Lemon peel (Citrus limon) and Lagundi (Vitex negundo)

Extracts as Potential Mosquito Larvicide

An official entry

to the

2019 Division Science and Technology Fair

Life Science

Team Category

Alexander Luke A. Luberas

Riejil Kent Y. Dumadiego

Louis L. Uy

Researchers

Mr. Christian Floyd I. Erad

Research Adviser

Mrs. Adaiah A. Opeña

Co-Adviser

Doña Hortencia Salas Benedicto National High School

La Carlota City
 I

Abstract

Mosquitos can transmit more diseases than any other group of anthropods and

affects millions of people throughout the world. WHO has declared the mosquitoes as

“public enemy number 1”. Mosquito borne diseases are prevalent in more than 100

countries across the world, infecting over 700,000,000 people every year globally. (World

Heatlh Organization, 2016). This study aims to determine the effect of Lemon peel (Citrus

limon) and Lagundi (Vitex negundo) extracts as potential mosquito larvicides. The Lemon

(C. limon) were bought at the Agora Public Market while the Lagundi (v. negundo) were

gathered from a farm located athda. Remedios, Brgy. Balabag. The Lemon (C. limon) and

Lagundi (V. negundo) were washed with running water then they were pounded using

mortar and pestle. There are 4 treatments in this study; Treatment A (Lemon peel extracts),

Treatment B (Lagundi extracts), Treatment C (positive control) and Treatment D (negative

control). There are 4 mosquitoes in each replicate with the total of 12 mosquito larvae per

treatment. The One-Way Analysis Of Variance was used to determine the differences

among the 4 treatments respectively. Based on the results, Treatment A (lemon peel

extracts) were more effective than Treatment B (lagundi extracts) on the otherhand

Treatment C (positive control) were able to exterminate all mosquito larvae while

Treatment d (negatice control) did not record any number of mosquito larvae terminated.
 Table of Contents
Pages

Abstract………………………………………………………………………………...I

Forms……………………………………………………………………………………

Research Plan…………………………………………………………………………i-v

Introduction……………………………………………………………………………1

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

Statement of the Problem……………………………………………………….1

Objectives of the Study…………………………………………………………2

Hypothesis………………………………………………………………………2

Significance of the Study……………………………………………………….2

Scope and Limitations…………………………………………………………..3

Review of Related Literature………………………………………………………...4-11

Methodology…………………………………………………………………………...12

Materials………………………………………………………………………..12

Equipments……………………………………………………………………..12

Collection………………………………………………………………………12

Preparation of Treatments……………………………...……………………...13

Preparation of Lemon (Citrus limon) ………………...……………………….13

 Pages

Preparation of Lagundi (Vitex negundo) ………………...……………………13

Preparation of Set-ups………………...…………………………………….....14

Application of Treatments………………...………...…………………………14

Disposal………………...……………………………………...………………14

Data and Data Gathering Procedures………………...……………………….15

Results………………...……………………………………...………………………16-18

Table 1.1………….……...……………….…………….……………………...16

Table 1.2……….………...………………….…………….…...………………17

Table 1.3………………...….…………….….…….…………...………………18

Discussion………………...……………………………………...……………………..19

Summary………………...……………………………………...………………. .....19-20

Conclusion………………...……………………………………...…………………...20

Recommendations………………...……………………………………..……..……20-21

Appendices………………...……………………………………...…………………..

Bibliography………………...……………………………………...…………………

Acknowledgement………………...……………………………………...……………
 i

Lemon peel (Citrus limon) and Lagundi (Vitex negundo)

extracts as potential mosquito larvicide

Research Plan

A.Problem Being Addressed:

Mosquitoes can cause sickness and death through the diseases that they can carry.

Mosquitoes are mainly known for the disease they carry called as “Dengue”. Dengue is a

disease that debilitates a viral disease and viral fever and acute pains in the joints.

The Department of Health (DOH) has expressed alarm over the rising number of dengue cases

in Eastern Visayas (Region 8) this year, with 19 deaths and 4,809 confirmed cases. (Department of

Health, 2019) Mosquitoes can transmit many infectious diseases that are caused by a

variety of microorganisms to people, but these diseases are mostly confined to

certain geographical areas. In the Philippines, the mosquito-borne diseases include

dengue, Chikungunya, Japanese encephalitis, malaria, and filariasis. Dengue,

chikungunya, and Japanese encephalitis are caused by viruses

while malaria and filariasis are caused by parasites. (Gonazales,Eduardo, 2016).

B. Goals/Expected Outcome/Hypothesis:

This study aims to create a potential mosquito larvicide that is environment-

friendly using the extracts of Lemon peel (Citrus limon) and Lagundi (Vitex negundo).

This study also aims to make an alternative for commercial products such as Spike

Larvicide.
 ii

C. Methodology:

Sample Collection

Lemon (Citrus limon) will be bought Agora Public Market. Lagundi (Vitex

negundo) will be collected at Hda. Remedios, Brgy. Balabag. Mosquito larvae will be

collected at La Paz St., La Carlota City at the New Printing Press Beside the fishpond.

Spike larvicide and distilled water will be bought at Lopues La Carlota Branch.

Preparation of Treatment

Lagundi (Vitex negundo) will be pounded using a mortar and pestle the pounded

variables will be placed in a clean cloth and squeezed until extracts could be collected.

The extracts collected will be placed in a sterilized beaker. Lemon (Citrus limon) will be

peeled using a peeler and the lemon peel will be pounded using mortar and pestle the

pounded variables will be placed in a clean cloth and squeezed until the extracts could be

collected. The extracts collected will be placed in a sterilized beaker. The Lemon peel

(Citrus limon) extracts will be transferred in a 50 ml sterilized test tube and will be

labelled as A (treatment 1). Lagundi (Vitex negundo) extracts will be transferred in a 50

ml sterilized test tube and will be labelled as B (treatment 2). The spike lavicide will also

be placed in a 50 ml sterilized test tube and will be labelled as C (treatment 3) the water

will also be in a 50 ml sterilized test tube and will be labelled as D (treatment 4).
 iii

Preparation of Setup

Treatments R1 R2 R3

A. Lemon peel (Citrus

limon) extracts

(100%)

B. Lagundi (Vitex

negundo) extracts

(100%)

C. Spike larvicide

(100%)

D. Distilled Water

(100%)

Application of Treatment

Treatment A. 50ml of Lemon peel (Citrus limon) extracts

Treatment B. 50ml of Lagundi (Vitex negundo) extracts

Treatment C. (Positive Control) 50ml of Spike larvicide

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Treatment D. (Negative Control) 50ml of Distilled water

The application will be conducted once. Treatment A applied with 15 ml of Lemon

peel (Citrus limon) extracts per replicates, Treatment B will be conducted once with 15 ml

of Lagundi (Vitex negundo) extracts per replicates. Treatment C with positive control will

be conducted once with 15 ml of Spike larvicide per replicates. Treatment D with negative

control conducted once with 15 ml of water per replicates. Each replicate will be containing

a total of 4 mosquito larvae.

Waste disposal

The used petri dish, beaker, mortar and pestle will be washed using running water

and will be reused for future experimental uses. The left over extracts will be stored in

compatible containers with closed and fitted tops. The useless remains will be properly

disposed in the garbage dump.

Data and Data Gathering Procedures

The effectiveness of Lemon peel (Citrus limon) and Lagundi (Vitex negundo)

extracts based on the mosquito larva motality rate, survival and time of death were recorded

and compared among the four treatments having the same amount of extracts to which

revealed the highest result.

The data obtained from the study will be subjected to the following

inferentialstatistical methods using Package for Social Science (SPSS) Software.

 v

 Mean- to determine the average mosquito larva killed.

 One Way ANOVA (Analysis of Variance)- to show if there is significant

difference

Between means set at 0.05 level of significance

 Tukey test- to test which treatment is significantly effective than the other.

Bibliography

Gonazales, Eduardo. The disease that mosquitoes can cause, Manila Bulletin. Retrieved

on July 16, 2019 at http://www.pchrd.dost.gov.ph/index.php/news/library- health-

news/5689-the-mosquito-menace-in-the-philippines

Department Of Health. DOH the increasing Dengue Related Death Caused By

Mosquitoes. Retrieved on August 28, 2019 Retrieved from

https://www.pna.gov.ph/articles/1074779

Report of the WHO Informal Consultationon the Evaluation on the Testing of insecticides,

CTD/WHO; 1996. World Health Organization; p. 69.

 1

Introduction

1.0 Background of the study

Mosquitoes can transmit more diseases than any other group of arthropods and

affect million of people throughout the world. WHO has declared the mosquitoes as “public

enemy number one”. Mosquito borne diseases are prevalent in more than 100 countries

across the world, infecting over 700,000,000 people every year globally and 40,000,000 of

the Indian population .they act as vector for most of the life threatening diseases like

malaria, yellow fever, filariasis, encephalitis, West Nile virus infection, etc. , in almost all

tropical and subtropical countries and many other parts of the world. (World Health

Organization, 2015).

In this study, the researchers used Lemon Peel (Citrus limon) and Lagundi (Vitex

negundo) extracts as Potential Mosquito Larvicide

1.1 Statement of the problem

Mosquitoes act as vector for most of the life-threatening diseases like malaria,

yellow fever, filariasis, encephalitis, West Nile virus infection, etc. The major tool in

mosquito control operation is the application of synthetic larvicide such as organochlorine

and organophosphate compounds. due to lack of larvicide, high cost of synthetic larvicide,

concern for environmental sustainability, harmful effect on human health, and other non-

target populations, their non-biodegradable nature, higher rate of biological magnification

through ecosystems, and increasing insecticide resistance on a global scale (Anupam et.

al., 2012).
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1.2 Objectives of the study

General

To determine the effects of Lemon Peel (Citrus limon) and Lagundi (Vitex

negundo) extracts as potential mosquito larvicide

Specific objectives

1.2.1 To determine the effect of Lemon Peel (C. limon) extracts as potential mosquito

larvicide

1.2.2 To determine the effect of Lagundi (V. negundo) extracts as potential mosquito

larvicide

1.3 Hypothesis

 There is a significant effect using Lemon Peel (C. limon) extracts as potential

mosquito larvicide

 There is a significant effect using Lagundi (V. negundo) extracts as potential

mosquito larvicide

1.4 Significance of the study

Environmental safety is considered to be the paramount importance. An insecticide

does not need to cause high danger on the mortality rate on target organism in order to be

acceptable but should be eco-friendly in nature. Phytochemicals may serve as these are

relatively safe, inexpensive and readily available in many parts of the world
 3

1.5 Scope and Limitations of the study

This study is limited only of using Lemon Peel (C. limon) and Lagundi (V.

negundo) extracts as pontential mosquito larvicide

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Review of related literature

2.2 Lemon (Citrus limon) and Ginger (Zingiber officinale) extracts as potential
mosquito larvicide

2.2.1 Lemon (C. limon)

(Graf, 1986) Medium-sized tree, open and spreading; almost thornless. 2 000 mg/kg

of lemon oil in grapefruit juice suppressed growth and toxin production of A.

parasiticus after an incubation period of seven days. 300 mg/kg of oil in a glucose-yeast

extract medium were need to achieve the same result.Lemon uses natural and generally

non-toxic ingredients to repel or get rid of pests including ants, wasps, mites, moths, flies

and other insects. Citrus, like lemons, oranges, or grapefruit and harnesses the power of d-

limonene — the acidic oil found in the peels. This oil is toxic to ants (so it will kill them

on contact) and it messes up their trail, so live ones won’t be able to find the food source.

“Ants follow a pheromone trail when they find food, so if you can disrupt the trail, it will

confuse them and break them up,” says Scot Svenheim, an associate certified entomologist

in the training and e-learning department of Truly Nolen Pest Control.

2.2.2 Effects of Lemon (C. limon)

Lemon has been celebrated for centuries for its amazing internal and external uses

that include antiseptic, cleansing agent, beauty aid and stain remover. Among lemon's

many home uses is as an insect repellent. Moth balls, ant hotels, flea collars and bug sprays

all contain toxic chemicals, so try using lemon as a cheap and natural way to get rid of

unwanted pests around the home.Many gardeners look for natural and cheap alternatives

to the sometimes expensive, chemical pesticides found in most garden centers. Often, you
 5

don't need to go any farther than your pantry to find such alternatives. Lemon juice, for

example, can sometimes help control small infestations of garden pests, such as ants,

aphids and leaf beetles.Lemon juice acts as a natural herbicide in the garden and could

inadvertently burn the leaves of your wanted plants.

2.2.3 Citrus

Citrus crops, mainly sweet oranges, provide the main part of the world’s essential

oil production. Important quantities of essential oils are also extracted from other citrus

cultivars like tangerines, lemons, grapefruits, limes, and grapefruits. Citrus are damaged

by a broad range of pests and diseases and chemical phytosanitary products are commonly

applied in order to protect the crop. Essential oils are utilized in food, pharmaceutical and

cosmetic industries resulting in a very low tolerance for chemical residues. Not only the

legislations are becoming more stringent, but increasingly the industry, including citrus

producers, processors and end users are driving the demand for chemical free products. To

meet this demand non-chemical pest control strategies are required, that need to be both

efficient and environmental friendly as well as guarantee similar production yields.

Biological pest control, alone or as a part of an Integrated Pest Management (IPM) strategy,

represents such an alternative to chemical pesticides. As far as citrus essential oils are

concerned, recent studies demonstrate that essential oils produced without chemical

pesticides are not only residue free but also are of higher quality. The presentation will

describe the biological control and IPM principles and applications with special emphasis

on the citrus crop. Finally, Biobest’s program for the implementation of an IPM strategy

in citrus will be discussed.In a recent study by Di Bella et al. 2010, the presence of
 6

organophosphorus and organochlorine pesticides in essential oils extracted from citrus

originating from various countries (120 samples from Italy, and 70 samples from Brazil,

Argentine, South Africa and Spain) were investigated. According to the results, pesticides

from both categories were found in the majority of the samples examined. The largest

quantities of pesticides were found in essential oils from Brazil and Spain. The presence of

these pesticides in the citrus essential oils is probably associated with the insecticides used

for pest control.

2.2.4 D-limonene

d-Limonene (p-mentha-1,8-diene) is a monocyclic monoterpenoid found in citrus

oils. The spectrum of insecticidal activity of d-limonene was examined by using the

German cockroach, Blattella germanica (L.), house fly, Musca domestica L., rice weevil,

Sitophilus oryzae (L.), and western corn rootworm, Diabrotica virgifera virgifera leconte.

bioassays were performed to determine topical, fumigant, oral, repellent, residual, ovicidal,

and larvicidal activities. The material was slightly toxic topically to German cockroaches

and house flies and was synergized with piperonyl butoxide.High concentrations of vapors

caused mortality in German cockroaches and rice weevils. Oral administration did not

result in mortality to either adult or nymphal cockroaches but accelerated growth in

nymphs. Repellent activity against German cockroaches was noted at high concentrations.

No residual activity was observed on any of four surface types exposed to adult German

cockroaches. d-Limonene inhibited western corn rootworm egg hatch at high

concentrations and showed moderate toxicity in soil against third-instar western corn
 7

rootworm larvae. These findings indicate that the insecticidal properties of d-limonene are

limited.

2.2.5 Citrus peels

pesticides may be found on the skin of many fruits, including citrus fruits, but there

are official regulatory efforts to keep the levels low in the American market and ways to

remove most of the chemicals before the fruit is consumed, said Lambert H. B. Kanga,

professor of entomology at Florida A&M University in Tallahassee.Some study revealed

that essential oil from citrrus peels exhibited strong insecticidal activity against C.

maculatus, S. oryzae and T. confusum. ... The fumigant toxicity and repellent properties of

essential oils from citrus peels on agricultural pests and insects has also been

investigated.citrus peels are safe for use as insecticides. Many companies make flea

shampoos, medications and insecticides for gardens using the ingredients in citrus fruit.

Can also be used as homemade spray, cleaner or compost to rid pets, homes and gardens

of pests without using harsh or dangerous chemicals. Citrus fruit repels ants, fleas, fungus,

gnats, aphids and other pests.

2.2.6 Properties of Lagundi (V. negundo)

Lagundi is considered as anti-inflammatory, astringent, antibacterial, antifungal,

analgesic, alterant, depurative, rejuvenating, stomachic. Its Roots are considered tonic,

febrifuge, anti-rheumatic, diuretic and expectorant. The Leaves and seeds are considered

vulnerary. The Leaves are also considered aromatic, bitter, anti-inflammatory, bronchial

smooth muscle relaxant, lactagogue, emmenagogue, insecticide, and vermifuge. The

 8

Flowers are astringent, carminative, hepatoprotective, digestive, vermifuge and febrifuge.

While its Fruit is considered nervine, cephalic, aphrodisiac, emmenagogue and vermifuge.

Studies have suggested antivenom. anti-inflammatory, antibacterial, analgesic, antifungal,

insecticidal, anticonvulsant, larvicidal, anthelmintic, antioxidant, anxiolytic,

hepatoprotective, gastroprotective, antiamnesic, antidiabetic, antieosinophilic,

2.2.7Studies on Lagundi (V. negundo)

Vitex negundo Linn. has been reported to have various advantageous uses, and

therefore, this has pushed scientists and researchers to test and further explore the

capabilities of this plant. Anti-cancer potential was tested by Diaz et al. (2003), on the

cytotoxicity of flavones isolated from the chloroform extract of Vitex negundo leaves.

Vitexicarpin, a flavone was investigated for its cytotoxic action in human cancer cell line.

Antimicrobial capabilities were also evaluated. Rideout et al. (1999) reported antibacterial

& antifungal activity of Vitexilactone & Casticin from the chloroform extract of Vitex

negundo leaves against Staphylococcus aureus, Pseudomonas aeruginosa, Candida

albicans and Aspergillus niger using agar plate method. Alagarsamy et al. (1999) reported

antibacterial & antifungal activity of Vitex negundoleaves. Antibacterial & antifungal

activity of chloroform, methanol and water extracts were evaluated by Agar cup plate

method at concentrations of 10 mg/ml, 20 mg/ml and 30 mg/ml against E.coli, P.

aeruginosa, S. aureus & Candida albicansemploying co-trimoxazole & amphotericin as a

reference standards for said screeningsAmancharla et al. (1999) also tested mosquito

repellent activity of aqueous extract of Vitex negundo leaves. A new chemical ‘rotundial’

was tested for the said activity. The chloroform fraction of the aqueous extract of the fresh
 9

leaves of Vitex negundo by bioactivity guided isolation yielded a pure compound rotundial

which has shown mosquito repellent activity.

2.2.8 Lagundi (V. negundo)

(Lagundi) Vitex negundo Linn. (lagundi) is a large aromatic shrub or sometimes a

smaller slender tree with quadrangular, densely whitish tomentose branchlets up to 4.5-5.5

meters in height. The plant is used in a variety of means throughout its distribution points

as an astringent, cephalic, stomachic, antiseptic, alterant, thermogenic, depurative,

rejuvenating, ophthalmic, anti-gonnorhoeic, anti-inflammatory, antipyretic and useful in

bronchitis, asthma, and the enlargement of the spleen. Its roots are primarily used as a tonic,

febrifuge, antirheumatic, diuretic, expectorant, and are useful as a demulcent in dysentery,

in cephalalgia, otalgia, colic, uropathy, wound and ulcers. The bark is also useful in

odontalgia, verminosis and opthalmopathy. The flowers are mainly used as an astringent,

carminative, hepatoprotective, digestive and are useful in hemorrhages and cardiac

disorders. The leaves are chiefly utilized as astringents, anodyne, anti-inflammatory,

antipyretic or febrifuge, tranquilizer, bronchial smooth muscle relaxant, anti-arthritic,

antihelmintic and vermifuge. The leaves (leaf oil) are also reported to have repellent action

against stored product pests (Tandon, 2005). Vitex negundo Linn. is a large aromatic shrub

or small slender tree of about 3 meters in height with quadrangular branches. It is mostly

found in moist areas, scattered all throughout Mediterranean countries and Central Asia. It

is a plant of the Verbenaceae family and is commonly known as five leaved chaste tree.
 10

The plant is found throughout India, Ceylon- Afghanistan, tropical Africa, Madagascar,

China and Philippines (Kirtikar and Basu, 2008). The plant occurs in Bengal, Southern

India and Burma also (Nadkarni, 2002). It is common in waste places around villages, river

banks, moist localities and in the deciduous forests (Sharma et al., 2005)

2.2.9 Lagundi (V. negundo)

Lagundi, Vitex negundo L., is one of the ten medicinal plants being promoted by the

Department of Health due to its antitussive and anti-inflammatory properties. It is a

member of the family Verbenaceae, alongside other economically and medically important

plants such as alagaw (Premna odorata), lemon verbena (Lippia citriodorata) and common

vervain or “mosquito plant” (Verbena officinalis). It is an erect, branched shrub with five-

foliate, opposite-arranged leaves . It is indigenous to China, India and Malay Peninsula.

However, it is known to be widely distributed in the Philippines, growing in low to medium

altitudes and even in thickets and waste places. The leaves, bark, roots, and seeds of V.

negundo have long been used in traditional medicine to heal wounds, cleanseulcers,

prevent insect bites, treat snake bites, and relieve rheumatism. Its oil has also been used to

relieve sores and sinuses. Recent studies have proven its antiseptic, antitussive, as well as

its anti-inflammatory properties. It has been found to have antibacterial effects against

Bacillus subtilis, Escherichia coli, and Staphylococcus aureus. Other studies have also

shown its efficacy in reducing coughing and relaxing the bronchial smooth muscles

(Galvez Tan 2010). These properties make it an effective drug for coughs and

bronchopulmonary disorders such as brochial asthma and acute bronchitis.

 11

2.2.10 Components of Lagundi (V. negundo)

Vitex negundo (Lagundi) contains antioxidant phenolic compounds and flavonoids

such as casticin, chrysoplenol D, luteolin, iso-orientin, and luteolin-7-0-glucoside. Other

compounds such as epicatechin, quercetin, catechin, myricetin, tocopherol, β-carotene and

lycopene were also found to be present in lagundi leaf extracts.

 12

Methodology

I. Materials

 Lemon (Citrus limon)

 Lagundi (Vitex negundo)

 Mosquito Larvae

 Spike Larvicide

 Distilled Water

II. Equipments

 Mortar and Pestle

 Peeler

 Petri Dishes

 Beaker

 Test Tubes

Collection

Lemon (Citrus limon) were purchased at Agora Public Market. While Lagundi

(Vitex negundo) were gathered from a farm located at Hda. Remedios, Bryg. Balabag.

Mosquito larvae were collected at La Paz St., La Carlota City at the New Printing Press

Beside the fishpond. Spike larvicide was bought at Lopues La Carlota Branch. The

Distilled was bought at Lopues La Carlota Branch

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Preparation of Treatments

There were four (4) treatments in this study each having different variables. Each

treatment will have three (3) replicates.

Preparation of Lemon peel (Citrus limon) extracts.

The bought Lemon (Citrus limon) were washed thoroughly with clean water. The

Lemon (C. limon) were peeled using a peeler. The collected lemon peel was cut into smaller

pieces so that pounding it would be easier. The lemon peel was then pounded using mortar

and pestle the pounded peelings were placed in a clean cloth and squeezed until the extracts

could be collected. The extracts collected will be placed in a labeled and sterilized beaker.

Preparation of Lagundi Extracts (Vitex negundo)

The collected Lagundi (V. negundo) were washed thoroughly with clean water.

Then the leaves were separated from its stem. After Separating the stem healthy leaves

were chosen while the damaged one were disposed. The leaves were pounded using a

mortar and pestle the pounded, the pounded leaves were placed in a clean cloth and

squeezed until extracts could be collected. The collected extracts were placed in a sterilized

and labeled beaker.

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Preparation of Set-ups

Treatment A contains 50ml of Lemon peel (Citrus limon) extracts.The treatment B

contains 50ml of Lagundi (Vitex negundo) extracts. Treatment C the positive control

contained 50ml of Spike larvicide.Treatment D the negative control contained 50ml of

distilled water. Each replicate were assigned with 4 mosquitoes.

Application of Treatment

The applications of treatments were conducted once. Treatment A with 15ml of

Lemon peel (Citrus limon) extracts per replicate. Treatment B with 15ml of Lagundi (Vitex

negundo) extracts per replicate. Treatment C the positive control was applied with 15ml of

Spike larvicide per replicate. Treatment D the negative control was applied with 15ml of

water per replicate.

Disposal

The used container, beaker, graduated cylinder, mortar and pestle will be washed

using running water and will be reused for some experimental purposes. The left-over

extracts will be stored in compatible containers with closed and fitted tops. The useless

remains will be properly disposed in the garbage dump. All glasswares were autoclaved,

sterilized and dried.

 15

Data and Data Gathering Procedures

The data obtained from the study will be subjected to the following

inferentialstatistical methods using Package for Social Science (SPSS) Software

 Mean- to determine the average mosquito larva killed.

 One Way ANOVA (Analysis of Variance)- to show if there is significant

difference

Between means set at 0.05 level of significance

 Tukey test- to test which treatment is significantly effective than the other.
 16
Results

Table 1.1.

Mortality Rate of Mosquito Larvae

Replicates
Treatment Total Mean
1 2 3
Lemon peel (Citrus limon)
4 4 4 12 4.0
Extracts
Lagundi (Vitex negundo)
4 3 4 11 3.66
Leaves Extracts
Spike Larvicide 4 4 4 12 4.0
Distilled Water 0 0 0 0 0.0

The table 1.1. shows that the number of mosquito larvae exterminated was total of

12 in treatment A which is Lemon peel (Citrus limon) extracts. The total number of

mosquito larvae exterminated in treatment B which the Lagundi (Vitex negundo) extracts

was 11. The number of mosquito larvae killed the treatment C which is the spike larvicide

(positive control) was a total of 12. The treatment D which is the Distilled water (negative

control) did not record any number of mosquito larvae exterminated.

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Table 1.2.

One-way Analysis of Variance (ANOVA) for the mortality rate of the Mosquito Larvae

after the application of treatments.

Degree of
Sum of Squares Mean Square F Sig.
Freedom
Between Groups 34.250 3 11.417 137.000 .000

Within Groups .667 8 .083

Total 34.917 11
The table 1.2. shows that there is a significant difference in the mean of the

difference of mosquito larvae exterminated of the 4 treatments with F-value of 137.000.

With p-value of <0.05 giving the researchers the reason to reject Null hypothesis and

accept Alternative hypothesis which hypothesized that there is a significant effect of

using Lemon peel (Citrus limon) extracts and Lagundi (Vitex negundo) extracts in

exterminating mosquito larvae. As showed by the table above, it is certain that the

treatments have statistically significant effect against mosquito larvae at 0.05 or 5% level

of significance
 18

Table 1.3.

Dependent Variable: Mortality Rate

Tukey HSD
95% Confidence
Mean Interval
Difference Lower Upper
(I) Treatment (J) Treatment (I-J) Std. Error Sig. Bound Bound
Lemon peel Lagundi .333 .236 .525 -.42 1.09
Spike Larvicide .000 .236 1.000 -.75 .75
4.000* .236 .000 3.25 4.75
Distilled Water
Lagundi Lemon peel -.333 .236 .525 -1.09 .42
Spike Larvicide -.333 .236 .525 -1.09 .42
Distilled Water 3.667* .236 .000 2.91 4.42
Spike Lemon peel .000 .236 1.000 -.75 .75
Larvicide
Lagundi .333 .236 .525 -.42 1.09
Distilled Water 4.000* .236 .000 3.25 4.75
Distilled Lemon peel -4.000* .236 .000 -4.75 -3.25
Water
Lagundi -3.667* .236 .000 -4.42 -2.91
Spike Larvicide -4.000* .236 .000 -4.75 -3.25
*. The mean difference is significant at the 0.05 level.

The table 1.3. shows that Lemon peel (C. limon) extracts is significantly effective

than the Lagundi (V. negundo) and the negative control which is the Distilled water, and

equally significant with the positive control which is the spike larvicide. Lagundi (V.

negundo) is less effective than the the positive control which is the spike larvicide but the

spike larvicide is just slightly more effective than Lagundi (V. negundo)
 19

Discussion

Based on the observation, Treatment A killed 4 mosquito larvae in each replicate

totaling to 12 deaths while Treatment B exterminated 4 larvae in replicates 1 and 3; and 3

larvae in replicate 2. Treatment C, which is the positive control, killed 4 mosquito larvae

with a total of 12 and Treatment D which is the negative control put none of the larvae to

death.

As it was shown in the tables Treatment A has better potential than Treatment B in

terms of Mosquito Larvae exterminated. Treatment A killed all of the larvae in the

replicates while Treatment B nearly killed all the larvae but there was 1 left. The Lagundi

(V. negundo) extracts is statistically significant to be used as a mosquito larvicide

but only lesser capabilities than the positive control (spike larvicide) and the treatment A

which is the Lemon peel (C. limon) extracts.

Summary

This research was conducted to determine the effect of Lemon Peel (C. limon) and

Lagundi (Vitex negundo) Extracts as potential mosquito larvicide. This was conducted

from July 2019 to August 2019 at Dona Hortencia Salas Benedicto National High School,

Negros Occidental.

There are four mosquitoes in each replicate with the total of twelve mosquitoes

every treatment. There are four treatments: Treatment A which is Lemon peel (Citrus
 20

limon) extracts, Treatment B which the Lagundi (Vitex negundo), Treatment C which is the

spike larvicide (positive control) and Treatment D which is the Distilled water (negative

control)

In Treatment A, four mosquito larvae were killed in each replicate with the total of

12 mosquito larvae exterminated while in Treatment B, only 11 mosquito larvae were slain,

In Treatment C, four mosquito larvae were put to death in every replicate totaling to 12

mosquito larvae deaths. Treatment A and Treatment C killed the mosquito larvae in 30

minutes, in Treatment B, it took 2 hours to kill 11 mosquito larvae, while in Treatment D,

no mosquito larvae kills were recorded.

Conclusion

Therefore we conclude that Treatment A, which is Lemon Peel (C. limon) Extracts

is more effective than Treatment B, which is Lagundi (V. negundo) and D which is the

negative control (Distilled Water). There is no significant relationship between the positive

and negative control. The results show that the study achieved its objectives.

Recommendations

Based on the results of the research, the following recommendation are presented

for possible further and more profound study

 Study further before experimentations begin and gather information specifically

about the study

 21

 The use of Lemon Peel (C. limon) and Lagundi (Vitex negundo) extracts should

also be considered

 Use the oil of the Lemon Peel (C. limon) Extracts rather than just the extracts

because the concentration of d-limonene is much dense

 Gathering of information through research should be intensified

 Bibliography

Gonazales, Eduardo. The disease that mosquitoes can cause, Manila Bulletin. Retrieved

on July 16, 2019. Retrieved from http://www.pchrd.dost.govph/index.php/

news/library- health- news/5689-the-mosquito-menace-in-the- philippines

Department Of Health. DOH the increasing Dengue Related Death Caused By

Mosquitoes. Retrieved on August 28, 2019 Retrieved from

https://www.pna.gov.ph/articles/1074779

Report of the WHO Informal Consultationon the Evaluation on the Testing of insecticides,

CTD/WHO; 1996. World Health Organization; p. 69.

Anupam et. al. Plants extracts as potential mosquito larvicide, Retrieved on August 14,

2019 Retrieved from www.semanticscholar.org/44e1/f1453952a0323559fee8e

1154f18f91de155

Creative Home making. Pesticidal effects of Lemon. Retrieved on August 14, 2019.

Retrieved from https://creativehomemaking.com/cleaning/general/lemon-peel-as-

a-pesticide/

Hunker. How to use Lemon as insect repellent. Retrieved on August 14, 2019. Retrieved

from https://www.hunker.com/13421047/how-to-use-lemon-as-insect-repellent
Pesticide Science Society of Japan. Properties of D-limonene Retrieved on August 14,

2019. Retrieved from https://www.researchgate.net/publication/275625878

_Insecticidal_ Properties_of_ d-Limonene

Tandon, V. R & Gupta, R.K. (2011). Properties of Lagundi. Retrieved on August 14,2019

Retrieved from http://www.stuartxchange.org/Lagundi.html

Ganhinhin C.M. & A.P. Kintanar(2013). Studies on Lagundi. Retrieved on August 8, 2019

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Ganhinhin C.M. & A.P. Kintanar. Larvicidal Activity Of Vitex negundo (Lagundi) Against

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Bautista, N., Cubos A.F. and Lim, M.C. 2015. Genetic Diversity of Vitex negundo L.

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11%20Bautista-fullpaper
 Acknowledgement

First of all this study will not be successful without the guidance and protection of

our Father God Almighty that blessed us and gave us strength, knowledge and wisdom to

finish and complete this task. We would like to thank him for his faithfulness and we offer

our success to him. To him we bring back the honor and glory.

Secondly, we would like to thank our research teacher Mrs. Adaiah A. Opeña who

has been our guide in finishing this project step by step and the one who gave us the golden

opportunity to do this meaningful project on the topic Lemon peel (Citrus limon) and

Lagundi (Vitex negundo) extracts as potential mosquito larvicide.

We would like to thank our ever supportive and loving families, To our friends and

classmates who gave their full support and motivation helped us a lot in finalizing this

project within the limited time frame.

Furthermore we would also like to acknowledge with much appreciation the crucial

role of Mrs.Mavil C. Moncada , who gave the permission to borrow all required equipment

and the necessary materias for us to successfully conduct the experiment Last but not least,

many thanks go to our project adviser Mr. Christian Floyd I. Erad who have invested his

full effort in guiding and leading us to the right path in having the best possible outcome

for our the study.