ORIGINAL PAPER

Phytoextraction of cadmium and zinc by Salix from soilhistorically amended with sewage sludge

A. P. Maxted Æ C. R. Black Æ H. M. West ÆN. M. J. Crout Æ S. P. Mcgrath Æ S. D. Young

Received: 7 August 2006 / Accepted: 23 October 2006 / Published online: 8 December 2006� Springer Science+Business Media B.V. 2006

Abstract Short rotation coppice (SRC) such as

Salix spp. can be grown as an energy crop and

offers some potential for economic and practical

phytoextraction of marginally contaminated ara-

ble soil. This study tested various soil amend-

ments intended to increase soil metal availability

to Salix, investigated the distribution of metal

between different tree fractions and assessed the

viability of phytoextraction using SRC on arable

soils. Several Salix genotypes were grown in field

trials over 4 years. Cd and Zn concentrations

were generally ranked in the order leaves >

bark > wood. Metal concentrations in wood

increased towards the top of the willow stems,

whereas concentrations in leaves showed the

opposite trend. None of the amendments signif-

icantly increased uptake of Zn by willow. How-

ever, in response to a range of soil HCl

treatments, mean Cd concentrations in stems

and leaves were 112% and 130% of control

values. Data from the current experiment, and

previous studies, were combined to develop a

predictive model of Cd and Zn stem uptake by

Salix. The minimum biological concentration

factor (BCF) required to achieve a prescribed

soil metal target was also calculated based on

typical proportions of bioavailable Cd in sludge-

amended soils for a 25-year Salix rotation. The

best Salix genotypes investigated achieved less

than 20% of the uptake rate required to remove

one third of the soil Cd content (equivalent to the

average isotopically exchangeable Cd fraction in

soils at the study site).

Keywords Biomass energy � Cadmium � Salix �Sewage sludge � Short rotation coppice � Zinc

Introduction

The preferred disposal route for sewage sludge in

the UK is application to agricultural land (DETR

2000). However, this approach may add poten-

tially toxic metal contaminants to the soil (Allo-

way and Steinnes 1999) which, over time, can

pose a potential risk to human health through

consumption of food crops. Increased concentra-

tions of potentially toxic metals in crops grown on

land used for sewage sludge disposal has been

demonstrated (Keller et al. 2001) and legislative

A. P. Maxted � H. M. West � N. M. J. Crout �S. D. Young (&)School of Biosciences, Biology Building, UniversityPark, Nottingham, Nottinghamshire NG7 2RD, UKe-mail: scott.young@nottingham.ac.uk

C. R. BlackSchool of Biosciences, Sutton Bonington Campus,Loughborough, Leicestershire LE12 5RD, UK

S. P. McgrathRothamsted Research, Harpenden, HertfordshireAL5 2JQ, UK

123

Plant Soil (2007) 290:157–172

DOI 10.1007/s11104-006-9149-5

limits for metal concentrations in foodstuffs and

soil have been set by the EU (Commission

Regulation 2001). Problems with zinc (Zn) are

most likely to arise from phytotoxicity whereas

cadmium (Cd) poses a risk, even at low concen-

trations, due to potential violation of precaution-

ary soil and food standards.

Although metal concentrations in sewage

sludge have declined during recent decades

(Rowlands 1992; Gendebien et al. 1999), all

applications are accumulative in topsoil. There-

fore, the use of land management strategies to

maintain metal concentrations in soils and crops

below legislative limits may be required where

sludge application is practiced. Current options

include liming to maintain a high soil pH, on-farm

blending of grain and selecting crop varieties with

low uptake properties (Hough et al. 2003). How-

ever, removal of metals from historically contam-

inated arable land would be an attractive option if

cost-effective techniques were available. Tradi-

tional approaches to land remediation such as

‘excavation and burial’ are clearly inappropriate

where topsoil conservation is essential.

Phytoextraction of metals into the harvestable

components of plants may provide a low-cost

remediation approach (Schmidt 2003). Various

strategies have been investigated during the past

two decades. These fall into three categories

including the use of: (i) hyperaccumulator plants

which naturally possess metal accumulating prop-

erties; (ii) arable crops induced to accumulate

metals following application of metal mobilizing

agents to the soil; (iii) rapidly growing tree species

used for bio-fuel (McGrath et al. 2001). In view of

the requirements of sustainable arable land man-

agement, an effective strategy must leave the soil

structurally, chemically and biologically undam-

aged, be inexpensive to implement and utilize

existing land management skills; it must also fit

into a rotational system or, if applied for an

extended period, provide some financial return.

The use of high-yielding, fast-growing trees for

phytoextraction may provide an economic and

practical solution for marginally contaminated

arable soils (Klang-Westin and Eriksson 2003;

Dickinson and Pulford, 2005), if the land can be

dedicated to this purpose for at least 10–20 years.

Short rotation coppice (SRC) such as Salix spp.

can be used as a CO2-neutral energy crop

(Hammer et al. 2003; Demirbas 2005) and thus

help to meet emission targets set by the 1997

Kyoto Protocol (Kirschbaum 2003). Furthermore,

SRC production would contribute to the UK

Government’s goal of generating 10% of domes-

tic electricity from renewable sources by 2010

(DEFRA 2002). It is recognized that establishing

SRC on brownfield land reduces metal export

from the site through erosion (Hammer et al.

2003). Considerable effort has been directed to

studies of metal uptake by Salix, and variation in

metal concentrations has been observed between

species and clones (Landberg and Greger 1996;

Riddell-Black et al. 1997). Unfortunately, re-

ported values of BCF (biological concentration

factor) for Salix spp. are generally low. McGrath

and Zhao (2003) suggested that, for high biomass

crops (ca. 20 t ha–1 y–1), BCF values greater than

10 are required to halve soil metal concentrations

within 10 years. However, the BCF values for

Salix spp. reported by Dickinson and Pulford

(2005) were below 3 in six of the seven studies

reported; increasing metal uptake by Salix spp. is

therefore an important objective. SRC is a

perennial crop with a growing period of up to

25 years (Klang-Westin and Eriksson 2003). Thus

it may be possible to make repeated small

applications of low-cost mineral acids to increase

metal uptake following minor, and possibly tran-

sient, reductions in soil pH. However, the appli-

cation of chelate-assisted phytoremediation to

perennial crops such as SRC-willow is unlikely to

succeed because of phytotoxicity. Other problems

associated with the use of chelates (risk of

leaching, zootoxicity, cost) are well documented

and may also apply, to some extent, to repeated

application of mineral acids.

The current study aimed to assess the potential

of Salix spp. for the phytoextraction of Cd and Zn

from arable soils utilized for the disposal of

sewage sludge. This paper reports results from

field trials established at a dedicated sewage

sludge disposal facility operated by a major UK

water company. The objectives were to: (i) assess

the influence on biomass and metal off-take of

‘management decisions’ in relation to coppicing,

age at the time of harvest, removal of leaves,

choice of variety and use of soil amendments and

158 Plant Soil (2007) 290:157–172

123

(ii) develop predictive algorithms for Cd and Zn

uptake by Salix to assess the viability of phytoex-

traction of arable soils using SRC.

Materials and methods

Site description and soil analysis

The study site was located at a dedicated sewage

sludge disposal facility near Nottingham, UK.

The site consists of >630 ha of agricultural land

and is operated by a major UK water company

under the guidelines governing ‘dedicated sites’

set out by the 1989 UK Sludge Regulations (SI

1989). Sewage sludge has been applied to parts of

the site for approximately 100 years. One loca-

tion, ‘Field 8’, was chosen for the trials because of

the extended period over which sludge has been

applied; this field was originally used as a lagoon

for de-watering sludge but more recently has

received sewage sludge applications through sub-

surface injection. Soil samples were collected

from the plough layer (0–25 cm) throughout the

experimental site, using a W-transect, air-dried

and sieved to <2 mm for chemical analysis. Soil

samples were also collected at 0–10, 10–20, 20–30,

30–40, 40–60 and 60–80 cm depth intervals from

five auger borings to determine the distribution of

metals within the soil profile. Concentrations of

Cd and Zn were determined by flame atomic

absorption spectrophotometry (FAAS) following

Aqua-Regia digestion. Loss on ignition (LOI)

was determined for soil samples ignited at 550�C

in a muffle furnace. Available phosphate was

determined by extraction with 0.5 M sodium

bicarbonate and pH was determined in deionized

water (1:2.5 w/v). Concentrations of Cd and Zn in

soil pore water were determined by Graphite

Furnace AAS following extraction of solution

from moist soil using Rhizon soil solution sam-

plers (Hough et al. 2005).

Plant establishment

Three Salix plantations were established, the first

two in 2000 and the third in 2003. The genotypes

used and plot details are summarized in Table 1;

for convenience, the common name listed for

each genotype is used hereafter. The site was

ploughed to a depth of 25 cm and power har-

rowed to a depth of 20 cm. All trials were

established using 250 mm un-rooted cuttings,

hand-planted in a randomized block design with

four-fold replication. Where different Salix geno-

types were established in the same plantation,

they were planted in separate plots. Foliar treat-

ments of Mn ‘‘Jett’’ (1.5 l ha–1) and Fe solution

‘‘Ferrosol’’ (3 l ha–1) were applied regularly dur-

ing May–July between 2000 and 2004 to combat

severe leaf chlorosis, possibly induced by large

soil Zn concentrations. Hand-weeding was car-

ried out regularly, with some supplementary use

of ‘‘Roundup’’ (3 l ha–1) containing glyphosate

(broad spectrum herbicide) outside the experi-

mental plots.

The six Salix genotypes used were selected for

their ability to accumulate relatively high con-

centrations of Cd and Zn and their suitability for

metal-enriched arable soil. S. dasyclados

SW890129 (Loden) was recommended as an

accumulator of Cd by SW Seed, a commercial

grower in Sweden, following screening of 90 Salix

varieties and clones (Nils-Ove Bertholdsson, pers.

comm.). Two clones of S. viminalis (Sv-78101 and

Sv-78198) were identified as Cd accumulators

from a screening experiment of 103 Salix clones

by Landberg and Greger (1994). Clone Sv-78198

has already been used in a 5-year metal-uptake

study in Switzerland (Kayser et al. 2000; Hammer

et al. 2003). The remaining genotypes (Caloden-

dron, Rosewarne White and Spaethii) achieved

the greatest uptake of Cd and Zn in a screening

experiment of 20 Salix genotypes at the experi-

mental site used in the present study (Riddell-

Black et al. 1997; Pulford et al. 2002).

Experimental design

A range of treatments was applied to individual

plots within the three plantations (Trials 1–3;

Table 2) and samples were harvested for analysis

at various times during the growth period

(Table 1). Trials 1, 2 and 3 examined the effect

of chemical treatments on metal uptake (Table 2).

Trials 1 and 2 used trees from Plantation 1

(Calodendron) in 2001 and 2004, respectively.

Trial 3 used trees from Plantation 2 (Calodendron

Plant Soil (2007) 290:157–172 159

123

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160 Plant Soil (2007) 290:157–172

123

and Loden) in 2001. Trial 4 compared metal

uptake by six Salix varieties and clones, without

soil treatment, established in Plantation 3 in 2003

and harvested in 2004 (Table 1).

Effect of mobilizing agents on Cd and Zn

accumulation

Sub-plots (1.5 m by 3 m) were established within

all plots for application of chemical treatments.

Soil amendments, applied as single or multiple

doses, included a chelating agent (EDTA) and

HCl; the latter was intended to enhance Cd

uptake, through acidification and chloro-com-

plexation. Treatments were calculated for the 0–

20 cm soil profile and are summarized in Table 2;

treatments for Trial 1 commenced during May

2001, 14 months after planting. In all treatments,

100 l of solution was applied to each plot using

irrigation piping, equivalent to 2.22 cm of rainfall.

Chemical treatments were supplied in 50 l aliqu-

ots with a further 50 l of water being applied to

improve permeation into the topsoil. Control

plots received 100 l of water. Treatments in Trial

2 commenced during June 2004 and an additional

100 l of water was applied to all plots prior to

each chemical application to offset dry topsoil

conditions. The treatments used in Trial 3,

applied in August 2001, were as described for

Trial 1 (Table 2).

Plant harvest and analysis

The age and coppicing history of the trees are

given in Table 1. Harvest dates were timed to

occur as late in the year as possible before

significant leaf fall took place. In Trial 1, four

trees were harvested from all control and treat-

ment plots by cutting the stems 15 cm above

ground-level. All leaves were removed, mixed

and sub-sampled. One complete stem was se-

lected at random from each harvested tree,

Table 2 Description of chemical treatments applied in experimental Trials 1, 2 and 3

PlantationNo.

ExperimentalTrial No.

Plant age at sampling Chemical treatments applied

Description Application dates

1 1 17-month trees, 17-monthshoots

C17: Control –HS17: 10 mmol HCl kg–1; single

application22–23 May 2001

ES17: 2 mmol EDTA kg–1;single application

15–16 August 2001

HS17 + ES17 combined –EM17: 0.5 mmol EDTA kg–1; 4

applications24 May, 22 June, 20 July & 14

Aug 2001HS + EM –

1 2 53-month trees, 12-monthshoots after coppicing inprevious year

Ccop12: Control –HScop12: 10 mmol HCl kg–1;

single application8–9 June 2004

HMcop12; 10 mmol HCl kg–1; 5applications at fortnightlyintervals

8–9 June, 22–23 June, 6–7July, 20–21 July & 3–4August 2004

1 2 53-month trees, 36-monthshoots after coppicing 3 yearspreviously

Ccop36: Control –HScop36: 10 mmol HCl kg–1;

single application8–9 June 2004

HMcop36: 10 mmol HCl kg–1; 5applications at fortnightlyintervals

8–9 June, 22–23 June, 6–7July, 20–21 July & 3–4August 2004

2 3 17-month trees, 17-monthshoots

C17: Control –ES17: 2 mmol EDTA kg–1;

single application23 August 2001

Treatment codes include: C (control), H (HCl), E (EDTA), S (single application), M (multiple applications), cop(previously coppiced)

Subscript numbers denote sample age (months)

Plant Soil (2007) 290:157–172 161

123

shredded using an AL–CO Kober Silent Power

3500 shredder, thoroughly mixed and a sub-

sample (ca. 10%) was retained. To measure metal

concentrations in the bark and wood fractions,

one further stem was selected for each harvested

tree; a 10 cm length of stem was removed 50 cm

from the base and the bark and wood were

separated. Fresh weights were determined for

each complete stem and all associated sub-sam-

ples. Duplicate sub-samples were dried at 80�C,

milled and digested in concentrated HNO3 prior

to analysis for Cd and Zn by flame-AAS.

In Trials 2 and 3, one tree was randomly

harvested for analysis from each plot. Stems were

categorized into ‘small’, ‘medium’ and ‘large’

diameter groups and the median stem sampled

from each group; basal stem diameters were

measured using micrometer calipers. Each stem

was subdivided into three equal length sections:

‘base’, ‘centre’ and ‘top’. All leaves from each

stem section were processed as described above.

A 20 cm sub-sample was taken from the centre of

each of the three stem sections and the bark and

wood were separated and processed as described

above. In Trial 2, stem samples were processed

without separating the bark and wood. In Trial 4,

only two stems were taken from each harvested

tree, i.e. the median diameter stem from the

smallest and largest groups.

Effect of sample washing on tissue metal

concentration

The effect of washing sub-samples of leaves and

stems was investigated using samples of Loden

collected from Plantation 2 in October 2004. The

sub-samples were cut into two 10 cm sections and

either triple-washed in deionized water or left un-

washed prior to processing and analysis.

Statistical analysis

One-way analysis of variance (ANOVA) was

undertaken using GenStat (version 8.1) to estab-

lish treatment effects. The least significant differ-

ence (LSD) was determined to test the

significance of the differences between samples

means.

Predicting Cd and Zn uptake by willow

A predictive model of Cd and Zn uptake by

willow was derived using a combination of data

from the current experiments and several pub-

lished studies. In the absence of information on

metal bioavailability in previous studies, such as

‘extractable’ Cd or the concentration of free Cd2+

ions in pore water, uptake (Cdstem, mg kg–1) was

presented as an asymptotic function of total soil

Cd content (Cdsoil, mg kg–1; Eq. 1).

Cdstem ¼k1Cdsoil

1 þ k2Cdsoilð1Þ

Equation 1 was optimized for Cd and Zn

uptake by non-linear minimisation using the

‘Solver’ function in Microsoft Excel.

Results and discussion

Soil characteristics and contamination depth

Average topsoil characteristics for Field 8 (soil

F8) are summarized in Table 3; the distributions

of Cd and Zn to a depth of 80 cm within the

profile are shown in Fig. 1. Contamination ex-

tended below the plough layer following applica-

tions of sewage sludge over many decades and

possibly also due to the original practice of

lagooning the field with sludge. Although rooting

depth was not measured, it is likely that the

majority of the rhizosphere was exposed to

reasonably uniform soil conditions. Although

the rooting depth of Salix can extend to several

metres, Crow and Houston (2004) showed that

75–95% of Salix roots were within 36 cm of the

soil surface while Volk et al. (2001) suggested that

the majority of fine roots are commonly located in

the top 20 cm.

Effect of washing leaf and bark samples

No significant differences were found between

washed and unwashed leaf and bark samples of

Loden collected from Plantation 2 in October

2004. This suggests that the contribution to metal

off-take from soil particles adhering to leaves and

162 Plant Soil (2007) 290:157–172

123

bark was negligible and washing prior to analysis

was unnecessary.

Effect of mobilizing agents on biomass

production and metal uptake by Salix

Biomass production in Trial 1 (17-month-old

shoots) did not differ significantly between

treated and untreated plots (Table 4) and the

values obtained are consistent with those re-

ported for first year growth by Pulford et al.

(2002) and Hammer et al. (2003). No significant

differences in biomass were observed between

treated and untreated plots in Trial 2 (Table 4)

for shoots of the same age (12 or 36 months old).

In Trials 1 and 2, HCl added in single or

multiple (5) applications of 10 mmol kg–1 incr-

eased Cd concentrations in both the stems and

leaves of willow shoots harvested at 12, 17 or

36 months in all cases. For treatments HS17,

HScop12, HScop36, HMcop12 and HM36 (Table 4)

the Cd concentrations in stems and leaves were,

on average, 112% and 130% of their respective

controls. ANOVA revealed that only increased

Cd concentrations in leaves in Trial 1 and stems in

Trial 2 were statistically significant (P < 0.01).

Addition of 2 mmol EDTA kg–1 in Trial 1 had no

significant effect on Cd uptake. None of the

treatments in Trials 1 and 2 significantly increased

Zn uptake by stems or leaves.

The small increases in leaf and stem Cd

concentrations resulting from applications of

HCl in Trials 1 and 2 may have originated from

a number of factors. A transient decrease in pH

within the rhizosphere may have increased Cd

solubility, although no detectable change in bulk

soil pH was apparent in Trial 1 following tree

harvest in September. Alternatively, chloro-com-

plexation may have increased uptake of CdCl+

and CdCl20 species, or roots may have been

damaged by HCl application, thereby increasing

their permeability to metal ions. Hammer et al.

(2003) observed no significant increase in metal

concentrations in Sv-78198 following application

of elemental S to reduce soil pH, during a 5-year

growth period and suggested that frequent appli-

cations may be necessary to induce significant

increases in metal concentrations. However,

reductions in biomass were reported for trees

grown on S-treated soils which would offset, at

least partially, the potential benefits of any

increase in metal uptake.

Trial 3 repeated the EDTA treatment tested in

Trial 1 (ES17: 2 mmol EDTA kg–1) except that

this was applied to a mixed plantation containing

Calodendron and Loden (Plantation 2, Table 2).

Again, there was no significant increase in Cd or

Table 3 Selected topsoil characteristics at the field site

Soil Soil metal content (mg kg–1) Soil solution metal concentration (lg l–1) Soil pH(1:2.5 in water)

LOI (%)a AvailableP (mg kg–1)b

Cd Zn Cd Zn

F8 41.6 (0.58) 2418 (81.8) 20.9 480 6.40 (0.03) 35.0 (0.50) 224 (10.6)

Values in parenthesis are the standard errors of meansa LOI, Loss on ignition. b Bicarbonate-extractable phosphate

0

10

20

30

40

50

60

70

80

0 5 10 15 20 25 30 35 40

Cd concentration (mg kg-1)

So

il D

epth

(cm

)

0

10

20

30

40

50

60

70

80

0 500 1000 1500 2000 2500

Zn concentration (mg kg-1)

So

il D

epth

(cm

)

Fig. 1 Distribution of Cd and Zn in the soil profile. Soilsamples were collected from the field site in March 2001.Error bars are ± standard errors

Plant Soil (2007) 290:157–172 163

123

Zn concentrations in either leaves or stems as a

result of chelate treatment (results not shown).

Effect of coppicing and shoot age on metal

uptake

In Trial 2, all trees were 53 months old at harvest,

but had previously been coppiced once or twice,

thus allowing comparison between 12- and 36-

month-old shoots (Tables 2 and 4). The trees left

uncoppiced for 36 months produced a greater

annual biomass than those with shoots grown for

only 12 months (P < 0.005; Table 4). Annual

biomass production for Salix spp. is expected to

increase in the second and third year of growth

(DEFRA 2002). However, in the same Trial, stem

Cd and Zn concentrations were greater in 12-

month-old shoots (P < 0.001) irrespective of

chemical treatment (Table 4). Comparison of

Cd concentrations from control (un-amended)

plots for all harvests of Calodendron between

2001 and 2004 (Table 5) shows that values fell

within a relatively narrow range. Pulford et al.

(2002) compared metal uptake by 12-month-old

coppiced and uncoppiced trees of 20 varieties of

Salix at the experimental site used in the present

study. Stem metal concentrations were greater in

uncoppiced trees (1996 harvest) for most varieties

studied; however, by the second harvest (post-

coppicing, 1997), concentrations had decreased to

an average of 65% of the previous harvest for Cd

and 56% for Zn. Stem concentrations were

generally lower in 3-year-old shoots than in 2-

year-old shoots in a study by Sander and Ericsson

(1998). However, this effect may have resulted

from differences in stem length between the two

shoot ages as samples were collected at a fixed

height above the ground; the samples analysed for

2-year-old shoots would therefore have been

located closer to the stem apex.

The leaf:stem Cd concentration ratio (Fig. 2)

showed a slight, but non-significant, increase with

stem age; a more pronounced trend was observed

for Zn during the early stages of growth, although

this was again not significant. The concentration

ratios were also highly consistent for samples of

17-month-old stems collected from Plantations 1

and 3 (Fig. 2).

Table 4 Summary of the impact of chemical treatments on metal concentrations and overall biological concentrationfactors for Cd and Zn in Calodendron grown at the field site between 2000 and 2004

Trial no. Chemical treatmentidentifier

Annual Biomass(t ha–1)

Combined leaf metalconcentrations(mg kg–1)

Combined stem metalconcentrations(mg kg–1)

Biological concentrationfactor (BCF) forleaves + stems

Stems Leaves Cd Zn Cd Zn Cd Zn

1 C17 8.48 1.80 8.86 430 8.20 129 0.19 0.07HS17 9.40 1.94 10.7 405 9.20 121 0.22 0.07ES17 8.37 1.80 10.2 441 8.45 136 0.22 0.08HS17 + ES17 6.99 2.17 12.1 489 10.4 145 0.25 0.08EM17 8.22 2.00 9.71 387 9.20 141 0.22 0.07HS17 + EM17 8.22 1.95 11.7 448 10.6 134 0.25 0.08

2 Ccop12 5.60 1.32a 10.3b 539b 9.80 210 0.23 0.07Ccop36 12.0 2.83a 9.94b 563b 8.20 163 0.20 0.05HScop12 8.15 1.92a 13.3b 601b 10.9 216 0.27 0.07HScop36 10.9 2.58a 12.8b 732b 8.22 173 0.21 0.06HMcop12 5.77 1.36a 13.0b 551b 11.0 195 0.27 0.06HMcop36 8.08 1.91a 14.5b 573b 10.1 171 0.26 0.06

Treatment codes include: C (control), H (HCl), E (EDTA), S (single application), M (multiple applications), cop(previously coppiced)

Subscript numbers denote sample age (months)a Leaf biomass values for Trial 2 are estimated and assume a contribution to biomass from leaves which is consistent withTrial 1b Leaf metal concentrations for Trial 2 are for leaves sampled from the top third of the stem only

164 Plant Soil (2007) 290:157–172

123

Differences in Cd and Zn uptake between

genotypes of Salix

The six Salix genotypes used in Trial 4 demon-

strated substantial variation in Cd and Zn con-

centrations in leaves, bark and wood (P < 0.001);

there was approximately a 4-fold variation across

the genotypes studied (Figs. 3 and 4). Values of

BCF (plant:soil ratios of metal concentration)

were similar for Loden, Calodendron and Spa-

ethii for both Cd and Zn (Table 6). However, the

two S. viminalis clones (Sv-78101 and Sv-78198)

consistently produced the lowest Cd and Zn

concentrations (P < 0.001). Substantial variation

in metal concentrations between Salix genotypes

have been demonstrated previously (Landberg

and Greger 1996; Riddell-Black et al. 1997;

Greger and Landberg 1999; Pulford et al. 2002).

The latter study, in particular, revealed substan-

tial variation in Cd concentration in the stems of

20 Salix genotypes grown at the same site as the

current study.

Distribution of metals within trees

Metal concentrations in leaves, wood and bark

Concentrations of Cd and Zn were generally

ranked in the order leaves > bark > wood

(Figs. 3 and 4), consistent with previous findings.

Klang-Westin and Eriksson (2003), Riddel-Black

(1994) and Robinson et al. (2000) all reported

greater metal concentrations in leaves than in

stems. Mean leaf metal concentrations were 1.9

times greater than stem concentrations for Cd and

4.0 times greater for Zn for the six Salix

genotypes grown in Trial 4 of the present study

(Table 6). Adler et al. (2005) and Laureysens

et al. (2004) reported greater metal concentra-

tions in bark than wood, although Pulford et al.

(2002) reported some variation in relative bark

and wood metal concentrations for different

elements, suggesting that metal uptake properties

may vary between Salix genotypes.

Concentrations of Cd and Zn in leaves and

stems and harvestable biomass are compared for

each variety or clone in Table 6. The percentage

of total shoot biomass contributed by leaves

varied from 15 to 36% between Salix genotypes.

The proportion of above-ground biomass contrib-

uted by leaves may vary with variety and growing

Table 5 Metal concentrations in Calodendron grown at the field site between 2000 and 2004 for different ages of leaves andstems

Plantation No. Sampling dates and age Coppiced(Y/N)

Combined leaf metalconcentrations(mg kg–1)

Combined stem metalconcentrations(mg kg–1)

Cd Zn Cd Zn

1 Sept 2001; 17-month trees, 17-month shoots N 8.86 430 8.20 1293 Sept 2004; 17-month trees, 17-month shoots N 11.4 550 10.7 1591 Oct 2004; 53-month trees, 12-month shoots Y 10.3 540 9.80 2101 Oct 2003; 41-month trees, 24-month shoots Y 10.0 569 8.10 1561 Oct 2004; 53-month trees, 36-month shoots Y 9.94 563 8.20 163Mean ± (Standard Error) 10.1 (0.40) 530 (26) 9.00 (0.53) 163 (13)

0

0.5

1

1.5

2

2.5

3

3.5

4

0 5 10 15 20 25 30 35 40

Age of stem (months)

Co

nce

ntr

atio

n r

atio

(L

eaf:

Ste

m)

Fig. 2 Relationship between Leaf:Stem metal concentra-tion ratio for Zn (h) and Cd (D) and age of stems forCalodendron grown at the field site between 2000 and 2004(see Table 5 for origins of Salix material)

Plant Soil (2007) 290:157–172 165

123

conditions. Thus, Dickinson and Pulford (2005)

reported that leaves comprised 25% of total

above-ground biomass, while Greger and Land-

berg (1999) and Robinson et al. (2000) reported

values of 29% and 49% respectively; the latter

study was based on findings from a pot experiment

and so may not reliably reflect field-grown trees.

The percentage contribution to total metal off-

take provided by leaves ranged from 22 to 56%

for Cd, and from 35 to 77% for Zn in the current

study. The Salix genotypes were ranked in the

order Sv-78101 > Sv-78198 > Loden > Caloden-

dron > Rosewarne White and Spaethii for both

Cd and Zn. Published values for the proportion of

Cd off-take by Salix derived from leaves range

between 20 and 40% (Dickinson and Pulford

2005); Greger and Landberg (1999) also reported

a value of 40%. When grown as a renewable

biofuel, SRC is normally harvested during the

winter, after the leaves have been lost, to mini-

mize the water content of the biomass obtained.

This would result in a substantial return of metals

to the soil in litter and thereby limit the effec-

tiveness of the phytoextraction process. Such

losses could be avoided by harvesting the trees

earlier; in practice, we harvested Salix prior to

leaf fall and regeneration was always vigorous in

the following season.

0

5

10

15

20

25

30

35

40

0

5

10

15

20

25

0

2

4

6

8

10

12

Cd-Leaves

Cd-Wood

Cd-Bark

Cd

conc

entr

atio

n (m

g kg

-1)

Sv-78101 Sv-78198 Loden Calodendron Rosewarne White Spaethii

Salix Type

Fig. 3 Concentration ofCd in leaves, bark andwood for six genotypes ofSalix grown at the fieldsite between 2003 and2004 (Trial 4; Tables 1and 5). Trees wereplanted in April 2003 andshoots were harvested inSeptember 2004. Black,Grey and Whitehistograms correspond tothe Base, Centre and Topsections of stems.Histogram columns arepaired, with the medianstem from the smallestand largest classes ofstems on the left and rightrespectively. Error barsshow SED value forgenotype · heightinteraction (leaves) andSalix genotype effect(bark and wood)

166 Plant Soil (2007) 290:157–172

123

0

25

50

75

100

125

150

175

200

0

100

200

300

400

500

600

700

800

900

1000

0

50

100

150

200

250

300

350

400

Zn-Leaves

Zn-Wood

Zn-Bark

Zn

conc

entr

atio

n (m

g kg

-1)

Sv-78101 Sv-78198 Loden Calodendron Rosewarne White Spaethii

Salix Type

Fig. 4 Concentration ofZn in leaves, bark andwood for six genotypes ofSalix grown at the fieldsite between 2003 and2004 (Trial 4; Tables 1and 5). Trees wereplanted in April 2003 andshoots were harvested inSeptember 2004. Black,Grey and Whitehistograms correspond tothe Base, Centre and Topsections of stems.Histogram columns arepaired with the medianstem from the smallestand largest classes ofstems on the left and rightrespectively. Error barsshow SED value forvariety/clone heightinteraction (leaves) andvariety/clone effect (barkand wood)

Table 6 Summary of metal concentrations and biological concentration factors of Cd and Zn for six Salix types (un-coppiced and grown for 17 months, April 2003–September 2004)

Experimental trial no. Salix type Biomass(t ha–1)

Combined leafmetalconcentrations(mg kg–1)

Combined stemmetalconcentrations(mg kg–1)

Biologicalconcentration factor(BCF) for leaves andstems

Stems Leaves Cd Zn Cd Zn Cd Zn

4 Sv-78101 4.80 1.48 3.23 178 2.80 82.3 0.06 0.04Sv-78198 5.70 1.47 6.02 216 3.00 66.6 0.08 0.04Loden 20.1 3.50 18.4 705 8.51 125 0.24 0.08Calodendron 2.85 0.84 11.4 549 10.7 161 0.26 0.10Rosewarne White 13.3 3.37 13.9 430 5.00 90.8 0.16 0.06Spaethii 11.2 4.17 18.7 588 7.90 112 0.26 0.09

Plant Soil (2007) 290:157–172 167

123

Vertical distribution of Cd and Zn in six Salix

genotypes

Sander and Ericsson (1998) reported a general

increase in metal concentrations towards the

stem apex in 2-, 3- and 5-year-old shoots of Sv-

78183, although increases in Cd were significant

only for 5-year-old stems. They suggested that

this effect resulted from an increase in the

proportion of bark with stem height. Consistent

with this view, Fig. 5 shows that the ratio of bark

to wood biomass declined sharply with increasing

stem diameter in all six Salix genotypes investi-

gated in Trial 4. However, analysis of the same

stems revealed a significant (P < 0.001) increase

in the concentrations of Zn and Cd in wood

(bark removed) towards the top of the stem

(Figs. 3 and 4), suggesting an independent effect

of stem height. This may have resulted from the

‘‘xylem gradient effect’’ reported by Laureysens

et al. (2005) for poplar trees. The extent of the

gradients of metal concentrations along the

length of tree stems may vary between elements;

for example, Laureysens et al. (2005) found that

Al, Cd, Co and Cu concentrations in wood

increased toward the stem apex, whereas there

was no consistent trend for Mn, Na, Ni and V.

No consistent height trend in metal distribution

was observed for Cd and Zn concentrations in

the bark (Figs. 3 and 4), again consistent with

the findings of Laureysens et al. (2005) for

poplar.

Bark and wood samples were also analysed

separately for Calodendron trees in Plantation 1

in 2003 (41-month-old trees, 24-month-old shoots;

Table 1). Again, a significant height effect

R2 = 0.5833

Stem Diameter (mm)

Bar

k: W

ood

Rat

io (

Sam

ple

Wei

ght

g)

R2 = 0.5546

0

0.2

0.4

0.6

0.8

1

1

1.2

1.4

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0

0.2

0.4

0.6

0.8

1.2

1.4

1

0

0.2

0.4

0.6

0.8

1.2

1.4

0 5 10 15 20 25

0 5 10 15 20 25

0 5 10 15 20 25

0 5 10 15 20 25

0 5 10 15 20 25 0 5 10 15 20 25

R2 = 0.3383

R2 = 0.5139 R2 = 0.1866

R2 = 0.3359

Sv-78101 Sv-78198

Loden Calodendron

Rosewarne White Spaethii

Fig. 5 Relationshipbetween bark:wood dryweight ratio and stemdiameter for sixgenotypes of Salix(uncoppiced and grownfor 17 months betweenApril 2003 and September2004)

168 Plant Soil (2007) 290:157–172

123

(P < 0.001) was observed for wood, demonstrat-

ing that this trend was also apparent in older plant

material. Mean Cd concentrations in wood from

the base, centre and top of stems were 3.8, 3.8 and

6.0 mg kg–1 respectively; the corresponding val-

ues for Zn were 66, 82 and 158 mg kg–1. As the

inner and outer bark fractions were not sepa-

rated, differences in metal concentrations be-

tween the phloem and periderm could not be

distinguished.

The trend for metal concentrations in leaves

was the reverse of that for wood, as the values

generally decreased with height (P < 0.001;

Figs. 3 and 4). Laureysens et al. (2004) also found

that leaf metal concentrations were ranked in the

order senescing leaves > mature leaves > young

leaves; Dinelli and Lombini (1996) reported

similar findings. In the current study leaves were

categorized by height but, as a greater proportion

of young leaves were located towards the stem

Cd

0

5

10

15

20

0. 1 1 10 100

Soil Cd (mg kg-1)

Ste

m C

d (

mg

kg

-1)

Model line

All clones

R-B 1994

P etal 2002(I)

P etal 2002(II)

G&L 1999

K-W&E 2003

R etal 2003

V etal 2003

H et al 2003

Current study

Zn

0

50

100

150

200

10 100 1000 10000

Soil Zn (mg kg-1)

Ste

m Z

n (

mg

kg

-1)

Model line

R-B 1994

P etal 2002

R etal 2003

V etal 2003

H etal 2003

Current Study

Fig. 6 Cadmium and zincconcentrations in Salixstems as a function oftotal soil metal content.Average published dataare shown as discretesymbols; these includestudies by Riddell-Black(1994) (R-B 1994);Greger and Landberg(1999)(G & L 1999);Pulford et al. (2002) (Pet al. 2002); Klang-Westinand Eriksson (2003) (K-W & E 2003); Rosselliet al. (2003) (R et al.2003); Vervaeka et al.(2003) (V et al. 2003);Hammer et al. (2003) (Het al. 2003) and thecurrent study. Values forindividual Salix genotypesin all trials are shown assmall open circles. Thecurves represent anoptimized fit of Eq. 1 tothe averaged data: for Cdk1 = 1.87 and k2 = 0.194(RSD = 1.51 mg kg–1);for Zn k1 = 1.00 andk2 = 6.19 · 10–3

(RSD = 33.9 mg kg–1)

Plant Soil (2007) 290:157–172 169

123

apex, these results again appear consistent with

previous findings.

Previous studies have also reported seasonal

variation in metal concentrations in Salix; for

example, metal concentrations in leaves may

increase prior to senescence (Riddell-Black

1994; Dinelli and Lombini 1996). Variation in

metal concentrations in wood have also been

reported for samples collected in August and

November from the same plantation (Laureysens

et al. 2005). Although investigations of seasonal

variations in metal concentrations in Salix may

help determine the ideal time for SCR biomass

harvests, the need to include leaves limits the

period available for harvesting.

Predicting Cd and Zn uptake by willow stems

Equation 1 was fitted to a combined dataset for

Cd and Zn concentrations in the stems of willow,

averaged across all Salix genotypes examined in

the present study and previous investigations

(Riddell-Black 1994; Greger and Landberg 1999;

Pulford et al. 2002; Klang-Westin and Eriksson

2003; Rosselli et al. 2003; Vervaeka et al. 2003;

Hammer et al. 2003; Fig. 6). The predictive

equation relates metal concentrations in willow

stems to soil metal concentrations and provides a

good fit to the average Cdstem data from each

study; the ‘Langmuir equation’ suggests an

asymptote for Cd concentrations in willow stems

of around 10 mg kg–1. However, it is also clear

that a major determinant of uptake of Cd and Zn

by willow is the choice of variety or clone and this

gives rise to considerable scatter around the

average values for each study. This is particularly

well shown by the datasets of Pulford et al.

(2002), represented by the substantial vertical

spread of data at the right-hand end of the x-axis

in Fig. 6, where the range of Cdstem values for 20

willow varieties grown on the same soil spanned

an order of magnitude over the course of two

sequential harvests in 1996 and 1997. The values

obtained in the present study match the range

obtained for the 1997 harvest by Pulford et al.

(2002), particularly for Zn. Another limitation to

the general use of the predictive equation is that it

was only possible to use total soil metal concen-

tration as the determining factor for the com-

bined dataset due to lack of published

information on ‘available’ metal or metal solubil-

ity. However, the goodness of fit to the average

Cdstem data and the large degree of scatter shown

by the different genotypes suggests that improved

estimates of metal ‘bioavailability’ would provide

limited additional benefit when attempting to

predict uptake by unknown genotypes. An alter-

native ‘Freundlich’ equation provided a margin-

ally better fit to the Cd data but a considerably

worse fit to the Zn data.

Assessing the viability of phytoremediation

using SRC willow

To assess the phytoextraction potential of SRC

willow, it is necessary to consider the minimum

‘Biological Concentration Factor’ (BCFtarget)

required to achieve a prescribed target for soil

metal concentration. This can be approximated

by applying Eq. 2 applied over the period of SRC

growth:

Cdsoil;tþ 1 ¼ Cdsoil;t � BCFtargetCdsoil;tWwillow

Wsoil

� �

ð2Þ

where Wsoil and Wwillow are the mass of topsoil

and annual harvested biomass of willow (t ha–1),

respectively; Cdsoil,t is the concentration of Cd in

the soil at time ‘t’(y). A reasonable value for the

mass of a ‘hectare slice’ of topsoil (Wsoil) is 2400 t

ha–1 based on a bulk density of 1.2 g cm–3 and a

topsoil depth of 20 cm. Although willow biomass

production exceeding 30 t ha–1 y–1 has been

reported (Labrecque et al. 1998; Hammer et al.

2003; Fischer et al. 2005), values between 10 and

20 t ha–1 y–1 are more common, with new varie-

ties out-performing older ones (DEFRA 2002);

it is therefore reasonable to assume that

Wwillow = 20 t ha–1 y–1. In a recent survey of the

entire sewage disposal farm used in this study, the

average fraction of ‘reactive Cd’, measured by

isotopic dilution with 109Cd (Young et al. 2000),

was 32% (n = 122; SD = 18%; unpublished data);

thus, provisionally, this proportion may be taken

as a reasonable target for removal from arable

soils in receiving sewage sludge. The typical

rotation period for SRC is 25 years. Application

170 Plant Soil (2007) 290:157–172

123

of Eq. 2 over this period suggests that 32% of the

soil Cd would be removed with a BCFtarget value

of 1.8. Comparison of this value with the mea-

sured BCF values shown in Tables 4 and 6 (for

stems and leaves combined) indicates that all

Salix genotypes investigated fall below the level

required, and that the best ones only achieved

approximately 15–20% of the uptake rate re-

quired. Although this may be perceived as a

disappointing performance it is important to

recognize that although complete removal of the

(notionally) bioavailable pool of metal is an ideal

goal there are undoubtedly operational advanta-

ges in lesser targets. For example, Salix-based

phytoextraction may be applied to arable soils

which require only minor ‘polishing’ in order to

meet arable soil standards or produce metal

concentrations in edible crops which do not

violate risk-based hazard quotients. Thus apply-

ing Eq. 2 to the more modest target of depleting

soil Cd from marginally above to just below the

current UK sludge regulation of 3.0 mg Cd kg–1

soil (eg 3.1–2.9 mg Cd kg–1) would be achieved

with a BCFtarget value of only 0.3 which is within

reach of the best genotypes examined here.

Furthermore, the substantial variation seen be-

tween genotypes (Fig. 6, Cd) gives considerable

cause for optimism that conventional breeding

approaches may provide clones which can achieve

more realistic values of BCFtarget in future.

Clearly this will always depend upon the specific

scenario to which Salix-based phytoextraction is

applied.

Acknowledgements We wish to thank Maria Greger ofStockholm University, Nils-Ove Bertholdsson and StigLarsson of SW Seed Ltd for providing willow cuttings andstaff on the Salix Project at Cardiff University for usefuladvice and guidance. We gratefully acknowledge financialsupport from the Lawes Agricultural Trust and the U.K.Biotechnology and Biological Sciences Research Councilunder their Bioremediation Link Programme (projectBiorem 11). We also thank our industrial partners fortheir assistance.

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