Use of Populus specie as a potential Rhizofiltration

plant against Trichloroethylene in contaminated soil

By

GROUP # 04

Noor Ul Sama (075)

Preeti Kumari (076)
Shanza Mushtaq (090)
Syeda Zer e Zehra (098)
Ujala (102)
Ume Habiba (103)
Minal Fatima (116)
 ACKNOWLEDGEMENT
In the name of Almighty Allah, the most merciful and beneficial who gave us strength and
inspiration to complete this report. Without blessing of Allah, it would not be possible for us to
complete this task.

We are extremely grateful to our teacher Dr. Kifayatullah Dawar for his best and timely guidance
and constant encouragement throughout the task. Without the support of our teacher, it would
have been impossible for us to write this report.

We are also obliged to all class fellows for their cooperation, support and constant patience
which helped us to complete this project.
 Table of Content
1. INTRODUCTION: .................................................................................................................. 2

DESCRIPTION OF TCE ............................................................................................................ 2

PHYSICAL AND CHEMICAL PROPERTIES OF TCE .......................................................... 2

1.2 BACKGROUND ................................................................................................................. 3

CHARACTERISTICS OF POPULUS ....................................................................................... 3

PRESENT STATUS ................................................................................................................... 4

1.3 FUTURE WORK ...................................................................................................................... 5

2. AIMS AND OBJECTIVES ..................................................................................................... 6

3. METHADOLOGY .................................................................................................................. 7

RHIZOFILTERATION:.............................................................................................................. 7

RHIZOFILTARTION OF TCE USING POPULOUS: .............................................................. 8

OVERVIEW OF MECHANISM ................................................................................................ 8

EXPERIMENT............................................................................................................................ 9

4. FINDINGS: ........................................................................................................................... 12

5. CONCLUSION: .................................................................................................................... 13

6. REFERENCES ...................................................................................................................... 14

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 1. INTRODUCTION:

Phytoremediation is an emerging technology which uses plants and their associated rhizospheric

microorganisms to remove, degrade, or contain chemical contaminants located in the soil,

sediments, groundwater, surface water, and even the atmosphere. Researchers have found that

plants can be used to treat most classes of contaminants, including petroleum hydrocarbons,

chlorinated solvents, pesticides, metals, radionuclides, explosives, and excess nutrients. Plant

species are selected for phytoremediation based on their potential to evapotranspirate groundwater,

the degradative enzymes they produce, their growth rates and yield, the depth of their root zone,

and their ability to bio accumulate contaminants.

DESCRIPTION OF TCE

Trichloroethylene (TCE) is a chlorinated hydrocarbon used as a solvent in several degreasing and

dry cleaning operations and is one of the most extensive contaminants of soil and groundwater.

Remediation of TCE is precedence because it is a suspected carcinogen known as a mutagen from

laboratory studies, chronic inhalation exposure has been found to cause liver, kidney, and neural

reactions in animals. Vinyl chloride, breakdown product of TCE, is considered even more toxic.

TCE tends to enter soil in relatively small area from leaking drums, pipes or landfills and to diffuse

outward through unsaturated zones in the soil. While it continues to move through the soil, it

reaches downward to underlying aquifers. Because TCE is denser than water, it is then "collects

in small pools of contaminant" at the bottom of aquifers. Therefore, TCE is difficult to locate and

bring to the surface.

PHYSICAL AND CHEMICAL PROPERTIES OF TCE

The physical characteristics of TCE, which are listed in Table below, make it difficult to remove

from the soil using traditional technologies.

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The World health organization estimates that drinking 1 part per million (ppm) TCE in water over

a lifetime will cause cancer cases in 32 people in 100,000, while drinking 1 parts per million (ppm)

vinyl chloride will cause 9,570 cases in 100,000.

PROPERTY VALUE
Molecular weight 131.5
Boiling Point 87 C
Melting point -73 C
Specific gravity 1.4642 at 20 C
Solubility in water 1,000 mg/liter
Log octanol/ water partitioning coefficient 2.29
Vapor pressure 60 mm Hg at 20 C
Vapor density 4.53

Physical and Chemical properties of TCE

1.2 BACKGROUND

CHARACTERISTICS OF POPULUS

Populus is a member of the Salicaceae family and there are around 30 species of Populus

distributed throughout the Northern Hemisphere. Populus sp. have the ability to cross within the

genus both in the wild and through controlled breeding, so there are a large number of potential

hybrids.

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 • Greater than 25 species worldwide

• Fast growing (3 to 5 meters/year)

• High transpiration rates (100 liters/day optimally for 5 year old tree)

• Not part of food chain

• Trees can be used for paper production or as biomass for energy

• Long lived (25-30 years)

• Grow easily from cuttings

• Can be harvested and then regrown from the stump

Advantages of Populus sp. in Phytoremediation

Recently, phytoremediation has been suggested as an approach for treating TCE contamination.

In the case of TCE contamination, hybrid poplars (Populus trichocarpa × Populus deltoides) and

Eastern cottonwoods (Populus deltoides) have acquired most of the attention. These are said to

be as phreatophytic species, meaning that their deep roots draw water from the water table.

Additionally, poplars and cottonwoods have a fast growth rate and have demonstrated an ability

to take up TCE from both soil and water.

PRESENT STATUS

✓ Currently researches have been able to clarify some of the predicted mechanisms for TCE
phytoremediation. Early studies fascinating on the degradation of TCE by ex-planta

enzymes (those that are released by the plant into the root zone).

✓ Dehalogenases have been recognized as enzymes that degrade TCE; however their

degradation capacities are not fully understood. Degradation capacity depends not only on

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 the production of dehalogenases and their capability to degrade trichloroethylene, although

also on their rate of release, the nature of the soil, and the concentration of

trichloroethylene, areas that have not yet been addressed.

Some countries where Populus are used to treat TCE contamination:

The United States military is presently testing TCE phytoremediation in the field at the Edward

Sears Site in New Jersey and at the Aberdeen Proving Grounds in Maryland hybrid poplars are

being used on these sites to treat TCE contamination at depths of around 2.5 m (8 ft.). At the

Aberdeen site, the poplars have been planted as hydraulic pumps, in a pattern intended to try to

prevent TCE from moving to a nearby marsh. The major military demonstration site is at the Naval

Air Station in Ft. Worth, Texas (USA), formerly Carswell Air Force Base. Eastern cottonwoods

(Populus deltoides) have been planted at the Ft. Worth site to treat TCE contamination in the

groundwater plume at depths of 1.2 to 2.5 m (4 to 8 ft.)

1.3 FUTURE WORK

• As phytoremediation of TCE does not have a long way record of successful use, so there

is a need for more fundamental research, field study and innovation.

• Plant scientists, engineers and regulators must work together to develop standards for

sampling, analyzing, managing and monitoring TCE phytoremediation. Realistic systems

models need to be developed to include: input parameters such as contaminant

concentration, bioavailability, and plant uptake and degradation; output parameters such as

metabolic end points, rate and extent of remediation; events within the soil, plant, air, water

matrix such as diffusion, mass balance, mass flow and costs, including true capital and

operating expenses.

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 • Further research will focus on plant selection and development. Plants to be selected for

TCE phytoremediation must develop substantial biomass rapidly, take up water at

significant rates and elongate roots to deep levels. Plants other than poplars and

cottonwoods should be found; Willows (Salix spp.), eucalyptus (Eucalyptus spp.), black

locust (Robinia pseudoacacia) are some species being considered.

• In addition, genetic engineering is seen as a promising area for plant research, as genetic

tools may enable researchers to design plants better equipped to locate and metabolize TCE

in particular ecosystems. Hybrid technologies are observed as another area of promising

research. These technologies combine the most effective aspects of two or more strategies.

2. AIMS AND OBJECTIVES

The purpose of this report is to briefly summarize the current state of phytoremediation

technology, and then focus on the use of poplar trees to degrade (TCE) in contaminated soil.

o The chemical and physical properties have shown difficulty in its removal by traditional

technologies. As the TCE is denser then water, pump and treat methods can be employed.

Many scientists and hazardous waste site managers have revealed that phytoremediation

should be considered in this case.

o Our aim is to remove TCE from the contaminated soil because of it toxicity to animals and

plants. In animal, it has shown its mutagenic behavior. It can also damage to liver, kidney

and nervous system due to its inhalation. While in plants it causes electrolyte leakage.

o Outsets to our objective we will be using Populus species for this purpose. The rate of

transpiration is high and it is not part of food chain. It tress are easily grown and have long

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 life. Poplar is phreatophytic plants, which means that they can extend their roots to the

water table.

o We have used Populus, as fast-growing tree utilized as an ornamental or in afforestation

and reforestation. It also have resistance to drought, salt and borers. This make its ideal for

use in the process of cleaning of contaminated soil.

o Primary goals can only be achieved by performing an experiment that help us to conclude

that Populus have been used for the purification of soil and removal of TCE from it.

3. METHADOLOGY

The following sections will discuss the phytoremediation mechanism that have been reported

when poplars are used to treat TCE. However, the use of these plants to degrade TCE is still a

relatively new idea, so not all of the mechanisms are clearly elaborated at this time.

RHIZOFILTERATION:

A phytoremediation technology which implements the use of aquatic and land plants for water

treatment by different methods such as absorbing, concentrating, precipitating heavy metals from

the waste water. Rhizofiltration is a hydroponic –based green technology.

Rhizofiltration of soil:

The technique is used for the remediation of waste water through aquatic or land plants. Pb, Cd,

Cu, Ni, Zn and Cr, these are heavy metals but can be extracted by the use to Rhizofiltration

process. Plants which are employed for Rhizofiltration are populous plans, sunflower, tobacco,

spinach etc. terrestrial plants can be used for this technique as they have fibrous root systems

with fast growth.

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RHIZOFILTARTION OF TCE USING POPULOUS:

Researchers have been researching about the possibility of using trees in the genus populous to

contain it hydraulically and remediate plumes of TCE in ground water. Wastes sites have planted

populous in effort to treat groundwater contaminated with chlorinated solvents. TCE is a

common contaminant of many of the hazardous sites. In superfund national priority list NPL, it

can be found around 50% there. It above action levels in groundwater of 17% and soils of 16%.

TCE used become prevalent primarily through its use as an industrial degreasing agent. Its use as

an solvent for dry cleaning, an aesthetic for medical and dental use and as an ingredient in paints,

inks, cosmetics and cleaning fluids etc.

OVERVIEW OF MECHANISM

The cells were capable of metabolizing TCE to produce trichloroethanol, di- and trichloroacetic

acid. Some of the carbon from TCE was found in insoluble, non-extractable cell residue, and

small amounts were mineralized to CO2. Poplar cuttings grown in soil and exposed to TCE

produced the same metabolites. In field trials, trees were planted in soil in test cells and exposed

to TCE via underground water injection during the growing season. During the growing season,

at least 95% of the TCE was removed from the influent water stream in cells containing trees.

Mass balance studies conducted in the laboratory indicated that 70 to 90% of the TCE was

transpired; however, greenhouse and field study results showed that less than 5% of the total

TCE taken up by the plants is transpired. These results show that significant TCE uptake and

degradation occur in poplars. Poplars appear to be useful for in situ remediation of TCE-

contaminated sites under proper conditions

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EXPERIMENT

Cuttings of the two hybrid clones noted above were rooted in PVC (polyvinyl chloride) pipes,

20.5 cm diameter, containing a30-cm bottom layer of sand with 60 cm soil (Sultan silty clay

loam) overlay. The plants were dosed with water alone, or with 50 mg TCE/l added directly to

the sand layer through an inner watering tube.

A total of 8 g TCE was added over a period of 8 months. During the period of growth,

transpiration was assayed by enclosing the leaves in plastic bags and pulling the air through a

charcoal filter for 0.5 hr. The results were highly variable and indicated transpiration values from

undetectable to about with an average of approximately 1.0 pg. TCE/leaf/hr.

After 8 months, the plants in PVC pipes were harvested and various morphologic measurements

were taken the major difference noted was that the density of the fine roots in the sand layer of

TCE exposed plants was less than that of the controls. The plant tissues were analyzed for

metabolites of TCE using the same methods that were used for cell analyses.

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The nature and levels of the chlorinated metabolites suggest that the TCE is oxidized as it moves

from bottom roots to the aerial sections of the plant, especially the leaves. These results, together

with the products derived from the axenically cultured poplar cells, strongly argue for a role of

the plant in the metabolism of TCE, in addition to the well-known degradation of TCE by soil

microorganisms.

Mass Balance Studies

The mass balance of TCE in hybrid poplar trees was determined in a bioreactor. The leaves, stem

and roots of the plant were enclosed in separate sections of the chamber to allow the study of

plant uptake, incorporation, transpiration, and rhizosphere degradation without interference from

soil volatilization.

The major problems we faced were due to the volatility of TCE and its suspected metabolites,

and the absorption of these materials by commonly used laboratory materials such as rubber,

tygon, and various sealants. The chamber is shown in It is constructed of glass, aluminum foil,

Teflon, and inert inorganic materials. Note that the separate sections for roots, stems, and leaves

are independently aspirated to prevent volatile transpirates from leaking into other chambers.

Controls without plants show only 0.03% leakage of the TCE into the foliage chamber.

Small rooted poplar cuttings (approximately 20 cm tall) were planted in a peat moss/vermiculite

mixture in the root section and TCE was added to the root section to a final concentration of 5

mg/l and spiked with 2 x 106 dpm 14C-TCE. After 7 days approximately 0.8% of the added 14C

was detected in the transpirate. Several conclusions can be drawn from these three laboratory

experiments. First, TCE can be taken up by the poplars.

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Second, the plants transpire some of the material, and some is metabolized to known extractable

metabolites, as found in. Finally, some material is fixed in tissue; the nature of incorporation in

tissues is under investigation. We believe that the contribution of each of these pathways towards

the destruction of TCE depends upon variables such as concentration of TCE, size of tree, nature

of soil microflora, type of soil, temperature, light intensity, relative humidity, wind velocity, etc.

Control field trials controlled field trial was set up in collaboration with the Occidental

Chemical Corporation. Double-walled cells, 3.7 m x 6.1 m X 1.5 m deep, were constructed of

60-gauge high-density polyethylene sheeting. The bottoms had about 0.3 m sand overlaid with

1.1 m Sultan silt loam. In order to ensure a uniform flow of input water, a T-shaped input pipe

was used at the bottom of the influent pipe and a 1/40 slope was oriented toward the effluent

well. Four cells were each planted with fifteen 30-cm plants of poplar clone H 11-1 1. Two of

these cells were dosed with water containing 50 mg/I TCE; the other two cells received only

water. The fifth cell, which had no vegetation, also received water containing 50 mg/l TCE.

Each of the cells received the same volume of liquid; however, variable amounts of liquid were

pumped out of the cells on a daily basis to maintain a level of about 20 cm of liquid in the

bottom of the cells. About 9 weeks after injection of TCE into the-cells, breakthrough of TCE

and related metabolites, particularly cis-1,2- dichloroethylene, occurred in the cells that did not

contain trees. The occurrence of this compound is indicative of anaerobic bacterial

dehalogenation of TCE, perhaps because of ethanol, which was initially used as a solvent for the

TCE in the stock solutions.

The use of ethanol was discontinued, and the concentrations of these compounds decreased. In

cells with trees, very little TCE or metabolites could be detected in the effluent until after leaf

drop (Figure 3). Throughout the course of this first year of the experiment, a total of 957 mmol

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TCE was added to each dosed cell. Of this amount a total of 366 mmol was recovered from the

unplanted cell (cell 8, 38% recovery), while 21 and 61 mmol of TCE were recovered from cells 3

and 5, which were planted with hybrid poplars (2 and 6%, respectively). The stems, roots, and

leaves of the trees exposed to TCE showed the same products of oxidative metabolism which

were seen in the greenhouse studies and tissue culture experiments (Tables 1, 2). The 1-year-old

trees (approximately 3.5 m tall) were very effective in removing TCE from the input water.

4. FINDINGS:

As a result of 2 more years of operation of the experimental field trial, it can be found that

approximately 99% of the TCE is removed from influent water. Virtually there is no detectable

transpiration of TCE on the parts of trees that are more mature and are now 40 feet tall. An

increase in chloride ion, obtained from TCE dechlorination, appears in the soil surrounding the

roots of the trees.

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 5. CONCLUSION:

The conclusion of the experiments shows that TCE has multiple fates in poplars. Although the

mass balance data would have us believe that most of the TCE taken up by the plant is

transpired, the field and greenhouse studies show that very little is transpired under field

conditions. The compound also can undergo oxidation to chloromethyl derivatives such as

trichloroethanol, trichloroacetic acid, trichloroacetic acid, or complete mineralization to C02,

which are oxidative metabolites found in mammalian livers .Variable amounts of 14C from TCE

are found fixed in insoluble, non-extractable residues. Plant uptake coupled with TCE

metabolism serves to remove TCE from groundwater and from the soil environment. Details

about these reactions and methods for increasing the amount of mass flowing along these

pathways are still under the state of investigation. Enzymes poplars are being introduced to

increase their tolerance to higher levels of TCE and to increase the fraction of TCE that is

mineralized to CO2. These experiments show that roots of actively metabolizing poplars are able

to intercept a plume of TCE-contaminated water and metabolize most of the TCE taken up. The

system shows promise where there is sufficient space to plant trees and when the roots can reach

the contaminated region.

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 6. REFERENCES

The superfund innovative technology evaluation program: Technology profiles. US Environmental

Protection Agency, office of solid waste and emergency response, Washington, DC 20460.
EPA/540/5-88/003. (1991). Remediation Journal, 1(2), 248-248.
doi:10.1002/rem.3440010217
Compton, H., L. Erickson, L. Newman, P. Cichy, and C. Hartnett. Summary of the Remediation
Technologies Development Forum, Phytoremediation of Organics
Cooke, M.T. 1999. Phytoremediation: Using Plants to Remediate Groundwater Contaminated
with Trichloroethylene (TCE). State College, PA: Pennsylvania State University.
JJ, Mello JW, Thomas RF. The groundwater supply survey. J Am Water Works Assoc 5:52-59
(1984). 2.
National Cancer Institute. Carcinogenesis Bioassay of Trichloroethylene. NIH Publ no 76-01-6.
Bethesda, MD:National Institutes of Health, 1976.
Burken, J.G. 2001. Uptake and volatilization of chlorinated solvents by poplars at field-scale. In:
Phytoremediation, Wetlands, and Sediments, pp. 113-120. (Leeson, A., Foote, E.A.,
Banks, M.K., and Magar, V.S., Eds.). Columbus, OH, Battelle.
Neuman, D.S., Wagner, M., Braatne, J.H., and Howe, J. 1996. Stress physiology - abiotic. Part
II, Ch 17, In: Biology of Populus and Its Implications for Management and Conservation, pp.
423-458. (Stettler, R.F., Bradshaw, H.D. Jr., Heilman, P.E., and 68 Hinckley, T.M., Eds.). NRC
Research Press, National Research Council of Canada, Ottawa, ON
(PDF) Phytoremediation of trichloroethylene with hybrid poplars. Retrieved from
https://www.researchgate.net/publication/13580785_Phytoremediation_of_Trichloroethyl
ene_with_Hybrid_Poplars

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