Ecological Engineering 90 (2016) 2534

Contents lists available at ScienceDirect

Ecological Engineering
journal homepage: www.elsevier.com/locate/ecoleng

Vetiver grass: An environment clean-up tool for heavy metal

contaminated iron ore mine-soil
Ritesh Banerjee a,1 , Priya Goswami a,1 , Khanindra Pathak b , Anita Mukherjee a,
a
Cell Biology and Genetic Toxicology Laboratory, Centre of Advanced Study, Department of Botany, University of Calcutta, 35 Ballygunge Circular Road,
Kolkata 700019, India
b
Department of Mining Engineering, IIT, Kharagpur, 721302 India

a r t i c l e i n f o a b s t r a c t

Article history: Aims: Vetiver grass Chyrsopogon zizanioides (L.) Roberty is a known plant tolerant to heavy metals and
Received 31 July 2015 its use as an alternative method for rehabilitation of iron ore mine-soil has been investigated.
Received in revised form 5 November 2015 Methods: A pot experiment was performed over a period of three months and the performance of vetiver
Accepted 26 January 2016
grass on heavy metal-rich soil from iron ore mine was assessed. The presence of the following heavy
Available online 11 February 2016
metals Fe, Mn, Zn, Cu, Pb, Ni, Cr and Al were assessed in mine-soil and their uptake by vetiver grass
system was evaluated. In addition, oxidative and genotoxic stress were used to monitor the changes over
Keywords:
the period.
Genotoxicity
Heavy metals
Results: Physico-chemical characterization and metal analysis revealed that vetiver plant is a good
Iron ore mine-soil phytostabilizer. The plant is a potential accumulator of heavy metals and the root parts of the plant
Phytoremediation accumulated higher metal concentrations than the shoot. The high proportions of metals inhibited
Vetiver system the chlorophyll content of leaves, but stimulated the accumulation of proline and lipid peroxidation.
Enhanced activities of catalase (CAT), guaiacol peroxidase (GPOD) and glutathione (GSH) implied that
different mechanisms existed to detoxify reactive oxygen species (ROS) in the shoot of the plants. Geno-
toxicity assays demonstrated the absence of genetic instability or DNA damage in the plants.
Conclusion: Vetiver can be used as an excellent candidate for remediation and restoration of iron-ore
mine spoil-dumps.
2016 Elsevier B.V. All rights reserved.

1. Introduction
India is one of the leading countries in the production of iron ore.
Surface mines are the major source of iron ore in India. Over the last
four decades, as a result of mining activity, a large amount of iron
mine spoil-dumps are deposited in these mines. A number of mines
are now on the verge of completing their mining life. Thus after the
closure of the mine, generation of alternative economic activities in
the mining areas and enhancing the environmental condition near
the mining site is a demanding problem to be addressed under the
corporate social responsibility.
Various steps for reclaiming the mine sites have been under-
taken by the environmental management. However, there are still
certain problems like stabilization of spoil-dump slope, control of
soil erosion, sedimentation loads on the catchment areas of the
river system near the mining area and enhancing the soil quality of
Corresponding author.
E-mail address: anitamukherjee28@gmail.com (A. Mukherjee).
1
These authors contributed equally to this work.
the broken areas near mining site. The iron ore spoil-dump often
poses a major management problem due to its volume and the pres-
ence of pollutants, mainly heavy metals like Fe, Mn, Cr, Cd, Pb, Zn,
Ni, Cu etc. (Verma et al., 2012).
Phytoremediation technology is one of the best and ecofriendly
alternative management to vegetate the dumpsite area. This can
serve the purpose of both stabilization of mine spoil-dump site as
well as provide a pleasing landscape. Selection of appropriate plant
species for successful site reclamation and in phytoremediation
efforts is important. Plants are known to remove toxic heavy metals
and other elements from contaminated soil via their accumulation
in harvestable shoot parts (i.e. phytoextraction) or to immobilize
heavy metals in soils or sediments by root uptake and transloca-
tion into shoots (i.e. phytostabilization) (Ahmad et al., 2010; Gupta
and Sinha, 2009; Pedron et al., 2011).
Vetiver grass is regarded as one of the most versatile crops
of the millennium based on its innumerable novel and mar-
velous qualities (Khan, 2006). Vetiver grass Vetiveria zizanioides L.
recently reclassied as C. zizanioides (L.) Roberty, belonging to the
family Poaceae is a dense, bunch-type grass with stiff stem, and
an extremely strong root system (up to 4.6 m deep), and grows to

http://dx.doi.org/10.1016/j.ecoleng.2016.01.027
0925-8574/ 2016 Elsevier B.V. All rights reserved.
26 R. Banerjee et al. / Ecological Engineering 90 (2016) 2534
the height of over 2 m (The Wealth of India, 1976). India is the pri-
mary center of origin and dispersion of vetiver (Lavania, 2000). It
grows on all continents in the tropical and subtropical regions, tol-
erates a wide range of soil pH and low fertility. The plant is highly
tolerant to elevated levels of heavy metals such as arsenic, cad-
mium, copper, chromium, lead, mercury, nickel, selenium and zinc
(Roongtanakiat et al., 2007; Truong and Baker, 1998; Truong and
Hart, 2001). Vetiver plants growing on y-ash soil amendments (0,
25, 50 and 100%), over a period of 18 months, were capable of sta-
bilizing the metals lead, arsenic and cadmium in the roots (Ghosh
et al., 2015).
The present study is a part of a eld experiment to restore and
revegetate the iron ore mine site using biological interventions. It
involves the use and assessment of Vetiver system in phytore-
mediating iron ore mine-soil in a controlled laboratory condition.
For better understanding and interpretations of results, elemental
analysis, uptake of heavy metals by vetiver grass system, oxidative
and genotoxic stress were carried out.
2. Materials and methods
2.1. Collection of sample
Soil sample was collected from the spoil-dumpsite at Joda East
Iron mine (22 0 11 N, 85 26 28 E, Fig. 1), Odisha, India, in plastic
drums, dried under sunlight and stored in the laboratory at room
temperature (28 1 C). Vetiver plants (variety CIMAP Khus-40)
were collected from the Central Institute of Medicinal and Aromatic
Plants (CIMAP), Lucknow, India, and grown in the experimental
garden of the department of Botany, University of Calcutta, for
acclimatization and multiplication.
2.2. Treatment conditions
Disease free, healthy vetiver plants were grown on mine soil in
earthen pots (size diameter 20 cm; height 17 cm) for a period of
three months (July to September, 2014). Plastic vessels were placed
below the earthen pots to collect the water that seeped out from
the pots.
Vetiver plants grown on garden soil served as a control set. Two
plants were planted per pot and three sets were prepared for each
treatment. Plants were allowed to grow under normal environmen-
tal conditions.
2.3. Estimation of heavy metals in mine and garden soil; roots
and shoots of vetiver plants
Air-dried sample of mine soil was ground and passed through a
2 mm sieve. Five grams of soil sample were digested in HNO3 (70%)
in a microwave digestion system (Ethos D Microwave Labstation,
Milestone Inc., USA) and analyzed for Fe, Mn, Zn, Cu, Pb, Ni, Cr and
Al by Inductively Coupled Plasma Atomic Emission Spectrom-
eter (ARCOS, Spectro, Germany). The metal concentrations were
expressed as mg/kg.
At the end of three months, the plants were taken out of the
pots, washed thoroughly in running tap water, EDTA and deminer-
alized water. Five grams of plant tissue was cut into small pieces,
and ash was prepared at 500 C after drying in oven at 80 C. The
ash was digested in HNO3 (70%) in a microwave digestion system
(Ethos D Microwave Labstation, Milestone Inc., USA) and analyzed
for Fe, Mn, Zn, Cu, Pb, Ni, Cr and Al by Inductively Coupled Plasma
Atomic Emission Spectrometer (ARCOS, Spectro, Germany). Metal
concentrations in the plant sample were calculated as g/g dry
weight.
2.4. Phytoextraction ability
Two important parameters, translocation factor (TF) and
bioaccumulation factor (BF) were used for evaluating the phytoex-
traction ability of vetiver according to the method of (Qihang et al.,
2011).
TF = [Metal]shoot /[Metal]root
BFshoot = [Metal]shoot /[Metal]soil
BFroot = [Metal]root /[Metal]soil
2.5. Performance of the vetiver plants under the inuence of mine
soil
The performance of the vetiver plants under the inuence
of mine-soil was assessed by estimating the total chlorophyll
content and anatomical changes in the root and shoot of the
plants.
Total chlorophyll content was determined according to the
method of Arnon (1949). Fresh leaves (200 mg) were cut and kept
overnight in 80% acetone at 4 C. The extract was centrifuged at
10,000 g for 5 min. Absorbance of the supernatant was read at
645,663 nm using a spectrophotometer. Total chlorophyll content
was calculated by the following formula:
Total chlorophyll mg/g tissue = [20.2(A645 ) + 8.02(A663 )]
[V/(1000 W )]
where A = absorbance at specic wavelength; V = nal volume of
chlorophyll extract in 80% acetone; W = fresh weight of tissue
extracted.
Transverse sections of leaf and root were processed to observe
the anatomical changes and localization of iron under light micro-
scope. Perls stain was used to locate the uptake of iron in the root
and leaf tissue according to the method of Stacey et al. (2008). The
transverse sections were vacuum inltrated with equal volumes of
4% (v/v) HCl and 4% (w/v) K-ferrocyanide (Perls stain solution) for
15 min and incubated for 30 min at room temperature. Excess stain
was removed by washing with distillated water the sections were
observed under light microscope.
2.6. Biochemical stress markers
2.6.1. Lipid peroxidation
Lipid peroxidation was measured as the amount of MDA pro-
duced by the thiobarbituric acid (TBA) reaction as described by
Dhindsa et al. (1981). Briey, fresh leaves were homogenized in
a reaction mixture containing 20% (w/v) trichloroacetic acid (TCA),
and 0.5% (w/v) TBA. The homogenate was incubated at 95 C for
30 min. The sample was centrifuged at 13,000 g for 10 min. The
absorbance of the resulting supernatant was recorded at 532 and
600 nm. The non-specic absorbance at 600 nm was subtracted
from that recorded at 532 nm. The concentration of MDA was cal-
culated using an extinction coefcient ( = 155 mM1 cm1 ) and
expressed in mol g1 FW (fresh weight).
2.6.2. Activities of antioxidant enzymes
Catalase (CAT, EC 1.11.1.6), guaiacol peroxidase (GPOD, EC.
1.11.1.7) and reduced glutathione (GSH) enzymes were analyzed
in the leaves.
CAT Fresh leaves (100 mg) were homogenized in 50 mM Tris
buffer (50 mM Tris, 1 mM EDTA, 1 mM magnesium chloride, 1.5%
PVP; pH 7.8). The homogenate was centrifuged at 10,000 g for
 R. Banerjee et al. / Ecological Engineering 90 (2016) 2534
Fig. 1. (a) Geographical location of the collection site (b) iron mine spoil-dump site.
10 min at 4 C. Catalase activity was measured according to the
method of Aebi (1984). Briey, enzyme extract was added to a
3 ml reaction mixture containing 50 mM sodium phosphate buffer
at pH 7.0 and 10 mM of H2 O2 . The decrease in absorbance as
a result of H2 O2 ( = 39.4 mM1 cm1 ) degradation was recorded
at 240 nm and expressed in mol of H2 O2 oxidized min1 g1
protein.
GPOD activity was determined in the enzyme extract by the
method of Chance and Maehly (1955). The formation of tetra gua-
iacol was measured in a 3 ml reaction mixture containing 50 mM of
sodium phosphate buffer (pH 7.0), 10 mM H2 O2 , and 0.5 mM gua-
iacol. The activity was calculated as the increase in the absorbance
at 470 nm expressed in mol of H2 O2 reduced min1 g1 protein.
GSH activity was determined in the enzyme extract by the
method of Smith et al. (1990).
Fresh leaves (100 mg) were homogenized with 0.02 M EDTA on
ice. The reduced glutathione (GSH) content of the homogenates
were measured using Elmans reagent 5, 5 -dithio-bis-(2-
nitrobenzoic acid) (DTNB). Briey, 5 ml of the homogenate was
mixed with 4 ml of distilled water and 1 ml of 50% TCA. After
incubating for 15 min on ice, the samples were centrifuged at
10,000 rpm for 15 min. The reaction mixture was prepared by
adding 2 ml of supernatant, 4 ml of 0.4 M Tris buffer (pH 8.9) and
0.1 ml of DTNB. Absorbance of the reaction mixture was read within
5 min at 412 nm ( = 14,150 M1 cm1 ) and expressed as nmol mg1
protein.
27
2.6.3. Proline content
Proline content was determined according to the method of
Bates et al. (1973). Leaves were chopped and transferred to 0.5%
aqueous toluene. After an hour, the extract was ltered. An equal
volume of ltrate, ninhydrin, and glacial acetic acid (1:1:1) was
added and incubated at 100 C for 1 h. The reaction was arrested in
an ice bath and the chromophore was extracted with 4 ml toluene.
The absorbance was read at 520 nm using a spectrophotometer. The
proline content was determined from a standard curve and calcu-
lated. The values were expressed as mol g1 FW (fresh weight).
2.6.4. Protein extraction
One gram of leaf was homogenized with 2 ml of 50 mM TrisHCl
buffer at pH 7.2, containing 0.1 mM ethylenediamine-tetra acetic
acid (EDTA), and 1% (w/v) polyvinyl-polypyrrolidone at 4 C. The
homogenate was centrifuged at 10,000 g for 15 min at 4 C and
the resultant crude supernatant was collected and stored at 20 C
for the estimation of protein. Soluble protein content in the super-
natant was determined according to the method of Bradford (1976)
with bovine serum albumin as the standard.
2.7. Assessment of genomic instability
2.7.1. Determination of DNA damage by the alkaline comet assay
The DNA damage studies were carried out on nuclei isolated
from the leaves of vetiver following the method of Mukherjee and
28 R. Banerjee et al. / Ecological Engineering 90 (2016) 2534

Gichner (2009) with modications (Chakraborty and Mukherjee, Table 1

Physico-chemical characteristics of mine-soil.
2010). For each slide, 25 randomly chosen nuclei were analyzed
from 3 slides per experimental set. A computerized image analysis Parameters Values
system (Komet version 5.5, Kinetic Imaging Ltd., Andor Technology, Conductivity 174.70 S
Nottingham, UK) was employed. For analysis the median values pH 6.04
of comet parameter-tail DNA (%) were scored from each slide and Total dissolved solids (TDS) 165.60 mg/l
expressed as means for each treatment group. The parameter of Salinity (NaCl content) 162.60 mg/l
Resistivity 3.03 
percentage of tail DNA was particularly selected because it was
Fe 1725.28 48.32 mg/kg
considered the most meaningful and easy to conceptualize among Al 627.99 23.85 mg/kg
the parameters available for study (Kumaravel and Jha, 2006). Cu 45.25 10.04 mg/kg
Zn 24.49 9.01 mg/kg
Cr 7.61 1.93 mg/kg
2.7.2. DNA fragmentation assay
Mn 26.48 3.89 mg/kg
DNA was isolated from vetiver leaves using a modied CTAB Ni 2.43 0.08 mg/kg
method (Doyle and Doyle, 1987). Purity was determined by mea- Pb <0.01 mg/kg
suring the absorbance of the diluted DNA solution at 260 and Values are mean of 3 samples SD.
280 nm. The isolated DNA samples were resolved on 2.5% agarose
gel in 1 TAE (Tris-acetate-EDTA) buffer at 100 V, for 90 min at 4 C.
Lane A was loaded with 100 bp DNA ladder for reference. DNA was
stained with aqueous solution of ethidium bromide, visualized and
photographed under a gel documentation system (E-gel Imager, slightly acidic (6.04). Amongst the metals estimated, concentration
Life Technologies, California, USA). of Fe was highest, followed by Al, Cu, Zn, Cr, Mn and Ni.

2.7.3. Random Amplied Polymorphic DNA (RAPD) analysis

A total of 20 random primers were used for RAPD analysis. The
PCR reaction was performed in a Veriti 96-Well Thermal Cycler
(Applied Biosystems, Carlsbad, CA, USA). Initial denaturation was
performed at 94 C for 5 min. This was followed by 40 cycles of
1 min at 94 C, 1 min at 37 C and 2 min at 72 C. The nal exten-
sion was performed at 72 C for 10 min. The amplied products
were resolved in 1.5% agarose gel, stained with aqueous solution of
ethidium bromide and photographed in gel documentation system
(E-gel Imager, Life Technologies, California, USA).
2.8. Statistical analyses
Statistical analyses of the data were performed using the statis-
tical program SigmaStat 3.0 (SPSS Inc., Chicago, IL, USA). Students
t-test was done for the comet parameter percentage of tail DNA,
lipid peroxidation and different antioxidant enzymes between
exposed and control sets. The level of signicance was established
at P 0.05.
3. Results
3.1. Physico-chemical characterization and metal content
The physico-chemical parameters and heavy metal content in
iron ore mine-soil are presented in Table 1. The pH of the soil was
3.2. Performance of the vetiver under the inuence of mine-soil
As compared with the control (garden soil) a signicant decrease
in shoot and root length was observed in vetiver plants grown on
mine-soil (Fig. 2a). The average shoot length of vetiver grown on
garden soil and mine-soil was 140.01 11.81 and 116.25 7.16 cm
and that of roots were 91.84 4.95 and 67.97 4.29 cm respec-
tively. To observe the effect on root system, the plants were
taken out of the pot at the end of the experiment. The dense
mesh-like growth of roots held the mine-soil together which
is considered as an ideal character of a good phytostabilizer
(Fig. 2b, c).
The anatomical changes in the leaves and roots of vetiver plants
growing on mine-soil were studied by preparing their transverse
sections (Fig. 3). The root and leaf sections of plants grown on mine-
soil do not show any abnormalities. Distribution of Fe in roots and
leaves of the plant was detected by Perls iron staining procedure
(Stacey et al., 2008). The epidermal and cortical region of roots and
lower epidermal region and vascular bundle of leaves show blue
deposition of iron.
Chlorophyll content correlates to the photosynthetic potential,
physiological and metabolic health of the plant (Gaborcik, 2003). A
signicant decrease in the total chlorophyll content was observed
in the leaves of vetiver grown on mine-soil than that grown on
garden soil (Fig. 4).

Fig. 2. (a) Shoot and root length of vetiver plants after three months of exposure; [*P 0.05]; (b) root system entangling the soil; (c) dense, mesh-like growth of roots after
removal of the soil.
 R. Banerjee et al. / Ecological Engineering 90 (2016) 2534
Fig. 3. Transverse sections of shoots of vetiver plant grown on: (a) garden soil (b) mine-soil; roots of vetiver plant grown on: (c) garden soil, (d) mine-soil. Arrows point the
localization of iron; scale bar = 50 m.
3.3. Phytoextraction of heavy metals
The phytoextraction or phytoaccumulation of the heavy metals
was estimated. Metals like Fe, Al, Cu, Zn, Cr, Mn, Ni and Pb in shoot
and root of vetiver plants were estimated by ICP-AES (Table 2). The
amount of Fe was highest, followed by Al, Cu, Mn, Zn, Cr and Ni in
the roots and that in shoots Cu was the second highest of the metals
in the plants grown on mine-soil.
To study the transfer of heavy metals from the soil to the plant,
bioaccumulation and translocation factors (BF and TF respectively)
are very useful parameters. The translocation factor (TF) is the
phytoavailabilty of metal from root to shoot part within a plant
and expressed by the ratio of [Metal]shoot /[Metal]root (Maiti and
Nandhini, 2006). The data of the present study indicate that, except
Cu, the other metals (Fe, Al, Zn, Cr, Mn and Ni) accumulated by the
plants were largely retained in the roots, as shown by general TF
Fig. 4. Total chlorophyll content in shoots of vetiver plants after three months of
exposure; [*P 0.05].
29
values <1 (Table 3). The bioaccumulation factor (BF) reveals
efcient uptake of the metals by root and shoot of the
plant. It is expressed as the ratio of [Metal]shoot /[Metal]soil or
Metal]root /[Metal]soil . In general, the root part of the plant accu-
mulated higher concentrations of metal than the shoot part. The
acidic pH of the soil may have helped for the high Al, Fe, and Cr
accumulation in the roots.
3.4. Biochemical stress markers
3.4.1. Changes in proline content
A signicant increase in proline content was observed in the
leaves of vetiver plants grown on mine-soil (Fig. 5).
3.4.2. Changes in lipid peroxidation
Oxidative damage to membrane and cellular lipids were esti-
mated by lipid peroxidation assay. Lipid peroxidation expressed
in terms of malondialdehyde (MDA) formed in leaf tissues of the
plants increased (41%) signicantly when compared to the vetiver
plants grown on garden soil (Fig. 6a). Increased MDA content indi-
cated imbalance of ROS equilibrium as a result of stress-induced by
mine-soil.
3.4.3. Changes in antioxidant enzymes
The activities of catalase (CAT), guaiacol peroxidase (GPOD) and
reduced glutathione (GSH) were estimated in shoots and are pre-
sented in Fig. 6bd. A signicant increase in CAT and GPOD activity
was observed in vetiver plants grown on mine-soil. The CAT values
were 1.309 0.011 and 0.453 0.007 mol/g protein for vetiver
grown in mine-soil and in garden soil, respectively. The GPOD val-
ues increased from 0.244 0.006 mol/g protein in control plants
to 0.485 0.009 mol/g protein in the shoots of the plants grown
on mine-soil. The GSH activity of vetiver leaves was 5.107 0.237
and 4.299 0.107 nmol mg1 protein in plants grown on mine-soil
and garden soil respectively (Fig. 6d).
30 R. Banerjee et al. / Ecological Engineering 90 (2016) 2534

Table 2
Estimation of metal contents (g/g) in shoot and root of vetiver plants grown on garden soil and mine-soil.

Iron Aluminum Copper Zinc Chromium Manganese Nickel Lead

Garden soil
Shoot 5.13 1.94 4.40 1.08 1.92 0.85 1.73 0.67 2.52 1.74 1.70 0.10 1.90 0.23 <0.01
Root 8.45 3.91 6.81 1.41 1.97 0.92 6.99 0.55 3.28 1.89 8.22 0.06 1.41 0.05 <0.01
Mine soil
Shoot 88.26 12.58 34.52 4.37 53.29 9.77 4.07 0.61 6.02 0.96 4.19 0.21 1.05 0.15 <0.01
Root 504.34 83.42 179.5 22.83 32.85 2.8 9.46 1.48 7.20 1.26 13.10 2.18 2.27 0.58 <0.01

Values are mean of 3 samples SD.

Table 3
Relative translocation and bioaccumulation of heavy metals in vetiver plants grown on mine-soil.

Iron Aluminum Copper Zinc Chromium Manganese Nickel

TF 0.17 0.19 1.62 0.43 0.83 0.32 0.46

BFshoot 0.05 0.05 1.17 0.17 0.79 0.16 0.43
BFroot 0.29 0.28 0.73 0.39 0.95 0.49 0.93

3.5. Assessment of genomic instability
In addition to reclamation and phyto-stabilization, phytoreme-
diation can be used in reducing toxic potential of contaminants
including complex mixtures of heavy metals, and hence, in addition
to metal estimation, genotoxicity was used to monitor the changes
over the study period.
of 118 RAPD fragments of 1501800 bp in molecular size were
recorded (Table 4). RAPD proles neither showed alterations in
band intensity nor any gain or loss of bands. This conrmed strong
genomic DNA template stability (GTS).
4. Discussion

In the present study, the efcient use of vetiver system growing

3.5.1. Genotoxicity
3.5.1.1. Comet assay. The shoots of vetiver plants grown on mine-
soil show an increase in DNA strand breaks leading to greater DNA
migration out of the nucleus into the tail of the comet. The increase
in Tail DNA (%) scored was 7.87 2.69 in plants grown on mine-soil
whereas in the control (garden soil) it was 4.82 0.84%. The values
were not statistically signicant (P 0.05) (Fig. 7a).
3.5.1.2. DNA fragmentation assay. DNA fragmentation assay was
carried out to assess the changes in the genomic DNA of the plants.
The results showed undamaged genomic DNA represented by a
thick band on the agarose gel in both control and experimental
plants (Fig. 7b).
3.5.2. Random Amplied Polymorphic DNA (RAPD) analysis
To study the genetic stability of the vetiver plants grown
on mine-soil RAPD analysis was carried out. 20 arbitrary RAPD
primers were initially screened, among which 16 produced clear
and scorable amplication products (Fig. 8a, b). A total number
Fig. 5. Proline content in shoots of vetiver plants after three months of exposure;
[*P 0.05].
on iron ore mine-soil was monitored over a period of 3 months.
The different parameters studied provide a comprehensive under-
standing of the potential of vetiver grass as an alternative mean to
vegetate metal-contaminated soil.
The physico-chemical parameters and heavy metal content in
iron ore mine-soil demonstrated that the concentration of Fe was
highest, followed by Al, Cu, Zn, Cr, Mn and Ni. The overburden (OB)
and tailings from iron ore mines located in the state of Jharkhand,
in India revealed that the OB samples showed high concentration
of Fe along with Mn, Pb and Zn as a typical characteristic of iron
ore (Verma et al., 2012). We have taken the advantage of Perls iron
staining procedure to detect Fe distribution in roots and leaves of
the plant (Stacey et al., 2008). The epidermal and cortical region
of roots and lower epidermal region and vascular bundle of leaves
show a blue deposition of iron. This conrmed the uptake of iron
by the roots that was translocated to the leaves of vetiver plants
growing on mine-soil.
After a period of 3 months the vetiver plants grown on mine-
soil manifested a signicant decrease in shoot and root length and
a decrease in chlorophyll content. There was a signicant increase
in proline and malondialdehyde (MDA) levels in leaf tissues of the
plants. Accumulation of proline in plant tissues is a clear marker of
environmental stress (Jaleel et al., 2007; Routley, 1966), believed
to play an adaptive role in plant stress tolerance (Verbruggen and
Hermans, 2008). Similar increases in proline concentrations were
observed in both leaves and roots of vetiver plants growing on met-
alliferous mine wastes (Pb/Zn tailings) (Pang et al., 2003). Increased
MDA content indicated imbalance of ROS equilibrium as a result of
stress-induced by the mine-soil. Previous studies by Patnaik et al.
(2013) in onion, Rai et al. (2004) in Ocimum tenuiorum and Reddy
et al. (2005) in horsegram and Bengalgram have shown an increase
in the levels of lipid peroxidation under heavy metal stress.
The activities of antioxidant enzymes catalase (CAT), guia-
col peroxidase (GPOD) and reduced glutathione (GSH) were
estimated in shoots. A signicant increase in CAT, GPOD and
GSH activity was observed in vetiver plants grown on mine-soil.
Our results are in agreement with those reported by Pang et al.
(2003) that Pb/Zn mine tailings could enhance the activities
of POD, SOD and CAT, activities in vetiver. It is well known
 R. Banerjee et al. / Ecological Engineering 90 (2016) 2534 31

Fig. 6. (a) Lipid peroxidation (LPO); (b) catalase (CAT); (c) guaiacol peroxidase (GPOD); (d) total glutathione (GSH) activity in shoots of vetiver plants after three months of
exposure; [*P 0.05].

that exposure of plants to heavy metals induces the generation
of reactive oxygen species (ROS), which is harmful to plants
(Zenk, 1996). Rennenberg (1995) reported that GSH plays an
active role in protecting membranes against free radical dam-
age. Major enzymes that are responsible for participating in
mechanisms to scavenge the toxic ROS, play an important role
in plant defense mechanism (Anderson and Davis, 2004). In the
present study the adverse effects of mine-soil enhanced the activ-
ities of major antioxidant enzymes in vetiver plants to detoxify
ROS.
Excess of ROS can damage cellular components, such as DNA,
proteins, and lipids (Lopez et al., 2006). All primary compo-
nents of DNA (sugar residues, phosphodiester linkages, purine and
pyrimidine bases) can suffer from damage. The resulting DNA-
lesions that vary from innocuous molecular changes to highly
mutagenic or genotoxic alterations in the genome may often lead to
genomic instability or genotoxic stress (Saha et al., 2015). Although
most DNA damages are repaired, error-prone DNA repair or DNA
damage beyond repair triggers programmed cell death (Bray and
West, 2005). Earlier report from our laboratory Chakraborty and
Mukherjee (2010), conferred that the DNA damage in vetiver plants
grown on y ash for three months was not signicantly different
from that of the plants grown in garden soil. In the present study,
the shoots assessed for genotoxicity, induced a 2-fold increase in
DNA strand breaks in plants grown on mine-soil when compared
to that of plants grown in control (garden soil). The values were
however not statistically signicant (P 0.05). Absence of dam-
age in the nuclear DNA of vetiver implies its long-term survival on
the mine soil. In addition, DNA fragmentation assay and the RAPD
proles of the genomic DNA in vetiver plants grown on mine-soil

Fig. 7. DNA damage in shoots of vetiver plants after three months of exposure: (a) comet assay; (b) agarose gel electrophoresis of DNA isolated from shoots (Lane L 100 bp
DNA ladder, Lane A garden soil, Lane B mine-soil).
32 R. Banerjee et al. / Ecological Engineering 90 (2016) 2534

Table 4
List of primers, their sequences and size of the amplied fragments generated by 16 RAPD (Sl. No. 116) primers.

Sl. No Primer Primer sequence (5 -3 ) G + C content (%) No of scorable bands Size range (bp) approx.

1 OPY-15 AGTCGCCCTT 60 8 4001100

2 OPY-18 GTGGAGTCAG 60 12 3001800
3 OPZ-6 GTGCCGTTCA 60 8 2801400
4 OPH-4 GGAAGTCGCC 70 2 350700
5 OPH-5 AGTCGTCCCC 70 4 3501400
6 OPH-6 ACGCATCGCA 60 8 4001600
7 OPH-18 GAATCGGCCA 60 10 2801200
8 OPI-12 AGAGGGCACA 60 7 2501500
9 OPI-13 CTGGGGCTGA 70 12 2001200
10 OPI-14 TGACGGCGGT 70 10 4001200
11 OPI-18 TGCCCAGCCT 60 8 3201200
12 OPA-7 GAAACGGGTG 60 2 150600
13 OPA-9 GGGTAACGCC 70 10 2801400
14 OPA-13 CAGCACCCAC 70 7 4001550
15 OPL-14 GTGACAGGCT 60 5 300800
16 OPL-17 AGCCTGAGCC 70 5 4501400

Total 118 1501800

established genomic stability without any alterations in band
intensity nor any gain or loss of bands. Recently, molecular tech-
niques have proven to be a potential tool for eco-toxicological
studies (Wolf et al., 2004). According to the previous reports, DNA
ngerprinting offers a useful biomarker assay in the toxicology of
soil. Changes in RAPD banding patterns induced by pollutants like
heavy metals can be regarded as changes in genomic DNA tem-
plate stability (GTS) that can be directly correlated with alterations
in other parameters (Atienzar et al., 2000; Liu et al., 2005). RAPD
analysis is capable of detecting temporary DNA changes that may
not nally manifest themselves as mutations and that is why it is
preferred over classical genotoxicity assays (Labra et al., 2003).
To observe the effect on the root system, the plants were taken
out of the pot at the end of the experiment. The dense mesh-like
growth of root that held the mine-soil together is considered as an
Fig. 8. (a) Random Amplied Polymorphic DNA (RAPD) amplication pattern
obtained from plants grown on garden soil (1a, 2a, 3a, 4a, 5a, 6a, 7a) and mine-soil
(1b, 2b, 3b, 4b, 5b, 6b, 7b) generated by 7 primers viz. 1. OPY-15, 2. OPY-18, 3. OPZ-6,
4. OPH-4, 5. OPH-5, 6. OPH-6, 7. OPH-18, L. 100 bp DNA ladder. (b) Random Ampli-
ed Polymorphic DNA (RAPD) amplication pattern obtained from plants grown on
garden soil (1a, 2a, 3a, 4a, 5a, 6a, 7a, 8a, 9a) and mine-soil (1b, 2b, 3b, 4b, 5b, 6b,
7b, 8b, 9b) generated by 9 primers viz. 1. OPI-12, 2. OPI-13, 3. OPI-14, 4. OPI-18, 5.
OPA-7, 6. OPA-9, 7. OPA-13, 8. OPL-14, 9. OPA-17, L.100 bp DNA Ladder.
ideal character of a good phytostabilizer. This is particularly impor-
tant because the dense root system and vegetative cover may also
help to retard the formation of hazardous leachate from the soil
(Chakraborty and Mukherjee, 2010).
Besides performance, phytoextraction of metals by the plant is
an important criterion for the success of phytoremediation. When
plants are grown in a heavy metal-contaminated soil, the roots are
the primary site of heavy metal accumulation. Plants are capable of
absorbing and accumulating metals in their tissues from contami-
nated soils, sediments and water at high concentrations (Porter and
Peterson, 1975). Such a process is called phytoextraction or phy-
toaccumulation (USEPA, 2000). In our study, a considerable amount
of heavy metals was absorbed by the roots of vetiver but the same
was not translocated to the shoot system above. The amount of Fe
was highest followed by Al, Cu, Mn, Zn, Cr and Ni in the roots and
in shoots Cu was the second highest of metals in the plants grown
for 3 months on mine-soil. Roongtanakiat and Chairoj (2001) con-
ducted pot experiments to investigate the uptake potential of three
ecotypes of vetiver grass. They found that the uptake and retention
of Mn, Zn, Cu, Cd and Pb in shoot and root were dependent on the
period and ecotype of the vetiver grass harvested. In contrast to
the shoot, vetiver grass harvested at 120 days had higher heavy-
metal root concentration than the 60-day harvest. At the 60-day
harvest, for all three vetiver ecotypes, heavy-metal concentrations
were found to be higher in shoots than in roots.
Bioaccumulation and translocation factors (BF and TF respec-
tively) are very useful parameters to study the soil to plant transfer
pattern of heavy metal accumulation (Rezvani and Zaefarian, 2011;
Scragg, 2005). The translocation factor (TF) is the phytoavailabilty
of metal from root to shoot part within a plant and expressed by the
ratio of [Metal]shoot /[Metal]root (Maiti and Nandhini, 2006). BF and
TF values greater than unity are indicative of the fact that the plant
is a potential heavy metal accumulator and is effective in transloca-
tion of the metals from root to harvestable parts (Tu and Ma, 2002)
respectively. Thus BF and TF determine the overall phytoremedi-
ating potential of the plant. The data of the present study indicate
that except Cu, the other metals (Fe, Al, Zn, Cr, Mn and Ni) accumu-
lated by the plants growing on the mine-soil were largely retained
in the roots, as shown by general TF values <1. The bioaccumulation
factor (BF) reveals efcient uptake of the metals by root and shoot
of the plant. It is expressed as the ratio of [Metal]shoot /[Metal]soil
or [Metal]root /[Metal]soil . In general, the root parts of the plant
accumulated higher metal concentrations than the shoot. A sim-
ilar trend in heavy metal accumulation and translocation in root
and shoot has been observed in vetiver grown on iron ore mine
tailings (Roongtanakiat et al., 2008).
 R. Banerjee et al. / Ecological Engineering 90 (2016) 2534
5. Conclusion
In the present study vetiver plants growing on mine-soil act as a
good phytostabilizer. The dense mesh-like growth of root that held
the mine-soil together can prevent from erosion and also help to
retard the formation of hazardous leachate from the soil. The plant
is a potential accumulator of heavy metals and the root parts of the
plant accumulated higher metal concentrations than the shoot. The
adverse effect of mine soil in vetiver plants induced a decrease in
photosynthetic activities with enhanced activities of major antiox-
idant enzymes to detoxify the ROS generated under stress. Absence
of damage in the nuclear DNA of vetiver implies its long-term sur-
vival on the mine-soil.
The results of the present study points to the success of uti-
lizing vetiver plants in phytoremediating iron ore mine-soil in a
controlled laboratory conditions. For the restoration and revegeta-
tion of iron ore mine site further study should be carried out on
site as this will help in addressing a challenging problem under the
corporate social responsibility.
Conict of interest
The authors declare that there is no conict of interest.
Ritesh Banerjee and Priya Goswami contributed equally in all
experiments performed.
Acknowledgements
The authors would like to acknowledge the Council of Scien-
tic and Industrial Research, New Delhi, Govt. of India for nancial
support (Sanction No. 38 (1367)/13/EMR-II dt. 01.10.2013 Inves-
tigation on iron ore mine site restoration and species performance
in spoil dumps slope stabilization with vetiver system technology).
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