Journal of Environmental Chemistry and Ecotoxicology Vol. 4(5), pp.

103-109, 2 March, 2012

Available online at http://www.academicjournals.org/JECE
DOI: 10.5897/JECE11.078
ISSN-2141-226X ©2012 Academic Journals

Full Length Research Paper

Ethylenediaminetetraacetate (EDTA)-Assisted
phytoremediation of heavy metal contaminated soil by
Eleusine indica L. Gearth
Garba, Shuaibu Tela1*, Osemeahon, Akuewanbhor Sunday2, Maina, Humphrey Manji2 and
Barminas, Jeffry Tsaware2
1
Department of Chemistry, P. M. B. 1069. University of Maiduguri, Borno State.Nigeria.
2
Department of Chemistry, P. M. B. 2076. Federal University of Technology Yola (FUTY), Adamawa State, Nigeria.
Accepted 2 February, 2012

This study was designed to assess the natural and chemically enhanced phytoextraction ability of
Eleusine indica (grass). Three sets of laboratory pot experiment were conducted. Viable seeds of the
grass were seeded into one kilogram of the experimental soil placed in each plastic pot. The shoot, root
and the experimental soil around root were analyzed for the preliminary levels of the heavy metals:
Copper (Cu), Cadmium (Cd), Chromium (Cr), Cobalt (Co) and Lead (Pb). The preliminary levels of Cu,
Cd, Cr, Co and Pb in soil, root and shoot of the grass are: soils: 104.5, 5.1, 36.4, 13.3, 14.4 μg/g; root:
164.2, 4.3, 153.9, 11.5 and 24.7 μg/g and shoot of the grass are: 111.5, 2.9, 51.2, 11.1, and 60.7 μg/g
respectively. The phytoextraction ability was assessed in terms of its metal transfer factors; Enrichment
Coefficient (EC) and Translocation Factor (TF). Copper, Chromium and Lead had the highest EC of 1.07,
1.41 and 4.22 respectively. The levels of the elements in the roots and shoots of the grass at the end of
the laboratory experiment shows that more than the bioavailable pool of Cu, Cd, Cr Co and Pb were
taken up in the roots with slow translocation of Pb to the shoot: t1Cu 236.0 to 108.2 μgg-1 root-shoot;
t2Cu 137.5 to 316.8 μgg-1 root to shoot; t1Cr 228 to 84.3 μgg-1 root-shoot; t2 Cr 242.6 to 94.2 μgg-1 root to
-1 -1
shoot; t1Pb 54.8 to 176.2 μgg root to shoot and t2 Pb 96.0 to 326.0 μgg root-shoot. Inductively Coupled
Plasma to Optical Emission Spectroscopy - ICP-OES (for Pb determination) and X-ray fluorescence
(XRF) (for Cu, Cr, Cd and Co determination) were used for heavy metals determination in this study.
The grass showed relatively good response to EDTA application and the higher levels of Cu and Cr
concentration in the root suggested that the grass may be a good metal excluder with the possibility of
extracting Pb from contaminated soils.

Key words: Phytoextraction, phytostabilization, pollution, soil, grass, cadmium, cobalt, copper, lead and
chromium.

INTRODUCTION

Our environment has always been under natural stresses polluting substances including heavy metals.
but its degradation was not as severe as it is today. The Environmental pollution by heavy metals is now a
importance of the study of environmental hazards and global issue that requires considerable attention. Soils
their impact on living beings needs no emphasis. The contaminated with heavy metals usually lack established
human use of soil can lead to its deterioration by the vegetation cover either due to the toxic effects of the
degradation of soil organic matter and the lowering of its heavy metal or to the incessant physical disturbances
fertility due to erosion and the introduction of various such as erosion (Salt et al., 1995). Most heavy metals are
emitted from anthropogenic sources such as industries
and transportation. Manure and herbicides as well as,
sewage silt used in agriculture are also sources of heavy
*Corresponding author. E-mail: ms.tg13@yahoo.com. Tel: 070 metals in the environment (Fargasova, 1999).
30352060. Persistence of these heavy metals in soils and
104 J. Environ. Chem. Ecotoxicol.

continuous exposure to them can directly or indirectly (Nowack et al., 2006; Evangelou et al., 2007). Many
lead to their accumulation in plants, animals and chemical amendments, such as ethylenediaminetetraacetic
subsequently humans. acid (EDTA), Hydroxyethylene-diaminetriacetic acid
Trace amount of some heavy metals such as Cu, Zn, (HEDTA), Nitrilotriacetic acid (NTA) and organic acids
Fe, and Co are required by living organisms, however, have been used in pot and field experiments to enhance
any excess amount of these metals can be detrimental extraction rates of heavy metals and to achieve higher
(Berti and Jacobs, 1996). Non-essential heavy metals phytoextraction efficiency (Blaylock et al., 1997; Wu et
include arsenic, antimony, cadmium, chromium, mercury, al., 2006). There is much evidence confirming that EDTA
lead, etc; these metals are of particular concern because is one of the most efficient chelating agents in enhancing
they cause air, soil and water pollution (Kennish, 1992). Pb phytoavailability in soil and subsequent uptake and
Decontamination of such soils has therefore, become translocation in shoots (Chen and Cutright, 2001; Shen et
imperative for the safety of animals and humans. A al., 2002).
number of techniques have been developed to remove Majority of studies on phytoremediation was based on
metals from contaminated soils. However, many sites pot experiments and hydroponic culture, and only a few
remain contaminated because of economic and reports evaluated the phytoextraction potential of
environmental costs of the available technologies. hyperaccumulators or high biomass crops under field
Techniques such as excavation and disposal of conditions (McGrath et al., 2006; Zhuang et al., 2007).
contaminated soils in landfills are not environmentally Only a few attempts have been made to evaluate the
friendly and may serve as secondary pollution sources. possibility of metal removal in response to modifications
Therefore, new environmentally friendly and less of agronomic practices (Marchiol et al., 2007). Some
expensive techniques are required. weeds of the grass family have been experimented to be
Phytoremediation of heavy metal contaminated soils is suitable for phytoremediation because of their multiple
an emerging technology that extracts or inactivates ramified root systems.
metals in soils. It is defined as the engineered use of In this study, we assessed the natural and chelated
green plants (including grasses, shrubs and woody phytoextraction potential of the native tropical grass:
species) to remove, concentrate, or render harmless Eleusine indica, and when chemically enhanced with
such environmental contaminants as heavy metals, trace EDTA, to evaluate the ability of the grass to remediate
elements, organic compounds, and radioactive soils contaminated with multiple heavy metals.
compounds in soil or water (Hinchman et al., 1996). It is
environmentally friendly, of low cost, in situ applicable MATERIALS AND METHODS
technique for the clean-up of sites contaminated with
toxic metals or organic pollutants. Depending on the Sampling
degree of contamination and the size and volume of the
Four samples of soil and grass were collected from a refuse
polluted area, different technologies can be used to dumping site along Gombe road at the outskirt of Maiduguri
achieve the desired goals (Henry, 2000; McGrath, 1998; metropolis (Figure 1). Fresh plant samples were collected in the
Salt et al., 1998). Phytoextraction seems to be the most morning by pulling carefully from the soil to avoid damage to the
promising technique and has received increasing roots and washed with tap water. They were then separated into
attention from researchers since it was proposed by shoots and roots. Soil samples were collected from the surface to
subsurface portion of the soil around the plant roots (Rotkittikhum et
Chaney (1983) as a technology for reclaiming metal
al., 2006) at a range interval of 20 to 30 square meter apart.
polluted soils. Several approaches have been used but
the two basic strategies of phytoextraction, which have
finally been developed are: Chelate or chemically Sample preparation and analysis
assisted phytoextraction or induced phytoextraction, in Both the soil and plant samples collected were dried at 60°C to a
which artificial chelates are added to increase the mobility constant weight, grounded into fine powder, sieved with 2 mm wire
and uptake of metal contaminant and continuous or mesh and analyzed for the preliminary levels of the heavy metals:
natural phytoextraction which measures the natural ability Cd, Cu, Co, and Cr were analyzed using X-ray fluorescence (XRF)
of the plant to remediate soil. Only the number of plant while Pb was determined using inductively coupled plasma optical
emission spectroscopy (ICP-OES) following aqua- regia digestion
growth repetitions is therefore controlled (Salt et al., (McGrath and Cunliffe, 1985). The dried soil sample was also
1995, 1998). characterized for its physicochemical properties (Lombi et al.,
In view of the fact that the rate of bioremediation is 2001). The concentration of Cd, Cu, Co, and Cr in the shoots and
directly proportional to the plant growth rate and the total roots of the grass samples were also determined by X-ray
amount of bioremediation is correlated with plant’s total fluorescence (XRF) while ICP-OES was used to determined the
level of Pb. Using 0.5 g of the powdered sample, digested with
biomass, the integration of specially selected high
HNO3 and HClO4 acid (Lombi et al., 2000).
biomass crops with improved plant husbandry and
innovative soil management practices is a promising
alternative strategy towards achieving high biomass and Physicochemical analysis of soil samples
metal accumulation rates from contaminated soil Soil texture was determined by the Bouyoucos hydrometer method.
 Garba et al. 105

Table 1. Physicochemical properties of experimental soil.

Soil parameter Mean ±S.D

Clay (%) 25.90 ±1.80
Silt (%) 21.70 ±2.50
Sand (%) 50.40 ±2.80
pH 7.80 ±0.10
Organic matter (%) 4.15 ±0.05
Nitrogen (%) 0.05 ±0.02
C EC (mol/ 100 g soil) 11.27 ±0.76
EC (mS/cm) 464.00 ±0.10
Potassium (μg/g) 22.73 ±2.63
Moisture content (%) 34.00±1.80
Measurements are averages of three replicates ± S.D (Standard deviation); CEC: Cation
exchange capacity; EC: Electrical conductivity.

The moisture content of soil was calculated by the weight difference < 0.05) was used throughout the study.
before and after drying at 105°C to a constant weight. The pH and
electrical conductivity (EC) were measured after 20 min of vigorous
mixed samples at 1: 2.5. Solid: deionized water ratio using digital RESULTS
meters (Elico, Model LI-120) with a combination pH electrode and a
1-cm platinum conductivity cell respectively. Total nitrogen was The taxonomic classification of the experimental soil
determined according to the standard methods of the American
Public Health Association (1998). Cation exchange capacity (CEC)
(Table 1) was sandy loam with pH of 7.8, EC of 464
was determined after extraction with ammonium acetate at pH 7.0 mS/cm. The high pH level of the soil is generally within
and the organic carbon was determined by using Walkley–Black the range for soil in the region; soil pH plays an important
method (Jackson, 1973). role in the sorption of heavy metals, it controls the
Three sets of controlled and artificial laboratory experiment were solubility and hydrolysis of metal hydroxides, carbonates
conducted. Plastic pots were used for the experiment. 0.5 to 1.00
and phosphates and also influences ion-pair formation,
kg soils of known chemical composition were placed into each of
the pots and viable seeds of grass were seeded to soil. Soils of solubility of organic matter, as well as surface charge of
known chemical concentration were contaminated with various Fe, Mn and Al-oxides, organic matter and clay edges
grams of the metals; Cu, Co, Cr, Cd and Pb. The contaminated soil (Tokalioglu et al., 2006).
received the metals Cd as Cd (NO3)2; Co as CoCl2; Cr as Chromic The preliminary concentration levels of Cr and Co
acid; Cu as CuCl2 and Pb as Pb (NO3)2 at the concentration of 50, observed in experimental soil are 36.4 and 13.30 μg/g
150, 250, 250 and 150 mgkg-1 respectively. EDTA was applied to
respectively. Maiduguri metropolitan highway road
another soil of known chemical composition, amended with the
same level of the said elements. This was done at the rates of one networking has been characterized with high level of Cu
gram per kilogram (2.7 mmolkg-1 of soil), four weeks after (Garba et al., 2007). It is specifically adsorbed or fixed in
germination of the grass. soils, making it one of the trace metals (Baker and Senft,
Experiments were exposed to natural day and night 1995). Hence, the level of Cu observed in experimental
temperatures. Since humidity is one of the factors ensuring the soil used in this study (104.50 μg/g) was the highest of all
growth of plants and the necessary physiological processes, the
experimental grass in the pots were watered every 5 days with 200
the five metals studied. The level of Pb in the soil was
ml of deionized water (Lombi et al., 2001). To prevent loss of found to be 14.4 μg/g. Cadmium is considered to be
nutrients and trace elements out of the pots, plastic trays were mobile in soils but is present in much smaller
placed under each pot and the leachates collected were put back in concentrations (Zhu et al., 1999). This could explain why
the respective pots. This was done for a period of three months. the level of Cd (5.10 μg/g) observed in the experimental
Four replicates for each pot of grass were planted for statistical soil used in this study was the lowest when compared to
data handling. The samples of grass collected at the end of the
experiment, were separated into roots and shoots, dried at 60oC to the other metals (Table 2). It has been reported that the
a constant weight, grounded into fine powder, sieved with 2 mm level and impact of heavy metals on the environment is
wire mesh and analyzed using X-ray fluorescence (XRF) for the greatly dependent on their speciation in soil solution and
level of the metals; Cu, Co, Cr, Cd, while ICP-OES was used to solid phase which determine their environmental
determine the level of Pb. availability, geochemical transfer and mobility pathways
(Pinto et al., 2004).
Statistical analysis

All statistical analyses were performed using the SPSS 17 package. Uptake and accumulation of metals by the grass
Differences in heavy metal concentrations among different parts of plant E. indica
the grass were detected using One-way ANOVA, followed by
multiple comparisons using Turkey tests. A significance level of (p Table 2 shows the preliminary naturally desorbed
106 J. Environ. Chem. Ecotoxicol.

Table 2. Preliminary concentrations (μg/g) of Cu, Cd, Cr, Co and Pb observed in the roots, shoots and the
experimental soil samples from the sampling site.

Sample Root Shoot Soil

Element Mean ±SD Mean ±SD Mean ±SD
k c d
Cu 164.20 ± 2.93 111.50 ±1.61 104.50 ±1.94
h b e
Cd 4.30 ±0.88 2.90 ±1.94 5.10 ±1.03
g q 0f
Cr 153.90 ± 3.18 51.20 ±2.16 36.4 ±2.68
w b
Co 11.50 ±2.87 11.10 ± 2.42 13.30a±2.36
Pb 24.70n±2.59 60.70x±2.57 14.40a±2.09
The mean differences of elements in the same column with same letters are not significant at (p<0.05). (n=4).

Table 3. Enrichment coefficient (EC) and Translocation factor (TF) of the metals by the grass.

Sample
Translocation factor (TF) Enrichment coefficient (EC)
Element
Cu 0.68 1.07
Cd 0.67 0.57
Cr 0.33 1.41
Co 0.97 0.83
Pb 2.46 4.22
TF is calculated by the relation: - ratio of concentration of metal in the shoot to the concentration of metal
in the roots (Cui et al., 2007). EC is given by the relation: - The ratio of the concentration of metal in the
shoots to the concentration of metal in the soil (Chen et al., 2004).

concentration of the metals observed in the grass root absorbed by the grass. Translocation factor is a measure
and shoot of this study. In the roots, the levels of Cu, Cr, of the ability of plants to transfer accumulated metals
Pb, Co and Cd observed are: 164.20; 153.90; 24.70, from the roots to the shoots. It is given by the ratio of
11.50 and 4.30 μg/g respectively. And in the shoot the concentration of metal in the shoot to that in the roots
levels for Cu, Cr, Pb, Co and Cd are 111.5, 51.2, 60.7, (Cui et al., 2007; Li et al., 2007). The TF observed for Pb
11.10 and 2.90 (μg/g) respectively. Most of the metals was 2.46, the only element that has TF greater than one.
(Cu, Cr and Cd) were found at higher level greater in root The enrichment coefficient (EC) was used to evaluate the
than the shoot. It has been reported that most grass ability of plant to accumulate heavy metals in the root.
specie are known to concentrate heavy metals in the Enrichment coefficient was given by the ratio of the
roots, with only very low translocation to the shoot (Speir concentration of metal in the shoots to the concentration
et al., 2003; Bennett et al., 2003). Several studies have of metal in the soil (Chen et al., 2004). In this study, the
demonstrated that the concentration of metals in plant EC of 1.07, 1.41 and 4.22 were observed for the
tissue is a function of the metal content in the growing elements: Cu, Cr and Pb respectively. Plants of high EC
environment (Grifferty and Barrington, 2000). The results greater than one, accumulates metals in the root with
indicated that accumulation of Pb, Cu, Cd, Co and Cr in less or poor translocation to the aerial parts (shoot), they
the roots can be arranged in the order: mainly restrict metal in their roots.
Cu>Cr>Pb>Co>Cd. However, the levels of the elements
in either the root or the shoot of the grass plant; E. indica
cannot determine its hyperaccumulating potential. Soil-to- Effect of EDTA application on metal uptake by the
plant metal transfer ratio is an important component of grass
phytoextraction, it determines which part of a plant, root
or shoot that accumulate in terms of translocation factor Most metals in soils exist in unavailable forms, thus, soil
(TF) and enrichment coefficient (EC) (Frissel, 1997). conditions have to be altered to promote phytoextraction
since the phenomenon, depends on a relatively abundant
source of soluble metal for uptake and translocation to
Metal transfer coefficients shoots. Table 4 gives the level of the metals desorbed
when EDTA was applied. The observed level of Pb was
Table 3 shows the enrichment coefficient (EC) and found to increase higher in the shoots, 326.0 μg/g than
translocation factor (TF) of the elements naturally the root compared to what was observed preliminarily.
 Garba et al. 107

Table 4. Mean concentration (μg/g) of the metals in roots and shoots of the grass from the
laboratory pot experiment.

Sample Root Shoot

Element Mean ±SD Mean ±SD
t1 236.00±3.72 108.20±2.12
Cu
t2 137.50±4.22 316.80±2.82

t1 228.10±4.39 84.30±4.42
Cr
t2 242.60±2.57 94.20±2.57

t1 54.80±3.57 176.20±1.75
Pb
t2 96.00±3.22 326.00±4.26
t1=soil contaminated with heavy metal concentrations, t2 =soil amended with EDTA and SD=
Standard deviation. The mean differences of the elements were found significant at (p<0.05; n=4).

The concentration level of Cu on the other hand, was Although, adverse effects of Cr on plant height and
also found to increase in the shoot 316.8 μg/g. The shoot growth has been reported (Rout et al., 1997); a
application of EDTA to the experimental soil, increased significant reduction in plant height in Sinapsis alba when
the level of Cr in the root (242.6 μg/g) with less or poor Cr was given at the rates of 200 or 400 mg kg-1 soil has
translocation of the element to the shoot (94.2 μg/g). been reported (Hanus and Tomas, 1993). Wenger et al.
Plant uptake of metal in soil solution has been observed (2003) reported that the critical toxicity level of Cu in the
to depend on a number of factors: physical processes shoots of crop plants is greater than 20 to 30 mg kg-1. But
such as root intrusion, water and ion fluxes; biological no sign of toxicity at all was expressed by the grass E.
parameters, including kinetics of membrane transport, ion indica in this study.
interactions, and the ability of plants to adapt Ethylenediaminetetraacetate (EDTA) has been
metabolically to changing metal stress in the environment reported to be the most effective amendment in
(Cataldo and Wildung, 1978). phytoextraction research. It has been successfully utilized
for instance, to enhance phytoextraction of lead and other
metals from contaminated soils (Cunningham and Ow,
DISCUSSION 1996; Chen et al., 2004). In this study, EDTA was found
to enhance the bioavailability and to improve the uptake
Uptake of contaminants from the soil by plants occurs and translocation of Cu and Pb to the shoot. Huang et al.
primarily through the root system in which the principle (1997) showed that EDTA was the most efficient chelator
mechanisms of preventing contaminant toxicity are found. for inducing the hyperaccumulation of Pb in pea plants
The root system provides an enormous surface area that shoots. Vassil et al. (1998) also found that Indian mustard
absorbs and accumulates the water and nutrients that are exposed to Pb and EDTA in nutrient solution
essential for growth, but also absorbs other non-essential accumulated 11,000 mg kg-1 Pb in dry shoot tissue. The
contaminants (Arthur et al., 2005) such as Pb and Cd. poor translocation of Cr to the shoots despite the addition
Naturally the grass was found to accumulate most of the of EDTA could be due to sequestration of most of the Cr
elements of interest in the root. The heavy metals: Cu, Cr in the vacuoles of the root cells to render it non-toxic
and Cd were found at high levels in the root than the which may be a natural toxicity response of the plant
shoot with no sign of toxicity. Cadmium, for instance has (Shanker et al., 2004). Phytoextraction is a long-term
been reported in many studies to be accumulated at remediation practice. It requires many cropping cycles to
higher concentrations in the roots than in the leaves decontaminate contaminated sites to an acceptable level
(Boominathan and Doran, 2003). Pulford et al. (2001), in favourable for human use. It has been reported that for
a study with temperate plants confirmed that Cr was phytoremediation, grasses are the most commonly
poorly taken up into the aerial tissues but was held evaluated plants (Ebbs and Kochian, 1998; Shu et al.,
predominantly in the root. Similarly the grass E. indica in 2002). The large surface area of their fibrous roots and
this study expressed high level of Cr in its roots. One of their intensive penetration of soil reduces leaching,
the mechanisms by which uptake of metal occurs in the runoff, and erosion via stabilization of soil and offers
roots may include binding of the positively charged toxic advantages for phytoremediation.
metal ions to negative charges in the cell wall (Gothberg
et al., 2004) and the low transport of heavy metal to Conclusion
shoots may be due to saturation of root metal uptake,
when internal metal concentrations are high. This study therefore has proved the possibility of using
108 J. Environ. Chem. Ecotoxicol.

the grass E. indica for phytoremediation especially Garba ST, Ogugbuaja VO, Samali A (2007). Public Health and The
Trace Elements: Copper (Cu), Chromium (Cr) and Cobalt (Co) in
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