1

TOXICITY OF CARICA PAPAYA SEED

Life Science - Team

Aleck Josef C. Aquino

Ma. Christinne D. Avanzado
Aaron Clarence F. Dizon
Proponents

Jeffrey Magtibay
Franz Kevin Manalo
Research Advisers

San Pablo City Science High School

Brgy. San Jose, San Pablo City Laguna
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Toxicity of Carica papaya seed

Aleck Josef C. Aquino, Ma. Christinne D. Avanzado, Aaron Clarence F. Dizon
San Pablo City Science High School, San Pablo Laguna

ABSTRACT
This study was attained in order to determine if there is a possible toxic effect out of
ethanolic extract from Carica papaya.To do this, a bioassay using Artemia salina, commonly
known as brine shrimp, is used. The Carica papaya seeds were extracted using ethyl alcohol and
left out for 5 weeks. The treatments were added with varying amounts of ethyl alcohol to further
diffuse the extract. The Artemia salina was hatched in 24 hours in artificial saline water. The
treatments were mixed with a number of brine shrimps and the results were recorded.

In analyzing the results, One-way ANOVA is used. It showed that the there is no significant
effect on the mortality rate of Artemia salina In conclusion, the study revealed that the
concentration of Carica papaya seed extract has no toxic effect on brine shrimps

Keywords: Artemia salina, Carica papaya,ethanolic, bioassay, saline

 ACKNOWLEDGEMENTS

This whole Research has been a challenge for us here in San Pablo City Science High

School and we would like to express our gratitude and appreciation to the people who have

helped and continued to encourage us until the end of our Research Project.

First of all we would like to thank God for giving us strength, guidance, and knowledge

to finish this project.

We would like to thank our parents for their support especially financial support and

moral support. We would not finish this research without their help.

We would like to thank Sir Franz Kevin B. Manalo for encouraging us to finish our

research. Thank you for staying with us the whole time.

We also would like to thank our research adviser, Sir Jeffrey Magtibay, for doing all his

best to help us in our research project and patiently waiting for the outcome of this study.

Lastly, we would like to thank our classmates for giving us all their support and

encouraging us through the hard times.

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TABLE OF CONTENTS

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

Abstract ………………………………………………………………………………………. ii

Acknowledgement …………………………………………………………………………… iii

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

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

Statement of Objectives ……………………………………………………………... 1

Hypotheses……………………………………………………………………………………. 2

Significant of the Study ………………………………………………………………. 2

Scope and Limitations ………………………………………………………………... 2

Review of Related Literature…………………………………………………………………. 3

Definition of Terms …………………………………………………………………………... 7

Methodology …………………………………………………………………………………. 8

Results and Discussion ……………………………………………………………………….. 11

Summary and Conclusion …………………………………………………………………….. 14

Recommendations …………………………………………………………………………….. 15

Bibliography ………………………………………………………………………………….. 16

Appendices …………………………………………………………………………………… 21

Appendix A …………………………………………………………………………... 21

Appendix B …………………………………………………………………………… 22

Appendix C …………………………………………………………………………… 23

Appendix D …………………………………………………………………………… 24
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INTRODUCTION

Background of the Study

Cultural medicine has always been linked to herbal medicines and other obscure tactics

are based on different cultural folk belief and the effects of medicinal plants. Moreover, due to

tropical climate of the Philippines, the diversity and natural resources of it are mildly abundant.

Certain folk beliefs concern the different medicinal and other effects of Carica papaya. Thus,

effects may vary from being medicinal or maybe even used in some researches concerning

insecticides. The study was done in order to identify if Carica papaya actually has toxic

properties.

Brine shrimps are commonly used in bioassays concerning the toxicity of a material due

to their resilience to different environments. They are commonly used as fish feed in some places

and they are sold in pet shops. The study will use a bioassay that uses brine shrimps that will be

exposed to the extract.

Statement of Objectives

This study aims to determine the possible toxicity rate of Carica papaya on the number

of the brine shrimp processed through the Bioassay.

Moreover, this study is aimed to:

 Evaluate if the Carica papaya extract will have a toxicity rate

 Asses if the concentration of Carica papaya extract will have an effect to the

mortality rate.

 Evaluate which concentration level will kill the most/least number of brine shrimp
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Hypothesis:

1. The ethanolic extract from Carica papaya will not have a significant effect on the

mortality rate of brine shrimps.

Significance of Study

- To determine if the effect of Carica papaya seeds to the brine shrimp by the use of

Bioassay.

- To determine if Carica papaya seeds can potentially be used as a toxin

- To upgrade the economic importance of Carica papaya from a food product to

industrial and medicinal

Scopes and Limitation

The study will be only concerning the possible lethality rate of Carica papaya seed

extracts to the Artemia Salina.

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Review of Related Literature

Papaya

Carica papaya also known as papaya belongs to the Caricaceae family. The word Carica

came from the Latin name for a kind of fig (Du Puy & Telford, 1993). This species in the genus

of Carica of the plant family of Caricaceae, is a large tree-like plant, with a single stem with

spirally arranged leaves and has numerous seeds. Papaya stem, when wounded, white milky latex

oozed to the wound.

Papaya is grown in tropical countries. According from the data given by the Food and

Agriculture Organization (2007), Philippines is the 10th country that produces and exports

papaya, averaging of 132 kilo tonnes. Traditionally, papaya is cultured for its edible fruits and

industrial uses.

Papaya contains lots of nutrients, the ripe papaya fruit is a good source of a powerful

antioxidant of Vitamin A, C and E; magnesium, calcium and potassium and other minerals that

can be found in papaya. The fruit is an excellent source of beta carotene that prevents damage

caused by free radicals that may cause some forms of cancer.

According to the International Journal of Food Science and Nutrition, papaya can halt

breast cancer. Papaya has an abundance of cancer fighting lycopene. Additionally, lycopene can

induce the death of cancer cells. Also, Isothiocyanates was found in this plant to refresh the cycle

of the cell to eliminate the cancer. Under the Isothiocyanates is the organo which protects against

different cancer. Isothiocyanates shown the capability of inhibiting the development and

formation of cells through multiple mechanism and pathways (International Journal of Oncology,

2008)
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Papaya has an enzyme called proteolytic enzyme used in protein-digesting and can help

getting rid of parasites including to this are papain and chymopapain. Papaya proteinase I, also

known as papain, is an enzyme that is used to dissociate cells that holds the cells together. Along

with papain, chymopapain used to reduce the inflammation and improve the healing time from

burns and wounds. Carpaine, an alkaloid, which moderates the blood pressure and can kill

worms and amoebas.

Carica papaya seeds averages up to 600 seeds that is 5 millimeter in diameter each seeds.

Papaya seeds help to heal cirrhosis in our liver. Also, when having food poison, papaya seeds

can help to keep away the bacteria and helps in detoxification. Papaya seeds are not only for

medication, it can also be lined up in beauty purposes like prevent aging, wrinkle-free, and

remedying acne that the seeds are rich in amino acids that is good for the skin.

Toxicity

Toxocity is the degree to which a substance can harm human or animals. Toxicity can

refer to the effect on a whole organism such as animal, bacterium, or plant, as well as the effect

on a substructure of the organism,such as cell (cytotoxicity) or an organ such as the liver

(hepatotoxicity). By extension, the word may be metaphorically used to describe toxic effects on

larger and more complex groups, such as the family unit or society at large. Sometimes the word

is more or less synonymous with poisoning in everyday usage.

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Types of Toxicity

Toxicity is the degree to which a chemical substance or a particular mixture of substances

can damage an organism. Acute toxicity involves harmful effects in an organism through a single

or short-term exposure. Subchronic toxicity is the ability of a toxic substance to cause effects for

more than one year but less than the lifetime of the exposed organism. Chronic toxicity is the

ability of substance or mixture of substances to cause harmful effects over an extended period,

usually upon repeated or continuous exposure, sometimes lasting for the entire life of the

exposed organism.

Brine Shrimp

Artemia salina is a type of aquatic crustaceans also known as brine shrimps. They can

coexist with different aquatic animals such as fishes and can also live in waters with high salinity

levels. Their prominent characteristic are the possession of three eyes and small resilience to

toxic environments.

They are resilient to their environment and can tolerate to saltwater with 50% salinity

level. Their color mainly depends on the salinity level of their environment with higher

concentrations making them to have a slight red color. They mainly feed on green algae and they

can die when put to freshwater.

Their resilience both to varying salinity levels and toxicity makes them ideal specimen

for researches or studies concerning toxicity. They are widely used in experiments due to the fact
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that they can easily reproduce and when they do, they produce a large number of offspring.

Moreover, the eggs of their species can survive for years.

Artemia salina are found in about 500 natural salt lakes and man-made salt lakes

scattered throughout the tropical, subtropical, and temperate climatic zones. They cannot migrate

from one saline biotope to another because they depend on their adaptations on the waters with a

high salinity level to avoid predation and competition with other animals that feed on green

algae.

Their adaptations such as resilience to high salinity levels provide an efficient ecological

defense to predators. They also possess the capacity to synthesize their efficient respiratory

pigments to cope with environments that have a low oxygen level. As a result of their adaptation

to such salinity levels, they cannot cope with a non-existing salinity saturation.

Among the diets of fish and shellfish also consists of Artemia salina. As their eggs are

available year round, they are widely used as “on demand” live feed to such fishes. At present,

Artemia salina are used and exploited in over 5 countries.

Their eggs are called “cysts” that float on water surfaces and can be swept by waves and
winds. This is the only natural way of their propagation of seeds. These cysts are metabolically
inactive and do not further develop as long as they are kept dry.
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DEFINITION OF TERMS

These following terms are the most common terms that you could encounter in

this study. The following are defined operationally as how it is used in the study.

Abbot’s Mortality – where x is the number of dead Brine shrimps and y as the number of

currently dead Brine shrimps in a treatment. This is used to correct the percentage mortality rate.

Artemia salina – an aquatic crustacean that coexist with other aquatic animals. Can live in

saltwater with 50% salinity level. Also known as brine shrimp.

Brine Shrimp Lethality Assay – it is conducted to measure to toxicity rate of the seed extract of

a fruit and can provide the result of effectiveness of a seed extract to have toxicity effect

Carica Papaya – an oval-shaped fruit with numerous seeds. Simply, it is called as papaya.

Cysts – It is the eggs of the Artemia salina. A demand as a food for some aquatic animals.

Toxicity – degree to which a substance can harm animals. It is to describe toxic effects on larger

and more complex groups like society or community.

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Methodology

Gathering of Materials

The papaya (Carica papaya) and salt was bought from the public market of San Pablo City,

Laguna. 70% Ethyl alcohol, jar, plastic container, air pump, and 3 liter of distilled water was

bought from the grocery store. The mortar and pestle, weighing scale and beakers was borrowed

from the school’s laboratory.

Extraction of Papaya Seeds

Using the outdoor drying method, the papaya seeds will be dried in a container and

afterwards it will be pulverized using the mortar and pestle. The pulverized seeds will be put in a

jar together with the 70% ethyl alcohol and will be placed in room temperature for 5 weeks.

Preparation of Treatments

A modified dilution procedure making use of an alcoholic type of extraction will be used

in the preparation of the concentrations. In the Brine Shrimp Lethality Assay, varying amounts of

the seed extracts will be used with the 100 mL of artificial seawater. The varying concentrations

are shown on the table below.

Treatment 1 Treatment 2 Treatment 3 Positive control

5 mL seed extract 10 mL seed extract 15 mL seed extract

500 mL water
15 mL ethyl alcohol 10 mL ethyl alcohol 5 mL ethyl alcohol
9
2 Replications 2 Replications 2 Replications 2 Replications

Brine Shrimp Lethality Assay

 The Brine Shrimp Lethality Assay will be conducted to assess the toxic effect of the

Carica papaya seed extract. The bioassay, once conducted, will provide adequate results on the

effectivity of the Carica papaya seed extract to have a toxic effect.

The artificial saltwater solution will be prepared using 22.0 grams of sodium chloride

with 1 liter of distilled water. More than 200 brine shrimp eggs will be allowed to hatch on the

artificial saltwater solution under constant aeration and illumination from a fluorescent lamp for

24 hours. After hatching, number of hatched brine shrimps will be put in different plates and will

be applied with the varying concentrations of the seed extract with the use of a micropipette. The

inactivity of Artemia salina for 15 seconds and the lying of the brine shrimp on the bottom of the

plates will be the basis for the lethality of the extract. The percentage mortality (%M) will be

calculated by dividing the number of dead brine shrimps to the total number of brine shrimps,

then multiplied to 100. If the number of dead brine shrimps within 18 hours will exceed that of

the ones measured on the previous measurements, Abbott’s mortality formula will be used to

correct the percentage mortality rate.

Abbott’s Mortality formula

% mortality due to treatment =

Wherein x = no. of dead brine shrimps in the treatment prior to

y = no. of currently dead brine shrimps in the treatment

Data Analysis
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Comparison between different amount of papaya seed extracts by means of One-Way

Analysis of variance (ANOVA). Difference with P<0.05 between experimental and control

groups will be considered.

Risk Assessment

There are minimal expected hazardous or toxic effects from the use of the chemicals that will be

used. In inclusion, the researchers will be orientated of the different procedures to be familiarized

with the processes that will be performed.

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Results and Discussion

The treatments were added with varying amount of ethyl alcohol in order to diffuse it.

The brine shrimps are determined to be dead if they exhibit no sign of movement for 15 seconds.

The brine shrimps are put in a container with 500 mL of distilled water that will be mixed with

the treatments. The results are as follows.

First replication:

Treatment Results

Treatment 1 Alive – 8

15 mL ethyl alcohol, 5 mL extract Dead -28

Treatment 2 Alive – 12

10 mL ethyl alcohol, 10 mL extract Dead -23

Treatment 3 Alive – 13

5 mL ethyl alcohol, 15 mL extract Dead -22

Negative control Alive – 30

500 mL distilled water Dead -2

Second replication:
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Treatment Results

Treatment 1 Alive – 11

15 mL ethyl alcohol, 5 mL extract Dead – 21

Treatment 2 Alive – 33

10 mL ethyl alcohol, 10 mL extract Dead – 57

Treatment 3 Alive – 35

5 mL ethyl alcohol, 15 mL extract Dead -43

Negative control Alive – 52

500 mL distilled water Dead – 4

The objective of this study was to determine the effect of the concentration of the Carica papaya

seed extract to the number of brine shrimp killed. Here are the hypotheses:

2. The ethanolic extract from Carica papaya will have an effect to the mortality rate

of brine shrimps.

3. The ethanolic extract from Carica papaya will not have a significant effect on the

mortality rate of brine shrimps.

After the extraction of the Carica papaya seed extract, it was teste and the results were recorded.
In analyzing the results One-way ANOVA is used. One-way ANOVA was used to determine
whether the independent variables will significantly affect the dependent variable.
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First Replication:

Std.
Mean n Dev  
15 mL ethyl alcohol, 5 mL distilled
18.0 2 14.14 water
10mL ethyl alcohol, 10 mL
17.5 2 7.78 distilled water
5mL ethyl alcohol, 15 mL distilled
17.5 2 6.36 water
16.0 2 19.80 Negative control
17.3 8 9.98 Total

ANOVA
table
p-
Source SS df MS F value
Treatment 4.50 3 1.500 0.01 .9987
693.0 173.25
Error 0 4 0
697.5
Total 0 7      

Second Replication:

Std.
Mean n Dev  
15 mL ethyl alcohol, 5 mL distilled
16.0 2 7.07 water
10mL ethyl alcohol, 10 mL distilled
45.0 2 16.97 water
5mL ethyl alcohol, 15 mL distilled
39.0 2 5.66 water
28.0 2 33.94 Negative control
32.0 8 18.91 Total

ANOVA
table
p-
Source SS df MS F value
Treatment 980.00 3 326.667 0.86 .5312
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Error 1,522.00 4 380.500

Total 2,502.00 7      
Summary and Conclusion

Ethanolic extract from Carica papaya seeds was investigated in this study to identify

whether it may contain toxic properties. The dried papaya seeds were pulverized and were mixed

with ethyl alcohol for 5 weeks to get the extract needed for the study. The extracts were then

mixed with ethyl alcohol to get the desired concentration needed. Only two replications are done

for each concentration in the experiment have more reliable results. To analyse the results, One-

way ANOVA is used.

The study revealed that among the concentrations of Carica papaya seed extract, with 15

mL extract and 5 mL alcohol treatment killed the least number of brine shrimp. It is

hypothesized that this may be due to the higher concentration of ethyl alcohol in other

treatments.

Furthermore this study showed that the concentration of Carica papaya seed extract does

not affect the mortality rate of brine shrimp. One-way ANOVA showed that the concentration of

Carica papaya seed extract does not affect the number of brine shrimp killed.
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Recommendations

To further improve the results of the study, the following recommendations are suggested.

1. For the proceeding researchers with a similar topic of this paper, test the setups with more

replicates for a more precise result.

2. Use a water based extraction instead of ethanolic based extraction to further reduce the

mortality rate of brine shrimps.

3. Use a different species of test subject on testing the toxicity of the extract.
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APPENDIX A

Experimental Design Diagram

Title: Toxicity of Carica papaya seed

Question: Is there a significant effect on the Carica papaya seed extracts to the brine shrimp?

Hypothesis: There is no significant effect on the Carica papaya seed extracts to the number of

brine shrimp

Dependent Variable:

 Number of brine shrimp

Constant Variable:

 Type of water

 Number of trials

 days of culturing

 Amount of water

 Type of salt solution applied

 Amount of salt applied

 Hatching time

 Shrimp used (Artemi salina)

 APPENDIX B

First replication:

35

30

25

20
Alive
15 Dead

10

0
Treatment 1 Treatment 2 Treatment 3 Positive control

Second Replication:

23
60

50

40

30 Alive
Dead

20

10

0
Treatment 1 Treatment 2 Treatment 3 Negative control
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Appendix C

Flow Chart

Prepation of
Materials

Extraction of
Papaya Seeds

Preparation of
Treatments

Brine Shrimp
Lethality Assay

Data Gathering
and Analysis
 Appendix D

Acquiring papaya seeds Pulverizing the papaya seeds

after drying the seeds in 5 days

Pulverized seeds
will be added in
450 mL ethyl
alcohol

Preparation of treatments

Hatching of Brine shrimps

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26

First treatment, first replicate: The 15 mL ethyl

alcohol with 5 mL seed extract. The Brine
shrimps were observed after the extracts were
applied

Second treatment, first replicate: The 10 mL ethyl

alcohol with 10 mL seed extract. The Brine shrimps
were observed after the extracts were applied

Third treatment, first replicate: The 5 mL ethyl

alcohol with 15 mL seed extract. The Brine shrimps
were observed after the extracts were applied
Negative control, first replication: The Brine shrimps
were placed in 500 mL distilled water and after that
the Brine shrimps were observed
27

Third treatment, second replicate: 5 mL ethyl alcohol

with 15 mL seed extract. The Brine shrimps were
observed after the extracts were applied
First treatment, second replicate: 5 mL ethyl alcohol
with 15 mL seed extract. The Brine shrimps were
observed after the extracts were applied

Negative control, first replication: The Brine shrimps

were placed in 500 mL distilled water and after that
the Brine shrimps were observed
Second treatment, second replicate: 10 mL ethyl
alcohol with 10 mL seed extract. The Brine shrimps
were observed after the extracts were applied