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 wit
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,
2
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
3
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 ElShehawi, 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’
4
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
5
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,
6
1979; NRCC, 1983). These activities include accidental oil spills, municipal
and industrial effluents discharge, and disposal of wastes containing PAHs
(Jackson, Patterson, Graham, Bahr, Bélanger, 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 nonrenewability, 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
7
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).
8
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
9
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.
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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.
11
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.
12
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
13
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
14
hydrocarbons (PAHs) have not received considerable attention in
environmental management thereby undermining their lethal and sublethal 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
15
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-
16
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.
1.9 Limitations of Study
a. Micronucleus (MN) profile of aquatic life increases with time. The
effect of time or years on micronucleus formation in these fish
was not studied due to time frame.
b. Finance was a major limiting factor. If not for the financial
constraint, the researcher would have loved to go further in
evaluating the water quality of the rivers discharging into
Anambra River and the various wastewater effluents. Chemical
pollutants other than heavy metals and PAHs would have been
assessed if not for financial incapacitation to determine their
aquatic genotoxicity. Complex mixture of pollutants might be
involved (Payne, Mathieu, Melvin, and Fancey, 1996)
c. Time was equally a constraint limiting the research to only rainy
and dry season.
17
d. The equipment required for these analyses were scarce. The
researcher had to travel long distances to carry out the research.
e. Reluctance and uninviting expressions of some respondents
created delay in collation of public survey data. The investigator
was many times confronted with threats.
f. We could not obtain values for predicted environmental
concentration (PEC) and the predicted environmental no effect
concentration (PNEC) of the pollutants studied. In the case of
PEC, calculations are based on known rates of release and
dilution factors in the environment. If for example, a chemical is
used on an industrial process, the level of the industrial effluent
is measured or calculated. This figure is then divided by the
dilution that occurs in receiving waters (e.g., River) to obtain a
value for the PEC. The PNEC can be estimated by dividing LC50 or
EC50 for the most sensitive species tested in the laboratory by an
arbitrary safety factor (often 1000). This is to allow for the great
uncertainty in extrapolating from laboratory toxicity data for one
species to expected field toxicity to other species;
PEC
= risk quotient
If this value is <1, the risk is low, if it is 1 or >1, there is
substantial risk.
g. The conclusions drawn from this study were based on the results
of analyses and field surveys made during the research. Lack of
detailed scientific data on freshwater of Anambra River resulted
in a considerable degree of uncertainty in assessing pollution
loads. For example, where data on pollution concentrations are
available, data on volumes of discharges are lacking. Where
information on types of contaminants is available, no information
PNEC
18
on transport pathways exists. It is also clear that many of the key
sources of pollution are very closely linked, e.g., effluents, sewage
and nutrients. Knowledge on interaction and synergies between
different land-based pollutants in the freshwater is insufficient.
We had no access to any baseline report of water quality of the
river prior to the commencement of industrial and other
anthropogenic waste discharges to measure deviations. This
made it difficult to determine the change in levels of pollutants
with time or because of change in land use. Consequently, few
literature reports were obtained on heavy metal and non on PAH
pollution of the river.
1.10 Conceptual Framework
This research work is built on the General Model of Toxicity and based on
Biomarker Strategy.
General Model
The fate of a xenobiotic in an individual organism is represented in Figure
1.1a. In this figure, an integrated picture is given of the movements,
interactions, and biotransformation that occur after an organism has been
exposed to a xenobiotic. It should be stressed that this highly simplified
model identifies those processes, which are important from a toxicological
point of view. The interplay between them will determine the toxic effect of
a pollutant. For any particular chemical, interspecific differences in the
operation of these processes will lead to corresponding differences in
toxicity between species (selective toxicity).
The model identifies five types of sites – sites of uptake, metabolism,
action, storage, and excretion, and the arrows identify the movements of
chemicals between them. The overall model will now be considered in
outline.
19
Figure 1.1a: General Model Describing the Fate of Xenobiotics in Living
Organisms. Reproduced from Walker, C.H (Chapter 9) in Hodgson and Levi
(1994) and Walker et al. (2003)
Once a chemical has entered an organism, four types of sites, which it
may reach, are identified, as follows:
1. Sites of (toxic) action. Here, the toxic form of a pollutant interacts
with an endogenous macromolecule (e.g. protein or DNA) or
structure (e.g. membrane) and this molecular interaction leads to
the appearance of toxic manifestations in the whole organism (The
chemical acts upon the organism).
2. Sites of metabolism. These are enzymes, which metabolize
xenobiotics. Usually metabolism causes detoxication, but in a small
yet highly significant number of cases, it causes activation (The
organism acts upon the chemical).
Excretion
Sites of
Action
Sites of
Metabolism
Sites of
StorageUptake
20
3. Sites of storage. Here, the xenobiotic exists in an inert state from the
toxicological point of view. It is not ‘acting upon the organism’;
neither is it being ‘acted upon’.
4. Sites of excretion. Excretion may be of the original pollutant, or of a
biotransformation product (metabolite or conjugate). After terrestrial
animals have been exposed to lipophilic xenobiotics, excretion is
very largely of biotransformation products, not of original
compounds.
In this simple model, a single box is shown for each of the categories of
sites. In reality of course, there may be more than one type of site in any
particular category- and more than one location in the body for any type of
site. Thus, a xenobiotic may be stored both in fat depots and in inert
membranes. Also, a target site for a neurotoxin (e.g. cholinesterase) may
exists in both the central and the peripheral nervous system.
After uptake, pollutants are transported to different compartments of the
body by blood and lymph (vertebrates) or haemolymph (insects). Movement
into organs and tissues may be by diffusion across membranous barriers
or, in the case of extremely lipophilic compounds, by transport with lipids.
Uncharged molecules, which have a reasonable balance between oil and
water solubility, tend to move across membranous barriers by passive
diffusion. This happens if they are not too large (mol. wt < 800), and have
an optimal octanol-water partition coefficient (Kow) for doing so. Some very
lipophilic compounds are transported ‘dissolved’ in lipoproteins. After
partial degradation, fragments of lipoprotein are taken into cells such as
hepatocytes by endocytosis, carrying the associated lipophilic molecules
with them. Most xenobiotics are distributed throughout the different
compartments of the body after uptake.
21
The organic pollutants discussed in this work are highly lipophilic
(hydrophobic), i.e. they will be stored in fat depots or in other lipophilic
sites such as membranes or lipoproteins. Such storage of potentially toxic
lipophilic xenobiotics may be protective in the short term. In the long term,
however, release from storage may occur, and this may lead to toxic effects
in the organism. Delayed toxicity may be observed some time after initial
exposure to the xenobiotic, as in the case of organochlorine insecticides
such as dieldrin. Because of their marked tendency to move into
hydrophobic locations (e.g. membranes, fat depots), xenobiotics with high
Kow values are not directly excreted in the faeces or urine of terrestrial
organisms to any important extent. Their efficient elimination is dependent
upon biotransformation to water-soluble metabolites and conjugates,
which are then readily excreted in faeces and/or urine. Thus, the thick
arrow through the middle of Figure 1.1a emphasizes the importance of this
process to terrestrial animals. With aquatic organisms, however, loss by
direct diffusion into the ambient water (e.g across gills of fish) represents a
very important mechanism of excretion for lipophilic xenobiotics.
The model can be subdivided into two parts. The processes of uptake,
distribution, and metabolism constitute the ‘toxicokinetic’ component.
Molecular interactions at the site of action are part of the toxicodynamic
component. The operation of toxicokinetic processes determines how much
of a toxic compound reaches the site of action (this may be the original
xenobiotic or an active metabolite of same). By contrast, the nature and
degree of interaction between the toxic compound and the site of action
will determine the toxic response that is produced (toxicodynamic
component). In genotoxicity, the target molecular interaction is always the
DNA. There is a sequence of events between the first interaction of
xenobiotic with DNA and consequent mutation, which may be divided into
four broad categories as given by Walker et al. (2003). The first stage is the
formation of adducts (covalent binding of the pollutant to DNA). At the
22
next stage, there maybe secondary modifications of DNA, such as strand
breakage or an increase in the rate of DNA repair. The third stage is
reached when the structural perturbations to the DNA become fixed. At
this stage, affected cells often show altered function. Finally, when cells
divide, damage caused by toxic chemicals can lead to the creation of
mutant DNA, subsequent cytoplasmic chromatin materials (micronucleus
formations) and consequent alterations in gene function.
Sometimes, it is convenient to consider these two elements (toxicokinetic
and toxicodynamic components) separately when investigating the
mechanisms that underlie toxicity.
Biomarker Strategy
Biomarker Strategy is the approach that allows linkage to be made
between the different levels of organization; from molecules to physiologies
to populations, right through to ecosystems (Walker et al., 2003). This is
the underlying basis for the Biomarker Strategy, which seeks to measure
sequence of responses to pollutants from the molecular level to the level of
ecosystems (Figure 1.1b). Biomarkers have been classified as markers of
exposure, markers of effect, and markers of susceptibility (NAS/NRC,
1989; Walker et al., 2003).
23
Figure 1.1b: Schematic Relationship of Linkages between Responses at
Different Organizational Level (Biomarker Strategy).
Source: Walker et al. (2003)
It could be argued that the most crucial task for ecotoxicologist is to
ensure that the structure and function of ecosystems are preserved. It is
also the most difficult (Walker et al., 2003). The linkages between
biochemical, physiological, individual, population and community
responses to pollutants are shown in Figure 1.1b. The dilemma is that as
the importance of a change increases so does the difficulty of measuring it
and relating it to a specific cause. Linking physiological effects to
population effects (e.g. fish population decline) is a particular feature of
this research. Studying changes in communities or ecosystems could be
structural or functional. Structural changes relate to changes in
composition. Thus, heavy metal and organic pollution have affected whole
Ecosystems
Community
composition
Population
changes
Whole organism
responses
Physiological
responses
Biochemical
changes
Pollutant
Increasing response time
Increasing difficulty of linkage to specified chemicals
Increasing importance
24
ecosystems, sometimes with dramatic consequences for the population
within them (Hodgson and Levi, 1994; Walker et al., 2003). In
ecotoxicology, the ecosystem response is studied at all levels.
Biodiversity-rich freshwater ecosystems are currently declining faster than
marine or land ecosystems making them the world’s most vulnerable
habitats (World Wide Fund for Nature, 2008); their sustainability being
threatened by anthropocentrism (Botkin and Keller, 1998; WHO, 1997;
2002; UNFPA, 2003). Anthropogenic activities such as industrial,
agricultural, domestic activities and urbanization processes give rise to
pollutants, which are introduced into the surface waters through point
and non-point sources and mechanisms (UNFPA, 2003) and much of the
world still do not have access to clean, safe water (Clark and King, 2006;
Hoekstra, 2006).
In genotoxic pollution of freshwater, the toxicants like heavy metals and
polycyclic aromatic hydrocarbons are mostly introduced into the water
bodies through anthropogenic activities such as industrial, agricultural,
domestic and urban activities and due to ecological-level interactions, the
health of the biota that depend on it is adversely compromised through
contact with hazardous chemicals capable of damaging the DNA and
perpetuating the irreversible effects evidenced by micronuclei formations.
These micronuclei serve as useful marker for environmental biomonitoring
of the aquatic chemical contaminants. This damage tends to be
irreversible and continues manifesting in future generations through
heredity. The species diversity of the impacted ecosystem would be
drastically reduced and humans occupying higher trophic levels become
threatened through sufficient biomagnification along the food chain. These
pollutants have similar genotoxic effects on the human biological systems
as they can induce chromosomal rearrangements and aneuploidy (change
in chromosome number). This study looks at chemical pollutants
25
responsible for aquatic eco-genotoxcity through biochemical changes,
frequency of micronucleus formations in response to them; makes
assumptions using the population changes (fish diversity decline) and
susceptibility of the resident user populations of the river to the aquatic
damage.
1.11 Description of the Study Area
The study area is Anambra River in Anambra State of Nigeria. Anambra
State lies between latitudes 50 40’N and 60 45’N and between longitude 60
35’E and 70 21’E. The climate is tropical with average annual rainfall of
2000mm and mean temperature of 270C (Anyanwu, 2006).
1.11.1 Location and Extent of Anambra River
The Anambra River spatially lies between latitudes 60 00’N and 60 30’N and
between longitudes 60 45’E and 70 15’E. The river on the other hand is
located in the South Central region of Nigeria, just close to the east of the
Niger River into which it empties (Awachie and Hare, 1977). Anambra
River is approximately 207.4km to 210km in length (Odo, 2004; Shahin,
2002), rising from the Ankpa hills (ca. 305-610m above sea level) and
discharging into River Niger at Onitsha (Odo, 2004). The entire River basin
draining an area of 14014km2 (Awachie and Hare, 1977), see Figure 1.2.
1.11.2 Geology
Anambra River is geologically underlain by cretaceous sedimentary rock
(Awachie and Hare, 1977). The basin is situated at the Southern extremity
of the Benue Trough of Nigeria (Figure 1.3), bounded on the West by the
Precambrian Basement Complex Rocks of Western Nigeria and on the East
by the Abakaliki Anticlinoruim (Uma and Onuoha, 1997).
26
Figure 1.2: Map of Anambra River Showing Localities Interacting with it
27Figure 1.3: Geology of Anambra Basin
Source: Uma and Onuoha (1997)
1.11.3 Climate
There are two main seasons, the dry season (October/November-March)
and the rainy season (April-September/October) approximately
corresponding to the dry and flood-phases, respectively, of the hydrological
regime (Odo et al., 2009). It is affected by the movement of the intertropical convergence zone (ITCZ), the boundary zone between the dry
continental air mass of the Sahara and the moist maritime air mass from
the Atlantic Ocean. Seasonal shifts in the position of this boundary zone
are responsible for the cycle of rainy and dry season weather observed in
this area. Generally temperature is highest and rainfall lowest from
January to March. In addition, temperatures tend to be higher and rainfall
and humidity lower as one moves North in the river basin. This is due to a
combination of increasing distance from the maritime air mass over the
28
Atlantic Ocean and increasing elevation (Awachie and Hare, 1977). The
water temperature and Secchi disc reading in the river ranges from 240C
to 310C and 5cm to 85cm, respectively (Odo, 2004). The mean annual
rainfall of the river is between 150cm and 200cm (Ilozumba, 1989).
1.11.4 Hydrology
Anambra River rises from the Ankpa Hills and discharges into River Niger
(Odo, 2004; Awachie and Hare, 1977; Odo et al., 2009). The river receives
many river tributaries that form its river basin as shown in Figure 1.4,
forming a dendritic drainage pattern.Figure 1.4: Anambra River Showing its Network of Tributaries
Source: Awachie and Hare (1977)
29
1.11.5 Landuse and Landcover
Anambra Basin is one of the richest, if not the richest area for Agricultural
and fishery production in the Nigerian Lower Niger (Mutter, 1973;
Awachie, 1976; Awachie and Walson, 1978). Principal crop products
include a wide variety of large yams (Dioscorea spp), sweet potatoes,
cassava, and rice; while clariids, Gymnarchus, and mormyrids dominate
fish production, which are available throughout the year (Awachie and
Hare, 1977).
The river has fifty-two species belonging to seventeen families; 171, 236
and 169 individuals at Ogurugu, Otuocha and Nsugbe stations,
respectively. Two families, Characidae, 11.5% and Mochokidae, 11.8%,
constitute the dominant fish families in the river. The dominant fish
species were Synodontis clarias 6.9%, Macrolepidotus curvier 5.7%, Labeo
coube 5.4%, Distichodus rostrtus 4.9% and Schilbe mystus 4.5% (Odo et
al., 2009).
Table 1.1 Diversity of Fish Fauna in the Anambra River
Sample Ogurugu Otuocha Nsugbe
Station
Number of samples 44 44 44
Number of species 51 52 51
Number of individuals 171 236 169
Species Richness (d) 3.19 3.01 2.97
General diversity (H) 0.82 1.10 0.78
Evenness (E) 0.56 0.70 0.49
Source: Odo et al. (2009)
Members of Ardeidae aquatic animal family were the most abundant and
they were followed by Acciptridae while Sirenia were the least occurring in
the river. The most abundant animals utilizing the river was the Ardea
30
cinora, with 22.2% occurrence and this was followed by Caprini spp. with
13.5% and Varanus niloticus with 10.04%. The least abundant animals
utilizing the river were Chephalophus rufilatus and Erythrocebus petas
with 0.58% of occurrence each (Odo et al., 2009).
In addition to the fish species found in the river, there are some other
forms of aquatic fauna. The crab, sudanonantes african occurs in large
quantity, as well as snails, crocodiles, and snakes. 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).
Fish eating birds were always the most abundant species confined largely
to the vicinity of River Anambra and Shoreline. Domestic animal
populations are on the increase. The moist and easily saturated soil
condition for some months of the year encourages growth of herbaceous
grasses and forbs, which could serve as fodder to the livestock. In fact,
more than 250 domestic animals were counted during the dry season
utilizing the floodplain.
1.11.6 Sources of Freshwater, Pollutants and their Distribution
The Anambra River by virtue of the various uses is put to navigation,
boating, fishing, excavation of sand and gravel, extraction of cooling water
and the fact that densely populated areas and commercial activities
surround it, makes it a convenient dumping site for numerous industrial
and domestic wastes.
Tributaries entering the Anambra River contribute additional inputs of
freshwater. By and large, these tributaries genotoxic pollutant
concentrations have been considered relatively high and thus, not
31
rigorously monitored (Figure 1.4). The River receives a number of major
rivers and streams resulting in the basin draining approximately
14010km2 of the Nigerian hinterland (Awachie and Hare, 1977), which
transport varied industrial, domestic and agricultural wastes including
pesticides, hydrocarbons and heavy metals daily into the Anambra River.
Certainly, the concentrations of all persistent pollutants including PAHs
and heavy metals will continually rise by unknown amounts annually in
the freshwater sediment, water and biota since the sources of the
contaminants are in a state of continuous flow, a fact that justifies
continuous monitoring or evaluation of all priority pollutants in the
principal media (sediment, water and biota) of the river. But the river has
not received considerable attention as the main source of water for the
state when compared with some aquatic resources in the country such as
Lagos Lagoon. River Oyi, a tributary of Anambra has been an active site of
municipal and industrial wastewater effluent discharge including solid
wastes (Figure 1.2). At Abalata Nsugbe, where the River Oyi mouth is
located, over 106 tank dislodgers were seen or counted discharging
municipal sewage to industrial effluents in the river. Other tributaries are
considered to be under similar environmental stress.
The Anambra freshwater area also receives sewage/wastewater effluents
and solid wastes directly from major industries in the state. There is
paucity of data on their sites, locations and discharge within the river but
residents reported the activities to have lasted for 18years. And this has
been implicated to be the major source of pollutant loading in the river
because of the point source pollution of admixture of wastes emanating
from the various industries with obscure origin. But easily identified
industries discharging virtually all their production wastes into the river
were the various rice mills located haphazardly along the fringes of the
river from Otuocha to Enugu Otu, which is at the outskirts of the state
and extreme of the river (Figure 1.2) and forms the major artery of rice
32
production in the state and around the river. The wastes include rice
husks, petroleum products (used in operation of machineries), polymeric,
metal scraps, synthetic chemicals, and other burning materials used in
parboiling of rice during processing. The river forms the repository of the
rice mills wastes.
Industrial effluents and domestic wastes have since been recognized as
one of the most important sources of heavy metal and other pollutants in
the Anambra River as well as similar water bodies all over the world
(Obodo, 2004; Igwilo et al., 2006 and Odo et al., 2009). Sewage sludge
have been reported to contain considerable amounts of heavy metals
(Clark, 1992) and PAHs, therefore, considering the high volume of
discharge of sewage materials in the freshwater of Anambra River placed
at 18 years now, the contribution of these pollutants is very likely to be
quite significant and urgently needs to be quantified and controlled. It is
noteworthy that the dumping of untreated sewage into the Anambra River
will pose environmental consequences that extend beyond the biological
damage potential of heavy metals and PAHs. This is so because, the
dumping of sewage will tremendously increase the organic load in the
water body with a corresponding reduction in dissolved oxygen (Jenkins,
1982) and nutrient enrichment (Fodeke, 1979); which may bring about
eutrophication (Anyanwu, 2006) with its attendant limiting problem to
aerobic organisms. The combined effects of all these environmental
pollutants may be reduction in population densities and species diversity,
whereas, increases may be observed in a few opportunistic species that
take advantage of the polluted environment, representing a change in
prevailing conditions (Margalef, 1961; Fay, 1982).
Because of the burgeoning population and rapid urbanization around the
river areas, there is a beehive of commercial activities surrounding them.
Major Markets such as Otu Nsugbe and Otuocha Markets are located on
33
the banks of the river. These activities are occasioned by indiscriminate
dumping of wastes in the river and along its banks. The waste comprises
of glassware, worn-out tyres, waste papers, cattle dungs and poultry litter,
wood and furniture wastes, metal scraps and aluminium foils, polymeric
materials and agrochemical containers, and vegetable and food residues.
Residents dig and excavate sands for sale at the floodplains and areas of
sand deposition, especially at Onono and Onitsha axes. These activities
lead to the contamination of the water and soil around it. There are heavy
road constructions with asphalt and coal tar, networking the various
towns and markets bordering the Anambra River. This results in
imperviousness of the surroundings and hydrological activity of the
surfaces, so that surface runoff carries enormous amount of these
dangerous construction materials into the river, especially during the
rainy season. These materials comprise of PAH organics (RDRC, 1987;
Tecsult, 1989; Vandermeulen, 1989) and heavy metals (Ogbuagu, 1999).
Agricultural activities were noticed to be carried out along the fringes and
banks of the river. Indiscriminate use of pesticides and fertilizers, both
chemical and organic fertilizers was a common practice. Fishery activities
are concentrated on the Niger/Anambra floodplain; here most fish are
taken during the flood season. In the dry season, catches drop sharply in
the river channels but are maintained in the floodplain ponds and pools
(Awachie and Hare, 1977).
Set nets appear to be the most popular type of fishing gear in the river.
Other common methods include cast-nets, longlines (hook and line) and
traps, while drawnets (Seines) and spears are less common (Awachie and
Hare, 1977). Generally, as one moves up the river, the fishing gear
becomes less sophisticated, tending more towards the traditional spears
and traps. Traditional, man-powered, dugout canoes are the most
commonly used fishing craft. Fishermen operating on a part-time basis
34
tend to do so without the use of fishing vessels. Secret uses of poisonous
chemicals in fishing by the commercial residents were observed. Some of
the chemicals commonly use include gamalin-20 (of PAH concentrations)
and other toxic organics and synthetic chemicals. Domestic activities and
food processing were sighted in the river, enlarging the pollution level.
Indiscriminate bush burning of the surrounding river flora was on the
increase during the dry season; wood preservations, polishing and
carpentry works, and smoke drying of harvested fish at commercial level
was observed. These activities have been reported to increase the PAH level
of freshwater ecosystems (NRCC, 1983; Westerholm, Alsberg, Frommelin,
Strandell, Ranney, Winquist, Grigoriadis, and Egebäck, 1988; Bjørseth
and Ramdhal, 1985; Slooff et al., 1989; Wan, 1991; 1993; Jackson et al.,
1985; van Coillie, Bermingham, Blaise, Vezeau, and Lakshuminaraganan,
1990).
The Anambra River serves as the main source of potable water in the areas
surrounding it, as it is common for residents to fetch drinking water there.
There is currently no treatment of this source of drinking water, either
done by government agencies or by individuals in their private homes
before usage (Igwilo et al., 2006). In addition, according to recent geological
surveys, crude oil has been located in this valley. Dirt and hazardous
wastes are transported into the river through non-point sources, thus
polluting the river and the soil around it. Hence, water, food and soil
contamination are serious health problems for the communities in
Anambra State (Igwilo et al., 2006). With the anticipated drilling of crude
oil in the area, the risk of contamination of this water source will be
increased, with grave health and economic consequences, unless standard
risk assessment and water quality-assurance programs are initiated and
sustained. Following the accumulation of these monitoring data, it is
expected that the relevant regulatory bodies would use them effectively to
establish or modify existing or proposed effluent limitation standards and
35
water quality criteria for the protection of resident aquatic lives in the
Anambra River and similar bodies of water in the sub-region.
1.12 Plan of Study
The thesis was organized into five chapters:
Chapter 1: This is the introduction, which is a general overview of the
research. It is made up of the brief highlight of the topic,
problem description, research aim and objectives and
description of the study area.
Chapter 2: This chapter reviews the previous and current studies on the
subject related to the research on Anambra River.
Chapter 3: Shows the research methodology employed. It comprises of the
data needs and sources, research design, experimental site,
data collection and analysis, and statistical techniques for
data analysis.
Chapter 4: This chapter presents the data obtained from the research
using various statistical techniques.
Chapter 5: Discusses the results obtained, draws conclusions, and makes
recommendations in forestalling further pollution and
recommending further research.
Reference: Provides the literature reports and other relevant materials
consulted.
Appendix: Finally, statistical calculations, tables and relevant deductions
and data are shown in the appendices.