Table of Contents

Introduction

Review of Literature 3
Problem Statement

Experimental Design 10
Data and Observations

14

Data Analysis and Interpretation

Conclusion

19

29

Acknowledgements

33

Appendix A: Randomization 34
Appendix B: Jar Preparation 35
Appendix C: Microscope Preparation36
Appendix D: Pesticide 1 Preparation 37
Appendix E: Pesticide 2 Preparation 38
Appendix F: Table 1 Calculations

40

Appendix G: Statistical Test Calculations

Works Cited

43

41

Introduction
Home and garden pesticides are used by 78 million US households. Jay Feldman stated in his
article titled Lawn Pesticide Facts and Figures that of 30 commonly used lawn pesticides: 16 are toxic
to birds, 24 are toxic to fish and aquatic organisms, and 11 are deadly to bees. These can cause
significant harm to humans and the environment.
Pesticides often enter the water supply through runoff, vapor drift, or other ways, and can harm
aquatic organisms just as they harm the pests they are intended to kill. Protozoa are one type of such
organism. Protozoa are an essential part of an aquatic environment because they consume bacteria,
eliminating pathogens from the water and slowing the spread of disease. When pesticides enter the
water supply, they can harm these small organisms and thus have a detrimental effect on the
environment.
In the research and writings of Louis Hom and Christine A. Bahlai and other researchers, it was
shown that it is important to analyze the use of organic pesticides through statistical analysis rather than
simply classification, because organic pesticides are often assumed safer than synthetic pesticides
because they are natural. The research detailed in this experiment expanded on their writings by
examining which home remedy pesticide, neem oil or insecticidal soap spray (also referred to as pepper
spray), would be less detrimental to the environment through the destruction of the population of
protozoa in a water sample.
Neem oil and insecticidal soap spray were selected for the experiment because they are two of
the most commonly used home remedy pesticides. Suburban lawns and gardens receive more
pesticide applications per acre than agriculture, so it is likely that significant amounts of neem oil and
insecticidal soap spray leak into the water supply. By analyzing the effect of these pesticides on the
population of protozoa, it is possible to gain insight as to how harmful these pesticides truly are to an
environment. When pesticides enter the water supply, they can affect the organisms there directly,

and/or they can affect humans directly and indirectly. If a human were to swim in water contaminated
with pesticides, it is in direct contact with their body causing the same or similar effects the pesticide
have on the pests. If the human were to consume the water or an organism living in it, then it would
also affect them similarly to how it affects the pests it is designed to harm (Belanger). With this
research, agriculturalists can determine which pesticides are more environmentally harmful and avoid
their use in their work. The reduction of the use of harmful pesticides can aid in protecting organisms
and preventing negative effects on human populations.

Review of Literature
All across the world, people from farmers to backyard gardeners make use of special chemicals
to protect their plants from harmful animals. These special chemicals are any substance or mix of
substances that protect the plants by having an effect on the life cycle of a pest (WhatWork?). The
EPA states that these substances, known as pesticides, can be synthetic, natural, or even bacterium.
Pesticides are comprised of bactericides, baits, fungicides, herbicides, insecticides, lures, rodenticides,
and repellents (WhatWork?). This research focuses on insecticides, which are designed to kill,
harm, suppress, or diminish a variety of species of insects (Insecticides) (WhatWork?).
Pesticides work by invading the nervous system and interrupting the information being sent by
neurotransmitters to the synapses, parts of the brain that control the functions of the body (Myers), in
turn causing new messages to be sent creating muscles to relax and no longer function (Gerber).
Pesticides rid plants of pests by either repelling them or killing them. Organic pesticides come
from living organisms and are often chemicals that they use to protect themselves from parasites,
predators, and pathogens (Griffin). Organic pesticides repel insects using things such as pepper, garlic,
or essential oils of herbs. Products with a citrus-fruit peel base or those that contain citrus oils, such as
Orange Guard, kill insects on contact by destroying their respiratory system (BuyersControl).
They may also cause the pest to no longer eat, suffocate them, or disturb reproduction or how their
bodies process water (Griffin). Synthetic pesticides, however, are poisonous chemicals or
combinations of chemicals that were developed from mustard gas used in World War II (Synthetic
Pesticides). They kill pests by destroying their nervous systems, causing paralysis and death
(Pyrethroids and Pyrethrins).
Pesticides can contaminate water sources through runoff, vapor drift, and other methods. They
can also contaminate the food people eat when the pesticides are applied on or near the plant that
produces the food (Jakuboski). Pesticides present many risks to both humans and the environment.

Synthetic pesticides in particular cause serious health concerns for humans. According to Organic
Valley, synthetic pesticides can cause birth defects, damage to the nervous system; disruption of
hormones and endocrine systems; respiratory disorders; skin and eye irritations; and various types of
cancers. Pesticides slowly poison the body from where they are stored in the colon. They are also
linked to cancer, Alzheimers disease, ADHD, and damage to a developing fetus (Jakuboski).
As for the environment, pesticides often kill insects that are helpful (or at least not harmful) to
crops. This, in turn, hurts the crop yield, which is the main reason farmers use pesticides: to increase
their crop yield. In an experiment performed by six researchers from the Beekeeping Research and
Information Centre in Louvain la Neuve, Belgium, and the Plant Protection and Ecotoxicology Unit at
Walloon Agricultural Research Centre in Gembloux, Belgium, the researchers found a significant
correlationbetween the presence of fungicide residues and honeybee colony disorders (SimonDelso, et al.) A similar experiment was performed by two researchers from the Faculty of Agriculture
and Environment at The University of Sydney in Eveleigh, New South Wales, Australia, and the
National Institute for Environmental Sciences in Tsukuba, Ibaraki, Japan. Their research found that
pesticides pose a possible risk for bees through both their pollen and through direct contact (SanchezBayo & Goka). Honey bees are essential to our food supply, as they pollenate a large amount of our
crops. Without them, our food supply would dwindle. Pesticides not only affect humans and bees, but
they may also harm fish and other aquatic life when they enter the water supply.
Despite the dangers related to pesticides, they are still employed often. This research will put
emphasis on the home-remedied pesticides. There are many ways to create this type of pest repellent
because of the many different types of homemade pesticides. Two organic pesticides that will be
focused on are neem and an insecticidal soap spray. According to Dr. Edward F. Group III, a design to
concoct a neem pesticide is to mix water, organic liquid soap, and neem oil. One way to create an
insecticidal soap spray is to blend garlic, chile, and water and then strain into a container. Soap is then

added to the concentration and two tablespoons are used with one liter of water to make the final spray
(Belsinger & Wilcox).
To be more specific, the first pesticide mentioned is neem, which is found in the Azadirachta
indicaa tree in India (Grisak). For hundreds of years, neem oil has been employed as a pesticide
(Feldman), but since around the 1920s, research has shown neem oil to be an effective remedy for
repelling insects (Grisak). The insects take the chemicals into their bodies and it interferes with their
hormones, causing the insects to forget to eat and in turn reduces the reproduction of the insects
(Grisak) (Feldman). However, neem oil is almost completely non-toxic to birds, mammals, bees, and
plants; but slightly toxic to fish and aquatic organisms. The neem oil does not harm bees due to the fact
that in order for the oil to repel the pests, it must consume the pesticide. Bees are known pollinators, so
they do not actually eat the plants. The active ingredient most responsible for neem oils repellent
qualities is azadirachtin. According to an article from Extoxnet, azadirachtin may cause significant
deaths in fish communities if large concentrations of this ingredient enter waterways. Luckily, it breaks
down very rapidly and has a half-life of about 48-100 hours (Feldman) (Azadirachtin).
The second pesticide included in this research is an insecticidal soap. Since the insecticidal soap
only needs to be sprayed directly on to soft-bodied pests instead of being ingested, this remedy is often
used. The fatty acids of the concoction saturate the skin of the pest and take all of the moisture from the
body (Grisak). Joyce D. Ubl adds to this by stating that the soaps appear to disrupt the cellular
membranes of the insect and may also remove some of the protective waxes that cover the pests,
causing dehydration. Amy Grisak also shares that insecticidal soap is most effective when directly
applied to pests since after the soap dries it is no longer effective at warding off and killing the insects.
More benefits to employing insecticidal soap to ward off pests is that it is a safe, effective, and has a
low toxicity. Additional advantages are that these soaps are inexpensive, leave no harsh residues, they
are virtually non-toxic to animals and birds, and most beneficial insects are not harmed (Ubl).

This experiment will analyze the effect of each pesticide on the environment through the
population change of protozoa in a water sample. Protozoa are single-celled microorganisms of the
Protista family, and are eukaryotic (contain no nucleus) and heterotrophic (consume other organisms)
(Estapa). They are often found in bodies of water near the border between soil and water, or within very
moist soil. They primarily consume bacteria, but have also been found to consume other protozoa,
soluble organic matter, and fungi (Ingham). Protozoa help the bacteria population grow (and thus
increase decomposition rates and soil aggregation) when they feed on them. They also help reduce
disease by feeding on or consuming pathogens, which cause diseases (Ingham).
Trophozoite is a general term for the active, feeding, multiplying stage of most protozoa
(Yaeger). They multiply through asexual reproduction. The first stage, G1, is where the cell grows and
develops. The DNA replication occurs next in the S stage. Protozoa then go through the G2 stage where
they prepare for division, and then the M stage where nuclear and cytoplasmic division occur (Estapa).
In Pesticides in Organic Farming, Louis Hom, a molecular and cell biology graduate at
Berkeley University, states that there has been recent research on an organic pesticide versus a
synthetic one, but he also says that it is difficult to compare the two categories of pesticides because
scientists look at them differently. The effects of organic pesticides have not been the subject of many
studies due to the assumption that natural pesticides are safe for the environment simply because they
are natural (Hom). Many people assume that organic pesticides are more environmentally friendly
and leave less of a footprint on the Earth, simply because they are called organic; however, a study by
Christine A. Bahlai and other researchers, members of the School of Environmental Sciences and
Department of Plant Agriculture at the University of Guelph in Guelph and Ridgetown, Ontario,
Canada, suggests otherwise. They conclude that they reject the organic-conventional dichotomy and
instead stress that in order to know whether a pesticide is more eco-friendly it must be evaluated in
regards to data and analysis rather than arbitrary classifications (Bahlai, et al.) The research in this

paper furthered the information in Louis Homs writings by evaluating the effects of two homemade
organic pesticides on the aquatic organisms in the water samples, used to represent a portion of the
environment. In Bahlais paper Choosing Organic Pesticides over Synthetic Pesticides May Not
Effectively Mitigate Environmental Risk in Soybeans, the researchers determined that organic
pesticides needed to be tested based on data analysis rather than common ideas. They compared the
environmental impact of synthetic and organic insecticide on soybean aphid. Overall they found that
organic pesticide proved to have a similar or even a greater detrimental impact than the synthetic
pesticide. The methods in Bahlais paper were used as a base for the experimental design in this
research. The decision the researchers in Bahlais paper formed had been used in the formulation of the
conclusion in this paper.

Problem Statement
Problem:
Which home remedy pesticide, neem oil or insecticidal soap spray, will be least detrimental to
the environment through the destruction of the population of protozoa in a water sample?

Hypothesis:
The neem oil will be more detrimental to the population of protozoa in a water sample
measuring the number of individual protists present than the insecticidal soap.

Data Measured:
The experiment compared two pesticides as the independent variables, neem pesticide and
pepper pesticide. The dependent variable was the percent change in population of protozoa, measured
by a count of organisms from five randomly chosen samples of the beakers under a microscope with
400x magnification. A two-sample t-test was used to analyze the data. The statistical analysis allowed
the researchers to determine which home remedy pesticide had a greater negative impact on the
protozoa population. Three two-sample t-tests were performed to compare the neem pesticide to the
pepper pesticide, the neem pesticide to the control, and the pepper pesticide to the control. The control
is no application to the sample. There were five samples taken from each of six beakers, which resulted
in thirty samples per treatment, in order to have an appropriate number of data points to assume a
normal distribution.

Experimental Design
Materials:
250 mL Beakers (18)
1 L Glass Jars with River Contents (3)
Pipettes (6)
Microscope
Glass Slides (6)
Glass Cover Slides (6)
Fume Hood

Masking Tape
Pen or Marker
Pesticide 1
Pesticide 2
Disposable Plastic Serving Spoons (3)
Graphing Calculator
2 mL Graduated Cylinder

Procedures:
1.

Use the pen/marker and masking tape to label the beakers 1 through 18 and randomize the
treatment using the graphing calculator (Appendix A).

2.

After jar preparation (Appendix B), use the long-handled plastic spoon to fill each beaker with
100 mL of mud and then add water until the beaker is filled to 200 mL.

4.

Use a disposable plastic spoon to gently mix each beaker, and allow them to settle.

5.

Use the pipette to collect a small sample of water from the first location shown in Figure 1
below, just above the surface of the mud in the beaker.

6.

Use the pipette to place one drop of sample water in the center of a glass slide and cover with
glass cover slide. Discard the remaining water sample from the pipette.

7.

Secure the glass slide on the stage of the microscope and focus the microscope, see Appendix C.

8.

Observe and record the initial population of protozoa in one viewable area of the sample. If
population numbers are high, divide the viewable area into eight sections of equal area and
count the population in one area, then multiply that population by eight to estimate the total
population in the viewable area of the sample.

9.

Remove the glass slide from the stage of the microscope and clean it thoroughly.

10.

Repeat steps 5-9 for each sample taken, with one sample from each of the five locations shown
in Figure 1 on page 12.

11.

Repeat steps 5-10 for each beaker to collect final population counts. And turn off microscope
when completed.

12.

Use a pipette to transfer 2 mL of the appropriate pesticide(s) to the appropriate graduated

cylinder(s).

13.

Apply the designated amount of pesticide 1 (Appendix D) and pesticide 2 (Appendix E) to the
corresponding beakers.

14.

Use a separate disposable plastic spoon to gently mix each beaker, control and pesticides, and
then discard the spoons.

15.

Allow the beakers to sit undisturbed under the fume hood for 24 hours.

16.

Discard all used samples after both the initial and final population counts have

attained.

been

Diagrams:

Sample Location

Beaker

Dirt/Grass/Water
Figure 1. Beaker Above View\
Figure 1 above shows the diagram for the locations that the researchers chose the samples from
the beaker. The red crosses mark the sample locations. Each beaker was sampled five times from the
same location. These samples were looked at under the microscope and the population counted.

Water

Dirt/Grass

Figure 2. Beaker Side View

Figure 2 above shows the side diagram for the dirt and water levels in the beakers. Each beaker
1. mL
Graduated
Cylinders
was filled with 100 mL of dirt and grass, then filled to 200
with water.
2. Disposable Spoons
3. Jar with Dirt Sample
4. Pesticides
5. Beakers
6. Plastic Spoon
7. Microscope
8. Masking Tape/Marker
1
3
9. Calculator
5
2
6
7
4
10. Pipettes
11. Glass Slides/Cover Slides

8
10
Figure 3. Materials

11

Figure 3 displays all of the items from the materials list, excluding the fume hood. The most
important materials for this experiment are the pesticides, jars with dirt samples, and the microscope.

Data and Observations

Table 1
Collected Protozoa Population and Percent Change in Population
Beaker

Beaker

Treatment

Initial
Population
5

0.00

0.00

0.00

0.00

66.67

17

19

-85.71

-80.00

-80.00

10

-75.00

0.00

-42.86

33

-33.33

-77.78

-66.67

18

-80.00

50.00

25.00

39

13

0.00

-60.00

150.00

-75.00

-90.91

-50.00

17

20

-33.33

50.00

11

-50.00

-33.33

25.00

-33.33

22

-40.00

Control

Control

Pepper

Pepper

Neem

Neem

Treatment

Percent
Change in
Population

Final
Population

Initial
Population

Final
Population

Percent
Change in
Population

Average
Percent
Change in
Population

13.33

-64.14

-60.13

33.00

-39.85

-26.33

Average
Percent
Change in
Population

10

11

12

150.00

100.00

300.00

-50.00

-33.33

19

14

200.00

200.00

100.00

300.00

100.00

33.33

15

20

-66.67

-16.67

-80.00

33.33

-33.33

50.00

16

100.00

-33.33

0.00

100.00

50.00

-60.00

23

16

0.00

-33.33

100.00

50.00

0.00

0.00

10

10

-33.33

Neem

Control

Pepper

Neem

Control

Pepper

Beaker

Treatment

13

Neem

Initial
Population

Percent
Change in
Population

Final
Population
1

-60.00

-50.00

93.33

180.00

-19.33

16.67

11.33

23.33

Average
Percent
Change in
Population
-38.67

14

15

16

17

18

Control

Pepper

Pepper

Control

Neem

-33.33

-50.00

0.00

12

10

150.00

-33.33

200.00

33.33

100.00

0.00

10

100.00

100.00

0.00

-50.00

-50.00

0.00

16

10

200.00

0.00

0.00

-50.00

100.00

-66.67

10

16

0.00

0.00

-33.33

200.00

0.00

-66.67

0.00

90.00

30.00

30.00

-3.33

20.00

Table 1 shows the data collected during trials (initial and final populations) and the values
calculated from them (percent change in population and average percent change). See Appendix F for
sample calculations.
Table 2
Trial Observations
Beaker(s)
Observation
3, 5 Too much dirt after it settled
8 Lots of dead protozoa
16, 17, 18 Water was cold from being transported

Table 2 shows the observations made during experimentation. These observations may have
affected the data or been an effect of a factor, known or unknown, possibly altering the data collected.

Figure 4. During Trials

In Figure 4, first the sample of water was taken with the pipette (top left), then the sample was
placed on the glass slide (bottom left), next the cover slide was placed on top of water (top right), and
lastly the slide was placed on the microscope and the microscope was adjusted so the sample could be
observed (bottom right). From here the samples were looked at and the data was collected.

Figure 5. Example Protozoa

When observing the sample under the microscope the researchers took count of the protozoa
population. The protozoa from this research look like the one in Figure 5 (Datko).

Data Analysis and Interpretation

To determine the how successful the hypothesis was, a statistical analysis must be completed.
The likelihood of the neem pesticide having a greater negative effect on the protozoa percent
population change than the pepper pesticide was measured. In order to test the hypothesis, pesticides
were randomly allocated to trials using the TI-nspire graphing calculator to assist in reducing bias. In
addition, to further reduce bias, the researcher counting the protozoa population was blind, meaning
that they did not know which pesticide application they were observing, and multiple samples were
taken from each beaker to ensure replication would produce similar results. The statistical analysis tests
that best fit the data were three two-sample t-tests because the data collected compares the means of
two different factors, in this case, the control and the neem pesticides, the control and the pepper
pesticides, and the neem and the pepper pesticides. The pesticides are being compared to controls as
well as each other, rather than only comparing one pesticide to the other, making it possible to
determine how detrimental pesticides are on the environment relative to no pesticide, instead of only to
determine which pesticide is better or worse. If there are lurking variables, controls provide a basis to
show that the variables may affect all treatments and may not affect the significance of the treatments.
In order to conduct the test, the statistical analysis assumptions must be met. The assumptions
for this analysis are that the samples come from independent populations, the samples have been
selected using a simple random sample, and that either the sample size is greater than or equal to thirty
or the data is known come from a normal population. Also, alpha () is equal to 0.10. First, the samples
can be assumed independent because the pesticides are two separate mixtures and do not affect each
other because they do not interact. The pesticides have been selected using a simple random sample
because they were randomly allocated to each trial. The population means and population standard
deviations are unknown as not every protozoa can be recorded. The alpha value used in this statistical
analysis was increased from the standard value of 0.05 to 0.10 to adjust for error in experimentation.

Lastly, the assumption that the sample comes from a normal distribution has been met because the
sample size is greater than or equal to thirty, therefore by the Central Limit Theorem the sampling
distribution is normal, so it is safe to assume that the results from a t-test will be reliable. Five samples
were taken in every beaker in order to replicate data collection to reduce variability. Overall, controls,
randomization and replication were used to ensure accurate data that is representative of the overall
population.
The validity of this depends on how accurately the experiment was conducted and the
experience level of the researchers. The null hypothesis for control and neem sets the first mean of the

control,

H o : c =n

, equal to the neem,

, in order to see if the percent population change is the same,

. This means that the null hypothesis states that the mean value of the control population

is equal to the mean value of the neem population. The alternative hypothesis attempts to validate if the
control has a greater percent change, so the first mean of the control is set as greater than the second

mean of the neem,

null hypothesis is

H a : c> n

H o : c = p

. For the statistical analysis between control and pepper,

, the

, which says that the control percent change will be equal to the pepper

percent change. The alternative hypothesis is

H a : c> p

. This means that the control will have a

greater percent change than the pepper. Lastly, the analysis between neem and pepper has the null

hypothesis

H o : n= p

, and the alternative hypothesis

H a : n< p

. The alternative hypothesis

tests that the neem pesticide will have less of a percent change than the pepper, or in other words, less
growth, or a more detrimental effect on the population of protozoa.

300

200
200

100

33
38

Figure 6. Box Plots With Outliers

0
Figure 6 shows the box plots for the control, neem, and pepper data. The box plot on top is the
control data, the box plot in the middle is the neem data, and the box plot on the bottom is the pepper
-33 to be right skewed. They overlap for a large majority of their data. 100% of
data. All box plots appear

the pepper data overlaps the control data, with over 75% overlapping neem. Also, almost 100% of the
neem data overlaps the control data. 25% of the control data is higher than the neem and just less than
-86
25% is higher than
the pepper. The control data ranges from about -86 to 200, the neem data ranges

from around -91 to 100, and the pepper data ranges from about -80 to 150. The median for control and
pepper are at zero percent population change. For neem, it is located at approximately -40 percent
population change. The dotted line is the mean. For control, the mean is located around 38 percent
population change, for neem is at 4 percent, and pepper is at 6 percent. Since the means and medians of
neem and pepper are so close, there is probably no significant difference between them. There are
outliers for all three groups. For control the outlier is at 300; for neem the outliers are at 150, 200, and
300; and for pepper the outlier is at 200. Outliers were left in the data for calculations, however the

Control

outliers were removed and the statistical analysis were run again to show the effect of the outliers.

Percent population change

-33
-50
-91

4 25
100

150

200

300

-50

6
150

Pepper

-80

Figure 7. Probability Graph and Two Sample t-test Calculations for Control and Neem
Figure 7 displays the probability graph for the control and neem data. The t-value was found to
be 1.3927, and the p-value was found to be 0.0846. The t-value is the number of standard deviations
above or below zero on a t-distribution where zero represents no difference. The p-value is the
percentage of the time that data this extreme will be found under the assumption that the null
hypothesis is true. Also shown are all of the values used to calculate the t-value and the p-value. See
Appendix G for sample calculations. A confidence interval is obtained from two sets of sample data (in
this case, control and neem) and tries to estimate the true population mean (in this case, the true
population mean of the difference between the control group and the neem group). It gives the level of
80% confidence that the calculated interval will contain the true population mean (in this case, the true
population mean difference between the control group and the neem group). The degrees of freedom is
one less that the smallest sample size, so for control and neem the degree of freedom is 30 minus 1,
which equals 29.
For the control and neem analysis, the null hypothesis was rejected because the p-value of
0.0846 is less than the alpha level of 0.10. There is significant difference between control and the neem.
There is only an 8.46% chance of getting a difference in the sample mean scores this extreme by
chance alone, if the null hypothesis is true. There is enough evidence to show that the control does
create a larger percent population change than neem, meaning that the control does not kill as many
protozoa as the neem pesticide. The confidence interval, or estimated value for the population mean for

the difference in percent population change between the control and neem groups, was calculated to be
between 2.3241 and 65.0213. This interval supports the rejection of the null hypothesis because it does
not include zero, meaning the treatments may be significant. See Appendix G for sample calculations.

Figure 8. Probability Graph and Two Sample t-test Calculations for Control and Pepper
Figure 8 is the probability graph for the control and pepper data. The t-value was found to be
1.4335, and the p-value was found to be 0.0789. Figure 8 also shows all of the values used to calculate
the t-value and the p-value for control and pepper. The confidence interval was obtained from control
and pepper. It tries to estimate the true population mean of the difference between the control group and
the pepper group. It gives the level of 80% confidence that the true population mean difference between
the control group and the pepper group. The degrees of freedom is one less that the smallest sample
size, so for control and pepper the degree of freedom is 30 minus 1, which equals 29.
The control and pepper analysis rejected the null hypothesis because the p-value of 0.0789 is
less than the alpha level of 0.10. There is significant difference between control and the pepper. There
is only a 7.89% chance of getting a difference in the sample mean scores this extreme by chance alone,
if the null hypothesis is true. There is enough evidence to show that the control does create a larger
percent population change than pepper, meaning that the control does not kill as many protozoa as the
pepper pesticide. The confidence interval, or estimated value for the population mean for the difference
in percent population change between the control and pepper groups, was calculated to be between

2.9972 and 60.4435. This interval supports the rejection of the null hypothesis because it does not
include zero, meaning the treatments may be significant.

Figure 9. Probability Graph and Two Sample t-test Calculations for Neem and Pepper
Figure 9 is the neem and pepper probability graph. The t-value in this analysis was found to be
-0.0957, and the p-value was found to be 0.4621. This figure also shows all of the values used to
calculate the t-value and the p-value for neem and pepper. The confidence interval was obtained from
neem and pepper. It tries to estimate the true population mean of the difference between the neem
group and the pepper group. It gives the level of 80% confidence that the true population mean
difference between the neem group and the pepper group. The degrees of freedom is one less that the
smallest sample size, so for neem and pepper the degree of freedom is 30 minus 1, which equals 29.
For the neem and pepper analysis, the null hypothesis failed to be rejected because the p-value
of 0.4621 is greater than the alpha level of 0.10. There is no significant difference between neem and
pepper. There is about a 46.21% chance of getting a difference in the sample mean values by chance
alone, if the null hypothesis is true. There is not enough evidence to show that the neem does create a
larger percent population change than pepper, meaning that the neem may not kill more protozoa than

the pepper pesticide. The confidence interval, or estimated value for the population mean for the
difference in percent population change between the neem and pepper groups, was calculated to be
between -28.4259 and 24.5212. This interval supports the conclusion of failing to reject the null
hypothesis because it does include zero, meaning the treatments may not be significant.
0
The three two-sample
t-tests above were performed with the inclusion of the outliers seen in
33
-50 -19
150

-80

Figure 6 above. To see if the outliers had an influence on the data they were removed from the
calculations and the t-tests were done again, seen below. The outliers were not inaccurate data, meaning
they were mistakes, but they were just larger than the rest of the data. After the outliers were removed,
n was less than 30, so the data was required to be normal in order for the 2-sample t-test to give reliable

Control

results.
-42

29

100
200
Percent population change

-86

-91

50

100

Pepper

Neem

0
-50
-33-0.5

Figure 10. Box Plots without Outliers

Figure 10 shows the box plots for the control, neem, and pepper. The box plot on top is the
control data, the box plot in the middle is the neem data, and the box plot on the bottom is the pepper
data. All box plots appear to be right skewed. The box plots overlap for a large majority of their data.
100% of the pepper data overlaps the control data with over 75% overlapping the neem data. Also,
almost 100% of the neem data overlaps the control data. Over 25% of the control data is higher than the
neem and less than 25% is higher than the pepper. The median for control, pepper, and neem are all at

zero percent population change. The dotted line is the mean. For the control the mean is located around
29 percent population change, for neem is at approximately -0.5, and pepper is at approximately -19.
Despite having removed the outliers from the initial data, the removal of these data points revealed a
new outlier for the neem data. This outlier was left in the data for calculations, which were run again to
show the outliers effect.

Figure 11. Probability Graph & Two Sample t-test for Neem and Pepper without Outliers
Figure 11 is the neem and pepper normal distribution graph. The t-value in this analysis was
found to be -1.3013, and the p-value was found to be 0.0994. Figure 11 also shows all of the values
used to calculate the t-value and the p-value for neem and pepper. The confidence interval was obtained
from neem and pepper. It tries to estimate the true population mean of the difference between the neem
group and the pepper group. It gives the level of confidence that the true population mean of the
difference between the neem group and the pepper group.
For the neem and pepper analysis, the null hypothesis was rejected because the p-value of
0.0994 is less than the alpha level of 0.10. There is significant difference between the neem and the
pepper. There is about a 9.94% chance of getting a difference in the test scores this extreme by chance
alone, if the null hypothesis is true. There is enough evidence to show that the pepper does create a
larger percent population change than neem, meaning that the pepper does not kill as many protozoa as
the neem pesticide. However, this value is very close to 10%, or the alpha level of 0.1, meaning this
significance may be minor. The confidence interval, or estimated value for the population mean for the

difference in percent population change between the neem and pepper groups without outliers, was
calculated to be between -37.7001 and -0.0517. This interval supports the rejection of the null
hypothesis because it does not include zero, meaning the treatments may be significant.
The outliers do not influence the control-neem or control-pepper tests; however, they do cause
the neem-pepper tests to be rejected. This means that without the outliers, the difference between the
neem and pepper is significant. Therefore, the neem may have a more detrimental effect on the
protozoa population than the pepper.

Conclusion
Overall, the hypothesis that the neem pesticide would cause the least amount of protozoa
growth was accepted. The population percent change of protozoa after the application of neem
pesticide, pepper pesticide, or no pesticide to determine which of the three treatments would be most
detrimental to the protozoa population was tested. This determination was made using the collected
values and the results of the statistical analysis, comprised of a series of two sample t-tests, of the data.
The data analysis of the values showed that the null hypothesis of killing the same amount of protozoa
was rejected, because there were percentages below 10% chance that the computed values could be as
extreme as or more extreme than were calculated by chance alone, for control and neem, and control
and pepper. However, the neem and pepper analysis failed to reject the null hypothesis with a 46.21%
chance of getting a difference in test scores this extreme by chance alone, if there was no difference in
protozoa killed. With the removal of the outliers, the control and neem, and the control and pepper
analyses continued to be rejected. As for the neem and pepper, the analysis rejected the null hypothesis
with a 9.94% chance of getting results as extreme as this by chance alone if the null were true. This
close proximity to alpha may be due to error in the experiment preventing the data from having a more
significant difference.
These conclusions support the hypothesis because the neem pesticide yielded the smallest
population percent change than either the pepper pesticide or the control treatment. The control and
pepper data indicated that there was significant evidence that the control did produce a larger
population percent change, or killing fewer protozoa, than the pepper pesticide. There is not enough
evidence to show that the neem creates a larger percent population change than pepper before the
removal of the outliers, meaning that the neem may not kill more protozoa than the pepper pesticide.
However, without outliers, the neem pesticide did create a smaller population percent change than the
pepper. This outcome coheres with the scientific explanation of the treatments. The control would

allow for no hindrance in the population growth, leading to the expectation that the population would
be the largest of the three. The pepper pesticide works by dehydrating the pest and killing it (Ubl).
When the pepper pesticide was added to the water sample, it was anticipated that the pesticide would
become less effective since it would be diluted in water and unable to easily dehydrate the protozoa.
Neem, however, begins to kill the pest once it is consumed by interfering with its hormones and
causing the pest to forget to eat and in turn reducing its reproduction (Grisak) (Myers). While in the
water, the protozoa would consume the neem and the same effects would be applied to the protozoa as
the pest.
The results of this research agree with and build upon the information currently published in the
scientific community. In Pesticides in Organic Farming by Louis Hom, a molecular and cell biology
graduate at Berkeley University, Hom encourages the research of organic pesticides and their effects on
the environment by stating how little research has actually been conducted on them because they are
often assumed safer because they are natural (Hom). Additionally, research conducted by Christine
A. Bahlai and other researchers, members of the School of Environmental Sciences and Department of
Plant Agriculture at the University of Guelph in Guelph and Ridgetown, Ontario, Canada, suggests that
how eco-friendly a pesticide is should be determined by data analysis rather than arbitrary
classifications (Bahlai, et al.) The research detailed in the experiment at hand expanded on the research
and writings of Hom, Bahlai and others to support through careful data analysis that organic pesticides
used for agricultural or domestic purposes are, in fact, harmful to the environment.
If the experiment were repeated, flaws could be reduced. First, the amount of pesticide applied
should be manipulated to a value closer to that applied in farming environments to provide for a more
realistic interpretation. The beakers to which the pesticides were applied should have been larger in size
to achieve population values closer to the mean. This would be achieved by combining areas of high
and low population into one larger population where they may even out. When preparing such beakers,

the amounts of dirt, water, and vegetation should be closely monitored to ensure the beaker contents do
not vary. These amounts varied slightly in the experiment, which could have caused a change in the
total population a beaker could support, modifying the resulting data. More samples should be taken
from the field, as well as each beaker, to ensure data closer to the population mean, as it was observed
that populations in samples seemed somewhat dependent on the depth of the sample relative to the top
of the dirt. In addition, the experiment could be conducted over a larger span of time to allow the
population to grow to a larger initial value and to allow the pesticides a greater opportunity to affect the
population.
Further research should be conducted in order to gain the appropriate knowledge necessary to
truly judge how environmentally damaging a pesticide is. Future research could examine the effects of
other organic pesticides, possibly those that are less common home remedies, commercially available,
or used in commercial farms. It could also examine their effects on organisms other than protozoa, such
as those on aquatic plants, native species, land or water fauna, or even humans. Combined with the
elimination or decreasing of design flaws as stated above, this future research could provide necessary
insight into the real effects of organic pesticides. Some pesticides have been found to have a severe
negative impact on wildlife, in addition to contaminating humans food sources and causing health
problems. Similar research could expose these threats before it becomes too late to save populations of
flora and fauna.

Acknowledgements
The researchers wish to acknowledge several people who have aided them in carrying out their
research. Mr. Mark Estapa has helped the researchers by editing and proofreading sections of this paper
and supplying scientific knowledge relative to the research, laboratory space, and necessary materials.
Mrs. Rose Cybulski has assisted the researchers by educating them about the statistical test used to
analyze the data and editing this paper. The researchers also wish to thank Mrs. Christine Tallman for
aiding in the data analysis and editing and proofreading the section and Mr. Scot Acre for assisting with
any additional mathematical questions. Lastly, they wish to thank Mr. Rob Griffin for providing
materials and answering questions relating to pesticides, as well as Mr. David Szlek for providing
information as to the amounts of pesticides to apply.

Appendix B: Jar Preparation

Materials:
1 L Glass Jars with Lids (3)
Latex Gloves (1 pair)
Masking Tape

Pen or Marker
Small, Clean Garden Shovel
Rag or Paper Towel

Procedure:
1.

While near a shallow natural water source, put on the latex gloves.

2.

Open the glass jars and set the lids aside.

3.

Use the garden shovel to scoop dirt/mud from the bottom of the water source, filling each jar
1/3 of the way.

4.

One at a time, dip the glass jars into the water, filling the remaining 2/3 of each jar with pond
water. If possible, take the water from near the bottom of the body of water.

5.

Replace and secure the lids on the jars.

6.

Use the rag or paper towel to dry the outside of each jar.

7.

Remove and discard the latex gloves

8.

Use the pen or marker with the masking tape to label the jars Jar 1, Jar 2, and Jar 3.

Appendix C: Microscope Preparation

Materials:
Microscope
Depression Slides (6)

Microscope Lens (40x magnification)

Microscope Lens (100x magnification)

Procedure:
1.

Place the microscope on a clean, flat surface near an electrical outlet.

2.

Use the coarse adjustment wheel to lower the stage of the microscope.

3.

Secure both the 40x and 100x magnification lenses to the compound objective wheel. Then plug
in the microscope.

4.

When viewing a sample, first adjust the amount of light to what seems most visually
appropriate for that microscope and sample. Then use the coarse adjustment wheel to bring it
into focus, and use the fine adjustment wheel to refine the image detail.

Appendix D: Pesticide 1 Preparation

Materials:
Organic Neem Oil (1/2 oz, or 1 T)
Mild Organic Liquid Castile Soap (1/2 tsp)
Warm Water (8 c)
Measuring Cup
tsp Measuring Spoon

1 T Measuring Spoon
Medium Mixing Bowl
Plastic Stirring Spoon
1 L Spray Bottle (2)
Pen or Marker
Masking Tape

Safety Precautions:
Neem oil produces a strong unpleasant odor, so perform procedure in a well-ventilated area under a
fume hood.

Procedure:
1

Label spray bottles by writing on the masking tape, Pesticide 1.

Use 1 T measuring spoon to add the organic neem oil to medium mixing bowl.

Use the tsp measuring spoon to add the mild organic liquid castile soap and use the
measuring cup to add 8 cups of warm water in the mixing bowl.

Slowly stir contents with plastic stirring spoon.

Remove the top of a spray bottle and hold the funnel at the top of the bottle.

Pour the liquid through the funnel and in to spray bottles and use as noted in the experimental
design.

Appendix E: Pesticide 2 Preparation

Materials:
Large Garlic Cloves (10)
Hot Chile Peppers (4)
Water (6.5 c)
Liquid Castile Soap (1 tbs.)
Blender
Strainer
Coffee Filter
Liquid Measuring Cup
Jar with Plastic Lid (3 c)

Plastic Spoon
Spray Bottle (1L)
Masking Tape
Pen or Marker
Mixing Bowl (7 c)
Funnel
Paring Knife
Cutting Board

Safety precautions:
When working with chili peppers, take all precautions necessary to avoid contact with eyes. Wash
hands thoroughly after handling.

Procedure:
1.

Use the masking tape and pen or marker to label the 3c jar and the 1 L spray bottle Pesticide
2.

2.

Place the cutting board on a flat surface and use the paring knife to crush the garlic and cut off
the stems of the chile peppers.

3.

Put garlic, chiles and water into a blender and puree contents until foamy

4.

Let mixture stand for 24 hours. When mixture settles, it will be coral-colored with sediment at
the bottom.

5.

Place a coffee filter inside the strainer, and pour the mixture through it and into the mixing
bowl.

6.

Remove the lid from the jar with the plastic lid and hold the funnel at the opening of the jar.

7.

Pour the concentrate through the funnel and into the jar, add liquid castile soap, and stir with the
plastic spoon.

8.

Secure the plastic lid on the jar.

9.

Store in a cool, dark place until needed, up to a few months.

10.

When ready to use, place 2 tablespoons of the concentrate in the 1 L spray bottle and fill the rest
with water.

Appendix F: Table 1 Calculations

To calculate the percent change in population given a final and initial population value, use the
following formula.
finalintial
100
initial

53
2
100 100 66.67
3
3

Figure 12. Percent Change in Population

Figure 12 shows the formula used to calculate the percent change in population. Subtract the
initial population value from the final population value, then divide that by the initial value and
multiply it by 100. Then the values from sample five are substituted into the equation and the
percentage is found.
To calculate the average percent change in population given five data points from the same
beaker for the percent change in population , use the following formula.
n1+ n2 +n3 +n 4 +n5 (85.71 ) + (80.00 ) + (80.00 ) + (75.00 )+ 0.00

5
5
64.14

Figure 13. Formula for Average Percent Change in Population

Figure 13 shows the formula used to calculate the average percent change in population. Add
the five previously calculated values for percent change and divide that sum by 5. The result is the
average percent change in population. Sample calculation, given percent change values of -85.71,
-80.00, -80.00, -75.00, and 0.00.

Appendix G: Statistical Test Calculations

t=

t=

x 1 x 2

s 1 2 s 12
+
n2 n2

37.86534.1927
9864.8399 7673.0767
+
30
30

t=1.3927

Figure 14. Sample Calculation for Two-Sample t Test

Figure 14 shows the sample calculation for the two-sample t test. The values used were from
the control-neem test with outliers. The formula used has the first mean of control, u1, minus the second
mean of the neem, u2, all divided by the square root of the first standard deviation of control squared,
s1

, over the number of control trials,

the neem squared,

s2

n1

, plus the square root of the second standard deviation of

, divided by the number of neem trials,

n2

.The same process would be

completed for the trials involving other data. The result of this calculation, the t-value, is equal to
1.3927.
x
( 1x 2 )t

s 12 s 22
+
n1 n 2

( 37.86534.1927 ) 1.311

9864.8399 7673.0767
+
30
30

33.6726 31.6979
Figure 15. Sample Calculation for Confidence Interval

Figure 15 shows the sample calculation for the confidence interval. The values used were from
the control-neem test with outliers. The same process would be completed for the trials involving other
data. The result of this calculation, the estimated population mean, is between 1.9746 and 65.3705. This
example has been calculated by hand, estimating the degrees of freedom. However, when using the Tinspire software, the degrees of freedom is calculated differently. The Ti-nspire value for the confidence
interval results in a range between 2.3241 and 65.0213.

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