Chemosphere 57 (2004) 1213–1218

www.elsevier.com/locate/chemosphere
Short Communication

Cadmium accumulation in Allium schoenoprasum L. grown

in an aqueous medium
O. Barazani a, N. Dudai b, U.R. Khadka a, A. Golan-Goldhirsh a,*

a
Albert Katz Department of Dryland Biotechnologies, Desert Plant Biotechnology Laboratory, The Jacob Blaustein
Institute for Desert Research, Ben-Gurion University of The Negev, Sede Boqer Campus, 84990, Israel
b
Aromatic, Medicinal and Spice Crops Unit, Newe Ya’ar Research Center, Agricultural Research Organization,
Ramat Yishay 30095, Israel

Received 21 January 2004; received in revised form 14 July 2004; accepted 10 August 2004

Abstract

The ability of Allium schoenoprasum L. (chives) to accumulate and tolerate cadmium in aqueous Hoagland medium
at 50 lM and 250 lM was tested under continuous growth or several successive harvests of shoots. After 28 days of
continuous growth, chives accumulated the metal up to 0.2% and 0.5% of its dry weight, when grown in 50 lM and
250 lM, respectively. In experiments that the leaves were successively harvested every 16 days, there were no obvious
stress symptoms after six harvests during a period of 96 days at 50 lM Cd. At 250 lM, after 64 days and four harvests,
inhibition of growth occurred. In each treatment, a total of 1.2 g kg
1 DW and 2.4 g kg
1 DW was accumulated in the
leaves, respectively. Total SH compounds concentration in leaf was found significantly higher by 3 and 7.4 times in
plants treated with Cd at 50 lM and 250 lM in comparison to the control, respectively, while no difference in the con-
centration of glutathione (GSH + GSSG) was found. Thus, it is assumed that sulphur-containing compounds, yet
unknown, are involved in defensive mechanisms against heavy metals in chives. The results presented, point to chives
phytoremediation potential, but also on the potential risk in accumulation of heavy metals in a commonly edible plant.
Ó 2004 Elsevier Ltd. All rights reserved.

Keywords: Allium schoenoprasum L.; Cadmium; Glutathione; Phytoremediation; SH-compounds

1. Introduction and fast biomass production, and ability to translocate

Phytoremediation, refers to clean-up of polluted
sites, terrestrial as well as aquatic environments from
heavy metal and organic contaminants by plants. A
good phytoremediator plant should ideally have high
*
Corresponding author. Tel.: +972 8 6596753; fax: +972 8
6596742.
E-mail address: avigolan@bgumail.bgu.ac.il (A. Golan-
Goldhirsh).
the pollutants into the shoot (Cunningham and Ow,
1996). In the last few years, great efforts have been direc-
ted to the search of hyperaccumulator plants and the
study of their tolerance mechanisms (for reviews see,
Baker and Brooks, 1989; Salt et al., 1998).
Due to its high toxicity to animal as well as plants,
and its high solubility in water, cadmium (Cd) is consid-
ered to be one of the most problematic heavy metal
pollutants. Cadmium is considered biologically as a
non-essential heavy metal, although at low concentra-
tion it was found to have stimulatory effect on root

0045-6535/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2004.08.037
1214 O. Barazani et al. / Chemosphere 57 (2004) 1213–1218
growth of Allium sativum (Liu et al., 2003), at high con-
centration it is extremely toxic to most plants. Toxic ef-
fects of Cd include chlorosis, growth inhibition, damage
to root tips, reduction in water and nutrient uptake and
damage to proteins (for review see Das et al., 1997).
Nevertheless, a few terrestrial plant species, e.g. Thlaspi
sp. (Lombi et al., 2000) and Arabidopsis halleri (Bert
et al., 2002) were reported to accumulate high concen-
tration of Cd, higher than the hyperaccumulation crite-
rion, 0.01% of shoot dry matter (Salt et al., 1995). It was
shown that the semi-aquatic and aquatic Phragmites
australis, Nymphoides peltata, Nymphaeae sp., Eichhor-
nia crassipes and Lemna minor are fairly good accumula-
tors of Cd (Lavid et al., 2001; Ianneli et al., 2002; Wang
et al., 2002), supporting the use of plants for clean-up of
polluted aquatic environments and water reservoirs.
One of the mechanisms involved in plant tolerance to
Cd is the synthesis of the heavy metal binding ligand,
phytochelatin (PC) from a reduced glutathione (GSH)
(for review see Clemens, 2001). A few genetic studies
in yeast and higher plants have shown that GSH and
PCs deficient mutants are sensitive to Cd (e.g. Glaeser
et al., 1991; Howden et al., 1995), and expression of
these genes in Saccharomyces cerevisiae induced toler-
ance to Cd (Vatamaniuk et al., 1999). Cd, like other
environmental stresses, induces also antioxidant re-
sponses in plants. It was shown that the exposure to
Cd increased the activities of antioxidant enzymes in
Phragmites australis (Ianneli et al., 2002). GSH and its
oxidized state (GSSG) form an important antioxidant/
chelator system (for review see Noctor et al., 1998). Thus
suggesting that glutathione is also involved in defence
mechanisms against Cd toxicity.
Allium schoenoprasum L. (chives), Liliaceae, is a com-
mon edible plant. The long tube-shaped leaves are
mostly used fresh for spicing salads. In Israel, chives is
cultivated mainly on artificial media in greenhouses,
but observations of its growth on contaminated soil
led us to test its ability to accumulate cadmium. Chives
like most Allium species is highly rich in pungent sulphur
containing volatile compounds, which attributes the
unique flavour and aroma. To test whether sulphur
containing compounds are involved in heavy metal
detoxification, the content of glutathione and total
SH-containing compounds were measured.
2. Material and methods
2.1. Plant material
Seedlings of A. schoenoprasum were obtained from
Shorashim nursery (Ein Ha
Bsor, Israel), and were
grown in the greenhouse in germination trays
contain-
ing commercial pot soil mixture. Three to four-weeks-
old seedlings were taken out of the germination tray,
and were put to grow in buckets with 7 l aerated Hoa-
gland
s liquid medium. Each bucket contained 20
bunches of 20 seedlings each. The plants were grown
in a wet-mattress cooled greenhouse at average temper-
atures of 25 ± 3 °C and 16 ± 3 °C, day and night regime,
respectively. Natural light radiation was approximately
1000 lmol m
2 s
1. The Hoagland medium was changed
every seven days and the volume in each bucket was
checked daily throughout the experiments.
2.2. Cadmium treatments
After 7 days of growth in the liquid Hoagland med-
ium, Cd(NO3)2 Æ 4H2O was added at 50 lM and
250 lM. Two treatments were tested: (1) continuous
growth for 28 days; (2) continuous growth with succes-
sive harvests every 16 days and regrowth of the shoots.
The effect of Cd on plant growth was evaluated by meas-
urements of root and shoot length. Plant samples were
weighed after each harvest for determination of fresh
weight, and dry weight after oven drying at 70 °C for
72 h.
2.3. Heavy metal analysis
Plant samples were divided into roots and shoots and
washed thoroughly in tap, distilled and double distilled
water. Roots were washed also in 0.1 mM EDTA, dou-
ble distilled water and 80% ethanol. Samples were oven
dried as described above. The dried plant material was
ground to powder and samples (0.25 g) were digested
in 5 ml acid mixture of HClO4 (60%) and HNO3 (85%,
s.g. 1.42) (15:85 v/v) overnight. Digestion was completed
by gradual increase of temperature from 60 to 195 °C
(60 °C, 3 h; 100 °C, 1 h; 120 °C, 1 h; 150 °C, 30 min;
175 °C, 30 min; 195 °C, 2.5 h). After cooling, HCl (20%,
2.5 ml) was added, whirl mixed and warmed to 80 °C
for 30 min. The final volume was brought to 10 ml with
double distilled water and re-warmed to 80 °C for an-
other 30 min. Samples were filtered through cellulose fil-
ter (0.2 lM), and Cd concentration in the filtrate was
determined with an Atomic Absorption Spectrophoto-
meter (Perkin Elmer 110B).
2.4. Determination of total SH-containing compounds and
glutathione
Plant samples, 1 g of shoot or root, were homoge-
nized in 5% meta-phosphoric acid in a ratio of 1:5
(w/v), and centrifuged at 11 000g for 10 min. The supern-
atant was eluted and total SH-containing compounds
were determined according to Ellman (1959).
Glutathione (reduced and oxidized forms) was as-
sayed by an enzymatic recycling procedure (Anderson,
1985) in which GSH is sequentially oxidized by Ellman
s
reagent, 5,5 0 -dithiobis(2-nitrobenzoic acid) (DTNB) and
 O. Barazani et al. / Chemosphere 57 (2004) 1213–1218
reduced by reduced nicotine amide dinucleotide phos-
phate (NADPH) in the presence of glutathione reduc-
tase (GR). The extent of the thio-nitrobenzoic acid
thiol anion formation was monitored spectrophotomet-
rically at 412 nm for 4 min. For the assay of GSSG in
plant extract, GSH was blocked by derivatization with
2-vinylpyridine (2%) for 1 h at room temperature. For
determination of total glutathione (GSH + GSSG),
plant extracts, 50 ll from each sample was mixed in a
reaction mixture containing 100 mM potassium phos-
phate buffer pH 7.4, 2.5 mM EDTA, 1 mM DTNB,
and 0.2 mM NADPH, in a final volume of 1 ml. The
reaction was initiated by the addition of 0.25 U GR.
2.5. Data analysis
Analysis of variance (ANOVA) was determined at a
level of 95% confidence using Sigma Stat software (SPSS
Inc.).
3. Results and discussion
Treatment of A. schoenoprasum plants with cadmium
in the medium, either continuously or with successive
harvests of the shoot, led to accumulation of high metal
concentrations in shoots and roots (Figs. 1 and 2). Un-
der successive harvests, plants grown at 50 lM and
250 lM Cd for 96 and 64 days, accumulated a total of
2.5 g kg
1 DW and 5.4 g kg1 DW, respectively (Fig. 1).
Shoots of plants grown in 50 lM Cd, showed no stress
symptoms. At 250 lM, chlorotic stress symptoms and
growth inhibition were observed (data not shown). A
high concentration of Cd was accumulated in shoot
2500
Cd (mg kg-1 DW)
1000
500
50 250
Cd concentration in the medium (µM)
Fig. 1. Total cadmium accumulation in leaf and root of A.
schoenoprasum. Plants were grown in Hoagland medium
supplemented with Cd (50 lM and 250 lM) under successive
harvests (see Section 2.2). Leaf—black bars; root—gray bars.
1215
50 µM
2000
500
Cd (mg kg-1 DW)
250
250 µM
5000
4000
1000
1 7 14 21 28
Time (days)
Fig. 2. Cadmium accumulation in leaf and root of A. schoeno-
prasum. Plants were grown continuously in Hoagland medium
supplemented with Cd, 50 lM and 250 lM for 28 days. Leaf—
black bars; root—gray bars.
(1.2 g kg
1 DW and 2.4 g kg
1 DW) and in root
(1.3 g kg
1 DW, and 2.9 g kg
1 DW) when grown in
50 lM and 250 lM, respectively (Fig. 1).
Under continuous growth of A. schoenoprasum in Cd
treatments without successive reharvesting, it was shown
that after 1 day of growth at 50 lM, plants accumulated
the heavy metal in concentration of 24 mg kg
1 DW and
480 mg kg
1 DW in leaf and root, respectively (Fig. 2).
In 250 lM Cd treatment, plants accumulated 94 mg kg
1
DW and 1480 mg kg
1 DW of Cd, respectively (Fig. 2).
Jiang et al. (2001) reported that after 9 days growth in
Hoagland medium with Cd at 100 M, Allium sativum
accumulated about 17.4 mg kg
1 DW and 350 mg kg
1
DW of the metal in shoots and roots, respectively. Sug-
gesting that Allium schoenoprasum might be a better
hyperaccumulator of Cd. There was no significant in-
crease in the metal concentration in leaf and root be-
tween 7 and 28 days of growth in both treatments
(50 lM and 250 lM, Cd) (Fig. 2), indicating that after
1 week the rate of accumulation of the heavy metal
was proportional to the rate of growth. After 28 days
of growth in both 50 lM and 250 lM, the total accumu-
lation of the metal in plants increased by 3.7 and 3.5
folds in comparison to its accumulation after the first
day, respectively (Fig. 2). A. schoenoprasum hyperaccu-
mulated Cd up to 0.2% and 0.5% of dry weight in
1216 O. Barazani et al. / Chemosphere 57 (2004) 1213–1218

Table 1
Leaf and root fresh weight and dry weight of A. schoenoprasum grown in Hoagland medium containing Cd
Plant part Cd (lM) Days after treatment
7 14 21 28
Fresh weight* Leaf 0 240.1a 411.0a 950.7a 1301.0a
50 238.7a 427.1a 690.7b 772.2b
250 221.6a 288.9b 440.8c 464.4c

Root 0 38.0a 74.1a 156.1a 212.8a

50 25.4a 85.3a 137.2a 151.3b
250 47.2a 65.7a 96.6a 125.2b

Dry weight* Leaf 0 18.5a 33.9ab 77.4a 113.1a

50 21.1a 37.2a 61.2b 76.1b
250 20.8a 29.6b 42.0c 49.3c

Root 0 3.7a 6.6a 14.0a 21.5a

50 4.9b 7.9a 14.0a 16.3b
250 4.9b 7.5a 10.2b 14.2b
*
Different letters represent statistical differences (p < 0.05) among treatments in leaf and root fresh weight (mg) and dry weight (mg) at
each harvest time and in leaf and root separately.

50 lM and 250 lM treatments, respectively (Figs. 1 and

2, Table 1). It was previously suggested that hyperaccu-
mulator criterion for Cd is the ability of a plant to toler-
ate and accumulate Cd to more than 0.01% of shoot dry 35
Leaf length (cm)

weight (Salt et al., 1998). Until now only two Cd hyper-

accumulator species were characterized, both of the
Brassicaceae family, Thlaspi caerulescens and Arabidop- 30
sis halleri (Lombi et al., 2000; Bert et al., 2002). The data
presented here suggest that A. schoenoprasum could be
25
considered as a potential plant for phytoremediation
of Cd polluted water. Beside high heavy metal uptake
and translocation to the shoots, phytoremediation prac-
tice requires high biomass crops that are easily harvest-
able (Cunningham and Ow, 1996). Thus, traits as fast 20
growth rate and regrowth after many successive harvests
Root length (cm)

contribute to chives potential use for remediation. 18

During the 28 days of continuous growth of A. scho-
enoprasum, without successive harvests, some inhibitory 16
effects of Cd on plant growth were observed and meas-
ured (Table 1, Fig. 3). Significant differences (p < 0.05) 14
in fresh weight and dry weight of plants treated with
the heavy metal and that of the control were found after 12
14 days growth at 250 lM and after 21 days growth at
50 lM (Table 1). Between the 14th and 28th days of
7 14 21 28
exposure to Cd at 250 lM, fresh weight of shoots was re- Days
duced by 30–65% in comparison to the control (Table 1).
Similarly, significant differences (p < 0.05) in root and Fig. 3. Leaf and root elongation of A. schoenoprasum. Plants
leaf lengths were obtained after 14 days exposure to 
were grown in Hoagland medium ( ) supplemented with Cd at
250 lM Cd (Fig. 3). In 50 lM, fresh weight of the leaves 50 lM (s) and 250 lM (.).
was reduced by 27% and 41% after 21 and 28 days of
growth, respectively. Fresh weight of roots treated with
Cd at 50 lM was significantly reduced (29%) in compar- weight after 21 days growth at both Cd concentrations,
ison to control plants (Table 1). When comparing the and in roots after 21 and 28 days growth at 250 lM and
dry weights, significant differences were found in leaves 50 lM, respectively (Table 1). It was reported by Jiang
 O. Barazani et al. / Chemosphere 57 (2004) 1213–1218
et al. (2001) that Cd inhibited significantly whole plant
and root growth of A. sativum at 1 mM, while at lower
concentration it didn
t have any effect on plant growth.
Most plant species are sensitive to very low concentra-
tion of Cd (Pietrini et al., 2003) and toxic symptoms
as inhibition of plant growth, browning of root tips
can be easily seen (Das et al., 1997). Exposure of A.
schoenoprasum to Cd at 50 lM did not have any effect
on leaf nor root elongation (Fig. 3), thus pointing to
chives tolerance to the metal.
The concentration of total SH-group and glutathione
(GSH + GSSH) in root and leaf of A. schoenoprasum
were measured after 28 days of continuous growth
(Fig. 4). Total SH-group concentration in roots was
1.1, 5.4 and 4.5 times higher than in the shoots in the
control, 50 lM and 250 lM treatments, respectively
(Fig. 4). In roots, the concentration of SH in plants
grown in Cd in both treatments was significantly higher
than in root of plants grown in the control treatment (15
and 30 times, respectively). In leaf, SH concentration in
Cd treated plants was also significantly higher by 3 and
7.4 times, respectively, than in control treatment (Fig. 4).
Recently, it was shown that Cd induced antioxidative
activity including accumulation of glutathione in Phrag-
mites australis (Pietrini et al., 2003). In phytochelatin
deficient mutants of Arabidopsis thaliana low PC accu-
mulation was correlated with its sensitivity to Cd (How-
den et al., 1995). In A. schoenoprasum, significant
differences in glutathione concentration were found be-
tween roots of control plants and Cd treatments at
50 lM and 250 lM (Fig. 4). However, in comparison
to control there was a decline in glutathione concentra-
tion in roots (Fig. 4). Thus, these results suggest that
other SH-containing compounds, yet unknown, are in-
volved in chives defense against Cd toxicity.
A. schoenoprasum high tolerance to Cd as well as to
other toxic heavy metals (data not shown), fast regrowth
after several successive harvests and Cd hyperaccumula-
SH-GROUP (nmol g-1 FW)
9000
4000
2000
leaves roots leaves roots
SH-group Glutathione
Fig. 4. SH-containing compounds and glutathione accumula-
tion in root and leaf of A. schoenoprasum. Plants were grown
for 28 days in control medium (black bars) and with Cd at
50 lM (white bars) and 250 lM (gray bars).
1217
tion indicate that chives can be considered as a potential
candidate for phytoremediation of polluted water. How-
ever, chives is a commonly used food plant. Therefore,
great precaution should be given to its hyperaccumula-
tion trait.
Acknowledgment
This project was supported by a post-doctoral fellow-
ship to O. Barazani from the Blaustein Center for Scien-
tific Cooperation and by a graduate fellowship to U.R.
Khadka from Albert Katz International School for De-
sert Studies.
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