THE ALLIUM TEST AS A METHOD FOR WATER QUALITY MONITORING OF BALILI RIVER

Bantola, D.M.; Belen, A.J.; Castillo, G.; Juguilon, P.M.; Rayo, J.L.

ABSTRACT
The Allium test is a possible method to use in order to determine the water quality of a particular sample of water. The
assessment of genotoxic effects of the water of Balili River in Barangay Brookside, Baguio City was conducted. Three
setups were prepared: the positive control (mineral water with EDTA), the negative control (pure mineral water), and the
water sample from the river, with each setup bearing 7 equally sized onion bulbs. The scraped bottoms of the onions bulbs
were soaked in their respective water samples for four days. Microscopic analyses were done on the second day while
macroscopic analyses were done on the fourth day. The results showed that the positive control contained more
chromosomal aberrations than the negative control. The paired T-test showed that the water sample is not statistically
significantly different from the negative control. Therefore, the water quality of the Balili River based from the Allium test
is high. Errors in the experiment include miscounting of the cells in the Allium cepa slide.

INTRODUCTION 1938 when the effects of colchicine were investigated

(Fiskesjo, 1985). Since then, Allium sp. including Allium
Biological monitoring and assessment of cepa has been used as the standard test organism in
genotoxic effects caused by chemical treatments, usually detecting toxic chemicals in water samples found in the
in water samples, are performed and executed using an environment.
experimental system called the Allium test. The Allium
test is a simple yet effective method that studies water Young and developing root tips are measured to
samples up to the cytological level. It has many yield information on the direct effects of such toxic
advantages over other methods including it being cost- chemicals found in the sample, both on the cellular level
efficient, easy to handle, and good chromosome and the organism as a whole. In order to obtain unbiased
conditions for study (Fiskesjo, 1985). Conclusions are and reliable results, all other conditions including light
based on the effect of possible chemical contamination and temperature are kept at a standard for all setups to
of the water sample to cell division and chromosomal ensure that the only factor affecting the growth and
characteristics of the Allium root. This method is development of the Allium sample is water. This is
applicable in the assessment of natural bodies of water, usually done at room temperature and away from direct
drinking water, domestic and industrial wastewaters, and sunlight.
water-soluble or water-insoluble chemicals containing
solvents. In this activity, the assessment of genotoxic
effects of the water from Balili River, Barangay
The amount of a toxic chemical in a certain Brookside, Baguio City was analyzed using the Allium
medium is very crucial and can damage or mutate the test. Balili River is a large river system running from the
cells of living organisms when it reaches critical City of Baguio to La Trinidad, Benguet. It is situated
concentration levels. When a living cell is exposed to the within urban communities making it prone to pollutants
toxic chemical, it can acquire toxic effects that inhibit or and waste materials from households and vehicles
terminate the growth of cells and genotoxic effects like passing by. Through the years, the level of
chromosome breaks that can be inherited by the cells of contamination has increased to an alarming state.
future generations. Mutagens in eukaryotic nuclei can be Biological monitoring of the water quality is suggested
detected by cytological inhibition, disruption in to determine if the water is still safe or is already prone
metaphase, and induction of chromosomal aberrations to affect its surrounding community negatively.
(Tedesco, 2012). The Allium test is composed of both
macroscopic and microscopic setups that determine both The objectives of the activity include the
effects on the organism. determination of the genotoxic potential of the water
sample obtained from Balili River, enumeration of the
The basis for using Allium sp. as the test types of chromosomal aberrations present, and
organism is its great sensitivity to soil and water calculation of the mitotic index, leading to the
contaminants. It has proved to be the most useful test classification of the natural body of water and its effects
organism and has repeatedly been suggested as a on living cells of organisms.
standard test material. It was introduced by Levan in
 Further maceration was done by placing a coin on the
slide and adding equal pressure with the thumb. Excess
MATERIALS AND METHODS stain was wiped down after preparation.
The Allium test requires three set-ups including The mitotic index was scored from 300 cells
the water sample, the negative control and the positive given the equation:
control. 1000 millilitres of water obtained from Balili
River was used as the water sample. The negative and P+ M + A+T
positive controls both contained standard mineral water
Mitotic Index =
Total no . of cells
with a pH of 7.0 and containing 50 to 70 mg of calcium
(Ca) and magnesium (Mg). No toxic ions should be Figure 1. Mitotic Index Equation
found in the mineral water sample. For the positive
The Mitotic Index was used in order to
control, an additional 1 mg of
determine the significant differences between the control
ethylenediaminotetraacetic acid (EDTA) was added to
groups and the water sample. The Paired T-test was
1000 ml of the mineral water.
employed to determine if the two sample means are the
7 equally sized onion bulbs were used for each estimates of the same population mean. The software
setup. All in all, 21 onions were collected and prepared SPSS by IBM was used for statistical analysis of the
for the test. All onion bulbs were placed in clean and data.
empty Yakult® bottles that were filled to the rim with
Aside from the mitotic index of the cell,
their corresponding water samples and all tubes were
chromosomal aberrations were also observed and noted.
labelled properly.
Such chromosomal aberrations include C-mitosis,
Before the experiment proper, all the outer laggard chromosomes, sticky chromosomes, and bridges
scales and brownish bottoms of the onion bulbs were and fragments. The comparison of the probable
removed using a blade in order to expose the ring of the chromosomal aberrations in the water sample were
root primordial that will give rise to the young root compared to that of the chromosomes found in the cells
systems of the onion. After which, the bulbs were placed of the root tip of Allium cepa in the positive control
on the brim of the tube ensuring that only the bottom of setup. Otherwise, the absence of chromosomes could be
the onion touches the water sample. The setups were compared to the negative control setup containing
checked from time to time to see if some of the water mineral water treated with EDTA.
has evaporated and requires replenishing. Standard setup
The remaining 4 bulbs per setup were used for
conditions include room temperature and protection
the macroscopic parameters performed on day 4 or two
against direct sunlight. All test liquids were stored in the
days after the conduction of the microscopic parameters.
refrigerator and brought to room temperature before use.
For the macroscopic parameters, the root length, root
The Allium samples were allowed to grow for form, and shoot length of individual bulbs were
two days with replenishing of water when necessary. On observed and noted. The root formed was described
the second day, the microscopic parameters were based on the presence or absence of twists or crochet
observed. Slides for the microscopic study were hooks, swellings or C-tumors, broken root tips, color,
prepared. For each setup, 3 bulbs were obtained. For and turgidity. The macroscopic parameters were
each bulb, 2 slides were prepared and fixed for compared from among the bulbs per setup and the
observation under the microscope. overall comparison of Allium cepa bulbs.

A standard protocol for mitosis in Allium cepa

root tip was used to prepare the slides. First, the root tip
RESULTS AND DISCUSSION
of the Allium bulb was obtained using a cutter or razor
blade. Then, the roots were fixed in Farmer’s solution Various steps in the experiment are first
which was comprised of 1 part glacial acetic acid and 3 discussed in order to determine the importance of the
parts absolute ethyl alcohol. Prior to maceration, the methodology in the experiment.
roots were then soaked in HCL for about an hour until
they were soft. Once they were soft, the cells were rinsed EDTA is a polyamino carboxylic acid. It acts as
ain distilled water to remove excess acid. After which, a chelating agent that deionizes metal ions, such as
about 0.5mm of the root tip was positioned on the slide calcium and magnesium, in water. It acts as a “water
and a drop of aceeto-orcein stain was added. The size of softener” eliminating metal ions present in the sample.
the root tip was considered and must be the smallest In analytical chemistry, it acts as a compound used to
possible in order to obtain just one field of view and to standardize solutions used in titration. It also restricts the
avoid overlapping of cells. After staining, a cover slip access of any other species to the metal (Robinson,
was placed on top of the onion root tip and squashed. 2015). The reason for having negative and positive
controls is to have a form of basis for whether or not 2 5 100 0 All
chromosomal aberrations are present in the water sample white
of choice. The positive control is predicted to cause 3 5.75 100 0 All
aberrations to the Allium root cells because of its mineral white
contents which may be toxic to the living cells of the 4 6.13 87.5 12.5 All
onion. On the other hand, the negative control will white
render little to no mutations in the Allium root tip cells
because the minerals were eliminated by the addition of
EDTA as a solute. Table 2. Root Form of the Bulbs Exposed to EDTA
Solution
For the Allium bulbs, all should be of equal size Bulb Root Form (%)
so as to avoid multiple results. They must be relatively Numbe Norm Hoo Wav Broke Swelling
young and not bearing green leaves which indicate r al k y n s
maturation. 1 56.25 18.7 0 12.5 12.5
The protocol followed in the Allium test 5
indicated important steps to follow: First, the root tip of 2 50 25 0 0 25
the Allium bulb was obtained using a cutter or razor 3 0 0 0 25 75
blade. Then, the roots were fixed in Farmer’s solution 4 50 12.5 0 0 37.5
which is comprised of 1 part glacial acetic acid and 3
parts absolute ethyl alcohol. This was so that the root
tips will be preserved and further growth and Even in small concentrations, chemicals like
development would be terminated. The roots were then EDTA affect the growth of Allium sp. samples.
soaked in HCL for about an hour until they were soft. Differences in the root length, color and root form
The role of hydrochloric acid was to disintegrate single among the samples exposed to different growth
cells from one another for easier and more accurate conditions were observable. When the bulbs were
count and observation of cells under the microscope. exposed to small amount of chemicals, the root length
Once they were soft, the cells were rinsed ain distilled was affected. However, exposure to large amount of
water to remove excess acid. After which, about 0.5mm chemicals had severe toxic effect to the samples and
of the root tip was positioned on the slide and a drop of influenced the shape, form and color of the root tips.
aceeto-orcein stain was added. The function of the stain Table 1 and 2 show the observed results from
was to distinguish the cells and their chromosomal the experiment. It can be seen that the measured mean
contents from the background. After staining, a cover length of the roots of the sample was shorter than usual.
slip was placed on top of the onion root tip and squashed If growth of the roots decreases over 45% compared to
in order to spread out the individual cells of the Allium usual, it indicates the presence of toxic substances
root. The coin was simply used to macerate the root.
[ CITATION Kha13 \l 13321 ]. Also, the presence of
The three setups—positive control, negative EDTA affected the turgidity of the roots. Some roots
control, and the water sample—are discussed according became flaccid or soft.
to their macroscopic and microscopic analyses. Besides
However, the presence of different colored
this, each of the setups are subjected to the T-test, in
roots (yellow, brown) should be observed when the plant
order to statistically determine if the two sets of data—
sample is exposed to chemicals. In the obtained results,
particularly the positive control and the water sample,
it was seen that the color of the roots were all white.
and the negative control and the water sample—are
significantly different from one another. Table 3. Number of Cells in Mitosis and Aberrant
Chromosomes in the Allium sp. Samples Exposed to
EDTA Solution
Positive Control Bulb 1 Bulb 2 Bulb 3
Normal 83 75 40
Table 1. Macroscopic Analysis of Four Sample Bulbs Prophase
Exposed to EDTA Solution Normal 1 2 0
Bulb Mean Turgescence (%) Color Metaphase
Number Root Turgid Flaccid of roots Normal 0 10 0
Length Anaphase
(mm) Normal 0 28 0
1 14.4 87.5 12.5 All Telophase
white Sticky 0 5 3
 Chromosomes chromosomes are chromosomes that moved ahead of
Laggard 0 0 6 from its chromosomal group toward poles. This
Chromosomes movement leads to the unequal separation of number of
Chromosome 0 0 0 chromosomes in the daughter cell. Ring chromosomes
breaks or result from the loss of chromosomes from the telomeric
fragments side. The presence of chromatin bridges happen during
Other 144 98 112 translocation of the unequal chromatid exchange. Lastly,
Aberrations delayed anaphase happens when the two anaphasic
(binucleated, chromosomal groups lie close to each other near the
decompressed equatorial plate. As the concentration of the chemicals
chromosomes) increase, the frequency of delayed chromosomes also
increase [ CITATION Kha13 \l 13321 ].
The roots have the highest sensibility to Table 4. Mitotic Index of the Roots from Bulbs Exposed
chemicals even at low concentrations. After 4 days of to EDTA
treatment with EDTA, it was observed that there was Bulb 1 Bulb 2 Bulb 3
swelling, bending/ hooked and broken roots. Root Root Root Root Root Roo
1 2 1 2 1 t2
In the positive control, it was observed that the Interphas 267 249 245 235 268 283
presence of EDTA caused chromosomal aberrations in e
the root cells of Allium sp. Chromosomal aberrations are Prophase 33 50 32 43 29 17
changed in either total number of chromosomes or the Metaphas 0 1 2 5 3 0
chromosomal structure of the normal chromosomes. e
There are two types of chromosomal aberrations: (1) the Anaphase 0 0 3 7 0 0
clastogenic aberration, which includes chromatin
bridges, ring chromosomes and chromosomal breaks, Telophase 0 0 18 10 0 0
and (2) the physiological aberration, which includes c-
mitosis, vagrants, sticky choromosomes, delayed TOTAL 300 300 300 300 300 300
anaphase and laggard chromosomes. EDTA influences Mitotic 11.0 17.0 18.3 18.3 10.6 5.67
the inhibition of DNA synthesis which may be due to the Index (%) 0 0 3 3 7
damage it caused to the enzymes responsible in DNA
replication. Based on early studies, it was shown that
EDTA causes morphological changes of chromatin and
Mitotic indices (MI) are used to measure how
chromosome structure in plant and animal cells. Some
fast the cells are dividing. The level of cytotoxicity of
evident changes are dispersion or swelling of
chemical or compound can be determined based on the
chromosomes and loss of interphase chromatin structure
increase or decrease in the MI. In the samples exposed to
(Heindorff et al., 1983).
EDTA, the MI computed are low, thus, the cells divide
The observed chromosomal aberrations in this slowly due to the presence of the chemical EDTA which
experiment were laggard chromosomes, sticky interfered to the DNA synthesis of the cell or the
chromosomes, binucleated cells and decompressed blocking in the G2 phase of the cell cycle [ CITATION
chromosomes. Sticky chromosomes are the result of the Lyl90 \l 13321 ]. It is said that a decrease below 22% of
increased chromosomal contraction and condensation. It the mitotic index of the positive control cause lethal
can also be due to the depolymerisation of DNA and effects on the plant sample tested while a decrease below
partial dissolution of nucleoproteins. Stickiness of 50% has sublethal effects. This is called the cytotoxic
chromosomes indicates the toxic effects of the substance limit value [ CITATION Kha13 \l 13321 ].
being tested. It is usually irreversible and leads to cell
death. On the other hand, laggard chromosomes are from
the failures of the chromosomes to get attached to the Negative Control
spindle fibre and to move to either of the two poles.
Binucleated cells are the result of the inhibition of
cytokinesis of the cells.
Water Sample
Other chromosomal aberrations which were not The potential cytotoxicity and genotoxicity of
observed in the samples were the presence of vagrant
the sample water from Brookside River using the Allium
chromosomes, chromatin bridges, chromatin breaks, ring cepa test was investigated by macroscopic and
chromosomes and delayed anaphase. Vagrant
microscopic parameters. For the macroscopic
parameters, the number of roots, mean root length, Normal
37 21 60
turgescence, color of roots, and root form of the onions Metaphase
from four water samples out of the seven water samples Normal
were evaluated. - - -
Anaphase
Normal
Table 8. Macroscopic Analysis of Four Sample Bulbs of - - -
Telophase
Water Sample
Sticky 0 0 0
Bulb Mean Turgescence (%) Color
Chromosomes
Number Root Turgid Flaccid of roots
Laggard 0 0 0
Length
Chromosomes
(mm)
Chromosome 0 0 0
1 11.125 62.5 100%
breaks or
37.5 white
fragments
2 5.5 100 0 100%
Other
white
Aberrations
3 4.5 100 0 100% (binucleated, 111 82 97
white decompressed
7 12.9 90 10 100% chromosomes)
white

It was observed that the number of cells in

Table 9. Root Form of the Bulbs of Water Sample Normal Prophase and those with aberrations are high.
Bulb Root Form (%) Only a few number of cells has Normal Metaphase,
Numbe Norma Hoo Wav Broke Swelling while no cells were observed to have undergone either
r l k y n s Normal Anaphase or Telophase. Chromosomal
1 62.5 12.5 0 25 0 aberrations induced in the onion root tip cells were
mostly decompressed chromosomes and binucleated
2 100 0 0 0 0 cells. The findings under these microscopic analysis
3 75 25 0 0 0 indicate that there are toxic chemicals present in the
water sample which are responsible for the observed
7 100 0 0 0 0 genotoxic effects on the onion root tip cells. For further
analysis, the mitotic indices of two roots in each onion
from the three water samples were evaluated.
It was observed that the macroscopic parameters
of the onions from each of the four water samples vary Table 11. Mitotic Index of the Roots from Bulbs of
in the number of roots, mean root length, turgescence, Water Sample
and root form. In terms of the color of the onion roots, Bulb 1 Bulb 6 Bulb 7
all were observed to be white. The cytotoxic effects on Root Root Root Root Root Root
the roots tips showed growth retardation as observed in 1 2 1 2 1 2
all the tested water samples, with some of it being Interphase 153 166 125 134 117 133
flaccid. Also, the root tips of onion bulbs grown in the
water samples were characterized by hooks and broken Prophase 128 111 101 126 98 102
roots. The induction of root malformations in A. cepa Metaphase 19 23 22 17 31 46
has been shown to be useful signs of toxicity in previous
studies (Bakare, 2009). Anaphase - - - - - -
Telophase - - - - - -
For the microscopic parameters, the cells in TOTAL 300 300 248 277 246 281
mitosis and aberrant chromosomes as well as the mitotic Mitotic 49 45 41 48 43 49
indices of two roots were observed in the onions from Index (%)
three water samples out of the seven water samples.
Table 10. Number of Cells in Mitosis and Aberrant MI is a measure for the proliferation status of a
Chromosomes in the Allium sp. Samples of Water cell population. Most of the number of cells was in the
Sample Interphase and Prophase stage of Mitosis, while there
Bulb 1 Bulb 6 Bulb 7 were no cells observed under Anaphase and Telophase.
Normal Using these information on the number of cells, the
152 163 126
Prophase mitotic indices of all the roots of onions from the three
water samples were calculated as the number of dividing expected result based from the observations of the group
cells per 300 observed cells. The mitotic indices were all in the macroscopic analyses Moreover, the negative
calculated to be less than 50% because the cells were control and the water sample’s T value is 2.419, which is
inhibited in terms of growth. This decrease in the mitotic greater than 0.05. This implied that the means come
index of Allium cepa meristem cells can be considered from the same population, and that they are not
as a reliable method to determine the presence of statistically significantly different from one another.
cytotoxic agents in the water sample (Hilada, 2013). 
The T-test implies that the water quality in the
Statistical analysis must be conducted in order to
Balili River is not low, and is comparable to that of
back these claims. The paired T-test was used as it
compares two sets of quantitative data. If the two means mineral water.
of the sets of data are equal, it would imply that they Possible errors in the experiment may have been
came from the same population. the counting of the cells itself. The group had trouble
Table 12. Paired t-test for positive control and river counting the cells of the prepared slides of Allium cepa,
water sample and negative control and river water especially with the faulty compound microscopes. This
sample may have led to errors in the mitotic index, and,
possibly, into the paired samples t-test.
Paired Samples Test
Paired Differences t df Sig.
Me Std. Std. 95% (2- CONCLUSION
an Devi Error Confidence tailed
ation Mean Interval of )
The Allium Test was used in order to determine
the
Difference if a water sample is mixed with toxic substances. A
Lowe Uppe water sample taken from Balili River in Barangay
r r Brookside, Baguio City, was compared with a positive
Positive control (mineral water laden with EDTA) and a negative
P - -
Control - - -
ai
River
32. 7.37 3.012
40.07 24.58
10.
5 .000 control (pure mineral water). EDTA is toxic to onion
r 333 955 69 73
1
Water
33
770 897
2 cells, making it suitable to use for the positive control of
Sample
the Allium test.

P
River Results showed that aberrations microscopically
Water 13. and macroscopically were apparent in the positive
ai 13.8 5.648 -.854 28.18 2.4
Sample - 666 5 .060
r
Negative 67
3715 99 53 786 19 control. This was expected us the EDTA is toxic to the
2
Control growing onion root. The negative control showed less
macroscopic and microscopic aberrations. This was also
expected as the roots were simply exposed to the water
In this T-test, the positive and negative controls and minerals that were suitable for growth.
were both compared with the water sample from the
river. Based from the macroscopic analysis and the The water sample show abberations similar to
that of the positive control. However, the statistical
number of chromosomal aberrations in the positive and
analysis show that the water sample is not statistically
negative controls, it was expected that the t-test would
significantly different from the negative control. This
interpret in the same way as the group had predicted— implies that the water sample is similar to the negative
the positive control and the water sample would not have control, and that the water quality of the Balili River is
a statistically significant difference, while the negative high. Possible errors include the miscounting of the cells
control and the water sample would have a statistically in the slides.
significant difference. Being significantly different
would imply that the two data sets were not taken from
the same population mean. REFERENCES
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