GENOTOXIC EVALUATION OF ANAMBRA RIVER USING BIOMARKER

GENOTOXIC EVALUATION OF ANAMBRA RIVER USING BIOMARKER

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ABSTRACT

Genotoxicity of freshwater fish in Anambra River was studied by micronucleus (MN) assay, and the resultant micronucleus indices were used as biomarkers to estimate and predict pollution profile and possible danger of feeding on the aquatic species. The micronucleus profiles of the fish were measured from gill and kidney erythrocytes using microscopic technique. Season, breed, and location effects on micronucleus indices, together with their interactions, and the correlation between the pollutants in fish, water ecosystem, and the micronucleus profiles were also studied. Two major seasons (Rainy and Dry) and preponderant fish breeds in the river [Synodontis clarias -Linnaeus, 1758 and Tilapia nilotica -Linnaeus, 1757] were studied at five distinct locations that displayed differential environmental stresses. The study revealed that the micronucleus index of fish is an excellent biomarker for measuring the level of pollution in a freshwater habitat. This is more evident with regard to zinc and copper. Season, breed and location affect micronucleus profile adversely and strong correlations exist between zinc and copper in water and fish and micronuclei profiles. Disease outbreak among rural dwellers depending on the water for domestic and other uses is imminent and they lack knowledge on its health implication. Furthermore, the study maintained that the micronucleus in fish could be measured with higher efficiency from the gill than the kidney erythrocytes and Synodontis clarias is more vulnerable to genetic damage due to high zinc and copper pollutants than Tilapia nilotica. Consequently, the study recommends environmental sensitization of the resident population and regular monitoring (micronucleus tests) of edible aquatic life such as Synodontis clarias (catfish) in order to eliminate the danger of people feeding on toxic metals, some of which are carcinogenic.

CHAPTER ONE

INTRODUCTION

1.1       Background

Many toxic and potentially toxic chemical substances, some of which are of natural origin and others due to human activities are available in the fresh water ecosystem daily. It is difficult to practise even elementary hygiene without sufficient quantities of water free of these contaminants (UNFPA, 2001). As such, it is necessary to protect the water sources themselves from faecal, agricultural, and industrial contaminations (pollutants). In developing countries, 90 to 95 percent of all sewage and 70 percent of all industrial wastes are dumped untreated into surface water (UNFPA, 2001). Due to the increasing environmental exposure to these agents, the need for monitoring terrestrial and aquatic ecosystems, especially in regions compromised by chemical pollution is paramount (Mitchelmore and Chipman, 1998; Avishai, Rabinwitz, Moiseeva and Rinkevch, 2002; Silva, Heuser and Andrade, 2003; Matsumoto, Janaina, Mario, Maria, 2005).

Genotoxic pollution of aquatic ecosystem describes the introduction of contaminants with mutagenic, tertogenic and/or carcinogenic potentials into its principal media and genome of the resident organisms (Badr and El-Dib, 1978; Environ Health Perspect, 1996; Fagr, El Shehawi and Seehy, 2008). Genotoxicity is a deleterious action, which affects a cell’s genetic material affecting its integrity (Environ Health Perspect, 1996; WHO, 1997). Several genotoxic substances are known to be mutagenic and carcinogenic, specifically those capable of causing genetic mutation and of contributing to the development of human tumors or cancers (Black, Birge, Westerman and Francis, 1983; Hose, Hannah, Puffer and Landolt, 1984; Hose, 1985; Baumann and Mac, 1988; Shugart, 1988; Hayashi,


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Ueda, Uyeno, Wada, Kinae, Saotome, Tanaka, Takai, Sasaki, Asano, Sofuni and Ojima, 1998; Fagr et al., 2008). These include certain chemical compounds like heavy metals (Pruski and Dixon, 2002; Lee and Steinert, 2003; Matsumoto, 2003; Matsumoto et al., 2005; Igwilo, Afonne, Maduabuchi and Orisakwe, 2006) and polycyclic aromatic hydrocarbons (PAHs) (Santodonato, Howard and Basu, 1981; IARC, 1983; Black et al., 1983; Germain, Perron and Van Coillie, 1993). These genotoxicants have been reported to cause mutations because they form strong covalent bonds with deoxyribonucleic acid (DNA), resulting in the formation of DNA adducts preventing accurate replication (Varanasi, Stein and Nishimoto, 1989; Hartwell, Hood, Goldberg, Reynolds, Silver and Veres, 2000; Luch, 2005). Genotoxins affecting germ cells (sperm and egg cells) can pass genetic changes down to descendants (Hartwell et al., 2000) and have been implicated to be against sustainable development principles by WHO (1997; 2002) portraying them as significant factors in congenital anomalies, which account for 589,000 deaths annually.

Biomarkers are biological responses to environmental chemicals at the individual level or below demonstrating departure from normal status (NAS/NRC, 1989; Walker, Hopkin, Sibly and Peakall, 2003). Biomarker responses may be at the molecular, cellular or ‘whole organism’ level. An important thing to emphasize about biomarkers is that they represent measurements of effects (Biomarkers of effect), which can be related to the presence of particular levels of environmental chemical (Biomarkers of exposure); they provide a means of interpreting environmental levels of pollutants in biological terms. It is an indicator of an inherent or acquired limitation of an organism's ability to respond to the challenge of exposure to a specific xenobiotic substance (Biomarkers of susceptibility). It can be an intrinsic characteristic or pre-existing diseases or activities that may result in an increase in absorbed dose required for biological effectiveness, or a target tissue response (NAS/NRC, 1989). Fish are excellent subjects


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for the study of the mutagenic and carcinogenic potential of contaminants present in water. This is so because they can metabolize, concentrate, and store waterborne pollutants (Park, Lee and Etoh, 1993; Ali and El-Shehawi, 2007). Since fish often respond to toxicants in a similar way to higher vertebrates with fast responses on low concentrations of direct acting toxicants (Poele and Strik, 1975; Koeman, Poel and Sloof, 1977; Poele, 1977; Sloof, 1977; Badr and El-Dib, 1978), they can be used to screen for chemicals that are potentially teratogenic and carcinogenic in humans. The main application for model systems using fish is to determine the distribution and effects of chemical contaminants in the aquatic environment (Al-Sabti and Metcalfe, 1995).

Micronucleus (MN) assay is an ideal monitoring system that uses aquatic organisms to assess the genotoxicity of water in the field and in the laboratory. Research reports maintained that it can be applicable to freshwater and marine fishes and that gill cells are more sensitive than the hematopoietic cells to micronucleus inducing agents (Hayashi et al., 1998). Micronuclei are cytoplasmic chromatin-containing bodies formed when acentric chromosome fragments or chromosomes lag during anaphase and fail to become incorporated into daughter cell nuclei during cell division (Palhares and Grisolia, 2002; Fagr et al., 2008). This genetic damage arises as results of chromosome or spindle abnormalities leading to micronucleus formation. Recent research reports maintained that micronucleus formation in freshwater and marine fish is a function of water pollution caused primarily by heavy metals and polycyclic aromatic hydrocarbons. According to Hartwell et al. (2000) and Fagr et al. (2008), the incidence of micronuclei in fish and other aquatic lives serve as an index of these types of damage and counting of micronuclei is much faster and less technically demanding than scoring of chromosomal aberrations. The micronucleus assay has been widely used to screen for chemicals that cause these types of damage (Kligerman, 1982; De flora, Vigario, D’


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Agostini, Camoirano, Bagnasco, Bennecelli, Melodia and Arillo, 1993; De flora, Vigario, D’ Agostini, Camoirano, Bagnasco, Bennecelli, Melodia and Arillo, 1993; Campana, Panzeri, Moreno and Dulout, 1999; Palhares and Grisolia, 2002).

Ability of the water body to support aquatic life as well as its suitability for other uses depends on many factors among which are trace element concentrations. Some metals such as manganese, zinc, copper, nickel, when present in trace concentrations are important for the physiological functions of living tissue and regulation of many biochemical processes (Rainbow and White, 1989; Sanders, 1997). Generally, trace amount of metals are always present in freshwaters from the weathering of rocks and soils. In addition, industrial wastewater discharges and mining are other sources of metals in freshwaters. Through precipitation and atmospheric deposition, significant amounts also enter the hydrological circle through surface waters (Merian 1991; Robinson, 1996).

Some metals when available in natural waters at higher concentration in sewage, industrial effluent or from mining and refining operations can have severe toxicological effects on aquatic environment and humans (Merian, 1991; DWAF, 1996). In addition, heavy metal becomes toxic when a level is exceeded; it then damages the life function of an organism (Albergoni and Piccinni, 1983).

Various physical parameters such as temperature, pH, water hardness, salinity, and organic matter can influence the toxicity of metals in solution (Bryan, 1976; Dojlildo and Best, 1993; DWAF, 1996). Also, the lack of natural elimination process for metals aggravates the situation (Emoyan et al., 2006). As a result, metals shift from one compartment within the aquatic environment to another including the biota often with detrimental effects, through sufficient bioaccumulation. Food chain transfer also


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increases toxicological risk in humans (Rainbow, 1985; Mason, 1991). Bioconcentration or bioaccumulation of heavy metals over time in aquatic ecosystems has been reported by Koli, Canty, Felix, Reed and Whitmore (1978); Alabaster and Lloyd (1980); Spear (1981); Friberg, Elinder, Kjellstroem and Nordberg (1986); Fischer (1987); Clark (1992); and Kiffney and Clement (1993) in developed countries such as U.S.A, UK and Canada while Oyewo (1998); Otitoloju (2001); Groundwork (2002); Don-Pedro, Oyewo and Otitoloju (2004) and Aderinola, Clarke, Olarinmoye (2009) reported similar trend in Nigeria for various Lagos Lagoon epipelagic and benthic organisms and Obodo (2004) and Agboazu, Ekweozor and Opuene (2007) in fish (Synodontis membranaceus and Tilapia zili; and Synodontis clarias) from Anambra River and Taylor Creek, respectively. The distribution of heavy metals (Ni, Cd, Pb and Cu) in bank sediment and surface water column of Anambra River, Otuocha axis, has been investigated by Igwilo et al. (2006) in a single sampling period. According to Mason (1991), heavy metal pollution is one of the five major types of toxic pollutants commonly present in surface and ground waters. The environmental pollutants tend to accumulate in organisms and become persistent because of their chemical stability or poor biodegradability and that they are readily soluble and therefore environmentally mobile, forming one of the major contributors to the pollution of natural aquatic ecosystems (Purves, 1985; Sanders, 1997).

Polycyclic aromatic hydrocarbons (PAHs) are one of the most widespread organic pollutants (BBC News, 2001). As a pollutant, they are of concern because some compounds have been identified as carcinogenic, mutagenic and teratogenic (Larsson, 1983; IARC, 1983; Black et al., 1983; Germain et al., 1993). Though they occur naturally through such events as forest fires (NRCC, 1983), human activities can exacerbate their spread and are considered the major source of release of PAHs to the environment (Neff,






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