Current Topics in
Toxicology Vol. 9, 2013
Cytotoxic and genotoxic assessment of textile effluent using Allium assay Chibuisi G. Alimba1,*, Adebayo L. Ogunkanmi2 and Femi J. Ogunmola2 1 Cell Biology and Genetics Unit, Department of Zoology, University of Ibadan, Nigeria, 2 Department of Cell Biology and Genetics, University of Lagos, Akoka, Lagos, Nigeria
ABSTRACT Textile processes release various waste streams, gaseous, liquid and solid, into the surroundings and these are capable of contaminating the environment, posing health hazards to the biota. There is paucity of information on textile effluent induced cyto-genotoxicity in eukaryotic plant systems. This study investigated the cytotoxicity and DNA damaging effects of textile effluent using the Allium test. Twelve onion bulbs were grown in concentrations, 1, 2, 5, 10, 25, 50 and 100% (v/v; effluent/tap water) of the effluent (tap water serving as control), for 96 h. Daily root length inhibition for 4 days and cytogenetic analyses at 48 h were investigated. There was significant (p < 0.0001) root growth retardation with 50% effective concentration (EC50) values of 16, 35, 6.5 and 8% for the 24, 48, 72 and 96 h, respectively. Also a concentration dependent significant (p < 0.05) decrease in cell proliferation and increase in chromosomal aberrations compared to the control were observed. Cytological aberrations such as binucleated cells, sticky chromosomes, chromosome fragments and anaphase bridges were induced by the effluents in the root meristems. Fe, Cd, Mn, Ni, Cr, and other physicochemical parameters analysed in the samples may have induced the observed cytogenotoxic effects. This indicates that textile effluent is capable of inducing genome instability and cytotoxicity in *Corresponding author:
[email protected];
[email protected]
A. cepa. The findings may suggest environmental pollution and public health risk from textile effluent exposure. It is also useful in promulgating stringent rules discouraging illegal effluent discharge into the environment. KEYWORDS: Allium cepa, chromosome aberration, effective concentration, mitotic index, textile effluent, pollution 1. INTRODUCTION There are numerous environmental pollution issues worldwide, with some issues common in some regions or countries of the world. Pollution of the natural environment with xenobiotics from various industrial activities is the most common pollution issue, which will continue to increase in an unprecedented manner due to increase in population growth and accelerated industrialization. Textile industrial activities that involve the use of cotton and synthetic fibers and the related printing and dyeing operations, have emerged as a major pollution issue worldwide due to the accompanied release of xenobiotics into the environment. Solid, liquid and gaseous wastes are generated during the long sequence of wet processing stages that require the inputs of large volumes of water, chemicals and energy at each stage. Also, as garment designs require different colour combinations of fabric materials, the use of different fibers and chemicals are involved in textile processing. This will lead to the production of significant volume of wastes with different compositions. It has been well documented that in
66 textile industries, dyeing and printing operations involve the use of different varieties of organic dyes [1, 2], and different fabric processes like singeing, starching and fire retarding which utilize different chemicals including bleaching agents [3], followed by rinsing with large volumes of water to eliminate all residues from the operational stages so as to produce quality fibers. The volume of water used during the various operational stages is discharged as wastewater into the environment, and this has been reported to be highly toxic to living organisms mostly due to the presence of salts, surfactants (such as detergents, emulsifiers and dispersants), ionic metals and their metal complexes, toxic organic chemicals, biocides and toxic anions [4, 5]. In Nigeria, textile industries mostly located in industrialized cities are well known for the discharge of huge volumes of untreated effluent directly into the environment [3, 6-8]. This act is exacerbated by the ignorance on the part of many Nigerians on the effects of textile effluent on environmental degradation and its impacts on public health. Also, poor enforcement by the government of stringent regulations that prohibit illegal discharge of textile effluents is not favouring environmental conservation. This situation is further aggravated as seasonal vegetables and arable crops are cultivated in textile effluent-polluted environments or using textile effluents and/or effluent polluted water sources for irrigation. This is commonly observed in the northern parts of Nigeria where rivers and other water bodies receiving untreated textile effluent are used in irrigation farming systems and sometimes these effluents are used directly [7, 8]. This is a common observation during the dry season when rainfall is usually low and drought extends to between seven to eight months of each year. Vegetables and other crops produced through irrigation farming are important sources of food for humans and domesticated animals. It is possible that these crops bioaccumulate toxic compounds from the effluents and these substances biomagnify along the feeding pathway to affect human and other tertiary consumers. It is also plausible that these bioaccumulated toxic substances could alter the genetic compositions of these crops leading to loss of biodiversity.
Chibuisi G. Alimba et al. Therefore there is a need to investigate the DNA damaging potentials of textile effluent on plant somatic chromosome system. We had previously shown that textile effluent is capable of contaminating aquatic environment through the chemical and physical substances present in it. These xenobiotics induced micronucleus and nuclear abnormalities in peripheral erythrocytes of treated Clarias garipinus through oxidative stress [9]. Also, Awomeso et al. [6] showed that water bodies receiving untreated textile effluents in Lagos had higher physicochemical parameters and heavy metals than permissible limits in Nigeria. Ohioma et al. [7] similarly observed higher physicochemical parameters and heavy metals in textile effluent collected from Kaduna textile industry, Nigeria. Although, there is paucity of information on the cyto-genotoxicological impacts of textile effluents on plant test systems in Nigeria, Marwari and Khan [10] have reported that Lycopersicon esculentum var K21 (Tomato plants) grown in 20 and 30% concentrations of textile effluents from Sanganer, India, showed reduced chlorophyll, carbohydrate, protein and nitrogen contents than untreated plants. Also, root and shoot length, root and shoot dry weight and total dry weight of the plants were reduced and analysis of the fruits showed higher concentrations of Cr, Pb, Cu and Zn than the permissible limits. The authors concluded that the practice of growing vegetables using textile wastewater can enhance bioaccumulation and biomagnification of metals via food chain. Plant test systems are especially well suited for research in screening and environmental monitoring of xenobiotics [11]. Moreover, they are sensitive and simple in comparison with animal bioassays, and have been validated in international collaborative studies as efficient test systems for the cytotoxicity and genotoxicity monitoring of environmental pollutants [12]. The common onion (A. cepa) is a good experimental model for in vivo genotoxicity evaluation of substances and complex mixtures [13]. A. cepa bioassay can be employed in in situ and laboratory studies to measure phytotoxicity, cytotoxicity and genotoxicity [14]. It has been successfully used in both toxicity and genotoxicity screening for the presence of xenobiotics in
Cyto-genotoxicity of textile effluent using Allium test aquatic habitats, underground water, wastewater and solid waste leachates [15-17]. In this study, we utilized the Allium assay to evaluate the cytotoxicity and DNA damaging potentials of textile effluent. Some physicochemical characteristics and heavy metal content of the effluent were also determined in accordance with standard methods. 2. MATERIALS AND METHODS 2.1. Textile wastewater effluent collection
The textile wastewater effluent used for this study was collected from Woolen and Synthetic Textile Manufacturing Limited Ikeja, Lagos, where an underground pipe channels untreated effluent into the Lagos lagoon. A composite mixture of the collected sample was taken to the laboratory in a precleaned 4 L transparent container. It was filtered using 2.5 µm filter paper (Whatman® No. 42) and the pH, colour and odour were determined within 24 h of sample collection before refrigeration at 4 °C. 2.2. Physicochemical and heavy metal analysis of the effluent
Some of the physicochemical parameters of the effluent, biochemical oxygen demand (BOD), chemical oxygen demand (COD), dissolved oxygen (DO), total suspension solids, chloride, sulphate, hardness, alkalinity, nitrate, phosphate, and conductivity, were measured in accordance with the guidelines set by the American Public Health Association [18] and United States Environmental Protection Agency [19]. Iron (Fe), cadmium (Cd), chromium (Cr), manganese (Mn), aluminium (Al) and nickel (Ni) concentrations were also analysed in the effluent. 100 ml of the effluent was digested using 1.0 N concentrated HNO3. The concentrations of examined metals in the digested sample were analysed using PerkinElmer® A3100 atomic absorption spectrophotometer. 2.3. Allium cepa root growth inhibition and cytological analysis
Equal-sized onion bulbs (Allium cepa; 2n = 16), used for this study were obtained from Bariga, Lagos, Nigeria. They were air dried for 14 days before being used to evaluate the root growth inhibition and mitotic index (MI) (cytotoxicity indices), and in vivo induction of chromosomal
67 aberrations (genotoxicity index) [11, 20]. The outer dry, brown scales of the bulbs and the bottom plates (dead roots) of the onions were carefully removed, leaving rings of the primordial roots intact. Twelve onion bulbs were used for each of the following concentrations of the effluent: 1, 2, 5, 10, 25, 50 and 100% (v/v; effluent/tap water). They were placed directly on each concentration of the sample in 100 ml beakers at room temperature in the dark, as soon as the bottom plates were removed to prevent the primordial roots from drying up. Similar treatment was used to grow the bulbs in tap water to serve as control. Effluent for each concentration was changed daily to ensure constant exposure of the bulbs. The root lengths of ten bulbs were measured daily for 4 days using measuring rule. The average root length per bulb per concentration was recorded as mean ± SE. These values were used to determine the percentile root growth restriction in relation to the negative control and the EC50 for the effluent per day was evaluated. Two onion bulbs with good root growth were collected and 0.5-1 cm of the root tips of each root on each bulb was cut and fixed in ethanol:glacial acetic acid (3:1, v/v) for 24 hours and was used for the cytological analysis. The roots were hydrolyzed with 1N HCl at 60 °C for 5 minutes. They were then rinsed in distilled water, squashed on micro-glass slides and stained with acetocarmine for 10 minutes and cover slip was carefully lowered onto each slide to exclude air bubbles. The cover slip was sealed on the slide with finger nail polish. Five slides per onion were prepared for each concentration and 1000 cells/ slide were scored microscopically at 1000x magnification, for mitotic index analysis and induction of chromosome aberrations. The occurrence and frequency of aberrant cells were examined in all the stages of cell division and percentage aberrations were determined relative to the total number of dividing cells. The mitotic index (MI) was determined by counting the number of dividing cells per concentration and the control relative to the total number of cells scored. These scoring procedures were in accordance with Fiskejo [11]. 2.4. Statistical analysis
All statistical analyses were conducted with Graphpad prism 5.0® computer program. Data are
68 presented as mean ± SE. The percentage root growth, frequency of chromosomal aberrations and mitotic index in relation to the negative control were determined. One-way Analysis of Variance (ANOVA) was used to determine the differences among the means of treated and control groups. When the F-test for differences among means of the treated groups were significant pair wise, comparison between treated groups and negative control was determined using multiple comparison procedure of the Dunnett post-hoc test and the differences were considered significant at p < 0.05, p < 0.01 and p < 0.001 levels of significance. 3. RESULTS 3.1. Physicochemical characteristics
The physicochemical parameters and heavy metals analyzed in the textile effluent are presented in Table 1. The effluent had a slight irritating odour and brownish green colour, and was slightly alkaline (pH = 9.2). The values of most of the inorganic parameters and heavy metals analyzed in the effluent were above standard permissible limits. 3.2. Cytotoxicity
The cytotoxic effect of the textile effluent was evaluated from the root growth inhibition and cell proliferation data. There was good root growth in onions cultivated in tap water while the textile effluent induced a concentration-dependent significant (p < 0.05) root growth inhibition in A. cepa during the 96 h (Figures 1 - 4). The highest root growth was in 1% concentration while the least was in 100% concentration for each day. During the experiment, the root tips of onions cultivated in 25, 50 and 100% turned greenish brown. This was observed at days 3 and 4, and change in root tip colour was most intense in onions cultivated in 50 - 100%. EC50 for the effluent at days 1 - 4 are 16, 35, 6.5 and 8.0%, respectively (Figures 1 - 4). The effect of the effluent on the cell division in the roots of treated onions was determined through the mitotic index. There was concentrationdependent significant (p < 0.05) reduction in cell proliferation compared to the control (Table 2).
Chibuisi G. Alimba et al. Table 1. Physicochemical parameters and heavy metals analysis of textile effluent, and their national and international permissible limits. Textile effluent
NESREAa [46]
USEPAb [19]
9.2
6.0 - 9.0
6.5 - 8.5
Bluish green
-
-
16650.0
-
-
Salinity
12.76
-
-
Turbidity
80.0
-
-
Nitrate
64.16
10
10
BOD#
155.0
50
-
COD**
Parameters* H Colour Conductivity
290.0
90
-
***
3.0
-
-
Chloride
7260.8
250
250
Sulphate
330.0
250
250
Hardness
196
150
0 - 75
Alkalinity
2510
-
20
TSS##
12.0
-
-
9320.0
-
-
Iron
0.12
0.3
0.3
Manganese
0.03
-
-
Cadmium
0.12
0.2
0.05
Nickels
0.07
0.05
-
Chromium
11.05
0.05
0.1
Aluminium
ND
-
-
DO
TDS
*
*All values are in mg/L except pH, Conductivity (µScm-1), Salinity (%), Turbidity (FTU); # BOD - Biochemical Oxygen Demand; ## TSS - Total Suspended Solid; **COD - Chemical Oxygen Demand; ***DO - Dissolved Oxygen; a National Environmental Standards and Regulation Enforcement Agency (2009) (Nigeria) maximum permissible limits for wastewater. b United State Environmental Protection Agency (2006) (www.epa.gov/safewater/mcl.html).
Cyto-genotoxicity of textile effluent using Allium test 5
p=0.0001; F = 6.05; r2=0.57; EC50=16% (100)
0.6
(77.29)
*
(67.73)
0.4
*
(71.71)
*
(65.74)
* (27.89)
c
0.2
(23.51) (19.92)
c
c
0.0
3
(59.22)
c
(58.28) (58.18)
c
c
2
(34.07) (31.97)
c
c
(23.06)
c
1
(17.30)
c
10 .0 25 .0 50 .0 10 0. 0
5. 0
2. 0
Ta p
w
1. 0
at er
.0
.0
10 0
50
.0 25
0 5.
.0
0 2.
10
0 1.
at er w Ta p
Day 1
Figure 1. Root growth (Mean ± SE) of A. cepa at different concentrations of the textile effluent on day 1 of exposure. Means of treated are significantly different from the control (tap water) as determined by Dunnette Multiple Posthoc Test (*p > 0.05, cp < 0.001). Parentheses: percentage of root inhibition of treated compared to control.
Day 3
Concentration (% , v/v)
Figure 3. Root growth (Mean ± SE) of A. cepa at different concentrations of the textile effluent on day 3 of exposure. Means of treated are significantly different from the control (tap water) as determined by Dunnette Multiple Posthoc Test (cp < 0.001). Parentheses: percentage of root inhibition of treated compared to control.
p=0.0008; F = 4.85; r 2=0.51; EC50=15%
5
(100)
p 0.05, ap < 0.05, bp < 0.01, cp < 0.001).
3.3. Genome instability
The induction of chromosomal aberrations in the effluent treated onions compared to the tap water was used as the genotoxicity index. There was concentration-dependent significant (p < 0.05) increase in the percentage chromosomal aberrations in the treated onions than in the control (Table 2). Figure 5 presents photomicrographs of some cells observed in the slides. Figure 5a shows normal metaphase and Figure 5b normal anaphase observed in the untreated onion roots. The chromosome aberrations observed in the treated onions include C-mitosis (Figure 5c), vagrant (Figure 5d), binucleated cells with the two nuclei at different phases of cell division (Figure 5e) and anaphase bridge (Figure 5f). It was observed that some cells (at 50 and 100% treatment) showed some degree of apoptosis, as their nuclear materials tend to fragment (Figure 5g), while some cells, observed mainly at 100%, had total loss of the nuclear materials (without nucleus; Figure 5h). 4. DISCUSSION Environmental pollution effects and toxicity of textile effluents vary considerably due to their
composition. These in turn depend on the differences in fabric production processes. In Nigeria, effluents are usually discharged through drainage channels into both terrestrial and aquatic environments untreated. This will not only alter the water quality but may also affect both fauna and flora populations of the ecosystems. Physicochemical analysis and heavy metal characterization of effluents and dye residues produced from textile industries are the common methods of assessing the toxicity of textile wastewater. These methods usually do not provide information on the total toxic chemicals present in the effluents and the possible synergistic and antagonistic interactions of the chemicals in biological systems. The Allium assay is a universally recognized toxicological assay for assessing the effects of individual and complex mixtures of chemicals on root growth, mitotic depression and DNA damage, due to its short cycle duration, sensitivity, simplicity and cost effectiveness [20, 21]. Data from studies utilizing the Allium test may indicate the presence of cytotoxic, genotoxic and/or mutagenic agents that may pose direct or indirect risks to living
Cyto-genotoxicity of textile effluent using Allium test
71
Figure 5. (a) Normal metaphase; (b) normal anaphase. Others are different aberrations observed in the Allium cepa meristematic cells treated with textile effluents: (c) C-mitosis; (d) vagrant chromosomes; (e) binucleated cells with different stages of nuclear division; (f) anaphase bridge; (g) vacuolated nucleus at interphase; (h) cells with eroded nuclei. Magnification 1000x.
organisms. Allium assay has extensively been shown to have good correlation with different in vitro and in vivo mammalian cell line studies [22-24]. We had previously shown that the Allium assay was sensitive to the cytotoxic and genotoxic effects of xenobiotics present in e-waste and incinerated bottom ash leachates [16, 25]. The results of this study showed that textile effluent induced concentration-dependent significant decrease in root growth and mitotic index, and increase in percentage of chromosomes with aberrations in treated A. cepa compared to the tap water (control). Environmental pollution from textile effluents have been the subject of much thought and research in recent times [7, 8, 10, 26-28], and it is expected that these studies will eventually lead to the promulgation and enforcement of regulations that will mitigate pollution of the natural environment by textile industries. The inhibition of mitotic index (MI) by the effluent reflects its cytotoxicity and may explain its direct effect on root elongation (Figures 1 - 4). Reduction in mitotic index below 22% when compared with the control may indicate that the effluent can cause lethal effects on organisms [29]. In our study, there was concentration-dependent decrease in the mitotic index induced by the tested textile effluent on the
onions compared to control, although only 50 and 100% concentrations of the effluent were below 22%. This suggests that at these concentrations severe toxicity may be inflicted on organisms in case of exposure. It is possible that the analyzed physicochemical parameters, heavy metals and unidentified organic components of the effluents may have induced the observed mitotic index inhibition. These xenobiotics are also capable of inducing apoptosis which may lead to cell death in plants [30]. This may account for the presence of cells with vacuolated and empty nuclei (Figures 5g and 5h) in the treated onions, mostly at 50 and 100% concentration. Metals are also capable of distorting A. cepa cell cycle and/or inducing chromatin dysfunction during interactions with DNA, leading to decreased mitotic index [31]. It is also possible that xenobiotics in the effluent caused disturbances in DNA synthesis or halted metabolic processes, thus preventing cells from dividing during mitosis, which resulted in the decreased mitotic index [32, 33]. This has been similarly reported in A. cepa treated with e-waste leachates [15, 16], bottom ash hospital waste leachate [25], municipal landfill leachates [34], and industrial effluents from metal and dye manufacturing plants [30]. Also, textile effluents have been shown to induce root growth inhibition in Celossia argentea [26] and two vegetables,
72 Lagenarian siceraria (Lauki) and Abelmoschus esculentus (Lady Finger) irrigated with textile effluent [35]. The induction of chromosomal aberrations observed in the root cytology of treated A. cepa compared to control showed the potential genotoxic effects of the textile effluent. Most of these aberrations are lethal and can lead to death while others may cause genetic defects that could be expressed as congenital abnormalities in organisms including humans, or be transferred from one generation to another if germ cells are affected [36, 37]. It can also lead to loss of biodiversity by reducing the selective advantage or fitness of these plants to environmental selective pressures. These findings from this study further showed that the constituents of the tested effluent (Table 1) interacted with the nuclear materials possibly through different mechanisms. It is possible that metals present in the effluent distorted the spindle fibers and caused chromosome disturbances during mitosis, cross-linking with DNA and/or proteins causing direct chromosome break or exchange [30]. For instance, Cr and Ni analyzed in the effluent have been shown to affect mitotic spindles that lead to chromosome aberrations [38]. It is also plausible that metals and other physicochemical parameters in the effluents induced reactive oxygen species formation that caused DNA strand breaks through lipid damage. This was inferred following the studies of Odjegba and Bamgbose, [26] wherein they exposed Celossia argentea to different concentrations of textile effluents and observed significant increase in malondialdehyde (MDA) concentrations in the leaves compared to control (an index of lipid peroxidation). Ayoola et al. [9] similarly showed that textile effluent induced micronucleus and nuclear abnormalities (genotoxicity) in C. gariepinus through significant alterations in the antioxidant status of the fish. Also, Radetskia et al. [39] showed that mixture of chemicals in incinerator bottom ash leachates induced genotoxic effects in Vicia faba through reactive oxygen species formation as determined by assessing the antioxidant stress enzyme (catalase, superoxide dismutase, peroxidase and glutathione reductase) activities in the root tissues of the plant. Besides free radical generation that
Chibuisi G. Alimba et al. induces oxidative damage, metals in the textile effluent are also capable of binding to phosphate and nitrogenous bases altering DNA primary and secondary structures [40], while sulphates and nitrates can interfere with protein structure and function and cause DNA damage [41]. The presence of sticky and anaphase bridge chromosomes are due to chromatin dysfunction, chromatid breaks and spindle failure and this suggests the presence of clastogens and aneugens in the effluent [42]. Sticky chromosomes when formed can lead to the inhibition of cytokinesis which will cause the formation of binucleated cells (Figure 5e). The presence of sticky chromosome also signifies high toxicity of the effluent which will lead to irreversibility of chromosome aberrations and cell death (Figures 5g and 5h). Also, the presence of vagrant chromosomes may indicate risk of aneuploidy [11]. Besides the cytogenotoxic effects of the textile effluents, colour dyes in the effluents could obscure visibility in aquatic environments, affecting the aesthetic value of water bodies. It may be responsible for low water transparency (turbidity) recorded in the effluent and can lead to poor gaseous solubility in aquatic environment [43]. The presence of toxic metals as observed in the effluent can enhance the depletion of dissolved oxygen and destabilize the ability of the water to reduce microbial loads leading to autopurification [3]. The analyzed metals in the effluent could be deposited as particulates in the aquatic environment, become bioaccumulated in aquatic forms and pose health risk to humans. Low levels of dissolved oxygen in the effluent could indicate an increased anaerobic condition of the effluent which could affect respiration in aquatic species. Hydrogen sulphide is usually formed during conditions of oxygen deficit in water bodies with high organic materials and sulphate [44], and this is usually deleterious to aquatic health. High BOD concentration corroborates the low DO and suggests increased pollution strength of the effluent. Similarly, high COD level suggests higher concentrations of harmful organic compounds in the effluent [45]. The data obtained from this study indicate that the untreated effluents from the textile industry that are directly discharged into the Ibeshe River
Cyto-genotoxicity of textile effluent using Allium test
73
contain toxic compounds. These compounds may contaminate the surface water, thereby making it unfit for irrigation and drinking. Therefore, indiscriminate discharge of textile wastewater into water bodies should be prohibited and proper treatment of effluents before discharge into the environment should be enforced. Moreover, laws regulating pollution from all forms of anthropogenic activities should be enforced by appropriate authorities to mitigate its consequences on both plant and animal species. The continual discharge of untreated textile effluents into the environment will undoubtedly cause threat to the ecosystem and to human health.
5.
5. CONCLUSION
11. 12. 13.
Textile effluent induced mitotic inhibition, poor root elongation and various chromosomal aberrations in A. cepa. The observed physicochemical parameters, heavy metals and possibly unanalyzed organic components of the effluent, which are deleterious to biological organisms, induced the observed cytotoxicity and genome instability in the A. cepa. It is suggested that adequate treatment of effluents and proper monitoring of their discharge be instituted to avert environmental pollution and possible health risk due to exposure to chemical mixtures in the effluents. ACKNOWLEDGEMENTS We thank Mr. Obu, Technical staff, Department of Cell Biology and Genetics, University of Lagos for his technical assistance on the A. cepa slide preparation.
6. 7. 8. 9. 10.
14. 15. 16. 17. 18.
19.
CONFLICT OF INTEREST STATEMENT The authors declare no conflict of interest. REFERENCES 1. 2. 3. 4.
Weltrowski, M., Patry, J. and Bourget, M. 1996, Advanced Chitinous Sci., 1, 462. Achwal, W. B. 1997, Colourage, 44, 31. Ademoroti, C. M. A., Ukponmwan, D. O. and Omode, A. A. 1992, Int. J. Environ. Studies, 39, 291. Dorn, P. B., Salanitro, J. P., Evans, S. H. and Kravetz, L. 1993, Environ. Toxicol. Chem., 12, 1751.
20. 21. 22. 23. 24.
Lin, S. H. and Peng, C. F. 1994, Water Res., 28, 277. Awomeso, J. A., Taiwo, A. M., Gbadebo, A. M. and Adenowo, J. A. 2010, J. Applied Sci. Environ. Sanitation, 5(4), 353. Ohioma, A. I., Luke, N. O. and Amraibure, O. 2009, J. Toxicol. Environ. Healt. Sci., 1(2), 34. Yusuff, R. O. and Sonibare, J. A. 2004, Global Nest: The Int. J., 6(3), 212. Ayoola, S. O., Bassey, B. O., Alimba, C. G. and Ajani, E. K. 2012, Pakistan J. Biol. Sci., 15(17), 804. Marwari, R. and Khan, T. I. 2012, J. Environ. Biol., 33, 849. Fiskesjo, G. 1985, Hereditas, 102, 102. Türkoğlu, Ş. 2009, Caryologia, 62(3), 171. Lerda, D., Biagi Bistoni, M., Pelliccioni, P. and Litterio, N. 2010, Plant Biol., 12(4), 685. Yildiz, M. and Arikan, E. S. 2008, Caryologia, 61(1), 45. Bakare, A. A., Adeyemi, A. O., Adeyemi, A., Alabi, O. A. and Osibanjo, O. 2012, Caryologia, 65(2), 94. Bakare, A. A., Alabi, O. A., Gbadebo, A. M., Ogunsuyi, O. I. and Alimba, C. G. 2013, Challenges, 4, 169. Kong, M. S. and Ma, T. H. 1999, Mutat. Res., 426, 221. American Public Health Association, 1998, Standard Methods for the Examination of Water and Wastewater, American Public Health Association (APHA, AWWA, WPCF), 20th Ed., Washington, DC. United State Environmental Protection Agency (USEPA). Drinking water contaminants. Washington, DC, USA, 2009, Available online: http://water.epa.gov/drink/contaminants/ index.cfm#List (accessed on 5 February 2010). Nielson, M. H. and Rank, J. 1994, Hereditas, 121, 249. Evseeva, T. I., Geras’kin, S. A. and Shuktomova, I. I. 2003, J. Environ. Radioactivity, 68, 235. Fiskesjo, G. 1981, Hereditas, 95, 155. Gerstner, H. B. and Huff, J. F. 1997, J. Toxicol. Environ. Healt., 2, 491. Vig, B. K. 1978, Environ. Healt. Perspect., 27, 27.
74 25. 26. 27. 28.
29. 30. 31.
32. 33. 34. 35. 36.
Chibuisi G. Alimba et al. Akinbola, T. I., Adeyemi A., Morenikeji, O. A., Bakare, A. A. and Alimba, C. G. 2011, Toxicol. Industrial Healt., 27(6), 505. Odjegba, V. J. and Bamgbose, N. M. 2012, Afri. J. Environ. Sci. Technol., 6(11), 438. Roopadevi, H. and Somashekar, R. K. 2012, J. Environ. Biol., 33, 167. Sharma, S., Kalpana, Arti, Shweta, Suryavathi, V., Singh, P. K., Ramesh, Shipra and Sharma, K. P. 2007, Australasian J. Ecotoxicol., 13, 81. Antonsiewicz, D. 1990, Folia Histochem. Cytobiol, 28, 79. Chandra, S., Chauhan, L. K. S., Murthy, R. C., Saxena, P. N., Pande, P. N. and Gupta, S. K. 2005, Sci. Total Environ., 347, 46. Glińska, S., Bartczak, M., Oleksiaka, S., Wolska, A., Gabara, B., Posmyk, M. and Janas, K. 2007, Ecotoxicol. Environ. Safe, 68, 343. Yuzbasioglu, D., Unal, F., Sancak, C. and Kasap, R. 2003, Caryologia, 56, 97. Metin, M. and Bürün, B. 2010, Caryologia, 63(4), 367. Bakare, A. A. and Wale-Adeyemo, A. R. 2004, Natl. Environ. Pollut. Technol., 3, 455. Matuhur, N., Bhatnagar, P. and Verma, H. 2006. J. Environ. Sci., 18(5), 964. Swierenga, S. H. H., Heddle, J. A., Sigal, E. A., Gilman, J. P. W., Brillinger, R. L., Douglas, G. R. and Nestmann, E. R. 1991, Mutat. Res., 246(2), 301.
37. 38. 39. 40. 41. 42. 43.
44.
45. 46.
Özmen, A. 2010, Caryologia, 63(2), 157. Grover, I. S. and Kaur, S. 1999, Mutat. Res., 426, 183. Radetskia, C. M., Ferrarib, B., Cotellec, S., Masfaraudc, J. F. and Ferard, J. F. 2004, Sci. Total Environ., 333, 209. Bridgewater, L., Manning, F., Woo, E. and Patierno, S. 1994, Mol. Carcinogen., 9, 122. Resit, M., Jenner, P. and Halliwell, B. 1998, FEBS Letter, 43, 231. Leme, D. M. and Marin-Morales, M. A. 2009, Mutat. Res., 682, 71. Knasmuller, S., Zohrer, E., Kainzbauer, E., Kienzl, H., Colbert, B., Lamprecht, G. and Schulte-Hermann, R. 1993, Mutat. Res., 299, 45-53. WHO, 2002, Water Pollutants: Biological Agents, Dissolved Chemicals, Non-dissolved Chemicals, Sediments, Heat, WHO CEHA, Amman, Jordan. Sawyer, C. C. and McCarty, P. L. 1978, Chemistry for Environmental Engineers, McGraw Hill, New York, pp. 331-514. National Environmental Standards and Regulation Enforcement Agency (NESREA) 2009, (Federal Republic of Nigeria Official Gazette), National Environmental (Sanitation and Waste Control). Federal Government of Nigeria Printer, Abuja, Nigeria; FGP 112/102009/L000 (OL54), No.60 (96), pp. 1057-1102.
