2.0 Materials and methods:
2.1: Experimental plant description:
Catharanthus roseus (Periwinkle) is a species of
Catharanthus genus and Apocynaceae family native
to Madagascar. Synonyms include Vinca rosea (the
basionym), Ammocallis rosea, and Lochnera rosea;
other English names occasionally used include Cape
Periwinkle, Rose Periwinkle, Rosy Periwinkle, and
"Old-maid". It is also widely cultivated and is
naturalized in subtropical and tropical areas of the
world. As an ornamental plant, it is appreciated for
its hardiness in dry and nutritionally deficient
conditions, popular in subtropical gardens where
temperatures never fall below 5°C to 7°C, and as a
warm-season bedding plant in temperate gardens. It
is noted for its long flowering period, throughout the
year in tropical conditions, and from spring to late
autumn, in warm temperate climates. Full sun and
well-drained soil are preferred. Numerous cultivars
have been selected, for variation in flower colour
(white, mauve, peach, scarlet and reddish-orange),
and also for tolerance of cooler growing conditions
in temperate regions. Notable cultivars include
'Albus' (white flowers), 'Grape Cooler' (rose-pink;
cool-tolerant), the Ocellatus Group (various colours),
and 'Peppermint Cooler' (white with a red centre;
cool-tolerant). (Huxley, 1992). It is an evergreen sub�shrub or herbaceous plant. The flowers are white to
dark pink with a darker red centre. The fruit is a pair
of follicles 2–4 cm long and 3 mm broad. As an
ornamental plant, it is appreciated for its hardiness
in dry and nutritionally deficient conditions, popular
in subtropical gardens. It is noted for its long
flowering period, throughout the year in tropical
conditions. Numerous cultivars have been selected,
for variation in flower colour (white, mauve, peach,
scarlet and reddish-orange), and also for tolerance of
cooler growing conditions in temperate regions.
(Gamble, 2008).
Photo: Photograph of Catharanthus roseus, the
experimental plant
2.2: Sample collection and metal analysis:
Catharanthus roseus plants were grown in pots
filled with garden soil. The seedlings were collected
from the uncontaminated soils. All the selected
seedlings were of uniform size and free of any
disease symptoms. Nickel and Lead were selected
for the study , the uptake was estimated in root,
stem and leaves for every 20 days for a total period
of 60 days. In addition a set of control blank
experimental pots was also maintained. The metal
solutions prepared by dissolving in distilled water to
prepare stock solution of 1000 ppm for each metal.
The calibration curves for each metal were also
prepared. A blank reading was taken to incorporate
necessary correction factor. The heavy metal
solutions of 5mg/L was prepared from the stock and
administered to the plants and care was taken to
avoid leaching of water from the pots. The metal
uptake was estimated once in every 20 days. The
sample plants were removed from the pots and
washed under a stream of water and then with
distilled water. The collected plants were air dried,
then placed in a dehydrator for 2-3 days and then
oven dried for four hours at 100 ºc. The dried
samples of the plant were powdered and stored in
polyethylene bags. The powdered samples were
subjected to acid digestion. 1gm of the powdered
plant material were weighed in separate digestion
flasks and digested with HNO3 and HCl in the ratio of
3:1. The digestion on hot plate at 110ºc for 3-4 hours
or continued till a clean solution was obtained. After
filtering with Whatman No. 42 filter paper the
Universal Journal of Environmental Research and Technology
468
Subhashini and Swamy
filtrate was analyzed for the metal contents in AAS.
(Simarzdu 6800).
3.0 Results and Discussion
In the present investigation, Catharanthus roseus
plant accumulated both the metals. By 20th day Lead
content was high in roots and low in leaves. While in
stem it was 67.31 mg/kg biomass. There was no
change in lead accumulation in leaf after 40th day.
Stem concentration increased to 68.09 mg/kg and
root concentration was increased to 88.74 mg/kg
biomass much change observed in the roots (Table
1). In the 60th day only minimum change was
observed in leaf, stem and root. Finally, after the
total experimental period it was concluded that root
accumulation was higher compared to stem and
leaves.
Table 1: Total accumulation of Lead (mg/kg) in Catharanthus roseus during the experimental
period
Fig 1: Accumulation of lead in the experimental plants
0
50
100
150
200
LEAF
STEM
ROOT
TOTAL
ACCUMULATION
Control 20th day 40th day 60th day Total accumulation
Accumulation of Lead in Catharanthus roseus in the experimental period
Concentration (mg/kg)
Plant part Control 20th
day
40th
day
60th
day
Total
Accumulation
Leaf 24.03±0.41 24.53±0.15 24.5±0.16 24.95±0.08 0.92
Stem 60.69±0.16 67.31±0.18 68.09±0.08 69.49±0.17 8.8
Root 21.47±0.16 84.32±0.15 88.74±0.08 88.81±0.17 67.34
Total
Accumulation 106.19 176.16 181.33 183.33 77.06
Universal Journal of Environmental Research and Technology
469
Subhashini and Swamy
Table 2: Total accumulation of Nickel (mg/kg) in Catharanthus roseus during the
experimental period
Plant part Control
20th
day
40th
day
60th
day
Total
Accumulation
Leaf 2.09±0.18 3.87±0.15 6.18±0.16 12.58±0.08 10.49
Stem 5.39±0.49 9±0.18 9.21±0.08 22.02±0.17 16.63
Root 4.65±0.16 5.94±0.15 7.41±0.08 25.28±0.17 20.63
Total
Accumulation 12.12 18.82 22.8 59.87 47.75
Fig 2: Accumulation of nickel in the experimental plants
In the 20th day Nickel accumulation was observed
that it was increased than the control plants. In this
period high accumulation was observed in the stem
part than roots and leaves. After 40th day root, stem
and leaf accumulations was increased and high
accumulation difference was observed in the leaf
part. After 60th day high accumulation was
observed in the leaf, stem and root almost doubled
than the 40th day accumulations, a high
accumulation difference observed in the root part.
(Table 2). After the experimental period nickel
accumulated in the order roots > stem > leaves.
Bioconcentration factor (BCF) and
Translocation factor (TF):
The Bioconcentration Factor (BCF) of metals was
used to determine the quantity of heavy metals that
is absorbed by the plant from the soil. This is an
index of the ability of the plant to accumulate a
particular metal with respect to its concentration in
the soil (Ghosh and Singh, 2005a) and is calculated
using the formula:
Metal concentration in plant tissue
BCF = _________________________________
Initial concentration of metal in
substrate (soil)
0
10
20
30
40
50
60
LEAF
STEM
ROOT
TOTAL
ACCUMULATION
Control 20th day 40th day 60th day Total accumulation
Accumulation of Nickel (mg/kg) in Catharanthus roseus during the experimental period
Concentration(mg/kg)
Universal Journal of Environmental Research and Technology
470
Subhashini and Swamy
The higher the BCF value the more suitable is the
plant for phytoextraction (Blaylock et al., 1997). BCF
Values > 2 were regarded as high values. To
evaluate the potential of plants for phytoextraction
the translocation factor (TF) was used. This ratio is
an indication of the ability of the plant to
translocate metals from the roots to the aerial parts
of the plant (Marchiol et al., 2004). It is represented
by the ratio:
Metal concentration (stems + leaves)
TF = ___________________________
Metal concentration (roots)
Metals that are accumulated by plants and largely
stored in the roots of plants are indicated by TF
values < 1 with values > 1 indicating that the metals
are stored in the stems and leaves. Determination
of hyperaccumulator and excluder plant species is
based on strict criteria. A plant is classified as a
hyperaccumulator for heavy metal (s) when it meets
four criteria; (a) shoot/root quotient (level of heavy
metal in the shoot divide by level of heavy metal in
the root) > 1, (b) extraction coefficient (level of
heavy metal in the shoot divide by total level of
heavy metal in the soil) > 1; extraction coefficient
gives the proportion of total heavy metal in the soil
which is taken up by the plant shoot/aerial part of
the plant (Harrison and Chirgawi 1989, Rotkittikhun
et al. 2006), (c) higher levels of heavy metals of 10 –
500 times the levels in normal plants
(uncontaminated plants) according to Allen (1989)
(d) more than 1000g/g of copper, lead, nickel,
chromium; or more than 100g/g of cadmium or
more than 10000g/g of zinc (Shen and Liu 1998,
Ginocchio and Baker 2004, , Rotkittikhun et al.
2006). Furthermore, a plant which has high levels of
heavy metals in the roots but with shoot/root
quotients less than 1 is classified as a heavy metal
excluder (Boularbah et al. 2006). According to Baker
and Walker (1990) an indicator plant species is the
one of which the levels of heavy metals in the
tissues are similar to those in the surrounding
environment; soil.
The rate of metal translocation from the root to the
shoot may depend on metal concentration in the
root (Hardiman et al., 1984). The movement of the
heavy metal from the polluted sediments into the
roots of the plant and the ability to translocate the
metals from roots to aerial parts were assessed
correspondingly by means of Bioconcentration
Factor (BCF) and the Translocation Factor (TF).
Bioconcentration factor is an index of the ability of
plant to accumulate a particular metal with respect
to its concentration in the sediment (Ghosh and
Singh, 2005). Bioconcentration factor (BCF) was
calculated as a ratio of concentration of heavy metal
in plant roots to that of soil. Translocation factor is
the ratio of metal concentration in the shoots to the
roots. The ability of plants to tolerate and
accumulate heavy metals is useful for
phytoextraction and phytostabilization purpose
(Yoon et al., 2006). Plants with both
bioconcentration factors and translocation factors
greater than one (TF and BCF> 1) have the potential
to be used in phytoextraction. The higher the BCF
value higher the suitability of the plant for
phytoextraction (Blaylock et al., 1997). This ratio is
an indication of the ability of the plant to
translocate metals from the roots to the aerial parts
of the plant (Marchiol et al., 2004). Based on the
results bioconcentration factor and translocation
factor values were calculated. Bioconcentration
factor was calculated using soil lead and nickel
background concentrations. Lead and Nickel
backgrouned concentration was 9.47mg/kg and
8.43mg/kg respectively. Lead bioconcentration
factor was 7.1 and translocation factor was 0.14.
Nickel bioconcentration factor value was 2.44 and
translocation factor value was 1.31. Based on these
values the plant species was highly accumulated
these metals in the roots part, finally it was very
useful for phytostabilization.
Identify dependent variable and independent variable (if provided)? Was there a control?
The independent variables in this study are the concentrations of Nickel and Lead administered to the Catharanthus roseus plants, as well as the duration of exposure (20, 40, and 60 days). The dependent variables are the accumulation of Nickel and Lead in different parts of the plant (leaves, stems, roots), as well as the total accumulation over the experimental period.
A control was maintained in the study, with plants grown in uncontaminated soil without the addition of Nickel and Lead. This control was used for comparison to determine the effects of the heavy metal exposure on the plant.