This article was downloaded by: [RMIT University]On: 25 August 2014, At: 08:12Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

International Journal ofPhytoremediationPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/bijp20

Comparison of EDTA- and Citric Acid-Enhanced Phytoextraction of HeavyMetals in Artificially Metal ContaminatedSoil by Typha AngustifoliaDawood Muhammad a , Fei Chen a , Jing Zhao a , Guoping Zhang a &Feibo Wu aa Institute of Crop Science, College of Agriculture and Biotechnology,Huajiachi Campus , Zhejiang University , Hangzhou , PR ChinaPublished online: 01 Apr 2009.

To cite this article: Dawood Muhammad , Fei Chen , Jing Zhao , Guoping Zhang & Feibo Wu (2009)Comparison of EDTA- and Citric Acid-Enhanced Phytoextraction of Heavy Metals in Artificially MetalContaminated Soil by Typha Angustifolia , International Journal of Phytoremediation, 11:6, 558-574,DOI: 10.1080/15226510902717580

To link to this article: http://dx.doi.org/10.1080/15226510902717580

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the“Content”) contained in the publications on our platform. However, Taylor & Francis,our agents, and our licensors make no representations or warranties whatsoever as tothe accuracy, completeness, or suitability for any purpose of the Content. Any opinionsand views expressed in this publication are the opinions and views of the authors,and are not the views of or endorsed by Taylor & Francis. The accuracy of the Contentshould not be relied upon and should be independently verified with primary sourcesof information. Taylor and Francis shall not be liable for any losses, actions, claims,proceedings, demands, costs, expenses, damages, and other liabilities whatsoever orhowsoever caused arising directly or indirectly in connection with, in relation to or arisingout of the use of the Content.

This article may be used for research, teaching, and private study purposes. Anysubstantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &

Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

Dow

nloa

ded

by [

RM

IT U

nive

rsity

] at

08:

12 2

5 A

ugus

t 201

4

International Journal of Phytoremediation, 11:558–574, 2009Copyright C© Taylor & Francis Group, LLCISSN: 1522-6514 print / 1549-7879 onlineDOI: 10.1080/15226510902717580

COMPARISON OF EDTA- AND CITRIC ACID-ENHANCEDPHYTOEXTRACTION OF HEAVY METALS IN ARTIFICIALLYMETAL CONTAMINATED SOIL BY TYPHA ANGUSTIFOLIA

Dawood Muhammad, Fei Chen, Jing Zhao, Guoping Zhang,and Feibo WuInstitute of Crop Science, College of Agriculture and Biotechnology, HuajiachiCampus, Zhejiang University, Hangzhou, PR China

A pot experiment was conducted to study the performance of EDTA and citric acid (CA)addition in improving phytoextraction of Cd, Cu, Pb, and Cr from artificially contaminatedsoil by T. angustifolia. T. angustifolia showed the remarkable resistance to heavy metaltoxicity with no visual toxic symptom including chlorosis and necrosis when exposed tometal stress. EDTA-addition significantly reduced plant height and biomass, compared withthe control, and stunted plant growth, while 2.5 and 5 mM CA addition induced significantincreases in root dry weight. EDTA, and 5 and 10 mM CA significantly increased shootCd, Pb, and Cr concentrations compared with the control, with EDTA being more effective.At final harvest, the highest shoot Cd, Cr, and Pb concentrations were recorded in thetreatment of 5 mM EDTA addition, while maximal root Pb concentration was found at the2.5 mM CA treatment. However, shoot Cd accumulation in the 10 mM CA treatment was36.9% higher than that in 2.5 mM EDTA, and similar with that in 10 mM EDTA. ShootPb accumulation was lower in 10 mM CA than that in EDTA treatments. Further, root Cd,Cu, and Pb accumulation of CA treatments and shoot Cr accumulation in 5 or 10 mM CAtreatments were markedly higher than that of control and EDTA treatments. The resultsalso showed that EDTA dramatically increased the dissolution of Cu, Cr, Pb, and Cd in soil,while CA addition had less effect on water-soluble Cu, Cr, and Cd, and no effect on Pblevels. It is suggested that CA can be a good chelator candidate for T. angustifolia used forenvironmentally safe phytoextraction of Cd and Cr in soils.

KEY WORDS CA (citric acid), EDTA, heavy metal, phytoextraction, Typha angustifolia

INTRODUCTION

Heavy metal contamination of soil, mainly originating from former or current miningactivities, agronomic practices, industrial emissions, and the application of sewage sludge,poses great threats to sustainable agriculture and human health (Shen et al., 2002; Wu,Zhang, and Dominy, 2003; Wu et al., 2005; Pilon-Smits, 2005; Chen et al., 2007; Zenget al., 2008). The remediation of metal contaminated soils is imperative because metalswill persist almost indefinitely in the environment due to its stability (Alkorta et al., 2004a

Address correspondence to F.B. Wu, Institute of Crop Science, College of Agriculture and Biotechnology,Huajiachi Campus, Zhejiang University, Hangzhou 310029, PR China. E-mail: wufeibo@zju.edu.cn

558

Dow

nloa

ded

by [

RM

IT U

nive

rsity

] at

08:

12 2

5 A

ugus

t 201

4

HEAVY METAL SOIL PHYTOEXTRACTION 559

and 2004b; Bachir et al., 2004; Wu et al., 2004b; Dong et al., 2007a). Phytoextraction, aphytotechnology developed to extract heavy metals from contaminated soils using metalhyperaccumulators has received increasing attention due to its cost-effective, non-intrusive,and aesthetically pleasing nature (Alkorta et al., 2004a; Bachir et al., 2004; Dong et al.,2007b). However, available hyperaccumulators are generally not efficient in extractingheavy metals because of low biomass production (McGrath et al., 2001; Alkorta et al.,2004b). Meanwhile, under many circumstances, low bioavailability of heavy metal in soilprevents efficiency of phytoextraction (Alkorta et al., 2004b; Pilon-Smits, 2005). Thus,chelate-induced phytoextraction was developed with the objective of facilitating plantheavy metal absorption and translocation (Lombi et al., 2001; Shen et al., 2002), making itpossible to use high biomass-producing species with low uptake ability of toxic elementsas an alternative to hyperaccumulators (Luo, Shen, and Li, 2005). Synthetic chelators andlow molecular weight organic acids (LMWOA), capable of modifying the bioavailabilityof heavy metals in soils, are the most common chemical amendments that have been usedin chemically assisted phytoextraction of metals from soils (Evangelou et al., 2007).

Ethylenediaminetetraacetic acid (EDTA) is probably one of the most efficientsynthetic chelators improving shoot accumulation of heavy metals (Cunningham andOw, 1996; Blaylock et al., 1997; Huang et al., 1997; Vassil et al., 1998). However, itsslow degradation rate and long persistence in soil increase leaching risk, which may leadto groundwater contamination, and make it unsuitable for practical use (Lombi et al.,2001; Wu, Luo et al., 2004; Meers et al., 2005). Alternatively, LMWOA, being easilybiodegradable as the natural products of root exudates, microbial secretions, and plantand animal residue decomposition in soils, may find greater social acceptance (Dinget al., 2005). Huang et al. (1998)) found that citric acid (CA) significantly increasedmetal availability and enhanced uranium accumulation in the shoots of selected plants. doNascimento et al. (2006)) also reported that gallic and citric acids enhanced removal of Cd,Zn, Cu, and Ni by Indian mustard (Brassica juncea) from multi-metal contaminated soils.Some authors, however, have found lower effectiveness of aliphatic LMWOA such as citricand oxalic acids in inducing metal accumulation in plants compared to synthetic chelators(Lombi et al., 2001; Kos and Lestan, 2004; Wu, Luo et al., 2004). Some studies have alsoshown reduced bioavailability of heavy metals in contaminated soils treated with organicacids (Khan et al., 2000; Zhou et al., 2001; Liao and Xie, 2004). This inconsistency couldbe attributed to the difference of plant species used in the experiments, and partially reflectsdifferences in adsorption and desorption behavior of heavy metals from soils with diversemetal concentration and soil properties (Puls et al., 1991). More detailed information istherefore required about LMWOA- enhanced phytoextraction of heavy metals in metalcontaminated soil.

Typha angustifolia L. (narrow-leaved cattail), a perennial macrophyte, is charac-terized by its fast growth, high productivity and remarkable resistance to high levels ofheavy metals in the soil (Demirezen et al., 2004; Panich-Pat et al., 2004; Dong et al.,2007b). However, no study has been performed on this plant species to show whether theLMWOA such as citric acid (CA) could efficiently improve its phytoextraction of heavymetals in metal contaminated soil, despite the fact that this plant is a reasonable candidatefor induced phytoextraction of metal polluted soils (Lan, Chen, and Li 1992; Wen, Xiu,and Mao, 1999a; Wen, Wen, and Wu, 1999b). The objectives of the present study wereto examine the potential use of T. angustifolia in EDTA and CA chemically enhancedphytoextraction of heavy metals (Cd, Cu, Pb, and Cr) from artificially contaminated soils;and to compare the efficacy of EDTA and CA on metal accumulation by T. angustifolia.

Dow

nloa

ded

by [

RM

IT U

nive

rsity

] at

08:

12 2

5 A

ugus

t 201

4

560 D. MUHAMMAD ET AL.

The solubilization processes of heavy metals in the soil and as affected by the applicationof EDTA and CA was also assessed in this study.

MATERIALS AND METHODS

Soil Collection and Artificial Heavy Metal Contamination

Agricultural soil was collected from the experimental farm (depth 0–15 cm) onHuajiachi Campus of Zhejiang University, Hangzhou, China. The soil was air-dried underroom temperature and mixed daily to 8% water content. Air-dried soil was ground,sieved through 2-mm mesh and some basic physicochemical properties were measuredwith routine analytical methods [Agrochemistry Commission and Soil Science Societyof China (ACSSSC, 1983); Table 1]. Soil particle size distribution was estimated by thehydrometer method after pre-treating soil with H2O2 and dispersing overnight in sodiumhexametaphosphate. Cation exchange capacity (CEC) was determined using the ammoniumacetate saturation method. Soil organic C and total N were determined by dichromateoxidation and Kjeldahl digestion–distillation, respectively (Nelson and Sommers, 1982;Bremner and Mulvaney, 1982). Soil available P was extracted by shaking 2.5 g of air-driedsoil for 30 min with 50 ml of 0.5 M NaHCO3 (pH 8.5). Available K was extracted with 1 MNH4OAc and determined using atomic absorption spectrometry (ACSSSC, 1983). EDTA(ethylenediaminetetra-acetic acid) extractable heavy metal concentrations were determinedby the atomic adsorption spectrophotometry (AAS, SHIMADZU AA-6300; Wu and Zhang,2002). Soil pH was conducted at a 1:1 (w/v) soil:distilled water ratio using a pH meter.

Four kg air-dried soil was weighed and loaded into a plastic pot (5 L, 20 cm high).The soil was then artificially contaminated by adding solution containing heavy metals, andmixed thoroughly: Pb (20 mg kg−1 soil) as Pb (NO3)2, Cd (10 mg kg−1 soil) as CdCl2, Cu(50mg kg−1 soil) as CuSO4, and Cr (10 mg kg−1 soil) as K2Cr2O7. After addition of metalsolutions, the soil was allowed to equilibrate for 30 d in a greenhouse. The equilibrationinvolved undergoing seven cycles of saturation with deionized water, air drying and blended,before being vegetated.

Plant Materials and Chelator Treatments

The pot experiment was carried out in a greenhouse under natural light conditionduring May to September, 2006. Seeds of T. angustifolia were germinated and the seedlingswere grown in trays of the substratum of 1/2 compost, 1/4 vermiculite and 1/4 sand (v/v/v),and irrigated every day with tap water. After 9 weeks, uniform, healthy seedlings withsimilar biomass were selected and transplanted into the above mentioned metal-amendedpots. There were 9 seedlings in each pot. Seedlings were allowed to grow for 20 d in thepots before EDTA and CA application. During this period, soils in the pots were kept humid(80–100% water holding capacity).

At the 20th day after transplanting, 200 ml of 0 (control), 2.5, 5, and 10 mM EDTAor CA were added to each pot at the rate of 50 ml chelator solution per kg soil, and thesoil was kept humid throughout the growth period (80–100% field water-holding capacity).All reagents were analytical grade and all stock solutions were made with deionized water.The experiment was laid in a randomized block design with 4 replicates. Each replicateconsisted of 9 plants, and 2 plants from each pot were marked for final harvest.

Dow

nloa

ded

by [

RM

IT U

nive

rsity

] at

08:

12 2

5 A

ugus

t 201

4

Tabl

e1

Bas

icph

ysic

oche

mic

alpr

oper

ties

ofth

eso

ilus

edfo

rth

est

udy

Soil

text

ure

(silt

loam

)A

vail.

heav

ym

etal

conc

entr

atio

n(m

gkg

−1)

Sand

(%)

Silt

(%)

Cla

y(%

)pH

(H2O

)kg

−1)

Org

anic

C(g

kg−1

)To

talN

(gkg

−1)

Ava

il.P

(mg

kg−1

)A

vail.

K(m

gkg

−1)

Cd

Cu

PbC

r

65.0

±0.

928

.8±

1.2

6.2

±0.

66.

8±

0.1

12.1

±0.

315

.8±

1.3

2.4

±0.

138

.2±

1.3

31.5

±2.

60.

15±

0.02

3.53

±0.

019.

63±

0.94

1.67

±0.

28

561

Dow

nloa

ded

by [

RM

IT U

nive

rsity

] at

08:

12 2

5 A

ugus

t 201

4

562 D. MUHAMMAD ET AL.

Sampling and Measurements

At 1, 5, 10, and 15 d after chelator application, 2 shoots from each pot were cut0.5 cm above the surface of soil, washed thoroughly with deionized water, and oven driedas the sample for determination of metal uptake. At final harvest (at 25 d after chelatorapplication), previously-tagged plants (2 plants from each pot) were gently removed fromsoil, separated into shoots and roots (including undeveloped rhizomes), washed with tapwater and then rinsed in distilled water. Plant tissues were then oven dried at 70◦C for 48h to a constant weight. The dry weight was determined and the samples were powderedwith a mortar and pestle. Powdered samples were then ashed at 550◦C for 12 h. The ashwas digested with 5 ml of 30% HNO3, and diluted using deionized water (Cheng et al.,2004), and then metal concentration was measured with an atomic absorption spectrometry(SHIMADZU AA-6300; Fang, 1991).

Soil Metal Dissolution and As Affected by Chelators

To evaluate the relative efficiency of EDTA and CA in enhancing metal solubilization,a soil metal dissolution experiment was carried out as described by Luo et al. (2005). Eight gof above-mentioned contaminated soil were weighted into a 50-ml polypropylene centrifugetube, and then 4 ml deionized water or 10 mM EDTA/CA was added to each tube. Therewere 27 replicates (total 81 tubes). After 0, 0.5, 1, 2, 4, 6, 8, 10, and 15 d, deionizedwater was added to the soil (at a soil-to-water ratio of 1:5) and the suspension was shakenfor 30 min. After centrifugation, the supernatant was filtered through 0.45 µm filter paper[Whatman, (Maidstone, UK) 42], and analyzed for different metal concentrations by atomicabsorption spectrometry (Shimadzu, AA 6300), as mentioned above.

Statistical Analysis

All data were processed by SAS (version 9.0). Values reported here are means of 4and 3 replicates, respectively, for the pot and soil metal dissolution experiments. Data weretested at a significance level of P < 0.05 using Duncan is Multiple Range Test.

RESULTS AND ANALYSES

Effect of Chelators on Plant Biomass of T. angustifolia

At final harvest, the plants were at the vegetative growth stage without inflorescenceand rhizome development, so only shoots, and roots were examined for experimentalparameters. There were no visual symptoms of metal toxicity such as chlorosis or necrosison T. angustifolia shoots 25 d after chelator application. However, a significant reductionin plant height (8%, 15%, and 24%) and biomass production (22%, 40%, and 46%) wereobserved in the 2.5, 5, 10 mM EDTA treatments, as compared with that of the control (Table2). In contrast, 2.5 and 5 mM CA addition increased root dry weight by 27% and 24% overthe control, respectively; and had no significant influence on shoot dry weight.

Effect of Chelators on Tissue Metal Concentration of T. angustifolia

Cd concentration. The dose- and time-responses for shoot Cd concentration in T.angustifolia are summarized in Figure 1A. Compared with the control, addition of EDTA

Dow

nloa

ded

by [

RM

IT U

nive

rsity

] at

08:

12 2

5 A

ugus

t 201

4

HEAVY METAL SOIL PHYTOEXTRACTION 563

Table 2 Plant height and dry weight of the T. angustifolia plants 25 d after chelator addition

Dry weight (g plant−1)

Treatments[1] Plant height (cm) Shoot Root Total

Control 138 a[2] 2.42a 0.33b 2.75aEDTA 2.5 mM 128 bc 1.90b 0.27c 2.16bEDTA 5 mM 118 c 1.42c 0.25cd 1.67cEDTA 10 mM 106 d 1.28c 0.22d 1.49cCA 2.5 mM 137 ab 2.39a 0.42a 2.81aCA 5 mM 127 c 2.42a 0.41a 2.83aCA 10 mM 120 c 2.28a 0.37b 2.65a

[1]Treatments of control, EDTA 2.5, EDTA 5, and EDTA 10 correspond to 0, 2.5, 5, and10 mM EDTA, respectively, and CA 2.5, CA 5, and CA 10 correspond to 2.5, 5, and 10 mMCA

[2]Means for the same measurement in a particular column followed by the same letterare not significantly different according to Duncan’s multiple range test at p = 0.05.

and CA significantly increased shoot Cd concentration, with EDTA being more obviousthan CA. However, on day 10, CA addition did not change shoot Cd concentration relativeto the control, and on day 1, 2.5 mM EDTA/CA induced a slight but not significant increase(Figure 1A). EDTA performed better when applied at the concentration of 5 mM, ascompared with the other two treatments. At 10 d with 5 mM EDTA application, shoot Cdconcentration reached 25.0 mg kg−1 DW, being 5-folds higher than that of the control, andafter that trended to decrease (day 15). On day 1 and 5 after 5 and 10 mM CA application,shoot Cd concentration significantly increased, showing the maximum value of 11.5 mgkg−1 DW on day 5 after 10 mM CA application.

Figure 2A presents shoot and root Cd concentrations at the final harvest on 25 dafter chelator application. In shoots, the highest Cd concentration (21.0 mg kg−1 DW) wasrecorded in the 5 mM EDTA treatment, with 10 mM EDTA treatment having a similarconcentration, but 10-fold higher than that of the control. CA addition also significantlyenhanced shoot Cd concentration, although less effective than EDTA. The maximum rootCd concentration was found in 10 mM EDTA treatment, followed by 5 mM EDTA, and 5and 10 mM CA treatments, but all treatments were significantly higher than the control.

Cu concentration. The application of 2.5 and 5 mM EDTA significantly increasedshoot Cu concentration of T. angustifolia (Figure 1B). The highest Cu concentration wasfound in 5 mM EDTA at 5 d after application, being 2.5-fold significantly higher than thecontrol. However, CA application was not effective in moving Cu into shoots and there wasno significant difference between CA treatments and the control at 5, 10, and 15 d afterapplication, while lower on the first day.

At 25 d after chelator application, maximum Cu concentration in shoots (39.2 mgkg−1 DW) and in roots (181.1 mg kg−1 DW) was found in the 10 mM EDTA treatment,being 2- and 1.56- fold higher than the control, respectively. Whereas, CA amendmentsshowed no significant effect on Cu concentration in roots and shoots (Fig 2B). Furthermore,the Cu concentration of the control was lower at harvest than at other day (1–15 d; Figures1 and 2); this may due to the dilution effect induced by plant growth and decrease in Cuuptake during day 15–25.

Dow

nloa

ded

by [

RM

IT U

nive

rsity

] at

08:

12 2

5 A

ugus

t 201

4

564 D. MUHAMMAD ET AL.

Pb concentration. EDTA was much more effective in improving shoot Pbconcentration than CA. At 1 d after chelator application, EDTA induced a slight increase inshoot Pb concentration, but CA addition had no effect. The highest shoot Pb concentrationwas detected in the plants treated with 5 mM EDTA, which were higher than those

Figure 1 Effect of EDTA and CA addition on metal concentration (A: Cd; B: Cu; C: Pb; D: Cr) in shoots of T.angustifolia seedlings at different exposure times. Error bars represent ± SE. Means within the same samplingdate followed by the same letter are not statistically significant at P<0.05. Treatments of control, EDTA 2.5,EDTA 5, and EDTA 10 correspond to 0, 2.5, 5, 10, and mM EDTA, respectively, and CA 2.5, CA 5, and CA 10correspond to 2.5, 5, and 10 mM CA. (Continued)

Dow

nloa

ded

by [

RM

IT U

nive

rsity

] at

08:

12 2

5 A

ugus

t 201

4

HEAVY METAL SOIL PHYTOEXTRACTION 565

Figure 1 Continued

treated with both 2.5 and 10 mM EDTA. At 10 d after 5 mM EDTA application, shootPb concentrations reached 60.3 mg kg−1 DW, being 6-fold higher than the control. Theaddition of 5, and 10 mM CA caused an increase in shoot Pb concentration at 5 and 15 d,while no statistical difference in the other two sampling dates compared with the control(Figure 1C).

At 25 d after chelator application, EDTA and 5, and 10 mM CA significantly enhancedshoot Pb concentration (Figure 2C), with EDTA being more effective than CA. The highestshoot concentration was recorded in 5 mM EDTA (cf. 8-fold over the control), whilemaximal root Pb concentration was found in the 2.5 mM CA treatment.

Cr concentration. As shown in Figure 1D, application of EDTA and 5, and 10mM CA had a significant effect in increasing shoot Cr concentration in T. angustifolia,EDTA showed a greater increase than CA addition. Shoot Cr concentration increasedconsistently up to 10 d EDTA application, with the highest concentration in 2.5 mM EDTAtreatment. Above that level Cr concentration tended to decrease. The effect of CA on shootCr concentration was less prominent, nevertheless showed an increase in comparison to thecontrol (Figure 1D).

At final harvest, Cr concentrations in the shoots of T. angustfolia under all chelatortreatments, except for 2.5 mM CA, were significantly (p < 0.05) higher than the control(Figure 2D). Maximum shoot Cr concentration (17 mg kg−1) was noted in 10 mM EDTA,being 3-fold higher than that of the control. Meanwhile, EDTA and CA addition increasedCr concentrations in roots compared with the control.

Effect of Chelators on Metal Accumulation by Plants

At final harvest (25 d after chelator application), Cd and Pb accumulation in shootsof T. angustifolia grown in EDTA and CA amended soil were significantly higher than thatof the control. Cadmium accumulation of 10 mM CA treatment was 36.9% significantlyhigher than that of 2.5 mM EDTA, and similar with 10 mM EDTA, but 20.9% lower thanthat of 5 mM EDTA. Furthermore, root Cd/Pb accumulation of CA treatments was markedlyhigher than that of the control and EDTA treatments (Figure 3A and C). However, shoot

Dow

nloa

ded

by [

RM

IT U

nive

rsity

] at

08:

12 2

5 A

ugus

t 201

4

566 D. MUHAMMAD ET AL.

Figure 2 Effect of EDTA and CA on shoot (left) and root (right) metal concentrations (A: Cd; B: Cu; C: Pb;D: Cr) at final harvest (25 d after chelators application). Error bars represent ± SE. Treatments of control, EDTA2.5, EDTA 5, EDTA 10, CA 2.5, CA 5, and CA 10 are the same as Figure 1.

Dow

nloa

ded

by [

RM

IT U

nive

rsity

] at

08:

12 2

5 A

ugus

t 201

4

HEAVY METAL SOIL PHYTOEXTRACTION 567

Pb accumulation in CA treatments was significantly lower than EDTA treatments (Figure3C).

For shoot Cr accumulation, 5 and 10 mM CA was more efficient, being 86.2%, 18.3%,24.9%, 26.5%, and 111.9%, 34.6%, 42.2%, 44.0% higher, respectively, than control, 2.5,5 and 10 mM EDTA treatments (Figure 3D). Addition of CA also increased root Craccumulation compared with the control and 5, and 10 mM EDTA treatments.

For shoot Cu accumulation, there was no statistical difference among all chelatortreatments and the control. Whereas, root Cu accumulation in CA was statistically higherrelative to the control and EDTA treatments (Figure 3B).

Effects of Chelator Addition on Metal Solubility in Soil

The concentrations of water-soluble metals in soil were examined to assess the relativeefficiency of EDTA and CA in improving the solubilization of metals from soil. Dramaticeffects were detected in the presence of EDTA on the dissolution of Cu, Cr, Pb and Cdrelative to CA (Figure 4). At 2 d after EDTA application, highly significant increase inthe concentrations of water-soluble Cd, Cu, Pb and Cr in soil was noted, which were 118,18, 94 and 4-fold higher than those in the control soil (treated with H2O). After 2 d, theconcentration of water-soluble metals, except for Cr, remained relatively constant near theend of the study (up to day 15). However, water-soluble Cr still increased up to day 6, andthen remained almost constant (Figure 4D).

As shown in Figure 4B, a similar trend was observed for water-soluble Cu with CAaddition, i.e. Cu concentration in soil increased sharply over the first 0.5–2 d CA, andafter that Cu level tended to be constant, although markedly lower than that in the EDTAtreatment. Addition of CA also slightly increased water-soluble Cd and Cr concentrationscompared with the control, with the maximal levels at day 6 and 1, respectively (Figure4A, D), while still dramatically less effective than EDTA. In addition, CA had almost noeffect on water-soluble Pb (Figure 4C).

DISCUSSION

Soil heavy metal contamination is a major environmental hazard to terrestrial plants,and is increasingly aggravated due to anthropogenic activities (Wu et al., 2004a, 2005;Lim, Chui, and Goh, 2005; Meers et al., 2005). Improvement of the capacity of plants toaccumulate metals by the application of chelating agents to the soil has proved to open upnew possibilities for phytoremediation (Blaylock et al., 1997; Huang et al., 1997). It is aprerequisite that plants be tolerant to metal toxicity in addition to showing rapid growthwith a reasonably high biomass in the field (Garbisu et al. 2002; Liphadzi et al., 2003). It istherefore crucially important to identify metal resisting/tolerant, high biomass plant speciesand feasible chelators for effectual remediation of contaminated soils. In our previous study,we showed that T. angustifolia is resistance to Cr stress (Dong et al., 2007bb). In the presentstudy, no visual symptoms of metal toxicity, chlorosis, or necrosis on the shoots of this plantgrown in artificially multi-metals polluted soil were observed, which added evidence forits high tolerance to heavy metal toxicity. This plant also possesses a number of desirablecharacteristics as a phytoremediator, e.g., it is a rapid growth perennial macrophyte andcould harvest for 2 or 3 times each year, with a profuse tuberous root system and reasonablyhigh biomass in the field especially after its second growth season (Panich-Pat et al., 2004).

Dow

nloa

ded

by [

RM

IT U

nive

rsity

] at

08:

12 2

5 A

ugus

t 201

4

568 D. MUHAMMAD ET AL.

Figure 3 Effect of EDTA and CA on total shoot (left) and root (right) metal content per plant (A: Cd; B: Cu;C: Pb; D: Cr) at final harvest (25 d after chelator application). Error bars represent ± SE. Treatments of control,EDTA 2.5, EDTA 5, EDTA 10, CA 2.5, CA 5, and CA 10 are the same as Figure 1.

Dow

nloa

ded

by [

RM

IT U

nive

rsity

] at

08:

12 2

5 A

ugus

t 201

4

HEAVY METAL SOIL PHYTOEXTRACTION 569

Figure 4 Water-soluble Cd (A), Cu (B), Pb (C), and Cr (D) concentrations in soil with time and as affected by10 mM EDTA and 10 mM CA. Error bars represent ± SE.

Dow

nloa

ded

by [

RM

IT U

nive

rsity

] at

08:

12 2

5 A

ugus

t 201

4

570 D. MUHAMMAD ET AL.

Enhancing the accumulation of trace pollutants in harvestable plant tissues is aprerequisite for the chelator to be practically applicable. It has been reported that syntheticchelators such as EDTA could assist plants in extracting high percentages of heavy metalsfrom contaminated soils (Wu, Luo et al., 2004; Chen and Cutright, 2001; Luo et al., 2005;Meers et al., 2005; Lim et al., 2005; Komarek et al., 2007). Results of the present studyshow that EDTA was more efficient in enhancing shoot and root metal concentrationsas compared with CA. At 25 d, shoot Cu and Cr concentration increased with EDTAlevel, while for Cd and Pb concentration, the highest level recorded in 5 mM EDTAtreatment and being 10 and 8 -fold higher, respectively, over the control. Root Cd andPb concentrations in 5 mM EDTA were 2.5 and 1.7-fold higher relative to the control. Inaddition, the transfer factor (TF, the ratio of metal concentration in shoots to that in roots)can also be used to evaluate the capacity of a plant to translocate metals from roots toshoots. In the present study, increased metal transfer factor was found in EDTA amendedtreatments, and for Cd in CA amended treatments (Table 3). Therefore, it may be assumedthat EDTA is beneficial to improve metal translocation to the shoots, and CA addition wouldfacilitate Cd translocation. CA addition also significantly enhanced shoot/root Cd, Pb, andCr concentrations, but was less effective compared with EDTA (Figures 1 and 2), whereas,CA amendments proved to have no significant effect on Cu concentration in roots and shoots(Figures 1B and 2B). The effectiveness of EDTA and CA -enhanced metal uptake in plantswas consistent with the ability of the two chelators to solubilize metals in soil. As shownin Figure 4, EDTA was dramatically more effective than CA on increasing water-solubleCd, Cu, Pb, and Cr, which were consistent with the earlier studies (Chaney et al., 1997;Gramss, 2004; do Nascimento et al., 2006; Evangelou et al., 2006). This also indicated thatEDTA addition could increase leaching risk and may lead to groundwater contamination,although it is efficient in improving shoot heavy metal accumulation (Vassil et al., 1998;Lombi et al., 2001; Wu, Luo et al., 2004; Meers et al., 2005). We also investigated theeffect of chelator (EDTA and CA) addition on plant biomass of T. angustifolia and its metalaccumulation. Results showed that EDTA-addition significantly reduced plant growth, e.g.markedly reduced plant height and biomass production as shown in Table 1. This agreedwith the results reported by Chaney et al. (1997)) and Blaylock et al. (1997)) that EDTAposed adverse effects on plant biomass. Reduction in plant growth after EDTA addition isprobably due to the toxicity of EDTA itself and EDTA-metal complexes (Chen and Cutright,2001; do Nascimento et al., 2006). However, more studies under laboratory-controlledconditions are required. It is also interesting to note that 2.5 and 5 mM CA addition inducedsignificant increases in root dry weight over the control, and had no significant effect onshoot dry weight in the 3 CA treatments was found, despite of the reduction in plantheight. Therefore, total Cd accumulation in shoots of 5 and 10 mM CA treatments washigher than 2.5 mM EDTA and similar with the 10 mM EDTA treatments (Figure 3A), Craccumulation was also simultaneously higher than EDTA treatments (Figure 3D), althoughshoot Cd/Cr concentrations were lower than that of EDTA treatments (Figure 2). The lowerCd accumulation in 10 mM EDTA treatment was due to the adverse effects of EDTA onplant biomass. In addition, CA has the advantage of being readily biodegradable and nottoxic to fish, daphnia and soil fungi, and poses little risk from the leaching of metals ongroundwater (Strobel, 2001; Krishnamurti et al., 1997; Romkens et al., 2002). The resultssuggest that CA might be a chelator candidate for T. angustifolia used in phytoextraction ofCd and Cr in soils. Further research, however, is needed to determine the optimum methodsof effective utilization.

Dow

nloa

ded

by [

RM

IT U

nive

rsity

] at

08:

12 2

5 A

ugus

t 201

4

HEAVY METAL SOIL PHYTOEXTRACTION 571

Table 3 The metal transfer factor (TF) of T. angustifolia 25 d after chelator application

Treatment[1] Cd Cu Pb Cr

Control 0.028 d[2] 0.167c 0.062d 0.113cdEDTA 2.5 0.069 b 0.276a 0.220c 0.090dEDTA 5 0.104 a 0.249ab 0.253b 0.168abcEDTA 10 0.081 b 0.213bc 0.291a 0.222aCA 2.5 0.028 d 0.168c 0.055d 0.084dCA 5 0.045 c 0.152c 0.067d 0.135bcdCA 10 0.049 c 0.174c 0.071d 0.186ab

TF is defined as the ratio of metal concentration in plant shoot to that in plant root.[1]Treatments of control, EDTA 2.5, EDTA 5, and EDTA 10 correspond to 0, 2.5, 5, and 10 mM EDTA,

respectively, and CA 2.5, CA 5, and CA 10 correspond to 2.5, 5, and 10 mM CA.[2]Means for the same measurement in a particular column followed by the same letter are not significantly

different according to Duncan’s multiple range test at p = 0.05.

ACKNOWLEDGMENTS

This project was funded by the National Natural Science Foundation of China(30671256). We appreciate Miss Fang Wang, Miss Jing Dong, and Mr. Yue Cai, AgronomyDepartment of Zhejiang University, for their helpful assistance during the experimentalwork.

REFERENCES

Agrochemistry Commission and Soil Science Society of China (ACSSSC). 1983. Routine Methodsfor Soil and Agrochemical Analysis, Beijing, China, Science Press.

Alkorta, I., Hernandez-Allica, J., Becerril, J.M., Amezaga, I., Albizu, I., and Garbisu, C. 2004a.Recent findings on the phytoremediation of soils contaminated with environmentally toxicheavy metals and metalloids such as zinc, cadmium, lead, and arsenic. Rev. Environ. Sci.Biotechnol. 3, 71–90.

Alkorta, I., Hernandez-Allica, J., Becerril, J.M., Amezaga, I., Albizu, I., Onaindia, M., and Garbisu, C.2004b. Chelate-enhanced phytoremediation of soils polluted with heavy metals. Rev. Environ.Sci. Biotechnol. 3, 55–70.

Bachir, D.M., Wu, F.B., Zhang, G.P., and Wu, H.X. 2004a. Genotypic difference in effect of cadmiumon development and mineral concentrations of cotton. Commun. Soil Sci. Plan. Anal. 35,285–299.

Blaylock, M.J., Salt, D.E., Dushenkov, S., Zakharova, O., Gussman, C., Kapulnik, Y., Ensley, B.D.,and Raskin, I. 1997. Enhanced accumulation of Pb in Indian mustard by soil-applied chelatingagents. Environ. Sci. Technol. 31, 860–865.

Bremner, J.M. and Mulvaney, C.S. 1982. Total nitrogen. In: Methods of Soil Analysis, pp. 595–662.(Page, T., Miller, R.H., and Keeney, D.R., Eds.) Madison, WI, Soil Science Society of America.

Chaney, R.L., Malik, M., Li, Y.M., Brown, S.L., Brewer, E.P., Angle, J.S., and Baker, A.J. 1997.Phytoremediation of soil metals. Curr. Opin. Biotechnol. 8, 279–284.

Chen, F., Wu, F.B., Dong, J., Vincze, E., Zhang, G., Wang, F., Huang, Y., and Wei, K. 2007. Cadmiumtranslocation and accumulation in developing barley grains. Planta 227, 223–232.

Chen, H. and Cutright, T. 2001. EDTA and HEDTA effects on Cd, Cr, and Ni uptake by Helianthusannuus. Chemosphere 45, 21–28.

Dow

nloa

ded

by [

RM

IT U

nive

rsity

] at

08:

12 2

5 A

ugus

t 201

4

572 D. MUHAMMAD ET AL.

Cheng, W.D., Zhang, G.P., Yao, H.G., Dominy, P., Wu, W., and Wang, R.Y. 2004. Possibility ofpredicting heavy-metal contents in rice grains based on DTPA-extracted levels in soil. Commun.Soil Sci. Plant Anal. 35, 2731–2745.

Cunningham, S.D. and Ow, D.W. 1996. Promises and prospects of phytoremediation. Plant Physiol.110, 715–719.

Demirezen, D. and Aksoy, A. 2004. Accumulation of heavy metals in Typha angustifolia (L.) andPotamogeton pectinatus (L.) living in Sultan Marsh (Kayseri, Turkey). Chemosphere 56,685–696.

Ding, Y.Z., Li, Z.A., and Zou, B. 2005. Low-molecular weight organic acids and their ecologicalroles in soil. Soils. 37, 243–250 (in Chinese).

do Nascimento, C.W., Amarasiriwardena, D., and Xing, B. 2006. Comparison of natural organic acidsand synthetic chelates at enhancing phytoextraction of metals from a multi-metal contaminatedsoil. Environ. Pollut. 140, 114–123.

Dong, J., Mao, W.H., Zhang, G.P., Wu, F.B., and Cai Y. 2007a. Root excretion and plant tolerance tocadmium toxicity—a review. Plant Soil Environ. 53(5), 193–200.

Dong, J., Wu, F.B., Huang, R.G., and Zang, G.P. 2007b. A chromium-tolerant plant growing inCr-contaminated land. Int. J. Phytoremediat. 9, 167–179.

Evangelou, M.W.H., Ebel, M., and Schaeffer, A. 2006. Evaluation of the effect of small organic acidson phytoextraction of Cu and Pb from soil with tobacco Nicotiana tabacum. Chemosphere 63,996–1004.

Evangelou, M.W.H., Ebel, M., and Schaeffer, A. 2007. Chelate assisted phytoextraction of heavymetals from soil. Effect, mechanism, toxicity, and fate of chelating agents. Chemosphere 68,989–1003.

Fang, R. 1991. Application of Atomic Absorption Spectroscopy in Sanitary Test, pp. 148–158, Beijing,Beijing University Press.

Garbisu, C., Hernandez-Allica, J., Barrutia, O., Alkorta, I., and Becerril, J.M. 2002. Phytoremediation:a technology using green plants to remove contaminants from polluted areas. Rev. Environ.Health 17, 173–188.

Gramss, G., Voigt, K.D., and Bergmann, H. 2004. Plant availability and leaching of (heavy) metalsfrom ammonium-, calcium-, carbohydrate-, and citric acid-treated uranium-mine-dump soil.J. Plant Nutr. Soil Sci. 167, 417–427.

Huang, J.W., Blaylock, M.J., Kapulnik, Y., and Ensley, B. 1998 Phytoremediation of uranium-contaminated soils: Role of organic acids in triggering uranium hyperaccumulation in plants.Environ. Sci. Technol. 32, 2004–2008.

Huang, J.W., Chen, J.J., Berti, W.R., and Cunningham, S.D. 1997. Phytoremediation of lead-contaminated soils: role of synthetic chelates in lead phytoextraction. Environ. Sci. Technol.31, 800–805.

Khan, A.G., Kuek, C., Chaudhry, T.M., Khoo, C.S., and Hayes, W.J. 2000. Role of plants, mycorrhizaeand phytochelators in heavy metal contaminated land remediation. Chemosphere 41, 197–207.

Komarek, M., Tlustos, P., Szakova, J., Chrastny, V., and Ettler, V. 2007. The use of maize and poplar inchelant-enhanced phytoextraction of lead from contaminated agricultural soils. Chemosphere67, 640–651.

Kos, B. and Lestan, D.,2004. Chelator induced phytoextraction and in situ soil washing of Cu. Environ.Pollut. 132, 333–339.

Krishnamurti, G.S.R., Cieslinski, G., Huang, P.M., and van Pees, K.C.J. 1997. Kinetics of cadmiumrelease from soils as influenced by organic acids: implication in cadmium availability. J.Environ. Qual. 26, 271–277.

Lan, C.Y., Chen, G.Z., and Li, L.C. 1992. Use of cattails in treating wastewater from a Pb/Zn mine.Environ. Manag. 16, 75–80 (in Chinese).

Liao, M., Lan, C.Y., Chen, G.Z., Li, L.C., and Xie, X.M. 2004. Cadmium release in contaminatedsoils due to organic acids. Pedosphere 14, 223–228.

Dow

nloa

ded

by [

RM

IT U

nive

rsity

] at

08:

12 2

5 A

ugus

t 201

4

HEAVY METAL SOIL PHYTOEXTRACTION 573

Lim, T.T., Chui, P.C., and Goh, K.H. 2005. Process evaluation for optimization of EDTA useand recovery for heavy metal removal from a contaminated soil. Chemosphere 58, 1031–1040.

Liphadzi, M.S., Kirkham, M.B., Mankin, K.R., and Paulsen, G.M. 2003. EDTA-assisted heavy-metaluptake by poplar and sunflower grown at a long-term sewage-sludge farm. Plant Soil 257,171–182.

Lombi, E., Zhao, F.J., Dunham, S.J., and McGrath, S.P. 2001. Phytoremediation of heavy metal-contaminated soils: natural hyperaccumulation versus chemically enhanced phytoextraction.J. Environ. Qual. 30, 1919–1926.

Luo, C., Shen, Z., and Li, X. 2005. Enhanced phytoextraction of Cu, Pb, Zn and Cd with EDTA andEDDS. Chemosphere 59, 1–11.

McGrath, S.P., Zhao, F.J., and Lombi, E. 2001. Plant and rhizosphere processes involved inphytoremediation of metal-contaminated soils. Plant Soil 232, 207–214.

Meers, E., Ruttens, A., Hopgood, M.J., Samson, D., and Tack, F.M. 2005. Comparison of EDTAand EDDS as potential soil amendments for enhanced phytoextraction of heavy metals.Chemosphere 58, 1011–1022.

Nelson, D.W. and Sommers, L.E. 1982. Total carbon, organic carbon and organic matter. In: Methodsof Soil Analysis, pp. 539–577. (Page, A.L., Miller, R.H., and Keeney, D.R., Eds.)., Madison,WI, Soil Science Society of America.

Panich-Pat, T., Pokethitiyook, P., Kruatrachue, M., Upatham, E.S., Srinives, P., and Lanza, G.R. 2004.Removal of lead from contaminated soils by Typha angustifolia. Water Air Soil Pollut. 155,159–171.

Pilon-Smits, E. 2005. Phytoremediation. Annu. Rev. Plant Biol. 56, 15–39.Puls, R.W., Powell, R.M., Clark, D., and Eldred, C. J. 1991. Effects of pH, solid/solution ratio, ionic

strength, and organic acids on Pb and Cd sorption on kaolinite. Water Air Soil Pollut. 57–58,423–430.

Romkens, P., Bouwman, L., Japenga, J., and Draaisma, C. 2002. Potentials and drawbacks of chelate-enhanced phytoremediation of soils. Environ. Pollut. 116, 109–121.

Shen, Z.G., Li, X.D., Wang, C.C., Chen, H.M., and Chua, H. 2002. Lead phytoextraction fromcontaminated soil with high-biomass plant species. J. Environ. Qual. 31, 1893–1900.

Strobel, B.W. 2001. Influence of vegetation on low-molecular weight carboxylic acids in soil solution-a review. Geoderma 99, 169–198.

Vassil, A.D., Kapulnik, Y., Raskin, I.I., and Salt, D.E. 1998. The role of EDTA in lead transport andaccumulation by indian mustard. Plant Physiol. 117, 447–453.

Wen, Z.L., Wen, Y.M., and Wu, X.F. 1999b. The application of cattail plant in sewage disposal.Environ. Devel. 14, 28–30.

Wen, Z.L., Xiu, H.N., and Mao Y.F. 1999a. The utilization and exploitation of cattail plant inenvironmental protection. Environ. Protec. 10, 39–40, 42.

Wu, F.B., Chen, F., Wei, K., and Zhang, G.P. 2004a. Effect of cadmium on free amino acid, glutathioneand ascorbic acid concentrations in two barley genotypes (Hordeum vulgare L.) differing incadmium tolerance. Chemosphere 57, 447–454.

Wu, F.B., Dong, J., Qian, Q.Q., and Zhang, G.P. 2005. Subcellular distribution and chemicalform of Cd and Cd-Zn interaction in different barley genotypes. Chemosphere 60, 1437–1446.

Wu, F.B., Wu, H.X., Zhang, G.P., and Bachir, D.M.L. 2004b. Difference in growth and yield inresponse to cadmium toxicity in cotton genotypes. J. Plant Nutri. Soil Sci. -Zeitschrift FurPflanzenernahrung Und Bodenkunde 167, 85–90.

Wu, F.B. and Zhang, G.P. 2002. Genotypic variation in kernel heavy metal concentrations in barleyand as affected by soil factors. J. Plant Nutr. 25, 1163–1173.

Wu, F.B., Zhang, G.P., and Dominy P. 2003. Four barley genotypes respond differently to cadmium:lipid peroxidation and activities of antioxidant capacity. Environ. Exp. Bot. 50, 67–78.

Dow

nloa

ded

by [

RM

IT U

nive

rsity

] at

08:

12 2

5 A

ugus

t 201

4

574 D. MUHAMMAD ET AL.

Wu, L.H., Luo, Y.M., Xing, X.R., and Christie, P. 2004. EDTA-enhanced phytoremediation of heavymetal contaminated soil with Indian mustard and associated potential leaching risk. Agr.Ecosyst. Environ. 102, 307–318.

Zeng, F.R., Chen, S., Miao, Y., Wu, F.B., and Zhang, G.P. 2008. Changes of organic acid exudationand rhizosphere pH in rice plants under chromium stress. Environ. Pollut. 155, 284–289.

Zhou, D.M., Wang, S.Q., and Chen, H.M. 2001. Interaction of Cd and citric acid, EDTA in red soil.J. Environ. Sci. 13, 153–156.

Dow

nloa

ded

by [

RM

IT U

nive

rsity

] at

08:

12 2

5 A

ugus

t 201

4