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CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE

Junior High School Research Paper

Contact Toxicity of Ginger (Zingiber officinale) Essential Oil and Coconut (Cocos
nucifera) Shell Liquid Smoke against mango stem borer larvae

Pauline Ciara Lou B. Flores


Ashley Nicole B. Macabeo
Ralph EJ Jabez G. Garingo
Sofia May B. Castro
Iyanna Eunice B. De Ocampo

April 2019

Science, Technology, and Engineering Program


Science and Technology Department
Mater Dei Academy
Santa Maria, Bulacan
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE ii

Abstract

Seeing as the mango stem borer is a very problematic pest, the study aims to

produce an eco-friendly and cost-friendly larvicide by testing its contact toxicity against

the stem borer. Ginger essential oil (GEO) and coconut liquid smoke (CLS) were the two

primary treatments as larvicide, because of its abundance, and its cheapness. The study

tested the contact toxicity of ginger essential oil, coconut liquid smoke and mixture of

both formulations (GEO and CLS). Tested on 4 treatment sets with 10 larvae each,

including ethanol treatment as the control group. The larvae were checked in 1 hour

intervals, checking for activity and responses when tapped, and also the time it took for

the larvae to die. It was seen that the mixed treatment was the most effective in killing

larvae, killing 9/10 larvae, with the first larva dying within the 1st hour, followed by the

coconut liquid smoke, which killed 8/10 larvae, with the first larva also dying within the

1st hour. It is then followed by the ethanol control group, which killed 4/10 larvae, and

killing the first larva at 4th hour, and then the ginger essential oil, which only killed 3/10

larvae, and taking 12 hours to kill the first larva.

Keywords: ginger essential oils, liquid smoke, stem borer larvae.


CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE iii

Approval Sheet

This research paper hereto entitled “CONTACT TOXICITY OF GINGER


(Zingiber officinale) ESSENTIAL OIL, COCONUT (Cocos nucifera) SHELL
LIQUID SMOKE AGAINST MANGO STEM BORER LARVAE”, prepared and
submitted by Pauline Ciara Lou B. Flores, Sofia May B. Castro, Ralph EJ Jabez G.
Garingo, Ashley Nicole B. Macabeo, Iyanna Eumice B. De Ocampo in partial
fulfillment of the requirements for the subject Science Research II, is hereby
recommended for acceptance.

MS. BEVERLY T. BAYO MR. FRANCIS DON C. JUAN


Panel Member Panel Member

MR. JOHN MARK N. FRANCISCO MS. ERIKA STEPHANIE R. VILORIA


Panel Member Panel Chair

MR. IVAN HUBERT C. JUAN


Research Adviser
Science Paper Director

This Research Paper is accepted in partial fulfilment of the requirements for the
Science Research II:

MS. MARIBEL R. GAITE, PhD.


President, Mater Dei Academy

_______________________
Date
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE iv

Acknowledgements

First and foremost, we would like to thank God Almighty for giving us the

knowledge, skills and guidance they needed undertaking this research.

We would also like to thank our research adviser, Mr. Ivan Hubert C. Juan, for his

limitless patience, encouragement and guidance, which helped us get through this study.

Our sincerest gratitude to the thesis panel for giving us their insights on our

research and comments to help us,

To our classmates in 10 – Queen of All Saints, our class adviser Mr. Christian

Felix, for giving us the support and motivation to finish these dreams of ours, and

especially Lance Sheridan Inoncillo for helping us in our research study,

To our family and relatives who have given us support through this journey,

ensuring we continue and finish this,

To Mr. Manny Correa, for giving us a safe journey throughout our research

appointments.

To Engineer Agriculturist Agapito Pascual, for giving us advices to better our

paper and helping us obtain samples of larvae,

To the Chemicals & Energy Division of DOST-ITDI and its staff, Mr. Romulio

Estrella, Mr. Peter Rubio, and Ms. Evelyn Manongsong, who assisted in and facilitated

the extraction of the ginger essential oil, giving way for our study,
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE v

To the people in the Forest Products Research Development Institute, Engr. Gino

S. Pasco, Engr. George, Mr. Rodelio and Dr. Jennifer Tamato, who also guided us in our

study,

And Mater Dei Academy, for providing the education and the curriculum they

administered. It is because of our Alma Mater that we were taught on how to conduct the

processes in the research study.

We would also like to thank anyone and everyone who have directly or indirectly

helped us. We express our sincere gratitude.

Castro, Sofia May B.

De Ocampo, Iyanna Eunice B.

Flores, Pauline Ciara Lou B.

Garingo, Ralph EJ Jabez G.

Macabeo, Ashley Nicole B.


CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE vi

List of Tables

1 Result of four formulations to each set of larvae ...................................................24

2 Result of Ethanol to one set of larvae for 24 hours................................................35

3 Result of GEO to one set of larvae for 24 hours ....................................................36

4 Result of CLS to one set of larvae for 24 hours .....................................................37

5 Result of mixed GEO and CLS to one set of larvae for 24 hours ..........................38
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE vii

List of Figures

1 Schematic diagram of the research process ...........................................................18

2 Gathering of materials and collection of mango stem borer larvae .......................19

3 Extraction of Ginger Essential Oil .........................................................................20

4 Pyrolysis .................................................................................................................21

5 Emulsion Process ...................................................................................................21

6 Investigation of Contact toxicity of each formulation against mango stem borer

larvae ......................................................................................................................22

7 Larvae sprayed with different formulation ............................................................25

8 (Top) Machine used for the extraction; (Bottom) Extracted ginger essential oil...39

9 (Top) Signing of forms for the extraction of ginger essential oil in ITDI-CED

DOST; (Bottom) Researchers after getting the ginger essential oil .......................40

10 Cleaning of coconut shells before undergone Pyrolysis ........................................41

11 (Top, Left) Apparatus used in Pyrolysis process; (Top, Right) Produced coconut

shells liquid smoke; (Bottom) Researchers with the staffs of FPRDI. ..................42

12 Emulsification Process and Emulsified Formulations ...........................................43

13 Collection of mango stem borer larvae ..................................................................44


CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE viii

List of Appendices

A Accomplishment Report of Extraction ..................................................................31

B Memorandum of Agreement ..................................................................................32

C Proof of Guidance ..................................................................................................33

D Experimental Results .............................................................................................35

E Photo Documentation.............................................................................................39

F Abott’s Formula .....................................................................................................45

G Receipts ..................................................................................................................46

H Work Plan...............................................................................................................49

I Budget Plan ............................................................................................................50


CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE ix

Table of Contents

Title Page ............................................................................................................................. i

Abstract ............................................................................................................................... ii

Approval Sheet ................................................................................................................... iii

Acknowledgements ..............................................................................................................iv

List of Tables .......................................................................................................................vi

List of Figures ....................................................................................................................vii

List of Appendices ............................................................................................................ viii

CHAPTER

1 INTRODUCTION......................................................................................1

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

Hypotheses ...................................................................................................3

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

Scopes and Limitations ................................................................................4

Definition of Terms......................................................................................5

2 REVIEW OF RELATED LITERATURE AND STUDIES ...................6

Integrated Management of Mango Stem Borer (Batocera rufomaculata

Dejan) in Nepal ............................................................................................7

Diversity and nature of damage of mango insect pests at Kaliachak-II

Block of Malda, West Bengal, India ............................................................8


CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE x

Alcohol dehydrogenase activities and ethanol tolerance in Anastrepha

(Diptera, Tephritidae) fruit-fly species and their hybrids ............................8

Larvicidal activity of Tagetes patula essential oil against three mosquito

species……………………….. ..................................................................10

Effect of Pyrolysis temperature and Distillation on character of coconut

shell liquid smoke…………. .....................................................................11

Activity of Coconut-shell liquid smoke as an insecticide on the rice brown

planthopper (Nilaparvata lugens) ..............................................................12

Larvicidal activities of ginger (Zingiber officinale) against

Angiostrongylus cantonensis .....................................................................14

Contact Toxicity, Feeding Reduction, and Repellency of Essential Oils

from Three Plants from the Ginger Family (Zingiberaceae) and their

Major Components Against Sitophilus zeamais and Tribolium

castaneum ...................................................................................................15

Transgenic sugarcane plants resistant to stem borer attack .......................16

3 MATERIALS AND METHODS ........................................................................18

Research design .........................................................................................18

Phase 1: Gathering of materials and collection of mango stem borer

larvae….. ....................................................................................................19

Phase 2: Extraction of ginger essential oil and production of coconut shell

liquid smoke ...............................................................................................20

Extraction .......................................................................................20
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE xi

Pyrolysis .........................................................................................20

Phase 3: Emulsion Process (Mixing of GEO, Ethanol, and distilled water)

and (Mixing of GEO, CLS, and distilled water) ........................................21

Phase 4: Investigation of Contact toxicity of each formulation against

mango stem borer larvae ...........................................................................22

Contact toxicity of ginger essential oil ..........................................22

Contact toxicity of coconut shell liquid smoke..............................22

Contact toxicity of mixed formulations of GEO and CLS ............23

Contact toxicity of Control sample (Ethanol) ................................23

4 RESULTS AND DISCUSSION ..........................................................................24

Results ........................................................................................................24

Discussion ..................................................................................................24

5 CONCLUSION AND RECOMMENDATIONS ...............................................26

Conclusion .....................................................................................................

Recommendations ..........................................................................................

REFERENCES .................................................................................................................27

APPENDICES.. ............................................................................................................…31
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 1

Chapter 1

Introduction

The larvae of stem borer, causes widespread damage against mango trees. This is

a considerable problem because Philippines is one of the leading global exporters of

mango. The country has established itself as a leading producer and exporter of processed

mangos, particularly in the dried mango category (Duke CGGC, 2017). Locally, mango is

the third most important fruit both in terms of quantity and value of production after

bananas and pineapples. Thus, the mango industry plays an important role in the

economy of the country, providing a source of livelihood to about 2.5 million farmers

(DOST-PCAARRD, 2011).

Mango stem borer is one of the major pests of mango orchards. The mango stem

borer feeds internally and thus becomes difficult to control it once the larvae enter the

mango stem/shoot. This polyphagous pest attacks both living and dead trees, wherein

severe infestation affects the whole shoots and tree looks like burned trees and causes

heavy reduction in yield (Upadhyay et al., 2013).

Ginger (Zingiber officinale) is a flowering plant whose rhizome, ginger root or

simply ginger, is widely used as a spice or a folk medicine. Previous studies were

conducted about ginger in which many pharmacological properties of ginger have been

identified. This includes essential oil, zingiberol, zingiberone, zingiberene, and pungent

components such as gingerol and shogaol (Goto et al., 1990; Shoji et al., 1982; Connell

and Sutherland, 1969). The essential oil of Zingiber officinale (GEO) is proven that it has

larvicidal activity against Cx. Tritaeniorhynchus and An. subpictus mosquito species
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 2

(Govindarajan, 2011). It was also found to have larvicidal activity against Cx.

quinquefasciatus with LC50 of 50.78ppm (Pushpanathan et al., 2008).

Coconut shell is a common wood waste material in Philippines. For the reason

that the Philippines is also known as the world’s second largest producer of coconuts

according to World Atlas. Coconut shell composition is composed of lignin and cellulose,

which makes it a good chip in making liquid smoke (CLS).

Liquid smoke is a solution of the result of condensation of vapor of wood smoke

which is burned with limited air at high temperatures (Yulistiani, 1997). It contains many

compounds that are formed due to the pyrolysis of three components of wood,

specifically cellulose, hemicellulose and lignin. It contains a variety of compounds

including phenol, carbonyl, acids, furans, alcohols, lactones, hydrocarbons, and polycylic

aromatic that are anti-microbial and toxic to insect pests (Thamrin, 2007). The study will

use coconut shells and ginger essential oil to produce a liquid smoke larvicide targeting

the larval stage of stem borers.

Statement of the Problem

The study investigated on what is more applicable solution against stem borers

and the accuracy of the quantity of killed pests with each solution (with GEO only, with

CLS only, and with mixed solution of GEO and CLS.) The following questions were

answered:

1. Which formulation of larvicide (GEO, CLS, GEO and CLS) will have the fastest

reaction against larvae in terms of kill time in killing larvae of each set(10 larvae)

after 24 hours?
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 3

2. Which formulation of larvicide (GEO, CLS, GEO and CLS) will have the most

number of killed larvae after 24 hours?

Research Hypotheses

1. The mixed formulation of GEO and CLS would have the fastest reaction against

larvae in terms of kill time in killing larvae of each set(10 larvae) after 24 hours.

2. The mixed formulation of GEO and CLS would have the most number of killed

larvae after 24 hours.

Significance of the Study

The stem borer is the most damaging pest because the larvae of the pest live for

long time (about 1 Year) and hibernate in winter inside the dry shoot, when climate

warms up they activate and pupate inside then adults emerge and start egg laying from

July-August. When the larvae matured it starts to bore too deep in the stem/shoot

(Upadhyay et al., 2013).

It can destroy one of the abundant crop in our country at any stage of the plant for

the grub is damaging stage and damages by cutting and chewing of newly twigs and

shoots. Once grub enters in the shoot creates tunnel inside the stem and damages the stem

resulting in drying of shoots (Upadhyay et al., 2013).

The study aims to eliminate these stem borer larvae by targeting it in its larval

stage. By addressing the problem of stem borers against mango trees, liquid smoke can be

used as an alternative to synthetic pesticides.

The study targets to deal with the use of organic materials abundant in our country

and liquid smoke concentrations as a natural larvicide that is effective for killing stem
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 4

borers in mango trees. It also aims on recycling coconut shells, for it is one of the major

wood wastes that has not been utilized properly and will make use of ginger essential oil

in the field of larviciding stem borer larvae.

Also, one of the objectives of the study is to investigate in which solutions and

expected time is most applicable for better results.

The study can benefit the community by accomplishing the following goals:

address problems regarding stem borers against mango trees and the use of liquid smoke

as an alternative to synthetic pesticides, since the synthetic ones can cause negative

environmental impacts (destruction of biodiversity, runoff, increases pest resistance,

pollution); and raising environmental awareness by recycling wood waste material into

organic larvicide.

The study will attempt to produce a pesticide (larvicide specifically) that is

environmental friendly. The information of larvicide that this study will create can

contribute to the elimination of stem borers, one of mainly harmful pests in plants.

Scope and Limitations

The study examined the larvicidal effectiveness and properties of Zingiber

officinale essential oil and Cocos nucifera shell liquid smoke towards stem borers.

The study also investigated what solutions (GEO: CLS: 1:1 GEO and CLS) has

the best results in terms of the time it took to kill a group of stem borer larvae and in

terms of the quantity of killed stem borer larvae after 24 hours.

The study was limited to the analysis that the said larvicide has the same

effectiveness in terms of the quantity of killed larvae and how fast it kills larvae against
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 5

other mango tree pest. The effects of other limitations such as effectiveness of the

larvicide on other species aside from stem borer, and effects of it in mango trees was not

included in the study.

Definition of Terms

Larvicide. An insecticide that targets larvae in breeding habitat before they can mature

into adult mosquitoes and disperse.

Liquid smoke. A substance produced from wood carbonization process at a high

temperature in the absence of oxygen. It has a potential chemical as biopesticide

and wood preservative due to the uniqueness of its complex structure and

chemical composition.

Pyrolysis. Refers to the thermal decomposition of biomass occurring in the absence of

oxygen. The products of biomass include biochar, bio-oil and gases including

methane, hydrogen, carbon monoxide, and carbon dioxide.

Emulsion. Refers to the mixture of two or more liquids that are normally immiscible.

Stem borer. Any insert larva, or arthropod, that bores into plant stems. However the term

most frequently refers among the Coleoptera to the larva of certain longhorn

beetles such as Dorysthenes buqueti and those of the genus Oberea, and among

the Lepidoptera to certain moths of the Crambidae, Castniidae, Gelechiidae,

Nolidae, and Pyralidae families.

Shoot. A young branch or sucker springing from the main stock of a tree or other plant.
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 6

Chapter 2

Review of Related Literature and Studies

Mango (Mangifera indica L.) a member of family Anacardiaceae is known as

king of fruits, asking of fruits for its sweetness, excellent flavor, delicious taste and high

nutritive value. This important tropical fruit is being grown in more than 100 countries

(Sauco, 1997). It is also valuable environmental and shade tree, which contributes to the

protection of soil against erosion and different medicinal virtues (D Almeida, 1995). The

Philippines holds a relatively significant position in the mango GVC (Global Value

Chain). The country has been an important player in the global market since the 1980,

with exports taking off in the 1990s.

The Life Cycle study was done at RARS, Tarahara (Horticulture research unit) in

borer infested Amrapali mango tree. The standing plant was covered by cloth net

(mosquito net) and no any treatment was applied upon plant. The observation was taken

at 15 days interval. Study was started from July 2011 to October 2012. The study showed

that the mango stem borer adult starts egg laying upon the bark and dead shoots of mango

tree from July – August and egg hatching takes place within 10- 15 days. Newly hatched

larvae of the pest start feeding upon the newly flushed leaves and start boring onward.

Once larva enters the shoot, it starts cutting and boring the shoots.

The mango stem borer (Batocera rufomaculata) a species of beetle in the family

Cerambycidae. The adult starts egg laying upon the bark and dead shoots of mango tree

from July –August and egg hatching takes place within 10- 15 days. Newly hatched

larvae of the pest start feeding upon the newly flushed leaves and start boring onward.
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 7

Once larva enters the shoot, it starts cutting and boring the shoots. This pest damages the

young shoots of mango turning dry and no flowering and fruiting. It is also because that

dry shoot does not carry any type of pesticides towards the dry shoot where larva remains

(Upadhyay, 2013).

Integrated Management of Mango Stem Borer (Batocera rufomaculata Dejan) in

Nepal

In this study conducted by Upadhyay, Chaudhary, & Sapkota, Mango (Mangifera

indica L.) a member of family Anacardiaceae is known as king of fruits, asking of fruits

for its sweetness, excellent flavor, delicious taste and high nutritive value. This important

tropical fruit is being grown in more than 100 countries (Sauco, 1997). It is also valuable

environmental and shade tree, which contributes to the protection of soil against erosion

and different medicinal virtues (D Almeida, 1995). Mango stem borer is one of the major

pests of mango orchards. The farmers are destroying the orchards in ETR due to the

heavy infestation of this pest. A survey in Siraha and Saptari districts reported mango

fruit fly, mango hopper and mango fruit borer as prevailing insect pests of mango (Regmi

et al., 2004).

The survey and field observation of the orchards indicated that Mango stem borer

is a serious pest of mango in Saptari district and also shifting to Siraha and Udaipur

districts respectively. A detailed farm and farmers' survey was conducted among 30

farmers in each district in ETR of Nepal during 2010-11 by using pre-tested semi-

structured questionnaire. The survey assessed the information on insect pest incidence,

their nature and extent of damage (Symptoms, plant-parts infested, time of attack and
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 8

extent of damage due to insect pests) in different varieties of mango fruit and available

management practices. Among eight treatments, Imidacloprid 17.8% SL, Thiamethoxame

25% WG and Trizophos 40% SL performed best in management of mango stem borer.

Among the treatments, Imidacloprid 17.8% SL, Thiamethoxame 25% WG was found

best in management of mango stem borer.

Diversity and nature of damage of mango insect pests at Kaliachak-II Block of

Malda, West Bengal, India

In this study presented by Chowdhury, The aim of this present study is to focus

yield loss of mango fruit by different mango insect pests and their effect on economic

condition of mada peoples as well as national and international trading system. Northern,

eastern, western and southern regions of India are recognized as distinct mango growing

regions, though some cultivars are grown over wider areas (Yadav and Rajan, 1993). The

insects along with their immature and mature stages were collected by traditional

methods of hand picking. The adults and nymphs were collected and preserved in 75%

alcohol in vials for later identification in Laboratory. Grubs feed inside the stem boring

upward making irregular tunnels which results in interruption of nutrient and water

transport in the tissue. Drying of terminal shoot in early stages and severe symptoms

causes wilting of branches or entire tree.

Alcohol dehydrogenase activities and ethanol tolerance in Anastrepha (Diptera,

Tephritidae) fruit-fly species and their hybrids

In this study conducted by Carvalho, Solferini and Matioli, The alcohol

dehydrogenase enzyme system (ADH) of Drosophila is a classical model used in


CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 9

understanding the question of the evolutionary relevance of enzyme polymorphism. An

important element is the ADH system, which degrades 90% of the total ethanol in

Drosophila melanogaster (Heinstra et al., 1987; Geer et al., 1993). ADH action per se is

enough to convert a toxic exogenous substance (ethanol) into a common endogenous one

(acetate) (Kapoun et al., 1990; Chakir et al., 1993; Geer et al., 1993). Third instar larvae

were exposed to ethanol. The exposure was carried out in Petridishes sealed with PVC

film, each with a cellulose sponge soaked in a solution containing ethanol at different

concentrations, 0.15 M NaCl (to maintain the osmotic equilibrium) and 1% glucose (to

minimize the use of ethanol as a source of energy or carbon).

This experiment was carried out at 25 °C in the absence of light. In each

exposure experiment and after the first 12 h of exposure, the larvae were transferred to

new Petri dishes with fresh solutions at the same ethanol concentration. The

concentration required to kill half of the larvae exposed during a given time was called

the Lethal Concentration 50 (LC 50). The survival of larvae exposed to ethanol (whose

toxicity was placed in evidence by our model) was dependent on several factors.

Regarding the effects of the genetic background, data from LC analysis suggest that there

was hybrid superiority, thus characterizing a heterosis effect, although there was no clear

statistical significance for this statement. Through this rare and informative model, in

which the crossing of species with differences in genetic constitution, as in phenotypic

traits, is made possible, we demonstrated that the rearegenetic factors acting on the

enzymatic activity of ADH and on ethanol tolerance as well.


CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 10

Larvicidal activity of Tagetes patula essential oil against three mosquito species

In this study that was conducted by Dharmaggada, Naik, Mittal and Vasudevan,

Mosquitoes are well known vectors of several disease causing pathogens. The synthetic

insecticides are toxic and adversely affect the environment by contaminating soil, water

and air. There is a need to find alternatives to these synthetic pesticides. Botanical

pesticides are promising in that they are effective, environment – friendly, easily

biodegradable and also inexpensive. The steam carried the volatile organic compounds

(essential oil) present in the plant material into the vapor phase. Condensation decreased

the solubility of essential oil constituents in the vapor and the essential oil was easily

separated from the water and collected.

Fourth instar larvae, which is the fourth larval stage in the life cycle of mosquito

after which the larva finally becomes a pupa, were exposed to sub lethal concentrations

of 6.25, 12.5, 25, 50, 100 and 150 ppm of the oil in distilled water for 24 h according to

standard WHO procedure (1981). As the essential oil does not dissolve in water it was

first dissolved in ethanol (99.8%). The test medium was prepared by adding 1 ml of

appropriate dilution of essential oil in ethanol and mixed with 249 ml of water to make up

250 ml of test solution. The oil – ethanol – water solution was stirred for 30 s with a glass

road. After about 15 min 25 larvae taken on a strainer with fine mesh were transferred

gently to the test medium by tapping. Two replicates, with 25 larvae in each, were taken

for each mosquito species in each solution and the control of ethanol – water mixture.

The dead larvae were counted after every hour and percentage mortality is reported from

the average for the two replicates taken together. The diviation between the replicates is
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 11

±10 e. Observations were also made on the larval behavior, morphological changes and

adult emergence.

Effect of Pyrolysis temperature and distillation on character of coconut shell liquid

smoke

A research on effect of coconut shell pyrolysis temperature and liquid smoke

distillation on character of resulted liquid smoke has been done. Objective of the research

is to identify effect of coconut shell pyrolisis temperature and liquid smoke distillation on

character of coconut shell liquid smoke distillate. Composition of liquid smoke is

affected by raw material type, duration of burning and burning temperature. Liquid

smoke is used commonly by agent providing aroma, texture and taste of food product,

such as mead, fish and cheese. Washed coconut shell with its dimension is reduced was

put into pyrolisis reactor and closed well. Reactor was heated in high temperature (150-

450°C) with heating rate of 30°C per minutes. After reaching desired temperature, it was

let 2 hours and smoke flow through evaporation pipe, cooled through cooling media.

Then its liquid was discharged through pipe placed in container. After there is no liquid,

the heating is stopped and reactor is cooled. Condensate and charcoal was weighted to

determine concentration. Condensate concentration is mix of liquid smoke and tar

calculated as % volume/weight and charcoal is calculated as % weight/weight. Charcoal

concentration was 28.75% (m/m). Characteristic (pH, acid level and phenol level) of

liquid smoke before and after purification process and distillation are various. Highest pH

(6.2) was D-I, while the lowest (4.1) at D-VI. The highest acid level (58.40%) was at D-

VI, while the lowest (1.86%) was at D-I. at D-I liquid smoke does not contain phenol,
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 12

while the highest phenol content (3.85%) was in D-VI and the lowest was liquid smoke

residue (1.93%).

Activity of Coconut-shell liquid smoke as an insecticide on the rice brown

planthopper (Nilaparvata lugens)

In this study conducted by F.X. Wagiman, Arik Ardiansyah and Witjaksono, they

used liquid smoke as a form of pesticide to the rice brown planthopper. Since coconut

shell composition is composed of lignin and cellulose and is suitable for pyrolysis

process, they used it as a chip in making the liquid smoke pesticide. Laboratory trial

proved that the genuine of coconut-shell liquid-smoke grade II was very toxic against the

rice brown planthopper (Nilaparvata lugens) and phytotoxic to the rice plants.

The genuine liquid smoke is very acid (pH 1.2). It could be neutralized (pH 6, 46) by

adding it with calcium oxide at rate of 7 grams per 100 ml. LC50 of the neutral coconut-

shell liquid-smoke grade II against N. lugens at 24, 48, and 72 hours after treatment were

12.89, 11, and 9.94%, respectively. The concentration rate of 12.5% was feasible to be

developed into application dosage at which this concentration rate of neutral liquid smoke

was not toxic to the plant.

Insecticidal properties of Zingiber officinale and Piper cubeba essential oils against

Tribolium castaneum Herbst (Coleoptera: Tenebrionidae)

Essential oils are the complex mixture of volatile organic compounds produced as

secondary metabolites whose functions are other than nutrition. Essential oils and its

constituents are known to have repellent and insecticidal activities against several stored

grain insect pests. In the present study essential oils from Zingiber officinale rhizome and
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 13

Piper cubeba berries have been isolated and its insecticidal activities against red flour

beetle Tribolium castaneum have been explored. Repellency assay was carried out in

glass petri dishes (diameter 8.5 cm and height 1.2 cm). Test solutions of serial dilution of

Z. officinale and P. cubeba essential oils were prepared in acetone. Whatmann filter paper

was cut into two equal halves and each essential oil solution was applied to filter paper

half as uniform as possible using micropipette. The other half of filter paper was treated

with acetone only.

Twenty adults of T. castaneum were released at the centre of filter paper disc and

then petri dishes were covered and kept in dark. Four replicates were set for each

concentration of essential oil solution. Number of insects on both treated and untreated

halves was recorded after 4 h in mild light. Fumigant toxicity of Z. officinale and P.

cubeba essential oils was tested against larvae and adults of T. castaneum. Ten

adults/larvae taken from the laboratory culture were placed with 2 g of wheat flour in

petri dishes (diameter 8.5 cm and height 1.2 cm). A filter paper strip (4 cm2) treated with

essential oil solutions of different concentration prepared in acetone, was pasted on the

under cover of petri dishes. Oviposition inhibitory activity of Z. officinale and P. cubeba

essential oils was tested against T. castaneum by fumigation method.

According to results, Z. officinale and P. cubeba essential oils are repellant and

toxic to growing larvae and adults of T. castaneum. These essential oils repel adult

beetles significantly even at very low concentration.


CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 14

Larvicidal activities of ginger (Zingiber officinale) against Angiostrongylus

cantonensis

In this study, it is investigated that the anthelmintic activity of [6]-gingerol, [10]-

shogaol, [10]-gingerol, [6]-shogaol and hexahydrocurcumin, a constituent isolate from

the roots of ginger (Zingiber officinale), for the parasite Angiostrongylus cantonensis.

This study found that the above constituents killed A. cantonensis larvae or reduced their

spontaneous movements in a time- and dose-dependent manner. The larvicidal effect or

ability to halt spontaneous parasite movement of [10]-shogaol, [6]-gingerol, [10]-

gingerol, [6]- shogaol and hexahydrocurcumin at various concentrations was reached

from 24 to 72 h, respectively. Further investigation to determine minimal effective doses

of [10]-gingerol and hexahydrocurcumin revealed [10]-gingerol to have a greater

maximum larvicidal effect and loss of spontaneous movements than hexahydrocurcumin,

mebendazole and albendazole. These constituents of ginger showed effects against DPPH

and peroxyl radical under larvicidal effect. Together, these findings suggest that these

constituents of ginger might be used as larvicidal agents against A. cantonensis.

Mosquito larvicidal activity of isolated compounds from the rhizome of Zingiber

officinale

The larvicidal activity of a petroleum ether extract of Zingiber officinale Roscoe

(Zingiberaceae) was evaluated against Aedes aegypti L. and Culex quinquefasciatus

(Diptera). Bioassay-guided fractionation led to the isolation of 4-gingerol (1), (6)-

dehydrogingerdione (2) and (6)-dihydrogingerdione (3); the latter has not previously been

reported from Z. officinale. The structures were established from infrared (IR), ultraviolet
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 15

(UV), 1H-nuclear magnetic resonance (NMR), 13C-NMR and mass spectral data.

Following a 24 h exposure, compounds 1–3 exhibited larvicidal activities against fourth

instar larvae of A. aegypti (LC50 4.25, 9.80, 18.20 ppm) and C. quinquefasciatus (LC50

5.52, 7.66, 27.24 ppm), respectively. The results show that the most effective compound

was 4-gingerol.

Contact Toxicity, Feeding Reduction, and Repellency of Essential Oils from Three

Plants from the Ginger Family (Zingiberaceae) and their Major Components

Against Sitophilus zeamais and Tribolium castaneum

The essential oils from rhizomes of Alpinia conchigera Griff, Zingiber zerumbet

Smitt, Curcuma zedoaria (Berg.) Roscoe; their major compounds (camphene, camphor,

1,8-cineole, -humulene, isoborneol, α -pinene, β -pinene and terpinen-4-ol); and synthetic

essential oils comprised of mixtures of major pure compounds in the same ratios as the

extracted essential oils were tested for contact, feeding reduction, and repellency against

Sitophilus zeamais Motschulsky and Tribolium castaneum (Herbst) adults.

Several essential oils are also toxic to stored product insects (Huangetal. 2000

Negahban and Moharramipour 2007 Rajendran and Sriranjini 2008). Therefore, the

essential oils could also be used as alternative sources to control stored-product insects,

due to their low toxicity to warm-blooded mammals (Shaaya et al. 1991, 1997; Kim et al.

2003). The objective of this study is to determine whether the essential oils from three

Thai plants from the Zingiberaceae-A. conchigera, Z. zerumbet, and C. zedoaria – are

active against two stored-product insects, S. zeamais and T. castaneum. Pure Compounds

and Synthetic Essential Oils. Chemical constituents of the essential oils were determined
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 16

using gas chromatography-mass spectrometer (GC-MS; Shimadzu QP 5050A) as

described in Suthisut et al. (2011). Contact Toxicity. Aliquots of 0.5

ml of three essential oils, eight pure compounds or three synthetic oils at different

concentrations(0,10,20,30,40,or50%ofoils or compounds diluted with ethanol) were

applied topically onto the pronotum of S. zeamais (average weight of insect, 2.305 mg) or

T. castaneum (average weight of insect, 2.004 mg).

Transgenic sugarcane plants resistant to stem borer attack

The commercial sugarcane variety Ja 60-5 was obtained from the germplasm

collection of the Exper-imental Sugarcane Station, Jovellanos, Cuba. Sug-arcane

embryogenic calli were used in the electro-poration experiment according to the

methodology reported by Payan et al. Briefly, meristematic tissue from sugarcane was

excised, disinfected and placed on callus induction medium (P) containing MS salt, 100

mg/ml myo-inositol, 0.8 mg/l thiamine-HCl, 500 mg/l casein hydrolysate, 20 g/l

sucrose,4 mg/l 2,4-D, 7 g/l agar-agar, pH 5.6. The culture was maintained in the dark at

25 C for one month. Subsequently, friable embryogenic calli were transferred to fresh

medium for an additional subculture and used for transformation experiments.

SCSB colonies were established using semi-artificial diet: 33 g/l maize flour, 45

g/l wheat germ powder, 16.5 g/l sugarcane leaf powder, 42.5 g/l dry yeast,4.3 g/l ascorbic

acid, 1.32 g/l boric acid, 3 g/l methyl parabene, 10% formaldehyde. Soluble proteins from

E.coliex pressing the tcryIA(b)gene were assayed for lethality to neonate SCSB larvae by

incorporation into the diet. CryIA(b) concentrations were determined by DAS-ELISA.

The evaluation of larvicidal activity was carried out against 1-day old, third-instar SCSB
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 17

larvae by putting them onto the artificial diet. Ten larvae were used for each toxin dose. A

diet, supplemented with lysates obtained from pUC 19-transformed E. coli culture, was

used as negative control. Mortality was scored after 7 days and LC50 was calculated.
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 18

Chapter 3

Materials and Methods

This chapter presents the research processes that are going to be used, equipment

that were utilized, as well as the techniques that we were used in the treatment of data

gathered.

Research Design

This research has 4 phases which is shown in Fig. 1 below

Fig. 1. Schematic Diagram. Rectangles on the left demote major phases of the study,

rectangles on the right denote steps involved in each phase.


CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 19

Research Methodology

Phase 1 – Gathering of materials (ginger and coconut shells) and collection of stem

borer larvae. The first phase of the study entails the gathering of raw materials,

consisting of rhizome of ginger (30 kilograms) and coconut shells (six sacks) that were

gathered in wet markets of Pulong Buhangin and Poblacion in Santa Maria, Bulacan and

Bicutan in Taguig City.

Also, the proponents went to mango farms in Santa Maria and Norzagaray,

Bulacan where the stem borer larvae are collected.

Fig. 2. Gathering of materials and collection of mango stem borer larvae. 36-kilogram

sample of ginger (top, left), gathered six sacks of coconut shells (top, right), collected
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 20

mango stem borer in the stem (bottom, left), and the mango stem borer removed from the

stem (bottom, right).

Phase 2 – Extraction of ginger essential oil and production of coconut shell liquid

smoke (pyrolysis)

Extraction. The collected gingers weighing 30 kilograms were passed to Industrial

Technology Development Institute (CED – DOST Chemicals and Energy Division) for

extraction of essential oil in Taguig which cost Php9, 336.00.

Fig. 3. Extraction of ginger essential oil. Sliced ginger (top, left), Oil pressing machine

(top, right), and the extracted ginger essential oil (bottom).

Pyrolysis. The collected six sacks of coconut shells (crushed) were passed to Forests

Products Research and Development Institute (FPRDI) in Los Banos, Laguna.

The coconut shells were placed to the combustion tube with a temperature of

350°C. Every hour for 4 hours, the combustion tube was refilled with new set of coconut

shells.
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 21

Fig.4. Pyrolysis. The combustion tube and tank condenser used for Pyrolysis (left), and

the coconut shell liquid smoke (right).

Phase 3 – Emulsion Process (Mixing of GEO, Ethanol, and distilled water) and

(Mixing of GEO, CLS, and distilled water). The extracted ginger essential oil and

produced coconut shell liquid smoke were mixed with ethanol and distilled water. For the

formulation of GEO only, 37.5µL of ginger essential oil, 5055µL of ethanol and distilled

water. For the formulation of mixed GEO and CLS, same values used in GEO and

ethanol and CLS were mixed with a total of 250 mL.

Fig. 5. Emulsion Process. The mixed GEO with Ethanol ( left), the Ethanol as control
sample (center), and the mixed GEO and CLS with Ethanol ( right).
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 22

Fig. 6. Investigation of Contact toxicity of each formulation against mango stem borer.

The produced formulations used to kill stem borer larvae (left and right).

Phase 4 – Investigation of Contact toxicity of each formulation against mango stem

borer larvae

Contact toxicity of ginger essential oil

The parameter that was investigated is the formulation of GEO against mango

stem borer larvae. The investigation was done through spraying method (100 sprays) to

the set of larvae (10 larvae) and observing the time of death of each larva within time

ranges. Then, the study got the number of killed larva/larvae per hour and the number of

killed larva/larvae in each set under the formulation of GEO.

Contact toxicity of coconut shell liquid smoke

The parameter that was investigated is the formulation of CLS against mango

stem borer larvae. The investigation was done by spraying 10 mL (100 sprays) of CLS to

the set of larvae (10 larvae) and was observed for 24 hours. Then, the study got the

number of killed larva/larvae per hour and the number of killed larva/larvae in each set

under the formulation of CLS.


CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 23

Contact toxicity of mixed formulations of GEO and CLS

The parameter was investigated is mixed formulation was done by spraying the

mixed concentrations of GEO (37.5 µL) and CLS emulsified in 5,055 µL of ethanol with

a total amount of 250 ml (100 sprays) to the set of larvae (10 larvae). The parameter that

was investigated was observed for 24 hours. Then, the study got the number of killed

larva/larvae per hour and the number of killed larva/larvae in each set under the

formulation of GEO and CLS.

Contact toxicity of Control sample (Ethanol)

The parameter that was investigated is the control sample for the study, ethanol

against mango stem borer larvae. The investigation was done through spraying method to

the set of larvae (10 larvae) using 10mL (100 sprays) of ethanol and observing the time of

death of each larva within time ranges using 1 hour time intervals. Then, the study got the

number of killed larva/larvae per hour and the number of killed larva/larvae in each set

under the control sample.


CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 24

Chapter 4

Results and Discussion

Results

Formulation Responding Not Responding


Ethanol 6 4
GEO 7 3
CLS 2 8
Mixed GEO and CLS 1 9

Table 1. Shows the results of four different formulations sprayed on 4 different sets

containing 10 larvae each set for 24 hours.

Discussion

The result of contact toxicity of Ethanol being the control sample against mango

stem borer was during the fourth hour of observation, the control sample started to affect

the larvae, killing one larva. From the seventh hour until twelfth hour, there were another

larvae killed. In 13th hour until 16th, a total of three larvae were killed. On the next hour

(17th hour) until 24th hour there was another larva killed resulting to 4 killed larvae out of

10 larvae alive for the contact toxicity of the controlled sample.

For the result of contact toxicity of GEO against mango stem borer, during the

12th hour after the GEO was sprayed to larvae, 1 out of 10 larvae was killed. For the next

4 hours (sixteenth hour), another two larvae were killed. In twenty-third hour up to the

last hour of observation, another larva was killed having a total of 3 killed larvae out of

10 larvae alive in the formulation of GEO. To summarize, for the whole 24-hour

observation of larvae with sprayed GEO, there were 3 out of 10 larvae killed.
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 25

For the contact toxicity of CLS against mango stem borer, for the first hour of

observation, there were already six larvae killed and this was the observation until

seventh hour. For the eighth hour up to ninth hour, another larva was killed and finally,

for the tenth hour up to the twenty-fourth hour of observation, another larva was killed

again. Over the 24-hour observation, there were 8 out of 10 larvae that were killed.

For the last observation of contact toxicity against mango stem borer with the

formulation of mixed GEO and CLS, during the first hour of observation, there were

already five dead larvae. During the third hour, another larva was killed. In the fourth

hour until tenth hour of observation, a total of seven were killed larvae. In 11th hour up to

17th hour, another larva was killed as well as in the 18th hour until 24th hour having a total

of a 9 larvae killed out of 10 larvae for the whole observation.

Fig. 7. The larvae sprayed with different formulation. Ethanol (top, left), mixed GEO and
CLS (top, right), GEO (bottom, left), and CLS (bottom, right).
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 26

Chapter 5
Conclusions and Recommendations

Conclusions

The researchers have concluded that the mixed formulation of CLS and GEO

have the highest contact toxicity among the other formulation (GEO and CLS) for it has

the fastest reaction against mango stem borer larvae. It had killed five larvae after the first

hour (larvae does not respond after tapping; straight in body shape but have minimal dark

spots). Also, it has the most number of killed larvae in set after 24 hours (9 over 10)

Recommendations

The researchers recommend to the future researchers to discover the real-life

application of the formulations (Mixed, GEO, and CLS) against Batocera rufomaculata.

Also, for them to try different concentrations of the said formulations, to determine things

such as:

If ginger essential oil has really low contact toxicity to mango stem borer larvae,

or if there is a possibility that it is effective in such specific concentration and emulsifier.

The result to the ratio of mixed formulation such as (1:1), (1:2), and (2:1).
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 27

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CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 31

Appendix A

Accomplishment Report of Extraction


CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 32

Appendix B

Memorandum of Agreement
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 33
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 34

Appendix C

Proof of Guidance
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 35

Appendix D

Experimental Results

Number of larvae that show response


Time Dead
Before tapping After tapping
Starting time 10 10 0
1st hour 7 10 0
2nd hour 5 10 0
3rd hour 8 10 0
4th hour 8 9 1
5th hour 5 9 1
6th hour 6 9 1
7th hour 7 8 2
8th hour 3 8 2
9th hour 5 8 2
10th hour 4 8 2
11th hour 5 8 2
12th hour 7 8 2
13th hour 5 7 3
14th hour 5 7 3
15th hour 3 7 3
16th hour 4 7 3
17th hour 5 6 4
18th hour 4 6 4
19th hour 6 6 4
20th hour 6 6 4
21st hour 5 6 4
22nd hour 5 6 4
23rd hour 5 6 4
24th hour 6 6 4

Table C1. Shows the effects of the control sample (Ethanol) to 10 larvae for 24 hours.
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 36

Number of larvae that show response


Time Dead
Before tapping After tapping
Starting time 10 10 0
1st hour 8 10 0
2nd hour 8 10 0
3rd hour 8 10 0
4th hour 8 10 0
5th hour 8 10 0
6th hour 8 10 0
7th hour 8 10 0
8th hour 8 10 0
9th hour 8 10 0
10th hour 8 10 0
11th hour 8 10 0
12th hour 7 9 1
13th hour 7 9 1
14th hour 7 9 1
15th hour 7 9 1
16th hour 5 7 2
17th hour 5 7 2
18th hour 5 7 2
19th hour 5 7 2
20th hour 5 7 2
21st hour 5 7 2
22nd hour 5 7 2
23rd hour 5 7 3
24th hour 5 7 3

Table C2. Shows the effects of the GEO to 10 larvae for 24 hours.
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 37

Number of larvae that show response


Time Dead
Before tapping After tapping
Starting time 10 10 0
1st hour 0 4 6
2nd hour 1 4 6
3rd hour 1 4 6
4th hour 1 4 6
5th hour 1 4 6
6th hour 1 4 6
7th hour 1 4 6
8th hour 1 3 7
9th hour 1 3 7
10th hour 1 2 8
11th hour 1 2 8
12th hour 1 2 8
13th hour 1 2 8
14th hour 1 2 8
15th hour 1 2 8
16th hour 1 2 8
17th hour 1 2 8
18th hour 1 2 8
19th hour 1 2 8
20th hour 1 2 8
21st hour 1 2 8
22nd hour 1 2 8
23rd hour 1 2 8
24th hour 1 2 8

Table C3. Shows the effects of the CLS to 10 larvae for 24 hours.
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 38

Number of larvae that show response


Time Dead
Before tapping After tapping
Starting time 10 10 0
1st hour 2 5 5
2nd hour 2 5 5
3rd hour 2 4 6
4th hour 2 3 7
5th hour 2 3 7
6th hour 2 3 7
7th hour 2 3 7
8th hour 2 3 7
9th hour 2 3 7
10th hour 2 3 7
11th hour 2 2 8
12th hour 2 2 8
13th hour 2 2 8
14th hour 2 2 8
15th hour 2 2 8
16th hour 1 2 8
17th hour 1 2 8
18th hour 1 1 9
19th hour 1 1 9
20th hour 1 1 9
21st hour 1 1 9
22nd hour 1 1 9
23rd hour 1 1 9
24th hour 1 1 9

Table C4. Shows the effects of the mixed GEO and CLS to 10 larvae for 24 hours.
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 39

Appendix E

Photo Documentation

Fig. D. Machine used for the extraction (top). The extracted ginger essential oil (bottom).
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 40

Fig. D2. Signing of forms for the Extraction of ginger essential oil in ITDI-CED DOST

(top). The researchers after getting the ginger essential oil (bottom).
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 41

Fig. D3. Cleaning of coconut shells before undergone Pyrolysis


CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 42

Fig. D4. The apparatus used in Pyrolysis process (top, left).The produced coconut shells

liquid smoke (top, right). The researchers with the staffs of FPRDI (bottom).
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 43

Fig. D5. The emulsification process of GEO and Ethanol (top, left); GEO and CLS with

Ethanol (top, right). The emulsified formulations (bottom).


CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 44

Fig. D6. Collection of mango stem borer larvae.


CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 45

Appendix F

Abbott’s Formula

Formula

Let x= the percentage living in the check

Let y=the percentage living in the treated plant

Then x-y=the percentage killed by the treatment and the percentage killed by the

treatment (x-y) divided by the percentage living in the check (x) gives the control or

expressed by an equation:

𝑥−𝑦
(100) = percentage control
𝑥

Ethanol:

4
10

CLS:

6−2
(100) = 66.67%
6

GEO:

6−7
(100) = −16.67%
6

Mixed:

6−1
(100) = 83.3%
6
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 46

Appendix G

Receipts
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 47
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 48
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 49

Appendix H

Work Plan
CONTACT TOXICITY OF GINGER OIL AND LIQUID SMOKE 50

Appendix I

Budget Plan

Services Budget Requested Definition of Expenditures


The ginger and coconut
Gathering of ginger and shells wExtractionere
Php2,550.00
Collection of coconut shells gathered and used in the
experiment
Extracted the needed
Extraction of ginger
Php9,336.00 (6kg) formula for the mango stem
essential oil
borer larvicide
Converted Coconut shells
Pyrolysis Php150.00
into liquid smoke
Mixed with water and
Ethanol Php250.00 mixed with GEO and CLS;
Control sample
The Memorandum of
Notary Php500.00 Agreement papers were
signed by the attorney
TOTAL Php12,786.00