Genotoxic effects of bilge water on mitotic activity in Allium cepa L.

Abstract

Bilge water from oceanic vessels is usually discharged through the bilge wells into the Nigerian marine environment indiscriminately as there is no strong indication of compliance with International Maritime Organization (IMO) regulations relating to its exchange and treatment. In this study, the genotoxic effect of bilge water on root meristems of Allium cepa L. was investigated. Exposure of the onion roots to the wastewater at 1, 5, 10, 25 and 50% (v/v; wastewater/tap water) showed strong concentration-dependent root growth inhibition with an EC₅₀ value of 52.6% at 72 h and induced a variety of chromatid and chromosome structural aberrations at 6, 12, 24 and 48 h. The bilge water was effective in the disturbance of the spindle fibre apparatus at all the concentrations used and this was probably due to the heavy metals (Cu, Mn, Pb, Fe, Cd, Cr, Ag, Ni and Zn) present in the wastewater. In comparison with tap water (negative control), the bilge water was also mitodepressive, causing lower mitotic indices. The findings in this study indicate that the toxic chemicals present in the bilge water are responsible for the observed genotoxic effects, which may contribute to the toxicological assessment of the risk associated with its indiscriminate discharge into the environment.

Keywords:

• Allium cepa
• chromosome aberration
• mitotic index
• bilge water
• root growth

Introduction

In spite of the indisputable relevance of the marine environment in the socio-economic development of nations, the maritime industry has contributed significantly to the pollution of coastal waters and the attendant adverse effects on the ecosystem through indiscriminate discharge of oily bilge waste water from ocean-going vessels. Depending on the design and function of the ship, the bilge water so formed is a mixture of substances such as fresh water, seawater, lubricants, oil, detergents and metals that accumulate in the bilge wells (Karakulski et al. 1998).

Cargo throughput handled in Nigeria’s ports is high. Statistics for August 2012 issued by the Nigerian Ports Authority showed that cargo throughput stood at 6,872,901 t, which showed a 4.9% increase over the same period of August 2011 (NPA 2013). Although Nigeria is a signatory to the Memoranda of Understanding (MOU) on Port State Control in the West and Central African sub-region, the MOU has been ineffective in reducing, controlling and preventing marine pollution in the country (Igbokwe 2001); a direct consequence of this is that wastewaters from oil tankers are discharged into the nation’s environment, disregarding the International Convention for the Prevention of Pollution from Ships (MARPOL 73/78) (International Maritime Organization, 2011). Untreated oily bilge water discharged directly into the ocean can damage marine life and a number of cruise lines have been charged with environmental violations in this regard elsewhere (Abowei et al. 2011).

In spite of the level of environmental contamination and human exposure to bilge water discharged from ships in Nigeria, there is no available literature on the toxicological effects and genotoxicity of the wastewater in plant systems. Allium cepa, a plant species commonly used for cyto-genotoxicity evaluation, has been used to investigate the cytotoxic and genotoxic effects of surface and ground waters (de Almeida Grippa et al. 2012), electronic waste leachates (Bakare et al. 2012, 2013), industrial wastewater/sludge (Sudhakar et al. 2001; Hossain et al. 2002; El-Shahaby et al. 2003; Olorunfemi et al. 2011) and ballast water from oceanic vessels (Olorunfemi, Duru, et al. 2012). The Allium test is a simple, sensitive and rapid bio-tool and it shows high sensitivity to toxic compounds. In addition, this plant has large and few chromosomes, increasing their application for cytogenetic studies. This study was carried out to investigate the effects of bilge water on cytogenetic parameters on the root meristem cells of Allium cepa L. to ascertain the extent of pollution and the possible health implications of the wastewater.

Materials and methods

The bilge water used was collected freshly from Green Nova, a multipurpose vessel from Mauritania and berthed at canal in Warri Port in Delta State, Nigeria (5°30′N, 5°41′E). The wastewater and,a control (tap water) were analysed for a number of standard physico-chemical properties, including total dissolved solids (TDS), sulphates, phosphates, nitrates, turbidity, conductivity, biochemical oxygen demand (BOD), ammonia and chloride according to methods described by APHA (2005). Nine metals – lead, copper, cadmium, chromium, iron, zinc, nickel, silver and manganese – were analysed in the water samples according to standard analytical methods (USEPA 1996; APHA 2005) using an atomic absorption spectrophotometer (AAS) (PerkinElmer A Analyst 100, Waltham, MA, USA). The metal standards were prepared to known concentrations, labelled, and kept inside plastic bottles that were pre-cleansed with concentrated nitric acid and distilled water. The absorbance of the standards, effluent samples and control was taken in triplicate. Graphs of the concentrations against the absorbance of each of the standards for the metals were plotted. Thereafter, the concentrations of the metals in the wastewater were interpolated from their respective graphs.

Equal-sized onion (Allium cepa L.) bulbs (2n = 16) were purchased from a local market in Benin City, Nigeria (6°15′N, 5°25′E) and the same batch of bulbs were used throughout. The onions were sun dried for two weeks and those attacked by fungi were discarded at the beginning of the experiment. The outer scales were carefully removed, without tampering with the primordial root ring. Several onion bulbs were utilized per treatment to account for a number of bulbs in the population that would be naturally slow or poor growing, as in previous studies (Olorunfemi, Duru, et al. 2012). The concentrations of the test samples used were 1, 5, 10, 25 and 50%% (v/v; wastewater/tap water). As suggested by Fiskesjö (1985), the negative control was set up using pollutant-free tap water obtained from UNIBEN Enterprises, University of Benin, Benin City, Nigeria. The tap water was ascertained to be of good quality by having a pH around 7 and has relatively high hardness (Ca + Mg = 50–70 mg l⁻¹) and free from any chlorine compounds and toxic ions. The bases of 12 onion bulbs were suspended in the wastewater and negative control inside 100 ml beakers containing about 75 ml of the test sample at 27 ± 1°C in the dark. Test samples were changed daily. For the evaluation of in vivo induction of chromosomal aberration, the test samples were cultivated for 6, 12, 24 and 48 h. Root tips from two onion bulbs were cut and fixed in ethanol:glacial acetic acid (3:1, v/v) inside universal bottles after the exposure periods and kept at 4°C for 24 h before use. The already fixed root tips were hydrolysed in 1 N HCl at 60°C for 5 min. The hydrolysed root tips were washed several times with distilled water. Two root tips were squashed on each slide and stained with aceto-orcein for 10 min. Excess stains were removed and the cover slip’s edges were sealed as suggested by Grant (1982). Five scorable slides were prepared per sample and examined for different mitotic stages and occurrence of aberrant cells at 1000× magnification using a Nikon Eclipse (E400) light microscope. The mitotic index and the percentage aberration relative to the total number of dividing cells were calculated (Olorunfemi, Omo-Ataga, et al. 2012).

For the evaluation of root growth inhibition, root lengths of 20 roots of 10 onion bulbs were removed with a forceps after 72 hours and measured using a metre rule. From the weighted averages for each sample and the control, the percentile root growth inhibition in relation to the negative control and EC₅₀ value was interpolated from a plot of root lengths as per cent of control against the log concentrations for the wastewater. Photographs of morphological changes induced by the wastewater on the A. cepa root tips were taken.

Quantitative data were summarized as means ± standard errors and percentages, which were then subjected to Duncan multiple comparison and Dunetts tests in a one-way ANOVA, using SPSS version 15.0 for Windows 2007, Chicago, IL, USA. The effects of the test samples and control on root growth, cell division and chromosome aberrations of A. cepa were compared. Significant differences were set at p ≤ 0.05.

Results

The average values of the physico-chemical characteristics of the bilge water samples (Table 1) revealed that the wastewater was light grey in colour, slightly acidic and contained relatively high amounts of total dissolved solids and chlorides. Compared with values obtained in the tap water (used as the negative control), the concentration of most of the inorganic and heavy metals were higher. Similarly, these parameters showed deviation from national (National Environmental Standards and Regulation Enforcement Agency; NESREA) and international (US Environmental Protection Agency; USEPA) specifications for maximum limit allowed for effluent discharge into water bodies for all categories of industries.

Table 1. Some physico-chemical parameters of bilge water sample obtained from Green Nova in Warri Port.

Parameter	Bilge water	Tap water (control)	NESREA^a	USEPA^b
Colour	Light grey	Colourless	—	—
pH	5.91	7.10	6–9	6.5–8.5
BOD5 @ 20°C	6.20	1.0	50	250
Turbidity	15 units	3 units	—	—
Conductivity	1400	120	—	—
TDS	114	78	500	500
Ammonia	15	0.1	1	0.03
Nitrates	99.5	3.0	10	10
Sulphates	107.5	0.01	250	250
Phosphates	100.5	1.45	2	5
Chloride	970.3	16	250	250
Copper	0.5	—	0.5	1.3
Manganese	3.9	0.3	0.2	0.05
Lead	0.3	—	0.05	0.02
Iron	5.7	0.22	—	0.3
Cadmium	0.1	—	0.2	0.01
Chromium	1.4	0.001	0.05	0.1
Silver	0.2	—	—	0.1
Nickel	0.3	0.01	0.05	0.005
Zinc	11.6	1.1	—	5

Notes: Values are means of three replicates. All values are in mg l⁻¹ except conductivity (μS cm⁻¹), turbidity (NTU) and pH with no unit.

BOD: biochemical oxygen demand. TDS: total dissolved solids. ^aNESREA (2009) (Nigeria) maximum permissible limits for wastewater. ^bUSEPA (2009). National recommended water quality criteria – correction.

The effect of bilge water on root growth of A. cepa is presented in Figure 1. Good root growth was recorded in the tap water while significant (p < 0.05) concentration-dependent root growth retardation was observed in the onion bulbs exposed to the wastewater. Mean root lengths obtained were lowest in the 50% concentration and the extrapolated EC₅₀ value was 52.6%. Roots of onion bulbs grown in the wastewater were characterized by the presence of malformations (scanty roots, swellings, coils and twists) at various concentrations (Figure 2).

Figure 1. Effect of bilge water on root growth of Allium cepa.

Figure 2. Morphological changes in A. cepa root tips induced by bilge water at various concentrations: (a) C-tumour roots at 50%; (b) short roots at 25%; (c) coils and twists at 1–10%.

Microscopic examination of squashed Allium cepa L. root tip meristem cells showed that mitotic activity, expressed as mitotic index, was progressively inhibited as the concentration of the bilge water increased and the period of treatment prolonged (Figure 3). The reduction in mitotic activity was accompanied with induction of a number of chromosomal abnormalities compared with the control. The percentage of abnormalities was dose and duration dependent (Table 2). The most common chromosomal abnormalities were C-mitosis, stickiness, bridges, vagrants, polar deviation and spindle disturbance (Figure 4).

Figure 3. Mitotic index of Allium cepa cells at various time intervals and concentrations of bilge water.

Table 2. Cytological effects of bilge water on cells of Allium cepa L.

Duration of treatment (h)	Concentration (%)	Number of observed cells	Mitotic abnormalities								Aberrant cells(% ± SE)
			Stickiness	C-mitosis	Fragments	Bridges	Vagrants	Laggards	Spindle disturbance	Polar deviation
6	0	1001	1	0	0	0	0	1	1	0	0.36 ± 0.01
	1	934	7	9	4	4	5	6	4	4	4.60 ± 0.96*
	5	916	9	10	5	5	5	7	4	4	5.30 ± 0.31*
	10	918	17	21	5	6	4	5	4	6	7.40 ± 0.51*
	25	1026	26	32	4	12	7	3	2	2	8.60 ± 0.75*
	50	996	34	41	10	7	6	2	1	5	10.60 ± 0.09*
12	0	976	0	2	0	0	0	0	1	1	0.39 ± 0.10
	1	917	9	10	5	5	5	7	4	4	5.30 ± 0.31*
	5	1086	9	11	8	11	6	6	6	1	5.40 ± 0.51*
	10	1002	24	33	5	11	8	2	2	3	8.80 ± 0.39*
	25	972	30	39	11	7	7	1	0	2	10.00 ± 0.11*
	50	1000	38	46	12	8	4	1	3	8	12.00 ± 0.22*
24	0	989	0	1	0	0	1	1	0	0	0.33 ± 0.02
	1	991	14	20	6	5	4	3	3	0	6.01 ± 0.06*
	5	998	16	23	7	6	4	5	4	5	7.00 ± 0.45*
	10	996	32	42	11	8	6	2	1	5	10.80 ± 0.86*
	25	1018	37	49	16	8	5	2	2	9	12.60 ± 0.96*
	50	1014	46	56	26	18	10	9	2	7	17.16 ± 1.35*
48	0	899	0	1	0	0	0	0	0	0	0.11 ± 0.01
	1	1001	20	28	4	9	9	2	0	1	7.21 ± 0.08*
	5	996	22	31	5	10	10	3	0	3	8.40 ± 0.87*
	10	1006	33	44	12	4	6	2	2	7	11.00 ± 1.14*
	25	962	40	51	16	10	5	2	2	10	14.16 ± 1.35*
	50	992	44	67	28	21	11	8	8	17	20.58 ± 1.59*

*Significant difference from negative control (p < 0.05).

Figure 4. Chromosomal aberrations observed in A. cepa root tips cultivated in bilge water: (a) C-mitosis; (b) stickiness at metaphase; (c) spindle disturbance; (d) multiple bridges; (e) vagrant chromosome; (f) polar deviation at anaphase.

Discussion

Heavy metals are widely distributed in the environment as a result of their multiple industrial, domestic, agricultural, medical and technological applications. They are dangerous because they tend to bioaccumulate and may become toxic when they are not metabolized by the living organism. Industrial exposure accounts for a common route of contamination for adults (Baby et al. 2010). Because of their high degree of toxicity, arsenic, cadmium, chromium, lead, and mercury rank among the priority metals that are of public health significance, even at low levels of exposure (Goyer 2001; Duruibe et al. 2007). Some heavy metals acting individually or synergistically or both have been implicated in impaired reproductive functions of animal life. They have been reported to affect cellular organelles and components such as cell membrane, mitochondrial, lysosome, endoplasmic reticulum, nuclei, and some enzymes involved in metabolism, detoxification, and damage repair in biological systems (Wang and Shi 2001). Results obtained from the physico-chemical analysis of the bilge water in this study indicate that the high levels of inorganic and heavy metals present in the wastewater are capable of inducing adverse affects on human life through occupational exposure to the wastewater or through the consumption of contaminated water or fish in the environment (Oschmann et al. 2005).

In a recent study undertaken with wastewater obtained from an ocean-going vessel, the authors showed the genotoxic effect induced by ballast water on the meristematic cells of Allium cepa root tips (Olorunfemi, Duru, et al. 2012). However, to our knowledge there have been no studies published to date on the genotoxic effect of bilge water. In this study, we examined the macroscopic effect of bilge water on A. cepa root meristems. Our data showed that the wastewater induced concentration-dependent root growth inhibition and alteration in root morphology. The root growth inhibition and morphological changes observed in the A. cepa root meristems could be attributable to the individual and/or complex interactions of heavy elements and other inorganic compounds present in the wastewater water at varying concentrations.

Mitotic index is an acceptable measure of cytotoxicity for all living organisms (Smaka-Kinel et al. 1996). In this study, bilge water decreased the mitotic index at all concentrations and at all treatment periods when compared with the control. The mitotic index is regarded as a dependable criteria to identify the presence of cytotoxic pollutants in the environment. Trace metals and other pollutants have been implicated in the diminished mitotic index of the A. cepa roots exposed to industrial wastewaters (Fatima and Ahmad 2006; Caritá and Marin-Morales 2008) and ballast water from an ocean-going ship (Olorunfemi, Duru, et al. 2012). This decrease in mitotic index is attributable to either disturbances in the cell cycle or chromatin dysfunction induced by metal–DNA interactions (Glinska et al. 2007).

The bilge water used in this study significantly increased the percentage of chromosomal aberrations at all concentrations and treatment periods in mitotic cell division. It has been shown by many investigators that several other domestic and industrial wastewaters induced chromosomal abnormalities on exposure to A. cepa (Sudhakar et al. 2001; Hossain et al. 2002; El-Shahaby et al. 2003; Fatima and Ahmad 2006; Babatunde and Bakare, 2006; Olorunfemi et al. 2011; de Almeida Grippa et al. 2012; Olorunfemi, Duru, et al. 2012). In this study, the most common abnormalities were C-mitosis, stickiness, bridges, vagrants, polar deviation and spindle disturbance in somatic cell division.

Chromosomal stickiness is characterized by chromosomal clustering during any phase of the cell cycle. Stickiness and clumping may be caused by genetic and environmental factors and several agents (Panneerselvam et al. 2012). The primary cause and biochemical basis of chromosomal stickiness are still unknown (Pagliarini 2000) but it has been suggested that sticky chromosomes result from the defective functioning of one or two types of specific non-histone proteins involving chromosome organization which are needed for chromatid separation and segregation (Gaulden 1987). The altered functioning of these proteins is caused by mutation in the structural genes coding for them or by the direct action of mutagens (Turkoglu 2007). C-mitosis is a direct consequence of inactivation of spindle apparatus connected with delay in the division of centromere (Mann 1977). The occurrence of several types of these chromosomal abnormalities in A. cepa L. root tip cells exposed to bilge water clearly shows that genotoxic and mutagenic capabilities of chemical substances in the wastewater could result in the inactivation of spindle formation, deformation of non-histone chromosomal proteins and mutation of the structural genes.

The results obtained in this study show that the bilge water contained a number of hazardous substances that are linked to cancer and developmental disorders. The tested wastewater induced genotoxicity at the chromosome level in A. cepa. This is of environmental and public health significance. Workers in ships, local residents in the environment, and fish farmers and consumers where the wastewaters are indiscriminately dumped are at increased risk of health effects through accidental ingestion, dermal exposure and dietary intake. The mechanism of genetic damage induced by wastewater in animal models as well as health effects in individuals occupationally exposed to bilge water would form the basis of further investigation.

Acknowledgements

The authors are grateful to Mrs Esther Duru for assistance with procurement of bilge water samples. We also acknowledge the contributions of Mr Emmanuel Oseke and Mr Romeo Ofomata for their technical assistance with physico-chemical analyses and the plant bioassay respectively.

Disclosure statement

No potential conflict of interest was reported by the authors.