MOLLUSCICIDAL EFFICACY OF ALKALINE-TREATED TAGPO

AGAINST GOLDEN APPLE SNAILS (Pomacea canaliculata Lam.)

______________________

A Bachelor’s Thesis

Presented to the Faculty of the

College of Arts and Sciences

Eastern Visayas State University

Tacloban City

________________________

In Partial Fulfillment

of the Requirements for the Degree

BACHELOR OF SCIENCE IN CHEMISTRY

______________________

SHERRIELYN M. BERINO

JEAN VALERIE C. CARSON

LARALIN B. MOLDEZ

S.Y. 2020-2021

i
 EASTERN VISAYAS STATE UNIVERSITY
Tacloban City

APPROVAL SHEET

This Thesis Entitled

“MOLLUSCICIDAL EFFICACY OF ALKALINE-TREATED TAGPO AGAINST

GOLDEN APPLE SNAILS (Pomacea canaliculata Lam.)”, prepared by
SHERRIELYN BERINO, JEAN VALERIE C. CARSON, and LARALIN MOLDEZ, in
partial fulfillment of the requirements for the degree of Bachelor of Science in Chemistry
has been examined and is recommended for acceptance and approval for ORAL
EXAMINATION.

GRECHELLE N. SOCIAS, RCh, LPT

Adviser

________________________________________________________________________

Approved by the committee on Oral Examination with a grade of PASSED on June 2021.

FE T. PIEDAD, RCh, MAIS

Chair

DARWIN C. GOMEZ, MSc ADORNE MADERA

Member Member

Accepted and approved in partial fulfillment of the requirements for the degree of
BACHELOR OF SCIENCE IN CHEMISTRY.

SHELDON IVES AGATON

Dean
College of Arts and Sciences

ii
 ACCEPTANCE SHEET

This thesis entitled MOLLUSCIDAL EFFICACY OF TAGPO EXTRACT AND

ALKALINE-TREATED TAGPO EXTRACT AGAINST GOLDEN APPLE
SNAILS (Pomacea canaliculata)”, prepared and submitted by SHERRIELYN M.
BERINO, JEAN VALERIE C. CARSON, and LARALIN B. MOLDEZ, in partial
fulfillment of the requirements for the degree of Bachelor of Science in Chemistry, is
hereby accepted.

FE T. PIEDAD, RCh, MAIS

Chair

DARWIN C. GOMEZ, MSc ADORNE MADERA

Member Member

Accepted in partial fulfillment of the requirements for the degree of BACHELOR OF

SCIENCE IN CHEMISTRY.

SHELDON
Dean
College of Arts and Sciences
EVSU, Tacloban City

Date

iii
 TABLE OF CONTENTS

Page
TITLE PAGE …………………………………………………………………….. i

TABLE OF CONTENTS

………………………………………………………... ii

LIST OF TABLES……………………………………………………………….. iv

LIST OF FIGURES……………………………………………………………… v

CHAPTER

I INTRODUCTION

Background of the Study ………………………….............. 3

Statement of the Problem …………………………………. 5

Null Hypothesis …………………………………………... 6

Significance of the Study …………………………………. 6

Scope and Delimitation …………………………….…….. 7

II REVIEW OF RELATED LITERATURE AND

CONCEPTUAL FRAMEWORK

Related Literature ……………………………………..….. 8

iv
 Related Studies ……………………………………………. 10

Conceptual Framework ………………………………..…. 12

Definition of Terms ………………………………………. 16

III METHODOLOGY

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

Laboratory Materials, Equipment and Apparatus ……...…. 18

Collection and Preparation of Plant Sample and

Niclosamide ……………………………………….. 19

Preparation of Stock Solutions and

Different Concentrations of Plant Extract and
Alkaline-Treated Tagpo Extract ………….............. 19

Acclimatization …………………………………… 20

Molluscidal Assay ……...…………………............. 20

Statistical Tool …………………………………….. 22

IV PRESENTATION, ANALYSIS AND

INTERPRETATION OF DATA

Molluscicide Activity of Tagpo Extract

against Golden Apple Snails ………………. 23

Molluscicidal Activity of
Alkaline-Treated Tagpo Extract against
Golden Apple Snails ………………………. 26

Lethal Concentration of Test Substances

against Golden Apple Snails ……………… 30
v
 Comparison Performance of
Tagpo Extract and
Alkaline-Treated Tagpo against
Golden Apple Snails ……………………….. 32

V SUMMARY, CONCLUSION AND RECOMMENDATION

Summary…………………………………………… 36
Findings ……………………………………………. 38
Conclusion ………………………………………… 39
Recommendation …………………………………. 39
BIBLIOGRAPHY ……………………………………………………….. 41

APPENDICES……………………………………………………………. 43

LIST OF TABLES

Table

1 Mortality Rate of
Different Concentration of
Tagpo against Golden Apple Snails…………………………. 23

2 Mortality Rate of
Different Concentration
of Alkaline-Treated Tagpo
Against Golden Apple Snail………………………………… 26

3 Lethal Concentrations of
Tagpo Extract and
Alkaline-Treated Tagpo…………………………………….. 31

3 Comparison of Lethal Concentration of

Tagpo Extract and Alkaline-Treated Tagpo………………… 34

vi
 LIST OF FIGURES

Figure

1 Schematic Diagram showing conceptualization of the study……… 15

2 Dose-Response Curve of
Tagpo Extract against
Golden Apple Snails……………………………………….. 24

3 Dose-Response Curve of the

Alkaline-Treated Tagpo against
Golden Apple Snails………………………………………. 28

4 Effective-Dose Comparison of
Lethal Concentration of Tagpo Extract and
Alkaline-Treated Tagpo……………………………………. 34

vii
viii
 ACKNOWLEDGEMENT

This thesis becomes a reality with the kind support and help of many individuals.

The researchers would like to express their deep appreciation and indebtedness

particularly to the following:

Foremost, the researchers want to offer this endeavor to GOD for letting them

through all the difficulties and for the wisdom He bestowed upon them, the strength,

peace of mind and good health in order to finish this thesis.

The researchers would like to express their gratitude towards their family for the

encouragement, moral, and especially financial support which contributed in the

completion of this paper and for their continuous support and understanding.

The researchers were highly appreciative and indebted to Prof. Darwin C.

Gomez for his guidance, advice and constant supervision as well as for providing

necessary information regarding this study and his support in completing this endeavor.

The researchers would like to express their special gratitude to their adviser, Prof.

Grechelle N. Socias for her guidance, advice and for imparting her knowledge and

experience in this study.

Distinguished members of the panel headed by Prof. Fe Piedad together with

Prof. Adorne Madera for the kind and understanding spirit during presentation and for

the approval of this study.

9
 The researchers expressed their thanks and appreciation to their colleague and

people who have willingly shared their knowledge and experience, making this

undergraduate paper possible.

S. M. B.

J. V. C. C.

L. B. M.

10
BERINO, SHERRIELYN, CARSON, JEAN VALERIE, AND MOLDEZ LARALIN B.,
“MOLLUSCICIDAL EFFICACY OF ALKALINE-TREATED TAGPO AGAINST
GOLDEN APPLE SNAILS (Pomacea canaliculta Lam.)”. Unpublished Bachelor’s
Thesis, Tacloban City: Eastern Visayas State University, June 2022.

Adviser: GRECHELLE N. SOCIAS, RCh

Faculty, Chemistry Department
College of Arts and Sciences

In this study, the tagpo fruit extract and the alkali-treated tagpo was evaluated for

its molluscicidal activity against Pomacea canaliculata Lam. also known as the golden

apple snails. In the alkalie-treated tagpo, a pulverized sample of the tagpo fruit was

treated with NaOH. The preparation of the solution was carried out at alkali solution pH

10, extraction temperature 60°C, extraction time 60 min, and acid precipitation pH 7,

which was based on the study of Yongjun et. al. in 2016. The result for the median (LC50)

and maximal (LC90) lethal concentrations of the tagpo fruit extract were 28.67 and 37.94

ppm respectively. At the same time, in alkaline-treated tagpo extract, the median (LC 50)

and maximal (LC90) lethal concentrations are 27.08 and 33.20 ppm respectively. The

result shows that both the tagpo fruit extract and the alkaline-treated tagpo generated

bioassay data that were dose-dependent. Moreover, in the negative control, zero mortality

rate indicates that the water used and the condition of where the experiment was

conducted did not influence in the mortality of the golden apple snails. The results in the

lethal concentrations shows that the alkaline-treated tagpo has a lower lethal

concentration value compared with the tagpo fruit extract untreated. Furthermore, the

11
comparison of lethal concentration of tagpo extract and the alkaline-treated tagpo have

estimated values of 1.06 and 1.14 ppm for the LC50 and LC90 respectively.

12
 1

CHAPTER I

Introduction

Background of the Study

Schistosomiasis is one of the most common tropical and sub-tropical parasitic

diseases that is caused by parasitic flatworms called schistosomes. Schistosomiasis

mansoni is one of the most crucial but often neglected tropical diseases transmitted by

host snails of the genus Biomphalaria. Around 240 million people are affected worldwide

and it is second most prevalent parasitic disease in the world after malaria. In Africa, 200,

000 deaths are recorded per year due to schistosomiasis and considered as the major

source of morbidity and mortality among the developing country in Caribbean, Africa,

South America, Middle East and Asia. People living in rural or impoverished urban

settings are prevalent to this disease due to limited access to clean water and inadequate

sanitation provision. This water borne parasitic infectious diseases are conciliated by both

ecological and socioeconomic processes and established an important public health

problem in developing countries (de Carvalho Augusto et al., 2020).

Golden apple snail (Pomacea Canaliculata) is an aquatic organism that can easily

found in and around rice paddy fields and widely distributed throughout Southeast Asia

including the Philippines. In year 1982 and 1984, the golden apple snail was introduced

in the Philippines to aid the source of food protein for low-income farmers and as a

source for protein-rich feed livestock. Golden apple snail has not become a major threat

in their native environment. Its physiological adaptability and ability to move a long

distance within a water system and their release in the environment led to heavily damage
 2

on rice farms and rapid multiplication resulted on threatening on rice production and food

security in the country (Joshi et. al 2008).

Numerous measures and strategies have been taken to control P. Canaliculata that

include biological approaches, water management, botanical and various chemical

approach such as the application of synthetic niclosamide (Liang et al., 2014). In the

agricultural farming, the application of synthetic molluscicidal formulations such as

synthetic niclosamide is widely use (Marwoto et al., 2020). The Department of Health

(DOH) recommended the use of niclosamide for the control of the snail and immediately

fast tracked for registration by the Fertilizer and Pesticide Authority (FPA) and later

recommended as molluscicide control for golden apple snail (Dela Cruz et al., 2018). The

invasion of snail may cover large area and the application of synthetic molluscicidal may

had an adverse effect on both human and environment. Synthetic molluscicides are

known for its toxicity and adverse effect on human health and environment. The high

costs of application have driven interest in search for natural plant derived molluscicides

that are toxicologically and environmentally safe.

Botanical pesticides have long been employed as an effective alternative to

chemical-base pesticides correlative due to its low toxicity to non-target organisms, less

expensive in their crude form and preventing the development of resistance against

phytochemical mixtures. Plant components are naturally and environmentally acceptable

because of its non-toxic and less harmful effects (Ravichandran et al., 2017).

In northern Argentina and the Philippines, a new molluscicide based on saponins

produced from the Andean plant Chenopodium quinoa Willd. (quinoa) has been

successfully tested to suppress P. canaliculata snails (San Martin R. et. al., 2008; Joshi
 3

R.C. et. al., 2008). The bidesmosidic saponins naturally found in quinoa husks have been

transformed to more hydrophobic saponin derivatives by an alkaline procedure to

improve their molluscicidal efficacy (San Martin R. et. al., 2008). The snails close their

operculum and stop feeding shortly after the chemical is applied, protecting the immature

rice plants. Under laboratory circumstances, with a high snail density (90 snails/m2), 30

ppm of product (about 10 ppm of saponin) protects 92-95 percent of immature rice

seedlings (Joshi R.C. et. al., 2008).

The combination of alkali solution and acid isolation method is a separation and

purification approach that uses the acid-alkali difference of components in a mixture to

extract proteins, flavonoids, and other compounds (Deng H. et. al., 2010; Sun Y. et. al.,

2014). The extraction rate of the desired product can be improved by using this method.

It also has benefits in terms of investment, ease of operation, pollution, nonsolvent

extraction, and so forth. Saponin from Camellia oleifera cake is a type of triterpenoid

saponin that is made up of a fat-soluble pentacyclic triterpenoid unit and a water-soluble

sugar unit connected by a carbon-oxygen link. A weak alkali solution can entirely

dissolve some acidic saponin components (such as tannin). After that, dropwise additions

of hydrochloric acid to the alkali solution extract are made until the pH value reaches a

desired level. The high polarity acid solution will precipitate the saponin with a

liposoluble pentacyclic triterpenoid unit (Yongjun L. et. al., 2016).

Ardisia japonica has been shown to have four triterpenoid saponins so far.

Researchers have conducted a chemical analysis on the complete plants of Ardisia as part

of an ongoing investigation into medicinal plants in the genus Ardisia and an interest in

the chemistry of bioactive triterpenoid saponins of A. japonica, yielding 21 triterpenoid

 4

saponins, including 11 novel triterpenoid saponins known as ardisianosides A, B (1, 2),

C-F (4-7), and G-K. (15-19) (Jia Z. et. al., 1999; Zheng Q. et. al., 2005). The complete

plants of A. japonica yielded 11 novel triterpenoid saponins, ardisianosides A (1), B (2),

C (4), D (5), E (6), F (7), G (15), H (16), I (17), J (18), and K (19), as well as 10

recognized saponins. Extensive 1D and 2D NMR and MS analyses, as well as chemical

degradations, were used to determine the novel saponins' structures. Saponins 1-21 have

been shown to have cytotoxic activity against three human cancer cell lines: HL-60

myeloid leukemia, KATO-III stomach adenocarcinoma, and A549 lung adenocarcinoma

(Chang X. et. al., 2007).

A previous study of Gomez and Anacta (2020) evidently revealed that the

aqueous extract of tagpo is a potential source of molluscicides. Their study shown that

the crude extract of tagpo fruit is ten times less effective than niclosamide. The ability of

tagpo extracts to decrease the water-escaping tendency among O. hupensis quadrasi

snails relative to that of niclosamide is noteworthy.

Statement of the Problem

This study aimed to determine the molluscicides efficacy of tagpo fruit

endosperm extract and alkaline-treated tagpo fruit extract against golden apple snails.

Specifically, the study aimed to answer the following questions:

1. What is the mortality rate of golden apple snails treated with different

concentrations of tagpo extract?

1.1 13.6 ppm

1.2 22.0 ppm

 5

1.3 26.4 ppm

1.4 30.7 ppm

1.5 35.0 ppm

1.6 40.0 ppm

2. What is the mortality rate of golden apple snails in different concentrations of

alkaline-treated tagpo extract?

2.1 13.3 ppm

2.2 17.6 ppm

2.3 22.0 ppm

2.4 26.4 ppm

2.5 30.7 ppm

2.6 35.0 ppm

3. What are the lethal concentrations in terms of LC 50 and LC90 of the following

molluscicides against golden apple snails?

3.1 Tagpo Fruit extract

3.2 Alkaline-treated Tagpo

4. Is there a significant difference on the lethal concentrations of tagpo extract

and alkaline-treated tagpo?

Significance of the Study

The outcome of this study will be a great benefit to the following:

Farmers. Tagpo extract and alkaline treated tagpo is an effective plant-derive

molluscicides product to eradicate golden apple snail (GAS) on farmlands. Rice farmers

would ultimately benefit from the results of this study.

 6

Department of Health. The adverse effects of synthetic molluscicides on human

and environment is a big problem nowadays. The potential products developed in this

study would help to produce a natural plant derived molluscicides that are toxicologically

and environmentally safe.

Department of Agriculture. The potential products developed in this study

would help to optimize plant derive molluscicides to help control of pests and lower the

cost production of rice.

Researchers. The results of this study may serve as the baseline information for

the future researchers.

Scope and Delimitation of the Study

The objective of this study is to evaluate the molluscicidal activity of the tagpo

fruit extract and the alkaline-treated tagpo fruit extract against golden apple snails. To

attain this objective, the researcher prepared a pulverized sample of the tagpo fruit to be

treated with NaOH. The preparation of the solution was carried out at alkali solution pH

10, extraction temperature 60°C, extraction time 60 min, and acid precipitation pH 7.

This preparation was based on the study of Yongjun et. al. in 2016. This solution was

used to treat 10 snails in each plastic bottle with respective concentrations of 13.3, 17.6,

22.0, 26.4, 30.7, and 35.0 ppm performed in 3 replicates in a simulated basin condition

exposed for 24-hour and with a 24-hour recovery. The non-treated tagpo fruit was

extracted with water stirred for 60 min. and was spin at 4000 rpm and decanted. The

concentrations used for tagpo fruit extract were 13.6, 22.0, 26.4, 30.7, 35.0, and 40.0

ppm. The calculation of concentrations was based on the total solid analysis of Ardisia

aqueous extract previously studied by Socias, where the weight of the dish, dish and
 7

residue, and the residue were identified and evaluated to get the total solids of the sample,

this was done in three replicates. The moisture content of the sample was also evaluated

and was reported to have a mean of 12.15% based on the study done by Socias. The

assessment of mortality in snails is limited to motility criterion. Mortality rates were used

to model the LC50 and LC90 values for each concentration using R statistical software.

The tagpo fruit sample was collected from Sta. Fe, Leyte as well as the snails used

in the experiment. All experiments were performed in Environmental Disaster Research

Laboratory, Eastern Visayas State University, Tacloban City.

 8

CHAPTER II

REVIEW OF RELATED LITERATURE AND STUDIES

This chapter discusses the related literature and studies gathered from journal,

articles, books, and other sources which are crucial for the study conceptualization.

Related Literature

Golden Apple Snails. Pomacea canaliculata are aquatic organisms and a

notorious agricultural pest in the Philippines and other countries in Asia (Mochida 1988,

1991; Naylor 1996). According to the Global Invasive Species Database (GISD), it is

considered as one of the top 100 worst invasive species in the world (Lowe et al., 2000).

P. canaliculata occurs over approximately 50% of the area devoted to the crop, losses in

yield, replanting, and pesticide use cost many millions of dollars annually. The

physiological adaptability and ability to move a long distance within a water system led

to heavily damage on rice farms and rapid multiplication resulted on threatening on rice

production and food security in the country (Joshi et. al., 2008).

Molluscicides are synthetic product designed to control invasive mollusks

including slugs and snails. In 1961, the World Health Organization (WHO) published

guidance on laboratory screening and field efficacy testing of molluscicides and later

recommended its use and other traditional method, such as water treatment, provision of

basic sanitation, chemotherapy and health related education. Later that year, the World

Health Organization (WHO) recommended the use of molluscicides in controlling

intermediate host snails that involved in the lifecycle of schistosomes.

 9

Tagpo or Ardisia species. Ardisia genus of plants that are commonly seen in

tropical and subtropical countries. This species is widely used and famous in China as

medical plants for more than 900 years. The species from the genus are so analogous that

it is difficult to discriminate them just by morphological characteristics. A study of

Ardisia eleptica, a different species of Ardisia, was evaluated for its antibiotic and

antiviral properties. The phytochemical compound contained in A. elliptica leaves are

Bauerenol, and amyrin and Bergenin. Bergenin is an isocoumarin found in various plant

species and exhibits a wide range of biological activites including hepatoprotective,

antifungal, anti-HIV, antiarrhythmic and hypolipidemic (Hideka and Elvira, 2004).

Niclosamide. The only chemical synthetic molluscicide product recommended by

the World Health Organization (WHO) because of its low toxicity impact, high efficiency

and low concerns of pesticide residue. The molluscicidal activity of niclosamide

displayed strong potency against different kind of snails. (He et. al. 2017). Molliscicides

are non-selective against the mollusk species. The activity of molluscicides is dependent

upon proper concentration, method and timing of application, and contact time or length

of exposure and may cause a negative impact to non-target aquatic organisms at use rates

that control mollusks.

Niclosamide is used as an anthelminthic drug for treating parasitic infections in

millions of people worldwide. In 1953, it was discovered in Bayer chemotherapy

laboratories originally developed as a vector control for schistosomiasis. In 1982, it was

approved by the US FDA for use in humans to treat tapeworm infection and later

included in the World Health Organization's list of essential medicines. Mounting

evidence has accumulated and beyond its approved medical use for parasitic disease
 10

treatment, niclosamide has demonstrated preclinical activity in many disease models,

ranging from cancer and metabolic diseases to multiple types of infections. The

underlying mechanisms associated with the drug actions of niclosamide are uncoupling

of oxidative phosphorylation, and modulation of Wnt/β-catenin, mTORC1, STAT3, NF-

κB and Notch signaling pathways.

Related Studies

The contingency on synthetic molluscicides has prompted the large-scale

synthesis of newer chemicals in averting the extensive invasion and infestation of golden

apple snail. Synthetic molluscicides created a value of effectiveness and convenience but

also possessed certain adverse effects including environmental degradation, health

hazards, bioaccumulation and high toxicity to non-target organisms. Therefore, many

studies were conducted to find an alternative way to aid in controlling the widespread

invasion and infestation of golden apple snail.

Achyranthes aspera (A. aspera) is a perineal herb in the family of

Amaranthaceous and was identified as a potential molluscicide. According to the

study, saponins are the principal molluscicidal compounds in the aqueous extract of this

plant and revealed that A. aspera has a concentration-dependent molluscicidal effect

against adult B. pfeifferi snail. The aqueous extract of this plant was used to test its

mollusicidal efficacy on adult snail Biomphalaria pfeifferi and Lymnaea natalensis. The

result of the study showed that the mortality rate of B. pfeifferi is 56.7 percent (17 out of

30) and L. natalensis is 53.3 percent (16 out of 30). The result of this study indicated the

efficacy and considered as molluscicidal agent in controlling snails (Mandefro et al.,

2017).
 11

Another study was conducted on the molluscicidal activity of Persea americana

(avocado) stem bark ethanolic extract against different life cycle stages of the freshwater

snail B. glabrata and exposed to different concentrations for 90 hours. Results indicated

that the LC50 of P. americana extract was lower for embryos (27.06 ppm) and newly-

hatched (30.60 ppm) compared to adult snails (55.55 ppm), demonstrating that the

toxicity of P. americana extract to B. glabrata is dependent on developmental stage.

Interestingly, the P. americana extract LC50 in embryos, newly-hatched and adult B.

glabrata was lower than the values observed for other plant extracts and derivatives

(Silva et. al., 2020).

In another study, the potential molluscicidal activity of Camellia oleifera seed

meal crude methanol extract was assessed against P. canaliculata within 24 hours. In that

study, 12.5 kg/ha were used in the experiment and resulted a 100% mortality rate within

24 hours and was recorded at the lowest concentration of oil seed camellia meal. This

result confirms the use of oil-seed camellia meal to control golden apple snail. In the year

2000, a new compound has been found in C. oleifera (tea-oil) tree by alkaline hydrolysis

and purification of the saponins extracted from the tea seed pomace, termed seed distilled

saponin (TDS). The technical materials of TDS were active against O. hupensis snails. In

2007, it was listed as a plant-derive molluscicide and registered as “Lou-Wei” as its

common name which means “snail threatener” by the authority of Minister of Agriculture

(MoA) of People’s Republic of China. The results of the study evidently demonstrate that

4% TDS is active against O. hupensis, B. alexandrina and B. truncates.

Massaguni and Latip (2015) conducted a study on the molluscicidal activity of

neem crude extract against the golden apple snail. Two parts of neem plant were used, the
 12

neem seeds and leaves with combined application of required concentrations rates of

0.75. 1.50, 2.25 percent and were carried out on two different sizes of golden apple snail.

The LC50 values of aqueous neem leaves extract after 96 hours exposure period resulted

to 2.34 times and 2.10 times more toxic than the aqueous neem seed extract for small and

large sizes of golden apple snails, respectively. In addition, the aqueous neem leaves

extract resulted to high snail mortality with 93.33% and 84.17% of small and large size of

golden apple snail compared to aqueous neem seed extract with 71.67% and 73.33%

respectively. The results that carried out through their entire study reflects that both plant

parts possessed potential in averting golden apple snail but a trace of effectiveness of

aqueous neem leaves extract was expressed as a potential molluscicides.

Null Hypothesis

There is no significant difference in median and maximal lethal concentration of

tagpo extract and alkaline-treated tagpo extract.

Conceptual Framework
Control of the environment host snails play a crucial role in preventing the

widespread invasion of schistosomiasis. In reference of impact, this disease is second

most prevalent parasitic disease in the world after malaria. According to the World

Health Organization (WHO 2010), almost 200 million people worldwide and 700 million

people who live in endemic areas were affected by schistosomiasis. In this case, the

World Health Organization administered strategies in controlling and eliminating

schistosomiasis by developing antiparasitic praziquantel. However, the development of

praziquantel does not hold full measure effects in most of the juvenile schistosomes,

since it only deters sphingomyelinase activity that will lessen the persistent extricate of
 13

schistosome eggs. Administering and averting morbidity in subjects using praziquantel

has not been entirely successful of confine transmission in high-risk areas as there have

been recent reports of schistosomes resistance.

The World Health Organization proposed the use of molluscicide (niclosamide) to

control the transference of schistosomiasis. The use of niclosamide is the only approved

and recommended molluscicides by the WHO in controlling intermediate host snails that

involved in the life cycle of schistosomes. The effects of niclosamide were visible in

killing the vector host of schistosomes. However, it leaves trace of adverse effects to the

non-target organisms.

Currently, the trend of testing new possible molluscicide in administering the

invasion and infestation of host snails brought a rapid increased of recognition by testing

the biological toxicity of plant extract as an alternative to chemical base molluscicides.

The existing studies of different plant extract as molluscicide had been stimulated issues

on environmental preservation, high cost and recurrent resistance of snails against

synthetic molluscicides. A series of analysis were accumulated from various researchers

on evaluation of molluscicide activity of various plants and showed an evidently

promising result. Phytochemicals compounds that can be found in different varieties of

plants that could kill specific mollusk were saponins, alkaloids, flavonoids, disterpenoids,

monoterpenoid, sesquiterpenses lactones and tannins (Singh et. al,.2009).

Ardisia tomentosa commonly known as Tagpo was tested for molluscicidal

efficacy and evidently displayed a potent molluscicidal activity against the golden apple

snails. In this study, the researchers aim to administered new product that asses the

molluscicidal efficacy of the tagpo extract and alkaline treated tagpo formulation against
 14

golden apple snail. The entire process of tagpo extract and alkaline treated tagpo

formulation to the scope of assay development including the evaluation of the mortality

of golden apple snail is presented in the schema of the conceptualization of the study in

Figure 1.

Definition of Terms

The following are the technical terms used in the study that are hereby defined

conceptually and how they were used:

Acclimatization. The process of becoming adjusted to a new environment that

promotes reproducible experiment result (UCI, 2020). In this study, it refers to the

process of stabilizing the test subject in a new environment by putting them in two

separate plastic basins with dechlorinated water.

Alkaline. The measures of how acidic or alkaline a substance at a certain pH

level. In this study, it refers to a developing substance in combination of tagpo extract to

formulate a possible molluscicide.

Concentration. It refers to the relative amount of substance or solute in each

given amount of solvent or solution. In this study, it refers the amount of tagpo extract

and alkaline-treated tagpo formulation in a defined amount of distilled water.

Maximal Lethal Concentration (LC90). Concentration of stimulus or active

component that kills 90 percent of the organisms in an experiment. In this study, it

pertains to concentration modeled to induce 90 percent snail mortality in a given period

of time.
 15

Median Lethal Concentration (LC50). A concentration of substance that will kill

50 percent of the test population and other 50 percent will still be alive. In this study, it

refers to the concentration to develop 50 percent snail mortality in a given period of time.
 16

Figure 1. Schematic Diagram showing conceptualization of the stud

Molluscicides. A chemical substance primarily to kill intermediate host snails

that involved in the cycle of schistosomes (WHO, 1992). In this study, it refers to the

property of tagpo and alkaline-treated tagpo formulation that are able to kill golden apple

snails.

Mollusk. An invertebrate animal with a soft body having a calcareous shell

(Claudi et. al.,1994). In the study, it refers to the golden apple snail as a test subject of

this experiment.

Mortality. The relative frequency of deaths in specific population that transpire in

a given period of time (Merriam-Webster). In this study, it refers to the number of deaths

of golden apple snail.

Replicate. It refers to multiple experimental runs used to characterize the

associated error of the mean value. In this study, it refers to the number of uniform set-

ups at various concentration of the tagpo extract and alkaline treated tagpo formulation.

Tagpo Extract. A substance from Ardisia that exerted phytochemicals such as

flavonoids polyphenols, phenolics, triterpenoid saponins, quinones and alkylphenols

(Joaquin-Cruz et. al.,). In this study, it refers to the substance that is used to formulate a

potential plant derived molluscicide.

 17

Operculum. It refers to corneous or horny operculum on the foot to seal off the

aperture when snails’ foot is drawn out into the shell. In this study, it refers the protective

materials of the snail to resist any activity that will disturb them.
 18

CHAPTER III

METHODOLOGY

This chapter presents the research design; the list of materials, equipment and

laboratory instruments; process for collecting and preparing extracts from tagpo and

niclosamide; preparation of alkaline treated tagpo extract test solutions; the

acclimatization of snails and statistical analysis

Research Design

This study used experimental method of research to test Tagpo Fruit Extract and

Alkaline-Treated Tagpo Fruit extract used as molluscicides against golden apple snails.

The experiment was done in 3 replicates paired with negative control in each replicate.

The objectives are met by employing a laboratory assay to assess the maximal lethal

concentration (LC50) and (LC90). To draw findings, this study used dose-response data. At

least six concentrations of each of the test chemicals are employed in the investigation,

and molluscicidal assays are done in two separate trials.

Laboratory Materials, Apparatus, and Equipment

The following list of laboratory materials, apparatus, and equipment that are used

in the study:

Analytical Balance Funnel Niclosamide powder

Aspirator Gloves Paper Clip

Basins Hot plate Pipette

Beaker Incubator Rubber Bands

Centrifuge tubes Lab Gown Scissors

 19

Cling wrap Magnetic Stirrer 1.5 Plastic bottles

Dropper Masking Tape Tissue Paper

Face mask Micropipette Tong

Filter Mortar and pestle Volumetric flask

Freezer Mosquito net Wash Bottle

Collection and Preparation of Tagpo and Niclosamide

Mature tagpo fruits were harvested locally from natural habitat located in Sta. Fe,

Leyte (11° 11`52” N, 124° 56`29” E, 43 m asl). At the Analytical Laboratory,

Department of Chemistry, Eastern Visayas State University, Tacloban City, fruit samples

were washed thoroughly using running water and rinsed with distilled water. The tagpo

powder were obtained by incubating at 60 °C for 24 hr. The endosperm is separated from

the shell and pulverized with a mortar and pestle. The powdered fruit samples were

stored in the refrigerator at room temperature until use.

Niclosamide is purchased domestically as a 70% wettable powder (Niclos M ,

70WP, Leads Agricultural Products Corp.)

Preparation of Tagpo Extract, Alkaline-Treated Tagpo Extract and Niclosamide

Stock Solutions

Tagpo stock solution was prepared by weighing 1.5064 grams on an Radwag AS

310.X2 analytical balance and mixed with 150.0 mL of distilled water. The mixture was

then put in a beaker and spun with magnetic stirrer for one hour. The mixture was set

aside for 30 minutes to allow any residue to settle to the bottom. The outer layer was

decanted into a 15 ml centrifuge tube and then centrifuged at 4000 rpm (Labnet, USA).

The centrifugate was decanted and stored in 50 mL centrifuge tubes.

 20

The alkali-treated Tagpo extract solution was prepared by weighing 1.5065 grams

on a Radwag AS 310.X2 analytical balance and mixing with 150.0 mL of distilled water.

The mixture was put in a beaker and spun with a magnetic stirrer for one hour. For the

Alkaline solution: 2 M sodium hydroxide was added dropwise to the prepared Tagpo

extract to reach a specified pH of 10, followed by centrifugal separation to remove solid

impurities. For Acid isolation, Hydrochloric acid (20% by volume) was added dropwise

to

centrifuge solution to reach a specified pH of 7.

Acclimatization of Snails

Approximately 1,200 snail samples were collected at Sta. Fe, Leyte (11°11’52” N,

124°56’29” E, 43 m altitude). Snails were transported to the laboratory in clean plastic

bucket half-filled with ground water. The snails were then acclimatized in the laboratory

for two days in 3 clean large plastic basins with half-filled dechlorinated water, each

containing 400 snails at room temperature. Snails were fed with kamote tops (Ipomea

batatas) ad libitum into each bucket every 24 hours and water was change in every 12

hours.

Molluscicidal Assay

Molluscicide testing was conducted on the third floor of SB 329 at Eastern Visayas State

University, Tacloban City. The test was carried out in 1.5-liter plastic bottles. Thirty-six

of the 60 bottles were used for the test substance (Tagpo extract and alkali-treated Tagpo

extract), while twelve plastic containers were used as a positive control (niclosamide) and

the remaining plastic bottles were used as negative controls (only dechlorinated water). In

this study, six concentrations of each of the tagpo extract and alkali-treated tagpo extract
 21

test substances were used, and the selection of the test concentrations is based on the

previous report by Gomez and Anacta (2020) and follows the methods proposed by

Yongjun Liu., et al. Concentrations used in concentration setup 1 and 2: 13.6, 22.0, 26.4,

30.7, 35.0, 40.0 (tagpo extract); 13.3, 17.6, 22.0, 26.4, 30.7, 35.0 (alkaline-treated Tagpo

Extract). The lowest concentrations, 13.6 and 13.3, respectively, were expected to result

in zero or less than 10% mortality, while the other two highest concentrations, 35.0, 40.0;

307, 35.0, have predicted slug mortality rates between ninety and one hundred percent.

To perform a test substance molluscicide assay, 36 plastic bottles were prepared and a

defined volume (500 mL) of dechlorinated water was added to each container. The

collected snails were randomly selected and assigned to multiple replicates. Ten snails

were added and randomly distributed in each clear plastic container containing

dechlorinated water. Added a desired number of concentrations, 13.6, 22.0, 26.4, 30.7,

35.0, 40.0 (tagpo extract); 13.3, 17.6, 22.0, 26.4, 30.7, 35.0 (alkaline-treated Tagpo

Extract). This means that the first setup of three replicates of plastic containers containing

10 snails has 13.6 ppm, as well in the second setup, and so on until the highest

concentration.

After 24 hours of exposure, plastic containers containing golden apple snails at a

specific concentration were checked. Dead snails were noted and confirmed with no

reaction to pin pricks, open operculum, pale colored foot. When the snails responded to

the toothpick and the operculum was closed, the snails were transferred to a plastic

container with 500 mL of dechlorinated water and recovered for an additional 24 hours.

The snails were checked again to record the final mortality rate.
 22

Statistical Analysis

All statistical analysis in this study were performed in R (The R core Team, 2017;

Ritz et al./ 2015, from dose response and for maximal lethal concentration (LC 50) and

(LC90).
 23

CHAPTER IV

PRESENTATION, ANALYSIS AND INTERPRETATION OF DATA

This chapter presents the results in terms of mortality rates of P. canaliculata

(Golden Apple Snail) and lethal concentrations of tagpo extract and alkaline-treated

tagpo. This chapter also presents the analysis, interpretation, and implication of research

findings.

Molluscicide Activity of Tagpo Extract against Golden Apple Snails

This study aimed to determine the mortality rate of golden apple snails exposed to

different concentrations of tagpo extract and the observation of the general behavior of

the golden apple snails upon the administration of tagpo extract. The dose-response curve

for tagpo extract against golden apple snails are presented in Figure 1.

TABLE 1

MORTALITY RATE OF DIFFERENT CONCENTRATION OF TAGPO

AGAINST GOLDEN APPLE SNAILS

Replicatesa Mean
Concentration Negative
Mortality
(ppm) R1 R2 R3 Controlb
Ratec (%)
17.6 10 0 10 0 6.7
22.0 0 0 20 0 6.7
26.4 0 30 40 0 23.3
30.7 70 40 70 0 60.0
35.0 80 100 90 0 90.0
39.3 100 100 90 0 96.7
a
10 snails per replicate in 500 mL test solution
b
10 snails in dechlorinated water, 500 mL
c
N = 30 per concentration; assessed after 48 hours

The mean mortality rate of golden apple snails is found in Table 1 which

extrapolates the effects of concentrations used on golden apple snails, each replicates
 24

contains 10 snails in 500 mL test solution. It was determined that when golden apple

snails were exposed to a concentration of 17.6, 22.0, 26.4, 30.7, 35.0, and 39.3 ppm of

tagpo extract, it resulted in 6.7, 6.7, 23.3, 60.0, 90.0, and 96.7 percent mean mortality

rate, respectively. In addition, shows zero mortality rate in the negative control which

explains that the water used in conducting the experiment shows no effect on the

mortality rate of the golden apple snails. The snail mortality rates varied dependently on

the concentration of tagpo extract, taking into account that the snail mortality increases as

the concentration increases. The snail mortality is concentration-dependent by looking at

the dose-response curve of tagpo extract against golden apple snails. These results are

depicted visually in Figure 1, where the data collected in experiments are drawn as solid

red diamonds and the modelled curve as solid black line. In terms of the behavioral

changes, the snails after 24-hour exposure to tagpo extract became weak, and those

considered dead exhibited discolored foot muscles. Dead snails were unreactive to any

external stimuli, and some had an open opercula.

LC50 = 28.67 ± 0.77a ppm

LC90 = 37.94 ± 1.78a ppm

Figure 2 Dose-Response Curve of Tagpo Extract against Golden Apple Snails.

a
Standard Error.
 25

In the assessment of the experiment, the snails were observed to have retracted

from sticking on the surface of the plastic bottle the moment the concentration was added

to the water, although most of the snails were still active after sinking to the bottom of the

plastic bottle. The snails exposed to 30.7, 35.0, 39.3 ppm concentrations of tagpo extract

showed a weak attachment on the wall and most of the snails withdrew into their shell,

closing the operculum while some of the snails were still thriving to go above water. This

behavior shows that the tagpo extract had an effect on snails, and after 24-hour exposure,

it was observed that tagpo extract could kill 7, 20, and 19 snails in total, exposed to

concentrations of 30.7, 35.0, 39.3 ppm of tagpo extract respectively.

To validate if the tagpo extract had effect on snails with tightly closed opercula,

another 24-hour recovery was conducted. It was observed that after another 24-hour

recovery stage, snails exposed to 35.0 and 39.3 ppm tagpo extract emitted a foul smell

and the water showed obvious discoloration compared to the water with snails that were

treated with only 17.6, 22.0, 26.4, and 30.7 ppm of tagpo extract. The final mortality

assessment after another 24-hour recovery resulted to a total of 60.0, 90.0, and 96.6

percent mean mortality. This further support the molluscicidal activity of tagpo extract

against golden apple snails. Studies on molluscicides discovered that plants with

phytochemical constituents; saponins, alkaloids, flavonoids, diterpenoids,

monoterpenoid, sesquiterpenes lactones, and tannins are effective in killing mollusks

(Singh et. al., 2009). Ardisia species have been reported to have active ingredients such

as flavonoids, saponins, tannins, and other secondary metabolites (Hussein and El-

Anssary,2018).
 26

In this study the application of tagpo extract as a molluscicide was based on the

gathered related studies that prove that some Ardisia species shows a molluscicidal

activity. The tagpo extract used in conducting this study was not examined and was only

an attempt to provide more data supporting the reports of Ardisia species’ molluscicidal

activity on golden apple snails.

The result shows that tagpo extract at certain concentrations can kill golden apple

snails and as shown in the dose-response curve, the mortality rate of the golden apple

snails is concentration-dependent.

Molluscicidal Activity of Alkaline-Treated Tagpo Extract against

Golden Apple Snails

This study also aimed to assess the mortality rate of golden apple snail exposed to

an alkaline-treated tagpo and the observation of the general behavior of golden apple

snails upon the administration of the alkaline-treated tagpo solution. The results are

presented in Table 2.

TABLE 2
MORTALITY RATE OF DIFFERENT CONCENTRATION OF
ALKALINE-TREATED TAGPO AGAINST GOLDEN APPLE SNAIL

Mean
Concentration Replicatesa Negative
Mortality
(ppm) Controlb
Ratec (%)
R1 R2 R3
13.3 0 0 0 0 0
17.6 0 10 0 0 3.3
22.0 0 10 10 0 6.7
26.4 10 70 40 0 40.0
30.7 70 80 100 0 83.3
35.0 90 90 100 0 93.3
a
10 snails per replicate in 500 mL test solution
b
10 snails in dechlorinated water, 500 mL
d
N = 30 per concentration; assessed after 48 hours
 27

It can be observed that when golden apple snails were exposed to 13.3, 17.6, 22.0,

26.4, 30.7, and 35.0 ppm concentration of the alkaline-treated tagpo solution, there were

0, 3.3, 6.7, 40.0, 83.3, and 93.3 percent mean mortality rate, respectively. The highest

concentration of the alkaline-treated tagpo achieved a 93.3 percent mean mortality rate at

35 ppm. Snails exposed to sublethal concentrations showed less to zero mortality rate.

Snails treated with 13.3 ppm concentration of the solution were still active and shows no

signs of mortality. Concentrations 17.6 and 22.0 ppm of solution shows less mortality

rate, still 10 snails in replicate 2 and 3 were affected with the solution, snails were not

able to retract back inside the shell and were unreactive, these snails were recorded as

dead. Based on the recorded mean mortality, the effectiveness of the alkaline-treated

tagpo against the golden apple snails can kill more than 90 percent of the snails at 35.0

ppm concentration. Moreover, the snail mortality rate in negative control indicates that

the mortality rate of golden apple snails varies dependently on the different

concentrations of alkaline-treated tagpo solution. The dose-response curve in Figure 2

further explains the relationship between the different concentrations of alkaline-treated

tagpo and the mortality rate of golden apple snails. Increasing level of alkaline-treated

tagpo concentration also increases the rate of mortality of the golden apple snails.
 28

LC50 27.08 ± 0.61a ppm

LC90 33.20 ± 1.24a ppm

Figure 3 Dose-Response Curve of the Alkaline-Treated Tagpo against Golden Apple

Snails. a Standard Error.

In the assessment of mortality in the case of the application of the alkaline-tagpo

extract, the snails that was exposed to sublethal concentration, was observed that the

snails were more active and tend to leave from the water treated with the solution. This

behavior is an effect that is associated to the water leaving tendency of the snail exposed

to sublethal concentration of the alkaline-treated tagpo. This reasoning was used by

Gomez and Anacta (2020) to explain their study’s findings based on the schistsomiasis

snails. In their study, they argued that the schistosomiasis snails react to molluscicidal

compounds by rapidly escaping from the solution, climbing out of the testing containers.

Also, this effect of snails to leave water upon application of the solution further explains

in Figure 2 and can be seen that, as the concentration of the solution decreases, so as the

mortality rate of the golden apple snails. The case was different at higher concentrations,

the snails at higher concentrations shows weak to slow behavior and some had retracted

back into the shell, while other snails was floating and shows no sign of activity. Snails
 29

exposed to 26.4, 30.7 and 35.0 ppm concentrations of the solution have shown an inactive

behavior after several hours. This behavior is an indication that as the alkaline-treated

tagpo solution increases in concentration, so as the efficacy of the solution to exhibit an

immediate effect to the golden apple snails causing a morbid behavior after. The snail

mortality after 24-hour exposure to each concentration of the solution are 0, 0, 1, 5, 17,

and 16 snails respectively. The total mortality rate was recorded after the recovery stage,

that is 93.3 percent mortality rate, which demonstrate the potency of the alkaline-treated

tagpo solution penetrating into the snails and resulted in snails’ mortality. The

discrepancy in mortality rate of golden apple snails between the solution of the tagpo

treated with alkali and the tagpo extract in terms of concentration at 35.0 ppm explains

the effectiveness of the alkaline-treated tagpo by a small amount. This can be the result of

the change in composition the moment the tagpo was treated with alkali, as the

experiment was conducted, the color change of the alkaline-treated tagpo was evident

that there has been a chemical change occurring. Studies in extraction of saponin using a

combination method of Alkali solution and acid isolation by Yongjun Liu et. al., has led

this research to attempt the same technique considering the average of saponin extraction

rate resulted in 76.12 percent. This technique has been reported to be optimal for

separation and purification making use of the different components in a mixture and

found to be effective in extracting the proteins, flavonoids, and other substances (Deng H.

et. al., 2010; Sun Y. et. al., 2014). Saponins were discovered to have molluscicidal

activity and a bioassay data was published that all of the isolated saponins was evident in

killing golden apple snails (Hostettmann K. et. al., 1982; Maraton A. et. al., 1985;

Mochida O. et. al., 1991; Naylor R. et. al., 1996; Lunn K. et. al., 1989; Cowie R. H. et.
 30

al., 2002; Albrecht E. A. et. al., 1996; Halwart M. et. al., 1994; Calumpang S. M. F. et.

al., 1995). In this study the pH value of the alkali solution and the acid isolation was fixed

at 10 and 7 respectively, thus resulted to at most 93.3 percent mortality rate. The tagpo

extract had been investigated to have contain saponin which is reported to have exhibited

molluscicidal activity against golden apple snails (Huang et al., 2003). Which led this

study to undertake extraction of saponins using alkali solution and acid isolation method

expecting to have a result that can compete with the efficacy of the tagpo extract. There

are many factors that can influence the extraction yield of saponins, including the

extraction temperature, the alkali solution pH, and the acid isolation pH. The technique

used in this study did not have much effect on the mortality rate of the golden apple

snails compared to the tagpo extract, this infer that the extraction process utilized in this

experiment did little effect on maximizing the yield of saponin which is the limitation of

this study.

Based on the result, treating tagpo with alkali could increase the mortality rate by

3.3 percent. This indicates that treating tagpo with alkali had somehow change the

composition and perhaps isolated that main component saponin. However, the difference

in effect is not far from the results in tagpo extract which limit the impact of the

technique if compared to the pure tagpo extract. It is highly recommended that the

heating, pH of the alkali solution and acid isolation should be modified to optimize the

molluscicidal efficacy of tagpo powder against the golden apple snails.

Lethal Concentration of Test Substances against Golden Apple Snails

Lethal concentrations of test substances were calculated using the R software

for statistical computing. The aim of this study is to obtain a constructed result from the
 31

data records throughout the experiment. This study presented only two data, for the

Tagpo Extract and for the Alkaline-treated extract. The lethal concentrations for the two

concentrations are presented in Table 3.

Table 3

LETHAL CONCENTRATIONS OF
TAGPO EXTRACT AND ALKALINE-TREATED TAGPO

Value Confidence Interval*

Type Molluscicide SE
(ppm) Upper Lower
LC50 Tagpo Extract 28.67 0.77 30.17 27.17
Alkaline-Treated Tagpo 27.08 0.61 28.28 25.88

LC90 Tagpo Extract 37.94 1.78 41.42 34.45

Alkaline-Treated Tagpo 33.20 1.24 35.63 30.76
SE – standard error
*evaluated at 95 percent confidence level

In tagpo extract the median (LC50) and maximal (LC90) lethal concentrations

are 28.67 and 37.94 respectively. In the alkaline-treated tagpo extract the median (LC 50)

and maximal (LC90) lethal concentrations are 27.08 and 33.20 respectively. It can be

interpreted that comparing the results for the LC 50 and LC90 the tagpo extract performs

less molluscicidal activity on golden apple snails than the alkaline-treated tagpo. As

shown in Table 3, the tagpo extract exhibited a higher value for LC 50 and LC90 which

imply that the higher the value of the lethal concentration, the lower its effects on the

golden apple snails at LC50 and LC90, in this case, the concentration of the tagpo extract

should be increase to obtain a higher mortality rate. On the other hand, in the alkaline-

treated tagpo, the median and maximal lethal concentrations are lower than the tagpo

extract, which imply that the alkaline-treated tagpo is more potent at that level of lethal

concentrations compared to the tagpo extract.

 32

The results reveal the difference in terms of lethal concentrations between the

two solutions, and it was revealed that alkaline-treated tagpo has a lower lethal

concentration value when compared with the tagpo extract. A study of Socias et. al.,

reported that the median lethal concentration of tagpo extract is 26.99 ± 0.71 mg L -1,

which is consistent with the result of the alkaline-treated tagpo obtained in this study.

This report shows that the alkaline-treated tagpo have a molluscicide activity against

golden apple snails, but reveal that it is not superior than the tagpo. The result of the

alkaline-treated tagpo, though appear to have a higher efficacy, it was not high enough to

be deemed better than the pure tagpo extract considering the preparation done to obtain

the solution.

The result of the lethal concentrations of the alkaline-treated tagpo explains

its efficacy as a molluscicide against golden apple snails. The efficacy of the solution

may have to do with the preparation of the solution. For future researchers, it is highly

recommended that the preparation should be modified according to the extraction

temperature, pH of alkali solution, and acid isolation pH to obtain a more optimal result.

Additionally, to investigate more on the efficacy of the alkaline-treated tagpo, it is

recommended that a further study or research on this type of method extraction should be

conducted, to compare the results based on the different ways of isolating saponin from

the tagpo powder.

Comparison Performance of Tagpo Extract and Alkaline-Treated Tagpo against

Golden Apple Snails

The aim of this study is to further compare the performance of Tagpo Extract

and Alkaline-treated tagpo against Golden Apple Snails. Table 4 displays the comparison
 33

of the LC50 and LC90 of both substances and the estimated value and confidence limit

evaluated at 95 percent confidence level.

TABLE 4

COMPARISON OF LETHAL CONCENTRATION OF TAGPO EXTRACT AND

ALKALINE-TREATED TAGPO

Lethal Confidence Limits*

Comparison of
Concentration Estimated Lower Upper Interpretation
LC50 and LC90
Ratios Limit Limit
Not
TE/ATTE: 50/50 28.67/27.08 1.06 0.99 1.13
significant
TE/ATTE: 90/90 37.94/33.20 1.14 1.01 1.28 Significant
*evaluated at 95 percent confidence level

The statistical computed values are 1.06 and 1.14 ppm corresponding for the

comparison of LC50 and LC90. In Table 4, the comparison for the LC50 values is

interpreted as not significant and for the LC90 values significant. This means that in terms

of median lethal concentration there was no significant difference in mortality rate of

golden apple snails both in tagpo extract and alkaline-treated tagpo. It was only in the

maximal lethal concentration did the mortality rate vary considering the LC 90 value of

tagpo extract is higher than the alkaline-treated tagpo. This difference in mortality rate in

LC90 is considered significant. Figure 3 further explains the comparison of the lethal

concentration of tagpo extract and the alkaline-treated tagpo. In Figure 3 the comparison

of dose-responses for both substances represent the relationship of the concentration and

the mortality rate. The black curve displays the dose-response curve of the alkaline-

treated tagpo, while the red displays the tagpo extract. By looking at the figure, it can be
 34

concluded that there is a difference of dose-responses. There may be an overlap at the

sublethal concentration showing almost same responses to the doses but as the

concentration gets higher the responses to both substances becomes evidently different.

red, TEa
black, ATTEb

Figure 4 Effective-Dose Comparison of Lethal Concentration of Tagpo Extract and

Alkaline-Treated Tagpo. aTagpo Extract. bAlkaline-Treated Tagpo.

The data revealed that the value in the alkaline-treated tagpo is lower in the

maximal lethal concentration than in the tagpo extract, which means that 90 percent of

snail population immediately felt the effect of alkaline-treated tagpo solution at lower

concentration compared to tagpo. While for the median lethal concentration, the

confidence limit for both the tagpo extract and alkaline-treated extract overlap, showing

no significant difference with each other.

 35

Hence, the null hypothesis stating that there is no significant difference in mean

mortality rate of tagpo extract and alkaline-treated tagpo was accepted in LC 50 and

rejected in LC90.
 36

CHAPTER V
SUMMARY, CONCLUSION AND RECOMMENDATIONS
This chapter discusses the summary, findings, conclusion drawn from the study
findings and recommendations offered.

SUMMARY

This study assessed the molluscicide activity of tagpo extract and alkaline-treated
tagpo extract against golden apple snails.
Specifically, this study sought answers to the following questions:
1. What is the mortality rate of golden apple snails in different concentrations of

tagpo extract?

1.1 13.6 ppm

1.2 22.0 ppm

1.3 26.4 ppm

1.4 30.7 ppm

1.5 35.0 ppm

1.6 40.0 ppm

2. What is the mortality rate of golden apple snails in different concentrations of

alkaline-treated tagpo extract?

2.1 13.3 ppm

2.2 17.6 ppm

2.3 22.0 ppm

2.4 26.4 ppm

2.5 30.7 ppm

2.6 35.0 ppm

 37

3. What are the lethal concentrations in terms of LC 50 and LC90 of the following

molluscicides against golden apple snails?

a. Tagpo

b. Alkaline-Treated Tagpo

4. Is there a significant difference on the lethal concentrations of tagpo extract and

alkaline-treated tagpo?

In this study, the molluscicidal properties were determined using different

concentrations of tagpo extract and alkaline-treated tagpo extract against golden apple

snail. The tagpo fruits were collected in Sta. Fe, Leyte, washed, incubated at 60°C for 24

hours, pulverized, and made into a solution.

Snails collected from Sta. Fe, Leyte was washed using running water and

acclimatized in dechlorinated water for 2 days. 10 snails were chosen per replicate and

were tested using 1.5 plastic bottles with 500 mL of dechlorinated water and was treated

with tagpo extract and alkaline-treated tagpo at different concentrations. After 24 hours

of exposure, snails were gently checked and assessed if they were dead. A dead snail is

unreactive when poke with a toothpick or pin, operculum is open, and has a pale muscle

foot. When the snails responded to the toothpick and retracted back into the shell, the

snails were then transferred to a plastic container with 500 mL of dechlorinated water and

recovered for an additional 24 hours. The snails were checked again and the final

mortality rate was recorded.

 38

FINDINGS

The Molluscicidal activities, lethal concentrations, and comparison performance

obtained from the evaluation of tagpo extract and the alkaline-treated tagpo against

golden apple snails are revealed in the following:

The mean mortality rates of golden apple snails treated with 17.6, 22.0, 26.4,

30.7, 35.0, and 39.3 ppm concentration of tagpo extracts are 6.7, 6.7, 23.3, 60.0, 90.0, and

96.7 percent mean mortality rate respectively. Whereas the mean mortality rates of

golden apple snails applied with 13.3, 17.6, 22.0, 26.4, 30.7, and 35.0 ppm concentration

of the alkaline-treated tagpo solution are 0, 3.3, 6.7, 40.0, 83.3, and 93.3 percent mean

mortality rate respectively.

For the lethal concentrations, in tagpo extract, the median (LC 50) and maximal

(LC90) lethal concentrations are 28.67 and 37.94 ppm respectively. At the same time, in

alkaline-treated tagpo extract, the median (LC 50) and maximal (LC90) lethal

concentrations are 27.08 and 33.20 ppm respectively. It was also observed in the

comparison of LC50 and LC90 of the tagpo extract and alkaline-treated tagpo that LC 90 is

not significant based on the statistical computation while the LC 50 shows significant

results getting an estimated value of 1.11 for both substances and (0.99, 1.26) lower and

upper limit for the confidence interval. The observed in the comparison of LC 50 and LC90

of the tagpo extract and alkaline-treated tagpo accepted the null hypothesis in LC 90 and

rejected the null hypothesis in LC50.

 39

CONCLUSION

On the basis of the foregoing findings, the following conclusions were drawn:

1. The mortality rates of golden apple snails treated with tagpo extract are

concentration dependent.

2. The mortality rates of golden apple snail applied with alkaline-treated tagpo

extract are concentration dependent.

3. The median (LC50) and maximal (LC90) lethal concentrations of tagpo extract are

28.67 and 37.94. The median (LC50) and maximal (LC90) lethal concentrations of

tagpo extract are 27.08 and 33.20.

4. There is a significant difference on the LC 90 of both the tagpo extract and the

alkaline-treated tagpo. There is no significant difference on the LC 50 of both the

tagpo extract and the alkaline-treated extract.

RECOMMENDATION

Based on the findings and conclusions of the study, the researcher hereby

recommends the following:

1. For future researchers, it is highly recommended that the preparation should be

modified according to the extraction temperature, pH of alkali solution, and acid

isolation pH to obtain a more optimal result.

2. To generate a more uniform and accurate bioassay, the size of the golden apple

snail should be sorted for standard sizes. The size of golden apple snail should be

measured according to its shell height which could provide convenience in

selecting snails of uniform size.

 40

3. To investigate more on the efficacy of the alkaline-treated tagpo, it is

recommended that a further study or research on this type of method extraction

should be conducted, to compare the results based on the different ways of

isolating saponin from the tagpo powder.

4. For future researcher, this technique of applying alkali to tagpo extract is

recommended for testing other organisms.

 41

BIBLIOGRAPHY
 42

JOURNALS AND PERIODICALS

Adalla, C.B., and Rejesus B.M., 1989. The Golden Apple Snail, Pomacea sp., a serious

pest of the lowland rice in the Philippines. In: Henderson I, editor. Slugs and

Snails in World Agriculture. British Crop Protection Council Monograph No. 41.

Thornton Heath: BCPC. p 417 -427.

Albrecht, E. A. et. al. A quantitative study of copulation and spawning in the South

American apple-snail, Pomacea canaliculata (Prosobranchia: Ampullariidae).

Veliger 1996, 39, 142-147.

Benchawattananon, R., and Boonkong, U., 2006. The toxicity of leave crude extract from

neem tree (Azadirachta indica Juss.) and Garlic (Allium sativom L.) on mortality

rate of golden apple snail (Pomacea sp.), 32nd Congress on Science and

Technology of Thailand. Queen Sirikit National Convention Center, Bangkok

Calumpang, S. et. al. Environmental impact of two molluscicidal: niclosamide and

metaldehyde in a rice paddy eco

Cowie, R. H. Apple snails (Ampullariidae) as agricultural pests: Their biology, impacts,

and management. Molluscs as Crop Pests; CABI Publishing: Oxfordshire, UK,

2002; pp 145- 192.

Dela Cruz, M.S., and Joshi, R.C., 2001. Efficacy of commercial molluscicide

formulations against the golden apple snail Pomacea canaliculata (Lamarck). In:

The Philippine Agricultural Scientist Vol. 84 No. 1 (Jan.-Mar. 2001).

Deng H. et. al., Guo, “Study on the optimization of extraction processing for Xanthoceras

sorbifolia bunge kernel protein by alkali-solution and acid isolation,” Science and

Technology of Food Industry, vol. 8, pp. 46–52, 2010.

 43

Gomez D.C. and Anacta N. (2020). A New Method to Test Molluscicides against the

Philippine Schistosomiasis Snail Vectors. Journal of Parasitology Research,

2020(),

He P. et. al., (2017) Molluscicidal activity and mechanism of toxicity of a novel

salicylanilide ester derivative against Biomphalaria species. Parasite Vector

10:383

Halwart, M. The golden apple snail Pomacea canaliculata in Asian rice-farmimg systems:

Present impact and future threat. Int. J. Pest Manage. 1994, 40, 199-206.

Hostettmann, K. et. al. Molluscicidal properties of various saponins. Planta Med. 1982,

44, 34-35

Huang, H.-Ch., et. al., 2003. Molluscicidal saponins from Sapindus mukorossi, inhibitory

agents of golden apple snails, Pomacea canaliculata. J. Agric. Food Chem. 51,

4916–4919

Jia, Z. et. al., 1994. Phytochemistry. 37, 1389-1396. (b) Jia, Z. et. al., 1994. Chem.

Pharm. Bull. 42, 2309-2314. (c) Jia, Z. et. al., 1994. Tetrahedron. 50, 11853-

11864. (d) Koike K. et. al., 1999. Chem. Pharm. Bull. 47, 434-435.

Joshi, R., C., 2005. Mini Review: Managing invasive alien mollusc species in rice.

Lale, N.E.S., and Abdulrahman, H.T., 1999. Evaluation of neem (Azadirachta indica A.

Juss) seed oil obtained by different methods and neem powder for the

management of Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) in stored

cowpea. Journal of Stored Products Research 35, p.135-143

Lum, K. A. and Kenny, J. S. The reproductive biology of the Ampullariid snail Pomacea

canaliculata (Muller). J. Moll. Stud. 1989, 55, 53-66.

 44

Maraton, A. and Hostettmann, K. Plant molluscicides. Phytochemistry 1985, 24, 639-

652.

Massaguni, R., and Latip, S.N.H Md, 2012. Neem Crude Extract as Potential

Biopesticide from Controlling Golden Apple Snail, Pomacea canaliculata.

Mochida, O. Spread of freshwater Pomacea snails (Pilidae, Mollusca) from Argentina to

Asia. Micronesica 1991, 3, 51- 62.

Naylor, R. Invasions in agriculture: Assessing the cost of the golden apple snail in Asia.

Ambio 1996, 25, 443-448.

Rejesus, H.M., and Punzalan, E.G., 1997. Molluscicidal action of some Philippine plants

on golden apple snail, Pomacea spp. Philipp. Ent. 11(1): 65-79

San Martin,R., et. al., 2008. Novel molluscicide against Pomacea canaliculata based on

quinoa (Chenopodium quinoa) saponins. Crop Protection 27 (2008) 310–319

Singh, A. & Singh, V. (2009). Molluscicidal acticity of Saraca asoca and Thuja orientalis

against the fresh water snail Lymnaea acuminata. Veterinary Parasitology, 164(5).

Sun Y. et. al., “Optimisation of the extraction conditions of natural colourant carthamin

from safflower (Carthamus tinctorius L.) by response surface methodology,”

International Journal of Food Science & Technology, vol. 49, no. 4, pp. 1168–

1174, 2014.

Yongjun L. et. al., (2016). Extraction of Saponin from Camellia oleifera Abel Cake by a

Combination Method of Alkali Solution and Acid Isolation. Journal of Chemistry,

2016(),

Zheng, Q. et. al., 2004. Nat. Prod. 67, 604-613. (b) Li W. et. al., 2005. Tetrahedron. 61,

2921-2929. (c) Fu H. et. al., 2005. Nat. Prod. 68, 754-758

 45

UNPUBLISHED RESEARCH

Hebres, C., & Pacaňot, I. A. (2017). Molluscicidal potential of Ardisia spp. and Solanum

spp. against Oncomelania hupensis quadrasi. Unpublished Bachelor’s Thesis:

Eastern Visayas State University, Tacloban City

 46

APPENDIX A
PLANT COLLECTION AND PREPARATION

a. Actual Plant

Tagpo Fruit

b. Preparation of Tagpo Fruit Samples and Processing of Tagpo Fruit Samples

PREPARATION OF TEST SOLUTIONS

 47

APPENDIX C
SNAILS COLLECTION AND ACCLIMATIZATION

Washed with dechlorinated water to removed mud

Feeding Golden Apple Snails with Camote Leaves

Covering with net

 48

APPENDIX D
MOLLUSCIDAL ASSAY
 49

APPENDIX E

mg Total Solids/L = (A -B) x 1000

mL sample

(35.16955 – 35.147325) x 1000

10mL
Total Solids Analysis of
Ardisia Aqueous Extract
= 2.2225 g/L

(0.03516955 – 0.035147325) X 1000

10mL

= 2.2225 x 103mg/L

1L mg
[(x) mL concentrations] 3mL x x 2222 = 6.666 mg
1000 mL L

6.666 mg 6.666 mg 1000 mL

x x = 13.3 mg/L
500+ x mL 503 mL 1L

24 hours 48 hours

Replicatesa Mean Replicatesa Mean

Concentration b
(ppm) N.C. Mortality N.C.b Mortality Ratec
Ratec (%) (%)
R1 R2 R3 R1 R2 R3
17.6 10 0 10 0 6.7 10 0 10 0 6.7
22.0 0 0 20 0 6.7 0 0 20 0 6.7
26.4 0 20 40 0 16.7 0 30 40 0 23.3
30.7 30 20 20 0 23.3 70 40 70 0 60.0
35.0 70 80 50 0 66.7 80 100 90 0 90.0
39.3 70 70 50 0 63.3 100 100 90 0 96.7
Table 1. Mortality Rate of Different Concentration of Tagpo Extract against Golden
Apple Snails
a
10 snails per replicate in 500 mL test solution
b
Negative Control, 10 snails in dechlorinated water, 500 mL
c
N = 30 per concentration; assessed after 48 hours
 50

24 hours 48 hours

Mean
Concentration Replicatesa Mean Replicatesa
Mortality
(ppm) N.C.b P.C.c Mortality N.C.b P.C.c
R Rated
R1 R2 Rated (%) R1 R2 R3
3 (%)
13.3 0 0 0 0 20 0 0 0 0 0 40 0
17.6 0 0 0 0 20 0 0 10 0 0 60 3.3
22.0 0 10 0 0 20 3.3 0 10 10 0 60 6.7
26.4 0 30 20 0 20 16.7 10 70 40 0 70 40.0
30.7 40 50 80 0 30 56.7 70 80 100 0 60 83.3
35.0 40 70 50 0 20 53.3 90 90 100 0 40 93.3
Table 2. Mortality Rate of Different Concentration of Alkaline-Treated Tagpo
Extract against Golden Apple Snails
a
10 snails per replicate in 500 mL test solution
b
Negative Control, 10 snails in dechlorinated water, 500 mL
c
Positive Control, 10 snails per replicates in 500 mL in 1.5 mg/L niclosamide
d
N = 30 per concentration; assessed after 48 hours
 51

APPENDIX F
RAW DATA INPUT IN R

>library(drc)
>library(Rmisc)
>library(ggplot2)
>stp3.tag <- read.table(“stp3.tag.csv”, sep=”,”, header=TRUE)
>stp3.tag.ll2 <- drm(dead/snail~conc, weights=snail, data=stp3.tag, fct=LL.2(),
type=’binomial’)
>stp3.tag
conc dead snail mrate
1 13.63 1 10 10
2 13.63 0 10 0
3 13.63 1 10 10
4 22 0 10 0
5 22 0 10 0
6 22 2 10 20
7 26.35 0 10 0
8 26.35 3 10 30
9 26.35 4 10 40
10 30.68 7 10 70
11 30.68 4 10 40
12 30.68 7 10 70
13 34.99 8 10 80
14 34.99 10 10 100
15 34.99 9 10 90
16 39.29 10 10 100
17 39.29 10 10 100
18 39.29 9 10 90

>stp3.tag.ll2.lc<-ED(stp3.tag.ll2, c(50,90), interval= 'delta')

Estimated effective doses
 52

Estimate Std. Error Lower Upper

e:1:50 28.66890 0.76621 27.16716 30.17063
e:1:90 37.93668 1.77648 34.45485 41.41852

>stp3.tag.data<-expand.grid(conc=exp(seq(log(13.63), log(39.29), length=100)))

>stp3.tag.pm<-predict(stp3.tag.ll2, newdata=stp3.tag.data, interval="confidence")
>stp3.tag.data$p<-stp3.tag.pm[,1]
>stp3.tag.data$pmin<-stp3.tag.pm[,2]
>stp3.tag.data$pmax<-stp3.tag.pm[,3]
>stp3.tag.se<-summarySE(stp3.tag, measurevar="mrate", groupvars=c("conc"))
>ggplot()+ geom_line(data=stp3.tag.data,aes(x=conc, y=p*100), colour="black", size=2)
+ theme(text=element_text(size=28), panel.border=element_rect(colour="black",
fill=NA, size=2), panel.background=element_rect(fill="white", colour="black")) +
geom_point(data=stp3.tag.se, aes(x=conc, y=mrate), colour="brown1", size=5,
shape=18) + xlab("Concentration,ppm") + ylab("Mortality Rate (%)") +
geom_point(data=stp3.tag.se, aes(x=conc, y=mrate), colour="brown1", size=5,
shape=18)+geom_line(data=stp3.tag.data, aes(x=conc, y=p*100), colour="black",
size=2)
>ggsave(file="stp3.tag.png", height=6, width=6)

>library(drc)
 53

>library(Rmisc)
>library(ggplot2)
>stp4.alk <- read.table(“stp4.alk.csv”, sep=”,”, header=TRUE)
>stp4.alk.ll2 <- drm(dead/snail~conc, weights=snail, data=stp4.alk, fct=LL.2(),
type=’binomial’)
>stp4.alk
conc dead snail mrate
1 13.25 0 10 0
2 13.25 0 10 0
3 13.25 0 10 0
4 17.63 0 10 0
5 17.63 1 10 10
6 17.63 0 10 0
7 22 0 10 0
8 22 1 10 10
9 22 1 10 10
10 26.35 1 10 10
11 26.35 7 10 70
12 26.35 4 10 40
13 30.68 7 10 70
14 30.68 8 10 80
15 30.68 10 10 100
16 34.99 9 10 90
17 34.99 9 10 90
18 34.99 10 10 100

>stp4.alk.ll2.lc<-ED(stp4.alk.ll2, c(50,90), interval= 'delta')

Estimated effective doses
Estimate Std. Error Lower Upper
e:1:50 27.08313 0.61305 25.88156 28.28469
e:1:90 33.19694 1.24169 30.76327 35.63060
 54

>stp4.alk.data<-expand.grid(conc=exp(seq(log(13.25), log(34.99), length=100)))

>stp4.alk.pm<-predict(stp4.alk.ll2, newdata=stp4.alk.data, interval="confidence")
>stp4.alk.data$p<-stp4.alk.pm[,1]
>stp4.alk.data$pmin<-stp4.alk.pm[,2]
>stp4.alk.data$pmax<-stp4.alk.pm[,3]
>stp4.alk.se<-summarySE(stp4.alk, measurevar="mrate", groupvars=c("conc"))
>ggplot()+ geom_line(data=stp4.alk.data,aes(x=conc, y=p*100), colour="black", size=2)
+ theme(text=element_text(size=28), panel.border=element_rect(colour="black",
fill=NA, size=2), panel.background=element_rect(fill="white", colour="black")) +
geom_point(data=stp4.alk.se, aes(x=conc, y=mrate), colour="brown1", size=5,
shape=18) + xlab("Concentration,ppm") + ylab("Mortality Rate (%)") +
geom_point(data=stp4.alk.se, aes(x=conc, y=mrate), colour="brown1", size=5,
shape=18)+geom_line(data=stp4.alk.data, aes(x=conc, y=p*100), colour="black",
size=2)
>ggsave(file="stp4.alk.png", height=6, width=6)

>library(drc)
> library(Rmisc)
 55

> library(ggplot2)
>stp3_4<-read.table("stp3_4.csv", sep=",", header=TRUE)
>stp3_4.ll2<-drm(dead/snail~conc, weights=snail, data=stp3_4, fct=LL.2(),
type='binomial')
> stp3_4
conc dead snail mrate X
1 13.63 1 10 10 1
2 13.63 0 10 0 1
3 13.63 1 10 10 1
4 22 0 10 0 1
5 22 0 10 0 1
6 22 2 10 20 1
7 26.35 0 10 0 1
8 26.35 3 10 30 1
9 26.35 4 10 40 1
10 30.68 7 10 70 1
11 30.68 4 10 40 1
12 30.68 7 10 70 1
13 34.99 8 10 80 1
14 34.99 10 10 100 1
15 34.99 9 10 90 1
16 39.29 10 10 100 1
17 39.29 10 10 100 1
18 39.29 9 10 90 1
19 13.25 0 10 0 2
20 13.25 0 10 0 2
21 13.25 0 10 0 2
22 17.63 0 10 0 2
23 17.63 1 10 10 2
24 17.63 0 10 0 2
25 22 0 10 0 2
26 22 1 10 10 2
 56

27 22 1 10 10 2
28 26.35 1 10 10 2
29 26.35 7 10 70 2
30 26.35 4 10 40 2
31 30.68 7 10 70 2
32 30.68 8 10 80 2
33 30.68 10 10 100 2
34 34.99 9 10 90 2
35 34.99 9 10 90 2
36 34.99 10 10 100 2

>stp3_4.ll2<-drm(dead/snail~conc, weights=snail, X, data=stp3_4, fct=LL.2(),

type='binomial')
> EDcomp(stp3_4.ll2, c(50,50), interval='delta')

Estimated ratios of effect doses

Estimate Lower Upper
1/2:50/50 1.05859 0.98593 1.13125
> EDcomp(stp3_4.ll2, c(90,90), interval='delta')

Estimated ratios of effect doses

Estimate Lower Upper
1/2:90/90 1.1427 1.0085 1.2769

> stp3_4.data<-expand.grid(conc=exp(seq(log(13.25), log(39.29), length=100)))

> stp3_4.pm<-predict(stp3_4.ll2, newdata=stp3_4.data, interval="confidence")
> stp3_4.data$p<-stp3_4.pm[,1]
> stp3_4.data$pmin<-stp3_4.pm[,2]
> stp3_4.data$pmax<-stp3_4.pm[,3]
> stp3_4.se<-summarySE(stp3_4, measurevar="mrate", groupvars=c("conc"))
 57

> ggplot()+ geom_line(data=stp3.tag.data,aes(x=conc, y=p*100), colour="red", size=1.5)

+ theme(text=element_text(size=28), panel.border=element_rect(colour="black",
fill=NA, size=2), panel.background=element_rect(fill="white", colour="black")) +
geom_point(data=stp3.tag.se, aes(x=conc, y=mrate), colour="green", size=5, shape=18)
+ xlab("Concentration,ppm") + ylab("Mortality Rate (%)") +
geom_point(data=stp4.alk.se, aes(x=conc, y=mrate), colour="purple", size=5,
shape=18)+geom_line(data=stp4.alk.data, aes(x=conc, y=p*100), colour="black",
size=1.5)
> ggsave(file="stp3_4.png", height=6, width=6)

CURRICULUM VITAE
 58

Name: SHERRIELYN M. BERINO

Email Address: shemberino010@gamil.com
Age: 25
Date of Birth: October 1, 1996
Residence: 515 San Roque St. Brgy. Lapu-Lapu Dist. 2 Dagami, Leyte
Birth Place: MacArthur, Leyte
Civil Status: Single
Religion: Roman Catholic
Father’s Name: Eduardo S. Berino
Occupation: Farmer
Mother’s Name: Mary Ann G. Morante
Occupation: Self- Employed
EDUCATIONAL BACKGROUND
Elementary Dagami, South Central School
Dagami, Leyte
Batch 2009
Secondary Saint Joseph High School of Dagami Inc.
San Jose Dagami, Leyte
2013
College Eastern Visayas State University
Tacloban City
Honors Received With Honor (Elementary)
With High Honor (Secondary)
SEMINARS/ WORKSHOP ATTENDED

 Virtual OJT Orientation on Work Ethics and Sexual Harassment in the

Workplace, given 2nd day of July, 2021.
 OJT Virtual Orientation on Personality Development, given this 5th day of July,
2021.
 59

 Webinar entitled; “Role of Safety in the Laboratory” held on the 5th day of July
2021.
 WEBINAR SERIES: GHS in the Chemical Laboratory, held on the 7th of July,
2021.
 Webinar entitled; “Occupational Safety” held on the 7th day of July 2021.
 Virtual OJT Orientation on Chemical Security in the Laboratory and Experiences
in Crafting a Laboratory Safety Manual, on July 09, 2021.
 Webinar series for BS Chemistry and BS Environmental Sciences OJT 2021 on
Safety in the Chemical Laboratory, entitled: "Elements in the Chemical LSM",
given this 9th day of July, 2021.
 60

Name: JEAN VALERIE C. CARSON

Email Address: jeanvalerie.carson@evsu.edu.ph
Age: 23
Date of Birth: April 16, 1999
Residence: Brgy. Macopa Jaro, Leyte
Birth Place: Jaro, Leyte
Civil Status: Single
Religion: Roman Catholic
Father’s Name: Greg B. Carson
Occupation:
Mother’s Name: Virginia C. Carzon
Occupation: Teacher
EDUCATIONAL BACKGROUND
Elementary Macopa Elementary School
Brgy. Macopa Jaro, Leyte
2009-2010
Secondary Notre Dame of Jaro
Jaro, Leyte
College Eastern Visayas State University
Tacloban, City
Honors Received Valedictorian (Elementary)
SEMINARS/ WORKSHOP ATTENDED

 Virtual OJT Orientation on Work Ethics and Sexual Harassment in the

Workplace, given 2nd day of July, 2021.
 OJT Virtual Orientation on Personality Development, given this 5th day of July,
2021.
 Webinar entitled; “Role of Safety in the Laboratory” held on the 5th day of July
2021.
 61

 WEBINAR SERIES: GHS in the Chemical Laboratory, held on the 7th of July,
2021.
 Webinar entitled; “Occupational Safety” held on the 7th day of July 2021.
 Virtual OJT Orientation on Chemical Security in the Laboratory and Experiences
in Crafting a Laboratory Safety Manual, on July 09, 2021.
 Webinar series for BS Chemistry and BS Environmental Sciences OJT 2021 on
Safety in the Chemical Laboratory, entitled: "Elements in the Chemical LSM",
given this 9th day of July, 2021.
 62

Name: LARALIN B. MOLDEZ

Email Address: lara.moldez@evsu.edu.ph
Age: 23
Date of Birth: December 28, 1998
Residence: Brgy. Lomonon, Palompon, Leyte
Birth Place: Palompon, Leyte
Civil Status: Single
Religion: Roman Catholic
Father’s Name: Hilario B. Moldez
Occupation: Seaman
Mother’s Name: Maria Linda B. Moldez
Occupation: Housewife
EDUCATIONAL BACKGROUND
Elementary Lomonon Elementary School
Brgy. Lomonon, Palompon, Leyte
2010-2011
Secondary Ace Learning Center, Inc.
Palompon, Leyte
College Eastern Visayas State University
Tacloban, City
Honors Received With Honor (Elementary)
With High Honor (Secondary)
SEMINARS/ WORKSHOP ATTENDED

 Virtual OJT Orientation on Work Ethics and Sexual Harassment in the

Workplace, given 2nd day of July, 2021.
 OJT Virtual Orientation on Personality Development, given this 5th day of July,
2021.
 63

 Webinar entitled; “Role of Safety in the Laboratory” held on the 5th day of July
2021.
 WEBINAR SERIES: GHS in the Chemical Laboratory, held on the 7th of July,
2021.
 Webinar entitled; “Occupational Safety” held on the 7th day of July 2021.
 Virtual OJT Orientation on Chemical Security in the Laboratory and Experiences
in Crafting a Laboratory Safety Manual, on July 09, 2021.
 Webinar series for BS Chemistry and BS Environmental Sciences OJT 2021 on
Safety in the Chemical Laboratory, entitled: "Elements in the Chemical LSM",
given this 9th day of July, 2021.