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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. Fajardo12, Pauline Angelic T. Roxas13
1
Institute of Chemistry, University of the Philippines Los Baños, Laguna, Philippines jannogironella@yahoo.com, 2normsfajardo@gmail.com, 3eflask91@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 (Gratão 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
2 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
3 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 100oC for 9 hours and then weighed. The oven-dried plant samples were ashed in a muffle furnace raising the temperature slowly to 450oC then maintaining this temperature for 16 hours. The ashes were then dissolved with 40 mL HCl/H2O (1:3) solution in crucibles. Five drops of concentrated HNO3 were subsequently added. The crucibles were placed in a hot plate maintained at 200oC 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 H2SO4 the evaporated (without boiling) until the volume was reduced to 50 mL. A volume of 5 mL concentrated HNO3 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
4 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 1.03 0.24 0.18 0.00 0.16 0.01 17.21 3 2.93 0.46 1.52 0.09 1.52 0.08 50.67 5 5.15 0.78 2.78 0.04 1.96 0.04 47.32 7 6.44 0.24 2.95 0.37 2.63 0.13 39.87 10 11.11 0.08 4.09 0.11 3.99 0.09 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 24.72 1.89 2.61 0.28 1.65 0.14 14.19 60.19 2 20.81 0.53 2.22 0.09 4.09 0.19 21.03 86.19 4 20.02 2.82 2.91 0.01 5.01 0.33 26.39 156.21 6 18.58 1.24 2.62 0.26 5.63 0.13 27.50 110.10 8 18.31 0.30 2.53 0.25 6.19 0.68 29.05 164.15 10 17.43 0.26 2.50 0.07 7.08 0.28 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 am o u n t o f Pb , 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 [Pb ], m g
length of exposure, no. of days
roots aerial tissue
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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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References
Brankovic S, Pavloc-Muratshapi D, Topuzovi M, Bankovic D and Stankovic M. 2011.
Environmetal study of some metals on several aquatic macrophytes. African Journal
of Biotecnology, vol.10(56), pp. 11956-65
Cho-Ruk, K., J. Kurukote, P. Supprung, and S. Vetayasuporn. 2006. Perennial plants in the phytoremediation of leadcontaminated soils. Biotechnology, vol. 5, no. 1, pp. 1–4.
Gratão P.L., M.N.V. Prasad, P.F. Cardoso, P. John and R.A. Azevedo. 2005.
Phytoremediation: Green Technology for the clean-up of toxic metals in the environment. Brazil Journal of Plant Physiology. 17(1): 53-64.
Gothberg, A. 2008. Metal fate and sensitivity in the aquatic tropical vegetable Ipomoea
aquatica. Department of Applied Environmental Science, Stockholm University.
Universitetsservice, US-AB, Stockholm.
Hale, M.G and Griffin, G.J. 1974. Effect of injury in exudation from immature and mature plant
fruits. Plants Physiol, Abstract 13
Hinchman, R. R., M. C. Negri, and E. G. Gatliff. 1995. Phytoremediation: using green
plants to clean up contaminated soil, groundwater, and wastewater. Argonne National Laboratory Hinchman, Applied Natural Sciences, Inc, http://www.treemediation.com/Technical/Phytoremediation 1998.pdf.
Jadia C.D and Fulekar M.H. 2009. Phytoremediation of heavy metals: Recent Techniques.
African Journal of Biotechnology Vol. 8(6), pp 921-928.
Lone M.I, HE Zhen-li, P.J. Stoffella, X. Yang. 2008. Phytoremediation of heavy metal polluted
soils and water: Progresses and perspectives.Journal of Zhejiang University
Science.9(3):210-220
Pilon-Smits E. 2005. Phytoremediation. Annu. Rev. Plant. Biol. 56:15-39.
Pivetz B.E. 2001. Ground Water Issue. Phytoremediation of Contaminated Soil and Ground
Water at Hazardous Waste Sites.United States Environmental Protection Agency. EPA/540/S-01/500
Rauser, W.E. 1999. Structure and function of metal chelators produced by plants: the case for organic acids, amino acids, phytin and metallothioneins. Cell Biochem Biophys. 31 pp 19-48
Shahandeh, H. and Hossner, L. R. 2000. Plant Screening for Chromium Phytoremediation, International Journal of Phytoremediation: Vol. 2, No. 1, pp. 31–51
Shtangeeva, I., J. V.-P. Laiho, H. Kahelin, and G. R. Gobran. 2004. Phytoremediation of
metal-contaminated soils. Symposia Papers Presented Before the Division of
Environmental Chemistry. American Chemical
Society.http://ersdprojects.science.doe.gov/workshoppdfs/california 2004/p050.pdf.
Susarla S, Medina VF, McCutcheon SC. 2002. Phytoremediation, an ecological solution to
organic contamination. Ecol. Eng. 18:647-58
Tangahu B.V, S.R.S. Abdullah, H. Basri, M. Idris, N. Anuar, and M. Mukhlisin. 2011. A
Review on HeavyMetals (As, Pb, and Hg) Uptake by Plants through Phytoremediation, International Journal of Chemical Engineering.Hindawi
Publishing Corporation. Volume 2011, Article ID 939161
Yoon, J. Cao, X., Zhou Q. and Lena, Q. 2006. Accumulation of Pb, Cu, and Zn in native plants growing on a contaminated Florida State. Ma. Science of the Total
Environment. 368:456-464
Weis J.S. and Weis P. 2004. Metal Uptake, transport and release by wetland plants,
