The effectiveness of the phytoremediation of dicofol using Lycopersiocon esculentum

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The effectiveness of the phytoremediation of dicofol using Lypersiocon esculentum

Research Paper 2009-2010

William John O’Brochta

Research Instructor: Mr. Steven Smith

Roanoke Valley Governor’s School for Science and Technology



Abstract

The purpose of this project was to determine if there were detrimental effects caused by

phytoremediation, as well as whether or not tomato plants can remove dicofol from the soil.

Phytoremediation is an emerging technology used to clean up soil contaminated with

environmental pollutants (Wiley, 2007). The experimental hypothesis was that the dicofol

should produce plants that grow shorter and are less healthy than regular tomato plants, though

phytoremediation would occur. Tomato plants were utilized in this experiment because they

grow quickly and have unknown phytoremediation properties (Busch, n.d.). Dicofol was

chosen due to the length of time it remains in the soil; it also causes problems in humans such

as learning difficulties and birth defects (Qiu, et al, 2005; Arms, 2004). Phytoremediation was

measured by using a gas chromatograph-mass spectrophotometer to detect the presence of

dicofol’s major degradation produce, dichlorobenzophenone, though this test has not been

completed due to a problem with the researcher performing these tests. Results using the height

and leaf area of the plants as an indicator of changes in health showed that the tomatoes grown

without dicofol grew at a faster rate, though the changes in height of all plants were smaller

over time. Leaf area supported the same conclusion, though both were statistically not

significant. Chlorophyll concentration tests supported the experimental hypothesis with

statistical significance, showing that the chlorophyll concentration of regular tomato plants was

0.5 milligrams per gram of leaf tissue greater than plants with dicofol.

Introduction

Every day, somewhere in the world, a large or semi-large chemical spill occurs,

destroying buildings, plant and animal life, and most importantly, ecosystems. This pollution



causes death and disease to spread among human and animal populations (Arms, 2004).

Commonly spilled chemicals vary widely from pesticides to lead, many causing possibly

harmful effects to people, such as birth defects and cancer (Arms, 2004). However, not all

pollution is man made. Heavy metals such as cadmium and mercury occur naturally in rocks

and dirt; they produce effects just as dangerous as those from man-made sources (Arms, 2004).

Cleaning up a chemical spill or dangerous concentrations of a certain element is extremely

costly and time consuming. In Canada, 200,000 well sites, usually on large farms, have built up

so much salt that the amount in the soil has become a serious problem (Burtt, 2009). A situation

like this merits immediate action, but governments are strongly opposed to spending the money

to clean the site correctly, instead resorting to digging up all of the soil and trucking it away

(Burtt, 2009).

Three current methods are used to solve soil contamination issues: landfills,

incineration, and phytoremediation. Use of landfills to transfer contaminated soil only prolongs

an already bad problem (Gardea-Torresdey, 2003). Incineration emits harmful ash that if

ingested can lead to breathing problems, making the method worse than using a landfill

(Gardea-Torresdey, 2003). Phytoremediation is the new potential solution for this 1.7 trillion

dollar problem (Gardea-Torresdey, 2003). Various types of plants are placed on soil that

contains either chemical pesticides or heavy metals. “Roots explore the soil, and where you

have roots, you see an increase in the microbial population of usually 100-fold, or as much as

10,000-fold” (Evans, 2002). Therefore, the addition of the roots allows for hydrocarbon

degradation, meaning that the amount of chemical is reduced (Evans, 2002). The reduction can

be drastic, as much as 75 percent in two to three years, compared to 45 percent using bio-



remediation (Evans, 2002).

Probably the greatest downfall for phytoremediation is not the effectiveness, but the

expense, time, and compatible plants and chemicals. In short, this method works with a few

plants on a few chemicals and metals very slowly. Potential spill chemicals and toxins can be

broken into two groups, heavy metals and chemical compounds (Cutraro and Goldstein, 2005).

Polycyclic aromatic hydrocarbons (PAH), polychlorinated biphenyls (PCB), and even

dichlorodiphenyltrichloroethane (DDT) can be removed to some degree with phytoremediation

(Eckley, 2001). Impacts from DDT have cause huge problems, though DDT is still used for

some agricultural applications (Eckley, 2001). All places have some kind of PAH

contamination caused by the degradation of organic compounds in the soil (Cutraro and

Goldstein, 2005). Thus PAH’s and Persistent Organic Pollutants (POP) are in the process of

being eliminated from exported pesticides (Smith, et al., 2008).

Plant type becomes the second biggest limitation of phytoremediation after chemical

effectiveness. The ideal plant for the type of contamination should be selected, though no list of

effective plants exists (Cutraro and Goldstein, 2005). Phytoremediation has produced

successful results in grasses (especially fescue), legumes, aquatic plants, and metal

hyperaccumulators (Gardea-Torresdey, 2003). A metal hyperaccumulator stores the metal in

the leaves of the plant, a feat few plants can perform (Cutraro and Goldstein, 2005). The first

application of phytoremediation used Saint Augustine grass and got effective results (Evans,

2002). This was probably more luck than proper plant choice. The Ford Motor Company is

trying the best method available at this time, planting many species of plant to test which work

best in their affected area (Evans, 2002). Researchers began with 55 plant species, which was



narrowed down to 22 (Evans, 2002). Each was tested on a portion of the contaminated land and

results were compared, producing the best plant for the site (Evans, 2002). This method is time

consuming and inefficient, discouraging the use of phytoremediation.

Time and money are also considerations when choosing to use phytoremediation and

can be presented as drawbacks. An oil spill cleaned using Saint Augustine grass took two entire

years to see a reduction of 75 percent of the pollutants (Evans, 2002). Phytoremediation does

not work on a schedule, and repeated trials never take the same amount of time (Evans, 2002).

The Ford project mentioned above is being implemented; however, it might have to be

supplemented with old incineration or landfill techniques because the phytoremediation is

taking longer than their four-year deadline (Evans, 2002). Though the cost of phytoremediation

is decreasing, it is still much more expensive than conventional methods (Cutraro and

Goldstein, 2005). The phytoremediation market now tops 214 million dollars per year (Evans,

2002). Even with these many problems, “phytoremediation is expected to solve the

environmental pollution problem” (Wiley, 2007).

The relatively new phenomenon of phytoremediation has been the subject of some

small-scale research, though no real consensus exists regarding appropriate plants or chemicals

to use. Interest lay, therefore, in determining if common plants can phytoremediate land

contaminated with pollutants. Additionally, little research has been done to indicate what

happens to plants during phytoremediation. The project’s purpose was to determine if

detrimental effects occur to a “regular” plant that attempts to phytoremediate a chemical or

heavy metal. This project has a practical application within the realm of phytoremediation.

Scientists could compile and narrow a list of appropriate plants to use with appropriate



chemicals to assure the desired phytoremediation results. No such list exists and the only way

to create one is to begin testing different chemicals on plants, as was done in this experiment.

Tomato plants and dicofol miticide (Kelthane) were used to complete this

phytoremediation test. Tomato plants are not known for their phytoremediation abilities (Bush,

n.d.). They represent, therefore, a common plant that is widely grown throughout the United

States and world. Kelthane 50W (or WSP) Agricultural Miticide is manufactured by Dow

AgroSciences Canada Inc. and is “a miticide that provides a high initial kill and good residual.

A white to gray powder, it has an odor of fresh cut hay” (MSDS: Kelthane, 2008). Kelthane is

composed of about 51 percent dicofol (Kelthane, 2005). Dicofol is “a nonsystematic acaricide

(poisonous to mites) used to control mites that damage cotton, fruit trees, and vegetables” (Qiu,

et al., 2005). Dicofol is similar in composition to DDT and, therefore, is classified a POP

(Eckley, 2001). DDT has caused huge environmental problems and was the basis for the

popular “Silent Spring” by Rachael Carson (Eckley, 2001).

This experiment involved dictated growing tomato plants and applying dicofol one time

to see how much phytoremediation occured and what the effects of the phytoremediation were

on the plants. The independent variable in the experiment was the application of dicofol on the

plants and soil. Dependent variables were how much phytoremediation occurs in the plants and

bio-remediation in the soil, and the effect of this phytoremediation on the growth of the plant.

Leaf area and chlorophyll content were analyzed post-experiment to determine if there was a

significant difference between average initial growth of the plants and average final growth.

Analysis from an outside company determined the amount of dicofol in the soil. The hypothesis

for this experiment focused on the ability of the tomato plants to phytoremediate: If 5 mg of



dicofol is added to growing tomato plants, then the plants with dicofol will have a height

significantly less than the plants grown without dicofol.

Similar experiments have been conducted using different plants and different chemicals.

Industry news (2002) extensively reports on National Science Foundation and Environmental

Protection Agency grants that allow for various projects pertaining to phytoremediation. Evans

(2002) also reports on some attempts to use phytoremediation in the real world. Applications

included the previously acknowledged Ford Motor Company project, the first phytoremediation

attempt in Texas, and a Connecticut community restoration program (Evans, 2002).

Universities are also in the process of performing studies pertaining to the effectiveness of

phytoremediation in plants from cottonwood to vegetables (Evans, 2002).

Materials and Methods

The experiment was set-up like a tent shaped greenhouse. The structure used a long

metal pole taped to two medium Quick-Grip clamps, clamps attached to a piece of wood (about

56 cm), blue plastic on top of the table being used, and clear plastic over the poles and on the

table being used. A metal chain (30 cm) was attached to the pole with a light fixture. C9 (one

strand) lights were wrapped around the fluorescent light fixture (sunlight bulb, 40 watts; 4 foot

tube). Forty 5 oz. (148 ml) plastic cups were used as pots with one 5/16 inch hole in the bottom

of each pot. Each pot was filled with 2/3 cup (158 ml) of soil that included fertilizer. In twenty

pots, three tomato seeds were planted ¼ inch below the soil. Twenty pots were left with just

soil. Rope lights (8 feet) wrapped around the pans and connected to a timer provided additional

heat. One concrete stone (16 by 16 cm), located in each pan, was used for heat retention. A

timer set for the hours of seven in the morning to eleven at night controlled the lighting for the



plants. The plastic cover remained closed to keep temperature constant. Temperature was

desired between seventy and eighty degrees Fahrenheit.

Tomato plants were grown with two to three plants in each pot for 120 total plants. Plants

were allowed to grow for at least three weeks before the beginning of this experiment. Plants

were watered with the same amount and the same container three days a week with 59 ml tap

water. Added to the ten or so pots of plants were multiple controls. Ten pots with soil and ten

pots with soil and pesticide were used. Plants and controls were brought into school.

Pesticide (dicofol 100 mg) was added at one time during normal watering in a certain

quantity, provided the opportunity for phytoremediation. Fourteen mL of water combined with

5 mg of dicofol created the final solution used in the experiment. Two hundred and eighty mL

of water and 100 mg dicofol made the stock solution. The solution was heated and 5 mL

ethanol and acetone were added to help dissolve the dicofol.

Each day, after the pesticide was added, plant height and health were recorded. Health was

recorded using photographs for comparison purposes only. Height was measured in cm from

the point where the stem meets the dirt to the last split off the main stalk of the plant. The

distance from where ruler starts to the zero point was subtracted to give actual height readings.

After the pesticide was added, a week went by until the plants were removed. Health was

again recorded with a photograph. Final height and leaf area were measured. Leaf area

measured using the below method, both at the beginning and at the end of the experiment.

Leaf area used the top leaf of the tomato or radish plant farthest from the stem of the plant.

Photographs were taken of the largest leaf, removing the end leaflet. A square reference block

was included in each photograph. This test used sticky notes with an area of 3 square inches or



7.6 square cm. Imported photographs were cropped to allow plant and block to be shown.

Adobe Photoshop Elements 6.0 software was used to find leaf area. The magnetic marquee tool

was used to select the perimeter of each leaf. In the pallet toolbar, the histogram was opened. It

had to be expanded and refreshed to give accurate readings. Leaf pixels were recorded for each

leaf. The block of known size was selected and the number of pixels was determined. The

following equation was used to determine the square centimeter area of the plant: {[(Plant

pixels total)/(Block Pixels)] x 7.6 sq cm}/(number of plants)=square centimeters of leaf area.

Chlorophyll content was analyzed to determine health. This required testing leaves from

every plant. A standard procedure using Arnon’s equation and a spectrophotometer was used.

Chlorophyll concentrations were compared.

The soil was supposed to have been analyzed via gas chromatography-mass spectrometry,

but, due to health issues with the scientist, it has not been performed. It would have shown how

much of the pesticide existed when compared to the control with just the miticide. Data was

compiled and statistical analysis performed to see changes in plant growth, leaf area, and

chlorophyll concentration.

Results

This experiment produced results that generally supported the experimental hypothesis:

that the plants grown with dicofol would be adversely affected through height, leaf area, and

chlorophyll concentration. Results cannot be completely correlated to the presence of dicofol in

the plant because the test requiring EPA Method Solid Waste 3550C and a gas chromatograph-

mass spectrometer have not been completed yet. In sum, differences between measured values

with the tomato plants and tomato plants grown with dicofol were detected; however, some



conclusions can still be drawn.

The plant height was measured every day and the differences between the heights of

plants were compared daily. Through this test, all of the statistics performed indicated that there

was no height difference between the average heights. Initial findings about the daily height

differences were promising; however, the P values did not statistically support this conclusion.

The plant with dicofol grew about 1.5 cm between the first two days, while the tomato plants

grew just 0.9 cm, with a P value of 0.28. Opposing this result was the difference between days

two and three that showed that the regular tomato plant grew higher. Overall, the results of the

plant height could not prove differences between the tomato plant with or without dicofol.

Leaf area produced similar results. This area was measured using a computer program

that provided accurate results. Initially, the leaf areas were statistically insignificant. This trend

continued to the final leaf area, which was not significant either. Leaf area varied widely from

leaves in the same test groups. No completely valid conclusions can be made from the data

since it was statically insignificant.

Chlorophyll concentration tests fully and statistically supported the hypothesis. Tests

were performed using a spectrophotometer. In this case, the P value was extremely small and

the difference between the amounts of chlorophyll was more than 0.5 mg per gram of leaf

tissue. These results were obvious when conducting the tests, as the solution containing the

tomato and dicofol leaves was noticeably lighter in color than that of the tomato leaves. The

tests support the experimental hypothesis, though it is impossible to say that phytoremediation

caused the difference in chlorophyll concentration.

Qualitative health comparisons were performed using the photographs shown in the



Appendix. These pictures were taken at the end of the experiment. From careful study of the

photographs, the experimenter cannot detect any difference in appearance between the two

groups of plants or the two groups of soil. Though these observations can only be used in

comparison, it was possible to say that the phytoremediation does not seem to harm plants,

supporting earlier conclusions.

Overall, there was no clear indication as to whether phytoremediation harms plants

because phytoremediation may or may not have occurred. Some results showed that there

might be harm, while other results suggested that no harm existed. These predictions can be

quantified when the soil is tested.

Discussion and Conclusions

The purpose of this experiment was to determine if phytoremediation occurred in

tomato plants using dicofol and what any harmful effects of this might be. Thus, the hypothesis

supported the conclusion that there would be harmful effects if phytoremediation occurred.

This hypothesis was not disproved; however, it was not completely supported either. As of this

time, it is impossible to determine if the hypothesis is valid until further tests are performed.

With this in mind, definitive conclusions are few. There was definitely an adverse effect

caused by the presence of dicofol, whether phytoremediation occurred or not. This was

especially true in the chlorophyll concentration tests, where a statistical difference existed. If no

phytoremediation occurred, this conclusion is not surprising, as dicofol has been proven to

destroy environments (Qiu, et al., 2005). However, if the level of dicofol in the soil with

tomatoes is significantly less than that in the soil alone, then definitive conclusions can be made

supporting the hypothesis. The leaf area and plant height tests, though rendered insignificant,



did show some variation in higher height for the plants grown without dicofol.

The hypothesis cannot be totally confirmed or disproven. Chlorophyll concentration

tests were supportive, of the experimental hypothesis, but not the other two tests. It would be

possible to draw the conclusion that the phytoremediation harmed the plants, if soil tests were

completed. Thus, further study is needed to support the hypothesis.

There was no current literature or precedents pertaining to using dicofol and tomato

plants (Bush, n.d.). Many other tests have shown that phytoremediation can occur with

common plants. However, no information could be found pertaining to the effect

phytoremediation has on the growth or health of the plants. Thus, this was a new type of

experiment. Still, there were no exact or very similar projects that could be found, eliminating

the option for a direct comparison between results.

Future ideas for study center around finishing the soil tests. These tests will allow

definitive results to be compared to the potential conclusions outlined above. There is also

interest in expanding the scope of this project to include different chemicals and multiple

plants. This can easily be accomplished if a reliable source of testing equipment is found. If the

conclusions made with tomato plants and dicofol are supported with other plants and

chemicals, it could be possible to develop a listing of compatible plants and chemicals. Other

research using dicofol could be performed, possibly on other plants or in different

concentrations. Because the concentration used in this experiment was not very high, results

that looked promising could become significant. A better source for dicofol or Kelthane would

need to be located, though, because the cost was too high to order more dicofol than was

utilized in this experiment.



Literature Cited

Arms, K. (2004). Environmental science. Austin, Texas: Holt, Rinehart, and Winston.

Burtt, B. (2009, October 27). UW firm uses plants to clean contamination. The Guelph

Mercury.

Bush, C. (n.d.). Stress tolerant plants. Retrieved from

http://arabidopsis.info/students/stress/stresshome.html.

Cutraro, J., & Goldstein, N. (2005, August 01). Cleaning up contaminants with plants. Bicycle,

46(8), 30.

Eckley, N. (2001). Traveling toxics. Environment, 43(7), 24.

Evans, LD. (2002). The Dirt on phytoremediation. Journal of Soil and Water Conservation,

57(1), 12A.

Gardea-Torresdey, JL. (2003, April 01). Phytoremediation: where does it stand and where will

it go? Environmental Progress.

Industry news: team to study phytoremediation. (2002, March 01). Waste Treatment

Technology News.

Kelthane 50W agricultural miticide. (2005). Dow AgroSciences Canada.

Material safety data sheet: Kelthane 50W agricultural miticide. (2008). Dow AgroSciences

Canada.

Qiu, X., Zhu, T., Yao, B., Hu, J., & Hu, S. (2005). Contribution of dicofol to the current DDT

pollution in China. State Key Joint Laboratory for Environmental Simulation and

Pollution Control.

Smith, C., Kerr, K., & Sadripour, A. (2008). Pesticide exports from U.S. ports, 2001-2003.



International Journal of Occupational and Environmental Health.

Willey, N. (2007). Phytoremediation: methods and reviews. Totowa, New Jersey: Humana

Press Inc.

Acknowledgments

The experimenter would like to thank his parents for their help and support during this entire

project. He would also like to recognize his teacher who has helped him a great deal during the

entire process.



Appendix

Figure 1: Soil Only Figure 2: Soil with Dicofol Figure 3: Tomatoes OnlyFigure 4: Tomatoes with

Dicofol

Tomato Plants at Conclusion of Experiment



Figure 6: Preparing to Take Leaf Area Pictures

Figure 7: Measuring Area in Adobe Photoshop

Figure 5: Photograph Used to Test Area

Method for Taking Leaf Area

Method for Taking Chlorophyll Concentration

Figure 8: Drilling Holes in Pots

e 9: Spec. 20 Spectrophotometer Figure 10: Samples to be Tested



Figure 11: Average Growth Per Day (cm) vs. Time



Figure 12: Table 1- Experimental Data

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The effectiveness of the phytoremediation of dicofol using Lycopersiocon esculentum