Ruelans Group Research Paper Ixodicide Activity of Citrus X Microcarpa Calamansi Peel and Juice Extract Against Dog Ticks 1 1

Ixodicide Activity of Citrus x Microcarpa (Calamansi) Peel and Juice Extract on Dog Ticks
St. Louise de Marillac College of Bogo

Sor Dorotea Rubio St., Bogo City, Cebu
Ixodicide Activity of Citrus x Microcarpa (Calamansi)
Peel and Juice Extract on
Dog Ticks
Researchers:
Ruelan, Christian John L.
Bojos, Rose Zael
Pilapil, Cy
Inot, Jacqueline Phoebe
Masong, Anche Marie Cueva
Serdoncillo, Abegail Dela Cruz
Torres, Meg
Villegas, Blessed Dawn

Abstract
Ticks are one of the vector-disease borne parasites that can be harmful to dogs. Calamansi is
prominently cultivated in the Philippines and is notorious for its acidic properties that could
potentially be used as an anti-tick agent. The purpose of this paper was to test, in the terms of
mortality rate and rapid action, of both calamansi essential oil and calamansi pulp juice against
dog ticks. The sample size was 45 ticks obtained from 5 different dogs. The calamansi peel
extract was purchased online while the calamansi pulp juice was obtained by squeezing the
calamansi. The ticks are tested on three concentrations; 50%, 75%, and 95%. All concentrations
that were tested on the calamansi extract showed a mortality rate of 100% at 15 minutes whereas
the calamansi juice extract showed the highest mortality rate of 80% at 45 minutes. We suggest
that calamansi peel extract performed faster and effective against ticks compared to the
calamansi juice extract.
Keywords: Ixodicide activity, dog ticks, acarines, calamansi, calamansi juice extract,
calamansi peel extract, rapid action

ACKNOWLEDGEMENTS
The following people deserve special thanks for their assistance in our research
completion.
We would like to acknowledge our research advisors, Ms. Michelle Marrie Tinapay and
Mr. Renz Letrell Lepon, for their support and encouragement. We would also like to
acknowledge the research consultants, Madame Trinidad Labastida and Engr. Florenie Rosaña
and Ms. Aien Lambujon for their extensive knowledge and research recommendations. Without
their suggestions, we would not have been able to conclude our research. Despite their hectic
schedules, our dear advisers and consultants provided us with thoughtful counsel and suggestions
that helped us at every stage of the thesis-writing process.
We would want to express our gratitude to our families for their unwavering support
during the thesis writing process. We want to thank Ms. Emely Abendanio, who collected the
ticks and organized the resources for the experimental research. We also want to thank the
individual who gathered ticks, Mrs. Zenith Ayn Pilapil.
Finally, we want to express our gratitude to Our God Almighty for providing us with the
wisdom and knowledge necessary to complete our research paper, which motivates us to
persevere in our intellectual endeavor.

TABLE OF CONTENTS
CHAPTER I: INTRODUCTION………………………………………………………................1
A. Background of the Study………………………………………………………………1
B. Statement of the Hypothesis …………………………………………………………..3
C. Research Objectives…………………………………………………………………...4
D. Statement of Hypothesis………………………………………………………………5
E. Significance of the Study……………………………………………………………...6
F. Scope and Delimitation……………………………………………………………......7
G. Definition of Terms……………………………………………………………………8
CHAPTER II: REVIEW OF RELATED LITERATURE………………………………………..9
A. Related Literature……………………………………………………………………...9
B. Related Studies………………………………………………………………………..13
Theoretical Framework…………………………………………………………………..16
Conceptual Framework…………………………………………………………………..17
CHAPTER III: METHODOLOGY……………………………………………………………...18
Research Design………………………………………………………………………….19
Subject of the Study……………………………………………………………………...20
Research Instrument to be Used…………………………………………………………21
Procedure of Data Gathering…………………………………………………………….22
Statistical Treatment……………………………………………………………………..24
Collection of Materials…………………………………………………………………..25
Preparation of Treatment………………………………………………………………...26
CHAPTER IV: PRESENTATION AND INTERPRETATION OF DATA…………………….27
CHAPTER V: DISCUSSION, CONCLUSION, AND RECOMMENDATIONS……………....31
SUMMARY OF FINDINGS…………………………………………………………….31
CONCLUSION…………………………………………………………………………..34
RECOMMENDATIONS………………………………………………………………...35
REFERENCES…………………………………………………………………………………..36
APPENDIX A: AN IMAGE OF ONE OF THE DOG TICKS………………………………….42
APPENDIX B: CALAMANSI PEEL EXTRACT ON DIFFERENT CONCENTRATIONS….43
APPENDIX C: CALAMANSI PEEL EXTRACT ON DIFFERENT CONCENTRATIONS
WITH DOG TICKS…………………………………………………………………………….44
APPENDIX D: CALAMANSI JUICE EXTRACT ON DIFFERENT CONCENTRATIONS...45

APPENDIX E: CALAMANSI JUICE EXTRACT ON DIFFERENT CONCENTRATIONS
WITH DOG TICKS……………………………………………………………………………46
APPENDIX F: CONTROL GROUP WITH DOG TICKS……………………………………...47
CIRRULUM OF VITAE………………………………………………………………………...48
Ixodicide Activity of Citrus x Microcarpa (Calamansi) Peel and Juice Extract on Dog Ticks 1
Chapter I
INTRODUCTION
A. Background of the Study
Ticks are considered as the enemy of many pet lovers, especially dogs. They are small
organisms that feed on blood from dogs. They are most likely found in the environment and on
other infected dogs. The problem is, these ticks can be bad for the health of the dogs.
According to N. Boulanger et al. (2019),”ticks are acarines that may be distinguished
from insects by the absence of a segmented body and by their spherical form.” There are over
900 species of ticks that have been identified. Tick that is mostly found in dogs is Rhipicephalus
sanguineus (Boulanger et al., 2019) but other types of ticks can still be found on dogs.
A single tick can be a transmitter of different diseases to both humans and dogs. These
ticks usually suck the blood from the body of its victims. These ticks can cause spotted fever
when transmitted to humans (Blanda et. al, 2017; Parola et al, 2013) and weakening the immune
system of the dog. In a study done in the Philippines, the diseases found in dogs' blood systems
are Anaplasma/Ehrlichia spp, Babesia/Hepatozoon spp., Ehrlichia canis, and Riskettsia infection
(Galay, 2018). Diseases that a dog may get from a tick are not transmittable to humans.
However, it is still possible to be infected with such diseases if a person is bitten by infected
ticks. Moreover, it would be harmful when ticks infest humans. Filipe Dantas-Torres (2010)
noted,
Recent studies have demonstrated that ticks exposed to high temperatures attach and feed
on humans and rabbits more rapidly. This observation suggests that the risk of human
parasitism by the brown dog tick (Rhipicephalus sanguineus) could increase in areas
experiencing warmer and/or longer summers, consequently increasing the risk of
transmission of zoonotic agents (e.g., Rickettsia conorii and Rickettsia rickettsii) (p. 14).
The regular use of tick control products could help prevent ticks from attaching to your
dogs. Thus, it would also help to regulate ticks infesting humans. The researcher has a hypothesis
that a certain fruit extract can be used to kill these ticks. The researchers will test on the juice and
peel extract of juice to kill the dog's tick.
Calamansi, scientifically known as Citrofortunella microcarpa, is a popular citrus fruit
source in the Philippines that can work as a natural insect repellent as well as a disinfectant for
various bacteria and diseases. Calamansi is a prolific naturally-grown citrus tree that may be
easily cultivated as it adapts to various environmental conditions and can be found all across the
country. Calamansi has citric acid of 5.52% (Chew et al. 2017; Morton, 2013). Calamansi is used
in cleaning products, a natural exfoliator, insecticides, and insect repellents. In addition, it is also
used to cure coughs and as a natural anti-inflammatory medication.
As ticks on the rise, a tick remover is an absolute necessity. Calamansi peel and juice
extract can be used as an alternative tick killer as it is cheaper. The researchers would test the
effectiveness of various calamansi extracts against dog ticks. The study can be applied to a
variety of tick removal methods and become a product such as spray solutions, shampoo, and
home remedies. Moreover, calamansi peel and juice extract contains antioxidative,
hypolipidemic, and hypoglycemic properties that may be effective in the removal of ticks.
B. Statement of the Problem
Ticks are blood sucking parasites that can transmit diseases on dogs. They are transmitted
by the environment such as the outside. These dog ticks can cause redness and irritation on dogs.
Most importantly, dog ticks can carry many diseases such as Lyme disease and spotted fever.
Furthermore, ticks can attach and feed on human skin which can pass different types of diseases
and cause skin irritation.
With the result of this study, it will produce a cheaper tick killer alternative. This will
help the health condition of many dogs.
This study was aimed to determine the effectiveness of juice and peel extract from
calamansi against dog ticks. Specifically, it aimed to answer the following:
1. What are the mortality rate of ticks of both calamansi juice and peel extracts?
2. In comparison with the control group, what are the significance results of both calamansi
juice and peel extracts based on the mortality rate and rapid action?
3. What is the best concentration for calamansi juice and peel extract to kill the ticks in
dogs? Based on the study of Armugam, Sundraraj, and Saleh (2016), the concentrations
used are:
b.) 50% of 10 ml
c.) 75% of 10 ml
d.) 95% of 10 ml
4. Which of the two extracts performs well as a dog tick killer?
5. How long do ticks last in the solution before they completely die?
C. Research Objectives
This study aims to determine and answer the following:
a) To add the dog ticks on the solution on both the juice and peel extract solution.
b) To analyze the time duration of the tick on the solution before it dies.
c) To investigate the effectiveness of different concentrations of juice and peel
extract of calamansi.
D. Statement of Hypothesis
The following are the null and alternative hypotheses:
Na (Alternative hypothesis): There is a significant result on peel and juice extract as an
alternative tick killer agent.
No (Null hypothesis): There is no significant result on peel and juice extract as an alternative tick
killer agent.
E. Significance of the Study
The study is deemed important specifically to the following:
● Dogs
- They will be able to live without any insects in their body. It will also help to keep
them healthy and away from diseases that the tick may carry.
● Humans
- They will not have to worry about their pets as they have a solution that will kill the
ticks from the dog. It will also keep the owner of the dog exposing themselves from
diseases that may come from the tick.
● Famers
- The production of calamansi will increase if the result shows that it is effective in
eliminating ticks. Thus, it will help the farmers with their occupation.
F. Scope and Delimitation
This study mainly focused on the effectiveness of the calamansi extract against dog ticks.
It is mostly to see whether the juice and peel extract of calamansi can be an alternative tick
removal agent. With the result of this study, the juice and peel extract of calamansi kills the ticks
of dogs which are beneficial on the health of both dogs and humans.
The process included were crushing, squeezing, and pounding of the calamansi to get the
extract used in the solution. Based on the study of Armugam, Sundraraj, and Saleh (2016), the
peel extract will be heated first and then crushed into small pieces. After that, the crushed peels
are then mixed with the distilled water. The researchers will separate the crushed peels from the
solution by using a filter paper or a cloth.
The study will be conducted in Bogo City, Cebu in mid-January to the last week of
February of 2022. If any changes due to some issues, the researchers will try the finish the
experiment before the month of April of 2022.
It would be a big limitation for the researchers to conduct the study without supervisors
that will facilitate them while doing the experiment such as the analyzation of different kinds of
dog ticks. Hence, it is the reason why the researchers did not choose a specific dog tick, for such
a reason of the lack of equipment and knowledge for identifying a tick. The best solution for this
problem is by asking a coordinator from St. Louise de Marrilac College of Bogo for any help
regarding the experiment.

G. Definition of terms
The following terms were defined according to how they are being used in the study:
Ixodicide activity – A test to see whether the given variables can kill ticks.
Dog ticks – Ticks are small blood-sucking parasites that can transmit diseases. It resembles like a
spider with 8 legs.
Acarines – A takon of arachnids that contains mites and ticks.
Calamansi – An acidic citrus fruit.
Calamansi juice extract – The liquid inside the pulp of the calamansi.
Calamansi peel Extract - The waste material; obtained by a Steam Distillation from the peels of
calamansi.
Rapid action: the time taken for the mortality of ticks.

CHAPTER II
REVIEW OF RELATED LITERATURE
A. Related Literature
Ticks
To control the increasing growth of ticks especially on pets, many solutions were applied
and some contain toxic chemicals that can be harmful to animals, hence why the researchers aim
to study and examine the acaricidal of the calamansi. This chapter contains the related literature
and the related study that supports the calamansi extracts being an alternative tick remover.
Ticks are acarines that can be distinguished from insects by looking at their bodies. Ticks
have a segmented and spherical form body. Ticks are hematophagous; they are dependent on a
host for their survival. They find a warm and humid spot on the skin where they secrete a
cement-like substance that helps them to stay firmly on the skin. After that, they pierce the skin
with their sharp blades and use their denticulated harpoon to settle. Mostly the female ticks are
the one who ingest the blood compared to the male ticks as they have hard dorsal scutum
covering one third of their body. They usually lay their eggs on the skin of the host (N.
Boulanger et al., 2019).
More than 900 species of mites have been confirmed. Common dog ticks in the U.S. are
the lone star tick, american dog tick, brown dog tick and rocky mountain wood tick (Centers for
Disease Control and Prevention [CDC], 2019). Ticks that are commonly found in dogs,
especially in the Philippines are Rhipicephalus sanguineus (Boulanger et al., 2019). These ticks
are found on dogs, doghouses, walls of houses, and humans. Climate change has a negative
impact on the vector behavior of the worldwide dog tick Rhipicephalus sanguineus. However,
different kinds of dog ticks can still be found as it depends on the habitat of the host. These ticks
are a carrier of life-threatening bacterias and diseases.
Diseases transmitted by Ticks are Crimean-Congo hemorrhagic fever, spotted fever,
Kyasanur, lyme disease, Omsk Hemorrhagic fever, tick borne encephalitis outside the U.S. while
Anaplasmosis, Babesiosis, Borrelia mayonii infection, Borrelia miyamotoi infection, Bourbon
virus, Colorado tick fever, Ehrlichiosis, Heartland virus, Powassan disease, Rickettsia parkeri
rickettsiosis, Rocky Mountain spotted fever, Southern tick-associated rash illness, Tick Borne
relapsing fever, Tularemia, 364D rickettsiosis (CDC, 2020). Based on the study done in the
Philippines (Galay, 2018), Anaplasma/Ehrlichia spp, Ehrlichia canis and Rickettsia infection, are
found to be in dogs.
After a bite, a person might suffer and develop symptoms within a week. The most
common symptoms are fever/chills, aches and pains, and rashes. There are cases of tick paralysis
which are able to weaken or paralyze the body of the host. It is advisable to go to a healthcare
provider so they could evaluate the tick and symptoms (CDC, 2021).
As ticks and tick-borne diseases are becoming more well-known as a source of disease in
dogs and humans all over the world (Zanet, 2020), the use of insectides and pesticides for ticks
are advised. These products are a mix of many other chemicals. One of the most common
chemical used in insecticides is Permethrin. In the study of Prose et al. (2018), the permethrin-
treated clothing showed a significant result on preventing bites from ticks like Ixodes scapularis,
Amblyomma americanum, and Dermacentor variabilis ticks. Based on different studies,
Permethrin is commonly used as a cloth repellent against ticks in the wilderness.

While there are great insecticides especially for ticks, citrus fruits are great alternatives
since it is cheaper and commonly found in the household. The extracts of citrus fruits such as the
peel, pulp, and seeds, are a great source for cleaning products because of their acidic behavior
(Alisha et al., 2019).
Calamansi
Calamansi or Calamondin (× Citrofortunella microcarpa) or “limau kasturi” in Malaysia,
(Danping et al., 2012) is the fruit crop of the Philippines. It can also be found in different parts of
the world, especially in Southeast Asia. In the U.S. and Europe, it is mainly grown as
ornamentals. Calamansi is known as a shrub or small tree with a height of 3 to 5 meters high.
Calamansi fruit resembles a small, spherical lime with a diameter of 4.5mm, but can reach 45mm
in some cases. The fruit is usually with dark green leaves on the surface and seedless or with 1 to
5 seeds within. When ripe, the center pulp and juice are the color of a tangerine, with a very thin
orange peel (Morte, 2017). With a 5.52% citric acid concentration and a good flavor, it could
produce products that can be syrup, juices, and a cleaning product (Chew, S. K., 2018; Morton,
2013).
Calamansi has flavonoids and alkaloids, which were found to have insecticidal effects.
Calamansi also contains phytochemical extracts from citrus fruits guarantee repellency against
insects generally. It was found to be an effective larvicide against mosquitos (Aedes aegypti
(Linn.)) with determined lethal concentration 50 and lethal concentration 90 at 581 and 1009 mg
l-1 respectively (De Villa et al., 2012; Hollingsworth, 2005). Previous studies on Citrofortunella
microcarpa (lemonsito) extract mixed with onion extract also revealed an effective cockroach
killer (Pansit, Avila & Calumba, 2018) and they concluded that the nature of calamansi such as
the flavonoids, alkaloids helped a lot. This is the reason why calamansi is used as a cleaning
product and a great substitute of insecticides as it is highly acidic.
Calamansi is known for Vitamin C. Therefore, the juice is mainly used for consumption
as it has a low sugar content (glucose, fructose, sucrose), rich in aromatic components, high in
ascorbic acid (44.5 mg / 100 g), dehydroascorbic acid (2.2 mg / 100 g), citric acid (3.6%)
Therefore, fruits are used as freshly brewed tea. The high acid content reflected in the high citric
acid content is one characteristic of Calamansi juice. That's why Calamansi beverages are
popular these days (Chen et al., 2013). Citrus fruits such as Calamansi are mainly used in the
pulp and juice, but only the pulp is squeezed and needed. The remaining fruit or squeezed pulp
and pulp coatings are considered to be the largest source of citrus waste as they are discarded
after the juice has been extracted. Recently the peels and the juice of the calamansi were
chemically analyzed for their acidity. Finding a way to recycle this waste is beneficial to the
environment. Chen et al. (2013) found that calamansi rinds were composed mainly of volatiles
and phenolic acids. The volatiles were predominantly monoterpenes such as limonene, β-
myrcene, germacrene D, β-pinene, α-pinene, β-phellandrene and sabinene. Meanwhile, the
calamansi juice (Giovanelli et. al., 2020) have the volatiles components such as the limonene, 3-
methyl-2,5-furandione, furfural, ethyl acetate, butyl methyl ether, 5-methyl furfural, myrcene, 2-
heptyl acetate, phenylacetaldehyde, n-octanol, linalool, 4 terpineol, α-terpineol, γ-terpineol, α-
cadinol, valerianol, and cryptomeridiol.
Calamansi peel, a natural waste product from the fruit calamansi. It is utilized as
traditional medicine, and several natural components of this plant offer medical properties such
as to the dengue virus (De Villa et al. 2012), as a source of quality essential oils, and as an
alternate material organic micro-bead in cosmetic formulations. The presence of key

components, such as limonene, has greatly contributed to the aroma's antibacterial, antifungal,
and insect repellent properties.
Calamansi contains the compound D-Limonene or the limonene, which is proven to have
insecticidal activities (Yee, 2014). Limonene is a monocyclic monoterpene with a molecular
formula of C10H16 and a constituent found in several Citrus peels (Yee, 2014). The presence of
major compounds, limonene, had contributed immensely to the aroma, antibacterial, antifungal
and insect repellent properties. The extraction of d-limonene and the limonene is either with
pressure or steam from the peels of the fruits. The mechanism of D-limonene and limonene is to
dissolve a protective wax layer from the exoskeleton of arthropods, causing them to die from
suffocation (Direct Chem, 2001, as cited in Prado-Rebolledo, 2017). Thus, it acts as the major
natural component of calamansi peel used for pesticides and a common natural chemical in
essential oils.
B. Related Studies
Foreign Studies
Calamansi is a part of the rue family, Rutaceae. It is a family of plants that produces
citrus fruits. The uses of these citrus fruits as essential oils for repelling and destroying dog ticks.
The quality of citrus essential oils is highly dependent on the composition of limonene in the
essential oils found in various citrus fruits. Calamansi extract shows a fairly high level of
larvicidal activity, indicating that calamansi can be considered a powerful alternative insecticide.
(De Villa, 2012).
A study was conducted to show the effectiveness of the lime and black pepper plant
essential oils on cattle ticks (Vinturelle et al., 2017). In the study, the researchers dissolve the
essential oils in an aqueous solution of 2% of dimethyl sulfoxide to get their experimental
solution to test on ticks. They have tested on the concentrations of 2.5, 5.0, and 10% of each
plant oils, respectively. On the combination of oils, both oils have the concentrations of 5%:5%,
2.5%:2.5%, and 1.25%:1.25%. The female ticks are the subject of the study. On their findings,
essential oil of lime showed a higher ixodicide activity on the female ticks with 100% mortality
rate and 96% on black pepper plant essential oil. Another study done on cattle ticks showed that
the essential oil containing citrus fruit, kaffir lime, have a higher ixodicide activity compared to
the West Indian lemon grass (Shezryna et al., 2020).
In the study of Armugam, Sundraraj, and Saleh (2016), they have used orange peel to test
whether it could kill ticks. They have put the peels into a conventional oven at a temperature of
50 ℃ and crushed it into small pieces which are used in the tincture of distilled water to gather
the acid from the peels. The researchers tested on 50%, 75%, and 95% concentrations. Then,
they purified it on a rotary evaporator under a temperature of 60°C. They found that the 95%
solution with heat compressed of peels is effective on killing ticks. The concentration of 95%
shows 98% of tick mortality rate with P < 0.05. It was also shown that the heat compressed
solution had a mortality rate of 78% in the time of the first 15 minutes and finished at 30 minutes
of application. They then concluded that their findings are similar to Kamal et al (2012), the
findings are that oils derived from plants materials that is dried in the oven had a better yield than
oils from fresh materials.
On the discussion section of all studies, they have discussed that the reason of why citrus
fruits perform better is because of the acids on the fruits. In the study of Vinturelle et al. (2017),
they have studied the chemical composition of both essential oils. Essential oil of lime showed
the chemical of limonene (50.3%), β-pinene (14.4%), γ-terpinene (11.7%). In the study of
Armugam, Sundraraj, and Saleh (2016), they also stated that the peel of orange has a higher
concentration of active compounds such as the d-limonene. In the study of the Shezryna, et al.
(2020), they have also stated that the reason why the citrus fruit performed better is because of
the acid also such as the limonene and α-pinene. The volatiles and phenolic acids found on the
citrus fruits might contribute on the mortality of ticks which calamansi has some natural
components that can kill ticks.
Tick-borne diseases remain the leading cause of illness and death worldwide. Many
chemicals have been used to control ticks. They are normally safe, but some of them may be
responsible for toxic reactions in animals, reduced effect in the long run due to development of
resistant strains of ticks, environmental pollution and human health hazards. Agricultural waste
such as Citrofortunella microcarpa peel and its pulp juices has been exploited and highly
potential as anti-tick. The purpose of this study is to assess the effect of Citrofortunella
microcarpa peel and juice extract against dog ticks that was presented in the related literature.
Theoretical Framework
Carry diseases that can affect the

Dog ticks health of the host (humans and
dogs).
The use of calamansi extract to

kill dog ticks.
Volatiles acids such as limonene
Compound that can be used as

tick killer.
Fig 1.1
Theoretical Framework of the Study
Dog Ticks are vectors of pathogens that can cause illness to the hosts. There are many
diseases that can affect the host such as anemia to dogs, Lyme diseases, spotted fever, and many
more. The solution is to kill the carrier to stop the spread of the tick-borne diseases with the help
of calamansi extract. Calamansi contains volatiles acids that have the ability to kill. After the
study, the compounds of the extract of calamansi peel and juice can be used as an agent to kill
tick.
Conceptual Framework
Input Process Output
 The nature of the  Testing the extracts  Calamansi peel

calamansi. of calamansi to and juice extract
substitute tick killer can be used as a
 Have an effective
solution. tick killer.
extraction method
of calamansi juice  To record the time it
from the pulp. takes to kill the

ticks.
 Bought calamansi
essential oils via
online.
Fig 1.2
Conceptual Framework of the Study
The model of Input, Process, and Output will served as the process that the researchers
would follow. The nature of the calamansi is the primary source of the experiment and will serve
as the input. As the experiment is conducted, the time duration and the extracts of calamansi peel
and pulp juice will be tested for the mortality of the ticks. The experiment will produce
calamansi peel and pulp juice extract as a dog tick killer.

CHAPTER III
METHODOLOGY
This chapter covers the research design and methodology, including the sampling,
population, establishing rigour during and after data collection, ethical considerations and data
analysis.
A. Research Design
The Experimental Research Design was used for this study where the researchers
manipulate the independent variable on the different product formulations and a control group
that does not used the formulations. In this case, the researchers have different concentrations of
calamansi peel and calamansi juice to kill the kicks and a control group that uses a commercial
tick killer brand. The dog ticks are dropped on different concentration of tick killers. The
experiment sought to obtain the mortality rate of ticks in a given calamansi extract solution. The
researchers would also test the duration of the mortality rate of ticks in the calamansi extract
solution. The experimental research design allows the researchers to see whether the extract of
calamansi peel and juice is effective on killing the ticks.

B. Subject of the Study
In this experimental study, six (6) dogs are selected that are infected with ticks. The ticks
are the subject of the experiment. The researchers would collect a total of thirty-five (35) ticks as
the total population of the study.

C. Research Instrument to be Used
Making a solution from calamansi extracts is the research instrument that will be used in
the study. To gather the data of the mortality rate and duration of the ticks on the solution, the
researchers would drop the ticks into the two experimental solutions and one on the commercial
tick killer brand. The researchers would also use a watch or a stopwatch on the phone to record
the duration of the effectiveness of calamansi extracts to kill the ticks.

D. Procedure of Data Gathering
The procedure of data gathering is done by the obtaining the dog ticks, extracting the
citrus fruits of its natural components, and the purchase of calamansi peel essential oil via an
online shop.
The ticks would be gathered and obtained by carefully going through the skin of the dogs.
The researchers would use their hands to take the tick away from the dog. Then, the ticks are
kept in a bag until the sample size has been achieved; a month before the experiment. After
acquiring the sample size, the ticks are then transported from Barangay Argawanon, San
Remegio, Bogo City to Barangay Cogon, A & B Subdivision, Bogo City, Cebu, Cebu for the
experiment and the evaluation of the ticks.
For the extraction of calamansi juice, the hands are used as an instrument to squeeze the
juice out from the pulp. For the calamansi peel essential oil, the researchers would purchase it via
online shops. The researchers would interview and interrogate the shop about the product
whether it is right for the experiment. The questions were:
1. Is the calamansi peel used for the essential oil?
2. Is it diluted with other substance?
The researchers would drop the ticks on the solution with different concentrations to test
if the calamansi extracts can kill the tick. The researchers would drop five (5) ticks on each
solution with different concentrations. The research would take the ticks out every couple of
minutes to check any movements from the ticks. The evaluation of ticks on the different
solutions would be based on any signs of movements. The ticks that are unable to move were
considered dead (Food and Agriculture Organization - FAO, 1984). The researchers would use a
stopwatch on a watch or on their phone to record the duration of mortality. All the data will be
written in a piece of paper to avoid the loss of information.

E. Statistical Treatment
The statistical treatment that the researchers will use is the percentage formula to find the
mortality of the ticks. The numerical data gathered from the experiment is converted into
percentages to form the conclusion of the study.
Percentage Formula:
F
P≡ x 100 %
N
Where in:
P = Percentage
F = the number of dead ticks
N = total number of ticks
100 = constant value

F. Collection of Materials
Materials:
1. 1kg of Calamansi
2. Cup and spoon
3. Calamansi Essential Oil
4. Commercial tick kill brand
The materials such as the cups, spoon, pen and paper will be bought at the Gaisano
located at P. Rodriguez St, Bogo City, Cebu, Philippines. If the researchers could not find the
material needed for the experiment, they would have to go to another shop or on the online
market. The calamansi essential oil would be bought via online stores.
Calamansi
The researchers will buy the 1kg of calamansi on the Bogo Public Market located at
Camtecson, Garian, Dakbayan sa Bogo, 6010 Lalawigan ng Cebu, Philippines.
Commercial brand of Tick killer
The researchers will one (1) or two (2) solution of tick killer in the market or in a
veterinarian shop within Bogo City, Cebu, Philippines.

G. Preparation of Treatment
The preparation of treatment will be the three (3) levels of the treatment on the solutions.
The researchers will have a three setups in this experiments. The first set is the calamansi peel
solution in the concentration of 50%, 75%, and 95% of 15ml, respectively. The second set is the
calamansi juice from the pulp in the concentration of 50%, 75%, and 95% of 15ml, respectively.
The third set is the commercial tick brand that will also be in the concentration of 50%, 75%, and
95% of 15ml.
The values of the three (3) concentrations:
50% of 10 ml = 5 ml
75% of 10 ml = 7.5 ml
95% of 10 ml = 9.5 ml
CHAPTER IV
PRESENTATION AND INTERPRETATION OF DATA
This chapter contains the data gathered from the experiment to provide the data needed.
A set of three levels of the treatment for the solution to provide the effectiveness of the
calamansi juice and peel extracts against dog ticks. Researchers obtained the best concentration
to kill the dog ticks and the most effective extract as dog ticks remover. The formula
P=f/n×100% is being used to get the mortality rate of the ticks.

GRAPH OF RAPID ACTION AND MORTALITY RATE OF TICKS
120
100
Calamansi juice: 50% concentra-

80 tion
tion
60 tion
Calamansi Essential Oil: 50%
concentration
40 Calamansi Essential Oil: 75%
concentration
Calamansi Essential Oil: 95%
concentration
20 Commercialize Tick Killer: 95%
concentration
0
S S S S S S
UTE UTE UTE UTE UTE UTE
IN IN IN IN IN IN
M M M M M M
15 30 45 60 75 90
Fig. 2.0
The bar graph shows the mortality rate at a given time frame. In 15 minutes, the
concentration of 50%, 75%, and 95%, calamansi juice extract showed a mortality rate of 20%
while the calamansi peel extract at each concentration and commercialized tick killer showed a
100% of mortality rate. In 30 minutes, only the concentration of 50% calamansi juice extract
showed a mortality rate of 40%. In 45 minutes, the 20% concentration of calamansi juice extract
showed a mortality rate of 40% while the concentration of 75% and 95% showed an 80% and
60% mortality rate, respectively. In the time frame of 60 and 75 minutes, there were no ticks that
died from the solution. Lastly, in 90 minutes, the 95% concentration of calamansi juice extract
showed a 20% mortality rate.

TABLE OF MORTALITY RATE AND RAPID ACTION OF TICKS
TYPE OF CONC NUMB NUM NUM NUM NUM NUM NUM TOTAL
EXTRAC ENTR ER OF BER BER BER BER BER BER MORTAL
T ATION TICKS FOR FOR FOR FOR FOR FOR ITY
/S INTRO TICK TIC TICK TIC TIC TIC
DUCE S TO KS S TO KS KS KS
D DIE TO DIE TO TO TO
IN 15 DIE IN 45 DIE DIE DIE
MINU IN 30 MINU IN 60 IN 75 IN 90
TES MIN TES MIN MIN MIN
UTE UTE UTE UTE
S S S S
Calamansi 5 ml 5 20% 40% 40% 0% 0% 0% 100%
Juice 7.5 ml 5 20% 0% 80% 0% 0% 0% 100%
9.5 ml 5 20% 0% 60% 0% 0% 20% 100%
Calamansi 5 ml 5 100% 0% 100%
Peel 7.5 ml 5
Essential
9.5 ml 5
Oil
Commerci 5 ml 5 100% 0% 100%
alize Tick 7.5 ml 5

Killer
9.5 ml 5
Fig 3.0
The table consists of types of extracts which are the commercialized tick killer,
Calamansi peel extract, and calamansi juice extract which are tested in three concentrations; 5ml,
7.5ml, and 9.5ml. The mortality rate of calamansi juice extract shows 20% with the
corresponding percentage on each concentration in the first 15 minutes. On the other hand, the
commercial tick killer and the calamansi peel extract showed a 100% mortality rate in the first 15
minutes. The 5ml of the calamansi juice concentration showed a mortality rate of 40% in both
time frames of 30 minutes and 45 minutes. While the 7.5ml concentration showed an 80%
mortality rate and 60% for the 9.5 ml concentration. In the time frame between 60 and 75
minutes, the mortality rate is zero because there were no zero ticks dead during that time frame.
Lastly, the 9.5 ml concentration of calamansi juice extract showed a 20% mortality rate at the 90
minutes mark.
CHAPTER V
DISCUSSION, CONCLUSION, AND RECOMMENDATIONS
SUMMARY
This is an experimental study on both calamansi peel and calamansi juice extracts to test
their ixodicide activity against dog ticks. The dog ticks are dropped into three concentrations of
both extracts: 50%, 75%, and 90%. The main purpose of the study is to test the ixodicide activity
of both calamansi peel and juice extracts against dog ticks in terms of rapid actions and mortality
rate.
FINDINGS
The following are the discussion of findings from the data collected:
Problem no.1: What are the mortality rate of ticks of both calamansi juice and peel extracts?
The calamansi juice and peel extract were effective in killing the ticks in under 90
minutes with a mortality rate of 100%. The calamansi peel extract achieved a 100% mortality
rate in 15 minutes whereas the calamansi juice achieved at 90 minutes.
The major reason for the effectiveness of both extracts is the presence of limonene.
According to the research study by Chen et. Al. (2013), the three major volatile components
present in the calamansi essential oil from the peel are limonene, myrcene, and germacrene D.
91.15-92.70%, 2.08-2.46%, and the 0.54-1.21% were the percentages of limonene, myrcene, and
germacrene D, respectively. Whereas the calamansi juice, the three major volatile components
are limonene, 3 methyl 2,5 furandione, and furfural. 26.1%, 17.8%, and 11.6% were the
percentage of limonene, 3 methyl 2,5 furandione, and furfural, respectively (Giovanelli et. Al.
2020).
Limonene is an olefin hydrocarbon that can occur in two optical forms. Limonene is used
in insect repellent and insecticide. Limonene dissolves the wax layer of the exoskeleton of the
arthropods causing the ticks to suffocate to death (Direct Chem, 2001, as cited in Prado-
Rebolledo, 2017). Thus, the reason for the effectiveness of both calamansi extracts.
Problem No. 2: In comparison with the control group, what are the significance results of both
calamansi juice and peel extracts based on the mortality rate and rapid action?
There are no significant results between the control group and the calamansi peel extract
where both solutions achieved a 100% mortality rate in 15 minutes. On the other hand, there are
significant results between the control group and the calamansi juice extract where the calamansi
juice extract took 90 minutes to achieve a 100% mortality rate.
The active ingredients used in the commercialized tick management product are Fipronil,
Radix stemonae extract, and glycerol. Both the Fipronil and Radix stemonae extract has been
studied on their ixodicide on dog ticks (Ibarra-Velarde et. al., 2020; Lan et. al., 2016). The
reason for the effectiveness of fipronil, a phenylpyrazoles, is that it targets the GABA-gated and
inhibits glutamate-activated chloride channels on insects (Narahashi et. al., 2010).
The concentration of each active ingredients are unknown. With both calamansi extracts,
limonene is the volatile components that has the ability to kill dog ticks. From the study of Chen
et. Al. (2013), peel extract derived from essential oil have the concentration of 90% while the
juice extract have 26% concentration of limonene (Giovanelli et. Al., 2020). Thus, 90%
limonene have a similar effect of the commercial tick killer product than the 26% calamansi
juice extract. Therefore, only the calamansi juice extract have a significant result compared with
the commercialized tick product. However, further studies needs to identify the cause between
the two calamansi extracts and the commercialized tick product.
Problem No. 3: What is the best concentration for the calamansi juice and peel extract to the
ticks in dogs? Based on the study of Armugam, Sundraraj, and Saleh (2016), the concentrations
used are:
a) 50% of 10ml
b) 75% of 10ml
c) 95% of 10ml
The best concentration for calamansi juice was 75% of 10 ml which achieved the highest
mortality rate in all time frames. On the other hand, the three concentrations (50%, 75%, and
95%) tested from calamansi peel extract were effective and achieved a mortality rate of 100% in
15 minutes.
From the study by Armugam, Sundraraj, and Saleh (2016), the best concentration was the
95% concentration. There is no connection between the study of Armugam, Sundraraj, and Saleh
with the calamansi juice extract apart from the calamansi peel extract which has the connection
with the 95% concentration. However, it still depends on the composition and concentration of
limonene present in the agent which is unknown based on the study of Armugam, Sundraraj, and
Saleh.
Problem No. 4: Which of the two extracts performs well as a dog tick killer?
In terms of rapid action and mortality rate, the calamansi peel extract performed better.
Calamansi peel extract is able to terminate the ticks in 15 minutes while the calamansi juice goes
on for 90 minutes. This is due to the concentration of limonene in the extracts. Limonene
obtained in steam distillation contains more than 90% limonene (Nikfar & Behboudi, 2014)
which is also present in the study of Chen et. Al. (2013). The calamansi juice contained a lower
concentration of more than 26% (Giovanelli et. Al. 2020). The difference in concentration of
limonene in an extract is responsible for one extract performing better than one extract.
Problem No. 5: How long do ticks last in the solution before they completely die?
The ticks last 15 minutes in the calamansi peel extract whereas the ticks in the calamansi
juice last over 60 minutes to show an ixodicide activity. With the different concentrations treated
on calamansi juice, the 50% and 75% concentrations took 45 minutes to kill the ticks. The 95%
concentration took 90 minutes to kill the ticks. All the concentrations treated on the calamansi
peel extract took 15 minutes to kill the ticks.
CONCLUSION
Both the calamansi extracts showed an ixodicide activity on dog ticks. Moreover,
Calamansi extract performed better in all concentrations than the calamansi juice extract. Further
research on the calamansi extracts to be widely used in anti-tick products.

RECOMMENDATIONS
The following recommendations are suggestions that the future researchers should
implement and improve in the research:
1. The researchers recommend the use and acquiring sets of equipment needed for the
making of essential oil. This would allow future researchers to modify the equipment and
ingredients of the essential oil.
2. The researchers recommend in future studies to increase the frequency of ticks to be
tested. Bigger sample size would increase the validity of the research.
3. The researchers recommend taking the volatile components on both extracts.

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APPENDIX A: AN IMAGE OF ONE OF THE DOG TICKS
Fig. 4.0: An image of one of the dog ticks used in the experiment.
APPENDIX B: CALAMANSI PEEL EXTRACT ON DIFFERENT CONCENTRATIONS
Fig. 5.0: An image of the calamansi peel extract that was treated with different concentrations.
APPENDIX C: CALAMANSI PEEL EXTRACT ON DIFFERENT CONCENTRATIONS
WITH DOG TICKS
Fig. 6.0: An image of dog ticks on the calamansi peel extract treated with different
concentrations.
APPENDIX D: CALAMANSI JUICE EXTRACT ON DIFFERENT CONCENTRATIONS
Fig. 7.0: An image of the calamansi juice extract that was treated with different concentrations.
APPENDIX E: CALAMANSI JUICE EXTRACT ON DIFFERENT CONCENTRATIONS
WITH DOG TICKS
Fig. 8.0: An image of dog ticks on the calamansi juice extract treated with different
concentrations.
APPENDIX F: CONTROL GROUP WITH DOG TICKS
Fig. 9.0: An image of dog ticks on the commercialized tick killer solution.
CURRICULUM VITAE
PERSONAL INFORMATION
Name: Serdoncillo, Abegail Dela Cruz
Birthdate: November 21, 2003
Birthplace: Bogo City
Age: 18
Status: Single
Nationality: Filipino
Religion: Roman Catholic
Address: Gairan, Bogo City, Cebu
EDUCATION
Elementary School: Bogo Central School ll
Junior High School: St. Louise de Marillac College of Bogo
Senior High School: St. Louise de Marillac College of Bogo
Name: Rose Zael C. Bojos
Birthdate: October 14, 2003
Birthplace: Banban, Bogo City, Cebu
Age: 18
Status: Single
Address: Purok San Jose, Banban, Bogo City, Cebu

EDUCATION
Elementary School: Cagtina-e Elementary School
Name: Pilapil, Cy, Fernandez
Birthdate: August 09, 2003
Birthplace: Bogo, Medillin Medical Center
Age: 18
Status: Single
Religion: Seven Day Adventist
Address: Argawanon, San remigio, Cebu
EDUCATION
Elementary School: BOMEDCO Elementary School
Name: Inot, Jacqueline Phoebe
Birthdate: July 26, 2001
Birthplace: Cebu provincial Hospital – Bogo City
Age: 20
Status: Single
Religion: Christianity
Address: M.h St. del Pilar, Bogo City, Ceby
EDUCATION
Elementary School: Bogo Central School 2
Name: Villegas, Blessed Dawn
Birthdate: May 19, 2003
Birthplace: Perpetual Succor Hospital
Age: 18
Status: Single
Religion: Catholic
Address: Don Virgillo Gonzales, Medellin, Cebu
EDUCATION
Elementary School: Don Virgillo Elementary School
Name: Ruelan, Christian John
Birthdate: December 03, 2003

Birthplace: Medellin Medical Hospital
Age: 18
Status: Single
Address: A & B Subdivision, Cogon, Bogo City, Cebu.
EDUCATION
Elementary School: San Roque College de Cebu-North Campus and Asian International Private
School
Junior High School: Asian International Private School and St. Louise de Marillac College of
Bogo
Name: Torres, Meg
Birthdate: September 27, 2003
Birthplace: Cebu City
Age: 18
Status: Single
Address: Pelaez Street, Bogo City, Cebu
EDUCATION
Elementary School: San Roque College de Cebu-North Campus
Junior High School: San Roque College de Cebu-North Campus

Name: Masong, Anche Marie Cueva
Birthdate: February 17, 2004
Birthplace: Margarita Birthing Center, Gorordo Avenue, Cebu City
Age: 18
Status: Single
Address: Gairan, Bogo City, Cebu
EDUCATION
Elementary School: Bogo Central School III

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Ixodicide Activity of Citrus x Microcarpa (Calamansi) Peel and Juice Extract on Dog Ticks

St. Louise de Marillac College of Bogo

Ixodicide Activity of Citrus x Microcarpa (Calamansi)

Peel and Juice Extract on

Ruelan, Christian John L.

Bojos, Rose Zael

Inot, Jacqueline Phoebe

Masong, Anche Marie Cueva

Serdoncillo, Abegail Dela Cruz

Villegas, Blessed Dawn

calamansi juice extract.

calamansi peel extract, rapid action

that helped us at every stage of the thesis-writing process.

individual who gathered ticks, Mrs. Zenith Ayn Pilapil.

persevere in our intellectual endeavor.

A. Background of the Study………………………………………………………………1

B. Statement of the Hypothesis …………………………………………………………..3

E. Significance of the Study……………………………………………………………...6

F. Scope and Delimitation……………………………………………………………......7

CHAPTER II: REVIEW OF RELATED LITERATURE………………………………………..9

CHAPTER III: METHODOLOGY……………………………………………………………...18

Subject of the Study……………………………………………………………………...20

Research Instrument to be Used…………………………………………………………21

Procedure of Data Gathering…………………………………………………………….22

CHAPTER IV: PRESENTATION AND INTERPRETATION OF DATA…………………….27

CHAPTER V: DISCUSSION, CONCLUSION, AND RECOMMENDATIONS……………....31

APPENDIX A: AN IMAGE OF ONE OF THE DOG TICKS………………………………….42

APPENDIX B: CALAMANSI PEEL EXTRACT ON DIFFERENT CONCENTRATIONS….43

APPENDIX C: CALAMANSI PEEL EXTRACT ON DIFFERENT CONCENTRATIONS

WITH DOG TICKS…………………………………………………………………………….44

APPENDIX D: CALAMANSI JUICE EXTRACT ON DIFFERENT CONCENTRATIONS...45

APPENDIX E: CALAMANSI JUICE EXTRACT ON DIFFERENT CONCENTRATIONS

WITH DOG TICKS……………………………………………………………………………46

APPENDIX F: CONTROL GROUP WITH DOG TICKS……………………………………...47

A. Background of the Study

According to N. Boulanger et al. (2019),”ticks are acarines that may be distinguished

experiencing warmer and/or longer summers, consequently increasing the risk of

peel extract of juice to kill the dog's tick.

Calamansi, scientifically known as Citrofortunella microcarpa, is a popular citrus fruit

used to cure coughs and as a natural anti-inflammatory medication.

B. Statement of the Problem

and cause skin irritation.

help the health condition of many dogs.

calamansi against dog ticks. Specifically, it aimed to answer the following:

4. Which of the two extracts performs well as a dog tick killer?

This study aims to determine and answer the following:

c) To investigate the effectiveness of different concentrations of juice and peel

The following are the null and alternative hypotheses:

Na (Alternative hypothesis): There is a significant result on peel and juice extract as an

alternative tick killer agent.

E. Significance of the Study

The study is deemed important specifically to the following:

diseases that may come from the tick.

F. Scope and Delimitation

solution by using a filter paper or a cloth.

experiment before the month of April of 2022.

regarding the experiment.

spider with 8 legs.

Acarines – A takon of arachnids that contains mites and ticks.

Calamansi – An acidic citrus fruit.

Rapid action: the time taken for the mortality of ticks.

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