This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution

and sharing with colleagues.

Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party

websites are prohibited.

In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information

regarding Elsevier’s archiving and manuscript policies areencouraged to visit:

http://www.elsevier.com/copyright

Author's personal copy

Phytoextraction of Pb and Cd from a contaminated agricultural soil usingdifferent EDTA application regimes: Laboratory

versus field scale measures of efficiency

Reinhard W. Neugschwandtner ⁎, Pavel Tlustoš, Michael Komárek, Jiřina Száková

Department of Agrochemistry and Plant Nutrition, Czech University of Agriculture in Prague, Kamýcká 129, 165 21 Prague 6 — Suchdol, Czech Republic

Received 15 February 2007; received in revised form 21 October 2007; accepted 28 November 2007Available online 15 February 2008

Abstract

Enhanced phytoextraction of heavy metals using chelating agents and agricultural crops is widely discussed as a remediation technique foragricultural soils contaminated with low mobile heavy metals. In this study, phytoextraction efficiency of Zea mays after single and split applications ofEDTAwas tested on the laboratory and the field scale. EDTA effectively increased the mobility of target heavy metals (Pb and Cd) in the soil solution.Split applications provided generally lower water-soluble levels of Pb andCd both in the pot and the field experiment. Therefore, the risk of groundwatercontamination may be reduced after split applications. Higher Pb and Cd mobilisation after single applications increased plant stress, phytotoxicity andreduced plant dry above-ground biomass production compared to corresponding split doses. Single doses enhanced plant uptake of Pb and Cd and thephytoremediation efficiency compared to corresponding split doses. Results of plant dry above-ground biomass and heavy metal uptake obtained fromthe pot experiment could be to some extent verified in the field experiment. Plant uptake of Pb and Cd was lower and biomass production dropped afterEDTA additions in the field experiment. Remediation factors in the field experiment were in general significantly lower than in the pot experimentmainly due to themuch highermass of soil per plant under field conditions. This highlights the limitations when going from the lab to the field scale. Thelow phytoremediation efficiency in the field and the mobilisation of high amounts of Pb and Cd down the soil profile maymake the use of EDTA and Z.mays not suitable for the remediation of severely heavy metal contaminated soils in a reasonable time frame and may result in substantial groundwaterpollution under used crop management.© 2007 Elsevier B.V. All rights reserved.

Keywords: Phytoextraction; EDTA; Lead; Cadmium; Zea mays; Contaminated soil

1. Introduction

Smelting industry is one of the main contamination sourcesof agricultural soils with heavy metals (Loska et al., 2004).The possible negative impacts on the environment and humanhealth demand the need for remediation of contaminated sites.Phytoextraction, the use of engineered metal-accumulating plantsto remove toxic metals from soil, could potentially be an environ-ment-friendly and cost-effective technology compared to con-ventional remediation techniques (Salt et al., 1995; Blaylocket al., 1997; Kos and Leštan, 2003).

Heavy metals are bound primarily to organic, oxide andresidual fractions in soils resulting thus into low mobility(Adriano, 2001). The low solubility and bioavailability of sometoxic metals (e.g., Pb) is the major limiting factor in inducedphytoextraction (Lasat, 2000) as the Pb exchangeable fraction isvery low in most soils (Maiz et al., 1997). Synthetic chelatingagents have the potential to remobilise metals and to form strongsoluble complexes (Nowack et al., 2001; Sun et al., 2001).Ethylenediaminetetraacetic acid (EDTA) proved to be the mosteffective chelating agent among several tested in increasing Pbdesorption from soils (Huang et al., 1997; Komárek et al., 2007).Increasing Me–chelant complexes in the soil solution promotethe uptake by plants and the translocation of heavy metals fromroots to shoots and their accumulation in the harvestable parts of

Available online at www.sciencedirect.com

Geoderma 144 (2008) 446–454www.elsevier.com/locate/geoderma

⁎ Corresponding author. Tel.: +420 22 438 2736; fax: +420 23 438 1801.E-mail address: reinhard@neugschwandtner.com (R.W. Neugschwandtner).

0016-7061/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.geoderma.2007.11.021

Author's personal copy

the plants (Blaylock et al., 1997; Huang et al., 1997; Epsteinet al., 1999; Grčman et al., 2001; Schmidt, 2003).

The phytoextraction efficiency of a plant is dependent on theheavy metal contents in the biomass and the biomass production(Blaylock et al., 1997). Because of the lack of large-scale culti-vation techniques for metal-hyperaccumulating plants and theirlow biomass, research has been focused on the use of chelatingagents and high yielding agricultural crops that can be cultivatedusing established agronomic practices (Raskin et al., 1997). Someagricultural crops (e.g., Brassica napus L., Brassica juncea L.,Cannabis sativa L., Helianthus annuus L., Phaseolus vulgarisL., Sinapis alba L. and Zea mays L.) were found to be effective inremoving metals under model conditions but their evaluation infield conditions is missing (Kos et al., 2003; Wu et al., 2004;Hajiboland, 2005; Li et al., 2005; Grispen et al., 2006; Komáreket al., 2007). The harvested biomass containing high concentra-tions of heavy metals would have to be removed from the site andstored at a landfill (Keller et al., 2005).

The main drawback of EDTA is its high persistence in theenvironment due to negligible biodegradation (Bucheli-Witscheland Egli, 2001). This can cause a high risk of metal leaching dueto the rapid mobilisation of metals and the subsequent slowdecrease of metal mobility in the soil solution (Grčman et al.,2001; Lombi et al., 2001; Wenzel et al., 2003; Wu et al., 2004).Therefore, there is a need to estimate the movement of metalsthrough the soil profile and to optimise agronomic practices tomaximise the cleanup potential of remediative plants and tominimise risks to humans and the environment (Lasat, 2000).

The aims of this studywere (i) to evaluate differentmanagementtechniques (single versus split applications of EDTA) on the lab andthe field scale for remediation of an agricultural soil contaminatedwith Pb and Cd originating from the smelting area of Příbram(Czech Republic) and (ii) to assess the possible risks of EDTAapplication on groundwater pollution under field conditions.

2. Materials and methods

2.1. Soil sampling and sample preparation

A site was chosen in the close vicinity of Příbram, ahistorical smelting and mining town located approximately60 km SW of Prague. This area belongs to the most pollutedones in the Czech Republic due to atmospheric deposition ofpotentially toxic elements from the Pb smelter (Šichorová et al.,2004). Soil samples were taken from the arable layer (0–25 cm)of an agriculturally used soil (N 49°42.441′; E 13°59.603′)approximately 1 km NE of the smelter. Samples used for soilcharacteristics determination, heavy metal contents, Pb and Cdfractionation and the incubation experiment were air-dried,homogenised and sieved through a 2-mm stainless sieve prior toanalyses. Soil used for pot experiments was air-dried, homo-genised and sieved through a 10-mm stainless sieve.

2.2. Basic physico-chemical soil characteristics

The pH was determined using deionised water or 0.2 M KCl(w/v=1:2.5). Cation exchange capacity was calculated as the sum

of Ca, Mg, K, Na and Al extractable in 0.1 M BaCl2 (w/v=1:20). Particle size distribution was determined by thehydrometer method. Total organic carbon (TOC) was determinedspectrophotometrically after the oxidation of organic matter byK2Cr2O7 (Sims and Haby, 1971). The approximate amount ofamorphous and poorly crystalline Fe-, Mn- and Al-oxides andhydroxides was determined by acid oxalate extraction (0.2 Mammonium oxalate/oxalate acid at pH 3) (Carty et al., 1998). Theoxalate extracts were analysed for Fe, Al and Mn using opticalemission spectroscopy with inductively coupled plasma (ICP-OES, Varian VistaPro, Australia). Available contents of nutrientswere determined by the Mehlich III soil extraction procedure(Zbíral, 2000) using flame atomic absorption spectroscopy(FAAS, VARIAN SpectrAA-300, Australia) (for Ca, K andMg) and ICP-OES (for P). Reference Material ISE 989 Riverclay(Wageningen Agricultural University, The Netherlands) wasused for quality assurance of analytical data.

2.3. Total heavy metal content and Pb and Cd fractionation

Total element contents in the soil were determined by a twostep procedure: (i) 0.5 g of soil samples were decomposed bydry ashing in Apion Dry Mode Mineraliser (Tessek, CzechRepublic) in a mixture of oxidising gases (O2+O3+NOx) at400 °C for 10 h; and (ii) the ash was then decomposed in amixture of HNO3+HF, evaporated to dryness on a hot plate at160 °C and dissolved in diluted aqua regia (Száková et al.,1999). Heavy metal contents (As, Cd, Cr, Cu, Ni, Pb and Zn) indigests were analysed using ICP-OES. Reference Material 7003Silty clay loam (Analytika Ltd., Czech Republic) was used forquality assurance of analytical data.

Pb and Cd fractionation in the soil was performed using thesequential extraction byRauret et al. (1999). Determined fractionswere: (i) FractionA— exchangeable and acid extractable (0.11MCH3COOH — extractable); (ii) Fraction B — reducible (0.1 MNH2OH·HCl — extractable); (iii) Fraction C — oxidisable(8.8MH2O2/1MCH3COONH4— extractable); and (iv) FractionD — residual, computed as the sum of fractions A, B, and Csubtracted from the total content. Heavy metal contents in digestswere analysed using ICP-OES.

2.4. Batch experiment

An incubation experiment was carried out to assess the effectof different EDTA concentrations (Disodium EDTA Dihydrate,N98.0% purity, Omikron, Germany) and application regimes(single and split applications) on the mobility of metals andnutrients. A mass of 50 g of air-dried and sieved (≤2 mm) soilwas put into acid-clean 250-ml polyethylene plastic bottles.Incubations were carried out using three replicates for 0.5–42 dat a constant temperature of 25 °C. Seven treatments with 3replicates were performed: control (deionised H2O), three singlerate doses (3, 6, 9 mmol EDTA kg−1) and three split rate doses(3×1, 3×2, 3×3 mmol EDTA kg−1).

An amount of deionised water and chelant solution, respec-tively, representing 60% of water holding capacity (WHC)(20 ml) was applied to the soil at single doses applications. First

447R.W. Neugschwandtner et al. / Geoderma 144 (2008) 446–454

Author's personal copy

split rate applications were performed on the same day as thesingle applications at a volume equivalent to 50% of WHC.Second and third applications followed in ten day intervals (withvolumes representing 10% of WHC). The pH values of thesolutions were between 4.8–4.9 (compared to pH 6.6 ofdeionised water). However, possible decrease of soil pH shouldnot affect metal mobility at such high extent as the effect ofEDTA. The bottles were aerated every 3 d using a mechanicalpump. Samples were extracted with 120 ml of deionised waterfor 1 h on a horizontal shaker at 30 rpm. Water-solubleconcentrations of elements Cd, Cu, Fe, Mn, P, Pb and Zn insolutions were determined using ICP-OES, the elements Ca, Mgand K were determined using FAAS.

2.5. Pot experiment

Samples of air-dried, homogenised and sieved (10-mm) soil(5 kg) were fertilised with 1 g N (NH4NO3), 0.16 g P and 0.4 g K(K2HPO4) and transferred to pots. Eight seeds of Z. mays cv.Rivaldo were sown in every pot and thinned to four plants threeweeks after sowing. Additional fertilisation was performed twiceduring the vegetation period (a total of 1 g N (NH4NO3), 0.16 g Pand 0.4 K g (K2HPO4) per pot). Pots were placed in an outdoorweather-controlled vegetation hall. Soil moisture was kept at 60%of WHC by watering daily with deionised water. Leachate wascollected and re-circulated to the system so that mass could beconserved (Tandy et al., 2006). Same EDTA treatments wereperformed as in the batch incubation experiment. Single applica-tions (3, 6, 9 mmol EDTA kg−1) and the first of the three splitapplications (3×1, 3×2, 3×3 mmol EDTA kg−1) were performed61 d after sowing. The following applications were added after 10 dand 20 d. Deionised water and chelant solutions were applied at avolume of 100 ml. Treatments were conducted in four replications.Soil solution was collected using suction cups (G. Wieshammer,Vienna, Austria) four days after EDTA applications and then in tenday intervals. Pots were watered with deionised water to full WHCand left for one day to reach equilibrium before sampling. Elementsin soil solution samples were analysed using ICP-OES (Pb, Cd, FeandMn). Plants were harvested 67 d after the first EDTA additionsat the dough-ripe stage.

2.6. Field experiment

The phytoremediation efficiency of Z. mays was investigatedin a precise field experiment (plot size: 10 m2). Z. mays cv.Rivaldo was sown after the pre-crop triticale (x triticosecale).Fertilising with slurry and mineral fertiliser to an aliquot of128 kg N kg ha−1 was performed.

Single and the first split applications were performed on thesame day 90 days after sowing of Z. mays, second and third singleapplications followed in two-week intervals. EDTA doses wereapplied to an equivalent of 300 kg of soil m−2 (arable layer depth:25 cm; bulk density: 1.2 g cm−3). Chelant solutions and waterwere applied at a volume of 10 1 m−2. Plants were harvested 40 dafter the first EDTA applications (at the dough-ripe stage).

Soil sampling was performed with soil probes to a depth of50 cm 58 d after the first applications of EDTA. Soil profile

samples were divided into 5 cm layers in order to assess thevertical mobilisation of heavy metals after EDTA applications.Composite samples consisting of five equally sized, discretesubsamples were taken per field for every layer from evenlyspaced, adjacent sampling points (U.S. EPA, 2005). Sampleswere dried at 70 °C. For determination of water-soluble Pb andCd, 5 g of sieved soil (≤2 mm) were extracted with 14 ml ofdeionised water on a horizontal shaker at 30 rpm for 1 h andcentrifuged.

2.7. Plant analyses

Harvested plants were gently washed with deionisedwater, dried at 60 °C to constant weight and then finelygrounded with a microfine grinder (MF 10 basic, KIKI-Werke,Germany). Total contents of elements in the plant samples weredetermined in mineral extracts obtained by dry decomposi-tion (Mader et al., 1998; Street et al., 2006). Samples weredecomposed first on a hot plate and then in a muffle furnacewith a stepwise increase of the ashing temperature to 500 °C.The ash was dissolved in aqua regia. The contents of Pb and Cdwere determined using ICP-OES. Certified Reference MaterialCTA-OTL-1 Oriental Tobacco Leaves (Polish Academy ofSciences and Institute of Nuclear Chemistry and Technology,Warsaw, Poland) was used for quality assurance of analyticaldata.

2.8. Phytoextraction efficiency

The phytoextraction efficiency of plants depends on theamount of heavy metals accumulated in the dry above-groundbiomass of the plants and the plant yields. The remediationfactor (RF) (Vysloužilová et al., 2003) which represents thepercentage of an element removed by the plant dry above-ground biomass from the total element content in the soil duringone cropping season was calculated as follows:

RF ¼ Pbplant � Bplant

Pbsoil � wsoil� 100 kð Þ ð1Þ

where Pbplant is the content of Pb in plant dry above-groundbiomass (mg kg−1); Bplant the plant dry above-ground biomassyield (g); Pbsoil the total content of Pb in soil (mg kg−1) and wsoil

the amount of soil in the pot (g).

2.9. Phytoextraction potential

The phytoextraction potential (PP) is representing the totalamount of heavy metals extracted per ha of soil in one singlephytoextraction cycle (Kos et al., 2003). It is calculated asfollows:

PP ¼ Pbplant � Bplant ð2Þ

where Pbplant is the content of Pb in plant dry above-groundbiomass (mg kg−1) and Bplant the plant dry above-ground matterbiomass yield (t ha−1).

448 R.W. Neugschwandtner et al. / Geoderma 144 (2008) 446–454

Author's personal copy

2.10. Statistics

Statistical analyses were performed using software STATIS-TICA Version 6.0 (StatSoft, USA). Analysis of variance(ANOVA) with subsequent Duncan test was performed at thesignificance level of pb0.05.

3. Results and discussion

3.1. Soil properties and total contents

Basic physico-chemical soil characteristics, total heavymetal contents and Pb and Cd fractionation in the studied soilare summarised in Table 1. The soil used was a loamy soilclassified as a Cambisol. High contents of Pb and Cd werefound in the soil due to the vicinity of the local Pb-smelter. Thecontents of the elements As, Cr, Cu, Ni and Zn were below thesoil limit values in the Czech Republic (Czech Regulation Nr.13/1994). Determination of the available form of macronu-trients in the soil showed a sufficient content of Ca and P andhigh K and Mg supply (Marschner, 2005). Amorphous Fe-oxides and hydroxides were predominant compared to those of

Al and Mn. They can present important sorption positions forheavy metals. Pb is mostly present in the reducible fraction (FB)and Cd in the residual fraction (FD). Only small portions of Pbare present in the exchangeable and acid-extractable fraction(FA), whereas there is a high share of Cd in this most “labile”fraction.

3.2. Effect of EDTA on heavy metal solubility in soil

The time evolution of water-soluble Pb and Cd in the batchincubation experiment is summarised in Fig. 1. EDTAapplications increased significantly water-soluble Pb and Cdconcentrations. EDTA effectively solubilised Pb and Cdwhereas water-soluble concentrations of the control remainedlow over time. The highest single dose of EDTA (9 mmol kg−1)resulted in a 148-fold increase of water-soluble Pb concentra-tions and a 203-fold increase of water-soluble Cd concentra-tions compared to the control. Thereby, the water-solublefractions of Pb and Cd were 65% and 33%, respectively. Adecrease of water-soluble metal concentrations was observedover time. The decrease was higher in the case of singleapplications with lower initial amended concentrations ofEDTA (especially for Cd concentrations where all three singledoses led to comparable levels at the start of the incubationexperiment) (Fig. 1a and c). The highest single dose (9 mmolEDTA kg−1) provided relative constant water-soluble Cd

Table 1Basic physico-chemical soil characteristics, total heavy metal contents and Pband Cd fractionation in the experimental soil

Soil

pHH2O 6.5pHKCl 5.7CEC (cmol kg−1) 13.1TOC (%) 1.74Particle size distribution (%)

Sand (%) 42.8Silt (%) 41.8Clay (%) 15.4Soil texture Loam

Oxalate extractable (g kg−1)Fe 2.78Mn 0.5Al 0.36

Available nutrients after Mehlich 3 (mg kg−1) (n=3)Ca 1688±71Mg 202±10K 264±37P 47.4±0.8

Total heavy metal contents (mg kg−1) (n=3)Pb 544±69Cd 4.99±1.72As 28.8±6.2Cr 34.9±2.7Cu 19.7±0.6Ni 17.0±2.9Zn 143±16

Pb fractionation (mg kg−1) (n=3)Fraction A (exchangeable and acid-extractable) 14.9±0.9 (2.7%)Fraction B (reducible) 369.8±23.3 (68.0%)Fraction C (oxidisable) 46.5±2.0 (8.5%)Fraction D (residual) 112.8 (20.7%)

Cd fractionation (mg kg−1) (n=3)Fraction A (exchangeable and acid-extractable) 1.10±0.12 (21.8%)Fraction B (reducible) 0.89±0.09 (17.8%)Fraction C (oxidisable) 0.29±0.01 (5.9%)Fraction D (residual) 2.72 (54.5%)

Fig. 1. Time evolution of water-soluble Pb (a, b), Cd (c, d) and Fe (e, f)concentrations following single and split applications of EDTA. Data shown aremeans±SD (n=3).

449R.W. Neugschwandtner et al. / Geoderma 144 (2008) 446–454

Author's personal copy

concentrations for about 24 days (Fig. 1c). The decrease ofwater-soluble Pb and Cd may be attributed to the continuousincrease of Fe in the water extract over time (Figs. 1e and f)which is originating from slowly dissolving Fe-oxides andhydroxides (Nowack and Sigg, 1997; Komárek et al., 2007). Feis becoming thereby the main competitor to metals due to thehigher stability of Fe–EDTA complexes (log KFe(III)–

EDTA=27.7) compared to other metal complexes (log KPb–

EDTA=17.9 and log KCd–EDTA=16.3) (Nowack et al., 2006; logK values are from Martell et al., 1998). Higher EDTA dosesresisted better the Fe competition.

Split doses resulted in lower initial mobilisation of heavymetals but provided generally a more constant amount of water-soluble Pb and Cd concentrations over time compared to singledoses (Fig. 1b and d). The maximal Pb mobilisation was lowerafter split applications, whereas maximal mobilisation of Cdwas higher following the highest split application (3×3 mmolEDTA kg−1) compared to the corresponding single application(9 mmol EDTA kg−1).

Concentrations of Pb and Cd in the soil solution during thepot experiment are summarised in Fig. 2. EDTA applicationsresulted in a high solubilisation of Pb and Cd. The mobilisationof Pb and Cd was more constant over time contrary to the batchincubation experiment. Single applications showed highermaximal concentrations of water-soluble Pb and Cd than splitones. Additional split doses resulted in an increase of water-soluble Pb and Cd concentrations. Water-soluble concentrationsof heavy metal showed a high variability after EDTAapplications in the pot experiment. Such variability was already

observed by Fischerová et al. (2006). The high remainingconcentrations of water-soluble heavy metals highlight the riskof groundwater contamination when EDTA is used for en-hanced phytoextraction.

Water-extractable Pb and Cd concentrations in the fieldexperiment measured 58 d after the first applications aresummarised in Fig. 3. Water-soluble fractions of heavy metalsincreased substantially following the EDTA applications inthe field. Mobilisation was the highest in the upper layers anddecreased gradually with increasing soil depth. Water-solublePb and Cd in the upper layer (0–5 cm) were 326-fold and 105-fold, respectively, higher compared to the control treatment 58d following the highest single dose (9 mmol EDTA kg−1).Thereby, the water-soluble fractions of Pb and Cd wereincreased to 73% and 67%, respectively. In the depth of 45–50 cm, water-soluble Pb and Cd were still 4.3-fold and 4.1-fold, respectively, higher compared to the variant un-treatedwith EDTA. Vertical distribution of water-soluble Pb and Cdconcentrations were lower after the split applications. Water-soluble Pb and Cd were still increased 3.8-fold and 2.3-fold,respectively, in a depth of 45–50 cm after split applications.This supports observations made by Shen et al. (2002) whosuggested that multidose chelate application may limit thesolubility and migration potential of heavy metals in soils. Sunet al. (2001) reported that the mode of EDTA addition is one ofthe main factors controlling the behaviour of metal leaching.Fig. 2. Pb (a, b) and Cd (c, d) concentrations in soil solution following single

and split applications of EDTA in the pot experiment. Data shown are means±SD (n=4).

Fig. 3. Vertical distribution of water-soluble Pb (a, b) andCd (c, d) concentrationsafter single and split applications of EDTA 58 d following first applications. Datashown are means±SD (n=3).

450 R.W. Neugschwandtner et al. / Geoderma 144 (2008) 446–454

Author's personal copy

Additionally, better plant growth following split applicationscould have limited leaching due to higher transpiration.

3.3. Effects of EDTA on plant dry above-ground biomassproduction of Z. mays

Z. mays was affected by visible phytotoxicity symptoms(wilting and plant necrosis) following all EDTA treatments.Plants treated with high single doses of EDTA (6, 9 mmol kg−1)showed visible signs of phytotoxicity within days causing deathof the plants before harvest. Plants receiving the lowest singleand split doses showed moderate damages but survived until theend of the experiment. The addition of several lower dosescompared to the same amount added once provides time forplants to initiate their adaptation mechanisms and to raise theirdamage threshold (Barocsi et al., 2003). Phytotoxicity is mainlyattributed to free EDTA (Vassil et al., 1998). However, Cooperet al. (1999) reported that phytotoxic effects occurred at leastpartly by the uptake of Pb after EDTA addition.

Phytotoxic effects caused a significant decrease of plant dryabove-ground biomass production of Z. mays for all EDTAtreatments (Fig. 4). This is in accordance with results fromHovsepyan and Greipsson (2005) and Li et al. (2005). Plant dryabove-ground biomass decrease was more pronounced withincreased EDTA doses. The highest EDTA dose (9 mmol kg−1)resulted in the highest dry matter production decrease comparedto the control (43% in the pot experiment and 77% in the fieldexperiment). Decrease of plant dry above-ground biomassproduction was generally significantly higher after singleapplications compared to corresponding split applications. Plantdry above-ground biomass decrease was much more pronouncedin the field experiment. These results may be explained by theoptimal nutrient and water supply in the pot experiment.

3.4. Pb and Cd contents in plant dry above-ground drybiomass of Z. mays

Heavy metal (Pb, Cd) contents in plant shoots of Z. mays afterthe application of EDTA are summarised in Fig. 5. EDTA sig-nificantly increased the uptake of heavy metals (especially of Pb)

(Hovsepyan and Greipsson, 2005; Huang et al., 1997). Pbcontents in the plant dry above-ground dry biomass of Z. maysincreased linearlywith the rate of applied single applications (3, 6,9 mmol EDTA kg−1) and split applications (3×1, 3×2,3×3mmol EDTAkg−1). Single applications showed a correlationof R2 =0.90 (pot experiment) and 0.97 (field experiment),respectively, and split applications showed one of R2=0.85 (potexperiment) and 0.97 (field experiment), respectively. The highestcontent of Pb (110.5±9.6 mg kg−1) was observed in the potexperiment following the highest single dose representing a 23.2-fold increase compared to the control (4.2±1.2 mg kg−1). Thehighest uptake of Pb (37.6±6.2 mg kg−1, 9 mmol EDTA kg−1) inthe field experiment was 6.0-times higher compared to the control(6.3±1.1 mg kg−1). Cd uptake was linearly increased after EDTAapplications. Single applications showed a correlation ofR2=0.97 (pot experiment) and 0.99 (field experiment), respec-tively, and split applications showed one of R2=0.48 (potexperiment) and 0.72 (field experiment), respectively. Cd contentsincreased by a factor of 3.1 (from 0.46±0.15 to 1.53±0.13 mgkg−1) in the pot experiment and by a factor of 1.7 in the fieldexperiment (from 0.39±0.00 to 0.66±0.08 mg kg−1) afteraddition of the highest single dose (9 mmol EDTA kg−1)compared to the control. Similar trends could be observed ingeneral in the pot and in the field experiment, although, heavymetal uptake was lower in the field. The higher differences oftreatments under controlled conditions in the pot experiment maybe attributed to the better penetration of the soil by roots (Frieslet al., 2006) and to the higher moisture content and homogeneousnature of the soil used (Conesa et al., 2006).

Fig. 4. Plant dry above-ground biomass yields of Z. mays following the additionof different concentrations of EDTA (0, 3, 6, 9, 3×1, 3×2, 3×3 mmol kg−1) inthe pot and in the field experiment. Data shown are means±SD (n=4). Data withthe same letter represent statistically identical values (pb0.05).

Fig. 5. Pb (a) and Cd (b) contents in plant dry above-ground biomass of Z. maysin the pot and in the field experiment following the addition of differentconcentrations of EDTA (0, 3, 6, 9, 3×1, 3×2, 3×3 mmol kg−1). Data shownare means±SD (n=4). Data with the same letter represent statistically identicalvalues (pb0.05).

451R.W. Neugschwandtner et al. / Geoderma 144 (2008) 446–454

Author's personal copy

Single doses of EDTA resulted generally in significantlyhigher Pb contents in shoots of Z. mays compared to correspond-ing split doses. A similar trend was observed for Cd, especially inthe pot experiment. Our findings are contradictory to findings ofShen et al. (2002) who observed a higher Pb accumulation incabbage (Brassica rapa) shoots following three separate dosescompared to one- and two-dose application and Wenzel et al.(2003) who reported generally higher concentrations of Pb incanola (Brassica napus) following split applications compared tosingle applications. Copper uptake of Agrostis tenuis L. wasincreased by applying 18 small doses of EDTA compared to twolarge ones (Thayalakumaran et al., 2003). However, our resultssupport the importance of physiological stress (evident asnecrosis) for the increased unrestricted uptake of Me–chelatecomplexes (Schaider et al., 2006). When EDTA is present, Pb istaken up and translocated in the plant as Pb–EDTA complexes(Vassil et al., 1998; Epstein et al., 1999). Shoot accumulation ofEDTA ismainly related to physiological stress. The concentrationof Me–EDTA complexes is significantly higher and theselectivity among solutes is lower in the xylem sap underconditions of stress (Vassil et al., 1998; Schaider et al., 2006).Jiang et al. (2003) reported that Cd toxicity stress induced byEDTA application might have stimulated the translocation of themetal from roots to shoots. Given the high competition of Fe totarget metals in the soil solution as shown in the batch incubationexperiment, high concentrations of the chelant are necessary toachieve the desired concentration of target metal complexes(Nowack et al., 2006).

3.5. Phytoextraction efficiency

The phytoextraction efficiency ofZ. mays in response to singleand split applications of EDTA in the pot and in the fieldexperiment is summarised in Table 2. The highest remediationfactor for Pb (0.30%) and Cd (0.51%) was obtained after theaddition of 9 mmol EDTA kg−1 in the pot experiment. In the fieldexperiment, the highest remediation factor (calculated as elementremoval from 300 kg soil m−2) for Pb (0.0075%) was obtainedafter additions of 3×2 mmol EDTA kg−1 and the highest re-mediation factor for Cd (0.0246%) was obtained in the controltreatment. In the case of Cd, the increase of metal translocationinto the shoots after EDTA application was not high enough to

compensate for the plant dry above-ground biomass decrease.The maximal phytoextraction efficiency was in the pot experi-ment 40-times higher for Pb and 25-times higher for Cd comparedto the field experiment. This can be mainly explained by thehigher plant/soil ratio as one plant rooted in 1.25 kg of soil in thepot experiment whereas there was just one plant for more than40 kg of topsoil in the field one. Our results confirmed that thephytoremediation efficiency is likely to be lower in the field thanin laboratory conditions as suggested by Kumar et al. (1995).Tlustoš et al. (2006) found evidence that the influence ofexperimental factors may bemore pronounced in pot experimentsthan in field ones due to the limited volume of soil and the welldeveloped contact of all roots with the whole mass of the soil.

Despite the substantial increase of Pb and Cd contents in theplant dry above-ground biomass, heavy metal uptake was nothigh enough to achieve extraction rates which would benecessary for practical use. Similar observations have beenmade by Kos and Leštan (2003) for the EDTA-enhanced uptakeof Pb by the Chinese cabbage (Brassica rapa). An accumulationof more than 1% of total heavy metal contents present in the soilinto the plant dry above-ground biomass should be achieved foran economically feasible use of phytoextraction (Huang et al.,1997). Calculating with the maximal obtained remediationfactors it would take approximately 300 cropping seasons toobtain Czech threshold values in heavy soils given for Pb(220 mg kg−1 soil) and approximately 260 cropping seasons forCd (1 mg kg−1 soil) in the pot experiment. Due to thesignificantly lower remediation factors in the field experiment,phytoextraction may not be suitable for field use under givenconditions in a reasonable time frame and currently due to therelatively high economic costs.

3.6. Phytoextraction potential

The phytoextraction potential of Z. mays in response to singleand split applications of EDTA in the pot and in the field experimentis summarised in Table 3. The Pb phytoextraction potential of Z.mays in one growing season was up to 122.6 (±10.4) g Pb ha−1

after addition of 3×2 mmol EDTA kg−1 of soil (compared to 58.9(±7.7) g Pb ha−1 extracted by the control). This is about one orderof magnitude less than values reported by Kos et al. (2003) for thephytoextraction potential of Z. mays (940–1380 g Pb ha−1). This

Table 2Phytoextraction efficiency of Z. mays in response to single and split applicationsof EDTA in the pot and in the field experiment

EDTAapplication(mmol kg−1)

Pb Cd

Pot (%) Field (%) Pot (%) Field (%)

Control 0.02±0.01 0.0036±0.0005 0.27±0.08 0.0246±0.00143 0.13±0.03 0.0060±0.0001 0.39±0.04 0.0178±0.00386 0.15±0.02 0.0058±0.0001 0.42±0.05 0.0114±0.00069 0.30±0.04 0.0049±0.0012 0.51±0.08 0.0094±0.00193×1 0.09±0.01 0.0045±0.0005 0.39±0.07 0.0182±0.00423×2 0.14±0.05 0.0075±0.0006 0.34±0.06 0.0205±0.00553×3 0.13±0.04 0.0065±0.0017 0.33±0.05 0.0198±0.0035

Results are expressed as means±SD (n=4).

Table 3Calculated dry matter yields per ha, concentrations of Pb and Cd in the harvestableparts ofZ.mays and phytoextraction potential (PP) per ha following single and splitapplications of EDTA in the field experiment

EDTAapplication(mmol kg−1)

Drymatter(t ha−1)

Pb Cd

Uptake(mg kg−1)

PP(g ha−1)

Uptake(mg kg−1)

PP(g ha−1)

Control 9.4±0.4 6.3±1.1 58.9±7.7 0.39±0.04 3.7±0.23 5.6±0.1 17.7±0.7 98.0±1.6 0.48±0.11 2.7±0.66 2.9±0.4 32.7±4.8 95.1±1.6 0.58±0.04 1.7±0.19 2.1±0.2 37.6±6.2 80.3±19.5 0.66±0.08 1.4±0.33×1 7.6±0.7 9.6±2.0 72.8±8.6 0.36±0.12 2.7±0.63×2 6.4±0.4 19.0±0.4 122.6±10.4 0.47±0.10 3.1±0.83×3 4.4±0.7 3.7±2.5 105.8±27.3 0.67±0.02 3.0±0.5

Results are expressed as means±SD (n=4).

452 R.W. Neugschwandtner et al. / Geoderma 144 (2008) 446–454

Author's personal copy

can be attributed to the fact that the authors used above-groundbiomass yields obtained from literature and metal uptake ratesobtained from pot experiments neglecting thereby the above-ground biomass decrease after EDTA addition and the possiblelower uptake rates under field conditions. However, taking intoaccount the total amount of Pb in the arable layer (1632 kg ha−1,calculated for 3000 tonnes of soil ha−1), the Pb phytoextractionpotential of Z. mays was very low under given conditions.

Cd phytoextraction potential actually decreased after theaddition of EDTA. The higher Cd phytoextraction efficiency bythe control treatment was due to the better plant growth. Slightlyincreased Cd concentrations were too low to compensate thereduced plant dry above-ground biomass production. Similarobservations have been made by Lesage et al. (2005) in EDTAamended sunflower (H. annuus). Consequently, the highestphytoextraction potential for Cd (3.7±0.2 g Cd per ha−1) in thecontrol treatment was considerably lower compared to valuesgiven by Kos et al. (2003) for Z. mays (50–82 g of Cd ha−1).

4. Conclusion

A comparison of split and single applications of EDTA andthe evaluation of phytoextraction from the lab to the field scaleare presented.

The assumption that a longer relatively constant level ofwater-soluble and a lower maximal solubility of Pb after splitapplications of EDTA could increase phytoremediation effi-ciency could not be confirmed from our results. Singleapplications proved to be generally more efficient due to higherplant uptake rates most likely enhanced by physiological stress.

Plant dry above-ground biomass and plant uptake resultsfrom the pot experiment were generally verified in the field. Itwas not possible to transfer phytoextraction efficiency resultshighlighting the limitations when going from the lab to the fieldscale. Phytoremediation efficiency was too low in the field forsuccessful remediation in a reasonable time frame under usedplant management. Furthermore, EDTA addition in the fieldresulted in a mobilisation of high amounts of Pb and Cd downthe soil profile (especially after single EDTA applications)which may result in substantial groundwater pollution.

Acknowledgements

This study has been funded by the Czech ScienceFoundation (GAČR— 521/06/0496) and Ministry of Educationof the Czech Republic (MSM 6046070901). The authors wishto thank the “Österreichische Forschungsgemeinschaft (ÖFG)”,Vienna, Austria for funding support (MOEL-Grant) awarded tothe first author.

References

Adriano, D.C., 2001. Trace Elements in the Terrestrial Environments:Biogeochemistry, Bioavailability, and Risks of Metals. Springer, New York.

Barocsi, A., Csintalan, Z., Kocsanyi, L., Dushenkov, S., Kuperberg, J.M.,Kucharski, R., Richter, P.I., 2003. Optimizing phytoremediation of heavy

metal-contaminated soil by exploiting plants' stress adaptation. InternationalJournal of Phytoremediation 5, 13–23.

Blaylock, M.J., Salt, D.E., Dushenkov, S., Zakhrova, O., Gussman, C.,Kapulnik, Y., Ensley, B.D., Raskin, I., 1997. Enhanced accumulation of Pbin Indian mustard by soil-applied chelating agents. Environmental Scienceand Technology 31, 860–865.

Bucheli-Witschel,M., Egli, T., 2001. Environmental fate andmicrobial degradationof aminopolycarboxylic acids. FEMS Microbiology Reviews 25, 69–106.

Carty, D.K., Moore, J.N., Marcus, W.A., 1998. Mineralogy and trace elementassociation in an acid mine drainage iron oxide precipitate; comparison ofselective extractions. Applied Geochemistry 13, 165–176.

Conesa, H.M., Robinson, B.H., Schulin, R., Nowack, B., 2006. Growth ofLygeum spartum in acid mine tailings: response of plants developed fromseedlings, rhizomes and at field conditions. Environmental Pollution 145,700–707.

Cooper, E.M., Sims, J.T., Cunningham, S.D., Huang, J.W., Berti, W.R., 1999.Chelate-assisted phytoextraction of lead from contaminated soils. Journal ofEnvironmental Quality 28, 1709–1719.

Epstein, A.L., Gussman, C.D., Blaylock, M.J., Yermiyahu, U., Huang, J.W.,Kapulnik, Y., Orser, C.S., 1999. EDTA and Pb–EDTA accumulation inBrassica juncea grown in Pb-amended soil. Plant and Soil 208, 87–94.

Fischerová, Z., Tlustoš, P., Száková, J., Šichorova, K., 2006. A comparison ofphytoremediation capability of selected plant species for given traceelements. Environmental Pollution 144, 93–100.

Friesl, W., Friedl, J., Platzer, K., Horak, O., Gerzabek, M.H., 2006. Remediationof contaminated agricultural soils near a former Pb/Zn smelter in Austria:batch, pot and field experiments. Environmental Pollution 144, 40–50.

Grčman, H., Velikonja-Bolta, Š., Vodnik, D., Kos, B., Leštan, D., 2001. EDTAenhanced heavy metal phytoextraction: metal accumulation, leaching andtoxicity. Plant and Soil 235, 105–114.

Grispen, V.M.J., Nelissen, H.J.M., Verkleij, J.A.C., 2006. Phytoextraction withBrassica napus L.: a tool for sustainable management of heavy metalcontaminated soils. Environmental Pollution 144, 77–83.

Hajiboland, R., 2005. An evaluation of the efficiency of cultural plants toremove heavy metals from growing medium. Plant Soil Environment 51,156–164.

Hovsepyan, A., Greipsson, S., 2005. EDTA-enhanced phytoremediation of lead-contaminated soil by corn. Journal of Plant Nutrition 28, 2037–2048.

Huang, J.W., Chen, J., Berti, W.R., Cunningham, S.D., 1997. Phytoremediadonof lead-contaminated soils: role of synthetic chelates in lead phytoextraction.Environmental Science and Technology 31, 800–805.

Jiang, X.J., Luo, Y.M., Zhao, Q.G., Baker, A.J.M., Christie, P., Wong, M.H., 2003.Soil Cd availability to Indianmustard and environmental risk following EDTAaddition to Cd-contaminated soil. Chemosphere 50, 813–818.

Keller, C., Ludwig, C., Davoli, F., Wocheke, J., 2005. Thermal treatment of metal-enriched biomass produced from heavy metal phytoextration. EnvironmentalScience and Technology 39, 3359–3367.

Komárek, M., Tlustoš, P., Száková, J., Chrastný, J., Ettler, V., 2007. The use ofmaize and poplar in chelant enhanced phytoextraction of lead fromcontaminated agricultural soil. Chemosphere 67, 640–651.

Kos, B., Leštan, D., 2003. Influence of a biodegradable ([S,S]-EDDS) andnondegradable (EDTA) chelate and hydrogel modified soil water sorptioncapacity on Pb phytoextraction and leaching. Plant and Soil 253, 403–411.

Kos, B., Grčman, H., Leštan, D., 2003. Phytoextraction of lead, zinc andcadmium from soil by selected plants. Plant Soil Environment 49, 548–553.

Kumar, N.P.B.A., Dushenkov, V., Motto, H., Raskin, I., 1995. Phytoextraction:the use of plants to remove heavy metals from soils. Environmental Scienceand Technology 29, 1232–1238.

Lasat, M.M., 2000. Phytoextraction of metals from contaminated soil: a reviewof plant/soil/metal interactions and assessment of pertinent agronomicissues. Journal of Hazardous Substance Research 2, 1–25.

Lesage, E., Meers, E., Vervaeke, P., Lamsal, S., Hopgood, M., Tack, F.M.,Verloo, M.G., 2005. Enhanced phytoextraction: II. Effect of EDTA and citricacid on heavy metal uptake by Helianthus annuus from a calcareous soil.International Journal of Phytoremediation 7, 143–152.

Li, H., Wang, Q., Cui, Y., Dong, Y., Christie, P., 2005. Slow release chelateenhancement of lead phytoextraction by corn (ZeamaysL.) from contaminatedsoil—a preliminary study. Science of the Total Environment 339, 179–187.

453R.W. Neugschwandtner et al. / Geoderma 144 (2008) 446–454

Author's personal copy

Lombi, E., Zhao, F.J., Dunham, S.J., McGrath, S.P., 2001. Phytoremediation ofheavy metal-contaminated soils: natural hyperaccumulation versus chemi-cally enhanced phytoextraction. Journal of Environmental Quality 30,1919–1926.

Loska, K., Wiechulła, D., Korus, I., 2004. Metal contamination of farming soilsaffected by industry. Environment International 30, 159–165.

Mader, P., Száková, J., Milholová, D., 1998. Classical dry ashing of biologicaland agricultural materials. Part II. Losses of analytes due to their retention inan insoluble residue. Analusis 26, 121–129.

Maiz, I., Esnaola, V., Millán, E., 1997. Evaluation of heavy metal availability incontaminated soils by a short sequential extraction procedure. Science of theTotal Environment 206, 107–115.

Marschner, H., 2005. Mineral Nutrition of Higher Plants, Second ed. AcademicPress, London.

Martell, A.E., Smith, R.M., Motekaitis, R.J., 1998. NIST SRD 46 CriticallySelected Stability Constants of Metal Complexes, Version 5.0. NIST,Gaithersburg, MD.

Nowack, B., Sigg, L., 1997. Dissolution of iron(III)(hydr)oxides by metal–EDTA complexes. Geochimica et Cosmochimica Acta 61, 951–963.

Nowack, B., Kari, F.G., Krüger, H.G., 2001. The remobilization of metals formiron oxides and sediments by metal–EDTA complexes. Water, Air, and SoilPollution 125, 243–257.

Nowack, B., Schulin, R., Robinson, B., 2006. Critical assessment of chelant-enhancedmetal phytoextraction. Environmental Science and Technology 40,5225–5236.

Raskin, I., Smith, R.D., Salt, D.E., 1997. Phytoremediation of metals: usingplants to remove pollutants from the environment. Current Opinion inBiotechnology 8, 221–226.

Rauret, G., López-Sánchez, J.F., Sahuquillo, A., Rubio, R., Davidson, C., Ure,A., Quevauviller, Ph., 1999. Improvement of the BCR three step sequentialextraction procedure prior to the certification of new sediment and soilreference materials. Journal of Environmental Monitoring 1, 57–61.

Salt, D.E., Blaylock,M., Kumar, N.P.B.A., Dushenkov, V., Ensley, B.D., Chet, I.,Raskin, I., 1995. Phytoremediation: a novel strategy for the removal of toxicmetals from the environment using plants. Bio/Technology 13, 468–474.

Schaider, L.A., Parker, D.R., Sedlak, D.L., 2006. Uptake of EDTA-complexedPb, Cd and Fe by solution and sand-cultured Brassica juncea. Plant and Soil286, 377–391.

Schmidt, U., 2003. Enhancing phytoextraction: the effect of chemical soilmanipulation on mobility, plant accumulation, and leaching of heavy metals.Journal of Environmental Quality 32, 1939–1954.

Shen, Z.G., Li, X.D., Wang, C.C., Chen, H.M., Chua, H., 2002. Leadphytoextraction from contaminated soil with high-biomass plant species.Journal of Environmental Quality 31, 1893–1900.

Šichorová, K., Tlustoš, P., Száková, J., Kořínek, K., Balík, J., 2004. Horizontaland vertical variability of heavy metals in the soil of a polluted area. PlantSoil Environment 50, 525–534.

Sims, J.R., Haby, V.A., 1971. Simplified colorimetric determination of soilorganic matter. Soil Science 112, 137–141.

Sun, B., Zhao, F.J., Lombi, E., McGrath, S.P., 2001. Leaching of heavy metalsfrom contaminated soils using EDTA. Environmental Pollution 113,111–120.

Street, R., Száková, J., Drábek, O., Mládkova, L., 2006. The status ofmicronutrients (Cu, Fe, Mn, Zn) in tea and tea infusions in selected samplesimported to the Czech Republic. Czech Journal of Food Science 24, 62–71.

Száková, J., Tlustoš, P., Balík, J., Pavlíková, D., Vanĕk, V., 1999. The sequentialanalytical procedure as a tool for evaluation of As, Cd and Zn mobility insoil. Fresenius Journal of Analytical Chemistry 363, 594–595.

Tandy, S., Schulin, R., Nowack, B., 2006. Uptake of metals during chelant-assisted phytoextraction with EDDS related to the solubilized metalconcentration. Environmental Science and Technology 40, 2753–2758.

Thayalakumaran, T., Robinson, B.H., Vogeler, I., Scotter, R., Clothier, B.E.,Percival, H.J., 2003. Plant uptake and leaching of copper during EDTA-enhanced phytoremediation of repacked and undisturbed soil. Plant and Soil254, 415–423.

Tlustoš, P., Száková, J., Hrubý, J., Hartman, I., Najmanová, J., Nedělník, J.,Pavlíková, D., Batysta, M., 2006. Removal of As, Cd, Pb, and Zn fromcontaminated soil by high biomass producing plants. Plant Soil Environment52, 413–423.

United States Environment Protection Agency (U.S EPA), 1995. EPAObservational Economy Series, Volume 1: Composite sampling. EPA-230-R-95-005.

Vassil, A.D., Kapulnik, Y., Raskin, I., Salt, D.E., 1998. The role of EDTA in leadtransport and accumulation by Indianmustard. Plant Physiology 117, 447–453.

Vysloužilová, M., Tlustoš, P., Száková, J., 2003. Cadmium and zincphytoextraction potential of seven clones of Salix spp. planted on heavymetal contaminated soils. Plant Soil Environment 49, 542–547.

Wenzel, W.W., Unterbrunner, R., Sommer, P., Sacco, P., 2003. Chelate-assistedphytoextraction using canola (Brassica napus L.) in outdoors pot andlysimeter experiments. Plant and Soil 249, 83–96.

Wu, L.H., Luo, Y.M., Xing, X.R., Christie, P., 2004. EDTA-enhancedphytoremediation of heavy metal contaminated soil with Indian mustardand associated potential leaching risk. Agriculture, Ecosystems and Envi-ronment 102, 307–318.

Zbíral, J., 2000. Determination of phosphorus in calcareous soils by Mehlich 3,Mehlich 2, CAL, and Egner extractants. Communications in Soil Scienceand Plant Analysis 31, 3037–3048.

454 R.W. Neugschwandtner et al. / Geoderma 144 (2008) 446–454