Larvicidal Activity of the Rind Essential Oils

of Citrus microcarpa Bunge and Citrus

maxima (Burm.) Merr. on Aedes aegypti L.

EMMA CZARINAH M. MAGNAYE

PAOLO L. MEDINA

A Thesis Submitted to the

Department of Biology

College of Arts and Sciences

University of the Philippines Manila

Padre Faura, Manila

In partial fulfillment of the requirements

for the degree of

Bachelor of Science in Biology

May 10, 2018

ii

Department of Biology

College of Arts and Sciences

University of the Philippines Manila

Padre Faura, Manila

Announcement of the

Undergraduate Thesis Presentation

EMMA CZARINAH M. MAGNAYE

and

PAOLO L. MEDINA

Entitled

Larvicidal Activity of the Rind Essential Oils of Citrus microcarpa Bunge and Citrus

maxima (Burm.) Merr. on Aedes aegypti L.

For the degree of

Bachelor of Science in Biology

8:30 A.M., 10 May 2018

Student Center

THESIS ADVISER

Josephine D. Agapito, DrPH.

Associate Professor

Department of Biology

College of Arts and Sciences

University of the Philippines Manila

Padre Faura, Manila

CO-ADVISER

Alicia G. Garbo

Science research specialist II

Head Entomology Section

Department of Science and Technology

Bicutan, Taguig City

THESIS READER

Melody Anne B. Ocampo, M. Sc.

Assistant Professor

Department of Biology

College of Arts and Sciences

University of the Philippines Manila

Padre Faura, Manila

Endorsed by:

Miriam P. De Vera, Ph. D.

Chair

Thesis Committee

Authorized by:

Jay T. Dalet, Ph. D.

Chair

Department of Biology

3

iv

Table of Contents

Page

TABLE OF CONTENTS…………………………………………………………………………iv

LIST OF TABLES………………………………………………………………………………..vi

LIST OF FIGURES……………………………………………………………………………...vii

LIST OF APPENDICES………………………………………………………………………..viii

ACKNOWLEDGMENT……………………………………………………………………...….ix

ABSTRACT…………………………………………………………………………………….…x

CHAPTER 1: INTRODUCTION

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

Statement of the Problem……………...……………………….…………….……………..2

Research Objectives…………...……………….…………………………………………...2

Hypotheses…………………….......………………...……………………………….……..3

Significance of the Study………………….………………..…………………….………....3

Scope and Limitations………………………………………………………………...…….4

Definition of Terms …….......................................................................................................4

CHAPTER 2: REVIEW OF RELATED LITERATURE

Citrus microcarpa………………...……….…....…………………………………………..6

Citrus maxima…………….……….......……………………………………………………8

Essential oil.......…………………….…………………………………………….……….10

Limonene…..........................................…………………………………...………......…..11

Aedes aegypti: Life Cycle and Diseases Carried..................................................................12

Plants As Aedes Mosquito Larvicides…….…………………………...…….….............…17

v

CHAPTER 3: METHODOLOGY

Collection and Identification of Plant..……………………………………………….……19

Essential oil Extraction………………………………………………………..……..…….19

Essential oil Components Determination………………………...……….…………..…...20

Rearing and Preparation of Mosquito Larva………………………………………………21

Larvicidal Bioassay………………………………………………….…………………….22

Data Analysis………………………………………………………………….……….….24

Biosafety and Waste Management……………………………..………………………….24

CHAPTER 4: RESULTS………………………………………………………………….…..…26

CHAPTER 5: DISCUSSION……………………….……………………………………...….…30

CONCLUSION .............................................................................................................................34

RECOMMENDATIONS…………….…………………………………......................................35

LITERATURE CITED…………………………………………………………..……...….……36

APPENDICES……………………………………………………………………………….…..42

vi

List of Tables

Page

Table 1. Aliquots added to 100 ml water to yield final concentration (WHO, 2005)...................23

Table 2. Preliminary Average Percent Mortalities of Aedes aegypti Larvae Treated with C.

microcarpa and C. maxima Rinds Essential Oils..........................................................................26

Table 3. Average Percentage Mortalities (± SD) of Aedes aegypti 3rd Instar Larvae, 24 Hours

after Treatment with C. microcarpa and C. maxima Rinds Essential Oils....................................26

Table 4. LC50 and LC90 (with 95% Confidence Limits) of C. microcarpa and C. maxima Rind

Essential Oils against Aedes aegypti 3rd Instar Larvae..................................................................27

Table 5. Average Percent Mortalities of Aedes aegypti Larvae Treated with Different

Concentrations of Abate 1SG mosquito larvicide.........................................................................27

Table 6. Percent Composition of the Essential Oil Components of C. microcarpa and C. maxima

Analyzed by Gas Chromatography................................................................................................29

Table 7. Summary of Results of Related Literature and of Present Study....................................30

vii

List of Figures

Page

Figure 1. Citrus microcarpa Bunge fruits......................................................................................19

Figure 2. Citrus maxima (Burm.) Merr. fruits...............................................................................19

Figure 3. C. microcarpa rinds........................................................................................................19

Figure 4. C. maxima rinds..............................................................................................................19

Figure 5. Water Distillation Apparatus..........................................................................................20

Figure 6. C. microcarpa and C. maxima rinds essential oil...........................................................20

Figure 7. Larvae Rearing Setup.....................................................................................................21

Figure 8. 4-5-day-old larvae in a water container..........................................................................21

Figure 9. 10,000 ppm stock solution of rinds essential oil............................................................22

Figure 10. Larvicidal Bioassay setup of C. microcarpa (6 replicates of 5 concentrations)..........23

Figure 11. Larvicidal Bioassay setup of C. maxima (6 replicates of 5 concentrations)................23

Figure 12. Schematic Diagram of Procedure of the Study............................................................25

Figure 13 Linear regression line equation of Probit Percentage Mortality to Different log10

concentrations of C. microcarpa Rinds Essential Oil....................................................................28

Figure 14 Linear regression line equation of Probit Percentage Mortality to Different log10

concentrations of C. maxima Rinds Essential Oil..........................................................................28

Figure 15. Linear regression line equation of Probit Percentage Mortality to Different log10

concentrations of Abate 1SG mosquito larvicide..........................................................................29

viii

List of Appendices

Page

Appendix 1. Larvicidal Activity of Citrus microcarpa Rinds Essential Oil (Initial Test) – Raw

Data………………………………………………………………………………………............42

Appendix 2. Larvicidal Activity of Citrus maxima Rinds Essential Oil (Initial Test) – Raw

Data…………………………………………………………………………………....................42

Appendix 3. Larvicidal Activity of Citrus microcarpa Rinds Essential Oil (Final Test) – Raw

Data………………………………………………………………………………...….................43

Appendix 4. Larvicidal Activity of Citrus maxima Rinds Essential Oil (Final Test) – Raw

Data…………………………………………………………………………………....................43

Appendix 5. Larvicidal Activity of Abate 1SG mosquito larvicide – Raw Data...................................43

Appendix 6. SPSS Probit Analysis Output....................................................................................44

6-A. Citrus microcarpa rind oil…………………………………………………………......44

6-B. Citrus maxima rind oil………………………………………………………………....45

6-C. Abate 1SG…………………………………………………………………….………..46

Appendix 7. Identification and Certification of Citrus microcarpa ………………………….…47

Appendix 8. Identification and Certification of Citrus maxima………………………...…….…49

Appendix 9. Accomplishment Report of Extraction of Citrus microcarpa Rinds Essential

Oil………………………………………………………………………………………….…….51

Appendix 10. Accomplishment Report of Extraction of Citrus maxima Rinds Essential

Oil……………………………………………………………………………………….……….52

Appendix 11. Gas Chromatography Test Report………………………………………………...53

Appendix 12. Certification of Laboratory-Reared Larvae……………………….………………54

ix

Acknowledgment

The authors are indebted to the people who made this investigative project possible.

Sincerest gratitude is given to the staff of the Department of Science and Technology-

Industrial Technology Development Institute (DOST-ITDI), specifically to those of the

Standards and Testing Division (STD) for providing the equipment, materials, and laboratory

space for this study. The authors are especially grateful to Ms. Alicia Garbo of the Entomology

Section of DOST-ITDI-STD for her supervision during the whole process of experimentation.

The authors are also thankful for the specific services from the following: Bureau of Plant

Industry (BPI), Chemistry Section of DOST-ITDI-STD, and Chemicals and Energy Division

(CED) of DOST-ITDI.

Lastly, yet most importantly, acknowledgement is accorded to their thesis adviser, Dr.

Josephine Agapito, and their thesis reader, Ms. Melody Ocampo, for all the guidance, advice and

information they have given in finishing the write-up.

x

ABSTRACT

Controlling mosquitoes at the larval stage is an effective measure to prevent mosquito-borne

diseases. Chemical larvicides are known to raise several environmental and public health

concerns; thus, non-toxic and biodegradable plant-based larvicides are becoming popular. The

present study tested the larvicidal activity of Citrus microcarpa (calamansi) and Citrus maxima

(suha) rinds against the third-instar larvae of Aedes aegypti by measuring their LC50 and LC90.

Simple distillation was employed for the extraction of the essential oils. Larvicidal bioassays

showed that Citrus maxima has a stronger larvicidal effect (LC50 of 26.13 ppm and LC90 of

49.34 ppm) than Citrus microcarpa (LC50 of 53.10 ppm and LC90 of 81.38 ppm). The

percentage compositions of their bioactive substances, analyzed by gas chromatography,

revealed limonene to be the dominant component in both essential oils, though higher in Citrus

maxima (95.5%) than Citrus microcarpa (91.6%). The stronger larvicidal property of Citrus

maxima can be attributed to the higher limonene content in its essential oil compared to Citrus

microcarpa.

Key words: essential oil, vector control, larvicidal activity, Aedes aegypti, limonene

0

1

Chapter 1

INTRODUCTION

Background of the Study

Mosquitoes are vectors for various diseases such as dengue fever, Zika virus infection,

malaria, filariasis, yellow fever and encephalitis (Ghosh et al., 2012). Nowadays, these diseases

are becoming more prevalent in different geographical areas. In the Philippines, dengue fever,

malaria and the recent outbreak of Japanese encephalitis contribute to this major health burden.

This is very alarming since the complications brought about by these diseases can be life-

threatening.

In the absence of a definitive cure for Aedes aegypti (L.)-borne diseases such as dengue

fever and Zika virus infection, and because of the recent controversy regarding Dengue vaccine

(Dengvaxia), vector control is an important measure in the prevention of mosquito-borne

diseases. Since larvicides act on the larval stage within breeding sites, its use is promising. In

the aquatic medium, the larvae are easily accessible for the larvicides to effectively disrupt their

growth (Malar et al., 2017). However, the extensive use of chemical insecticides raises several

environmental and public health concerns. Widespread emergence of resistance in vector

species, biological proliferation of toxic chemicals through the food-chain, and the untoward

side effects on non-target organisms developed with the prolonged application of synthetic

insecticides. Because of these drawbacks with chemical insecticides, plant-based larvicidals

which are non-toxic, environment-friendly, biodegradable, and easily available are becoming

popular (Ghosh et al., 2012).

Plants are known to possess unique bioactive substances used for the development of

safe, eco-friendly vector pest control products (Govindarajan, 2011). In most cases, the essential

oils extracted from different parts of plants are biologically active, and have antifungal,

antibacterial, larvicidal and repellent properties. The lipophilic nature of essential oils makes

them able to interfere with the basic functions of insects like metabolic, biochemical,

physiological and behavioral (Govindarajan, 2011). They also have monoterpenes,

sesquiterpenes and other volatile constituents that are attributed to these properties (Zapata &

Smagghe, 2010).

2

The plant samples investigated in previous studies include citrus fruits, like oranges,

citronella and lemon grass (Vargas, 2012). Citrus fruits have been found to exhibit repellent and

larvicidal activities against mosquitoes. Citrus microcarpa Bunge is a plant belonging to the

family Rutaceae, found in Southeast Asia, including the Philippines. The rinds contain

monoterpenes such as limonene, β-myrcene, β-pinene, α -pinene, β-phellandrene and sabinene

as revealed by previous studies (Cheong et al., 2012). Another plant, Citrus maxima (Burm.)

Merr., known in the vernacular as pomelo is indigenous to the tropical parts of Asia. Like C.

microcarpa, it is also part of the Family Rutaceae. Its edible part is small, and the rinds contain

flavonoids and phenolic acid (Oboh & Ademosun, 2011).

Statement of the Problem

To find an alternative larvicidal that is effective, safe, environment-friendly and

affordable, this study tested and compared the larvicidal activities of the rind essential oils of C.

microcarpa and C. maxima against A. aegypti, the vector of dengue fever and Zika virus.

Likewise, it determined and compared the percentage composition of C. microcarpa and C.

maxima rinds essential oils. Specifically, the study answered the following questions:

1. Will the essential oil of C. microcarpa exhibit higher larvicidal activity against A. aegypti

mosquitoes than the C. maxima essential oil?

2. Will the rinds essential oil of C. microcarpa and C. maxima show different percentage

composition of their bioactive substances?

Research Objectives

The study aimed to compare the efficacy of C. microcarpa and C. maxima rinds essential

oil as larvicides against the third instar larvae of A. aegypti by determining of the lethal

concentrations that produced 50% and 90% mortality of mosquito larvae, or the LC50 and

LC90, respectively. Furthermore, it was also the objective of this study to compare the

composition of the bioactive components of the rind essential oils from both plant samples.

3

Hypotheses

The study proved the following hypotheses:

Ho: The essential oil from C. microcarpa rinds will not show higher larvicidal activity against A.

aegypti mosquitoes than that of C. maxima rinds

Ha: The essential oil from C. microcarpa rinds will show higher larvicidal activity against A.

aegypti mosquitoes than that of Citrus maxima rinds.

Ho: The essential oil from C. microcarpa rinds and C. maxima rinds will not differ in the

percentage compositions of the components.

Ha: The essential oil from C. microcarpa and C. maxima rinds will differ in the percentage

compositions of the components.

Significance of the Study

Mosquito-borne Zika virus and Dengue fever have become important public health

concerns because of the increasing number of reported cases in the country. Hence, the

mosquito control remains to be an effective measure to prevent mosquito-borne diseases. Vector

control by arresting the earlier stages of the mosquito rather than the later adult stages is a

generally accepted method by using larvicides. However, the use of commercially-available

chemical larvicides raises several concerns related to the environment and public health. Thus,

alternative and naturally-occurring larvicides, mostly coming from plants, are becoming popular

since they are considered as safe and environmentally friendly. Generally, the rinds and peelings

of fruits produce a considerable amount of waste, which ultimately worsen the garbage problem

in the country. The utilization of these citrus fruit rinds for larvicidal purposes will minimize

garbage piles from kitchen wastes that are being sent to landfills for disposal. If the result of the

larvicidal bioassay show that these fruit rinds contain essential oils that are effective larvicides,

then more use can be derived from these fruits that are only used mostly as food. Furthermore,

knowledge of the components of these essential oils can serve as a basis for the development of

4

a larvicide that can be as effective as the commercial products but also retaining being safe and

environmentally friendly. This provides the consumers an alternative product to the

commercially available larvicidals since it is natural, safe, economical and environment

friendly.

Scope and Limitations

The study focused on testing the larvicidal effect of the rind essential oils from the fruits of

C. microcarpa and C. maxima against the 3rd instar larvae of A. aegypti. The rind essential oils

of the two plant samples was extracted through water distillation method. The larvicidal

bioassays were performed on laboratory-reared larvae in laboratory conditions and thus, results

may not be reproduced in field setting. In the determination of percentage composition of the rind

essential oils, only six possible essential oil components were available to be analyzed at the time

of the procedure.

Definition of Terms

Aedes aegypti (L.) is the species of mosquito whose females are the main vector for the

transmission of Dengue fever and Zika virus (Kliegman et al., 2007).

Citrus microcarpa Bunge is a variety of citrus fruit belonging to the Family Rutaceae, with

green to yellowish spongy covering (Dharmawan et al., 2009).

Citrus maxima (Burm.) Merr is a variety of citrus fruit belonging to the Family Rutaceae,

characterized as having small edible part and with pale yellow to yellowish-green, thick and

spongy covering (Oboh & Ademosun, 2011).

Dengue fever is a disease caused by a virus transmitted to man by the female Aedes aegypti,

with flu-like symptoms of high grade fever, headache, joint pains (Kliegman et al., 2007).

5

Essential oil is a natural oil extracted from plants through hydrodistillation, steam distillation,

dry distillation or mechanical cold compressing (Ragnault-Roger et al., 2011).

Instar is the age between two periods of molting in an insect larva’s development.

Larvae are the immature forms of an insect that undergo some metamorphosis.

Larvicidal are substances used in killing larval pests.

LC50 is the lethal concentration that produces 50% mortality of mosquito larvae (Manzoor et

al., 2013).

LC90 is the lethal concentration that causes 90% mortality of the exposed mosquito larvae

(Reegan et al., 2013).

Zika virus is a mosquito-borne disease with Aedes aegypti as the main vector, and where the

infected individual maybe asymptomatic or with only mild symptoms of fever and macula-

papular rashes, or symptomatic with more severe symptoms and may cause death of the

infected person (Ghosh et al., 2016).

6

Chapter 2

REVIEW OF RELATED LITERATURE

Citrus microcarpa

Fruits from the Genus Citrus are ancient crops from the Family Rutaceae, thought to have

originated from Southeast Asia, Southwest China, East India and North Burma, that are

considered world major fruit crops. Important fruits that can be classified as Genus Citrus

include oranges, mandarins, limes, lemons and grape fruits. Traditionally, citrus fruits are mainly

intended for production of dessert, juice and jam. Thus, to satisfy the high demands of agro-food

industry and exportation, the citrus varieties are frequently and widely cultivated (Morte &

Acero, 2017). Citrus fruits come in a range of sizes, from the small Citrus microcarpa fruits to

the large C. maxima fruits.

Citrus microcarpa Bunge is a member species of the Family Rutaceae. It is widely grown

in tropical and subtropical areas, including Taiwan, China, the Philippines, Vietnam, and

Malaysia. The fruit is most abundant from August to October in the Philippines but is

commercially available all year round. Also known to many by other names such as calamondin,

calamansi is considered a natural hybrid of C. reticulata (mandarin) and C. japonica (oval

kumquat). Its exotic nature has recently attracted worldwide attention (Lawrence, as cited in

Cheong et al., 2012a), and now the plant is grown in South and Southeast Asia, Central and

North America, the Caribbean and Hawaii (Cheong et al., 2012a).

The calamansi tree on the average has a height of 3 to 5 meters and is slender and erect. It

has dark green leaves on the surface, but yellowish beneath. It is densely branched, close to the

ground, having slight spines, with sweetly fragrant white flowers with 5 elliptic-oblong petals.

The fruit of this plant is small and round, averaging a diameter of 4.5 cm, and has a very thin

green colored peel or rind with noticeable pores. The calamansi fruit is likened to small unripe

lemon or orange, though much smaller in size. The inner flesh of calamansi fruit or the pulp is

colored yellow or bright yellow with 6 to 10 segments. The flesh is juicy, and is sweet and sour,

bright yellow in color has a unique and pleasant aroma, with 1-5 small ovoid green seeds, but

7

sometimes seedless (Morte & Acero, 2017). In the Philippines the calamansi fruit is extensively

used in various food and beverage preparations. It is used as a condiment for some famous dishes

and is one of the most common citrus fruits that is often used by Filipinos in seasoning dishes. In

addition, the juice extracted from calamansi is used as dipping sauce for some dishes, and is also

used in marinades, salad dressings, barbecue sauces, meat stews as well as in herbal tea

preparations (Chen et al. 2013). The fruit may be crushed and applied onto the scalp as hair shampoo;

it also relieves itchiness and is utilized to enhance hair growth. To eliminate irritation and itching, the

juice may also be applied and rubbed on mosquito bites. Moreover, it may also be used to remove freckles

and if applied on a regular basis is said to clear up pimples or acne vulgaris (Chen, et al. 2013).

Calamansi is one of the good sources of Vitamin C. The fruit is squeezed for juice.

Because of its low sugar content (glucose, fructose, and sucrose), its rich aroma components and

high levels of ascorbic acid (44.5 mg/100 g), dehydroascorbic acid (2.2 mg/100 g), and citric

acid (3.6%), the fruit is used as brewed tea. The high acidity reflected by the high citric acid

content characterizes the calamansi juice. Thus, calamansi drinks are popular nowadays (Chen et

al., 2013). Another use of calamansi juice is as fecal softener when diluted with warm water and

drunk. Furthermore, it can also be used as a cough remedy, to expel the phlegm when taken

orally with pepper (Morte & Acero, 2017).

Citrus fruits such as calamansi are utilized mainly for its pulp and juice, but only the pulp

was squeezed and is needed. The rest of the fruit or the pressed pulp, covering of pulp segment,

seeds, and the rinds and peels are unused and discarded after the extraction of the juice, and are

considered largest source of citrus waste. However, the peel/rind has been chemically analyzed

recently (Cheong et al., 2012a). Finding a way to utilize those wastes will help the environment.

Cheong et al. (2012a) found that calamansi rinds were composed mainly of volatiles and

phenolic acids. The volatiles were predominantly comprised of monoterpenes such as limonene,

β-myrcene, β-pinene, α -pinene, β-phellandrene and sabinene. Meanwhile, four types of phenolic

acids were found: caffeic acid, p-coumaric acid, ferulic acid, and sinapic acid.

The calamansi is just one of the common citrus fruits commercially available in the

Philippines. Another is the fruit of C. maxima or suha.

8

Citrus maxima

Another member of Family Rutaceae, Citrus maxima (Burm.) Merr. is known as pomelo,

suha, and shaddock in the vernacular. This species of citrus fruit is native to Southern China and

Malaysia and other parts of Southeast Asia, but now many semi-tropical and tropical countries

grow these large fruits. C. maxima fruit is spherical in shape, around 7.9 cm in diameter, fleshy,

and tastes sour and bitter, but is edible. The outer surface of fresh peel is greenish yellow in color

and has strong aromatic odor, when dried it turns to brownish yellow. The inside of fresh peel is

white, but when dried it turns to brownish color (Jadhav et al., 2013). The large size, thicker,

spongy rinds, milder and sweet flavor, and tough bitter membranes characterize C. maxima. The

fruits are also rich in vitamin C, just like the other citrus fruits. The medicinal uses of C. maxima

fruit are for treatment of coughs, fevers, and gastrointestinal disorders. In Vietnam, the aromatic

flowers are made into perfume (Oboh & Adenmosun, 2011).

The essential oils extracted from leaves or rinds of pomelos are popular because they

offer a variety of health benefits. The pharmaceutical industries and aromatherapy use this

essential oil. It has antioxidant, larvicidal, antibacterial, antifungal, anticancer, antiplatelet,

antidiabetic activities, and is hepatoprotective (Jadhav et al., 2013). The main compounds in the

pomelo essential oils consist of limonene, terpinolene, sabinine, α-pinene, β-pinene, myrcene, α-

terpinene, γ-terpinene and linalool (Oboh & Ademosun, 2011). However, Minh Tu et al. (2002)

did not detect γ-terpinene in pomelo when subjected to the cold-pressing essential oil extraction

method. While Njoroge et al. (2005) found that limonene was the major monoterpene

hydrocarbon compound with 94.8% followed by α-terpinene and α-pinene with 1.8% and 0.5%,

respectively; they also used the cold-pressing method. Meanwhile, Hosni et al. (2010) found that

β-pinene was the second major compound that followed limonene with 1.52%; they isolated the

pomelo rind essential oils using hydrodistillation.

Pomelo rind essential oils have been tested for their antioxidant, antimicrobial, and

acaricidal activity. The antioxidant property of pomelo rinds has been recently exhibited when

Chowdhury et al. (2015) treated rats with pomelo rind powder to prevent oxidative stress and

liver damage when exposed to CCl4. Oboh & Ademosun (2011) characterized this antioxidative

property to the phenolic extracts from the pomelo rinds. They demonstrated that the phenolics

9

from the rinds inhibited the production malondialdehyde in rat pancreas; malondialdehyde is an

oxidative stress marker.

Chanthapon, Chanthachum & Hongpattarakere (2008) found that the peel essential oil and

crude extracts of pomelo peels have low antimicrobial activity. They used two different methods

for extracting essential oil from the pomelo rinds: hydrodistillation and ethyl acetate extraction.

Using disk diffusion assay, they found that the ethyl acetate extracts have low to no antimicrobial

activity against Staphylococcus aureus and Escherichia coli. They also found that both the

hydrodistilled extract and ethyl acetate extracts have a relatively high MIC and MBC (>2.25

mg/ml) against all the bacteria they tested, namely Bacillus cereus, S. aureus, Listeria

monocytogenes, Salmonella sp., and E. coli. However, they found that the ethyl acetate extract of

pomelo peel had a significantly lower MIC (0.56 mg/ml) against the fungi species they tested

(Saccharomyces cerevisiae and Aspergillus fumigatus) indicating that pomelo peels might be

more of an antifungal agent rather than an antibacterial one.

Chungsamarnyart and Jansawan (1996) tested the larvicidal and acaricidal activity of

pomelo—among other citrus fruits—peel oil against the tick Boophilus microplus. They

mechanically pressed the citrus fruit peels to extract the oil. They dipped the larval and adult

ticks to appropriate ethanolic dilutions of the oil extract. They found after 1-2 hours that the

pomelo peel oil had the second highest larval mortality rate compared to the other citrus peel oils

with 95% following closely the 98% of C. sinensis peel oil. After 24 hours from dipping in

pomelo peel oil, 98% of the adult ticks died. Their study also found that limonene may not be the

active ingredient in pomelo peel oil as a larvicidal and acaricidal agent because when they also

conducted the same dipping method using pure limonene, it resulted in lower mortality than

pomelo peel oil with 76% larval mortality and 73% adult mortality.

Compared to that of C. microcarpa, the rind essential oil of C. maxima has been studied

more extensively. However, in terms of composition they are similar in having limonene as a

major component in their rind essential oils.

10

Essential Oil

Large quantities of oils from citrus peel can be obtained cheaply by cold pressing and

simple water distillation. Essential oils are produced in 17,500 aromatic species of higher plants

belonging mostly to a few families, including the Myrtaceae, Rutaceae, Lauraceae, Lamiaceae,

and Asteraceae. The production and collection of essential oils are related to the complex

secretory structures of the plant including glandular trichomes (in Lamiaceae), secretory cavities

(in Myrtaceae, Rutaceae), and resin ducts (in Asteraceae, Apiaceae). Essential oils are stored in

different plant organs, depending on the species being considered. Essential oils’ storage

includes plant organs like wood (sandalwood, Santalum spp.), roots (vetiver grass, Chrysopogon

zizanioides), leaves (lemon grass, Citronella spp.; eucalyptus, Eucalyptus spp.), flowers

(bergamot orange, Citrus bergamia), rhizomes (ginger, Zingiber officinale; turmeric, Curcuma

longa), seeds (nutmeg, Myristica fragrans), and fruits (anise Pimpinella anisum) (Regnault-

Roger, 2011). The function of the essential oils for these plants may have been discovered due to

the studies of their insecticidal activities.

Essential oils have repellent, insecticidal, and growth-retarding effects on a variety of

insects. Their constituents exert insecticidal effects or retard and disrupt the growth of insects at

several life stages. Because of the popularity of essential oils from aromatic plants with organic

growers and environmentally conscious consumers, their use as low-risk insecticides have

increased considerably recently (Regnault-Roger et al., 2011). Essential oil from various plants

has been found to be toxic against different mosquito species in the field of vector control. It is

presumed that essential oils interfere with basic metabolic, biochemical, physiological and

behavioral functions of insects. A significant vacuolation, swollen nuclei and elongated epithelial

cells of the mosquito larvae treated with essential oil (Ruiz et al., 2004). It is at the apical region

that said epithelial cells are disrupted. In addition, there are vesicle formation, lysis and leakage

of cytoplasm material into the gut lumen. (Mann & Kraufman, 2012). Likewise, the mosquito

larvae and pupae must come to the surface frequently to breathe because they are known to

breathe through spiracles located on the eighth abdominal segment. The plant essential oils block

the spiracles, thereby blocking the respiratory siphons, resulting to asphyxiation and hence death

of the larvae (Karufmann & Briegel, 2004).

11

Due to their rich source of bioactive compounds that may be biodegradable into nontoxic

products, plant oils may offer a potential benefit for mosquito control (Phukerd & Soonmera,

2013). Several plant oils have been studied and found to possess mosquito larvicidal and

pupicidal properties (Regnault-Roger, 2011). An effective method to reduce mosquito densities

before they emerge as adults is the use of larvicides, and synthetic insecticides have been used

for this purpose (Tiwary et al., 2007). The larvicides are best employed in small breeding places

because the water is stagnant. It is more difficult to kill adult mosquitoes than to disrupt the

growth and development of larvae in stagnant water (Ansari et al., 2005).

The larvicidal activity of monoterpenes affects multiple targets, thereby capable of

disrupting the cellular activity and biological processes of insects (Regnault-Roger et al., 2011).

The mode of action of these compounds include their activity on insects through neurotoxic

effects involving several mechanisms, such as through, gamma-aminobutyric acid (GABA),

octopamine synapses (Regnault-Roger, 2011). In addition, a review on the mechanism of action

of essential oils on the body of insects was documented and include the following: disruption of

the molecular events of morphogenesis, alteration in the behavior and memory of cholinergic

system. and inhibition of acetylcholinesterase, of these, the last one is the most important since it

is the enzyme acetylcholinesterase which is responsible for terminating the nerve impulse

transmission through synaptic pathway (Rattan 2010).

A proposed mechanism of action of the small-volatile molecules, acyclic or monocyclic

monoterpenes are known to be its involvement in the transmission of airborne signals from

plants to insects. Specialized odorant binding proteins (OBPs) which are in the sinsilla of insects

respond to volatile monoterpenes. An example is the trichoid sinsilla in the female silkworm

(Bombyx mori), which is responsive to linalool (Regnault-Roger, 2011). One small component

molecule but is the most represented one in citrus rinds is limonene.

Limonene

D-limonene (1-methyl-4-(1-methylethenyl) cyclohexane) is a monocyclic monoterpene

known to be the major constituent of citrus essential oils. Limonene is a clear colorless liquid at

12

room temperature. It is a naturally occurring substance known to possess various biological

activities and has been associated to have potential chemopreventive and antitumor effects (Sun,

2007). Limonene also exhibit repellent and larvicidal activities. Limonene is known to target the

nerve activity of insects through several neurotoxic mechanisms. These mechanisms include

action on gamma-aminobutyric acid (GABA) and octopamine synapses (Regnault-Roger, 2011).

Limonene is known to alter the insects’ behavior and memory and inhibit acetylcholinesterase,

which is the enzyme responsible for terminating the nerve impulse transmission through synaptic

pathway (Rattan, 2010). The transmission of airborne signals from plants to insects is said to be

affected by the small-volatile molecules, acyclic or monocyclic monoterpenes, particularly

limonene, which produces massive over-activation of motor nerve, thereby inducing convulsion,

paralysis and eventually death of the mosquitoes (Mwaiko, 1992).

Aedes aegypti: Life Cycle and Diseases Carried

The Aedes aegypti (L.) mosquito is the main vector for dengue fever and Zika virus. Unlike

other mosquitoes, the Aedes mosquito is usually an early morning feeder. They bite at dusk and

dawn, though at night under artificial light, they may also bite. Female Aedes aegypti bites

multiple people during each feeding period. They prefer breeding areas like containers filled with

stagnant water, wet shower floors, toilet tanks and discarded tires. It is the female mosquito that

bites because it needs blood to mature its eggs. Depending on conditions, the life span of an adult

mosquito is 2-4 weeks (Kliegman et al., 2007).

The whole life span or cycle (WLC) consists of four main stages: egg, larval, pupa, and

adult stages; it will take 10 days from egg to adult. Eggs are deposited singly by female adults in

stagnant water-filled containers. They do not have floats unlike Anopheline mosquitoes and are

uniquely resistant to dessication. The eggs are hatched when flooded with water. However,

development will cease in dry conditions and will commence when rains arrive. Larvae will

hatch from eggs typically 2-3 days from laying. Like all other mosquitoes, the larval stages of A.

aegypti consist of four instars, or stages of growth between molting. A. aegypti larvae are

characterized by resting at an angle below the surface of water. They have no palmate hairs like

other Culicine mosquitos. They are differentiated from Culex larvae by having only one pair of

hair tufts in their siphon or air tube. They filter feed on microorganisms until they pupate. The

13

pupae are comma-shaped active and have a pair of breathing tubes with narrow openings. The

pupal stage lasts for 2-3 days. Adults emerge quickly when the skin of the pupal thorax facing

the water surface splits, and fly away (Cox, 1993; Roberts, Schmidt and Janovy, 2009). A newly-

emerged female adult will seek for a blood meal, and after acquiring blood, the gonotrophic

cycle, which lasts for 2-3 days, will begin (Cox, 1993). The gonotrophic cycle (GC) will be

followed by the laying of eggs when the blood is fully digested.

According to Goindin (2015), under optical conditions, when a female A. aegypti adult

ingests flavivirus-infected blood (Zika and dengue virus for example), it is its offspring that will

become vectors of the virus not the parent. This is because the virus has an incubation period

within A. aegypti which lasts 12-13 days (the duration of one GC plus the duration of one WLC).

The F1 mosquitoes are the ones capable of transmitting diseases such as dengue fever.

Dengue fever is a mosquito-borne infection found in tropical and sub-tropical regions

around the world. In recent years, transmission has increased predominantly in urban and semi-

urban areas and has become a major public health concern worldwide. There are four distinct

serotypes of the virus, but closely related that cause dengue (DEN-1, DEN-2, DEN-3 and DEN-

4) (WHO, 2016).

It is through the bites of infected female Aedes aegypti mosquito that the dengue virus is

transmitted to humans. An infected mosquito can transmit the virus for the rest of its life after 4–

10 days incubation. Infected humans serve as the source of the virus for uninfected mosquitoes

since they are the main carriers and multipliers of the virus. After the patient’s first symptom

appear, they can transmit the infection for 4–5 days up to a maximum of 12 days via Aedes

aegypti mosquitoes (WHO, 2017).

Dengue fever is an illness that affects infants, young children and even adults. The patient

experiences flu-like symptoms such as fever (temperature around 40°C), severe headache,

muscle and joint pains, nausea and vomiting. Maculopapular rashes like those of Measles appear

in some patients. Symptoms usually last for 2–7 days, after an incubation period of 4–10 days

after the bite from an infected mosquito. The more severe form of dengue hemorrhagic fever is

14

one of the leading causes of hospitalization and mortality in the country, and is experienced by a

few number of patients, mostly pediatrics. Symptoms of dengue, hemorrhagic fever include high

grade fever, severe abdominal pain, drowsiness and lethargy. It is a life-threatening condition,

and internal bleeding may result because of very low platelet counts and plasma leakage.

Warning signs occur 3–7 days after the first symptoms in conjunction with a decrease in

temperature (below 38°C/100°F) and include: severe abdominal pain, persistent vomiting, rapid

breathing, bleeding gums, fatigue, restlessness, blood in vomitus. The next 24–48 hours of the

critical stage can be lethal; hospitalization and proper medical care is needed to avoid

complications and risk of death. A more advanced condition, the dengue shock syndrome causes

low blood pressure and is likewise life- threatening (Kliegman et al., 2007).

When a victim recovers from the infection it provides him lifelong immunity against that

specific serotype. However, cross-immunity to the other serotypes after recovery is only partial

and temporary, and subsequent infections by other serotypes increase the risk of having severe

dengue. Dengue cases have dramatically increased around the world in recent decades (WHO,

2017).

There is no specific treatment for dengue fever, and the management of patients focuses on

the symptoms. However, for severe dengue, medical care by physicians and nurses, who are

experienced with the effects and progression of the disease is of outmost importance as this can

save lives. It is necessary to maintain the patient’s body volume in this stage of dengue (WHO

2017).

Dengvaxia, the first dengue vaccine produced by Sanofi Pasteur was registered in the late

2015 and early 2016 for use in individuals 9-45 years of age living in endemic areas. However,

there is still uncertainty about this vaccine. There is a need for more research regarding its dose

and the recipients. There are important factors affecting the effectiveness of the vaccine,

including the age of the recipient, the type of dengue, and previous infection with dengue. A

controversy has arisen with this vaccine because of the reported deaths of some patients, which

are attributed to Dengvaxia, so that presently, the only method to control or prevent the

transmission of dengue virus is to combat vector mosquitoes through: environmental

15

modification and management by preventing mosquitoes from gaining access to egg-laying

habitats; spraying insecticides during outbreaks; employing the use of insecticides to outdoor

containers with stagnant water; using of personal household protection such as use of repellents,

wearing long-sleeved clothes, and installing window screens, coils and vaporizers; proper

disposing of solid wastes; covering, emptying and cleaning of household outside water storage

containers regularly; and continued monitoring and surveillance of vectors to determine the

effectiveness of vector control measures (WHO, 2016).

The Aedes aegypti mosquito is also the primary carrier of the virus that causes Zika virus

infection. Zika virus is a member of the Flavivirus genus within the Flaviviridae family from the

Spondweni group. This Flaviviridae family consists of a large group of enveloped viruses, which

includes dengue, yellow fever, West Nile, Japanese encephalitis. They have a single stranded

RNA genome of positive polarity, which serves as mRNA upon the infection of susceptible cells

(Hamel et al. 2015).

Unlike other genus of mosquitoes, Aedes bites during the day. Vector-mediated

transmission of ZIKV starts when a blood-feeding female Aedes mosquito bites a mammalian

host and thereby injects the virus into the host’s skin. Infection via specific receptors of

permissive cells, like, skin immune cells, including dermal fibroblasts, epidermal keratinocytes,

and immature dendritic cells, follows the intital stage (Hamel et al. 2015).

Sexual transmission has been reported in several occasions. Other modes of transmission

of Zika virus include: maternal-fetal probably through trans-placental transmission and most

likely during delivery by an infected mother, transmission through blood transfusion, and organ

transplantation.

The incubation period is usually 3-12 days after the bite of an infected mosquito. Around

80 % of the infected patients were found to be asymptomatic. The remaining 20% of those

afflicted with disease generally manifested with milder symptoms such as an acute onset of

fever, macula-papular rashes, non-purulent conjunctivitis, arthralgia, headache or myalgia. The

infection is usually self-limiting and short lived, usually lasting for 2-7 days without serious

complications. Other patients experienced dizziness, anorexia, constipation, diarrhea and

abdominal pain (WHO, 2016).

16

Among pregnant women infected with Zika virus, microcephaly and fetal losses were

reported during several recent outbreaks of Zika virus infection. A not so usual increase of

incidence of Guillain–Barré syndrome were reported to coincide with the Zika virus outbreaks in

2014 in several countries in the Americas and French Polynesia (Rubin et al., 2016).

Just like Dengue virus infection, there is no curative treatment for Zika virus infection. In

addition, there is no vaccine yet for this disease (Ghosh et al., 2016). The management of

infected patients remains symptomatic and includes maintaining adequate hydration, giving pain

reliever, and antihistamine for itchiness. However, the giving of aspirin and non-steroidal anti-

inflammatory drugs should be avoided if the possibility of Dengue has not yet been ruled out

because of the possibility of bleeding. Treatment with Aspirin is also not recommended in

children and teenagers because of the risk of Reye’s syndrome. Although the use of antivirals

during acute infection represents a challenge, such therapies would be impractical because the

illness is only self-limiting. But because the development of vaccines takes years, measures to

reduce transmission through mosquito vector control deserves attention. Hence effective

prophylactic measures are of outmost importance (Nabel & Zerhouni, 2016).

Primary prevention is mainly based on protection against mosquito bites, and personal

protection should be employed all day, more so during mid-morning to twilight, when the

highest biting activity of Aedes aegypti mosquitoes is observed (Nabel & Zerhouni, 2016).

Measures to reduce mosquito breeding places both indoor and outdoor, like in the case of

Dengue, merit attention. These include: discarding containers with stagnant water, elimination of

adult mosquitoes using aerial insecticide spray, using repellents. Pregnant women should avoid

or at least postpone travelling to places where there is Zika virus outbreak. However, political

will, adequate economic resources, improved diagnostic technology, manufacturing capacity,

clinical infrastructure, and health care delivery systems are necessary to improve protection,

thereby ensuring the public that measures are applied systematically, consistently, with local

flexibility (Nabel & Zerhouni, 2016). Another preventive measure is the use of larvicides. Since

Goindin (2015) found that the development of these viruses into an infective form occurs during

the larval stages of the mosquito, arresting the mosquito development at these stages might be

effective in the prevention of the spread of disease. However, commercially-available larvicides

have proven to be problematic in many ways: resistance in vector species, adverse effects on

17

environment, biological magnification of toxic substances through the food chain, and toxic

effects non-target organisms including human health (Ghosh et al., 2012). And thus, many

studies have been done on plant extracts being tested as larvicides as alternatives to these

synthetic larvicides.

Plants as Aedes Mosquito Larvicides

Various plant materials have been tested against A. aegypti. Among them was Abutilon

indicum, an invasive medicinal plant species in the Malvaceae family (Arivoli & Tennyson,

2011). It was found that its LC50 against 3rd instar larvae after 24 hours of exposure was 261

ppm. Meanwhile, Adeleke et al. (2013) used two different species of seed oils against the same

stage of A. aegypti, namely, Pterocarpus santalinoides and Tropical Manihot Species (TMS)

30572. Their LC50 were both statistically similar (p>0.05) and lower than the result of Arivoli &

Tennyson (2011) at 104 ppm and 114 ppm, respectively, meaning these two are more potent

larvicides than A. indicum. Even lower was the result of El-Sheikh et al. (2016) when they tested

Tribulus terrestris also against 3rd instar A. aegypti larvae with an LC50 of 64.6 ppm. Other

studies used 4th instar instead of 3rd instar A. aegypti larvae. Such as Reegan et al. (2013), when

they tested the methanolic extract of Cliona celata against the said larval stage and found that its

LC50 was 158.40 which was statistically like both results of Adeleke et al. (2009). However,

since those two results were against 3rd instar, the 3rd instar LC50 of C. celata might be lower

than the LC50 those two plants (P. santalinoides and TMS 30572). Another study used 4th instar

larvae and also used two plants in one study (Kamalakannan, 2011). A unique aspect of this

study is the combination of the two plant materials after testing each against 4th instar A. aegypti

larvae. The two plants were Achyranthes aspera and Acalypha indica indica, with LC50 at 409

and 420 ppm, respectively. When combined, the LC50 lowered to 277 ppm making it

statistically similar to the result of Arivoli & Tennyson (2011) which tested on 3rd instar larvae,

so the combined A. aspera and A. i. indica LC50 might be actually lower when tested on 3rd

instar larvae. The LC50 result of El-Sheikh et al. (2013) is the lowest among the non-citrus

plants tested against A. aegypti at 64.6 ppm.

Citrus fruits have been tested as mosquito larvicides around the world. These studies

oftentimes test citrus fruits that are available in their local markets. Akram et al. (2010) tested

seed extracts from ten Citrus plants against the 4th instar larvae of another dengue-carrying

18

mosquito species, Aedes albopictus. The test plant samples were seeds from C. aurantium, C.

grandis, C. pseudolimon, C. paradisi, C. reticulata, C. limon, C. mitis, C. jambhiri, and C.

sinensis. The results of their study revealed the most effective among the tested plant samples

against the larvae of A. albopictus is the seed extract of C. jambiri with the lowest LC50 of 120

ppm after 24 hours. One of the least effective was C. sinensis seed extract with an LC50 of 1389

ppm which is statistically (at 0.05 significance level) significantly higher than the LC50 of 437

ppm result of Murugan et al. (2012) when they tested the peel extracts of C. sinensis against A.

aegypti 4th instar larvae. This result of Murugan et al. (2012) was statistically (at 0.05

significance level) like the result of Warikoo et al. (2012) which found that the LC 50 of leaf

extracts of C. sinensis against A. aegypti 4th instar larvae was 447 ppm. These results show that

seed extracts may not be as toxic to Aedes sp. 4th instar larvae as the leaf and peel extracts of C.

sinensis. Meanwhile, Kumar et al. (2012) tested C. limetta peel extracts against also the 4th instar

larvae of A. aegypti which resulted in an LC50 of 96.15 ppm which is statistically significantly

lower than all the results above other than the result of C. jambhiri seed extract of Akram et al.

(2010). However, the lowest LC50 results were from Torres et al. (2016) with 1.11 ppm from the

peel extract C. grandis from Davao, Philippines against 3rd instar A. aegypti larvae. This is

statistically significantly lower than the C. grandis (harvested from Pakistan) seed extract result

(LC50 = 335 ppm) of Akram et al. (2010) against 4th instar A. aegypti larvae. This suggest that

geographical location may affect the larvicidal activity of some plant materials. Another

Philippine study that also used a citrus fruit is De Villa et al. (2012). Their C. microcarpa peel

extract bioassay against 3rd instar larva of A. aegypti resulted in an LC50 of 451 ppm which is

statistically like the results of Murugan et al. (2012) and Warikoo et al. (2012) said above.

However, since these two results were against 4th instar larvae, the LC50 of these two plant

materials against 3rd instar larvae might be lower; thus, it might be said that C. microcarpa peel

extract is a less effective larvicide than C. sinensis peel and seed extracts.

Comparing the lowest values of LC50 from the non-citrus plants and from the citrus

plants, it can be said that the genus Citrus contain the most potent natural larvicide. It can also be

predicted just from related literature that the plant materials tested in the current study may be

highly effective agents against mosquito larva.

19

Chapter 3

METHODOLOGY

Collection and Identification of the Plant Material

Figure 1. Citrus microcarpa Bunge fruits Figure 2. Citrus maxima (Burm.) Merr. fruits

Citrus microcarpa Bunge fruits were collected on December 5, 2017 in the province of

Batangas in the Philippines, and Citrus maxima (Burm.) Merr. fruits were collected on October

29, 2017 in the province of Nueva Vizcaya. The fruits were brought to the Bureau of Plant

Industry, Department of Agriculture, Malate, Manila, 1004, Philippines for necessary

identification and certification on December 12, 2017 (Appendices 7-8). The rinds of the fruits

were processed for essential oil extraction and larvicidal bioassay.

Essential oil Extraction

Figure 3. C. microcarpa rinds Figure 4. C. maxima rinds

20

Figure 5. Water Distillation Apparatus

The essential oil extraction for both species of fruits was performed at the Chemicals and

Energy Division of the Industrial Technology Development Institute (ITDI), Department of

Science and Technology (DOST), Taguig City, 1631, Philippines (Appendices 9-10). 1.2 kilos

of C. microcarpa and 2 kilos of C. maxima air-dried rinds were subjected to water distillation

using a simple distillation apparatus (Figure 5). The percentage oil yield of the 2 plant samples

were computed in terms of volume per weight percent. The resulting moisture-free essential oils

were stored in dark colored bottles in a refrigerator.

Essential oil Components Determination

Figure 6. C. microcarpa and C. maxima rinds essential oil

21

Analysis of the components of each of the two essential oils from the rinds of the two

species was performed at the Standards and Testing Division of ITDI, DOST, Taguig City

(Appendix 11). Gas chromatography was performed to analyze these components. These

components included myrcene, caryophyllene, phellandrene, limonene, linalool, and β-pinene. A

Shimadzu GC-2010 gas chromatograph was used in the analysis to determine the percentage

composition of each component.

Rearing and Preparation of Mosquito Larvae

Figure 7. Larvae Rearing Setup.

Figure 8. 4-5 day-old larvae in a water container.

Third instar larvae were used. Aedes aegypti L. eggs were obtained from the Entomology

Laboratory of ITDI, DOST, Taguig City (Appendix 12). The eggs were attached to the adhesive

side of masking tapes that served as artificial egg rafts. The egg rafts were put in basins

containing distilled water with a pellet of brewer’s yeast as larval food to let them hatch. To

22

ensure third instar larvae were available at the scheduled time for the larvicidal bioassay

procedure, the egg rafts were put in the water-containing basins 4-5 days before the scheduled

bioassay procedure.

Larvicidal Bioassay

The larvicidal bioassay was performed in the Entomology Laboratory of ITDI, DOST,

Taguig City following the method of the World Health Organization (WHO, 2005). Twenty (20)

third instar larvae were transferred using Pasteur pipettes from the hatch basins to polypropylene

plastic cups, each containing 100 ml of distilled water, as test containers.

Figure 9. 10,000 ppm stock solution of rinds essential oil.

Stock solutions were prepared by diluting 0.25 mL of essential oil extract in 25 mL of

95% ethanol. From the resulting 1% stock solutions, certain volumes of aliquots were added to

the cups containing 100 mL of distilled water and 20 third instar larva. These certain volumes

were added in 100 mL to obtain the desired final test concentrations. Data from Table 1. were

used to determine certain volumes of aliquots that should be added to 100 mL water to obtain the

desired final concentration of the essential oil. Twenty-four hours after the addition of the

aliquots to the test containers, dead larvae were counted in each container. A larva was

considered dead if it cannot be induced to move when probed by a needle, cannot rise to the

surface, and/or cannot dive to the bottom of the cup.

23

Table 1. Aliquots added to 100 ml water to yield final concentration (WHO, 2005).

Initial Solution Aliquot Final Concentration

in % in ppm in ml in ppm

1.0

10 000 1.0

0.5

0.1

100

50

10

0.1

1 000 1.0

0.5

0.1

10

5

1

Successive preliminary bioassays were performed for both rind essential oils of C.

microcarpa and C. maxima. Each test consisted of a different set of 5 test concentrations with 3

replicates for each essential oil. The purpose of the initial test was to roughly determine the

concentration that would kill 50% of the third instar larvae. Also, each initial bioassay procedure

included three replicates of negative control group with no included aliquot from the 1% stock

solutions from both species.

Figure 10. Larvicidal Bioassay setup of C. microcarpa (6 replicates of 5 concentrations).

Figure 11. Larvicidal Bioassay setup of C. maxima (6 replicates of 5 concentrations).

24

After having determined an estimate of the test concentration which killed half of the

larvae tested, an additional bioassay was conducted as confirmatory test. A five-concentration

range having equal intervals that contained the probable 50% lethal concentration in the middle

was tested with 6 replicates for each Citrus species. Also, another 5 x 6 (concentration x

replicate) bioassay for Abate 1SG mosquito larvicide were conducted as positive control.

Data Analysis

Mortality data (expressed in percentages, calculated using the formula below) from the

three confirmatory tests—one for each species and one for positive control—were used to

calculate LC50 and LC90 from a log concentration-probit mortality regression line using SPSS

25.0.

% 𝑚𝑜𝑟𝑡𝑎𝑙𝑖𝑡𝑦 = 𝑛𝑜. 𝑑𝑒𝑎𝑑 𝑙𝑎𝑟𝑣𝑎𝑒

20× 100%

Biosafety and Waste Management

All mosquito larvae used in the study were certified laboratory-reared and safe by the

Department of Science and Technology (Appendix 12). After every larvicidal bioassay

procedure, all containers that contained the larvae—hatch basins and test containers—were

poured with boiling water to ensure no living larva will be released to the environment. The dead

larvae were buried in the soil.

25

Figure 12. Schematic Diagram of Procedure of the Study.

Collection and Identification of Plant Materials

Essential oil Extraction

Essential Oil Components Determination

Rearing and Preparation of Mosquito Larvae

Larvicidal Bioassay

Data Analysis

Biosafety and Waste Management

26

Chapter 4

RESULTS

The results of the preliminary test are shown in Table 2. For the rind essential oil of C.

microcarpa, the concentration that caused the closest to 50% mortality was 60 ppm, while that of

C. maxima was 30 ppm.

Table 2. Preliminary Average Percent Mortalities of Aedes aegypti Larvae Treated with C. microcarpa

and C. maxima Rinds Essential Oils.

Average % mortality at different concentrations (in ppm)/100 mL solutions after 24 h

Essential Oil 10 20 30 40 50 60 70 80 100 120

C. microcarpa - 0.00 - 18.33 - 51.67 - 90.00 100.00 100.00

C. maxima 0.00 11.67 56.67 83.33 90.00 100.00 100.00 - - -

Based on the above results, the following concentrations of C. microcarpa were used for

the confirmatory test: 20 ppm, 40 ppm, 60 ppm, 80 ppm and 100 ppm. For C. maxima, 10 ppm,

20 ppm, 30 ppm, 40 ppm and 50 ppm were used.

Table 3 shows the percentage larval mortality results of the confirmatory test that was also

used for the log concentration-probit mortality regression that was used to determine the LC50

and LC90 of both rind essential oil. At the same concentration of 20 ppm, C. maxima induced

24.17% mortality which is almost 30 times the percentage mortality (0.83%) induced by C.

microcarpa. Meanwhile, at 40 ppm, C. maxima induced 82.5% mortality which is about 4 times

the percentage mortality (19.17%) induced by C. microcarpa.

Table 3. Average Percentage Mortalities (± SD) of Aedes aegypti 3rd Instar Larvae, 24 Hours after

Treatment with C. microcarpa and C. maxima Rinds Essential Oils.

Average % mortality (± SD) at different concentrations (in ppm)/100 mL solutions after 24 h

Essential Oil 10 20 30 40 50 60 80 100

C. microcarpa - 0.83±2.04 - 19.17±5.85 - 60.83±12.01 90±8.37 98.3±4.08

C. maxima 5.83±3.76 24.17±5.85 55±5.48 82.50±5.24 94.17±5.85 - - -

The lethal concentrations (LC50 and LC90) of the essential oils from C. microcarpa and

C. maxima rinds are shown in Table 4. They were calculated from a log concentration-probit

mortality regression line using Statistical Package for the Social Sciences (SPSS) 25.0. Their

27

efficacy demonstrated the remarkable report regarding Citrus fruits potential as alternative

larvicides. Since there is no overlap between the 95% confidence limits of the median lethal

concentrations (LC50) of C. microcarpa and C. maxima rind essential oils against 3rd instar

larvae, it can be said that the essential oil from C. maxima rinds have a significantly lower

(p<0.05) LC50 of 26.130 ppm than 53.100 ppm which is the LC50 of the essential oil from C.

microcarpa rinds (Table 4). However, the LC90 of the essential oil from C. maxima rinds

(49.344 ppm) is not statistically significantly lower than that of the essential oil from C.

microcarpa rinds (81.381 ppm) because of the overlap in their 95% confidence limits.

Table 4. LC50 and LC90 (with 95% Confidence Limits) of C. microcarpa and C. maxima Rind Essential

Oils against Aedes aegypti 3rd Instar Larvae.

95% Confidence Limits

Essential Oil Lower Limit LC50 Upper Limit Lower Limit LC90 Upper Limit

C. microcarpa 50.187 53.100a 55.918 76.172 81.381c 88.412

C. maxima 20.888 26.130b 31.547 39.287 49.344c 78.325

Same or different superscript letters indicate statistically significant differences.

The response of the larva to different concentrations of the Abate 1SG mosquito larvicide

is shown in Table 5. The mean percentage larval mortality per concentration are as follows: 10%

for 0.3 ppm, 27.5% for 0.6 ppm, 59.17% for 0.9 ppm, 87.5% for 1.2 ppm, and 97.5% for 1.5

ppm. From the probit analysis of these results, Abate 1SG has an LC50 of 0.715 ppm and an

LC90 of 1.385 ppm which are considerably lower than the LC50 and LC90, respectively, of the

two rind essential oils.

Table 5. Average Percent Mortalities of Aedes aegypti Larvae Treated with Different Concentrations of

Abate 1SG mosquito larvicide.

Larval mortality (%)

0.3 ppm 0.6 ppm 0.9 ppm 1.2 ppm 1.5 ppm

mean 10 27.5 59.17 87.5 97.5

SD 5.48 6.89 5.85 4.18 4.18

Figures 13-15 show the log concentration-probit mortality regression line that was used

in probit analysis in SPSS 25.0. The statistical software log10-transformed the independent

concentration variable and transformed the dependent percentage mortalities into probit. The

figures also show the equations of the regression lines and the R2 value that indicates linearity of

28

the relationships. All three treatments demonstrated a direct almost-linear dose-dependent

mortality (after data transformation). The three treatments showed that the C. microcarpa

regression line is the most linear followed by C. maxima. Regression analysis also illustrated a

statistically significant relationship (p<0.05) between the mortality rates of the third-instar A.

aegypti larvae and concentrations of the two plant samples.

Figure 13. Linear regression line equation of Probit Percentage Mortality to Different log10 concentrations

of C. microcarpa Rinds Essential Oil.

Figure 14. Linear regression line equation of Probit Percentage Mortality to Different log10 concentrations

of C. maxima Rinds Essential Oil.

29

Figure 15. Linear regression line equation of Probit Percentage Mortality to Different log10 concentrations

of Abate 1SG mosquito larvicide.

In Table 6, the results of the gas chromatography essential oil component analysis

showed that limonene is the main component for both Citrus rind oils. However, C. maxima rind

essential oil contained a greater percentage (95.5%) of limonene than the C. microcarpa rind oil

(91.6%). The other components such as Myrcene, Linalool, β-pinene, and Caryophylline are higher

in C. microcarpa while Phellandrene is higher in C. maxima.

Table 6. Percent Composition of the Essential Oil Components of C. microcarpa and C. maxima.

Analyzed by Gas Chromatography.

Components Citrus

microcarpa

Citrus

maxima

Limonene 91.600 95.500

Myrcene 1.990 1.940

Linalool 0.659 0.511

β-pinene 0.453 0.198

Caryophyllene 0.096 0.034

Phellandrene 0.086 0.213

30

Chapter 5

DISCUSSION

The study revealed that the tested concentrations of the two essential oils caused mortality

of mosquito larvae at 0.05 level of significance, in comparison to those in the control group

(ethanol treated), wherein no mortality was observed (Appendices 1-4). Variations of the

mortality percentage of mosquito larvae between the two essential oils were noted. Comparing

the essential oils of the two plant samples investigated, that of the Citrus maxima rinds provided

a higher toxicity against the third instar larvae of Aedes aegypti, with a lower lethal concentration

(LC50 of 26.130 and LC90 of 49.344) after 24 hours of exposure, whereas Citrus microcarpa

with a higher LC50 of 53.1 and LC90 of 81.381. Table 7 shows a comparison of results from

previous studies, and the results of the present study.

Table 7. Summary of Results of Related Literature and of Present Study. Study Test Material Larva LC50 (ppm) LC90 (ppm)

Murugan et al. (2012) C. sinensis 3rd instar A. aegypti 320.38 524.57

Arivoli & Tennyson (2011) Abutilon indicum 3rd instar A. aegypti 261.31

Kamalakannan et al. (2011) Achyranthes aspera 4th instar A. aegypti 409.00

Acalypha indica indica 4th instar A. aegypti 420.00

Combined A. aspera

and A. indica indica

4th instar A. aegypti 277.00

Adeleke et al. (2009) Pterocarpus

santalinoides

3rd instar A. aegypti 104.00 184.50

TMS 30572 3rd instar A. aegypti 113.50 201.20

Warikoo et al. (2012) C. sinensis 4th instar A. aegypti 446.84 370.96

El-Sheikh et al. (2016) Tribulus terrestris 3rd instar A. aegypti 64.60

Kumar et al. (2011) C. limetta 4th instar A. aegypti 96.15

Reegan et al. (2013) Cliona celata 4th instar A. aegypti 158.40 780.16

De Villa et al. (2012) C. microcarpa 3rd instar A. aegypti 451.00 628.00

Torres et al. (2016) C. grandis 3rd instar A. aegypti 1.11 3.32

This study (2018) C. microcarpa 3rd instar A. aegypti 53.10 81.38

C. maxima 3rd instar A. aegypti 26.13 49.34

The report of Murugan et al. in 2012 on the larvicidal effect of C. sinensis against the same

test mosquito (3rd instar larvae Aedes aegypti) resulted to LC50 value of 320.38 and LC90 of

524.57. Arivoli and Tennyson (2011) reported that the hexane extract of Abutilon indicum leaves

was effective against A. aegypti larvae with LC50 value of 261.31 ppm after a period of 24 hour.

Kamalakannan et al. in 2011 demonstrated the larvicidal activity of the methanol extract of

Achyranthes aspera and noted a LC50 of 409 ppm against the 4th instar A. aegypti, and Acalypha

indica indica with a LC50 of 420 ppm. When combined, the two plant extracts exhibited a

31

stronger larvicidal activity (LC50 = 277), which is still higher than the results obtained by the

present study. Adeleke et al. in 2009 found the seed oils of Pterocarpus santalinoides and

Tropical Manihot species (TMS 30572) to be both toxic to the 3rd instar A. aegypti. The oil of P.

santalinoides showed a stronger toxicity to the larvae with LC50 of 104.0 ppm and LC90 184.5

ppm than the oil of TMS (LC50 113.5 and LC90 201.2). Warikoo et al. (2012) investigated the

larvicidal properties of hexane extract of the leaves of Citrus sinensis against the early 4th instar

of A. aegypti and reported a 50% mortality at 446.84 ppm. LC90 value obtained was 370.96 ppm.

El-Sheikh et al. (2016) reported that the petroleum ether extract of Tribulus terrestris was lethal

to A. aegypti larvae at LC50 of 64.60 ppm. The study of Kumar et al. in 2011 on the larvicidal

activity against the 4th instar A. aegypti with hexane extracts of Citrus limetta resulted in LC50

96.15 ppm. Reegan et al. (2013) reported the larvicidal activity of methanol extract of Marine

Sponge Cliona celata Grant extracts against A. aegypti at LC50 and LC90 values 158.40 and

780.16 ppm respectively All the above mentioned studies reported larvicidal activities against

mosquito larvae of A. aegypti but the results obtained by the present study were remarkably

better considering that the essential oil of C. microcarpa rinds afforded mortality of the tested

larvae at LC50 of 53.10 and LC90 of 81.38 values lower than the previously reported results. C.

maxima rind essential oil was lethal to the tested mosquito larvae at LC50 value of 26.130 and

LC90 of 49.344, much lower than those mentioned.

Local researches have also been done to explore the larvicidal potentials of citrus plants in

the Philippines. De Villa et al. in 2012 found that the peels of C. microcarpa showed the highest

larvicidal activity against A. aegypti among the 4 plant samples tested (peels of C. microcarpa,

seeds of Nephelium lappaceum, flowers of Jasminum sambac and leaves of Chromolaena

odorata), with LC50 of 451 and LC90 of 628. The result of the current investigation is again

better compared to this study. Another study by Torres et al. (2016) reported that the variety of

Citrus grandis from Davao showed the highest toxicity against the 3rd instar larvae of Aedes

aegypti (LC50 of 1.11 and LC90 of 3.32) among the different varieties tested (from Palawan,

Benguet, Antique, South Cotabato and Iloilo). The C. maxima used in this study were obtained

from Nueva Vizcaya. According to Torres et al. (2016), some factors like type of soil,

geographic and seasonal differences where the plant samples were gathered can affect the

larvicidal activity of the plant. Those factors may account for the stronger larvicidal activity

32

exhibited by the same plant sample (C. grandis) from Davao compared to our sample. The

strength of the insecticidal effect of a particular plant sample is not constant, but differs with

mosquito species, plant species, part of the plant used, geographical varieties, solvent used

during extraction and the method of extraction selected (de Morais et al., 2007).

This study employed gas chromatography which allowed for the determination of six (6)

compounds as main constituents of the essential oils from rinds of C. microcarpa and C. maxima

(Table 6). Limonene is the major component of both essential oils; however, it is more dominant

in C. maxima (95.5%) than in C. microcarpa (91.6%). The result is in conformity with the report

of Javed et al. (2013) that of limonene being identified as the major component of 5 Citrus peel

oils (Malta, Mousami, Tangerine, Mandarin and Grapefruit). Sulthanont et al. (2010) found out

in their study that Citrus reticulata peel oil contains mostly d-limonene (62.39%). Cheong et al.

in 2012 reported the major constituent in C.microcarpa are monoterpenes, which include

limonene, β- myrcene, β-pinene, α-pinene, β-phellandrene and sabinene. This is in congruence

with the result of the study of Amusan et.al in 2005 establishing the genus Citrus (family:

Rutaceae) larvicidal activity, with the compound a-tepinoline, a monoterpene that is similar in

action to d-limonone, present in Citrus sinensis. Limonene was noted as the bioactive compound

responsible for the larvicidal property. The high level of limonene of the essential oil from the

rinds of C. microcarpa and C. maxima accounted for the high mortality rate they afforded on the

3rd instar larvae of A. aegypti. This may also support the finding that the LC50 of the rind

essential oil of C. maxima was statistically significantly lower than that of C. microcarpa;

however, it is not conclusive since no results in this study can prove that a 3.9% difference in

limonene composition can cause that significant difference in LC50.

Plant chemical components found in essential oil of C. microcarpa and C. maxima fruit

rinds, particularly limonene, was the major bioactive compound. This conformed to past reports

that limonene was present in copious amount in citrus fruit rinds (Cheong et al., 2012a;

Chungsamarnyart & Jansawan, 1996; Oboh & Ademosun, 2011). This agrees with the findings

of some researchers, which reported that limonene in citrus fruit rinds was known to be the

contact poison when insects are exposed to (Mwaiko, 1992). The mode of action of limonene

includes its impact on insects’ nerve activity through several mechanisms which are neurotoxic.

33

These mechanisms include action on gamma-aminobutyric acid (GABA) and octopamine

synapses (Regnault-Roger, 2011). Moreover, Rattan in 2010 made a review on the mechanism of

action of citrus essential oils on the body of insects and reported the following mechanisms:

disruption of the molecular events of morphogenesis, alteration in the behavior and memory of

cholinergic system and inhibition of acetylcholinesterase. It is said that the most important of

said mechanisms was the effect on acetylcholinesterase, the enzyme responsible for terminating

the nerve impulse transmission through synaptic pathway. The transmission of airborne signals

from plants to insects is said to be affected by the small-volatile molecules, acyclic or

monocyclic monoterpenes, particularly limonene, which produces massive over-activation of

motor nerve, thereby inducing convulsion, paralysis and eventually death of the mosquitoes

(Mwaiko, 1992).

The rind essential oils of C. microcarpa and C. maxima were both found to have larvicidal

activity against 3rd instar A. aegypti larvae. Since the LC50 and LC90 results of most of the

previous studies on larvbicidal assay of natural products against the same species of mosquito

larvae were significantly higher than the present results, the rind essential oils from the two (2)

plant samples can be considered promising as larvicides. They are highly effective, safe and

environment-friendly. The statistically significant difference between the LC50 of the rind

essential oils of C. microcarpa and C. maxima cannot conclusively be attributed to the difference

in limonene percentage composition; however, after comparing them with the results of other test

materials, and after reviewing the action of limonene on insects, the larvicidal activities of both

rind essential oils can be correlated to their limonene percentage composition.

34

CONCLUSION

This study has demonstrated the larvicidal activity of rind essential oils from Citrus

microcarpa and Citrus maxima against 3rd instar larvae of Aedes aegypti mosquito by the

determination of LC50 and LC90 using probit analysis. It showed that the LC50 of the rind

essential oil of C. maxima (26.13 ppm) is significantly lower than the LC50 of the rind essential

oil of C. microcarpa (53.1 ppm. On the other hand, the LC90 of the rind essential oil of C.

maxima (49.24 ppm), though not statistically significantly different, is lower than the LC90 of

the rind essential oil of C. microcarpa (81.38 ppm). Thus, the rind essential oil from C. maxima

was more toxic to the 3rd instar larvae of A. aegypti than that of C. microcarpa. The study also

demonstrated through gas chromatography that limonene is the predominant composition in both

rind essential oils. The higher percentage composition of limonene in the rind essential oil of C.

maxima (95.5%) than that of C. microcarpa (91.6%) could have caused the higher larvicidal

activity of C. maxima rind essential oil. The results of this study suggest that the limonene of

essential oil from the rinds of the two (2) plant samples is promising as larvicides. Although

essential oils from citrus fruits are less potent than synthetic larvicides against the 3rd instar

larvae of Aedes aegypti, their safety, natural biodegradation and remarkable activity on

laboratory-reared mosquitoes make them good candidates for further study in mosquito-borne

disease control program.

35

RECOMMENDATIONS

It is recommended for future researchers to add more concentrations per test sample to

attain a narrower 95% confidence range for LC90. In addition, the authors recommend that the

investigation be extended to isolate, and to identify the specific active component involved, its

mechanism of action, and the effects on the environment and non-target organisms. Likewise,

it is recommended to investigate the effects of combining the two essential oils to determine

any possible synergism for enhanced potency and stability, which could be used in formulating

an alternative larvicide like the results of Kamalakannan et al. (2011). Furthermore, the authors

suggest taking into account environmental factors by performing analyses on the environment

of each source of the plant material, like the study of Torres et al. (2016). These would be of

help in the development of more efficient mosquito control strategies.

36

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42

APPENDICES

APPENDIX 1

Larvicidal Activity of Citrus microcarpa Rinds Essential Oil (Initial Test) – Raw Data.

% mortality at different concentrations/100 mL solutions

Replicates 20 ppm 40 ppm 60 ppm 80 ppm 100 ppm 120 ppm control

1 0 20 50 80 100 100 0

2 0 20 55 100 100 100 0

3 0 15 50 90 100 100 0

Mean 0 18.33 51.67 90 100 100 0

APPENDIX 2

Larvicidal Activity of Citrus maxima Rinds Essential Oil (Initial Test) – Raw Data.

% mortality at different concentrations/100 mL solutions

Replicates 10 ppm 20 ppm 30 ppm 40 ppm 50 ppm 60 ppm 70 ppm control

1 0 0 50 85 85 100 100 0

2 0 25 40 70 85 100 100 0

3 0 10 80 95 100 100 100 0

Mean 0 11.67 56.67 83.33 90 100 100 0

43

APPENDIX 3

Larvicidal Activity of Citrus microcarpa Rinds Essential Oil (Final Test) – Raw Data.

% mortality at different concentrations/100 mL solutions after 24 h

Replicates 20 ppm 40 ppm 60 ppm 80 ppm 100 ppm control

1 0 25 50 85 100 0

2 0 10 55 85 100 0

3 0 25 50 100 100 0

4 5 20 70 80 90 0

5 0 20 80 100 100 0

6 0 15 60 90 100 0

Mean 0.83 19.17 60.83 90.00 98.33333 0

SD 2.04 5.85 12.01 8.37 4.08 0

APPENDIX 4

Larvicidal Activity of Citrus maxima Rinds Essential Oil (Final Test) – Raw Data.

% mortality at different concentrations/100 mL solutions after 24 h

Replicates 10 ppm 20 ppm 30 ppm 40 ppm 50 ppm control

1 5 25 60 85 90 0

2 10 30 55 80 95 0

3 0 30 55 75 85 0

4 5 15 60 80 95 0

5 10 20 55 90 100 0

6 5 25 45 85 100 0

mean 5.83 24.17 55 82.5 94.17 0

SD 3.76 5.85 5.48 5.24 5.85 0

APPENDIX 5

Larvicidal Activity of Abate 1SG mosquito larvicide – Raw Data.

Larval mortality (%)

Replicates

0.3

ppm

0.6

ppm

0.9

ppm

1.2

ppm

1.5

ppm

1 10 25 65 90 100

2 15 20 55 85 90

3 0 35 50 90 95

4 10 35 60 90 100

5 10 20 60 80 100

6 15 30 65 90 100

mean 10 27.5 59.17 87.5 97.5

SD 5.48 6.89 5.85 4.18 4.18

44

APPENDIX 6

SPSS Probit Analysis Output

6-A.Citrus microcarpa rind oil.

Parameter Estimates

Parameter Estimate Std. Error Z Sig.

95% Confidence Interval

Lower Bound Upper Bound

PROBITa micro_conc 6.911 .552 12.529 .000 5.830 7.992

Intercept -11.923 .977 -12.201 .000 -12.900 -10.945

a. PROBIT model: PROBIT(p) = Intercept + BX (Covariates X are transformed using the base 10.000 logarithm.)

Chi-Square Tests

Chi-Square dfb Sig.

PROBIT Pearson Goodness-of-Fit

Test

3.719 3 .293a

a. Since the significance level is greater than .050, no heterogeneity factor is used

in the calculation of confidence limits.

b. Statistics based on individual cases differ from statistics based on aggregated

cases.

Confidence Limits

Probability

95% Confidence Limits for micro_conc 95% Confidence Limits for log(micro_conc)a

Estimate Lower Bound Upper Bound Estimate Lower Bound Upper Bound

PROBIT .500 53.100 50.187 55.918 1.725 1.701 1.748

.900 81.381 76.172 88.412 1.911 1.882 1.947

a. Logarithm base = 10.

45

6-B. Citrus maxima rind oil.

Convergence Information

Number of

Iterations

Optimal Solution

Found

PROBIT 15 Yes

Parameter Estimates

Parameter Estimate Std. Error Z Sig.

95% Confidence Interval

Lower Bound Upper Bound

PROBITa maxi_conc 4.642 .359 12.934 .000 3.938 5.345

Intercept -6.578 .526 -12.504 .000 -7.104 -6.052

a. PROBIT model: PROBIT(p) = Intercept + BX (Covariates X are transformed using the base 10.000 logarithm.)

Chi-Square Tests

Chi-Square dfb Sig.

PROBIT Pearson Goodness-of-Fit

Test

8.670 3 .034a

a. Since the significance level is less than .050, a heterogeneity factor is used in the

calculation of confidence limits.

b. Statistics based on individual cases differ from statistics based on aggregated

cases.

Confidence Limits

Probability

95% Confidence Limits for maxi_conc 95% Confidence Limits for log(maxi_conc)b

Estimate Lower Bound Upper Bound Estimate Lower Bound Upper Bound

PROBITa .500 26.130 20.888 31.547 1.417 1.320 1.499

.900 49.344 39.287 78.325 1.693 1.594 1.894

a. A heterogeneity factor is used.

b. Logarithm base = 10.

46

6-C. Abate 1SG.

Convergence Information

Number of

Iterations

Optimal Solution

Found

PROBIT 14 Yes

Parameter Estimates

Parameter Estimate Std. Error Z Sig.

95% Confidence Interval

Lower Bound Upper Bound

PROBITa abate_conc 4.463 .336 13.287 .000 3.805 5.122

Intercept .650 .076 8.548 .000 .574 .726

a. PROBIT model: PROBIT(p) = Intercept + BX (Covariates X are transformed using the base 10.000 logarithm.)

Chi-Square Tests

Chi-Square dfb Sig.

PROBIT Pearson Goodness-of-Fit

Test

17.635 3 .001a

a. Since the significance level is less than .050, a heterogeneity factor is used in the

calculation of confidence limits.

b. Statistics based on individual cases differ from statistics based on aggregated

cases.

Confidence Limits

Probability

95% Confidence Limits for abate_conc 95% Confidence Limits for log(abate_conc)b

Estimate Lower Bound Upper Bound Estimate Lower Bound Upper Bound

PROBITa .500 .715 .478 .958 -.146 -.320 -.019

.900 1.385 1.018 3.309 .141 .008 .520

a. A heterogeneity factor is used.

b. Logarithm base = 10.

47

APPENDIX 7

Identification and Certification of Citrus microcarpa.

48

49

APPENDIX 8

Identification and Certification of Citrus maxima.

50

51

APPENDIX 9

Accomplishment Report of Extraction of Citrus microcarpa Rinds Essential Oil.

52

APPENDIX 10

Accomplishment Report of Extraction of Citrus maxima Rinds Essential Oil.

53

APPENDIX 11

Gas Chromatography Test Report.

54

APPENDIX 12

Certification of Laboratory-Reared Larvae.