Philippine Christian University i

Senior High School

“Cytotoxicity of Leach Collected from Manila Dumpsite using

Macroscopic Analysis of Allium Cepa as

Parameter”

A Research Paper Presented to the Faculty of the Philippine Christian

University Union High School of Manila General Malvar Street, Malate,

Manila

In Partial Fulfillment of the Requirements in Research

12-Stephen

Researchers:

De Angel, Marc Ramon Emmanuel

Favorito, Marl Zyrenne

Gravador, Dianah

Lomibao, Allen Ray

Masangkay, Jyrrianne

Matic, Kim

Palmos, Samantha Nicole

Pasco, Ezekiel

Poblete, Rapahela Mae

Rapelo, Calista Rickhart

 Philippine Christian University ii
Senior High School

Abstract

The cytotoxicity of leach from Manila dumpsite was determined

using Allium Cepa as parameter and its biomarkers. Since a dumpsite has

toxic wastes such as plastics, styrofoams, bottle with chemicals, or heavy

metals it can affect the soil and flow of water that is happening

underground. Thus, contaminating the water of neighboring houses near the

dumpsite, especially if they are using ‘poso’.Macroscopic Analysis of

Allium Cepa is what the researchers used. Allium Cepa is a standard in

environmental monitoring. It has many advantages such as it is low cost,

fast growth, easy to handle, good chromosome conditions for the study of

chromosome damage or disturbance. Some studies showed that Allium

Cepa test can be considered as an indicator in testing chemical that may be

harmful to the humans and to the environment. The researchers used

Macroscopic Analysis to observe the changes in the Allium Cepa caused by

the leach. As the onion was cleaned and the primordial roots were scraped,

the Allium Cepa was placed in five test tubes per site containing the leach.

The experiment lasted for 3 days. To get the results the roots were counted

and measured using a tape measure. Based on the research done, the results

showed that the leach from site 1, 2, and 3 are not toxic as it reached the

average number, length, and color of roots of the Negative Control. The

Manila dumpsite is not that toxic to contaminate water and put to risk our

environment and us, human beings.

 Philippine Christian University iii
Senior High School

Acknowledgement

We the researchers would like to express our deepest gratitude to the

Philippine Christian University and especially to the Department of Public

Services for sharing both of their knowledge and their resources for the

fulfillment of the study. We also want to extend our gratitude to Mr.

Angelito Alfonso for giving us the permission to make this research

happened, especially to our research adviser Ms. Virgines Mendoza, for

helping us and for seeing the study through and through. Finally, the

authors extend their deepest gratitude to the Almighty God, that in all

things, He may be praised.

 Philippine Christian University iv
Senior High School

Table of Contents

Title Page…………………………………………………………………...i
Abstract…………………………………………………………………….ii
Acknowledgment………………………………………………………….iii

Table of Contents………………………………………………………….iv
Chapter 1: The Problem and its Background………………………………1
1.1 Background of the Study……………………………………………….1
1.2 Statement of the Problem………………………………………………2
1.3 Hypothesis……………………………………………………………...2
1.4 Significance of the Study………………………………………………3
1.5 Scope and Limitation…………………………………………………..3
1.6 Definition of Terminologies……………………………………………4
Chapter 2: Review of Related Literature and Studies……………………...6

Foreign Related Literature ………………………………………………...6

Local Related Literature…………………………………………………...7
Foreign Related Studies……………………………………………………8
Local Related Studies……………………………………………………..15
2.2 Synthesis………………………………………………………………17
2.3 Theoretical Framework and Conceptual Framework…………………18
Chapter 3: Methodology…………………………………………………..19
3.1 Research Design………………………………………………………19

3.2 Research Locale………………………………………………………20

3.3 Data Gathering Procedure…………………………………………….20
3.4 Sources of Data……………………………………………………….21
3.5 Statistical Analysis……………………………………………………21
Chapter 4: Presentation, Results, and Discussions………………………..23
Chapter 5: Summary of Findings, Conclusion, Recommendation………..27
 Philippine Christian University
Senior High School

References………………………………………………………………...30
Appendices………………………………………………………………..33 1

CHAPTER I

THE PROBLEM AND ITS BACKGROUND

1.1 Background of the Study

Within the past few years we have been suddenly awakened to the

dangers caused by the mismanagement of wastes. We are now faced with

dealing with past waste accumulations, as well as with tremendous task of

creating new guidelines and solutions to fight with ever-increasing

quantities of waste. There are various types of waste but municipal waste

can be properly managed without causing any pollution. Municipal solid

waste is now called ‘solid waste’ (Bharti, Sisngh, 2017).

Different hazards are associated with waste dump sites, such as surface

water contamination, soil water contamination, bad smell or odor,

greenhouse gas release, accidental fire hazard, slope instability, vegetation

loss, soil contamination and bird-hitting, etc. These problems are due to the

lack of liners, the lack of cover and poor treatment (Yadav, Kumar, et.al.,

2018). Harmful chemicals from plastic bottles breaks down and releases

DEHA (Bis (2-ethylhexyl) adipate), a type of carcinogen that can cause

health problems. These types of chemicals can leach into the soil and cause
2
contamination.

The Allium Cepa test has been used for monitoring the genotoxic,

 Philippine Christian University
Senior High School

cytotoxic, and mutagenic nature of different test chemicals. The use of the

Allium cepa test system is advantageous since its main component is a

vascular plant, making it an excellent genetic model for assessing

pollutants in the environment.

This study aims to measure the level of toxicity and contamination of

leach collected from Manila dumpsite with the use of Allium Cepa as a

parameter. The following biomarkers will be the number of roots

germinated, length of roots, and the color of roots. “Macroscopic” analysis

of a product refers to an evaluation of the substance through the use

unaided sense of an individual. This method of analysis uses different

biomarkers (i.e. number of roots germinated, length of roots, and color of

roots) of the parameter in determining the environment pollutant.

1.2 Statement of the Problem

This research aims to observe the cytotoxicity of macroscopic

parameter of leach collected from Manila dumpsite using Allium Cepa.

Specifically, it seeks to find the answer to these questions:

1. What is the level of toxicity of leach collected from Manila

dumpsite?

2. Is there a significant difference on the level of toxicity of leach

collected from the site 1, site 2, and site 3 using Allium Cepa in

terms of the number of roots germinated, length of roots, and color

of roots?

1.3 Hypothesis

There is no significant difference between the level of toxicity of leach

 Philippine Christian University
Senior High School

collected from site 1, site 2, and site 3 using Allium Cepa in terms of the

number of roots germinated, length of roots, and color of roots.

1.4 Significance of the Study

The results of the study will benefit the following people:

 Environmentalists. Data will be served as baseline to create an

action plan to lessen or reduce the risk of soil contamination or

water contamination.

 Department of Environment and Natural Resources

(DENR). The organization may create a program or a guideline

regarding the environmental problem near the site raise

awareness.

 Non-Government Organizations. This research may serve as a

basis on what action must be done to be able to improve the

condition of environment.

 Future researchers. The result will be a useful guide and

reference for the future researchers in terms of finding

information about the topic.

1.5 Scope and Limitations

This study will conduct an experiment of Macroscopic Analysis of

 Philippine Christian University
Senior High School

Leach collected from Manila dumpsite using Allium Cepa. The possible

effects of leach to the Allium Cepa can be seen through the length of roots,

number of roots germinated, and its color. The sample soil will be collected

from Manila dumpsite. The Allium Cepa (onion)

1.6 Definition of Terminologies

 Allium Cepa- Commonly known as onion. Used as a bioindicator of

environmental pollution (Bagatani et al. 2009; Leme&Marin-

Morales, 2009)

 Color of roots- It is one of the biomarkers that can indicate the level

of toxicity of leach.

 Cytotoxicity- Determines the potential toxicity of a substance

including plant extracts or biologically active compounds isolated

from plants (McGaw, L. &Eloff, J., 2014). In this study, the

cytotoxicity of leach will be measured using Allium Cepa’s

biomarkers.

 Dumpsite- It is a facility in which solid waste from municipal

and/or industrial sources is disposed; sanitary landfills are those that

are operated in accordance with environmental protection standards

(Tse, A.C. &Adamu, C. I., 2012). This is where the leach that will

be used in the experiment will be collected.

 Philippine Christian University
Senior High School

 Germinated roots- It is the number of roots germinated when the

experiment is performed.

 Leaching- The process of extracting substances from solid by

dissolving them in a liquid, naturally (Ricardson, 2016).

 Length of roots- The size of roots in length that grows when the
5
Allium Cepa is planted on the leach of soil.

 Macroscopic Analysis- Method of observation, description, and

analysis of the macroscopic features (Cium, Shuaijian, et al., 2019).

In this study it is defined as observed in Allium Cepa’s biomarkers

such as number of roots germinated, length of roots, and color of

roots.

 Parameter- It is used as data for a wide range of published

ecological and hydrological model (Breuer, L. &Frede, H.G., 2003).

The parameter used in this study is Allium Cepa to determine the

different biomarkers that shows the level of toxicity.

 Philippine Christian University 6
Senior High School

CHAPTER II

REVIEW OF RELATED LITERATURE AND STUDIES

Foreign Related

Allium Cepa, commonly known as onion, belonging to the order Liliales

Literature

and family Liliaceae. The maximum diversity of Allium species is found in

a belt from the Mediterranean basin to Iran and Afghanistan, i.e. Iran, north

Iraq, Afghanistan, Soviet middle-Asia and West Pakistan, indicating the

primary centre of origin. The earliest record comes from Egypt where

onions appear as carvings on pyramid walls and in tombs from the third

and fourth dynasties (2700 BC). Allium Cepa is used as a potential

biomarker of genotoxic studies (Firbas and Amon, 2013). Widely used as a

bioindicator of genotoxicity from the different aquatic environs. This test

helps to evaluate mutagens and detecting toxic substances found in the

environment (El-Shahaby et al., 2003). Also, it is known as a fundamental

biomarker to evaluate environmental pollution (Bagatini et al., 2009;

Leme and Marin-Morales, 2009). The Allium Cepa test has been used by

many researchers mainly as a bioindicator of environmental pollution

(Bagatini et al. 2009; Leme & Marin-Morales, 2009), testing crude extracts

of cyanobacteria

(Laughinghouse, 2007), as well as to evaluate the genotoxic potential of

medicinal plants (Fachinetto & Tedesco, 2009; Dalla Nora et al. 2010),

because this test uses a model that is adequately sensitive to detect

innumerous substances that cause chromosomal alterations.

 Philippine Christian University
Senior High School
7

8
Local Related Literature

Phytochemicals are substances obtained from plants that provide

health benefits. Onions, scientifically named Allium Cepa Lin., of the

Liliaceae family, have been known to contain phytochemicals (Jaiswal et

al., 2013; Gregorio et al., 2010). Red Allium cepa Lin. var. ascalonicum

was obtained from three different regions in the Philippines, namely

Region I (Pangasinan), Region II (Nueva Vizcaya), and Region III (Nueva

Ecija). Onions from the three sources were ground separately, and 80%

ethanol was used for extraction. Crude ethanolic extract was then purified

through column chromatography using ethyl acetate:formic acid:water

(8:1:1) as solvents. Eluates were tested for the presence of flavonoid using

Magnesium Turning-Wilstatter Cyanidin test as screening method and Thin

Layer Chromatography as confirmatory test (Guevarra et al., 2005). Ferric

chloride and potassium ferricyanide were used as spray reagents to

visualize the chromatogram. Phenotype varieties of onions, whether red,

white, yellow or golden, have differences in their flavonoid content

(Yamada et al., 2004). On the other hand, growing condition, whether

grown hydroponically or in potting soil, showed no significant difference in

the onion composition (Lapointe et al., 2006). According to the National

Onion Association, approximately 87% of the onions produced in the

United States are yellow, with about 8% red onions and 5% white onions.

While in the Philippines, the most common onion varieties found in the
 Philippine Christian University
Senior High School

marketplace are the red onions and the white onions (Agribusiness, D.A.

2003). Only the red onions are included in this study. The effect of

flavonoids present in the crude ethanolic extract of red Allium Cepa Lin.

was identified.

The Allium Test is suggested as a standard in environmental

monitoring, e.g. as a part of a test battery. Background, the method

including suggested parameter for standard use, results from various

application areas and comparison with a number of other test systems are

presented. The Allium test is a short term test with many advantages. Low

cost, easy to handle, good chromosomes conditions for the study of

chromosome damage or disturbance of cell division including the

evaluation of aneuploidy. The ability of the root cells to activate

promutagens (The MFO system) further widens the application areas of the

Allium test. The use of series of onions for each concentration values are

obtained. The Allium test, lastly, a sensitive test showing good correlations

to other test systems. Thus, positive result in the Allium test should be

considered as a warning and also indication that the tested chemical may be

a risk to human health and to our environment. (g. Fisk esjo 2008)
9
Foreign Related Studies

Based on Bioindicator of Genotoxicity: The Allium cepa Test.

The Allium cepa test has been used by many researchers mainly as a

bioindicator of environmental pollution (Bagatini et al. 2009; Leme &

Marin-Morales, 2009), testing crude extracts of cyanobacteria

(Laughinghouse, 2007), as well as to evaluate the genotoxic potential of

 Philippine Christian University
Senior High School

medicinal plants (Camparoto et al. 2003; Knoll et al. 2006; Fachinetto et al.

2007; Lubini et al. 2008; Fachinetto et al. 2009; Fachinetto & Tedesco,

2009; Dalla Nora et al. 2010), because this test uses a model that is

adequately sensitive to detect innumerous substances that cause

chromosomal alterations. The Allium cepa test is important since it is an

excellent model in vivo, where the roots grow in direct contact with the

substance of interest (i.e. effluent or complex medicinal mix being tested)

enabling possible damage to the DNA of eukaryotes to be predicted.

Therefore, the data can be extrapolated for all animal and plant

biodiversity. The analysis of chromosomal alterations can be equal to the

test of mutagenicity mainly for the detection of structural alterations;

however, it is possible to observe numerical chromosomal alterations, as

10
well.

The Allium Cepa test consists in obtaining onion bulbs cultivated

without the application of herbicides or fungicides. After obtaining the

bulbs, they should be scraped at the root to promote the emergence of new

roots. Other bioassays can be performed with Allium cepa seeds placed to

germinate in a BOD (biochemical oxygen demand) incubator with

controlled temperature, which are used for allelopathy testing and also

genotoxicity assessment. To set-up the experiment allowing rootlets to

grow, all bulbs should be placed initially in a small 50 mL plastic cup

(Figure 1), containing distilled or tap water (being that it is potable), for

approximately 03 to 04 days so rootlets can emerge. After this period, the

bulbs should be transferred to other clean and dry containers including the
 Philippine Christian University
Senior High School

treatments. The plastic cups for water control treatment can be reused, to

minimize the effect of plastic on the environment. In general, we use 5

groups of bulbs of Allium cepa for each treatment, with one being a

negative control in water and another for the positive control in

methylsulfonylmethane (MMS) or glyphosate. The issue of positive control

is cited by Rank (2003) using methylsulfonylmethane and by Souza et al.

(2010) using glyphosate. Through the studies developed to date at the

Laboratory of Plant Cytogenetics and Genotoxicity (LABCITOGEN) of the

Department of Biology at the Federal University of Santa Maria, various

concentrations of glyphosate have shown chromosomal changes directly to

the rootlets. Its residual effect on the environment after us is not proven.

After rooting, the bulbs of the two control groups should remain in the

water (negative control) and in the respective positive control, and the

remaining should be transferred to the chosen treatments, which can be

solutions of essential oil, leaf extracts by infusion, root or stem extracts by

decoction, or samples of industrial and/or hospital effluents. These should

11
remain in the dark for exactly 24 hours.

It is emphasized that in a case where a product such as ethanol PA is

used for the dilution of the oil in which the rootlets of Allium cepa will be

immersed, the same should consist of one of the treatments. The main idea

is to minimize the error, allowing the results to express the effect of the

substances that are interacting. Therefore, when possible, test them

separately. After the rootlets are submitted for 24 hours in the individual

treatments, they should be collected and immediately fixed in ethanol:

 Philippine Christian University
Senior High School

acetic acid (3:1), also for 24 hours. Afterwards, the rootlets should be

removed from the fixing solution and transferred to ethanol 70% and be

kept under refrigeration (4°C) until used. It is important to emphasize that

all glassware used to keep the rootlets should be identified with a number

or sample name and/or treatment, as well as date, using small tags written

in pencil on the inner and outer part of the glass, thus avoiding any kind of

mistake with the samples. The technique of bulb use is described by

Fiskesjö (1994) and adapted by researchers who use the test, such as Knoll

et al. (2006) and Fachinetto et al. (2009). In the next stage, slides are

prepared, analyzing 1000 cells per bulb, totaling 5000 cells per treatment or

variations of these values, such as 500 cells per bulb, totaling 2500 cells. It

has already been proven that one rootlet is enough for observing the

damage caused to the DNA of Allium cepa, observing the cells after the
12
treatment with mutagenic agents.

The disposal of municipal waste in landfills or dumpsites may pose

an environmental problem. The product of the decomposition of these

residues generates large volumes of leach, which may present high toxicity.

The aim of this study was to assess the toxic and genotoxic effects of a

sample of untreated leachate in fish (Leporinusobtusidens) and onions

(Allium cepa). The leach was collected in a landfill located in the region of

Vale do Rio dos Sinos, southern Brazil. The fish were exposed to raw

leachate, at concentrations of 0.5%, 1.0%, 5%, 10% and 20% for 6 days,

while the bulbs of Allium Cepa were exposed to concentrations of 5%,

10%, 25%, 50% and 100% for 48 h. For fish, the concentrations of 5%,
 Philippine Christian University
Senior High School

10% and 20% were lethal, thus indicating high toxicity; however, sublethal

concentrations (0.5% and 1.0%) showed no genotoxicity by micronucleus

test when compared with the control group. Base on the observation and

bioassay involving onions, high toxicity was observed, with significant

reduction of root growth and mitotic index in bulbs exposed to the 100%

concentration of the leachate. An increase in the frequency of chromosome

abnormalities in the Allium Cepa root cells in anaphase-telophase was

observed in accordance with the increase in the concentration of leachate

(5%, 10%, 25% and 50%), with values significantly greater than the

control, at the highest concentration. The results showed that the leach

collected contains toxic and genotoxic substances, thus representing a

major source of environmental pollution if not handled properly. (Klauck

13
C. R., Rodrigues, M. A., et. al., 2013)

Based on Assessment of genotoxic effects of pesticide and

vermicompost treated soil with allium cepa (Datts, S. et al, 2018). Soil

forms a huge reservoir of nutrients that sustains life on earth.

Anthropogenic and natural impacts have led to degradation of land which

declines the overall quality of soil, water or vegetation. The present study

involves comparison of genotoxicity of soil procured from two different

agricultural sites, pesticide treated soil (PTS) and vermicompost treated soil

(VTS). The soil was physico-chemically characterized and showed

significant differences in terms of cytotoxicity (root length; mitotic index)

and genotoxicity (chromosomal aberrations) in Allium cepa test. The

mitotic index of the control after 24 and 48 h was found to be 26.1 ± 1.6
 Philippine Christian University
Senior High School

and 26.1 ± 1.3 respectively. Mitotic index was reduced to 10.3 ± 0.9 and

9.7 ± 0.6 in 100% PTS and 24.4 ± 1.7 and 25.4 ± 0.8 in 100% VTS after 24

and 48 h of exposure, respectively. Clastogenic aberrations were found to

be highest (54.5%) in 100% PTS which was significantly different from

VTS extract. The PTS extracts incurred significantly more cytotoxic and

genotoxic effects on A. cepa in comparison to VTS. The result indicates

that addition of vermicompost in agriculture field acts as soil ameliorator

and plays an important role in promotion of cell division and proliferation,

14
hence good for the plant health and crop productivity.

According to “Assessment of Heavy Metal Composition and

Cytogenotoxic Risk Potential of Dumpsite Soil and Water Collected

from Kogi State University Student Halls of Residence” (Onoja, A.O et.

Al, 2019) Waste disposal is a vital environmental problem in both

developed and developing countries. Nigeria generates an estimate of 29.78

x 10 9 kg waste annually which decomposes to produce toxicants with high

concentrations which pose serious ecological and health risks to organisms.

This research was carried out to evaluate the heavy metal composition as

well as cytogenotoxic risk potential of soil and water samples collected

from the halls of residence of Kogi State University, Nigeria. The

concentration of heavy metals (Copper (Cu), Iron (Fe), Lead (Pb), Zinc

(Zn), Nickel (Ni), Arsenic (As), Cadmium (Cd), and Chromium (Cr)) in

soil samples were determined using Atomic Absorption Spectroscopy

(AAS) and pollution index (PI) was assessed by considering the combined

contributions of all studied metals while cytogenotoxic potential of soil and

 Philippine Christian University
Senior High School

water samples were assessed by using the Allium cepa test. The average

concentration of metals in the dumpsite soils followed the trend

Fe>Zn>Cu>Ni>Cr>Cd>Pb>As. Pollution index (PI) result revealed all

dumpsite soil samples were within the range of moderate to strong

pollution indicating a potential risk to human health. The samples induced

significant inhibition of Allium cepa root growth, decreased Mitotic index

(MI) down the groups and induction of cell abnormalities compared to the

control. The observed cytogenotoxic effects were assumed to be instigated

by the heavy metals present in the samples. These results suggest that the

investigated samples may be contaminated and may be of health risk to the

human population in case of exposure through dermal contact and direct

ingestion. A serious and urgent introspection of these sites is therefore

15
required by the stakeholders to mitigate this threat.

Based on Relationship Between Land Use and Water Quality

Obtained For the Evaluation Of Genetoxic Effects in Plant

Bioindicator. (N. Bone, P. Filno, et. Al 2019). Anthropic activities as well

as land use and occupation are closely linked to water and soil quality. An

alternative to assess their influence on water quality in bodies is through

biomonitoring using Allium Cepa as bioindicator organism. This technique

allows the detection of cytogenetic abnormalities in Allium Cepa not

merismatic cells after contract with water analyzed samples. Thus, we

evaluated the genetoxic potential water samples from the Rio DasAntas

(Antas River) long the urban perimeter of the city of Frati, Parana, Brazil.

With the aid of Geo Eyesatelite high-resolution fused orbital images, we

 Philippine Christian University
Senior High School

searched for possible relationships between the use of and occupation of

land around this river. Biomonitoring was performed at three different

sampling point. Statistical equality between the negative control and Point

1 and between Point 2 and 3 was obtained during Fisher’s test and a

Principal Coordinate Analysis (PCoA). The former presented low

frequency of abnormalities chromosomes and later presented larger

averages. In addition, Point 1 had strong influence of tree vegetation, Point

2 and 3, demonstrated a strong influenced of urbanized area, with

significant degradation of permanent preservation areas. The result shows

remarkable anthropogenic interference to the ecosystem. Furthermore, this

indicates importance of an APP (Areas de Preservacao Permanente) or

permanent preservation areas, watercourses functioning to preserve the

quality of the water resources.

Local Related Studies

According to C. Cabuga et al, 2017 Allium Cepa test: An

Evaluation of Genetoxcity, the importance of Allium cepa test contributes

knowledge in preventing toxicity in the environment. This test widely used

in determining genotoxic and cytotoxic substances found in the water

system. In this study, the genotoxic effects of H2O2 (Hydrogen Peroxide)

and CH2O (Formalin) were determined using Allium cepa root tip cells

while the water samples collected from the mining areas of Sorex Barobo,

Surigao del Sur and Rosario, Agusan del Sur were also tested for

genotoxicity. One-Way ANOVA shows that water samples treated with

H2O2 were statistically significant (P<0.05) when compared to CH2O

 Philippine Christian University
Senior High School

while the water sample collected from the mining areas shows statistically

non-significant. A concentration-dependent increased were observed among

the dividing cells and aberrant cells of the treated and collected water

samples. The observed abnormalities were seen into its roots morphology

indicating genotoxicity. Thus, the obtained results in this study show that
16
Allium cepa test is useful as bio-indicator to detect genotoxicity.

Based on Toxic effects of environmental pollutants:

Comparative investigation using Allium cepa L. and Lactuca sativa L

(Silveira, G. L. et al, 2017). Studies that help understand the mechanisms

of action of environmental pollutants are extremely important in

environmental toxicology. In this context, assays using plants as models

stand out for their simplicity and low performance cost. Among the plants

used for this purpose, Allium cepa L. is the model most commonly applied

for cytogenotoxic tests, while Lactuca sativa L., already widely used in

phytotoxic investigations, has been gaining prominence in cytotoxic

analyses. The present study aimed to compare the responses of A. cepa and

L. sativa via macroscopic (root growth) and microscopic analyses (cell

cycle and DNA fragmentation via TdT-mediated deoxy-uracil nick and

labeling (TUNEL) and comet assays) after exposure of their roots to

environmental pollutants with known cytogenotoxic mechanisms. Both

species presented sensitive and efficient response to the applied tests after

exposure to the DNA-alkylating agent Methyl Methanesulfonate (MMS),

the heavy metal Cadmium, the aluminum industry waste Spent Potliner

(SPL) and the herbicide Atrazine. However, they differed regarding the
 Philippine Christian University
Senior High School

responses to the evaluated endpoints. Overall, A. cepa was more efficient in

detecting clastogenic changes, arising from DNA breakage, while L. sativa

rather detected aneugenic alterations, related to chromosome segregation in

mitosis. In the tests applied to verify DNA fragmentation (comet and

TUNEL assays), A. cepa presented higher sensitivity. In conclusion, both

models are efficient to evaluate toxicological risks of environmental

pollutants. 17

According to Galarpe, 2017, The Republic Act (RA) 9003-

Ecological Solid Waste Management (ESWM) Act of 2000 of the

Philippines provides the mandate and framework for solid waste

management in the country. The implementation however on the local

government units reflects the lack of institutional arrangements for waste

management. This has been reflected through the utilization of unregulated

dumpsites and landfills exhaustively although other alternatives can be

considered. Primary reasons were drawn from inadequate technical and

financial resources, lack of political will, unwillingness of stakeholders,

and minimal local awareness. Consequently, there is absence of

comprehensive monitoring scheme of dumpsites and landfills operation in

the country. Present studies reviewed in this paper evidenced the threat that

disposal sites may pose to the environment from potential leaching of

hazardous chemicals due to dumped wastes.

2.2 Synthesis

Allium Cepa is used as a bioindicator in environmental monitoring. It was

 Philippine Christian University
Senior High School

used as a parameter in assessing the cytotoxicity of leach that is collected

from Manila dumpsite through its biomarkers.

18

2.3 Theoretical Framework

Manila Dumpsite

Site 1 Site 2 Site 3

Leach Negative Control

Allium Cepa

Conceptual Framework
 Philippine Christian University
Senior High School

Input Process Output

 Manila  Collection  There is no

Dumpsite  Cleaning significant
 Leach  Extraction difference
 Allium Cepa  Planting between the
 Harvesting level of
toxicity Site
1, 2, and 3
using Allium
Cepa
19

CHAPTER III

METHODOLOGY

3.1 Research Design

A Quantitative type of research will be done in this study. A

quantitative research design is defined as a systematic phenomena by

gathering quantifiable data and performing statistical, mathematical, or

computational techniques. Quantitative research collects information from

existing and potential customers using sampling methods and sending out

online surveys, online polls, questionnaires, etc., the results of which can

be depicted in the form of numerical. (Bhat, A., 2019). In the process of

conducting the experimentation there are certain number of set ups that will

be used and biomarkers of the parameter will be measured. The results of

this study will undergo statistical treatment.

While an experimental research design is any research conducted

with specific approach, where a set of variables are kept constant while the
 Philippine Christian University
Senior High School

other set of variables are being measured as the subject of the experiment.

Experimental research can gather a lot of data that can help you make

better decisions (Bhat, A., 2019). The experiment of this study will be done

in the cytotoxicity of leach collected from Manila dumpsite using

macroscopic parameter of Allium Cepa.

20

3.2 Research Locale

The study of this research will be conducted in Cabrera Pasay City.

3.3 Data Gathering Procedure

The research procedure shows how the researchers will conduct the

experiment.

3.3.1 Collection

The Leach will be collected in February 27, 2020 at the Manila

Dumpsite and will be stored in a laboratory glass bottle. The Allium Cepa

will be bought at the Pasay Public Market on March 14, 2020 and will be

stored in a plastic container.

3.3.2 Extraction

Adapted from the method by H. Brower (2018) Leaching the Soil,

the soil sample to be taken from the dumpsite is where the leach will be

extracted. The sample soil will be stored inside a plastic container. The pure

water will be poured down on the sample soil and the leach that has been
 Philippine Christian University
Senior High School

carried by the water will be the leach that the researchers will use to irrigate

the onion crops soaked on test tubes.

21
3.3.3 Planting

The Allium Cepa test consists in obtaining onion bulbs cultivated

without the application of herbicides or fungicides. After obtaining the

bulbs, they should be scraped at the root to promote the emergence of new

roots. With the same uniform size weighing about 3-3.5 kg, small onion

bulbs (Allium Cepa L.) were peeled exposing its outer scales so that the

root primordial were immersed into the different tested liquids with

different level of leach collected.

3.3.4 Harvesting

The exposure time of the experiment will be done in 72 at 22 C

where they are protected against direct sunlight within 3 to 7 days.

Macroscopic parameters include the root length and other parameters such

as usual root form, number, color, and turgidity after cultivation for 72

hours.

3.3.5 Materials and Specimen

Specimen:

-Leach

-Allium Cepa

Apparatus:

-Test Tubes

-Laboratory Glass bottles

- Sack Cloth or katya

 Philippine Christian University
Senior High School

(Quantitative) 3.4 Sources of Data

We will be able to know how much toxicity the dumpsite is based

on the changes on the Allium cepa. We should be able to define their level

of toxicity based on its biomarkers specifically; length of roots, number of

roots germinated, colour of roots.

3.5 Statistical Analysis

The Statistical Analysis tool will be used to determine the soil

toxicity from the leach collected using the macroscopic parameter

22
of Allium cepa.

Mean – it implies average and it is the sum of a set of data divided

by the number of data. It can prove to be an effective tool when

comparing different sets of data; however this method might be

disadvantage by the impact of extreme values.

Formula: ∫ χ÷Ν

Where:

∫= Sum of (add up)

χ = Values

Ν = number of values

ANOVA – It is one type of inferential statistics. It is used to

determine whether there is significant difference between the means of two

groups.
Philippine Christian University
Senior High School
 Philippine Christian University 23
Senior High School

CHAPTER IV

PRESENTATION, RESULTS, AND DISCUSSIONS

This chapter presents, analyze, and interprets data gathered in

this research study. The results are presented in the succeeding tables with

corresponding discussions and explanations.

Table 1. Number of Roots Germinated

NEGATIVE
SITE 1 SITE 2 SITE 3
CONTROL
TEST
28 17 6 11
TUBE #1
TEST
14 3 7 15
TUBE #2
TEST
11 19 15 15
TUBE # 3
TEST
13 14 17 14
TUBE #4
TEST
24 19 10 13
TUBE #5
x= 18 x= 14.4 x= 11 x= 13.6
Table 1

Table 1. Shows the average number of roots germinated. Data

shows that Site #1 had the highest roots that germinated and Site #3 shows

the lowest number of roots.

24

Table 2. Number of Roots Germinated

SUM OF MEAN OF
SOURCES DF F – RATIO
SQUARES SQUARE
 Philippine Christian University
Senior High School

BETWEEN 125.35 3 41.783

WITHIN 510.4 16 31.9 1.3098
TOTAL 635.75 19
Table 2

Table2 shows the computed value using the ANOVA is

1.3098 that is lower than the tabular data 3.24 at 0.05 level of significance.

Therefore, the null hypothesis is accepted, there is no significant difference

between the number of roots germinated in site 1, 2, and 3.

Table 3. Length of Roots Germinated

SITE 1 SITE 2 SITE 3 NEGATIVE

CONTROL
TEST 32mm 39mm 26mm 38mm

TUBE # 1
TEST 15mm 1mm 43mm 31mm

TUBE #2
TEST 19mm 16mm 57mm 16mm

TUBE #3
TEST 14mm 15mm 42mm 33mm

TUBE #4 25
TEST 25mm 32mm 51mm 12mm

TUBE #5
x= 21 x= 20.6 x= 43.8 x= 26.2
Table 3

Table 3 shows the length of roots in each site per Allium Cepa

measured by a tape measure using mm or millimeter measuring the longest

to the shortest of roots. Data shows that Site #3 has the longest number of

roots, while Site #2 has the shortest number of roots.

Table 4. Length of Roots

SOURCES SUM OF DF MEAN OF F – RATIO

 Philippine Christian University
Senior High School

SQUARES SQUARES
BETWEEN 1718.2 3 572.733
WITHIN 2235.6 16 189.725 4.099
TOTAL 3953.8 19
Table 4

Table4 shows the computed value using the ANOVA is 4.099 that is higher

than the tabular data 3.24 at 0.05 level of significance. Therefore, the null

hypothesis is rejected, there is a significant difference between the length

of roots in site 1, 2, and 3.

Table 5. Color of Roots

NEGATIVE
SITE 1 SITE 2 SITE 3
CONTROL
TEST White-
TUBE White White ish White
#1 Brown
TEST White- White- 26
TUBE ish ish White White
#2 Brown Brown
TEST White-
TUBE White ish White White
#3 Brown
TEST White-
TUBE White White ish White
#4 Brown
TEST White White- White-
TUBE and ish ish White
#5 Brown Brown Brown
Table 5

Table 5 shows the data of the color of the roots germinated in Allium Cepa

in site 1, 2, 3 and the Negative Control using five test tubes in each. The

common color in Site #1, Site #2 and Site #3 is White-ish brown.

Philippine Christian University
Senior High School
 Philippine Christian University 27
Senior High School

CHAPTER V

SUMMARY OF FINDINGS, CONCLUSION, AND

RECOMMENDATIONS

Summary of Findings

This research was conducted for the purpose of determining the

toxicity and contamination of leach collected in Manila dumpsite using

Allium Cepa as parameter. The method of research used was Macroscopic

Analysis. In using experimental research they were able to conduct the

experiment. It is a type of a true experimental research. Based on the results

of the research the following findings are made.

The level of toxicity of leach collected from Manila dumpsite

was derived from the selected biomarkers: Number of Roots Germinated,

Length of Roots and Color of Roots.

The level of toxicity of leach based on the number of roots

germinated showed that site 3 has the most toxic leach with an average of

11. In terms of length of roots, site 2 has the shortest roots with an average

of 20.6. The color of roots of site 2 and site 3 have equal results in which
28
there were 3 whitish-brown in each test tube.

In the number of roots germinated, the negative control has an

average of 13.6, site 3 has a lower average of 11 defines that when it is

below the negative control mean, which means site 3 can be considered as

toxic. While site 2 has an average of 14.4 and site 1 has an average of 18,

bigger value of average defines that it cannot be considered as toxic. The

 Philippine Christian University
Senior High School

computed value using ANOVA is 1.3098 that is lower than the tabular data

3.25 at 0.05 level of significance.

In the length of roots germinated, the negative control has an

average of 15-3, longest to shortest length measured using a tape measure

using mm or millimeter. Site 1 has an average of 26-1, Site 2 has an

average of 34-2, and Site 3 has an average of 54-3, bigger average than the

negative control signifies that it can be considered not toxic. The computed

value using ANOVA is 4.099 that is higher than the tabular data 3.24 at

0.05 level of significance.

The colors of roots germinated, negative control is having white

color of roots. Site 1 has a color of white to white-ish brown to having both

brown and white colors, having three white colors, one of white-ish brown

and white and brown, which indicates that it is slight toxicity since it has a

three color of white which is the negative control. Site 2 has a color of

white to white-ish brown, having two white and three of white-ish brown,

which indicates a slight toxicity. Site 3 has a color of white to white-ish

brown, having two white and three white-ish brown, which indicates a

slight toxicity.
29
Conclusion

Allium Cepa test shows emphasis on determining

environmental problems such as soil pollution and soil contamination. The

study conducted was made to determine the contamination and toxicity of

the leach extracted from the soil collected on Manila Dumpsite. Based on

the findings using One-Way ANOVA, the number of roots germinated on

 Philippine Christian University
Senior High School

Allium Cepa on site 1 has the most number of roots, while the least in

number of roots germinated is in Site 3 . In terms of length, Site 3 has the

longest roots and Site 1 has the shortest. Lastly in the color of roots, the

most common is having white-ish brown roots. Within the 3 days of

experimentation, there is a minor differences on data collected and

observed.

The response of Allium Cepa on the experiment shows the

capability of it to determine the potential soil contamination. This kind of

test is essential on determining different environmental condition.

Recommendation

Experimental results shows no difference of each site to the

negative control. Further research is recommended to the future researchers

by using Microscopic Analysis rather than Macroscopic Analysis, just like

what has been used in this study. Aside from seeing the physical changes of

Allium Cepa, through the Microscopic Analysis, the cell cycle and DNA of

the Allium Cepa will better determine the changes from the leach being

used. The researchers suggest in looking for a location in getting leach such

as rivers or streams. For the Department of Environment and Natural

Resources or DENR, the researchers recommend to raise awareness to the

people living near a dumpsite to avoid any risks that may affect their

health. Lastly for the NGO’s or Non-Government Organization to

implement movements that will help improving our environment such as

helping those people near dumpsites to have a clean surrounding.

 Philippine Christian University
Senior High School

30

References

1. Asemave, K. and Anhwange, B. (2013). Evaluation of Heavy

Metals.

2. Bakare, A.A., (2012). Cytogenotoxic effects of electronic waste

leachate in Allium Cepa. Volume 65, 2012 - Issue 2

3. Belen, M.A., et al. (2019). Assessment of the impact of open waste

dumpsite on groundwater quality: A case study of the Payatas

dumpsite in Quezon City, Philippines. IOP Conf. Ser.: Earth

Environ. Sci. 344 012050

4. Bone, N., et. al. (2019). Relationship Between Land Use and Water

Quality Obtained For the Evaluation Of Genetoxic Effects in Plant

Bioindicator. vol.14, n.2, e2299. ISSN 1980-993X.

5. Cabuga, C., et al. (2017). Allium Cepa test: An Evaluation of

Genetoxcity.

6. Datts, S. et al. (2018). Assessment of genotoxic effects of pesticide

and vermicompost treated soil with Allium Cepa. Sustainable

Environment Research Volume 28, Issue 4, Pages 171-178.

7. De la Calzada, G. & Pineda, M.R. (2013). Effect of Philippine Red

Allium cepa Lin. (Sibuyas na Pula) on Serum LDL-Cholesterol

 Philippine Christian University
Senior High School

Level. Vol 1 (3), pp, 014-020 31

8. Fiskesjo, G. (2008). The Allium test as a standard in environmental

monitoring, Volume: 102, Issue. 1, pp 99-112

9. Galarpe, V.R. (2017). Review on the Impacts of Waste Disposal

Sites in the Philippines, Sci.Int.(Lahore),29(1),379-385.

10. Laughinghouse, H.D. & Tedesco, S.B. Bioindicator of

Genotoxicity: The Allium Cepa Test IV: 137-153

11. Nagarajan, R., et al. (2012). Impact of leachate on groundwater

pollution due to non-engineered municipal solid waste landfill sites

of erode city, Tamil Nadu, India. J Environ Health Sci Engineer 9,

35. https://doi.org/10.1186/1735-2746-9-35.

12. Naveen, B.P., et al. (2018). A study on contamination of ground and

surface water bodies by leachate leakage from a landfill in

Bangalore, India. International Journal of Geo-Engineering volume

9, Article number: 27

13. Njagi, J.M., et al. (2016). Heavy Metal Pollution of the

Environment by Dumpsites: A Case of Kadhodeki Dumpsite, Int. J.

Life. Sci. Scienti. Res., VOL 2, ISSUE 2, pp: 191-197

14. Onoja, A.O et el. (2019). Assessment of Heavy Metal Composition

and Cytogenotoxic Risk Potential of Dumpsite Soil and Water

 Philippine Christian University
Senior High School

Collected from Kogi State University Student Halls of Residence”.

International Journal of Innovative Research & Growth 4(11):1-11 32

15. Pattanamahakul, P., et al. (2017). Pollution Status and Potential

Ecological Risk Assessment of Heavy Metals in Soils from a

Municipal Solid Waste Open Dumpsite in Thailand

16. Sarvesh, J.S., et al. (2009). Toxicity evaluation of pesticide waste

leachate by Allium Cepa root (growth and cytogenetic) assay.

International Journal of Applied Environmental Sciences 4(1):71-82

17. Silveira, G. L. et al, (2017). Toxic effects of environmental

pollutants: Comparative investigation using Allium cepa L. and

Lactuca sativa L. Chemosphere 178:359-367

 Philippine Christian University
Senior High School

33

Appendices

Solution of ANOVA in Chapter 4

Below is the solution in finding the computed value for the number of roots
germinated:
Number of Roots Germinated
N=20, n=5, a=4
Dfbetween = 4-1 = 3
Dfwithin = 20-4 = 16
Dftotal = 20-1 =19
Critical Value = (3,16)
= 3.2389 or 3.24
SSbetween= (Σ(Σai) /n) – (T2/N)
2
F= MSbetween / MSwithin
= (82 + 302 + 102 + 282 / 5 ) – (762/20) = 26.93 / 7.65
= (64+900+100+784 / 5 ) – (5776) / 20) = 3.52
= (1848/5) – 288.8
= 369.6 – 288.8
= 80.8

SSwithin= Σy2 – (Σ(Σai)2 /n)

= Σy2 – 369.6
= (72+02+02+02+12) + (62+42+62+92+52) + (02+02+32+02+72) + (22+ 82+32+72+82)
– 369.6
= (49+1) + (36+16+36+81+25) + (9+49) + (4+64+9+49+64) – 369.6
= (50) + (194) + (58) + (190) – 369.6
= 492 – 369.6
= 122.4

SStotal= Σy2 - (T2/N) MSbetween= 80.8 / 3

= 492 – 288.8 = 26.93
= 203.2 MSwithin= 122.4 / 16
= 7.65

Below is the solution in finding the computed value for the longest length
of roots:
N=20, n=5, a=4
Dfbetween = 4-1 = 3
Dfwithin = 20-4 = 16
Dftotal = 20-1 =19
Critical Value = (3,16)
= 3.2389 or 3.24
SSbetween= (Σ(Σai)2 /n) – (T2/N)
= (902 + 722 + 552 + 682 / 5 ) – (2852/20)
 Philippine Christian University
Senior High School

= (8100+5184+3025+4624 / 5 ) – (81225 / 20)

= (20933/5) – 4061.25
= 4186.6 – 4061.25
= 125.35 34

SSwithin= Σy2 – (Σ(Σai)2 /n)

= (282+142 +112+132+242) + (172+32 + 192+142 + 192) +
(6 +7 +152+172+102) + (112+152 +152+142+132) - 4186.6
2 2

= (784+196+121+169+576) + (289+9+36+196+361) +
(36+49+225+289+100) + (121+225+225+196+169) - 4148.6
= (1846)+(1216)+(699)+(936) – 4186.6
= 4697- 4186.6
= 510.4

SStotal= Σy2 - (T2/N)

= 4697 – 4061.25
= 635.75

Below is the solution in finding the computed value for the shortest length
of roots:
N=20, n=5, a=4
Dfbetween = 4-1 = 3
Dfwithin = 20-4 = 16
Dftotal = 20-1 =19
Critical Value = (3,16)
= 3.2389 or 3.24
SSbetween= (Σ(Σai) /n) – (T2/N)
2

= (132 + 112 + 42 + 72 / 5 ) – (352/20)

= (169+121+16+49 / 5 ) – (1225) / 20)
= (355/5) – 61.25
= 71 – 61.25
= 9.75
SSwithin= Σy2 – (Σ(Σai)2 /n)
= Σy2 – 71
= (22+112) + (42+22+22+1.52+1.52) + (1.52+2.52) = (22+ 12+12+22+12) –
71
= (4+121) + (16+4+4+2.25+2.25) + (2.25+6.25) + (4+1+1+4+1) – 71
= (125) + (28.5) + (8.5) + (11) – 71
= 173 – 71
= 102

SStotal= Σy2 - (T2/N)

= 173 – 61.25
= 111.75
Philippine Christian University
Senior High School