(1) PROJECT PROFILE

Project Title: LARVICIDAL EFFICACY OF CHICOS (Manilkara zapota) PEEL

AND SEEDS ETHANOLIC CRUDE EXTRACTS AGAINST
DENGUE VECTOR MOSQUITO (Aedes aegypti) LARVAE

Name of Project Proponent: Cordova, James Paul O.

Region: VII – Central Visayas Division: Cebu Province
School: Bantayan Science High School Grade Level: 12
Project Duration: Two (2) months
Email: cordovajamespaul.o@gmail.com Contact number: +63 949 168 0528

(2) CATEGORY OF RESEARCH (4) THEME

Physical Science ____ Food Safety
✓ Life Science ____ Water Conservation
Robotics and Intelligent Machines ____ Renewable Energy
Mathematics and Computational ____ Cyber Security
Sciences ____ Traffic/Road Congestion
(3) Health
✓ Individual ____ Disaster Mitigation
____ Team ✓ Agriculture and
Environment
____ Others (please specify)

(5) EXECUTIVE SUMMARY

The research project is aimed to assess the larvicidal efficacy of the ethanolic crude
extracts of the seeds and peel of the chicos fruit (Manilkara zapota) against dengue vector
mosquito (Aedes aegypti) larvae. The materials will be tested as potential larvicide candidates
using a larvicidal bioassay experiment based on the guidelines set by the World Health
Organization. An experimental research design will be used in the study with two experimental
treatments consisting of the chicos seeds and peel ethanolic crude extracts and two control
treatments, a negative control treatment with 1% ethanol and a positive control treatment with
Abate 1SG commercial larvicide. The study will use an ethanolic extraction methodology based
on a study by Alibo et al. (2021). Moreover, experimental treatments will be tested for bioactivity
in different concentrations determined by a preliminary test. A probit model will be used to model
the concentration-mortality relationship in the study that will be justified with the chi-squared
goodness of fit test. Standard toxicology values such as the LC50 and LC90 values of the materials
will be calculated using probit analysis. The significance of the results will then be measured using
the LC50 Ratio Test as recommended by Wheeler et al. (2006).
(6) INTRODUCTION

(6.1) RATIONALE
Mosquitoes are notorious for being one of the deadliest animals on the planet. Over
one million people die from mosquito-borne diseases every year. Aedes aegypti is the
primary vector that transmits dengue and other diseases such as yellow fever and malaria.
Vector control is one of the few effective ways of controlling the transmission of dengue.
However, the overuse of artificial insecticides has developed resistance among
mosquitoes, decreasing their effectiveness. These substances also tend to be toxic to the
environment, which raises concerns for the health of non-target populations including
humans. Alternative methods include the use of plant-based larvicides, which aim to
reduce the transmission of mosquito-borne diseases by targeting the young stages of
mosquitoes, the larval and pupal stages, resulting in a lower adult population (Choi et al.
2019).
Manilkara zapota, known commonly as sapodilla and locally as chicos, is a long-
lived evergreen tree cultivated in the Philippines for its sweet egg-shaped fruit. The chicos
fruit has a brown, rough skin and large black seeds. These parts are considered byproducts
and are thrown away or composted after consumption (Lim 2013).
Previous studies have reported the presence of phytochemicals on several parts of
the chicos tree. Phytochemicals are essential compounds synthesized by plants to fend
against threats in their environments such as pests, microorganisms, and herbivores.
Preliminary phytochemical screening on studies by several researchers found that there
are significant amounts of phytochemicals found in the parts of the chicos fruit (Pravin
and Shashikant 2019; Shanmugapriya et al. 2011; Gomathy et al. 2013; Rakholiya et al.
2014). With all these considered, this study will be aimed to assess the larvicidal efficacy
of the ethanolic crude extracts of the chicos peel and seeds to fill gaps in literature and
explore the potential of these materials as a potential alternative to artificial larvicides.

(6.2) THEORETICAL FRAMEWORK

According to the book by Frank and Ottoboni (1991), entitled The Dose Makes the
Poison: A Plain-Language Guide to Toxicology, the toxicity of a substance is dependent
on many factors. However, the dose-time relationship to toxicity is the most relevant. The
prevailing theory in toxicology explains toxicity with the amount of the substance that is
involved (dose) and the period of exposure (time). Both of these factors have a direct
relationship with toxicity and thus mortality; that is, the increase of one of the factors will
cause an increase in the toxicity and therefore an increase in the mortality.
Contrary to what is expected, the dose-mortality relationship is not a simple linear
model. However, as consistent with many toxicology studies, the relationship between
dose and mortality can be transformed and modeled using a sigmoid model, a monotonic
S-shaped curve called the dose-response curve (Robertson et al. 2017). This makes sense
because below a certain threshold, a substance will not be potent enough to kill an
organism. Moreover, as the dose increases, it is observed that the change in mortality rate
will decrease until above a certain threshold where it will be potent enough to kill all of
the organisms in a population. The dose is synonymous with the concentration in solutions,
which is the factor that the current research will be studying. From these theories in the
literature, the use of the probit analysis procedure is justified for the current study as
consistent with other toxicology studies. Furthermore, it is also stated that analyzing
toxicities between larvicide candidates involves comparing LC50 and other toxicological
dose descriptors. In general, the lower the LC50 and LC90 values, the higher the toxicity
and larvicidal efficacy of a larvicide candidate. The researcher used the theories to
establish the appropriate data analysis and interpretation models that will be used in the
study.
The presence of phytochemicals in plants give them the ability to fend off foreign
intruders. Larvicidal efficacy is attributed to the presence of phytochemicals in several
studies (Jawale 2014; Alibo et al. 2021) According to Sanjeev and Sushil (2022), the
biological effects of various plant larvicides vary with the species of plant, the plant part
extracted, and the solvent used in extraction, end even the geographical source of the plant
material (Sanjeev and Sushil 2022; Sukumar et al. 1991). According to Siskos et al. (2007),
the maturity of fruit samples will also cause variability in response among experimental
units in a bioassay. It was determined that extracts of ripe fruit had a higher toxicity
compared to extracts of unripe fruit. This became the basis for the treatments used in the
study where the researcher will solely test the difference in larvicidal efficacy between
two different parts of the chicos fruit—the chicos seeds and the chicos peel. Other
mentioned sources of variability in response are controlled.
According to Robertson et al. (2017), on the book entitled Bioassays with
Arthropods, there are several factors that influence population response to toxic
substances. A source of variation in response in bioassays is the use of different species of
insects. Larvicides will have varying toxicities on different insects (Sukumar et al. 1991).
Another source of variation in response to larvicides is the body weight and the
developmental stage of the larvae. In general, larvae in younger developmental stages with
smaller body weights are more vulnerable to larvicides when compared to older
developmental stages (Robertson et al. 1981; Biddinger, Hull, and Rajotte. 1998).
Moreover, the temperature, relative humidity, and the presence of light in a larvicidal
bioassay have been determined as situational variables that affect response among
experimental units and cause a significant difference on LC50 and LC90 values. However,
the direction and magnitude of the effects of these variables on toxicity varies between
chemicals (Reichenbach and Collins 1984; Sukumar et al. 1991).
 Figure 1: Conceptual Framework

(6.3) OBJECTIVES
The main goal of this study will be to determine the larvicidal efficacy of the
ethanolic crude extracts of the peel and seeds of the chicos fruit (Manilkara zapota) on the
larvae of Aedes aegypti mosquitoes. Specifically, it will seek answers to the following
questions:
1. What is the percent mortality on Aedes aegypti larvae after 48 hours of
exposure to various concentrations of the ethanolic crude extracts of the chico
peel and chico seeds?
2. How well does the probit model fit the concentration-mortality relationship
among the treatments derived from the larvicidal bioassay?
3. What is the lowest concentration of chico peel and chico seed crude extracts
that would yield the mortality rates in the larval populations equal to:
3.1. Fifty percent (LC50)?
3.2. Ninety percent (LC90)?
4. Is there a significant difference between the larvicidal efficacy of the chico
peel and chico seed crude extracts, and the commercial larvicide (positive
control) in terms of:
4.1. LC50 values?
4.2. LC90 values?
(6.4) SCOPE AND DELIMITATIONS
The objective of the study will be to determine the larvicidal efficacy of the
ethanolic crude extracts of the Manilkara zapota peel and seed against Aedes aegypti
larvae. Specifically, the study will determine the mortality rates from each of treatments
with varying concentrations to calculate the LC50 and LC90 values for each of the
samples. The study will look at the effect the various treatments in the study will have on
the mortality of the target organisms and how changes in concentration would modify
these relationships. The study will utilize a four-group experimental design that uses the
larvicidal bioassay method as recommended by the World Health Organization (2005).
Moreover, statistical methods such as the chi-square goodness of fit test, probit analysis
and the LC50 ratio test will be used by the researcher to analyze the data.
The specific solvent that will be used for extraction is ethanol, yielding the
ethanolic crude extracts from the samples. For the larvicidal bioassay, thirty Aedes aegypti
larvae of random developmental stages will be used in the study as experimental units for
each treatment. Although unconventional, the researcher is limited to the availability of
larvae and the difficulties in identifying the specific developmental stages because of the
lack of specialized equipment for identification and temporal problems with logistics. This
is justified as the study will replicate real life compositions of larvae populations where
larvae of homogenous characteristics are rarely observed. The bias that will result from
this variation is minimized using randomization in the experimental design. Moreover, the
researcher will not conduct qualitative or quantitative phytochemical screenings for the
larvicide candidates due to financial and instrumental limitations. The researcher will also
not look into the toxicity of these samples to non-target organisms.

(7) REVIEW OF LITERATURE

The researcher will conduct a study on the larvicidal efficacy of the ethanolic crude
extracts from Manilkara zapota seeds and peel against Aedes aegypti larvae. The
following sections will summarize the relevant literature that will establish the
significance of conducting research in this field, what has been done in the field and the
gap in the literature that it can potentially fill, and the methodology that various authors
have devised and how they compare to each other. However, it should be noted that there
is a scarcity of literature and studies surrounding the Manilkara zapota fruit and anything
related to its larvicidal efficacy. Therefore, this study will be conducted primarily to test
for potential natural larvicide candidates and fill the gap in literature.
1. Plant Insecticides and Phytochemicals
The use of larvicides has been explored in mosquito population control in past
decades because of its effectiveness. This is attributed to the fact that larvae are less mobile
than mosquito adults and they eliminate disease vectors before they can transmit disease
(Sanjeev and Sushil 2022). The overuse of insecticides in the past few years has been
controversial due to its effects on non-target organisms and the development of resistance
among mosquito populations. Insecticide resistance is caused by evolutionary pressure on
mosquitoes where mosquitoes with alleles for insecticide resistance will be represented
more in the next generation after the application of a larvicide that will wipe out most of
the mosquito population, causing an accidental artificial selection of insecticide resistant
mosquitoes (Liu 2015).
The high biodegradability exhibited by plant larvicides makes them better and eco-
friendly replacements for synthetic larvicides. According to Shalaan et al. (2005), the field
of phytochemical-based larvicides is still in its early stages and there are a lot of studies
that are needed to be done to discover new promising candidates. Moreover, it was also
concluded that the potential of biological larvicides as alternatives to synthetic larvicides
should not be disregarded.
Phytochemicals are compounds synthesized by plants to overcome temporary or
continuous external threats such as pests, fungi, and pathogens. In small doses, these
phytochemicals are not toxic enough to affect larger organisms such as mammals.
(Molyneux et al. 2007). Moreover, in specific doses, these phytochemicals can be used to
kill insects. Specifically, as relevant to this study, there has been several studies that
investigated the various negative effects phytochemicals can have on mosquito larvae.
According to Alibo et al. (2021), different phytochemicals have a range of biological
effects on mosquito larvae. The study discussed that saponins can reduce the action of
mosquito larvae’s digestive enzymes and target its cuticle membrane. Furthermore, they
also discussed that flavonoids affect the neurons and respiratory systems of mosquito
larvae. Tannins are also found to have negative effects on the digestive system of mosquito
larvae. Previous research has found that the amount of saponins and tannins in plant
extracts is responsible for the plant extract’s larvicidal effectiveness. Moreover, it has also
been found that total phenolic content and larvicidal efficacy are correlated linearly (El-
Hela et al. 2013).
2. Larvicidal Bioassays
According to Sanjeev and Sushil (2022), the best way to determine the larvicidal
efficacy of phytochemical-based larvicides is through the measurement of the Lethal
Concentration 50 or LC50. It is defined to be the concentration of larvicide in a solution
that will kill 50% of the target population. Similarly, other values such as the LC90 and
LC99 are calculated. These are defined to be the concentrations of larvicide in a solution
that will kill 90% and 99% of the target population respectively. This method of measuring
larvicidal efficacy is consistent with the fact that the dose-mortality curves are usually S-
shaped. To compare several larvicide candidates to each other, the LC50 values of the
samples are compared in magnitude. The lower the LC50 value, the more effective a
larvicide candidate is in becoming a larvicide.
There is one predominant method used in testing the larvicidal efficacy of plant
extracts—the larvicidal bioassay. Most studies base their methodology on the guidelines
published by the World Health Organization (2005) called Guidelines for Laboratory and
Field Testing of Mosquito Larvicides. The guidelines are used by the researcher of the
current study with modifications that consider the limitations of the study.
3. Characteristics of Manilkara zapota
3.1. Phytochemical Content of Manilkara zapota
There have been several studies that measured the phytochemical content of the
various parts of the Manilkara zapota tree as preliminary screening for toxicology and
pharmacological studies using qualitative and quantitative methods. Preliminary
phytochemical screening on studies by several researchers found that there are significant
amounts of phytochemicals found in the parts of the chicos tree (Islam et. al. 2013; Pingale
and Dash 2015; Ganguly and Rahman 2015). According to Pravin and Shashikant (2019),
the fruits of the chicos tree are a good source of phenolic compounds and antioxidants in
general. The qualitative phytochemical screenings done by Shanmugapriya et al. (2011)
showed that various fractions of the Manilkara zapota seed contain traces of alkaloids,
flavonoids, saponins, tannins, and phenols. Notably, the ethanolic extracts of the
Manilkara zapota seed showed the most amounts of phytochemicals compared to other
fractions most notably the aqueous fraction. As for the Manilkara zapota peel, the
phytochemical screenings done by various researchers have revealed the presence of
alkaloids, flavonoids, saponins, tannins, and other phytochemicals (Gomathy et al. 2013;
Rakholiya et al. 2014). There has been evidence that seeds contain more phytochemicals
than peels (Bhandary et al. 2012). Consistent with this observation is the analysis done by
Tamsir et al. (2020), which found that chicos seeds contain more phenols and tannins than
chicos peels.
3.2. Toxicology Studies on Manilkara zapota
There are currently two electronically available studies on the larvicidal efficacy
of the Manilkara zapota peel and seeds. The first one is a study by Trisnawati and Azizah
(2019) titled “Comparison of Larvicide Effectiveness of Peel and Pulp Manilkara zapota
Fruit on Mortality of Aedes aegypti Mosquito”. However, there are noticeable gaps in the
study. First, the study only used ten (10) Aedes aegypti larvae per replicate, which is lower
than the recommended value by the World Health Organization (2005). Second, the study
did not calculate the LC50 values of the plant larvicides, which is essential for comparison
and replicability. Lastly, the researchers did a regression analysis on the data. However,
this is on the assumption that the model is linear, which will yield non-reliable results since
concentration-response models are known to be S-shaped in nature rather than a straight
line. The current study will fill the gaps of this research by replicating the analysis of the
larvicidal efficacy of the Manilkara zapota peel but using the appropriate and standardized
methods of the World Health Organization (2005).
The other study is on the study of Parthiban and Ramanibai (2017) titled
“Entomotoxicity Properties of Eco-Friendly Crude Protein Extract From Manilkara
zapota Seed against Asian Tiger Vector Aedes aegypti”. This study assesses the larvicidal
efficacy of the crude protein extract of chicos seeds. This is different from the current
study due to the difference in the use of solvents in the extraction process. The researchers
used a specific methodology that would yield the crude protein extracts from the chicos
seeds meanwhile the current study will use ethanol to extract the chicos seeds. The results
of the study showed that the crude protein extract from the chicos seeds showed larvicidal
activity against all four instars of Aedes aegypti larvae. They also found that the LC50 of
the crude protein extract was 3.45 mg/mL or 3450 PPM and the LC90 was 7.75 mg/mL or
7750 PPM against instar III mosquitoes. This means that chicos seeds do have larvicidal
effects against mosquito larvae. Lastly, the researchers conducted a non-toxicity assay that
found that the crude protein extract of the chicos seed is not toxic to non-target organisms.
4. Ethanolic Extraction
The purpose of plant extraction is to separate the soluble plant metabolites such as
phytochemicals from the insoluble solid plant residue. Maceration is an extraction
technique where the plant material is soaked in a specific solvent for a long period of time
with frequent agitation. Solvents used in maceration will play a huge role in determining
what substances will be dissolved in the final yield (Handa et al. 2008). Ethanolic and
other hydroalcoholic extracts seem to show the most presence of phytochemical content
in studies involving plant material. (Al-Mansoub, Asmawi, and Murugaiyah 2014; Arya,
Thakur, and Kashyap 2012)
According to an article by Azwanida (2015), entitled “A review on the extraction
methods use in medicinal plants, principle, strength and limitation”, the phytochemical
content of plant extract yields varies with several factors. Dried samples are preferred over
fresh samples because of time limitations in the experimental design. It is also shown that
there is no significant difference between the total phenolic content of dried and fresh
samples although flavonoid content is higher in dried samples. Moreover, it has also been
found that the particle size of the ground sample also significantly affects the yield of the
extraction. In general, smaller particle sizes will have more surface area for extraction and
thus will cause in a greater yield.
5. Dose-response, Probit Analysis and the LC50 Ratio Test
Dose-response relationships are studied in the field of toxicology to determine the
response of an organism to stimuli, typically used to test for toxicity. The dose-response
relationship relevant to larvicidal bioassays are binary response models, which involves a
response that only has two values—alive or dead. The sigmoid curve is commonly used
to model dose-response relationships. In fact, it is universally recognized as an appropriate
approximation for most biological dose-response relationships (Altshuler 1981). In the
current study, the stimuli are the concentrations of the tested larvicides, and the response
is the mortality of Aedes aegypti larvae. Since this study involves a dose-response
relationship, the concentration and mortality are expected to have a relationship described
by the sigmoid curve.
Probit analysis is a statistical technique used extensively in the field of Toxicology.
The method was first published by Chester Itnerr Bliss in 1934 to determine the toxicity
of pesticides against pests that fed on grape leaves. Since then, the method has been used
in bioassays of substances such as larvicides, insecticides, fungicides and other chemicals
that are tested to produce a response in populations (Greenberg, 1980, as cited in Vincent,
2008). This statistical method is used for studies that have a binary response to stimuli. In
our study, the binary response is whether the larvae are alive or dead. Moreover, it is the
analytical technique recommended by the World Health Organization to determine the
larvicidal activity of different substances (World Health Organization [WHO], 2005). It is
a kind of regression that determines the LC50 and LC90 values of a larvicide candidate.
Moreover, there is a need to determine the statistical significance of these LC50
and LC90 values. Because concentration-response models are not normally distributed,
conventional high school hypothesis tests such as the Student T-test and the ANOVA are
not applicable for these types of studies. For decades, toxicologists used overlapping
confidence interval tests to determine the statistical significance of LC50 and LC90 tests.
However, as Wheeler et. al. (2006) argued, this statistical test has less power in
determining the statistical significance of the lethal values compared to the alternative
method. The alternative method is called the LC50 Ratio Test. It is a simple statistical test
that involves calculating the ratio between the LC50 values of different larvicides.

(8) METHODOLOGY
1. Research Design

Figure 2: Research Design

 The study will use an experimental design with two experimental groups and two
control groups. The study will be aimed to measure the larvicidal efficacy of the two
experimental groups—the ethanolic crude extract of chico peel and seeds. There are two
control groups: a commercial larvicide that will act as the positive control and an untreated
water solution with 1% ethanol that will act as the negative control for observation only.
The methodology is adapted from the modified version of the methodologies prescribed
by the World Health Organization (2005) and Alibo et al. (2021). Statistical analysis of
data includes the use of the chi-square goodness of fit test and the probit analysis method
with its corresponding ratio test for significance.

2. Research Procedure
The procedure that will be used in the study is based on the World Health
Organization (2005) guidelines for laboratory and field testing of mosquito larvicides.
Some parts of the methodology are adapted from the modified methodology of a similar
study done by Alibo et. al. (2021). The experiment will be conducted in the Bantayan
Science High School Biology Laboratory located in Barangay Ticad, Bantayan, Cebu
(11°10'20''N, 123°43'16''E). Proper laboratory practices will be observed throughout the
study.
Collection and Preparation of Materials
First, the researcher will collect chicos fruits one week before each
experiment. Mature chicos fruits will be collected from a property of the
researcher’s family located in Barangay Patao, Bantayan, Cebu (11°13'00"N,
123°42'11"E). Other materials and instruments needed for the study will be
purchased, rented, or borrowed.
The peel and seeds of the fruit will be separated, mashed, and ground to
ease the drying process. The samples will be dried using an oven on low heat until
most of the water is evaporated. The dried samples will then be ground using a
blender to yield a fine powder.
Extraction of Ethanolic Crude Extract
One hundred grams of each of the samples will then be mixed and
macerated in one liter of 95% ethanol in sterilized gas bottles. The samples would
then be left for 24 hours with frequent stirring.
The solid parts of the suspension will then be filtered out using a grade
0905 crepe filter paper. The remaining liquid from filtration will then be subjected
to rotary evaporation to remove most of the ethanol. At this point, the researcher
should have the ethanolic crude extracts of the chicos peel and seeds.
Preliminary Testing
To determine the test concentrations that will be used for the actual
bioassay, preliminary tests will be done on a wide range of concentrations until a
narrower set of five concentrations that will yield mortalities from 10% to 95% of
the larval population after 48 hours.
Making of Stock Solution
For the preliminary tests and the actual larvicidal bioassay stock solutions
will be created. Note, however, that the needed concentration for the stock solution
will change throughout the research study after final test concentrations are
determined from the preliminary tests. For the preliminary tests, creating the stock
solution for the plant-based larvicides, 2.0 g of the crude extract will be mixed with
2.5 mL ethanol and 17.5 mL water to yield 20 mL of a 100,000 PPM solution for
each of the samples. The main stock solution will be serially diluted three times by
adding 2 mL of the last solution to 18 mL of 95% ethanol in a graduated cylinder,
yielding 20 mL solutions with concentrations of 100000 PPM, 10000 PPM, 1000
PPM, and 100 PPM.
Similarly, a stock solution would then be made for the commercial larvicide
by dissolving 1.0 gram of the Abate 1SG larvicide with 1000 mL of water to yield
100 mL of a 1000 PPM solution. The stock solution will be serially diluted three
times to yield 20 mL solutions with concentrations of 1000 PPM, 100 PPM, 10
PPM and 1 PPM.
Larvicidal Bioassay

Figure 3: Larvicidal Bioassay

A larvicidal bioassay will then be set up that follows the guidelines set by
the World Health Organization (2005) with modifications. The experiment will be
done with three replicates, each done on a Saturday, a week apart from each other.
For each replicate, there will be three treatments with five sub-treatments each, and
one observation group with one sub-treatment and three replicates. Each sub-
treatment will have a different concentration from the concentrations determined
by the preliminary tests. Disposable cups with 200 mL distilled water will be filled
with thirty (30) Aedes aegypti larvae of various stages in development to be
 acquired from the Department of Science and Technology – Industrial Technology
Development Institute (DOST-ITDI) Entomology Section Insectary. Weak or dead
larvae will be removed and replaced. For the randomization of the experimental
units, the available larvae are grouped according to their body size. Each group will
be allocated equally among the prepared disposable cups. A random number
generator will be used to randomize the treatment and sub-treatment received by
each cup in the bioassay. Lastly, proper amount of aliquot from the stock solutions,
as specified in the Annex 2 of the World Health Organization (2005) guidelines for
laboratory and field testing of mosquito larvicides, would then be added to each
cup to yield a specific concentration. The larvicidal bioassay will be conducted on
the Bantayan Science High School Biology Laboratory on a photoperiod of 24
hours in light and 24 hours in darkness.
Data Collection and Disposal of Waste
In 12-hour intervals, data from the larvicidal bioassay such as the dead and
moribund larvae will be counted and recorded. Each replicate will have a duration
of 48 hours. The appropriate data analysis specified in the statistical treatment
section will be carried out.
The waste generated from the larvicidal bioassay will then be stored in
sealed containers and disposed in the laboratory disposal facility for biohazardous
waste.
3. Statistical Treatment

Mortality in each replicate of the larvicidal bioassay will be recorded in 12-hour

intervals for 48 hours. A larva will be counted as dead or moribund if it does not show
signs of locomotion after the researcher shakes the container slightly several times. The
mortality rate will then be calculated using the following equation for each replicate.

𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑑𝑒𝑎𝑑 𝑜𝑟 𝑚𝑜𝑟𝑖𝑏𝑢𝑛𝑑 𝑙𝑎𝑟𝑣𝑎𝑒

𝑚𝑜𝑟𝑡𝑎𝑙𝑖𝑡𝑦 𝑟𝑎𝑡𝑒 = × 100
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑙𝑎𝑟𝑣𝑎𝑒

In cases when there is observed mortality in the control group, the mortality rates
will be adjusted using a formula from Abbott (1925).

% 𝑚𝑜𝑟𝑡𝑎𝑙𝑖𝑡𝑦 𝑜𝑛 𝑡𝑟𝑒𝑎𝑡𝑚𝑒𝑛𝑡 − % 𝑚𝑜𝑟𝑡𝑎𝑙𝑖𝑡𝑦 𝑜𝑛 𝑐𝑜𝑛𝑡𝑟𝑜𝑙

𝑚𝑜𝑟𝑡𝑎𝑙𝑖𝑡𝑦 (%) = × 100%
1 − % 𝑚𝑜𝑟𝑡𝑎𝑙𝑖𝑡𝑦 𝑜𝑛 𝑐𝑜𝑛𝑡𝑟𝑜𝑙

The mean and standard deviation of the mortality rates for each test concentration
of the treatments will be calculated and then illustrated using bar graphs. The LC50 and
LC90 values with their respective 95% confidence intervals for each of the treatments
will then be calculated by probit analysis using the Microsoft Excel® for Microsoft 365
software with a toxicology calculator created by Lei and Sun (2018). The statistical
significance between the respective LC50 and LC90 values of the treatments is then
determined using the LC50 Ratio Test as recommended by Wheeler et al. (2006).

Research Hypotheses
The study will use a four-group experimental design that has a categorical
independent variable (type of larvicide) and a discrete dependent variable (mortality,
mortality rate). Since the researcher chose a moderator variable (concentration) for this
study, conventional test statistics will not be applicable to the study. However, Wheeler et
al. (2006) recommended the use of an LC50 ratio test to determine the statistical
significance of the findings. The use of the probit model is justified using the Chi-square
Goodness of Fit test. An alpha level of α = 0.05 will be used for all statistical tests.

Chi-square Goodness of Fit Test

Ho: The probit model adequately fits the mortality rates observed from the
larvicidal bioassay.
Ha: The probit model does not adequately fit the mortality rates observed from the
larvicidal bioassay.

LC50 Ratio Test

LC50 values
Ho: There is no significant difference between the LC50 values of the ethanolic
crude extracts of chico peel, chico seeds and the commercial larvicide.
Ha: There is a significant difference between the LC50 values of the ethanolic
crude extracts of chico peel, chico seeds and the commercial larvicide.
LC90 values
Ho: There is no significant difference between the LC90 values of the ethanolic
crude extracts of chico peel, chico seeds and the commercial larvicide.
Ha: There is a significant difference between the LC90 values of the ethanolic
crude extracts of chico peel, chico seeds and the commercial larvicide.

(9) EXPECTED OUTPUTS AND POTENTIAL IMPACTS

The effectiveness of insecticides has been decreasing due to the increasing
resistance of mosquitoes against these products. This posed a problem for researchers since
insecticides were the most effective way of deterring the spread of dengue for years.
Recent developments suggested that targeting the young of these dengue vectors through
plant-based larvicides may be an effective alternative. Along with the increasing
consciousness for environmental and social safety, there is a need for cheap and eco-
friendly substitutes for commercially available artificial larvicides. This study will be
aimed to fill that gap by exploring the potential of the peel and seeds of local fruit chicos
(Manilkara zapota) as a plant-based larvicide. Specifically, this study will potentially
benefit the following beneficiaries:
• Community: The outcome of this research is expected to be helpful to the
community since it will potentially discover a way to decrease the population of the
 vector that spreads dengue that is safer for humans than their commercial and
artificial counterparts.
• Environment: This study is also expected to reduce the frequent use of synthetic
insecticides and larvicides by providing a natural alternative that is safer for the
environment and organisms.
• Stakeholders and manufacturers: This study will be useful to people that are
interested in making programs and policies on the use of natural plant-based
larvicides to control mosquito populations. It will also be helpful to larvicide
manufacturers that seek new innovations for their products.
• Field of Toxicology and future researchers: This study will fill an existing gap in
research. Relatedly, it will serve as a reference for future researchers that wish to
conduct a related study.

(10) WORK PLAN AND TARGET DELIVERABLES

TIME FRAME ACTIVITY

1 week Reading relevant literature
Formulating the research design
1-3 weeks Requesting and buying of Aedes aegypti larvae from DOST-ITDI
or starting the process of rearing Aedes aegypti larvae (tentative)
1 week Collecting the samples, making the stock solution, and preparing
the environment and set-ups for the preliminary experiment
Conducting a preliminary experiment (48 hours) to determine the
activity range of the samples studied and the test concentrations
that will be used in the final larvicidal bioassay
3 weeks Collecting the materials for the final experiment, making the stock
solution, preparing the larvicidal bioassay, and acquiring the test
subjects from logistics
Conducting the three replicates for the larvicidal bioassay for three
weeks. Each replicate has a duration of 48 hours, with additional
hours allotted for data collection.
Data analysis, descriptive statistics, and probit analysis for the
collected data and conducting corresponding test statistics to
evaluate hypotheses
1 week Writing of research report

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