Table of Contents
Introduction ..........................................................................................................................1
Review of Literature ............................................................................................................3
Problem Statement ...............................................................................................................9
Experimental Design ..........................................................................................................10
Data and Observations .......................................................................................................14
Data Analysis and Interpretation .......................................................................................19
Conclusion .........................................................................................................................29
Acknowledgements ............................................................................................................33
Appendix A: Randomization .............................................................................................34
Appendix B: Jar Preparation ..............................................................................................35
Appendix C: Microscope Preparation ................................................................................36
Appendix D: Pesticide 1 Preparation .................................................................................37
Appendix E: Pesticide 2 Preparation .................................................................................38
Appendix F: Table 1 Calculations .....................................................................................40
Appendix G: Statistical Test Calculations .........................................................................41
Works Cited .......................................................................................................................43
Kirby-Koury 1
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
Kirby-Koury 2
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.
Kirby-Koury 3
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 (“What…Work?”). 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
(“What…Work?”). This research focuses on insecticides, which are designed to kill,
harm, suppress, or diminish a variety of species of insects (“Insecticides”)
(“What…Work?”). 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 (“Buyer’s…Control”). 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
Kirby-Koury 4
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, Alzheimer’s 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 correlation…between
the presence of fungicide residues and honeybee colony disorders” (Simon-Delso, 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 (Sanchez-Bayo & Goka). Honey bees are essential to our food
Kirby-Koury 5
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 indica—a 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 oil’s repellent qualities is azadirachtin. According to an article from Extoxnet,
Kirby-Koury 6
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
Kirby-Koury 7
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 Hom’s writings by evaluating the effects of two homemade organic
pesticides on the aquatic organisms in the water samples, used to represent a portion of
Kirby-Koury 8
the environment. In Bahlai’s 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 Bahlai’s paper were used as a base for the experimental design in this
research. The decision the researchers in Bahlai’s paper formed had been used in the
formulation of the conclusion in this paper.
Kirby-Koury 9
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.
Kirby-Koury 10
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
Kirby-Koury 11
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
been attained.
Kirby-Koury 12
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 was filled with 100 mL of dirt and grass, then filled to 200 mL with water.
Kirby-Koury 13
1
2
3
4
5
6
7
8
9
10
1. Graduated Cylinders
2. Disposable Spoons
3. Jar with Dirt Sample
4. Pesticides
5. Beakers
6. Plastic Spoon
7. Microscope
8. Masking Tape/Marker
9. Calculator
10. Pipettes
11. Glass Slides/Cover Slides
11
Figure 3. Materials
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.
Kirby-Koury 14
Data and Observations
Table 1
Collected Protozoa Population and Percent Change in Population
Beaker
1
2
3
4
5
6
Treatment
Control
Control
Pepper
Pepper
Neem
Neem
Initial
Population
Percent
Change in
Population
Final
Population
5
5
0.00
3
3
0.00
4
4
0.00
2
2
0.00
3
5
66.67
17
19
-85.71
7
1
-80.00
5
1
-80.00
10
2
-75.00
8
2
0.00
3
3
-42.86
33
9
-33.33
7
4
-77.78
3
2
-66.67
18
4
-80.00
6
2
50.00
5
1
25.00
39
13
0.00
4
6
-60.00
4
5
150.00
2
2
-75.00
5
2
-90.91
2
5
-50.00
17
20
-33.33
4
1
50.00
11
1
-50.00
2
1
-33.33
3
2
25.00
2
3
-33.33
22
8
-40.00
Average
Percent
Change in
Population
13.33
-64.14
-60.13
33.00
-39.85
-26.33
Kirby-Koury 15
Beaker
7
8
9
10
11
12
Treatment
Neem
Control
Pepper
Neem
Control
Pepper
Initial
Population
Percent
Change in
Population
Final
Population
4
2
150.00
3
2
100.00
4
5
300.00
3
2
-50.00
5
3
-33.33
19
14
200.00
2
5
200.00
3
6
100.00
1
4
300.00
6
3
100.00
3
2
33.33
15
20
-66.67
1
3
-16.67
1
3
-80.00
2
4
33.33
1
4
-33.33
1
2
50.00
6
16
100.00
3
4
-33.33
6
2
0.00
6
5
100.00
5
1
50.00
3
4
-60.00
23
16
0.00
3
2
-33.33
2
3
100.00
1
2
50.00
3
2
0.00
1
1
0.00
10
10
-33.33
Average
Percent
Change in
Population
93.33
180.00
-19.33
16.67
11.33
23.33
Kirby-Koury 16
Beaker
13
14
15
16
17
18
Treatment
Neem
Control
Pepper
Pepper
Control
Neem
Initial
Population
Percent
Change in
Population
Final
Population
1
2
-60.00
2
3
-50.00
5
2
-33.33
1
1
-50.00
3
2
0.00
12
10
150.00
1
2
-33.33
2
3
200.00
1
1
33.33
2
2
100.00
3
2
0.00
9
10
100.00
5
2
100.00
2
1
0.00
3
2
-50.00
2
1
-50.00
4
4
0.00
16
10
200.00
2
5
0.00
3
2
0.00
1
3
-50.00
3
4
100.00
1
2
-66.67
10
16
0.00
1
1
0.00
1
2
-33.33
1
2
200.00
1
1
0.00
2
1
-66.67
6
7
0.00
Average
Percent
Change in
Population
-38.67
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.
Kirby-Koury 17
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.
Kirby-Koury 18
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).
Kirby-Koury 19
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 (α) is equal to 0.10. First, the samples can be assumed independent because
Kirby-Koury 20
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, 𝜇𝑐 , equal to the neem, 𝜇𝑛 , in order to see if the percent
population change is the same, 𝐻𝑜 : 𝜇𝑐 = 𝜇𝑛 . 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, 𝐻𝑎 : 𝜇𝑐 > 𝜇𝑛 . For the statistical analysis between control and pepper, 𝜇𝑝 , the
null hypothesis is 𝐻𝑜 : 𝜇𝑐 = 𝜇𝑝 , which says that the control percent change will be equal
to the pepper percent change. The alternative hypothesis is 𝐻𝑎 : 𝜇𝑐 > 𝜇𝑝 . This means that
the control will have a greater percent change than the pepper. Lastly, the analysis
Kirby-Koury 21
between neem and pepper has the null hypothesis 𝐻𝑜 : 𝜇𝑛 = 𝜇𝑝 , and the alternative
hypothesis 𝐻𝑎 : 𝜇𝑛 < 𝜇𝑝 . 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.
Control
-33
-33
Neem
100
-86
100
-91
-50
-80
200
300
150
200
300
150
200
4 25
-50
Pepper
38
0
0
6
33
Percent population change
Figure 6. Box Plots With Outliers
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 data. All box plots appear to be right skewed. They overlap for a
large majority of their data. 100% of 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 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.
Kirby-Koury 22
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 outliers were
removed and the statistical analysis were run again to show the effect of the outliers.
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
Kirby-Koury 23
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 pvalue 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
Kirby-Koury 24
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.
Kirby-Koury 25
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
Kirby-Koury 26
failing to reject the null hypothesis because it does include zero, meaning the treatments
may not be significant.
The three two-sample t-tests above were performed with the inclusion of the
outliers seen in 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 results.
0
Control
-42
Neem
100
200
-50
0
-33 -0.5
-91
50
0
-50
Pepper
29
-86
-80
-19
100
33
150
Percent population change
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
Kirby-Koury 27
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 pvalue 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.
Kirby-Koury 28
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.
Kirby-Koury 29
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
Kirby-Koury 30
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
Kirby-Koury 31
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,
Kirby-Koury 32
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.
Kirby-Koury 33
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.
Kirby-Koury 34
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”.
Kirby-Koury 35
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.
Kirby-Koury 36
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
½ 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”.
2.
Use 1 T measuring spoon to add the organic neem oil to medium mixing bowl.
3.
Use the ½ 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.
4.
Slowly stir contents with plastic stirring spoon.
5.
Remove the top of a spray bottle and hold the funnel at the top of the bottle.
6.
Pour the liquid through the funnel and in to spray bottles and use as noted in the
experimental design.
Kirby-Koury 37
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.
Kirby-Koury 38
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.
Kirby-Koury 39
Appendix F: Table 1 Calculations
To calculate the percent change in population given a final and initial population
value, use the following formula.
𝑓𝑖𝑛𝑎𝑙−𝑖𝑛𝑡𝑖𝑎𝑙
𝑖𝑛𝑖𝑡𝑖𝑎𝑙
∙ 100 →
5−3
3
∙ 100 →
2
3
∙ 100 → 66.67%
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.
(−85.71) + (−80.00) + (−80.00) + (−75.00) + 0.00
𝑛1 + 𝑛2 + 𝑛3 + 𝑛4 + 𝑛5
→
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.
Kirby-Koury 40
Appendix G: Statistical Test Calculations
t=
x̅1 − x̅ 2
√
t=
s1 2 s1 2
+
n2
n2
37.8653 − 4.1927
√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, n1 , plus
the square root of the second standard deviation of the neem squared, s2 , 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.
(𝑥̅1 − 𝑥̅2 ) ∓ 𝑡 ∗ ∙ √
𝑠1 2 𝑠2 2
+
𝑛1
𝑛2
(37.8653 − 4.1927) ∓ 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 Ti-nspire software,
Kirby-Koury 41
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.
Kirby-Koury 42
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