Abstract: This communication present the potential genetoxicty effects of simulated leachate from semi-urban waste dumps in the Niger Delta, Nigeria, on Allum cepa. The root of Allium at about 2-5 cm long was treated with 1, 2.5, 5, 10, 25 and 50% concentration of the leachate for 24 h. These were then used to prepare for observation of chromosomal aberrations and frequency of mitotic division. Different types of chromosomal abberations was induced (sticky chromosome, disturbed spindle, analphase bridge etc.). There was significant (p≥0.05) reduction in mitotic index at all levels of treatment compared to the control. The observed genetoxic effect was due to presence of certain chemical compounds inherent in the semi-urban waste leachate.
INTRODUCTION
Management of solid wastes is often a problem in urban and sub-urban environment in Nigeria. Most urban and sub-urban areas in Nigeria do not have waste disposal facilities; most at times domestic and agricultural wastes are allow to decompose in the open waste dumps. These open dumps do not have geoliner or concrete walls that could prevent the leachate from percolating into the soils. Leachate and/or runoff from such dumps could contaminate the adjoining surface water bodies and groundwater which form the major sources of drinking water. The various water drinking water sources are abstracted without any form of further treatment. Cytotoxicity assessment of the leachate is therefore necessary to safeguard these communities from the effects of hazardous chemicals found in the waste dumps. Leachates are produced as a result of the action of water on decomposing organic materials. Leachate can be characterized as extremely complex mixture containing numerous inorganic as well as organic compounds; the complexity makes it almost impossible to carry out a hazardous assessment base on chemical analysis.
The Allium test employing rooted bulbs of Allium cepa has been used as standard short-term test in environmental monitoring and as well as tool for evaluating and ranking chemical with reference to their toxicity by various agencies (Mishra, 1993). This test has been frequently used by many researchers (Fiskesjo, 1981, 1985; Grant, 1982; Mishra, 1993; Rank and Nielsen, 1994; Bakare et al., 1999a, 2001).
Allium cepa test is easy to handle, low cost and good correlation with mammalian test system (Fiskesjo, 1985). The test system comprises of rapid and sensitive method at microscopic level by measuring growth inhibition of roots as well as at microscopic level comprising harmful qualitative and quantitative effects on cells and tissues (Mishra, 1993). The microscopic level also include the Allium micronucleus assay which has been used in studying genetoxicity and bioavailability of solid deposits (containing mercury) from chloralkali plant (Dash et al., 1988; Panda et al., 1989, waste water (Amin, 2002) and urban and rural dumps leachates (Bakare et al., 1999a, 1999b, 2001).
The present study was undertaken to assess cytotoxic effects of simulated leachate from sub-urban communities in the Niger Delta. This will provide information for assessment of the hazardous effects of the chemicals from solid waste dumpsites.
MATERIALS AND METHODS
For leachate simulation, solid wastes were colleted from Abraka, Eku and Obiaruku (all in Delta State of Nigeria) in June 2005. Simulation was done using the extraction procedure of the American Society of Testing and Material (ASTM) method (Perkel et al., 1982) with slight modification. From the initial sample of 1 kg, 700 g of the wastes were shredded and extracted with a volume of distilled water four times the waste sample. The waste mixture was mixed thoroughly and allowed to standard for 48 h at room temperature and stirring was done manually at regular interval of 2 h. At 48 h the solid and liquid portions were separated and the pH is recorded and stored at 4°C (Bakare et al., 1999a).
The physicochemical properties of the leachate samples were examined following the method described in APHA (1992). The heavy metals was determined by atomic absorption spectrophotometer after extraction with Ammonium pyrrolidine dithiocarbamate-Methyl isobutyl ketone (APDC-MIBK) (APHA, 1992).
Allium cepa Assay
Equal-size bulbs were chosen from a commercial variety of Allium cepa
(2n = 16). The outer scale of the bulb and brownish plate were removed. The
root of Allium cepa were generated by suspending the bulb over 50 mL
beaker containing distilled water for 48 h. When the roots were about 2-3cm
long, they were placed directly in 1, 2.5, 5, 10, 25 and 50% concentrations
of the simulated leachate of 2.4 h. Onion root generated in distilled water
served as negative control (Bakare et al., 1999a,b). The experiment was
performed at about 25±2°C and was protected against sunlight. Root
from treated bulbs and those from the control were harvested and fixed in ethanol:
acetic acid (3:1) V/V for 24 h. After fixing the root were hydrolyzed with I
M HCl at 65°C for 3 min.
Two root tips were then squashed in each slide, stained with acetocarmine for 10 min and cover slips were carefully lowered on them to exclude air bubbles. The cover slips were sealed on the slides with clear fingernail polish as suggested by Grant (1982). Five slides were prepared for each treatment and 100 cells were scored for each treatment and control. The data were expressed in terms of mitotic index, number and percentages of chromosome aberrations.
RESULTS AND DISCUSSION
The results for the physicochemical analysis of the simulated leachate from sub-urban waste dumps is presented in Table 1. Table 2, 3 and 4 present the results of genetoxicity of simulated leachate on Allium cepa.
Leachate contains a variety of chemical agents and their interaction and reactions
under changing environmental condition further complicate the issue. The synergistic
and antagonistic effects among the chemicals are inevitable. As shown in Table
1, the concentration of heavy metals we found in this study exceeds the
WHO limits for drinking water. Some of the metals (Cd, Pb and Ni) are known
to be carcinogens (Fowler et al., 1994; Haugen et al., 1994; Elinder
and Jarub, 1996). Leachate containing significant amount of these metals as
part of it content can induce cancer. For example lead was reported to be a
strong clastogen, which break chromosome in Chinese hamster ovary cells (Bauchinger
and Schmid, 1972), in bone marrow erythrocytes of rat and in cells of Allium
cepa (Lerda, 1992; Bakare, 2001).
| Table 1: | Physicochemical and heavy metal characteristic of simulated leachates |
| *Mean of triplicate analysis: CV = <5% | |
Nickel produces highly selective damage to heterochromatic in Chinese hamster genomes (Costa et al., 1994). While cadmium (Elinder and Jarub, 1996), lead (Fowler et al., 1994) and nickel (Haugen et al., 1994) have been reported to induce of variety of tumors in animal studies.
The Biochemical Oxygen Demand (BOD) of leachate reported herein is greater than 40 mg L-1 in all samples (Table 1). Samples with such high BOD values have been demonstrated to have corresponding high mutagenic activities (Omura et al., 1992). Thus high activities as seen with the induction of various types of aberration are associated with high BOD. This is inline with observation of Bakare et al. (2001). Table 2-4 present the results of microscopic studies. The suppression of mitotic activities was often used for tracing cytotoxicity (Smaka-Mkinel et al., 1996). This is usually accompanied by an increase in the fraction of cells with c-mitosis, multi groups, sticky and abnormal chromosome orientation (Amin and Mighid, 2000; Amin, 2002). At each treatment level for all leachate samples, there was a significant decrease in mitotic index compared to the control. Mitotic index decreases with increasing concentrations of leachate, which indicates that, the mixture was mitodepressant. For leachate samples the lowest mitotic index observed was 2.80 (for Obia), 4.50 (for ABK) and 5.58 (for Eku). The lowest mitotic index was observed at 50% concentration in all samples. In the present study, a decrease in mitotic index was found to be significant with increase in the concentrations of the leachate. The suppression of mitotic index was probably due to either the blocking of G1, suppressing cells from DNA synthesis (Amin, 2002) or blocking of G2, preventing cells from entering mitosis (Amin, 2002). This occurred probably through the uncoupling of respiration processes and carbohydrate metabolism (Andrew et al., 1983) leading to low ATP content, which is essential for progress of mitosis.
Sticky chromosomes indicate highly toxic effect, usually not reversible and
probably leading to cell death (Fiskesjo, 1985; Bakare et al., 1999a).
The highest amount of sticky chromosome was observed at 50% concentration of
leachate from Abraka (ABK) and Obiaruku while 25% concentration gave the highest
amount of sticky chromosome for leachate from Eku. Chromosome stickness is caused
probably through intermediate reaction of DNA during its inhibition period causing
DNA-DNA or DNA-protein cross linking (Ashour, 1998; Amin, 1991; 2002) or through
reactions with the lysosomal system altering the physicohemical properties of
nucleic acid and/or nucleiprotein, liquidification of the chromatin materials
(Adam and Farah, 1989), which consequently lead to hindrance of normal chromosome
separation at anaphase giving rise to sticky chromosome bridges.
| Table 2: | Frequency and spectrum of cytological effects of treatment with different concentrations of (EKUSL) simulated leachate |
| Table 3: | Frequency and spectrum of cytological effect of treatment with different concentrations of Abraka Simulated Leachate (ABK SL) |
Sticky bridges might also result from incomplete replication of chromosomes by defective or less active replication enzymes (Sinha, 1979) or late replicating DNA sequences of the felomeric heterochromatin (Amin, 2002). If heterochromatin blocks did not finish DNA replication when the nucleus is ready to divide, bridge formation would occur (Kaltisikes et al., 1984).
Chromosomal aberrations occur due to lesion in both DNA and chromosomal and
spindle protein causing genetic damage. The different types of aberrations observed
in our study were probably due to the effects of genotoxic compound found in
waste dumps (e.g., household chemicals such as chlorophenol, heavy metals etc.).
This caused drastic changes in chromatin, spindle apparatus and centromeres,
leading to impairment of chromosome alignment onto metaphase plate, abnormal
spindle orientation, abnormal chromosome movement and c-mitosis. The abnormal
chromosome orientation was due to the altered quality and quantity of kinetochore
heterochromatin (Jannifer et al., 1988). In the present study spindle
disturbances occur high frequency in Abraka, Eku and Obiaruku leachate samples.
| Table 4: | Frequency and spectrum of cytological effect of treatment with different concentrations of Obiaruku simulated leachate (OBIA SL) |
There was no observable trend in the frequency of spindle disturbances with increasing concentration of the leachates. The mechanistic background to spindle disturbances with compounds might be partially due to partitioning of some household chemicals into hydrophobic components of the cell (Onfelt, 1987a, b; Amin, 2002).
The results of this study agrees previous reports from animal studies (Bakare, 1999,b) where the raw lechate from the dumpsite induced abnormally shaped sperm head in albino mice. Further more, Bakare et al. (1999a, b, 2000, 2001) reported the clastogenic, mutagenic and cytotoxic effects of raw and simulated leachate from institutional, domestic, municipal and industrial waste dumpsites in south west Nigeria. Similarly, Cabrera et al. (1999) and Cabrera and Todriguez (1999) reported the genotoxic effect of landfill leachate and extracts from the organic and total municipal garbage in Tradescantia and A. cepa.
The genomic disturbance recorded in this study presents damage to the DNA ranging from point of aberrations to chromosomal mutations. The effect of this to the present and future generations of the communities in the vicinity of the dumpsites could be grievous. This is because the dumpsites were not properly sited and do not have geoliner or concrete walls that could prevent the leachate from perculating into the groundwater or diffusing into the nearby river. Communities in vicinity of these dump depend solely on water from boreholes and river water for domestic and commercial purposes. This is confirmed by the reports of several researches who presented different forms of genetic abnormalities e.g., Hen et al., (1988) reported that water from a well near a waste dumpsite induced chromosomal alterations in human lympocytes and Gonsebatt et al. (1995) reported significantly high frequencies of chromatid and chromosomal delections in individual at landfill for harzadous wastes. Increased incidences of bladder and gasrointestinal cancers (Griffith et al., 1989) reproductive and congenital malformations (Goldman et al., 1985) has reported for people living close to hazardous waste dump sites.
Bakare (2001) suggested that considering the high correlation between mutagenicity and carcinogenicity, it may be pertinent to add that organisms that are predisposed to cancer and when exposed to landfill leachate may be at a very high risk of developing the disease. This is because of the possibility that the leachate contains chemicals possessing initiation, promotion and progression properties working in concert to bring about neoplastic transformation.
This study has shown that sub-urban refuse leachate induced chromosomal aberration in Allium cepa through interaction with DNA and protein leading to chromosome stickness, mitotic disturbances and/or cell damage. In addition, semi-urban waste dumps leachate contained pollutant compounds causing high rate of chromosomal abnormalities. Therefore, it is high recommended that adequate leachate treatment process and concrete liners or geoliner be provided for these waste dumps to prevent the leachate from percolating into the subsurface or diffusing to adjoining water body.
ACKNOWLEDGMENT
The authors are grateful to Mr. Godday Itedje, and Mr. Remi Akinboboye for carrying out the cytological observations.