phytoremediation: to mutate or not to mutate?

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Phytoremediation: to mutate or not to mutate? Research Paper 2010-2011 A Continuation of 2009-2010 Research Paper: The effectiveness of the phytoremediation of dicofol using Lycopersiocon esculentum William John O’Brochta Research Instructor: Mrs. Cindy Bohland Roanoke Valley Governor’s School for Science and Technology

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Research found that mutant tomato plants were better than non-mutated plants at removing plant chemicals, but soil microbe bacteria were most effective. Written by William O'Brochta, Roanoke Valley Governor's School.

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Page 1: Phytoremediation: to mutate or not to mutate?

Phytoremediation: to mutate or not to mutate?

Research Paper 2010-2011

A Continuation of 2009-2010 Research Paper:

The effectiveness of the phytoremediation of dicofol using Lycopersiocon esculentum

William John O’Brochta

Research Instructor: Mrs. Cindy Bohland

Roanoke Valley Governor’s School for Science and Technology

Page 2: Phytoremediation: to mutate or not to mutate?

Abstract

The purpose of this project was to determine whether brt mutated tomato plants

phytoremediate more than non-mutated tomato plants and if phytoremediation has any

detrimental health effects to the mutated plants. The hypothesis was that tomato plants, mutated

to increase root length and size, would phytoremediate more effectively, with greater negative

health effects, when 5 mg of dicofol is applied, than non-mutated tomato plants.

Phytoremediation ability was measured using a mustard bioassay and laboratory analytical

testing. Plant health was determined by measuring chlorophyll concentration, leaf area, and plant

height tests. Results showed that phytoremediating did not significantly affect plant health of

mutant or wild-type plants. The average chlorophyll concentration of the mutant was 1.4353 mg,

while the non-mutated tomato had a value of 2.628 mg. Neither value was statistically

significant. The bioassay and GC/MS both showed that phytoremediation did not occur in either

type of tomato plant. There was 0.63 mg/kg concentration remaining in regular plants, but 0.29

mg/kg in mutated plants, showing that the mutated plants had the least amount of dicofol

remaining in the soil. However, the soil controls had only 0.52 mg/kg and 0.19 mg/kg. Roots

remaining in the soil after plants were removed may explain this finding. More dicofol was

removed in the mutated tomato plants when compared to non-mutated plants. A potential reason

for this phenomenon is the branching and quantity of roots in the mutated tomatoes. Another

possibility is that mutated roots contain sucrose or more organic transport molecules that could

aid phytoremediation.

Introduction

Pollution causes death and disease to spread among human and animal populations, even

in the developed world (Arms, 2004). Commonly spilled chemicals vary widely from pesticides

Page 3: Phytoremediation: to mutate or not to mutate?

to lead, many causing possibly harmful effects to people, such as birth defects and cancer (Arms,

2004). Heavy metals such as cadmium and mercury occur naturally in rocks and dirt; they

produce effects just as dangerous as those from human-made sources (Arms, 2004). How can

this situation be rectified? 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). Landfills combine many hazardous pesticides together to

create a high concentration of dangerous chemicals and leach into groundwater, causing further

contamination. Incineration emits harmful ash that if inhaled 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

cause an increase in the number of pesticide digesting microbes by as much as 10,000 fold

(Evans, 2002). Therefore, the addition of the roots allows for pesticide 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 (the use of soil

microbes to digest the pesticide) (Evans, 2002). In a study on contaminated soil sites, Crane

(2009) notes that phytoremediation removes between 33 and 46 percent of an oily contaminant,

Page 4: Phytoremediation: to mutate or not to mutate?

confirming conclusions that phytoremediation is definitely an effective clean-up method (Crane,

2009).

The phytoremediation used in these experiments involved rhizodegradation, enhanced

phytoremediation abilities in plant roots, and phytoextraction, chemical accumulates in the

leaves of plants (Russell, 2005). Rhizodegradation involves increases in the amount of bacteria

present in the rhizosphere area of the root (near the top) (Zobel, et. al., 2005). This type of

phytoremediation is most common; however, some rhizosphere bacteria can harm the plant and

environment, due to phytotoxicity (Zobel, et. al., 2005). Effects of rhizodegradation can include

increased nutrient uptake and increased water uptake, both important in phytoremediation ability

(Zobel, et. al., 2005). Phytoextraction works when phytoremediated compounds are too heavy to

be released and are slowly degraded in the plant (Gerhardt, et. al., 2009). Plants that phytoextract

can be removed and incinerated or left in the soil (Gerhardt, et. al., 2009).

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

types of plants on a few chemicals and metals over a very long period of time. A typical

phytoremediation application can cost up to $694,000 (Russell, 2005). The Environmental

Protection Agency notes that the amount of time for phytoremediation to occur depends greatly

on the type of plants and amount of dangerous pesticide present (U.S. EPA, 2001).

Potential spill chemicals and toxins that may be removed by phytoremediation 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). All places have some kind of PAH contamination caused by the degradation of

Page 5: Phytoremediation: to mutate or not to mutate?

organic compounds in the soil (Cutraro and Goldstein, 2005). Thus PAH’s and Persistent

Organic Pollutants (POP), chemicals defined by the EPA as having the longest half-life, are in

the process of being eliminated from exported pesticides; however, removal of these chemicals

from the soil will be a problem for years to come (Smith, et al., 2008). Plant type becomes the

second biggest limitation of phytoremediation after chemical type. 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 such as alpine pennygrass

(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 to remediate former auto manufacturing plants where the soil is

contaminated with oil, planting many species of plant to test which work best in their affected

area (Evans, 2002). Researchers began with 55 plant species, 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 reduced 75 percent

of the pollutants in two years (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

Page 6: Phytoremediation: to mutate or not to mutate?

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 which chemicals

might be best suited for phytoremediation. 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 plant that attempts to phytoremediate a

chemical, in this case a pesticide. The above objective is the same as a previous research project,

except the goal has changed to testing mutated plants that exhibit characteristics especially

helpful to phytoremediation. This project has a practical application within the realm of

phytoremediation. Specific mutations can be identified that improve phytoremediation abilities.

These mutations, like the one tested in this experiment, will allow companies to apply

phytoremediation with fewer plants and greater effectiveness, making the technology much more

attractive to companies.

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.). Research showed that mutated tomato plants may phytoremediate more effectively than

regular tomato plants (Buch, n.d.). This may be due to modified root structure and veins. A

bushy root variety was selected for this experiment under the rationale that plants with larger

Page 7: Phytoremediation: to mutate or not to mutate?

roots could take up more chemical (Chetelat, 2010). Remediation depends solely on the root

length and depth (Russell, 2005).

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 (long lasting

effectiveness). 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 (Figure 1) and,

therefore, is classified a Persistent Organic Pesticide (Eckley, 2001). These two pesticides are

often used interchangeably and results in a dicofol experiment should apply to DDT (Garber and

Peck, 2009).

DDT has caused huge environmental problems and was the basis for the popular “Silent

Spring” by Rachael Carson (Eckley, 2001). It has also been linked to causing over fifty percent

of breast cancer cases in women when it was in use (Watts, 2008). Dicofol is also extremely

present in soil after long periods of treatment, with a half-life of 2-15 years (Garber and Peck,

2009; Russell, 2005). However, after only a short period of exposure to dicofol, initial

degradation is somewhat exponential (Garber and Peck, 2009). This is not uncommon, though

significant pesticide initially degrades; the rate of degradation slows after little additional time,

but still meets or exceeds legal regulations in Italy (Cabras, et. al., 1985). Still, dicofol remains a

huge problem because of its toxicity to many fish, causing mutations and decreased survival

(Garber and Peck, 2009). DDT also bioaccumulates, or builds up. As predators eat prey, the

concentration of DDT increases significantly (Withgott and Brennan, 2008).

Page 8: Phytoremediation: to mutate or not to mutate?

Similar experiments have been conducted using different plants and different chemicals

from this experimenter and others. A phytoremediation experiment in 2005 using rye grass to

remove DDT was extremely effective (Greenberg, 2006). In fact, 30% of the DDT was removed

within 90 days, but it is noted that there is know way to know “whether DDT is being degraded

in the soil or in the plants,” an important consideration (Greenberg, 2006). Initially,

phytoremediation of DDT was deemed impossible, but was proven possible in 1977 (Russell,

2005). 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). Many

of the researchers and professors that the experimenter spoke to are also working on

phytoremediation and genetic mutation analysis. The experimenter also performed previous

research on this topic, using regular tomato plants to perform a similar test.

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

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

on the plants. The experiment was a simulation of environmental conditions where dicofol was

present in the soil and tomato plants were added. The independent variable in the experiment was

the application of dicofol or Kelthane 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. This data was compared to previous research that

Page 9: Phytoremediation: to mutate or not to mutate?

focused on regular tomato plants to determine whether mutated tomato plants are more effective

at phytoremediating. 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 company tested soil from both the experiment using mutated and regular tomato plants.

Later, the experimenter conducted another test to verify the results from the laboratory. This test

used a bioassay of the soil and a base test using mustard seeds. The hypothesis for this

experiment focused on the ability of the mutated tomato plants to phytoremediate: Tomato plants

that have been genetically mutated to increase root length and size will phytoremediate more

effectively, with fewer health effects when 5 mg of dicofol is applied than regular tomato plants

that have not been mutated.

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; 122 cm tube).

This structure was used for the first half of the experiment and then transferred to school. At

school, the plants were placed in two racks with the same fluorescent light fixtures.

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

of each pot for drainage. Each pot was filled with 155 ml of soil that included fertilizer. In

twenty pots, mutated tomato seeds (bushy root variety, treated with 15% hydrochloric acid for 1

minute) were planted 0.635 cm below the soil. These seeds were obtained from a university, but

Page 10: Phytoremediation: to mutate or not to mutate?

they had limited quantity, so some pots received two seeds and some received three. Twenty pots

were left with just soil.

Rope lights (244 cm) wrapped around the pans and connected to a timer provided additional

heat. 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 21.1 and 26.6 degrees Celsius and it was recorded daily through the use of a

digital thermometer in the enclosure.

Tomato plants were grown with one plant in each pot. Many seeds did not germinate and test

groups were combined to produce the most relevant results. Plants were allowed to grow for at

least three weeks before the beginning of this experiment.

Plants were watered in the same amount with the same container on Monday, Wednesday,

and Friday with 59 ml tap water. Watering schedule was adjusted based on plants water needs,

but watering was constant across all test groups.

Test groups included: six pots of plants receiving pesticide, six pots of soil, six pots of soil

receiving pesticide, and two pots of plants. These numbers were restricted due to the availability

of mutated tomato seeds and the large number of mutated tomato seeds that did not germinate.

Pesticide (Kelthane 50 WSP miticide 200 mg or dicofol 100 mg) added at one time during

normal watering in a certain quantity, provided the opportunity for phytoremediation. The

dilution was 1:10 Kelthane to water, based on typical pesticide dilutions for farm applications

(Kelthane, 2005). Two hundred and fifty ml of Kelthane was ordered. This Kelthane was

combined with 350 ml of water to create a stock solution. The solution was heated and 5 ml

ethanol and acetone were added to force the solution to combine. The ethanol and acetone

Page 11: Phytoremediation: to mutate or not to mutate?

evaporated. Fourteen point six ml of the solution was applied to each pot that was designated to

receive pesticide. Additional stock chemical solution was reserved for use in bioassay testing.

Initial testing measurements on the day the chemical was added included: height, health, and

leaf area using below explained methods. 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 petiole on the

stem of the plant. The distance from where ruler starts to the zero point, when subtracted from

the height, gave accurate 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 below method. Height measured using above method.

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

Photographs were taken of the largest leaf on the highest petiole, removing the end leaflet.

Include a square reference block in each photograph. This test used sticky notes with an area of

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. Using the magnetic marquee tool,

select the perimeter of each leaf. In the pallet toolbar, open the histogram. Expand and refresh.

Leaf pixels should be recorded for each leaf. Select the block of known size and determine the

number of pixels. Use the following equation 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. These calculations were performed using Microsoft Excel.

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

every plant. Cut leaf into small pieces, not using the major veins. Weigh about 100 mg of leaf,

record weight, use hole punch, scissors, or similar to remove parts of leaf. Put tissue into a

Page 12: Phytoremediation: to mutate or not to mutate?

mortar and add 10 ml 91% isopropyl alcohol. Pulverize tissue with a pestle; result is the leaf

homogenate. Filter the leaf homogenate through filter paper (F3 was used). Trash the retentate

(extra pulp). The extract was collected in the test-tube. A clean cuvette was obtained for the

spectrophotometer. Wipe the bottom of the cuvette to make sure there are no watermarks.

Additional alcohol was added to obtain 10 ml of solution in the cuvette. This alcohol change was

recorded. Obtain a cuvette with only alcohol and place in the spectrophotometer. Cover cuvette

chamber and set to zero absorbance with the blank in place at 663 nm. Remove blank and save

for next measurement. Swirl the first extract in the test-tube. Wipe and place in the

spectrophotometer and close the hatch. The spectrophotometer should show the absorbance at

663 nm (A663). Record and repeat with other extracts. Change wavelength to 645 nm. Reinsert

the blank cuvette and re-zero the spectrophotometer at the new wavelength. Remove the blank

and insert a cuvette containing the first extract. Read and record A645. Repeat for other extracts.

Calculate using Arnon’s equation to convert absorbance measurements to mg Chl g-1 leaf tissue.

Equation used: Chl a (mg g-1) = [(12.7 x A663)-(2.6 x A645)] x (ml alcohol / mg leaf tissue). Chl b

(mg g-1) = [(22.9 x A645)-(4.68 x A663)] x [ml alcohol / mg leaf tissue]. Total Chl=Chl a+Chl b.

Chlorophyll concentrations were compared and equation was computed using Microsoft Excel.

The soil was analyzed to see how much of the pesticide exists when compared to the control

with just the miticide. Two methods were used: a bioassay and a quantitative test.

An analytical company performed the quantitative test. They were sent four samples of soil,

two from previous research and two from current research. In each set, one sample had soil

where plants had been grown with pesticide and one sample had only soil and pesticide. Fifty mg

of soil were required to complete the testing, so two pot of soil were combined. The company

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used EPA Methods Solid Waste 3550B and 8081A to test the soil. The result was the

concentration of dicofol remaining in the soil in mg/kg units.

The technique of using a bioassay was instrumental in the completion of this experiment.

A bioassay was the main method of testing the amount of dicofol remaining in soil samples to

quantitatively determine how much dicofol remained and how effective tomato plants were at

phytoremediating. There is little available research about the method of bioassays. Orcutt (2010)

cautions that there is not much literature that dictates proper bioassay method (Orcutt, 2010).

Thus, part of this experiment was determining a proper bioassay method (Orcutt, 2010). A

simple definition of a bioassay is “a method for estimating the potency of a drug or material…by

utilizing the reaction caused by its application to experimental subjects” (Govindarajulu, 2001).

The bioassay is a new method of testing, developed in the 1940’s (Govindarajulu, 2001). Key to

successful bioassays is creating a standard data set with known amounts of chemical for which to

compare the sample data sets (Govindarajulu, 2001). Thus, the bioassay is an inexpensive and

easy method of testing soil, though it is imprecise, meaning that additional testing is required to

create truly quantitative results.

To prepare the bioassay, a baseline test was conducted. Pots of soil were prepared as

described above. Each pot was given varying amounts of dicofol, from 0 mg to 7 mg, with two

groups of 0 mg and increasing by 0.5 mg starting from 1 mg. Twenty mustard seeds were added

to each pot. Mustard seeds were chosen because they have been known to be effective indicators

of DDT (extremely similar to dicofol) (Orcutt, 2010). The number of plants was measured for

ten days. The results were compiled and averaged and one equation that was representative of the

data was found to allow for estimation of the amount of dicofol in soil with relation to the

number of seeds that germinated. Similar testing was repeated with samples from current and

Page 14: Phytoremediation: to mutate or not to mutate?

past research. Germination of mustard seeds was recorded and using the equation found above,

an average estimated amount of dicofol remaining in the soil was obtained. This value was

compared with other values to determine if the amount was significantly different than other

samples.

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

area, chlorophyll concentration, and mustard seed germination. Averages were performed on

appropriate data sets. T-tests and error analysis was also completed. A logistic function was used

to fit the bioassay results.

There were many constants used in the project. They included the amount of light, amount of

water, temperature, amount of soil, number of seeds, amount of chemical, method of height,

area, chlorophyll content, and analysis methods. The independent variable included the presence

of chemical in grass or in the soil. Growth of the resulting tomato plants, the amount of

phytoremediation that occurred, the amount of chemical in plant, the amount of chemical in soil,

the height of the plant, the health of plants recorded using photographic comparison, and the leaf

area of plants are some examples. In order to keep the experiment controlled, three groupings:

tomato plants without added chemical, soil with no chemical, and soil with added chemical were

used.

Results

The hypothesis that more phytoremediation would occur in mutated tomato plants when

compared to regular tomato plants, but the mutated plants would experience adverse health

effects was partially supported. Data showed that no phytoremediation occurred in either test of

tomato plants. Slightly less dicofol was left in brt mutated plants, but there was no detectible

difference between the health of the two types of plant. Table 1 shows that the leaf area and

Page 15: Phytoremediation: to mutate or not to mutate?

height of the test groups were not statistically significant. Figures 2 through 5 show testing that

was performed to determine the health of the mutated tomato plants. This data was compared to

results of the same testing from the previous year. However, chlorophyll concentration was

significant in the regular tomato, but not in mutated tomato. Overall, the portion of the

hypothesis dealing with the health of plants was unsupported. In Figure 6, mutated tomato plants

with dicofol and without dicofol are shown. Anecdotally, there was no visible difference

between these plants and the quantitative data supports this conclusion.

Through bioassay and analytical testing, the phytoremediation portion of the hypothesis

was slightly supported. These results are shown in Table 2, with the bioassay analysis, and Table

3, with the lab tested analysis. Samples of tomato plants had higher concentrations of dicofol

remaining, indicating that phytoremediation may not have occurred. The mutated tomato plants

did indeed have less dicofol than regular tomato plants, but both were less than microbe

remediation in the soil. The bioassay used soil that was stored in a freezer from 2009 tests

(Figure 7). Figure 8 shows the bioassay testing. In the bioassay, the amounts of dicofol used

were an issue. Concentrations calculated from the bioassay were between 0 mg and 1 mg of

dicofol, the smallest amounts in the test. To make these conclusions, Graph 1 was generated of a

baseline test with known amounts of dicofol. A standard logistic fit was used on these points to

generate Graph 2. The logistic fit equation is shown and explained in Table 4. Graph 2 was used

to read the predicted amount of dicofol present in the soil to a precision of 0.01 mg. These results

are shown as an average of the dicofol values over the entire experiment in Row 1. Rows 2 and 3

of Table 2 show values calculated from Row 1.

Table 3 Row 3 shows the values given from an analytical laboratory about the amount of

dicofol in the soil. As shown, conclusions from Table 3 support Table 2 conclusions. However,

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the amounts of dicofol present in the soil were very small. There was more microbial remediated

dicofol than phytoremediated dicofol in these tests. The major issue with the results is the one

hundred-fold difference in the concentration remaining (mg/kg) between bioassay and analytical

testing. Figure 9 shows the testing set-up used to perform the analytical testing.

The experimenter decided to attempt to locate where the brt gene was mutated on the

tomato chromosomes because Zobel (2010) does not remember how he mutated the plant in

1971. This was an extension to the project that was not in the hypothesis. He used information

available from Tomato Genetic Cooperative Reports (Report, 1951-2010) to isolate the brt gene

to tomato chromosome 12. Additional data about related mutations further isolated the gene to

about 19.8 cM (unit of length of chromosome) or 95.8 cM on chromosome 12. This was

discovered using the fd gene and the aud gene, located very close together. Fd was located on the

long arm of the chromosome and aud on the short arm, so the genes must be located near where

the two arms intersect. The point of intersection is 57.8 cM. Aud is 38 cM from brt, so brt could

either be at 19.8 cM or 95.8 cM. Using tomato chromosome information, a gene was located at

95.8 cM, but not at 19.8 cM. Thus, the brt gene is likely located at 95.8 cM. There were four

DNA sequences at 96 cM. Each was read in forward and reverse sequences into a protein

translation service. The best match was gene TG296, located at 96 cM on chromosome 12.

Forty-five amino acids matched a protein sequence using the forward strand from 3’ to 5’. A

BLAST search located potential proteins for this sequence. The search resulted in a match to a

Lysr transcriptional regulator protein, which aligned fairly well. This protein was found in

Acinetobacter lwoffi (a bacteria). The bacteria protein was also matched to plant proteins that are

similar; the best match was the SDS degradation transcriptional activation protein, found in the

castor bean. Because castor beans and tomatoes share a similar lineage (both dicots) this protein

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may be the one that was mutated to create the brt tomato plant. The activator in this protein may

cause different genes to be expressed because of transcriptional changes, leading to longer and

more highly branched roots. This finding is important because it could allow a scientist to mutate

the tomato plant again to create a mutation with known location.

Discussions and Conclusions

Conclusions from this experiment are many and varied. First, bioassay analysis worked

effectively, but the method of testing could have been improved. The method used came

exclusively from Orcutt (2010) because of the limited research available on this subject. Smaller

increments of dicofol concentration were needed to be more precise in measurement. More

mustard seeds were also needed, again to help testing precision.

The effect of dicofol on health was shown to be negligible. This result is not surprising,

due to similar results with mutated and regular tomato plants. Reasons for differences in

chlorophyll concentration are unknown. These tests suggest that some internal damage may

occur in phytoremediating plants. Russell (2005) supports this conclusion and notes that plants

must have phytotoxicity, or ability to withstand the presence of dicofol. Weaver (2010) warns

that tomato plants are usually fairly phytotoxic and are used as bioindicators. Low levels of a

pesticide are usually tested with tomato plants to make sure that the test groups will not be killed

by the phytoremediation (Rose, 2010). While this experiment does not support such a drastic

conclusion, there is some evidence to suggest a potential phytotoxic property in regular tomato

plants. The mutated plants, however, exhibit no chlorophyll concentration significance, so their

phytotoxic abilities may have been impaired by the mutation.

The major finding from this experiment was that effective phytoremediation did not

occur in tomato plants. There was, however, less dicofol remaining in mutated tomato plants

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when compared to non-mutated plants. This could indicate that some phytoremediation may

have happened, but it was not as effective as bioremediation. There are many potential reasons

for this phenomenon. Zobel (1971) provides the only available research into the brt tomato

mutant used in this experiment. He notes, “few mutants…have as poor a background as brt”

(Zobel, 2010). Various scientists are currently attempting to successfully identify how the mutant

was formed (Thompson, 2010; Benedito, 2010). “The root system is very highly branched…the

root system branches profusely within one day after emergence, in contrast to normal roots,

which branch only after several days of growth” (Zobel, 1971). Zobel also notes that brt mutated

tomato plants germinate more slowly than non-mutated plants (Voland and Zobel, 1988).

The experimenter spoke with many researchers about the possibilities for

phytoremediation in the brt mutant and found multiple different potential theories. This mutant

also displays increased colonization of fungus on its roots (Zsogon, et. al., 2008). Increased

fungus presence could contribute to phytoremediation abilities because of the plant’s growing

need for nutrients (Zsogon, et. al., 2008). Another theory could be that there are more microbial

enzymes in the roots (Benedito, 2010). Peres (2010) noted that he observed an increased

concentration of Brix (sucrose) on the roots. Zobel (2010) confirms this observation by stating

that there is an increase in starch at the base of the roots that could be duplicated by the presence

of sucrose. This sucrose is likely located on the microbial chelators, which are known to deliver

nutrients to the plant, while sucrose probably is located on the top of the rizosphere (root shoot)

(Gerhardt, et. al., 2009). Levels of Auxin and Gibberellin (plant growth hormones) increased in

the brt mutant, when compared to non-mutated plants (Sidorova, et. al., 2002). These results

were observed in pea plants with the same mutants, so the results should be similar for tomato

plants (Sidorova, et. al., 2002). However, the same researcher showed that Auxin levels were

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actually decreased when compared to the control in a later experiment (Sidorova, et. al., 2010).

Thus, the plant growth hormone levels cannot effectively be compared to phytoremediation

ability. Root nodulation was observed as statistically the same as the control, which could

explain why the phytoremediation abilities were similar (Sidorova, et. al., 2002).

It cannot be said, however, that the tomato plants were more effective in removing the

dicofol when compared to soil microbe bioremediation. This finding is surprising, considering

the relative effectiveness of phytoremediation when compared to bioremediation. All of the

studies that the experimenter read identified phytoremediation as 30 percent or more effective

when compared to bioremediation (Greenberg, 2006). Aerobic bacteria have been known to

biodegrade many pesticides and metals and oxygen can oxidize some petroleum products

(Thieman and Palladino, 2009). There may simply have not been enough dicofol present in the

soil to distinguish a large difference between the microbe remediation and phytoremediation.

The soil used was not sterilized because microbe remediation was being compared to

phytoremediation, but it would be useful to autoclave soil in a future experiment to determine

whether microbes in the soil were more active, causing the difference between the microbe and

phytoremediation of the soil.

Why the regular tomato plants did not phytoremediate and the mutated tomato plants

contained less dicofol is unknown. The most likely correct theory is that there is a modification

in the mutant to the root genes that work to take up organic molecules (Benedito, 2010). This

mutation could help bring more nutrients into the plant, therefore, causing some

phytoremediation to occur, especially when compared to regular tomato plants. The brt mutant

also has modified root architecture, increasing root branching and surface area, causing more

phytoremediation (Zobel, 1971). Another possibility is that the microbes in the soil have

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undergone ecological secession from 2009 to 2010 that results in more microbe activity and

remediation (Thieman and Palladino, 2009). This could explain why remediation was more

effective in 2010 than 2009. Tomato plants may have actually competed with soil microbes for

the dicofol, reducing phytoremediation and microbe remediation abilities. The last possibility is

that the dicofol simply evaporated when applied. This is extremely unlikely, however, because

dicofol was applied near the roots and the pesticide is known to be persistent and not degrade

that quickly.

Further research centers on determining if sterilized soil exhibits any remediation of

dicofol. This would show if microbe remediation played such a large part in the experiment as

suggested by the data. It would also be useful to take research from Benedito (2010) about

membrane transporters for organic material in tomatoes and attempt to identify which microbes

are present in the brt mutant that allow it to phytoremediate more than regular tomato plants.

Combining a metal hyperaccumulator with a species that have increases root branching and mass

may provide the greatest phytoremediation abilities (Zobel, et. al., 2005). Thus, combining the

brt mutated tomato plant’s genes with those from a metal or pesticide hyperaccumulator such as

alpine pennygrass could be the best phytoremediation situation (Zobel, et. al., 2005). Increasing

the concentration of dicofol may produce more significant results; however, this project was

based off of typical pesticide applications. Increasing bioassay capability by using more mustard

seeds is also important and could warrant a project in itself to determine the most effective

testing method (Orcutt, 2010).

Literature Cited

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

Benedito, V. "Tomato phytoremediation of dicofol." Message to researcher. 2010. E-mail.

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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.

Cabras, P., Cabitza, F., Meloni, M., & Pirisi, F.M. (1985). Behavior of some pesticide residues

on greenhouse tomatoes. 2. fungicides, acaricides, and insecticides. Journal of

Agriculture and Food Chemistry, 33, 935-937.

Chetelat, R. (2010). Revised list of monogenic stocks. Davis, CA: C.M. Rick Tomato Genetics

Resource Center, Department of Plant Sciences: University of California, Davis.

Crane, C. (2009, September 21). Cleaning up soiled sites. Science World, 66(2), 6.

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.

Garber, K., & Peck, C. Office of Pesticide Programs, Environmental Fate and Effects Division.

(2009). Risks of dicofol use to federally threatened California red-legged frog (rana

aurora draytonii). Washington, D.C.

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

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Govindarajulu, Z. (2001). Statistical techniques in bioassay. Basel, Switzerland: S. Karger.

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Greenburg. (2006). Newsletter for the NSERC CRD multi-process phytoremediation system.

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Orcutt, D. "Testing plant samples." Message to researcher. 2010. E-mail.

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

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Russell, K. U.S. Environmental Protection Agency, Office of Solid Waste and Emergency

Response: Technology Innovation and Field Services Division. (2005). The use and

effectiveness of phytoremediation to treat persistent organic pollutants. Washington, D.C.

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Sidorva, K.K., Shumny, V.K., Vlasova, E.Yu., Glyanenko, M.N, Mishehenko, T.M. (2002). The

brt (branched roots) and lrt (long roots) genes control the development of roots in peas

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intense nitrogen fixation by the example of pea. Вестник ВОГиС, 14(2), 357-374.

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Zobel, R.W., Wright, S.F., Al-Amoodi, L.K., Barbarick, K.A., Roberts, C.A., & Dick, W.A.

(Ed.). (2005). Roots and soil management: interactions between roots and the soil.

Madison, WI: American Society of Agronomy, Inc.; Crop Science Society of America,

Inc.; Soil Science Society of America, Inc.

Zsogon, A., Lambais, M.R., Benedito, V.A., de Oliveria Figueria, A.V., & Peres, L.E.P. (2008).

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Agricola, 65(3), 259-267.

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Acknowledgements

The experimenter would like to acknowledge many researchers and professors from

various institutions that provided great advice for this project. This includes the staff of

Brookside Laboratories, Inc. and Ms. Kari Long for helping me test the soil samples. Dr.

Jonathan Watkinson and Roanoke College were both extremely helpful in obtaining the correct

mutant tomato seeds. Great insight and advice was received from the following people: Dr.

David Orcutt, Dr. Richard Zobel, Dr. Roger Chetelat and the C. M. Rick Tomato Genetics

Center at U.C. Davis, Mr. Darren Cribbes, Dr. Michael Weaver, Mr. Keith Rose, Dr. Bernard

Glick, Dr. Saleh Shah, Mr. Barry Robinson, Mr. Dennis Anderson, Mr. David Richert, Dr.

Vagner Benedito, Dr. Andrew Thompson, Ms. Patty Webb, Dr. Victoriano Gutiérrez, Dr. Lazaro

E.P. Peres, Mr. Paul Foran and Dow AgroSciences, Ms. Linda Fiedler, Dr. Priscilla Gannicott,

Dr. Donald Mullins, Ms. Tricia Stoss, Dr. J.O. Rogers, Mr. Greg Evanylo, Mr. Wythe Morris,

Dr. Kari Benson, Dr. Jim Westwood, Dr. Darwin Jorgensen, and many others. Special thanks to

my parents and research instructor who were instrumental in the success and funding of this

project.

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Appendix

Graph 1: Logger Pro Generated Graph of Number of Mustard Seeds Germinated (number) vs. Amount of Dicofol (mg/pot). Error bars of 5% error are shown. Curve was automatically fit and then tweaked so that it fit the data better. Point (4.5, 12) was stricken from the curve fitting because it was viewed as an outlier.

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Graph 2: Logger Pro Generated Graph of Number of Mustard Seeds Germinated (number) vs. Amount of Dicofol (mg/pot) Through Logistic Curve Fit by Generating Points Fitting the Equation For Every 0.01 mg/pot. This curve was used to estimate the amount of dicofol remaining in every soil sample after phytoremediation occurred.

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Figure 1: Comparison Of Chemical Structures-Dicofol On Left, DDT On Right-To Show Their Similarities (Drawings-PubChem)

Figure 2: Chlorophyll Concentration Testing On 2010 Tomato Leaf Samples

Figure 3: Measuring Chlorophyll Concentration At A663 And A645 In A Spectrometer

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Figure 4: Leaf Area Pictures With Reference Block

Figure 5: Using Adobe Photoshop To Determine Leaf Area

Figure 6: Mutated brt Plants -Health Is Comparable Between Tomatoes With and Without Dicofol

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Figure 7: Tomato Soil Stored From 2009 Experiments in Freezer For Preservation

Figure 8: Number Of Mustard Seeds Germinated Was Counted Each Day

Figure 9: Gas Chromatograph-Mass Spectrometer At Brookside Laboratories Used To Analyze Soil Samples Packaged In Ball Jars (Picture-Brookside Labs)