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Environmental Pollution 151 (2008) 27e38www.elsevier.com/locate/envpol

The use of poplar during a two-year induced phytoextractionof metals from contaminated agricultural soils

Michael Komarek a,*, Pavel Tlustos a, Jirina Szakova a, Vladislav Chrastny b

a Department of Agrochemistry and Plant Nutrition, Czech University of Agriculture in Prague, Kamycka 129, 165 21, Prague 6, Czech Republicb Department of Applied Chemistry and Chemistry Teaching, University of South Bohemia, Studentska 13, 370 05, �Ceske Budejovice, Czech Republic

Received 12 January 2007; received in revised form 5 March 2007; accepted 12 March 2007

Application of mobilizing agents is not optimal during a two-year phytoextractionof metals from severely contaminated soils using poplars.

Abstract

The efficiency of poplar (Populus nigra L.� Populus maximowiczii Henry.) was assessed during a two-year chemically enhanced phytoex-traction of metals from contaminated soils. The tested metal mobilizing agents were EDTA (ethylenediaminetetraacetic acid) and NH4Cl. EDTAwas more efficient than chlorides in solubilizing metals (especially Pb) from the soil matrix. The application of chlorides only increased thesolubility of Cd and Zn. However, the increased uptake of metals after the application of higher concentrations of mobilizing agents wasassociated with low biomass yields of the poplar plants and the extraction efficiencies after the two vegetation periods were thus comparableto the untreated plants. Additionally, the application of mobilizing agents led to phytotoxicity effects and increased mobility of metals. Higherphytoextraction efficiencies were observed for Cd and Zn compared to Pb and Cu. Poplars are therefore not suitable for chemically enhancedphytoextraction of metals from severely contaminated agricultural soils.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Populus spp.; EDTA; Chlorides; Phytoextraction; Metals; Soils

1. Introduction

The contamination of agricultural soils with metals belongscurrently to the most important environmental issues. The ele-vated concentrations of metallic elements in soils originatemainly from anthropogenic activities, such as the mining andsmelting industry, sewage sludge application, the use ofmineral fertilizers and, to some extent, from the former useof leaded petrol (Adriano, 2001). Traditional remediationmethods of such contaminated soils (e.g., soil excavation anddumping, vitrification, stabilization and soil washing/flushing)are generally costly and harmful to soil properties (Mulligan

* Corresponding author. Tel.: þ420 2 24382740; fax: þ420 2 34381801.

E-mail addresses: komarek@af.czu.cz (M. Komarek), tlustos@af.czu.cz

(P. Tlustos), szakova@af.czu.cz (J. Szakova), vladislavchrastny@seznam.cz

(V. Chrastny).

0269-7491/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.envpol.2007.03.010

et al., 2001). Phytoextraction, the use of plants for extractingcontaminants from soils, therefore presents currently an alter-native for soils polluted with metals. However, the low mobilityand bioavailability of some metallic elements (e.g., Cr and Pb)limit the efficiency of the phytoextraction process. For this rea-son, synthetic chelating agents, such as ethylenediaminetetra-acetic acid (EDTA), have been introduced as amendmentscapable of solubilizing metals into the soil solution and en-hancing the root-to-shoot translocation efficiency (Blaylocket al., 1997; Vassil et al., 1998). Chemically enhanced phytoex-traction of metals using crop species with high biomass yields(e.g., Brassica juncea L., Zea mays L., Helianthus annuus L.)and fast growing trees (e.g., Salix spp., Populus spp.) has there-fore attracted much attention as a perspective and cost effectivemethod of soil remediation (Chen and Cutright, 2001; Liphadziet al., 2003; Wu et al., 2004; Luo et al., 2005; Baum et al.,2006; Fischerova et al., 2006). Unfortunately, due to their

28 M. Komarek et al. / Environmental Pollution 151 (2008) 27e38

high persistence, the formed metaleEDTA complexes in soilscan leach through the soil profile and contaminate the groundwater (Romkens et al., 2002). For this reason, several al-ternatives to EDTA have been proposed. Among many, thebiodegradable structural isomer of EDTA, (S, S ) N, N0-ethyle-nediaminedisuccinic acid (EDDS), has proved to be efficient inenhancing the solubility and phytoextraction of some metals(especially Cu) (Kos and Lestan, 2003; Meers et al., 2005;Tandy et al., 2006). However, for soils contaminated predomi-nantly with Pb, the extraction efficiency of EDTA remainssignificantly higher compared to EDDS (Tandy et al., 2004;Komarek et al., 2007). Xiong and Feng (2001) introduced chlo-ride salts as another alternative to chelating agents. Highly sol-uble chloride salts dissociate in soil solution providing cationscapable of exchanging the adsorbed metals and ligands neededto form water-soluble mobile complexes (such as CdClþ).Other studies (e.g., Smolders et al., 1998) also proved the pos-itive influence of chloride salts on metal (especially Cd) accu-mulation in plants. The phytoextraction efficiency is mostlyinfluenced by two parameters: (i) metal concentrations in planttissues, and (ii) high biomass yields of the plants. As freeEDTA is toxic to plants (Vassil et al., 1998), the applicationof chloride salts does not negatively influence, in such extent,plant biomass production (Xiong and Feng, 2001).

To the best of our knowledge, many recent phytoextractionstudies (e.g., Xiong and Feng, 2001; Lai and Chen, 2004; Hov-sepyan and Greipsson, 2005) are focused on one-year experi-ments with soils spiked with a single metal in a form (e.g.,Pb(NO3)2) that does not represent the geochemical positionof the metals in field conditions. The main aim of the pre-sented study is to evaluate the metal (Pb, Cd, Zn and Cu) phy-toextraction efficiency of a hybrid poplar (Populus nigraL.� Populus maximoviczii Henry.) grown on two differentcontaminated soils originating from a polluted smelting andmining area during a two-year (two vegetation periods) phy-toextraction experiment after the application of differentEDTA and NH4Cl concentrations.

2. Materials and methods

2.1. Study area and soil sampling

Two agricultural soils (A and B) with different physico-chemical charac-

teristics and different degrees of metal contamination (Table 1) were chosen

in the severely contaminated mining and smelting area of Prıbram, Czech Re-

public. The first site (soil A), representing an area contaminated predominantly

by smelting activities, is located in the close vicinity of a secondary Pb

smelter, operational for more than 200 years. The second site (soil B) repre-

sents an agricultural soil contaminated predominantly by historical mining ac-

tivities in the area (Rieuwerts et al., 2000). The sampling sites were chosen in

accordance with other works from the area dealing with contamination of for-

est and agricultural soils (Rieuwerts et al., 2000; Ettler et al., 2005a; Komarek

et al., 2006). The soils are classified as Gleyic Cambisols developed on a Pro-

terozoic volcano-sedimentary rock complex belonging to the Prıbram ore dis-

trict. The soils in the studied area are used for cultivation of wheat (Triticumaestivum L.), corn (Z. mays L.), barley (Hordeum sativum L.), rye (Secale ce-

reale L.), rape (Brassica napus oleifera L.) and horse bean (Faba vulgaris

Moench.). Only manure and NPK fertilizers are regularly applied to the soils

without any liming.

Samples were collected from the arable layer (0e20 cm) of the agricultural

soils. Samples used for the determination of basic physico-chemical character-

istics, metal concentrations and chemical fractionation were air-dried, homog-

enized and sieved through a 2-mm stainless sieve prior to further processing.

Soil used for pot experiments was air-dried, homogenized and sieved through

a 10-mm stainless sieve.

2.2. Basic physico-chemical characteristics of the studied soils

Soil pH was measured in suspension using a 1:2.5 (w/v) ratio of soil and

deionized water/0.2 M KCl. Cation exchange capacity (CEC) was determined

as a sum of basic cations and Al extracted with 0.1 M BaCl2 solution and the

extractable acidity. Soil acidity was measured by back titration with 0.05 M

NaOH. Particle size distribution was determined by the hydrometer method.

Total organic carbon (TOC) contents were determined by catalytic oxidation

(1250 �C) using ELTRA (Neuss, Germany) Metalyt CS1000S elemental ana-

lyzer. Available forms of soil nutrients (Ca, K, Mg and P) were determined us-

ing the Mehlich 3 soil extraction procedure (Zbıral, 2000). The approximate

water holding capacity of soils was determined by subtracting the weight of

a water-logged soil sample from the weight of an air-dried soil sample.

2.3. Total concentrations of metals and theirchemical fractionation in soils

Total metal concentrations in soils were determined using a two-step de-

composition procedure. Soil samples were decomposed using the dry ashing

procedure in a mixture of oxidizing gases (O2þO3þNOx) at 400 �C for

10 h in Dry Mode Mineralizer Apion (Tessek, Czech Republic). The ash

was then decomposed using HNO3 and HF, evaporated to dryness at 160 �Cand dissolved in diluted aqua regia. The standard reference material CRM

Light Sandy Soil 7001 (Analytika Prague, Czech Republic) was used for the

evaluation of measurement precision and accuracy. In order to determine metal

fractionation in studied soils, the sequential extraction technique by Quevau-

viller (1998) was used. Extraction solutions were prepared using chemicals

Table 1

Physico-chemical soil characteristics and total metal concentrations in the

studied soils

Soil A Soil B

pHH2O 4.82 6.60

pHKCl 3.85 5.77

CEC (mmol kg�1) 113 134

TOC (%) 1.9 2.6

Particle size distribution (%)

Sand 53.3 43.6

Silt 40.7 47.5

Clay 6.0 8.9

Oxalate-extractable (g kg�1)

Fe 3.69 3.04

Mn 1.56 0.69

Al 0.89 0.59

Available nutrients after

Mehlich 3 (mg kg�1) (n¼ 3)

Ca 182� 25 1058� 30

K 164� 4 392� 1

Mg 26.4� 0.9 121� 5

P 190� 2 162� 0.1

Total metal concentrations

(mg kg�1) (n¼ 3)

Pb 1360� 10 200� 2

Cd 4.86� 0.33 1.61� 0.18

Cu 76.3� 13.2 61.9� 9.2

Zn 266� 31 169� 3

29M. Komarek et al. / Environmental Pollution 151 (2008) 27e38

of analytical grade (Lachema, Czech Republic and Merck, Germany) and de-

ionized water. The following fractions were determined: FA (Fraction A) e

exchangeable and acid-extractable (0.11 M CH3COOH-extractable); FB

(Fraction B) e reducible (0.1 M NH2OH$HCl-extractable); FC (Fraction C) e

oxidizable (8.8 M H2O2/1 M CH3COONH4-extractable); FD (Fraction D) eresidual, computed as the sum of fractions A, B, C subtracted from the total

concentration. Metal concentrations in extracts were determined using ICP-

OES (Vista Pro, Varian, Australia).

2.4. Extraction efficiency of the mobilizing agents

Twenty-four-hour extraction experiments were carried in order to deter-

mine the approximate amounts of metals mobilizable by EDTA and NH4Cl.

An aliquot part of 50 g of air-dried soil was placed into acid-clean polyethyl-

ene bottles. An amount of 15 ml of the mobilizing solutions, resulting thus into

a w60% water holding capacity, was added to the soil samples. The concen-

trations of the mobilizing solutions used were 3, 6, 9 mmol EDTA kg�1 and

10, 20, 30 mmol NH4Cl kg�1, respectively, as the total concentrations used

in the pot experiment. The control variant was treated with 15 ml of deionized

water. After 24 h, soil samples were extracted using 125 ml of deionized water

on an end-over-end shaker at 30 rpm and centrifuged. The water-soluble con-

centrations of selected metals (Pb, Cd, Zn, Cu, Fe, Mn, Ca Mg and K) were

determined using ICP-OES and a flame atomic absorption spectrometer

(FAAS) (SpectrAA 300, Varian, Australia). The results were further used for

speciation modeling.

2.5. Pot experiments

The metal phytoextraction efficiency of the hybrid poplar was assessed

during a two-year phytoextraction process using pot experiments. The pots

were kept in an outdoor weather-controlled vegetation hall. Poplars were cho-

sen because it is a fast growing tolerant tree species capable of extracting sig-

nificant amounts of metals from soils (Robinson et al., 2000; Liphadzi et al.,

2003). The species used was a hybrid poplar (P. nigra L.� P. maximowiczii

Henry.), which has been already tested for its phytoextraction potential by sev-

eral authors (Fischerova et al., 2006; Komarek et al., 2007). Cuttings with

a similar diameter originating from one specimen were chosen for the exper-

iment. Each pot contained 5 kg of air-dried and sieved (10-mm sieve) soil.

Two poplar cuttings were planted in each pot. Each pot was fertilized at the

beginning of each vegetation period with 0.5 g of N as NH4NO3; 0.16 g of

P and 0.4 g of K as K2HPO4. Due to the lack of Mg in soil A (Table 1), poplars

grown on this soil were additionally fertilized five times with 0.2 g of Mg as

Mg(NO3)2). Pots were watered twice per day using only deionized water in

order to maintain w60% of the water holding capacity.

The metal mobilizing amendments applied to poplars were: (i) EDTA as

a highly effective Pb-complexing chelating agent (Luo et al., 2005; Komarek

et al., 2007) and (ii) NH4Cl as an inorganic mobilizing amendment (Xiong and

Feng, 2001). The EDTA and NH4Cl treatments were split into three 15-ml

doses (3� 1, 3� 2, 3� 3 mmol EDTA kg soil�1 and 3� 3.3, 3� 6.6,

3� 10 mmol NH4Cl kg soil�1, respectively) in order to achieve higher phy-

toextraction efficiency by reducing the phytotoxicity effects together with min-

imizing the risks associated with leaching of the mobilized metals (Shen et al.,

2002). The first application of the mobilizing agents was added to plants after

100 days of growth. The following applications were added after 10 and 20

days. The control variant was treated with deionized water only. Each

EDTA and NH4Cl treatment as well as the control treatment was conducted

in quadruplicates. The above-ground biomass was harvested after 10 days

from the last EDTA and NH4Cl addition (i.e., 130 days after planting).

Dead poplar plants were removed and new cuttings were planted into pots be-

fore the second vegetation period. The applications of fertilizers and mobiliz-

ing amendments followed the same pattern during the second vegetation

period with the only exception that mobilizing amendments were applied

only to half of the pots. The second half remained untreated. Soil samples

were collected from pots after each vegetation period in order to evaluate

changes in metal mobility during the phytoextraction process.

During harvests, poplar stems and leaves were carefully separated. The

biomass was washed carefully using deionized water and dried at 60 �C until

constant weight and finely ground prior to decomposition. Samples were de-

composed using the dry ashing procedure. An aliquot part of the plant samples

was decomposed in a mixture of oxidizing gases (O2þO3þNOx) at 400 �Cfor 10 h in Dry Mode Mineralizer Apion. The ash was dissolved in 1.5%

HNO3 (Analytika, Czech Republic). Metal concentrations in digests were de-

termined using ICP-OES. The standard reference material DC73350 Leaves of

Poplar (China National Analysis Centre for Iron and Steel, China) was used

for evaluating the measurement precision.

2.6. Speciation calculations

In order to estimate the speciation of metals in soil extracts after the appli-

cation of mobilizing agents, the PHREEQC-2 speciation/solubility code (Par-

khurst and Appelo, 1999) version 2.13.0 for Windows, was used together with

the Minteq.v4.dat thermodynamic database (derived from MINTEQA2 code,

version 4, U.S. EPA, 1999). Metal concentrations (Pb, Cd, Zn, Cu, Fe, Mn,

Ca, Mg and K) obtained from the EDTA and NH4Cl extraction experiments

and pH values of the water extracts were used for the calculations.

2.7. Statistical evaluation

All statistical analyses were performed using analyses of variance (ANOVA)

with consequent Tukey test (software Statistica 6.0, StatSoft). For each set of

sorted means obtained from repeated measurements, the probability was

assessed under the null hypothesis. The results were evaluated on the basis of

homogenous groups at a given significance level ( p< 0.05).

3. Results and discussion

3.1. Characteristics of the studied soils

Basic physico-chemical characteristics of the studied soilsare summarized in Table 1. Due to the relatively low pH ofsoil A, leaching of the bases occurred and significantly loweramounts of available nutrients (Ca, K and Mg) are present inthis soil (Table 1). The chemical fractionation of metals inthe soils is given in Fig. 1. Data obtained from the chemicalfractionation of the studied soils suggest that Pb, as the maincontaminant, is predominantly bound to the reducible (FB)and oxidizable (FC) fractions of the soil complex dependingon the contents of TOC and Fe, Mn, Al oxides and hydroxides(Table 1). Only small portions of Pb are present in the most

Soil A

Pb Cd Zn Cu

%

0

20

40

60

80

100Soil B

Pb Cd Zn Cu

Fraction CFraction AFraction B Fraction D

Fig. 1. Chemical fractionation of Pb, Cd, Cu and Zn in studied soils: Fraction

A, exchangeable and acid-extractable; Fraction B, reducible; Fraction C, oxi-

dizable; Fraction D, residual.

30 M. Komarek et al. / Environmental Pollution 151 (2008) 27e38

‘‘labile’’ fractions (FA) of both soils. Due to the lower pH andhigher total concentrations of Pb, a higher amount of Pb in FA

was found in soil A (5.4%) compared to soil B (0.6%). Becauseof high contents of anthropogenic Pb, only a small portion (1.4and 5.3%) was bound to the residual fraction of the soils. Ahigh concentration of Cd in soils (26 and 48%) was, as ex-pected, present in the mobile phase (FA), suggesting a high mo-bility of Cd in the studied soils. The majority of Zn and Cuwere present in the residual fraction (FD) (49 and 53% of Zn;66 and 81% of Cu). A higher portion of 10 and 14% fromthe total Zn concentration in both soils is exchangeable andacid-extractable (FA). Similar to Pb, just a small amount ofCu in both soils was present in FA in both soils (2.2 and2.9%). The higher portion of easily extractable (more mobile)metals in soil A compared to soil B is probably due to the lowerpH of soil A (Table 1). Additionally, metals originating fromthe more recent smelting activities are usually more mobilethan metals originating from the older mining activities, mainlydue to their different mineralogical and geochemical nature(Rieuwerts et al., 2000; Ettler et al., 2005b).

3.2. The effect of NH4Cl and EDTA onmetal mobility in studied soils

The changes of water-soluble metal (Pb, Cd, Cu, Zn) con-centrations after the application of NH4Cl and EDTA are sum-marized in Table 2. The application of NH4Cl to both soils didnot increase water-soluble Cu and Pb concentrations. Chloridesadded to the soils actually reduced the water-soluble Pb con-centrations. However, these decreases were not statistically sig-nificant. The PHREEQC-2 speciation modeling showed thatthe most abundant species of Pb after the chloride addition isPbCl4

2�, which is most probably sorbed under the low pH(4.8) of the soil A. The abundance of this species is evenmore emphasized at higher chloride concentrations. Addition-ally, the low mobility of Pb in soils (Fig. 1) plays an importantrole here as well. The same pattern was observed for Cu. Theaddition of chlorides does not mobilize Cu in soils, which is

present mostly as free Cu2þ in the soils, even after the chloridesalt addition. The most abundant species of Cd and Zn areCdClþ and ZnClþ, respectively. The highest chloride concen-tration in the solution leads to the preferential formation ofCdCl3

� and ZnCl42�. The addition of NH4Cl helps to mobilize

mostly Cd (up to a 20-fold increase compared to the control)and Zn (up to a ninefold increase) from soil A with a low pHvalue (Tables 1 and 2). In the case of soil B (with a near-neutralpH of 6.6), a slightly different pattern was observed. The addi-tion of NH4Cl did not positively influence the mobility of anymetal (Table 2). This is probably caused by the higher pH of thesoil extract, which favors sorption of the positively chargedmetal ions to the soil complex.

As expected, the chelating agent EDTA was much more ef-ficient than NH4Cl in solubilizing and complexing metalsfrom the soil matrix. Increasing the EDTA concentrationincreased the soluble concentrations of all metals. Higher ex-traction efficiencies were observed in soil B, mostly becauseof the higher pH of the soil (the vast majority of the metaleEDTA complexes are present as negatively charged [MeeEDTA]2� complexes). The addition of the highest EDTAdose (9 mmol kg�1) to soils led to a 120-fold (soil A) anda 180-fold (soil B) increase of soluble Pb compared to the con-trol after one day. This increase was even more pronounced inthe case of Cd in soil B (up to a 290-fold increase). Complexesof EDTA with Pb, Fe and Cu are the predominant species. Ironis mainly oxidised and forms [FeeEDTA]� and [FeOHeEDTA]2� species. The role of [FeOHeEDTA]2� complexesis more emphasized in soil B due to the higher pH of thesoil extract. The competition of Fe during EDTA complexa-tion plays an important role in soils. The addition of EDTAleads to a slow dissolution of Fe oxides and hydroxides andthe subsequent formation of FeeEDTA complexes (log K¼27.2) controls the metal extraction efficiency of EDTA(Nowack et al., 2006). This was proved, in our previous study,by the increasing concentrations of solubilized Fe anddecreasing concentrations of solubilized Pb in soils after theEDTA addition during 28 days (Komarek et al., 2007).

Table 2

The extraction efficiency of the tested mobilizing agents

Pb (mg kg�1) Cd (mg kg�1) Cu (mg kg�1) Zn (mg kg�1)

Soil A

Control 3.62a� 0.44 0.02a� 0.003 0.08a� 0.02 0.57a� 0.07

10 mmol NH4Cl kg�1 0.70a� 0.01 0.14b� 0.002 0.07a� 0.01 2.49b� 0.03

20 mmol NH4Cl kg�1 0.53a� 0.04 0.25c� 0.01 0.08a� 0.01 3.80c� 0.16

30 mmol NH4Cl kg�1 0.67a� 0.04 0.39d� 0.006 0.09a� 0.01 5.09d� 0.07

3 mmol EDTA kg�1 95.6b� 2.2 0.57e� 0.01 2.23b� 0.18 11.7e� 0.1

6 mmol EDTA kg�1 284c� 3 1.36f� 0.02 10.5c� 0.1 25.2f� 0.3

9 mmol EDTA kg�1 440d� 7 2.01g� 0.03 15.1d� 0.2 34.9g� 0.4

Soil B

Control 0.56a� 0.06 0.004a� 0.0008 0.10a� 0.02 0.47a� 0.12

10 mmol NH4Cl kg�1 0.21a� 0.05 0.004a� 0.001 0.12a� 0.02 0.42a� 0.03

20 mmol NH4Cl kg�1 0.11a� 0.02 0.004a� 0.001 0.10a� 0.03 0.37a� 0.01

30 mmol NH4Cl kg�1 0.06a� 0.03 0.004a� 0.001 0.15a� 0.03 0.40a� 0.01

3 mmol EDTA kg�1 66.0b� 0.2 1.09b� 0.004 1.69b� 0.03 18.3b� 0.01

6 mmol EDTA kg�1 86.5c� 0.01 1.15c� 0.01 2.31c� 0.09 19.3c� 0.1

9 mmol EDTA kg�1 101d� 0.3 1.17c� 0.004 3.02d� 0.10 19.7d� 0.1

Data shown are means� SD (n¼ 3). Data with the same index represent statistically identical values ( p< 0.05).

31M. Komarek et al. / Environmental Pollution 151 (2008) 27e38

3.3. The effect of NH4Cl and EDTA onbiomass yields of poplar

The biomass yields of poplar plants (stems and leaves)grown on both soils during the two vegetation periods afterthe application of mobilizing agents are summarized inFig. 2. The significantly lower biomass yields were obtainedfor poplars grown on soil A, predominantly due to the lowerpH of the soil, and to the higher concentrations of mobilemetals (Table 1; Fig. 1). Additionally, poplars grown on soilA treated with 6 and 9 mmol EDTA kg�1 died during the firstvegetation period and the newly planted cuttings did notgrow further during the second year. Similarly in the case ofsoil B, the further addition of 9 mmol EDTA kg�1 hinderedplant growth to such extent that this additional applicationcaused plant death. This was probably caused not only by thehigh concentrations of metals in soil solution, but to someextent, by the possible presence of free EDTA, which provedto be more phytotoxic to plants than metaleEDTA complexes

(Vassil et al., 1998). Higher biomass yields were obtained dur-ing the first vegetation period compared to the second year.Furthermore, the repeated addition of chelating agents duringthe second year led to lower biomass production compared topots untreated in the second year. Poplars grown on the morecontaminated soil A exhibited leaf chlorosis and necrosissymptoms, which were probably caused by the low pH, lackof available Fe and Mg and phytotoxicity of mobilized metalspresent in soils. Increasing the concentration of chlorides led toa decrease of biomass yields, especially on soil B (Fig. 2). Drybiomass yields of poplar stems did not significantly changeafter any EDTA addition to both soils during the first year.However, the addition of 6 and especially 3 mmol EDTA kg�1

significantly increased leaf biomass of poplars grown on theless contaminated soil B (Fig. 2). This is probably due to thefact that the added chelating agent helped the poplars to acquirelacked nutrients (especially Fe) from the soil and reducedthe phytotoxicity of some free ions (e.g., Zn2þ and Cd2þ) pres-ent in the soil solution in relatively high concentrations by

Soil A

0

10

20

30

40

50

1st year leaves

2nd year leaves U

2nd year leaves T

Soil B

Soil BSoil A

0

10

20

30

40

1st year stems

2nd year stems U

2nd year stems T

g po

t-1g

pot-1

Contro

l

mmol NH4Cl kg-1 mmol EDTA kg-1

3×6.6

3×3.3 3×

10 3×1

3×2

3×3

Contro

l

mmol NH4Cl kg-1 mmol EDTA kg-1

3×6.6

3×3.3 3×

10 3×1

3×2

3×3

Fig. 2. Biomass yields of poplar stems and leaves during the two-year phytoextraction process. U, plants untreated with mobilizing agents during the second

vegetation period. T, plants additionally treated with mobilizing agents during the second vegetation period. Data shown are means� SD (n¼ 4).

32 M. Komarek et al. / Environmental Pollution 151 (2008) 27e38

complexing them and making them less available to plants(Wallace, 1980).

3.4. The effect of NH4Cl and EDTA onmetal uptake by poplar

Metal (Pb, Cd, Cu and Zn) concentrations in poplar stemsand leaves after the application of mobilizing agents are sum-marized in Figs. 3 and 4. Higher concentrations of metals inpoplar stems and leaves were observed on soil A. The concen-trations of Pb and Cu in control (untreated) plants were similaror lower during the second vegetation period, suggesting thatthe already low bioavailable pool of Pb and Cu was depletedafter the first year. The increase of Cd and Zn concentrationsin untreated poplar plants after the second vegetation periodcompared to the first one could be explained by the decreaseof soil pH (from 4.8 to 4.6 in soil A and from 6.6 to 4.8 insoil B) after the second year of plant growth. This is in accor-dance with other works that observed that trees can mobilizemetals through progressive soil acidification (Mayer, 1998).Additionally, the application of fertilizers to the pots couldhave led to a slight pH decrease as well. However, concentra-tions of metals in leaves of fast growing tree species cultivatedin the field are usually much lower than in trees grown in lab-oratory conditions (Dickinson, 2006).

The addition of NH4Cl did not significantly enhance Pb andCu uptake to poplar plants during the first year, mostly due tothe low extraction efficiency of the chloride salt. These resultsare, therefore, contradictory to the work of Xiong and Feng(2001), where equilibrium between the artificially added Pb(as highly soluble Pb(NO3)2) and the soil constituents couldnot have been established due to a short time between thePb addition and Pb phytoextraction. However, adding NH4Clto soil significantly increased Cd and Zn concentrations inleaves, especially in the case of soil B, proving thus a strongtranslocation rate of Zn and Cd within poplar plants (Figs. 3and 4). This suggests that the formation of mobile CdClþ

and ZnClþ favors plant uptake (Smolders et al., 1998). Thiscorresponds well to the similar biogeochemical behavior ofthese two metals. Increased concentrations of Zn and Cd inpoplar plants were already described by other authors (e.g.,Mertens et al., 2004). During the second vegetation period,no significant differences between treated and untreated vari-ants were observed. Increasing further the concentration ofNH4Cl (to up to 60 mmol kg�1 in total) would have rather neg-ative effects on the soil properties. Surprisingly, the highestconcentrations of Cd (35.6 mg kg�1) and Zn (509 mg kg�1)in poplar leaves from soil A were observed after the two appli-cations of the lowest NH4Cl dose (2� 10 mmol NH4Cl kg�1).In the case of soil B, the highest Cd (23.7 mg kg�1) and Zn(926 mg kg�1) concentrations were observed after the additionof 2� 30 and 1� 30 mmol NH4Cl kg�1. However, as men-tioned before, the differences between the variants treatedonce and twice during the two-year process were not statisti-cally significant. Interestingly, higher Zn concentrations wereobserved in poplar leaves grown on soil B compared to soilA during the second year (Figs. 3 and 4). This is probably

associated with the steeper decrease of pH of the soil B afterthe first vegetation season (by 0.2 units in soil A and by up to1.8 units in soil B).

The addition of EDTA significantly enhanced the uptake ofPb especially into poplar leaves, proving thus a strong translo-cation rate of the PbeEDTA complexes. Because there is nospecific Pb transporter known for selective Pb uptake, the trans-location of Pb into plant shoots is most likely in the form of PbeEDTA complexes through a passive uptake mechanism (Epsteinet al., 1999; Sarret et al., 2001; Schaider et al., 2006). Theformed PbeEDTA complexes may enter the root system at dis-ruptions of the endodermis, root tips and at the Casparian strip(Haynes, 1980). EDTA significantly enhanced the phytoextrac-tion efficiency only for Pb. For example, NH4Cl was more effi-cient than EDTA in enhancing the uptake of Cd into leaves(Figs. 3 and 4). However, this will be the case only for multi-metal contaminated soils where EDTA preferentially complex-es Pb due to the higher Pb concentrations in the soils. Duringthe first vegetation season, the highest Pb concentration(266 mg kg�1) was observed in poplar leaves after the applica-tion of 9 mmol EDTA kg�1. The additional application ofEDTA to the soils during the second vegetation period hinderedplant growth and in the case of higher EDTA doses, led to dyingof plants. However, the additional split application of 3 and6 mmol EDTA kg�1 further increased the uptake of Pb intoleaves (to up to 240 mg Pb kg�1 in soil A and 102 mg Pb kg�1

in soil B; Figs. 3 and 4). The application of EDTA helped tomaintain a relatively high portion of available Pb in soil Beven during the second year, which led to higher concentrationsof Pb in the poplar biomass compared to the first year. The ex-traction efficiency of EDTA is not only influenced by the stabil-ity constant (log K ), but also by the concentrations of the metalsand the ligands. Therefore, the phytoextraction efficiency ofEDTA will be mostly controlled by Pb (present in the soils inhigh concentrations) and Fe (from the dissolved Fe oxides)(Komarek et al., 2007). Only high concentrations of EDTA(9 mmol kg�1) affected significantly the uptake of Cd and Cu.

3.5. Phytoextraction efficiency and associatedenvironmental hazards

The phytoextraction efficiency of plants does not only de-pend on metal concentration in aboveground biomass, but toa great extent, on the biomass yields of the plants. In orderto evaluate the efficiency of the phytoextraction process duringseparate vegetation periods and after different treatments withNH4Cl and EDTA, the remediation factor (RF) was calculatedas follows (Vyslou�zilova et al., 2003):

RF ð%Þ ¼MeplantBplant

Mesoilwsoil

100 ð1Þ

where Meplant is the concentration of a metal in plant dry bio-mass (mg g�1); Bplant the dry weight plant biomass yield (g);Mesoil the total concentration of a metal in soil (mg kg�1)and wsoil the amount of soil in pot (g). The RF, therefore, re-flects the amount of metals extracted by plants from the soil

33M. Komarek et al. / Environmental Pollution 151 (2008) 27e38

mg

Cd

kg-1

0

5

10

15

20

25

mg

Zn k

g-1

0

100

200

300

mg

Cu

kg-1

0

5

10

15

20

mg

Pb k

g-1

0

50

100

150

200

2501st year stems2nd year stems U2nd year stems T

mg

Cd

kg-1

0

10

20

30

40

mg

Zn k

g-1

0

100

200

300

400

500

mg

Cu

kg-1

0

10

20

30

mg

Pb k

g-1

0

50

100

150

200

250

300

3501st year leaves2nd year leaves U2nd year leaves T

Contro

l

mmol NH4Cl kg-1 mmol EDTA kg-13×

6.63×

3.3 3×10 3×

13×

23×

3

Contro

l

mmol EDTA kg-13×

6.63×

3.3 3×10 3×

13×

23×

3

Contro

l

mmol NH4Cl kg-1 mmol EDTA kg-13×

6.63×

3.3 3×10 3×

13×

23×

3

Contro

l

mmol NH4Cl kg-1 mmol EDTA kg-13×

6.63×

3.3 3×10 3×

13×

23×

3

Contro

l3×

6.63×

3.3 3×10 3×

13×

23×

3

Contro

l

mmol NH4Cl kg-13×

6.63×

3.3 3×10 3×

13×

23×

3

stems

leaves

Fig. 3. Metal (Pb, Cd, Cu, Zn) concentrations in poplar stems and leaves grown on soil A during the two-year phytoextraction process. U, plants untreated with

mobilizing agents during the second vegetation period. T, plants additionally treated with mobilizing agents during the second vegetation period. Data shown are

means� SD (n¼ 4).

34 M. Komarek et al. / Environmental Pollution 151 (2008) 27e38

mg

Cu

kg-1

0

5

10

15

20

25

mg

Pb k

g-1

0

20

40

60

801st year stems2nd year stems U2nd year stems T

mg

Cd

kg-1

0

2

4

6

8

10

12

14

mg

Zn k

g-1

0

100

200

300

400

mg

Cu

kg-1

0

5

10

15

20

25

mg

Pb k

g-1

0

20

40

60

80

100

120

1st year leaves2nd year leaves U2nd year leaves T

0

5

10

15

20

25

30

mg

Cd

kg-1

mg

Zn k

g-1

0

200

400

600

800

1000

Contro

l

mmol NH4Cl kg-1 mmol EDTA kg-13×

6.63×

3.3 3×10 3×

13×

23×

3

Contro

l

mmol NH4Cl kg-1 mmol EDTA kg-13×

6.63×

3.3 3×10 3×

13×

23×

3

Contro

l

mmol NH4Cl kg-1 mmol EDTA kg-13×

6.63×

3.3 3×10 3×

13×

23×

3

Contro

l

mmol NH4Cl kg-1 mmol EDTA kg-13×

6.63×

3.3 3×10 3×

13×

23×

3

stems

leaves

Fig. 4. Metal (Pb, Cd, Cu, Zn) concentrations in poplar stems and leaves grown on soil B during the two-year phytoextraction process. U, plants untreated with

mobilizing agents during the second vegetation period. T, plants additionally treated with mobilizing agents during the second vegetation period. Data shown are

means� SD (n¼ 4).

35M. Komarek et al. / Environmental Pollution 151 (2008) 27e38

during one vegetation period. The calculated RFs for separateyears and different mobilizing amendments are given in Table 3.

The results obtained from RF calculations suggest thatchemically enhanced phytoextraction of Pb from such contam-inated soils with several metals is not a suitable remediationmethod. The highest Pb RFs after the two years of the phytoex-traction process were obtained on soil B and reached only0.17% (6 and 3þ 3 mmol EDTA kg�1 treatments) and 0.18%(6þ 6 mmol EDTA kg�1 treatments). The Pb RF values ob-tained on soil A are very low (maximum 0.04%), due to thehigh total concentration of Pb in the soil. Better results wereobtained for Cd: the RF values are more than 1% for all treat-ments during the first vegetation period. The lower Cd RFsafter the second vegetation period are mostly due to lower bio-mass yields (especially on soil A). Interestingly, the highest CdRF values (2.22%) for soil A after the two-year phytoextractionprocess were obtained for the untreated control variant, mostlydue to the higher biomass yields obtained during the secondyear. Again, higher Cd phytoextraction efficiencies wereobtained on soil B where the highest RFs after the two yearswere found for 10þ 10 mmol NH4Cl kg�1 (10.8%) and for20 mmol NH4Cl kg�1 (10.4%). However, the control variantgave a relatively high RF value (8.13%) as well. The Cu RFvalues are very low and all the treatments (including thecontrol) showed similar results. Promising results for Znafter two years (RF¼ 4.48%) were obtained after the applica-tion of a single dose of 10 mmol NH4Cl kg�1 to soil B.However, the Zn RF values obtained after two years from theuntreated control plants were high enough (3.80%) as in thecase of Cd. Therefore, the application of these mobilizing agentsis not needed to enhance the uptake of either Cd or Zn by poplar.

In order to evaluate the changes in mobility of metalsduring the two-year phytoextraction process, the results fromthe first step of the sequential extraction procedure (easily mo-bilizable metals obtained from the 0.11 M CH3COOH extrac-tion) were compared after separate vegetation periods. The

results are given in Fig. 5. Without any addition of mobilizingagents an increase in the metal mobility occurred throughoutthe two-year process, which is associated with the loweringpH (by up to 1.8 units in soil B) and the presence of root ex-udates capable of mobilizing nutrients and metals. The addi-tion of 9 mmol EDTA kg�1 maintained a high portion of Pband Cu (up to 500 mg Pb kg�1 in soil A) in the labile fractionof the soils throughout the two vegetation periods. The in-creased metal concentrations are associated to a great extentwith the dissolution of the Fe- and Mn-oxides and hydroxidesafter the application of EDTA and the remobilization of thesorbed metals (Nowack et al., 2006). The addition of lowerconcentrations of NH4Cl did not significantly enhance the mo-bility of metals compared to the controls during the two yearsas did EDTA. Therefore, the application of EDTA, which isa persistent chelating agent, can lead to unwanted leachingof the chelates throughout the soil profile and thus presentan important environmental hazard.

Another phytoremediation strategy involving poplars is themethod of phytostabilization (Mertens et al., 2004). However,in our experiment, a lowering of pH occurred after the two-year phytoremediation process leading to an increased mobil-ity of the metals. Furthermore, increased concentrations offoliar Cd in control plants could lead to its redistribution insoils by leaf degradation and could thus introduce Cd intothe food chain. This poplar clone is therefore not suitablefor phytostabilization on such contaminated soils.

4. Conclusions

Phytoextraction of metals from contaminated soils belongscurrently to one of the most studied methods of environmen-tally friendly and cost effective soil remediation. In order tobe feasible, a successful chemically enhanced phytoextractionprocess must meet the following requirements: (i) the metalcontents in soils must be reduced to an acceptable level in

Table 3

Remediation factors (RF) in % obtained after two subsequent cropping seasons

Soil A e 1st year Soil A e 2nd year

Pb Cd Cu Zn Pb Cd Cu Zn

Control 0.01 1.06 0.04 0.17 0.01 1.16 0.04 0.31

10 mmol NH4Cl kg�1 0.01 1.01 0.04 0.27 0.01 (0.01) 0.87 (0.69) 0.04 (0.03) 0.24 (0.16)

20 mmol NH4Cl kg�1 0.01 1.07 0.04 0.30 0.01 (0.01) 0.74 (0.65) 0.02 (0.03) 0.20 (0.15)

30 mmol NH4Cl kg�1 0.01 1.23 0.04 0.32 0.01 (0.01) 0.53 (0.47) 0.02 (0.02) 0.12 (0.12)

3 mmol EDTA kg�1 0.03 1.27 0.05 0.33 0.01 (0.02) 0.79 (0.77) 0.04 (0.04) 0.20 (0.18)

6 mmol EDTA kg�1 0.03 1.26 0.05 0.33 n.a. n.a. n.a. n.a.

9 mmol EDTA kg�1 0.04 1.06 0.05 0.31 n.a. n.a. n.a. n.a.

Soil B e 1st year Soil B e 2nd year

Control 0.01 2.96 0.15 1.01 0.02 5.17 0.15 2.79

10 mmol NH4Cl kg�1 0.01 4.59 0.17 1.55 0.02 (0.02) 5.55 (6.18) 0.15 (0.15) 2.93 (2.76)

20 mmol NH4Cl kg�1 0.01 4.33 0.14 1.40 0.01 (0.01) 6.10 (5.13) 0.12 (0.08) 2.44 (1.87)

30 mmol NH4Cl kg�1 0.01 3.51 0.13 1.23 0.01 (0.004) 3.86 (2.47) 0.07 (0.04) 1.52 (0.75)

3 mmol EDTA kg�1 0.09 4.06 0.18 1.56 0.03 (0.08) 4.02 (2.18) 0.16 (0.10) 1.65 (1.80)

6 mmol EDTA kg�1 0.15 4.91 0.19 1.83 0.02 (0.03) 0.83 (0.37) 0.04 (0.02) 0.27 (0.19)

9 mmol EDTA kg�1 0.14 3.64 0.16 1.52 0.01 (n.a.) 0.24 (n.a.) 0.01 (n.a.) 0.11 (n.a.)

Data in parentheses represent variants treated with additional doses of mobilizing agents during the second vegetation period.

36 M. Komarek et al. / Environmental Pollution 151 (2008) 27e38

mmol NH4Cl kg-1 mmol EDTA kg-1 mmol NH4Cl kg-1 mmol EDTA kg-1

mg

Pb k

g-1

0

100

200

300

400

500

600Original soil1st year2nd year U2nd year T

mg

Cd

kg-1

0

1

2

3

4

5

mg

Zn k

g-1

0

20

40

60

mg

Cu

kg-1

0

2

4

6

8

10

12

14

16

Contro

l3×

6.63×

3.3 3×10 3×

13×

23×

3

Origina

l soil

Contro

l3×

6.63×

3.3 3×10 3×

13×

23×

3

Origina

l soil

mg

Pb k

g-1

0

10

20

30

40

50

mg

Zn k

g-1

0

50

100

150

200

mg

Cu

kg-1

0

2

4

6

mg

Cd

kg-1

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

Contro

l

mmol NH4Cl kg-1 mmol EDTA kg-1

3×6.6

3×3.3 3×

10 3×1

3×2

3×3

Origina

l soil

Contro

l

mmol NH4Cl kg-1 mmol EDTA kg-1

3×6.6

3×3.3 3×

10 3×1

3×2

3×3

Origina

l soil

Original soil1st year2nd year U2nd year T

soil A

soil B

Fig. 5. Evolution of metal (Pb, Cd, Cu, Zn) concentrations in soil Fraction A (exchangeable and acid-extractable) during the two-year phytoextraction process. U,

pots untreated with mobilizing agents during the second vegetation period. T, pots additionally treated with mobilizing agents during the second vegetation period.

Data shown are means� SD (n¼ 4).

37M. Komarek et al. / Environmental Pollution 151 (2008) 27e38

a reasonable time frame; (ii) the hazards associated with theapplication of metal mobilizing agents (e.g., chelate leaching)must be minimized. From our results it is possible to state thatthe use of poplar during the two-year chemically enhancedphytoextraction of metals from severely contaminated soils(such as soil A) is not a suitable method for soil remediationdue to its low efficiency (low remediation factors) and dueto the risks associated with the application of the metal mobi-lizing agents, such as the possible leaching of the chelatesto groundwater. Especially, in the case of Pb (the maincontaminant in the area), the phytoextraction efficiencies onboth studied soils were very low for a successful phytoreme-diation process. Satisfying results were obtained during thetwo-year phytoextraction of Cd especially from soil B (mainlybecause of the significantly lower Cd concentration in thesoil). The application of NH4Cl proved to be more efficientthan EDTA during the phytoextraction of Cd and Zn. Never-theless, remediation factors for Cd and Zn obtained from thecontrol (untreated) variants were high enough and therefore,the application of mobilizing agents would have rather nega-tive effects. Additionally, the application of EDTA increasedthe mobility of the metals after the two-year phytoextractionprocess. Furthermore, this poplar clone is not suitable forphytostabilization due to the high concentrations of foliar Cdin untreated plants and its subsequent possible introductioninto the food chain.

Acknowledgements

The presented study was supported by the research projectsMSM 6046070901 (Ministry of Education of the Czech Re-public) and GA�CR 526/06/0418 (Czech Science Foundation).The authors thank Mr. Jan Weger (VUKOZ, Czech Republic)for providing poplar cuttings and two anonymous reviewersfor improving the quality of the manuscript.

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