Journal of Geoscience and Environment Protection, 2014, 2, 52-59
Published Online April 2014 in SciRes. http://www.scirp.org/journal/gep
How to cite this paper: Olorunfemi, D. I. et al. (2014). Genotoxicity Assessment of Contaminated Drinking Water Sources in
a Rural Community in Edo State of Nigeria. Journal of Geoscience and Environment Protection, 2, 52-59.
Genotoxicity Assessment of Contaminated
Drinking Water Sources in a
Rural Community in Edo State
D. I. Olorunfemi1*, O. P. Olorunfemi2, I. E. Agbozu3
1Department of Plant Biology and Biotechnology, University of Benin, Benin City, Nigeria
2Department of Animal and Environmental Sciences, University of Benin, Benin City, Nigeria
3Department of Environmental Sciences, Federal University of Petroleum Resources, Effurun, Nigeria
Email: *ud anfem @g mail.c om
Received Dec emb er 2013
In most rural settlements in Nigeria, provision of potable water for drinking and domestic pur-
poses is a big challenge; therefore analysis of drinking water is of great importance as contami-
nated water jeopardizes both the physical and social health of all people. Water samples were ob-
tained during the dry and wet seasons from a borehole and a man-made lake constructed through
self-help effort in Obazuwa community in Ovia North East Local Government Area of Edo State, Ni-
geri a. The y were analyzed for physicochemical parameters and subjected to cyto-genotoxic
evaluation using the Allium cepa assay. Results of the physicochemical analysis showed that most
of the parameters (pH, chromium, copper, chlorides, nickel, iron, zinc, cadmium, lead and manga-
nese) of the lake water in both seasons exceeded World Health Organisation (WHO) permissible
limits. Total heterotrophic bacteria and E. coli were present with dry season water samples having
higher amounts. Compared to the control, the mitotic index decreased significantly (p < 0.05) in
the water samples and were characterized by a number of chromosomal aberrations notably
bridges, fragments, sticky chromosomes, disoriented chromosomes, and micronuclei in significant
amounts and these were more pronounced in water samples obtained during the dry season. The
findings in this study are of public health relevance as access to safe water is a fundamental hu-
man need and therefore, a basic human right.
Man-Made Lake; Obazuwa Community; Ph ysicoch emi stry; Microbial Load; Ge no tox ici ty
One of the primary goals of the World Health Organization (WHO) and its Member States is that “all people,
D. I. Olorunfemi et al.
whatever their stage of development and their social and economic conditions, have the right to have access to
an adequate supply of safe drinking water” (WHO, 2011). Although Nigeria is known to be endowed with
abundant water resources, the availability of potable water is a problem in many parts of the country. The provi-
sion of pipe borne potable water is the primary responsibility of the Federal, State and Local Governments but
unfortunately, access to drinking water is grossly inadequate both in quantity and quality (Onokerhoraye, 1995).
As a result, most rural dwellers result to streams, hand-dug wells, rivers, lakes for their drinking water supply.
These water sources are under threat from pollution from either human life style manifested by the low level of
hygiene practiced or through non-point source when already polluted water in the area enters into the ground
water body by lateral or side movement (Wright et al., 2004). Obazuwa community is a rural settlement located
within latitude 5°E, 6°E and longitude 5°N, 7°N, in Ovia North-East Local Government Area of Edo State whose
main sources of drinking water are a man-made lake and an unreliable borehole, therefore the community de-
pends mainly on the lake constructed by the inhabitants through self-help. The community is characterized by
two major tropical climates marked by two seasons (rainy and dry) which run from April to September and Oc-
tober to March respectively.
The most effective way to protect the quality of drinking water is through consistent monitoring of the drink-
ing water supply (Yassi et al., 2001). The complexity of contaminated water makes it almost impossible to carry
out a hazard assessment based on physicochemical analysis alone, therefore, a comprehensive approach involv-
ing the use of plant bioassays alongside chemical and microbial analysis has been advocated for hazard assess-
ment in toxicity screening of contaminated water (Arkhipchuk et al., 2000). The Allium test, a higher plant ge-
netic assay has been accepted as an excellent and alternative first–tier indicator for safety evaluation of cytoge-
netic and mutagenic effects of drinking water and environmental pollutants (Ma et al., 1985; Fiskesjö, 1993,
1997). Currently, there is no documentation on the physicochemical, microbial and cytogenotoxicological
evaluation of the drinking water sources in this rural community, therefore, this study was undertaken to inves-
tigate the efficiency of the combined methods in monitoring the drinking water sources in the rural settlement.
2. Materials and Methods
2.1. Collection of Water Samples
Water samples for the study were collected in March and July 2012. Plate 1 (a-c) shows the site of water collec-
tion: lake water during the dry season, lake water during the wet season and borehole water respectively. The
lake water samples were collected at different depths from five random points within the lake. For the borehole,
sampling protocols described by Claaasen (1982), Barcelona et al. (1985), and APHA (2005) were strictly ad-
hered to during sampling collection. The nozzle of the borehole was swabbed with cotton wool soaked in 70%
(v/v) ethanol and flamed for 2 - 3 min. Samples were collected using washed and sterilized plastic containers
after running water to waste for 4 - 5 min. In both cases, samples were taken in triplicates from each sampling
point aseptically into plastic containers and kept in an ice chest and stored in the refrigerator at 4˚C and analysed
within 24 h of collection.
2.2. Physicochemical Analysis of the Water Samples
Water samples, together with control, were analysed for a number of standard physicochemical properties, in-
cluding nitrates, chlorides and phosphates, according to methods described by APHA (2005). Nine metals
namely lead, copper, cadmium, chromium, iron, zinc, nickel, magnesium and manganese were analysed in the
water samples according to standard analytical methods (USEPA, 1996; APHA, 2005) using an atomic absorp-
tion spectrometer (AAS) (PerkinElmer A Analyst 100). The metal standards were prepared to known concentra-
tions, labelled, and kept inside plastic bottles that were pre-cleansed with concentrated nitric acid and distilled
water. For microbial analysis, the techniques employed were estimation of total heterotrophic bacteria (THB) by
plate count technique and estimation of coliform bacilli by MPN presumptive test (APHA, 1998).
2.3. Allium cepa Assay
The purple variety of average sized onion bulbs, Allium cepa L. (about 30 g, 15 - 22 mm diameter) were pur-
chased from a local market in Benin City and the same batch of bulbs was used throughout. They were sun-dried
for two weeks and the dried roots present at the base of the onion bulbs were carefully shaved off, with a sharp
D. I. Olorunfemi et al.
razor blade to expose the fresh meristematic tissues. The bulbs were then placed in freshly prepared distilled
water to protect the primordial cells from drying up. To account for a number of bulbs in the population that
would be naturally slow or poor growing, seven replicate bulbs were used for each test sample and control (tap
water) and the best five bulbs were chosen for examination. The bulbs were removed from the distilled water
and placed on a blotting paper to remove excess water.
For the root growth inhibition evaluation, seven onion bulbs were utilized for each water sample and the con-
trol (tap water). The base of each of the bulbs was suspended on the water sample inside 100 ml beakers in the
dark for 72 h. Test samples were changed daily. At the end of the exposure period, the roots of five onion bulbs
with the best growth were removed with a forceps and their lengths measured (in cm) with a metre rule. The
percentage root growth inhibition in relation to the negative control was then calculated.
For the evaluation of induction of chromosomal aberration, five onion bulbs were suspended in the water
samples and the control for 48 h., at the end of which root tips from these bulbs were cut and fixed in etha-
nol:glacial acetic acid (3:1, v/v). These were hydrolyzed in 1N HCl at 65˚C for 3 min after which they were
was hed in distilled water. Two root tips were then squashed on each slide, stained with acetocarmine for 10 min
and cover slips carefully lowered on to exclude air bubble. The cover slips were sealed on the slides with clear
fingernail polish to prevent drying out of the preparation by the heat of the microscope. Six slides were prepared
for each water sample and the control out of which five (at 1000 cells per slide) were analyzed at ×1000 magni-
fication for induction of chromosomal aberrations. The mitotic index and the frequency of aberrant cells (%)
were calculated following methods of previous studies (Olorunfemi et al., 2011).
2.4. Statistical Analysis
The means, with 95% confidence limits and the standard errors for results of the root inhibition and chromo-
some aberrations of each water sample were calculated. Data were expressed as Mean ± Standard Error of Mean
(SEM). Differences between the control and the different water samples were analyzed by means of the Stu-
dent’s unpaired t-test. P values of ≤ 0.05 were considered to be statistically significant. All statistical analyses
were carried out using SPSS®14.0 statistical package.
3. Results and Discussion
3.1. Physicochemical Analysis
The results of the physical and chemical analysis of the test water samples collected are presented in Table 1. The pH
value was slightly basic (7.59) in the borehole during the wet season and acidic (5.15) in the lake during the dry sea-
son. The results revealed that the level of parameters such as turbidity, lead, nickel, chromium, in the lake water in the
dry season compared with international (WHO) and national (SON) standards exceeded permissible limits.
The consumption of unsafe water has been implicated in a number of health risks including cancer of the di-
gestive and urinary tract, human gastrointestinal irritation and laxative effects (Okonko et al., 2008; Sia Su, 2007)
and chromosome aberration and DNA damage enhancing genetic changes in somatic cells that can result in de-
creased cell survival or transformation and eventual reproductive abnormalities (Shugart et al., 1992, Fawole et
al., 2008, Olorunfemi et al., 2013). All the water samples were turbid with elevated levels of heterotrophic bac-
teria and E. coli, some of which could produce toxins. Coupled with the acidic nature of the lake water in both
seasons and the slightly alkaline nature of the borehole water in the wet season with the high levels of inorganic
compounds and heavy metals, the water sources would not only be unpalatable in taste but could also pose sig-
nificant health risk to this community as have been found elsewhere (Gupta et al., 2001; Sia Su, 2005).
3.2. Cytogenotoxic Evaluation of Allium Test
Compared to the control, there was gross inhibition of root growth in the water samples in significant (p < 0.05)
amounts. Higher root growth rates were recorded in the water samples obtained in the wet than those collected
in the dry season, for example, the mean root length of A. cepa grown in the lake water sample during dry sea-
son was 4.36 ± 0.55 cm and 4.71 ± 0.64 cm in the wet season while the mean root length was 3.40 ± 0.54 cm and
4.85 ± 0.77 cm for borehole water samples during dry and wet seasons respectively (Figure 1). Comparative analysis
of microscopic parameters shows that the mitotic indices of the test water samples during wet season
Table 1. Physicochemical properties of water samples obtained from the study site (Obazuwa) during dry and wet seasons.
D. I. Olorunfemi et al.
Parameter s Lake water
(Dry season) Lake water
(Wet season) Borehole water
(Dry season) Borehole Water
(Wet season) WHO (2006)
Limit SON (2007)
pH 5.15 5.80 6.45 7.59 6.5-9.5 6.5-8.5
Turbidity 13.1 10.5 1 1. 3 10. 1 - -
Conduc tivi t y 1350 1540 1420 1040 - -
Total Hardness 263.33 326.66 2 36 .66 216 .66 5 00 1 50
Nitrates 100.9 99.3 50.8 2 9. 0 50 50
Phos p h ates 145 .25 1 23 .25 80. 05 9 7.65 - -
Magnesium 163.9 170.8 59.5 71. 3 - 0.20
Chloride 798.75 7 63 .25 745 .50 3 05 .75 - 250
Man ganese 4.1 5.9 - 0.1 - 0.20
Nic kel 0.2 0.3 - - 0.02 0.02
Cadmium 0.1 0.2 - - 0.003 0.003
Lead 0.2 0 .1 0 .1 - 0.01 0.01
Iron 1 1. 1 1 5. 3 - 1.3 - 0.3
Zinc 21.6 3 0. 5 2 .2 2.6 0.01 3.0
Chromium 2.4 3 .0 - - 0.05 0.05
Copper 2 .8 1 .8 1 .8 0 .1 - 0.1
Silver 0.2 0 .1 - - - -
TH Bacteria 5.4 2 .6 4 .1 3 .3 0 0
Coliform Organisms 10 2.0 6.4 4 .6 0 0
Plate 1 (a-c) shows the site of water collection: lake water during the dry season, lake water during the wet season and borehole water respectively.
Plate 1. The study site: Obazuwa man-made lake during the (a) dry
season (b) wet season (c) the only borehole located in Obazuwa
community from where water samples were collected. Photo Credit:
Dr. D. I. Olorunfemi.
D. I. Olorunfemi et al.
Figure 1. Percentage root growth of Allium cepa roots exposed to
the test water samples during dry and wet season.
was greater than those of the dry season in the water samples obtained from both the lake and borehole while the
percentage aberrant cells were greater during the dry season (Figures 2 & 3). Chromosomal aberrations were
induced on exposure to all the test water samples. The most frequent aberrations were chromosome stickiness
and anaphase bridges (Plate 2).
There is a linear relationship between macroscopic and microscopic parameters for all the water samples. In A.
cepa, whenever there is root growth inhibition, there is always reduction in the number of dividing cells; the
lower the mitotic index, the more toxic the waste water, chemical or pollutant (Fiskesjö, 1997; Babatunde &
Bakare, 2006). The direct relationship between the macroscopic growth and microscopic parameters in this
study may infer that the heavy metals in the water samples are responsible for the aberrations observed in the
root tip cells of the onion. The presence of trace metals (Cr, Fe, Mn, Zn) and other inorganic compounds at
varying concentrations in the water samples may be responsible for the root growth inhibition and chromosome
aberrations observed in the A. cepa root meristems. The individual and/or complex interactions of these metals
may have caused the observed cytogenotoxic effects. The inhibition of mitotic index has been attributed to the
effects of environmental chemicals on DNA/protein synthesis of the biological systems (Chauhan et al., 1998).
Anderson (1985) reported that binary mixture of Cr and Ni affected mitotic spindles leading to chromosomal
aberration in exposed A. cepa. More so, some of these metals (Fe, Cr and Zn) in their combined states have been
reported to induce high percentage of micronucleated red blood cells and nuclear abnormalities in newt larvae
(Godet et al., 1993).
Most of the observed cytotoxic/genotoxic effects in the root meristems in A. cepa were possibly induced by
the chemicals and microorganisms in the water samples and this may present a direct or indirect risk to living
organisms. The presence of laggards and stickiness and chromosome bridges are regarded as mitotic irregulari-
ties induced by pollutants (Zhang & Yang, 1994). Sticking of chromosomes probably occurs due to degrada-
tion or depolymerization of chromosomes DNA (Grant, 1982) or as a result of DNA condensation and stickiness
of inter-chromosome fibres (Schneiderman, 1971). Stickiness reflects high toxicity of substance as well as ire-
versibility of the change while acentric fragments in anaphase is the result of chromosome or chromatids inter -
ruptions, indicating interference with DNA. Bridges probably occur by the interruption and joining chromo-
somes or chromatids (Turkoglu, 2007), or as a result of chromosome stickiness, or it may be ascribed to unequal
translocation or inversion of chromosome segments (Gömürgen, 2005).
In conclusion, although the study did not obtain documented evidence on the incidence of major water borne
disease outbreak in the community, notwithstanding, the concentrations of physicochemical constituents and
microbial load of the water samples were higher than permissible national and international limits. Furthermore,
the classical Allium chromosomal aberration conventional test used in this study to detect environmental clasto-
gens revealed reduced mitotic index and chromosome aberrations in the water samples. The observed frequen-
cies of chromosomal bridge, sticky and laggard chromosomes are indicators of chromosome damage. The com-
bination of these methods for evaluating drinking water quality in a developing country like Nigeria is relatively
Gro wt h
D. I. Olorunfemi et al.
Plate 2. Chromosomal abnormalities induced in A. cepa root tips
grown in the water samples (a) sticky chromosome (b) multiple
bridges (c) polar deviation (d) vagrant chromosomes with breaks.
Figure 2. Effect of water samples on mitotic index of A. cepa.
Figure 3. Effect of water samples on chromosomal aberration in A.
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risk to human health and therefore need proper purification treatments before they become safe for consumption.
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