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.



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,

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 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’

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 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, 1979; NRCC, 1983). These activities include accidental oil spills, municipal and industrial effluents discharge, and disposal of wastes containing PAHs (Jackson, Patterson, Graham, Bahr, BĂ©langer, Lockwood, and Priddle, 1985). These organic pollutants can accumulate in freshwater organism. Bioconcentration factors (BCF) have been reported in some organisms (Lu, Metcalfe, Plummer and Mandel, 1977; Casserly, Davis, Downs, and Guthrie, 1983; Mailhot, 1987) and effects detected using limited number of biomarkers (Shugart, 1988; Hose, 1985). Physical factors such as temperature, pH, dissolved oxygen, and hardness have also been documented to enhance the toxicity of PAHs in freshwater organisms (Finger, Little, Henry, Fairchild and Boyle, 1985; Black et al., 1983; Trucco, Englehardt and Tracey, 1983; Call, Brooke, Harting, Poirier and MacCauley, 1986; Oris, Tilghman and Tylka, 1990). Organic pollutants considered here are examples of xenobiotics (foreign compounds). They play no part in the normal biochemistry of living organisms.

However, apart from the adverse biodiversity effects imposed by the aquatic chemicals, changes are much more important from a human perspective, where human demands are placed on the aquatic system. Potable water in residential user communities around Anambra River is essential for human survival. Freshwater supply for human consumption should not only be safe but also wholesome (Kapoor, 2001), free from harmful chemical substances, pleasant in appearance, odour, taste and usable for drinking purposes (Kapoor, 2001). Pathetically, in rural communities, potable water is collected from unprotected streams and rivers that are distant and prone to various material loadings that affect its quality, biota, and health of the dependent population. In view of the growing scarcity of water resources and its recently acknowledged non-renewability, it is becoming important to plan its sustainability, safeguard and improve human conditions and enhance development. Currently, the situation is perhaps far-fetched as the ignorant pollution and consumptions of freshwater resources are almost becoming acceptable trends, which potentially predispose human population to possible disease outbreak and ecological damage.

1.2       Statement of the Problem
Rivers are highly prone to material loadings that can result in pollution. According to Odo (2004), Anambra River is a shallow and fragile ecosystem that has suffered drastic changes in the past years from pollution of its waters. The River has secchi disc ranging from 25cm to 85cm (Odo, 2004). Its setting in a tropical humid environment with potential hydrological instability makes the river very vulnerable to degradation. It receives mean annual rainfall of 150cm-200cm (Awachie and Hare, 1977; Ilozumba, 1989). This together with point source pollution from industries and surrounding urban areas and non-point sources from agricultural lands has brought serious environmental concerns of genotoxic pollution and the sustainability of this resource.

There is a strong evidence of the serious reduction in local biodiversity of the river as a result of pollution. Ndakide (1988) and Odo et al. (2009) maintained that very low number of fish species recorded at Nsugbe, Otuocha and Ogurugu stations of the river has been as a result of synergistic effects from the various industries and growing population impact. These effects arise as a result of discharge of municipal wastes/sewage and individual pollutants (Odo, Didigwu and Eyo, 2009). Toxic effect of detergents, petroleum products, and household factories had been documented (Omoregie, 1995). Both the numbers and distribution of large mammals in the river have been greatly reduced due to increased human influence such as hunting and burning (Ndakide, 1988). The present fauna in the river is dominated by weed associated meso-predators (Welcome, 1979).

The water quality of the rivers discharging into Anambra River is the main determining factor of the water quality status of the River. For example, Oyi River discharging in Anambra River is the main collecting medium of municipal sewage, industrial effluents and human domesticates for more than seventeen years now.

Reconnaisance tour to various regions surrounding the river revealed crop agricultural and fishery production within the zone including the floodplains. About 15 percent of all irrigated cropland suffers from waterlogging and possibly, salinization due to drainage problems, thereby resulting in reduced crop yields. Soil fertility improvement is mostly based on application of inorganic fertilizer, especially during the dry season while natural spontaneous flooding takes care of crop yield during the rainy season along the floodplains, an earlier observation also documented by Anyanwu (2006). According to the author, the river is gradually becoming eutrophic. Use of agrochemicals was also evident. Despite the campaign against the use of lethal chemicals in fishing, strong empirical evidence abounds that fishermen use poisonous chemicals especially gamalin-20 in fishing. Because of early decaying potentials of such treated fish, they are often smoke-dried immediately after harvesting beside the river and at their organized camps. The inefficient use of fertilizers and pesticides is also a major cause of pollution of both surface and ground waters (FAO, 2002). Indiscriminate dumping of wastes, industrial, domestic and marketing activities are common practices at the river. Two major markets (Otuocha and Otu Nsugbe) are located on the bank of the river.

The residents around the river complained of their source of drinking water being polluted through effluent discharge and other activities, fish diversity declining with resultant adverse effects on the bio-economic values of the area such as occupations of the fishermen and local food menu. Consequently, the thrilling part was that they excluded their agricultural, marketing, and domestic activities as agents adversely influencing the river. However, the truth remains that the inhabitants are outrightly ignorant of long time health implications that could arise from the consumption of water and aquatic edibles of the river. The residents erroneously quoted and believed that anything in water does not kill, a primitive juggernaut maintained by them throughout the eco-survey. In addition, aquaculture, which is the major source of animal protein for the rural dwellers and beyond is not safe. For sustainability, there is need to itemize the chemical pollutants of this river; the toxicity of the aquatic life; the possibility of disease outbreak among the users and possible precautionary measures. Such study can easily be carried out using fish micronucleus biomarkers.

Ozouf-Costaz et al. (1990) reported that the Karyotype and chromatin materials of Clarias gariepinus (Burchell, 1822) are very stable. They observed no detectable Karyotypic differences among the species derived from three different geographical areas. Similarly, karyological and chromosomal analysis of the same species by Okonkwo and Obiakor (2009) confirmed uniformity in Karyotypic polymorphism. However, they reported chromosomal aberrations among the resident Clarias gariepinus of the Anambra River sourced from different locations. These observations implied that chemical pollutants of genotoxic potentials have been introduced into the physiological functions of these native species (Okonkwo and Obiakor, 2009). Hence, this work was designed to identify these pollutants, which have genotoxic potentials.

1.3       Research Questions
At the end of the study, answers would have been provided for the following questions:

1.                  What are the preponderant pollutants in Anambra River and aquatic lives?

2.                  What is the effect of seasonal changes and location on the availability and magnitude of the pollutants in the river?
3.                  Among Tilapia nilotica (Linnaeus, 1757) and Synodontis clarias (Linnaeus, 1758), which breed is more vulnerable or susceptible to chromosomal damage due to pollutants?

4.                  What is the relationship between the micronucleus profile in the fish and water and the pollutants detected in them?

5.                  What is the differential genotoxicity with its attendant mortality response of the prominent heavy metals in the river, acting singly and jointly against some test animals?
6.                  What is the indication that the level of pollution of the river can lead to disease outbreak among the user population?

7.                  What is the extent of knowledge about the health implication of using the river among the residents?

8.                  What are the possible remedies and recommendations for the management of the river?

1.4       Research Aim and Objectives
The aim of the study is to evaluate the genotoxic pollution of the Anambra River, Anambra State of Nigeria using micronucleus assay in fish genome.

The specific objectives are:

1.                  To determine the heavy metal and polycyclic aromatic hydrocarbon (PAH) contents of the river and two fish species (tilapia and catfish) using atomic absorption spectrophotometer and gas chromatography.

2.                  To test effect of season and location on the heavy metal and PAH contents of the River and fish.

3.                  To test the breed effect of these chemical pollutants.

4.                  To establish the relationship between the micronuclei indices of the fish and heavy metals and PAHs detected in them and water.

5.                  To investigate the differential genotoxicity/mortality of heavy metals found to be most prominent in the Anambra River, acting singly and jointly against the test animals based on ratios of individual 96hLC50 values.

6.                  To investigate the indication that the level of pollution of the Anambra River can lead to disease outbreak among the user population.

7.                  To investigate the level of awareness among the user population about the health implication of using the river.

8.                  To recommend measures for the management of this resources of multiple uses.

1.5       Research Hypothesis
The work tested the following research hypotheses;

Hypothesis 1
HO – There is no significant indication that the level of pollution of the Anambra River can lead to disease outbreak among the user population.

Hypothesis 2
HO – The level of knowledge/ awareness among the population about the health implication of using the Anambra River is high and effective.

1.6       Justification of Study
The aquatic environment makes up the major part of our environment and resources. Therefore, its safety is directly related to the safety of our health and food security. The most compelling reason for using biomarkers in environmental risk assessment is that they can give information on the effects of pollutants. Thus, the use of biomarkers in biomonitoring is complementary to the more usual monitoring involving the determination or prediction of residue level. Biomarkers and bioindicators using fish micronucleus assay in eco-genotoxicology offers several types of unique information not available from other methods. These include:

-          early warning on environmental damage;

-          the integrated effect of a variety of environmental stresses on the health of an organism and the population, community, and ecosystem;
-          relationships between the individual responses of exposed organisms to pollution and the effects at the population level;

-          early warning of potential harm to human health based on the responses of wildlife to population; and

-          the effectiveness of remediation efforts in decontaminating waterways (Villela, De Oliveira, Da Silva and Henriques, 2006).

Why use biomarkers in hazard assessment? One important reason lies in the limitations of classic hazard assessment. The basic approach of classic hazard assessment is to measure the amount of the chemical present and then relate that, via animal experimental data, to the adverse effects caused by this amount of chemical. The limitation of this approach is that only for a very few compounds has it been possible to define the levels of a chemical that are critical to an organism (Walker et al., 2003). Under real life situations, a wide variety of organisms is exposed to complex and changing levels of mixtures of pollutants. Biological and chemical monitoring systems should be complementary to each other. It is important to know both what is there and what it does.

The first question that biomarkers can be used to answer is ‘are environmental pollutants present at a sufficiently high concentration to cause an effect? If the answer is positive, further investigation to assess the nature and degree of damage and the casual agent or agents is justified. If negative, it means that additional resources do not have to be invested (i.e. it is an early warning system). The role of biomarkers in environmental assessment is envisaged as determining whether or not, in a specific environment, organisms are physiologically normal. A suite of tests can be carried out to see whether the individual is healthy. It is necessary to select both the tests and the species to be tested. It is important to see that the main trophic levels are covered and not to rely completely on organisms at the top of the food chain. In the selection of tests, the specificity of the test to pollutants and the degree to which the change can be related to harm need to be considered. The use of biomarkers to measure responses to the chemical in individual organisms can provide a casual link between exposure to a chemical and a change at the population level (e.g. population decline, decline in reproductive success or increased mortality rate) as would be explained in this research with vulnerability effects to other organisms (e.g. resident human population).

An exciting feature of eco-genotoxicology is that it represents a ‘molecule to ecosystem’ approach, which relates to the ‘genes-to-physiologies’ approach originally identified by Clarke (1975) and extensively developed in North America in the 1980s (see for example Feder, Bennett, Burggren, and Huey, 1987). Freshwater pollution due to heavy metals poses serious problem because of its high toxicity and of the bioaccumulation ability of these agents. Priority organic pollutants (POP) like polycyclic aromatic hydrocarbons (PAHs) have not received considerable attention in environmental management thereby undermining their lethal and sub-lethal effects. These pollutants have been reported to be eco-toxic in developed countries (Germain et al., 1993). However, little or no information exists in Nigerian Rivers, particularly Anambra River. Studies of this type would invariably establish the heavy metal status and PAHs concentrations within the river and call for proactive measures in control.

Genotoxic evaluation of the Anambra River is a key mechanism for translating the principle of sustainable development into action. Genotoxic pollutants have been associated with gene mutation (mutagenic) and proliferation of tissue (carcinogenic potential). These chemicals are capable of transforming the future generations if unchecked since it can affect the genetic materials of the future population. According to Okpokwasili (2009), though fish are dying first due environmental pollution, next is human.

1.7       Significance of Study
The research would be of immense benefit to the following categories; 

Medical Practitioners and Epidemiologists: Due to the increasing environmental exposure to many toxic and potentially toxic chemical substances, the study will provide essential tools to clinical personnel on particular outbreak of congenital anomalies and diseases.

Resident Population: The indigenes around the area will be sensitized by this work on their various inactions and negative influences on the river and ultimately be educated on the permanency of the health effects of these activities.

Socio-economy: Aquaculture will be maintained and sustained –following the recommended management approaches in this work. The fish species, which form the major food in the diet of the resident population and beyond would be made safe and support the burgeoning population indefinitely.

Government: The quality state of the major river of the state will be portrayed to the government, for stricter regulations and monitoring of the state freshwater systems for the protection of aquatic life and forestall water quality decay.

Environmental Managers: The bio-techniques employed in this work will form major eco-tools for eco-managers in monitoring and predicting impacts of aquatic pollution and at the population level.

The work will provide a baseline data for the assessment of the status of priority organic pollutants (POP); determination of management mechanisms and ultimately, of regulatory measures of freshwater resource of Anambra River aimed at the protection of its habitat and astronomical improvement of fishery resources of the river.

The methodology applied for this research would serve as a fundamental procedural step in evaluation of the genotoxic potentials of other aquatic bodies.

1.8       Scope of Study
This study was designed to evaluate the genotoxic pollution status of the Anambra River. The two preponderant fish species were examined for micronuclei profiles. The values obtained served as indices of chemical pollution of the river and contamination of the aquatic life. The water and fish samples were also analyzed for metal ions and polycyclic aromatic hydrocarbons (PAHs) contents known to be genotoxic by atomic absorption spectrophotometer and gas chromatographic (GC) technique. The physico-chemical characteristics were measured to determine the factors that enhance the environmental mobility and bioavailability of these pollutants.

The work spanned between rainy and dry seasons to determine the effect of seasonal changes on the above parameters. Also, the breed and location effects were evaluated to measure the susceptibility difference between the preponderant fish species and the locations with significant degree of the chemical pollution impacts. It was limited to the stretch of Anambra River excluding its tributaries. Differential genotoxicity/mortality of two heavy metals found to be most prominent in the Anambra River, acting singly and jointly against the test animals based on ratios of individual 96hLC50 values were also evaluated. Public survey was embarked upon to ascertain the level of awareness on health implication and susceptibility/ indication that the level of pollution could lead to disease outbreak among the resident population operating at, and using the ecologically stressed river.

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