Thesis draft on fish

Chapter One

1. INTRODUCTION

1.1. Back ground of the title

Fishes and fish products have a high source of animal protein of

high quality essential amino acids that contains sulpher bond.

Such amino acids are cystine, lysine& methionine which are rare

in other animal products (Co´rdova Murueta et al., 2007).Besides to

essential nutrients for human diets it is important sources of

income and high market value. Fish is also a cheapest source of

animal protein in third World Countries. The consumption of fish

is related to health factor because of a rich content in proteins

of high quality; minerals, vitamins and distinguishing lipids. It

should be considered that fish tissue presents high nutritional

significance and therefore is a particularly optional dietary

module. In addition, fish are a good source of micro and macro

elements such as calcium, phosphorus, selenium and manganese.

Fish lipids are well known to be rich in long-chain W-3

polyunsaturated fatty acids (LC W-3 PUFA), especially

eicosapentaenoic acid (EPA) and docosahexaenoic acid. Therefore

studying fishes for heavy metals contamination would benefit

individuals living in a country surrounded by water body

(R.VigneshandM.Srinivasan, 2012). The term “heavy metals” refers

to any metallic element

that has a relatively high density and is toxic or poisonous even

at low concentration (Lenntech, 2004). “Heavy metals” is a

1

general collective term, which applies to the group of metals and

metalloids with atomic density greater than 4 g/cm3, or 5 times

or more, greater than water (Duruibe et al., 2007).

Heavy metals pollution in water bodies has become a major water

quality issue in many fast growing cities over the last few

decades. This is because heavy metals pose threats to public

water supplies and can also cause health hazard to human

consumption of fish resources (Akoto et al., 2008). Metals enter

rivers and lakes from a variety of sources, such as rocks and

soils that are directly exposed to surface waters, fallout of

atmospheric particulate matter, and from man’s activities,

including the discharge of treated and untreated wastes into

water bodies. Excess amounts of these metals entering into the

aquatic ecosystem may pollute the environment and also affect the

food chain and ultimately pose serious human health risks to

those who depend directly or indirectly on the water body for the

supply of fish and water (Akoto, 2014). The

biota that inhabits contaminated sites is generally exposed to

very high concentrations of these pollutants because many of them

process sediment as a food source and thus can be susceptible to

bioaccumulation can potentially threaten the health of many

species at the top of the food chain, especially birds, fish and

humans (Edward J. B. et al., 2013). Heavy metals at high

concentrations can cause harmful effects on metabolic,

2

physiological, and biochemical systems of fishes (Kumar Parvathi

et al., 2011).

Among different types of fish Nile Tilapia (Oreochromis niloticus) and

African Catfish (Clarias gariepinus) are the commonest commercially

important fish species for diets in tropical and sub tropical

areas. Tilapias are the most important fishes cultivated both in

tropical and sub tropical countries. They are chosen for the fact

that they have good consumer acceptance, economically viable and

are in low fat content. Tilapias are a good source of essential

amino acids, fatty acids, vitamins and minerals (Judilyn M.

Solidum et.al., 2013). Catfish (Clariasgariepinus) is an important

protein food in tropics, like Tilapia, and it constitute 40% of

animal protein. (John A. Daramola et.al, 2013).

1.2. Statement of the problem

In our country Ethiopia, the population is being aggressively

increased in all dimension of the country; in the other hand, the

country is being grown economically. Due to the progress of

industrial plants and less tight rule and regulation, still, some

of the plants are constructed nearby water bodies to discharge

their byproducts & effluents to local rivers, lakes and oceans.

Most of these byproducts contain physical, biological and

chemicals hazards. But the chemical hazards such as heavy metals

are relatively difficult to control. Aquatic plants (macrophytes)

3

absorb these chemicals and accumulate in their body, then large

phytoplankton could be eaten by small animals such as zoo

plankton in turn eaten by other animals like fish, finally fishes

are being eaten by human beings. As a result, accumulation of

heavy metals via food chain can be toxic when reached significant

level (Susanta Nathet.al, 2013). Hence, bioaccumulation of heavy

metals in water, Zooplankton as well as in fish has direct

consequences to the health of human and ecosystems.

1.3. Significance of the study

The main rationale of this study will be to assess and determine

the heavy metals especially those which are toxic and hazardous

to human health and its bio accumulation in Zooplankton, two fish

species ( Nile Tilapia and African Catfish), which are commonly

edible by human beings and water samples obtained from two types

of lakes. The lakes from which these fishes obtained are

classified as chemically polluted by industrially avoided

byproducts and being absorbed by aquatic plants& animals through

food chain system and the heavy metals will accumulate to above

recommended level to cause some chronic disease to human being.

So, determining and recognizing the level and toxicity of these

metals in our water bodies is essential to take an action and

make decision. It also enabled one to compare and contrast the

amount of heavy and toxic metals in these two fish species ,water

samples and Zooplanktons obtained from lakes polluted very

4

seriously with lakes that are not exposed to pollution so that

societies will have awareness on environmental pollution on

edible aquatic animals. The aim of this work is to determine and

compare the concentration of heavy metals (Co. Pb, Cd, Hg, Cr, Zn

and Cu) in tilapia ,catfish,Zooplankton and water samples in two

rift valley lakes of Ethiopia (koka and Zeway ). The high level

of pollution caused by heavy metals and their threat they pose to

consumers and public health cannot be over emphasized. So, this

work will create awareness on the harmful effect of heavy metals

consumption and suggest ways by which pollution by heavy metals

can be reduced.

1.4. OBJECTIVES

1.4.1. General objective

To determine the status of the lake ecosystems and heavy

metal toxicity level of samples and provide safeguard

mechanism for the end users.

1.4.2. Specific objectives

To determine heavy metals (Pb, Cd, Hg, Cr, As and Se) in

tilapia and catfish, Zooplankton & water sample using

graphite furnace atomic absorption spectroscopy (GFAAS).

To determine the bioaccumulation of heavy metals in water

sample, zooplankton and fish species.

To determine the toxicity of each metal in comparing with

the standard label of toxicity of heavy metal

5

Chapter Two

2. Literature Review

2.1. Heavy metals as toxic elements

Heavy metals are metals which have relatively high density and

toxic even at low concentration. These metals are e.g.

Arsenic(As), lead(Pb), Mercury(Hg), Cadmium(Cd)

Chromium(Cr),Nickel(Ni) and Selenium(Se) (P. Govind et al, 2014).

Majority of the known metals are very toxic to living organisms

and even those considered as essential, can be toxic if present

in excess. Concentrations of several toxic metal and metalloids

have been largely increased as a result of human activities. They

can disturb important biochemical processes, constituting an

important threat for the health of plant and animals. Plants and

animals absorb these elements from soils, sediments, and water by

contact with their external surfaces, through ingestion and also

from inhalation of airborne particles and vaporized metals (Nidhi

Madaan et.al, 2010).

The other cause of heavy metal toxicity comes from rapid

population growth, urbanization, intensive agricultural and

industrial production and these entire gives rise to increased

levels of emissions of organic and inorganic pollutants into the

environment ( Tole et al.,2003). Out of these environments water

bodies such as rivers, lakes, wetlands and oceans are the one

that could be affected by these pollutants. Some of these

6

pollutants are directly discharged by industrial plants and

municipal sewage treatment plants and others from polluted runoff

in urban and agricultural areas and some are the result of

historical contamination (Begum et al., 2009).

The pollution of the aquatic environment with heavy metals has

become a worldwide problem and they are of particular concern due

to their potential toxic effect and ability to bioaccumulate in

aquatic ecosystems (Öztürk et.al, 2009). Among the different

metals analyzed, lead (Pb), cadmium (Cd), mercury (Hg), chromium

(Cr) and arsenic (As) are classified as chemical hazards and

maximum residual levels have been prescribed for humans.

Essential metals, such as copper (Cu) and zinc (Zn), have normal

physiological regulatory functions, but may also bioaccumulate

and reach toxic levels (Zheng Zhang et.al, 2007). These heavy

metals are commonly found in natural waters in small quantities

and some are essential to living organisms, yet they may become

highly toxic when present in high concentrations. The rate of

bioaccumulation of heavy metals in aquatic organisms depends on

the ability of the organisms to digest the metals and the

concentration of such metal in the river. Aquatic animals

including fish bioaccumulate trace metals in considerable amounts

and stay over a long period. Age of fish, lipid content in the

tissue and mode of feeding are significant factors that affect

the accumulation of heavy metals in fishes. They are finally

7

transferred to other animals including humans through the food

chain (S. Eneji etal., 2011).

The most common Potential Toxic Elements (PTE) listed by the

United State Environmental Protection Agency (USEPA) are mercury

(Hg), arsenic (As), cadmium (Cd), chromium (Cr), copper (Cu),

nickel (Ni), lead (Pb) and zinc (Zn). Some of these are essential

for the metabolic activities of living organisms. Potential toxic

elements such as Cr, Cu, Ni and Zn are required by organisms at

low level and become toxic at some higher levels. Non- essential

elements including Hg, As, Cd and Pb are toxic and not required

by organisms at any level. Heavy metals toxicity can result in

damaged or reduced mental and central nervous function, lower

energy levels, and damage to blood composition, lungs, kidneys,

liver, and other vital organ (Zenebe, 2011). Once these metals

enter the environment their potential toxicity is determined by

the form of existence. These metals could be ingested directly

through drinking or indirectly by consumption of plants

cultivated along the bank of the water body and through the

consumption of aquatic animals such as fishes (Adekola etal.,

2003). In aquatic environment, larger animals such as fish have

been exposed to heavy metals as a direct consequence of

biomagnifications. The danger is that heavy metals even at low

concentrations in fish and water have a particular significance

in ecotoxicology and their toxic effects have been widely

8

published for a number of water bodies. Among aquatic species,

fishes are the inhabitants that cannot escape from the

detrimental effects of heavy metal pollution. This is because of

their very intimate contact with water that carries the heavy

metals in solution or suspension and also fish have to extract

oxygen from water by passing water over their gills (Muiruri

etal., 2013).

The gills, skin and digestive tract are potential sites of fish

for absorption of water borne chemicals. Once the chemical is

absorbed it is transported by the blood to either a storage

point, such as bone or to the liver for transportation. If

transported by the liver it may be stored there and excreted in

bile, or passed back into the blood for possible excretion by

kidney or gills or stored in extra hepatic tissues such as fat.

It is known that heavy metals have toxic effects even at places

away from the source of pollution as they have the ability of

biological accumulation (Usmani and Javed, 2011).

2.2. Health Impact of heavy metals

Due to formation of toxic soluble compounds, certain heavy metals

become toxic. However, some metals are without any biological

role or they are not needed by the body and they become poisonous

only in specific forms (Pandey Govind and Madhuri., 2014). People

exposed to heavy metals may cause neurobehavioral disorders such

9

as fatigue, insomnia, decreased concentration, depression,

irritability, gastric symptoms, sensory symptoms, and motor

symptoms. Exposure to heavy metals has been linked with

developmental retardation, various cancers, kidney damage,

autoimmunity, and even death in some instances of exposure to

very high concentrations and intake of toxic heavy metals through

fish and aquatic foods has a considerable effect on human health

(Prabhat Kumar Rai, 2008). Some of the toxic heavy metals and

their health impact are described bellow as follows.

2.3. Heavy metals

2.3.1. Lead(Pb)

Lead is one of the most toxic metals and mostly it is obtained

from the sulphide ore called galena, by roasting process (Dudka

and Adriano.,1997). The two commonly known stable form of lead

are +2 and +4 and from these states the free +2 ion is more toxic

than inorganic complexes and there fore any factor which

increases complexation and decreases the concentration of the

free ion is found to affect lead toxicity (Sadiq,1992;Forsyth et

al., 1991).

Lead is a persistent and common environmental contaminant like

other commonly found, toxic metals such as mercury, arsenic, and

cadmium. It damages cellular material and alters cellular

genetics. The mechanism all of these toxic metals have in common

involves oxidative damage. Toxic metals increase production of

10

free radicals and decrease availability of antioxidant reserves

to respond to the resultant damage (Patrick, 2005).

Lead toxicity affects several organ systems, including the

nervous, hematopoietic, renal, endocrine, and skeletal, depending

on the age of the subject and the size of the dose, but the

effect of major concern today is the impairment of cognitive and

behavioral development in infants and young children in the

general population (Goyer, 1997). Regarding to exposure

opportunity , children are more sensitive than adults that their

developing brains and nervous system are very sensitive to lead

and childhood lead exposure associated with higher absenteeism in

high school, lower class rank, poor vocabulary grammatical

reasoning and longer reaction time.

(AgencyforToxicSubstancesandDiseaseregistery, 2012)

The general body of literature on lead toxicity indicates that,

depending on the dose, lead exposure in children and adults can

cause a wide spectrum of health problems, ranging from

convulsions, coma, renal failure, and death at the high end to

subtle effects on metabolism and

intelligence at the low end of exposures Children appear to be

particularly vulnerable to the neurotoxic effects of lead.

(McCally, 2002). According to Codex standard 193-1995, the level

of lead in fish should be 0.3mg/Kg (FAO and WHO, 2011).

11

The Joint FAO/ World Health Organization Expert Committee on Food

Additives (JECFA) established a provisional tolerable weekly

intake (PTWI) for lead as 0.025 mg/kg body weight (bw) (JECFA,

2004). The WHO provisional guideline of 0.01 mg/L has been

adopted as the standard for drinking water (WHO, 2004a). (Simone

Morais et al., 2012)

2.3.2. Cadmium (Cd

Cadmium appears in the earth’s crust mainly in association with

ores containing zinc, lead, and copper (in the form of complex

oxides, sulfides, and carbonates). Elemental cadmium is a soft,

silver-white metal, which is recovered as a by-product of zinc

mining and refining. The average terrestrial abundance of cadmium

is 0.1–0.2 mg/kg, although higher concentrations are found in

zinc, lead, and copper ore deposits. Naturally occurring cadmium

levels in ocean water range, on average, from < 5 to 110 ng/L.

(ATSDR, 2008; UNEP, 2008).

Because tobacco leaves naturally accumulate large amounts of

cadmium (Morrow, 2003) , cigarettes are a significant source of

cadmium exposure for the smoking general population. It has been

estimated that tobacco smokers are exposed to 1.7 μg cadmium per

cigarette, and about 10% is inhaled when smoked (Morrow, 2001;

NTP, 2005). The non-smoking general population is exposed to

cadmium primarily via ingestion of food and, to a lesser extent,

12

via inhalation ambient air, ingestion of drinking-water,

contaminated soil or dust. For the US population, the geometric

mean daily intake of cadmium in food is estimated to be 18.9

μg/day. In most countries, the average daily intake of cadmium in

food is in the range of 0.1–0.4 μg/kg body weight (ATSDR, 2008;

EFSA, 2009).

Certain compounds of cadmium (Cd) are highly toxic to humans and

it is a cumulative toxicant and carcinogenic heavy metal that

affects kidneys, generates various toxic effects in the body,

disturbs bone metabolism and deforms reproductive tract as well

as endocrine system. Cadmium and cadmium compounds are known

human carcinogens and it causes lung cancer, kidney disease and

fragile bones (Mudgal et.al, 2010). Cadmium accumulates in the

human body affecting negatively several organs: liver, kidney,

lung, bones, placenta, brain and the central nervous system

(Castro-González & Méndez-Armenta, 2008).

There are large differences in the concentrations of cadmium in

different kinds of food (milk, 1μg/kg; meat, fish and fruit 1–

50μg/kg; wheat, rice, potatoes and leafy vegetables 10–300μg/kg;

kidney, liver and oysters 100–1000μg/kg). These figures indicate

that people in population groups consuming excessive amounts of

specific food items (mussels, kidney, liver, leafy vegetables)

have a higher risk of cadmium exposure (WHO, 2007). The EPA

maximum contaminant level for cadmium in drinking water is 0.005

13

mg/L whereas the WHO adopted the provisional guideline of 0.003

mg/L (WHO,2004). (Simone Morais et al., 2012).

2.3.3. Mercury (Hg)

Mercury is an element of special concern because its inorganic

form is biologically transformed in aquatic environments into

methyl mercury (MeHg), a lipophilic organic compound that

bioaccumulates and biomagnifies as it move up the aquatic food

chain (Olmedo et.al, 2013). It is a known human toxicant and the

primary sources of mercury contamination in man through eating

fish. Biotransformation of mercury and methyl mercury formation

constitutes a dangerous problem for human health (Ghazi-Khansari

et.al, 2004). The concentration of mercury biomagnifies along the

food chain i.e. it increases with trophic position in the food

chain (Ermias Deribe et al., 2014).

Mercury and its compounds affect the central nervous system,

kidneys, and liver and can disturb autoimmune processes; cause

tremors, impaired vision and hearing, paralysis, insomnia, and

emotional instability. Mercury compounds cross the placental

barrier and can cause developmental deficits during fetal

development, and attention deficit and developmental delays

during childhood (Strategy, 2006).

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According to EPA 2ppb of Hg in drinking water is permissible and

only 1ppm of Hg will be permissible in sea foods ( Martin and

Griswold, 2009). Because of the extreme health effects associated

with mercury exposure, the current standards for drinking water

were set by EPA and WHO at the very low levels of 0.002 mg/L and

0.001 mg/L, respectively (WHO,2004a) (Simone Morais et al.,

2012).

2.3.4. Arsenic (As)

Arsenic is a metalloid. It is rarely found as a free element in

the natural environment, but more commonly as a component of

sulphur-containing ores in which it occurs as metal arsenide’s

(Simone Morais et al., 2012). It is found to be one of the most

toxic elements. Humans may be exposed to arsenic through food,

water and air. Exposure may also occur through skin contact with

soil or water that contains arsenic. Levels of arsenic in food

are fairly low, as it is not added due to its toxicity. But

levels of arsenic in fish and seafood may be high, because fish

absorb arsenic from the water they live in and fish that contain

significant amounts of inorganic arsenic

maybeadangertohumanhealth.(http://www.lenntech.com/periodic/eleme

nts/as.htm#ixzz39FP53z1f)/.

Data on arsenic occurrence in food shows that fish and seafood

account for over 90% of total exposure to arsenic in food

15

(Duruibe et al., 2007). Arsenic txicity causes arsenicosis

depending on the amount of intake of arsenic and the higher the

concentration of arsenic above the maximum permissible level

(0.05mg/l) or higher the amount of daily intake, the symptom will

appear within six months to two years (Guha, 2000). Following a

thorough review and in order to maximize health risk reduction,

the USEPA in 2001 decided to reduce the drinking water maximum

contaminant limit (MCL) to 0.010 mg/L, which is now the same as

the WHO guidelines (USEPA, 2005).

2.3.5. Chromium (Cr)

Chromium chemicals are used in a variety of applications. The

largest amount is consumed to manufacture pigments for use in

paints and inks. Other applications include leather tanning,

metal corrosion inhibition, drilling muds, textile dyes,

catalysts, wood and water treatment

( Bielicka et al., 2004). Severe and often deadly pathological

changes are associated with excessive intake of Cr (VI) compounds

(A. Bielicka et al., 2004).

Chromium is an essential trace nutrient that is required in small

amounts for carbohydrate metabolism, but becomes toxic at higher

concentrations. The most bioavailable and therefore most toxic

form of chromium is the hexavalent ion Cr+6. Therefore, water

quality standards for protection of aquatic life are stricter for

hexavalent chromium than for trivalent chromium (Cr+3) (Solomon,

16

2008). Chromium (VI) compounds have been found to be mutagenic

and carcinogenic (Kumar Parvath et al., 2011)

2.3.6. Nickel(Ni)

2.4. Bioaccumulation of Heavy metals in fish

Fishes have been recognized as a good accumulator of organic and

inorganic pollutants(Eneji et al., 2011). Aquatic organisms such

as fish and shell fish accumulate metals to concentrations many

times higher than present in water or sediment. They can take up

metals concentrated at different levels in their different body

organs. Thus heavy metals acquired through the food chain as a

result of pollution are potential chemical hazards and

threatening consumers (U.Sani, 2011). At low levels, some heavy

metals such as copper, cobalt, zinc, iron and manganese are

essential for enzymatic activity and many biological processes.

Other metals, such as cadmium, mercury and lead have not known

essential role in living organisms and are toxic even at low

concentrations. The essential metals also become toxic at high

concentrations. The consequence of heavy metals pollution can be

hazardous to man through his food (U.Sani, 2011).

Fish is the most susceptible of the aquatic animal to these

metals, it is cheap, easy to get and it is consumed in different

forms such as boiling, frying in deep oil, smoking, sun drying

17

amongst others. The physical and chemical environment in which

the fish resides appears to influence the rate of bioaccumulation

of trace elements in fish. Fish is generally appreciated as one

of the healthiest and cheapest source of protein and it has amino

acid compositions that are higher in cysteine than most other

source of protein. The effects of exposure to any hazardous

substance depend on the dose, the length of time, the mode of

exposure, personal habits, traits, and whether other chemicals

are present (Adekola et al., 2003). Therefore it is important to

monitor heavy metal in aquatic environment, especially in fish.

Increases loads of heavy metals in waste water may increases the

risk of ground water contamination. The major anthropogenic

sources of heavy metals include waste water, run-off from roads

and industrial wastes from mining, manufacturing and metal

finishing plants.

2.5. Distribution Of Heavy Metals In Aquatic Environment

The distribution and behavior of heavy metals in the marine

environment, as well as their impact upon marine organisms and

human health, are of great concern due to their persistent, non

biodegradable and toxic properties. In this context, the present

study was aimed to study the heavy metals accumulation in water,

Zooplankton and fish of two selected lake areas. (V. Chinnaraja et al,

2011).

Heavy metals are normal constituents of marine environment that

occur as a result of pollution principally due to the discharge

18

of untreated wastes into rivers by many industries (Murtala et

al., 2012). Phyto- and zooplanktons are micro-organisms at the

first and second lower trophic levels which are capable of

extracting, and bio-concentrating, heavy metals inside their

cellular tissues. Fish, as a consequence of being predators on

these and other microorganisms, accumulate higher levels of these

metals than phyto- and Zooplanktons. Eventually, man at the apex

of the food chain pyramid, being a seafood consumer, is

susceptible to the potential harm of toxic metals pollution

resulting from metal enrichment in edible marine species, at the

higher trophic levels (bio-magnification). Metals are found to

accumulate to variable levels in different organs of the same

individual species. The accumulation also depends upon age, size,

and developmental stage in the life cycles of individual species.

Sites of metal accumulation, storage and impact also vary widely

among different organs of a marine organism. The degree of

bioaccumulation of metals varies widely from one organism to

another, from one metal to another, and from one set of ambient

conditions to another. The ambient conditions include abiotic

physical factors such as temperature, solar energy, and depth of

the water column, in addition to the general hydrographic

characteristics of the region (Saad Al-Sulami et al., 2002)

19

Chapter three

3. MATERIALS AND METHODS

20

3.1. Study area Lake Koka is an artificial lake in central Ethiopia. It was

created in 1960 by the construction of the Koka dam across the

Awash River to provide hydro electric power (Dsikowitzky et al.,

2012). It is situated at the geographical co-ordinates of N

08021’ 55.1” E 0380 01’ 13.7” and has an altitude of 1,540meters

above sea level. The total area of the lake is 200 square

kilometers.

the GPS co-ordinates for the start of shore length is 37P0502385

UTM 0924954 and the GPS coordinates for the end of the shore

length is 37P0502758 UTM 0925702. (E. Mihretet.al, 2013)

Lake Zeway is the third largest of the Rift Valley Lakes which is

very rich in water birds compared to other birds which excels it

in size. It has a total area of 442 sq. km found in southern part

of the country 160 km from Addis Ababa. (E.Mihret et.al, 2013)

Geographycally it islocated (7052’-808’N and 38040’-38056’E) is

found within down faulted structural basins, which is surrounded

by lands that are under continuous cultivation throughout the

year. It is relatively fresh-water lake with two big tributaries

and one out flowing river (W.Taddese, 2004).

3.2. Study method3.2.1. Collection of fish samples:

The fish samples from two kinds of fish species Nile Tilapia

(Oreochromis niloticus) and African Catfish (Clarias gariepinus) were

21

purchased from two lakes (Zeway and Koka,) of fisher men. These

were purchased from lake Koka of Tannery and Amudie sites and

Zeway of Korekonch and Menafesha site. Their muscles were

filleted using stainless steel knife, the filleted muscle was

packed in to plastic bag , put in to cool ice box and was taken

to the laboratory for further analysis. The fillet fish collected

was put in sterile polyethene bags and kept in the laboratory

deep freezer (-20ºC) to prevent deterioration till further

analysis (Shandilet.al, 2011). The fish obtained from each

species was packed in ice and was brought to the laboratory.

Tissues of the examined fishes will be dissected, using clean

equipment and put in Petri dishes and transferred into an oven

set to 60 °C to dry for 72 hr. Drying continued until all the wet

tissues reached to a constant weight. Dried tissue samples were

grounded with pestle and mortar, sieved with 1mm sieve and

weighed for digestion. The weighed powder (triplicate each 1 g)

was put into digestion flasks followed by addition of 10 ml

nitric acid and heated at 180 °C for 1hr and 30minutes. Then it

is semi-cooled for till the yellowish color turned to white and

again 10ml conc. Nitric acid and 4ml30% H2O2 was added and heated

for another 1hr 1nd 30minute. Finally,the digestion is completed

and the semi-cooled sample was rinsed with 10% conc. Hcl and the

solution was transferred to the measuring flask of 50ml through

filter paper. After digestion, the digested samples will be

diluted with de-ionized water, filtered and completed, using de-

22

ionized water to 50 ml. The resulting solutions will be analyzed,

using graphite furnace atomic absorption spectrophotometer

(Ahmed El Nemret.al, 2003).

3.2.2. Collection of Zooplankton samples:

Zooplankton samples were collected from two lakes named as koka

of two sites (Tannery & Amudie) and Zeway of Menafesha & Gellila

area by pulling a 50micro meter mesh net in surface. The samples

from each lake was rinsed with distilled water, stored in plastic

bottles and taken to the food Science laboratory and stored in

deep freezer for further analysis. (Tinna Tulonenet et al, 2006).

For heavy metals determination, two aliquots of approximately 500

mg of each homogenized dried sample was digested in 5 ml of

suprapure 14 N nitric acid at 600C on a hot plate until the

solution was clear. After evaporation, the residue was dissolved

in 10 ml of 0.3N suprapure nitric acid. The metals were

determined by graphite furnace Atomic Absorption

Spectrophotometer (AAS) (P. Bocher et al., 2003).

3.2.3. Collection of water sample:

Filtered (0.45 What man filter paper) of water samples was

collected and put (in triplicate) in 500 ml. Plastic bottles that

was prewashed in metal-free HNO3, and preserved with 2 ml.Conc.

HNO3. Graphite furnace Atomic Absorption Spectrophotometer will

23

be used to determine the concentrations of Pb, Cd, Se, Hg, Cr and

As in the water. (Muhalulukhu Shitsama, 2003).

Materials and chemicals:

Chemicals; All chemicals are used according to analytical

grade: concentrated nitric acid will be used for digestion

purpose (conc.HNO3), Concentrated sulfuric acid (conc.H2SO4),

acid(HClO4), formaldehyde, hydrogen per oxide (H2O2 ) and de-

ionized distilled water (K.Chalapathi, 2012). Materials; Drying

oven, Graphite furnace atomic absorption spectrophotometer

(GFAAS) (Perkin-Elmer, 4100 and Polyethylene sampling bottles

24

4. ReferenceAgencyforToxicSubstancesandDiseaseregistery. (2012). Lead Toxicity.

Agency for Toxic Substances and Disease Registry.

Ahmed El Nemr, Amany El-Sikaily and Azza Khaled (2003). Heavy

metals concentration in some fish

tissues from south Mediteranean waters,Egypt. Egypt. J. Aquatic

Biology & Fish , pp:155 -172.

Akoto, O., Bruce, T. N., & Darko, G. (2008).

Heavy metals pollution profiles in streams serving

the Owabi reserv.

African Journal of Environmental Science and Technology, 2(11),354-359.

25

Alemayehu Hailemicael Mezgebe, Ethiopia and A.J. Solomon Raju

Andhra University, India (2011, Desember). Towards

sustainable Utilization of Lake Chamo Biodiversity

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1. INTRODUCTION................................................1

1.1. Back ground of the title.................................1

1.2. Statement of the problem.................................2

1.3. Significance of the study................................2

1.4. OBJECTIVES...............................................3

1.4.1.General objective.....................................3

2. Literature Review...........................................4

2.1. Heavy metals as toxic elements...........................4

2.2. Health Impact of heavy metals............................6

2.3. Heavy metals.............................................6

2.3.1.Lead(Pb)..............................................6

2.3.2.Cadmium (Cd...........................................7

2.3.3.Mercury (Hg)..........................................8

2.3.4.Arsenic (As)..........................................9

2.3.5.Chromium (Cr)........................................10

2.3.6.Nickel(Ni)...........................................10

32

2.4. Bioaccumulation of Heavy metals in fish.................10

2.5. Distribution Of Heavy Metals In Aquatic Environment.....11

Chapter three.................................................13

3. MATERIALS AND METHODS......................................13

3.1. Study area..............................................13

3.2. Study method............................................13

3.2.1.Collection of fish samples:..........................13

3.2.2.Collection of Zooplankton samples:...................14

3.2.3.Collection of water sample:..........................14

4. Reference..................................................16

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