Screening of Asa River water in Ilorin, Kwara State, Nigeria for available pollutants and its effects on mitosis and chromosomes morphology in Allium cepa cells

Improper disposal of wastes as an environmental problem is common in African and other developing countries of the world, and it raises concerns due to its potential threats to the life of organisms in both terrestrial and aquatic ecosystems. In this study, Asa River in Ilorin, Nigeria was evaluated for cytogenotoxicity at 25.0 %, 50.0 %, 100.0 % following the Allium cepa assay. Water samples were collected from three points tagged A, B, C, and each point was 500 m apart from each other. The water samples were used to grow A . cepa for microscopic and macroscopic toxicities screenings. Heavy metals and volatile organic pollutants in the water were elucidated following the Atomic Absorption Spectroscopy and Gas Chromatography-Mass Spectroscopy. The Water samples induced higher mitotic index values, except the sample C which induced smaller mitotic index value than the negative control. Root growth in the exposed A . cepa was signicantly promoted at 25.0 % of the water samples, while signicant reduction was obtained at 50.0 % and 100.0 % of the sample C, and 100.0 % of the sample A. The absolute water sample A induced highest percentage chromosomal aberrations, as the water samples B and C induced higher percentage chromosomal aberration than the negative control. Cadmium was detected at a concentration higher than its permissible limit in drinking water unlike Zinc, Iron, Manganase. Poly aromatic hydrocarbons, Aromatic amines, Acridine dye, Phenolic and Polychlorinated compounds were detected in the water sample. These pollutants may be responsible for the observed proliferative, inhibitory, cytotoxic and genotoxic effects of the water samples on A . cepa cells. Our results suggest that Asa River is polluted, having potential to inict different adverse effects on human, animals and plants utilizing it along its course.


Introduction
Water is most essential in the life of living organisms perhaps due to the fact that cell (s) that gives life to living organisms contains water to a large extent. The nourishment of cells that form unicellular or multicellular organisms requires water apart from other essentially required nutrients in both macro and micro quantities. The functions of water are best derived from it when it is in a state of purity. However, pollution makes water impure thereby making it to lose its expected functions when used up through chemical reactions. Rivers, lakes and ponds are sources of freshwater providing water for drinking, irrigating farmlands and other human activities globally. Pollution of surface water source occurs through release of industrial wastewater, agricultural and domestic wastes into water body according to Guan et al. [1] Hussein et al. [2] observed that contamination and pollution of aquatic environment has become a serious matter of concern globally due to their adverse effects on the ecological balance including loss of aquatic biodiversity. Although natural contamination of freshwater sources may occur at gradual processes through chemical weathering and soil leaching, but the release from anthropogenic sources is rapid and takes place at alarming rate as reported by Ogunkunle et al. [3]. Therefore, it should be of paramount importance that freshwater sources are regularly screened in order to ensure that drinking water and agricultural products being watered with water from these sources are free of contaminants and pollutants that might nd their way into water through series of anthropogenic activities.
Asa river has its source in Oyo state, south west, Nigeria, while in Kwara state, it is located within Asa and Ilorin west local governments. The river is 56 km long, about 100 m wide supplying water to majority of people of Ilorin through building of dams such as Asa, Agba, Unilorin and Sobi lake dams according to Ogunkunle et al. [3] and Balogun and Ganiyu [4]. Asa river is a reservoir to a number of contaminants and pollutants from industrial, municipal and agricultural sources. Poor solid wastes management is a problem common to both rural and urban cities in Nigeria and other developing countries in Africa.
Ebikapade and Jim [5] observed that solid wastes are usually dumped on open lands along major highways and riverbanks. Asa river is not exempted in this unhealthy practice as its riverbanks serve as a refuse dumpsite to different kinds of wastes from various sources. These contaminants and pollutants are chemicals compounds and heavy metals capable of in icting various harmful effects on organisms at their cellular level according to Opasola et al. [6] and Olatunji et al. [7]. Chemical compounds found in water body from different sources have been reported to be cytotoxic, mutagenic and genotoxic as said by Pellacani et al. [8]. Assessment of quality of this water body is necessary in order to ensure that its use for different purposes is not associated with adverse effects in humans, animals and plants. Genetic toxicological assessment of water is an important indispensable quality assurance screening of water before its nal consumption. According to Iqbal et al. [9] it reveals possible genetic effects of tested water samples on cell division and chromosomes behavior during mitosis and meiosis in the test model.
Allium cepa is one of the higher plants that is usually employed as an excellent genetic model for assessing cytogenotoxic effects of environmental contaminants and pollutants on eukaryotic cells due to its good chromosomes condition characterized by large metacentric chromosomes [10,11]. It is easy to carryout and cheap in term cost implication. Interestingly, it produces results that are comparable with results from animal genetic assays. Bhat et al. [12] stated that it can be used to screen cytogenotoxicity of single and complex chemicals in industrial wastes. The Allium cepa assay was employed to evaluate cytogenotxicity of Sungai Dua river water in Pulau Pinang, Malaysia and Guaribas river water in Piauí, Brazil [13,14]. Other genetic models such as Ames test, SOS/Umu test, comet assay and micronucleus test have also been employed in studying potential genotoxicity and mutagenicity of Organochlorine Pesticides (OCPs), their degradation products, and Polycyclic Aromatic Hydrocarbons (PHAs) in river surface waters in Central Chile, Shanghai, China and Adige river basin [1,15,16,17]. Previous studies on cytogenotoxicity of heavy metals and their accumulation in the organs of some selected sh species in Asa river water body and Apodu reservoir in Malete Ilorin, Kwara State, Nigeria employed micronucleus assay and Ames test which can detect only clastogenic effect and point mutations caused by environmental contaminants in this water body as reported by Hussein et al. [2] and Anifowose et al. [18].
This present study was undertaken on Asa river water samples to screen for potential cytotoxic and genotoxic effects of its available chemicals contaminants / pollutants following the Allium cepa assay, and their identi cation using Gas Chromatographic-Mass spectroscopy and Atomic Absorption Spectrophotometric techniques.

Study Area
Asa river has its source in Oyo State Southwestern Nigeria and ows through Ilorin, the capital of Kwara State, Nigeria in the South-North according to Balogun and Ganiyu [4]. It is located in Ilorin at the Southwestern part of Kwara state, surrounded by Moro local government to the North, Oyun and Offa local governments to the South and Ilorin local government to the East. Asa river covers an area of 5036.625km 2 and lies between latitudes 4°12 N and 4°29 N and longitudes 8°7 E and 8°42 E [19,20]. (Figure 1).
The major occupation which Kwara State's economy depends on is Agricultural practices from which farmers produce cassava, sorghum, yam, cowpea, maize, groundnut and rice as reported by [21]. Olatunji et al. [7] reported that the river receives domestic wastes including sewage and agricultural wastes run off along the bank of the river.

Collection of water samples
Water samples were collected from three points in Asa river in Ilorin, Kwara state. The collection points were selected and tagged as sampling point A which is close to a refuse dump site at the river bank, point B which is ve hundred meters away from point A, point C which is ve hundred meters away from point B and this is where farmers who have farms beside the river bank usually pump water to irrigate their farms or gardens along the course of Asa River (Plate 1).
The collected water samples were kept in different three clean 4 liters capacity glass bottles and were immediately transferred for refrigeration at 4°C in the new Biology laboratory complex, LAUTECH, Ogbomoso, Oyo state, Nigeria, until further analyses in line with the study reported by Climents et al. [16].

Allium cepa Assay on water samples
Onions were purchased from Ipata market, Ilorin, Kwara state, Nigeria. They were sun dried for 1 week in order to reduce moisture content to facilitate root growth [23]. Ten onions per concentration were prepared by removing dried outer scales and scraping dried old roots of the primordial root ring for the onions to sprout new roots as previously described by Akinboro et al. [24] and Ahlem et al. [25]. Each of the water samples A, B, and C (absolute at 100%) was diluted with clean borehole water to obtain 25.0% and 50.0% concentrations. Negative and positive controls were borehole water and 0.1% hydrogen peroxide. Ten onions were planted on water sample in 100 ml capacity beakers at each of the selected concentrations and placed in a cupboard for 48 hours and 72 hours in order to evaluate effects of the water on cell division and chromosomes structures (microscopic evaluation) and on root growth (macroscopic evaluation), respectively [26]. The water samples in the beakers were replaced with fresh one after every 24 hours and the sprouted onions were placed in the fresh water sample, and thereafter put back into the cupboard for root growth to continue. When it was 48 hours of onions root growth in the water sample, root tips from four onions were cut with a scissors and xed in ethanol acetic acid xative (3:1). The xed root tips were rinsed with distilled water and then hydrolyzed in 1N HCL for 10 minutes at 60°C. Slides were prepared and observed under the light microscope (CH-China) using oil immersion objective lens to score dividing cells and chromosomal aberrations as previously described by de Castro e Sousa et al. [14] and Akinboro et al. [27]. Root length from the remaining six onions was measured with a ruler and recorded in centimeter to determine toxicity level of the water samples to A. cepa roots as described before by Akinboro and Bakare [28], Feretti et al. [29] and Verma and Srivastava [30].

Heavy metals analysis of water sample
Fifty milliliters of water sample collected from a point close to the refuse dump site at the River bank was added with 5 ml nitric acid and the mixture was subjected to heat in order to digest the sample completely in line with the method reported by Aloke et al. [31]. Complete digestion of the sample was achieved when the sample became light colour which remained unchanged even with continued heating of the mixture of the water sample and nitric acid [32,33]. The digested solution was ltered through a Whatman® lter paper (No 1, Qualitative Circles 110 mm Ø, Cat No 1001 110) into a 50 ml conical ask, and the ltrate was diluted with deionized water up to the calibrated line of the ask according to Wold et al. [34]. The analysis of the diluted ltrate for Cadmium (Cd +2 ), Copper (Cu +2 ), Nickel (Ni +2 ), Zinc (Zn +2 ), Lead (Pb +2 ), Manganase (Mn +2 ) and Iron (Fe +2 ) was carried out using each of the standards of the heavy metals to determine their concentration in the water sample using an auto-sampler Atomic Absorption Spectrophotometer (Agilent technology, Series A, USA). The reading was taken in triplicate and the mean concentration of each heavy metal was recorded.

Gas chromatography-Mass spectroscopy (GC-MS) analysis on water sample
The volatile organic chemical compounds of the water sample were extracted by successive liquid-liquid extraction using analytical grade n-hexane as the solvent. Water sample A was subjected to Gas Chromatography coupled with Mass Spectrometer (Agilent Technologies, Model GC7890A/5975MS, USA) available at the Central Research Laboratory, Federal University of Technology, Akure, Nigeria. The analysis was performed with the equipment interfaced with HP Chemstation data processor tted with a quartz Agilent J &W DB -5ms. Ultra inert GC capillary column 30m × 0.53mm I.D., 5.0um lm thickness (Hewlett Packard, Palo Alto, USA) was used. The analysis was run setting the column temperature at 40°C for 6min, increased by 2°C/min up to 100°C. The injector temperature was set at 225°C. Detector temperature for the Mass Spectrometer was set at 250°C. Helium was the carrier gas with pressure 5.0328 psi and split ratio 1:10. The sample was diluted in hexane and ethanol and 10µL was injected.
The column inlet temperature was 280°C, the ow rate was 1.0mL/min. The ionization energy for recording mass spectra was 70ev. Individual chemical components of the water sample were identi ed compared with the NIST Library and the percentage match of the identi ed chemicals was recorded according to Akinboro et al. [26,35].

Statistical analysis
Obtained data were summarized as means and standard deviations of the measured parameters. These were transformed into percentage and the mean values from the treatment groups were compared with that of the negative control using Duncan multiple range comparison in one way ANOVA of the SPSS (version 17.0). Signi cantly different value was considered at P < 0.05.

Results
The water samples from points A, B and C caused varying degrees of effects on the root growth, cell division and chromosomes structure in Allium cepa. At the tested concentrations 25.0%, 50.0% and 100.0%, water sample A which was collected at the point close to the refuse dump site at Asa River's bank induced more dividing cells than the negative control (borehole water). The mitotic index (MI) value obtained at 100.0% of the water sample A was 1.70% which was signi cantly higher (P < 0.05) than the MI value of 1.40% induced by the negative control. Water sampled from the point B also induced higher MI value of 1.50% at each of the selected concentrations than the negative control. However, these were not signi cantly different from that induced by the negative control (P > 0.05). The water sample from point C had the same MI value of 1.40% at 25.0%, while at 50.0% and 100.0% concentrations, the MI values were 1.0% and 0.9%, respectively. These were not signi cantly different from MI induced by the negative control, except at 100% (Table 1). Water samples from the three points A, B, and C induced normal stages of mitosis and different types of chromosomal aberrations (CA) namely; disturbed spindle, bridge anaphase and sticky metaphase (Plate 2). Table 1 shows the percentage of CA induced at 100.0% water sample A was 2.2% and the highest. It was signi cantly different from the CA caused by the negative control (P < 0.05). Similarly, other tested concentrations of the water samples A, B, and C induced percentage chromosomal aberrations that were signi cantly different from CA induced by the negative control (P < 0.05) Effects of the water samples on root growth of A. cepa are presented in Table 2. The water sample A at 25.0% and 50.0% concentrations respectively induced 127.60% and 106.60% root lengths which were higher than 100% root length obtained with the negative control. However, there was inhibition of root growth resulted to 90% root length obtained at 100% concentration of the water sample. Water samples at 25.0% concentration of the samples B and C, and 50.0% concentration of the sample B caused higher percentage root lengths than that of the negative control. There was root growth inhibition in the onions exposed to 100% of the water sample B, as well as 50% and 100% concentrations of the water sample C. The root growth inhibitions caused by the water sample C were signi cantly different from the negative control. The positive control caused the least root length and this was signi cantly different from that of the negative control (P < 0.05). Assessment of the water sample A from Asa River to detect and quantify heavy metals in it revealed as presented (Table 3). Heavy metals such as Cadmium (Cd +2 ), Zinc (Zn +2 ), Nickel (Ni +2 ), Copper (Cu +2 ), Manganase (Mn +2 ), Lead (Pb +2 ) and Iron (Fe +2 ) were detected in the water sample in varying concentrations. Cd was detected at a higher concentration of 0.022 mg/L than its WHO and EPA permissible limit of 0.003 mg/L in drinking water. The concentrations of Zn, Mn and Fe detected in the water sample were lower than their permissible limits in drinking water according to the WHO and FAO standards. However, Ni, Cu and Pb were present below zero level. The results of analysis of chemical compounds in the water sample following the Gas Chromatography -Mass Spectroscopy technique (GC-MS) are presented in Table 4. Twenty two different chemicals compounds representing 5 groups of aromatic compounds belonging to Polycyclic Aromatic Hydrocarbons (PAH), polychlorinated compounds, aromatic amines, phenolic compound and acridine dye were revealed by the GC-MS technique. The percentage abundance of the detected chemical compounds in the water sample as revealed by the library of the chemicals station of the instrument ranged between 12 -43%.

Discussion
The importance of water to living organisms is enormous that it forms the major constituent of the ecosystem. No wonder its availability is always being ensured by man through its various possible sources such as rain, river, dams, lakes, glaciers and ground water [36]. Water is used for sustainability of aquatic life and for domestic, industrial, agricultural, recreational and commercial purposes [37]. The e ciency of water depends on its good quality, achievable in its state of being free from contamination or / and pollution. Assessment of water quality is necessary and important in order to ensure safe consumption and utilization of water.
Evaluation of Asa river water samples for toxicity to cell division and chromosomes morphology in the root tip cells of Allium cepa showed varying degrees of toxicity at different sampling points. Induction of signi cantly higher mitotic index (MI) value at 100.0% of the water from the sampling point A (a point close to refuse dump site by the River bank), and non-signi cantly different but higher MI values recorded at 50.0, 25.0% of the water sample A and at all the tested concentrations of the water from the sampling point B compared to the negative control suggests non-cytotoxicity, but rather indicates proliferative potency of the water samples. Similarly, the water sample C was not cytotoxic at 25.0% and 50.0% except at 100.0%. The observed proliferative and cytotoxic effects of the water samples from Asa River indicate that the water body was contaminated and polluted. Our results are different from over 40% decrease in MI value obtained when industrial e uents collected near the Sava River in Croatia were tested for genotoxicity using the A. cepa assay by Radić et al. [38]. Mitotic index (MI) as the total number of dividing cells in a cell cycle has been severally employed to evaluate toxic levels of different kinds of agents in A. cepa cells. Both signi cant increase and decrease in MI values observed with a tested substance compared with the MI value of the negative control are important indices in monitoring environmental pollution. This parameter reveals possibilities of the tested water samples to induce uncontrolled proliferation of cells, leading to tumour formation, or / and stunted growth in biological organisms having similar eukaryotic cells and genetic constituents with A. cepa cells [10]. Antimitotic activity of chemical compounds in A. cepa cells is attributed to prevention of DNA synthesis and G2 from entering M-phase, chemical (s) mitodepressive activity, inhibition of DNA polymerase due to inactivity of speci c nuclear proteins, changing of mitotic stage duration and reactive oxygen species, causing disturbance of homeostasis in a cell cycle as reported by Liman et al. [39].
Induction of signi cantly different chromosomal aberrations (CA) such as disturbed spindle, bridged anaphase and sticky chromosomes at the tested concentrations of the water samples from that recorded with the negative control further corroborates that Asa River water was contaminated or /and polluted. Chromatid breaks develops to anaphase bridge, disturbed spindles must have been caused by the genotoxic action disrupting the tubulin that is cytoskeleton holding the chromosomes together, while sticky chromosomes is an irreversible CA induced by dissolution and deploymerization of histone protein and chromatin bres binding subunits of the chromosomes together [25,30,40]. Indcution of these forms of chromosomal aberrations by the tested water samples in this study implies that Asa River water possesses clastogenic, aneugenic and severe genotoxic effects on A. cepa cells. Interestingly, the obtainment of highest percentage of CA at 100.0% of the water sample A further con rms its toxic potential in possibly causing tumour and cancer since it signi cantly promoted uncontrolled proliferation of cells and caused signi cantly high frequency of CA necessary for the initiation stage of cancer development to be established in a multicellular organisms.
The results of root length measurement in the exposed A. cepa to the water samples corroborated the results of microscopic screening. The induction of smaller root lengths by the absolute water samples from points A, B, and C than that recorded for the negative control implied root growth inhibitory effect of the Asa River water body. However, induction of longer root lengths at lower concentrations of the water samples than that induced with the negative control is in support of the proliferative activity of these water samples, and further suggests the ability of the water samples especially the one from points A and B to induce uncontrolled proliferation of cells, while, signi cant reduction of root length caused by the water sample C further suggests its phytotoxicity.
Our results in this study for the rst time using the A. cepa plant genetic assay have further con rmed pollution of Asa River water body as previously reported in the study on heavy metals accumulation in some species of sh in this water body, and another one following micronucleus and Ames tests in sh, Salmonella typhi tester strain TA100 and SOS chromotests (Escherichia coli PQ37) [2,41]. It is now established that the use of polluted Asa River water to water farmlands along the River bank could cause stunted growth of crops based on the effects of the water sample C on A. cepa root growth. Furthermore, the effects of water sample C on A. cepa cells and root growth in this study may logically be expected in the crops (along the River bank) that are being watered with water from the sampling point C through bioaccumulation and biomagni cation. These effects may be further transferred to consumers of these crops including humans along the food chain as reported by Guan et al. [1]. This extrapolation of the outcomes of this study to predict similar effects in animal cells is based on the fact that there is oxidase enzyme in A. cepa cells which makes it suitable to evaluate genotoxicity of promutagens which requires conversion to mutagenic or genotoxic metabolites, although at a lesser rate compared to how it is carried out by P-450 enzyme in the liver cells of higher animals. Furthermore, chemicals that are able to cause chromosome damage (genotoxicity) in plant cells can also pose danger to animal cells since the damaged material is DNA which is common to both plant and animal cells [10].
The observed proliferative, root growth promoting and inhibitory, cytotoxic and genotoxic effects of the water samples along Asa River's course might have been caused by the types and concentration of polycyclic aromatic hydrocarbons (PAH), aromatic amines, acridine dye, polychlorinated and phenolic compounds, and heavy metals detected in the water samples following the Gas Chromatography Mass Spectrophotometry (GC-MS) and Atomic Absorption Spectroscopy (AAS) techniques. Heavy metals viz a viz; Cadmium (Cd +2 ), Zinc (Zn +2 ), Manganase (Mn +2 ) and Iron (Fe +2 ) were detected in the water sample at various concentrations as previously reported by Hussein et al. [2] and Anifowose et al. [41]. Heavy metals such as Hg, Cd, As, Pb, Sb, Cr, and Sr are very toxic even at low concentrations because they are nonbiodegradable leading to their bioaccumulation in the human body to cause damage to nervous system and internal organs according to Ogunkunle et al. [3]. Leme and Marin-Morales [10] reported that generally, at various concentrations they have been associated with mitotic inhibition and chromosomes abnormalities in A. cepa cells. They are toxic agents capable of causing DNA damage through interference with the enzymatic processes and DNA repair mechanism in exposed organisms and this may lead to cancer development according to Mouna et al. [42] and Matos et al. [43]. Apart from the refuses dumped at the river bank of Asa River, fertilizer and pesticide application by the farmers along the river bank may be the source of heavy metals into the water body [21]. Heavy metals are accumulated not only in the soil but also in plants. The concentration of Cd higher than its permissible limit in drinking water according to the WHO and USEPA standards further implies that the water body is polluted and un t for consumption in any form. High concentration of Cd in water sources could be possibly due to the waste disposal method, natural processes, human /anthropogenic activities, agricultural practices, closeness of water body to the roads with high tra c density, metal melting and electroplating, coal re ning and oil red power stations [31]. The concentration of Cd (0.022 mg/L) in this study was higher than 0.3 -1.36 µg/L recorded for wastewater treatment plant in the city of Guelma in Algeria [42].
Environmental exposure to Cd at a high concentration is injurious to health as it can cause kidney damage, being an important organ of homeostasis that rst displays sign of toxicity to Cd, neurotoxicity and bone damage according to Oluyemi and Olabanji [44] and Chaitali and Jayashree [45]. Rajappa et al. [46] reported that it causes a painful disease called Itai-itai.
Polycyclic aromatic hydrocarbons (PAHs), aromatic amines, acridine dye, phenolic and polychlorinated compounds are well known cytotoxic and genotoxic agents causing mitotic inhibition and various kinds of chromosomal aberrations in A. cepa cells and different types of point mutations in the Ames Salmonella/microsome assay [47,48,49,50,51]. In detecting the presence of PAHs as pollutants in any tested samples, A. cepa uses its ethoxyresoru n-O-deethylase (EROD), a subunit of dependent mixed function oxidases of cytochrome P450-(CYP-). PAH ligands activate the hydrocarbon receptor (AhR) on the cell membrane and this is translocated into the nucleus and then activates the CYP1A1 genes according to Claudia Ramos deRainho et al. [52]. PAHs are indirect-acting mutagens which need metabolic activation in order to exert their effects [1]. Polycyclic aromatic hydrocarbons among hydrocarbon compounds in the crude oil are the most dangerous environmental contaminants due to their harmful effects such as toxicity, mutagenicity and carcinogenicity on different living organisms. The presence of these aromatic organic compounds and heavy metals in Asa River as we report in this study makes its use unhealthy to water farmlands along the river bank, as sources of underground and surface drinking water through construction of dams and digging wells. These practices portend danger to human, animals and plants coming in contact with this water.

Conclusion
The present study on quality assurance tests on Asa River water using the A. cepa genetic assay, GC-MS and AAS techniques has established that the water body was polluted with heavy metals such as Zinc, Manganase, Cadmium and Iron of which the concentration of Cadmium was above the permissible limit according to the WHO and USEPA standards. Furthermore, the water also contained polycyclic aromatic hydrocarbons, aromatic amines, acridine dye, polychlorinated and phenolic compounds which are responsible for induction of higher and reduced number of dividing cells (MI) and root lengths, and various types of chromosomal aberrations (CA) that were signi cantly different from those recorded for the negative control at different sampling points. These observed effects on A. cepa cells suggest that Asa River water body was polluted and its use as source of drinking water to surface or underground waters, as well as to irrigate farmland along the river bank may be associated with adverse effects in human, animals and plants that utilize it. For the rst time, our results have suggested possible effects of this water body on plant cells including its potential to cause different types of chromosomal aberrations using the A. cepa assay in addition to the outcomes of the previous studies that have reported the pollution status of Asa River water body.

Declarations
Availability of data and materials -Not applicable Competing Interest -Not applicable