Utilization of MRAV (Marigold (Tagetes erecta L) Extract, Red Rose (Rosa)

Extract, and Aloe Vera (Aloe barbadensis miller) gel) Mixture as Tick

(Rhipicephalus sanguineus) Spray Pesticide for Aspin Dogs (Canis lupus

familiaris): In Vitro Study

Researchers:

JULIANA KEIRA C. QUINTO

REYMARK C. SAN JOSE

JUSTIN LLOYD B. SANCHEZ

Submitted to:

MS. ROSE ANN G. BAÑAS

MR. OLIVER JOHN M. TRES REYES

In-Partial Fulfillment of the Requirements in Inquiries Investigation and Immersion

(III)

and Capstone Project

Senior High School

Camarines Sur National High School

June 26,2023
 i

APPROVAL SHEET

The subject research hereto attached, entitled Utilization of MRAV

(Marigold (Tagetes erecta L) Extract, Red Rose (Rosa) Extract, and Aloe Vera (Aloe
barbadensis miller) gel) Mixture as Tick (Rhipicephalus sanguineus) Spray Pesticide
for Aspin Dogs (Canis lupus familiaris): In Vitro Study prepared and submitted by
Justin Lloyd B. Sanchez, Juliana Keira C. Quinto, Reymark C. San Jose in partial
fulfilment of the requirements for Inquiries, Investigations and Immersion and Capstone
Project is hereby accepted.

MICHELLE D. LEE RAYNE JULLIANE M. LAGRIMAS

Panelist Panelist

_____________________ _____________________
Date Signed Date Signed

ANCHOY G. BRAZAL
Chairperson

_____________________
Date Signed

Approved as partial fulfilment of the requirements for Inquiries, Investigations and

Immersion and Capstone Project

ROSE ANN G. BAÑAS OLIVER JOHN M. TRES REYES

Research Adviser Capstone Project Adviser

_____________________ _____________________
Date Signed Date Signed

Respectfully submitted to the Assistant Principal of Senior High School for

approval.
MELISSA B. BOBOS
Assistant Principal II, SHS

_____________________
Date Signed

SULPICIO C. ALFEREZ III, PhD

Principal IV

_____________________
Date Signed
 ii

CERTIFICATION

This is to certify that the subject research entitled Utilization of MRAV

(Marigold (Tagetes erecta L) Extract, Red Rose (Rosa) Extract, and Aloe Vera (Aloe

barbadensis miller) gel) Mixture as Tick (Rhipicephalus sanguineus) Spray Pesticide

for Aspin Dogs (Canis lupus familiaris): In Vitro Study prepared and submitted by

Justin Lloyd B. Sanchez, Juliana Keira C. Quinto, Reymark C. San Jose has been

examined, edited and found to be in accordance with the suggestions and

recommendations made by the panel of evaluators.

ROSE ANN G. BAÑAS

Research Adviser

_____________________
Date Signed

OLIVER JOHN TRES REYES

Capstone Project Adviser

_____________________
Date Signed
 iii

ACKNOWLEDGEMENT

The completion of this research could not have been accomplished without the

participation and cooperation of numerous individuals, whose names may not all be

enumerated. The researchers sincerely appreciate and gratefully acknowledge their

contributions. The researchers would like to express their deepest gratitude, particularly

to the following:

First and foremost, the researchers extend their gratitude to the Almighty God for

the enduring grace, guidance, and protection bestowed upon them throughout this

research project.

The researchers would also like to express their sincere appreciation to their

research adviser, Ms. Rose Ann G. Bañas, for her unwavering support, patience,

valuable suggestions, motivation, and immense knowledge. Her guidance has been

instrumental in every aspect of the research.

The researchers would like to thank their parents, who have consistently supported

them financially in accomplishing this study and have provided guidance,

encouragement, and inspiration throughout their academic journey. The researchers

offer a heartfelt thank you for their unwavering presence and constant guidance.

The researchers are also grateful to their fellow classmates and friends for

engaging in stimulating discussions, offering valuable suggestions, providing moral

support, and enduring sleepless nights as they worked together to meet deadlines, while

also creating cherished memories.

 iv

The researchers extend their thanks to Mr. Oliver John Tres Reyes and Mrs.

Michelle D. Lee for their advice, support, and guidance throughout the course of the

study, as well as for their valuable suggestions and direction.

Special thanks are due to Ms. Charlene C. Esmenda, Ms. Angel Grace Barra, and

Ms. Samantha Ysabelle T. Saballo for providing tick samples required for the study. The

researchers also appreciate Ms. Meriam Perlyn R. Quinto for providing them with

equipment. Furthermore, the researchers would like to express their gratitude to Mr. Nel

Ajee B. Mendioro for lending them a digital vernier caliper, which was used to measure

the sizes of the tick samples. Lastly, the researchers are grateful for the voluntary

participation of Ms. Alexis Nicole B. Ayson and Mr. Dunkan T. Mapa in the experiment,

as well as Ms. Maya Alexandra A. Rapi for her assistance in documenting the

experiment.

The researchers apologize to any individuals whose names may have been

inadvertently omitted but who were instrumental in their research project. Their

contributions are sincerely appreciated.

Once again, the researchers express their deepest gratitude to all those mentioned

above for their valuable contributions, support, and guidance, which have significantly

enhanced the outcome of this research project.

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TABLE OF CONTENTS

Page No.

Title Page

Approval Sheet…………………………………………………………………….........i

Certification……………………………………………………………………….……ii

Acknowledgment………………….…………………………………………...….…...iii

Table of Contents……………………….…………………………...…….……..…......v

Abstract…………………...……………………………………………....…….….....viii

The Problem and Its Setting

Introduction………...……………………………………………..………...…......1

Statement of the Problem……………………………...…….……….…….……...6

Hypotheses………...……………………………….…………………..……….....7

Significance of the Study…………………………….………..…..…………….....7

Scope and Delimitation………………………………………………..…..………9

Locale of the Study……………………………………………...…….…….........10

Risk Assessment………………………….………..………………….….……....11

Conceptual Framework………………………………...…..……….…..….…….13
 vi

Methodology

Research Design……………………………………………..…………..……….14

Research Instruments…………………………………………..……...……...…..16

Experimental Procedure………………………………….........………..………..16

Data to be Gathered………………………………………….…………….……..21

Statistical Analysis………………….…………………...……….……….……...24

Data Presentation, Interpretation and Discussion………………………….…………25

Summary of Findings, Conclusions and Recommendations………...………..….......30

BIBLIOGRAPHY…………………………………………………………….……….34

APPENDICES

A. Data and Statistical Analysis…………………………………....….…………36

B. Documentation………………...………………...…………..………………...42

C. Communication Letter……………………………..……..…………………...53

D. Financial Report………………...……………………………..……………....62

E. Journal………………...……………….……………………………...…….....66
 vii

ABSTRACT

This study investigated the effectiveness of marigold, rose, and aloe vera

ethanolic extracts as alternative pesticides for ticks. The aim of the research was to

evaluate the mortality rate and motility rate of a tick pesticide known as MRAV. The

findings indicated that Treatment 1, which involved a 100% concentration of MRAV

extract, was the most successful in eradicating dog ticks (Rhipicephalus sanguineus),

with a mortality rate of 100% and an average motility time of 3.276 minutes. Treatment

2, utilizing a 75% concentration, also demonstrated significant efficacy, resulting in a

mortality rate of 100% and an average motility time of 5.88533 minutes. Similarly,

Treatment 3, using a 50% concentration, exhibited a mortality rate of 100% and an

average motility time of 5.692 minutes. Treatment 4, with a 25% concentration, showed

an 86.67% mortality rate and an average motility time of 4.09667 minutes. The positive

control treatment resulted in a mortality rate of 46.67% and an average motility time of

5.48133 minutes.

In summary, the utilization of marigold, rose, and aloe vera treatments displayed

a moderate effect in reducing the mortality rate of dog ticks (Rhipicephalus sanguineus).

These treatments proved to be more effective in reducing tick mortality compared to the

control group, indicating a significant difference. However, their impact on tick motility

was minimal, suggesting that the treatments did not significantly affect the movement

of the ticks. Among the treatments, the 100% concentration of MRAV extract

demonstrated the highest effectiveness in terms of both mortality and motility rate.
 viii

Statistical analysis confirmed a significant difference in the mortality rate

between the treatments and the control group. However, no significant difference was

observed in the motility rate. Therefore, the study recommends conducting rigorous

testing and exploring essential oils as potential alternatives to ethanolic extracts.

This research significantly contributes to the understanding of natural tick

pesticides and provides valuable insights for the development of effective strategies

against tick infestations. The findings underscore the potential of MRAV extract as a

promising solution for tick control and highlight the importance of conducting

comprehensive testing and comparisons with commercial products to ensure credibility

and validity. The study's significance lies in its contribution to the field of pest control,

offering safer and more environmentally friendly alternatives to conventional

insecticides. Further research in this area is warranted to fully explore the potential of

these natural extracts as tick control agents and to establish standardized testing

protocols.

Keywords: Rose (Rosa), Aloe Vera (Aloe barbadensis miller), Marigold (Tagetes

erecta L), Pesticide, Brown Dog Tick (Rhipicephalus Sanguineous), Extract, Aspin

Dogs (Canis lupus familiaris),

 1

CHAPTER 1

THE PROBLEM AND IT'S STATEMENT

Introduction

Marigold (Tagetes) is native to America, and its cultivation currently extends

to other countries in Africa, Asia, and Europe. Many species of this genus, such as T.

minuta, T. erecta, T. patula, and T. tenuifolia, are cultivated as ornamental plants and

studied for their medicinal properties on the basis of their use in folk medicine. Different

parts of the Tagetes species are used as remedies to treat various health problems,

including dental, stomach, intestinal, emotional,and nervous disorders, as well as

muscular pain, across the world. [1]

Furthermore, these plants are studied in the field of agriculture for their

fungicidal, bactericidal, and insecticidal activities. The phytochemical composition of

the extracts of different Tagetes species parts are reported in this work. These

compounds exhibit antioxidant, anti-inflammatory, and enzyme inhibitory properties.

Cultivation and the factors affecting the chemical composition of Tagetes species are

also covered. [1]

In the current work, available literature on Tagetes species in traditional

medicine, their application as a food preservative, and their antimicrobial activities are

reviewed.[1]

Rose (Rosa), on the other hand, is used as an ornamental plant in the gardens,

indoors, and mostly on sites where occasions, such as wedding ceremonies are held.

Rose has been also used for commercial perfumery, commercial cosmetics, and

commercial cut flower crop. Rosa is used for medicinal uses as well, such as antiseptic,

and antioxidant, and is a rich source of vitamins A, B3, C, D, and E. Rose emits a sweet

smell which is an instant deterrent for insects.[2]

 2

Aloe vera (Aloe barbadensis miller) has a variety of healthful compounds,

mainly found in its gel which includes most of the important bioactive chemical

compounds in the plant, including vitamins, minerals, amino acids, and antioxidants.

Aloe vera has some antifungal properties that may help clear up certain skin conditions.

Aloe vera helps soothes dry and itchy skin.[3]

According to numerous research, Aloe vera has been linked to wound healing

and prevention. Aloe vera can be applied topically to dermal injuries (such as burns,

frostbite, skin infections, surgical wounds, inflammation, herpes ulcers, diabetic foot

ulcers, pressure sores, and chronic wounds) to help with healing and prevent ulcers from

forming. Aloe vera is an excellent choice for use in wound dressings. The majority of

studies focused on burn injuries. The traditional treatment for burns is aloe vera. Burn

wound healing was examined in five trials. Aloe vera outperformed silver sulfadiazine

1% ointment, petroleum jelly gauze dressing, and framycetin cream in these

investigations. Additionally, it sped up healing, avoided wound infection, and stopped

itching and redness.[3]

Lyme disease in dogs has been reported in every state in the United States,

although some regions are significantly more likely than others to have ticks that spread

the bacterium. Tick infection rates might range from zero to fifty percent. The northeast

of the country, particularly the New England states, the upper Midwest, and the Pacific

coast are where it most frequently occurs. The town of Lyme, Connecticut, where an

early outbreak was first reported, is really where the disease gets its name. Europe and

Asia are also affected by Lyme disease. People are now more at risk of contracting Lyme

disease. The number of cases reported to the Centers for Disease Control has roughly

doubled over the past 20 years, reaching about 30,000 cases per year. Although the rate
 3

of occurrence of the disease in a geographic area is similar in both animals and people,

animals, especially dogs, are at significantly higher risk because of their greater exposure

to ticks. [4]

Numerous animals can have Lyme disease and not exhibit any symptoms. The

most typical symptoms in dogs are fever, appetite loss, painful or swollen joints,

intermittent lameness, swollen lymph nodes, and lethargy. Kidney, neurological system,

and heart problems can result from untreated Lyme disease. The second most prevalent

illness in dogs is kidney-related Lyme disease, which is typically fatal. The illness type

affecting the nerve system has been known to cause facial paralysis and seizure

problems. Heart illness is infrequent in its heart-related form.[4]

The majority of dogs in the region harbor important ectoparasites for public

health. The government must prioritize the need for an integrated multidisciplinary

approach involving veterinary and medical personnel as well as public health officials

to implement the necessary intervention measures (through health education and the

provision of reasonably priced veterinary clinics) to reduce the risk of zoonotic

transmission.[5]

Tropical brown dog ticks (Rhipicephalus sanguineus) are parasites that live in

warm climates. This tick species is unique among ticks in that it can complete its entire

lifecycle indoors. Tropical brown dogs feed on mammals, but dogs are their preferred

home and eat their blood. On a dog, the preferred attachment sites are the head, ears,

back, toes, and axilla. Tropical brown dog ticks can transmit a variety of diseases while

feeding, including Ehrlichia canis (dog dengue) and Babesiosis. It can also spread

rickettsii, the bacteria that causes Rocky Mountain spotted fever in humans in the

Southwest United States. Furthermore, tropical brown dog ticks secrete venomous
 4

venom that can paralyze their hosts. Tick bites can cause secondary bacterial infections.

Severe tick infestations can cause anemia and death. [6]

In one study, it is confirmed that the tropical brown dog tick (R. linnaei) and the

cat flea (C. felis) are the most prominent tick and flea species parasitizing dogs in the

Manila Metro area in the Philippines. The canine VBPs R. felis and B. clarridgeiae were

confirmed by demonstration of their DNA in ectoparasites collected from dogs in Manila

Metro. Fleas and ticks remain significant pathogens for urban-owned dogs in Metro

Manila implying that prevention.[7]

With progressive research related to exterminating ticks, humans have found

solutions to eliminate ticks by using different methods such as using soap, vaccines,

spray and other forms of treatment in eliminating dog ticks. Common tick eradication

solutions, remedies, and commercial products found in our local area include such as

Bayopet soap, PetPals shampoo, the Detick vaccine, Nexguard tablets, and VetCore

spray.

Bayopet soap contains 90g of the active ingredient Coumaphos, a nonvolatile,

fat-soluble phosphorothioate with ectoparasiticide properties that kills insects and mites.

Ticks, fleas, and lice are effectively killed and controlled in dogs by Bayopet, a tick and

flea soap.

PetPals Flea and Tick Pet Shampoo, which also stops flea eggs from hatching for

up to 28 days and contains rosemary, neem, and madre cacao oil as main ingredients.

Fipronil Tick Buster spray has an active ingredient which is fipronil, which is the

only anti-tick therapy that works both on and off the dog. All stages of ticks, fleas, and

lice are killed by this product. Ticks are killed in the immediate region. All flea phases

are prevented, and the killing force is protected for 3-4 weeks (12 hours) Ticks and fleas
 5

can be killed both on and off the dog using this product, which only eradicates the

infestation of ticks.

Detick 1cc and 2cc, which also contains fipronil, that kills dog fleas, cat fleas,

and mites. They are fast acting, killing fleas in 24 hours and ticks in 48 hours. For dogs

and cats older than eight weeks, they keep cats flea- and tick-free for up to five weeks

and up to two months, respectively.

Vet Core Pet Spray contains Citronella, Lemongrass, Eucalyptus, and

ethnobotanical extracts. Vet Core+ Nature's Advance kills ticks, fleas, flea larvae, and

flea eggs for up to two weeks after a single application and naturally repels mosquitoes

that carry heartworms.

The researchers came up with the idea in creating the solution to test the

ingredients as an organic solution for exterminating tick dogs, which may have a

potential as an alternative tick pesticide depending on the result of the experiment

compared with the commercial products.

The researchers also encourage for the study to be continued by the future

researchers, since marigold, rose, and aloe vera are prominent and can be found locally

in Naga City. Another reason is the concern for growing in amount of domestication of

dogs, where it would be needed to treat any health concerns of dogs, especially the ticks,

which may possibly affect the health of human beings

 6

Statement of the Problem

Generally, the purpose of this study is to evaluate the efficiency of red rose

extract, marigold extract, and aloe vera gel solution to exterminating Tropical brown dog

tick. This also considers the significant difference between the different concentrations

of solution spray in its effectiveness in exterminating ticks and the allergic reaction of

the solution on aspin dogs.

Specifically, this study aims to answer the following:

1.) What is the effect is the MRAV solution as a tick pesticide in terms of:

a. Mortality rate

b. Motility rate

2.) What is the level of effectiveness of MRAV Solution as pesticide on dog ticks in

the following treatments?

a. 100% (20 mL MRAV Solution : 0 mL Distilled Water)

b. 75% (15 mL MRAV Solution : 5 mL Distilled Water)

c. 50% (10 mL MRAV Solution : 10 mL Distilled Water)

d. 25% (5 mL MRAV Solution : 15 mL Distilled Water)

3.) Is there a significant difference between the MRAV solutions and the controlled

treatment in terms of:

a. Mortality Rate

b. Motility Rate
 7

Hypothesis

Ho: There is no significant difference between the MRAV Solution and the controlled

tick pesticide in terms of mortality rate.

Ha: There is a significant difference between the MRAV Solution and the controlled tick

pesticide in terms of mortality rate.

Ho: There is no significant difference between the MRAV Solution and the controlled

tick pesticide in terms of motility rate.

Ha: There is a significant difference between the MRAV Solution and the controlled tick

pesticide in terms of motility rate.

Significance of the Study

This study aims to determine the efficiency of the spray solution for

exterminating ticks and its potential as an alternative tick pesticide. The following will

benefit from this research:

Department of Agriculture

This research will help the Department of Agriculture in learning more about the

benefits of Marigold, Rose, and Aloe Vera as tick pesticide.

 8

Bureau of Animal Industry

This research will help the Bureau of Animal Industry in developing ideas for

improving the production process of alternative organic spray solutions.

Business Industry

This research will also help the Business Industry in developing ideas for how a

Marigold, Rose, and Aloe Vera mixture may be used as an alternative organic spray

solution as they improve the manufacturing process.

Communities

The communities will gain from this study because it will enable them to avoid

using sprays sold commercially that may expose their health to potentially harmful

chemicals.

Environment

This study will benefit the environment and communities by encouraging pet

owners to use organic spray instead of purchasing commercially produced spray in the

market that may potentially contain harmful chemicals.

Pet Owners

This study will help pet owners to know the benefits of utilizing organic

ingredients, specifically marigold, rose and aloe vera and its effeciency against ticks.
 9

Veterinarian

This study will provide them with the information about cost-efficient, organic

plant- based medication and benefits of using organic or natural products like MRAV

spray solution rather than products with chemical ingredients that may potentially harm

dogs.

Researchers

The outcome of the study will be beneficial for both the current researchers and

future researchers. This may serve as a reference for other researchers who want to

conduct related research in the future.

Scope and Delimitation

The purpose of the study is to determine the efficacy of marigold and red rose

extract and aloe vera gel solution to exter motility minate Tropical brown dog tick, with

the researchers considering the mortality rate, rate, and the time of effect. The

researchers also test which of the different concentration MRAV solution spray is the

best, in which commercial tick pesticide, that serves as a controlled positive treatment,

that has been tested along and compare the results to the MRAV solution, to see which

of the treatments is effective

The MRAV Solution contains 20 mL Marigold Extract, 20 mL Rose Extract, and

20 mL Aloe Vera Gel, with an overall total of 60 mL. The solutions are divided into four

20 mL spray bottles with a predetermined amount of distilled water to each spray bottle.

Distilled water to control the concentration of the solution. The formulation of the

solution is of the following where:

 10

● Treatment 1 (100%) = 20 mL MRAV Solution : 0 mL Distilled Water

● Treatment 2: (75%) = 15 mL MRAV Solution : 5 mL Distilled Water

● Treatment 3: (50%) = 10 mL MRAV Solution : 10 mL Distilled Water

● Treatment 4: (25%) = 5 mL MRAV Solution : 15 mL Distilled Water

● Control (+) = 0 mL MRAV Solution : 0 mL Distilled Water

The study contains 4 treatments and one controlled variable, each consists of 5

replicates. The duration of this study takes for about 2 months, starting from acquiring

the materials needed for the study until the end of data gathering, which was be

conducted within the first and second semester of the school year 2022-2023. The

experimentation took 1 hour with a 10-minute break for each trial for time allotment in

recording data.

Any other types of ingredients are not be included in the study besides Marigold

(Tagetes erecta L) ethanolic extract, red rose (Rosa) ethanolic extract, Aloe vera (Aloe

barbadensis miller) gel, Ethanol, and Distilled Water. Anything not included or

mentioned in the study limits the scope of the research.

Locale of the Study

The collection of dog ticks was conducted in the one of the researchers, located

in Concepcion Grande, Naga City, wherein the dog ticks was collected.

The experimental procedure of Marigold, red rose, and Aloe Vera was held at

DOST Laboratory in Camarines Sur National High School Liboton Annex, in Liboton,

Naga City.
 11

Risk Assessment

In the study, the researchers practices safety precautions before, during, and after

conducting the experiment. The researchers also assess the possible risks when

conducting the experiment.

The researchers should practice the following throughout the whole study:

● The researchers must wear PPE (Personal Protective Equipment) such as masks,

goggles, earplugs, and gloves while conducting the study to protect themselves

from hazardous chemicals found in the production of the product and to avoid

contaminating the product.

● The researchers request assistance from an adult or someone with authority to

supervise and guide them while experimenting.

● The researchers should sanitize their hands before and after conducting the

experiment.

● The researchers should sanitize the laboratory apparatuses before and after

conducting the experiment.

● The researchers are required to practice proper handling of laboratory

apparatuses to avoid external injuries.

The possibility of risk during acquisition of materials and collection of ticks:

● The researchers may potentially risk allergic reaction when collecting marigold,

rose and aloe vera, so the researchers are required to fully wear the PPE.

● The researchers may inhale the chloroform, where the researchers are required

to properly handle the chloroform and wear a double mask.

 12

● The researchers may get harmed when applying a dog muzzle, in which it would

require 1-2 people to assist the researcher who collects the ticks.

● The researchers may get harmed by the dogs when collecting ticks, where it

would require 1-2 people to assist the researcher who collects the ticks.

● The researchers may risk harming the dogs’ skin, so the researchers are required

to practice carefully picking ticks from dog skin, and request assistance from an

adult or a person with knowledge in proper way of removing ticks.

The possibility of risk during execution of the experiment:

● The researchers could risk being accidentally spilled by the product, where the

researchers are required to wear PPE.

● The researchers could risk getting caught fire from the alcohol lamp, where the

researchers should prepare a fire distinguisher or nearby source of water.

Disposal

● Laboratory waste was disposed of in recycling bins, trash cans, laboratory

glassware (sterilized) disposal bins, sharps containers, or regulated medical

waste containers.

● Empty plastic, glass, and bottle containers was all recycled. Before recycling,

empty chemical and solvent bottles that were used in the experiment must be

triple rinsed and dried.

● Waste has been separated into biodegradable and non-biodegradable items

before putting it in the trash bin from the general work area or laboratory.
 13

● Experimental ticks were disposed of through burial. It was first wrapped in a

plastic bag, then placed in a box. After that, the ground was excavated for 91.44

cm deep, and the box was then buried.

Conceptual Framework

Utilization of MRAV (Marigold (Tagetes erecta L) Extract, Red Rose (Rosa) Extract,
and Aloe Vera (Aloe barbadensis miller) gel) Mixture as Tick (Rhipicephalus
sanguineus) Spray Pesticide for Aspin Dogs : In Vitro Study

Independent Variable: Dependent Variable:

Different Concentrations of MRAV Effectiveness of MRAV Solution as

Solution: as tick pesticide in terms of:
• Treatment 1 (100%) – 1:0 • Mortality Rate
• Treatment 2 (75%) – 3:1 • Motility Rate
• Treatment 3 (50%) – 1:1
• Treatment 4 (25%) – 3:1
• C(+) – 0:0

Figure 1. Conceptual Paradigm

 14

CHAPTER 3

METHODOLOGY

This chapter presents the research design and materials that was be used in the

study. This chapter also includes the experimental procedure the statistical tool used.

Research Design

Quantitative experimental research design was be used in this study. The study

needs experimental procedure in order to determine the feasibility of the Marigold

(Tagetes erecta L) ethanolic extract, Red Rose (Rosa) ethanolic extract, and Aloe Vera

(Aloe barbadensis mill) gel, or MRAV Solution Spray in eradicating ticks

(Rhipicephalus sanguineus) in Canis lupus familiaris in terms of the motility and

mortality rate.

Four groups are going to be used as the experimental treatment which are the

mixture of MRAV with different concentrations that was be compared to the commercial

pesticide which serve as the control treatment.

 15

Figure 2. Procedural Flowchart

Research Instruments

The researchers used observational forms to record data that was observed during

the experiment.
 16

Acquisition of Materials

The researchers gathered all the material needed such as 30ml spray bottle

(5pcs), 60g aloe vera, 70g marigold, 87g red rose, 75 tropical brown dog ticks, 500 mL

ethanol, and 30mL distilled water. Marigold ethanolic extract, red rose ethanolic extract,

and aloe vera gel as the main ingredients used to decimate the ticks. Distilled water was

used to control the concentrations of the spray solution. Spray bottles was used as a

container for every solution with different percentages of concentrations. The

researchers also use Fipronil to serve as the positive control treatment.

Furthermore, the researchers intend to purchase marigold, red roses, aloe vera

and cotton, and 1 roll aluminum foil in Naga City People's Mall, in Naga City, Camarines

Sur.

Phenylpyrazole Insecticides spray-treatment (100mL), 4pcs spray bottle (30mL),

and dog muzzle was be bought online.

The chemicals that were used in the study are chloroform (1 dip), ethanol (500

mL) and distilled water (30 mL), which can be obtained at the DOST laboratory in

CSNHS Liboton Annex.

The apparatus that wad be used in the study are; vernier caliper, 5pcs tweezer, 1

box whatman paper no.1, 2pcs funnel, weighing scale, 3pcs breaker (300mL), 2pcs

erlenmeyer flask (300mL), 3pcs 25 mL Graduated cylinder, 1pc 100 mL Graduated

Cylinder, 15 pcs petri dish, 5pcs dissecting needle, 2pcs stirring rod, and 1 set hot plate,

2 pcs iron clamp, 1 pc thermometer which all can be obtained at the DOST laboratory

in CSNHS Liboton Annex. Blender was acquired at Sampaloc, Gainza, Camarines Sur.
 17

KN95 Face Mask (10pcs), Nitrile Gloves (10 pairs), and specimen bottles

(15pcs), wasvobtained at Concepcion Pequeña, Naga City. The researchers rovided their

respective protective clothing, including a lab gown, and safety goggles.

Extraction of Marigold Petal, Red Rose Petal and Aloe Vera Gel

The extraction procedure that used by the researchers is the maceration process.

The acquired marigold and red rose flowers was thoroughly washed with running water

to remove unwanted dirt and chemicals, and was dried in the process of air drying for

10-14 days.[8][9][10]

After air drying, the flower petals were collected and then finely chopped using

a blender. The two pulverized petals respectively were immersed in two air-tight sealed

jars consisting each with 00mL ethanol. The solutions were then kept in a dark place

with a temperature around 23°C - 32°C for 72 hours. The solution was shaked for 1

minute for every 2 hours within 8 hours. After leaving it for 3 days, the ethanolic extracts

was poured in a beaker and filtered using Whatman paper no.1. After filtering the

ethanolic extract of marigold and sunflower, the mixture undergoes a water bath between

50°C - 61°C, for 15 minutes or until the solution is reduced. After the water bath, the

ethanolic extract of marigold and rose was collected.[8][9][10]

The aloe vera leaves was peeled using a scalpel, and the gel was collected by

using a spoon. The amount of collected aloe vera gel is expected to be at least 20 mL or

more. After collecting the aloe vera, it was liquidized by using a blender.
 18

Formulation of the Solution

In this study, there are be four treatments; Treatment 1 - 100% (6.66 mL of

Marigold, Rose and Aloe Vera: 0mL Distilled Water), Treatment 2 - 75% (5 mL of

Marigold, Rose and Aloe Vera: 5 mL Distilled Water) , Treatment 3 - 50% (3.33 mL of

Marigold, Rose and Aloe Vera: 10 mL Distilled Water) , Treatment 4 - 25% ( 1.66 mL

of Marigold, Rose and Aloe Vera: 15 mL Distilled Water), and the positive group

Fipronil Tick Buster Spray Treatment (No presence of Marigold, Rose, and Aloe Vera

and Distilled Water). Before proceeding with the concentration process, the extracts of

marigold, and rose, and aloe vera gel are combined to form a solution. The extracts of

marigold, red rose, and aloe vera gel should each have 20 mL for the total mixture of 60

mL solution.

The MRAV Solution contains 20 mL Marigold Oil Extract, 20 mL Rose Extract,

and 20 mL Aloe Vera Gel, with an overall total of 60 mL. The solution was be then

divided into four 20 mL spray bottles with a predetermined amount of distilled water to

each spray bottle. Distilled water was used to control the concentration of the solution.

Collection of Ticks

The ticks were collected from one of the researcher's homes in the barangay of

Concepcion Grande. The researchers used a dog mouth muzzle to prevent the risk of

having dogs bite the researchers during the collection of ticks. The researchers used

tweezers to collect the ticks from the dogs. To immobilize the ticks and allow for

accurate measurement, the ticks first inhale a drop of chloroform with a weight of 0.0239

g in a cotton. The collected tick was then measured if it is an adult tick, where its length
 19

should be ranging from 2.28 to 3.18 mm and 1.11 to 1.68 mm in width by using the

vernier caliper.[3]

The gathered ticks are kept in a mosquito net-covered specimen bottle. Each

specimen bottle are labeled according to the assigned label of treatments and should

consist each with 5 ticks.

Acclimatization of Ticks

The ticks were placed inside a specimen bottle with mosquito-net in order to

ventilate air. The ticks are then resting for 120 minutes Temperature is expected to be

around 25°C to 30°C.

Preparation of Set-ups

The researchers divided the MRAV Solution in four 30 mL spray bottles, each

with a containng a 20 mL solution. The researchers used distilled water to produce the

required concentration for the four different treatments. In total, there are four identical

spray bottles that are labeled as T1 (100%), T2 (75%), T3 (50%), T3 (25%), and the C+

(Positive Control) respectively. T1 contains 20mL MRAV solution, with no distilled

water present. T2 contains 15 mL MRAV solution, and 5 mL distilled water. T3 contains

10 mL of both MRAV solution and distilled water. T4 contains 5 mL MRAV Solution

and 15 mL distilled water. The positive control variable (Phenylpyrazole Insecticides)

has its own formulation. Each of the treatments have a petri dish that are labeled within

its corresponding treatment.

 20

Testing of the Product

The experimental group and control group have three trials. Each treatment

contains five replicates of ticks (R. sanguineus) placed in a petri dish. The subjects are

sprayed 3 times with a distance of 6 inches above the petri dish. The timer was set right

after the spraying of each specimen bottle. The spray bottle of treatments has a nozzle

with a size of 145 mm.

Observation and Gathering of Data

The data was gathered during the observation. There are three trials done in each

treatment. The motility and mortality rate of the ticks was observed for 12 minutes.

Trial 1 Trial 2 Trial 3 Total x̄ Kill Rate

Ticks
Decimated

Treatment
1

Treatment
2

Treatment
3

Treatment
4

C+
Table 1. Mortality Rate

The mortality rate of the replicates are checked for every interval of 4 minutes to

see how many ticks have been decimated in the said interval. The mortality of the ticks
 21

was checked using a dissecting needle when the tick loses its motility. The headcount of

the dead ticks are recorded in accumulative manner after confirming that there is no sign

of movement in the tick. The mortality rate and mean of headcount are computed and

gathered to determine which treatment is the most efficient in deterring ticks

(Rhipicephalus sanguineus).

The mortality rate was calculated by taking all the number of accumulated dead

ticks in a given treatment and dividing it into the total number of the tick population per

treatment. (See Table 8, page 38)

 22

Treatment / T1 T2 T3 T4 C (+)
Replicate

Replicate Trial 1
1
Trial 2

Trial 3

Replicate Trial 1
2
Trial 2

Trial 3

Replicate Trial 1
3
Trial 2

Trial 3

Replicate Trial 1
4
Trial 2

Trial 3

Replicate Trial 1
5
Trial 2

Trial 3

x̄

Table 2. Motility Rate

Five adult ticks are subjected to the motility rate of the replicates and was

checked for every time one of the replicates is immobilized. The motility rate of the ticks
 23

was recorded through laps by using a stopwatch every time a tick shows no sign of

motility. Same with the mortality rate, the motility rate and mean of time are computed

and gathered to determine which specific concentration is the most efficient in deterring

ticks (Rhipicephalus sanguineus).

The motility rate was calculated by taking all the number of immobilized ticks

in a given treatment and dividing it into the total number of the tick population. The

mean of time was calculated by adding all the time in seconds in each replicate in a given

treatment and divide it by the number of replicates. (See Table 9, page 39)

Statistical Tool

The values input in the table as the results are the mean value in each treatment,

the data gathered are put through in a statistical analysis using one-way analysis of

variance (ANOVA) that determines the significant difference in the activity of the

MRAV solution with different concentrations of 100%, 75%, 50% and 25% to the

positive control (Phenylpyrazole Insecticide), using p<0.05. Descriptive Statistics used

on how effective are treatments 1 to 4 vase on a certain percentage. Cohen’s D was also

utilized to determine the level of effectiveness of the treatments.

 24

CHAPTER 4

DATA PRESENTATION, INTERPRETATION, AND DISCUSSION

This chapter presents the data acquired and interpreted throughout the

experiment. It shows the effects of Marigold (Tagetes erecta L) Extract, Red Rose

(Rosa) Extract, and Aloe Vera (Aloe barbadensis miller) gel) Mixture as Tick

(Rhipicephalus sanguineus) Spray Pesticide for Aspin Dogs (Canis lupus familiaris).

Objective 1.A : What is the effect of the MRAV solution as a tick pesticide in terms of

mortality rate?

Trial 1 Trial 2 Trial 3 Total x̄ Kill Rate

Ticks
Decimated

T1 5 5 5 15 5 100%

T2 5 5 5 15 5 100%

T3 5 5 5 15 5 100%

T4 5 4 4 13 4.33 86.47%

C+ 0 3 4 7 2.33 46.67%

Table 3. Mortality Rate of Spray Pesticides Under 12 Minutes

Table 3 displays how effective each of the treatments are based on their mortality

rate. Treatment 1, 2 and 3 have a mean of 5, which shows that these treatments with the
 25

best effect. Treatment 4 have a mean of 4.33, and Positive Controlled Treatment have a

mean if 2.33.
 26

Objective 1.B : What is the effect of the MRAV solution as a tick pesticide in terms of

motility rate?

Treatments / T1 T2 T3 T4 C(+)
Replicate

Replicate Trial 1 0.72 0.95 1.38 2.53 12

1
Trial 2 0.95 3.57 2.03 1.82 0.27

Trial 3 0.62 0.33 2.15 1.02 0.23

Replicate Trial 1 0.97 5.77 2.3 2.78 12

2
Trial 2 1.08 7.85 3.32 3.7 3.08

Trial 3 1.13 0.93 3.67 1.2 0.32

Replicate Trial 1 1.12 6.1 3.48 4.7 12

3
Trial 2 1.7 8.55 4.67 3.52 3.25

Trial 3 3.42 1.18 5.77 3.52 1.23

Replicate Trial 1 10.65 6.45 6.48 7 12

4
Trial 2 1.63 9.12 10.43 5.45 4.53

Trial 3 4.55 9.12 7.38 4.17 1.73

Replicate Trial 1 11.37 8.52 8.97 7.05 12

5
Trial 2 3.06 9.92 11.58 6.57 4.75

Trial 3 6.17 9.92 11.77 6.42 2.83

x̄ 3.28 5.89 5.69 4.10 5.48

Table 4. Motility Rate of Spray Pesticides Under 12 Minutes

 27

Table 4 displays how effective each of the treatments are based on their motility

rate. The lower the value of mean shows how fast the immobilization took effect.

Treatment 1 have a mean of 3.28, which is the most effective among the treatments in

terms of motility rate. Treatment 4 have a mean of 4.09. Positive Controlled Treatment

have a mean of 5.48. Treatment 3 have a mean of 5.69. Treatment 2 have a mean of 5.89

which is the least effective among the treatments.

Objective 2: What is the level of effectiveness of MRAV Solution and Positive Control

as Pesticide on Dog Ticks in the following treatments?

Treatments Mortality Rate Evaluation Motility Rate Evaluation

Treatment 1 100% Very Effective 3.28 mins Very Effective

Treatment 2 100% Very Effective 5.89 mins Effective

Treatment 3 100% Very Effective 5.69 mins Effective

Treatment 4 86.67% Very Effective 4.10 mins Very Effective

Table 5. Level of Effectiveness of MRAV Solution as Pesticide in terms of

Mortality and Motility Rate

Table 5 displays how effective each of the treatments are based on their mortality

and motility rate. Treatment 1 and 4 are very effective in terms motility rate, having a

rate of 3.28 minutes and 4.10 minutes respectively. Treatment 2 and 3 are effective in

terms motility rate, having a rate of 5.89 minutes and 5.69 minutes respectively.
 28

Treatments 1 to 4 are very effective in terms of mortality rate, with Treatment 1, 2, and

3, having a 100% rate and Treatment 4, having a 86.675 rate.

Objective 3: Is there a significant difference between the MRAV solution and the

controlled treatment in terms:

A. Mortality Rate

Source of SS df MS F P-value F-crit

Variation

Between 8.45556 4 2.11389 14.23303 0.00039 3.47805

Groups

Within 1.4852 10 0.14852

Groups

Total 9.94076 14

Table 6. Statistical Analysis between Each Treatments in Different Parameters

(Mortality Rate)

This table shows the statistical analysis between the different treatments and

the positive treatments’ headcount. Generally, the computed F statistic of 14.23303 is

higher than the F critical value of 3.47804 at 0.05 level of significance. It was

interpreted as significant hence, null hypothesis has successfully rejected.

 29

Mortality Rate

𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑝𝑝𝑝𝑝−(𝑑𝑑𝑑𝑑𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔)((𝑀𝑀𝑀𝑀 𝑤𝑤𝑤𝑤𝑤𝑤ℎ𝑖𝑖𝑖𝑖 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔)

𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪′𝒔𝒔 𝑫𝑫 =
𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔 + 𝑆𝑆𝑆𝑆𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡

8.45556−(4)(0.14852)
𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪′𝒔𝒔 𝑫𝑫 =
2.11389 + 9.94076

Cohen’s D = 0.65215

Motility Rate

𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑝𝑝𝑝𝑝−(𝑑𝑑𝑑𝑑𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔)((𝑀𝑀𝑀𝑀 𝑤𝑤𝑤𝑤𝑤𝑤ℎ𝑖𝑖𝑖𝑖 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔)

𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪′𝒔𝒔 𝑫𝑫 =
𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔 + 𝑆𝑆𝑆𝑆𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡

78.26807−(4)(13.38120)
𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪′𝒔𝒔 𝑫𝑫 =
19.65703+ 1014.95195

Cohen’s D = 0.02392

Figure 3. Cohen's D Value of Mortality and Motility Rate

The data shows that the Cohen's d value of 0.65215 implies that there is a

moderate effect size in terms of the differences between the groups being compared.

Meaning that the differences between the group means are noticeable and meaningful.
 30

B. Motility Rate

Source of SS df MS F P-value F-crit

Variation

Between 78.26807 4 19.56703 1.46228 0.22297 2.50266

Groups

Within 936.68388 70 13.38120

Groups

Total 1014.9519 74

Table 7. Statistical Analysis between Each Treatments in Different Parameters

(Motility Rate)

This table shows the statistical analysis between the different treatments and

the positive treatments’ headcount. Generally, the computed F statistic of 1.46228 is

lower than the F critical value of 2.50266 at 0.05 level of significance. It was

interpreted as not significant hence, null hypothesis has been failed to be rejected.

The data shows that the Cohen's d value of 0.02391558736 implies that there is

a very small effect size in terms of the differences between the groups being compared.

This suggests that there’s a minimal difference between the means of the two groups

being compared. (Refer to page 30, Figure 3)

 31

CHAPTER 5

SUMMARY OF FINDINGS, CONCLUSIONS, AND RECOMMENDATIONS

This chapter summarizes the research work undertaken, the conclusion drawn, and

the recommendations made as an outgrowth of this study. This study determines the

effectiveness of marigold, rose and aloe vera ethanolic extract as alternative tick

pesticide.

In this study, the researchers determined the effectiveness of a MRAV tick

pesticide in terms of its mortality rate, and motility rate.

Objective 1: What is the effect of the MRAV solution as a tick pesticide in terms

of mortality and motility rate?

Findings: Treatment 1: 100% concentration of ethanolic extract was shown to be

the most efficient concentration of MRAV Solution in exterminating dog ticks,

with a with a mean of 5, with a motility rate of 3.276 minutes.

Conclusion: It is therefore concluded that Treatment 1: 100% concentration of

MRAV extract is the most effective treatment in exterminating Rhipicephalus

sanguineus.

Recommendation: The researcher recommends strict control in testing all

parameters such as the area of spray, distance of the spray from the sample, and

to avoid significant conflicts in the possible results. The researchers also

recommend testing in using essential oil in replacement for ethanolic extract, to

see whether an essential oil can manage to the dog ticks.

 32

Objective 2: What is the level of effectiveness of MRAV Solution (100%, 75%, 50%,

and 25% Concentration) as Pesticide on Dog Ticks in the following treatments?

Findings: A higher rate indicates a more effective, while a lower rate indicates a

less effective. Treatment 1: 100% concentration of ethanolic extract was shown to

be the most efficient concentration of MRAV Solution in exterminating dog ticks,

with a mortality rate of 100%, with a time of 3.276 minutes.

Conclusion: It is therefore concluded that Treatment 1: 100% concentration of

MRAV extract is the most effective treatment in exterminating Rhipicephalus

sanguineus.

Recommendation: According to the researchers, future researchers should

thoroughly analyze or keep a watch on the results required to compare the various

amounts. A single error in data observation might have an influence on the entire

set of information, resulting in erroneous outcomes.

Objective 3: Is there a significant difference between the MRAV solutions and the

controlled treatment?

Findings: Using a One-Way ANOVA, the statistics of the motility rate for the

computed F statistic of 14.23303 is higher than the F critical value of 3.47805 at

0.05 level of significance. The statistical analysis between the different treatments

and the positive control treatments’ headcount computed the F statistic of 1.46228

is lower than the F critical value of 2.50266 at 0.05 level of significance.

 33

The data about Cohen’s D shows the effectiveness of the Marigold (Tagetes erecta

L), Rose (Rose) ethanolic extract, and Aloe Vera gel as spray p against dog ticks

(Rhipicephalus sanguineus) in terms of mortality and motility rate. Regarding the

effectiveness of the treatments, there is moderate effect size in terms of mortality

rate with a Cohen’s D value of 0.65215. On the other hand, motility rate has a

Cohen’s D value of 0.02392, suggesting that it is considered to be negligible or

trivial, indicating that the groups being compared are practically indistinguishable

in terms of the variable being measured.

Conclusion: It is therefore concluded that there is a significant difference between

the different treatments/concentrations and the control positive treatment in terms

of mortality rate, since the F statistic is higher than the F critical value.

It is also concluded that there is no significant difference between the different

treatments/concentrations and the control positive treatment in terms of motility

rate, as the F statistic is lower than the F critical value.

It is therefore concluded that Marigold (Tagetes erecta L), Rose (Rosa) ethanolic

extract, and Aloe Vera gel treatments have a moderate effect on reducing the

mortality rate of dog ticks (Rhipicephalus sanguineus). The Cohen's d value of

0.65215 suggests a noticeable and meaningful difference in the mortality rate

between the treatment groups and the control group, indicating the effectiveness

of these treatments. However, the motility rate of the ticks shows a negligible

effect size, with a Cohen's d value of 0.02392, indicating that the treatments did

not significantly affect the movement or motility of the dog ticks. Overall, the
 34

treatments are found to be effective in terms of reducing tick mortality, but their

impact on tick motility is minimal.

Recommendation: Based on the findings, the researchers advise future

researchers that when assessing the significant difference between MRAV tick

pesticide and Phenylpyrazole Insecticides against dog ticks (Rhipicephalus

sanguineus), other factors that were overlooked in the study should be considered.

Furthermore, while comparing the efficacy of the produced solution to commercial

products, future researchers should ensure that the commercial product they will

be utilizing is similar to or has the same function as what they have formulated,

according to the researchers. The results of the study would be credible and valid

only if the product they received matched the ones they'd designed.
 35

BIBLIOGRAPHY

[1] Salehi et al. "Tagetes spp. Essential Oils and Other Extracts: Chemical

Characterization and Biological Activity" MDPI, 2018, https://www.mdpi.com/1420-

3049/23/11/2847

[2] Salman, Erbas, "Contact and repellency effects of Rosa damascena mill essential oil

and its two major constituents against Tetranychus urticae Koch (Acari:

Tetranychidae)" Researchgate, December 2014

https://www.researchgate.net/publication/286214514_Contact_and_repellency_effects

_of_Rosa_damascena_miller_essential_oil_and_its_two_major_constituents_against_

Tetranychus_urticae_Koch_Acari_Tetranychidae

[3] Hekmatpou et al., "The Effect of Aloe Vera Clinical Trials on Prevention and Healing

of Skin Wound: A Systematic Review" Iran Journal of Medical Sciences, January 2019,

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6330525/

[4] Straughbinger, Reinhard K. "Lyme Disease (Lyme Borreliosis) in Dogs" MSD

Veterinary Manual, June 2018, https://www.msdvetmanual.com/dog-owners/disorders-

affecting-multiple-body-systems-of-dogs/lyme-disease-lyme-borreliosis-in-dogs

[5] Abdulkareem et al., "Parasite Epidemiology and Control" Elsevier, March 22, 2018,

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6324013/#!po=1.42857
 36

[6] Tian et al., "common name: brown dog tick scientific name: Rhipicephalus

sanguineus Latreille (Arachnida: Acari: Ixodidae)" University of Florida, December

2018, https://entnemdept.ufl.edu/creatures/urban/medical/brown_dog_tick.htm

[7] Marquez et al., “Vector-Borne Pathogens in Ticks and Fleas of Client-Owned Dogs

in Metro Manila, Philippines” MDPI, November 18, 2021,

https://www.mdpi.com/2673-6772/1/4/26/pdf

[8] Altemini et al., "Phytochemicals: Extraction Methods, Identification, and Detection

of Bioactive Compounds from Plant Extracts" Journal of Pharmacognosy and

Phytochemistry, September 22, 2017, https://www.mdpi.com/2223-7747/6/4/42

[9] Azwanida, NN. "A Review on the Extraction Methods Use in Medicinal Plants,

Principle, Strength, and Limitation" Medical & Aromatic Plants, July 6, 2015,

http://dx.doi.org/10.4172/2167-0412.1000196

[10] Pandey A, and Tripathi S. "Concept of Standardization, Extraction, and Pre-

Phytochemical Screening Strategies for Herbal Drug" Journal of Pharmacognosy and

Phytochemistry, https://www.phytojournal.com/archives/2014.v2.i5.255/concept-of-

standardization-extraction-and-pre-phytochemical-screening-strategies-for-herbal-drug
 37

APPENDIX A- DATA & STATISTICAL ANALYSIS

Time passed Total

Treatment / Accumulate
Replicate 4 mins 8 mins 12 mins d
Headcounts

Treatment 1 Trial 1 3 3 5 5

Trial 2 5 5 5 5

Trial 3 3 5 5 5

Treatment 2 Trial 1 4 4 5 5

Trial 2 2 3 5 5

Trial 3 2 3 5 5

Treatment 3 Trial 1 1 3 5 5

Trial 2 2 3 5 5

Trial 3 2 2 5 5

Treatment 4 Trial 1 1 2 5 5

Trial 2 1 4 4 4

Trial 3 1 2 4 4

Positive Trial 1 - - - -
Control (C+)
Trial 2 1 2 3 3

Trial 3 2 2 4 4

Table 8. Raw Data for Mortality Rate

 38

Treatment / Treatmen Treatmen Treatmen T4 C(+)

Replicate t1 t2 t3

Replicate Trial 1 0.72 0.95 1.38 2.53 12

1
Trial 2 0.95 3.57 2.03 1.82 0.27

Trial 3 0.62 0.33 2.15 1.02 0.23

Replicate Trial 1 0.97 5.77 2.3 2.78 12

2
Trial 2 1.08 7.85 3.32 3.7 3.08

Trial 3 1.13 0.93 3.67 1.2 0.32

Replicate Trial 1 1.12 6.1 3.48 4.7 12

3
Trial 2 1.7 8.55 4.67 3.52 3.25

Trial 3 3.42 1.18 5.77 3.52 1.23

Replicate Trial 1 10.65 6.45 6.48 7 12

4
Trial 2 1.63 9.12 10.43 5.45 4.53

Trial 3 4.55 9.12 7.38 4.17 1.73

Replicate Trial 1 11.37 8.52 8.97 7.05 12

5
Trial 2 3.06 9.92 11.58 6.57 4.75

Trial 3 6.17 9.92 11.77 6.42 2.83

Table 9. Raw Data for Motility Rate

 39

Treatments Mortality Rate Evaluation Motility Rate Evaluation

Treatment 1 100% Very Effective 3.276 mins Very Effective

Treatment 2 100% Very Effective 5.88533 mins Effective

Treatment 3 100% Very Effective 5.692 mins Effective

Treatment 4 86.67% Very Effective 4.09667 mins Very Effective

C+ 46.67% Somehow 5.48133 mins Effective

Effective

Table 10. Descriptive Statistics on Effectiveness of MRAV Tick Pesticide

According to Mortality Rate and Motility Rate

 40

Legend

Mortality: Motility:

81 - 100% - Very Effective Under 4 mins - Very Effective

61 - 80% - Effective Under 8 mins - Effective

41 - 60% - Somehow effective Under 12 mins - Somehow effective

40 and below - Not effective 12 mins and beyond - Not effective

So SS df MS F P-value F-crit

urce of

Variation

Between 8.45556 4 2.11389 14.23303 0.00039 3.47805

Groups

Within 1.4852 10 0.14852

Groups

Total 9.94076 14

Table 11. Statistical Analysis between Each Treatments in Different Parameters

(Mortality Rate)
 41

Source of SS df MS F P-value F-crit

Variation

Between 78.26807 4 19.56702 1.46228 0.22297 2.50266

Groups

Within 936.68388 70 13.38120

Groups

Total 1014.9519 74

5466667

Table 12. Statistical Analysis between Each Treatments in Different Parameters

(Motility Rate)

Mortality Rate

𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑝𝑝𝑝𝑝−(𝑑𝑑𝑑𝑑𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔)((𝑀𝑀𝑀𝑀 𝑤𝑤𝑤𝑤𝑤𝑤ℎ𝑖𝑖𝑖𝑖 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔)

𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪′𝒔𝒔 𝑫𝑫 =
𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔 + 𝑆𝑆𝑆𝑆𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡

8.45556−(4)(0.14852)
𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪′𝒔𝒔 𝑫𝑫 =
2.11389 + 9.94076

Cohen’s D = 0.65215

Motility Rate

𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑝𝑝𝑝𝑝−(𝑑𝑑𝑑𝑑𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔)((𝑀𝑀𝑆𝑆 𝑤𝑤𝑤𝑤𝑤𝑤ℎ𝑖𝑖𝑖𝑖 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔)

𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪′𝒔𝒔 𝑫𝑫 =
𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔 + 𝑆𝑆𝑆𝑆𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡

78.26807−(4)(13.38120)
𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪′𝒔𝒔 𝑫𝑫 =
19.65703+ 1014.95195

Cohen’s D = 0.02392
 42

Interpretation:

More than 0.8 = practical/significant effect

0.8 = large effect

0.9 0.6 = medium effect

0.2 = small effect

Less than 0.2 = trivial/negligible effect

 43

APPENDIX B - DOCUMENTATION
44
45
46
47
48
49
50
51
52
53
 54

APPENDIX C - COMMUNICATION LETTERS

Figure 46. Parental Consent and Waiver Form of the Student Researcher 1
 55

Figure 47. Parental Consent and Waiver Form of the Student Researcher 2
 56

Figure 48. Parental Consent and Waiver Form of the Student Researcher 3
 57

Figure 49. First page of Communication Letter for the Principal

 58

Figure 50. Second page of Communication Letter for the Principal

 59

Figure 51. Third page of Communication Letter for the Principal

 60

Figure 52. First page of Communication Letter for the Science Department

Head
 61

Figure 53. Second page of Communication Letter for the Science Department

Head
 62

Figure 54. Third page of Communication Letter for the Science Department

Head
 63

APPENDIX D - FINANCIAL REPORT

MATERIALS QUANTITY COST PER UNIT TOTAL COST

Ethanol (500 mL) 2pcs 350 ₱700

Red Rose 7 dozens 300-350 ₱1,500

Petri Dish 15pcs 12 ₱180

Tweezer 3pcs 20 ₱60

Safety Goggles 5pcs 25.6 ₱128

Dog muzzle 1pc 43.23 ₱43.23

Soy Lecithin 1pc 115 ₱115

Phenylpyrazole 1pc 133 ₱133

Insecticide

Spray bottle 10pcs 9.3 ₱93

(30mL)

Aluminum Foil 1 roll 12 ₱12

TOTAL ₱2,964.23

Table 13. Cost of Materials Utilized in the Study

64
65
66
 67

APPENDIX E - JOURNAL

Figure 64. Journal Title Page

 68

Figure 65. Journal Entry no.1

 69

Figure 66. Journal Entry no. 2

 70

Figure 67. Journal Entry No. 3

 71

Figure 68. Journal Entry No. 4

 72

Figure 69. Journal Entry No. 5

 73

Figure 70. Journal Entry No. 6

 74

Figure 71. Journal Entry No. 7

 75

Figure 72. Journal Entry No. 8

 76

Figure 73. Journal Entry No. 9

 77

Figure 74. Journal Entry No. 10

 78

Figure 75. Journal Entry no. 11

 79

Figure 76. Journal Entry no. 12

 80

Figure 77. Journal Entry no. 13

 81

Figure 78. Journal Entry no. 14

 82

Figure 79. Journal Entry no. 15

 83

Figure 80. Journal Entry no. 16

 84

Figure 81. Journal Entry no. 17

 85

Figure 82. Journal entry no. 18

 86

Figure 83. Journal Entry no. 19

 87

Figure 84. Raw Data

 88

Figure 85. Journal Entry No. 20

89
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91
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95
96
 97

TIMETABLE
Research Itinerary
DATE ACTIVITIES EXPECTED OUTPUT
• Working and
October 5, 2022 – January Finished Chapter 1 to 3 of
Revision of
15, 2023 the Research Paper
Chapter 1 to 3
• Creation of
PowerPoint Finished Powerpoint for
January 13 -15, 2023
presentation for Title Defense
Title Defense
Panelists-reviewed
research paper, summary
January 16, 2023
• Title Defense of comments, suggestions,
(Monday)
and recommendations
from panelists
• Formulate
Research Itinerary
Itinerary;
• Creation of
Accomplished
Communication
Communication
Letter
February 12 – March 14, Letter; Accomplished
• Accomplishment
2023 Communication
of ISEF Forms
Letter; Acquired
• Acquisition of Materials; Consent
Materials Letter Distributed
• Distribution of
Consent Letter
Formulation of the Solution
• Air Drying of
February 28 – March 13,
Marigold and
2023
Roses
• Grinding of
Marigold and
Rose Flowers
March 14 –17, 2023
• Maceration of
Marigold and
Rose Flowers
March 17, 2023 • Filtration of MRAV Spray Solution
(Thursday) Extract
• Water Bath of
March 21, 2023 Collected
(Tuesday) Marigold and
Rose Extract
March 22, 2023 • Aloe Vera
(Wednesday) Extraction
March 28, 2023 • Formulation of
(Tuesday) MRAV Spray
 98

• Solution (4
Treatments)
Collection of Ticks
• Collection and
March 25, 2023
Acclimatization of
(Saturday)
Ticks
Collected Ticks Verified
• Collection and
March 28,2023 Acclimatization of
(Tuesday) Ticks (New Batch)
• Measuring of ticks
• Preparation of Set-
up (5 Treatments :
March 29, 2023 Experimentation Results
3 trials == 15
(Wednesday) (Raw Data)
trials)
• Data Gathering
• Working and
Revision of
Chapter 1-3
• Finishing Paper Finished Research Paper
from Chapter 4 to (Chapter 1 – 5);
May 27 -31, 2023
5 Finished Powerpoint for
• Creation of Final Defense
PowerPoint
presentation for
Final Defense
Panelists-reviewed
research paper, summary
June 02, 2023
• Final Defense of comments, suggestions,
(Friday)
and recommendations
from panelists
• Design and Print
Tarpaulin for
June 03 – 05, 2023 Tarpaulin created for
Capstone Project
exhibit; presented study to
Exhibit
Judges
June 06, 2023 • Capstone Project
(Tuesday) Exhibit
• Checking and
Working on
Revisions for
Final Paper
• Accomplished Finalized and approved
June 23 - 26, 2023 Required Forms manuscript of research
and Collected paper
Signatures for
Certification,
Approval Sheet,
etc.
 99

• Passing of
June 30, 2023
Hardbound Hardbound Manuscript
(Friday)
Manuscript
 100

BIOGRAPHY SKETCH

"Happiness is a choice. Optimism is a choice. Whatever choice you make makes

you. Choose wisely, kaya mag Lady's Choice"

Juliana Keira C. Quinto is a 17-year-old female student who resides in

Concepcion Grande, Naga City, Camarines Sur. Her birthday is on August 2, 2005, and

she can be contacted at [email protected].

In 2017, she completed her education at Concepcion Grande Elementary School

and continued her studies as a junior high school FFC (Family Farming Curriculum)

student at Camarines Sur National High School. Currently, she is a senior high school

STEM student at the same institution, with aspirations of becoming a medical

technologist in the future. Her dedication and perseverance in her studies have

contributed to the thriving of this research. For her, conducting this study is not only a

requirement but also an opportunity to expand her knowledge of science, develop

critical thinking skills, and enhance her professional abilities.

Juliana Keira C. Quinto

 101

BIOGRAPHY SKETCH

"Dreams don't work unless you do"

Reymark C. San Jose is a 18-year-old male student residing in Sampaloc, Gainza,

Camarines Sur. Born on November 12, 2004, Reymark is a determined and ambitious

individual who aspires to achieve his dreams. He is the son of Rico Deleon San Jose

and Eva Clavecilla San Jose, and he has a supportive younger brother named Regie C.

San Jose. Reymark can be contacted at [email protected].

Reymark completed his elementary education at Tabuco Naga City and continued

his high school journey at Camarines Sur National High School. Passionate about

architecture and civil engineering, he is currently pursuing his senior high school

education in the STEM program, actively engaging in activities to enhance his skills

for his desired career path.

Reymark C. San Jose

 102

BIOGRAPHY SKETCH

“Embrace the unknown journey of life with optimism as your guiding light,

unraveling the essence of living through uncharted territories and inspiring hope

with every step.”

Justin Lloyd B. Sanchez is an 18-year-old male student residing in Carolina, Naga

City, Camarines Sur. Born on November 01, 2004, Justin is a determined and highly

motivated individual with a strong drive for success.

Justin completed his elementary education at Naga Central School 2 and is

currently pursuing his senior high school education in the esteemed STEM program at

Camarines Sur National High School. With a profound interest in computer engineering

and multimedia design, he continuously enhances his skills and knowledge for future

success in these fields. His dedication, work ethic, and commitment to academic

excellence make him an outstanding student in the making.

Justin Lloyd B. Sanchez

 103

LISTS OF TABLES

Page No.

Table 1. Mortality Rate..…….……………………………………………..…..…….21

Table 2. Motility Rate..…….…………………………………………..………..........22

Table 3. Mortality Rate of Spray Pesticides Under 12 Minutes…..……..………...…24

Table 4. Motility Rate of Spray Pesticides Under 12 Minutes…..…..…..…...………26

Table 5. Level of Effectiveness of MRAV Solution as Pesticide in terms of Mortality

and Motility Rate……………………………………………………………………..27

Table 6. Statistical Analysis between Each Treatments in Different Parameters

(Mortality Rate)……………………………………………………………………....28

Table 7. Statistical Analysis between Each Treatments in Different Parameters

(Motility Rate)…….……………………………………………………………...…..30

Table 8. Raw Data for Mortality Rate..…………………………..………..……........37

Table 9. Raw Data for Motility Rate..……..………..……………………...………...38

Table 10. Descriptive Statistics on Effectiveness of MRAV Tick Pesticide According

to Mortality Rate and Motility Rate…………………...………………...……….…..39

Table 11. Statistical Analysis between Each Treatments in Different Parameters

(Mortality Rate) ………………………………...…..…………...……….…………..40

Table 12. Statistical Analysis between Each Treatments in Different Parameters

(Motility Rate)…………………………………………………………………....…..41
 104

Table 13. Cost of Materials Utilized……………………..…...........…….….……….63

 105

LIST OF FIGURES

Page No.

Figure 1. Conceptual Paradigm..…….……………………………………..…….…13

Figure 2. Procedural Flowchart..…….…………………………………….....……..15

Figure 3. Cohen's D Value of Mortality and Motility Rate..…..………..……..........29

Figure 4. Roses (Rosa) …….………………..……………………….……..…........43

Figure 5. Air drying of Roses…….……………………………….....…….……......43

Figure 6. Air Drying of Marigold (Tagetes erecta L.) …….…………….........…....43

Figure 7. Using mosquito net for catching falling rose petals………..……...……..43

Figure 8. Aloe Vera……………………………………………………..…………..44

Figure 9. Air-Dried Roses being weigh………………….…………...…….…........44

Figure 10. Air-Dried Marigold………………...………...…………………………44

Figure 11. Air-Dried Roses…………………………………..……...….....…….....44

Figure 12. Air-Dried Marigold being weigh………..…….…….……….……........45

Figure 13. Air-Dried Roses being weigh……………………………….…….…....45

Figure 14. Materials and Equipments 1…………………………….……..….…...45

Figure 15. Materials and Equipments 2…………………………………………....45

Figure 16. Pulverizing Flowers using Blender……………………….….………...46

Figure 17. Weighing Ai-Dried Roses after Pulverizing..………………………….46

 106

Figure 18. Weighing Air-Dried Marigold after Pulverizing……………………........46

Figure 19. Mixing Property of Solvent and Solution………………..……………….46

Figure 20. Soaking of Pulverized Flower in Ethanol…………..……….…..……......47

Figure 21. Preparation of Filtration………………………………………………....,.47

Figure 22. Filtration of Rose and Marigold Extract (side view) ………….…....……47

Figure 23. Filtration of Rose and Marigold Extract (top view) …………….…….…47

Figure 24. Filtered Extract…………………………………….………….………….48

Figure 25. Hot Plate……………………………………………......…...….…...........48

Figure 26. Water Bath…………………………………..……..…………..…...….....48

Figure 27. Reduced Ethanol Content……………………………...….……...…..….48

Figure 28. Peeling of Aloe Vera Leaves……………………….…….……...….…....49

Figure 29. Blending Aloe Vera Gel…………….…………………………...…….....49

Figure 30. Marigold, Rose and Aloe Vera Extract……………….....………....….....49

Figure 31. Measuring amount of Extract………………..……….…….…..…...……49

Figure 32. Preparation of Equipment for Tick Collection…………….……..…....…50

Figure 33. Sample bottle for Ticks…………..…………………….………..…….....50

Figure 34. Collection of Tick Assisted by an Adult Supervisor..…….….…………..50

Figure 35. Preparation of Materials for Data Gathering..…..……………………..…50

 107

Figure 36. Mixing Marigold, Rose and Aloe Vera Extract……..……………....……51

Figure 37. Formulation of Treatment……………..……………..…….….…….…....51

Figure 38. Different Concentration of MRAV Tick Repellent Solution..………..…..51

Figure 39. Immobilizing Ticks Using Chloroform……….………………...…..........51

Figure 40. Data Gathering…………………………………….………..….................52

Figure 41. Recording Data…………………………………………….…...…...........52

Figure 42. Phone used for time lapping of Motility of Ticks………………………...52

Figure 43. Collection of Experimented Sample………………………………. …….53

Figure 44. Adding Container for the Samples………………………....……….........53

Figure 45. Disposal of Samples………………………………………………..….....53

Figure 46. Parental Consent and Waiver Form of the Student Researcher 1.…….....54

Figure 47. Parental Consent and Waiver Form of the Student Researcher 2…..........55

Figure 48. Parental Consent and Waiver Form of the Student Researcher 3….….....56

Figure 49. First page of Communication Letter for the Principal….…….…….……57

Figure 50. Second page of Communication Letter for the Principal..……………….58

Figure 51. Third page of Communication Letter for the Principal…………. ..…......59

Figure 52. First page of Communication Letter for the Science Department Head...60
 108

Figure 53. Second page of Communication Letter for the Science Department Head.

………………………………………………………………………………………..61

Figure 54. Third page of Communication Letter for the Science Department .......….62

Figure 55. Receipt for Ethanol………………………………......……………..…….64

Figure 56. Receipt for 1 dozen of Rose……………………,...…..……….……..…...64

Figure 57. Receipt for 4 dozen of Roses……………………………..…………..…..64

Figure 58. Receipt for Petri Dish………………………………………..…..….…....64

Figure 59. Receipt for Dog Muzzle……………………….………………………....65

Figure 60. Receipt for Spray Bottles………………….………………..…....……....65

Figure 61. Receipt for Phenylpyrazole Insecticide…….………..……..…………….65

Figure 62. Receipt for Soy Lecithin…………………………………....…..………..66

Figure 63. Receipt For Safety Goggles……………………………..……………….66

Figure 64. Journal Title Page…………………………………………………….….67

Figure 65. Journal Entry no.1………………………………………………..………68

Figure 66. Journal Entry no.2……………………………………………..…………69

Figure 67. Journal Entry no.3………………………………………..……………....70

Figure 68. Journal Entry no.4……………………………………………..…………71

Figure 69. Journal Entry no.5.…………………………………………..…………..72

 109

Figure 70. Journal Entry no.6…………………………………………………....…..73

Figure 71. Journal Entry no.7………………………………………………….….....74

Figure 72. Journal Entry no.8……………………………………………….…….....75

Figure 73. Journal Entry no.9………………………………….…………….....…....76

Figure 74. Journal Entry no.10………………………………………..………...…...77

Figure 75. Journal Entry no.11……………………………………………......……..78

Figure 76. Journal Entry no.12…………………………………….……...…..……..79

Figure 77. Journal Entry no.13……………………………………………..….….....80

Figure 78. Journal Entry no.14……………………………………………...…….....81

Figure 79. Journal Entry no.15……………………………...…………………….....82

Figure 80. Journal Entry no.16…………………………………………...………….83

Figure 81. Journal Entry no.17…………………………………………………...….84

Figure 82. Journal Entry no.18…………………………………………………...….85

Figure 83. Journal Entry no.19………………………………………………...…….86

Figure 84. Raw Data……………………………………………………………….....87

Figure 85. Journal Entry no.20…………………………………………………...….88