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JOSON MEMORIAL HIGH
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DETERMINATION OF EFFICACY OF CALAMANSI (citrofortunella microcarpa) PEEL
EXTRACT AS MOSQUITO REPELLENT AGAINST ADULT MOSQUITO
Bajalah, Nike April A.
Bobis, Tyron Jan Ray B.
Dela Cruz, Chloe Beyonce F.
Dela Merced, Jared Matthew F.
Jose, Adrian C.
Lacanilao, Sean Matthew M.
Researchers
Harvey V. Valdez
Research Adviser
ELJMHS
2023
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CHAPTER 1
THE PROBLEM AND ITS BACKGROUND
Background of the Study
Insect repellents are substances used to repel and prevent harmful insects such as
mosquitoes from landing or biting. Insect repellents are in high demand in the Philippines these
days due to the prevalence of mosquito-borne diseases such as dengue, malaria, chikungunya,
and zika virus. From January 1 to December 17, 2022, there were 220,705 dengue cases reported
in the country. Central Luzon recorded the largest number of dengue cases, with 44,030
(Department of Health, 2023). N,N-diethyl-meta-toluamide (DEET) is the main component in
many repellents. It is frequently used to ward off biting insects like ticks and mosquitoes. DEET-
containing products are presently available to the general public in a range of liquids, lotions,
sprays, and impregnated materials (United States Environmental Protection Agency, 2022).
According to Koren, Matsui, and Bailey (n.d.), ingestion of DEET may lead to
hypotension, seizures, and coma within as little as 1 hour. A high concentration of DEET has
been reported to affect the nervous system in rare cases, with symptoms including seizures,
tremors, and slurred speech (Environmental Working Group, n.d.). DEET has been found in
waste water and may also affect some animals (Jacskon et al., 2008). Also, According to the
Centers for Disease Control and Prevention (2017), there have been instances of people suffering
negative reactions after using repellents containing DEET excessively.
Insect repellents can be chemical or natural. Many families use commercial insect
repellents that contain chemicals, not knowing that they have been proven to be detrimental to
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human health and the environment. Furthermore, these commercial repellents are considered to
be costly for the average household, and many families are unable to afford them. As a result,
utilizing natural substances as an alternative in developing safe and new eco-friendly repellents
might be an effective strategy to reduce the negative impacts on the environment and human
health (Paul, 2022).
Calamansi is a member of the Rutaceae family and of the genus Citrus. It is well known
as "Kalamansi" in the Philippines and is used as a condiment. Only the pulp, which was pressed,
was needed; the peels were simply thrown (Morte, 2017). Calamansi peel contains d-limonene,
a bitter flavor that irritates insects by damaging the wax covering of the insect's respiratory
system, making it an effective repellant (Capili, Cruz, & Cunanan, 2019).
In parallel to the stated problem above, Kripena (2020) stated that citrus fruits contain
chemicals that can kill mosquito larvae. In addition, it also shows that some citrus scents repel
adult mosquitos.
The aim of this study is to determine the effectiveness of calamansi peel extract as a
mosquito repellent against mosquito. This study will be conducted on response to the use of
chemical or synthetic insect repellents containing DEET.
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Objectives of the Study
This study aims to determine the efficacy of calamansi peel extract as mosquito repellent.
Specifically, it aims to:
1. Produce a mosquito repellent made up of Citrofortunella microcarpa peel extract.
2. Determine the quality of Calamansi peel extract mosquito repellent in terms of:
a. Protection Time
b. Repellency
3. Know if there is a significant difference in the effectiveness of the following in terms of
repelling mosquito:
Treatment 1: 100% calamansi peel extract
Treatment 2: 75% calamansi peel extract
Treatment 3: 50% calamansi peel extract
Treatment 4: Commercial mosquito repellent
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Research Framework
Test Subjects
Start T1. 100% calamansi peel
extract
T2. 75% calamansi peel
extract
Review related T3. 50% calamansi peel
studies to provide a extract
foundation of
knowledge on the topic T4. 40% DEET commercial
mosquito repellent
No
Is the review of
related literature Are the No
enough to support treatments
the study? completely
formulated?
Yes
Yes
Mosquito
Collection
Experimentation
Application of
treatments to
mosquitoes
Are
there enough No
mosquitoes
collected?
Is there
enough data No
Yes collected?
Calamansi Peel
Collection
Yes
Data Analysis
Is there No
enough peel
collected?
Is the data No
analysis
Yes
okay?
Calamansi Peel
Extraction
Yes
Report Writing
Is there
enough No
extract
collected?
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End
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Scope and Delimitation
The general interest of this study is to know the efficacy of Citrofortunella microcarpa
peel extract as mosquito repellent against Mosquito. Steam distillation will be used to extract the
essential oils from calamansi peel (Dewiani et al., 2022). The Calamansi (Citrofortunella
microcarpa) peel extract will only be utilized through spraying. In each of the three confined
spaces, the researchers will place three mosquitoes. In the confined spaces, treatments such as
100%, 75%, 50% calamansi peel extract will be sprayed. As a control, a commercial insect
repellent containing 40% DEET will be utilized. The data needed for the study will be acquired
through observation.
This study is limited to the use of calamansi extract as an insect repellent and its
effectiveness against mosquito. The identification of the calamansi peel extract's mosquito-
repelling or mosquito-weakening properties is not included in this study. This study will be
conducted in Brgy. Sta. Clara, Quezon, Nueva Ecija. The study will be carried out with limited
amount of financial resources and time framework.
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Significance of the Study
As many years go by, people around the world have been still using synthetic insecticides
despite the fact they are already aware of its harmful effects to the environment and the humans.
Prolonged exposure to synthetic insecticides can be really bad for your health. Also, synthetic
insecticides can be harmful to the environment due to the different toxic chemicals combined in
its ingredients. Calamansi is a widely available citrus fruit in many tropical countries, and its use
as a mosquito repellent could encourage the use of other natural products for pest control, which
could reduce the dependence on synthetic chemicals and promote sustainable agriculture. This
study will be conducted for the benefit of the following:
Students. The study might encourage students to pursue careers in the fields of science and
research. They could learn about the importance of mosquito control and disease prevention.
This could also lead them to be more aware and informed with the benefits and nutrients that can
be found in a native fruit that is grown locally, which can encourage them to also do a study
about another fruit or vegetable as well. Students can learn about the scientific method and
research process.
Parents. The study could provide information and data to gain more knowledge and have a
deeper understanding to the use of organic products. Parents could learn more about natural
alternatives to synthetic insect repellents while ensuring that their children are protected from
mosquito. Upon knowing that natural pest control products are supported by science may give
parents more confidence to use them.
Community. The study could contribute to the development of more environmentally friendly
and sustainable pest control solutions, which could benefit future generations. Organic products
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are more affordable and environmental-friendly than synthetic products, making it easily
accessible to those in need. The study could raise awareness of the importance of effective
mosquito control strategies and the risks associated with mosquito-borne diseases. The people
from the community could learn about the potential benefits of using locally available and
sustainable resources for pest control, eventually making it as their source of income.
Environment. The most common active ingredient in insect repellents is DEET, which is
harmful to the environment. It can be extremely toxic and pollute bodies of water such as rivers
and oceans. Whereas, the use of calamansi as a mosquito repellent could encourage the use of
other natural products for pest control, which could reduce the dependence on synthetic
chemicals and promote sustainable agriculture. The study could promote the use of locally
available and sustainable resources as an alternative to synthetic chemicals. The study could
contribute to the growing body of knowledge on natural products as potential sources of insect
repellents, which could have implications for the development of new and more sustainable pest
control strategies.
Future researchers. The study could encourage further research and development of natural
products as insect repellents. The results of the study could serve as a starting point for further
investigations on the use of other plant extracts as mosquito repellents. This could lead to the
discovery of new and effective natural products for pest control, which could have applications
in agriculture, public health, and other fields.
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Definition of Terms
Citrus - a genus of plants in the rue family that produces pulpy fruits with thick skins.
DEET - an acronym for N, N-Diethyl-meta-toluamide, a synthetic chemical used as a common
active ingredient in insect repellents to protect against mosquito, tick, and oather insect bites.
Dengue - a viral disease transmitted by mosquitoes of the Aedes genus that can cause fever, joint
pain, and in severe cases, hemorrhagic fever, shock, and death.
D-limonene - a colorless liquid compound commonly found in citrus peels and used in
fragrances, cleaners, and insecticides.
Detrimental - causing harm or damage.
Efficacy - the ability of a substance or treatment to produce a desired effect or result.
Implications - the potential consequences or effects of a particular action, decision, or situation.
Ingestion - the act of consuming or taking in a substance, such as through eating, drinking, or
inhaling.
Larvae - are the mosquito's most active aquatic stage. They play an important function in the
mosquito's life cycle and are hence highly suited to their role.
Peel - the outer layer of a fruit, such as the skin or rind, which is typically removed before
consumption but may have various uses.
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Repellent - a substance used to prevent insects, animals, or other organisms from approaching or
attaching to a surface or individual.
Rutaceae - a family of plants that includes citrus fruits, such as oranges, lemons, and grapefruits,
as well as other species with aromatic compounds, including D-limonene.
Sustainable - referring to practices or systems that are able to be maintained over the long term
without depleting natural resources or causing harm to the environment. Sustainable practices
aim to promote ecological balance and social equity while also meeting human needs.
Synthetic - referring to something that is artificially created or produced, often in a laboratory or
factory, rather than occurring naturally in the environment.
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CHAPTER II
REVIEW OF RELATED LITERATURE
This chapter presents the relevant literature and studies that the researcher considered in
strengthening the claim and importance of the present study. These related literature and studies
were gathered from electronic media such as the internet and other online literary works.
Related Literature
Calamansi is a condiment used in a wide variety of Filipino meals, as well as a
component in a number of beverages, such as calamansi juice. Calamansi peels are considered
garbage (Patrimonio, 2022).
According Atilano (2020), as the demand for calamansi juice expanded, so did the
amount of waste produced. The peel accounts for the bulk of the waste, although it contains
essential oils that may be utilized in a number of ways and be profitable, such as cleaning
products and others.
Charoenphun et al. (2023) stated that calamansi has several beneficial properties and is a
great source of vitamin C, D-limonene, and essential oils. It also has anti-inflammatory, anti-
cancer, anti-diabetic, anti-angiogenic, and anti-cancer qualities that benefit the immune system.
Pectin, a source of dietary fiber, is also present in good amounts. Additionally, the juice includes
bioactive substances such as phenolics and flavonoids, which may have antioxidant qualities.
The calamondin fruit's juice, pulp, seeds, and peel may all be utilized for a variety of purposes,
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demonstrating its adaptability and special qualities in both food stuff such as juices, powders,
and sweets as well as non-food uses in herbal medicine and cosmetics.
Mosquito-borne infections are a serious public health concern in all tropical and
subtropical nations. Malaria, filariasis, yellow fever, Japanese encephalitis, and dengue fever are
among the diseases spread. Over the years, several approaches for combating mosquito-borne
illnesses have been tested (Bora, Kalita, & Sharma, 2013).
Using repellents, as reported by Da Silva and Junior (2020), is one of the most effective
strategies to avoid mosquito bites. The practice of utilizing plants as insect repellents has
increased drastically. Sprays and lotions are the most often sold natural repellent formulations.
Among the traditional commercially available formulas, citronella essential oil was the most
commonly utilized.
According to Ruggeri (2019), given the fact that DEET is the most efficient insect
repellent, research indicates that it may have severe adverse effects in specific scenarios. DEET
exposure may cause allergic responses and even anaphylaxis in some persons. Ingestion of
DEET might cause seizures in certain people. Seizures caused by DEET have also been reported
in children. There is some evidence that DEET includes carcinogenic qualities that, when inhaled
or applied to the skin, might cause harmful consequences.
Natural essential oils that were produced from citrus fruit peels showed excellent
mosquitocidal action, suggesting that they might be a new, environmentally friendly option in
insect control operations (Science Daily, 2017).
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It was reported in a previous study by Dachriyanus, Husni, and Yeni (2021) that the main
components of the essential oil in the calamansi peels, according to GC-MS data, are D-limonene
(29.52%), (R)-(+)-citronellal (13.76%), 3-isopropenyl-5,5-dimethyl-cyclopentene (8.88%), y-
terpinene (7.30%), citronellol (6.90%), and -terpineol (4.61%). While the citronellal, citronellol,
3-carene, and -phellandrene present in calamansi leaves total (25.74%), (12.94%), (8.43), and β-
phellandrene (4.89%,) respectively.
D-limonene levels in calamansi peel were found to be high. D-limonene is a naturally
occurring oil ingredient known as terpenes that is most commonly found on citrus fruit peels. D-
limonene is often found in commercial insect repellents. Terpenes such as d-limonene are
responsible for the unique aroma of citrus fruits (Santos, 2020).
Calamansi has the component limonene in its S-oxide, which has been discovered. The
chemical D-limonene has the ability to dissolve an insect's protective wax covering. The
respiratory tracts of insects can also be harmed by S-oxide, which eventually repels them. Insect
repellents made from calamansi rind extract have a substantial impact on households since they
are a low-cost, all-natural insecticide (De Castro et al., 2022).
According to Palma et al. (2019), d-limonene is the predominant ingredient of calamansi
peel essential oil, accounting for at least 92.67% of the total oil determined using the qualitative
analytical results of gas chromatography-mass spectrometry. The essential oil of calamansi was
extracted by steam distillation and examined using Attenuated Total Reflectance-Fourier
Transform Infrared Spectroscopy and Gas Chromatography.
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Limonene is a terpene, a group of chemicals whose powerful aromas protect plants by
repelling predators. Limonene is used as a botanical insecticide due to its powerful aroma. It is
an active component in a variety of insecticide treatments, including eco-friendly insect
repellents. Limonene is extracted via hydrodistillation, which involves soaking fruit peels in
water and heating them until the volatile molecules are released via steam, condensed, and
separated (Sobel, 2019).
The essential oils of eight different citrus species, including calamansi, were investigated
for their effectiveness against mosquito vectors. The most effective essential oils against Aegypti
were those from key lime and calamansi, with mean protection periods of 65.0±22.9 minutes,
1.5% biting rate, and 98.5% protection and 61.7±2.9 minutes, 1.6% biting rate, and 98.4%
protection, respectively (Soonwera, 2015).
Citrus-based EOs are primarily produced from citrus fruit peels, which are typically
thrown away as waste and have an adverse effect on the environment. Citrus essential oils may
be extracted from discarded peels and utilized for a variety of purposes, such as food
preservation, in addition to helping the environment (Baral et al., 2019).
Steam distillation was used to extract the calamansi peel's essential oil. Starting with
5000 grams of fresh calamansi fruit that have been divided into the peel and meat, the essential
oil distillation process for calamansi peel begins. The essential oil from calamansi peel was
extracted using steam distillation for a full hour. The essential oil from the calamansi peel was
extracted using a separatory funnel (Dewiani et al., 2022).
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According to a research conducted by Dumilon, Estacio, Menil, and Tamaray (2020) on
the use of sampaguita and calamansi as mosquito repellents, both the calamansi and sampaguita
individually and in combination showed to be effective insect repellents, easily outperforming
the efficacy of citronella. The efficacy of each repellent, individually and in combination, was
measured by monitoring the number of mosquitos that landed on the human arm for more than
10 seconds.
According to Capili, Cruz, and Cunanan (2019), the calamansi peel extract had the
highest mortality of all the extracts, followed by the seed extract, and the carpel extract had the
lowest mortality effect on rice weevils. This was based on the determined mortality throughout
the experiment. The results showed that the extracts were capable of killing rice weevils and that
there was a significant difference between the extracts in terms of their insecticidal activity. The
researchers established that C. Rice weevils can be effectively controlled using microcarpa
extracts.
Calderon, Paculdo, Restrivera, and Sugatan (2017) demonstrate that calamansi peel
extract could potentially be utilized as a substitute for commercial termiticide because there is no
significant difference between calamansi peel extract and commercial termiticide.
Acabal, Caugma, Del Mundo, and Gerolaga (2020) argue that calamansi peel extract is a
promising insecticide against brown planthoppers and should be used as an organic plant-based
insecticide. It is an ecologically safe and cost-effective method of regulating brown
planthoppers as a water-based therapy. The results revealed that increasing the concentration of
C. microcarpa peel extract results in a higher percent mortality of brown planthoppers.
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In a study of De Castro et al. (2022) on the efficacy of calamansi peel extract as a house
fly repellent, various concentrations of calamansi extract, including 30%, 60%, and 90%, were
utilized. The metrics were used to determine which one was more effective. These measurements
were necessary for organic product investigations. The researchers tested the extract in the same
cage and fabric to avoid any bias in the results of the trial. Thus, the 90% calamansi extract was
determined to be the best performing house fly repellant when compared to the 30% and 60%
calamansi extracts.
Cornelio, Cruz, Gecala, and Senorio (2020) conducted a study on the feasibility of
calamansi and chili extract as cockroach killers. They used four treatments, including treatment
A (25 ml of calamansi, 15 ml of chili fruit extract, and 10 ml of water) and treatment B (20 ml of
calamansi, 20 ml of chili fruit extract, and 10 ml of water). Treatment C contains 15 mL of
Calamansi, 25 mL of chili fruit extract, and 10 mL of water, whereas treatment D has merely 40
mL of water. The study found that treatment A (25ml of calamansi extract, 15ml of chili fruit
extract, and 10ml of water) killed the most cockroaches.
Avila, Calumba, and Pansit (2018) found that when papaya (Carica papaya) and
calamansi (Citrofortunella microcarpa) were used as larvicides, calamansi exhibited a
concentration dependant impact and significantly greater larvicidal activity at the 0.05 level of
significance compared to papaya. They also proved that calamansi had significantly stronger
potential larvicidal activity against Aedes aegypti mosquito larvae, even exceeding the larvicidal
activity of a commercial larvicide at the concentration utilized in the experiment. This larvicidal
activity is due to the abundance of well-known insecticidal and larvicidal phytochemicals in both
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extracts of plants. In addition, fatty acid was found in calamansi extract, which may have an
additive effect with other abundant phytochemicals, resulting in increased larvicidal activity.
Carigaba, Garbo, Leonida, Masculino, and Mediodia (2020) conducted Probit Analysis to
assess data on larval mortality using calamansi peel extract after 24 hours of exposure. The
results showed that calamansi peel extract in 95% ethanol has a high larvicidal potential. This
suggests that calamansi peel EO is effective against third and early fourth instar Ae. aegypti
mosquito larvae at low concentrations and might be utilized as a possibly safer and alternative
biolarvicide.
Cheong et al. (2012) investigated the volatiles, aroma profiles, and phenolic acids in
calamansi peel from Malaysia, the Philippines, and Vietnam. The calamansi peel from the
Philippines has the highest amount of total phenolic acids. Additionally, p-Coumaric acid was
the most abundant free phenolic acid, whereas ferulic acid was the most abundant bound
phenolic acid.
Multiple studies has been conducted to find plant phenolics that have insecticidal
activities. It implies that phenolics have a detrimental effect on insects and are useful for the
control of insect pests (Golawaska, Kapusta, Lukasik, & Wójcicka, n.d.).
Rodriguez (2018) revealed that, among setups, calamansi peel-made candles got the most
favorable ratings for ability to repel, duration of repellency, and smell. To determine which
combination was the most effective, three different setups were used: 100% lemon grass, 100%
calamansi, and a combination of the two. Using ANOVA single factor, the data were analyzed. It
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can be concluded that the 100% Calamansi setup may serve as an effective alternative mosquito
repellent.
As stated by Abantes et al. (2012), C. microcarpa is safe to use. It is an environmentally
friendly approach of reducing dengue mosquito wrigglers that does not harm individuals or other
non-target organisms.
CHAPTER III
METHODOLOGY
Locale of the Study
The locale of this research was the researcher's house located in Sta. Clara, Quezon,
Nueva Ecija, Philippines. The choice of this specific locale was made to provide a controlled and
natural environment for conducting the research. Sta. Clara, Quezon, Nueva Ecija, is a residential
area situated in the province of Nueva Ecija. It offers a convenient and comfortable setting for
conducting research, allowing the researchers to have easy access to the study site without the
need for external facilities or travel. The research took place during the second semester of the
academic year 2022-2023.
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Research Design
The goal of this study was to determine how different concentrations of calamansi peel
extract differed in their ability to repel mosquitoes. These concentrations are as follows: 50%
Calamansi peel extract mixed with 50% water, 75% Calamansi peel extract mixed with 25%
water, 100% pure Calamansi peel extract, and lastly, a commercially sold mosquito repellent
containing DEET.
In this study, experimental design was used. The number of mosquitoes that land and rest
on the hand was the dependent variable in the study, whereas the mosquito repellent was the
independent variable.
Research Instrument
The researchers conducted an observation to gather the data for the study. The chosen
instrument was observation because it offers extensive data collection and gathering from test
subjects, which is required for accurate findings.
The observation was performed by the researchers. A box containing 10 mosquitoes was
observed after the calamansi peel extract solutions were sprayed on the hand and arm. This was
performed in order to determine how many mosquitoes would land on the test subject's hand
during 20 minutes of observation.
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A. Data Collection
Collection of Calamansi
3 kilograms of Calamansi were purchased in the Quezon Public Market in Barangay
Bertese, Quezon, Nueva Ecija.
Collection of Mosquitoes
A bucket containing 2 liters of stagnant water was placed in the garden of one of the
researchers' houses in Barangay Villarosa, Licab, Nueva Ecija. Female mosquitoes had finally
laid eggs in the bucket after 3 days. Within two weeks, the eggs had matured and underwent
different larval stages, including larva and pupa, before turning into adult mosquitoes. In this
study, 10 adult mosquitoes will be needed for each treatment, for a total of 40 mosquitoes, and
there will be three replications, for a total of 120 adult mosquitoes.
B. Extraction Process
Calamansi peel was extracted using an improvised steam distillation method. The 3 kg of
calamansi were washed and soaked in clean water before being peeled, producing 13 cups of
calamansi peel. The materials include a gas stove as a heat source, a pressure cooker, a 56-inch
clear hose, a tub of cold water to cool and condense the steam, a beaker, tape, the calamansi peel,
and 15 cups (3.5 liters) of clean and filtered water. The clear hose was placed into the pressure
cooker and then taped inside and out to prevent it from falling. The clear hose was then passed
through the cooling tub, with the open end of the hose positioned over the beaker.
After completing the setup, the calamansi peel was placed into the pressure cooker,
followed by 15 cups (3.5 liters) of filtered water and the pressure cooker was sealed. The
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distillation was then started with a low heat. After an hour of distillation, 80 ml of extract was
collected, 220 ml after 2 hours, and 240 ml after 3 hours, for a total of 540 ml of calamansi peel
extract obtained from 3 hours steam distillation.
Figure 1: Improvised Steam Distillation Method
Figure 2: Extracted product from calamansi peel
C. Preparation of Concentration
For the concentration, three 50 ml clear empty spray bottles were utilized. The first one
was filled with 50 ml of pure calamansi peel extract (100%). The next contains 12.5 ml (25%) of
filtered water and 37.5 ml (75%) of calamansi peel extract. The last container is for the 50%
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concentration and contains 25 ml of filtered water and 25 ml of calamansi peel extract. To
prevent spills, the mixtures were transferred to the spray bottle using a small plastic funnel.
D. Experimental Procedures
Transparent
storage/shoe box
Arm-insertion
Mosquito- stocking
insertion lid
Figure 4: Experimental Setup
The experimental setup was based on the experimental setup used in the study of
Dumilon, Estacio, Memil, and Tamaray (2020) on the effectiveness of sampaguita and calamansi
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extract as mosquito repellent. But there are few modifications to the setup because some
materials are not readily available. A shoe box, stocking, and mosquito net were utilized in the
above setup.
Inside the setup, 10 adult mosquitoes were placed. All treatments were sprayed in the arm
and hand in equal amounts of 5 sprays, except for the commercial mosquito repellent. The
sprayed hand will be inserted into the box through the arm insertion stocking for each treatment.
The number of mosquitoes that landed on the hand within 20 minutes was counted.
E. Statistical Analysis
The data collected from the experiment will be analyzed to compare the mosquito
repellency of calamansi peel extract with different concentrations and the commercial mosquito
repellent against adult mosquito. To test the repellency rate, the following formula will be used:
No. of mosquitoes that landed on the arm/hand
Repellency ( % ) =1− x 100 %
Number of mosquitoes
The ANOVA Single Factor will be utilized as a statistical tool for the experiments to see
if there is a significant difference between the treatments. Single factor ANOVA examines the
means of the groups in which you are interested to see if any of those means are statistically
significantly different from one another.
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