1

NNONAH CHIEDOZIE CHISOM

PG/M.SC/06/40885

CONCENTRATIONS OF SOME HEAVY METALS IN

NIGERIAN PORK SAMPLES

Pure and Industrial Chemistry

A DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF

THE REQUIREMENT FOR THE AWARD OF MASTER OF

SCIENCE DEGREE IN ANALYTICAL CHEMISTRY

Webmaster

Digitally Signed by Webmaster‟s Name

DN : CN = Webmaster‟s name O= University of Nigeria, Nsukka

OU = Innovation Centre

2009

UNIVERSITY OF NIGERIA

2

CONCENTRATIONS OF SOME HEAVY METALS IN

NIGERIAN PORK SAMPLES

A DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE

REQUIREMENT FOR THE AWARD OF MASTER OF SCIENCE

DEGREE IN ANALYTICAL CHEMISTRY

BY

NNONAH CHIEDOZIE CHISOM

PG/M.SC/06/40885

DEPARTMENT OF PURE AND INDUSTRIAL CHEMISTRY

UNIVERSITY OF NIGERIA

NSUKKA

MARCH, 2009

3

TITLE PAGE

CONCENTRATIONS OF SOME HEAVY METALS IN

NIGERIAN PORK SAMPLES

A DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE

REQUIREMENT FOR THE AWARD OF MASTER OF SCIENCE

DEGREE IN ANALYTICAL CHEMISTRY

BY

NNONAH CHIEDOZIE CHISOM

PG/M.SC/06/40885

DEPARTMENT OF PURE AND INDUSTRIAL CHEMISTRY

UNIVERSITY OF NIGERIA

NSUKKA

MARCH, 2009

4

CERTIFICATION

This is to certify that the researcher NNONAH CHIEDOZIE CHISOM has

fulfilled the requirements to be awarded with Master of Science (M.Sc.)

Degree in Analytical Chemistry in the Department of Pure and Industrial

Chemistry in Faculty of Physical Sciences.

Signed

…………………………. ……………………

DR. C. O. B. OKOYE DATE

(PROJECT SUPERVISOR)

…………………………… ……………………

DR. P. O.UKOHA DATE

(PROJECT SUPERVISOR)

………………………… ……………………….

DR. P. O.UKOHA DATE

(HEAD OF DEPARTMENT)

………………………… ………………………..

EXTERNAL EXAMINER DATE

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DEDICATION

This project is dedicated to Almighty God

and the entire family of Nnonah

6

ACKNOWLDGEMENT

I must acknowledge the help I received in writing this project. First, I

must thank Dr. C. O. B. Okoye who guided my thought on this work, and who

is a man of great chemical insight.

My thanks must also go to Dr. P.O. Ukoha for his suggestions and

direction during the course of this work. I am also indebted to Mr. Obidegwu

of Crop Science Department (UNN)for his assistance during the course of my

work,

My unquantifiable gratitude goes to my entire family for their maximum

support.

Finally, my thanks goes to Arc. Engr. A. O. Nwagbara, Chikodili

Nwodo, Iboro Udofia, Louis Chukwuma and others who I have not mentioned

here but who played useful role in making this project a success.

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ABSTRACT

The concentrations of some heavy metals namely; Zinc (Zn), Iron (Fe), Copper

(Cu), Chromium (Cr), Nickel (Ni), Lead (Pb) and Cadmium (Cd) were

determined in some Nigerian pork (muscles) samples namely pork muscles

from free ranger pigs and confined pigs. The determination was carried with

an atomic absorption spectrophotometer. Results obtained showed that all the

metals analysed for were present except lead and cadmium which were below

the detectable limits. However, the results showed significant difference

between the concentrations of metals found in free ranger pork (muscles) and

confined pork (muscles).

The mean concentrations for free ranger pork showed 82.80µg/g Zn;

135.61µg/g Fe; 2.24µg/g Cu, 1.52µg/g Cr, 0.79µg/g Ni, <0.004µg/g Pb, and

<0.002µg/g Cd while confined pork (muscles) showed 28.27µg/g Zn; 88.06 Fe,

2.01µg/g Cu; 1.40µg/g Cr; 0.67µg/g Ni; < 0.004µg/g Pb and < 0.002µg/g cd.

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TABLE OF CONTENTS

Title page i

Certification ii

Dedication iii

Acknowledgement iv

Abstract v

Table of content vi

CHAPTER ONE: INTRODUCTION

1.1 Heavy metals 1

1.2 Heavy metal pollution 2

1.3 Essential trace metals 4

1.4 Toxic trace metals 4

1.5 Objectives 6

CHAPTER TWO: LITERATURE REVIEW

2.1 Origin and Domestication of Swine (pigs) 7

2.2 Breeds of pigs 9

2.2.1 Berkshire 9

2.2.2 Chester White 9

2.2.3 Duroc 10

9

2.2.4 Hampshire 10

2.2.5 Poland China 11

2.2.6 American Land Race 11

2.2.7 Tamworth 11

2.2.8 Yorkshire 12

2.2.9 Feeds for Pigs 12

2.3.0 Biological functions and health effects associated with a

deficiency or excess of heavy metals 12

2.3.1 Zinc (Zn) 13

2.3.2 Iron (Fe) 15

2.3.3 Copper (Cu) 16

2.3.4 Chromium (Cr) 17

2.3.5 Nickel (Ni) 18

2.3.6 Lead (Pb) 20

2.3.7 Cadmium (Cd) 21

2.4.0 Methods of Analysis 24

2.4.1 Atomic Absorption Spectroscopy (AAS) 25

2.4.2 Sample Digestion Methods 27

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CHAPTER THREE: EXPERIMENTAL

3.1 Apparatus 31

3.2 Reagents and Reference Solutions 31

3.3 Sample Collection and Preservation 31

3.4 Determination of Moisture Content 32

3.5 Sample Digestion 32

CHAPTER FOUR

4.1 Results and Discussion 34

4.2 Conclusion 41

References 42

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LIST OF TABLES

Table 2.1 Health effects associated with a deficiency or excess of

heavy metals 23

Table 4.1 Moisture and Dry matter content in muscle of free ranger pigs 34

Table 4.2 Moisture and Dry matter content in muscle confined pigs 35

Table 4.3 Concentration (µg/g) of Heavy metals in muscle of free Ranger

pigs 36

Table 4.4 Mean and Range (µg/g) of Heavy metals in muscle of free Ranger

pigs 37

Table 4.5 Concentration (µg/g) of Heavy metals in muscle of confined pigs

38

Table 4.6 Mean and Ranges (µg/g) of Heavy metals in muscle of confined

pigs 39

Table 4.7 WHO Guideline values of Heavy metals in pork 41

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CHAPTER ONE

1.0 INTRODUCTION

1.1 HEAVY METALS

Heavy metals are metals having density greater than 5g/cm3 (or

5kgm-3

). These are found mostly in groups III – V of the periodic table

(1). Many different definitions have been proposed – some based on

density, some on atomic number or atomic mass and some on chemical

properties or toxicity. (1) The term heavy metal has been described as

„meaningless and misleading‟ in an International Union of Pure and

Applied Chemistry (IUPAC) technical report due to the contradictory

definitions and its lack of a “coherent scientific basis” (1). There is an

alternative term, toxic metal, for which no consensus of exact definition

exists either. Depending on context, heavy metals can include elements

lighter than carbon and may exclude some of the heaviest metals. One

source defines “heavy metal” as ….common transition metals such as

copper, lead and zinc. These metals cause environmental pollution

(heavy-metal pollution) from a number of sources, including lead in

petrol, industrial effluents and leaching of metal ions from the soil into

lakes and rivers as well as acid rain (2).

13

In medical usage, heavy metals are loosely defined (2) and include

all toxic metals irrespective of their atomic weight. “Heavy metal

poisoning” can possibly include excessive amount of iron, manganese,

aluminum or beryllium (the fourth lightest element) or such semimetal

as arsenic. This definition excludes bismuth, the heaviest of stable

element because of its low toxicity.

1.2 HEAVY METAL POLLUTION

Living organisms require varying amounts of some heavy metals”.

Such as iron, cobalt, copper, manganese, molybdenum and zinc (3).

Other heavy metals such as mercury, plutonium, and lead are toxic

metals that have no known vital or beneficial effect on organisms, and

their accumulation over time in the bodies of animals can cause serious

illness. Certain elements that are normally toxic are, for certain

organisms or under certain conditions, beneficial (4). Examples include

vanadium, tungsten, and even cadmium (3,4).

Analysis for trace heavy metals in food has become increasingly

important in medical, ecological and pollution studies due to the toxicity

of these metals. The environmental contamination from heavy metals is

of concern because they exhibit behaviour consistent with persistent

14

toxic chemicals unlike many organic contaminants that lose their toxicity

as a result of biodegradation. Metals cannot be degraded further and

their toxic effects can be long lasting (5). Whilst the concentrations in

biota can increase through bioaccumulation, heavy metals are also

known to have toxic effect at low concentration (6).

Heavy metal pollution can arise from many sources but most

commonly arises from the purification of metals, e.g. the smelting of

ores and the preparation of nuclear fuels. Electro plating is the primary

source of chromium and cadmium. Though precipitation of their

compounds or by ion exchange into soils and muds, heavy metals

pollutants can localize and lay dormant. Unlike organic pollutants,

heavy metals do not decay and thus pose a different kind of challenge for

remediation (5.6).

Advancement in technology has led to high levels of

industrialization leading to the discharge of effluents containing heavy

metals into out environment(7). Various activities by man in recent

years have increased the quantity and distribution of heavy metals in the

atmosphere, land and water bodies. The extent of this widespread, but

generally diffused contamination has caused concern about its possible

hazard on plants, animals and human beings.

15

The overall role of trace elements in living organisms is directly

related to the interactions within all environmental, geological,

biological, or marine systems. For example, the trace element

composition of soil may significantly influence the elemental

composition of the vegetation, which in turn influences that of animal or

human tissues or fluids via the food chain (8).

1.3 ESSENTIAL TRACE METALS

Some elements such as; copper, iron, nickel, chromium, zinc,

cobalt, iodine etc are essential in very low concentrations for the survival

of all forms of life, and are rather known as essential trace elements (9).

However, in higher concentrations, these essential trace elements can

also be quite toxic.

In addition to the essential elements, there are several others

which are always found in body tissues and fluids but for which no proof

of essentiality has been established. These elements are often referred to

as non-essential, e.g. lithium, boron, etc.

1.4 TOXIC TRACE METALS

Some elements such as cadmium, mercury and lead are

prominently classified as toxic. This is because of their detrimental

16

effect even at low levels (8,9). It should be noted, however that all trace

elements are predominantly toxic when their levels exceed the limits of

safe exposure. These limits vary widely from one element to another.

There is a range of normal background concentration of these elements

in soils, sediments, water and living organisms. Pollution therefore is

the addition of a substance by human activity to the environment which

can cause injury to human health or damage to natural ecosystems (20).

Ingestion of heavy metals in food stuff contributes a substantial

proportion to total contamination in humans. Heavy metals are ingested

through the food chain directly or indirectly by humans and partially

accumulate in the human body. Exceeding toxic threshold values can

affect heath. For example cadmium disturbs kidney functions and has

cancerous effect (10). High levels of lead in children blood have been

known to have an inhibiting effect on certain enzymes (11).

World Health Organization and Food and Agricultural

Organization (WHO/FAO) have set some standards for heavy metals in

food stuffs. Therefore, constant analysis / investigation of food stuffs is

necessary to evaluate the magnitude and impact of trace element

contamination of the environment and to ensure compliance with the set

standards.

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1.5 OBJECTIVES

i. Determine the concentration of heavy metals in pork.

ii. Determine the safety of pork sold in Nsukka and Enugu for human

consumption by comparing the levels of heavy metals in the muscle

with the WHO/FAO heavy metals standard for meat.

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CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 ORIGIN AND DOMESTICATION OF SWINE (PIGS)

Nomadic people could not move swine about with them easily as

they could move cattle, sheep or horses. More over, close confinement

was invariable accompanied by the foul odors of the pig sty. For this

reason, the early keepers of swine were regarded with contempt. This

may have been the origin of the Hebrew and Moslem dislike of swine

later fortified by religious precept (12). As swine do not migrate great

distance under natural conditions and the early nomadic peoples could

not move them about easily, there developed in these animals, more than

in most livestock, a differentiation into local races that varied from place

to place. It also appears that swine were domesticated in several

different regions and that each region or country developed a

characteristic type of hog (12).

The present species of domesticated pigs are descendants of a

species group of wild pigs, of which the European representative is sus

scrofa and the Eastern Asiatic representative is sus vittatus, the banded

pig (12, 13). As in the case of cattle, pigs were not domesticated before

the permanent settlements of Neolithic Agriculture. There is definite

19

evidence for their domesticity by about 2500 B.C. in what is now

Hungary, and in Troy (12). Although pigs are represented on pottery

found in Jericho and in Egypt, dating from earlier periods, these were

wild varieties. The animal had become of considerable importance for

meat by Greco-Roman times, when harms were salted ad smoked and

sausages manufactured. About 150 years ago European pigs began to

change as they were crossed with imported Chinese animals drives from

the sus vittatus Species (13). These pigs had short, fine boned legs and a

drooping back. Then in 1830, Neopolitan pigs, which had better back,

and hams, were introduced (13). It was customary in the past to classify

British pigs by their color- white, brown and black.

The improvement of pigs has not been continuous in one

direction, but has been related to changing requirements at different

periods. Of the improved breeds of pig now in use in the world the

majority originated in British stock (14). The first breed to be brought to

a high standard was the Berkshire (14). It is said to produce more

desirably shaped and sized dors muscles than any other breed. Berkshire

pigs, crossed with the warren country breed of U.S.A, helped to establish

the Poland China in that country a century ago.

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2.2 BREEDS OF PIGS

2.2.1 BERKSHIRE

The Berkshire is one of the oldest of the improved breeds of pigs

(15). The striking style of the carriage of the Berkshire has made it

known as the aristocrat among the breeds of swine. The native home of

the Berkshire is in South Central England, principally in the counties of

Berkshire and Wiltshire (15). The distinct peculiarity of the Berkshire is

the short up-turned nose. The face is dished, and the ears are erect but

inclined slightly forward. The color is black with six white points four

white feet, some white in face, and a white switch on the tail. The

typical Berkshire is long bodied, with a long deep side, moderately wide

across the black, smooth throughout, well balanced and medium in

length of legs. The meat is exceptionally fine in quality, well streaked

with lean, and has no heavy covering of fat (12).

2.2.2 CHESTER WHITE

The Chester White breed is very popular in northern part of the

United States. As the name indicates, the breed is white in color,

although small bluish spots, called freckles, are sometimes found on the

skin. Chester white sows are very prolific and exceptional mother. The

21

pigs are good feeders and grazers, they mature early and finished

barrows are very popular on the market (16).

2.2.3 DUROC

The Duroc is the leading breed of swine than any other breed.

The Duroc is red in color, with shades varying from light to dark (16).

Although a medium cherry red is preferred by the majority of breeders,

there is no particular discrimination against lighter or darker shades so

long as they are not too extreme.

2.2.4 HAMPSHIRE

The Hampshire is one of the youngest breeds of swine, but its rise

in popularity has been rapid. It is widely distributed throughout the Corn

Belt and the South of United State. The most striking characteristic of

the Hampshire is the white belt around the shoulders and the body. The

black color with the white belt constitutes a distinctive trade mark, the

jowl is trim and light, the head refined, the ears erect, the shoulders

smooth and well set, and the back well arched.

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2.2.5 POLAND CHINA

No other breed of pig has been subjected to such radical shifts in

type as the Poland China. Likewise, no other breed has swung from

such heights of popularity or fallen so low in disrepute. Poland Chinis

are black in color with six white points – the feet, face, and tip of tail –

but prior to 1872, they were generally mixed black and white and spotted

(16).

2.2.6 AMERICAN LAND RACE

The land race breed is white in color, although black skin spots or

freckles are rather common. The breed is characterized by its long deep

side, square ham: relatively short legs, trim jowl; heavy lop ears;

sometimes low back and frequently weak pasterns (13).

2.2.7 TAMWORTH

The tamworth is one of the oldest and probably one of the purest

of all breeds of hogs (15). It is also recognized as the most extreme

bacon type than any breed. The color of the breed is red varying from

light to dark. The conformation may be described as that of extreme

bacon type. The individuals are rather long-legged, with long smooth

sides, and strong backs. The head is strikingly long and narrow with a

23

long snout and fairly large ears that are carried some what erect (15).

The Tamworth carcass produces bacon of the finest quality. The sows

are polific and careful mothers, and the pigs are excellent foragers.

2.2.8 YORKSHIRE

In its native home, England, the Yorkshire breed is known as the

large white. Yorkshire should be entirely white in color. Although black

pigment spots called freckles, do not constitute a defect, they are

frowned upon by breeders. Yorkshire sows are noted as good mothers.

They not only farrow and raise large litters, but they are great milkers

(15). The pigs are excellent foragers and compare favorably with those

of any other breed in economy of gain and the loins are large; but some

times the harms lack depth and plumpness.

2.3.0 FEEDS FOR PIGS

Through out the world, pigs are raised on a variety of feeds,

including numerous by-products. Except when on pasture or when

ground dry forage is incorporated in the ration, they eat relatively little

roughages (16). In Nigeria, corn, palm-kernel cake, and pig production

have always been closely associated. Normally more than one half of

the corn crop is fed to the pigs. The diet of the pig is readily adapted to

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the feeds produced locally. Thus, in most section of the world, pigs are

fed predominantly on home-grown feeds (16).

2.3.0 BIOLOGICAL FUNCTIONS AND HEALTH EFFECTS

ASSOCIATED WITH A DEFICIENCY OR EXCESS OF HEAVY

METALS

2.3.1 ZINC

Zinc is an essential trace element for plants, animals and humans

as it is associated with many enzymes and with certain other proteins

(17). Zinc is relatively more abundant in the earth‟s crust than some

other metals (e.g. Copper); however, there are not many minerals that

contain zinc. There is only one common sulfide (ZnS). Clay minerals in

soils can adsorb some zinc. Anthropogenic sources of zinc in the

environment include printing processes, construction materials, metals

(iron, steel and brass coated with zinc), fertilizer, batteries, sewage

sludge, animal wastes in the form of manure (dairy, feedlot, swine or

chicken), zinc-containing pesticides (e.g zineb, mancozed and Ziram),

atmospheric deposition and coal combustion (17). The latter can

contribute to zinc input through deposition of atmospheric emission and

when the residue of coal combustion (furnace ash and fly ash) are

25

disposed of in landfills (20). The distribution of zinc in our food stuffs

has much in common with copper. It is an essential component of the

active sites of many enzymes, and it is therefore not surprising to find it

at high levels in animal tissues such as lean meat and liver (21). Zinc

concentration in soils typically range from 1 to 2000 mg kg-1

, (20) but at

some sites levels as high as 100,000mgkg-1

have been reported (20).

The major health concern of zinc in the general population is

marginal or deficient zinc intake rather than its toxicity. Zinc is

generally considered as being of low toxicity due to the wide margin

between usual environmental concentrations and toxic levels. However,

high levels of zinc are undesirable as it may lead to copper deficiency by

inhibiting copper absorption (18). Deficiency of zinc causes growth

retardation; hypogonadism, mental lethargy; poor appetite; skin lesions

(8,18,19). An excess intake of zinc causes Nausea, anaemia, and

neutropenia (19). The daily dietary allowances for zinc as recommended

in different countries and by the International Commission on

Radiological Protection (ICRP) are as follows (in mgd-1

): USA (adult

and growing children) 10, U.K,14.3, Japan, 14.4, India 16.1, Italy, 4.7 –

11.3; ICRP, 13.0 (20).

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2.3.2 IRON

Iron constitute 5% of the earth‟s crust. It ranks fourth in

abundance after oxygen, silicon and aluminum. Of all the metals iron is

probably the one which the layman is most aware of as nutrient and also

the one which is potentially in short supply in the diet. One reason for

this is that most people are familiar with the need for iron in the blood

even though a rather smaller number of people will know what it is

doing there, in the myoglobin of muscle and in respiratory enzyme

systems generally (21). Iron is the most important transition metal in the

animal body, where it occurs almost entirely in elaborate co-ordination

compounds based on the porphyrin nucleus, notably the haem pigments,

which carry oxygen (22). A deficiency of iron is always manifested as

anemia i.e. abnormally low blood hemoglobin level.

Iron is generally abundant in most food stuffs, of plant as well as

animal origin. Lean meat contains between 2 and 4mg per 100g, (22)

mostly as myoglobin, so that the relative redness of different cuts is a

fair guide to the relative abundance of the metal. Liver has rather more;

around 9mg per 100g (20). Leafy green vegetables, legumes, nuts and

whole cereal grains all have between 2 and 4 mg per 100g (22). Most

fruits, potatoes, and white fish such as cod have between 0.3 and 1.2mg

27

per 100g (22). A deficiency of iron is manifested as anaemia, while

excess iron intake may lead to cirrhosis of liver and haemochromatosis

(8,9). The recommended daily dietary allowance for iron is 18mg for

adults and 10 – 15mg for children (23).

2.3.3 COPPER

Copper constitutes about 10-4%

of the earth‟s crust. It occurs in the

metallic state as uncombined metal as well as in compounds e.g. cuprous

oxide (Cu20), cupric oxide (CuO). The basic carbonates of copper are

malachite, (CuCO3, Cu(OH)2, and azurite (2CuCO3) Cu(OH)2. The

complex sulphide (CuFeS2) occurs as chalcopyrite (copper pyrites).

Copper is a component of many enzymes, e.g. cytochrome

oxidase, dopaminehydroxylase, superoxide dismutase and Lysol oxidase.

Iron metabolism is closely dependent on copper and in animals it is

difficult to distinguish between the anemia arising from deficiencies of

the two elements “Menkes” syndrome is a rare genetically determined, a

failure to keratinize hair, which becomes kinky, hypothermia, low

concentration of copper in plasma and liver, skeletal changes and

degenerative changes in aortic elastica (24). Excess ingested copper is

deposited in tissue, mainly in the liver and basal nuclei of the brain.

28

This leads to cirrhosis of the liver and brain disturbances e.g. coarse

tremor and personality changes (25). The recommended daily dietary

allowance for copper is 3mg for adults and 1-2mg for children (23).

2.3.4 CHROMIUM

Chromium is one of the less abundant metals of the earth‟s crust.

It‟s principal ore is the chromite (FeCr2O4). The major industrial sources

of chromium in the environment are from steel works, organic chemicals

and petrochemicals, paper and pulp production, petroleum refining,

power plants, textile mills, leather, electroplating, cement, fertilizers,

asbestors, paints, dyes, fungicides, etc. It is released into the

environment either directly during various manufacturing and treatment

processes, or when products containing chromium are disposed.

Naturally occurring chromium is ubiquitous in soils and

vegetation, although concentrations are generally very low. Chromium

is toxic to animals and humans, but less to plants. Inhalation and

ingestion are the main routes of human exposure to chromium.

Chromium deficiency leads to a reduction rate of removal of

ingested glucose due to a low sensitivity of peripheral tissues to insulin.

There are also impaired lipids metabolism, neuropathy and brain

29

disorders. In some patients with impaired glucose tolerance especially

children with protein energy malnutrition appears to be associated with

chromium deficiency (26,27).

Excessive amount of chromium leads to eczema and linked to

cancer. The recommended daily dietary allowance is 0.05-0.2mg for

adults and 0.02-0.12mg for children (23).

2.3.5 NICKEL

Nickel is a major constituent of the earth‟s crust. Nickel ores are

principally sulfide and silicates mixed with other metals. The principal

minerals are pentlandite and nickel ferrous pyrrohite (mixed sulfides of

iron and nickel) and garnierite (a mixed silicate of magnesium and

nickel).

Nickel emissions to the atmosphere are mostly anthropogenic,

industrial sources account for more than 80% of the total emission.

Nickel levels in soils are generally between <50 and 100mgkg-1

, but very

high levels may be found in some areas, particularly in soils over

serpentine deposits (20). Plants seem to be more sensitive to nickel

toxicity than animals, although both can be affected by nickel pollution.

Nickel carbonyl, Ni(CO4), is extremely toxic and any emission is

30

especially hazardous to mammals and human. Nickel pollution from

metals smelting has been reported in Canada, Russia, Australia, Cuba

and other countries (20).

Nickel is introduced into the territorial environment as solid waste

from metallurgical industries or as deposition of atmospheric emissions.

Although major source of Ni pollution is sewage sludge when applied to

land. In spite of Ni accumulation in soils, uptake by plants is not

sufficient to be of concern in the food chain. Application of phosphate

fertilizers to cultivated land is also a source of nickel and this could lead

to elevated concentrations since nickel, together with other heavy metals,

is found in phosphate minerals in variable amounts.

Nickel is present in trace amount in foods, human and animal

tissue (28). Ni is a component of the enzyme ureas present in a wide

range of species, functions as a redox metal in several types of enzymes

of aerobic hydrogen and anaerobic bacteria. Ni deprivation affects

growth, reproductive performance and plasma glucose concentration and

distribution and proper functioning of other nutrients including cobalt,

iron, zinc etc (29). Contact dermatitis is the most important clinical

effect of excessive nickel exposure (29). Nickel salts exert toxic action

by gastrointestinal irritation and not by inherent toxicity, an oral dose of

31

nickel as nickel sulfate as low as 0.6mg produces a positive skin

reaction in nickel sensitive individuals (30).

2.3.6 LEAD

Lead is undoubtedly the metal that springs to mind first when the

question of metal contamination of food is raised.

Average concentrations of Pb in soil are between 15 and 25mgkg-

1(20). Lead is naturally present in galena (PbS). The history of

elemental lead contamination began with the development of

metallurgical science over 5000 years ago (31).

The discovery of cupellation, a process of economic and industrial

changes that resulted in an exponential increase in the release of

anthropologenic lead into the environment. Environmental lead

contamination began with the first mining and smelting of lead ores (31).

The first person that developed the cupellation process was probably the

first individual to suffer from lead toxicity. Major anthropogenic source

of lead include the use of lead as a petrol additive, Pb mining and

smelting, printing, Pb paint flakes, sewage sludge and the use of

pesticide containing Pb compounds (e.g. Lead arsenate) (32).

32

Lead contamination of livestock and poultry is primarily derived

from atmospheric lead. Grazing animals take up lead from forage and

feed. Lead concentrations in forage are related to the atmospheric

deposition rate, as in food crops. Forage grown adjacent to heavily

utilized roads may contain more than 950µPb/g (31).

Lead is a well-known poison, but the effects of exposure to lower

levels have been contentious. There is growing evidence of sub-clinical”

Pb poisoning especially among young children who play in polluted

parks, gardens and street. Exposure to low lead levels can cause system

disorders, hyperactivity, hypertension, behavioural changes and learning

difficulties in children (33). Some have gone as far as to blame anti-

social behaviour and criminality on sub-clinical Pb poisoning, although

the evidence is tenuous.

2.3.7 CADMIUM

Cadmium is a very rare element, its average concentration of 0.1 –

0.2µg/g makes it the sixty-seventh element in the earth‟s crust in terms

of abundance (31). The baseline concentration of cadmium in most

environmental media are also notoriously low, some of the most

common-values being 2-20ng/1 dissolved in lakes, 5-15ng/1 dissolved in

33

rivers <5ng/m3 in the atmosphere, and 0.35 – 0.62ng/g in soils (31).

Therefore, the background reservoirs of cadmium in the different

segments of any ecosystem should be small; suggesting that the

biogeochemical cycling of cadmium can be altered significantly by small

contributions from anthropogenic sources (35).

The epidemic of cadmium poisoning (itai-itai disease) was a clear

manifestation of the wanton discharge of cadmium into the local

environment and its subsequent transfer to the local food chain (36).

There is now growing evidence to suggest that the levels of cadmium in

air, water and soils in many part of the world have increased several fold

as a result of emission from industrial activities and that the natural

biogeochemical cycle of cadmium has been overwhelmed. Cadmium

pollution is being transferred to human, resulting in elevated cadmium

levels in our diets. Long term exposure to the elevated levels of

cadmium in the environment has apparently increased the accumulation

of cadmium in certain body organs (notably the kidney and liver). The

transfer of cadmium into human foods is through air, water and soil.

Of all the toxic metals released in large quantities to the

environment, cadmium is generally regarded as the most likely to

accumulate in human food chain. The pollutant cadmium is selectively

34

concentrated by certain food crops, notably the root crops, leafy

vegetables, and tobacco plants.

Cadmium is extremely poisonous and toxic to humans. When

inhaled it causes acute bronchitis, pneumonitis and inflammation in the

liver (36). Cadmium toxicity causes a disease known as itai-itai (ouch-

ouch) resulting in death and physical deformities that sometimes extend

to children born by affected mothers. This occurred in Japan, to people

who ingested cadmium from eating rice grown in paddy fields flooded

by water from a contaminated river (37). High intake of cadmium also

leads to kidney failure (38).

Table 2.1

Health effects associated with a deficiency or excess of heavy metals (8).

Trace

Element

Health effect

Deficiency Excess Fe Anaemia Cirrhosis of liver,

haemochromatosis

Cu Anaemia and changes in

ossification

Wilson‟s disease

Zn Growth retardation, mental

lethargy, poor appetite

Neutropenia, anaemia

Cr Impaired glucose tolerance,

brain disorders

Eczema and linked to cancer

Ni Affects growth, reproductive

performance

Contact dermatitis

Cd - Hypertension, renal damage,

anosmia (no sense of smell)

Pb - Impaired mental activity,

reproductive and development

problems, headaches

35

2.4.0 METHODS OF ANALYSIS

The ideal analytical technique for measuring trace elements in

environmental samples must offer:

(a) Very low detection limits

(b) A wide linear dynamic range

(c) Simple interference-free data

(d) Qualitative, semi quantitative and quantitative analysis

(e) Possible simultaneous multi-element capability

(f) Simple sample preparation

(g) High through put and low cost per determination (8)

In practice, whilst manufactures or salesmen may claim that their

technique is superior and ideal for environmental analysis; in reality

there is no universal analytical technique for environmental analysis. All

the main contenders, including atomic absorption spectrometry (flame or

electro thermal) AAS, ETAAS); Atomic fluorescence spectrometry

(ATS), inductively coupled plasma (optical or atomic) emission

spectrometry (ICP-OES or ICP – AES), neutron activation analysis

(NAA), X-ray fluorescence (XRF), proton-induced X-ray emission

36

(PIXE); spark source or isotope dilution mass spectrometry (SSMS,

IDMS), electrochemical (anodic stripping voltammetry and

polarography) or inductively coupled plasma mass spectrometry (ICP-

MS) have their advantages and disadvantages (8).

The main limitations for most of these analytical methods are

sensitivity or precision problems due to interferences and sample matrix

effects. In some cases these problems have been reduced or eliminated

by the use of pre-analysis separation schemes, matrix matching of

reagent blanks, calibration standards and samples, the inclusion of

instrument background correction (AAS) or the coupling of hydride

generation (HG), electro thermal vaporization ETV, cold vapor (CV),

flow injection (FI), ultrasonic nebulisation (US) or laser ablation (LA)

devices to the trace element detector (8).

2.4.1 SAMPLE DIGESTION METHODS

The majority of trace element analytical techniques require the

sample to be in solution. There is no universal procedure for all types of

sample. The most desirable features of such procedures are:

1. The ability to dissolve the sample completely (no insoluble residue)

2. Reasonably quick and always safe

37

3. No possible sources of sample loss through volatility, adsorption into

the wall of the vessel,

4. Elimination of sample contamination from the reagent used in the

dissolution processes.

The majority of dissolution procedures involve dry ashing or wet

digestion using one or a combination of concentrated mineral acids (8).

Muffle ashing at 500-5500C will decompose most organic matter,

although problems can occur through volatilization of Hg, As, Sn, Se,

Pb, Ni, and Cr. Wet digestion is often the preferred method for oils,

sediments, biological tissues and blood (8). For trace and ultra trace

element analysis the reagent blank is very important. The mineral acids

used in wet digestion. Procedures can be sources of many elements,

especially Al, As, Mn, Cr, Ni and Zn. Only ultra pure or Artista grade

purity acids should be used. For ultra trace element analysis distillation

of acids in a quartz sub-boiling still is necessary. Soil, sediment, and

sludges can be readily digested using perchloric acid, or nitric-

hydroflouric acid mixtures (1:1 HNO3/HF), with 0.1-0.5g of sample

attacked by 10ml concentrated acid solution in a polypropylene squat

beaker, heated by water or sand bath placed in a special fume cupboard.

Care must be taken for elements that form volatile chlorides or fluorides

38

and unstable nitrate complexes. Aqua regia (3:1 HCl/HCO3) is often

used for solid digestion. Biological tissues and fluids (0.25-1.0g or 1-

5ml) can be digested in micro kjeldahl digestion vessels, with controlled

temperature heating mantles using nitric acid and H2O2 (8). However,

wet digestion using an open vessel always is subjected to possible

element volatility problems. Pressurized decomposition with nitric acid

in Teflon digestion bombs eliminates this problem and has the added

feature of increased digestion efficiency through smaller aid volumes

and pressure digestion.

Microwave digestion provides both a closed system method and

shorter digestion times optimal conditions depend on the sample

(weight, composition, volume of digestion reagents, reaction

temperature, pressure and time), and the digestion system (especially

power ratings).

Both bomb and microwave digestion systems require strict

attention to general safety rules in order to prevent explosive type

reactions. In particular:

1. Digestion of very fine powder samples must be restricted to less than

500mg (ideally 100-200mg) of sample, add only a maximum of 2ml

39

HNO3 and start with the minimum amount of power and the shortest

possible time (microwave) (8,39).

2. Avoid the digestion of biological material containing more than 20%

fat content,

3. Digestion of organic material generates a large amount of vapour, so

after digestion, be careful and never open a warm vessel (8).

2.4.2 ATOMIC ABSORPTION SPECTROSCOPY (AAS)

This has become one of the most improved techniques in the

qualitative determination of metals in foodstuffs. It has gained favour

with analysts because it is both specific and sensitive. The principle of

atomic absorption is by no means new. In 1860, Kirchhoff concluded

that black lines in the spectrum of the sun were due to absorption by

elements in its outer atmosphere. Little practical application was made

of Kinchhoff‟ work until mid 1950s when Alan Walsh, the Australian

Physicist developed the first practical atomic absorption spectrometer

(39).

In principle, the technique is relatively simple. When a beam of

light of specific wavelength is passed through a population of atoms of

metals of interest, a proportion of this light is absorbed by the atoms and

40

the absorption is proportional to the number of atoms present. The

source of light is usually a hollow cathode lamp (39, 40). The sample

solution of the element in question are nebulised into a flame which has

only sufficient energy to produce groundstake atoms and not to excite

them to higher energy levels where they will emit light. The light

emerging from the flame is selected, detected and amplified so that it can

be measured (41,42).

In practice, the foodstuff under examination is digested or asked to

remove organic matter. The remaining mineral matter is dissolved and

the resultant solution is aspirated into the flame of the instrument. The

metal‟s hollow cathode lamp is fitted and the requisite beam of light is

passed through the population of the atoms. For e.g. if lead hollow

cathode lamp is fixed only lead atoms will absorb the light emitted by it.

Part of the energy will be absorbed by the specific atoms, which absorb

at that wavelength e.g. 283.3 for lead. The absorption is compared to

that obtained when standard solutions of the metal in question are

aspirated so as to determine the concentration (42). This is done by

preparing a series of standard solutions and successively aspirating them

into the flame. The absorbances are then obtained. The absorbance

values are plotted against concentration. As with other spectroscopic

41

techniques, the Beer-Lambert‟s law is obeyed in that absorbance is

proportional to the concentration of atoms in the flame and hence to the

concentration of the element in the aspirated solution. If a linear

calibration curve results, the slope of the calibration curve can be

obtained and use made of equation A=mc, where A, is absorbance and

C, is concentration, to calculate the concentration of the unknown

solution (42). It is important to remember that at high absorbance levels,

the relationship between absorbance and concentration may depart from

linearity. Additions should be of approximately the same concentration

as that anticipated for the dilute sample solutions (39).

42

CHAPTER THREE

EXPERIMENTAL

3.1 APPARATUS

A GBC Avanta Version 2.02 atomic absorption spectrophotometer

equipped with deuterium background corrector. Single-element

intensitron hollow cathode lamps were employed throughout this study.

A premix design, 10cm, titanium, single-slot burner head was used. The

fuel was acetylene and the oxidant air. The instrument settings were

those recommended by the manufacturer.

A block digestor system was used for the digestion of the samples.

3.2 REAGENTS AND STANDARD SOLUTIONS

Pechloric acid 72% and Nitric acid 70% were used for the

digestion of pork muscles. Stock solution of each element (1,000 ppm)

were used to prepare dilute standard solutions of various concentrations.

All solutions were prepared with deionized water.

3.3 SAMPLE COLLECTION AND PRESERVATION

Lumps of pork muscles were purchased from the meat of 30

different pigs during various sampling trips to Nsukka Central Market.

15 of the 30 samples were free ranger pigs and the other 15 from

43

confined pigs. The pork samples were labeled pi-p30. The free ranger

was identified with F marked on the sample bottles. The samples were

carefully cut using a plastic knife. They were later oven dried,

comminuted and preserved in polythene containers and stored in a

desicator.

3.4 DETERMINATION OF MOISTURE CONTENT

Each lump of muscle sample was placed in preweighed clean and

dry porcelain dish. The dish together with the content were weighed

again and then oven dried at 1050C. They were weighed at intervals

until a constant weight was obtained. The loss in weight was noted as

the moisture content. The moisture content was calculated and

expressed in percentage (%).

Moisture content (%) = Weight loss x 100

Sample weight 1

Dry matter (%) = 100 - % moisture content

3.5 SAMPLE DIGESTION

The outer portions of the dried lumps of pork muscles were cut off

using a plastic knife in order to avoid probable contamination by heavy

44

metals from the butchers knife. The inner portion was ground in a

porcelain mortar and stored for trace metal analysis.

About 1g of each oven dried sample was placed in a 25ml kjeldahl

digestion flask. 5ml HNO3 (concentrated) was carefully added and left

for a while before addition of 2ml concentrated perchloric acid (HCIO4),

to avoid explosion (39,40,42). The mixture was swirled gently and

digested at moderate heat. The mixture was digested for about 15

minutes after the appearance of white fumes. The flask was left to cool

and the flask was made up of the 25ml mark with de-ionized water (42).

The blank was prepared in the same manner but the pork muscle was

omitted. All the digestion were performed in a fume cupboard.

45

CHAPTER FOUR

4.1 RESULTS AND DISCUSSION

TABLE 1: MOISTURE AND DRY MATTER CONTENT IN

MUSCLE OF FREE RANGER PIGS

SAMPLE MOISTURE

CONTENT (%)

DRY MATTER

CONTENT (%)

1 74.42 25.58

2 78.02 21.98

3 76.09 23.91

4 77.35 22.65

5 76.12 23.88

6 72.33 23.67

7 67.05 32.95

8 68.16 31.84

9 75.90 24.96

10 74.21 25.79

11 74.05 25.95

12 75.65 24.35

13 75.85 24.15

14 75.94 24.06

15 80.93 19.07

46

TABLE 2: MOISTURE AND DRY MATTER CONTENT IN

MUSCLE OF CONFINED PIGS

SAMPLE MOISTURE

CONTENT %

DRY MATTER

CONTENT (%)

1 75.68 24.32

2 75.30 24.70

3 78.95 21.05

4 76.26 23.74

5 74.65 25.35

6 76.22 23.78

7 72.81 27.19

8 75.75 24.25

9 71.04 28.96

10 80.92 19.08

11 70.08 29.92

12 59.51 40.49

13 75.88 24.12

14 79.10 20.09

15 77.39 22.61

47

TABLE 3: CONCENTRATIONS (µg/g) OF HEAVY METALS IN

MUSCLE OF FREE RANGER PIGS

Sample Zn Fe Cu Cr Ni Pb Cd

1 231.08 40.60 1.65 0.98 1.37 < 0.01 < 0.01

2 93.58 173.10 3.00 5.00 0.72 < 0.01 < 0.01

3 116.08 135.60 0.89 2.60 0.45 < 0.01 < 0.01

4 98.58 108.10 1.67 1.87 1.02 < 0.01 < 0.01

5 38.30 103.10 3.46 0.75 0.65 < 0.01 < 0.01

6 77.00 97.55 2.28 3.02 0.50 < 0.01 < 0.01

7 46.79 137.05 2.13 1.60 0.57 < 0.01 < 0.01

8 56.41 37.80 1.23 1.10 0.33 < 0.01 < 0.01

9 42.22 250.05 2.85 0.82 0.92 < 0.01 < 0.01

10 72.20 117.05 1.48 1.00 0.95 < 0.01 < 0.01

11 34.64 130.05 2.03 0.40 0.20 < 0.01 < 0.01

12 112.04 205.80 4.23 1.20 0.87 < 0.01 < 0.01

13 91.44 233.05 1.83 1.82 0.85 < 0.01 < 0.01

14 60.31 76.05 1.63 0.55 1.35 < 0.01 < 0.01

15 71.43 189.30 3.35 0.15 1.10 < 0.01 < 0.01

48

TABLE 4: MEAN AND RANGES (µg/g) OF HEAVY METALS IN

MUSCLE OF FREE RANGER PIGS

Zn Fe Cu Cr Ni Pb Cd

Mean 82.80+48.63 135.61+64.13 2.24+0.93 1.52+1.24 0.79+0.34 <0.01 <0.01

Range 34.64-231.08 37.80-250.05 0.89-4.23 0.15-5.00 0.20-1.37 <0.01 <0.01

49

TABLE 5: CONCENTRATIONS (µg/g) OF HEAVY METALS IN

MUSCLE OF CONFINED PIGS

Sample Zn Fe Cu Cr Ni Pb Cd

1 19.15 42.10 3.66 0.09 1.07 < 0.01 < 0.01

2 19.85 30.88 2.33 1.80 0.82 < 0.01 < 0.01

3 42.50 46.00 2.42 4.13 1.32 < 0.01 < 0.01

4 24.98 68.10 4.12 1.93 1.62 < 0.01 < 0.01

5 31.68 105.10 0.82 2.23 0.60 < 0.01 < 0.01

6 20.16 122.30 2.35 2.85 1.27 < 0.01 < 0.01

7 22.24 195.30 4.65 0.95 0.75 < 0.01 < 0.01

8 38.46 54.80 0.60 1.37 0.05 < 0.01 < 0.01

9 28.30 106.30 3.83 1.92 0.50 < 0.01 < 0.01

10 19.28 95.05 1.58 0.72 0.12 < 0.01 < 0.01

11 28.48 133.80 2.63 0.60 0.95 < 0.01 < 0.01

12 30.47 86.05 1.03 0.12 0.35 < 0.01 < 0.01

13 22.33 116.05 1.75 0.17 0.10 < 0.01 < 0.01

14 29.53 56.05 0.78 0.70 0.32 < 0.01 < 0.01

15 46.45 75.05 1.08 1.47 0.27 < 0.01 < 0.01

50

TABLE 6: MEAN AND RANGES (µg/g) OF HEAVY METALS IN

MUSCLE OF CONFINED PIGS

Zn Fe Cu Cr Ni Pb Cd

Mean 28.27+8.64 88.06+43.16 2.01+1.13 1.40+1.12 0.67+0.47 <0.01 <0.01

Range 19.15-46.45 30.88-195.30 0.60-4.65 0.09-4.30 0.05-1.62 <0.01 <0.01

51

Table 1 and 2 shows the moisture and Dry matter content in muscles of free

ranger pigs and confined pigs respectively. All the samples have moisture

content above 50% and the Dry matter content was below 40%. This result

shows that these pigs are not malnourished. Also that the rate of hydration in

these pigs is greater than the rate of dehydration.

Table 3 and 4 show the concentrations, mean and ranges (µg/g) of heavy

metals in free ranger pig muscles. Zinc had a mean value of 82.80ug/g and

range of 34.68 to 231.08µg/g. Iron had he highest mean concentration of

135.6µg/g with range of 37.80 – 250.05µg/g. The mean value of copper was

2.25µg/g and range was 0.89-4.23µg/g. Chromium was detected in all the

samples with a mean value of 1.52µg/g and range of 0.15 – 5.00µg/g. Nickel

had a mean concentration value of 0.79µg/g and range of 0.20-1.37µg/g Lead

and Cadmium were below their detection limits of 0.004µg/g and 0.002µg/g

respectively in all samples.

However, table 5 and 6 illustrate the concentration; mean and ranges

(µg/g) of heavy metals in confined pigs (muscles). From the tables, zinc had a

mean value 28.27µg/g and a range of 19.15-46.45µg/g. More so, Iron had the

highest mean concentration of 88.06µg/g and range was 30.88-195.30µg/g.

Copper, chromium and nickel had mean values of 2.24µg/g, 1.40µg/g and

52

0.67µg/g respectively. The ranges were 0.60 – 4.65µg/g for copper, 0.09-

4.13µg/g for chromium and nickel had 0.05-1.62µg/g.

These results from table 3,4,5, and 6 show that free ranger pigs

(muscles) tend to accumulate slightly higher concentrations of the various

heavy metals than the confined pig (muscles). This might be due to their

feeding habits and their free movement.

The results fell in line with reports of F. Fagioli and S. Landi (43). Finally,

the results are within the permissible limits as proposed by Food and

Agricultural Organization (FAO) and most countries standards.

Guideline values for Zn, Fe, Cu, Cr, Ni, Pb, and Cd in Pork recommended

by World Health Organization (WHO) in (µg/g) (44)

Zn Fe Cu Cr Ni Pb Cd

150 320 14 2.6 12 0.25 0.1

Source: E Merian (1991)

53

4.2 CONCLUSION

The levels of heavy metals found in the muscles of the pigs do not

pose problems for human consumption. More so, the data baseline

procured are useful for future studies on pollution and bio accumulation

in pigs.

54

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