POTENTIAL OF GARLIC (Allium sativum) OIL AS BIOPESTICIDE AGAINST

LARVA MOSQUITO

CHAN, ALEXIS L.
FLORES, CHERISH B.
PADUA, CHERRY JOY M.
VILLAREAL, JANECEL G.

A thesis presented to the faculty of Education Department,

Aurora State College of Technology, Baler, Aurora
In partial fulfillment of the requirements
for the degree

BACHELOR OF SECONDARY EDUCATION

(Major in Science)

MAY 2023

i
 Republic of the Philippines
AURORA STATE COLLEGE OF TECHNOLOGY
School of Education
Baler, Aurora

Undergraduate Thesis Manuscript

Approval Sheet

Name of Students: ALEXIS L. CHAN, CHERISH B. FLORES, CHERRY JOY

M. PADUA, JANECEL G. VILLAREAL

Degree/Course: BACHELOR OF SECONDARY EDUCATION MAJOR IN

SCIENCE

Title of Research: POTENTIAL OF GARLIC (Allium sativum) OIL AS A

BIOPESTICIDE AGAINST MOSQUITO LARVAE

Research Number: ____________________

Name Signature Date

Approved: KARMINA B. VALETE

Adviser
Approved: EDEN ROSE B. OLAZO
Member
Approved: REY ANGELYN S. GONGOB
Member
Approved: NIKITA PAOLA A. DEL MUNDO
Statistician
Approved: ELIZABETH D. LOPEZ
English Critic
Approved: DR. ROWEL G. OLILA, EdD
Director
Approved: FLORDELIZA S. GUTIERREZ
College Registrar

ii
 BIOGRAPHICAL SKETCHES

The first researcher was born on the 28th day of July 2002 in Barangay Nonong Sr.

San Luis, Aurora. She is the youngest child of Mr. Primitivo D. Chan and Mrs. Maricel L.

Chan. Her father is a tricycle driver, and her mother is a housewife. She is happy and

grateful for her supportive parents.

She finished her elementary education at Nonong Sr. Elementary School in 2014.

She completed her junior and senior high school years at Mount Carmel School of San Luis

in 2020 with an honor and loyalty award. She is currently taking up a Bachelor of

Secondary Education major in Science at Aurora State College of Technology. I can do all

things through Christ, who gives me strength. - Philippians 4:13

ALEXIS L. CHAN

iii
 The second researcher was born on the 31st day of March 2002 in Salapingao East,

Dagupan City, Pangasinan. She is the fifth child of Mr. Gene B. Flores Sr., a kagawad at

Brgy. Dianawan, Maria Aurora, and Mrs. Trinidad B. Flores, a housewife. She is forever

grateful to her parents and older siblings for their endless love and support.

She finished her elementary education at Francisco Q. Dagyapen Memorial

Elementary School in 2014, completed her junior high school at Dianawan National High

School in 2018, and graduated senior high school at Wesleyan University-Philippines,

Aurora, in the year 2020 with honors. She is currently taking up a Bachelor of Secondary

Education with a major in Science at Aurora State College of Technology. For surely there

is a future, and your hope will not be cut off. - Proverbs 23:18

CHERISH B. FLORES

iv
 The third researcher was born on the 17th day of July 2002 in Barangay

Manggitahan, Dilasag, Aurora. She is the oldest daughter of Mr. Astrobal A. Padua and

Mrs. Cherryl M. Padua. Her father is a farmer, and her mother is a housewife. She is

thankful for having hardworking and supportive parents.

She finished her elementary education at Villa Santiago Elementary School in 2014.

She completed her junior and senior high school years at Manggitahan National High

School in 2020 with an honor and loyalty award. She is currently taking up a Bachelor of

Secondary Education major in Science at Aurora State College of Technology. Trust the

Lord with all your heart, and do not lean on your understanding. In all your ways,

acknowledge Him, and He will make your paths straight. - Romans 15:13

CHERRY JOY M. PADUA

v
 The fourth researcher was born on the 28th day of January 2001 in Barangay

Obligacion Baler, Aurora. She is the youngest child of Mr. Jonie V. Villareal and Mrs.

Merced G. Villareal. Her father is a farmer, and her mother is a housewife. She is happy

and grateful for her supportive parents.

She finished her elementary education at Obligacion Integrated School in 2014 and

junior high school in 2018 at the same school. She completed senior high school at

Wesleyan University Philippines - Aurora Campus in 2020 with honors. She is currently

taking up a Bachelor of Secondary Education major in Science at Aurora State College of

Technology. Commit to the Lord whatever you do, and he will establish your plans. -

Proverbs 16:3

JANECEL G. VILLAREAL

vi
 ACKNOWLEDGEMENTS

First and foremost, the researchers would like to express their sincere gratitude to

the following for helping them complete this research successfully:

Mrs. Karmina B. Valete, their Research Adviser, for her support, valuable

assistance, patience, comments, suggestions, and sharing of her insights and ideas

throughout the conduct of this study and to further improve this paper.

Dr. Rowel G. Olila, their Dean, for the approval of the request to conduct this

research in the laboratory at Aurora State College of Technology.

Ms. Nikita Paola A. Del Mundo, their Statistician, for being approachable,

willingly sharing her mathematical expertise, and helping in the analysis and interpretation

of the research data.

Ms. Elizabeth D. Lopez, their English Critic, for her support, effort, time, and

patience in checking and editing this manuscript.

Ms. Eden Rose B. Olazo and Ms. Rey Angelyn S. Gongob, their Panelists, for

their encouragement, insightful comments, suggestions, and recommendations to improve

this study.

The parents, for their financial and moral support, understanding, and patience

during the study; and

To Almighty God, for guidance, strength, and wisdom bestowed upon the

researchers to complete the research successfully.

vii
 TABLE OF CONTENTS

TITLE PAGE i

APPROVAL SHEET ii

BIOGRAPHICAL SKETCHES iii

ACKNOWLEDGEMENTS vii

TABLE OF CONTENTS viii

LIST OF TABLES xi

LIST OF FIGURES xi

LIST OF APPENDICES xii

LIST OF EXHIBITS xiii

ABSTRACT xiv

CHAPTER I 1

THE PROBLEM AND ITS SETTING 1

Introduction 1

Conceptual Framework 2

Objectives of the Study 4

Significance of the Study 4

Scope and Delimitations of the Study 5

Time and Place of Study 6

Definition of Terms 6

viii
CHAPTER II 8

REVIEW OF RELATED LITERATURE AND STUDIES 8

Description of Allium Sativum 8

Allium Sativum 8

Aedes mosquito 9

Vector Control 10

Biopesticide 12

Garlic as Biopesticide 13

CHAPTER III 14

MATERIALS AND METHOD 14

Collection of Sample 14

Preparation of Distillation 14

Preparation of Sample 15

Insect rearing 16

Larvicidal Assay 17

Data Collection Procedures 18

Data Gathered 18

Data Analysis 19

CHAPTER IV 20

RESULTS AND DISCUSSION 20

Nature of Mosquito Larvae 20

Garlic essential oil composition 20

ix
 Larvicidal Activity against Mosquito larvae 22

Statistical Analysis 23

Morphological analysis of larvae 24

Morphological analysis of larvae after contact with garlic oil 24

CHAPTER V 26

SUMMARY, CONCLUSION, AND RECOMMENDATION 26

Summary 26

Conclusion 28

Recommendation 29

REFERENCES 30

APPENDICES 34

EXHIBIT 44

x
 LIST OF TABLES

TABLE TITLE PAGE

1 Categories and Examples of vector control methods 11

2 Garlic oil composition 21

3 Garlic oil treatments and mortality rates 22

4 Model Summary 39

5 Analysis of Variance 23

6 Coefficients 39

LIST OF FIGURES

FIGURE TITLE PAGE

1 Conceptual Framework of the Study 3

2 Fresh garlic sample 8

3 Steam Distillation Setup 15

4 Garlic oil in griffin beaker 16

5 Mosquito larvae under light microscope after 12h 25

xi
 LIST OF APPENDICES

APPENDIX TITLE PAGE

1 Collection and preparation of raw material 34

2 Preparation of distillation 35

3 Preparation of Sample 35

4 Insect rearing 36

5 Larvicidal Assay 37

6 Mosquito larvae after garlic oil treatments 38

7 Statistical Analysis Tables 39

8 Letter of Request to borrow Laboratory glassware. 41

9 Letter of Request to access Dengue cases information at Aurora Memorial

Hospital 42

10 Dengue Cases in Baler, Aurora 43

xii
 LIST OF EXHIBITS

EXHIBIT TITLE PAGE

1 Fresh Garlic 44

2 Garlic Peeling 44

3 Garlic Chopping 45

4 Weighing 100 g of garlic 45

5 Tap water (200 ml) 46

6 Salt (6 g) in water 46

7 Preparing the Distillation Setup 47

8 Preparation of Sample 47

9 Heating and boiling 48

10 Maintaining the temperature (95℃) 48

11 Liquid-liquid extraction using separatory funnel. 49

12 Garlic oil 49

13 Insect rearing 50

14 24 hours water conditioning 50

15 Garlic oil treatments and replicates 51

16 Monitoring mortality rates 51

xiii
 ABSTRACT

CHAN, ALEXIS., L, FLORES, CHERISH., B PADUA, CHERRY JOY., M

and VILLAREAL, JANECEL G., School of Education, Aurora State College of
Technology, Baler, Aurora, May 2023. POTENTIAL OF GARLIC (Allium sativum) OIL
AS BIOPESTICIDE AGAINST LARVA MOSQUITO.

Adviser: Karmina Barrogo – Valete

This research study is entitled Potential of Garlic (Allium sativum) Oil as a

Biopesticide Against Mosquito Larvae. It was conducted at Aurora State College of

Technology, located in Barangay Zabali, Baler, Aurora. This study aimed to determine the

potential of garlic oil as a biopesticide against mosquito larvae. The researchers prepared

the water and salt and put them in a round bottom flask together with the garlic cloves for

the oil extraction using the steam distillation process. Larvicidal assays were performed

following the WHO guidelines' recommendations. The tests were carried out in three larval

batches, with garlic oil determined at three concentrations (25%, 50%, and 75%). A total

of 225 larval mosquitoes were exposed to three (3) replicates of each concentration. Based

on the results of the study, the Allium sativum oil that was used as a biopesticide against

mosquito larvae has potential and is effective. The application of oil with the respective

concentration formed a thin film over the surface of the water, suggesting that the oil film

could prevent the further exchange of gases, thereby causing the death of the larvae by

asphyxia. Furthermore, garlic contains allicin, which is toxic to parasites and bacterial

cells, and diallyl sulfide has bactericidal and flavonoid properties that act as respiratory

inhibitors for insects. Mosquito larvae treated with different garlic oil concentrations

showed morphological changes, including but not limited to destruction of the gut,

xiv
shrinkage of the abdominal region, loss of lateral hairs, and extrusion of hemolymphatic

content. Based on the results of the statistical analysis, the three concentrations have no

significant differences in terms of effectiveness. All treatments were effective but differed

in how long they affected the mosquito larvae. Among the three concentration levels

present, treatment 3 (75%) has the fastest mortality rate. Therefore, the more concentrated

the garlic was in the solution, the more active substances were present.

xv
 CHAPTER I

THE PROBLEM AND ITS SETTING

Introduction

Dengue is a mosquito-borne viral disease that is spread to people through the bite

of an infected Aedes species mosquito (Ae. aegypti or Ae. albopictus) (Centers for Disease

Control and Prevention, n.d.). It infects up to 50–200 million people annually, putting over

3.6 billion people living in tropical regions at risk and causing up to 20,000 deaths

(Murugesan & Manoharan, 2020).

104 °F (40 °C) rapid fever, aching muscles and joints, rash, nausea, vomiting, sore

throat, headache, and red eyes are just a few of the moderate symptoms of the widespread

viral virus dengue. Abdominal discomfort, at least three vomits in 24 hours, bleeding from

the nose or gums, blood vomited, restlessness or irritability, and temperature swings from

extremely hot to extremely cold are all considered severe symptoms (Paddock & Murphy,

PA-C, 2022).

According to data gathered by the Epidemiology Bureau (EB), Department of

Health (Philippines), there has been a six percent (6%) decrease from 27,010 last year to

25,268 this year for the same period. However, the reported 11,435 dengue cases from

March 20 to April 30, also known as Morbidity Weeks 12 to 17, were 94% higher than the

cases reported during the same period in 2021 (5,901) (Department of Health, Philippines,

2022).

1
 The expansion of dengue contributed to factors such as the modern dynamics of

climate change, globalization, travel, commerce, socioeconomics, settlement, and also

viral evolution (Murugesan & Manoharan, 2020).

Prior to 1940, global vector control relied heavily on environmental management,

and larval control was based on a thorough understanding of pathogen transmission but

was eventually replaced by insecticide-based vector control (Wilson, et al., 2020).

However, the use of synthetic repellents has raised several concerns in terms of

environmental and human health risks and safety (Lee, 2018).

This paper therefore presents research on how oils extracted from garlic are used

as a biopesticide against mosquito larvae. The paper will discuss the method and processes

of oil extraction from the organic plant and determine what concentration of garlic oil is

the most effective.

Conceptual Framework

The variables in this study are shown in the study's conceptual framework. The

dependent variable is assumed to be affected by the independent variable.

The independent variable in the study is garlic, which will be analyzed for its

concentration and mortality. The dependent variable of the study is the mosquito larvae.

2
 INPUT PROCESS OUTPUT

Garlic (Allium Larvicidal activity

The researchers used a quasi-
sativum) of the garlic oil.
experimental design where a batch

of 25 mosquito larvae in the

containers were treated with

Other ingredients:
selected concentration. The test
• Garlic (100 g)
was carried out in three different
• Salt (6 g)
larval batches in three replicates.
• Water (200 ml)
• Treatment 1 – 183 ml of

water and 25% (17 ml)

garlic oil.

• Treatment 2 – 167 ml of

water and 50% (33 ml)

garlic oil.

• Treatment 3- 150 ml of

water and 75% (50 ml)

garlic oil.

Figure 1. Conceptual Framework of the Study

The conceptual framework of the study employs the input-process-output approach.

The input includes fresh garlic, which was bought in the town market, as well as salt (6 g).

200 ml of tap water was used in the distillation of 100 g of fresh garlic material.

3
 The process shows that a quasi-experimental design was used in the larvicidal

activity of garlic oil extracted using steam distillation process. The test was carried out in

three different batches, each with three replicates.

The output shows the best garlic oil treatment among the three concentrations (25%,

50%, 75%) used in the experiment.

Objectives of the Study

The general objective of the study is to determine whether garlic (Allium sativum)

oil can be used as a biopesticide against larva mosquito.

The specific goals of the study are as follows:

1. Extract garlic (Allium sativum) oil using the process of steam distillation.

2. Identify which oil concentration treatment among all concentration levels (T1.

25%; T2. 50%; T3. 75%) has the highest mortality rate.

3. Determine any morphological changes that occurred in the morphology of

mosquito larvae after garlic oil treatments.

Significance of the Study

Mosquitoes and garlic are two common things that can be found in the Philippines.

Eradicating a pest does not necessarily need chemicals; alternatives can be found in the

environment. This study will investigate the potential of garlic oil as a biopesticide against

larvae mosquitoes. The results of this study will benefit the following:

4
 The Community. This study will benefit the community by providing information

about how garlic oil can be used as an alternative for preventing dengue. The study’s

findings will aid in stimulating and advancing work on disease prevention and control.

The Researchers. This study will provide researchers with knowledge and

understanding of the characteristics of garlic that give it mosquito-repellent properties,

which can be used to investigate other potential plants for use as a biopesticide.

The Future Researchers. This study will be used as a reference for future research

related to this study and may serve as a guide for further developing the research. This

study can also serve as a background of how biopesticides can be used to eradicate pests.

Scope and Delimitations of the Study

The main focus of this study is to determine whether garlic oil, which was locally

produced and bought at the local market, can be used as a biopesticide against mosquito

larvae that were reared and raised in one of the dengue-infested barangays in Baler,

Barangay Pingit, following the WHO guidelines’ recommendations for the larvicidal assay

procedure.

Vector control methods are categorized into chemical and non-chemical-based

tools. Chemical-based tools include chemical larvicides, while non-chemical-based tools

include habitat manipulation and modification (Wilson, et al., 2020).

The garlic oil treatments that was distilled through steam distillation process with

a 1:2 garlic to water ratio and a 3% NaCl concentration was used as a larvicide against

manually reared mosquito larvae.

5
 This study was carried out with limited resources (materials and funds) and in a

short timeframe.

Time and Place of Study

The collection of mosquito larvae from the reared larvae took place at Barangay

Pingit, Baler, Aurora. The garlic oil was extracted at the Chemistry Laboratory of Aurora

State College of Technology, as well as the conduct of experiments where we tested

whether garlic oil could be used as a biopesticide.

Definition of Terms

Aedes – any of a genus (Aedes) of mosquitoes including the vector of yellow fever,

dengue, and other diseases.

Allicin – a pungent oily liquid with antibacterial properties, present in garlic.

Allium sativum – garlic, frequently used as a seasoning in cooking, but it has also

been utilized as a medicine for both the prevention and treatment of a wide range of

illnesses throughout ancient and contemporary times.

Antifeedant – a naturally occurring substance in certain plants which adversely

affects insects or other animals which eat them.

Biopesticides – are pesticides generated from natural sources such as animals,

plants, microbes, and minerals. Less dangerous to individuals and the environment.

6
 Dengue – a viral infection transmitted to humans through the bite of infected

mosquitoes.

Endemic – a disease outbreak is endemic when it is consistently present but limited

to a particular region.

Epidemic – an outbreak of disease that spreads quickly and affects many

individuals at the same time.

Morbidity – is the state of being unhealthy for a particular disease or situation.

Mosquito – The word “mosquito” is Spanish for “little fly”. Mosquitoes have a

slender segmented body, one pair of wings, one pair of halteres, three pairs of long hair-

like legs, and elongated mouthparts.

Pathogen – is usually defined as a microorganism that causes, or can cause, disease.

Repellent – something that repels, as a substance that keeps away insects.

Steam Distillation – the most widely used method for extracting and isolating

essential oils from plants for use in natural goods.

Vector – an organism (such as an insect) that transmits a pathogen from one

organism or source to another.

Viral evolution – refers to the heritable genetic changes that a virus accumulates

during its lifetime, which can arise from adaptations in response to environmental changes.

7
 CHAPTER II

REVIEW OF RELATED LITERATURE AND STUDIES

Description of Allium Sativum

Kingdom: Plantae

Class: Liliopsida

Order: Asparagales

Family: Amaryllidaceae

Genus: Allium L

Figure 2. Fresh garlic sample Species: Allium sativum L (Cultivated garlic)

Allium Sativum

Garlic (Allium sativum) is one of the best-known herbs in the world. This perennial

plant annually produces edible bulbs composed of several cloves (Mahr, n.d.). Garlic is a

short, upright herb that can grow up to 70 cm high. The bulbs can grow up to 7 cm and

around 1 to 15 cloves each. Most Filipinos know Garlic as a spice - white, cream, pinkish

or brownish cloves - a kitchen mainstay (Sister, et al., 2018).

Besides being called the stinking rose with its pungent and not pleasing aroma,

Garlic has a long medicinal history in various traditions across the world and is also known

as ‘cure-all’ for its ability to treat illnesses of the nervous system, circulatory system,

8
digestive system, reproductive system, and the respiratory system (Ayurvedicoils.com,

2022).

Among the members of the onion family, garlic has by far the highest

concentrations of initial reaction products, making garlic much more potent than onion,

shallot, or leeks. Although many humans enjoy the taste of garlic, these compounds are

believed to have evolved as a defensive mechanism, deterring animals such as birds,

insects, and worms from eating the plant. Because of this, people throughout history have

used garlic to keep away pests such as mosquitoes and slugs (Afakhry.com, 2022).

Aedes mosquito

The different species of Aedes mosquitoes are native to temperate and tropical

habitats worldwide. Some species, however, have spread beyond their native ranges, either

as a result of having been introduced into new areas by humans or as a result of changing

environmental influences. Of particular importance has been the expansion to the Americas

of A. aegypti from Africa and A. albopictus from Asia, which has facilitated the spread of

certain debilitating infectious diseases, notably chikungunya fever, dengue fever, and Zika

fever (Rogers, 2019).

Aedes aegypti, the important carrier of the virus responsible for yellow fever, has

white bands on its legs and spots on its abdomen and thorax. This domestic species breeds

in almost any kind of container, from flowerpots to discarded car-tire casings (Britannica,

T. Editors of Encyclopaedia, 2022).

9
Vector Control

The Philippines has a long history of fighting with dengue. The first recorded

dengue epidemic in Southeast Asia occurred in Manila in 1954, and dengue has since

remained endemic. In 2019 there were 437,563 cases recorded in the Philippines,

contributing to the highest number dengue cases ever recorded globally (Ong, Obeles, Ong,

& Tantengco, 2022).

Vector-borne diseases (VBDs) such as dengue, Chagas disease, human African

trypanosomiasis (HAT), leishmaniasis, and malaria exert a burden of morbidity and

mortality worldwide. The principal method by which these diseases are controlled is

through vector control (Wilson, et al., 2020).

In 1993, the Philippine government’s National Dengue Prevention and Control

Program was established to address the increasing problem. The program includes case and

vector surveillance, case diagnosis and management, integrated vector management,

outbreak response, health promotion and advocacy, and research (Ong, Obeles, Ong, &

Tantengco, 2022).

Unfortunately, the program encountered roadblocks that prevented it from

accomplishing its goals. One of the difficulties encountered was the task of eradicating

local breeding sites, which are primarily water-holding containers. Residents of areas with

intermittent access to piped water store it in these containers and commonly keep a variety

of containers repurposed or even sold for a profit (Ong, Obeles, Ong, & Tantengco, 2022).

A wide range of vector control tools exist, which can be broadly classified into

chemical- and non–chemical-based tools.

10
Table 1. Categories and Examples of vector control methods (Wilson, et al., 2020)

Categories Maturity Vector control methods Description

Chemical Immature Chemical larvicides Contact pesticides affecting insect

nervous system or endocrine system

Adult IRS Spraying of residual insecticides indoors

Topical repellent Chemicals applied to the skin to reduce

vector biting.

Spatial repellent Transfluthrin / metafluthrin passive

emanators or coils

Nonchemical Immature Habitat modification Coverage of large water storage

containers with a material that is

impenetrable to mosquitoes.

Habitat manipulation Water-level manipulation, flushing of

streams, drain clearance, and source

reduction.

Regulatory measures Removal of man-made aquatic habitats

and appropriate waste disposal.

Adult House improvement and Closing eaves, door, and window

screening screening

Removal trapping Solar-powered mosquito trapping system

for malaria control and sticky adulticidal

oviposition traps for Aedes-borne

diseases

11
 Although vector control was considered the most effective approach to eliminate

dengue, it does not guarantee complete success. Thus, plant essential oils (Eos) with good

repellent properties and low toxicities to the environment and humans have been

considered as alternative to conventional synthetic insecticides (Lee, 2018).

Biopesticide

Plant extracts were most likely the first agricultural biocontrols, with nicotine being

used to control plum beetles as early as the 17th century. Experiments with biological

controls for insect pests in agriculture date back to 1835, when Agostine Bassi

demonstrated that the white-muscadine fungus (Beauveria bassiana) could be used to

infect silkworms. Mineral oil experiments as plant protectants were also reported in the

nineteenth century. During the early twentieth century's rapid institutionalization of

agricultural research, an increasing number of studies and proposals for biocontrols were

developed (University of Arkansas, the Ohio State University, U.S. EPA., 2022).

About 80% of pesticides applied enters various environmental resources as a result

of run-off, exposing animals, and farmers as well as consumers of the agricultural produce

to severe health problem. Natural pesticides or “reduced risk” pesticides are natural

compounds that effectively control insect pests, with low toxicity to nontarget organisms

such as humans, animals and natural enemies and the environment (Oguh, et al., 2019).

12
Garlic as Biopesticide

Garlic is widely cultivated and easy to grow in field, garden, or backyard. It is

appreciated as a seasoning or condiment for cooking and due to its medicinal properties.

Its value as a pesticide is also appreciated particularly in organic farming and cultivation

in the backyard gardens. Garlic has anti-feedant (insect stop feeding), bacterial, fungicidal,

insecticidal, nematicidal and repellent properties. Garlic is reportedly effective against a

wide range of disease-causing pathogens and insects at different stages in their life cycle

(egg, larvae, adult) (infonet-biovision.org, n.d.).

Garlic contains elements of chemical compounds such as essential oils, allyl

disulfide, and allicin, effective for repelling various pests. Compounds in allicin garlic,

saponins, and flavonoids are chemicals that can function as insecticides, especially in

eradicating pests and insects that are safe for health and the environment (Hardiansyah,

Ridho, & Nurhidayat, 2020).

13
 CHAPTER III

MATERIALS AND METHOD

This chapter presents the methods that were used in the study. It contains the

materials and statistical tools that were utilized in analyzing the data in the experimental

method of research.

Collection of Sample

The fresh garlic (Allium sativum) was procured at the Baler Public Market in

Población, Baler, Aurora and the same variety was used for all the experiments. Fresh garlic

samples were subjected to separation of cloves by peeling, slicing, paste making, and

scaling through digital weighing balance. Essential oil of garlic was extracted by steam

distillation which involves the diffusion of essential oil into hot water through the plant

membrane (Khan, Keshavalu, & Amaresh, 2017).

Preparation of Distillation

The distillation process that was used in the study is steam distillation. The steam

distillation was processed analogous with the Step-by-Step Procedures for Steam

Distillation (Nichols, 2020).

The plant material extracted was placed into a round-bottomed flask with a 24/29

joint and a 1:2 solid-to-liquid ratio, no more than half full. Rubber bands were used as an

alternative to the extension clamp to secure the large flask to the ring stand. The flask was

14
connected to the condenser by the three-way adapter. The water that runs from the inlet to

the outlet cools down the steam. The garlic extract was received by the Griffin beaker.

Figure 3. Steam Distillation Setup

Preparation of Sample

A 100-gram sample of sliced fresh garlic was put in a distillation flask. To that, 200

ml of water was added. With a further increase in the solid-to-solvent ratio, the oil yield

decreases (Sikdar & R, 2017). Heat is supplied to the distillation unit by the alcohol lamp.

The material-to-water ratio was 1:2, and NaCl concentration was 3.0% (Hanyue, et al.,

2022). The salt was added to the plant material before distillation and had no considerable

effect on the quality but an increase in the oil yield (Shamspur, Mohamadi, & Mostafavi,

2012).

One of the good ways to shorten the time of distillation is to make faster evaporation

of compounds without additional heating increased. It is possible to achieve by adding of

electrolytes i.e., a salt, into distilling pot which modifies the relative volatility of the

components (Mehra, 2009). Relatively small concentrations of salt are capable to increase

considerably the relative volatility of the more volatile component of the liquid solution to

15
be distilled. This behavior, known as salt effect, is due to the preferential solvation of the

ions (formed when the salt dissociates in solution) by the less volatile component of the

liquid solution. In such a case, the more volatile component is salted out from the liquid to

the vapor phase (Kumar & N., 2010).

The experiment was carried out at a constant temperature of 95oC for a 60-minute

time period. Further increase in time has no effect on oil yield (Sikdar & R, 2017). The

distillate was collected in a Griffin beaker. This distillate has two layers, one dense layer

and the other less dense layer. This was then separated using a separating funnel. The less

dense upper layer is the garlic oil. This oil was then stored in a glass bottles shown in Figure

4. The experiments continued, keeping distillation time and solid to solvent ratio constant

(Sikdar & R, 2017).

Figure 4. Garlic oil in griffin beaker

Insect rearing

Mosquito larvae were raised and reared at one of the four most dengue-infested

barangays in Baler, Aurora, Philippines, Barangay Pingit, by the report obtained from

Aurora Memorial Hospital, courtesy of Dr. Teh, PHO.

16
 The researchers stored a container filled with tap water under a kitchen sink to

provide a dark and moisture-rich environment suitable for mosquito breeding (Farnesi,

Barbosa, Araripe, & Bruno, 2018).

The mosquito eggs were deposited on the water as egg rafts and hatched 1-2 days

after deposition. They require no conditioning and cannot be dried (Department of

Communicable Disease Control, Prevention and Eradication, 2005). After the eggs had

hatched, each rearing tray was drained, and the larvae were removed by straining the

medium through a mesh screen (Morlan, Hayes, & Schoof, 1963).

Larvicidal Assay

Larvicidal assays were performed following the WHO guidelines'

recommendations. The water-filled plastic containers were given at least 24 hours for

conditioning or aging. A batch of 25 mosquito larvae that was tested were released into

each container.

After 2–3 h of larval acclimation, the containers were treated with selected

concentrations. The containers were covered with nylon mesh screens or solid covers to

prevent other mosquitoes or other insects from laying eggs and to protect the water from

falling debris. The water levels in the containers were sustained.

The tests were carried out in three different larval batches. The garlic oil was

determined at 3 concentrations (25%, 50%, and 75% of 100 mL garlic oil) in every 200 mL

of water. A total of 225 larvae were exposed to garlic oil in three replicates. The number of

dead larvae was recorded. Larvae with no movement after mechanical or luminous

17
stimulation were considered dead. This process continued until no mortality was noted

(Department of Communicable Disease Control, Prevention and Eradication, 2005).

Data Collection Procedures

A quasi-experimental design was used in the study to identify a comparison group,

that is as similar as possible to the treatment group in baseline characteristics (White &

Sabarwa, 2014). The independent variable, garlic oil, was distilled and prepared at Aurora

State College of Technology Chemistry Laboratory. The dependent variable, mosquito

larvae, was reared and collected at Barangay Pingit, Baler, Aurora. The study was

conducted and observed until no mortality was reported.

Data Gathered

The data gathered in this study were the following:

a. Preparation and distillation process of fresh garlic materials.

b. Essential oil and garlic extract was extracted from garlic (Allium sativum)

through steam distillation.

c. The potential of the three garlic oil concentration treatments as biopesticide

based on mortality rate and hours of exposure.

18
Data Analysis

The data obtained were statistically analyzed using Microsoft Excel 365. The

statistical tool used to analyze the data collected in this study was a one-way ANOVA. One-

way ANOVA has been used for the means of two or more groups that are significantly

different (Kent State University Libraries, 2023).

19
 CHAPTER IV

RESULTS AND DISCUSSION

This chapter was a presentation and analysis of data that were gathered in this study.

Nature of Mosquito Larvae

The larvae of most mosquito species hang suspended from the water surface

because they need air to breathe. An air tube, called a siphon, extends from the larva’s

posterior to the water surface and acts as a snorkel. As a defense mechanism, when alarmed,

the larvae can dive deeper into the water by swimming in a characteristic “S” motion, which

has earned them the nickname “wigglers” or “wrigglers” (VDCI Mosquito Management,

2023) .

While conducting the experiment, the application of oil with the respective

concentration formed a thin film over the surface of the experimental cups, suggesting that

the oil film could prevent the further exchange of gases in to the medium of experimental

cups thereby causing death of the larvae by asphyxia (Elumalai, et al., 2021).

Unlike conventional insecticides which are based on a single active ingredient,

insecticides of plant origin comprised of spectrum of chemical compounds which act

concertedly in many processes by disturbing the insect’s physiology or morphology

(Elumalai, et al., 2021).

Garlic essential oil composition

A total of 14 compounds from the garlic essential oil were obtained, 10 compounds

were identified and 4 unknown which accounted for 97.54% of the total composition.

20
Table 2. Garlic oil composition

Peak
Compound Formula MM RI Ri Rt m/z
number

1 Diallyl sulfide C6H10S 114 17.70 849 5.650 114.05

2 Methyl allyl disulfide C4 H8 S2 120 4.60 911 7.850 122.00

3 Dimethyl trisulfide C2H6S3 126 38.40 972 9.950 127.90

4 Diallyl disulfide C6H10S2 146 5.40 1099 14.90 145.95

5 Diallyl tetrasulfide C6H10S4 210 16.10 1601 15.56 147.95

6 Unknown C6H10S2 146 14.9 1099 16.11 145.95

7 Methyl allyl trisulfide C4 H8 S3 152 5.10 1128 17.42 110.90

8 3-vinyl-[4H]-1,2-dithiin C6 H8 S2 144 26.40 1134 21.18 157.85

9 Allyl trisulfide C6H10S3 178 4.90 1350 25.03 114.05

10 Unknown C4 H8 S2 120 8.60 911 28.45 183.85

11 1,4-Dimethyl tetrasulfide C6H10S4 210 6.60 1601 34.83 209.85

12 Diallyl trisulfide C6H10S3 178 11.80 1350 43.26 113.05

13 Unknown C6H10S3 178 5.40 1350 44.85 146.95

14 Unknown C6H10S3 178 13.40 1350 49.91 186.95

The primary compounds of the garlic essential oil were dimethyl trisulfide

(19.86%), diallyl disulfide (18.62%), diallyl sulfide (12.67%), diallyl tetrasulfide

(11.34%), and 3-vinyl- [4 H]-1,2-dithiin (10.11%), followed by diallyl trisulfide (5.74%),

allyl trisulfide (4.41%), 1,4- dimethyl tetrasulfide (4.06%), allyl disulfide (3.95%), methyl

allyl disulfide (3.87%), and methyl allyl trisulfide (3.76%) (Plata-Rueda, et al., 2017).

21
Larvicidal Activity against Mosquito larvae

The larvicidal activity of the garlic oil in this study was shown on the table below.

Table 3. Garlic oil treatments and mortality rates

Treatment Average Mortality rate (%) Hours of exposure

Treatment 1 3 1

12 2

23 3

29 4

33 5

Total 100 5

Treatment 2 12 1

21 2

35 3

32 4

Total 100 4

Treatment 3 21 1

35 2

44 3

Total 100 3

Table 3 showed the average mortality rate of the three replicates of each

concentration. The data presented showed that Treatment 3, 2, and 1 (75%, 50%, 25% of

100 ml garlic oil concentration) achieved a 100% mortality rate after 5, 4, and 3 hours,

respectively.

22
 The larvicidal activity of the garlic oil was evident at 75% concentrations and

caused a maximum mortality rate of 44% after 3 hours of treatment. The other

concentrations also showed significant differences with respect to the duration of exposure.

Statistical Analysis

The table of interest shown below provided the detail about the characteristics of

the model.

Table 4. Analysis of Variance

ANOVAa
Model Sum of Squares df Mean Square F Sig.

1 Regression 5.556 1 5.556 48.000 .091b

Residual .116 1 .116

Total 5.671 2

a. Dependent Variable: AMR

b. Predictors: (Constant), Treatment

This table indicates that the regression model predicts the dependent variable

significantly well. The "Regression" row and the "Sig.”, indicate the statistical significance

of the regression model that was run. Here, p < .91, which is more than 0.05, indicates that,

overall, the regression model statistically does not significantly predict the outcome

variable (Laerd.com, 2018).

23
Morphological analysis of larvae

The larvae of most mosquito species hang suspended from the water surface

because they need air to breathe. An air tube, called a siphon, extends from the larva’s

posterior to the water surface and acts as a snorkel (VDCI Mosquito Management, 2023)

While conducting the experiment, the application of oil with the respective

concentration formed a thin film over the surface of the water on the experimental plastic

containers, thereby causing the death of the larvae by asphyxia (Elumalai, et al., 2021).

Unlike conventional insecticides which are based on a single active ingredient,

insecticides of plant origin comprised of a spectrum of chemical compounds that act

concertedly in many processes by disturbing the insect’s physiology or morphology

(Elumalai, et al., 2021).

The active substances contained in the garlic solution, which play a role in

larvicidal mechanisms are allicin and flavonoids. Allicin will damage the larval cell

membrane, which will cause lysis and diallyl sulfide has a bactericidal and flavonoid that

act as a respiratory inhibitor in insects (Rahmah, Ashari, Suryadi, & Chairani, 2018).

Morphological analysis of larvae after contact with garlic oil

Random live mosquito larvae were observed under a light microscope before garlic

oil treatment. Dead mosquito larvae exposed to treatments with different concentrations

were mounted on glass slides for further observation and comparison.

24
 A B C D

Figure 5. Mosquito larvae under light microscope after 12h; A = live mosquito; B =

dead larvae (T1); C = dead larvae (T2); D = dead larvae (T3).

Microscopic analysis showed morphological changes throughout the chest of

mosquito larvae after contact with garlic oil treatments. Mosquito larvae showed major

structural damage such as shrinkage, including the abdominal region, the thorax shape

changed, the midgut was damaged, and there was loss of lateral hairs, anal gills, and

brushes (Gunathilaka, de Silva, Dunuweera, & Rajapakse, 2021), destruction of the gut and

extrusion of hemolymphatic content (Rocha, et al., 2015).

25
 CHAPTER V

SUMMARY, CONCLUSION, AND RECOMMENDATION

This chapter provides an overview of the study’s summary, conclusion, and

recommendations.

Summary

This study aimed to determine the potential of garlic (Allium sativum) oil as a

biopesticide against mosquito larvae. The study utilized a quasi-experimental

methodology. The garlic samples that were picked were uniform in size and freshness and

were subjected to steam distillation, wherein the plant material was submerged in water.

The garlic oil extraction process was repeated until 300 ml of garlic oil was

extracted. The oil was pale yellow to almost white and floated above the denser garlic

extract. The garlic oil was divided into three treatments with three replicates each. Each

replicate has 17 ml, 33 ml, and 50 ml for Treatments 1, 2, and 3, respectively, which tallied

up to 100 ml per replicate.

The control group (mosquito larvae) was manually reared and treated with different

dosages after 2-3 hours of larval acclimation. The larvae of most mosquito species hang

suspended from the water surface because they need air to breathe.

The analysis was conducted on April 21, 2023. The researchers followed the

larvicidal assay procedure of the WHO guidelines’ recommendations.

The tests were carried out in three larval batches, with garlic oil determined at three

concentrations (25%, 50%, and 75%). A total of 225 mosquito larvae were exposed to three

26
(3) replicates of each concentration. The number of dead larvae was recorded starting from

the first hour of exposure until no mortality was recorded. Larvae with no movement after

mechanical or luminous stimulation were considered dead.

The application of oil with the respective concentration formed a thin film over the

surface of the water. The active substances contained in the garlic solution, which play a

role in larvicidal mechanisms are allicin and flavonoids. Allicin will damage the larval cell

membrane, which will cause lysis and diallyl sulfide has bactericidal and flavonoid that act

as a respiratory inhibitor to insects.

Mosquito larvae treated with different garlic oil concentrations showed

morphological changes, including but not limited to destruction of the gut, shrinkage of the

abdominal region, loss of lateral hairs, and extrusion of hemolymphatic content causing

dead larvae to appear lighter in body color.

Based on the results of the study, the Allium sativum oil that was used as

biopesticide against mosquito larvae is effective. However, the statistical analysis

calculated that the three concentrations have no significant differences in terms of

effectiveness. This indicates that while all treatments were effective, they varied in how

long before they influenced the mosquito larvae.

Among the three concentration levels present, treatment 3 (75%) has the fastest

mortality rate, indicating that the more concentrated the garlic was in the solution, the more

active substances were present. The researchers therefore concluded that the higher the

concentration, the faster it affects the mosquito larvae.

27
 This study aims to provide a resource for future research and discoveries about the

advantages of garlic and other plant materials that may also be able to get rid of pests,

insects, and larvae, as well as to help the community by identifying effective alternatives

to chemical pesticides and cutting-edge pesticidal methods, particularly larvicidal ones.

Conclusion

Based on the study’s findings, conclusions have been drawn.

1. The extraction of Allium sativum oil as a biopesticide was successful, and

the effectiveness of the treatment was evaluated by the mortality rate of

mosquito larvae.

2. Among the three concentration levels present, treatment 3 (75%) has the

fastest mortality rate indicating that the more concentrated the garlic was in

the solution, the more active substances were present.

3. Mosquito larvae treated with different garlic oil concentrations showed

morphological changes for each treatment.

3.1. Treatment 1 has damaged larvae the least, with slight hemoplymphatic

content extrusion, as it showed a lighter color compared to live larvae.

3.2. Treatment 2 showed an altered thorax of the dead larvae, a damaged

midgut, and the loss of some lateral hair, and hemolymphatic content

extraction much more than treatment 1.

3.3. Treatment 3 has the most apparent shrinkage, a thinner thorax,

damaged midgut, and loss of lateral hairs, anal gills, and brushes, and

28
 it appeared lighter to almost transparent due to huge hemolymphatic

content extrusion.

Recommendation

Based on the results of the study, the researchers recommend the following for

further study:

1. To utilize other methods of garlic oil extraction

2. To test the effectivity of garlic oil as a biopesticide on other life stages of

mosquitoes.

3. To use other parts of garlic, such as chives, as biopesticides against mosquito

larvae; and finally

4. To test whether raw garlic has the potential to be a biopesticide against

mosquito larvae.

29
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33
 APPENDICES

Appendix 1. Collection and preparation of raw material

Fresh Garlic material Cloves-peeling

Chopping Weighing

34
Appendix 2. Preparation of distillation

Distillation Setup

Appendix 3. Preparation of Sample

Sample material placed in Adding 200 ml Heat supplied by

a distillation flask. water alcohol lamp.

35
 Temperature of Separating the oil in Garlic Oil

Distillation 95oC garlic extract.

Appendix 4. Insect rearing

Deposited water for larvae

rearing.

36
Appendix 5. Larvicidal Assay

24 hours water conditioning T1, T2, T3 with 3 replicates each

Monitoring larvae mortality Dead Larvae after 24 hours

37
Appendix 6. Mosquito larvae after garlic oil treatments

Live mosquito larvae Treatment 1

25% garlic oil concentration

Treatment 2 Treatment 3

50% garlic oil concentration 75% garlic oil concentration

38
Appendix 7. Statistical Analysis Tables

Table 5. Model Summary

Model Summary b
Adjusted R Std. Error of the
Model R R Square
Square Estimate

1 .990a .980 .959 .34021

a. Predictors: (Constant), Treatment

b. Dependent Variable: AMR (Average Mortality Rate)

This table provides the R and R2 values. The R value represents the simple

correlation and is .990 (the "R" Column), which indicates a high degree of correlation. The

R2 value (the "R Square" column) indicates how much of the total variation in the

dependent variable, AMR can be explained by the independent variable, treatment. In this

case, 98% can be explained, which is very large (Laerd.com, 2018).

Table 6. Coefficients

Coefficientsa
Unstandardized Standardized 95.0% Confidence

Coefficients Coefficients Interval for B

Model t Sig.
Lower Upper
B Std. Error Beta
Bound Bound

1 (Constant) 3.194 .520 6.147 .103 -3.409 9.798

Treatment 1.667 .241 .990 6.928 .091 -1.390 4.723

a. Dependent Variable: AMR

39
 The Coefficients table provides us with the necessary information to predict AMR

from treatment, and to determine whether treatment contributes statistically significantly

to the model (by looking at the "Sig." column). Furthermore, we can use the values in the

"B" column under the "Unstandardized Coefficients" column, as shown above (Laerd.com,

2018).

40
Appendix 8. Letter of Request to borrow Laboratory glassware.

41
Appendix 9. Letter of Request to access Dengue cases information at Aurora

Memorial Hospital

42
Appendix 10. Dengue Cases in Baler, Aurora

43
 EXHIBIT

Exhibit 1. Fresh Garlic

Exhibit 2. Garlic Peeling

44
 Exhibit 3. Garlic Chopping

Exhibit 4. Weighing 100 g of garlic

45
Exhibit 5. Tap water (200 ml)

Exhibit 6. Salt (6 g) in water

46
Exhibit 7. Preparing the Distillation Setup

Exhibit 8. Preparation of Sample

47
 Exhibit 9. Heating and boiling

Exhibit 10. Maintaining the temperature (95℃)

48
Exhibit 11. Liquid-liquid extraction using separatory funnel.

Exhibit 12. Garlic oil

49
 Exhibit 13. Insect rearing

Exhibit 14. 24 hours water conditioning

50
Exhibit 15. Garlic oil treatments and replicates (T1 -25%, T2-50% and T3-75%)

Exhibit 16. Monitoring mortality rates

51