The Phytotoxicity of Nanoparticles

Joshua Ahearn and Tahrim Choudhury

Macomb Mathematics Science Technology Center

Chemistry

12B

Mrs. Hilliard / Mrs. Cybulski / Mr. Acre / Mrs. Tallman

9 December 2016
 The Phytotoxicity of Nanoparticles

The purpose of this experiment was to test the level of phytotoxicity contained in

zinc oxide and silicon dioxide nanoparticles. It was hypothesized that the zinc oxide

nanoparticles would contain the highest phytotoxicity. The objective of understanding the

phytotoxic properties of nanoparticles is a crucial concept for the improvement of

agriculture, and because this is a new field of research, every new observation is critical

to the comprehension of the concept.

To carry out the experiment, mung beans were placed into one of three different

solutions: distilled water, a solution of distilled water with silicon dioxide nanoparticles,

or a solution of distilled water with zinc oxide nanoparticles. Four beans were placed into

eight petri dishes per group, and the nanoparticle solutions were added to each. The beans

were then placed into a growth chamber and monitored over a seven-day growing period.

After the seven-day growing period, the sprouts were removed from the petri dishes and

measured using the Logger Pro software.

The average length of the sprouts was used as the data in the ANOVA test and the

three two-sample t tests that were conducted. The null hypothesis for the ANOVA test

was that the mean sprouts lengths was the same for all treatments, and the alternative was

that the mean sprout lengths were different for all treatments. The null hypothesis was

rejected after running the ANOVA test. The null hypothesis for the t tests was that the

zinc oxide would yield a lower average sprout length compared to the silicon dioxide and

distilled water. After conducting these two-sample t tests, it was determined that the

original hypothesis that zinc oxide would yield the lowest sprout length, therefore

containing the highest amount of phytotoxicity, was accepted.

 Table of Contents

Introduction..........................................................................................................................1

Review of Literature............................................................................................................3

Problem Statement...............................................................................................................6

Experimental Design...........................................................................................................7

Data and Observations.......................................................................................................10

Data Analysis and Interpretation.......................................................................................15

Conclusion.........................................................................................................................26

Appendix A: Sterilization..................................................................................................31

Appendix B: Picture Analysis............................................................................................32

Appendix C: ANOVA Sample Calculations......................................................................33

Appendix D: Two-Sample t Test Sample Calculation.......................................................37

Appendix E: Table B for determining P-Value from t-Score.............................................38

Appendix F: Two-sample t Interval Sample Calculation...................................................39

Works Cited.......................................................................................................................40
 Ahearn Choudhury 1

Introduction

Those not familiar with nanotechnology are truly living in a pre-1980s world.

Nanotechnology is the study, usage, and application of nano-sized particles that can be

used in every field of science. These particles were not discovered until 1981, when the

development of scanning tunneling microscopes allowed scientists to see individual

atoms. This is when the study of nanoparticles truly began (What is Nanotechnology).

Nanoparticles are used by many popular companies, including Apple, General Motors,

and GAP for everything from phone screens to car exteriors to khaki pants (Williams).

Since nanoparticles are still a relatively new concept within the scientific

community, a large amount of research is still being conducted on them. Some

nanoparticles are known to help cure cancer, while others are known to be toxic. This

experiment aimed to help gain understanding on which nanoparticles were known to be

toxic to plant life. To carry out this experiment, solutions of silicon dioxide and zinc

oxide nanoparticles were created. Mung beans were then treated with these solutions or a

distilled water solution and monitored over a seven-day growing period. The sprout

lengths for these beans were then recorded and used to determine which nanoparticles

were proven to contain the highest amount of phytotoxicity, or toxicity to plant life.

This experiment hoped to create a better understanding of which nanoparticles are

known to contribute to a high amount of phytotoxicity and which nanoparticles contribute

to little to no phytotoxicity, which was accomplished when analyzing the results. This

information is useful in the scientific community, which is still researching new

properties of nanoparticles each and every day. This experiment helps to validate existing

research on which nanoparticles are harmful to plant life. Agriculturists can also apply
 Ahearn Choudhury 2

this information when choosing a pesticide to apply to plants, since some pesticides

contain nanoparticles as well. These farm owners would want to ensure that the pesticide

of their choice is free of any toxic nanoparticles, which could affect their average plant

yield, resulting in a decreased food supply for the general public. Furthermore, since

many manufacturers produce waste that contains nanoparticles that may be harmful to

plant life, this research can be used as an informative tool to notify industrialists of the

harmful effects of their actions.

As a whole, this topic of research, with the constant development of

nanotechnology, will only expand with time. Delving into and comprehending the

fundamental properties of nanoparticles is only the first step to developing a better

understanding of the effects and uses of these particles.

 Ahearn Choudhury 3

Review of Literature

The usage of nanoparticles is growing at an exponential rate. From medicine to

cosmetics, nanoparticles can be found in many of the manufactured goods people use

from day to day. Nanoparticles are defined as a material or particle with any external

dimension in the nanoscale range (1 nm to 100 nm) or having internal structure or surface

structure in the nanoscale (United States, Department of Toxic Substances Control).

However, not all nanoparticles are created the same. The California Nanosafety

Consortium of Higher Education differentiates nanoparticles into two categories:

naturally occurring and engineered. The European Commission also defined

nanoparticles ...on the basis of the size of the particles within a material, without any

given regard to whether they are a hazard or a risk (Milmo). Nanoparticles are known to

react differently than regularly sized particles, with the nano counterpart being more

highly-reactive. Since these particles are so miniscule, it is extremely easy for them to

leak into water supplies, food and crops, and even find their way into human bodies

through pores and other crevices. If these nanoparticles are proven to be toxic, it could be

fatal for any crop or animal that comes into contact with them.

Phytotoxicity is another important topic in this research experiment. Phytotoxicity

is defined as Plant injury (phytotoxicity)...when chemicals are employed to protect

plants from pests, fertilize plants, regulate plant growth, etc (Phytotoxicity) (Plant

Diseases). According to Pennsylvania State Extension, phytotoxicity can occur if a

material (such as a pesticide) is improperly applied to a plant; if a material from one crop

blows off and travels via wind to another, more sensitive crop; if a runoff of chemicals

contaminates the soil; or if a material is applied in adverse environmental conditions. It

 Ahearn Choudhury 4

lists symptoms of phytotoxicity as resulting in poor or no germination of seeds,

misshapen leaves or fruits, and dead spots on leaves or between the veins of the leaves.

Figure 1. Phytotoxicity of Fungicides on Glycine max (Allen)

Figure 1 above shows an example of the phytotoxicity of fungicides on a Glycine

max (soybean) leaf. The leaf has several apparent dead spots, showing how harmful the

effects of phytotoxicity really are to plant life.

Nanoparticles can also play a role in the phytotoxicity of plants. Since these

particles are so miniscule, they are able to easily travel through a plant via water or soil.

An example of this is illustrated below:

Figure 2. Nanoparticles on Root Surface of F. esculentum (Lee)

Figure 2 shows a comparison of F. esculentum (buckwheat) roots under a control

group (left) and treated with zinc oxide nanoparticles (right). It can be seen that the small
 Ahearn Choudhury 5

size of the nanoparticles allows them to enter the surface of the roots easily, facilitating

their path to cause potential destruction.

This experiment was designed to be able to test how easily nanoparticles mixed

into a water solution are imbibed by a seed. It also aimed to test the toxicity levels of

different types of nanoparticles, since not all nanoparticles are proven to be toxic. This

experiment was based heavily on past research published in the Journal of Chemical

Education to test the phytotoxicity of nanoparticles on mung beans. The research, run by

multiple professors at the University of California and Sacramento State College, showed

that nanoparticles of zinc oxide were proven to be toxic, since the beans they treated

germinated very little over a two-week period (Ross). This experiment aimed to replicate

the research done by the professors at the University of California and Sacramento State

College. However, since that research was executed at a university, the quality of

materials they had access to was likely higher than the quality of materials that were

available to use in this experiment. Nevertheless, the experiments carry the same central

purpose and therefore yielded similar results.

 Ahearn Choudhury 6

Problem Statement

Problem:

To determine the phytotoxicity of zinc oxide and silicon dioxide nanoparticles on

Vigna radiata to be measured in millimeters over a seven-day growing period.

Hypothesis:

Zinc oxide will produce the least amount of growth of Vigna radiata roots,

therefore yielding the most phytotoxicity.

Data Measured:

The independent variable in this experiment was the type of nanoparticle the

seeds were treated with (zinc oxide or silicon dioxide), measured in milligrams and

mixed with distilled water, measured in milliliters. The dependent variable was the length

that the seeds roots grew over a seven-day period, measured in millimeters. This was

completed in 96 trials, 32 per group (including control). This was analyzed by using an

Analysis of Variance (ANOVA) to determine whether the means of root length were

significantly different, and a two-sample t test to determine which group was most

significantly different from the control mean. These tests were appropriate, seeing as the

means of the zinc oxide and silicon dioxide treatments were compared to the mean of the

control group.
 Ahearn Choudhury 7

Experimental Design

Materials:

(96) Vigna radiata, mung bean seeds Thermometer

(24) 90 mm 15 mm Petri dishes 1 mL Bleach
(3) 1000 mL beaker Logger Pro Vernier software

(3) 65 mm pipettes Camera

(2) Weigh boats Ruler, cm
20 mg Zinc oxide, ZnO nanoparticles (10-30 nm) 250 mL beaker
20 mg Silicon dioxide, SiO nanoparticles (20-30 nm)
2 Growth chamber
3000 mL Distilled water Timer, 12 hr
Analytical balance (0.0001 g precision) Saran Wrap
Magnetic stirrer

Procedure:

1. Make sure all lab materials are sterile (see Appendix A).
2. Plug in growth chamber into timer and set timer to 12-hour cycle (6am to 6pm).
3. Prepare a one-part bleach to 100-part water solution in a 1000 mL beaker and soak
the mung beans within it for 15 minutes.

4. Label one 1000 mL beaker ZnO and the other beaker SiO using permanent
2

marker and tape.

5. Label the 250 mL beaker Distilled.

6. Wear gloves and use a weigh boat to mass 20 mg zinc oxide and add it to the beaker
labeled ZnO.

7. Mass 20 mg silicon dioxide and add it to the beaker labeled SiO . 2

8. Add distilled water to each beaker until 1000 mL is reached.

9. Place each beaker on a magnetic stirrer and stir for 15 minutes.
10. Meanwhile, split the 24 petri dishes into three groups of eight.
11. Add four mung beans into each petri dish, making sure to keep them spread out.
12. Using the pipette, add distilled water to one group of petri dishes until the beans are
submerged, but the petri dish is not overflowing.

13. Place covers on the dishes and carefully move this group to the growth chamber and
use permanent marker and tape to label each dish Control and a number 1-8.

14. Using the pipette, add the ZnO solution to one group of petri dishes until the beans
are submerged, but the petri dish is not overflowing.

15. Place covers on the dishes and carefully move this group to the growth chamber and
use the permanent marker and tape to label each dish ZnO and a number 1-8.
 Ahearn Choudhury 8

16. Using the pipette, add the SiO solution to one group of petri dishes until the beans are
2

submerged, but the petri dish is not overflowing.

17. Place covers on the dishes and carefully move this group to the growth chamber and
use the permanent marker and tape to label each dish SiO and a number 1-8.
2

18. Place thermometer inside of the growth chamber to ensure constant temperature.

19. Cover any remaining solutions of ZnO or SiO with saran wrap and place in a safe
2

location.

20. Check on the beans each day and if the solution is running low, stir again and add
more.

21. After 7 days, take a picture of each sprout with a ruler placed next to it.
22. Analyze pictures and record data (see Appendix B).

Diagram:

Figure 3. Setup

Figure 3 on the previous page shows the setup for the experiment. All petri dishes

with beans were placed under a growth chamber set for a 12-hour cycle.
 Ahearn Choudhury 9

Figure 4. Materials

Figure 4 shows the materials used throughout the experiment.

 Ahearn Choudhury 10

Data and Observations

In this experiment, data was collected using the Logger Pro software. The data

for the distilled water, silicon dioxide, and zinc oxide were put into tables, and the

averages and standard deviations were calculated. These tables, along with any

significant observations made, are shown below.

Table 1
Distilled Water (Control) Sprout Length
Trial Length (cm) Trial Length (cm)

1 9.969 17 6.221

2 7.779 18 8.431

3 8.888 19 9.664

4 6.959 20 6.387

5 8.489 21 10.088

6 9.354 22 6.176

7 7.919 23 6.253

8 9.471 24 6.707

9 9.848 25 7.342

10 6.967 26 9.237

11 7.326 27 10.27

12 7.627 28 8.381

13 9.349 29 6.625

14 9.617 30 9.201

15 10.508 31 6.293

16 6.256 32 0.000

Average 7.925
 Ahearn Choudhury 11

Standard Deviation 2.026

Table 1 on the previous page shows the recorded sprout lengths for the control

group of mung beans, along with their average and standard deviation in centimeters.

Table 2
Silicon Dioxide Sprout Length
Trial Length (cm) Trial Length (cm)

1 5.641 17 9.601

2 7.712 18 5.059

3 7.376 19 8.679

4 8.893 20 9.341

5 9.668 21 6.635

6 8.859 22 5.013

7 7.183 23 7.270

8 9.680 24 0.000

9 9.482 25 9.933

10 9.654 26 9.260

11 8.473 27 9.078

12 5.163 28 8.975

13 7.102 29 9.375

14 9.058 30 6.628

15 9.304 31 9.282

16 9.889 32 4.7545

Average 7.876

Standard Deviation 2.170

 Ahearn Choudhury 12

Table 2 on the previous page shows the recorded sprout lengths for the group of

mung beans treated with silicon dioxide solution, along with their average and standard

deviation in centimeters.

Table 3
Zinc Oxide Sprout Length
Trial Length (cm) Trial Length (cm)

1 5.876 17 5.458

2 6.209 18 5.113

3 6.353 19 5.651

4 0 20 0.000

5 7.517 21 5.978

6 5.891 22 5.807

7 7.097 23 5.169

8 7.351 24 6.422

9 6.507 25 5.424

10 6.221 26 5.677

11 6.219 27 5.524

12 5.035 28 5.812

13 6.470 29 4.995

14 6.318 30 5.582

15 6.737 31 5.754

16 1.786 32 0.000

Average 5.311

Standard Deviation 1.986

Table 3 shows the recorded sprout lengths for the group of mung beans treated

with zinc oxide solution, along with their average and standard deviation in centimeters.
 Ahearn Choudhury 13

Table 4
Observations
Date Observation

10/25 24.4C, control groups almost sprouted

10/26 Control sprouted, SiO and ZnO almost sprouted, ZnO has least growth
2

10/27 21.1C, some ZnO still not sprouted

Control group 6 had brown water and was dry, one seed in control group 8 did
10/31
not grow

One seed in SiO group 1 did not grow, SiO group 3 had a brown spot, one seed
2 2

in SiO group 6 molded, one seed in ZnO group 1 did not grow, one seed in
11/1
2

ZnO group 4 barely grew, one seed in ZnO group 5 molded, one seed in ZnO
group 8 did not grow

Table 4 above shows the observations of growth made during the experiment, and

notable observations made during data collection.

Figure 5. Seeds Before Treatment

Figure 5 above shows the mung beans placed in the appropriate solutions on the

first day of experimentation. It can be seen that there had not yet been any growth.
 Ahearn Choudhury 14

Figure 6. Seeds After Treatment

Figure 6 above shows the mung beans after 7 days of germination. It can be seen

that most of the seeds had grown substantially.

Figure 7. Measuring Roots

Figure 7 above shows a root accurately measured using Logger Pro Vernier

software. This is how data was collected and recorded. Refer to Appendix B for the

detailed procedure.
 Ahearn Choudhury 15

Data Analysis and Interpretation

The data from this experiment was quantitative, continuous, and univariate data.

For each trial, the length of the sprout was measured to the nearest thousandth of a

centimeter.

The data collected in this experiment is valid because it follows the principles of a

comparative experiment which are control, randomness, and replication. As a control,

data was collected by treating mung beans with pure distilled water. This then allowed the

data of the beans treated with nanoparticle solution to be compared to a standard. A

control was used so that lurking variables would then affect all trials equally and

confounding could be reduced. Randomness was achieved by randomizing the trials so

that not all the same treatments were done at the same time, which was therefore used to

reduce bias. Replication was met by doing 32 trials of each treatment, which was used to

ensure that variability was reduced as much as possible.

To analyze the distribution and spread of the data, a box plot was created for the

sprout lengths of the three groups of treatments.

Figure 8. Box Plots for All Three Treatments

 Ahearn Choudhury 16

In Figure 8 on the previous page, box plots of the sprout length for all three

treatments were plotted on the same graph. It can be seen that the range and interquartile

range of the box plots do vary for each treatment. This shows that there is some

variability in the data between each treatment, yet the ZnO treatment is shown to have

lower Q1, median, and Q3 values than SiO and the control group, which have similar
2

values. Similarly, the control group and SiO each have one outlier, whereas the ZnO trial
2

has two outliers. These outliers could affect the mean of the data when performing the

ANOVA test and two-sample t tests, so these outliers were removed when both the two-

sample t-test and ANOVA tests were conducted to remove any errors they may have

given. Because of these outliers, the means appear to be lower than the medians on the

box plot for all groups. This is due to the fact that means are affected greatly by outliers,

while medians are not. The box plots for the control treatment and ZnO do not appear to

have any extreme skewness to them, indicating that the data comes from normally

distributed populations. However, the SiO treatment does appear to be skewed left, but
2

since 32 trials were run for each treatment, the Central Limit Theorem states that the data

comes from a population with a normal distribution, the sampling distribution.

The box plots help determine that the data collected in the experiment is a good

representation of the sprout lengths. The data is assumed normal, and an ANOVA test can

be carried out to compare the means of the three groups of beans more carefully to

conclude that the treatments did make a significant difference in their sprout lengths.

For this data, the statistical test that was the most appropriate to use was an

ANOVA test because it compares the means of three or more populations and how far

apart they are relative to the variability between individual observations. To correctly
 Ahearn Choudhury 17

conduct an ANOVA test, three conditions must be met. The first condition was met,

which was having independent simple random samples for the given number of

populations - in this experiment, three. Since the box plot depicted a normal distribution

for the data overall, the second condition was also met. Also, because there were 32 data

points for each category, the Central Limit Theorem says the samples come from normal

sampling distributions, thus the sampling distribution of the sample mean is close to a

normal distribution. However, since outliers were removed to perform the tests, the ZnO

group only had 28 trials to analyze. Since the box plot shows that ZnO appears to be

normally distributed, the tests were still run. In addition, the rule of thumb was satisfied

as shown below:

N > 10n

N being the population of all mung beans in the universe, and n being the number

of data points in this experiment, which even times 10 is still less than the overall

population. Lastly, the sample standard deviations of the populations were similar,

fulfilling the third condition for this test.

H o : 1=2=3

H a : Not all 1 , 2 , 3 are equal

Figure 9. Null and Alternative Hypotheses

The variable in Figure 9 represents the mean of the control group, represents
1 2

the mean of the silicon dioxide group, and represents the mean of the zinc oxide group.
3

This tests null hypothesis states that the mean lengths for the control, silicon dioxide, and

zinc oxide roots are all equal. However, the alternative hypothesis states that not all
 Ahearn Choudhury 18

means are equal. More detailed procedures of the ANOVA test can be found in Appendix

C.

Figure 10. TI-Nspire ANOVA test

Figure 10 above shows the results of the ANOVA test for all three groups of the

beans. As shown above, the F statistic turned out to be approximately 24.83. A fairly

large F statistic such as this one would correspond to a small p-value, which turned out to

be about 2.938 x 10-9. The null hypothesis which stated that the mean sprout lengths for

the control, silicon dioxide, and zinc oxide treated beans would be equal was rejected

because the p-value of 2.938 x 10-9, which is essentially zero, is below the 0.05 alpha

level of significance. This means that there is strong evidence that the mean sprout

lengths for the control, silicon dioxide, and zinc oxide treated beans are not equal. There

is almost a 0% chance of getting an F statistic this extreme by chance alone if the null

hypothesis was true.

Since the ANOVA test results indicated that the means of all three groups were

not equal, two-sample t tests were conducted between all of the different treatments to

determine the mean difference between them. This experiment met all the conditions that

a two-sample t test requires. The data is from a randomized experiment. All of the
 Ahearn Choudhury 19

different treatments had 32 trials which meets the condition that n 30; n 30 to
1 2

make sure that the data comes from a normal sampling distribution by the Central Limit

Theorem. However, since outliers were removed to perform the tests, the ZnO group only

had 28 trials to analyze. Since the box plot shows that ZnO appears to be normally

distributed, the tests were still run. Also, the condition that N 10n; N 10n was met
1 2

because population of all mung beans in the universe is larger than ten times the sample

of mung beans used in this experiment. All observations and groups were independent of

each other.

Figure 11. Control Sprout Length and SiO Sprout Length T-Test Hypothesis
2

Figure 11 shows the null and alternate hypotheses for this two-sample t test. In

these hypotheses, stands for the mean of the sprout length for the control treatment,
1

and stands for the mean of the sprout length for the SiO treatment. The null hypothesis
2 2

was that the sprout length would be the same for both treatments. The alternative

hypothesis was that the sprout length for the control treatment would be greater than the

sprout length for the SiO treatment.

2
 Ahearn Choudhury 20

Figure 12. Control Sprout Length and SiO Sprout Length T-Test Results and P-Value
2

Graph

Figure 12 shows the results of the two-sample t test using the TI-Nspire student

software. The same results can be found using a formula (see Appendix D). The null

hypothesis, H , can not be rejected because the p-value of 0.4488 is greater than the alpha,
o

, level of 0.05. The p-value can also be calculated by using the t value, 0.1293 in this

case, and Table B (see Appendix E).

There is no significant evidence that the sprout length of the control group is

greater than the sprout length of the SiO treatment. If H was true, that sprout lengths of
2 o

both the control treatment and SiO treatment were the same, then there would be a
2

44.88% chance of getting a difference in sprout length this extreme by chance alone.

Since this is likely to happen almost half of the time, the null hypothesis was not rejected.
 Ahearn Choudhury 21

Figure 13. Control Sprout Length and SiO Sprout Length T-Interval
2

Figure 13 displays the results of the two-sample t interval. The same results can

be found by using a formula (See Appendix E). Using the information above, one may be

95% confident that the true population mean will fall between -0.7376 and -0.8359. It can

also be determined that if this experiment were to be run in the future, 95% of the

differences in sprout length would capture the true population mean.

Figure 14. Control Sprout Length and ZnO Sprout Length T-Test Hypothesis

Figure 14 shows the null and alternate hypotheses for this two-sample t test. In

these hypotheses, stands for the mean of the sprout length for the control treatment,
1

and stands for the mean of the sprout length for the ZnO treatment. The null hypothesis
2

was that the sprout length would be the same for both treatments. The alternative

hypothesis was that the sprout length for the control treatment would be greater than the

sprout length for the ZnO treatment.

 Ahearn Choudhury 22

Figure 15. Control Sprout Length and ZnO Sprout Length T-Test Results and P-Value

Graph

Figure 15 shows the results of the two-sample t test using the TI-Nspire student

software. The same results can be found using a formula (see Appendix D). The null

hypothesis, H , can be rejected because the p-value of 1.107 x 10 is less than the alpha, ,
o
-9

level of 0.05. The p-value can also be calculated by using the t value, 7.560 in this case,

and Table B (see Appendix E).

There is significant evidence that the sprout length of the control group is greater

than the sprout length of the ZnO treatment. If H was true, that sprout lengths of both the
o

control treatment and ZnO treatment were the same, then there would be an almost 0%

chance of getting a difference in sprout length this extreme by chance alone. Since this is

not likely to happen, the null hypothesis was rejected.

 Ahearn Choudhury 23

Figure 16. Control Sprout Length and ZnO Sprout Length T-Interval

Figure 16 displays the results of the two-sample t interval. The same results can

be found by using a formula (See Appendix F). Using the information above, one may be

95% confident that the true population mean will fall between 1.595 and 2.755. It can

also be determined that if this experiment were to be run in the future, 95% of the

differences in sprout length would capture the true population mean.

Figure 17. SiO Sprout Length and ZnO Sprout Length T-Test Hypothesis
2

Figure 17 shows the null and alternate hypotheses for this two-sample t test. In

these hypotheses, stands for the mean of the sprout length for the SiO treatment, and
1 2 2

stands for the mean of the sprout length for the ZnO treatment. The null hypothesis was

that the sprout length would be the same for both treatments. The alternative hypothesis

was that the sprout length for the SiO treatment would be greater than the sprout length
2

for the ZnO treatment.

 Ahearn Choudhury 24

Figure 18. SiO Sprout Length and ZnO Sprout Length T-Test Results and P-Value Graph
2

Figure 18 shows the results of the two-sample t test using the TI-Nspire student

software. The same results can be found using a formula (see Appendix D). The null

hypothesis, H , can be rejected because the p-value of 3.363 x 10-8 is less than the alpha,
o

, level of 0.05. The p-value can also be calculated by using the t value, 6.600 in this

case, and Table B (see Appendix E).

There is significant evidence that the sprout length of the SiO treatment group is
2

greater than the sprout length of the ZnO treatment. If H was true, that sprout lengths of
o

both the SiO treatment and ZnO treatment were the same, then there would be an almost
2

0% chance of getting a difference in sprout length this extreme by chance alone. Since

this is not likely to happen, the null hypothesis was rejected.

 Ahearn Choudhury 25

Figure 19. SiO Sprout Length and ZnO Sprout Length T-Interval
2

Figure 19 displays the results of the two-sample t interval. The same results can

be found by using a formula (See Appendix E). Using the information above, one may be

95% confident that the true population mean will fall between 1.473 cm and 2.774 cm. It

can also be determined that if this experiment were to be run in the future, 95% of the

differences in sprout length would capture the true population mean.

 Ahearn Choudhury 26

Conclusion

The purpose of this experiment was to test the phytotoxicity of various

nanoparticles on mung beans. This was tested by preparing solutions of either zinc oxide

nanoparticles, silicon dioxide nanoparticles, or distilled water. Four seeds were then

placed into a petri dish containing one of the three solutions and monitored over a seven-

day growing period. At the end of this experiment, it was determined which nanoparticles

contained the highest amount of phytotoxicity.

It was hypothesized that zinc oxide would produce the least amount of growth of

Vigna radiata roots, therefore yielding the most phytotoxicity. This hypothesis was

accepted after determining that the mung beans soaked in the zinc oxide solution did have

a mean sprout length significantly lower than the silicon dioxide or distilled water beans.

This conclusion was reached after performing an Analysis of Variance (ANOVA) test and

three two-sample t tests to compare the mean sprout lengths of each treatment to each

other. The p-value of the ANOVA test was 2.938 x 10-9, proving that not all of the

treatment groups had equal sprout lengths. The p-value of the t test comparing the silicon

dioxide group and the zinc oxide group was 3.363 x 10-8, and the p-value of the t test

compared the control group and zinc oxide group was 1.017 x 10-9, which showed a

significant difference in sprout length between the zinc oxide beans and the silicon

dioxide and distilled water beans. An illustration of this can be seen in Figure 8.

The zinc oxide treatment yielded the shortest sprouts compared to the other two

groups, which means that they possessed the most phytotoxicity. One reason why

nanoparticles in particular negatively affect plant growth is because of their exceptionally

miniscule size. This property allows the particles to easily break into cellular membranes
 Ahearn Choudhury 27

of plants and cause damage (Walter). Other factors that contribute to the toxicity of

nanoparticles are properties such as high surface area to volume ratio, chemical

composition, crystallinity, electronic properties, inorganic or organic coatings, solubility,

shape, and aggregation behavior. Combined, these characteristics of foreign substances

may interfere with respiration or cause plant cells to behave abnormally, become

inflamed, and become damaged or even die. Consequently, the sprout will not grow as

fully or as healthily as a normal root (Schrand et al.).

Scientifically speaking, the results garnered from this experiment agree with

current research in this field. A similar experiment published in the Journal of Chemical

Education conducted by The University of California and Sacramento City College

showed that bean plants exposed to zinc oxide nanoparticles did in fact grow significantly

less than the other treatment groups (Ross). The information collected by these

institutions is consistent with the results of this experiment: the bean plants exposed to

zinc oxide yielded the shortest sprout lengths of the three treatments. This experiment

also has ties to research conducted at Korea University where rats were topically exposed

to silicon dioxide nanoparticles and then sacrificed and tested for signs of toxicity or

other health problems. No health issues were found on the rats, and they remained

healthy until they were sacrificed (Ryu). Consistent with the results of this experiment,

silicon dioxide presented very little effect on the sprout lengths in comparison to distilled

water. Furthermore, the results of this experiment are consistent with a book published

called Nanotechnology and Plant Sciences: Nanoparticles and Their Impact on Plants by

Manzer H. Siddiqui, which shows that silicon dioxide nanoparticles play a nonsignificant

role on the germination of seeds (Siddiqui et al.). However, it does conflict with certain
 Ahearn Choudhury 28

parts of the publication which state that zinc oxide nanoparticles increase plant growth

and development. This could be due to the fact that different species were used in the

experiment, but this also contradicts other publications such as the Journal of Chemical

Education and Iranian Biomedical Journal, which have shown zinc oxide to play a

negative effect on the germination of seeds of various types.

Since nanoparticles and their effects on plant life and animal life are still being

researched to this day, this research can be beneficial to the scientific community. This

experiment validates current research that is being done to test the phytotoxicity of

various nanoparticles. It can also be applied to future research on this topic as a basis for

what nanoparticles have been tested to contain a high phytotoxicity.

There were many advantages to the experimental design used. All instruments

were sterilized, greatly reducing the chances of contamination. This includes the seeds,

which were rinsed with a bleach solution before treatment. Furthermore, a growth

chamber was used to maximize the exposure to light in a controlled environment. All of

these factors were beneficial to the experiment as a whole.

Although the experiment was successful, improvements could still be made to

better the quality of the experiment. Since this experiment was modelled after a pre-

existing experiment, flaws from that experiment may have easily carried over into this

one. The experiment in the Journal of Chemical Education that this experiment was

modelled after used nanopure water as a control in the experiment. Since a nanopure

filtration system was not available to use, distilled water was chosen as a replacement for

the control. Using nanopure water may have resulted in a larger difference in the mean

sprout length between the control and silicon dioxide sprout length, which were almost
 Ahearn Choudhury 29

identical. The experiment in the Journal of Chemical Education also sonicated their

nanoparticles when mixing them into water to ensure complete mixing. A sonicator,

which uses sound energy to completely incorporate particles into a solution, was not

available for use and a magnetic stirrer was used to mix the nanoparticles into solution

each time they were used. This may have resulted in separation of nanoparticle and water

in the growing phase, however the nanoparticle powder was not observed in any of the

petri dishes at the end of the growing period.

More research could be done on phytotoxicity by germinating other seeds, such as

radishes or tomatoes, and testing the overall vegetable quality of the zinc oxide and

silicon dioxide vegetables to the distilled water vegetables to see what effect the

phytotoxicity in these nanoparticles plays on overall plant quality, such as size, shape,

and color. A plant exposed to these toxic nanoparticles may have difficulties growing

correctly, resulting in discoloration of leaves and deformities of the fruits produced.

Another interesting factor that could be further researched would be to use regular tap

water as a control instead of distilled water, since most people would opt to water their

plants with a garden hose, in ground sprinkler system, or some other large scale sprinkler

system that is hooked up to a water main. Using this as a control would provide insight to

the phytotoxicity of the nanoparticles in a real-world scenario, where the plants are

watered using tap water, and nanoparticles are introduced to the plant as a part of a

pesticide or natural factors bringing it there. This would be useful since a laboratory

setting does not capture the methods in which nanoparticles are instituted to plants in the

real world.
 Ahearn Choudhury 30

Overall, this experiment can assist agriculturalists, manufacturers, and

subsequently, all of humankind. With the help of this research, farmers can avoid using

pesticides containing nanoparticles that are harmful to plants. Furthermore,

manufacturers can learn to be more aware of where they dump waste containing

phytotoxic nanoparticles, which could harm plants if exposed to them. With this

knowledge, plant life can be preserved, resulting in the overall improvement of human

life.
 Ahearn Choudhury 31

Appendix A: Sterilization

Materials:

Bleach
Ethanol
(2) Spray bottles

Procedure:

1. Gather all lab materials

2. Prepare one of the spray bottles by filling it completely with ethanol
3. Prepare the other spray bottle with one-part bleach to nine parts water.
4. Sterilize the lab bench by spraying the bleach solution onto it and waiting for five
minutes before wiping it down.

5. Sterilize the lab materials by spraying them with ethanol and then drying them off.
 Ahearn Choudhury 32

Appendix B: Picture Analysis

Materials:

Logger Pro Vernier software

Procedure:

1. Open Logger Pro Vernier software.

2. Upload an image of the petri dish and ruler (InsertPicturePicture with Photo

Analysis).
3. Click Set Scale (yellow ruler), draw a line from one centimeter mark to another,
and set the scale to 1 cm.

4. Click Photo Distance (yellow ruler with red arrow), trace the length of the root, and
sum the distances.

5. Record length, in cm, in a data table.

6. Repeat steps until measurements of all roots are recorded.
 Ahearn Choudhury 33

Appendix C: ANOVA Sample Calculations

The statistical analysis method used in this experiment was the ANOVA test. The

statistics and sample calculations for the test are shown below.

Table 5
ANOVA Statistics
Treatment n x x s x

Control 31 8.181 1.444

SiO 2 31 8.130 1.652

ZnO 28 6.006 0.6589

Table 5 above shows the statistics for the ANOVA test. The sample sizes, sample

means, and sample standard deviations are provided.

The first formula needed in order to conduct this test was the mean, x, which is

the number of observations in each sample times the mean of each sample - like weighted

means.

x =(n1 x 1 +n2 x2 +n 3 x 3)/ N

For this formula, the sample sizes and means of each individual population must

be known, including the total observations in all samples. In the formula, n stands for

each sample size and x stands for each sample mean. The total observations are

represented by N.

x =(n1 x 1 +n2 x2 +n 3 x 3)/ N

x =(318.181+318.130+286.006)/96

x 7.019

Figure 20. Mean

 Ahearn Choudhury 34

Figure 20 on the previous page shows a sample calculation for the mean of an

ANOVA test.

Next, the mean square group, the MSG, must be found. This is the variation

among the sample means between each population.

MSG=[ n1 ( x1 x ) + n2 ( x 2x ) +n3 ( x 3x ) ] /(I 1)

2 2 2

For this formula, the sample sizes and means of each individual population must

be known. The number of populations is also required and the x-value found in the

previous step.

MSG=[ n1 ( x1 x ) 2+ n2 ( x 2x )2 +n3 ( x 3x )2 ] /(I 1)

MSG=[ 31 ( 8.1817.019 )2+31 ( 8.1307.019 )2+ 31 ( 6.0067.019 )2 ] /(31)

MSG 54.43

Figure 21. Mean Square Group

Figure 21 above shows a sample calculation for the mean square group, or MSG

of an ANOVA test.

After finding the MSG, the MSE needs to be calculated, which stands for mean

square error. This measure the variation among individuals in all samples of each

population. Each sample variance does this job and is then weighted by one less than the

number of observations it represents.

MSE=[ ( n1 1 ) s 1 + ( n21 ) s 2 + ( n31 ) s3 ] /(N I )

2 2 2

For this formula, the sample sizes and standard deviations of each population

must be known. The number of observations in all samples including the number of

populations must also be known.

 Ahearn Choudhury 35

MSE=[ ( n1 1 ) s 12 + ( n21 ) s 22+ ( n31 ) s32 ] /(N I )

( 311 ) 1.4442 + ( 311 ) 1.6522+ ( 281 ) 0.65892 /(963)

MSE=

MSE 1.679

Figure 22. Mean Square Error

Figure 22 above shows a sample calculation for the mean square error, or MSE of

an ANOVA test.

Finally, the F Statistic for the ANOVA test is calculated using the MSG and MSE.

MSG
F=
MSE

The MSG, which is the variation among the sample means between each

population, is divided by the MSE, the variation among individuals in the same sample

within each population. When the F Statistic is large, the corresponding p-value is small.

MSG
F=
MSE

54.43
F=
1.679

F 32.42

Figure 23. F Statistic

Figure 23 above shows a sample calculation for the F Statistic of an ANOVA test.

The F Statistic is specified by a numerator degrees of freedom and denominator

degrees of freedom.

I 1
Degrees of Freedom :
N 1

For this, the number of populations and the number of observations in all samples

must be known.
 Ahearn Choudhury 36

I 1
df =
N 1

31
df =
963

df =293

Figure 24. Degrees of Freedom

Figure 24 above shows a sample calculation for the degrees of freedom in an

ANOVA test.
 Ahearn Choudhury 37

Appendix D: Two-Sample t Test Sample Calculation

Figure 25. Two Sample t Test Equation

Figure 25 shows the equation used to calculate a two-sample t test. First, the

sample mean from the second population, x 2 , is subtracted from the sample mean

from the first population, x 1 . Then, the standard deviations of both samples, s 1 and

s 2 , are squared, divided by their sample sizes, n1 and n2 and added together.

The square root of that product is then taken and the result of the two sample means

being subtracted is divided by the answer from the square root.

Figure 26. Two-Sample t Test Sample Calculation

The calculation above is a sample of the equation shown in Figure 26. The

substitutions for the variables in the equation were taken from the data for control sprout

length and SiO sprout length. The t value calculated was 0.1293, which is the same t
2

value as shown in Figure 12.

The p-value, assuming the null hypothesis is true, is the probability that the test

statistic would take a value as extreme or more than that observed by chance alone. This

can be found using the t value. The tail probability, p, can be approximated by using
 Ahearn Choudhury 38

Table B, a table of t distribution critical values, and locating the t value by the given

degrees of freedom and tracing upwards.

Appendix E: Table B for determining P-Value from t-Score

Figure 27. Table B to Find P-Value

Figure 27 shows the table that is used to find the p-value from the t-value. The

first digit of the t-score is located on the leftmost side of the table, and the following

digits are located among the top.

 Ahearn Choudhury 39

Appendix F: Two-sample t Interval Sample Calculation

Figure 28. Two-sample t Interval Equation

Figure 28 is the equation that is used to find a Two-sample t test interval. First, the

sample mean from the second population, x 2 , is subtracted from the sample mean

from the first population, x 1 . Then, the t value is multiplied by the square root of the

standard deviations of both samples, s 1 and s 2 , which are squared, divided by their

sample sizes, n1 and n2 and added together. The difference between the two sample

means is then both added and subtracted to the t value product.

Figure 29. Two-sample t Interval Sample Calculation

Figure 29 above is a sample of the equation shown in Figure 26. The substitutions

for the variables in the equation were taken from the data for control sprout length and

SiO sprout length. The confidence interval calculated was between -0.7376 cm and
2

0.8359 cm, which is the same confidence interval as shown in Figure 13. The confidence

interval is used to determine where the true population mean would fall.
Ahearn Choudhury 40
 Ahearn Choudhury 41

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