International Journal of Scientific Research in Environmental Sciences, 2(10), pp.

379-388, 2014
Available online at http://www.ijsrpub.com/ijsres
ISSN: 2322-4983; 2014 IJSRPUB
http://dx.doi.org/10.12983/ijsres-2014-p0379-0388
379
Full Length Research Paper
EDTA Enhanced Phytoextraction Capacity of Scirpus Maritimus L. Grown on Pb-Cr
Contaminated Soil and Associated Potential Leaching Risks
Mahdieh Ebrahimi
1
*, Fariba Shahsavand
2

1
Assistant Professor, Department of Range and Watershed Management, University of Zabol, Iran
2
Ph.D. graduate of Range Management, Science and Research Branch, Islamic Azad University, Iran
*Corresponding Author: Email: maebrahimi2007@uoz.ac.ir
Received 01 August 2014; Accepted 11 September 2014
Abstract. A pot experiment was conducted to study phytoextraction efficiency of Scirpus maritimus L. for lead and chromium
and to determine EDTA (ethylenediaminetetraacetic acid) enhancement of the mobility and phytoextraction of Pb and Cr and
the potential for leaching of metals. The results revealed that the bioconcentration factors of underground organs were
relatively higher and metals concentrations in the plant organs decreased in the order of roots rhizomes leaves stems. Thus,
the plant species would be applicable for Pb and Cr phytostabilization. Addition of EDTA (2.5, 5, 10 mmol kg
1
) to polluted
pots significantly enhanced the mobility of soil metals and led to elevated soil solution concentrations of Pb and Cr. Positive
correlation coefficients were found between treatment time and metals concentrations in the plant organs. Optimum
phytoextraction was observed when 5 mmol kg
-1
EDTA was added in single dosage 60 days after the plant cultivation and
consequently soil metals concentration decreased with the passage of time. It can be concluded that of Scirpus maritimus L can
remediate Pb-Cr contaminated soils and EDTA (at low doses) had potential to promote the uptake of Pb and Cr for the plant
species in eco-friendly manner.
Keywords: Chelating agent, heavy metal, leaching, phytoremediation, Scirpus maritimus L.
1. INTRODUCTION
Recently phytoremediation, as a potential method for
removal of metals from polluted areas, has attracted
great attention. It is an emerging technology driven by
solar energy that uses green plants to remove toxic
metals from contaminated aquatic and terrestrial sites
(Suresh and Ravishanker, 2004). The main features of
this green technology are that it is cost-effective and
environmentally sound unlike the chemical
remediation technology (Macek et al., 2000).
In present study, Scirpus maritimus L. was
evaluated to investigate lead and chromium
phytoremediation in the Lia contaminated area (35
27N-48 50E), an important industrial area of Qazvin
(Northern Iran). It has a total area of 108 ha with 151
industrial factories. The main human activities are
agriculture especially greenhouse cultivation,
smelting, sewage sludge treatment and electronic
industries. Industrialization in this area has exposed
the soil to various effluent inputs including heavy
metals. Several locations of Lia area suffer from
metals contamination (Ebrahimi, 2014).
S. maritimus L. is a facultative halophyte. This
plant belongs to the family Cyperaceae and it is a
metal-accumulator plant, which can accumulates
elevated values of heavy metals in its rhizomes, leaves
and stems (Carranza-lvarez et al., 2008).
Although phytoremediation can be applied for the
reclamation of elevated concentrations of heavy
metals present in contaminated soils, just a fraction of
soil metal content is readily available for plant uptake.
Therefore, in order to enhance both the availability of
metals in the soil and translocation from root to shoot,
many chelants have been studied (Arwidsson et al.,
2007). Among them, EDTA
(ethylenediaminetetraacetic acid) has been widely
examined which was found as the most efficient in
increasing the concentration of water-soluble heavy
metals. When EDTA is applied to the soils, a large
fraction of the total metals is dissolved and becomes
available for phytoremediation without inducing a
strong acidification of the growth medium (Evangelou
et al., 2006). Furthermore, as compared to other
chemical compounds, EDTA can solubilize metals
with fewer undesirable effects on the soil physico-
chemical properties (Steele and Pichtel, 1998). This
offers great prospects as a less invasive alternative for
Ebrahimi and Shahsavand
EDTA Enhanced Phytoextraction Capacity of Scirpus Maritimus L. Grown on Pb-Cr Contaminated Soil and
Associated Potential Leaching Risks
380
conventional ex situ soil washing procedures.
However, due to its high level of persistence, EDTA
has been disfavored by several scientists (Arwidsson
et al., 2007).
In recent years, the use of EDTA has been
disfavored. EDTA has been shown to persist for
extended periods in soils because of its poor
degradability (Nowack, 2006). It therefore increases
the activity of trace metals in the soil solution for
extended periods and might cause enhanced toxicity
towards plants and soil organisms. Moreover,
accelerated, uncontrolled release and leaching of
metals from sparingly soluble metal compounds is
likely to increase groundwater pollution (Grman et
al., 2001; Wenzel et al., 2003).
Little work has been done on the extent of EDTA
limitations. Therefore to avoid possible metals chelate
movement into groundwater and the effect of
remaining EDTA on the soil microorganisms, the
amount and method of chelate application are
important to novel irrigation technique and time
control of chelate application.
The objectives of this study were: (1) to investigate
the remediation ability of S. maritimus L. in Pb-Cr
contaminated soils; (2) to identify influence of
application of different concentrations of EDTA on
phytoextraction efficiency of the plant species and
recognize optimum of chelator dosage; and (3) to
consider effect of treatment time and application
mode on the phytoextraction of Pb and Cr
contaminated soils.

2. MATERIALS AND METHODS

2.1. Soil source characterization and treatments

The soil used in this study was taken from Lia area.
Uncontaminated soil was used as the control
throughout the study. The results of heavy metals
concentration and physico-chemical properties of the
soil such as Total N (Kjeldahl method) (Black, 1965),
total P (molybdenum blue method) (Olsen and
Sommers, 1982), total K (Flame photometry method)
(Berry et al., 1946), pH (1:1 soil/ water ratio, Model
691, Metrohm AG Herisau Switzerland) (Thomas,
1996), EC (solid: deionized water =1:2 w/v, Model
DDS-307, Shanghai, China) (Rhoades, 1996), organic
carbon (Walkley-Black method) (Nelson and
Sommers, 1996) and CaCO
3
equivalent (Black et al.,
1965) were depicted in Table 1.
After sieving (4mm), 5 kg of dried soil were stored
in plastic pots (diameter 20diameter 15height
60cm). Seeds of S. maritimus L. were obtained
commercially in Pakanbazr Company, Esfahan, Iran.
Seeds of the plant were buried evenly throughout each
pot at least 1 to 2 cm from the edge and pots placed in
the greenhouse with the environmental conditions,
temperature 245C (day) to 205C (night) without
supplementary light, humidity 60% and moisture
content 70% water-holding capacity until roots and
shoots had developed, then the seedlings were
harvested (30 days of growth) at the end of growing
trail, the plant dry weight, tolerance index (dry weight
of the plants grown in heavy metal solution/dry
weight of the plants grown in control solution)
(Wilkins, 1978), length of the shoot and length of the
root were determined, and the changes in these
parameters were used to evaluate heavy metals
toxicity.
Plant organs were washed before analysis and
samples were baked at 70C to a constant weight for
approximately 48h and ground into fine powder in an
agate mortar. Metals were analyzed after
mineralization of 400mg dry plant materials in a
microwave oven (MEMMERT UNB 400) with 5ml of
nitric acid (69% v/v), 5ml deionized water and 2ml
H
2
O
2
(30% v/v). The digest was made to 25ml final
volume with deionized water, filtered (0.45mm,
Millipore) and then analyzed for Pb and Cr using
ICP/OES (GBC Avanta, Australia). Dried soil
samples were passed through a 2mm diameter sieve.
About 100mg dry soil was digested with HNO
3
and
HCl (3:1) in a microwave oven (MEMMERT UNB
400). After mineralization, the samples were diluted,
filtered and analyzed using ICP/OES (GBC Avanta,
Australia). Metals concentrations of the soil samples
were measured as described for the plant samples (Du
Laing et al., 2003).
In second step, to recognize effect of EDTA on
phytoremediation efficiency of S. maritimus L.
seedlings of the plant were placed throughout each pot
and chelator solution was added to the soil. EDTA
(disodium salt dehydrate of EDTA, C
10
H
14
N
2
Na
2

O
8
.2H
2
O) solutions were prepared at concentrations of
2.5, 5, 10mmolkg
-1
soil. The control pots
(contaminated soil) prepared with no EDTA (C).
Plants were harvested after 30 days of adding chelator
solutions and dissected in root, leaf, stem, and
rhizome organs to recognize the different
bioaccumulation capability and optimum of chelator
dosage.
In third step (treatment time dependent
experiment), the plant was treated with the most
optimum dosage of chelating agent for the highest
heavy metal uptake for 30, 60 and 90 days,
respectively and at the end of each period, the plants
were harvested and metals analysis in the plants was
performed with ICP/OES (GBC Avanta, Australia).
In forth step (addition methods dependent
experiment), optimum dosage of EDTA (5 mmol kg
-1
)
was added to the pots in three different ways: single (5
mmol/kg) at day1, double (2.25 mmol/kg soil each) at
International Journal of Scientific Research in Environmental Sciences, 2(10), pp. 379-388, 2014
381
days 1 and 7 and triple (1.66 mmol/kg soil each) at
days 1, 7 and 14 successive doses. Finally, after the
experiment, the plants were harvested 60 days after
the first application of EDTA and the soil was
removed from 4/5 of length of the pots below the
surface, air-dried, ground to 0.2mm, and analyzed to
investigate changes in total metals concentrations
under different methods of application.
The methodology for metals concentrations in the
soil was referenced using the SRM 2711 (Institute of
Standard and Technology, USA) and methodology for
metals concentration in the plant was referenced using
BCR-060 (Institute for Reference Materials and
Measurements, Belgium). All the analyses were
performed in five replicates.

2.2. Determination of bioconcentration factor
(BCF) and translocation factor (TF)

Bioconcentration factor (BCF), represents the ratio of
metal concentration dry weight (DW) in the plant to
the metal concentration DW in the soil. BCF is an
indication of the magnification of contaminants from
a lower to a higher trophic level. For plants, the BCF
has been used as a measure of the metal accumulation
efficiency, whereby value greater than 1 is an
indication of plants potential to phytoextraction (Yoon
et al., 2006; Santillan et al., 2101). To analyze the
total metal concentration in DW taken by the upper
parts of the plants from ground level, translocation
factor (TF) was calculated (Yoon et al., 2006;
Santillan et al., 2101). In the current study, the BCF
and TF values for Pb and Cr are given by:
soil
C
root
C
root
BCF (1)
soil
C
leaf
C
leaf
BCF (2)
soil
C
rhizome
C
rhizome
BCF (3)
soil
C
stem
C
stem
BCF (4)
root
C
stem
C
stem/root
TF (5)
root
C
leaf
C
leaf/root
TF (6)
Where C
root
, C
leaf
, C
rhizome
and C
stem
are the metal
concentrations in the root, leaf, rhizome and stem,
respectively, and C
soil
is the metal concentration in the
soil (Yoon et al., 2006).



2.3. Statistical analysis

Parametric statistical tests require the data to be
normally distributed. Therefore, data were log-
transformed where needed, using the natural log (ln)
to attain normal distribution. The statistical processing
was mainly conducted by one-way analysis of
variance (ANOVA). For one-way analysis of
variance, Duncan t-test between means was calculated
only if F-test was significant at the 0.05 level of
probability. Correlation coefficients between
treatment time, mode of EDTA application with heavy
metals contents in plant organs and soil were also
calculated through the Pearsons r coefficient. A
probability of 0.05 or lower was considered as
significant. All statistical calculations were performed
using SPSS release 19.0.

3. RESULTS

3.1. Metals accumulation and plant tolerance

Results shown in Table 2 indicated the reduction for
the measured growth parameters. For S. maritimus L.
root elongation was more sensitive to Pb and Cr than
the rate of shoot growth or plant dry weight. The root
length reduction was 13.90% whilst the shoot
reduction was 10.21%. The tolerance index of the
plant species was 100% in the control treatment,
whereas it was only 80% in the contaminated soil.
Decrease in shoot growth and dry weight in Pb-Cr
contaminated soil was evident as compared to the
control treatment.
Heavy metals concentrations in the organs of S.
maritimus L. are shown in Table 3. The plant species
had root concentrations of metals that were greater
than concentrations in leaves, stems, or rhizomes. In
general, the metals level decreased in the order of:
roots rhizomes leaves stems.
Bioconcentration factors in the studied plant were
from 0.54 to 4.62 for Pb and 0.37 to 3.19 for Cr
(Table 3). In this study, metals in S. maritimus L. were
accumulated in the root with concentrations greater
than was found in adjacent soil with BCF of 1.
The results showed that the plant species had
BCF
root
values of 1 indicate high efficacy in the
phytostabilization of metal contaminated soils and the
plant has adopted an exclusion strategy and there is
less risk of metals entering the food chain if the plant
is consumed. The results evaluated that the plant
species had TFs values 1 for Pb and Cr, indicating
that accumulation of heavy metals in the roots is
higher than in the shoots (Table 4).

Ebrahimi and Shahsavand
EDTA Enhanced Phytoextraction Capacity of Scirpus Maritimus L. Grown on Pb-Cr Contaminated Soil and
Associated Potential Leaching Risks
382
Table 1: Primary physical and chemical properties of the soil samples
Treatments Texture N% P% K% OC% EC
(dS/m)
pH CaCO
3

(%)
Pb
mg /kg
Cr
Mg/kg
Contaminated soil
Control
Clay loam
Clay loam
0.15
0.12
0.51
0.47
0.37
0.38
0.15
0.15
3.67
3.54
8.30
8.00
12.34
13.17
57.30
ND
(0.02)
79.50
ND
(0.002)
*ND= NOT Detected/Below detectable range

Table 2: Morphological characteristics for S. maritimus L. at the end of growing trail (30 days of growth) in Pb-Cr treatments
Tolerance
index
Shoot length
(cm)
Root length
(cm)
Dry weight
(g)
Treatment
0.800.00
b
19.000.11
b
14.730.10
b
17.000.04
a
Contaminated soil
1.000.00
a
21.160.13
a
17.110.14
a
19.500.06
a
Control
*Values (SE) within a column followed by different letter are significantly different

Table 3: Total concentration of Pb and Cr in plant organs (mg/kg DW) and bioconcentration factors (BCF)
Plant
organs
Pb
mg/kg
Cr
mg/kg
BCF
Pb
BCF
Cr
Root
Stem
Leaf
Rhizome
95.651.16
a

44.761.10
d

53.141.10
c

77.221.11
b

74.111.00
a

42.200.90
c

48.180.90
c

59.090.94
b

4.620.07
a

0.540.03
c

0.760.03
c

2.200.06
b

3.190.05
a

0.370.02
c

0.390.02
c

1.660.03
b

*Values (SE) within a column followed by different letter are significantly different (p0.05, Duncan test)

Table 4: Translocation factors (TF) for S. maritimus L.
Metal TF
leaaf/root


TF
stem/root


Pb
Cr
0.550.03
a

0.650.03
a

0.470.03
a

0.560.03
a

*Values (SE) within a column followed by the same letter are not significantly different (p0.05)

3.2. The effect of EDTA on heavy metals
accumulation

The pH values after EDTA addition are shown in
Table 5. A gradual increase in EC and available
metals content were observed with increasing
concentration of EDTA (Table 5). pH did fall slightly,
from a weakly alkaline (8.00) pH to a weakly acid
(6.76) pH (Table 5).
A decrease in pH increases Pb desorption from soil
constituents resulting in increased Pb concentration in
the soil solution. In C treatment, levels of Pb and Cr
were below the set detection limits and the increase in
the level of metals uptake was quite significant from
C to 10EDTA but insignificant between 5EDTA and
10EDTA, showing that 5EDTA was enough to avoid
possible metal chelate movement into groundwater
and the effect of remaining EDTA on the soil
microorganisms.
EDTA exhibited heavy metals extraction
efficiency. The lead BCFs of the plant species varied
between 0.57 and 3.56, with the lowest BCF in
2.5EDTA for stem and the highest BCF in10EDTA
for the root (Table 6). BCF values for Cr in S.
maritimus L. 0.45 were the lowest and 2.23 the
highest. TFs of the metals are presented in Table 6.
The highest values (root to leaf) were recorded in
10EDTA with around 0.30 and 0.28 for Pb and Cr
respectively.
3.3. The correlation between treatment time and
extractable soil metals and the metals contents in
the plant species

The results showed a significant correlation between
treatment time and heavy metal concentrations in the
plant organs (Table 7). Metals concentration in the
plant organs of S. maritimus L. increased significantly
(p0.05) with passage of time. The most significant
increase in Pb and Cr concentration in the plant
organs occurred on 90
th
days of EDTA application.
However the maximum Pb and Cr in the plant organs
was observed on 90th day of chelating application,
there was no significant difference between
concentrations of heavy metals in the plant tissues on
60d and 90d (Table 7).
The soil Pb and Cr concentrations were negatively
related to harvest time which indicated that harvest
time had positive influences on the soil metals
reduction. The maximum reduction was measured on
90d but it could be seen that after 60 days of harvest,
no significant decrease was observed between day of
90th and 60th.
At present study, treatment time dependent
experiment showed that harvesting the shoots of the
plant on 60 day after the first harvest could achieve
highest phytoextraction efficiency. Consequently,
early harvest may not be effective in terms of
removing maximum amount.
International Journal of Scientific Research in Environmental Sciences, 2(10), pp. 379-388, 2014
383

Table 5: Physio-chemical analysis of soil after treatment by EDTA
Cr
mg/kg
Pb
mg/kg
EC
dS/m
pH Treatments
ND (0.002)
73.421.220
b

121.341.43
a

132.461.56
a

ND (0.02)
120.291.22
b

139.071.45
a

143.711.72
a

3.600.21
c

4.460.22
b

4.760.22
b

5.420.30
a

8.000.01
a

7.600.01
ab

7.420.01
bc

6.760.01
c

C
2.5 EDTA
5 EDTA
10 EDTA
*ND= NOT Detected/Below detectable range. Values shown are the meansSE. Values within a column followed by different letter are significantly different
(p0.05, Duncan test)

Table 6: Pb and Cr extraction efficiency
Metal Treatments BCF
root
BCF
stem
BCF
leaf
BCF
rhizome
TF
leaf/root
TF
stem/root

Pb C - - - - - -
2.5 EDTA 1.240.10
b
0.570.01
b
0.650.01
b
1.040.10
b
0.210.01
b
0.150.01
b

5 EDTA 2.500.15
b
0.640.01
b
1.440.07
a
1.700.10
a
0.270.01
ab
0.200.01
a

10 EDTA 3.560.20
a
1.110.07
a
1.590.09
a
2.020.22
a
0.300.01
a
0.250.01
a

Cr C - - - - - -
2.5EDTA 0.920.01
b
0.450.01
b
0.570.01
b
0.770.03
b
0.180.01
b
0.150.04
b

5EDTA 1.420.01
a
0.720.02
ab
1.130.06
a
1.330.05
a
0.230.01
ab
0.230.04
a

10EDTA 2.230.01
a
0.800.02
a
1.720.06
a
1.500.06
a
0.280.01
a
0.270.04
a

*Values shown are the means SE. Values within a column followed by different letter are significantly different (p0.05, Duncan test)

Table 7: Effect of treatment time of 5EDTA on heavy metals uptake and correlation coefficients
90d 60d 30d
Metal
Content (mg/kg)
r
2
Metal
Content (mg/kg)
r
2
Metal
Content (mg/kg)
r
2
Metals
186.423.20
a

105.831.84
a

119.042.55
a

169.662.98
a

81.622.00
b

0.59
0.47
0.74
*

0.91
**
-0.85
*

173.552.94
a

97.761.76
a

110.422.31
a

154.072.45
a

93.002.54
b

0.76
*

0.76
*

0.90
**

0.85
**

-0.69
*

96.342.77
b

53.211.56
b

62.181.78
b

87.542.17
b

130.462.94
a

0.82
**

0.85
**
0.86
**

0.90
**
-0.90
**

Root
Steam
Leaf
Rhizome
Soil
Pb

132.092.46
a

85.561.45
a

95.482.21
a

117.321.61
a

64.121.60
b

0.90
**
0.91
**
0.86
*

0.92
**
-0.75
*

117.662.32
a

74.701.33
a

82.161.54
a

100.131.33
a

70.661.32
b

0.96
**

0.90
**

0.88
**

0.96
**

-0.79
*

79.641.24
b

49.321.14
b

57.681.17
b

70.341.09
b

111.422.42
a

0.85
**
0.76
*

0.88
**
0.93
**
-0.90
**

Root
Steam
Leaf
Rhizome
Soil
Cr
Values shown are the means SE. Values within a row followed by different letter are significantly different (p0.05, Duncan test)
*The probability level of p0.05; **The probability level of p0.01

3.4. Effect of EDTA treatment methods on heavy
metals uptake

The relations between the application methods of
EDTA (5mmol/kg) and heavy metals concentrations
are shown in Table 8. Generally, the correlation
coefficients between the application method of EDTA
and Pb concentration in the plant organs and the soil
are better than those in Cr.
Negative correlations were obtained between Pb
and Cr concentrations in the plant organs of S.
maritimus L. and methods of application, which
indicated that there was reduction in the metals
content in the plant organs according to different
application methods (p0.05). The maximum Pb and
Cr concentration in the organs of the plant species was
calculated at single dosage. It was seen that during
three separate application methods, metals content in
the plant tissues reached at its minimum
concentration, while Pb concentration in the plant
organs did not vary considerably when single and
double dosages were added (p0.05).
The liner relationship between the soil
concentrations of heavy metals and the treatment
mode was positive (Table 8). The amounts of soil Pb
and Cr reached at their maximum at triple dosage. The
results from the application methods of EDTA on the
soil Pb and Cr concentration (Table 8) indicated that
under single dosage application, heavy metals content
in the soil reached at its minimum concentration. Pb
and Cr content increased by 39.36 % and 27.01%
respectively when double dosage was added. This
increase was calculated in triple dosage 56.83% and
69.86% for Pb and Cr respectively. However,
different application methods influenced the Pb
uptake, there was no significant effect between double
and triple dosage on the Pb content of the soil. The
results indicated that the chelate application method
could limit the solubility and migration potential of
heavy metals in the soil.

Ebrahimi and Shahsavand
EDTA Enhanced Phytoextraction Capacity of Scirpus Maritimus L. Grown on Pb-Cr Contaminated Soil and
Associated Potential Leaching Risks
384
Table 8: Effect of 5EDTA application methods on the concentration of Pb and Cr in tissues of S. maritimus L.
Metals r
2
Single r
2
Double r
2
Triple
Pb


Root
Steam
Leaf
Rhizome
Soil
-1.00
**

-0.83
**

-0.65
*

-0.72
*

1.00
**

170.093.00
a

90.391.43
a

103.181.54
a

144.122.64
a

54.901.00
b

-0.76
*

-0.61
*

-0.57
-0.72
*

0.79
*

166.162.44
a

88.451.26
a

96.331.43
a

132.282.39
a

76.511.14
a

-0.51
-0.45
0.63
*

-0.50
0.47
121.572.12
b

60.771.17
b

74.321.39
b

110.072.21
b

86.101.22
a

Cr

Root
Steam
Leaf
Rhizome
Soil
-0.70
*

-0.70
*

-0.60
*

-0.71
*

1.00
**

108.182.21
a

66.091.14
a

74.871.31
a

98.661.47
a

42.281.03
c

-0.72
*

-0.53
-0.60
*

-0.78
*

0.62
*

82.092.00
b

54.241.09
b

52.231.07
b

74.391.48
b
53.701.12
b

-0.60
*

-0.42
-0.44
-0.58
0.64
*

67.621.34
c

43.391.12
c

42.091.12
c

51.521.32
c

71.821.29
a

Values shown are the means SE. Values within a row followed by the same letter do not differ significantly (p0.05, Duncan test)
*The probability level of p <0.05

4. DISCUSSION

Results indicate the reduction for the measured
growth parameters and the root elongation was more
sensitive to metals. An excess of particular metal
above the critical level of accumulation may impair
the health of plant or may even kill it. Metals would
have a toxic effect on the normal metabolism of the
plant. Plant growing on soils with higher metals value,
exhibit signs of unhealthy growth (Rout and Das,
2003). The morphology of the plants differs to some
extent from the plants growing on normal soil. Leaves
of these plants are greenish yellow in color, less
glossy, more fragile and have crenulations of higher
amplitudes along the leaf margins compared to the
leaves of the similar species growing on soils without
any metal enrichment. The roots of these plants that
grow on high metals content soil are smaller
dimensions with relatively thinner barks and in lesser
quantities then from stems of those plants that
growing on normal soils (Ashraf et al., 2011). It seems
that damage to the plasmalemma of roots cells
constitutes the first effect of metals toxicity, causing a
loss of ions, such as K, and other solutes (Woolhouse
and Walker, 1981). Thus, the degree of metals
tolerance may depend on the capacity of the plant to
prevent these effects and one of the explanations for
roots to be more responsive to toxic metals in
environment might be that roots were the specialized
absorptive organs so that they were affected earlier
and subjected to accumulation of more heavy metals
than any of the other organs. This could also be the
main reason that root length was usually used as a
measure for determining heavy metal tolerant ability
of the plant (Xiong, 1998).
In this study, decrease in shoot growth and dry
weight in contaminated soil was evident. Peralta et al.
(2004) reported that reduction in chlorophyll could
diminish the growth of above ground organs and
decreasing in dry biomass might be due to toxic
metals decrease water absorption in plant tissues
causing undesirable impacts in plant growth.
S. maritimus L. accumulates metals in root. In this
study, the fact that roots showed high accumulation of
elements could imply relatively high availability in
the soil. Although higher root metals contents were
expected, as the dominant uptake pathway of the
metals from the soil is via the rhizosphere system. It is
generally known that most metals tend to accumulate
in the roots rather than in the shoots, which suggests
that the plants adopt either external or internal
exclusion mechanisms to hinder translocation of
metals to the aerial tissues (Hansel et al., 2001). On
the other hand, stems (which consist mainly of
vascular tissues) exhibit lower metabolic activity than
leaves and, therefore, it is expected that they
accumulate metals to a lesser extent than leaves. The
relatively low accumulation of heavy metals in
aboveground tissues was probably due to the need of
plant to prevent toxicity to the photosynthetic
apparatus as suggested by other authors (Macek et al.,
2000).
In this study, metals in S. maritimus L. were
accumulated in the root with BCF of 1. Plants can
tolerate high heavy metals concentration from soil by
two basic strategies. The first strategy is called
accumulation strategy where metal can accumulate in
plants at both high and low concentration from soil.
These plants are capable of rendering the metals in
various ways, for instance by binding them to cell
walls, compartmentalizing them in vacuoles or
complexing them to certain organic acids or proteins
(Reeves and Baker, 2000). The second strategy is
called exclusion strategy, where transport of heavy
metals in shoots and leaves is limited over a wide
range of metal concentrations in the soil. Some of the
plants make stable metal complexes in the root cells to
prevent metal translocation from the roots to
aboveground tissues (Tordoff et al., 2000).
The results showed that the plant species had
BCF
root
values of 1 indicate high efficacy in the
phytostabilization of metal contaminated soils and the
plant has adopted an exclusion strategy and there is
less risk of metals entering the food chain if the plant
is consumed.
An important characteristic as a hyperaccumulator
is the translocation factor (TF). Usually, it can
indicate the ability of metal transferring from the roots
International Journal of Scientific Research in Environmental Sciences, 2(10), pp. 379-388, 2014
385
to shoots of a plant. Plants with TF values 1 are
classified as high-efficiency plants for metal
translocation from the roots to shoots (Yoon et al.,
2006).
The roots of plant species are mainly responsible
for heavy metals phytoextraction and plants species
with TF 1 have the potential for phytostabilization
(Yoon et al., 2006), because in this process the metal
tolerant plant species immobilize heavy metals
through absorption and accumulation by roots,
adsorption onto roots or precipitation within the
rhizosphere (Wong, 2003). This process also
decreases metal mobility and reduce the likelihood of
metals entering into the food chain. Therefore, the use
of metal tolerant native flora represents an
inexpensive long-term solution.
A gradual increase in EC and available metals
content were observed with increasing concentration
of EDTA and pH did fall slightly. Soil pH is an
important parameter in determining the effectiveness
of applied EDTA in enhancing metals uptake (Bareen
and Tahira, 2010). Therefore, the efficiency of EDTA
on the metal solubilization and accumulation can be
enhanced by lowering the soil pH (Bareen and Tahira,
2010). Bareen and Tahira (2010) studied efficiency of
seven different cultivated plant species for
phytoextraction of toxic metals from tannery effluent
contaminated soil using EDTA and showed that
addition of EDTA to the soil at dose of 10mmol/kg
had highly significant effects on soil pH and EC.
Metal-extraction efficiency depends of several
factors, such as the matrix characteristics (i.e.,
substrate structure, chemical composition, texture,
grain size, etc.), metal properties, leachant
characteristics (i.e., concentration, binding power,
solubility, etc.), and, of course, the conditions of the
process itself (e.g., pH, temperature, phase ratio,
agitation, extraction time) (Liphadzi and Kirkham,
2005). Among these factors, pH is one of the most
important parameters since it governs speciation,
complexation and solubility as well as bioavailability
and transport of heavy metals (Nowack et al., 2006),
therefore metal uptake can be affected by application
of EDTA due to low acidity.
EDTA exhibited heavy metals extraction
efficiency. Wu et al. (1999) evaluated the effects of
different EDTA dosages (0, 3, 6, 12mmol/kg) on
leaching of different heavy metals (Cd, Cu, Pb, Zn).
The leaching of these heavy metals increased with
increasing concentration of EDTA. Soil analyses
showed a 4-78% loss in total Pb after application of
EDTA at the different rates. A maximum loss of Pb
from the soil was observed at a dose of 12mmol/kg
EDTA.
The results showed a significant correlation
between treatment time and heavy metal
concentrations in the plant organs and metals
concentration in the plant organs of S. maritimus L.
increased with passage of time. Harvest time had
positive influences on the soil metals reduction.
In general, harvest time as suitable dose of
chelating agents is a crucial factor in the effectiveness
of phytoextraction and there is still a lack of
information about the exact timing of the harvest after
application of chelating agents (Wang et al., 2009).
Wang et al. (2009) reported that the shoots of
Sedum alfredii on 14th day for low Pb soil and on
10th day for high Pb soil could achieve highest
phytoextraction effects. The authors cited EDDS
addition may affect plant growth significantly with the
passage of time, especially for high Pb soil because of
higher available Pb in soil.
The amounts of soil metals reached at their
maximum at triple dosage and under single dosage
application, heavy metals content in the soil reached
at its minimum concentration. The results indicated
that the chelate application method could limit the
solubility and migration potential of heavy metals in
the soil.
Increasing metal solubility is the major purpose of
applying chelants to the soil, and a precondition for
enhanced metal uptake by plants. On the other hand,
potential metal leaching associated with the
application of chelants may be of concern for the
chemical assisted phytoextraction.
Grman et al. (2001) reported that single dose of
2.9g EDTA/kg enhanced 105-fold Pb accumulation in
cabbage (Brassica oleracea L.) grown in a
greenhouse, as compared with a 44-fold increase if the
same amount of EDTA was split and added in four
intermittent doses. The authors reported if a soil has a
high Pb retention capacity, application of EDTA in
multiple doses could be ineffective in mobilizing and
enhancing root to shoot translocation. In these
conditions, application of the full rate of chelant in a
single dose could constitute the more effective
approach. Wenzel et al. (2003) assessed the effects of
dosage (up to 2.01g/kg) and mode (single vs. split) of
EDTA application on leaching of Cu, Pb and Zn
during and after the harvest of Brassica napus L. They
reported that the metals concentrations in the
leachates were related to the amount of EDTA
applied, but the authors found no difference between
applications of the same amount of EDTA in single or
split doses.

5. CONCLUSION

The results revealed that heavy metals concentrations
in S. maritimus L. depend significantly on the kind of
plant organs. Underground organs showed a greater
capacity of accumulation as compared to the shoots.
Ebrahimi and Shahsavand
EDTA Enhanced Phytoextraction Capacity of Scirpus Maritimus L. Grown on Pb-Cr Contaminated Soil and
Associated Potential Leaching Risks
386
The significantly different levels of Pb and Cr found
in the various organs may imply low metal mobility
from roots to rhizomes and to shoots (leaves and
stems) and the plant species would be applicable for
Pb and Cr phytostabilization because it had BCF
root

values 1 and a relatively low TF value. Considering
metals uptake, EDTA was efficient for extraction of
Pb and Cr from the tissues of the plant species but the
optimum dose of chelator for chelate-assisted
phytoextraction must be investigated before the
application of this technique. Present study concludes
that EDTA should be added at the concentration of
5mmol/kg a single dosage for 60 days in Pb and Cr
contaminated soils.

ACKNOWLEDGEMENTS

The authors are grateful to the Department of Range
and Watershed Management, University of Zabol
(Iran) for providing necessary facilities to undertake
this study.

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388








Dr. Mahdieh Ebrahimi, is an assistant professor in Department of Range and Watershed
management, University of Zabol, Iran. She obtained M.Sc. and Ph. D degrees in Range
management from University of Tehran, Iran in 2006 and 2011 respectively. She has published
books, national and international research papers. Her field of interest includes Range
management and phytoremediation.











Dr. Fariba Shahsavand is a Ph.D graduate in field of range management at Science and
Research Branch, Azad University, Iran. She received her degree in Master of Science in range
management from Azad University Iran in 2005. Her current research is focuses on range and
watershed management. To date, she has published several scientific articles related to
reclamation of rangelands.