EDTA and HEDTA printed - Các phức EDTA và HEDTA, vai trò của chúng trong môi trường

EDTA and HEDTA eects on Cd, Cr, and Ni uptake by
Helianthus annuus
Hong Chen, Teresa Cutright
*
Department of Civil Engineering, The University of Akron, Akron, OH 44325-3905, USA
Received 26 October 2000; received in revised form 16 January 2001; accepted 18 January 2001
Abstract
Phytoremediation has shown great potential as an alternative treatment for the remediation of heavy-metal-con-
taminated soils and groundwater. However, the lack of a clear understanding pertaining to metal uptake/translocation
mechanisms, enhancement amendments, and external eects on phytoremediation has hindered its full-scale applica-
tion. The objective of this research was to investigate the ability of synthetic chelators for enhancing the phytoreme-
diation of cadmium-, chromium- and nickel-contaminated soil. Ethylenediaminetriacetic acid (EDTA) and
N -(2-hydroxyethyl)-ethylenediaminetriacetic acid (HEDTA) were applied to the soil at various dosages to elevate metal
mobility. Uptake into and translocation within Helianthus annuus was determined. It was found that EDTA at a rate of
0.5 g/kg signi®cantly increased the shoot concentrations of Cd and Ni from 34 and 15 to 115 and 117 mg/kg, re-
spectively. The total removal eciency for EDTA was 59 lg/plant. HEDTA at the same application rate resulted in a
total metal uptake of 42 lg/plant. These research demonstrated that chelator enhancement is plant- and metal-speci®c
and is subjective to inhibition when multiple heavy metals are present. Results also showed that chelator toxicity re-
duced the plant's biomass, thereby decreasing the amount of metal accumulation. Ó 2001 Elsevier Science Ltd. All
rights reserved.
Keywords: Chelators; EDTA; HEDTA; Helianthus annuus; Phytoremediation; Metals
1. Introduction
Phytoremediation is a process that uses living green
plants for the in situ risk reduction for contaminated
soil, sludge, sediments, and groundwater through con-
taminant removal, degradation, or containment
(Anonymous, 1998). It has been shown to be more ad-
vantageous than conventional technologies for remedi-
ating heavy-metal-contaminated soils. The advantages
include large-scale application; growing plants is rela-
tively inexpensive; plants provide an aesthetic value to

the landscape of contaminated sites, and concentrated
hazardous wastes require smaller disposal facilities, and
the potential exists to recover metals from the biomass
(Saxena and KrishnaRaj, 1999).
At sites contaminated with heavy metals, phyto-
remediation can be applied as dierent strategies based
on the speci®c site condition. They may include phy-
toextraction, where metals are transported from the soil
into the harvestable shoots (Salt and Blaylock, 1995),
rhizo®ltration, where plant roots or seedlings grown in
aerated water precipitate and concentrate toxic metals
(Raskin et al., 1997), phytovolatilization, in which plants
extract volatile metals (e.g., Hg and Se) from soil and
volatilize them from the foliage (Salt and Blaylock,
1995), and phytostabilization, in which metal-tolerant
plants are used to reduce the mobility of heavy metals
(Raskin et al., 1997). For sites contaminated with both
heavy metals and toxic organics, phytoremediation is
Chemosphere 45 (2001) 21±28
www.elsevier.com/locate/chemosphere
*
Corresponding author. Tel.: +1-330-972-4935; fax: +1-330-
972-6020.
E-mail address: (T. Cutright).
0045-6535/01/$ - see front matter Ó 2001 Elsevier Science Ltd. All rights reserved.
PII: S 0 045-6535(01)00031-5
still applicable (Saxena and KrishnaRaj, 1999) because
the rhizosphere association between plants and soil mi-
croorganisms can be utilized to degrade or transform
complex organic±metal mixtures. This process has been

called phytotransformation or phytodegradation.
All plants have the potential to absorb a wide variety
of metals from the soil. For the most part, plants tend
to only absorb those metals that are essential for their
survival and growth. The most remarkable exception to
this general rule is a small group of plants that can
tolerate, uptake, and translocate high levels of certain
heavy metals that would be toxic to any other known
organism. Such plants are termed ``hyperaccumula-
tors''. According to Brown et al. (1995), hyperaccu-
mulator species are those plants whose leaves may
contain >100 mg/kg Cd, >1000 mg/kg Ni and Cu, or
>10 000 mg/kg Zn and Mn (dry weight) when grown in
metal-rich soils. With this extraordinary ability, these
plants can be used in future environmental remediation
activities, however, full-scale applications have yet to be
achieved. One important reason for this lies with the
lack of thorough knowledge on the biological processes
involved in metal acquisition, transport, and shoot
accumulation.
Salt et al. (1998) proposed that in the process of
acquiring metal ions from soil, plants have evolved
several strategies for increasing the metal bioavailability
due to the high binding capacity for metallic micronu-
trients by soil particles. The ®rst strategy is the plants'
ability to produce metal-chelating compounds (phytos-
iderophores) such as mugenic and avenic acids to
mobilize metal compounds from soil (Vonwiren et al.,
1996). The second approach involves the solubilization
of metals by exuding protons from roots to acidify the

rhizosphere soil (Crowley et al., 1991). Alloway (1995)
further suggested that the roots possess a signi®cant
CEC due to the presence of carboxyl groups, which
might help to move ions through the outer part of the
root to the plasmalemma where active absorption
occurs.
In addition to natural plant adaptations, the addi-
tion of synthetic chelators, soil acidi®ers, or commer-
cial nutrients can enhance phytoremediation. Several
studies have documented the success of pH adjustments
for mobilizing metals (Salt et al., 1998; Entry et al.,
1996; Chaney et al., 1997; Huang et al., 1997). Al-
though soil acidi®cation increased metal mobility, it
decreased the microbial activity of the surrounding
area (Cornish et al., 1995; Salt et al., 1998; Chen,
2000). Only the addition of synthetic chelators has been
shown to increase both the metal mobility within the
soil as well as the uptake (and translocation) through
the plant tissue without being irreversibly toxic to mi-
crobial activity. For instance, Huang and Cunningham
(1996) tested N -(2-hydroxyethyl)-ethylenediaminetri-
acetic acid (HEDTA) on Pb accumulation enhance-
ment and found that 1 week after transplanting, the
shoot Pb concentration was increased from 40 to
10 600 mg/kg. In addition to shoot concentration, the
shoot to root Pb content was increased from 0.2 to 1.2.
Blaylock (1997) showed that chelator supplements in-
creased the uptake of Pb, Cd, Cu, Ni, and Zn. Huang
et al. (1997) further reported that among the ®ve che-
lators, Ethylenediaminetriacetic acid (EDTA) was the

most ecient in increasing shoot Pb concentration in
both pea and corn, followed by HEDTA. They found
the order of the eectiveness in increasing Pb accu-
mulation to be EDTA > HEDTA > diethylenetrinitril-
opentacetic acid (DTPA) > ethylenegluatarotriacetic
acid (EGTA) > Ethylenediaminedinitrilodiac acid
(EDDTA). Dushenkov et al. (1999) demonstrated that
to increase
137
Cs bioavailability, of the 20 amendments
tested ammonium salts had the greatest eect.
Although chelators may increase the eectiveness of
phytoremediation by means of increasing the removable
metal concentrations, not all studies agree. Robinson
et al. (1999) reported that in their study, neither calcium
and magnesium carbonates, nor the addition of syn-
thetic chelating agents were eective in increasing metal
uptake by Berkheya coddii on serpentine soils. Bennett
et al. (1998) also found that their attempt to enhance
nickel uptake in B. coddii by adding EDTA and citric
acid to the substrates actually caused a decrease in nickel
uptake, despite causing an increase in the concentration
of soluble nickel.
The objective of this research was to investigate the
ability of synthetic chelators to enhance the phyto-
remediation of cadmium-, chromium- and nickel-con-
taminated soil. The preferred source (EDTA or
HEDTA) and dosage required to elevate metal mobility
and subsequent uptake and translocation within the
plant tissues were determined.

2. Experimental methods
2.1. Soil sources and characterization
An agricultural soil was collected from a clean resi-
dential garden center in northeastern Ohio. The soil was
air-dried under room temperature and mixed daily until
an 8% water content was reached. Soil was characterized
for soil texture, soil pH, ®eld capacity, cation-exchange
capacity (CEC), organic matter content (OM), and
contaminant background concentrations. Soil texture,
pH, and ®eld capacity were measured by the procedures
described by Tan (1995). CEC was determined by the
method proposed by Gillman (Nedelkoska and Doran,
2000). OM and TOC were analyzed with a Shimadzu
total organic carbon analyzer (TOC-5000) equipped
with a solid sample module (SSM-5000A). The back-
ground concentrations of total sorbed Cd, Cr, and Ni
22 H. Chen, T. Cutright / Chemosphere 45 (2001) 21±28
were determined with EPA method 3050 (Dramer et al.,
1996) (Table 1).
2.2. Soil preparation
To initiate the experiments, air-dried soil was
weighed and loaded into 21 Al pans (0.32 m  0.25
m  0.04 m). Each pan contained 1.5 kg soil DW per
pan. The soil was then rehydrated with a standard nu-
trient solution containing 250 mg N (NH
4
NO
3
,60mg
Mg (MgSO

4
, 109 mg P (KH
2
PO
4
, and 207 mg K
(KH
2
PO
4
+K
2
SO
4
 per kg soil DW (Senden et al.,
1990). Two days later, the appropriate metal solution
was spiked into the soil in Al pans and mixed thor-
oughly. For the ®rst experimental set, the solution
contained 50 ppm Cd
2
, (as CdCl
2
Á 2.5H
2
O), 50 ppm
Cr
3
(CrCl
3
Á 6H

2
O and 50 ppm Ni
2
(NiSO
4
Á 6H
2
O) for
a total metal concentration of 150 mg/kg. The metal
concentration corresponds to the individual metal ele-
ment content and not the overall compound. The second
experimental set had a reduced concentration of 30 mg/
kg for each metal. After the metal solution was added,
the soil was allowed to equilibrate for a period of 10
days in the greenhouse. The equilibration involved un-
dergoing three cycles of saturation with DI water and air
drying, before being remixed and vegetated (Muller and
Kordel, 1993). At day 12, pans were amended with ei-
ther EDTA or HEDTA (Sigma Chemical) at a concen-
tration of 1 or 2 g/kg. Chelator selection was based on
the previous work by Huang et al. (1997).
A control and blank pan were also prepared with
supplemental nutrients and/or metals and were subjected
to the saturation cycles as outlined above. The control
contained non-metal-spiked-vegetated soil for the in-
tention of obtaining data related to background activi-
ties, such as the plant accumulation of background
heavy metals and biomass growth in uncontaminated
soil. The four blanks consisted of the same levels of
spiked metal concentrations as treatments with an ex-

ception that no plants were grown in the soil. The pur-
pose of the blanks was to determine the vegetation eect
on metal mobility in the contaminated soil and to ensure
accuracy and precision in the analyses.
2.3. Cultivar source and seedling preparation
Cultivar selection was based on the plant's ability to
achieve hyperaccumulator status for at least one metal.
The dwarf sunspot sun¯ower, Helianthus annuus, has
been proven to be eective at removing heavy metals
and is capable of extracting higher than average
amounts of several radionuclides (Cooney, 1996; Gal-
lego et al., 1996; Dushenkov et al., 1997; Gouthu et al.,
1997; Sun and Shi, 1998; Chen, 2000; Zavoda et al.,
2001).
Seeds of H. annuus were obtained from USDA/ARS
Plant Introduction Station of Iowa State University.
They were initially sown in commercial potting soil
(SCHULTZ Professional Potting Soil Plus, SCHULTZ
Company) in a greenhouse illuminated with natural
light. Supplementary light was provided for maintaining
15-h photo-period daily. Greenhouse temperature was
28°C in the daytime and 15°C at night. After 2 weeks of
growth in the potting soil, seedlings with similar biomass
were transferred to the metal-spiked soil and the ex-
periment was initiated. Nine seedlings were used per
pan. Unless otherwise speci®ed, seedlings were harvested
4 weeks later.
2.4. Plant harvest and analysis
During harvest, plants were gently removed from soil
and washed until free of soil. Roots, leaves, and stems

were further separated with scissors and dried in a
convection oven at 70°C for 3 days (Page, 1982). Tissues
were milled with mortar and pestle and digested fol-
lowing the procedure outlined by Zheljazkov and Er-
ickson (1996). One g of milled plant matter was soaked
in 20 ml of concentrated nitric acid. After 6 h, the
mixture was boiled to 50% of its original volume. Then,
4 ml of perchloric acid was added and the mixture ref-
luxed for 90 min. The solution was ®nally diluted with
DI water to 25 ml of total volume and analyzed with
¯ame atomic absorption spectroscopy (Buck 200 AA).
2.5. Analysis of total metal and mobile metal fractions in
the soil
For this manuscript, the mobile metal fraction is
de®ned as the fraction that is not tightly bound to soil
and is mobile without the addition of chelators. The
Table 1
Physical and chemical characteristics of agricultural soil used in
this study
Soil properties Agricultural
soil
Sand (%) 16.62 Æ 0.87
a
Silt (%) 42.10 Æ 0.69
Clay (%) 41.28 Æ 1.13
Soil texture Silt clay
loam
Soil pH (1:1 soil/1 M KCl ratio) 5.51 Æ 0.21
Field capacity (%) 22.56 Æ 0.49
CEC (meq/100 g) 9.56 Æ 0.19

TOC (%) 1.53 Æ 0.10
OM (%) 3.06 Æ 0.07
Background Cd
2
level (mg/kg soil DW) 2.35 Æ 0.25
Background Cr
3
level (mg/kg soil DW) 8.05 Æ 1.57
Background Ni
2
level (mg/kg soil DW) 11.60 Æ 1.67
a
Mean Æ SE (each analysis was performed in duplicate).
H. Chen, T. Cutright / Chemosphere 45 (2001) 21±28 23
total metal concentration is the summation of the bound
and mobile fractions. In order to dierentiate between
the mobile and sorbed fractions, two dierent extraction
methods were used. The concentration of the total Cd,
Cr, and Ni was determined via an EPA acid digestion
method 3050 (Carter, 1993).
Approximately 10 ml of 1:1 HNO
3
was added to 2 g
of air-dried soil (<1 mm) in a 500-ml ball-shaped ¯ask
and heated at 95°C for 15 min. Five ml concentrated
HNO
3
was added and the solution was re¯uxed for an
additional 30 min at 95°C. This was repeated once and
the ®nal solution obtained was reduced to 5 ml. Once

cooled, approximately 25 ml of 30% H
2
O
2
was added to
the solution in 1-ml increments, followed by the addition
of 5 ml of concentrated HCl. The digestate was ®ltered
through a Whatman
â
No. 42 ®lter paper and the solu-
tion was diluted to 50 ml with DI water. The solution
was analyzed by FAAS (Buck 200 AA).
To extract the mobile metal fraction in the soil, a
procedure proposed by Maiz et al. (1997) was followed.
Two grams of air-dried soil sample was transferred into
a capped 40 ml heavy-duty PRYEX centrifuge tube,
mixed with 20 ml 0.01 M CaCl
2
solution, and agitated in
a rotary shaker at 200 rpm for 2 h. After 2 h, the soil
suspension was centrifuged at 2500 rpm for 15 min and
the supernatant was collected for FAAS analysis.
2.6. Statistical analysis
The experiments were designed as a two-stage nested
design with two types of chelators as the primary factors.
For each factor, two dierent concentrations were used.
The dierence between speci®c pairs of means was
identi®ed by Student±Newmans±Keuls (SNK) test
P < 0: 05. Statistical analysis of the data was performed
by using SigmaStat 2.0 (SPSS Science, Chicago, IL).

2.7. Results and discussion
2.7.1. Chelator eect on plant growth
Adding HEDTA and EDTA led to a severe yield
reduction in the biomass across the treatments. In the
®rst experimental set with higher metal concentration
and chelator (1 and 2 g/kg) doses, plants appeared to be
chlorotic and showed signs of wilting 1 day after the
experiment was initiated. Within 1 week, all plants were
dead. Therefore, the metal concentration was lowered
to 30 mg/kg per metal and the chelator additions low-
ered to 1.0 and 0.5 g/kg for the next set of experiments.
Lowering both metal concentrations and chelator ad-
ditions extended plant growth to some degree but a
large number of plants still died within 2 weeks. Plants
grown in 0.5 g/kg EDTA-treated soil exhibited better
growth rate and higher biomass was obtained. This was
supported by the visual observations where more than
half of the plants grown in the soil amended with
0.5 g/kg EDTA maintained vigorous growth through-
out 4 weeks. However, growth was still severely
retarded in comparison to non-chelator treatments. For
example, plants subjected to 30 mg/kg per metal with-
out chelators had less than a 10% reduction in biomass,
and none of the plants died. Furthermore, control pans
containing only chelator additions (i.e., no metals pre-
sent) did not exhibit a severe biomass reduction.
Therefore, the severe reduction in growth was attrib-
uted to the combination of heavy metal concentration
and chelator addition.
As compared with the control plants, the average

shoot biomass of the treatment plants decreased by
more than 75% for the 150-ppm contaminated soil (Fig.
1(a)) and more than 50% for the 90-ppm soil (Fig. 1(b)).
Plants in HEDTA-amended soil exhibited approxi-
mately the trend in biomass reduction. This indicated
that the levels of HEDTA added or the metal±HEDTA
compounds formed in soil were already too high and
therefore, toxic to the plants. Addition of EDTA ap-
peared to be less toxic to plants compared to HEDTA
as shown by a higher biomass. However, the yields be-
tween EDTA and HEDTA were not statistically dier-
ent. A possible reason was due to the dierent toxicity
of the two chelators and/or their metal±chelator com-
pounds formed. As a whole, this study demonstrated
that synthetic chelator addition had a signi®cant adverse
eect on plant growth.
Fig. 1. Eect of adding chelators on shoot biomass of 9 plants
grown in heavy-metal-contaminated soil. (a) Cd, Cr, and Ni at
50 mg/kg of each, (b) Cd, Cr, Ni spiked at 30 mg/kg of each.
Bars marked with (*) are statistically dierent with the control
P < 0:05. Error bars represent ÆSE of (n  3).
24 H. Chen, T. Cutright / Chemosphere 45 (2001) 21±28
2.7.2. Eects of chelators on mobile fractions of Cd, Cr,
and Ni in soil
As anticipated, chelator addition signi®cantly in-
creased the mobile fractions of Cd, Cr, and Ni as com-
pared with control (Fig. 2). Cr had the greatest increase
as its mobile fraction was raised by approximately 40-
fold in HEDTA-treated soil and 60-fold in EDTA-
amended soil (Fig. 2(b)). Cd and Ni were also increased

by more than 4- and 2-fold, respectively (Figs. 2(a) and
(c)).
The mobile fractions of Cd and Ni were shown to
increase with increasing levels of HEDTA and EDTA
added to the soil (Fig. 2). For chromium, however, in-
creasing of mobile fraction was more strongly dependent
on chelator species than on chelator concentration.
Since its mobile fraction did not increase when chelator
levels were increased to 1.0 g/kg, it may indicate that the
chelator level of 0.5 g/kg was high enough to elevate the
bioavailable Cr to the maximum level. Compared with
HEDTA, EDTA had approximately the same capability
to increase the mobile fraction of Cd and Ni while it was
more ecient at mobilizing Cr than HEDTA. The
general order of bioavailable metal concentrations as a
result of chelator addition in each treatment was
Cr > Cd  Ni.
While enhanced metal mobility can increase the up-
take into plants, the potential for movement into the
groundwater is also increased. An increase in metal
migration to the groundwater would have a detrimental
impact on the environment. Therefore, care should be
taken when selecting the ®nal chelator addition for ®eld
applications. The dosage must be high enough to mo-
bilize the metals to the root zone without being too high
to cause toxicity or elevated groundwater concentra-
tions.
2.7.3. Impact of chelator amendments on metal accumu-
lation in plants
Fig. 3 contains the shoot and/or root tissue accu-

mulation for each metal that resulted from the dierent
chelator amendments. Adding chelators signi®cantly
Fig. 2. Eects of HEDTA and EDTA additions on the mobile
fractions of (a) Cd, (b) Cr, and (c) Ni in soil for individual metal
concentrations of 30 mg/kg. For comparing chelator treatments
with the control, bars marked with a (*) are statistically dif-
ferent (P < 0:05). For comparing dierent chelator treatments,
the mean value followed by dierent capital letters are statis-
tically dierent (P < 0:05). Error bars represent ÆSE of n  3.
Fig. 3. Eect of adding HEDTA and EDTA on the tissue
concentrations of the dwarf sunspot sun¯ower. (a) Cd, (b) Cr,
and (c) Ni with individual spiked concentration of 30 mg/Kg.
For a given plant tissue, bars denoted with (*) are statistically
dierent from the control. Error bars represent ÆSE of n  3.
H. Chen, T. Cutright / Chemosphere 45 (2001) 21±28 25
enhanced shoot concentrations of Cd and Ni (Figs. 3(a)
and (c)). The shoot content of Cd and Ni were increased
by more than 2-fold and 4-fold as a result of the in-
creased mobile fractions of Cd and Ni in soil. In contrast
to shoot concentrations, root levels of Cd and Ni were
decreased by a small fraction as compared to the con-
trol. Therefore, they may be translocated to the shoot to
a greater extent than the non-chelated complexes. As a
result, the root concentrations of Cd and Ni were
slightly lowered.
Fig. 3(b) indicates that chelator additions, regardless
of the source or concentration, did not increase the
shoot concentration of Cr. This was surprising since the
mobile fraction of Cr surged from 0.47 to over 15 mg/kg
as shown in Fig. 2(b). In contrast, the root content of Cr

was enhanced. The root concentration increase was ap-
parently due to the increase of bioavailable Cr in soil.
The Cr-chelator compound may have dierent physio-
chemical properties as compared with Cd- and Ni-
complexes, therefore, it could not be translocated to
shoots.
Analysis of Fig. 3 indicated that the 0.5 g/kg
HEDTA dose had the best performance in enhancing
the concentrations of Cd and Ni in shoot and the
concentration of Cr in root tissue. However, it should
be noted that the enhanced high tissue concentration as
a result of chemical amendment might not necessarily
produce a high removal eciency for the target metal
contaminant since biomass change is another deter-
mining factor.
Some researchers (Huang and Cunningham, 1996;
Blaylock, 1997; Huang et al., 1997) have reported that
chelators such as HEDTA and EDTA may enhance the
shoot concentration of Pb by more than 100-fold.
However, in this study, these chelators demonstrated
only limited capability to improve the shoot accumula-
tions of Cd, Cr, and Ni. This is because most of the
current chelator studies focus on single metals like Pb.
Therefore inhibition from other metals would not im-
pede uptake and translocation. Moreover, dierent
plant species have been used in their studies. As a result,
it is believed that chelator enhancement is plant- and
metal-speci®c and is also subject to the interaction and
subsequent inhibitory eects when multiple heavy metals
are present.

2.7.4. Eect of chelator addition on total metal accumu-
lation
As compared with the control, the addition of che-
lators decreased heavy metal accumulation by plants
(Fig. 4). This was due to the severe biomass reduction.
If phytoremediation enhancement with chelators is go-
ing to succeed, a strategy that may protect plant bio-
mass from heavy loss is necessary. In this study, EDTA
at 0.5 g/kg appeared to be the best addition of the four
treatments with a total removal rate of 59 lg/plant (535
lg/pan), even though it caused a decrease in compari-
son with the control which was 103 lg/plant (927 lg/
pan). HEDTA at the application rate of 0.5 g/kg had
the highest metal concentration increase, yet it only
resulted in a total metal uptake of 42 lg/plant (376.7
lg/pan). These results indicate that the 0.5 g/kg chelator
dosage may be still be too high.
2.8. Conclusions and recommendations
EDTA and HEDTA both signi®cantly enhanced the
metal concentration in plant tissues, however, they re-
sulted in a severe biomass loss of more than 50%. As a
result, the total amount of metals removed by plants was
decreased. The study also determined that the eect of
synthetic chelators on phytoremediation is subject to the
in¯uence of multiple metal interactions and speci®c
plant species. With decreasing biomass aside, the che-
lator additions resulted in bioavailable metal order of
Cr > Cd  Ni.
For this study, the 0.5 g/kg EDTA application
achieved the best results. However, at this application

rate the use of chelators may not be economically
Fig. 4. Eect of adding HEDTA and EDTA on the total metals
accumulated by nine plants. (a) total Cd accumulation, (b) total
Cr accumulation and, (c) total Ni accumulated. Error bars
represent ÆSE of n  3.
26 H. Chen, T. Cutright / Chemosphere 45 (2001) 21±28
competitive with other technologies. Future studies will
focus on identifying the lowest, cost-eective chelator
addition that will enhance metal mobility and uptake
without posing a detrimental impact on groundwater
quality.
Acknowledgements
This work was conducted under the funding of
University of Akron Faculty Research Grant #1425.
The authors wish to extend their appreciation to Dr.
Randy Mitchell of the Department of Biology in Uni-
versity of Akron who provided greenhouse space for our
phytoremediation studies.
References
Anonymous, 1998. A citizen's guide to phytoremediation. EPA
542-F-98-011, Technology Innovation Oce.
Alloway, B.J. (Ed.), 1995. Heavy Metal in Soils, second ed
Chapman & Hall, London.
Bennett, F.A., Tyler, E.K., Brooks, R.R., Gregg, P.E.H.,
Stewart, R.B., 1998. Fertilization of hyperaccumulators to
enhance their potential for phytoremediation and phyto-
mining. In: Brooks, R.R. (Ed.), Plants that Hyperaccumu-
late Heavy Metals. CAB International, Wallingford.
Blaylock, M.J., 1997. Enhanced accumulation of Pb in Indian
mustard by soil-applied chelating agents. Environ. Sci.

Technol. 31, 860±865.
Brown, S.L., Chaney, R.L., Angle, J.S., Baker, A.J.M., 1995.
Zinc and cadmium uptake by hyperaccumulator Thlaspi
caerulescens grown in nutrient solution. Soil Sci. Soc. Am. J
59, 125±133.
Carter, M.R. (Ed.), 1993. Soil Sampling and Methods of
Analysis, Canadian Society of Soil Science, Lewis Publish-
ers, 1993.
Chaney, R.L., Malik, M., Li, Y.M., Brown, S.L., Brewer, E.P.,
Angle, J.S., Baker, A., 1997. Phytoremediation of soil
metals. Current Opinion Biotechnol. 8, 279±284.
Chen, H. 2000. Phytoremediation of Cd(II), Cr(III), and Ni(II)
contaminated soil, Masters thesis, The University of Akron.
Cooney, S.M., 1996. Sun¯owers remove radionuclides from
water in ongoing phytoremediation ®eld tests. Environ. Sci.
Technol. 30 (5), 194.
Cornish, J.E., Goldberg, W.C., Levine, R.S., Benemann, 1995.
Phytoremediation of soils contaminated with toxic elements
and radionuclides. In: Hinchee, R.E (Ed.), Bioremediation
of Inorganics. Battelle Press, Columbus, pp. 55±64.
Crowley, D.E., Wang, Y.C., Reid, C.P.P., Szaniszlo, P.J., 1991.
Mechanisms of iron acquisition from siderophores by
microorganisms and plants. Plant Soil 130, 179±198.
Dramer, U., Cotter-Howells, J.D., Charnock, J.M., Baker,
A.J.M., Smith, A.C., 1996. Free histidine as a metal chelator
in plants that accumulate nickel. Nature 379, 635±638.
Dushenkov, S., Kapulnik, Y., Fleisher, D.K.C., Ensley, B.,
1997. Removal of uranium from water using terrestrial
plants. Environ. Sci. Technol. 31 (12), 3468±3474.
Dushenkov, S., Vasudev, D., Gleba, D., 1999. Phytoremedi-

ation of radiocesium-contaminated soil in the vicinity of
Chernobyl, Ukraine. Environ. Sci. Technol. 33 (3),
469±475.
Entry, J.A., Vance, N.C., Hamilton, M.A., Zabowski, D.Z.,
Watrud, L.S., Andriano, D.C., 1996. Phytoremediation of
soils contaminated with heavy metals. Water, Air, and Soil
Pollut. 88, 167±176.
Gallego, S.M., Benavides, M.P., Tomaro, M.L., 1996. Eect of
heavy metal ion excess on sun¯ower leaves: evidence for
involvement of oxidative stress. Plant Sci. 121, 151±159.
Gouthu, S., Arie, T., Ambe, S., Yamaguchi, I., 1997. Screening
of plant species for the comparative uptake abilities of
radioactive Co, Rb, Sr, and Cs from soil. J. Radioanal.
Nucl. Chem. 222 (1±2), 247±251.
Huang, J.W., Cunningham, J.D., 1996. Lead phytoextraction:
species variation in lead uptake and translocation. New
Phytol. 134, 75±84.
Huang, J.W., Chen, J.J., Berti, W.B., Cuningham, S.D., 1997.
Phytoremediation of lead-contaminated soils: role of syn-
thetic chelates in lead phytoextraction. Environ. Sci. Tech-
nol. 31, 800±805.
Maiz, I., Esnaola, M.V., Millan, E., 1997. Evaluation of heavy
metal availability in contaminate soils by a short sequential
extraction procedure. Sci. Total Environ. 206, 107±115.
Muller, J., Kordel, W., 1993. Occurences and fate of phthalates
in soil and plants. Sci. Total Environ. (Supplement).
Nedelkoska, T.V., Doran, P.M., 2000. Hyperaccumulation of
cadmium by hairy roots of Thlaspi caerulescens. Biotechnol.
Bioeng. 67, 607±615.
Page, A.L. (Ed.), 1982. Method of Soil Analysis Pt 2: Chemical

and Microbiological Properties. American Society of
Agronomy, Madison WI, pp. 218±228.
Raskin, I., Smith, R.D, Salt, D.E., 1997. Phytoremediation of
metals: using plants to remove pollutants from the environ-
ment. Curr. Opinion Biotechnol. 8, 221±226.
Robinson, B.H., Brooks, R.R., Clothier, B.E., 1999. Soil
amendments aecting nickel and cobalt uptake by Berkheya
coddii: potential use for phytomining and phytoremediation.
Annu. Botany 84, 689±694.
Salt, D.E., Blaylock, M., 1995. Phytoremediation: a novel
strategy for the removal of toxic metals from the environ-
ment using plants. Biotechnology 13, 468±474.
Salt, D.E., Smith, R.D., Raskin, I., 1998. Annu. Rev. Plant
Physiol. Plant Mol. Biol. Phytoremediation 49, 643±668.
Saxena, P.K., KrishnaRaj, S., 1999. Phytoremediation of heavy
metal contaminated and polluted soil. In: Prasad, M.N.V.
(Ed.), Heavy Metal Stress Plants. Springer, Berlin,
pp. 305±329.
Senden, M.H.M.N., Van-Paassen, F.J.M., Van-Der-Meer
A.J.G.M, , Wolterbeek, H.T., 1990. Cadmium±citric acid±
xylem cell wall interactions in tomato plants. Plant Cell
Environ. 15, 71±79.
Sun, G., Shi, W., 1998. Sun¯ower stalks as adsorbents for the
removal of metal ions from wastewater. Ind. Eng. Chem.
Res. 37, 1324±1328.
Tan, K.H., 1995. Soil Sampling, Preparation, and Analysis.
Marcel Dekker, New York.
Vonwiren, N., Marschner, H., Romheld, V., 1996. Roots of
iron-ecient maize also absorb phytosiderophore-chelated
zinc. Plant Physiol. 111, 1119±1125.

H. Chen, T. Cutright / Chemosphere 45 (2001) 21±28 27
Zavoda, J., Cutright, T., Szpak, J., Fallon, E., 2001. The
uptake, selectivity, and inhibition of heavy metals, radio-
nuclides and phthalates treated hydroponically by plants. J.
Environ. Eng. (in press).
Zheljazkov, V.D., Erickson, N.E., 1996. Studies on the eects
of heavy metals (Cd, Pb, Cu, Mn, Zn, and Fe) upon the
growth, productivity, and quality of lavender production.
J. Essential Oil Res. 8, 259±274.
28 H. Chen, T. Cutright / Chemosphere 45 (2001) 21±28