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CHAPTER I

INTRODUCTION

Background of the Study

Golden Apple Snails are highly invasive and cause damage to rice crops. Also, sn

ails' species are also associated with transmission parasitic disease as intermediate host. S

chistosomiasis is one of the 17 diseases classified as neglected tropical diseases by the W

orld Health Organization (Keenan et al, 2013; De Vlas et al, 2016). In the Philippines, the

endemic species is S. japonicum to which 2.5 Filipinos are directly exposed. It was broug

ht up that Golden Apple Snails has undesirable effects to humans health and agricultural

sector.

Due to Golden Apple Snail as destruction to agriculture and vectors of parasitic

diseases, the researchers are searching an aide to the problem. The researchers decided to

find a good solution to the problem that is nature friendly and obtainable to the

environment. The researchers preferred to find a natural alternative molluscicide which is

cheaper and safer way to use by the farmers in order to solve the rising population of

golden apple snail which is a carrier of a parasitic disease and reduces rice yield.

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Statement of the Problem

This study aims to determine the molluscicidal activity of Century Plant Extracts

(Agave Americana) against the Golden Apple Snail (Pomacea canaliculata). Specifically,

it seeks to answer the following question:

1. What are the components present in Century Plant extract that makes it a

potent Golden Apple Snail Exterminator?

2. At what level of concentrations of the Century plant extract is most effective

in exterminating Golden Apple Snail in relation to time?

3. Is there a statistical difference between the Century plant extract and the

commercial molluscicide in exterminating Golden Apple Snail?

Objective of the Study

The study was conducted to prove determine the molluscicidal activity of Century

Plant Extracts (Agave Americana) against the Golden Apple Snail (Pomacea

canaliculata). Specifically, it aims to:

1. identify the components or properties present in Century plant extract that

make it a potent Golden Apple Snail exterminator

2. determine what level of concentrations of Century plant extract is most

effective in exterminating Golden Apple Snail in relation to time.

3. test if there is astatistical difference difference between the Century plant

extract and the commercial molluscicide

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Research Hypothesis

1. There are no components or properties present in Century Plant extract that make

it a potent Golden Apple Snail exterminator.

2. None of the different levels of concentration of Century Plant extract is most

effective in exterminating Golden Apple Snail in relation to time.

3. There is no statistical difference between the Century Plant extract and the

commercial molluscicide.

Significance of the Study

This study is a significant endeavour in promoting environmental friendly and

manages agricultural pests. It is a potential for the future studies in developing safer and

organic molluscicide which has benefits in increased profits for farmers and the

prevention of diseases. All because of its molluscicidal activity at a very cheap cost the

plant is also available in locality but are used as ornamental only and without specific

purposes. Therefore, this is a helpful contribution to mankind considered its efficacy in

exterminating golden apple snail due also to its organic nature. Combating molluscs in

fields will produce larger quantities of food and prevent the spreading of Schistosomiasis.

Scope and Limitation

This study focuses to determine the molluscicidal activity of Century Plant

Extracts (Agave Americana) against the Golden Apple Snail (Pomacea canaliculata). It is

limited to the use of the extract from Century Plant that was gathered from Surigao del

Norte National High School Campus to exterminate Golden Apple Snail that was taken

from rice fields in Brgy. San Roque, Surigao City. It focuses on the molluscicidal activity

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of Century Plant Extracts (Agave Americana) against the Golden Apple Snail (Pomacea

canaliculata).

Conceptual Framework

Experimental Set-Up

Level of Concentration
Controlled Set-up
T1 50% Extract; 50% Water
Commercial Molluscicide
T2 75% Extract; 25% Water

T3 100% Extract; 0% Water

Dependent Variable

Mortality rate of the Golden Apple


Snail

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Review of Related Literature

Agave americana, common names sentr

y plant, century plant, maguey, or American alo

e, is a species of flowering plant in the family A

gavaceae, native to Mexico, and the United Stat

es in New Mexico, Arizona and Texas. Today, i

t is cultivated worldwide as an ornamental plant

. It has become naturalized in many regions, including the West Indies, parts of South A

merica, the Southern Mediterranean Basin, and parts of Africa, India, China, Thailand, an

d Australia. Although it is called the century plant, it typically lives only 10 to 30 years. It

has a spread around 610 ft. (1.83.0 m) with gray-green leaves of 35 ft. (0.91.5 m) lon

g, each with a prickly margin and a heavy spike at the tip that can pierce deeply. Near the

end of its life, the plant sends up a tall, branched stalk, laden with yellow blossoms that m

ay reach a total height up to 2530 ft. (89 m) tall. Agave is a tropical plant with about 30

0 species. The leaves are fibrous, growing upward from the ground forming a massive ros

ette. Both sides of the leaves are smooth, with prickly edges and thorny tips.

Study yielded a new steroidal saponin: a bisdesmosidic spirostanol saponin.

Studies on chemical composition of leaves yielded high amount of total dietary

fiber (38.40%), total sugars (45.83%), and protein (35.33%), with a relatively low ash

content (5.94%) and lipid (2.03%). Agave inulin showed significant differences when

compared to commercial inulin. (Mohamed Ali Bouaziz et al., 2014)

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Study of leaves isolated tigogenin, hecogenin, 9-dehydrohecogenin,

rockogenin,12-epirockogenin, gitogenin, chlorogenin and manogenin. (CHEN YEN-YUNG,

TSUNG PU-CHU, HUANG LIANG.,1975)

The sap of Agave americana contains calcium oxalate crystals, acrid oils, saponin

s, among other compounds. Despite the known irritants, dermatitis is only rarely reported.

(Ricks MR, Vogel PS, Elston DM, Hivnor C.,1999)

The Century Plant contains calcium oxalate crystals which can cause abrasive inju

ries to the eyes or mucosal linings. It can also cause skin inflammation. The sap is the mo

st toxic part of the plant although other parts such as the thorns can also cause symptoms.

Century plants form six-sided raphides that contain saponins. Saponins are compo

unds with steroid cores to which one or more sugars are attached. They are also known as

steroidal glycosides, and they are found in many plant families in addition to the Agavace

ae family. Some of these saponins, including those in century plant are extremely poisono

us when consumed, but others may cause sever

e skin irritation.

There are more than 100 species of app

le snail that exists. Two species, Pomacea cana

liculata and Pomacea maculata, commonly kno

wn as Golden Apple Snails, are highly invasiv

e and cause damage to rice crops. They were i

ntroduced to Asia, from South America, in the 1980s as potential food for people, but it u

nfortunately became a major pest of rice.

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Golden apple snails have muddy brown shell and golden pinkish or orange-yellow

flesh. They are bigger and lighter in color compared to native snails. Its eggs are bright pi

nk in color. Snails are able to spread through irrigation canals, natural water distribution p

athways, and during flooding events. When water is absent, apple snails are able to bury t

hemselves in the mud and hibernate for up to six months. When water is re-applied to fiel

ds, snails may emerge. They damage direct wet-seeded rice and transplanted rice up to 30

days old. Once the rice plant reaches 3040 days, it will become thick enough to resist th

e snail. Golden apple snails eat young and emerging rice plants. They cut the rice stem at

the base, destroying the whole plant. The golden apple snail is considered a major proble

m of rice. If no control measure is taken, they can completely destroy 1 m2 of field overn

ight. This damage could lead to more than 50% yield loss.

Definition of Terms

Saponins - are glucosides with foaming characteristics.

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Molluscicides - also known as snail baits and snail pellets, a pesticides against molluscs,

which are usually used in agriculture or gardening, in order to control gastropod pests

specifically slugs and snails which damage crops or other valued plants by feeding on

them.

Calcium Oxalate Crystals - are the most common constituent of human kidney stones,

and calcium oxalate crystal formation is also one of the toxic effects of ethylene glycol

poisoning.

Raphide - a needle-shaped crystal of calcium oxalate occurring in clusters within the

tissues of certain plants.

Alkaloids - any of a class of plant origin that have pronounced physiological actions on

humans.

Glycosides - a compound formed from a simple sugar and another compound by

replacement of a hydroxyl group in the sugar molecule.

Carbohydrates - are one of the main types of nutrients.

Amino Acids - are organic compounds containing amine and carboxyl functional groups,

along with a side chain specific to each amino acids.

Flavonoid - any of a large class of plant pigments having a structure based on or similar

to that of flavone.

Tannins - a yellowish or brownish bitter-tasting organic substance present in some galls,

barks, and other plant tissues, consisting of derivatives of gallic acid, used in leather

production and ink manufacture.

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CHAPTER II

METHODOLOGY

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Materials

45 Golden Apple Snails

Century Plant

Equipment

5 Spray bottles 15 containers

Clean cloth/ Gauge pad Funnel

Strainer Pair of scissors

Graduated Cylinder Grater

Pair of gloves and masks Water

Beaker

Treatment/General Procedure

The materials and equipment will be ready for use.

1. Preparation of Century Plant extract

A pair of gloves was worn in order to avoid skin contact and ingestion of the

plants toxic components. The leaves will be washed thoroughly with running

water so as to get rid unwanted foreign substance like dirt to affect the process.

With a pair of scissor; the leaves were sliced into smaller pieces. The grinding

process was followed using grater until its totally extracted. The extracted leaves

were then manually placed into a strainer and finally placed into the beaker.

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2. Preparation of the sprays

The liquid material that was gathered from the extraction process will be poured

into beakers then in graduated cylinder to measure each different concentration

(50%, 75% and 100%) measuring 50 ml. Each of these spraying bottles will be

labelled Treatment 1, Treatment 2 and Treatment 3 with corresponding level of

concentration. The fourth spraying bottle will be filled with the 50 ml of positive

concentration (Commercial Pesticide) while fifth spraying bottle will be filled

with 50 mL of negative concentration (Distilled Water)

Treatment 1- 25 ml leaf extract level of concentration; 25 ml distilled water

Treatment 2- 35 ml leaf extract level of concentration; 15 ml distilled water

Treatment 3- 50 ml leaf extract level of concentration; 0 ml distilled water

Treatment 4- Positive concentration (commercial pesticide)

Treatment 5- Negative concentration (distilled water)

3. Testing

45 Golden Apple Snail with controlled characteristics will be placed in five

different small plastic containers. These containers will be placed with 3 Golden

Apple Snail. The three different concentration of Century Plant extract was

sprayed into Golden Apple Snail placed in the three groups of containers. On

fourth and fifth group of containers, the distilled water and commercial

molluscicide were sprayed.

Set-up A (50% Concentration)

Container A1 3 Golden Apple Snail

Container A2- 3 Golden Apple Snail

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Container A3- 3 Golden Apple Snail

Set-up B (75% Concentration)

Container B1 3 Golden Apple Snail

Container B2- 3 Golden Apple Snail

Container B3- 3 Golden Apple Snail

Set-up C (1OO% Concentration)

Container C1 3 Golden Apple Snail

Container C2- 3 Golden Apple Snail

Container C3- 3 Golden Apple Snail

Set-up D (Commercial Molluscicide)

Container D1 3 Golden Apple Snail

Container D2- 3 Golden Apple Snail

Container D3- 3 Golden Apple Snail

Set-up E (Distilled Water)

Container E1 3 Golden Apple Snail

Container E2- 3 Golden Apple Snail

Container E3- 3 Golden Apple Snail

4. Data Gathering Recording

The following critical data was gathered:

a. Number of Golden Apple Snail exterminated

b. Time in which extermination of Golden Apple Snail succeeded

c. Level of concentration most effective in exterminating Rice Black Bug

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5. Waste Disposal

At the end of the study, the samples, together with the containers

will be disposed properly in the compost pet and covered immediately with soil to

prevent other animals to be in contact with it. The used apparatus or glass will be

washed and cleaned using the antibacterial liquid soap and water, other equipment

will be sterilized.

Flow Chart

Figure 1. Flowchart of the General Procedures

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Preparation of Materials

Collection of Century Plant

Extraction of Century Plant

Preparation of different concentration

Collection of Golden Apple Snail

Spraying of different concentration to the container


with Golden Apple Snail

Observing the Mortality Rate of Golden Apple Snail

Collection of data

Disposal of Waste

Research Design

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Table 1. Research design of the Test for the mortality of Golden Apple Snail after

treatment.

Time Interval (Golden


No. of Apple Snails
Golden exterminated after
Set-ups Replicates treatment) AVERAGE
Apple
Snail 1-10 11-20 21-30
mins mins mins
A1
100% A2
A3
Average
B1
75% B2
B3
Average
C1
50% C2
C3
Average

D1
POSITIVE
CONTROL D2
D3
Average

E1
NEGATIVE
CONTROL E2
E3
Average

CHAPTER III

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RESULTS AND DISCUSSION

Table 2. Phytochemical Analysis of Century Plant

Preliminary phytochemical screening of A. Americana was carried out

ascertain the presence of some chemical constituents like alkaloids, glycosides,

carbohydrates, flavonoids, tanins and saponin. The extract was predicted to demonstrate

toxicity towards snails due to the presence of saponins (Hostettmann 1984; Sparg et al.

2004; Ojewole et al. 2004) and flavonoids (Macheix et al. 1990) in the extracts (Table 1).

The molluscicidal activity of saponin was due to its ability to effect the cell membrane

and its ability to lower the surface tension of water. The extract caused various responses

in the tested subjects as a result of direct contact between the tested organisms and the

saponin (Hostettmann et al. 1982; Mahato & Nandy 1991; Hutchings et al. 1996). The

tested P. canaliculata demonstrated a response to the molluscicidal action of saponin.

This response was initiated by the production and secretion of mucus in order to reduce

further contact of their body surfaces with the molluscicide (Brain et al. 1990).

Table 3.Tabular result of the mortality rate of Golden Apple Snail after spraying the

controlled set-ups.

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Time Interval (Golden
No. of Apple Snails
Golden exterminated after
Set-ups Replicates treatment) AVERAGE
Apple
Snail 1-10 11-20 21-30
mins mins mins
A1 3 0 2 1 3
100% A2 3 0 3 1 3
A3 3 0 1 2 3
Average 0 2 1.333
B1 3 0 1 2 3
75% B2 3 0 2 1 3
B3 3 0 2 1 3
Average 0 1.667 1.333
C1 3 0 1 2 3
50% C2 3 0 2 1 3
C3 3 0 1 2 3
Average 0 1.333 1.667
D1 3 3 0 0 3
POSITIVE
CONTROL D2 3 3 0 0 3
D3 3 3 0 0 3
Average 3 0 0
E1 3 0 0 0 0
NEGATIVE
CONTROL E2 3 0 0 0 0
E3 3 0 0 0 0
Average 0 0 0

The table 3 shows the mortality rate of the Golden Apple Snail against the

five set-ups. Based on the average of exterminated Golden Apple Snail of time interval,

the 100% level of concentration of Century Plant has an average of 2 which is greater

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than the average among the three different levels of concentration. Therefore, 100% level

of concentration is the most effective in exterminating Golden Apple Snail among the

three different levels of concentration. But considering the time, the Commercial

Molluscicide has exterminated the molluscs in all the containers in just 1-10 minutes time

interval. Thus, the commercial molluscicide has exterminated with great number with a

short period of time.

Figure 2. Graph of the Average result of the Mortality Rate of 100% Concentration of

Century Plant extract

2
Replicate 1
Replicate 2
1 Replicate 3

0
1-10 mins 11-20 mins 21-30 mins
Time Interval (Golden Apple Snails exterminated after
treatment)

Figure 3. Graph of the Average result of the Mortality Rate of 75% Concentration of

Century Plant extract

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3

Replicate 1
Replicate 2
1 Replicate 3

0
1-10 mins 11-20 mins 21-30 mins
Time Interval (Golden Apple Snails exterminated after
treatment)

Figure 4. Graph of the Average result of the Mortality Rate of 50% Concentration of

Century Plant extract

Replicate 1
Replicate 2
1
Replicate 3

0
1-10 mins 11-20 mins 21-30 mins
Time Interval (Golden Apple Snails exterminated
after treatment)
Figur

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e 5. Graph of the Average result of the Mortality Rate of Positive Control (Commercial

Molluscicide)

2.5

1.5 Replicate 1
Replicate 2
1
Replicate 3
0.5

0
1-10 mins 11-20 mins 21-30 mins
Time Interval (Golden Apple Snails exterminated after
treatment)

Figure 6. Graph of the Average result of the Mortality Rate of Negative Control

(Distilled Water)

Replicate 1
Replicate 2
1 Replicate 3

0
1-10 mins 11-20 mins 21-30 mins
Time Interval (Golden Apple Snails exterminated after
treatment)

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Table 4. Statistical Analysis of 1oo% Level of Concentration and Commercial

Molluscicide

t-Test: Paired Two Sample for Means

1 Commercial
Mean 1.111111 1
Variance 1.111111 2.25
Observations 9 9
Pearson Correlation -0.79057
Hypothesized Mean
Difference 0
df 8
t Stat 0.137686
P(T<=t) one-tail 0.446946
t Critical one-tail 1.859548
P(T<=t) two-tail 0.893891
t Critical two-tail 2.306004

Table 5. Statistical Analysis of 75% Level of Concentration and Commercial

Molluscicide

t-Test: Paired Two Sample for Means

0.75 Commercial
Mean 1 1
Variance 0.75 2.25
Observations 9 9
Pearson Correlation -0.86603
Hypothesized Mean
Difference 0
df 8
t Stat 0
P(T<=t) one-tail 0.5
t Critical one-tail 1.859548
P(T<=t) two-tail 1
t Critical two-tail 2.306004

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Table 6. Statistical Analysis of 50% Level of Concentration and Commercial

Molluscicide

t-Test: Paired Two Sample for Means

0.5 Commercial
Mean 1 1
Variance 0.75 2.25
Observations 9 9
Pearson Correlation -0.86603
Hypothesized Mean
Difference 0
df 8
t Stat 0
P(T<=t) one-tail 0.5
t Critical one-tail 1.859548
P(T<=t) two-tail 1
t Critical two-tail 2.306004

Based on the Statistical Analysis of data above, all of the t Stat is lesser than the t

Critical. Therefore, the second hypothesis is rejected so none of the different levels of

concentration of Century Plant extract is most effective in exterminating Golden Apple

Snail in relation to the time. But basing on the mortality rate of the molluscs in every set-

ups in different levels of concentrations and Commercial, both are effective in

exterminating all the number of molluscs in the containers.

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Table 7. Statistical Analysis of the three levels of concentration and commercial

molluscicide

Anova: Single Factor

SUMMARY
Groups Count Sum Average Variance
1 4 6 1.5 1.666667
4 7 1.75 2.25

4 6 1.5 1.666667
0.75 4 6 1.5 1.666667
4 6 1.5 1.666667

4 6 1.5 1.666667
0.5 4 6 1.5 1.666667
4 6 1.5 1.666667
4 6 1.5 1.666667
POSITIVE
CONTROL 4 6 1.5 3
4 6 1.5 3
4 6 1.5 3

ANOVA
Source of
Variation SS df MS F P-value F crit
Between Groups 0.229167 11 0.020833 0.010169 1 2.066608
Within Groups 73.75 36 2.048611

Total 73.97917 47

The table shows F value 0.011765 which is less than the F crit 2.03742

which means the null hypothesis is accepted, that there is no statistical difference

between the different levels of concentration and the commercial molluscicide. This

implies that the Century Plant is effective as an organic molluscicide.

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CHAPTER VI

SUMMARY, CONCLUSIONS, RECOMEMENDATION

Summary

Extract from Century Plant (Agave Americana) leaves were divided into

three levels of concentration, Treatment 1, 50 %; Treatment 2, 75%; and Treatment 3,

100%, and then compared to the controlled set-ups which are the commercial

molluscicide and distilled water. It was proven that different levels of concentration tests

conducted were showed a significant ability to exterminate Golden Apple Snail than the

negative control (distilled water) which showed insignificant ability to exterminate

Golden Apple Snail. However, in the set-ups the Treatment 3, 100% level of

concentration gave the best results in exterminating the Golden Apple Snail with a short

time interval.

Conclusions

Based on the findings of the study, the researchers have made the following conclusions:

1. The Century plant has a saponin which was predicted to demonstrate toxicity

towards snails and it also contains calcium oxalate crystals, toxic

components, called raphides which make it a potential molluscicide.

2. All the different concentrations of Century Plant leaf extract are effective

exterminating Golden Apple Snail. It is the treatment with the 100% level of

concentration is more effective among the other levels of concentration in

terms in time of mortality rate of the Golden Apple Snail.

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3. There is no significant difference between the Century Plant extract and the

commercial molluscicide.

Recommendations

Based on the conclusion stated thereof, the researcher recommends the

following especially for future studies in order to further develop its findings:

1. Test whether or not there are significant side effects of the said extracts to rice

plant, the environment, or to human and animals.

2. Study other pests that are also vulnerable to the leaf extracts.

3. Determine the level dose of the Century Plant extract towards the Golden Apple

Snail.

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APPENDICES

Documentation

Collection of Century Plant Leaves Measuring the extract of the


(Agave Americana) different levels of concentration

Golden Apple Snail in the container Different Set-ups


before the spraying of the extracts

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Spraying the different levels of concentration in
respective set-up

After spraying the different concentration in the


container with Golden Apple Snails

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Bibliography

Books:

STUDIES ON THE STEROIDAL SAPOGENINS OF AGAVE PLANT .ISOLA

TION AND IDENTIFICATION OF STEROIDAL SAPOGENINS FROM LEAV

ES OF AGAVE AMERICANA L. / CHEN YEN-YUNG, TSUNG PU-CHU, HU

ANG LIANG / Acta Chimica Sinica 1975, Vol. 33 Issue (2): 149-161

Journal:

Chemical Composition, Functional Properties, and Effect of Inulin from Tunisian

Agave americana L. Leaves on Textural Qualities of Pectin Gel / Mohamed Ali B

ouaziz, Rabaa Rassaoui, and Souhail Besbes / Journal of Chemistry, Volume 2014

(2014) / http://dx.doi.org/10.1155/2014/758697

Purpuric agave dermatitis. / Ricks MR, Vogel PS, Elston DM, Hivnor C. / J Am A

cad Dermatol. 1999 Feb;40(2 Pt 2):356-8.

Chemical Composition, Functional Properties, and Effect of Inulin from Tunisian

Agave americana L. Leaves on Textural Qualities of Pectin Gel / Mohamed Ali B

ouaziz, Rabaa Rassaoui, and Souhail Besbes / Journal of Chemistry, Volume 2014

(2014) / http://dx.doi.org/10.1155/2014/758697

Chemical Composition, Functional Properties, and Effect of Inulin from Tunisian

Agave americana L. Leaves on Textural Qualities of Pectin Gel / Mohamed Ali B

ouaziz, Rabaa Rassaoui, and Souhail Besbes / Journal of Chemistry, Volume 2014

(2014) / http://dx.doi.org/10.1155/2014/758697

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Pharmacognostic and Phytochemical Studies on Roots of Agave Americana

(Agavaceae) *Kadam P. V, Yadav K. N, Deoda R.S, Narappanawar N. S,

Shivatare R. S, Patil M. J Marathwada Mitra Mandals, College of Pharmacy,

Thergaon (Kalewadi), Pune-411033

Electronics: www.nebi.nlm.gov

www.stuartxchange.org/lingas.html

www.pinoyrice.com

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