arrived at a point in phytoremediation research history inwhich we need to distance ourselves from EDTA as a proposed soil amendment within the context of phy-toextraction. However, valuable lessons are to be learned from over a decade of EDTA-assisted phyto-remediation research when considering the implementation of more degradable alternatives inassisted phytoextraction practices.

� 2008 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12802. EDTA: a chelating agent. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12813. EDTA-assisted Pb phytoextraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1281

3.1. EDTA-assisted Pb dissolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12813.2. EDTA-assisted Pb uptake by plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1282

4. Factors affecting EDTA-assisted Pb phytoextraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12835. Potential risks associated with EDTA-assisted Pb phytoextraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1284

5.1. Plant growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12845.2. Soil biota . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12855.3. Leaching of heavy metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1285

6. Reducing potential risks associated with lead phytoextraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1286

ll rights reserved.

32 9 2646232.

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P

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H

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Pm

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1280 Saifullah et al. / Chemosphere 74 (2009) 1279–1291

7. Plant species facilitating and maximizing lead phytoextraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12878. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1287

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1288

1. Introduction

Lead is naturally present in soils. It is a trace constituent of com-mon rock-forming and readily weatherable minerals such as K-feldspar, plagioclase and mica, and a major constituent of varioussulphide, sulphate, oxide, carbonate and silicate minerals (e.g., ga-lena PbS, anglesite PbSO4, minium Pb3O4, cerussite PbCO3, alamo-site PbSiO3) locally found in Pb ores and their weatheringproducts (Reimann and de Caritat, 1998). In uncontaminated soils,Pb concentrations are generally below 50 mg kg�1 (Reimann andde Caritat, 1998). The Pb concentration in vegetation growing onthese soils is often less than 10 mg kg�1 dry mass.

Mining, industrial, and agricultural activities have lead to anaccelerated release of various metals including Pb into the environ-ment, creating potential hazards to ecosystems and human health(Lantzy and Mackenzie, 1979; Nriagu, 1979; Mushak, 1993). Anumber of trace metals are essential for plant growth. These in-clude copper, iron, manganese, nickel, zinc, and molybdenum.However, at high concentrations, any trace element may becometoxic to fauna and flora (Marschner, 1995; Schwab et al., 2005).

During the past century, industrialization and mining activitieshave resulted in an increasing number of metal-contaminatedsites. Strategies that are commonly used to remediate these sitesinclude removal of metals by leaching with acids and chelatorsor metal stabilization using soil amendments such as lime or flyash. Conventional clean-up technologies are costly and feasibleonly for small but heavily polluted sites where rapid and completedecontamination is required (Blaylock et al., 1997; Cooper et al.,1999; Shen et al., 2002). The increasing demand for uncontami-nated agricultural land fueled by population growth has increasedthe need for an effective clean-up of moderately contaminatedsoils as well.

Phytoremediation, especially phytoextraction, has receivedincreasing attention as a promising, cost-effective alternative toconventional engineering-based remediation methods (Salt et al.,1998). Phytoextraction can be generally classified as either naturalor chemically assisted (Table 1). A first approach involves the useof metal hyperaccumulating plant species (Baker et al., 1994;

able 1ain characteristics of the two strategies of phytoextraction of metals from soilsdapted from Nascimento et al., 2006).

atural phytoextraction Chemical-assisted phytoextraction

lants naturally hyperaccumulatemetals

Plants are normally metal excluders

low growing, low biomass production Fast growing, high biomass plantsatural ability to extract high amount

of metals from soilSynthetic chelators and organic acids areused to enhance metal uptake

fficient translocation of metals fromroots to shoots

Chemical amendments increase themetal transfer from roots to shoots

igh tolerance; survival with highconcentration of metals in tissues

Low tolerance to metals; the increase inabsorption leads to plant death

o environmental drawback regardingmetals

Risk of leaching of metal chelates togroundwater

lants are highly specific for metals Availability and uptake of almost alletals can be enhanced withamendments

o true Pb-hyperaccumulator has beenidentified so far

Very successful for enhancing Pbphytoextraction

Brown et al., 1994; Kumar et al., 1995). A plant is said to be ahyperaccumulating species if it accumulates metals in above-ground biomass above a given concentration, for example,100 mg kg�1 for Cd on a dry weight (DW) basis, 1000 mg kg�1 forAs, Co, Cu, Pb or Ni, or more than 10000 mg kg�1 for Mn or ZnDW (Brooks, 1998; Baker et al., 2000; Ma et al., 2001; McGrathet al., 2002; Schmidt, 2003). To date, over 400 vascular plants havebeen identified as hyperaccumulating species. Many of these werefound to develop on heavy metal-contaminated sites (Roosenset al., 2003). However, hyperaccumulators generally produce verylow biomass and are slow growing (Baker and Brooks, 1989). Manyof the identified hyperaccumulating plants have been shown to beunsuitable for use in phytoextraction, because of their limitedgrowth and low shoot biomass production (Evangelou et al.,2007; Japenga et al., 2007; Koopmans et al., 2007).

A second phytoextraction approach involves the use of highyielding plant species. These species lack inherent ability to takeup large concentrations of metals, but can accumulate elevatedamounts when cultivated on soils that have been chemically trea-ted with soil amendments to enhance metal phytoavailability andplant uptake (Meers et al., 2005b).

The success of phytoextraction is strongly determined by theamount of biomass, the concentration of heavy metals in plant tis-sues, and the bioavailable fraction of heavy metals in the rootingmedium (McGrath, 1998; Grcman et al., 2001). Pb is a widespreadmetal pollutant in soils (Mushak, 1993). However, it is usuallypoorly bioavailable (Miller, 1996; Raskin et al., 1997) due to theformation of insoluble precipitates (McBride, 1994; Blaylocket al., 1997; Ruby et al., 1999; Adriano, 2001; Shen et al., 2002).Pb contamination is usually accumulated in the uppermost hori-zons of soil profiles (Anderson, 1977; Wang et al., 1995; Abreuet al., 1998; Johnson and Petras, 1998; Sanchez-Camazano et al.,1998). Only a very minor fraction of the total soil Pb is in soil solu-tion (Davies, 1995; Maiz et al., 2000).

In order to enhance both the availability of Pb in soil and trans-location from root to shoot, synthetic chelating agents such asEDTA, diethylenetrinitrilopentaacetic acid (DTPA), nitrilotriaceticacid (NTA), pyridine-2,6-dicarboxylic acid (PDA), trans-l,2-diami-nocyclohexane-N,N,N0,N0-tetraacetate (CDTA), or ethylenediaminedisuccinate (EDDS) have been proposed (Huang et al., 1997; Coo-per et al., 1999; Kayser et al., 2000; Puschenreiter et al., 2001;Grcman et al., 2003; Wu et al., 2004; Li et al., 2005; Meers et al.,2005b). Among the chelating agents used to extract metals fromsoils, EDTA is regarded as the most effective in solubilizing soil-bound Pb (Huang et al., 1997; Hong et al., 1999; Kos et al., 2003;Alkorta et al., 2004; Nascimento et al., 2006; Yukselen and Gokyay,2006). It has therefore been extensively used in soil decontamina-tion technologies (Vassil et al., 1998; Cooper et al., 1999; Lasatet al., 2000). Moreover, EDTA has been shown to dissolve Pb ad-sorbed to soil particles (Means et al., 1978), increase Pb movementto roots via mass flow or diffusion, enhance metal uptake and trig-ger root to shoot translocation of heavy metals (Huang et al., 1997;Nascimento et al., 2006).

In recent years, the use of persistent APCAs such as EDTA hasbeen disfavored by several scientists (Alkorta et al., 2004; Meerset al., 2004). EDTA has been shown to persist for extended periodsin soils because of its poor degradability (Nörtemann, 1999; Now-ack, 2002; Meers et al., 2005a). It therefore increases the activity oftrace metals in the soil solution for extended periods and might

Saifullah et al. / Chemosphere 74 (2009) 1279–1291 1281

cause enhanced toxicity towards plants and soil organisms (Sillan-pää and Oikari, 1996; Dirilgen, 1998; Hu et al., 2003). Moreover,accelerated, uncontrolled release and leaching of metals from spar-ingly soluble metal compounds is likely to increase groundwaterpollution (Grcman et al., 2001; Römkens et al., 2002; Madridet al., 2003; Wenzel et al., 2003).

Despite these concerns, chelant-assisted phytoextraction hasthe potential to become an effective remediation approach forPb-contaminated soils. Careful management of soils and the appro-priate selection of plants and irrigation strategies are of paramountimportance (Chen et al., 2004), while the focus might need to shifttowards the use of more degradable alternatives, thus effectivelyreducing the risks implied with this technology (Meers et al.,2004). An overview of alternative soil amendments proposed forenhanced phytoextraction is provided by Meers et al. (2008).

Timing of amendment application (prior to harvesting) andmode of application (split doses) can significantly reduce the ad-verse effects associated with chelant-assisted phytoextraction(Barocsi et al., 2003). Recently, the development of slow-releasechelating agents such as solid EDTA encapsulated with a layer ofsilicate (Li et al., 2005) or polymer (Shibata et al., 2007) has beenproposed to slow down Pb mobilization in soils in order to matchwith the plant’s uptake capacity. More studies on a field scale areneeded to assess the usefulness of this and other techniques toovercome the leaching risks while maintaining a high uptake rateby plants.

The purpose of this review is to give a broad overview of the sci-ence behind EDTA-assisted phytoextraction of Pb. Following anintroduction to phytoextraction, the processes involved in mobiliz-ing heavy metals by EDTA, accumulation of metal–EDTA in roots,and translocation to shoots are reviewed. The benefits and limita-tions associated with this chemically assisted technology are criti-cally evaluated. Finally, innovative developments and strategiesthat have been proposed to reduce the undesirable effects withthe use of EDTA have also been discussed. Phytoremediationresearch has arrived at a cross-roads at which we need to questionthe validity of using recalcitrant compounds, such as EDTA, for en-hanced phytoextraction purposes. However, we do feel that thevast research efforts in the past which have focused on the use ofEDTA as well as on the requirements and technologies to addressthe risks associated with intentional metal mobilization, need tobe taken into account when directing the field of enhanced phy-toextraction towards more degradable alternatives. The many pub-lished work involving the use of EDTA for soil remediationpurposes serves as a valuable reference to compare the perfor-mance of future approaches.

2. EDTA: a chelating agent

The formation of a complex occurs when a metal ion is coordi-nately bonded to one or more electron donating groups that arecalled ligands. Many APCAs (ligands) can form biostable andwater-soluble complexes with metal ions. Ideally, complexingagents used for the remediation of metal-contaminated soilsshould be able to form a highly stable complex over a wide pHrange at a 1:1 ligand to metal molar ratio. The metal complexesformed should not be sorbed on soil surfaces, have a low toxicityand wide applicability, and be cost-effective. EDTA has been sug-gested to be very suited for use in the remediation of heavy me-tal-contaminated soils (Nörtemann, 1999; Nascimento et al.,2006; Evangelou et al., 2007). Metal–EDTA complexes are very sta-ble (Oviedo and Rodriguez, 2003). However, EDTA is highly persis-tent in the environment and its background concentrations rangefrom a few lg L�1 up to 100 lg L�1 (Fuerhacker et al., 2003). Thispersistence is a serious concern when considering wide field appli-

cation for enhanced phytoextraction purposes. This will be elabo-rated below.

3. EDTA-assisted Pb phytoextraction

For phytoextraction to be successful, plant should readily ex-tract and translocate heavy metals from the soil to above-groundbiomass within a reasonable period of time. Blaylock and Huang(2000) stipulate that plants should accumulate more than 1% ofthe target metal(s) in above-ground biomass and produce morethan 20 t ha�1 yr�1 of above-ground biomass. Under normal condi-tions, Pb uptake in plants is restricted due to the generally low sol-ubility of Pb in soils (Reeves and Baker, 2000; Adriano, 2001).Moreover, once metals are absorbed by roots, the efficiency oftranslocation to the above-ground harvestable biomass is oftenquite low (Jones et al., 1973; Lane and Martin, 1977; Ebbs and Ko-chian, 1997).

3.1. EDTA-assisted Pb dissolution

EDTA has been utilized in micronutrient fertilizers since the1950 s (Wallace et al., 1992; Bucheli-Witschel and Egli, 2001). Ithas recently been used as a supplement to soil washing techniques(Lim et al., 2004). It was Wallace et al. (1974) who first reportedthat metal–EDTA complexes could increase solubility and phyto-availability of metals in soils. In the case of Pb ions, EDTA has beenfound to be the most effective chelator among the studied naturaland synthetic complexing compounds (Davis and Singh, 1995;Blaylock et al., 1997; Huang et al., 1997; Evangelou et al., 2006;Kirpichtchikova et al., 2006; Liu et al., 2006). When EDTA is appliedto soils, a large fraction of the total metals is dissolved and be-comes available for phytoextraction (Elliott and Brown, 1989;Haag-Kerwer et al., 1999) without inducing a strong acidificationof the growth medium (Zeng et al., 2005; Evangelou et al., 2006).Furthermore, as compared to other chemical compounds (such asinorganic acids), EDTA can solubilize metals with fewer undesir-able effects on the soil physico-chemical properties (Ghestemand Bermond, 1998; Steele and Pichtel, 1998). This offers greatprospects as a less invasive alternative for conventional ex situ soilwashing procedures.

In the soil environment, EDTA can change the speciation of met-als, thereby influencing metal bioavailability. The ability of EDTA toincrease concentration of metals in soil solution is influenced by anumber of factors, including: concentration of metals and EDTA;presence of competing cations; metal species and their distributionamong soil fractions; soil pH; adsorption of free and complexedmetals onto charged soil particles and the formation constant ofmetal–ligand complexes. Because of its high binding affinity formetals, when applied at high concentrations, EDTA has the poten-tial to affect the release of metals from solid phases by forming dis-solved complexes. The formation of metal–EDTA complexes in soilsolution may shift precipitation and sorption equilibria toward in-creased dissolution of metals (Dushenkov et al., 1997). Alterna-tively, EDTA may interact directly with solid phases bycomplexing metal ions present in, and/or adsorbed onto primaryor secondary minerals (Gonsior et al., 1997).

A recent study by Nascimento et al. (2006) investigated the re-lease of Pb in soil solution after application of chelating agentsEDTA, DTPA, oxalic acid, citric acid, vanillic acid and gallic acid.EDTA was one of the most efficient chelating agents and signifi-cantly increased the concentration of Pb in soil solution 24 h upto 7 d after application. Furthermore, these authors noted a 217-fold increase of Pb concentration in the soil solution in EDTA-trea-ted pots (10 mmol kg�1 soil) as compared to control pots following7 d of application. The ability of EDTA to enhance the release of Pb

1282 Saifullah et al. / Chemosphere 74 (2009) 1279–1291

from insoluble or sparingly soluble compounds as compared toother chelating agents has been attributed to the higher bindingcapacity of EDTA for Pb as observed in numerous studies (e.g., Blay-lock et al., 1997; Huang et al., 1997; Wu et al., 1999). In an earlierstudy, Shen et al. (2002) reported EDTA was the most efficient che-lating agent in the release of soil-bound Pb. These authors reporteda 42-fold increase of Pb concentration in soil solution of the EDTA-treated soil (1.5 mmol kg�1 soil) as compared to the control, 3 dfollowing application.

Several authors have reported a linear relationship betweenEDTA concentration and metal removal from soils (Papassiopiet al., 1999; Hong and Jiang, 2005). For instance, Kim et al.(2003) studied the effects of solution:soil ratio, major cations pres-ent in soils and the EDTA:Pb stoichiometeric ratio on the extractionof Pb using different Superfund site soils. Extraction of Pb from Pb-contaminated soils was not affected by solution:soil ratio but in-stead was dependent on the quantity of EDTA present. Applicationof EDTA in sufficiently large amount (EDTA:Pb stoichiometric ratiogreater than 10) resulted in extraction of most Pb. In contrast,Peters and Shem (1992) and Elliott and Brown (1989) reportedthat EDTA, at concentrations above the stoichiometricallyrequired quantity, was not effective in the removal of Pb from soilsolution.

EDTA is a non-selective extracting agent that can form a strongcomplex with a variety of metals in soils including alkaline-earthcations such as Al3+, Ca2+, Fe2+ and Mg2+ and target heavy metal(s)such as Pb, Cd, Ni, Zn, Mn (Skoog et al., 1996; Zeng et al., 2005). Thepresence of competing cations (Ca2+, Mg2+, Al3+, Fe2+, Cu2+, Cd2+,Zn2+) and/or the nature of the soil matrix can substantially alterthe rate of complex formation predicted solely on the basis ofthe ratio of EDTA to the target heavy metal(s) (Manouchehriet al., 2006). In addition, the quantity of target heavy metal(s) ex-tracted using EDTA may reduce by up to 50% in calcareous soilscompared to non-calcareous soils. Therefore, it has been suggestedthat before initiating heavy metal leaching using chelating agents,the reagent concentration required to efficiently extract the targetheavy metal and the extractable competing elements present inthe sample must be established (Manouchehri et al., 2006). Forexample, Papassiopi et al. (1999) conducted leaching experimentsusing EDTA and reported that most of the EDTA (90%) was utilizedby calcite in soils, with less than 10% of the available EDTA utilizedto remove Pb and Zn. Furthermore, Papassiopi et al. (1999) exam-ined higher molar concentrations of tetrasodium EDTA (Na4-EDTA)salt (owing to its higher solubility) as compared to disodium EDTA(Na2-EDTA). These authors reported that the efficiency of Na4-EDTA salt in the removal of contaminants was significantly andsubstantially lower than that of Na2-EDTA salt. The reduction incontaminant removal efficiency of Na4-EDTA was attributed tothe alkaline pH of the chelating agent solution, which decreasedPb dissolution. In soils that have a higher active CaCO3 content ascompared to the degree of heavy metal contamination, the effi-ciency of EDTA is reduced due to the displacement of metals fromtheir EDTA complexes and the subsequent formation of CaH2EDTAand insoluble metal carbonates (Walker et al., 2003; Hong andJiang, 2005; Manouchehri et al., 2006).

Soil pH is an important factor for Pb adsorption and desorptionin soils: a decrease in pH increases Pb desorption from soil constit-uents resulting in increased Pb concentration in soil solution (Saltet al., 1995; Kayser et al., 2000; Yang et al., 2006). Therefore, theefficiency of chelating agents on metal solubilization and accumu-lation can be enhanced by lowering the soil pH. A study by Cuiet al. (2004) showed that the combined application of EDTA andelemental sulfur significantly enhanced solubilization of heavymetals (Pb and Zn). Similarly, citric acid in conjunction with EDTAhas been shown to enhance the solubilization of Pb and signifi-cantly increase the rate of metal translocation from root to shoot

in Fetuca arundianacea Scherb grown on Pb-contaminated soil(Begonia et al., 2005).

Remediation of polymetallic and aged polluted soils is difficultand challenging. The effectiveness of chelating agents in the removalof heavy metals is often highly variable. Lai and Chen (2005) re-ported that EDTA application at 5 mmol EDTA kg�1 significantly in-creased Pb concentrations in the soil solution of soils contaminatedboth in single and multiple metal-contaminated soils. However,EDTA application at 2 mmol kg�1 had not anymore effect on Pb sol-ubilisation. These authors attributed this to the fine clayey texture ofthe soil in addition to the competition of Zn and Cd for EDTA.

Recently, there have been many attempts to assess the effect ofEDTA on heavy metal remobilization from soil fractions (Lo andYang, 1999; Barona et al., 2001; Shen et al., 2002; Walker et al.,2003; Liu et al., 2006). The addition of EDTA increases the concen-tration of Pb in soil solution, generally at the expense of exchange-able, organic matter and carbonate bound fractions (Elliott andShastri, 1999; Kos et al., 2003). For example, Kirkham (2000) eval-uated the effects of EDTA on the solubility of Pb associated withdifferent soil fractions by using sequential extraction procedureand suggested that the addition of EDTA mobilized Pb associatedwith exchangeable and carbonate soil fractions.

The ability of EDTA to solubilize heavy metals from differentsoil fractions is dependent on the strength of adsorption of the me-tal to a particular solid phase. Lo and Yang (1999) evaluated the ef-fect of contact time (up to 120 min) and EDTA concentration (0.01,0.05, 0.10 M) on the extraction of Pb, Cu and Zn from four differentfractions (carbonate, Fe/Mn oxides, organic matter, and clay min-eral) of laboratory prepared metal-contaminated soils. Theseauthors reported that in general, the extraction reached equilib-rium within 20 min and indicated that the removal of Cu and Znfrom all the four fractions was similar at 0.01 and 0.05 M EDTAconcentrations. However, extraction of Pb from the organic matterand carbonate fraction was dependent on the EDTA concentration.The maximum removal of Pb from organic and carbonate fractionswas achieved when 0.10 M EDTA was used. However, it was only50% of the total Pb observed in these fractions. Efficiency of EDTAfor extracting Pb from the carbonate fraction was much lower thanfor extracting Cu and Zn. The lower efficiency of EDTA for Pb re-moval from carbonate fractions compared to Cu and Zn was attrib-uted to precipitation of Pb in the form of PbCO3(s) due to a small Ksp

(7 � 10�14) value of Pb carbonate.

3.2. EDTA-assisted Pb uptake by plants

Morel et al. (1986) reported that the majority of Pb taken up byroots is bound to carboxyl groups of mucilage uronic acids. Accord-ing to Jarvis and Leung (2002), Pb retention in roots is based on thebinding of Pb to ion exchange sites on the root cell walls and extra-cellular precipitation, mainly in the form of Pb-carbonates. Onceabsorbed by roots, Pb is rather immobile, showing very limitedtranslocation into above-ground foliage (Malone et al., 1974; Zim-dahl and Koeppe, 1977; Reeves and Brooks, 1983; Rudakova et al.,1988; Kumar et al., 1995; Zheljazkov and Nielsen, 1996; Brennanand Shelley, 1999; Salt and Kramer, 2000; Kabata-Pendias, 2001;Wilde et al., 2005). Some authors propose that application of phy-tostabilization might be more suited than attempts to artificiallyincrease phytoavailability of Pb for phytoextraction. Phytostabili-zation implies the use of excluder plant species which are intendedto stabilize metals within the soil matrix by reducing water perco-lation through increased evapotranspiration of the plant cover, andalso by reducing metal mobility through the increase of soil CECand soil organic matter (Vervaeke et al., 2003). This strategy is via-ble for sites for which no other use than nature development areenvisaged as the stabilization requires the permanent presence ofthe vegetation.

Saifullah et al. / Chemosphere 74 (2009) 1279–1291 1283

The efficiency of removing heavy metals using plant-basedremediation strategies depends on the availability of target heavymetals in the soil solution, also referred to as the bioavailable frac-tion. The bioavailability of heavy metals within these pools can beenhanced upon application of mobilizing agents such as organicacids (Elkhatib et al., 2001; Seuntjens et al., 2004; Nascimentoet al., 2006), synthetic chelators (Liphadzi et al., 2003; Madridet al., 2003; Wenzel et al., 2003; Begonia et al., 2004; Meerset al., 2005b; Zhuang et al., 2005; Ruley et al., 2006; Tandy et al.,2006), acidifying agents (Kayser et al., 2000; Puschenreiter et al.,2001; Cui et al., 2004), and humic substances (Halim et al., 2003;Evangelou et al., 2004; Turan and Angin, 2004).

Metal uptake by plants involves a series of processes such asmetal desorption from soil particles, transport of soluble metalsto root surfaces via diffusion or mass flow; metal uptake by rootsand metal translocation from roots to shoot. Chelators enhancedesorption of heavy metals from the soil matrix to the soil solution(Means et al., 1978; Stanhope et al., 2000; Nowack, 2002; Nasci-mento et al., 2006), facilitate metal transport into the xylem(Huang and Cunningham 1996; Blaylock et al., 1997; Huanget al., 1997) and increase metal translocation from roots to shoots(Barber and Lee, 1974; Hamon et al., 1995; Vassil et al., 1998; Gleb-a et al., 1999).

Several studies have observed an increase in metal uptake asmetal concentrations increase at the root surface (Blaylock et al.,1997; Collins et al., 2002; Moral et al., 2002; Shen et al., 2002; Liand Chen, 2006). Furthermore, total concentration of metals in soilsolution is considered a poor indicator of bioavailability as only se-lected chemical species such as free metal ions, soil soluble metalcomplexes and chelated species are believed to be effectively takenup by plants (Kabata-Pendias, 1995; Lasat et al., 2000).

Chelators were shown to induce deficiency of certain essentialelements such as Fe in solution culture studies with very low con-centrations of both metals and chelator. It was assumed that onlyfree metal ions can be absorbed by roots. Recent advances in ana-lytical techniques (ICP-MS, TEM, Extended X-ray Absorption FineStructure), and a greater interest in chelant-assisted phytoextrac-tion from metal polluted environments, has resulted in a numberof studies that have challenged the assumption that the activityof free metal ions in soil solution is an accurate indicator for metalbioaccumulation in plants (Smolders and McLaughlin, 1996;McLaughlin et al., 1998a; Sarret et al., 2001; Collins et al., 2002;Bell et al., 2003; Degryse et al., 2006; Tandy et al., 2006). Manystudies have indicated enhanced uptake and translocation of heavymetals following the application of chelators. There remains con-siderable controversy as to whether the metal–EDTA complexesare as such absorbed by plant roots, or dissociation of the complexis to take place first (Laurie et al., 1991; Vassil et al., 1998; Wuet al., 1999; Collins et al., 2002; Molas and Baran, 2004; Wengeret al., 2005).

Sarret et al. (2001) demonstrated that the mechanism of metalaccumulation in Phaseolus vulgaris induced by EDTA differed be-tween metals. In case of Zn, there was no difference betweenplants grown in Zn–EDTA and ZnSO4 solutions. Furthermore, Znin both treatments was predominately precipitated as Zn phos-phate in the roots and leaves. In contrast, cerussite was the majorform of Pb in the absence of EDTA, whereas in the presence ofEDTA, part of the Pb present in the leaves was complexed as Pb–EDTA. It was concluded that metal–EDTA complexes in soil solu-tion could be totally (Zn) or partially (Pb) dissociated when ab-sorbed by P. vulgaris. Recently, Wenzel et al. (2003) reported thatfollowing the addition of EDTA to soils, it entered into the plantroots in free form, where it bound metals and enhanced theirmobility within the plant.

For successful phytoextraction, following the absorption of met-als by plant roots, metal–EDTA complex must enter the xylem ves-

sels for translocation to above-ground harvestable plant parts.Translocation of complexes across the root cortex into the xylemcan be either symplastic (cell to cell) or apoplastic (e.g., at tips ofroots where the Casparian bands are not developed or at breaksin the endodermis) and depends on plant species as well as typeand concentration of ligand (Tanton and Crowdy, 1971; Collinset al., 2002; Jarvis and Leung, 2002). For example, Jarvis and Leung(2002) applied EDTA and HEDTA [N-(2-hydroxyethyl) ethylenedi-nitrilotriacetic acid] to Pinus radiata D. Don plants in Pb-contami-nated soils, and observed that Pb uptake was significantlyenhanced by the presence of chelating agents. Moreover, the ultra-structural studies employing TEM revealed that Pb uptake path-ways differed between chelating agents. In the case of HEDTA-chelated Pb, complexes were found around the intercellular spacesand within cell walls, indicating an apoplastic pathway of move-ment. Conversely, EDTA-chelated Pb followed a symplastic path,as most of the Pb in this form was found within structural tissuesin the cytoplasm.

4. Factors affecting EDTA-assisted Pb phytoextraction

EDTA-assisted Pb accumulation by plants depends on many fac-tors including the physical and chemical nature of the Pb-chelate insolution (Wu et al., 1999); concentration of Pb, other metals andEDTA (Vassil et al., 1998; Deram et al., 2000; Kayser et al., 2000; Col-lins et al., 2002; Begonia et al., 2004; Li and Chen, 2006), plant spe-cies (Ebbs and Kochian, 1998; Collins et al., 2002; Jordan et al.,2002;Shen et al., 2002; Chen et al., 2004; Zhuang et al., 2005), plant-ing density (Liphadzi et al., 2003), soil exposure time to contami-nants, time and mode of EDTA application (Barocsi et al., 2003;Grcman et al., 2003; Wenzel et al., 2003; Wu et al., 2003b; Begoniaet al., 2004; Meers and Tack, 2004; Luo et al., 2006a), and combinedapplication of EDTA with other amendments (Puschenreiter et al.,2001; Cui et al., 2004; López et al., 2005).

Phytoextraction efficiency of a crop strongly depends on thetranspiration rate of the vegetation and can be significantly en-hanced by increasing transpiration rate (Grifferty and Barrington,2000). The EDTA-assisted phytoextraction performance of a lineof Brassica juncea L. was substantially higher than a wild type B.juncea L. This was attributed to 130% higher transpiration rate inthe B. juncea L. line (Gleba et al., 1999). There is evidence thatlong-distance transportation of metal–ligand complexes fromroots to shoots is driven by the transpiration stream (Salt et al.,1995; Blaylock et al., 1997; Vassil et al., 1998).

Plant accumulation of metals is directly related to soil porewater concentrations. This pool can successfully be increased bythe application of chelating agents. Studies have shown that Pbuptake increases with increasing both the concentration of avail-able Pb and application rates of EDTA (Lai and Chen, 2005; Tandyet al., 2006). Vassil et al. (1998) reported that a threshold concen-tration of EDTA is required to induce accumulation of high levelsof EDTA or Pb–EDTA in shoots. High concentrations of Pb sur-rounding plant roots may also play a significant role in increasingits accumulation by plants either by destabilizing the physiologi-cal barrier to solute movement in roots (Harrison et al., 1979) orby increasing the activity of Pb–EDTA that can be absorbed byplants.

Ruley et al. (2006) studied the effect of type and concentrationof chelators (EDTA, DTPA, HEDTA, NTA, Citric acid at 0–10 mmolkg�1 soil) on Pb accumulation in Sesbania drummondii (Rydb.) Coryin soils contaminated with a high concentration of Pb (7.5 g kg�1).The effect of chelators on shoot accumulation of Pb was found to bestrongly concentration dependent. The highest uptake of Pb wasfound with EDTA application at 10 mmol kg�1. EDTA was the mostefficient chelant. Low EDTA rates have been reported to facilitate

1284 Saifullah et al. / Chemosphere 74 (2009) 1279–1291

the breakdown of barriers to the uptake of metals by plants (Chenet al., 2004; Meers et al., 2005c). High levels of EDTA applicationare detrimental or even lethal to plants because of high concentra-tions of free EDTA, and a decreases in the availability of essentialnutrients (Geebelen et al., 2002; Wu et al., 2004).

Plant species vary in their response to take up heavy metalsduring EDTA-assisted phytoextraction. The efficiency of phytoex-traction could be enhanced by the right combination of plant spe-cies and chelators. Dicotyledonous species (mung bean, sunflower,cabbage, buckwheat, pea and mustard) are more efficient in Pbuptake during EDTA-assisted phytoextraction than monocotyle-donous species (maize, sorghum, wheat, barley) (Chen et al.,2004; Meers et al., 2005c). Even the planting density of a cropcan affect the uptake of heavy metals from contaminated soils.Liphadzi et al. (2003) evaluated the effect of planting density onmetal accumulation by growing sunflower at two densities,20000 and 60000 plants ha�1. The EDTA was applied at 0, 0.5,1.0 and 2.0 g EDTA kg�1 soil. It was observed that for sunflower,1.0 g kg�1 rate of chelate addition resulted in maximum removalof three non-essential heavy metals (Cd, Ni, and Pb). Further, max-imum mass removal of heavy metals was observed for a density of60000 plants ha�1 as opposed to 20000 plants ha�1 because ofgreater biomass production at high planting density.

Because of the high persistence of Pb/Pb–EDTA in the soil envi-ronment, the toxicity of free EDTA on plants and increased leachingrisk of heavy metals, application of EDTA in several multiple dosesinstead of single full dose has been advocated (Shen et al., 2002;Wenzel et al., 2003). Recently, Barocsi et al. (2003) proposed amethod of EDTA application to increase plant uptake of Pb. The ap-proach aimed to reduce toxic effects due to free EDTA and/or highuptake of heavy metals. Instead of single dose treatment, the sameamount of EDTA was divided into multiple doses, which providedtime for plants to initiate their adaptation mechanism and raisetheir damage threshold. All the plots were exposed to the same to-tal EDTA rate (4 mmol kg�1 soil) but applied in various splits. Thoseplants that received EDTA in a single dose showed severe toxicitysymptoms, whereas plants exposed to EDTA in two and three dosesshowed intermediate and very little toxicity, respectively. Regard-ing Pb uptake, compared with the control (8 mg kg�1), the final Pbincreased to 364, 624, and 771 mg kg�1 of plant for plants receivingEDTA in one, two and three doses, respectively. On the other hand,if a soil has a high Pb retention capacity due to high carbonate, clayor organic matter content, application of EDTA in multiple dosescould be ineffective in mobilizing and enhancing root to shoottranslocation. In these conditions, application of the full rate ofchelant in a single dose could constitute the more effective ap-proach (Grcman et al., 2001).

Pb accumulation by plants was increased by a combined appli-cation of EDTA and EDDS compared to either alone (Luo et al.,2006b). This was attributed to (1) EDTA-induced mobilization ofsoil-bound Pb, and (2) EDDS-enhanced translocation of water-sol-uble Pb from roots to shoots.

The uptake of metals in chemically assisted phytoextractioncould substantially be increased by increasing the concentrationof total dissolved metals as well as breaking down the root exclu-sion mechanism (Nowack et al., 2006; Xu et al., 2007). The lattermay be achieved by root pretreatment with hot water (Luo et al.,2006c), combined application of herbicides and chelating agents,addition of chelants to soils at high concentrations (Vassil et al.,1998) or transplantation instead of direct sowing of a crop into acontaminated soil (Wu et al., 1999). Recently Luo et al. (2006a) re-ported that efficiency of chelators (EDTA and EDDS) in enhancingphytoextraction of heavy metals could significantly be improvedby adding these chelants in hot solution (90 �C) to soil in whichgarland chrysanthemum (Chrysanthemum coronarium L.) and whitebeans (P. vulgaris L.) were grown. The application of hot chelant

solutions was much more efficient than the application of coldsolutions (25 �C) in improving the uptake of heavy metals byplants.

Translocation of Pb from roots to shoots could also be dramat-ically increased by combined application of EDTA and indole aceticacid (IAA) (Liu et al., 2007). The Pb translocation from roots toleaves of alfalfa exposed only to EDTA at equimolar concentrationwas increased by about 300%, whereas the Pb concentration inleaves of plants exposed to EDTA/IAA increased by about 600%more compared to the EDTA-only treatment (López et al., 2005).Similarly, a recent study by Di Gregorio et al. (2006) showed syn-ergistic effect of combined application of a nonionic surfactant Tri-ton X-100 and an autochthonous plant growth promotingrhizobacterium (PGPR), Sinorhizobium sp. Pb002 inoculum for theimprovement of Pb phytoextraction in EDTA amended soil. TritonX-100 was used in order to damage the phospholipid membrane,which helped to increase root permeability to Pb–EDTA. Both Tri-ton X-100 and EDTA reduced the plant biomass but introductionof PGPR in rhizosphere significantly increased biomass and therebyincreased the Pb phytoextraction by 56% compared to the non-inoculated treatment.

Soil pH plays an important role in the availability of trace ele-ments including Pb to plants. With the exception of metals andmetalloids that form oxyanions (Cr, Se, and As), the metal retentionin soils is generally low at low soil pH (McLaughlin et al., 1998b). Inalkaline and neutral soils, EDTA-induced solubility and bioavail-ability of Pb to plants can be improved by lowering the pH byapplication of citric or acetic acid (Blaylock et al., 1997; Begoniaet al., 2002, 2005), physiological acidic fertilizers (Puschenreiteret al., 2001), and elemental sulfur (Kayser et al., 2000; Cui et al.,2004).

5. Potential risks associated with EDTA-assisted Pbphytoextraction

5.1. Plant growth

The efficiency of phytoextraction depends on the metal concen-trations in shoots and high biomass production (McGrath et al.,2001). In spite of reported successes in increasing the bioavailablefraction of heavy metals using EDTA, researchers have expressedconcerns about EDTA-assisted phytoextraction due to excessivelevels of heavy metals in soil solution and dissolution of soil-boundmetals including Pb. Plants exposed to high levels of both free Pband free EDTA produce low biomass due to low seed germination,leaf wilt, chlorosis and necrosis, abscission, shoot desiccation andreduced transpiration (Vassil et al., 1998; Lombi et al., 2001; Röm-kens et al., 2002; Grcman et al., 2003; Duo et al., 2005; Nascimentoet al., 2006), P-induced Fe deficiency during EDTA-assisted phy-toextraction (Hovsepyan and Greipsson, 2005). Reduction inabove-ground net primary production would lead to a decreasein the total amount of metals removed by plants (Quiroz et al.,2002). However, during EDTA-assisted Pb phytoextraction, thereare several factors that influence plant growth, among which themost important are: EDTA/Pb molar ratio (Vassil et al., 1998), modeand time of EDTA application (Chen and Cutright, 2001; Grcmanet al., 2003; Wenzel et al., 2003), plant species as well as typeand concentration of other heavy metals (Lombi et al., 2001).

The ratio of EDTA to Pb is one of the major factors determiningthe overall benefit of EDTA application for Pb phytoextraction. Atequimolar concentrations of Pb and EDTA, formation of 100% Pb–EDTA complex in the nutrient solution has been observed (Sarretet al., 2001) that could result in alleviation of the toxicity associ-ated with free EDTA and free Pb (Xu et al., 2007). However, in manynatural soils, high rates of EDTA application may be required to

Table 2Percent of total initial lead leached from soil during EDTA-assisted phytoextraction.

Total content of Pb in soil(mg kg�1)

Pb leached (%)

WithoutEDTA

WithEDTA

107 8.19 69 Wu et al. (2004)340 ND 0.35 Madrid et al. (2003)660 ND 36 Kos and Leštan

(2003)4130 ND 38 Grcman et al.

(2001)3280 0.05 3.5 Chen et al. (2004)4130 ND 23 Grcman et al.

(2003)204 0.001 0.60 Meers et al. (2005c)

ND = Not detectable.

Saifullah et al. / Chemosphere 74 (2009) 1279–1291 1285

complex all the Pb ions due to the presence of other cations like Ca,Fe, Al that compete with Pb to form EDTA complexes (Manouchehriet al., 2006).

Plant performance during EDTA-assisted phytoextraction maybe influenced adversely by both the direct action of EDTA andthe increased bioavailability of heavy metals in soil. The presenceof free EDTA is toxic to plants because it can negatively affect thebalance of minerals, e.g., Zn, Cu, Fe and Ca, leading to disturbancesin cell metabolism and destabilization of biological membranes(Ruley et al., 2004, 2006). Excess of free Pb(II) in growth mediumcan severely reduce cell division and plant growth (Johnson andPetras, 1998). However, in the presence of EDTA the cytological im-pacts of free Pb ions are eliminated. Recently, Ruley et al. (2006)evaluated the effects of Pb and chelates on the growth and photo-synthetic activity on Sesbenia durmmondii in a soil contaminatedwith 7.5 g kg�1 Pb(NO3)2. Application of chelators (EDTA, HEDTA,DTPA, NTA, and citric acid) mitigated the adverse effects associatedwith free Pb. Plant shoot and root weights in the presence of Pb andchelators were significantly higher than the plants grown in thepresence of Pb or chelators alone.

Both the mode of EDTA application and plant age has a pro-nounced influence on subsequent plant growth. Application ofEDTA in a single dose after the plants have attained sufficient bio-mass, or before transplanting or germination, is an option; alterna-tively, the same dose of EDTA can be added in several incrementsduring the entire growth period. Application of EDTA before germi-nation or transplanting and immediately after germination can se-verely inhibit plant growth through free EDTA itself or indirectlydue to enhanced dissolution of metals (Chen and Cutright, 2001;Meers et al., 2004). Addition of EDTA when plants have attainedsufficient biomass, can minimize its adverse effects on plantgrowth. However, EDTA-enhanced bioavailability of heavy metalsincluding Pb may inhibit plant growth in the next round of cropsplanted in the treated soil (Lombi et al., 2001). Incremental appli-cation of EDTA instead of a single dose could aid plants initiatetheir adaptation mechanisms and raise their damage threshold.Barocsi et al. (2003) found that when an EDTA dose of 4 mmol kg�1

was applied in several increments over time plants showed littlephytotoxicity but when it was applied in a single dose severetoxicity symptoms were displayed by B. juncea L.

In addition to direct effects on plant tolerance, chemicallyenhancing metal accumulation in plant shoots may also entail addi-tional indirect risks by introducing metals into the foodchain viaherbivory on accumulating crops. This aspect of ecotoxicologicalassessment of phytoextraction has been insufficiently highlightedto this date and requires additional attention when moving forwardwith enhanced phytoextraction as a remediation technique.

5.2. Soil biota

Soil biota plays important roles in mineralization and decompo-sition of organic matter, nitrification and denitrification, mainte-nance of soil structure, detoxification of hazardous chemicals(Filip, 2002). Solubilization of metals will increase the availabilityof metals to these organisms (Welp and Brummer, 1997). Excessivelevels of metals and chelating agents are expected to influence thediversity and activity of soil organisms. EDTA, DTPA, or EDA ap-plied at concentrations up to 3 mM were shown to inhibit nitrifica-tion rates by disrupting and depleting the cellular stabilizing ions(Ca2+ and Mg2+) from nitrifying bacterial cells. The order of inhibi-tion on molar-basis was EDA� EDTA > DTPA (Hu et al., 2003).Application of EDTA at 10 mmol kg�1 inhibited the developmentof arbuscular mycorrhiza, depressed the growth of soil fungi, bac-teria and actinomycetes. However, soil fungi were more sensitiveto EDTA or EDTA-mediated increases in metal bioavailability thansoil bacteria and actinomycetes (Grcman et al., 2001).

5.3. Leaching of heavy metals

Some researchers have expressed concerns regarding the in-creased risk of groundwater pollution associated with EDTA-as-sisted phytoextraction (Wu et al., 1999; Barona et al., 2001;Grcman et al., 2001; McGrath et al., 2002; Meers et al., 2004). In-creased leaching of Pb during EDTA-assisted phytoextraction hasmainly been attributed to the solubility of the Pb–EDTA complexes.It is further favored by the low sorption of Pb–EDTA complexes onsoil particles (Benyahya and Garnier, 1999; Chen et al., 2003; Wuet al., 2003a) compared to free ions. Release of soil-bound Pb couldincrease with an increase in EDTA concentration. Therefore, highlevels of soluble Pb in soil solution resulting from application ofEDTA may lead to contamination of ground water (Table 2). Theadverse effects of EDTA are not only related to the enhanced solu-bility of the metals but also to their low biodegradability. EDTAtherefore remains in soil for extended periods of time after treat-ment (Thomas et al., 1998; Nörtemann, 1999; Bucheli-Witscheland Egli, 2001; Meers et al., 2005b).

The extent of metal leaching during EDTA-enhanced phytoex-traction is affected by a number of factors. These include soil con-stituents and properties such as organic matter, amount and typeof clay, presence of carbonates and pH (Sun et al., 2001; Thayala-kumaran et al., 2003; Volger and Thayalakumaran, 2005), concen-tration of EDTA applied (Grcman et al., 2003; Kos and Leštan, 2003;Wenzel et al., 2003; Wu et al., 2004), mode (split vs. full dose) andtime (before and after germination or just prior to harvesting) ofapplication (Grcman et al., 2001; Shen et al., 2002), plant species(Madrid et al., 2003; Chen et al., 2004), amount of water applied(Grcman et al., 2001), root zone processes (Clothier and Green,1997) and extent by which solubilized metals are taken up byplants.

Information on leaching of heavy metals during EDTA-assistedphytoextraction is limited. Most of the studies restrict reportingto the fractions of total metals solubilized and absorbed by plants.Schmidt (2003) showed that metal solubility in soils was morestrongly enhanced than accumulation by plants, with only 1% ofEDTA-solubilized Pb effectively taken up by plants and accumu-lated in harvestable shoots. This was confirmed by Meers et al.(2005c) who observed that in a comparison between four differentcrops (Helianthus annuus, Zea mays, Cannabis sativa and Brassicarapa), less than 1.1% of mobilized metals were effectively accumu-lated in the plant shoots. This indicates that the risk for metalleaching can be substantial.

Time and method of chelant application may affect the extent ofleaching of metal chelates. It has been observed that solubilizationof soil Pb occurred in a short time (6 h) after application of EDTA(Shen et al., 2002) and it may continue to be enhanced for severaldays or even months (Meers et al., 2005b). The application of EDTA

1286 Saifullah et al. / Chemosphere 74 (2009) 1279–1291

prior to germination or just after germination, when there is no orinsignificant root mass to absorb metals from soil solution, will sig-nificantly increase the risks of ground water contamination. Plantswith deep roots may access metals from lower levels, and reducethe risk of groundwater pollution. Plants exhibiting high transpira-tion rates can decrease the risks of groundwater pollution byextracting more water from soil, thereby reducing downwardmovement of metals. Madrid et al. (2003) reported enhancedleaching of Pb and Cd in the presence of plant roots but the leach-ing of essential elements (Fe and Mn) during chelate-irrigated phy-toextraction was significantly and substantially lower than non-essential Pb. The authors attributed the enhanced leaching ofnon-essential elements (Pb, Cd) compared to essential (Fe, Mn) ele-ments to the differential absorption of essential and non-essentialelements by plants.

There can be considerable variation between different soil typesregarding leaching of heavy metals during EDTA-assisted phytoex-traction (Meers et al., 2005c). Soil texture, pH calcium carbonatecontent and CEC are important factors. Sun et al. (2001) evaluatedleaching of heavy metals (Cd, Cu, Pb, Zn) after addition of 14 gEDTA kg�1 to soils differing in calcium carbonate contents. Sevendays after EDTA application, Pb recovered in leachates was 3–30% of the total Pb in soil. The amounts of metals recovered inleachates reduced as the calcium carbonate content increased,either due to the competition of Ca2+ to form Ca–EDTA complexes(Brown and Elliott, 1992) or precipitation of Pb in the form ofPbCO3(s) (cerussite) (Morel, 1983; Lo and Yang, 1999).

The balance between release of soil-bound metals and metalabsorption by plants largely determines the amount of metalsleaching downwards in the soil during EDTA-assisted phytoextrac-tion. In a series of laboratory experiments, Wu et al. (2004) evalu-ated the effects of different EDTA dosages (0, 3, 6, 12 mmol kg�1)on leaching of different heavy metals (Cd, Cu, Pb, and Zn). Theleaching of these heavy metals increased with increasing concen-tration of EDTA. Soil analyses showed a 4–78% loss in total Pb afterapplication of EDTA at the different rates. A maximum loss of Pbfrom the soil was observed at a dose of 12 mmol kg�1 EDTA. Wen-zel et al. (2003) in a lysimeter study assessed the effects of dosage(up to 2.01 g kg�1) and mode (single vs. split) of EDTA applicationon leaching of heavy metals (Cu, Pb, and Zn) during and after theharvest of Brassica napus L. The metal concentrations in the leach-ates were related to the amount of EDTA applied, but the authorsfound no difference between applications of the same amount ofEDTA in single or split doses. They reported very low uptake of met-als compared to solubilization of metals after application of EDTA.For Pb, soil solution concentration increased about 1500-foldrelative to the control at the highest rate of EDTA application(2.01 g kg�1), while corresponding shoot Pb concentration in-creased by a factor of about 18.

Chen et al. (2004) performed phytoextraction experimentsusing short soil leaching columns (9 cm dia, 20 cm long) percolatedwith synthetic rainwater. They studied the effect of EDTA applica-tion (5.0 mmol kg�1 soil) on the leaching behavior of heavy metals(Cd, Cu, Pb, and Zn) and reported about 3.5%, 15.8%, 13.7%, and20.6% leaching of soil Pb, Cu, Zn, and Cd, respectively. Leachatescollected at the bottom of columns with sunflower (H. annuus)but without EDTA contained about 0.05% of the total initial content(3280 mg) of Pb. With EDTA, more than 3.5% of the initial Pb con-tent was leached from planted and unplanted columns. It wasfound that 177 times more Pb was leached than was absorbed bythe crop.

Optimization of plant efficiency to extract Pb by increasing bothbiomass and Pb concentration in plant tissues can minimize therisk of leaching from soil during EDTA-assisted phytoextraction.EDTA has the ability to disrupt cell membrane permeability. Itcould therefore increase metal translocation from roots to shoots,

but it will also decrease plant biomass production when appliedat too high concentration (Chen and Cutright, 2001). It has been re-ported that certain surfactants, when applied at high rates, candamage phospholipid membranes (Cserhati, 1995) This could in-crease their permeability to solutes. Some studies indicate thatcombined application of EDTA and surfactants may significantlyenhance phytoextraction of Pb (Elless and Blaylock, 2000; Di Greg-orio et al., 2006).

6. Reducing potential risks associated with leadphytoextraction

Several plant species can (hyper)accumulate metals such as Zn,Cu, Ni, and Cd without the addition of organic or inorganic agents.However, because of the low solubility and translocation of Pbfrom roots to shoots, phytoextraction of this element is very inef-fective without the application of metal mobilizing agents. Suitableselection of chelant as well as time, rate and method of application,combined with an appropriate selection of plants and soil manage-ment strategies are pre-requisites to achieve the desired benefitsand to reduce the negative side-effects of this technology. Persis-tent mobilizing agents such as EDTA may work well under labora-tory conditions, yet compound recalcitrance impairs field scaleapplication. Therefore, research must continue towards moredegradable alternatives (Meers et al., 2004, 2005b). Chelant appli-cation is advised shortly prior to harvest, when the standing crophas achieved maximum vegetative growth. Environmental risksof leaching need to be substantially reduced by selecting the opti-mal application rate (Epstein et al., 1999). It is imperative to choosethe right combination of chelating agent and plant species. Thespecies should be tolerant to elevated levels of Pb maintained insoil solution by the EDTA and should possess deep root systemsto minimize the risk of leaching.

The downward leaching of heavy metals in EDTA-assistedmethod of phytoextraction could effectively be reduced by grow-ing vetiver grass [Chrysopogon zizanioides (L.) Roberty]. This grasshas a long, massive and complex root system (Chen et al., 2004).Gradual application of small doses of EDTA during the growth per-iod can considerably reduce the phytotoxicity and environmentalproblems associated with its use (Barocsi et al., 2003; Wenzelet al., 2003). Shen et al. (2002) tested methods of EDTA application(1.5 mmol kg�1). Total EDTA was added to soil in one-, two- andthree-split applications. Application of EDTA in three splits re-sulted in the highest shoot Pb concentration in Brassica oleracea(cabbage), followed by the two and single-split procedures. Atthe conclusion of the experiment, soil analysis revealed the lowestconcentration of soluble Pb (80 mg Pb kg�1 soil) when EDTA wasapplied in three splits compared to 140 mg kg�1 in pots receivinga single split. The ratio between the amount of Pb taken up byplants (mg kg�1) and the amount of Pb solubilized by EDTA(mg kg�1) was 45 when EDTA was applied in three splits and only15 when EDTA was applied in single split. This calculation indi-cates that the ratio of Pb taken up by plants to that mobilized fromthe soil was lowest when EDTA was applied in a single dose. There-fore, application of EDTA in several increments rather than a singlesplit could reduce the risk of leaching.

Both cost and risk of leaching heavy metals can be also reducedby minimizing the amount of EDTA applied. Recently, Luo et al.(2006c) studied the effect of combined application of EDTA andEDDS on the balance of dissolved metals (Cu, Pb, Zn, and Cd) andmetal uptake by Z. mays L. EDTA and EDDS were applied to pots2 wk after germination of Z. mays L. in different modes: singleapplication of EDTA or EDDS alone (5 mmol kg�1 soil), and com-bined applications of EDTA and EDDS at ratios of 1:1, 1:2, and2:1. When EDTA and EDDS were applied at a ratio of 2:1, concen-

Saifullah et al. / Chemosphere 74 (2009) 1279–1291 1287

tration of soluble Pb in soil increased by a factor of only 1.2 com-pared to the treatment with EDTA alone. However, the concentra-tion in shoots increased by a factor 2.4. Thus a combinedapplication of EDTA and EDDS increased absorption of soluble Pb.

A sudden increase in concentration of metals by EDTA may en-hance the risk of leaching if absorption of metals does not coincidewith their release (Semer and Reddy, 1996; Barona et al., 2001;Moutsatsou et al., 2006). Li et al. (2005) developed a new, slow-re-lease chelating additive by coating solid EDTA granules with a layerof silicate which slows down the metal mobilization in soil so that itcan be matched by plant uptake. In a short-term pot experiment,these authors compared the effects of these slow-release EDTAgranules with uncoated EDTA granules, and EDTA solution. TheEDTA was applied at 13 mmol kg�1 in all cases and thoroughlymixed with soil. A control without EDTA was also included. Analysisof the leachates collected at the bottom of columns with and with-out EDTA revealed that EDTA was more slowly released from thecoated chelating agent than from the uncoated solid EDTA. The con-centration of Pb in leachates at the bottom of columns treated withcoated EDTA expressed as a percentage was lower than that fromcolumns treated with both solid uncoated EDTA and EDTA solution.The concentration of EDTA in all three EDTA treatments was similaryet the coated EDTA resulted in less leaching of Pb from the soil.This slow-release of EDTA from coated agents could effectively min-imize the risk of Pb leaching by (timing) its uptake by plants.

It has been reported that application of certain surface-activeagents like caprylic acid (C8H16O2), Triton X-100 (C14H22O(-C2H4O)n) or lauryl sulfate ([CH3(CH2)10CH2OSO3]�) may enhancemetal bioavailability (Maier et al., 2001; Mulligan et al., 2001),damage phospholipid membranes, and thus increase their perme-ability to solutes (Cserhati, 1995). Therefore, combined applicationof EDTA and certain surfactants may significantly enhance Pb phy-toextraction (Elless and Blaylock, 2000; Di Gregorio et al., 2006).This may allow to reduce the rate of EDTA application and thusminimize the leaching risk during EDTA-assisted phytoextraction.

7. Plant species facilitating and maximizing leadphytoextraction

There is considerable variation among plant species in their effi-ciency to take up and accumulate pollutants (Singh et al., 2003).Proper selection of the species for phytoremediation is an impor-tant step in the development of effective remediation methods,especially for low- or medium-polluted soil (Salt et al., 1995). Sev-eral plant species have been tested for application in EDTA-assistedPb phytoextraction (Table 3). Factors that should be consideredwhen selecting a particular plant species are (1) nature, amountand type of targeted metals and other contaminants present, (2)the soil and climate characteristics and (3) options available forprocessing and disposal of the metal-rich biomass. It has been sug-gested that a plant suitable for phytoextraction (1) should grow fastboth in single and multi-metal-contaminated soils, (2) can conve-niently be harvested by conventional agricultural methods, (3) isdeep-rooted with a well-branched root system, (4) is highly toler-ant to heavy metals including Pb as well as EDTA/Pb–EDTA com-plexes without significant inhibition to growth or reductions inbiomass, (5) can easily be propagated and can be grown continuallyor repeatedly (Robinson et al., 2000; Römkens et al., 2002; Kelleret al., 2003; Robinson et al., 2003; Ernst, 2005). To date, there areno plants available that possess all these characteristics. In general,the choice primarily depends on the removal potential, which isdetermined by shoot dry matter and Pb concentration in the shoots.

Phytoextraction without the application of any chemical agentto enhance availability of metals (natural phytoextraction) mustrely on natural hyperaccumulators. One of the main advantages

of hyperaccumulators is their high tolerance to metals. However,phytoextraction with the use of high biomass crops is relativelymore successful on soils with low and moderate concentration ofheavy metals (Berndes et al., 2004; Sebastiani et al., 2004; Ernst,2005).

In general monocotyledonous crops are more tolerant to metalsincluding Pb than dicotyledonous crop plants (Marschner, 1995).They also have a shallow, but highly branched root system in theupper soil layer, which makes them successful phytoextractors insoils with a shallow distribution of contaminants (Keller et al.,2003). However, during enhanced lixiviation and downward move-ment of Pb with application of EDTA, crops with shallow root sys-tem may fail to limit the leaching of Pb or Pb–EDTA complexes. Insuch cases, plants with a long, massive and complex root systemsuch as vetivar grass [C. zizanioides (L.) Roberty], which have theability to extract water and chemicals from lower soil depths (Chenet al., 2004), could be a viable option. Dicotyledonous species, de-spite being generally more sensitive than monocotyledonous spe-cies to growth depression, accumulated more metals (Cu, Pb, andZn) in their shoots after application of EDTA (Luo et al., 2006b). Thisgreater phytoextraction capacity is attributed to the heavier dam-age to physiological barriers by EDTA in dicotyledonous speciesthan monocotyledonous species.

Lead contamination tends to be mainly concentrated in theupper soil horizons. Therefore, annual plants having roots in theupper parts of the soil profile seem to be more suitable than peren-nial plants. However, in chelate-assisted phytoextraction the en-hanced mobility of metals down the soil profile increases the riskof contamination of deeper soil horizons and potentially aquifers.Under such circumstances, perennials might be an appropriate op-tion due to their ability to tap into deeper soil horizons (Deramet al., 2000).

8. Conclusions

Phytoextraction seems to have considerable potential fordecontamination of soils contaminated with heavy metals. How-ever, uptake of heavy metals by plants is limited by their low sol-ubility in soil solution especially in the case of Pb. Application ofEDTA has been proposed to increase Pb concentration in soil solu-tion through enhancing dissolution of sparingly soluble soil miner-als. Such enhanced concentration of Pb in soil solution occursmostly at the expense of exchangeable, organic matter and carbon-ate bound fractions. Effectiveness of EDTA to bring more Pb intosoil solution depends on its rate, contamination level of Pb as wellas complementary metals present in soils and method of its appli-cation. It has been proposed that the addition of EDTA onto soilsenhanced translocation Pb from roots to shoots by lowering theirbinding with cell walls. However, EDTA applied at larger ratescould result in contamination of ground water due to enhancedsolubilization and leaching of Pb and other metals as well as me-tal–EDTA complexes. Reduction of percolation risks by the use ofmore degradable alternatives to EDTA has been proposed over re-cent years. In fact, we have arrived at a cross-roads at which thescientific community is distancing itself from the concept of usingpersistent compounds such as EDTA in the context of field applica-tion to enhance phytoremediation and is turning its attention to-wards more degradable alternatives. After over a decade ofdedicated EDTA research, there is a need to clearly state thatalthough being an interesting bench mark model for enhancedphytoextraction research, EDTA itself as a compound has probablya low applicability in practical field application in the context ofphytoextraction due to unacceptable percolation risks associatedwith its environmental persistence. Discovery of and applied re-search towards degradable alternatives such as EDDS could help

Table 3Effect of EDTA on Pb concentration and its total uptake in harvestable parts of various crops.

Crop EDTA added(g kg�1 soil)

Concentration % increase overcontrol

Accumulationa % increase overcontrol

Toxicityb

Pb concentration and total uptake in plant shootsCabbage 3.72 9704 Biomass NA NA Kos and Leštan (2003)Turfgrass 3.72 3070 3465 No Duo et al. (2005)Canola 1.65 200 210 No Wenzel et al. (2003)Cabbage 1.11 3876 2977 No Shen et al. (2002)Indian mustard 2.9 1497 Biomass NA Yes Blaylock et al. (1997)Corn 1.86 2600 1904 Yes Luo et al. (2006b)Indian mustard 1.86 9502 2738 Yes Chen et al. (2004)Wheat 1.11 3212 2505 Yes Shen et al. (2002)Hemidesmus indicus 2.0 32 Biomass NA No Chandra Sekhar et al.

(2005)Brachiaria

decumbens0.39 88 23 No Santos et al. (2006)

Sunflower 0.37 360 259 Yes Lesage et al. (2005)Sunflower 1.11 2600 442 Yes Lesage et al. (2005)Corn 0.74 340 400–1000 Yes Meers et al. (2004)Sunflower 1.11 1457 2300 No Meers et al. (2005c)Hemp 1.11 750 750 No Meers et al. (2005c)Corn 1.11 450 396 No Meers et al. (2005c)Rapeseed 1.11 220 200 No Meers et al. (2005c)

NA = not available/not given.a Accumulation = concentration � shoot biomass.b Status of toxic effects during experiment.

1288 Saifullah et al. / Chemosphere 74 (2009) 1279–1291

to move enhanced phytoextraction, based on past EDTA based re-search, to the level of practical field scale application. Although agreat number of studies have been conducted during recent yearsto make assisted Pb phytoextraction an effective and low-risk tech-nology, it demands extensive investigations before drawingauthentic and generalized conclusions of commercial significance.Ultimately, the scientific community needs to determine whetherenhanced phytoextraction for Pb in general has a future as soilremediation technology based on observed removal efficiencyand the remaining associated risks as described in literature.Regardless of the debate whether EDTA itself should be abandonedas a potential amendment for phytoremediation due to the adverseeffects associated with its use, valuable lessons still remain to belearned from the vast historic research invested in the study of thiscompound throughout the world, as the scientific communitymakes the shift towards more degradable alternatives and safermeans and methods of incorporating the principle of intentionalincreasing metal phytoavailability for phytoextraction purposes.

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