The occurrence of heavy metals in water gills, muscles and liver of oreochromis niloticus in

Alode in Eleme Local Government Area of Rivers State.

0.79, 0.87, 0.88, 0.86, 0.90

CHAPTER ONE

INTRODUCTION

1.1 Background to the Study

Fishes in general irrespective of its broad mode of classification has contributed directly or

indirectly to the world economy. Fishes adds richness and nutrient value to diet as well as

improve palatable food (Stephen, 2019). Tilapia is a fish distributed in the tropical zones around

the world. It lives in fresh water and is sold in markets. The tropical freshwater fish, tilapia, is an

important cultural fish because it reproduces very easily and does not have feeding problems.

Stephen (2019) stated that the culture of tilapia has exceeded 800,000 metric tons and is ranked

under carps as the second most widely farmed fish group in the world. The tilapia fish is an ideal

species of organisms for an assessment study on effects of heavy metal contamination in

aquaculture ponds.

Tilapia can survive in bad environmental conditions because their resistance to disease is

physically powerful, and their respiratory demands are slight so that they can accept low oxygen

and high ammonia levels (Abdulali, et al., 2011). Fish are often at the top of aquatic food chain

and may concentrate large amounts of some metals from the water (Low, et al., 2010). Some

metals are essential to human health. Metals are naturally occurring elements that become

contaminants when human activities increase their concentrations above normal levels in the

environment. Heavy metal pollution is a serious and widespread environmental problem due to
their toxicity; heavy metals enter the environment through various natural methods and human

activities, and can accumulate in fish and other organisms (Amal, et al., 2022).

Fish is the final organism in the aquatic food chain and a significant food source for man.

Consequentially, heavy metals in aquatic environments are transferred throughout the web chain

into humans. It is well known that muscle of fish is not an active tissue to accumulate heavy

metals, but it was discovered that heavy metal levels in the edible portion (muscle) of some fish

in contaminated regions exceeded permissible levels (Mahmoud, et al, 2017). Therefore,

determination of heavy metal levels of fish is tremendously important for the health of human

beings. As observed by Amal, et al., (2022) metals are very toxic because, as ions or compound

form, they are soluble in water where the fish live and may be easily absorbed into the fish and

bind to structural proteins and enzymes. In humans, some metals can cause severe physiological

and health effects. Humans are exposed to different levels of heavy metals directly from the

water, air and food.

Fish consumers may be exposed to relatively higher levels of heavy metals by eating heavy

metals contaminated fish from rivers, ponds, lakes and seas. Edible fishes are often contaminated

with heavy metals as a result of agricultural technology, industrial pollution, sewage drainage

and other sources, which could affect human health and cause chronic diseases (Zhu, et al.,

2016). Heavy metals such as cadmium, zinc, mercury, chromium and copper cause heavy

pollution, particularly in the ponds, lakes and river systems in zones affected by effluents

released from industries, sewage and agricultural drains.

Among the animal species, fishes are inhabitants that cannot escape the detrimental effects of

these pollutants. The concentration of the essential elements like Zn and Cu are relatively higher

in the muscle and liver tissues than the non-essential metals like Pb and Cd (Abarshi, et al.,
2017). Different fish tissues can take heavy metals from surrounding environment, making

natural lakes and rivers major sources of fish contamination by heavy metals. This allows

humans to be exposed to pollution. Heavy metals will be studied through three different fish

organs because of the affinity between each of them. Rivers, ponds and lakes are major sources

of drinking water. However, the pollution of these natural waters is one of the most critical

environmental problems in recent years.

Observation by the researcher has shown that Alode in Eleme Local Government Area of Rivers

State has being highly polluted by elements such as mercury or lead, plastic, heavy metals like

gold, mercury and cadmium, these pollution are introduced into the river through domestic and

industrial wastes, or atmospheric sources and amplified by human activities like waste deposition

or mining. Mining loosens heavy metals buried in the earth, adding more of them to water. The

bunkry oil business going on within the catchments of the Imo River, have further worsened the

ecological condition of the river and made the water unsafe for human use.

The impact of illegal bunkry oil business on the Imo river cannot be over-emphasised. First, the

impacts have been felt on the river’s water quality, which has deteriorated. This has grave

implications for its biological diversity. Aside from the introduction of toxicants, the river, which

was once transparent enough for photosynthetic production, is now very turbid (cloudy) with a

characteristic gold colour. At extremely low water transparency, a river’s phytoplankton primary

production could be threatened, and by implication, its secondary (fish) production is also

threatened. It can also cause fish to die by blocking their gills and destroying their reproductive

sites. Another source of pollution in Imo river is human-generated waste that lands up in the

river. This is due to poor waste management practice in Oyigbo Local Government of Rivers

State.
Contamination with metals in aquatic systems has drawn particular attention due to their

persistence, toxicity and biological accumulation (Zahran, et al., 2015). Heavy metals enter the

human body through different paths, such as the food chain, and pose both non-carcinogenic and

carcinogenic health risks (Mohanta, et al., 2020). Fatal diseases such as renal tumor, nephritis,

osteoporosis, cancer, nasopharyngeal congestion, increased blood pressure associated with

cardiovascular diseases, and malfunctions of different body systems are caused by heavy metals

(Mohanta et al. 2020). Determination of heavy metal levels in environmental biota is an essential

process in assessing the human health risk resulting from the presence of these contaminants in

food.

1.2 Statement of the Problem

Fishes such as tilapia improves fertility value to human food and can survive even in river filled

by heavy metals because their resistance to water pollution and disease is physically powerful,

and their respiratory demands are slight so that they can accept low oxygen, but it has been

observed by the researcher that fish consumers may be exposed to relatively higher levels of

heavy metals by eating heavy metals contained fish from rivers could affect human health and

cause chronic diseases such as renal tumor, nephritis, osteoporosis, cancer, nasopharyngeal

congestion, increased blood pressure associated with cardiovascular diseases, and malfunctions

of different body systems.

Tilapia can take heavy metals from surrounding environment, making natural lakes and rivers

major sources of fish contamination by heavy metals. This allows humans to be exposed to

pollution. The pollution of these natural waters is one of the most critical environmental

problems in recent years.

1.3 Significance of the Study

It is acknowledged that fatal diseases such as renal tumor, nephritis, osteoporosis, cancer,

nasopharyngeal congestion, increased blood pressure associated with cardiovascular diseases,

and malfunctions of different body systems may be caused by tilapia? That has taken heavy

metals from surrounding environment. Hence, this study will assist in revealing the level of

heavy metals (cd, cr, cu, pb and zn) in the gills, muscles and liver of oreochromis niloticus in

Alode in Eleme Local Government Area of Rivers State, assess the non-carcinogenic and

carcinogenic risks associated with the consumption of in Alode in Eleme Local Government

Area of Rivers State and reveal the histopathological changes in the muscles, gills and liver of

oreochromis niloticus.

1.4 Scope of Study

This research will investigate the occurrence of heavy metals in gills, muscles and liver of

oreochromis niloticus in Alode in Eleme Local Government Area of Rivers State.

The study will look at the level of heavy metals in the gills, muscles and liver of in oreochromis

niloticus in Alode in Eleme Local Government Area of Rivers State, assess the non-carcinogenic

and carcinogenic risks associated with the consumption of oreochromis niloticus in Alode in

Eleme Local Government Area of Rivers State and reveal the histopathological changes in the

muscles, gills and liver of oreochromis niloticus. The study will be further limited to Alode in

Eleme Local Government Area of Rivers State.

1.5 Aim and Objectives of the Study

The aim of this research will be to investigate on the occurrence of heavy metals in gills, muscles

and liver of oreochromis niloticus in Alode in Eleme Local Government Area of Rivers State.
The objectives of the study are:

1. To determine the level of metals in the gills, muscles and liver of oreochromis niloticus

in Alode in Eleme Local Government Area of Rivers State.

2. To assess the non-carcinogenic and carcinogenic risks associated with the consumption

of oreochromis niloticus in Alode in Eleme Local Government Area of Rivers State.

3. To evaluate the histopathological changes in the muscles, gills and liver of oreochromis

niloticus.
 CHAPTER TWO

LITERATURE REVIEW

2.1 Definition of Heavy metals

Heavy metals are substances with high electrical conductivity, malleability and luster, which

voluntarily lose their electrons to form cations (Khlifi & Hamza-Chaffai, 2010). Heavy metals

are found naturally in the earth's crust and their compositions vary among different localities,

resulting in spatial variations of surrounding concentrations. Heavy metal distribution in the

atmosphere is monitored by the properties of the given metal and by various environmental

factors (Khlifi & Hamza-Chaffai, 2010).

Heavy metals are natural elements characterized by their high atomic mass and their high density

(Amal, et al., 2022). Although typically occurring in rather low concentration, they can be found

all through the crust of our planet (Khlifi & Hamza-Chaffai, 2010). Commonly, a density of at

least 5 g cm−3 is used to define a heavy metal and to differentiate it from other, light metals.

Other, broader definitions for “heavy metals” require an atomic mass higher than 23 or an atomic

number exceeding 20; these definitions are highly error prone and confusing. Both alternative

definitions cause the inclusion even of nonmetals; resorting to the atomic mass criterion, the

maximum number of elements classified as heavy metals rockets high to 99 out of the in total

118 building blocks of our universe. Looking at the periodic table of elements, we learn that

heavy metals sensu stricto (according to the density criterion) occupy the lion’s share, namely,

columns 3–16, of the periods 4 to 6, encompassing the transition metals, post-transition metals,

and lanthanides (Martin & Hosam, 2018).

As observed by Martin and Hosam (2018) some heavy metals like copper, selenium, or zinc are

essential trace elements, with functions indispensible for various biological processes also

driving the entire human metabolism. Heavy metals are generally defined as metals with

relatively high densities, atomic weights, or atomic numbers (Amal, et al., 2022). The criteria

used and whether metalloids are included, vary depending on the author and context (Amitabh,

et al., 2021). In metallurgy, for example, a heavy metal may be defined on the basis of density,

whereas in physics the distinguishing criterion might be atomic number, while a chemist would

likely be more concerned with chemical behaviour. As observed by Litasov and Shatskiy (2016)

heavy metal is any metallic chemical element that has a relatively high density and is toxic or

poisonous at low concentrations. Examples of heavy metals

include mercury (Hg), cadmium (Cd), arsenic (As), chromium (Cr), thallium (Tl), and lead (Pb).

Heavy metals are natural components of the Earth's crust (Amitabh, et al., 2021). They cannot be

degraded or destroyed. To a small extent they enter our bodies via food, drinking water and air.

As trace elements, some heavy metals (e.g. copper, selenium, zinc) are essential to maintain the

metabolism of the human body (Amitabh, et al., 2021). However, at higher concentrations they

can lead to poisoning (Martin & Hosam, 2018). Heavy metal poisoning could result, for instance,

from drinking contaminated wated (e.g. lead pipes), high ambient air concentrations near

emission sources, or intake via the food chain (Litasov & Shatskiy, 2016).

Heavy metals are dangerous because they tend to bioaccumulate. Bioaccumulation means an

increase in the concentration of a chemical in a biological organism over time, compared to the

chemical's concentration in the environment. Compounds accumulate in living things any time

they are taken up and stored faster than they are broken down (metabolized) or excreted. Heavy

metals can enter a water supply by industrial and consumer waste, or even from acidic rain
breaking down soils and releasing heavy metals into streams, lakes, rivers, and groundwater

(Litasov & Shatskiy, 2016).

Heavy metals are generally referred to as those metals which possess a specific density of more

than 5 g/cm3 and adversely affect the environment and living organisms (Järup, 2003). These

metals are quintessential to maintain various biochemical and physiological functions in living

organisms when in very low concentrations, however they become noxious when they exceed

certain threshold concentrations (Litasov & Shatskiy, 2016). Although it is acknowledged that

heavy metals have many adverse health effects and last for a long period of time, heavy metal

exposure continues and is increasing in many parts of the world. Heavy metals are significant

environmental pollutants and their toxicity is a problem of increasing significance for ecological,

evolutionary, nutritional and environmental reasons (Jaishankar et al., 2013). The most

commonly found heavy metals in waste water include arsenic, cadmium, chromium, copper,

lead, nickel, and zinc, all of which cause risks for human health and the environment (Lambert et

al., 2000).

Although these metals have crucial biological functions in plants and animals, sometimes their

chemical coordination and oxidation-reduction properties have given them an additional benefit

so that they can escape control mechanisms such as homeostasis, transport,

compartmentalization and binding to required cell constituents. These metals bind with protein

sites which are not made for them by displacing original metals from their natural binding sites

causing malfunctioning of cells and ultimately toxicity.

2.2 Ways by which heavy metals enter into the environment

A). Heavy metals as observed by Morais, et al., (2012) are introduced in aquatic systems as a

result of the weathering of soils and rocks, from volcanic eruptions, and from a variety of human

activities involving the mining, processing, or use of metals and/or substances that contain metal

pollutants. The most

common heavymetal pollutants are arsenic, cadmium, chromium, copper, nickel, lead and merc

y. There are different types of sources of pollutants: point sources (localized pollution), where

pollutants come from single, identifiable sources. The second type of pollutant sources

are nonpoint sources, where pollutants come from dispersed (and often difficult to identify)

sources. There are only a few examples of localized metal pollution, like the natural weathering

of ore bodies and the little metal particles coming from coal-burning power plants via

smokestacks in air, water and soils around the factory. The most common metal pollution in

freshwater comes from mining companies (Morais, et al., 2012). They usually use an acid mine

drainage system to release heavy metals from ores, because metals are very soluble in an acid

solution. After the drainage process, they disperse the acid solution in the groundwater,

containing high levels of metals (Morais, et al., 2012).

B). Heavy metals enter the surroundings by natural means and through human activities.

Various sources of heavy metals include soil erosion, natural weathering of the earth's crust,

mining, industrial effluents, urban runoff, sewage discharge, insect or disease control agents

applied to crops, and many others (Morais, et al., 2012).

River contamination with heavy metals is one of the major quality issues of many fast growing

cities, because maintenance of water quality and sanitation infrastructure has not improved along
with population and urbanization growth, especially in developing countries (Ahmad et al.

2010). Heavy metals from man-made pollution sources are continually released into aquatic

systems, and they pose a serious threat because of their toxicity, long persistence,

bioaccumulation and biomagnifications in the food chain (Amitabh, et al., 2021). Heavy metals

are known to distort structural or biological functions of biomolecules. Since metals act as

endocrine disrupters, they can interfere with metabolism, synthesis and transport of hormones or

receptors (Raphael, et al., 2021). Heavy metals are natural trace components of the aquatic

environment, but their levels have significantly increased due to domestic, industrial, mining and

agricultural activities. Both localized and dispersed metal pollution cause environmental damage

because metals are non-biodegradable. Unlike some organic pesticides, metals cannot be broken

down into less harmful components in the environment.

2.3 Ways by which Fishes take up heavy metals

Fish is widely consumed throughout the world as staple food and a rich source of proteins. It is

very likely that heavy metals present in water get incorporated in fish and enter the human body

via the food chain threatening human health. The term ‘heavy metal’ is somewhat imprecise, but

includes most heavy metals with an atomic number greater than 20, and excludes alkali metals,

alkaline earths, lanthanides and actinides. When the pH in water falls, metal solubility increases

and the metal particles become more mobile. That is why metals are more toxic in soft waters.

Metals can become ‘locked up’ in bottom sediments, where they remain for many years. Streams

coming from draining mining areas are often very acidic and contain high concentrations of

dissolved metals with little aquatic life.

Amal, et al., (2022) described two contrasting responses of an organism to heavy metal toxicity

with declining pH:

If there is little change in speciation and the heavy metal binding is weak at the biological

surface, a decrease in pH will decrease owning to competition for binding sites from hydrogen

ions. Where there is a marked effect on speciation and strong binding of the heavy metal at the

biological surface, the dominant effect of a decrease in pH will be to increase the heavy metal

availability.

Generally the ionic form of a heavy metal is more toxic, because it can form toxic compounds

with other ions. Electron transfer reactions that are connected with oxygen can lead to the

production of toxic oxyradicals, a toxicity mechanism now known to be of considerable

importance in both animals and plants. Some oxyradicals, such as superoxide anion (O2-) and the

hydroxyl radical (OH-), can cause serious cellular damage (Amal, et al., 2022)

Some inorganic pollutants are assimilated by organisms to a greater extent than others. This is

reflected in the Bioconcentration Factor (BCF), which can be expressed as follows:

BCF = concentration of the chemical in the organism / concentration of the chemical in the

ambient environment. The ambient environment for aquatic organisms is usually the water or

sediments. With inorganic chemicals, the extent of long-term bioaccumulation depends on the

rate of excretion. Toxic chemicals can be stored into tissues of species, especially fat tissues.

Bioaccumulation of cadmium in animals is high compared to most of the other metals, as it is

assimilated rapidly and excreted slowly. Also the sensitivity of individuals of a particular species

to a pollutant may be influenced by factors such as sex, age, or size. In general the concentrations

of metals in invertebrates are inversely related to their body mass. In fish, the embryonic and

larval stages are usually the most sensitive to pollutants.

Benthic organisms are likely to be the most directly affected by metal concentrations in the

sediments, because the benthos is the ultimate repository of the particulate materials that are

washed into aquatic systems (Amal, et al., 2022).

2.4 Metal tolerance

Some heavy metals, such as manganese, iron, copper, and zinc are essential micronutrients

(Lauren & McDonald, 1987). They are essential to life in the right concentrations, but in excess,

these chemicals can be poisonous (Lauren & McDonald, 1987). At the same time, chronic low

exposures to heavy metals can have serious health effects in the long run.

Tolerance to metals has also been recorded in invertebrates and in fish. After exposure for 24

hours to a copper concentration of 0.55 mg/l, rainbow trout showed a 55 per cent inhibition

of sodium uptake and a 4 per cent reduction in affinity for sodium, which resulted in an overall

decrease in total sodium concentration of sulphydryl-rich protein (Lauren & McDonald, 1987).

These low molecular weight proteins contain many sulphur-rich amino acids which bind and

detoxify some metals. The pretreatment of an organism with low doses of a metal may stimulate

metallothionein synthesis and provide tolerance during a subsequent exposure (Pascoe & Beattie,

1979).

Many rivers are polluted with heavy metals from old mine workings and some species of algae

become very tolerant to polluted conditions. A survey of 47 sites with different concentration of

zinc found the filamentous green alga 'Hormidium rivulare' to be abundant everywhere,

tolerating zinc concentrations as high as 30.2 mg Zn/l(Pascoe & Beattie, 1979).

Toxicity of metals

For the protection of human health, the maximum permissible concentrations for metals in

natural waters that are recommended by the Environmental Protection Agency (EPA),

S/ Heavy metal ions WHO’s permissible limit (mg L−1)

Se 0.02

Hg 0.001

Mn 0.02

Ag 0.1

Cd 0.05

Cr 0.003

Pb 0.01

Zn 3.00

Fe 0.30

Cu 0.02

As 0.01

Table 1: Permissible limit of heavy metal ions in water.

Source: Researcher
The presence of these heavy metals on the surface of the water can be due to natural or

anthropogenic activities. In natural activities, weathering of rocks that contain metals, an

eruption from volcanos, fires in the forest, and naturally occurring processes of weathering can

be included from these activities, metal enters the different sections of the environment

(Anubhav, et al., 2022). Heavy metals can be found in the forms of sulfates, hydroxides, oxides,

sulfides, phosphates, and silicates (Anubhav, et al., 2022). A huge amount of accumulation of

heavy metals into the water is mainly due to anthropogenic and natural activities. Some more

examples of natural source through which heavy metals contaminates water are wet and dry

deposition of atmospheric salts, water-rock interaction, or water interaction with the soil

(Anubhav, et al., 2022). While the sudden increase in urbanization and industrialization are an

example of anthropogenic sources through which water get contaminated.

Heavy metal ions are Copper (Cu), Zinc (Zn), Silver (Ag), Chromium (Cr), Arsenic (As),

Mercury (Hg) and Cadmium (Cd), while the common sources are fertilizers, tanning, and

photovoltaic cells, Soldering, cosmetics, and pigments, refining of copper, gold, nickel, zinc,

jewelry, and electroplating industries, leather industry, tanning, and chrome plating industries,

wooden electricity poles that are treated with arsenic-based preservatives, pesticides, fertilizers,

the release of untreated effluents, oxidation of pyrite (FeS) and arsenopyrite (FeAsS),

Combustion of coal, municipal solid waste incineration, and volcanic emissions and paints,

pigments, electroplated parts (Anubhav, et al., 2022).

2.5 Natural sources

Trace heavy metals are found in excess levels in the environment, they are formed by

geographical processes such as volcanic eruptions, weathering of rocks, and leaching into rivers,
lakes, and oceans due to the action of water (Anubhav, et al., 2022). The presence of heavy

metals in water depends on the local geology, hydrogeology, and geochemical characteristics of

the aquifer (Anubhav, et al., 2022). One of the main sources of pollution is weathering. The

weathering of the sedimentary rocks such as limestone or dolomite or shale makes the water

contaminated or polluted. When there is an interaction of water with rock element, it also leads

addition of these elements into the water; thus, contamination occurs. Examples of such elements

are granite, syenite, basalt, gabbro, nepheline, and andesite. Due to the particular ore or the

minerals, the element level increases. Elements examples are magnetite, hematite, goethite,

siderite, calcite, cuprite, malachite, azurite, chromite, kaolinite, montmorillonite, arsenic trioxide,

orpiment, arsenopyrite, calamine, smithsonite, pyrolusite, and rhodochrosite. The sulfide

deposition also increases as it is associated with the mineralization of the gold and hydrous iron

oxide ores (Anubhav et al., 2022).

2.6 Domestic sewage

Huge amount of untreated sewage from domestic is thrown into the river. This untreated waste from

domestic has the presence of toxins. These toxins are due to the presence of solid waste or from the

litter of plastic, or the contamination of bacteria due to the presence of these the water can get polluted

(Anubhav et al., 2022). Domestic untreated water is thrown directly into the water resource and this

majorly causes pollution inside the water and harms the ecosystem (Amal, et al., 2022). These pollutants

majorly depend upon what kind of industry has thrown those pollutants. When these toxic metals get

inside the water, they decrease the quality of the water. Around 25% of pollution inside the water is

caused only by these industries (Amal, et al., 2022). When the water gets contaminated, the water gets

enriched by the nitrogen and phosphorous elements. With the presence of these nutrients, the growth

rate of algae gets multiplied, and then it competed with the surrounding aquatic biota for the dissolved
oxygen in water. The presence of nitrite and nitrate anions leads to a major threat to the exposed

organisms; examples of such threats are methemoglobinemia. It is more common in small children, and

the symptoms caused by this are cyanotic color in the skin due to blood alterations. Water sources that

get deposited by this sewage also become anions rich, due to the presence of chlorine in urine, and NaCl

is used in the human diet. On the side of the sea, there is the presence of chloride in high concentrations

due to the leakage of salt into the sewerage system. It also may be increased by industrial processes

(Amal, et al., 2022).

2.7 Industrial source

Contamination of water by heavy metals in the aquatic environment is very harmful since these

elements cannot be degraded and they get accumulated inside the living organisms (Amal, et al.,

2022). Residue from the industry is the major source through which these heavy metals get into

the aquatic ecosystems, and their accumulation in water varies with the type of treatment used.

Effects known as deleterious can be observed when the metal particles are introduced into the

water system. In tissue, the concentration of several metals is slow and their concentration should

be kept in less range only as more concentration can be harmful to the biological development of

the pant. Through the food chain, fish contaminants can reach man. Effluent from industries,

water tank leakages, dumping beside marines, and due to radioactive waste and atmospheric

deposition, are some sources of water contamination (Amal, et al., 2022). Disposed of heavy

metals and waste from industries they get accumulated in rivers and lakes thus causing harmful

impacts on animals and humans. For suppression of the immune, reproductive failure and acute

poisoning toxins are responsible. Then there is direct damage to plant or animal nutrition at that

time human health is affected (Amal, et al., 2022). The pollutants that are polluting the water are
killing marine organisms such as mollusks, marine birds, fishes, and other organisms that live in

the sea (Amal, et al., 2022).

2.8 Urbanization

With an increase in the population has created many issues and one of the issues is the pollution

of water. An increase in the population leads automatically leads to more generations of solid

waste. Both solid waste and liquid waste are deposited into the water without any treatment

(Amal, et al., 2022). Human excreta also contaminate the water. Thus, contaminated water leads

to a generation of a large number of bacteria, which is a threat to human well-being (Amal, et al.,

2022). Government is unable to supply vital requirements to the People because of the increase

in the number of people. Facility for sanitization is more in urban areas as compared to rural

areas. Plastic bag and waste are a major contribution to pollution. People throw the waste in

plastic bags into water sources. From the research, it was found that around three people of the

population defecate in the open, while 77% population use flush and around 8% use the pit

latrines. Urbanization can cause many infectious diseases. Overpopulation, unhealthy conditions,

and dangerous drinkable water are these major health problems in urban areas. One-third of

urban people are vulnerable to disease (Anubhav et al., 2022).

2.9 Agriculture source

The population in rural areas is less but the use of fertilizers, pesticides, and eroded soil

contaminates the water (Amal, et al., 2022). When it rains the water from the surface runoff and

that rainwater enters the nearby water resource and thus pollutes the existing water (Amal, et al.,
2022). Agricultural runoff cases freshwater bodies’ eutrophication (Amal, et al., 2022). Half of

the lakes in the US are eutrophic. Phosphate has one of the major contributions to eutrophication,

and the high concentration of phosphates promotes cyanobacteria and algae growth which leads

to the excessive use of the biologically dissolved oxygen inside the water (Amal, et al., 2022).

Fertilizers that are too enriched with nitrogen decrease the dissolved oxygen in rivers and coastal

zones thus bringing hazardous effects to the biota. Since 2006, the nitrogen in fertilizers is being

controlled in America and Northwest Europe (Amal, et al., 2022). Like pesticides, which are

used as pest control, these pesticides leach into groundwater, thus polluting groundwater. The

pesticides that are water-soluble leach more and the sandy soil favors the process of leaching

(Anubhav et al., 2022).

2.10 Atmospheric source

Small pollutants particles which are present in the air, get into the surface water stream through

the rain, when it rains these particles come down and then with the flow of water enters into the

sea, thus polluting the water (Amal, et al., 2022). These pollutants that are present in the air

usually get from the burning of fossil fuels e.g. is CO 2, which combines with water and produces

sulfuric acid (Amal, et al., 2022). Sulfur dioxide, which is formed via volcanic eruption and from

industries, also gets attached to a water molecule to form the sulfuric acid. When coal is

combusted then also sulfuric dioxide is produced and it is also produced from petroleum

products. Just like this nitrogen dioxide also combines with the water and forms the nitric acid.

And with the help of rainwater, they enter the water resources (Anubhav et al., 2022).

2.11 Mining source

Heavy metals are present on the earth and thus they can enter the water system through various

pathways and one of them is through mining sources (Anubhav et al., 2022). When it rains or

through flowing water, it leaches heavy metals out from their geological formation. These

processes get disturbed when manmade economic activities such as mining are done (Anubhav et

al., 2022). Through these processes, the area that is already mined out gets exposed to water and

air and this leads to the acid mine drainage (AMD). The low pH conditions associated with

AMD mobilize heavy metals, including radionuclides where these are present (Anubhav et al.,

2022).

2.11 Heavy metal intake through water

Soil gets polluted with the presence of heavy metal on surface and underground water and the

pollution rises when mined ores are discarded on the ground surface for manual dressing

(Anubhav et al., 2022). Due to the dumping over the surface, the metals get exposed to air and

rain thereby generating huge AMD. If soil is polluted at that time, it gets into the plant tissue and

gets accumulated there. And when those plants are grazed by animals and water is used for the

drink from polluted waters, through there these heavy metals enter the body. Also, marine lives,

which reproduce in contaminated water, also have the presence of heavy metals inside their body

tissues, if they are lactating then inside their milk (Anubhav et al., 2022). As an overview, all

organisms within a given ecosystem are contaminated via these pollutants through their food

chains. When nutrition from these contaminated vegetables is taken, the presence of heavy

metals in those vegetables can lead to different chronic diseases. Toxic effects due to these heavy

metals usually depend on the amount of concentration and the oxidative state of the particular

heavy metals. Heavy metals have a very dangerous impact as they are non-biodegradable in
nature, have long biological half-lives, and have the potential to accumulate inside the body.

Also, there are some heavy metals that are extremely toxic only because of their solubility.

Fewer concentrations of heavy metals inside the food chain also show severe effects as there is

no particular procedure through which these heavy metals pollutants can be extracted from the

body of an organism (Anubhav, et al., 2022). Nowadays presence of these toxic heavy metals is

everywhere because of their extreme use in industries. In case of the wastewater, it contains a

huge concentration of heavy metals, which create various health-related problems (Anubhav, et

al., 2022).

Water from estuaries and freshwater is not polluted to some extent, but that water is also at threat

of being polluted in the long term due to metal deposition because of human past activities

(Anubhav, et al., 2022). The water in the river and lakes can be highly polluted depending on the

volume of flow and proximity to the point sources. Due to the human civilization, the element

content in water is raised. Such elements are cadmium, lead, mercury, zinc, and chromium

(Anubhav, et al., 2022). Unlike organic chemicals, there are some heavy metals that cannot be

converted into compounds with lesser toxicity, and one of its characteristics is the loss of

biodegradability. Once the heavy metals enter the water system it gets redistributed throughout

the column and gets accumulated in the sediments. The sediments constitute a partial

contribution to polluting the natural phenomena due to their activity and metal remobilization

processes. Metal residues that are present in the contaminated surroundings have the flexibility to

get bioaccumulated into the aquatic environment (Anubhav, et al., 2022). Growth in fish larvae

and juveniles is rapid. But when these heavy metals enter they might inhibit the growth rate. The

fish grows in length and bulk when given the right conditions, such as a specific temperature and

an acceptable amount of food. Fish growth, on the other hand, may be impeded in water
contaminated with toxicants, such as heavy metals. One of the most noticeable signs of metal

toxicity in fish larvae is growth inhibition. As a result, the length and bulk of fish are indications

of environmental conditions (Anubhav et al., 2022).

Heavy metals are introduced in liquid form and surface water constituents (carbonate, sulfate,

organic substances humic, fulvic, and amino acids) cause the formation of non-soluble salts or

complexes. Aquatic species are not expected to be harmed by these salts and compounds. Some

of them sink and collect in the sediments at the bottom (Anubhav, et al., 2022). A decrease in pH

of water either due to acid rain or any other acidic incidents, due to the heavy metal’s deposition

into the water column, causes aquatic biota to become poisonous. Low levels of heavy metals

can also make chronic stress, through fish might not get dead but can cause them to lose weight

and become smaller, reducing their capacity to compete for food and habitat. Pollution poses a

hazard to both freshwater and marine habitats (Anubhav et al., 2022).

Heavy metal poisoning of water is a significant environmental hazard that has detrimental

consequences for organisms who are exposed to it be that plant-animal or humans. Fish from

freshwater are majorly exposed to various heavy metals, which are added into the water bodies

through the different-different sources. Contamination of these heavy metals into aquaculture has

intensified global issues because it shows a risk to fish and has harmful impacts on fish buyers.

There are three different modes through which heavy metals enter the fish. These methods are

either through the gills of fish, by the body of the fish, or by the digestive tract of the fish. Heavy

metals immediately enter the fish body through the gills, while the body surface takes time for up

taking of these heavy metals through this mode (Anubhav, et al., 2022). Mostly the metals get

accumulated in the liver, kidney, and gills. In fishes, the muscles have most of the metals present

there as compared to the other body parts of the fish (Anubhav, et al., 2022). Too much
accumulation of these heavy metals inside the fish organ can cause lesions and operative

disturbances.

These heavy metals also interfered with the embryo’s shape and the metabolic processes of the

fishes. Structural and functional defects throughout the development of the embryo resulted in

fewer larvae hatching. Several freshly born larvae die shortly after hatching owing to lead and

copper absorption (Mahmoud, & Abd, 2017). Heavy metals get into the fish through three

routes: the first is via the fish gills, the second is through the digestive tract of the fish and the

last one is through the body of the fish. The gills of fish are the area that is known for the

primary metal intake from the contaminated water. On the fish gills, zinc accumulates. It

suggests a depressing influence on tissue respiration, which leads to hypoxia and mortality. Zinc

pollution also causes alterations in the structure of the lungs and heart (Mahmoud, & Abd, 2017).

Humans and fish are both affected by mercury. Brain damage, with postnatal and fetal problems,

leads to abortions, congenital deformity, and development differences in young fry due to

Monomethyl (Mahmoud, & Abd, 2017).

Minamata illness and Hg poisoning (via methyl Hg) both showed considerable neurotoxicity

(Amal, 2022). Anemia, eosinophilia, lymphocytosis, bronchial, and renal injuries can affect

chromium levels in the blood. Malformations in fish are caused by cadmium, nickel, mercury,

chromium, lead, and arsenic. The accumulation of these heavy metals in excessive amounts

causes a variety of physiological effects. Fin loss, gill underdevelopment, liver dysfunction, and

fin function in fingerlings were all prevalent findings in the studies. The harmful effects of heavy

metals have the greatest impact on the death rate, reproduction, individual development rates,

and physiological capacity of fish (Amal, 2022). There have been effects on physical functioning

and chemical parameters in the tissues and blood of fish living in water that is polluted via
metals. It has been reported that fish exposed to metals developed immune system defects,

making them more susceptible to infectious infections and increasing their chances of dying

(Amal, 2022).

One of the main sources of contamination of the water is heavy metals, as it overwhelms the

important species indirectly through biological chains or directly via chemical modifications in

water (Amal, el ta., 2022). By altering the normal activities of numerous enzymes and

metabolites, the accumulation of these heavy metals in the tissues causes significant biochemical,

physiological, and histological changes in fish and other freshwater fauna. Fish are one of the

most widely dispersed creatures in the aquatic ecosystem, and their susceptibility to metal

poisoning may indicate the extent of metal pollution’s biological impact (Amal, et ta., 2022).

Heavy metals, such as As, Cd, Cu, Cr, Fe, Pb, Mn, Hg, Ni, Zn, and tin (Sn), are major

contaminants that cause serious toxicity in fish (Amal, el ta., 2022). Due to the heavy metals, the

physiological and biochemical functions both in tissues and in blood Carpi can be altered. The

compounds of As and inorganic As, Cd, Ni, silica in its crystal form, beryllium, and its

compounds are considered to be chemical carcinogens, which results in the development of

cancer inside the fishes. The drop in hematological parameters indicated that the exposed fishes

had become anemic as a result of Cr exposure. This dangerous heavy metal was released into the

aquatic ecosystem via trash, causing severe anemia and changes in hematological parameters in

the Labeo rohita fish.

There are various studies on different fish such as Noemacheilus barbatulus, Perca

fluviatilis, Catostomus commersonii, Oreochromis mossambicus, and Oreochromis aureus, and

increased quantities of zinc, lead, cadmium, copper, mercury, and cadmium were found in the

gonads of fish, according to the study. This causes contamination of eggs and sperm, as well as a
reduction in fish fertility and embryonic development. The number of hatched larvae was

reduced due to anatomical and functional abnormalities during embryonic development. A

percentage of freshly born larvae died shortly after hatching as a result of lead and copper

absorption. The sensitivity of heavy metals inhibits estrogenic and androgenic secretion and

produces pathological differentiation in fish. The reproductive tissues of fish gonads are affected

by effluents such as industrial and agricultural waste, pesticides, and heavy metals. Zinc gets

accumulated over the gills of fish. It entails a depressing influence on tissue respiration, which

leads to hypoxia and death. Zinc contamination also causes abnormalities in the structure of the

ventilator and heart. Zinc is a toxicant that destroys gill tissue, disrupts acid–base and ion

balance, and causes hypoxia in fish.

More importantly, heavy metal for neurotoxicity is Hg. While other factors play a role in

neurotoxicity, mercury has a major impact on both fish and humans. Japan’s rivers have been

contaminated with mercury. Minamata illness and Hg poisoning (via methyl Hg) both showed

considerable neurotoxicity. Arsenic coagulates proteins, binds to coenzymes, and reduces the

production of adenosine triphosphate (ATP) during respiration. It can induce cancer in all of its

oxidation states in combination, and high-level articulation can result in mortality. Carcinogenic

metals such as cadmium, arsenic, nickel, and chromium can damage DNA by deleting base pairs,

mutating it, or attacking it with radical oxygen. Malformations in fish are caused by cadmium,

nickel, mercury, chromium, lead, and arsenic. When the accumulation of these heavy metals gets

excessive inside the water through this, many physical effects on fish can be seen. Fins get

shortened; gills are underdeveloped; and liver and fin functions of fingerlings were commonly

observed. The harmful effects of heavy metals have the greatest impact on the death rate,

individual growth rates, reproduction, and physiological capacity of fish. The effects of metal
pollution on physiological processes and biochemical parameters in the blood and tissue of fish

have been studied. Fish that are exposed to heavy metals reveal faults in the immune system and

thus are more exposed to diseases and chances of death increase (Stephen, 2019).

CHAPTER THREE

MATERIALS AND METHOD

3.1. Site description

The study will be carried out in Alode river, in Alode Eleme Local Government Area of Rivers

State. The Alode river flows 240 kilometers (150 mi) into the Atlantic Ocean. The Alode River is
located between longitudes 31°45’E and 32°22’E and latitudes 31°00’N and 31°35’N. Its

estuary is around 40 kilometers (25 mi) wide, and the river has an annual discharge of 4 cubic

kilometers (1.0 cu mi) with 26,000 hectares of wetland. The Alode river features an 830-metre

(2,720 ft).

Water temperature varied between (27.00- 28.00 ̊c), ph (6.3-6.6), turbidity (15-71 NTU),

electrical conductivity (19.9- 82.0μs/cm), dissolved oxygen (4.8- 7.05mg/l), biological oxygen

demand (2.10- 3.20 mg/l), total dissolved solids (19.9-30.30mg/l), total hydrocarbon (0.010-

0.031mg/l), total soluble solids (3.6- 9.1mg/l), nitrates (0.13-0.43mg/l), phosphates (0.07-

0.11mg/l), sulphates (0.99-7.00mg/l), and chlorides(0.01-0.08mg/l), the physicochemical

variability of the sediments that ph varied between (6.85-6.98),total

hydrocarbon(0.22-4.84mg/kg), nitrates (0.22-0.71mg/kg), phosphates (0.41-1.10mg/kg), and

sulphates (30.80- 65.20mg/kg).While, the microbiological variability of the sediments varied

between(2.30-2.80x102), total heterotrophic fungi (5.0-6.3x102), hydrocarbon utilizing bacteria

(1.40-1.95x102), and hydrocarbon utilizing fungi between (2.60-3.50x102).

Fig 4. View of Alode river

Source: Researcher 2023

Fig 5. Alode river

Source: Researcher 2023

3.2. Collection of samples

Fishing will be done late at night with the help of professional local fishermen from Alode in

Eleme Local Government of Rivers State. Three tilapia fish’s samples will be collected randomly

from each location, using fishing net to catch the fish from natural sites no. 2 and 3, while at site

1 the fish will be bought from fish sellers at the bank of the Alode river. The tilapia fish length

ranged between 20 and 22 cm and the weight ranged between 220 and 240 g. The tilapia fish will

be placed in clean polyethylene bags with ice and immediately taken to laboratory where the

samples will be deep frozen at -20°C until prepared and will be transferred cold to the laboratory

for analysis.

3.3. Analysis of heavy metals

Specimens of muscles and gills of different tilapia fish species will be dried in an oven at 105°C

for about 24 h. The dried specimens will be then ground to a fine powder. A representative

sample of 1 g dry weight of muscles or gills will be taken from tilapia fish specimens. The

samples will be digested according to the method described by Goldberg et al. (1983) in

Emmanuel (2022), during which concentrated nitric and perchloric acid (AR grade) in the 5:5

ratio will be use in Teflon beakers on a hot plate at 50°C for about 5 h till complete

decomposition of organic matter. The digested solutions will be cooled to room temperature,

filtered and diluted to a final volume of 50 ml with deionized distilled water. The concentrations

of Fe, Pb, Mn, Zn, Cu, Cd and Ni will be measured by an Australian GBC atomic absorption

reader (model Savant AA-AAS) with a GF 5000 graphite furnace and expressed in mg kg−1 dry

weight.
The precision of the analyzed metal values will be controlled by including triplicate samples in

analytical batches. Standard deviations for mean values of triplicate measurements will be up to

5%, which will be regarded as an acceptable precision. Practical quantitation limits for the

analysis of heavy metals will be in the range of 0.02–0.05 µg g−1.

3.4. Health risk assessment

In the present study, the muscles of tilapia species will be specifically selected for heavy metal

analysis because they are the only edible tissue; therefore the level of toxicants present in this

tissue will be particularly relevant.

3.5. Estimated daily intakes (EDIs)

The estimated daily intake (mg kg−1 body weight) for heavy metals will be calculated in fish

muscle samples using the following formula:

EDI = (FIR ˣ CM)/BW

where FIR is the food ingestion rate (g person−1 day−1; 57 g in Egypt, FAO 2014), Cm is the

metal concentration in fish muscle samples (mg kg−1), and BW is the body weight of an adult

assumed to be 70 kg (Shaheen, et al., 2016).

3.6. Non-carcinogenic risk

The non-carcinogenic risk for each metal in the muscles will be assessed by the hazard quotient

(HQ) (USEPA, 2012) using the following equation:

HD = (EF ˣED ˣFRM ˣCM/RfM ˣBW ˣAT) ˣ 10-3

where EF is the exposure frequency, ED is the lifetime exposure duration (70 years), RfD is the

oral reference dose (mg kg−1 day−1), AT is the averaging time for non-carcinogens (365 days

year−1 × 70 years) and 10−3 is the unit conversion factor. If the HQ is higher than 1, there is a

potential health risk (Islam, et al., 2017).

The overall potential for non-carcinogenic effects from all studied metals will be assessed using

the hazard index (HI). The hazard index will be expressed as the sum of the hazard quotients

(USEPA 2000) using the following equation:

❑
HI =∑ ❑= HQI
n =1

3.7. Carcinogenic risk

The carcinogen risk will be estimated using the target carcinogenic risk (United States

Environmental Protection Agency, 2012), which will be derived from the intake of Pb and Cd

using the following equation:

TRC=FE × ED × FIR × CM × CPSO /BW × AT × 10-3

where CPSo is the oral carcinogenic potency slope (mg kg−1 day−1). The CPSo values for Pb,

Cd and Ni were 0.0085 (OEHHA, 2011), 0.38 and 1.7 (USEPA, 2011), respectively. The US

Environmental Protection Agency set acceptable risk levels for carcinogens ranging from 10−4

to 10−6 (Atique-Ullah, et al. 2017; Miri, et al., 2017).

3.8. Relative risk

The relative risk (RR) of pollutants, defined by Yu, et al., (2014), can be helpful in identifying

the most harmful metals. The RR will be calculated according to the following equation:

RM= CM/RFD

Human health risk from fish consumption should increase as the relative risk values increase (Yu

et al. 2014).

3.9. Histological studies

Muscles, gills and liver of the fish oreochromis niloticus that will be collected from the area of

the study (Alode river) will be carefully removed and immediately fixed in 10% formalin for 48

h, dehydrated in ascending grades of alcohol and cleared in xylene. The fixed tissues will be

embedded in paraffin wax and cut with a Euromex Holland microtome into 4–6 microns.

Sections will be stained using the Harris haematoxylin and eosin (H&E) method (Saad, et al.,

2011). They will be then examined under a microscope and photos will be taken by a microscope

camera.

3.10. Statistical analysis

The results will be tested for significant differences for metals between different sites and species

using one-way ANOVA, and p < 0.05 will be considered statistically significant. In addition, the

relationships between the analyzed metals will be determined by calculating the Pearson

correlation index.
SAMPLE Heavy Metals
FISH Cd Pb Cr Zn Cu
Sample 1 0.021 0.011 0.41 7.42 4.34
Sample 2 0.023 0.011 0.43 7.45 4.33
Total 0.044 0.022 0.84 14.87 8.67
Mean 0.022 0.011 0.42 7.435 4.335
Standard 0.99 0.88 0.87 0.86 0.90
deviation

SAMPLE Heavy Metals

WATER Cd Pb Cr Zn Cu
Sample 1 1.18 1.66 0.71 31.31 1.56
Sample 2 1.19 1.60 0.72 31.34 1.60
Total 2.37 3.26 1.43 62.65 3.16
Mean 1.9 1.63 0.715 33 1.58
Standard 0.79 0.87 0.88 0.86 0.90
deviation

Reference

Abarshi, M.M., Dantala, E.O., & Mada, S.B. (2017). Bioaccumulation of heavy metals in some

tissues of croaker fish from oil spilled rivers of Niger Delta region, Nigeria. Asian Pac. J.

Trop. Biomed. 7(6), pp. 563–568.

Abdulali, T., Shuhaimi-Othman, M., & Ahmad, A.K. (2011). Heavy metals concentration in

different organs of tilapia fish (Oreochromis niloticus) from selected areas of Bangi,
 Selangor, Malaysia. [Unpublished Thesis] School of Environmental and Natural

Resource Sciences, Faculty of Science and Technology, University Kebangsaan

Malaysia.

Ajayi, M. E. (2022). Comparison of physico-chemical parameters with macroinvertebrate and

vertebrate fauna of Lake Ogelube and Lake Ojii, Opi-Agu, south-eastern Nigeria. African

Journal of Aquatic Science, 4(5). Pp. 13-21.

Amal, M. Y., Soaad, A. M., & Amaal, M. A. (2022). Accumulation of heavy metals in tilapia

fish species and related histopathological changes in muscles, gills and liver of

Oreochromis niloticus occurring in the area of Qahr El-Bahr, Lake Al-Manzalah, Egypt.

International Journal of Oceanography and Hydrobiology. 50 (1), pp. 1-5.

Atique-Ullah, A.K.M., Maksud, M.A., Khan, S.R., Lutfa, L.N. & Quraishi, S.B. (2017). Dietary

intake of heavy metal from eight highly consumed species of cultured fish and possible

human health risk implications in Bangladesh. Toxicology Reports 4(6). Pp. 574–579.

Emmanuel, O. A. (2022). Nigeria’s sacred Osun River supports millions of people - but pollution

is making it unsafe. https://theconversation.com/nigerias-sacred-osun-river-

supports-millions-of-people-but-pollution-is-making-it-unsafe-

191152#:~:text=Plastic%20pollution%20is%20a%20common,naturally%20in

%20the%20Earth's%20crust.

Goher, M.E., Abdo, M.H., Bayoumy, W.A. & Mansour, El-Ashkar T.Y. (2017). Some heavy

metal contents in surface water and sediment as a pollution index of El-Manzala Lake,

Egypt. Journal of Basic and Environmental Sciences 4(6). Pp. 210– 225.
Islam, M.S., Ahmed, M.K. & Habibullah-Al-Mamun, M. (2017). Heavy metals in sediment and

their accumulation in commonly consumed fish species in Bangladesh. Archives of

Environmental & Occupational Health 72(1). Pp. 26–38.

Low, K.H., Zain, S.M., & Abas, M.R. (2010). Evaluation of metal concentrations in red Tilapia

(Oreochromis Spp) from Three Sampling Sites in Jelebu, Malaysia Using Principal

Component Analysis. Food Anal Methods, 2 (1), pp. 1-10.

Mahmoud, S.A., & Abd, E. A.A. (2017). Eco- Toxicological studies of water and their effect on

fish in El Manzalah Lake. Research Journal of Pharmaceutical, Biological and Chemical

Sciences 8 (3), pp. 2497–2511.

Miri, M., Akbari, E., Amrane, A., Jafari, S.J., & Eslami, H. (2017). Health risk assessment of

heavy metal intake due to fish consumption in the Sistan region, Iran. Environ. Monit.

Assess. 4(6). Pp. 189: 583.

Saad, S.M.M., El-Deeb, A.E., Tayel, S.I. & Ahmed, N.A.M. (2011). Haematological and

histopathological studies on Clarias gariepinus in relation to water quality along Rossetta

Branch, River Nile, Egypt. Egypt. J. Exp. Bio. (Zool.) 7(2). Pp. 223–233.

Shaheen, N., Irfan, N.M., Khan, I.N., Islam, S., Islam, M.S. (2016). Presence of heavy metals in

fruits and vegetables: health risk implications in Bangladesh. Chemosphere 15(2). Pp.

431–438

The Office of Environmental Health Hazard Assessment (OEHHA) (2011). Air toxics hot spots

program technical support document for cancer potencies. Appendix B. Chemical-

specific summaries of the information used to derive unit risk and cancer potency values.
United States Environmental Protection Agency (USEPA). (2012). EPA Region III Risk-Based

Concentration (RBC) Table 2008 Region III, 1650 Arch Street, Philadelphia,

Pennsylvania 19103

Yu, Y., Wang, X., Yang, D., Lei, B., & Zhang, X. (2014). Evaluation of human health risks

posed by carcinogenic and non-carcinogenic multiple contaminants associated with

consumption of fish from Taihu Lake, China. Food and Chemical Toxicology 6(9). Pp.

86–93.

Zhu, H., Xu, Y., Yan, B., Guan, J., & Zhou, Q. (2016). Risk assessment of heavy metals

contamination in sediment and aquatic animals in downstream waters affected by

historical gold extraction in Northeast China. Human and Ecological Risk Assessment:

An International Journal 22(3), pp. 693–705.