Subject Area 5.

Ethidium Bromide

Subject Area 5.3: Phytoremediation and ecosystem restoration

Research Article

Phytoremediation Potentials of Selected Tropical Plants for Ethidium Bromide*

Raynato B. Uera1, Annie Melinda Paz-Alberto1 and Gilbert C. Sigua2**
1 Environmental

Management Department, Institute of Graduate Studies, Central Luzon State University, Science City of Muoz, Nueva Ecija, Philippines 3120 2 Subtropical Agricultural Research Station, Agricultural Research Service, United States Department of Agriculture, Brooksville, FL USA 34601 **Corresponding author (gcsigua@ifas.ufl.edu)
DOI: http://dx.doi.org/10.1065/espr2007.02.391 Please cite this paper as: Uera RB, Paz-Alberto AM, Sigua GC (2007): Phytoremediation Potentials of Selected Tropical Plants for Ethidium Bromide. Env Sci Pollut Res 14 (7) 505509 Abstract Background, Aims and Scope. Research and development has its own benefits and inconveniences. One of the inconveniences is the generation of enormous quantity of diverse toxic and hazardous wastes and its eventual contamination to soil and groundwater resources. Ethidium bromide (EtBr) is one of the commonly used substances in molecular biology experiments. It is highly mutagenic and moderately toxic substance used in DNA-staining during electrophoresis. Interest in phytoremediation as a method to solve chemical contamination has been growing rapidly in recent years. The technology has been utilized to clean up soil and groundwater from heavy metals and other toxic organic compounds in many countries like the United States, Russia, and most of European countries. Phytoremediation requires somewhat limited resources and very useful in treating wide variety of environmental contaminants. This study aimed to assess the potential of selected tropical plants as phytoremediators of EtBr. Materials and Methods. This study used tomato (Solanum lycopersicum), mustard (Brassica alba), vetivergrass (Vetiveria zizanioedes), cogongrass (Imperata cylindrica), carabaograss (Paspalum conjugatum), and talahib (Saccharum spontaneum) to remove EtBr from laboratory wastes. The six tropical plants were planted in individual plastic bags containing soil and 10% EtBr-stained agarose gel. The plants were allowed to establish and grow in soil for 30 days. Ethidium bromide content of the test plants and the soil were analyzed before and after soil treatment. Ethidium bromide contents of the plants and soils were analyzed using an UV VIS spectrophotometer. Results. Results showed a highly significant (p0.001) difference in the ability of the tropical plants to absorb EtBr from soils. Mustard registered the highest absorption of EtBr (1.40.12 g kg1) followed by tomato and vetivergrass with average uptake of 1.00.23 and 0.70.17 g kg1 EtBr, respectively. Cogongrass, talahib, and carabaograss had the least amount of EtBr absorbed (0.20.6 g kg1). Ethidium bromide content of soil planted to mustard was reduced by 10.7%. This was followed by tomato with an average reduction of 8.1%. Only 5.6% reduction was obtained from soils planted to vetivergrass. Soils planted to cogongrass, talahib, and carabaograss had the least reduction of 1.52% from its initial EtBr content. Discussion. In this study, mustard, tomato, and vetivergrass have shown their ability to absorb EtBr from contaminated soil keeping them from expanding their reach into the environment and preventing further contamination. Its downside, however, is that living creatures including humans, fish, and birds, must be prevented from eating the plants that utilized these substances. Nonetheless, it is still easier to isolate, cut down, and remove plants growing on the surface of the contaminated matrices, than to use strong acids and permanganates to chemically neutralize a dangerous process that can further contaminate the environment and pose additional risks to humans. Though this alternative method does not totally eliminate eventual environmental contamination, it is by far produces extremely insignificant amount of by-products compared with the existing processes and technologies. Conclusions. Mustard had the highest potential as phytoremediator of EtBr in soil. However, the absorption capabilities of the other test plants may also be considered in terms of period of maturity and productivity. Recommendations and Perspectives. It is recommended that a more detailed and complete investigation of the phytoremediation properties of the different plants tested should be conducted in actual field experiments. Plants should be exposed until they reach maturity to establish their maximum response to the toxicity and mutagenecity of EtBr and their maximum absorbing capabilities. Different plant parts should be analyzed individually to determine the movement and translocation of EtBr from soil to the tissues of plants. Since this study has established that some plants can thrive and dwell in EtBr-treated soil, an increased amount of EtBr application should be explored in future studies. It is suggested therefore that a larger, more comprehensive exploration of phytoremediation application in the management of toxic and hazardous wastes emanating from biotechnology research activities should be considered especially on the use of vetivergrass, a very promising tropical perennial grass.
Keywords: Ethidium bromide (EtBr); hazardous; mutagenic; phytoremediation; tropical plants

Background, Aims and Scope

Around the world, land, surface waters, and groundwater are increasingly affected by contamination from industrial, research experiments, military and agricultural activities either due to ignorance, lack of vision, carelessness, or high cost of waste disposal and treatment. The rapid build-up of toxic pollutants (metals, radionuclide, and organic contaminants in soil, surface water and groundwater) not only affects natural resources but also causes a major strain on ecosystems (Ona et al. 2006). Research and development has its own benefits and inconveniences. One of the inconveniences is the generation of enormous quantity of diverse toxic and hazardous wastes and its eventual contamination to soil and groundwater resources. Ethidium bromide (EtBr) is one of the commonly used substances in molecular biology experiments. It is highly mutagenic and moderately toxic substance used in DNA-staining during electrophoresis. It is a potent mutagen, capable of causing liv* ESS-Submission Editor: Professor Zhihong Xu (zhihong.xu@griffith.edu.au; www.griffith.edu.au/centre/cfhr)

Env Sci Pollut Res 14 (7) 505 509 (2007) 2007 ecomed publishers (Verlagsgruppe Hthig Jehle Rehm GmbH), D-86899 Landsberg and Tokyo Mumbai Seoul Melbourne Paris

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ing cell mutations and moderately toxic after an acute exposure. It can be absorbed through the skin and also an irritant to the skin, eyes, mouth, and upper respiratory tract. Although there is no evidence at this time of human carcinogenicity or teratogenicity, this material may still be considered a possible carcinogen or teratogen (Indiana University Fact Sheet 1996). Ethidium bromide is capable of disrupting efficient metabolism and protein synthesis in plants and could have had affected the growth and metabolism of the test plants during the 30 days exposure. The study of Zollinger and Morais (1979) showed that EtBr decreased the rate of growth of infected chick embryo cells and inhibited the synthesis of mitochondrial macromolecules. The DNA of a cell prevents the cell from accessing its genome to carry out its necessary protein synthesis to survive (Indiana University Chemical 1996). Interest in phytoremediation as a method to solve chemical contamination has been growing rapidly in recent years (Clemens et al. 2002, Dhanker et al. 2002, Bettis 1997). The technology has been utilized to clean up soil and groundwater from heavy metals and other toxic organic compounds in many countries like the United States, Russia, and most of European countries. Phytoremediation requires somewhat limited resources and very useful in treating wide variety of environmental contaminants. This study aimed to assess the potential of selected tropical plants as phytoremediators of EtBr. The technology has been utilized to clean up soil and groundwater from heavy metals and other toxic organic compounds in many countries like the United States and Russia. It requires limited resources and very useful in treating wide variety of environmental contaminants (Sigua 2005, Raskin and Salt 1997). Phytoremediation involves growing plants in a contaminated matrix, for a required growth period, to remove contaminants from the matrix, or facilitate immobilization (binding/containment) or degradation (detoxification) of the pollutants. The plants can be subsequently harvested, processed and disposed (Sigua et al. 2004b, Raskin and Salt 1997). Plants are unique organisms equipped with remarkable metabolic and absorption capabilities, as well as transport systems that can take up nutrients or contaminants selectively from the growth matrix, soil or water (Sigua et al. 2005, Sigua et al. 2004a, Sigua et al. 2004b, USEPA 2001, Raskin and Salt 1997). Phytoremediation of EtBr is relatively new area of research and not very many useful and relevant information is available in the scientific community. Alternative and more practical methods of treating and disposing hazardous wastes are highly needed. This study dealt with the possibility of utilizing some plants with phytoremediation properties, which could be potential alternative to a costly and dangerous hazardous waste treatment and disposal system of EtBr-contaminated materials in the Philippines and elsewhere. Finally, this study may pave the way for a larger and more comprehensive exploration of phytoremediation application utilizing plants in the management of toxic and hazardous wastes emanating from biotechnology research activities.
2
2.1

Subject Area 5.3

Agronomy and Soil Division, Philippine Rice Institute, Science City of Muoz, Nueva Ecija. The soil used had a medium acidity of pH 5.94, organic matter content of 3.1%, and a phosphorus content of 12.7 mg kg1. Ethidium bromide (EtBr)-stained agarose gels (2%) ready for disposal were obtained from the Molecular Genetics Laboratory of the Philippine Rice Research Institute. Soil materials (020 cm depth) were collected from actual agricultural fields planted with tomato and mustard. The same types of soil were collected from areas with vetivergrass, cogongrass, carabaograss, and talahib. Soil materials were air-dried, sieved, and mixed together (composite). Black polyethylene bags with 3 kg capacity were used in the experiment. The untreated soil (0% contamination) contained 2 kg of soil with no EtBrstained agarose gel. The treated soil (10% contamination), had 200 grams of EtBr-stained agarose gel (approximately 28.5 g kg1 EtBr) and 1,800 grams of soil in each bag. All treatments were replicated three times. All treatments were given approximately three liters of distilled water every four days to keep the moisture content of the soils at field capacity (21%). Distilled water was used in the experiment to make sure that water is free from impurities, chemicals and pollutants, which are potential sources of contamination. Individual bag was inspected almost daily for possible leakage that may compromise the results of the study.
2.2 Plant materials

This study used tomato (Solanum lycopersicum), mustard (Brassica alba), vetivergrass (Vetiveria zizanioedes), cogongrass (Imperata cylindrica), carabaograss (Paspalum conjugatum), and talahib (Saccharum spontaneum) to remove EtBr from laboratory wastes. Seeds of tomato and mustard were obtained from the Central Luzon State University Research, Extension and Training Center (CLSU-RET) for this study. Young seedlings of vetivergrass were secured from the Conservation Park of Environmental Management Institute, Central Luzon State University while young seedlings of cogongrass, carabaograss, and talahib were collected from a nearby city. Tomato, mustard, and vetivergrass are plants with phytoremediation properties. They can remove heavy metals and other organic materials from contaminated soil and water (Whitener 1993). Locally, cogongrass, carabaograss, and talahib can grow anywhere and are easily available. They are the most common and easily maintained grasses known to human in the tropics (Watson and Dallwitz 1992). Seeds of tomato and mustard were directly sown in straight rows in an 18"x 12"x 6" plastic trays containing pure soil. One-month old tomato and 21-day old mustard seedlings were transplanted into previously prepared soil media (see above). Young seedlings (approximately one-month old) of vetivergrass, cogongrass, carabaograss and talahib were transplanted in individual bags. All treatments in each plant were given 50 ml tap water every other day. No fertilizer was applied during the duration of the experiment.
2.3 Sample preparations for EtBr analysis

Materials and Methods

Soil materials and ethidium bromide treatment

2.3.1 Plant sample

The soil used in this study was collected in Barangay Abar 1st, San jose City, Nueva Ecija and was analyzed at the

The different test plants were harvested 30 days after exposure. The whole plants (roots, stems, and leaves) were used

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for the tissue analysis of EtBr. Plant samples were washed with running tap water until soils and other dirt particles were removed completely. Wet plant tissue samples were wiped with clean tissue paper and allowed to air dry. The samples were placed in properly labeled foil envelopes and placed inside plastic bags, then kept in freezer before laboratory analysis. Plant samples were extracted for EtBr following the extraction procedures developed by Washington State University, Chemical Wastes Section. The required amount of plant samples were weighed and treated with Tris-Borate-EDTA (TBE: Tris base + Boric acid + Na4EDTA). The plant tissue was heated at 65C for 10 minutes and then mixed thoroughly. Chloroform was added and the mixture was heated again for 10 minutes 65C. The solutions were filtered using a Whatman 342 filter paper. This was followed by the measurement of EtBr concentration using an UV VIS spectrophotometer (see section 2.3.3).
2.3.2 Soil samples

Ethidium Bromide

1.80

LEVELS OF EtBr IN PLANTS (u g kg-1)

1.60 1.40 b 1.20 1.00 0.80 0.60 0.40 0.20 0.00 TOMATO

MUSTARD

VETIVER

TALAHIB

CARABAO

COGON

TROPICAL PLANTS

Fig. 1: Average levels of EtBr in plants at harvest. Concentrations of EtBr among the different tropical plants are significantly different (p0.05) when superscripts located at top of bars are different

Soil samples from each treatment were thoroughly mixed until homogeneity was reached. A 20-gram composite soil sample was placed in a 50-liter corning tubes and were kept in a freezer before analysis. The concentration of EtBr in soils planted to the different test plants after trials was determined using an UV VIS spectrophotometer (see section 2.3.3). The reduction of EtBr in soil was computed by subtracting the final concentration of EtBr in soil from the initial concentration of EtBr in soil divide by the initial concentration of EtBr in soil multiply by 100.
2.3.3 Spectrophotometric analysis of EtBr

Analysis of the EtBr content was done using an UV VIS spectrophotometer at the Food Protection Laboratory of the Bureau of Post-Harvest Research and Extension. The wavelength was set at 317 nm. The calibration curve was set using a series of known concentration of EtBr solution set at 0.0, 0.2, 0.4, 0.6, 0.8 and 1.0 mg kg1. Calibration curve was done by plotting the absorbance against the concentration of EtBr standards. The concentration of the solution was computed based on Beer's Law of Concentration Determination.
2.4 Statistical design and data analysis

observed in tomato and vetivergrass (see Fig. 1). Different plants may have different responses to a variety of environmental stresses (e.g., EtBr treatment). Some plants can develop tolerance or resistance along the way, but others may be completely affected. A stress may change metabolism and may alter the morphology, therefore disrupting efficient growth and development (Hale and Orcutt 1978). Deficiency or abundance of an element can result from a number of conditions like the amount and concentration present in the soil, the form in which they exist, and the processes by which they become available (Clarkson and Hanson 1980). Hale and Orcutt (1987) claimed that slow rate of growth in plants is largely caused by environmental stress particularly nutrient deficiency and chemical toxicity. Clark (1982) declared that tolerance to a certain stress, be it chemical or physical is brought about by several factors. Some of these factors are: 1) the amount or concentration present in the soil is not enough to cause stress; 2) the length of exposure is very limited and not adequate to cause strain; 3) the plant has developed resistance or tolerance to the stress; and 4) inclusion of EtBr-stained agarose gel introduced not only the toxic and mutagenic component of EtBr, but also abundant supply of carbohydrates necessary in plant's energy production. Mustard, registered as the most active phytoremediator of EtBr in soil with 1.40.12 g kg1 after 30 days of exposure. This plant is known to remove large quantities of chromium, lead, copper, and nickel in soils (Wall Street 1996). This could be attributed to its well-developed root system as observed visually during sampling. Another factor could be its physiology for being an early maturing vegetable. As the length of exposure can affect the amount of uptake, it can also be influenced by the plant's maturity (Whitener 1993). In addition, mustard as a leafy vegetable has a biomass composed of nearly 80% leaves and 20% roots and stems. As such, mustard plant has more room for storing foods and other non-essential elements. Mustard can absorb lead, cadmium, chromium, nickel, zinc, and copper up to 3.5% of its dry body weight (Wall Street 1996). Tomato was second to mustard with 1.00.23 g kg1 of EtBr on their tissues (see Fig. 1). This could probably be due to its far-reaching root system observed visually during sampling and its excellent tolerance under the stress. Its stem structure is composed of 95% water and water-soluble materials enabling it to

This study was laid-out in a two-factor experiment in a completely randomized design. All treatments were replicated three times (SAS, 2000). Where the F-test indicated a significant (p0.05) effect, means were separated following the procedures of Duncan Multiple Range Test (DMRT).
3
3.1

Results and Discussion

Plant uptake of etbr and EtBr reduction in soils

Three out of the six plants screened as possible phytoremediator of EtBr-stained agarose gel in soil showed positive and remarkable results. These three were mustard, tomato, and vetivergrass (Fig. 1). Statistical analysis showed that the differences among the plant's accumulation of EtBr were highly significant (p0.001). A significant difference (p0.05) was noted between the means of mustard and vetivergrass. However, treatment means of mustard and tomato did not differ significantly at 5% level significance. The same was
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hold and store larger amount of micro and macronutrients. Tomato is also a known phytoremediator of chromium, lead, copper, and nickel in soil (Wall Street 1996). The inhibitory activity of toxic compounds is related to the amount absorbed and translocated to the different parts of the plants (Hale and Orcutt 1987, Fritz 1983). This may be the reason why mustard has accumulated the largest amount of EtBr in soil and exhibited cessation of growth among the other test plants. Decrease in growth rate of the root system or complete cessation of its growth in developed and fruit-bearing tomato plants is known phenomena. It was suggested that a limited supplyof carbohydrates to this organ, due to its relative weakness in competition with the flowers and developing fruitlets is the main cause for these disorders (Pressman et al. 1997). Conversely, growth responses of talahib, cogongrass and carabaograss were the least affected or not affected by EtBr treatment. Vetivergrass was able to absorb 0.70.17 g kg1 of EtBr from the soil (see Fig. 1). Roots and shoots of vetivergrass could accumulate five times more EtBr than the chromium and zinc levels in the soil (Troung 1999). This small amount of EtBr in vetivergrass might be a function of the length of exposure that affected the absorption rate. It is possible that vetivergrass being a perennial plant can still increase its uptake if its exposure to the soil had been prolonged much longer or until it reaches maturity. In general, mature (120 days of exposure) vetivergrass can absorb more heavy metals (Roongtanakiat 2001). Vetivergrass as compared with talahib and cogongrass is characterized by its massive finely structured, deep, and easily adoptive root system. In addition, vetivergrass is tolerant to extreme climatic variation such as prolonged drought, flood, submergence, and extreme temperature. It is also tolerant to wide range of soil pH, from 3.0 to 10.5, highly tolerant to soil salinity, sodicity, acidity, aluminum, and manganese toxicities, and heavy metals such as arsenic, cadmium, chromium, nickel, lead, zinc, mercury, selenium, and copper in the soil (Truong 1999). Carabaograss was observed to have a very strong and extensive root system, yet it was able to absorb very little amount of EtBr (0.20.06 g kg1) from the soil (see Fig. 1). Similar observation was found in talahib and cogongrass, which also registered 0.20.55 g kg1 of EtBr after 30 days of exposure (see Fig. 1). While a strong rooting system is one of the required characters of a good phytoremediator, it does not always mean it has inherent ability to accumulate some chemicals and nutrients available in the soil. Also, plant maturity period and type are to be considered. These grasses are perennial plants that may take longer time to mature and establish their rooting systems. Moreover, some plants can accumulate high concentrations of some elements while others may extrude them (Hale and Orcutt 1987). Absorption of materials by plants from the soil involves movement from soil to roots, from the exterior of roots to interior, and translocation within the plant. Furthermore, absorption and uptake in phytoremediation are governed by physicochemical properties of the compounds, and the compound's solubility to water (Raskin and Salt 1997). When inorganic nutrients and photosynthetic products are in water, these can easily be conveyed to various parts of the plant. This is also possible with EtBr, which is highly soluble in water and is most likely bioavailable to vascular plants.

Subject Area 5.3

10.0

PLANT UPTAKE OF EtBr (g kg )

8.0

-1

6.0

4.0

2.0 c c c

0.0 TOMATO MUSTARD VETIVER TALAHIB CARABAO COGON

TROPICAL PLANTS

Fig. 2: Average uptake of EtBr by the different tropical plants. Uptake of EtBr by different tropical plants are significantly different (p0.05) when superscripts located at top of bars are different

Mustard had the highest plant uptake of EtBr (7.1 g kg1) compared with 5.1 g kg1 by tomato and 3.1 g kg1 taken by vetivergrass. Talahib, cogongrass, and carabaograss recorded comparable uptake of 1.0 g kg1 EtBr from the soil (Fig. 2). Statistical analysis showed that the differences among the test plants regarding their ability to accumulate EtBr were highly significant at 1% level of significance. Comparison among treatment means also revealed that the reduction caused by mustard and tomato did not differ significantly (p0.05). The same was observed in tomato and vetivergrass. However, significant difference was noted between the means of mustard and vetiver grass (see Fig. 2). The determination of plants that can work most efficiently in a given application is the most important part in phytoremediation. Not all plant species can metabolize, volatilize, or accumulate pollutants in the same manner. A good candidate must grow quickly and consume large quantities of water in a short time. It should also be able to remediate more than one pollutant because pollution rarely occurs as a single compound. Moreover, it must also find a way to develop tolerance and must be able to absorb considerable amount of pollutants in the least possible time of exposure. Some studies were made on the subsequent result of plant absorption of organic pollutants from soil. Organic pollutants once absorbed by plants, can have multiple fates (Clemens et al. 2002, Dhanker et al. 2002, Clarkson and Hanson 1980). Many compounds are substantially bound into plant tissues in a form that is less biologically available and may be unavailable to normal chemical metabolism. This may mean that organic pollutants can be degraded or even mineralized by plants or their associated microorganisms. Also, some are lost with the aid of many physical factors such as temperature, moisture, pH, organic matter content, and sunlight. The reduction of EtBr content of soil was highest where mustard (10.7%) was planted (Table 1). This was followed by the soils planted with tomato (8.1%) and vetivergrass (5.6%). Soils planted with talahib, carabaograss, and cogongrass had the lowest reduction of EtBr (see Table 1). Statistical analysis showed highly significant differences (p0.001) among the different test plants to reduce EtBr in soil. The reduction of EtBr in soil by mustard, tomato, and vetivergrass were statistically different from one another (p0.05).
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Ethidium Bromide
vetiver grass, a very promising perennial grass. Finally, this study may have had paved the way for a larger and more comprehensive exploration of phytoremediation application in the management of toxic and hazardous wastes emanating from biotechnology research activities.
References
Betts K (1997): Phytoremediation project. Environmental Science and Technology 31 (8) 347355 Clark RB (1982): Plant response to mineral element toxicity and deficiency. pp 71142 Clarkson DT, Hanson JB (1980): The mineral nutrition of higher plants. Annu Rev Plant Physiology 31, 239298 Clemens S, Palmgren MG, Kramer U (2002): A long way ahead: Understanding and engineering plant metal accumulation. Trends in Plant Science 7 (7) 309314 Dhanker OM, Parkash II, Yujing R, Barry P, Shijin S, David S, Julie F, Sashti NA, Meagher RB (2002): Engineering tolerance and hyperaccumulation of arsenic in plants by combining arsenate reductase and y-glutamylcysteine synthetase expression. Nature Biotechnology 20, 11401145 Fritz GG (1983): Introductory plant physiology, 2nd edition. Prentice-Hall Inc., Englewood Cliffs, New Jersey, p 625 Hale MG, Dorcutt DM (1987): Physiology of plants under stress. Wiley and Sons Inc., Canada Indiana University Chemical Fact Sheet on Ethidium Bromide (1996): Indiana, USA Ona LF, Alberto AP, Prudente JA, Sigua GC (2006): Levels of lead in urban soils from selected cities in the rice-based region of the Philippines. Env Sci Pollut Res 13 (3) 177183 Pressman E, Bar-tal A, Shaked R, Osenfeld K (1997): The development of tomato root system in relation to the carbohydrate status of the whole plant. Annals of Botany 80, 533538 Raskin RD, Salt DE (1997): Phytoremediation of metals: Using plants to remove pollutants from the environment. Curr Opin Biotechnology 8 (2) 26 Roongtanakiat N (2001): Uptake potential of some heavy metals by vetivergrass. Kasetsart J Nat Sci 35, 4650 SAS (Statistical Analysis System) (2000): SAS/STAT User's Guide. Release 6.03. SAS Institute, Cary, North Carolina, 494 pp Sigua GC (2005): Current and future outlook of spoil and sludge materials in agriculture and environment. J Soils Sediments 5 (1) 5052 Sigua GC, Adjei MB, Rechcigl JE (2005): Cumulative and residual effects of repeated sewage sludge applications: Forage productivity and soil quality implications in South Florida, USA. Env Sci Pollut Res 12 (2) 8088 Sigua GC, Holtkamp ML, Coleman SW (2004a): Assessing the efficacy of dredged materials from Lake Panasoffkee, Florida: Implication to environment and agriculture. Part 1 Soil and Environmental Quality Aspect. Env Sci Pollut Res. 11 (5) 321326 Sigua GC, Holtkamp ML, Coleman SW (2004b): Assessing the efficacy of dredged materials from Lake Panasoffkee, Florida: Implication to environment and agriculture. Part 2 Pasture Establishment and Forage Quality. Env Sci Pollut Res 11 (6) 394399 United States Environmental Protection Agency (2001): A citizen's guide to phytoremediation. Accessed 12/11/2002, <http://www.cluin.org/products/citguide/phyto2.htm> Truong PN (1999): Vetivergrass system for environmental protection. Technical Bulletin no.1, Pacific Rim Vetiver Network, Bangkok, Thailand Wall Street (1996): Sunflowers bloom in tests to remove radioactive metals from soil and water. Wall Street Journal 6 (2) 2328 Watson L, Dallwitz MJ (1992): Grass genera of the world: Descriptions, illustrations, identification, and information retrieval; including synonyms, morphology, anatomy, physiology, phytochemistry, cytology, classification, pathogens, world and local distribution, and references Whitener K (1993): Phytoremediation. Environmental Science & Technology Magazine 93 (8) 3138 Zollinger M, Morais II (1979): Studies of the effect of chloramphenicol, ethidium bromide and camptothecin on the reproduction of rous sarcoma virus in infected chick embryo cells. Journal of General Virology 44, 323331
Received: January 24th, 2007 Accepted: Februay 28th, 2007 OnlineFirst: Februay 28th, 2007

Table 1: Levels of EtBr in soils and relative reduction of EtBr in soils after 30 days

Plants (treatments) Tomato Mustard Vetivergrass Talahib Carabaograss Cogongrass

a

Initial Level in Soil (g kg1) 19.7 19.7 19.7 19.7 19.7 19.7

Final Level in Soil (g kg1) 18.10.17 17.60.23 18.60.23 19.40.15 19.40.20 19.40.21

Percent Reduction in Soil 8.12b a 10.66a 5.58b 1.52c 1.52c 1.52c

Means in column followed by a common letter(s) are not significantly different from each other at p0.05

Conclusions

In this study, mustard, tomato, and vetivergrass have shown their ability to absorb EtBr in contaminated soil keeping them from expanding their reach into the environment and preventing further contamination. Its downside, however, is that living creatures including humans, fish, and birds, must be prevented from eating the plants with significant accumulation of these substances. Nonetheless, it is still easier to isolate, cut down and remove plants growing in the contaminated soils (e.g., EtBr in this study), than to use strong acids and permanganates to chemically neutralize a dangerous process that can further contaminate the environment and pose additional risks to humans. Though this alternative method does not totally eliminate eventual environmental contamination, it by far produces an extremely insignificant amount of by-products compared with other processes. Phytoremediation works in many favorable instances. It doesn't leave behind its own pollution; it doesn't require an unsightly treatment facility; it has no chemical add-ons; and the tools needed (usually quite ordinary plants) are readily available and inexpensive (Betts 1997). Also it is a safe, efficient and sound clean-up technology to manage and protect the environment. Because the sun acts as the engine, the pollutants themselves can supply some of the fuel, and green plants work like a well-oiled machine.
5 Recommendations and Perspectives

A more detailed and complete investigation of the phytoremediation potential of the different plants should be conducted in actual field experiments. Exposure of plants to EtBr should be prolonged until they reach maturity to establish the maximum response and maximum absorbing qualities of the plants to the toxicity and mutagenecity of EtBr. Also, time point sampling and analysis should be done to determine at what stage of the plant's development the uptake of EtBr should take place. Different plant parts should be analyzed individually to determine the movement and translocation of EtBr from soil to plant tissues. Since this study has established that some plants can thrive and dwell in EtBr treated soil, an increased amount of EtBr should be explored in future studies. It is suggested that a larger, more comprehensive exploration of phytoremediation application in the management of toxic and hazardous wastes emanating from biotechnology research activities should be considered especially the use of

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