Microchemical Journal 76 (2004) 39

Phytoremediation of lead using Indian mustard (Brassica juncea) with

EDTA and electrodics
Jae-Min Lim, Arthur L. Salido1, David J. Butcher*
Department of Chemistry and Physics, Western Carolina University, Cullowhee, NC 28723, USA
Accepted 14 October 2003

Abstract
The use of plants to remove toxic metals from soil (phytoremediation) is emerging as a cost-effective alternative to conventional
methods. Because contaminants such as lead have limited bioavailability in the soil, methods to facilitate its transport to the
shoots and roots of plants are required to the success of phytoremediation. Indian mustard (Brassica juncea) has been shown to
be effective at accumulating high tissue concentrations of lead when grown in contaminated soil with the addition of a chelating
agent, such as EDTA. The addition of an electric field around the plants is an alternative approach to increase the uptake of lead.
In this work, bench scale studies illustrated the application of electrodic phytoremediation with EDTA for lead using Indian
mustard. The effects of a range of parameters such as operating currentyvoltage with different concentrations, application time of
EDTA and electric potential, and daily application time of electric potential were studied. The accumulation of lead in the shoots
using 0.5 mmolykg EDTA with electric potential increased by two- to four-fold compared to the use of EDTA only. The maximum
lead accumulation in the shoots was obtained with the application of electric field 1 h per day for 9 days with EDTA.
2003 Elsevier B.V. All rights reserved.
Keywords: Soil; Lead; ICP-OES; Electrodics and electrokinetics; EDTA; Phytoremediation; Indian mustard (Brassica juncea)

1. Introduction
Soils may be polluted by a wide range of contaminants from industrial activities, sewage sludge disposal,
metal processing, and energy production. In many cases,
conventional remediation approaches, involving replacement of contaminated soil with clean soil, is both
expensive and intrusive to the ecosystem. Phytoremediation is the use of plants and plant processes to remove,
degrade, or render harmless hazardous materials present
in the soil or groundwater w1,2x. This emerging technology may offer a cost-effective, non-intrusive and safe
alternative to conventional soil cleanup techniques.
Phytoremediation developed from the study of heavy
metal tolerance in plants in the late 1980s. The discovery
of hyperaccumulator plants, which may accumulate levels of contaminants that are toxic to other plants,
*Corresponding author. Tel.: q1-828-227-3683; fax: q1-828-2277647.
E-mail address: butcher@email.wcu.edu (D. J. Butcher).
1
Present address: Department of Chemistry, Mercer University,
1400 Coleman Ave, Macon, GA 31207, USA.

prompted the use of these species to remediate soil.

Plants commonly employed for phytoremediation combine a high biomass plant, rapid growth, and high
hyperaccumulation, such as oat (Avena sativa), barley
(Hordeum vulgare), and Indian mustard (Brassica juncea) w35x.
Lead is a common and serious pollutant because of
its toxicity. It has limited solubility in soil and availability for plant uptake due to complexation with organic
matter, sorption on oxides and clays, and precipitation
as carbonates, hydroxides and phosphates w6x. Because
of its limited bioavailability, an approach to increase its
bioavailability is essential to the success of phytoremediation. Indian mustard (B. juncea) has been used for
the phytoremediation of lead and other metals w710x.
For more than 40 years, chelating agents, such as
EDTA, have been used to supply plants with micronutrients in soils and hydroponics, to increase the solubility
of metal cations in plant growth media, and to remove
toxic metals from contaminated soils in soil washing
remediation techniques w11x. In addition, these chemicals
solubilize metal cations from soil particles, and increase

0026-265X/04/$ - see front matter 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.microc.2003.10.002

J.-M. Lim et al. / Microchemical Journal 76 (2004) 39

employed in the orchard, including lead arsenate. In the

late 1980s and early 1990s, the property was subdivided
into residential housing. In 1999, extensive soil contamination by lead and arsenic was discovered on the home
sites, and the area was declared a Superfund site by the
Environmental Protection Agency (EPA) in late 1999.
The objective of this study is to investigate the
combination of EDTA and electrodic phytoremediation
for the removal of lead from contaminated soil from
Barber Orchard. The efficiency of phytoremediation was
optimized in greenhouse studies by investigation of
pertinent parameters including operating currentyvoltage
with different EDTA concentrations and application
times w23,24x. These results demonstrate that this
approach has considerable potential for the remediation
of lead from contaminated soils.
2. Experimental
2.1. Soil collection and treatment

Fig. 1. (a) Location and (b) configuration of the sampling site at

Barber Orchard, NC.

the availability of the metals to the plants w1113x.

EDTA has been the most widely used chelate because
of its relatively low cost and ability to increase the
solubility of many metals. Several publications have
demonstrated that the effectiveness of chelating agents
to significantly increase lead accumulation in Indian
mustard w3,12,14x.
Another approach to remove heavy metals, radionuclides, and organic contaminants from contaminated soil
and groundwater is electrodic and electrokinetic remediation w1522x. This technology employs the application of high-voltage, low-level direct current to the
polluted soil using electrodes placed in the ground to
remove contaminants. Electrodic and electrokinetic
remediation, which is based on the processes of electromigration of ionic species, electro osmosis, and electrophoresis, has been employed for heavy metals
w19,20x.
Barber Orchard is a housing development in Haywood
County, North Carolina, as shown in Fig. 1 w14x. The
area was used as a commercial apple orchard from 1908
to 1988. During this period, various pesticides were

Lead (Pb) and arsenic (As)-contaminated soil was

collected from a plot at Barber Orchard as shown in
Fig. 1. Samples were collected at an upper depth (015
cm) and a lower depth (1530 cm) (Fig. 1b). The soil
was screened to pass through a 1.0 cm sieve before
analysis.
For all phytoremediation experiments, a large quantity
of soil was thoroughly mixed to ensure homogeneous
concentration levels of lead and arsenic. The homogenized soil (ca. 1.2 kg) was subsequently transferred to
an 8 cm plastic pot. The pH of the homogenized soil
was between 5.0 and 5.2.
2.2. Cultivation practices
Indian mustard seeds (B. juncea) were germinated on
filter paper for 23 days before being transferred to
pots containing 1.2 kg of lead contaminated soil. The
plants were grown for 12 weeks, after transfer to the
pots, before phytoremediation experiments were initiated. They were watered daily and fertilized weekly
(Miracle-Gro). The plants were harvested 1 week following treatment by EDTA.
The plants were harvested by cutting the stem 1 cm
above the soil surface. The shoots and roots were washed
with soapy water, and rinsed with deionized water. The
plant samples were dried in an oven at 60 8C and
homogenized using a ball-mill (Spex 8000).
2.3. Experimental apparatus for electrodic phytoremediation
The electrodic phytoremediation system included an
array of electrodes, a power supply, EDTA, and plants
(Fig. 2) w16,17x. Copper plates were used for electrodes

J.-M. Lim et al. / Microchemical Journal 76 (2004) 39

Table 1
Operating conditions for ICP-OES
Model
Power
Frequency
Ar flow rates:
Nebulizer gas
Auxiliary gas
Plasma gas
Emission line:
Pb
As

Optima, 4100 DV, Perkin Elmer

1.3 kW
40 MHz
0.8 lymin
0.2 lymin
15 lymin
220.353, 217.000 and 283.306 nm
188.979, 193.696 and 197.197 nm

3. Results and discussion

3.1. Soil characterization
Fig. 2. Schematic diagram of the electrodic phytoremediation system.

because of their low cost and availability. Electrical

power was applied to the electrodes by DC power
supplies. Each supply was capable of delivering 400 V
and 1 A.
2.4. Sample preparation and analysis
Soil or homogenized plant tissue (shoots and roots of
Indian mustard) (ca. 0.2 g) were digested on a heating
block (lab manufactured) with 5 ml of concentrated
HNO3 followed by 1 ml of 30% H2O2. The digested
samples were diluted to 100 ml with distilled-deionized
water. Lead and arsenic concentrations were determined
by inductively coupled plasma optical emission spectrometry (ICP-OES) (PerkinElmer, Optima, 4100 DV).
Operating conditions for the ICP-OES are given in Table
1. A NIST (National Institute of Standards and Technology) standard reference material (SRM 2710, Montana Soil) was analyzed with the soil samples for quality
control purposes.

The concentrations of lead and arsenic were determined as a function of location and depth at the
sampling site at Barber Orchard (Fig. 1b). The results
are summarized in Table 2, where the values are
expressed as the mean"S.D. (ns3). The concentration
of lead with depth of sampling ranged from 71.1 to 473
mgykg in the upper soil (015 cm) and from 25.8 to
202 mgykg in the lower soil (1530 cm). Arsenic
concentrations varied from 32.9 to 282 mgykg in the
top soil and from 13.6 to 87.1 mgykg in the bottom
soil. The highest lead and arsenic levels were determined
at the same location. In general, higher lead and arsenic
concentrations occurred near the surface.
In order to evaluate the efficiency of phytoremediation, it was necessary to homogenize the soil. Fig. 3
shows that nearly identical concentrations levels of lead
and arsenic were present in each pot, demonstrating the
effectiveness of the homogenization procedure. The
average concentrations of lead and arsenic were determined to be 341"12 mgykg and 103"5 mgykg,
respectively.

Table 2
Determination of lead and arsenic in soil samples from the Barber Orchard lot in Fig. 1b
Element

Row

Concentration (mgykg)(mean"S.D.)
Column 1

Column 2

Column 3

Column 4

Column 5

Column 6

Column 7

Lead

Upper 1
Lower 1
Upper 2
Lower 2
Upper 3
Lower 3

179"21
49.5"6.3
125"9
25.8"1.9
473"17
102"6

184"8
54.1"2.4
254"7
70.5"2.4
256"16
31.9"1.5

71.1"6.9
29.5"2.6
396"17.6
137"13
107"14
32.6"3.8

149"15
132"9
130"10
152"5
266"17
202"12

291"25
33.9"5.8
569"26
45.1"2.4
205"18
161"6

287"16
40.1"2.6
285"14
190"15
273"26
189"18

154"11
49.7"7.4
138"13
35.6"6.4
99.3"8.2
104"10

Arsenic

Upper 1
Lower 1
Upper 2
Lower 2
Upper 3
Lower 3

51.0"8.8
19.3"0.7
50.0"2.1
14.4"0.2
282"9
51.7"2.7

62.3"8.6
22.5"1.1
85.3"6.8
27.6"3.5
96.8"6.4
20.9"1.6

34.8"4.9
15.5"1.8
164"14
59.7"8.6
46.3"3.5
17.8"2.4

52.5"2.9
49.1"4.8
46.1"2.6
50.7"5.1
88.2"1.6
83.7"5.6

104"8
16.7"0.7
229"17
22.0"1.3
103"5
87.1"7.2

95.5"12.5
18.0"2.9
126"6
85.1"6.8
90.3"4.1
67.9"9.7

54.1"2.4
20.4"1.8
42.0"3.7
13.6"0.7
32.9"3.2
36.6"5.9

J.-M. Lim et al. / Microchemical Journal 76 (2004) 39

Fig. 3. Evaluation of the homogeneity of soil based upon lead and

arsenic concentration in plastic pots. Values represent the mean"S.D.
of three replicates in each pot.

3.2. Effect of lead accumulation in Indian mustard by

EDTA

levels between 0 and 5 mmolykg were applied to the

controls in the absence of electrical potential. The
maximum accumulation of lead in shoots of Indian
mustard was obtained at the 5 mmolykg EDTA, which
is comparable to the previous EDTA-only experiment
(Fig. 4).
The use of electrical potential in combination with
EDTA, caused an enhancement in the amount of lead
accumulated in shoots of Indian mustard (Fig. 5a). The
maximum quantity of lead was obtained with 5 mmoly
kg EDTA and 3040 V of electric potential. Above 0.5
mmolykg EDTA, the combination of EDTA and electric
potential increased lead shoot accumulation by two to
four times compared to the use of EDTA only.
Fig. 5b shows a similar study performed on the roots
of Indian mustard. The combination of EDTA and
electric potential induced did not cause as large an
increase in lead accumulation in roots compared to the
use of EDTA only (Fig. 5b). Fig. 5c shows the relationship between the amount of EDTA added, applied

Two sequential harvests were conducted to optimize

the amount of EDTA for the phytoremediation of lead
from contaminated soils. The shoots and roots of Indian
mustard were harvested 1 week after application of
EDTA. Fig. 4 shows lead concentrations in shoots and
roots vs. added EDTA (0 to 5 mmolykg) for the first
of these harvests. The amount of lead in the shoots of
Indian mustard increased with the amount of EDTA
applied to the soil (Fig. 4a). At the highest amount of
added EDTA (5 mmolykg), lead concentrations in Indian mustard shoots had a maximum value of 677"102
mgykg.
However, the amount of EDTA added had a much
lower impact upon the uptake of lead by the roots of
Indian mustard (Fig. 4b). A significant difference was
only observed between very low and very high added
amounts of EDTA.
3.3. Optimization of electric potential and EDTA
concentration
Indian mustard plants, grown in homogenized Barber
Orchard soil, were treated with EDTA at concentrations
from 0 to 5 mmolykg with electric potential between 0
and 40 V. After the plants were allowed to grow for 12
weeks, EDTA was applied. Electric potential was then
applied for 1 h per day for 9 days. Current was measured
while the electric potential was applied. After 9 days,
the shoots and roots were harvested. This procedure was
repeated in two sequential harvests.
The concentration profiles of lead absorbed in the
shoots and roots, as well as current profiles, for the first
harvest are shown in Fig. 5. EDTA at concentration

Fig. 4. The effect of the amount of EDTA added upon lead accumulation in Indian mustard: (a) shoots and (b) roots. The plants were
harvested 7 days after the application of EDTA. Values represent the
mean"S.D. of three replicates from four plants.

J.-M. Lim et al. / Microchemical Journal 76 (2004) 39

potential, and the measured current. Comparison of Fig.

5a,c shows that an increase in current is accompanied
by an increase in lead accumulation in the shoots.
3.4. Effect of application time of EDTA and electric
potential
A study was performed to determine the optimum
time to harvest Indian mustard shoots after the application of EDTA and electric potential (Fig. 6). Indian
mustard plants were grown for 12 weeks. At this time
(times0), EDTA was applied to the plants, and electric
potential was applied for a 1 h period each day. The
results of this study are shown in Fig. 6. Minimal
accumulation of lead was observed in the absence of
both EDTA and electric potential, and in the presence
of electric potential only.
However, in the absence of electric potential and the
presence of EDTA, the maximum accumulation of lead
occurred 12 days from the beginning of the experiment.
After 12 days, the lead concentration decreased because

Fig. 6. The effect of EDTA and electric potential (1 hyday) upon

shoot lead accumulation in Indian mustard. The application of EDTA
and electric potential began at times0. Values represent the
mean"S.D. of three replicates from three plants.

Fig. 5. The effect of operating voltage and EDTA concentrations upon lead accumulation in Indian mustard: (a) shoots and (b) roots. The current
measured during application of the electric field is shown in (c). Values represent the mean"S.D. of three replicates from three plants.

J.-M. Lim et al. / Microchemical Journal 76 (2004) 39

EDTA. In summary, the combination of EDTA-enhanced

and electrodic phytoremediation provides significant
benefits compared to the use of EDTA alone.
Acknowledgments
This research was supported by Mr and Mrs Blanton
Whitmire. The ICP-OES instrument employed in this
research was partially funded by an award from the
National Science Foundation Division of Undergraduate
Education (NSF Award 995022 CCLI-A&I).

Fig. 7. The effect of daily application time and magnitude of electric

potential upon shoot lead accumulation in Indian mustard. Values represent the mean"S.D. of three replicates from three plants.

the mustard leaves fell off due to the toxicity of the

EDTA. The combination of EDTA and an electric field
induced maximum accumulation in the plants after only
9 days (Fig. 6). Moreover, the total amount of lead
remediated was approximately twice as large as the
study using EDTA only. Hence, the combination of
EDTA and electric potential increased the speed and
amount of lead remediated.
3.5. Effect of daily application time of electric potential
A study was performed to optimize the time that
electric field was applied to the plants each day. This
study was conducted because of the toxicity of EDTA
and electric field upon the plants, which decreases the
biomass and the amount of lead remediated. In this
experiment, either 30 or 60 min per day of electric
potential (0, 10 and 30 V) were applied to the Indian
mustard for 9 days with 2 mmolykg EDTA or without
EDTA (Fig. 7). The highest accumulation of lead in the
shoots occurred with an application time of 60 min per
day.
4. Conclusions
The combination of EDTA-enhanced phytoremediation with electrodics for lead in Indian mustard was
investigated. The addition of EDTA was shown to
significantly increase the accumulation of lead in Indian
mustard shoots. However, the use of electric potential
with EDTA caused increased phytoremediation by two
to four times compared to EDTA only. In addition, the
combined procedure increased the rate of remediation.
Optimum results were obtained using 5 mmol EDTAy
kg, 3040 V, an electric field application time of 1 hy
day, and a harvesting time of 9 days after application of

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