1

ASSESSMENT OF THE PHYTOREMEDIATIVE POTENTIAL OF

KAWAYANG TINIK (Bambusa blumeana) IN THE REMOVAL OF LEAD
FROM HYDROPONIC SYSTEMS

DOMINGO JANNO C. GIRONELLA JR.
1
, Dr. Norma N. Fajardo
12
, Pauline Angelic T.
Roxas
13


1
Institute of Chemistry, University of the Philippines Los Baos, Laguna, Philippines
jannogironella@yahoo.com,
2
normsfajardo@gmail.com,
3
eflask91@gmail.com



Abstract
The phytoremediative potential of "kawayang tinik" (Bambusa blumeana) in the
removal of lead, Pb, from hydroponic systems was evaluated using laboratory-
scale experiments. Lead was analyzed using Atomic Absorption Spectroscopy
(AAS).
Prior to the experiments, baseline Pb concentrations were generated for
both the plant samples and the medium in which they would be grown.
Preliminary analysis showed that within a 48-hour period, B. blumeana
could maximally absorb about 50% of the Pb in the medium which contained 3
mg Pb per liter. At this concentration of Pb in the medium, the growth of the
plants was not visibly affected.
Plant samples grown in medium containing 15 mg Pb per liter were
harvested at various time intervals. The roots and aerial parts were separately
analyzed for Pb absorbed. Pb remaining in the growth medium was also
determined. The amount of Pb absorbed by the plants continuously increased
during the 10-day monitoring period from 14.19% after Day 1 to 31.95% after
Day 10. B. blumeana absorbed 193.42 mg Pb/kg plant within the 10-day period.
The bioconcentration factor (BCF) and transfer factor (TF) were calculated
for B. blumeana. TF and BCF values indicate that this plant may be classified as
a Pb accumulator, though not a hyperaccumulator. Results suggest the potential
of B. blumeana for the phytostabilization of Pb-contaminated sites.


Introduction
The contamination of the environment by toxic metals poses a threat for
man and biosphere. In developed nations, this problem is being addressed and
solved to some extent using green technology which involves using metal
tolerant plants to clean up polluted soils and water. The huge potential of green
technology to address environmental problems makes it imperative to investigate
and understand how plants are able to tolerate toxic metals (Grato et al., 2005).
Heavy metals are among the most important contaminants in the
environment. Several methods are already being used to clean up the
environment from these kinds of contaminants, but most of them are costly. In
addition, it is difficult to get optimum results (Tangahu et al., 2011). Recent
concerns regarding environmental contamination have made essential the
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development of appropriate technologies to assess the presence and mobility of
metals in soil (Shtangeeva et al., 2004), water, and wastewater. Presently,
phytoremediation is recognized as one among the most effective and affordable
technological solutions used to extract or remove inactive metals and metal
pollutants from contaminated soil. Phytoremediation is the use of plants to clean
up contamination from soils, sediments, and water. This technology is
environment-friendly and potentially cost effective (Cho-Ruk et al., 2006).
Phytoremediation takes the advantage of the unique and selective uptake
capabilities of plant root systems, together with the translocation,
bioaccumulation, and contaminant degradation abilities of the entire plant body
(Hinchman et al., 1995).
The use of plant species for cleaning polluted soils and waters has gained
increasing attention since last decade as an emerging cheaper technology as
garnered from the many studies that have been conducted in this field.
Numerous plant species have been identified and tested for their traits in the
uptake and accumulation of different heavy metals. Mechanisms of metal uptake
at whole plant and cellular levels have been investigated. Progress has been
made in the mechanistic and practical application aspects of phytoremediation
(Lone et al., 2008).
Lead (Pb) is one of the very toxic heavy metals that have the ability to
affect the entire food chain and disrupt the health system of human beings,
animals and plants. Hence, proper treatment of lead from soil and industrial
wastewaters is very important. Several conventional methods are used for the
removal of heavy metals from wastewater but major drawbacks with such
treatments include production of large amounts of sludge. Many of the
processes have also been found to be ineffective or expensive. Hence, the
search for new, simpler, more effective and eco-friendly technologies for the
removal of toxic heavy metals from wastewater has directed attention towards
phytoremediaton.
Bambusa blumeana, Kawayang tinik in Filipino, also known as spiny
bamboo or thorny bamboo, is a tropical clumping bamboo that has been widely
introduced in Southeast Asia. This bamboo species grows in humid or dry
tropical areas along river banks, hill slopes, and freshwater creeks. In the
present work an attempt has been made to assess its potential use for
remediation of lead.
Using laboratory scale experiments, ability of B. blumeana to remove Pb
from hydroponic systems was evaluated. The baseline data generated included
the determination of the Bioconcentration Factor (BCF) and Transfer Factor (TF).


Materials and Methods
Collection of Sample
B. blumeana samples having approximately the same height, weight and
plant health status were obtained from the Ecosystems Research and
Development Bureau (ERDB), Los Banos. The plant samples were carefully
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uprooted to make sure that the root systems were not damaged. They were then
washed with tap water prior to their use in the experiments.
Water samples to be used in the growth medium were collected from the
faucet of the Environmental Chemistry Laboratory of the Institute of Chemistry at
the third floor of the Physical Sciences Building. Before samples were collected
from the distribution system, the line was flushed sufficiently to ensure that the
water sample is truly representative of the supply.

Determination of the Working Concentration for Lead
In order to determine the concentration of Pb that would be used in the Pb
absorption experiments, plant samples were immersed separately in containers
containing two (2) liters of water with lead concentrations of 0, 3, 5, 7 and 10 mg
Pb/L. The plants were allowed to stay immersed in the Pb-containing media for 2
days (48 hours).

Determination of the Lead Absorptive Capacity of B. blumeana
The concentration of Pb used in the monitoring experiments was 15 mg Pb/L.
Individual plants were immersed in separate containers containing two (2) liters
of water sample that was 15 mg/L in Pb. The plants were allowed to grow in the
Pb-containing medium for 1, 2, 4, 6 and 10 days. The experiment was performed
in triplicates.

Analysis of Samples for Pb
After harvesting the plants, the roots, culms and leaves were separated,
weighed and treated separately. Each portion was chopped, oven-dried at
100
o
C for 9 hours and then weighed. The oven-dried plant samples were ashed
in a muffle furnace raising the temperature slowly to 450
o
C then maintaining this
temperature for 16 hours. The ashes were then dissolved with 40 mL HCl/H
2
O
(1:3) solution in crucibles. Five drops of concentrated HNO
3
were subsequently
added. The crucibles were placed in a hot plate maintained at 200
o
C until the
remaining volume was about 10 mL. The digested samples were then filtered
and diluted to mark using deionized water in a 50-mL volumetric flask.
The growth solutions that remained were measured and then subjected to
wet acid digestion. The samples were acidified using 10 mL concentrated H
2
SO
4

the evaporated (without boiling) until the volume was reduced to 50 mL. A
volume of 5 mL concentrated HNO
3
was the added to the solution which was
then again evaporated until the volume was reduced to 25 mL. The digest was
filtered then diluted to 100 mL using deionized water.
The digested samples were analyzed for PB using Atomic Absortion
Spectroscopy (Varian SpectraAA 55B).


Results and Discussion
Preliminary Analysis of Plant and Water Samples
Preliminary analysis showed that the water sample to be used in the growth
medium had a Pb concentration of 0.012 mg Pb/L. The plant samples, having an
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average mass of 70.72 g on the other hand, were found to contain 4.82+0.07 mg
Pb (1.82+0.22 mg in roots and 3.0+0.05 mg in aerial tissues).

Analysis of Control Samples
In order to check on the accuracy of the method, solutions that were 15 mg Pb/L
in concentration were analyzed by AAS. Results showed that the method used
could recover an average of 14.0+0.1 mg/L from a 15 mg/L Pb solution indicating
a 93.1% accuracy at this Pb concentration.

Determination of Working Concentration for Pb

Table 1. Mean Pb amounts in the medium, roots and aerial parts of
B. blumeana after exposure to different Pb concentration
for 48 hours
concn,
ppm
Amount of Pb, mg
total Pb
removed by
plant, %
remained in the
medium
roots
culm and
leaves
1 1.030.24 0.180.00 0.160.01 17.21
3 2.930.46 1.520.09 1.520.08 50.67
5 5.150.78 2.780.04 1.960.04 47.32
7 6.440.24 2.950.37 2.630.13 39.87
10 11.110.08 4.090.11 3.990.09 40.36

Table 1 shows that the amount of Pb present in the roots and aerial tissues
generally increased as the concentration of Pb in the medium increased. Since
more metal species are available in the solution with higher metal concentration,
the plants tend to take up metal easily than in solutions with lower
concentrations.
The largest value for the ratio of Pb present in the plant tissue to the Pb
that remained in the medium was obtained with the 3 mg Pb/L solution. This
means that B. blumeana effectively absorbed Pb at this concentration for the
period of observation involved. Metal recovered from the plants amounted to
50.67% of the Pb added to the growth medium.
Figure 1 shows a plot of Pb absorbed by the roots and aerial biomass of
B. blumeana at different Pb concentrations in the growth medium. Generally, the
amount of Pb present in the roots is greater compared to that translocated to the
aerial part of the plant system. The roots contain organic acids that bind metals.
In addition, root exudates are very important agents that form complexes with
trace metals and affect their behavior in oxidation-reduction reactions (Rauser,
1999; Hale and Griffin, 1974).

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Figure 1. Mean amount of Pb in the roots and aerial biomass of
B. blumeana after exposure to different Pb concentration
for 48 hours.

During the 48-hour exposure of the plants to the metal, no wilting nor
yellowing of the leaves was observed in all plant samples. This suggests that B.
blumeana is capable of absorbing Pb at the concentration levels used without
any observable detrimental effect to the growth and development of the plant.
Since in the monitoring experiments longer exposure periods than 48
hours would be employed, it was decided that growth medium containing 15 mg
Pb/L would be used in evaluating the ability of B. blumeana to remove Pb from
hydroponic systems.

Monitoring of Phytoremediative Ability of Bambusa blumeana for Pb

Table 2. Mean amount of Pb in the medium, roots and aerial part of
Bambusa blumeana after exposure to 15 ppm Pb solution for
1, 2, 4, 6, 8 and 10 days.
Day
no.
Amount of Pb, mg
total Pb
removed
by plant, %
Absorptive
capacity,
mg Pb/kg
plant
remained in
the medium
roots
culms and
leaves
1 24.721.89 2.610.28
1.650.14
14.19 60.19
2 20.810.53 2.220.09
4.090.19
21.03 86.19
4 20.022.82 2.910.01
5.010.33
26.39 156.21
6 18.581.24 2.620.26
5.630.13
27.50 110.10
8 18.310.30 2.530.25
6.190.68
29.05 164.15
10 17.430.26 2.500.07
7.080.28
31.95 193.42

Plant samples grown in medium containing 15 mg Pb per liter were harvested at
various time intervals. The roots and aerial parts were separately analyzed for Pb
absorbed. Pb remaining in the growth medium was also determined. Table 2
0.00
1.00
2.00
3.00
4.00
5.00
1 3 5 7 10
a
m
o
u
n
t

o
f

P
b
,

m
g

[Pb] of growth medium, ppm
roots
leaves
and culm
6

shows that the amount of Pb absorbed by the plants continuously increased
during the 10-day monitoring period from 14.19% after Day 1 to 31.95% after
Day 10. Results also showed that large amounts of the metal remained in the
growth medium.
The absorptive capacity of B. blumeana was calculated as mg Pb
absorbed per kg of the plant sample. B. blumeana absorbed 193.42 mg Pb/kg
plant within the 10-day period.


Figure 2. Mean amount of Pb in roots and aerial tissue of Bambusa blumeana
after exposure to 15 ppm Pb solution for different number of days.

Due to binding of metals largely on the cell walls during metal
translocation through the plants, it was observed that there was a considerably
higher amount of Pb in the roots than in the aerial parts of the plant for Day 1.
However, Pb increased in the aerial parts as the monitoring progressed. Before
the metal can move from the growth medium into the plant, it must pass the
surface of the root. This can either be a passive process, with metal ions moving
through the porous cell wall of the root cell, or an active process by which metal
ions move symplastically through the cells of the root. This latter process
requires that the metal ions traverse the plasmalemma, a selectively permeable
barrier that surrounds cells (Pilon-Smiths, 2005). Special plant membrane
proteins recognize the chemical structure of essential metals; these proteins bind
the metals and are then ready for take up and transport. For root to shoot
transport, these elements are transported via the vascular system to the aerial
biomass (Jadia and Fulekar, 2008).
The decrease in the amount of Pb in the roots could be taken as an
indication that the metal absorbed was already taken up and transported. The Pb
taken up by the plant could also be released back to the external medium in a
process called metal cycling. Thus, the net accumulation of metal by a plant is
influenced by both the uptake and the release of such metal to the external
medium (Gothberg, 2008).



0.0000
2.0000
4.0000
6.0000
8.0000
1 2 3 4 5 6
[
P
b
]
,

m
g

length of exposure, no. of days
roots
aerial
tissue
7

Bioconcentration Factor and Transfer Factor of Lead in B. blumeana
One of the criteria used in identifying plants that can be used for
phytoremediation is the Bioconcentration Factor (BCF). This is calculated to
provide an index of the ability of the plant to accumulate the metal with respect to
the metal concentration in the substrate. According to Shahandeh and Hossner
(2000), the higher the bioaccumulation coefficient, the higher the metal uptake
from a contaminated medium.
Transfer Factor (TF) or the aerial part-to-root metal concentration ratio is
used to assess the ability of the plant to translocate the metals from roots to the
aerial part. Phytoremediative plants with TF greater than 1 are considered metal
accumulators while plants with TF less than 1 are called metal excluders
(Brankovic et. al, 2011).
Based on the computed BCFs, B. blumeana was most efficient in taking
up Pb after 10 days (BCF=12.89). The maximum calculated TF of 6.93 means
that B. blumeana can be considered a metal accumulator.
Although B. blumeana cannot be considered a hyperaccumulator (BCF
>1000), it has the potential for phytostabilization of Pb-contaminated sited.
Phytostabilization can be used to minimize the migration of contaminants in the
soil (Susarla et. al, 2002). This process uses the ability of plant roots to change
environmental conditions via root exudates. Plants can immobilize heavy metals
through absorption and accumulation by roots, adsorption onto the roots, or
precipitation within the rhizosphere. This process reduces metal mobility and
leaching into ground water, and also reduces metal bioavailability for entry into
the food chain. One advantage of this strategy over phytoextraction is that the
disposal of the metal-laden plant material is not required (Susarla et. al, 2002).
Metals accumulated in the roots are considered relatively stable as far as release
to the environment is concerned. Hyperaccumulator plants generally would not
be used due to their slow growth rate and propensity to accumulate metals
(Pivetz, 2001).
Studies are needed regarding the turnover of nutritive roots and the
potential release of metals from decomposing roots (Weis and Weis, 2004). Also,
effects of plant bacteria or plant mycorrhizae interactions that might affect the
metal uptake and translocation merit further investigation.

Summary and Conclusions
In this study the phytoremediative potential of B. blumeana in the removal
of lead from hydroponic systems was evaluated using laboratory-scale
experiments. The amount of Pb absorbed by the plants continuously increased
during the 10-day monitoring period. At the concentration of 15 mg Pb per liter in
the growth medium, the plant samples did not exhibit any wilting or change in leaf
color during the period of evaluation. It was estimated that B. blumeana absorbed
193.42 mg Pb/kg plant within the 10-day period.
The calculated TF and BCF values indicate that this plant may be
classified as a Pb accumulator, though not a hyperaccumulator. The ability of B.
blumeana to tolerate and accumulate Pb could find its application in
phytoremediation, specifically the phytostabilization of Pb-contaminated sites.
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