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THE EVALUATION OF GENOTOXICITY POTENTIAL OF WATER BODIES IN AND

AROUND METRO MANILA, PHILIPPINES USING ALLIUM CEPA METHOD.
*ZebaFarooqi Alam. Keith Alexius Wangkay and Saejin Oh. B...

Article  in  International Journal of Advanced Research · September 2016

DOI: 10.21474/IJAR01/xxx;

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Article DOI: 10.21474/IJAR01/xxx

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RESEARCH ARTICLE

THE EVALUATION OF GENOTOXICITY POTENTIAL OF WATER BODIES IN AND AROUND

METRO MANILA, PHILIPPINES USING ALLIUM CEPA METHOD.
*
ZebaFarooqi Alam. Keith Alexius Wangkay and Saejin Oh.
Biology Department, College of Science, De La Salle University, Taft Avenue, Manila, Philippines.
……………………………………………………………………………………………………....
Manuscript Info Abstract
……………………. ………………………………………………………………
Manuscript History Screening for hazardous chemicals and potential mutagens in water
bodies, which are often the recipients of contaminated and polluted
Received: xxxxxxxxxxxxxxxx substances, is being accepted as a routine method in environmental
Final Accepted: xxxxxxxxxxxx monitoring programs. In this study, Alliumcepa method was used to
Published: xxxxxxxxxxxxxxxx
evaluate the genotoxic potential of various water bodies namely- Pasig
and Marikina river and the Estero de Vitas located in and around Metro
Key words:- Manila, Philippines. Morphological modifications of the Alliumcepa
Alliumcepa Assay, Genotoxicity, roots, inhibition of root growth, mitotic index and the induction of high
Cytotoxicity, Pasig River, Marikina frequency of chromosomal aberrations comprising of chromosome
River, Estero de Vitas Chromosome
fragments, laggards, anaphase and telophase bridges and C- mitotic
Aberrations, Heavy Metals, Bio
Monitoring effect were observed in the Alliumceparoots exposed to water samples
from three sources tested as compared to the control. The observed
biological effects of the water samples appeared to be related to the
physicochemical characteristics studied using Atomic absorption
spectroscopy. The results of the investigation demonstrate that the
hazardous substances and pollutants present in the water bodies has
genotoxic and cytotoxic effects on Alliumcepa cells and there could be
a potential threat to the human health as well as to the water ecosystems
in and around Metro Manila. Hence the Alliumcepa test could be used
to get preliminary information about the presence of genotoxicand or
cytotoxic substances in the water bodies while performing the
environmental monitoring.

Copy Right, IJAR, 2016,. All rights reserved.

……………………………………………………………………………………………………....
Introduction:-
The pollution of the water bodies in and around urban areas all around the world including Philippines has become a
huge problem with the pollutants with genotoxic and mutagenic effects posing direct threat to the human health as
well as to the overall water ecosystems (O’Toole et al., 2009). The bodies of water in and around Metro Manila such
as Pasig and Marikina River are notorious for being polluted. The Pasig River System, which is composed of river
tributaries and several creeks and Esteros, was once inhabited by 25 varieties of fish and 13 types of aquatic plants.
Due to rapid industrialization, and the increase in the population of Metro Manila, only 6 fish species, which
includes Janitor fish (Pterygoplichthysdisjunctivus), Tilapia (Oreochromisniloticus), knife fish
(Apteronotusalbifrons), sea catfish (Ariidae), Bighead Carp (Hypophthalmichthysnobilis) and only 2 plant species
one of them being the water Hyacinth (Eichorniacrassipes), managed to survive in the polluted waters by the year

Corresponding Author:- ZebaFarooqi Alam.

Address:- Biology Department, College of Science, De La Salle University, Taft Avenue, Manila, Philippines.
ISSN: 2320-5407 Int. J. Adv. Res. 4(8), XX-XX

1997 (Helmer&Hespanhol, 1997). In spite of the efforts and initiatives taken by the government and various
organizations like thePasig River Rehabilitation Commission1, Asian Development Bank, Danish International
Development Agency (DANIDA), ABS-CBN Foundation Inc.etc.to clean and rehabilitate the Pasig River, a
worsening trend was reported with the presence of heavy metals (Greenpeace, 2001; Velasquez, 2002) along with
the decrease in Dissolved Oxygen from 1999 to 2006 (Asian Development Bank’s report, 2010).Between 1999-
2009, the continuous decline in the water quality with respect to the amount of Dissolved Oxygen (D.O) and
Biochemical Oxygen Demand (B.O.D.) continued (Gorme et al., 2010) and, the river never reached the Department
of Environment and National Resource’s (DENR) Class C standards (Table 1). Class ―C‖ standard refers to quality
of water that can be used for industrial purposes and recreational activities such as boating, fishing etc. Similar
trends were reported for the connecting Marikina River, where both dissolved Oxygen levels and Oil and Grease
concentrations did not meet Class C river standards of less than 5 mg/L and 2 mg/L respectively (Republic of The
Philippines Department of Public Works and Highways, 2011).

Further, the Payatas dumpsite (the newly relocated Smoky Mountain, Pier 18) drainage became the major source of
heavy metals such as chromium, copper, lead and zinc in the river, as well as other pollutants (Yoshimura et al.,
2014). The presence of these heavy metals is quite alarming as prolonged exposure to these can cause manyailments
including cancer, and mental retardation (Obligacion, 2015). Hence it is important to investigate if these polluted
rivers and water bodies have the potential to cause genotoxicity and cytotoxicity especially post rehabilitation efforts
by the Asian Development Bank and the Pasig River Rehabilitation Commission.

Since the pollution especially in the water bodies located in the urban areas can expose the environment and
populations to many health risks, there is a need for rigorous environmental monitoring, where the sensitive but
inexpensive biomonitorscan be used to alert the surrounding populations of environmental contamination and
pollutants. The merits of using Alliumcepa assay to detect environmental contamination are now well established.
The USEPA GeneTox Program has classified the Alliumcepa method as one of the most well established plant assay
systems for Environmental monitoring (Grant, 1982). Allium has small number of chromosomes (2n=16), relative
low cost, and easy to obtain root meristems. The test is also done in vivo which means data can be extrapolated to
other plants and even animals (Tedesco &Laughinghouse, 2012). Despite the typical use of it as a preliminary test,
the accuracy and legitimacy of using the Alliumcepa assay has been reported to be very accurate with 84%
correlation compared to carcinogenicity tests in rodents, and was even concluded to be more sensitive than the Ames
test to check for mutagenic potential of chemicals (Rank and Nielsen, 1998). The onion being in direct contact with
the substance of interest while growing is a major reason for its validity, especially in terms of measuring
cytotoxicity as well as genotoxicity(Vicentini et al., 2001).

Therefore, it is now being routinely used to evaluate the genotoxic, cytotoxic or mutagenic effects of chemical
compounds, nanomaterials, phytochemicals, pollutants and contaminants such as pesticides, herbicides, metals and
heavy metals using the induction of DNA damages, chromosomal aberrations and alterations in the mitotic index as
endpoints (Khallef et al., 2013; Klauck et al., 2013; Leme and Marin-Morales, 2008). Studies have indicated a
correlation between pollution (through chemical composition) and inhibition of root growth and decrease in mitotic
index (Radic et al., 2010).

Since the water bodies in and around Manila have been identified as reservoirs of all kinds of domestic waste and
contaminants hence, Alliumcepa test is being used for environmental biomonitoring of these water bodies and rivers
to assess the genotoxic potential of the environmental toxins if any present in the water.

Materials And Methods:-

Collection of Samples:-
Samples were taken from different locations along the rivers (Figure 1and 2), and the equal samples were mixed
together to get an average of the river’s water composition.

1
Presidential Decree Number 274, Pertaining to the Preservation, Beautification, Improvement, and Gainful Utilization of the
Pasig River, Providing for the Regulation and Control of the Pollution of the River and Its Banks In Order to Enhance Its
Development, Thereby Maximizing Its Utilization for Socio-Economic Purposes
ISSN: 2320-5407 Int. J. Adv. Res. 4(8), XX-XX

In addition, Estero de Vitaswhich is also connected to Pasig river, was also sampled being in a former dumpsite
known as Smokey Mountain (Figure 3). The estuary is now surrounded by land reclaimed areas, presumably made
with some of the garbage that used to accumulate there.

Laboratory Analysis:-
A. AlliumcepaTest:-
The standard Alliumcepa method was adopted to carry out the genotoxicity analysis as used in many similar studies
(Fiskesjo, 1997; Babatunde and Bakare, 2006). Briefly, medium sized (15-22 mm diameter) healthy bulbs of
common onion, Alliumcepa were purchased from the local market. The outer scales of the onions were removed and
had their roots cut without destroying the root primordial to allow the new roots to grow. The bases of the onions
were then submerged in equally sized containers with tap water for 48 hours. The lengths of the new rootlets were
measured. To test the effect of the river water samples, the onion roots were grown for 72 hours in treatments of
10%, 20%, 40%, 60%, 80% and 100% (v/v) concentrations of the sample waters collected. The treatment water
samples were replaced every 24 hours. Just as tap water was used as negative control, methylmethanesulfonate
(MMS, 10 ppm) was used as positive control. MMS is widely recommended as a positive control chemical in
mutagenicity testing (Rank and Nielsen, 1998; Caritá and Marin -Morales, 2008). For each treatment, a series of 5
replicates were set up.

Following the exposure to the various treatments, the root length of the onions was measured at 24, 48 and 72 hours
of growth. To study the chromosomal aberrations and Mitotic Index, the slides were prepared for microscopy using
the standard procedure. The roots, particularly the freshest and healthiest, were cut from each onion and were
hydrolyzed in 1 N HCL at 60 0C for 12 min and then stained with acetocarmine stain solution prepared in 45%
acetic acid. The microscope slides were prepared by placing the stained onion roots on the slide and squashing them
in 45% (v/v) glacial acetic acid. The slides were observed using optical microscope at an overall magnification of
100X.Calculation of the mitotic index was done using the standard formula:

The types of aberrations observed were anaphase bridges, chromosomal breakages, and laggards, stickiness (Figures
12). The standard formula used for calculating proportion of chromosomal aberration is as follows:

Where Pa, Ma, Aa and Ta are the abnormal mitotic cells, and P, M, A and T areall mitotic cells.

B. Physicochemical Analysis:-
Atomic Absorption Spectrometry was performed to determine the presences of heavy metals such as Lead (Pb),
Cadmium (Cd), Copper (Cu), Zinc (Zn), Iron (Fe), Nickel (Ni) and Mercury (Hg). Biochemical Oxygen Demand
and Dissolved Oxygen were also measured along with measurements for pH and turbidity.

C. Statistical Analysis:-
River treatments of different concentrations were compared to both positive and negative control through t-tests (for
root length) and z-tests (for mitotic index and aberration proportion). Any significance was determined at an alpha
of at least 0.05 (95% confidence). Linear regression was also done to determine if there were any trends between
root growth, mitotic index and aberration versus increasing concentrations of the treatments. PHSTAT 4.0 was used
for these statistical analyses.

Results And Discussion:-

Root Growth:-
The root growth for Pasig river water samples has a noticeable decreasing trend as the concentration of the treatment
increases, with a high R2 value of 0.8916 (Figure 4). This suggests a very good inverse correlation between root
growths versus concentration of Pasig river water samples tested. Marikina river samples on the other hand showed
a low R2 value of 0.0012 suggesting poor correlation (Figure 5). It’s noteworthy to point out that for both the Pasig
and Marikina treatments, the onion roots of the 10% concentrations grew better than the negative control itself. For
the Pasig river sample, there was a significant difference at an alpha of 0.05 whereas 10% Marikina water treatment
sample did not show the same significance (P > 0.05). This may be attributed to nutrients found in the river samples,
ISSN: 2320-5407 Int. J. Adv. Res. 4(8), XX-XX

which the tap water lacks. Human waste that goes into the rivers for example are rich in inorganic nitrate that is
readily absorbed by plant roots (Crawford, 1995) and at low concentrations, may have assisted in root growth.
Similar results have been reported where roots grew better in 20% sewage wastewater treatment than their negative
control (Shashank and Suresh, 2012).

All other concentrations tested, from 20% concentrations onwards were observed to have lower means than the
negative control, inducing significant inhibition of the root growth (Table 2). Significant differences were observed
in Pasig 80% and 100% water samples, and in Marikina 40% and 100% water samples (P < 0.05). These results
aggregately suggest that the river waters do inhibit root growth, starting from 20% concentration onwards with the
rotting of roots observed at 80% and 100% concentration of Pasig river and at 100%concentration of Marikina,
which is indicative of the cytotoxic effects of the water samples (Figure 7).

Estero de Vitas showed the best inverse trend of the three sample sites with a very high R 2 value of 0.9408. It should
be pointed out that the decaying of roots in the onions was observed starting at 60% concentration water sample.
The 80% and 100% concentrations were very highly toxic in particular (Figure 6), as the roots actually fell off due
to rotting causing the lower average root length after 72 hours compared to their initial length. The onions treated
with Estero de Vitas waters fared the worst over all of the three sample waters tested, with an average of 97.9%
inhibition in root length (46.3% for Pasig, 56.2% forMarikina).

B. Mitotic Index:-
The mitotic index for Pasig river samples showed a good trend with a very high R 2 value of 0.9259 (Figure 8).
Mitotic index was observed to decrease with the increasing concentrations of Pasig water samples. The Marikina
samples expectedly showed a low trend (R2 = 0.0812) for its mitotic index much like its root growth (Figure 9).
Both Pasig river water mitotic index and Marikina water mitotic index was found to be significantly lower than the
negative control at 20% concentrations onwards (P < 0.0001).

Estero de Vitas concentrations from 20% to 60% were also found to be very significantly lower, compared to the
negative control (P < 0.0001, Figure 11). The 80% and 100% Vitas treatments showed indeterminate mitotic indices
as cells in these concentrations were observed to be dead (Figure 10)

C. Chromosomal Aberrations:-
All concentrations of water samples from Pasig, Marikina River and Estero de Vitas tested, induced chromosomal
aberrations such as chromosomal fragments, anaphase and telophase bridges and laggards in the Alliumcepa root
cells. The lagging chromosomes are induced by a weak C- mitotic effect and are indicative of disturbance in the
mitotic spindle or the centromere (Fiskesjo, 1994).

Pasig river samples showed a low inverse trend at an R2 of 0.1122 (Figure 13). At 100%, the toxicity of the water
suppressed the mitotic index significantly and since there were no viable cells, no mitotic aberrations were observed
at all. (Figure 14).

Marikina river water samples showed good trend (R2 value of 0.6592) in terms of increase in aberrations with
increase in concentrations of water samples (Figure 15). This shift in correlations may be explained due to the
number of viable cells in Marikina treatments being comparable in amount as the root growths and mitotic index
were about equal, where at the same concentrations, the Pasig river samples had dying or dead cells.The low value
of chromosomal aberrations observed at Pasig 100% water treatment is indicative of the cytotoxicity of the water
sample to the onion root cells. Similar results were reported by Radic et al. (2010), who found thatcytotoxicity (cell
killing) was not necessarily correlated to genotoxicity (gene damaging) due to the presence of nitrate, nitrite,
ammonium and phosphate.Therefore, lower root growth/mitotic index does not necessarily mean higher
chromosomal aberrations rate. In 2010, Samuel, Osuala&Odeigah, using the same assay had similar results in terms
of correlation between concentrations versus mitotic index, where low concentrations had lower mitotic index or
even higher aberrations compared to higher concentrations. Regardless, overall there was significant difference
found at all concentrations showing the river water samples to have a genotoxic effect (P < 0.005). Estero de Vitas
water samples also showed significantly higher chromosomal aberrations compared to the negative control at 10%,
20%, 40% and 60% concentrations (P < 0.005, Figure 16). The presence of sticky chromosomes indicates that the
pollutants and hazardous substances present in the river water samples are affecting the organization of the
chromatin in the cells, which can ultimately lead to cell death. Many studies have reported the occurrence of sticky
ISSN: 2320-5407 Int. J. Adv. Res. 4(8), XX-XX

chromosomes in Allium roots after treatment with various heavy metals such as Hg , Ni and Cu (Fiskesjö, 1993,
1997; Egito et al., 2007).

Overall, the results using the Alliumcepa test show that the water samples from the Estero devitas caused maximum
genotoxicity as reported by other studies as well (Orozco &Zafaralla, 2012; Enguito, et.al., 2013). The human
activities have made these Esteros or creeks a major source of pollution with the waste dumped in these water ways
obstructing the smooth flow of water and causing major flooding during the rainy season (Gilbuena et al., 2013). To
improve the water quality of Pasig and Marikina river, it is important to clean these Esteros or creeks hence the
decision of the Pasig River Rehabilitation Commission to focus on cleaning one by one theseEsteros is a step in the
right direction. According to the Pasig River Rehabilitation Commission, efforts are on to clean all 48
tributaries/Esterosof Pasig river by 2019.

D.Physico-chemical Characteristics of Water Samples:-

The Atomic absorption spectroscopy carried out on the water samples collected from Pasig, Marikina rivers and
Estero de vitas locations indicate the presence of heavy metals such as Lead (Pb), Cadmium (Cd), Chromium (Cr),
Copper (Cu), Iron (Fe) and Mercury. Some of these metals such as Cadmium (Cd), Chromium (Cr), and lead (Pb)
were found to be present in excess of the standard accepted values set by the various local state agencies (Table 3).
The presence of heavy metals in the water samples is not surprising as heavy metals are among the most common
inorganic pollutants in water (Chandra et al., 2005). The source of heavy metals could be from the human waste
dumped by the informal settlers living all along the river banks as well as commercial and industrial establishments
operating near the river. Another possible source of the heavy metals could be the e waste leachates from some of
the formal and informal electronic waste dumpsites and recycling sites located in and around Manila. The studies
have indicated the presence of heavy metals in the soil and dust surface matrices from these electronic recycling
sites which due to rain and flooding can easily flow out into the water bodies in and around these dumpsites
(Fujimori et al., 2012). The presence of heavy metals such as Lead and Cadmium in excess of allowable limits set by
the United States Environmental Protection Agency was reported even in the community tap water samples of
Manila (Solidum and Solidum, 2012). The possibility of the leachates from the 19-hectare sanitary landfill in San
Mateo, Rizal being the source of hazardous chemicals in the Marikina river should also be investigated.

The Biological oxygen demand (BOD) at 29 mg/L was significantly higher than the DENR Class C DENR Standard
values of 7 mg/L. Similarly, the Dissolved Oxygen (DO) at 0.1 mg/L was much lower than the DENR Standard of 5
mg/L (Table 3). Though Pasig River Commission has been claiming steady improvement in the water quality of the
rivers however according to some reports, the water tested at 14 stations set up along the route of Pasig River, failed
to pass the DENR acceptable standards (Obligacion, 2015). The pH of the water samples from Pasig and Marikina
rivers was found to be with in the DENR standard limits however the water samples from Estero de vitas were
slightly acidic with pH of 6.7. The toxicity in terms of biological damage by complex mixtures is reported to
enhance by extreme pH values (Fiskesjo, 1985). There is correlation between our physico-chemical analysis results
of water samples and the genotoxicity observed in terms of inhibition of root growth and mitotic index and increase
in the chromosomal aberrations with increase in the concentrations of water samples tested.

Heavy metals as pollutants can cause DNA damage and thereby cause carcinogenic and mutagenic effects with
increase in the frequency of chromosome breaks and mutations in various test systems and also in humans (Minissi
et al., 1997; Ivanova et al., 2008). The studies have also shown that long-term exposure to even small concentrations
of heavy metals increases the risk of quite a few diseases, including cancers (Zhuang et al., 2009). Many studies
have indicated that a variety of ecotoxicity tests should be used to detect chemicals that are capable of causing
environmental mutagenesis as only chemical analysis is not sufficient to reveal their toxicity (Chen et al., 2004,
White et al., 2004., Fuentes et al., 2006). It has also been established that Alliumcepa is one of the useful test
systems for the detection of these potential genotoxic substances in water screening programs (Fiskesjo, 1997;
Ivanova et al., 2002a, 2002b). The results also conclude that the Alliumcepa test system can be used very effectively
to perform preliminary biomonitoring of contaminated water sources in the Philippines as well.
ISSN: 2320-5407 Int. J. Adv. Res. 4(8), XX-XX

Figure 1:- Location of samples collected for Pasig River (Red Dots)

Figure 2:- Location of samples collected for Marikina River (Red Dots)

Figure 3:- Location of sample collected for Estero de Vitas (Red Dot)
 ISSN: 2320-5407 Int. J. Adv. Res. 4(8), XX-XX

160%
140% R² = 0.891

Root Growth Inhibition

120%
100%
80%
60%
40%
20%
0%

Positive Control
Negative Control

Pasig 10%

Pasig 20%

Pasig 40%

Pasig 60%

Pasig 80%

Pasig 100%

(10ppm MMS)
Figure 4:- The Root growth inhibition with increase in Pasig River water concentration treatment
* – Lower root length at P < 0.05 compared to negative control. % Root Growth is length of growth after 72 hours.
% Relative Root Length is length compared to negative control. % Inhibition is negative control minus relative root
length.
140%
R² = 0.001
Root Growth Inhibition

120%
100%
80%
60%
40%
20%
0%
-20%

Positive Control
Negative Control

Marikina 10%

Marikina 20%

Marikina 40%

Marikina 60%

Marikina 80%

Marikina 100%

(10ppm MMS)
Figure 5:- Inhibition of Root length with increasing concentration of Marikina River Water Treatment

120%
R² = 0.940
Root Growth Inhibition

100%
80%
60%
40%
20%
0%
-20%
Positive Control
Negative Control

Vitas 10%

Vitas 20%

Vitas 40%

Vitas 60%

Vitas 80%

Vitas 100%

(10ppm MMS)

Figure 6:- Estero de Vitas Treatment – Inhibition of root growth with increase in the concentrations of the water
samples

Pasig 100% Marikina 100% Vitas 100% Negative Control

Initial

72 Hours

Figure 7:- Morphological comparison of onion roots and their growth after 72 hour
ISSN: 2320-5407 Int. J. Adv. Res. 4(8), XX-XX

5.00%

Mitotic Index
4.00% R² = 0.925
3.00%
2.00%
1.00%
0.00%

Positive Control
Negative Control

Pasig 100%
Pasig 10%

Pasig 20%

Pasig 40%

Pasig 60%

Pasig 80%

(10ppm MMS)
Figure 8:- Mitotic Index for Pasig River Water Treatments. Trend line is for concentrations from 20% to 100%
compared to negative and positive control samples.

4.50% R² = 0.081
4.00%
Mitotic Index

3.50%
3.00%
2.50%
2.00%
1.50%
1.00%
0.50%
0.00%

Positive Control
Negative Control

Marikina 100%
Marikina 10%

Marikina 20%

Marikina 40%

Marikina 60%

Marikina 80%

(10ppm MMS)
Figure 9:- Reduction in Mitotic Index for Marikina River Water Treatments. Trend line is for concentrations from
20% to 100% as compared to negative and positive control.

Figure 10:- Estero de Vitas 80% Water Sample Treatment with Root cells observed at 10X (Left) and 40X (Right).
Cytotoxicity with no viable cells was observed in all replicates at these concentrations.

4.50% R² = 0.963
4.00%
Mitotic Index

3.50%
3.00%
2.50%
2.00%
1.50%
1.00%
0.50%
0.00%
Positive Control
Negative Control

Vitas 10%

Vitas 20%

Vitas 40%

Vitas 60%

(10ppm MMS)

Figure 11:- Mitotic Index for Estero de Vitas Treatments. Concentrations at 80% and 100% were excluded due to
no observable viable cells.
ISSN: 2320-5407 Int. J. Adv. Res. 4(8), XX-XX

Figure 12:- Chromosomal aberrations observed in various treatments. A – C mitosis, B – Broken Spindle Fibers, C
– Sticky Chromosome, D – Anaphasic Bridge, E – Laggard Chromosome, F – Damaged Centriole

70.0%
Aberrations
60.0%
50.0% R² = 0.112
40.0%
30.0%
20.0%
10.0%
0.0%

Positive Control
Negative Control

Pasig 100%
Pasig 10%

Pasig 20%

Pasig 40%

Pasig 60%

Pasig 80%

(10ppm MMS)
Figure 13:- Aberration Proportion for Pasig River Treatments

Figure 14:- A – Normal cells observed. B – Interphase Cells at 100% Concentration (Pasig River water) ; Very
Dense Nucleus

100.0%
Aberrations

80.0%
R² = 0.659
60.0%
40.0%
20.0%
0.0%
Positive Control
Negative Control

Marikina 10%

Marikina 20%

Marikina 40%

Marikina 60%

Marikina 80%

Marikina 100%

(10ppm MMS)

Figure 15:- Aberration Proportion for Marikina River Treatments

ISSN: 2320-5407 Int. J. Adv. Res. 4(8), XX-XX

70.0%
60.0% R² = 0.999

Aberrations
50.0%
40.0%
30.0%
20.0%
10.0%
0.0%
-10.0%
-20.0%

Positive Control
Negative Control

Vitas 10%

Vitas 20%

Vitas 40%

Vitas 60%

(10ppm MMS)
Figure 16:- Aberration Proportion for Estero de Vitas Treatments Concentrations at 80% and 100% were excluded
due to no observable viable cells.

Table 1:- DENR Class C Fresh Water Classification

pH 6.5 – 8.5
D.O. (mg/L) 5.0
B.O.D. (mg/L) 7.0
Chromium (mg/L) 0.05
Oil and Grease (mg/L) 2.0
Lead (mg/L) 0.05
Mercury (mg/L) 0.002

Table 2:- Summary of Root Growth and Inhibition

Treatment % Root Growth % Relative Length % Inhibition
Negative Control 301 ± 106 100 —
Pasig 10% 401 ± 123 133 -33
Pasig 20% 303 ± 102 101 -1
Pasig 40% 158 ± 81 52 48
Pasig 60% 65 ± 9 22 78
Pasig 80%* 38 ± 35 13 87
Pasig 100%* 3 ± 20 1 99
Marikina 10% 372 ± 154 124 -24
Marikina 20% 64 ± 82 21 79
Marikina 40%* 42 ± 46 14 86
Marikina 60% 154 ± 67 51 49
Marikina 80% 162 ± 110 54 46
Marikina 100%* -4 ± 88 -1 101
Vitas 10% 96 ± 102 32 68
Vitas 20%* 36 ± 55 12 88
Vitas 40%* 1 ± 33 0.5 99.5
Vitas 60%* -9 ± 4 -3 103
Vitas 80%* -43 ± 4 -14 114
Vitas 100%* -46 ± 16 -15 115
Positive Control (MMS) 27 ± 22 9 91
ISSN: 2320-5407 Int. J. Adv. Res. 4(8), XX-XX

Table 3:- Comparison of Pasig and Marikina: Physico-chemical Characteristics versus different standards
Metal Pasig River Marikina River Estero de Vitas DENR Class C Standard
Cadmium (Cd) 0.0220 0.0275 0.0427 0.01
Chromium (Cr) 0.2266 0.2540 0.0130 0.05
Copper (Cu) 0.0217 0.0179 0.0203 0.05
Iron (Fe) 0.6360 0.6112 0.0424 -
Lead (Pb) 0.3176 0.4557 0.1608 0.05
Mercury (Hg) <0.001 <0.001 <0.001 0.002
B.O.D. 29 30 - 7
D.O. 0.10 1.10 - 60
pH 7.5- 8.04 7.2-8.2 6.7 6.5-8.5

Table 4:- Breakdown of Results for total Chromosomal abnormalities for Pasig and Marikina at Different
Concentrations of water samples tested
Treatme Total Total M.I. Propha Metapha Anaphas Telophas Abnormalities Total
nt Cells Mitosi (%) se (%) se (%) e (%) e (%) M P A T Abnormalit
Examine s ies
d
Negative 7257 277 3.82 1.60 0.74 0.52 0.95 0 0 7 0 7
Control
Positive 2925 25 0.85 0.27 0.44 0.10 0.03 2 5 3 0 10
Control
P10 5272 236 4.28 1.99 0.91 0.61 0.97 7 5 5 1 18b
P20 5609 193 3.33 1.59 0.68 0.50 0.68 6 8 6 3 23b
a
P40 3720 88 2.31 0.65 0.94 0.40 0.38 0 2 5 0 7b
a
P60 4431 80 1.77 0.59 0.41 0.41 0.41 3 3 13 6 25b
a
P80* 3538 59 1.64 0.54 0.54 0.31 0.28 0 0 3 1 4b
a
P100* 2466 4 0.16 0.16 0.00 0.00 0.00 0 0 0 0 0b
a
M10 3684 56 1.50 1.19 0.03 0.08 0.22 3 0 0 2 5b
a
M20 3312 30 0.90 0.45 0.12 0.18 0.15 2 1 2 0 5b
a
M40* 3725 80 2.10 0.45 0.12 0.18 0.15 7 5 2 0 14b
a
M60 3713 57 1.51 1.02 0.40 0.32 0.40 4 5 5 0 14b
a
M80 4597 93 1.98 0.78 0.30 0.30 0.16 5 6 9 0 20b
a
M100* 2283 6 0.26 0.04 0.18 0.00 0.04 0 3 0 0 3b
a
V10 1857 58 3.12 0.43 1.08 0.86 0.75 1 7 0 4 12b
a
V20* 2745 52 1.89 0.55 0.62 0.40 0.33 3 2 8 3 16b
a
V40* 2337 35 1.50 0.04 0.51 0.30 0.64 1 0 1 6 8b
a
V60* 1007 7 0.70 0.30 0.20 0.20 0.00 0 1 0 0 1b

Conclusion:-
The inhibition of root growth, the decrease in the mitotic index and the presence of chromosomal aberrations in the
Alliumcepa root cells exposed and grown in the water samples of Pasig river, Marikina river and Estero de Vitas is
indicative of the presence of hazardous substances and pollutants capable of causing genotoxicity in these water
bodies. Since the water samples of Marikina river and Estero de vitas have been also included in this study, the
results give more comprehensive information about the genotoxicity of the water bodies in and around Manila.
There are 48 esteros linked to the Pasig river and these have been identified as the major source of pollution to the
river due to human settlements around these tributaries. Only with the chemical analysis of water, it is not possible
to determine the eco-toxicological risk that chemicals present in it can cause, since such analysis alone does not
indicate toxicity (Fuentes et al., 2006). Therefore, ecotoxicity tests that can detect environmental mutagenesis, have
been proposed and applied to understand the genetic and physiological responses of exposed organisms (White et
al., 2004; Chen et al., 2004). The results clearly demonstrate that the Alliumcepa assay in combination with the
physicochemical analysis of the water bodies can be very effectively used as a preliminary screening test in the
environmental monitoring to evaluate the presence of genotoxic and cytotoxic pollutants and environmental
contaminants in the water bodies which will aid to safe guard the human populations as well as other organisms.
ISSN: 2320-5407 Int. J. Adv. Res. 4(8), XX-XX

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