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Lead Uptake and Translocation byWillows in Pot and Field ExperimentsOlena P. Zhivotovsky a , Yulia A. Kuzovkina a , Cristian P. Schulthess a

, Tom Morris a & Dawn Pettinelli aa Department of Plant Science and Landscape of Architecture,University of Connecticut, Storrs, Connecticut, USA

Available online: 26 May 2011

To cite this article: Olena P. Zhivotovsky, Yulia A. Kuzovkina, Cristian P. Schulthess, Tom Morris &Dawn Pettinelli (2011): Lead Uptake and Translocation by Willows in Pot and Field Experiments,International Journal of Phytoremediation, 13:8, 731-749

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International Journal of Phytoremediation, 13:731–749, 2011Copyright C© Taylor & Francis Group, LLCISSN: 1522-6514 print / 1549-7879 onlineDOI: 10.1080/15226514.2010.525555

LEAD UPTAKE AND TRANSLOCATION BY WILLOWSIN POT AND FIELD EXPERIMENTS

Olena P. Zhivotovsky, Yulia A. Kuzovkina,Cristian P. Schulthess, Tom Morris, and Dawn PettinelliDepartment of Plant Science and Landscape of Architecture, University ofConnecticut, Storrs, Connecticut, USA

Plant growth and lead (Pb) uptake by seven willow varieties were investigated in pot andfield experiments to assess the suitability of willows for phytoremediation of Pb at heavilycontaminated sites such as skeet ranges. Differences in uptake and translocation of Pbin Salix were observed between pot and field experiments. In the pot experiment, willowsgrown in Pb-contaminated field soil for 6 months showed tolerance to very high soil Pbconcentration (21360 mg kg−1), and with the addition of EDTA were able to take up andtranslocate more than 1000 mg kg−1 Pb into above-ground tissues. In the field experiment,all willow varieties showed tolerance to heterogeneously high soil Pb concentrations. Plantswere also able to take up and translocate Pb into above-ground tissues. However, after 4.5months, the lead concentration in the above-ground tissues of willows grown in soil amendedwith EDTA was less than 200 mg kg−1. The results from the pot experiment suggest thatSalix varieties have the potential to take up and translocate significant amounts of Pb intoabove-ground tissues using EDTA. However, to verify the phytoextraction abilities of Salixin the field, additional research is needed.

KEY WORDS phytoextraction, skeet range, Salix, EDTA, Pb

INTRODUCTION

In the United States alone, more than 50 000 metal-contaminated sites await remedi-ation (Ensley 2000), yet without remediation, harmful levels of contamination may remainin the soil up to 5000 years (Kumar et al. 1995). Lead is among the most widespread,persistent, and toxic soil contaminants. Short-term exposure to high levels of Pb can causebrain and kidney damage as well as gastrointestinal distress in humans, while long-termexposure can affect the central nervous system, blood, liver, and reproductive system (EPA2008). When present as a soil contaminant, Pb limits the ability of soil to sustain vegetationand a functioning ecosystem (Martin and Ruby 2004), and high available concentrationslead to plant death (Baker et al. 1976; Hoffmann 1983; Schaller and Diez 1991).

Address correspondence to Yulia A. Kuzovkina, Department of Plant Science and Landscape of Architecture,1376 Storrs Rd. Unit 4067, University of Connecticut, Storrs, CT 06269-4067, USA. E-mail: jkuzovkina@uconn.edu

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Because of the toxic nature of Pb, it is imperative to develop low-cost, effective,and sustainable methods to remove this heavy metal from the environment or to neutralizeit. Excavation and removal of polluted soil, the most commonly used cleanup method, isboth expensive and extremely environmentally disruptive (Huang and Cunningham 1996;Cooper et al. 1999; Garcia et al. 2004). Phytoremediation technology—the use of greenplants to extract, neutralize, or stabilize soil contaminants in situ (Salt et al. 1995)—holdspromise as a realistic, low-cost, ecologically sound, and sustainable alternative to excava-tion. One form of phytoremediation—phytostabilization—uses cover crops, or buffer strips,to stabilize contaminated sites but may involve another treatment over time or restrict fu-ture use of the site. Another form of phytoremediation—phytoextraction—is based on theability of some plant species to remove metals from the soil and concentrate contaminantsin the above-ground tissues (Kumar et al. 1995).

The use of Salix varieties for the remediation of metal-contaminated soils seemspromising, given their perennial nature and rapid growth, high resistance to many pollutants,long vegetative season, and high yields obtained on short rotations (Landberg and Greger1996; Tlustos et al. 2007; Kuzovkina et al. 2008). Further advantages of willows includethe ability to resprout after the harvest of aboveground biomass, significant transpirationrates, and their potential as a source of energy biomass. Recent research has investigatedwillows’ potential for the phytoextraction of heavy metals (Pulford et al. 2002; Watsonet al. 2003; Vandecasteele et al. 2004; Utmazian et al. 2007; Tlustos et al. 2007) andfor phytostabilization of degraded sites, including landfills (Maurice et al. 1999; Bungartand Huttl 2001; Vervaeke et al. 2003; Kuzovkina and Quigley 2005; Martin and Stephens2006).

Different species of willows, as well as some clones, vary considerably in their metaltranslocation patterns. Specific tolerance of plants to heavy metals is strongly correlatedwith the chemical characteristics of the soil and the level of contamination and is genotypespecific. Tolerance to Cd, Cu, and Zn has been documented for a few Salix species (Punshonand Dickinson 1997; Watson et al. 2003), but little is known about Salix tolerance tolead.

The objective of this study was to evaluate plant tolerance, metal mobility, andpatterns of lead translocation within the plant in Salix varieties using field soil in pot andfield experiments. Field experiments were conducted at a former skeet shotgun shootingrange in Sprague, CT, a site known to be heavily contaminated with lead.

MATERIAL AND METHODS

Site Description

The Mukluk site, in the town of Sprague, Connecticut (41◦38′N; 72◦ 06′W), is a4.9–5.7 hectare area highly contaminated with lead as a result of its former use as askeet shooting range. It is situated on steeply sloped terrain typical of the topography innortheastern Connecticut, and consists primarily of Paxton (coarse-loamy, mixed, active,mesic Oxyaquic Dystrudepts) and Woodbridge soils (coarse-loamy, mixed, active, mesicAquic Dystrudepts). Eighty-five to ninety percent of the area is forested with white oak(Quercus alba), ironwood (Carpinus caroliniana), sugar maple (Acer saccharum), andEastern white pine (Pinus strobus). The predominant herbaceous species at the site arelady fern (Athyrium filix-femina), cinnamon fern (Osmunda cinnamomea), Christmas fern

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(Polystichum acrostichoides), white wood aster (Eurybia divaricata), wild lettuce (Lactucacanadensis), and giant goldenrod (Solidago gigantea).

Soil Analyses

Soil analyses were conducted to assess the chemical and physical properties of thefield soil at the Mukluk site. Soil samples were taken from the experimental plot beforethe application of any soil amendments and again after plant harvest. Forty samples werecollected in 2007 from the A horizon (top 0–20 cm) within an area of 576 m2. Soil samplesfrom the A horizon of an adjacent uncontaminated site (213 m from the contaminatedarea) were collected to serve as a control. The samples from each area were screened topass through a 2 mm sieve and thoroughly mixed before analysis to ensure a homogenousconcentration of Pb.

A complete soil nutrient analysis was performed using the modified Morgan method(Wolf and Beegle 1995). Soil samples were analyzed for total metal content according toMiller (1991).

The sequential extraction technique. Soil was separated into six defined frac-tions following the procedure of Ma and Rao (1997), which represents a modified versionof the method described by Tessier et al. (1979). Two grams of soil were placed into a50-mL polypropylene copolymer tube with the following extraction steps. Water-solublefraction (F1): Soil extracted with 20 mL of deionized water (DI) for 2 h. Exchange-able fraction (F2): F1 residue extracted with 20-mL of 1 mol L−1 MgCl2, pH 7 for1 h. Carbonate-bound fraction (F3): F2 residue extracted with 20 mL of 1 mol L−1

NH4OAc, pH 5 for 5 h. Fe-Mn oxides-bound fraction (F4): F3 residue extracted with20 mL 0.04 mol L−1 NH2OH.HCl in 25% (v/v) HOAc at 96◦C with occasional agitation.Organic-bound fraction (F5): F4 residue extracted with 15 mL 30% H2O2 at pH 2 (adjustedwith HNO3) for 5.5 h (water bath, 85◦C). After cooling, 5 mL of 3.2 mol L−1 NH4OAcin 20% HNO3 was added and the solution was shaken for 30 min before final dilutionto 20 mL with DI water. Residual fraction (F6): (F5) residues digested using a HF–HCl/HNO3 (hydrofluoric/aqua regia) digestion procedure (Strelow and Jackson 1974; Sridharand Jackson 1974).

Aqua regia. Three mL of 12 mol L−1 of (HCl) 36–38% and 1 mL of 15 mol L−1

(HNO3) 69.3% were mixed just before use. F5 residue of 50 mg was extracted with 1 mLof aqua regia by adding it to the sample in a 250-mL polypropylene bottle along the insideof the bottle by using a pipette and further shaking for 10 min. After shaking, 10 mL ofHF was added and the bottle was closed immediately. Samples were shaken for 3 h, afterwhich time a saturated solution of boric acid was added to the samples to obtain a totalweight of 200 g for each bottle.

With the exception of F6, all of the solid phases were washed with 10 mL of DI waterbefore further extraction. The washes were collected and analyzed with supernatant fromthe previous fraction. After each extraction, the supernatant was separated by centrifugationfor 30 min. at 7500 ×g on a Sorvall RC-5B centrifuge. To verify the sum of metal recoveredin the sequential extraction steps, a separate total concentration of Pb was determined on thesubsample after the hydrofluoric/aqua regia digestion. Quality control was assured by theuse of procedural blanks. The recovery of trace metals in the sequential extraction steps waswithin 100 ± 10%. The total concentrations of metals in the supernatants from each stepwere analyzed using an inductively coupled plasma optical emission spectrophotometer

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Table 1 Willow varieties used in the pot and field experiments

Taxa Source

S. Allegany (S. purpurea L.) Double A Willow, NY, USAS. eriocephala Mich. (clone I.D.2005-40) native habitat, CT, USAS. nigra Marsh. (clone I.D.2005-30) native habitat, CT, USASV1 (S. dasyclados L.) Double A Willow, NY, USASX61 (S. sachalinensis L.) Double A Willow, NY, USASX64 (S. miyabeana L.) Double A Willow, NY, USASX67 (S. miyabeana L.) Double A Willow, NY, USA

(ELAN 6000 ICP-OES, Perkin-Elmer Corporation). The combined water-soluble and ion-exchangeable Pb fractions were considered to be available to plants.

Plant Material

Table 1 lists the 7 Salix varieties used in pot and field experiments. Cuttings of thesevarieties were obtained from native habitat in Connecticut and from the Double A Willownursery, New York.

Pot Experiment

Screened and thoroughly mixed field soil from the A horizon (0–20 cm) at the Mukluksite was used for the greenhouse pot experiment. Before planting, the total Pb concentra-tion of homogenized soil was determined with 5 replicates. Greenhouse temperature was15.5 ◦C night/ 18–21◦C day, with a high temperature limit of 24◦C. Twenty-centimeterdormant cuttings from one-year-old stems, selected for uniformity based on the diameterand the number of buds per cutting were planted in 400-mL plastic pots filled with amixture of 75% homogenized field soil and 25% perlite. The following 5 willow varietieswere planted one cutting per pot, and were grown for a period of 180 days from April toSeptember of 2008: S. nigra, S. eriocephala, SX61, SX64, and SX67. The experiment wasset in a completely randomized design with 4 replications and 3 treatments. The treatmentswere: uncontaminated field soil (control), contaminated soil (Pb), and contaminated soilwith EDTA (ethylenedinitrilotetraacetic acid, trisodium salt hydrate, 98%, Acros Organics,Belgium), (Pb +EDTA). The EDTA chelating agent was applied 7 days before harvestat a rate of 5 mmol kg−1 soil. To prevent the Pb and EDTA from leaching to drainage,individual plastic trays were placed under the pots. Throughout the growing season, plantswere watered as needed and fertilized once a month with 224 mg/L of nitrogen and235 mg/L of potassium. Metal-related phytotoxicity symptoms were monitored andrecorded throughout the experiment. At the end of the experiment all plants were har-vested and washed with deionized water and 1M HCl. Plants were separated into roots,wood (original cuttings), shoots (stems), and leaves. The tissues were cut into small piecesand dried in an oven for 48 hours at 70◦C. Dry weight was then recorded.

Field Experiment

Tree and litter removal. The contaminated field (576 m2) was cleared of vegeta-tion (90% of the woody plants and all the herbaceous plants cut at ground level), though

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WILLOWS’ LEAD TOLERANCE 735

tree stumps were left in the ground. Leaf litter was vacuumed in order to remove unburiedlead shot, but even after this effort, visible amounts of lead shot were still present on thesurface.

Preparation for planting. The site was fenced to prevent deer browsing. Surfaceroots of trees were cut and removed to facilitate planting. Where possible, the soil wastilled to a depth of 5–10 cm. Supplemental applications of synthetic urea (111 kg N ha−1)and potash (90 kg K ha−1) were applied before planting to stimulate plant growth.

A total trial size was 12.2 × 16.8 m, with 30.5 cm between rows, 61 cm betweentreatments, and 30.5 cm between plants. The total of 560 plants was used in the fieldexperiment. Based on site characteristics, which include slope and the shade from sur-rounding trees, identical RCBD experiments were set out on the contaminated site and ona non-contaminated control site (213 m from the contaminated site) with 4 replicates.

The EDTA (ethylenedinitrilotetraacetic acid, trisodium salt hydrate, 98%, AcrosOrganics, Belgium) treatments in both experiments were applied at a rate of 5 mmol kg−1

soil to evaluate its effect on the uptake and translocation of Pb in plant tissue. The limeapplication in the field study was used to increase the pH to 5.5 and assess its effecton the plant growth and Pb uptake. The contaminated site had the following treatments:contaminated soil (Pb), contaminated soil with lime (Pb+lime), and contaminated soil withEDTA (Pb+EDTA) and on uncontaminated site (control). Seven willow varieties were usedin this experiment: S. nigra, S. eriocephala, S. Allegany, SX61, SX64, SX67, and SV1.Twenty-centimeter dormant cuttings from one-year-old stems, selected for uniformity basedon the diameter and the number of buds per cutting were started in pots in the greenhousein April 2008, and on May 12 were planted at the Mukluk site. At this time the plants hadwell-developed root systems and growing shoots. After planting, each plant was mulchedindividually with cedar bark mulch to maintain soil moisture. The soil pH in the Pb+limetreatment was adjusted to pH 5.5 with hydrated lime (the level at which Pb availability is notcompromised and aluminium toxicity is avoided). The soil pH was monitored periodicallyand adjusted with acetate buffer to maintain the pH of 5.5. Ethylenediaminetetraacetic acid(EDTA) chelating agent was applied to the Pb+EDTA treatment one week before harvest atthe rate of 5 mmol kg−1 of soil. At the time of the EDTA treatment, plants continued to grow.Throughout the growing period, willows were monitored for the presence of metal-relatedphytotoxicity symptoms. On September 21, 2008, all plants were harvested by coppicingat the ground level and washed with dionized water and 1M HCl. Plants were separatedinto roots, wood (original cuttings), shoots (stems), and leaves. Plant tissues were dried inan oven for 48 hours at 70◦C, and dry weight was recorded.

Plant Tissue Analysis

Plant tissue samples were ground and ashed at 500◦C in a muffle furnace. The ashedsamples were digested with 10 mL 1M HCl solution and brought up to 50-mL volume. Metalconcentrations were determined in dry plant tissue using an Inductively Coupled PlasmaOptical Emission Spectrometer (ELAN 6000 ICP-EOS, Perkin-Elmer Corporation). TotalPb content in wood, shoots and leaves (mg plant−1) was calculated by multiplying the metalconcentration (mg kg−1) in a tissue by the total dry weight (kg plant−1) of this tissue. ABruker GADDS X-Ray Diffractometer was used to identify the silicon in the digest of theroot tissue.

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Statistical Analysis

Statistical analysis was performed using SAS software (version 9.2, SAS Institute,Cary, NC). The MIXED procedure for analysis of variance was used to determine statisticaldifferences (P < 0.05) among treatments and varieties. Initially, the data were tested forhomogeneity of variance and normality, however, violations were found. For both exper-iments, average dry weight of aboveground tissue, average Pb concentration in differentplant tissues and the aboveground tissue content in willows were measured. The data forthe field experiment were analyzed following procedures of Gomez and Gomez (1984) fordetermining treatment effects at different sites. The original data in the pot experiment werelog-transformed and for the field experiment the data were rank transformed to meet theassumptions. Original means are reported with transformed lettering according to Fisher’sLSD at P ≤ 0.05.

RESULTS

Soil Characteristics

The physical and chemical characteristics of the Mukluk soil used in the field and potexperiments are presented in Table 2. The results of modified Morgan and EPA 3050 meth-ods revealed relatively high (7–10%) soil organic matter (SOM); low soil pH (4.4–4.6) anddepleted nutrient content of potassium in the soil. The extractable phosphorus (P) concen-trations in the soil were elevated relative to the expected native P in a Paxton/Woodbridgesoils, but the concentrations were slightly below optimal level for plant growth (Griffinand Washko 1983). The average Pb concentration in the homogenized soil used in the potexperiment was 21,360 mg kg−1, and the Pb concentration in the field soil samples variedfrom 138 to 82,000 mg kg−1, with a mean of 21,303 mg kg−1.

Sequential extraction of the soil samples (Figure 1) indicated that Pb was predom-inantly contained in the SOM-bound F5 fraction (57%), with only 0.012% in the water-soluble F1 and 8.37% in the ion-exchangeable F2 fractions. The carbonate fraction F3contained 11.3%, followed by the Fe-Mn fraction F4 with 15%. The sum of the water

Table 2 Physical and chemical characteristics of control and contaminated soils in field and pot experiments

Parameter Control Contaminated

Sand (%) 63.6 67.6Silt (%) 31.3 28.3Clay (%) 5.1 4.1Organic matter (%) 10.2 7.3pH 4.4 4.6Calcium (mg kg-1) 23 41Magnesium (mg kg-1) 13 16Phosphorus (mg kg-1) 6 5Potassium (mg kg-1) 34 55Total soil [Pb mg kg-1] in pot experiment 13 21 360Total soil [Pb mg kg-1] in field experiment 12 21 303Water soluble Pb (mg kg-1) ND 3.52Ion exchangeable Pb (mg kg-1) ND 1788

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Extract

Water soluble

Ion exchangeble

Carbonate bound

Fe- Mn oxid

es

SOM bound

Residual

Pb

mg

kg

-1

0

2000

4000

6000

8000

10000

12000

14000

0.012%

8.37%11.33%

14.99%

57.5%

7.8%

Figure 1 Distribution of lead in various operationally defined chemical fractions (mg kg−1) in field soil. Percent-age of total Pb content is given inside the bars for each fraction.

soluble plus the ion-exchangeable fractions—8.38% of the total Pb—was considered to bepotentially available for plant uptake.

Pot Experiment

Biomass production. The dry weight of above-ground tissues (the sum of leaves,shoots and wood) is presented in Table 3. No visual symptoms of Pb toxicity were observedin leaves during the course of the experiment; however, the roots of Salix varieties in the Pbtreatment were short and small in diameter compared to those in the control. All varieties(F6, 45 = 63.07, P < 0.0001), treatment (F6, 45 = 3.19, P < 0.05), and species × treatmentinteractions effects (F12, 45 = 3.34, P < 0.0024) were significant. The Pb treatment had no

Table 3 Mean of dry weight (g) of all above-ground tissues of five willow varieties grown in pots using fieldsoil for 180 days

Varieties

Treatments SX64 S. eriocephala SX61 S. nigra SX67

Control 4.73a 2.93b 8.19a 1.82a 6.09a

Pb 5.34a 2.38b 4.7b 1.16b 5.81a

Pb+EDTA 5.48a 4.54a 4.09b 0.99b 4.83a

Means followed by the same letter in the same column are not significantly different, according to Fisher’sLSD, P = 0.05. Data presented with the original means and log-transformed lettering.

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Figure 2 Average Pb concentration (mg kg−1) in roots (A), wood (B), shoots (C), and leaves (D) of five willowvarieties after 180 days of growth in the pot experiment. Means within varieties followed by the same letter arenot significantly different (P > 0.05), Fisher’s LSD.

significant effect on dry weight of SX64, S. eriocephala, or SX67 compared to the control.However, there was a decrease in the dry weight of above-ground tissues of SX61 and S.nigra in the Pb treatment. The addition of EDTA increased the biomass of S. eriocephalabut did not change the above-ground dry weight of the other willow varieties. SX61 hadthe highest dry weight (8.19 g) in the control treatment, while SX67 had the highest dryweight in the Pb treatment (5.81 g) and SX64 in the Pb+EDTA treatment (5.64 g).

Metal concentrations in plant tissue. Pb concentration in different plant tissuesvaried considerably within varieties and treatments (P < 0.0001). In addition, we foundsignificant varieties × treatment interactions for roots (F8, 45 = 13.72, P < 0.0022), wood(F8, 45 = 9.18, P < 0.0001), shoots (F8, 45 = 5.38, P < 0.0001) and leaves (F8, 45 = 3.75,P < 0.0022). The average Pb concentrations in roots, wood, shoots, and leaves of all willowsare presented in Figure 2. For all varieties, the highest concentration of Pb was found in theroots. Pb concentration in roots ranged from 444 to 10134 mg kg−1 in different treatments,with the highest value in SX64 in the Pb+EDTA treatment. The concentration of Pb inwood tissues across all varieties and across all treatments ranged from 129 to 593 mg kg−1,with the highest concentration found in S. nigra in the Pb+EDTA treatment. In shoots, thehighest Pb concentration was in SX61 (558 mg kg−1) in the Pb+EDTA treatment and thelowest was in SX64 (14 mg kg−1) in the Pb treatment. Pb concentration in leaves was in therange of 368–1182 mg kg−1 for all varieties and treatments, with the highest value in SX67in the Pb+EDTA treatment. The Pb+EDTA treatment resulted in significantly increased

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WILLOWS’ LEAD TOLERANCE 739

Varieties

S. erio

cephala

S. nigra

SX61SX64

SX67

Ab

ove

gro

un

d P

b c

on

ten

t (m

g p

lan

t-1 )

0.0

0.2

0.4

0.6

0.8

1.0Control Pb Pb+EDTA

a

b

c

a

b

c

a

b

c

a

b

c

a

b

c

Figure 3 Aboveground tissue Pb content (mg plant−1) in five willow varieties grown in the pot experiment (n =4). Means within varieties followed by the same letter are not significantly different (P > 0.05), Fisher’s LSD.

Pb concentration in all plant organs for all varieties. The effect of EDTA application wasmore pronounced in leaves and shoots.

Total metal content in plant tissues. There were significant differences inmetal content across varieties and treatments, with significant interaction present in root(F8,45 = 12.08, P < 0.0001) and wood tissues (F8,45 = 2.82, P < 0.013). However, therewas no interaction effect for shoots and leaves in metal content. Whole plant total Pbcontent also revealed significant variation between varieties (F4,45 = 15.46, P < 0.0001),treatments (F2,45 = 90.07, P < 0.0001), and varieties × treatments interactions (F8,45 =7.37, P < 0.0001). An application of EDTA significantly increased Pb content in the wholeplant for all willow varieties. The Pb content of the above-ground tissues for varieties inPb and Pb+EDTA treatments ranged from 0.05 to 0.33 mg plant−1 and 0.26 to 0.78 mgplant−1 respectively (Figure 3). The highest Pb content in above-ground tissue was in S.eriocephala (0.78 mg plant−1) and SX67 (0.72 mg plant−1) in the Pb+ EDTA treatment(Figure 3).

Field Experiment

Biomass production. The dry weight of the above-ground biomass (the sum ofleaves, shoots, and wood) is presented in Table 4. Salix did not show any visual symptomsof Pb toxicity, but there were differences in plant growth in various treatments. In thecontrol treatment, SX64 had the highest dry weight (277.70 g), while in the Pb treatment S.eriocephala had the highest dry weight (237.76 g). The Pb+lime treatment had a significant

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Table 4 Mean dry weight (g) of all above-ground tissues of seven willow varieties grown in field experiment for130 days

Varieties

Treatments SX64 S. eriocephala SX61 S. nigra SV1 SX67 S. Allegany

Control 277.7a 231.9a 196.2a 189.4a 172.3a 122.5a 112.5bc

Pb+lime 277.9a 257.5a 191.5a 245.3a 171.6a 142.8a 142.8a

Pb+EDTA 232.7b 253.2a 187.4a 202.9a 119.5b 147.7a 151.8ab

Pb 226.1b 237.8a 189.3a 168.7a 128.5b 125.7a 87.2c

Means followed by the same letter in the same column are not significantly different. According to Fisher’sLSD, P = 0.05. Data were rank transformed prior to analysis. The data is presented with the original means andtransformed lettering.

increase on the dry weight of S. Allegany (142.78 g), SV1 (171.57 g), and SX64 (277.95 g)compared to the Pb treatment (87.20, 128.50, and 226.11 g, respectively). There was nosignificant effect of Pb+lime treatment on the dry weight of S. eriocephala, SX61, S. nigra,and SX67. Pb and Pb+EDTA treatments did not influence the dry weight of willows exceptSX64and SV1.

Metal concentrations in plant tissues. We found significant varieties × treat-ment interaction effects in wood (F18,59 = 2.57, P < 0.0033) and shoot tissues (F18,59 = 4.54,P < 0.0001). In addition, there were significant differences in Pb concentration in roots(F3,59 = 95.10, P < 0.0001) and leaves (F3,59 = 19.61, P < 0.0001) within treatments. Leadconcentrations in the wood and roots of the 7 willow varieties grown at the contaminatedsite were significantly higher than those grown in the control treatment (Figure 4). The Pbconcentrations in leaves of all willows grown in the control treatment were significantlylower than those of other treatments, except in the case of S. Allegany. The Pb+EDTAtreatment had a significant effect on the Pb concentration in shoots of S. Allegany andSX67. The Pb+lime treatment produced a significant increase in Pb concentration in woodtissue only in S. Allegany. The highest Pb concentrations across all willows were foundin roots with the range of 55–556 mg kg−1. Wood had the next highest concentration at8.27–84 mg kg−1, followed by shoots at 4.9–469.1 mg kg−1 and leaves at 7.8–00.9 mg kg−1.The highest Pb concentration in roots was observed in SX64 (604 mg kg−1) and S. nigra(556 mg kg−1). Salix eriocephala had the highest Pb concentration in the above-groundtissues in the Pb treatment (90.83 mg kg−1) which was calculated as the average of theconcentrations of Pb in leaves (68 mg kg−1), shoots (43.9 mg kg−1), and wood (48.6 mgkg−1) (Figure 4).

Total metal content in tissues. Pb content varied significantly across all treat-ments for all plant tissues (P < 0.0001). Total metal content in all above-ground tissuesdiffered significantly across treatments (F6,84 = 4.79, P < 0.0001) and varieties (F3,84 =57.04, P < 0.0001), with significant interaction of treatments and varieties (F18,84 = 1.55,P < 0.0492). The Pb+lime treatment had a significant increase on the above-ground Pbcontent in SV-1 compared to the Pb treatment (Figure 5). In addition, the Pb+EDTA treat-ment significantly increased Pb content in above-ground tissues in ‘SV1’ and SX67. In thePb treatment the above-ground Pb content was greatest for S. eriocephala (Figure 6), due toits higher biomass production and Pb concentration compared to the other willow varieties.

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Figure 4 Average lead concentration (mg kg−1) in dry roots (A), wood (B), shoots (C), and leaves (D) of willowvarieties grown in the field for 130 days (n = 4). Means within varieties followed by the same letter are notsignificantly different (P > 0.05), Fisher’s LSD.

DISCUSSION

Soil Characterization

The Mukluk site is highly contaminated, but the extreme variability of Pb concentra-tions (38–82000 mg kg−1) makes predicting plant uptake of heavy metals difficult. Plantsmay change the direction of their root growth to avoid highly concentrated areas of lead(Pulford and Watson 2003; Dickinson and Pulford 2005). In addition, the bioavailabilityof Pb to plants can considerably affect the success of phytoremediation. Sequential extrac-tion procedures provide qualitative information on the forms, associations, bioavailability,and potential harmful effects of soil metals on plant growth and assist in the choice ofremediation technologies (Iwegbue 2007).

Despite the low soil pH at the Mukluk site, 57% of the Pb was bound to the soilorganic matter and to Fe-Mn oxides (14%), thus making Pb much less available in soilsolution. This explains the low Pb uptake by willows in the Pb treatment compared tothe Pb+ EDTA treatment in the pot experiment. There are two main factors that affectthe distribution and availability of Pb in soil: organic matter and Fe-Mn oxides (Siposet al. 2005). Soil organic matter has high surface area and functional carboxyl and phenolgroups with which metals like Pb can form strong complexes (Strawn and Sparks 2000).Consequently, as soil organic matter increases, Pb adsorption largely increases. The type

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Varieties

Allegany

S. erio

cephala

S. nigra

SV-1SX61

SX64SX67

Ab

oveg

rou

nd

Pb

co

nte

nt

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)

0.0

0.1

0.2

0.3

0.4

0.5Control Pb+ lime Pb+ EDTA Pb

c

b

b

b

a

a

b

c b

a

a

a

a

a

b

c b

a

a

a

b

a

aa

c

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a

b

Figure 5 Aboveground Pb content (mg plant−1) of 7 willow varieties grown in the field experiment (n = 4).Means within willow varieties followed by the same letter are not significantly different (P > 0.05), Fisher’s LSD.

of complex formed between Pb and soil organic matter is stronger than that formed at themineral surface.

Plant Growth and Pb Accumulation

Pot experiment. The dry weights of SX61 and S. nigra decreased significantlyin the presence of Pb as compared to the control treatment, but the weights of SX64and SX67 did not significantly decrease. Interestingly, the dry weight of S. eriocephalaincreased in the Pb+EDTA treatment compared to the control and Pb treatments. Plant Pbconcentrations were lower in the Pb treatment than in the Pb+EDTA treatment. Adding5 mmol kg−1 EDTA to the soil significantly increased Pb concentration in all plant tissuesof all willows. The highest Pb concentrations in roots were in SX64 and in S. nigra.The highest Pb concentrations in wood and shoots were in S. nigra and SX61, while thehighest Pb concentration in leaves was observed in SX67. The addition of EDTA resultedin the highest concentrations of Pb in above-ground plant tissues ever reported for willows.The highest previously reported Pb concentration in leaves of white willow (S. alba) was27 mg kg−1 (Vyslouzilova et al. 2003), while the average Pb concentration in leaves ofseveral Salix clones was less than 5 mg kg−1 (Jensen et al. 2009). In general, the largestPb accumulation was in roots, followed by the wood, leaves, and shoots in Pb and Pb+EDTA treatments for all willow varieties. Generally, Pb has very low bioavailability in soil,and chelators such as EDTA have been used to increase the uptake and translocation of

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Varieties

S. Allegany

S. erio

cephala

S. nigra

SV1SX61

SX64SX67

Ab

oveg

rou

nd

Pb

co

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pla

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)

0.00

0.05

0.10

0.15

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0.25

a

b

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bb

b

b

Figure 6 Aboveground Pb content (mg plant−1) of 7 willow varieties grown in Pb treatment in field experiment(n = 4). Means with the same letter are not significantly different (P > 0.05) according to Fisher’s LSD.

this metal (Jorgensen 1993; Blaylock et al. 1999; Wenzel et al. 2003; Surat et al. 2008).EDTA dissolves carbonates, extracts metals from Fe oxides and hydroxides, and releasesorganically bound metals (Beckett 1989), thus increasing metal uptake by plants (Nowacket al. 2006). Soluble chelate Pb complexes are less toxic to plants than metal ions andare readily available for plant uptake and transport (Jorgensen 1993). According to Wuet al. (1999), EDTA increases plant transpiration, which is important in phytoextractionof Pb-chelate complexes from the soil. Application rates of EDTA are very important, asEDTA does not degrade under normal conditions and may persist for several years in soil.Moreover, leaching of toxic metals may occur even when small amounts of chelating agentsare used, as plants will take up only a fraction of the dissolved metals (Nowack et al. 2006).

Field experiment. Willow demonstrated the ability to grow on heterogeneouslyPb-contaminated soils (138–82,000 mg kg−1). Five of the willow varieties showed no dif-ference in dry weight between the Pb and control treatments. The highest Pb concentrationsof plants grown in the contaminated site were found in roots, followed by wood, shoots,and leaves. Since all these concentrations were higher than those found in plants grownin uncontaminated soil, this indicates that some uptake and translocation of Pb had takenplace. All varieties of willows varied considerably in their ability to uptake and translocatePb into above-ground tissues. The average Pb concentrations in all plant tissues of each wil-lows in Pb, Pb+lime, and Pb+EDTA treatments varied significantly. It has been previouslydocumented that Salix varieties vary greatly in their metal accumulation and translocationabilities (Punshon and Dickinson 1997; Sander and Eriksson 1998; Eriksson and Ledin1999; Greger and Landberg 1999, 2001; Klang-Westin and Eriksson 2003; Utmazian et al.2007). Other evidence has also shown that even willows grown in uncontaminated soils

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744 O. P. ZHIVOTOVSKY ET AL.

have a naturally high variability in uptake of essential metals (Nissen and Lepp 1997). Inaddition, uneven lead distribution at the Mukluk site, detected through soil tests, likelycontributed to the high variability in plant uptake of the toxic metal. Among seven Salixvarieties, S. eriocephala was the most efficient in lead uptake and translocation into above-ground tissues, with the highest lead concentration in the leaves. However, in the Pb+EDTAtreatment, SX67 had the highest Pb concentration in the shoots. The highest Pb contentin above-ground tissues of all willows and across all treatments was in S. eriocephala,followed by SX67.

In general, willows grown in the field accumulated lower metal concentrations thanknown hyperaccumulators (which accumulate >1000 mg kg−1), but much more than whatwould typically be found in plants in general (10 mg kg−1). They may therefore be consid-ered Pb accumulators according to Greger and Landberg (1999).

Pb concentrations in the field soil did not vary significantly before and after theplanting, most likely due to the low Pb uptake by willows and the continued release ofPb into the soil solution by lead shot. Due to the limited time span of this field study it isdifficult to make conclusions about willows’ efficiency in the phytoextraction of Pb. Willowstypically reach their peak rate of growth 3–4 years after planting, when root systems arewell established and more fixed carbon can be allocated to above-ground growth (Keoleianand Volk 2005). More field research is required to evaluate the growth potential of Salixover a period of 3–4 years (Klang-Westin and Perttu 2002; Lazdina et al. 2007).

Comparison of Pot and Field Experiments

Pb tolerance in willows and its relation to silicon dioxide. In both pot andfield experiments, willow (Salix) species showed tolerance to high Pb concentrations in thesoil, and were able to take up and translocate Pb into above-ground plant tissue. It is likelythat the Pb transport occurred through the Casparian strip in the root endodermis (Sharmaand Dubey 2005). This suggests that willows’ resistance to high Pb concentrations in thesoil is due to their tolerance of Pb accumulation in plant tissues, rather than to an avoidancestrategy of preventing metal uptake from the soil. As elevated concentrations of Pb in planttissues are toxic for plants, it is suggested that some detoxification mechanism is presentin willows. It has been suggested that cell wall phosphatases and phytochelatins may beinvolved in Pb detoxification in plants (Salt et al. 1995). However, Landberg and Greger(2004) found no presence of phytochelatins in five clones of S. viminalis, and concludedthat phytochelatins are not involved in heavy metal tolerance in Salix.

During the plant tissue digestion with 10 ml HCl, a white substance was recoveredfrom the root tissues of willows. Using the X-Ray Diffractometer, the substance wasidentified as a silicon dioxide (cristobalite), which could be involved in the tolerance ofwillows to Pb (Figure 7). The tolerance of several plant species to metal toxicity has beenimproved by the presence of silicon (Si) in plant tissues. This has been observed in the leafapoplast of Vigna unguiculata L. (Fecht-Christoffers et al. 2007) and in roots of Hordeumvulgare L. and Sorgum bicolor L. (Hammond et al. 1995; Hodson and Sangster 1993). Forexample, a part of the heavy metal tolerance mechanism in Cardaminopsis halleri is theformation of zinc-silicate precipitates on the leaf surface, in the intercellular spaces, thecell wall, and the cell wall thickenings of the xylem vessels (Neumann and Nieden 2001).The authors concluded that Zn in the cytoplasm is accumulated as silicate, which furtherdegrades to SiO2. In addition, they found that pinocytotic vesicles, formed by the plasmamembrane and the tonoplast, facilitate the translocation of Si and Zn from extracellular

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Angle (degree)

20 40 60 80 100 120 140

Inte

nsi

ty (

a.u

.)

0

1000

2000

3000

4000

5000

Si

Si

Si

Figure 7 XRD spectra of sample obtained from the root tissue of willows.

compartments into the vacuole. In rice seedlings, Cd tolerance and metal chelation withSi were involved in translocation of Cd from root to shoot tissues (Wang et al. 2000). Ina hydroponic study (Liang et al. 2005), Si reduced the toxicity of Cd in plants throughincreased Cd accumulation in the roots and restriction of metal transport to the shoots.Silicon increases the tightness and rigidity of the cell wall, thus creating a barrier againstthe transport of metal ions (Treder and Cieslinski 2005). Additional research is required toinvestigate the role of Si in the reduction of Pb toxicity in willows.

Assessment of Pb uptake in pot and field experiments. Although Salixshowed tolerance to a high concentration of Pb in soil, varieties differed in their ability totake up and translocate Pb to different plant tissues. In the pot experiment, the additionof EDTA resulted in the highest Pb content in aboveground tissues in S. eriocephala andSX67, while in the field experiment the highest Pb content in aboveground tissues wasin SX67. However, in the field Pb treatment, S. eriocephala had the highest Pb content.Based on the results of both experiments, S. eriocephala and SX67 may have potential asphytoextractors of Pb on highly contaminated soils. Salix nigra appears to be an efficientroot accumulator of Pb and may be suitable for rhizofiltration technology.

Willows in the pot experiment accumulated much more Pb than those in the fieldexperiment. In general, plants grown in pot experiments may contain higher concentrationsof heavy metals than those grown in the field (Fischerova et al. 2006). For example, tobaccoand sunflowers took up three times more Cd when grown in pots than when grown in thefield trial, while Indian mustard took up seven times more Cd (Kayser et al. 2000). TheEDTA treatment had significantly less effect in the field than in the pot experiment, mostlikely because plant roots in the pot are contained in a limited amount of space and soil,and therefore have more contact with soil amendments (Schmidt 2003).

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The higher metal uptake by Salix varieties in the pot experiment as compared to thefield experiment may be explained by several factors: (1) the heterogeneous soil contam-ination in the field rendered willows’ uptake of Pb less efficient, while homogenized soilin pots facilitated efficient Pb uptake; (2) in the field, plant roots tended to avoid high Pbconcentration spots, proliferating instead in uncontaminated soil zones; and (3) degradinglead shots in the field constantly released Pb, changing the metal concentrations in differentsoil fractions and thus affecting Pb availability for plant uptake.

The results indicate that willows have the ability to take up and translocate Pb intoabove-ground plant tissues, and that the remediation of Pb can be enhanced by the additionof EDTA. Although Salix showed high accumulation of Pb in plant tissues in the potexperiment, long-term field trials are required to further investigate the potential of willowsto remediate soils highly contaminated with Pb.

ACKNOWLEDGMENT

The project was funded by a USEPA Brownfields Cleanup Grant awarded to theTown of Sprague.

REFERENCES

Baker RS, Barrentine WL, Bowman DH, Hawthorne WL, Pettiet JV. 1976. Crop response and arsenicuptake following soil incorporation of MSMA. Weed Sci. 24: 322–326.

Beckett PHT. 1989. The use of extractants in studies on trace metals in soils, sewage sludges, andsludge-treated soils. Adv Soil Sci. 9: 143–176.

Blaylock MJ, Elless MP, Huang JW, Dushenkov SM. 1999. Phytoremediation of lead-contaminatedsoil at a New Jersey Brownfield site. Remediation. 9(3): 93–101.

Bungart R, Huttl RF. 2001. Production of biomass for energy in post-mining landscapes and nutrientdynamics. Biomass Bioenergy. 20: 181–187.

Cooper EM, Sims JT, Cunningham SD, Huang JW, Berti WR. 1999. Chelate-assisted phytoextractionof lead from contaminated soils. Journal of Environ Quality. 28:1709–1719.

Dickinson NM, Pulford ID. 2005. Cadmium phytoextraction using short-rotation coppice Salix: Theevidence trail. Environ Int. 31: 609–613.

Ensley BD. 2000. Rationale for use of phytoremediation. In: Phytoremediation if toxic metals- usingplants to clean up the environment. New York (NY): John Wiley and Sons, Inc. p. 1–12.

EPA. 1999. Phytoremediation resource guide. Washington (DC): U.S. Environmental ProtectionAgency. EPA 542–B–99–003.

EPA. 2000. Introduction to phytoremediation. Washington (DC): U.S. Environmental ProtectionAgency. EPA/600/R–99/107.

EPA. 2008. Addressing Lead at Superfund Sites; [cited 2 October 2011]. Available from: http://www.epa.gov/superfund/lead.

Eriksson J, Ledin S. 1999. Changes in phytoavailability and concentration of cadmium in soilfollowing long term Salix cropping. Water, Air, Soil Pollut. 114: 171–184.

Fecht-Christoffers P, Maier K, Iwasaki H, Braun P, Horst WJ. 2007. The role of the leaf apoplast inmanganese toxicity and tolerance in Cowpea (Vigna unguiculata L. Walp). In: Sattelmacher B,Horst WJ, eds. The apoplast of higher plants: Compartment storage, transport and reactions.Dordrecht, The Netherlands: Springer. p. 307–321.

Fischerova Z, Tlustos P, Szakova J, Sichorova K. 2006. A comparison of phytoremediation capabilityof selected plant species for given trace elements. Environ Pollut. 144: 93–100.

Dow

nloa

ded

by [

Uni

vers

ity o

f C

onne

ctic

ut]

at 0

7:42

05

July

201

1

WILLOWS’ LEAD TOLERANCE 747

Garcia G, Faz A, Cunha M. 2004. Performance of Piptatherum miliaceumn Smilograss in edaphiczinc phytoremediation over a short growth period. International Biodeterioration and Biodegra-dation. 54: 245–250.

Gomez KA, Gomez AA. 1984. Statistical procedures for agricultural research. 2nd ed. New York:John Wiley and Sons, Inc. p. 407.

Greger M, Landberg T. 1999. Use of willows in phytoextraction. Int J Phytorem. 1: 115–123.Greger M, Landberg T. 2001. Interclonal variation of heavy metal interactions in Salix viminalis.

Environ Toxicol Chem. 21: 2669–2674.Griffin GF, Washko WW. 1983. Computerized lime and fertilizer recommendations for agronomic

crops. Cooperative Extention Services College of Agriculture and Natural Resources. Univer-sity of Connecticut, Storrs.

Hammond KE, Evans DE, Hodson MJ. 1995. Aluminium/silicon interactions in barley (Hordeumvulgare L.) seedlings. Plant Soil. 173: 89–95.

Hoffmann, G. 1983. Relationships between critical levels of pollutants in soils, fodder, and crops.Landwirtsch Forsch Sonderh. 39: 130–152.

Hodson MJ, Sangster AG. 1993. The interaction between silicon and aluminium in Sorghum bicolor(L.) Moench: Growth analysis and X-ray microanalysis. Ann Bot. 72: 389–400.

Huang JW, Cunningham SD. 1996. Lead phytoextraction: species variation in lead uptake andtranslocation. New Phytology, 134: 75–84.

Iwegbue CMA. 2007. Fractionation, characterization and speciation of heavy metals in composts andcompost-amended soils. Afr J Biotechnol. 6: 067–078.

Jensen JK, Holm PE, Nejrup J, Larsen MB, Borggaard OK. 2009. The potential of willow forremediation of heavy metal polluted calcareous urban soils. Environ Pollut. 157: 931–937.

Jones Jr. JB, Wol B, Mills HA. 1991. High temperature oxidation: dry ashing. In: Plant AnalysisHandbook. A practical sampling, preparation, analysis, and interpretation guide. Athens (GA):Micro-Macro Publishing. p. 53–56.

Jorgensen SE. 1993. Removal of heavy metals from compost and soil by ecotechnological methods.Ecological Engineering. 2: 89–100.

Kayser A, Wenger K, Keller A, Attinger W, Felix HR, Gupta SK, Schulin R. 2000. Enhancementof phytoextraction of Zn, Cd, and Cu from calcareous soil: The use of NTA and sulphuramendments. Environ Sci Technol. 34: 1778–1783.

Keoleian GA, Volk TA. 2005. Renewable energy from willow biomass crops: Life cycle energy,environmental and economic performance. Crit Rev Plant Sci. 24: 385–406.

Klang-Westin E, Eriksson J. 2003. Potential of Salix as phytoextractor for Cd on moderately contam-inated soils. Plant Soil. 249: 127–137.

Klang-Westin E, Perttu K. 2002. Effects of nutrient supply and soil cadmium concentration oncadmium removal by willow. Biomass Bioenergy. 23: 415–426.

Kumar NPBA, Dushenkov V, Motto H, Raskin I. 1995. Phytoextration: The use of plants to removeheavy metals from soils. Environ Sci Technol. 29: 1232–1238.

Kuzovkina YA, Quigley MF. 2005. Willows beyond wetlands: Uses of Salix L. species for environ-mental projects. Water, Air, Soil Pollut. 162: 183–204.

Kuzovkina YA, Romero M, Charles J, Hurst S, Karp A, Labrecque M, McIvor I, Singh NB, Smart L,Teodorescu T, Trybush S, Volk T, Weih M. 2008. Salix: Botany and global horticulture. HorticRev. 34: 447–489.

Landberg T, Greger M. 1996. Differences in uptake and tolerance to heavy metals in Salix fromunpolluted and polluted areas. Appl Geochem. 11: 175–180.

Landberg T, Greger M. 2004. No phytochelatins found in Salix viminalis. Plant Physiol. 121: 481–487.

Lazdina D, Lazdins A, Karins Z, Kaposts V. 2007. Effect of sewage sludge fertilization in short-rotation willow plantations. J Environ Eng Landscape Manage. 15: 105–111.

Liang YC, Wong JWC, Wei L. 2005. Silicon-mediated enhancement of cadmium tolerance in maize(Zea mays L.) grown in cadmium contaminated soil. Chemosphere. 58: 475–483.

Dow

nloa

ded

by [

Uni

vers

ity o

f C

onne

ctic

ut]

at 0

7:42

05

July

201

1

748 O. P. ZHIVOTOVSKY ET AL.

Ma LQ, Rao N. 1997. Chemical fractionation of cadmium, copper, nickel and zinc in contaminatedsoils. J Environ Qual. 26: 259264.

Martin TA, Ruby MV. 2004. Review of in situ remediation technologies for lead, zinc, and cadmiumin soil. Remediation J. 14: 35–53.

Martin PJ, Stephens W. 2006. Willow growth in response to nutrients and moisture on a clay landfillcap soil. Bioresour Technol. 97: 449–458.

Maurice C, Ettala M, Lagerkvist A. 1999. Effect of leachate irrigation on landfill vegetation andsubsequent methane emissions. Water, Air, Soil Pollut. 113: 203–216.

Neumann D, Nieden UZ. 2001. Silicon and heavy metal tolerance of higher plants. Phytochemistry.56: 685–692.

Nissen LR, Lepp NW. 1997. Baseline concentrations of copper and zinc in shoot tissues of a rangeof Salix species. Biomass Bioenergy. 12: 115–120.

Nowack B, Rais D, Frey B, Menon M, Schulin R, Gunthardt-Goerg MS, Luster J. 2006. Influenceof metal contamination on soil parameters in a lysimeter experiment designed to evaluatephytostabilization by afforestation. Forest, Snow, Landscape Res. 80: 201–211.

Pulford ID, Riddell-Black D, Stewart C. 2002. Heavy metal uptake by willow clones from sewagesludge-treated soil: The potential of phytoremediation. Int J Phytorem. 4: 59–72.

Pulford ID, Watson C. 2003. Phytoremediation of heavy metal-contaminated land by trees: A review.Environ Int. 1032: 1–12.

Punshon T, Dickinson NM. 1997. Mobilisation of heavy metals using short-rotation coppice. AspectsAppl Biol. 49: 285–292.

Salt DE, Blaylock M, Kumar NPBA, Dushenkov V, Ensley BD, Chet I, Raskin I. 1995. Phytoreme-diation: a novel strategy for the removal of toxic metals from the environment using plants.Biotechnol. 13: 468–474.

Sander ML, Eriksson T. 1998. Vertical distribution of plant nutrients and heavy metals in Salixviminalis stems and their implications for sampling. Biomass Bioenergy. 14: 57–66.

Schaller A, Diez T. 1991. Plant specific aspects of heavy metal uptake and comparison with qualitystandards for food and forage crops (In German.) In: Sauerbeck D, Lubben S, editors. Germany:Der Einfluß von festen Abfallen auf Boden, Pflanzen. KFA, Julich. p. 92–125.

Schmidt U. 2003. Enhancing phytoextraction: The effect of chemical soil manipulation on mobility,plant accumulation, and leaching of heavy metals. J Environ Qual. 32: 1939–1954.

Sharma P, Dubey RS. 2005. Lead toxicity in plants. J Plant Physiol. 17: 35–52.Sipos P, Nemeth T, Mohai I. 2005. Distribution and possible immobilization of lead in a forest soil

(Luvisol) profile. Environ Geochem Health. 27: 1–10.Sridhar K, Jackson ML. 1974. Layer charge decrease by tetrahedral cation removal and silicon

incorporation during natural weathering of phlogopite to saponite. Soil Sci Soc Ame Proced.38: 847–851.

Strawn DG, Sparks DL. 2000. Effects of soil organic matter on the kinetics and mechanisms of Pb(II) sorption and desorption in soil. Soil Sci Soc Am J. 64: 144–156.

Strelow FEW, Jackson PFS. 1974. Determination of trace and ultra trace quantities of rare earthelements by ion exchange mass spectrography. Anal. Chem. 46(11): 1481–1486.

Surat W, Kruatrachue M, Pokethitiyook P, Tanhan P, and Samranwanich T. 2008. Potential ofSonchus arvensis for the phytoremediation of lead-contaminated soil. Int J of Phytorem. 10: 1–18.

Tessier A, Campbell P, Bison M. 1979. Sequential extraction procedure for the speciation of partic-ulate trace metals. Anal Chem. 51: 844–850.

Tlustos P, Szakova J, Vyslouzilova M, Pavlikova D, Weger J, Javorska H. 2007. Variation in theuptake of arsenic, cadmium, lead, and zinc by different species of willows Salix spp. grown incontaminated soils. Cent Eur J Biol. 2: 254–275.

Treder W, Cieslinski G. 2005. Effect of silicon application on cadmium uptake and distribution instrawberry plants grown on contaminated soils. J Plant Nutr. 28: 917–929.

Dow

nloa

ded

by [

Uni

vers

ity o

f C

onne

ctic

ut]

at 0

7:42

05

July

201

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WILLOWS’ LEAD TOLERANCE 749

Utmazian MNDS, Weishammer G, Vega R, Wenzel WW. 2007. Hydroponic screening for metalresistance and accumulation of cadmium and zinc in twenty clones of willows and poplars.Environ Pollut. 148: 155–165.

Vandecasteele B, Quataert P, De Vos B, Tack FG, Muys B. 2004. Foliar concentrations of volunteerwillows growing on polluted sediment-derived sites versus sites with baseline contaminationlevels. J Environ Monit. 6: 313–32.

Vervaeke P, Luyssaert S, Mertens J, Meers E, Tack FMG, Lust N. 2003. Phytoremediation prospectsof willow stands on contaminated sediment: A field trial. Environ Pollut. 126: 275–282.

Vyslouzilova M, Tlustos P, Szakova J, Pavlikova D. 2003. As, Cd, Pb and Zn uptake by Salixspp. clones grown in soils enriched by high loads of these elements. Plant Soil Environ. 49:191–196.

Wang LJ, Wang YH, Chen Q, Cao WD, Li M, Zhang FS. 2000. Silicon induced cadmium toleranceof rice seedlings. J Plant Nutr. 23: 1397–1406.

Watson C, Pulford ID, Riddell-Black D. 2003. Heavy metal toxicity responses of two willow (Salix)clones grown hydroponically; development of a resistance screening test. Environ GeochemHealth. 21: 359–364.

Wenzel WW, Unterbrunner R, Sommer P, Sacco P. 2003. Chelate-assisted phytoextraction usingcanola (Brassica napus L.) in outdoors pot and lysimeter experiments. Plant Soil. 249: 83–96.

Wolf A, Beegle D. 1995. Recommended Soil Testing Procedures for the Northeastern United States.Northeast Coordinating Committee on Soil Testing (NEC-67), ed. Northeast Regional Publi-cation No. 493. 2nd ed. Agricultural Experiment Station, University of Delaware, Newark, DE19717–1303. p. 94.

Wu J, Hsu FC, Cunningham SD, 1999. Chelate-assisted Pb phytoextraction: Pb availability, uptake,and translocation constraints. Environ Sci Technol. 33(11): 1898–1904.

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