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Digitally Signed by: Content manager’s Name
DN : CN = Weabmaster’s name
O= University of Nigeria, Nsukka
OU = Innovation Centre
ORJI ANN N.
Faculty of Physical Sciences
Department of Industrial Chemistry
DETERMINATION OF HEAVY METALS IN MUTTON AND EDIBLE
OFFAL OF SHEEP BRED IN NORTHERN NIGERIA.
Bello, Samuel Adeseye
PG/Ph.D/08/49678
2
DETERMINATION OF HEAVY METALS IN MUTTON AND EDIBLE OFFAL OF
SHEEP BRED IN NORTHERN NIGERIA.
BY
BELLO, SAMUEL ISIAKA ADESEYE
PG/Ph.D/08/49678
A THESIS PRESENTED TO THE DEPARTMENT OF PURE AND
INDUSTRIAL CHEMISTRY, FACULTY OF PHYSICAL SCIENCES,
UNIVERSITY OF NIGERIA, NSUKKA.
IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE
OF DOCTOR OF PHILOSOPHY OF THE UNIVERSITY OF NIGERIA,
NSUKKA.
April, 2014.
3
4
Certification
Bello, Samuel I. Adeseye, a postgraduate student in the Department of Pure and
Industrial Chemistry, and with registration no. PG/Ph.D/08/49678,has satisfactorily
completed the requirements for research work for the degree of Ph.D in
Environmental Analytical Chemistry. The work embodied in this thesis is original
and has not been submitted in part or full for any other diploma or degree of this or
any other university.
........................................................ ...............................................
PROF.. C.O.B OKOYE PROF. P.O. UKOHA
(Supervisor) (Head of Department)
Date:-....................................... Date:-......................................
5
Dedication
This thesis is dedicated to Pastor Samuel Omajali former State Overseer, Deeper Life
Bible Church, Taraba State, now for Bauchi State.
Acknowledgement
6
First and foremost I appreciate the Almighty God, the King of kings and the Lord of
lords, for His faithfulness, mercy and loving kindness, which I am enjoying as His
child, of a truth He has been good to me.
Sincerely, thanks is not enough and cannot adequately express my feelings of
gratitude and appreciation for my supervisor, Prof. C.O.B Okoye, who had made my
dream come true by providing an incentive and hope. Thanks for his tremendous help
and support all the time especially letting me realise and recognise my own potentials
and ambitions. His precious guidance, continuous encouragement, personal interest in
the work is highly commendable.
Many thanks to the Head of Department, Pure and Industrial Chemistry, Prof. P.O
Ukoha for his assistance. Dr (Mrs) Jane Ihedioha, was a ready hand in times of need
and Dr. L.N Obasi. To my late friend Dr. Ajali U. former Head of Department,
Pharmaceutical Chemistry, UNN, who equally encouraged me to enroll for the
programme, may his gentle soul rest in peace. I do not forget other members of staff
and colleagues in the Department of Pure and Industrial Chemistry, University of
Nigeria.
I would like to express my deepest gratitude to my parents; Alhaji(late) and Alhaja
T.Abioye Bello, who laid the foundation, my wife and companion, Sister Louiza
Ogechi Samuel Adeseye, my lovely and wonderful children; Samuel Jnr, Chioma
(Queen), Shalom and Ayodeji, and my in-laws for standing with me in prayers and
support.
I am highly indebted to my Pastor, Dr. Chukwudi G. Micheal of Taraba State
University who assisted in the final statistical analysis apart from his prayer and
concern. Dr and Mrs A.A.Hikko(former deputy provost C.O.E Jalingo), Mr Godwin
Adams Bambur of Federal University, Wukari. Dr. Ayuba Abarshi, Late Engr.
Danladi Suleiman, Dr. Habila Rimamsikwe, the Omojolas to mention a few. I cannot
even begin to express how much their love and continuous support meant to me. All
of you are my rock in the turbulent seas of life and you all played important roles in
my achievement and success in life.
7
Finally, I am also grateful to the Rector and members of staff, Taraba State
Polytechnic, Jalingo, TET-FUND and Pure Science Department. Members of my
Campus Church and the entire Deeper Life Campus Church, Taraba State and UNN
Chapter, thank you for your kind cooperation and prayer throughout my study.
My sincere thanks also go to Mr. Philip who analysed the samples and all the
veterinary doctors that assisted in the collection of the samples even at the peak of
“Boko Haram” crisis.
May the good Lord bless you all abundantly.
TABLE OF CONTENT
Title Page.. .. .. .. .. .. .. .. .. .. i
8
Approval page.. .. .. .. .. .. .. .. .. ii
Certification.. .. .. .. .. .. .. .. .. ..iii
Dedication.. .. .. .. .. .. .. .. .. ..iv
Acknowledgment.. .. .. .. .. .. .. .. .. v
Table of content.. .. .. .. .. .. .. .. ..vii
List of Tables. .. .. .. .. .. .. .. .. ..xi
List of Figures .. .. .. .. .. .. .. . ..xii
List of abbreviations .. .. .. .. .. .. .. .xiii
Abstract .. .. .. .. .. .. .. .. .. ..xiv
CHAPTER ONE
1.0 INTRODUCTION.. .. .. .. .. .. ..1
1.1 Environmental Contamination and Degradation .. .. .. 1
1.2 Human Exposure to Environmental Contamination.. ..4
1.3 Heavy metals pollution.. .. .. .. .. .. ..5
1.4 Environment.. .. .. .. .. .. .. ..6
1.4.1 Biosphere.. .. .. .. .. .. .. .. ..6
1.4.2 Lithosphere.. .. .. .. .. .. .. ..6
1.4.3 Hydrosphere.. .. .. .. .. .. .. ..6
1.4.4 Atmosphere.. .. .. .. .. .. .. ..6
1.5 Livestock Farming in Nigeria.. .. .. .. .. ..8
1.6 Nigeria Indigenous Sheep Breeds.. .. .. .. ..11
1.7 Classification of Heavy Metals.. .. .. .. .. ..12
1.7.1 Based on Importance.. .. .. .. .. .. ..13
1.8 Trace Elements in Meat .. .. .. .. .. ..14
1.9 Toxic Trace Metals .. .. .. .. .. .. ..16
1.10 Aims and Objective .. .. .. .. .. .. ..20
CHAPTER TWO
2.0 LITERATURE REVIEW.. .. .. .. .. ..21
9
2.1 Heavy Metals in The Soil .. .. .. .. .. ..21
2.2 Heavy Metals in Plant .. .. .. .. .. .. ..24
2.3 Heavy Metals in Food Chain.. .. .. .. .. ..25
2.4 Role of Trace Elements in Biological Cycles .. .. ..27
2.5 Toxicological Effects of Heavy Metals .. .. .. ..28
2.6 Environmentally Important Metals .. .. .. .. ..32
2.6.1 Chromium .. .. .. .. .. .. .. .. ..32
2.6.2 Iron .. .. .. .. .. .. .. .. .. ..35
2.6.3 Lead .. .. .. .. .. .. .. .. ..37
2.6.4 Zinc .. .. .. .. .. .. .. .. .. ..39
2.6.5 Copper .. .. .. .. .. .. .. .. ..40
2.6.6 Nickel.. .. .. .. .. .. .. .. ..41
2.6.7 Cadmium .. .. .. .. .. .. .. .. ..42
2.6.8 Manganese .. .. .. .. .. .. .. ..44
2.7 Bioaccumulation and Biomagnifications .. .. .. ..47
2.7.1 Bioaccumulation .. .. .. .. .. .. .. ..48
2.8 Analytical Techniques for Trace Metal Analysis .. .. ..50
2.8.1 Preparation of Biological Samples .. .. .. .. ..50
2.8.2 Drying ashing .. .. .. .. .. .. .. ..50
2.8.2.1 Ashing Technique.. .. .. .. .. .. ..50
2.8.2.2 Advantages of Ashing.. .. .. .. .. .. ..51
2.8.2.3 Disadvantages.. .. .. .. .. .. .. ..52
2.8.3 Wet digestion .. .. .. .. .. .. .. ..52
2.8.2.1 Wet Digestion With Single Acids .. .. .. .. ..54
2.8.2.2 Wet Digestion With Acid Mixture .. .. .. .. ..56
2.8.3 Microwave Digestion .. .. .. .. .. .. ..58
2.9 Detection of Trace Metals .. .. .. .. .. ..59
2.9.1 X-ray Fluorescence (XrF) .. .. .. .. .. ..60
2.9.2 Neutron Activation Analysis (NAA) .. .. .. .. ..61
2.9.3 Proton Induced X-ray Emission (PIXE) .. .. .. ..63
10
2.9.4 Atomic Spectroscopy .. .. .. .. .. .. ..64
2.9.4.1 Atomic (flame) Emission Spectrometry .. .. .. ..65
2.9.4.2 Inductively Coupled PlasmaMass Spectrometry (ICP-MS)..67
2.9.4.3 Atomic Absorption Spectrophotometry .. .. .. ..69
2.9.4.3.1 Technique .. .. .. .. .. .. .. ..69
2.9.4.3.2 Principle.. .. .. .. .. .. .. ..70
2.9.4.3.3 Instrumentation .. .. .. .. .. .. ..71
2.9.4.3.4 Atomizer .. .. .. .. .. .. .. ..71
2.9.4.3.5 Flame Atomizers .. .. .. .. .. .. ..71
2.9.4.4 Inductively Coupled Plasma Atomic Emission
Spectroscopy.. .. .. .. .. .. .. ..74
2.9.4.5 Spark and arc Atomic Emission Spectroscopy .. .. ..76
2.9.4.6 Graphite Furnace Atomic Absorption Spectrometry
(GFAAS) .... .. .. .. .. .. ..76
CHAPTER THREE
3.0 EXPERIMENTAL .. .. .. .. .. .. ..79
3.1 Map and Description of Study Area .. .. .. ..79
3.2 Sample Collection .. .. .. .. .. .. ..81
3.3 Cleaning of Glass wares .. .. .. .. .. ..82
3.4 Reagents and Glassware .. .. .. .. .. ..78
3.5 Preparation of Stock Solution for Heavy Metals .. ..82
3.6 Digestion of Samples – Metal recovery experiment .. ..83
3.7 Preparation of mixed standard solutions.. .. .. ..84
11
3.8 Sample Analysis.. .. .. .. .. .. .. ..84
3.9 Statistical Analysis.. .. .. .. .. .. ..86
CHAPTER FOUR
4.0 RESULT AND DISCUSSION .. .. .. .. ..87
4.1 Results.. .. .. .. .. .. .. .. ..87
4.1 .1 Moisture Content.. .. .. .. .. .. .. ..87
4.2 Discussion.. .. .. .. .. .. .. .. ..101
CHAPTER FIVE
5.0 CONCLUSION AND RECOMMENDATION.. .. ..111
5.1 Conclusion.. .. .. .. .. .. .. .. ..111
5.2 Contribution to knowledge.. .. .. .. .. ..112
5.3 Recommendations.. .. .. .. .. .. ..113
REFERENCE .. .. .. .. .. .. .. ..114
APPENDIXES .. .. .. .. .. .. ..134
List of Tables Page
1.1: Nigerian Livestock Population Estimate 9
1.2: Livestock import to Nigeria 10
12
1.3: Ruminant livestock population 11
3.1: Sampling sites and number of samples collected 81
3.2: Standard Analytical Conditions for Pb, Cd, Zn, Mn, Ni, Cu, Cr
and Fe using a GBC Avanta ver 2.02 AAS 85
4.1: The Moisture content of various organs of sheep from the sheep
breeding states in Northern Nigeria. 87
4.2: % Recovery of trace metals from meat samples 88
4.3: The Co-efficient of variation for the various metals (% RSD) 89
4.4 Mean Concentrations of Trace Metals in sheep from Kaduna State
(mgkg-1) 90
4.5 Mean Concentrations of Trace Metals in sheep from Katsina State
(mgkg-1) 91
4.5: Mean Concentrations of Trace Metal in sheep from Kano State
(mgkg-1) 92
4.6: Mean Concentrations of Trace Metal in sheep from Kebbi State
(mgkg-1) 93
4.7: Mean Concentrations of Trace Metal in sheep from Borno State
(mgkg-1) 94
4.8: Mean Concentrations of Trace Metal in sheep from Sokoto State
(mgkg-1) 95
4.9: Mean Concentrations of Trace Metal in sheep from Zamfara State
(mgkg-1) 96
4.10: Overall Mean Concentration of Metals in sheep(mgkg-1) 97
4.15: Comparison of Mean Elemental Concentration of Mutton
in present study with values in other Studies (mgkg-1) 98
List of Figures Page
13
1.1: Dose-response curves for essential elements and Non-essential elements. 16
1.2: Global production and consumption of selected toxic metals, 1850-1990 19
2.1: Food Chain and Movement of Heavy Metals 26
2.2: Atomic absorption spectrophotometer block diagram 71
3.1: Location and map of the study areas 79
LIST OF ABBREVIATIONS
14
Abbreviation/symbols Definition
% percentage
ºC degree Celsius
µg/g microgram per gram
µm micrometer
AAS atomic absorption spectrophotometer
ADD / ADHD Attention-Deficit Hyperactivity Disorder
Anova analysis of variance
ANZFA Australia New Zealand Food Authority
BAF bioaccumulation factors
BCF bioconcentration factors
BDL below detection limit
CRM certified reference material
DDW double distilled water
d.w dry weight
FEPA Federal Environmental Protection Agency
g Gram
GDP Gross domestic product
H2O2 hydrogen peroxide
HCl hydrochloric acid
HClO4 perchloric acid
HNO3 nitric acid
IUPAC International union of pure and applied chemist
IQ Intelligent quotient
mg/L milligram per liter
mL Millilitre
mm Millimetre
M Molar volume
M T Metallothioneins
MPI Metal Pollution Index
NH2OH.HCl Hydroxyl ammonium chloride
NH4CH3COO Ammonium acetate
No. Number
PLI pollution load index
ppm part per million
SPM suspended particulate matter
Abstract
15
The concentrations of essential and toxic metals in mutton and edible offal of sheep bred in
seven states in northern part of Nigeria, namely Kaduna, Katsina, Kano, Kebbi, Borno,
Sokoto and Zamfara states were determined. Forty one animals were each sampled for
intestine, kidney, liver and muscle in abattoirs in major sheep markets in these States, namely
Zaria, Katsina , Kano, Birnin Kebbi , Maiduguri, Sokoto and Gusau. The samples were put
in polythene bags treated with dilute HCl and stored in the refrigerator, and were later dried
in an oven at 105OC, pulverised using porcelain mortar and pestle and digested at room
temperature with 3:2 [HNO3 (65%, v/v), HClO4 (70%, v/v)] in corked plastic bottles. The
contents were gently swirled and allowed to stand overnight and then heated in a water bath
set at 70OC, with occasional swirling at 30 minutes intervals until a clear solution was
obtained. Analyses were carried out using GBC Avanta ver. 2.02 atomic absorption
spectrophotometer. One way analysis of variance (ANOVA) and Duncan’s multiple range
tests were conducted using SPSS version 17. The concentrations obtained were compared
among the States and with values reported in literature and set guideline values by World
Health Organization (WHO).Mean concentrations of the metals (mgkg,-1 fresh wt.) were: Pb
(0.799±0.224), Cd (0.182±0.172), Ni (0.484±0.230), Zn (5.527±0.876), Cu (0.607±0.115), Cr
(0.457±0.453), Mn (1.987 ± 1.464), and Fe (17.939±8.305) for intestine. For kidney, Pb
(0.916±0.226), Cd (0.259± 0.215), Ni (0.685±0.262),Zn(6.340±1.182), Cu (1.439±0.308), Cr
(1.005±0.975), Mn (0.793±0.371), Fe (19.857±5.030). Mean concentrations for liver were:
Pb (0.815±0.206),Cd(0.174±0.050),Ni (0.542±0.143),Zn(7.904±0.678),Cu (4.937±2.833), Cr
(0.529± 0.424), Mn (1.043±0.199), Fe (26.053±5.865), and for the muscle: Pb (0.724±0.168),
Cd (0.121±0.061), Ni (0.454±0.075), Zn (7.433±1.214), Cu(0.789±0.356),Cr
(0.484±0.406),Mn(0.473±0.122),and Fe (14.368±3.099).Pearson’s correlation showed Ni
significantly correlating with Cu (p<0.01) in the liver while Cr correlate positively with Ni in
muscle(p<0.05) and kidney(p<0.01). In the liver and kidney there were also strong
correlations(p<0.01) between Cr and Zn. All these are essential elements and their
correlations shows their areas of need. The toxic metals Pb and Cd did not show any
significant correlation (p<0.05). The concentrations of the metals in the various meat parts
(liver, kidney, intestine and muscle) were significantly different (p<0.05).The kidney samples
from Birnin Kebbi contained the highest concentrations of chromium, nickel and lead, while
the liver samples also from Birnin Kebbi had the highest copper concentrations. The highest
concentrations of iron, cadmium and zinc were found in intestine, kidney and liver samples
respectively, from Katsina. The highest concentrations of manganese in intestine were found
from Gusau samples. The intestine and muscle obtained from Maiduguri contained the lowest
16
concentrations of iron, nickel and lead, while the muscle and intestine from Kano had the
lowest concentrations of chromium and zinc respectively. The muscle samples from Sokoto,
Katsina and Zaria contained the lowest concentrations of copper, cadmium and manganese
respectively. The concentrations of lead, manganese, chromium and nickel were
higher(p<0.05) than the permissible limits set by Australia New Zealand Food Authority
(ANZFA) and WHO respectively; while the concentrations of zinc and copper were lower
than ANZFA. However, the determined concentrations compared favourably (p<0.05) with
values found in literature.
CHAPTER ONE
17
1.0 INTRODUCTION
1.1 Environmental Contamination and Degradation
The arrival of the new millennium became the occasion for taking stock in many
areas of human concern, including in particular, the global environment. The picture
of the state of our planet that emerged from a number of surveys is troubling. Four
global trends are of particular concern. These are:
(i) Population growth and economic development;
(ii) Decline of vital life- support ecosystems;
(iii) Global atmospheric changes and
(iv) Loss of biodiversity [1].
Concerns, about environmental integrity, degradation, alteration, and impairment
are steadily increasing. Environmental issues affect all life including mankind. The
patterns of consumption, toxic and hazardous waste, production and overpopulation in
both developed and developing countries are draining renewable and non-renewable
natural resources. As simply put “the contamination of the food chain by hazardous
elements and environmental chemical contaminants has become world wide public
health trepidation and also a leading cause of trade obstacles internationally” [2]. The
World Health Organization (WHO) has implemented the Global Environmental
Monitoring System/ Food Contamination Monitoring and Assessment programme
(GEMS/FOOD) [3], to inform and encourage governments of all countries, particular
those undergoing industrial and economic development, through a methodical
database of information to prevent food contamination, carrying out extensive diet
18
studies in order to lower human exposure to heavy metals and other industrial
contaminants. This is because these contaminants can promote or cause cancer, kidney
and liver dysfunction, hormonal imbalance, immune system suppression,
musculoskeletal disease, birth defects, premature birth, impeded nervous and sensory
system development, reproductive disorders, mental health problems, cardiovascular
disease, genital-urinary disease, old- age dementia and learning disabilities among
others [3].
Chemicals rarely disappear completely from the ecosystem, no wonder Nriagu
[4] said the effective management of contaminants in the environment is a complex
and challenging problem with worldwide ramifications. Numerous scientists
worldwide are supporting the view today that all life processes are being determined
by subtle electromagnetic and photon phenomena. All electrically active metals (ions)
and particularly, heavy metals can disturb the harmony of the electromagnetic
energies in the body, causing disharmony and disease, and can also increase the
production of free radicals million fold [3]. It has been stated that 90% of all chronic
and serious illnesses could be prevented if we were able to eliminate the 600 most
dangerous environmental toxins including heavy metals [5]. It was further stated that
every health practitioner is now fully aware of the devastating influence of heavy
metals and/or ionic metals can have on the mental, emotional and physical health and
well being. Until recently, most health care professionals and researchers assumed that
heavy metals had to be taken into account only when a patient showed definite
symptom, for instance, ‘poisoning’.
It has been stated that human health and well being is affected by much lower
19
levels of heavy metals than previously assumed. Health authorities constantly, correct
“permissible” maximum levels downwards. It is becoming more difficult to accurately
determine the appropriate drug profile in a given case, because the respective simile of
symptoms has undergone a shift due to the presence of heavy metal ions. In fact this
phenomenon may be observed for the majority of the classic Hahnemann remedy
profiles and it is fair to say that, at the present time the effectiveness of any anti-
oxidant therapy is significantly compromised by the presence of heavy metal ions. It is
therefore important to first identify the heavy metals in question and the degree of its
involvement.
However, hardly any appropriate treatment or diagnostic procedure is available
for cases of long-term heavy metal contamination. No satisfactory method exists for
the early recognition of heavy metal contamination. As a leading African nation and
an active member of the United Nation Organization (UNO), Nigeria is fully
conscious of her international obligations to ameliorate the impact of global climate
change through pollution abatement [6]. It is as a result of this that the Federal
Government of Nigeria established the ministry of Environment in 1999. The Ministry
has been charged with the responsibility of co-ordination, formulation and
implementation of the nation environmental policy [7]. The Nigerian environment has
been affected adversely by both natural disasters and human activities. There is
evidence that unregulated or unguarded exploitation and excessive consumption of
natural resources in Nigeria has inflicted severe damage to the environment [8].
1.2 Human Exposure to Environmental Contaminants
20
In Nigeria, just like in the rest of the world, rapid urbanization and
population growth have brought about a proportional increase in the amount of waste
that is generated. The inability to manage these wastes effectively in most developing
and some developed countries becomes an issue of great concern because apart from
the destruction of aesthetics of landscape by the waste dumpsites, some of the
municipal solid waste contain both organic and inorganic toxic pollutants (such as
heavy metals) that threaten the health of humans and the entire ecosystem [9].
Animal protein intake remains the surest way to furnish the body with a
complete assay of all the needed amino acids for proper tissue formation, growth and
repair. The common animal protein sources in Nigeria include fish, beef, goat, chicken
and mutton. The habitats of these animals are continually being contaminated with
heavy metals discharged from natural, domestic and industrial activities. These metals
find their ways into the food chain of these animals and consequently build up in these
animals and finally get to human beings who consume meat and other animal
products. It has been estimated that at the present time man’s load of these elements in
comparison to the last century has quadrupled . When animal products are consumed,
the heavy metals in them produce pathologies relative to quantity and the length of
time. This explains why the presence of heavy metals in animal products has
continued to receive a lot of attention from nutritionists and environmental scientists
[10].
1.3 Heavy Metals Pollution
21
Heavy metals contamination in ecosystems poses major environmental
problems worldwide with substantial economic consequences. Regulation of metals in
the environment presents many challenges. Meaningful characterization of effects of
metals on ecological receptors and humans requires understanding of biochemical,
physiological, and ecological processes that reflect an evolutionary history with ties as
far back as the origin of life.
Significant literature pertaining to biological/ ecological effects of metals in the
environment began in the mid-1800s and continues to build. The early focus (before
the 1960s) in terrestrial systems was primarily on nutrient requirements for plant,
livestock, and humans. The study of toxic effects gained prominence with the advent
of modern environmental law [11].
Population explosion, industrialization, urbanization and intensive agriculture
have caused tremendous damage to our environment. Man’s ignorance of laws of
nature and his over – exploitation of natural resources have further aggravated the
problem. Fortunately, during the last few years, the world has become more concern
and has began to make amends to prevent further degradation of the environment; a
number of national and international conferences have been held during the last
decade to debate the various issues involved. The most important and successful of
such meetings was the 1992 Earth Summit Rio de Janeiro, Brazil where more than
100 Heads of governments representing both developing and developed countries
participated. The result of the summit is that environment and development should be
treated as complementary to each other [11].
22
1.4 Environment means the surroundings in which we live. It is everything around
us that constitute the environment. When we consider the earth as an entity, we can
broadly categorize environment into four major parts, biosphere, atmosphere,
lithosphere and hydrosphere.
1.4.1 Biosphere:- this is the zone of living organisms which denotes mutual
interactions of organisms whether in the lithosphere, hydrosphere or atmosphere. It
represents the region relationships between living organisms about 10,000 meters
below and 6,000 meters above sea- level.[12].
1.4.2 Lithosphere:- this is the solid plane diaphragm. It is the mantle of rocks
constituting the earth crust.
1.4.3 Hydrosphere:- this is the region of the environment related to water. Eighty
percent of the earth’s surface is covered with water.
1.4.4 Atmosphere:- this is the region of gases, mainly air, which covers the earth to
a height of about 500 km from the earth’s surface. It is the protective thick gaseous
mantle, surrounding the earth, which sustains life on earth and saves it from
unfriendly radiation from outer space. It is subdivided into tour regions of varying
altitudes, viz; troposphere, stratosphere, mesosphere and thermosphere [11].
An environment is said to be polluted when matter or energy is accumulated in
it, in a quantity more than “normal”. Normal here refers to the natural quantity of that
matter or energy in that environment which does not constitute any problem or hazard
to the ecosystem [12].
23
Pollution is an undesirable change in the physical, chemical or biological
characteristics of air, water, and soil that may harmfully affect life or create a potential
health hazard for any living organism. It is thus, directly or indirectly a change in any
component of the biosphere that is harmful to any living component(s) and in
particular undesirable for man, affecting adversely the industrial progress, cultural and
natural assets or general environment [11].
Environmental pollution with heavy metals is a dangerous problem that is
recognized world wide. [13]. Litter is more than eyesore on city streets and along-side
highways. They pollute water ways and leaches toxic chemicals into soil and ground
water as it breaks down, the atmosphere is regarded as potential vehicle for
contamination of the hydrosphere and the earth’s surface. Heavy metals have recently
come to the forefront of dangerous substances and are considered as serious chemical
health hazards for man and animals [14]. The world wide production and use of
chemical compounds have increased tremendously since the Second World War.
Much of this growth can be attributed partly to the needs growing populations and
partly to the development of new compounds for the sake of “advancement”. The
environmental impact of these chemical interventions has only slowly become
apparent and is a cause for much concern. Thousands of chemical compounds are
released into the environment and many of these compounds resist decay and are
biologically non-degradable [11].
1.5 Livestock Farming in Nigeria
24
Livestock has historically constituted one of Africa’s major economic
resources in terms of livelihood of his population especially in West Central Africa
[15]. Investigation shows that livestock production alone has been about 12% of the
total agricultural gross domestic product (G.DP) [16], Nigeria was ranked as one of
the four leading livestock producers in the sub-Sahara region. Livestock production in
Nigeria was dominated by nomadic pastoralist long before the advent of the British
Colonial administration [17]. In Nigeria ruminant livestock are numerous and provide
substantial quantities of animal protein. These ruminants have a greater effect on
ecosystems than other animal species, especially, as their production is based on the
age-old husbandry systems. At present, cattle, goats and sheep production systems are
predominantly traditional or village systems, nomadic or pastoral systems, mixed
farming and the peri-urban and modern ruminant livestock husbandry. In general,
production and management systems vary from free range in less populated areas to
year round confinement and cut and carry feeding with grass to tie and browse in
densely populated areas.
In 1990, the livestock population in Nigeria comprised about 14 million cattle,
23 million goats and 13 million sheep [18]. However these figures have since
increased to 15.2 million cattle, 28 million goats and 23 million sheep. Other ruminant
livestock species of economic importance in Nigeria are asses, horses and camels.
Accurate statistics on livestock production and marketing are not easy to obtain and
therefore, detailed projections of the supply and demand of the livestock sub-sector
may be estimates [19]. It is noted that while beef and veal, goat and game meat
production have gradually increased, the production of sheep meat has doubled from
25
1995 to 2004. Large numbers of live cattle, sheep and goats are imported as well as
various milk products (to a value of US$ 250 M in 2003). [20].Table 1.1 shows the
livestock population estimates in Nigeria (2008) ; while Table 1.2 shows the data on
importation of livestock.
Table 1.1 Nigerian Livestock Population Estimate
Chicken 82,400,000 Other poultry* 31,900,000
Goats 34,500,000 Pigs 3,500,000
Sheep 22,100,000 Dogs 4,500,000
Cattle 13,900,000 Cats 3,300,000
Donkeys 900,000 Rabbits 1,700,000
Horses 200,000 Guinea pigs 500,000
Camels 90,000 Giant Rots 60,000
* Include: Pigeons, Ducks, Guinea fowl and Turkeys [20].
Table 1.2 Livestock import to Nigeria (1993- 1997)
26
Table (live) 1993 1994 1995 1996 1997
Cattle 205,058 68,597 28,684 853,350 853,350
Goat 377,131 31,209 87,861 966,385 966,385
Sheep 97,695 38,084 10,845 106,941 106,941
Pig 22,642 -------- 101 84,269 84,269
Camel 35,082 -------- 4,802 7,504 7,504
Horse 9,400 ---------- 2,740 4,021 4,021
Donkey 13, 111 -------- 41,738 48,753 48,753
Dogs 11,957 --------- 15,489 53,852 53,852
Source Fed. Min. of Agric and Water Resources [20]
Small ruminants, mainly goat and sheep, are found almost everywhere in
Nigeria. Goats and sheep are estimated to be a total of more than 51 million heads,
with goats out-numbering sheep. These animals are kept mostly for their meat and
skins (goatskin production was some 23,000 tonnes of fresh skins in 2004). Although
some seasonal movement of pastoral sheep does take place, the great majority of small
ruminants are kept in pens and feed and their patterns of distribution mirror those of
human settlement. The traditional system of feeding goats and sheep is based on the
use of kitchen wastes, agricultural by-products and browsing (scavenging).Table 1.3
shows data on the ruminant livestock population (1996 - 2005).
Table1.3: Ruminant livestock population (1996 - 2005) [19].
27
Livestock species
1996 1997 1998 1999 2000 2001 2002 2003 2004 2005
Cattle ('000)
15050 15073 15088 15103 15118 15133 15149 15164 15200 15200
Goats ('000)
25000 25500 25500 26000 26500 26500 27000 27000 28000 28000
Sheep ('000)
14000 19500 20000 20500 21000 21500 22000 22500 23000 23000
Asses ('000)
1000 1000 1000 1000 1000 1000 1000 1000 1050 1050
Horses ('000)
204 204 204 204 204 205 205 205 206 206
Camels 19105 18000 18000 18000 18000 18000 18000 18000 18000 18000
Sheep and goats have been neglected in animal production programmes
because they are looked upon as poor converters of food, slow growers and animals
that are destined to roam about in their country side subsisting on kitchen waste and
bush grazing. However, these small ruminants can subsist on low quality roughages
with mineral concentrate supplementation.
1.6 Nigerian Indigenous Sheep Breeds
Sheep play an important role in the socio-economic life of the people of Nigeria.
They also make a significant contribution to the national economy. There are four
main breeds of sheep native to Nigeria. These are Balami, Uda, Yankasa and West
African Dwarf. Balami and Uda are kept in the semi-arid regions of Northern Nigeria,
West African Dwarf are found in the South, while Yankasa is found throughout the
country [21]. These four breeds differ considerably in size, skin colour and other
characteristics. All indigenous breeds are hairy and can be broadly grouped into the
28
large, long-legged and the dwarf types. Sheep are the second most numerous pastoral
species, and small flocks accompany many cattle herds in a south - ward journey in
search of grazing grounds. A comparison of pastoral and village stock shows that
pastoral animals are generally more productive. The productivity of West African
Dwarf was found substantially lower than those of the other breeds[22].
All Nigerian sheep are used for wool, but are rarely milked. In the North they
are regularly eaten and form part of everyday protein supply, but there is also a
marked variation in demand coinciding with religions festivals. As a result there are
dramatic seasonal price fluctuations; and in some areas household fattening of sheep
for sale is a major economic activity.
1.7 Classification of Heavy Metals
Heavy metals are an ill-defined subset of elements that exhibit metallic properties
which would mainly include the transition metals, some metalloids, lanthanides, and
actinides. It is often used as group name of metals and semimetals (metalloids) that
have been associated with contamination and potential toxicity or eco - toxicity. The
term heavy metals have been described as meaningless 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” [23]. The term “heavy metal”
has never been defined by any authoritative body such as I.U.P.A.C.
Metals occur in widely different physiochemical forms (e.g oxidation states) in
different environmental components. They exist in the different environments as
aerosols and some such as Au, Fe and Hg, in elemental form as suspension or colloids,
29
some such as As, Hg, Pb and Sn can exist in gaseous form.
By definition, the term heavy metal refers to any metallic element that has a relatively
high density and is toxic or poisonous at low concentration. They are dangerous
because they tend to bioaccumalate [24].
Heavy metals are also called trace elements due to their presence in trace
(≤10mg kg-1) or in ultra trace (1mg Kg-1) quantities in the environmental matrices
[25]. A more meaningful classification of trace elements is difficult because the only
characteristics that they have in common is their occurrence in the tissues of plants,
animals and micro organisms in low concentrations [26].
1.7.1 Based on Importance
Heavy metals can be classified into four major groups on their health importance, as
follows:
(i) Essential: these include (Cu, Zn, Co, Cr, Mn, and Fe. These metals also called
micronutrient [27] and are toxic when taken in excess of requirements.
(ii) Non-essential: those that have beneficial metabolic effects but have not been
shown to be essential; Ba, AI, Li, Zr, Sb, Be, Pb, Hg, Ag and Sr;
(iii) Less Toxic: those that occur widely in living organisms but seem to be only
incidental contaminants, and are not known to be beneficial [27], for
example, Sn and AI and
(iv) Highly toxic: Hg, Cd and Pb.
An element is considered essential to an organism when reduction of exposure
30
to it below a certain limit would result consistently in a reduction of a physiologically
important function or when the element, is an integral part of an organic structure with
a vital function in that organism. Proof of essentiality of an element in one animal
species does not prove essentiality in another, but the probability that a function is
essential in any species including humans increases with the number of other species
in which essentiality has been proved. The definitions presented are therefore not
absolute; they depend on the judgment of what constitutes a “physiological important
function” or “consistent” functional impairment.
For humans, essential trace elements are those that need to be present in the
human diet to maintain normal physiological functions. Essentiality (a requirement for
normal organism metabolic function) of many metals is one of the primary factors that
differentiate risk assessment for metals and metal compounds from that of synthetic
organic chemicals [28]. Risk assessment of trace elements has examined two ends of
the toxicity-deficiency spectrum that associate with intakes that are too high and
results in toxicity and those that associate with intakes that are too low and result in
nutritional deficiency problems [29].
1.8 Trace Elements in Meat
Meat is known to be a source of trace elements but has, as well been discussed
in terms of accumulating heavy metals such as cadmium and lead. Several studies
have been carried out with respect to the contribution of meat consumption towards
fulfilling the requirements for several essential trace elements [30].
Trace elements such as cobalt, copper, iron, manganese, selenium,
31
molybdenum, and zinc are necessary for the normal development of plants and
animals. Extensive research on determination of essentiality occurred during the last
two centuries, with the focus on determining plant requirements for optimum growth
of crops. This involved determining the list of essential minerals, the form of uptake in
plant, threshold levels for sufficient and toxic levels. Review articles and books have
condensed the vast quantity of information into manageable units [31].
In many cases, these metals are added to animal feed and to pharmaceutical
products [32], just as the macronutrients potassium and phosphorous are added to
plant fertilizers. Other metals such as arsenic, cadmium, lead, and mercury, have no
known beneficial uses.
One of the biggest challenges faced by policy makers and risk assessors is how
to address concerns about the risks posed by toxic and inaccessible metal
concentrations without adversely affecting organism’ usage of metals that are known
to be essential or beneficial [32]. The essential and generally non-toxic “macro”
elements” calcium, magnesium, potassium, and sodium, as well as the “micro” or
“trace” element, iron [33] are required for proper organism growth and function.
Among the essential or beneficial elements that are metals, some are
recognized as macronutrients (i.e required in high concentration, such as Fe) and
others known as micro nutrients (i.e needed in very low concentrations such as Ni or
Mo). When present at high concentrations, even these metals (e.g cobalt, copper, iron,
magnesium, manganese, molybdenum, and selenium) are toxic [34].
The concept that many metals are required for organism health at one range of
32
concentrations and are toxic in quantities that may be either more or less than that
range has been referred to as the “window of essentiality” or the “optimal
concentration range” for essential elements [35].
Figure 1.1 Dose-response curves for essential elements and non-essential elements
[35].
1.9 Toxic Trace Metals
Today mankind is exposed to the highest level in recorded history of Pb,Hg, As, Al,
Cu, Ni, Sn, Sb, Br, Bi and Va. Levels are up to several thousand times higher than in
primitive men. Among the toxic heavy metals, lead (Pb) and cadmium (Cd) that are
most abundant toxic metals in the environment are emerging global concern due to
their potential deleterious hazard on public health [36].
33
The toxicity of a substance is its ability to cause harmful effects. These effects
can strike a single cell, a group of cells, an organ system, or the entire body. A toxic
effect may be visible damage, or a decrease in performance or function measurable
only by a test. All chemicals can cause harm. When only a very large amount of the
chemical can cause damage, the chemical is considered to be practically toxic. When a
tiny amount is harmful, the chemical is considered to be highly toxic.
The toxicity of a substance depends on three factors: its chemical structure, the
extent to which the substance is absorbed by the body and the body's ability to
detoxify the substance (change it into less toxic substances) and eliminate it from the
body.
A toxic metal is defined as that metal, which is neither essential nor has
beneficial effect. On the contrary, it displays severe toxicological symptoms at low
levels. With increasing industrialization, more and more metals are entering into the
environment. These metals stay permanently because they cannot be degraded from
the environment. They pass into the food chain and ultimately make their passage into
the tissue . This is due to their industrial use, the unrestricted burning of coal, natural
gas and petroleum, and incineration of waste materials worldwide. Toxic metals are
now everywhere and affect everyone on planet earth. They have become a major
cause of illness, aging and even genetic defects [37]. Toxic metals replace nutrient
minerals in enzyme binding sites. When this occurs the metals inhibit, over stimulate
or otherwise alter thousands of enzymes. An affected enzyme may operate at 5% of
normal activity. This may contribute to many health conditions. Toxic metals, such as
lead, cadmium and mercury may also replace other essential elements in some tissues
34
structures. These tissues, such as the arteries, joints, bones and muscles, are weakened
by the replacement process [38].
Toxic metals may also simply deposit in many sites, causing local irritation and
other toxic effects. They may also support development of fungal, bacterial and viral
infections that are difficult or impossible to eradicate until the cause is removed [38].
Replacement reactions, also called “fight for site”, occur when heavy metal grab the
biological spaces that should be filled by necessary minerals.
The toxic trace elements are generally regarded accidental contaminants,
although they are frequently found in minute amounts in the newborn [39], these
elements are translocated through the food chain to man and animals.
The distribution and localization of some heavy metals in the tissues of some
calf organs were detected, the most affected organs, which showed higher levels of
trace metals, were liver, kidney and small intestines [40].Toxic effects of metals have
been described in animals under relatively low levels of metal exposure. One of the
earliest effects is the disruption of trace element metabolism [41].
Toxic metals are natural components of the environment, but human
activities, notably industrial and mining processes, have been responsible for the
wider diffusion of these elements. They are accumulated in soils, and plants and
animal fed, with these plants will tend to accumulate toxic metals themselves [42].
Often, heavy metals are synonymous with toxic metals, but some light metals
such as beryllium also have toxicity Metals in an oxidation state abnormal to the body
35
may become toxic; chromium (iii) is an essential trace element, but chromium (vi) is
a carcinogen. Toxicity is a function of solubility. Insoluble compounds as well as the
metallic forms often exhibit negligible toxicity. The toxicity of any metal depends on
its ligands. In some cases, organometallic forms, such as dimetyl mercury;[(CH3)2Hg
]and tetraethyl lead;[(C2H5)4Pb] can be extremely toxic. In other cases, organometallic
derivatives are less toxic such as the cobaltocenium cation(Cc+).
Toxic metals can bioaccumlate in the body and in the food chain. Therefore, a
common characteristic of toxic metals is the chronic nature of their toxicity. This is
particularly notable with radioactive heavy metals such as thorium which imitates
calcium to the point of being incorporated to human bone, although similar health
implications are found in lead or mercury poisoning. The exceptions to this are barium
and aluminum, which can be removed efficiently by the kidneys.
Heavy metals production has soared since 1850, as can be seen in Figure 1.2
C :\Users\SAM\Documents\Heav y metals and health World Resources Institute_files\chart_w r9899_fg0207.gif
Figure 1.2 Global production and consumption of selected toxic metals, 1850- 1990. [43].
Therefore, the need to study the levels of some toxic and essential metals in
36
sheep cannot be over emphasized. Free - ranging animals are good indicators of
environmental heavy metals load. In Nigeria, sheep breeders move them from place to
place to graze.
1.10 Aims and Objective.
The aim of this study is to determine the effect of heavy metals on the various
tissues of sheep namely; intestine, kidney, liver and muscle, as it affects man.
To achieve the stated aim, the following objectives are put forward, which are to:-
(i) Determine the levels of Cu, Zn, Mn, Ni, Fe, Cr, Pb and Cd, in sheep meat
namely muscle, liver, kidney and intestine.
(ii) Use the data generated to serve as pollution indicator for the environment.
(iii) Contribute to the base-line data in environmental metal levels in Nigeria.
(iv) Compare the results among the sheep breeding states in Nigeria and see area
of greater concern with respect to toxic metals contamination.
(v) Correlate the levels of heavy metals in mutton in order to identify likely point
source(s) contamination.
CHAPTER TWO
37
2.0 LITERATURE REVIEW
2.1 Heavy Metals in the Soil
Soil pollution by metals differs from air or water pollution because heavy
metals persist in soil much longer than in other compartments of the biosphere. Heavy
meals concentrations in soils are associated with biological and geochemical cycles
and are influenced by anthropogenic activities such as metalliferous mining and
smelting, metallurgical industries, sewage sludge treatment, warfare and military
training, waste disposal sites, agricultural fertilizers and electronic industries [44].
Contamination and subsequent pollution of the environment by toxic heavy metals
have become an issue of global concern due to their sources, widespread distribution
and multiple effects on the ecosystem. Heavy metals are generally present in
agricultural soils at low levels. Due to their cumulative behavior and toxicity,
however, they have a potential hazardous effect not only on crop plants but also on
human health [45].
Small amount of heavy metals especially cadmium, copper, nickel and zinc
occur in soil solution because they are tightly held by the soil surface. The largest treat
to surface waters is from soil erosion rather than leaching of these metals to the
ground water. Soils with large amount of hydrous oxides and phyllosilicates are best
to adsorb these metals and reduce the chance of leachate problem [46].
Cadmium and mercury are adsorbed less intensely and pose a larger threat to
movement and availability. Cadmium is a slightly soluble metal that behaves like to
calcium in soil solution. Above a pH of 7, cadmium will precipitate, which will limit
38
solubility and mobility [46].
There are four main processes for the solution phase concentration of trace
elements (fate of trace elements in soil).
(i) Ion exchange on layer silicate:- Layer silicates in the soil provide permanent
charges and pH dependent charges which retain trace metal cations by non specific
electrostatic forces. These elements compete with Ca and Mg for the available
exchange sites. Trace elements are retained to higher concentrations when in lower pH
when metal hydrolysis is more prevalent and is the dominant reaction. Cd, Cu, Cr, Hg,
Pb, Ni and Zn are the main trace elements that are of greater concern on a waste
treatment facility [47].
(ii) Precipitation reactions:- when an element undergoes a precipitation reaction, a
certain sequence happens in the process as the concentration of the element in solution
increases. (a) Elements are adsorbed on the particle surfaces
(b) The reactants are supersaturated in the reaction
(c) Crystal growth.
The major classes of precipitates found in soils are silicates oxides, carbonates,
phosphates and sulphates. These precipitates form salts in the system which are not
beneficial to the soil system or the waste treatment system. Precipitate reactions are
completely reversible and still have some dissolution properties even in stable solid
phase.
(iii) Sorption to Hydrous Oxide surfaces:-This process involves the altering of the
39
surface charge using adsorption or chemisorptions. Trace elements in the form of
cations and anions will form short directional bonds with oxide surfaces.
(iv) Complex formation with soil organic matter :- Soil organic matter has many
functional group contained in it that can serve as exchange site. Most of the
compounds in question are functional group high in oxygen. Groups found in soil
organic matter and react with trace elements will most likely contain either – COOH
or –OH group. These functional groups help derive the complex reaction in the
organic matter. Trace metal compounds are tied up by the highly reactive oxygen
groups which hold the metals in place [48].
Availability of heavy metals in soil and heavy metal uptake by plants do not
only depend on the total metal content in the soil but also upon a variety of interacting
soil and plant factors e.g soil pH, soil organic matter, cation exchange capacity (CEC)
and plant species [49].
Soils are considered as sinks for trace elements, and therefore they play an
important role in the environmental cycling of elements. The principal components of
soil are inorganic materials: sand, silt and clay. Clay minerals may contain low levels
of trace elements as structural components but their surface properties play a vital role
in regulating the buffer and sink properties of soils.
Soil organic materials is around 2-5% of the total soil mass and plays an
important role in regulating water- holding capacity of the soil, it’s ion-exchange
capacity, and binding of metal ions. In fact, one of the most important properties of
soil and nutrient availability to plants.
40
2.2 Heavy Metals in Plant
Excessive uptake of both essential and non-essential metals may result in
adverse effects on soil biota; plants can transfer via the food chain, on mammals, birds
and human consumers [50]. Potential hazards associated with trace elements pertain to
their accumulation in soils which may lead to a plant toxicity condition or result in
increased uptake of metals into the food chain. Many of the trace metals are amplified
in the food chain.
The chemical composition of plants is generally related to the elemental
contact of nutrient solutions or soils. Absorption processes are very complex; the main
pathway of trace elements to plants is via the roots. Foliar uptake can occur but this is
only a major pathway in relation to aerial sources of pollutants. Root uptake is
dependent upon the dissolved forms in the soil solution (ionic, chelated, complexes)
pH, the presence of other ions, redox potential and temperature. There is a wide
variability in the bioacummulation of trace elements among different plants species.
Some elements such as B,Cd, Rb, Cs are readily taken up, where as Fe and Se are only
slightly available to plants. Trace elements absorption by plants roots is also
influenced by mycorrgizal fungi, which enhance uptake from soil solution in exchange
for carbohydrates from the host plants. Evolutionary changes have also resulted in
metal tolerant plant species which are able to accumulate very high concentrations of
specific metals (Ni,Zn, Cr, Co, Se, Cu, Hg).
Accumulation of elements in a plant can have major effects on key plants
metabolic processes such as respiration, photosynthesis and fixation or assimilation of
major nutrients.
41
2.3 Heavy Metals in Food Chain
Plant uptake of trace elements is generally the first step of their entry into the
agricultural food chain. Plant uptake is dependent on (a) movement of elements from
the soil to the plant root, (b) elements crossing the membrane of epidermal cells of the
root, (c) transport of elements from the epidermal cells to the xylem, in which a
solution of elements is transported from roots to shoots, and (d) possible mobilization,
from leaves to storage tissues used as food (seeds, tubers, and fruit), in the phloem
transport system. After plant uptake, metals are available to herbivores and humans
both directly and through the food chain. The limiting step for elemental entry to the
food chain is usually from the soil to the root [51].
Besides soil and water, food is also contaminated with trace metals by the
introduction of mechanized farming, ever increasing use of chemicals, sprays,
preservatives, food processing and canning. In order to get the minimum adverse
impact, it is important to measure and continuously monitor their levels in various
food items, total diet, water and inhaled air [52]. Heavy metals are ubiquitous in the
environment as a result of both natural and anthropogenic activities, and humans are
exposed to them through various pathways, especially food chain.
Food consumption had been identified as the major pathway of human
exposure to heavy metals, accounting for more than 90% compared to other ways of
exposure such as inhalation and dermal contact. Hence, the accumulation of heavy
metals in the environment is of increasing concern due to the food safety issues and
potential health risks [53]. Plants are important component of ecosystem as they
transfer elements from abiotic into biotic environments. The primary sources of
42
elements from the environment to plants are: air, water and the soil. Crops can take up
toxic elements through their roots from contaminated soils, and even leaves can
absorb toxic elements deposited on the surface [54], thereby transferred in to primary
consumers. In addition to the potential they accumulate in different animal source
foods such as meat and milk [55].
Figure 2.1 Food Chain and Movement of Heavy Metals
In the picture above it can be observed the way heavy metals follow, from the
first step of the pollution to the final step in the human body by means of food.[56].
A variety of exposure routes allow toxic heavy metals, predominantly lead and
cadmium to enter the food chain of animals , the commonest being the contaminated
animal feed and water [57], atmosphere deposition [58], land application of inorganic
43
fertilizers, biosolids, agro chemical, industrial effluents and animal manures [59].It
has been discovered that the trace element composition of animal and human tissues
or fluid is directly related to the trace element content and bioavailability in the soil or
sediment - plant – animal - human food chain. Many common components of food in
a given diet will influence both the amount of trace elements and their chemical form,
for example, carbohydrates, free fatty acids, fibre, protein, phytatae organic acids, etc
can affect trace element utilization in the body. Also complex inter element
interactions will affect both absorption and metabolism of specific trace elements e.g
excess dietary Ca, Pb and Zn will affect Zn, Fe and Cu respectively.
2.4 Role of Trace Elements in Biological Cycles
In recent years, there has been an increase in the realization of the importance
of trace elements in biological systems. The study of life processes shows that many
vital functions are dependent on the presence of a specific trace element because trace
elements are one of the important nutrient factors for the growth and maintenance of
human and animal life [60].
Heavy metals often have direct physiologically toxic effects and are stored or
incorporated in living tissues, sometimes permanently [61]. The properties of trace
elements which feature in their therapeutic activities are incomparision by macro
molecules such enzymes, nucleic acids etc with disturbance of biological function and
interaction with other elements [62]. Both animal and plant life depend for their
existence on appropriate amount of various trace elements, albeit in very small
amount. The significance of the biochemical and nutritional roles of trace elements is
44
widely recognized, since metals are found as constituent components of many
metallo- proteins and metallo- enzymes. Some trace elements such as, copper acts as
co-factors against hepatic fibrosis in chronic liver disease, particularly in the
biosynthesis of collagen. [62]. In recent years, there has been an increase in the
realization of the importance of the role of trace elements in biological systems. The
study of life processes shows that many vital functions are dependent on the presence
of a specific trace element. Because of that, trace elements are one of the important
nutrient factors for the growth and maintenance of human and animal life.
2.5 Toxicological Effects of Heavy Metals
A toxic metal is not defined as the metal which neither is essential nor has
beneficial effect; on the contrary, it displays severe toxicological symptoms at low
levels. With increased industrialization, more and more metals are entering the
environment. They pass into the food and from food they ultimately make their
passage into the tissue [63].
In a strict sense, most measured effects used to assess toxicity of metals are not
unique. Measurements of mortality/survival, growth, reproduction, and various
biochemical / physiological endpoints are common to all toxicity tests. Metals-
specific endpoints in animals are limited to a few biochemical or physiological
observations such as altered, delta aminolevulinic acid dehydratase(ALAD) which is
an enzyme that catalyzes the asymmetric addition of two molecules of ALA to form
prophobilinogen in heme synthesis. Lead interferes with the normal functioning of the
enzyme and triggers a cascade of physiological responses including elevated blood Pb
45
levels and lowered levels of ALA in plasma activity in response to lead, “staggers” in
response to selenium poisoning, or diagnostic lesions on foliage in response to nickel.
Plants exhibit characteristic patterns of discoloration and malformation of leaves in
response to metals [64]. Ion regulatory disturbances in fish and other aquatic
organisms are found in association with various metals. For example, disturbances of
sodium and chloride regulation have been reported to result from exposure to elevated
levels of copper or silver [65],and disruption of calcium regulation has been attributed
to exposure to cadmium or zinc [66]. These effects are frequently observed in
association with a variety of other, subtler biochemical and physiological effects [67]
provides a detailed review of effects on fish that result from waterborne exposure to
metals. Despite the limited number of metal –specific toxicity responses, there are
features of metal that require special consideration as toxic responses are interpreted.
Constructs that were developed to evaluate toxicity of synthetic organic compounds,
such as persistence and bioaccumulation, are not fully satisfactory when addressing
metals [67]. It is important to consider the evolutionary linkage organisms have with
metals in the environment.
The crustal abundance of elements and the physio-chemical properties of
metals are related to physiological responses such as essentiality, toxicity, and
tolerance [68]. With few exceptions, those elements that appear in greatest abundance
incorporate into enzymes, electron transport chains, and structural features of
primitive organisms. Homeostatic regulation of intracellular (or intraorganella)
concentrations of metals evolved to cope with existing conditions. It was observed
that, generally, the required nutritional levels of essential elements for plants tend to
46
be approximately one order of magnitude less than the average crustal concentration
and phytotoxic levels tend to be approximately one order of magnitude greater than
the average crustal concentration. This make good sense from an evolutionary
perspective-if nutrient requirements were greater than might typically occur, such
species would be restricted to isolated mineralized areas; if phytotoxicity occurred at
lower concentrations, species would be relegated to mineral-poor areas [69].
Toxicity occurs at the point where the capacity of an organism to regulate the
internal concentration of metals is lost, resulting in loss of function required for
normal growth or to sustain life. Generally, this occurs at one or more internal cellular
location or may affect an entire organ. For plants toxicity may also occur at the root
surface without the substance ever entering the internal portions of the plant.
Similarly, with microbes, toxic effects may occur at the external membrane surface.
Metals may also disrupt extracellular enzyme function.
The site of toxic action could be an enzymes membrane, or a co-factor critical
to some biochemical pathway. Often, multiple sites of action for a particular substance
might exist, and toxicity could be manifested in different ways, depending on how the
primary mode of action and the cascade of secondary effects are linked. For many
toxicity endpoints, such as reduced growth, fecundity, yield, or survival, multiple
disruptions of biochemical functions are likely to occur. For example, a phytotoxic
response of reduced growth might be a result of impaired photosynthetic function,
impaired respiration, and impaired water uptake by roots. Impaired electron transport
or neuro-transmission may lower the capacity of an animal to escape predation.
47
Toxicity thresholds refer to concentrations above which organism exhibit
adverse effects such as reduced growth or increased mortality data from a single
experiment or from several studies ( either laboratory or field observations) are used
to identity thresholds. Literature reviews of toxicity studies are often aimed at
identifying the lowest concentration of a substance at which adverse effects were
reported [69]. These values are useful in attempting to find an environmental
concentration protective of all species. However, many physicochemical
characteristics of soil alter the concentration-response relationships. Most commonly,
pH, organic matter content, soil texture, and relative amounts of other substances (eg.
calcium, iron, etc.) influence bioavailability and therefore the threshold concentration
of a particular field situation. Also, the chemical form of the substance can be very
significant. Because of this, some literature reviews have emphasized ranges of
toxicity threshold concentrations. Other disciplines, such as phytoremediation, have
focused on the most tolerant species. Though generally there are insufficient data to
describe the distribution of species across the range from most sensitive to most
tolerant, knowledge of this range will help in anticipating likely responses among
diverse groups of species to concentrations in particular field setting. As
environmental concentrations increase, it becomes more likely that many more species
will be harmed, and the magnitude or severity of the responses will increase.
Metallothioneins (MT) and phytochelatins are small proteins in animals and
plants respectively, which regulate and detoxify many metals with in the organism.
This mechanism of regulation is very effective when the organism is exposed to
background or even moderately elevated levels of many metals. The regulation of
48
metals within the organism has limits, however, and plants have demonstrated that the
interactive effects of cadmium and arsenate were concentration-dependent and ranged
from non-additive to synergistic, as concentrations increase. At high concentrations
the ability of the plant to regulate metals collapsed and phytochelatin levels dropped
[70]. An alternative protective mechanism, the formation of metal granules, has been
demonstrated in invertebrates. Toxic metals sometime imitate the action of an
essential element in the body, interfering with the metabolic process to cause illness.
[71].
2.6 Environmentally Important Metals
These are metals which are either essential or toxic
2.6.1 Chromium
Chromium (Cr) was first discovered in the Siberian red lead ore (crocoite) in
1798 by the French chemist Vauquelin. It is a transition element located in the group
VI-B of the periodic table with a ground-state electronic configuration of Ar 3d54s1.,
atomic number 24 (24Cr) present in the environment primarily in two oxidation state,
as trivalent chromium (iii), and hexavalent chromium (vi) chromium (vi) is readily
reduced to Cr (iii) although there are various other valence states which are unstable
and short lived in biological systems. Cr(VI) is considered the most toxic form of Cr,
which usually occurs associated with oxygen as chromate (CrO42-) or dichromate
(Cr2O72-) oxyanions. Cr(III) is less mobile, less toxic and is mainly found bound to
organic matter in soil and aquatic environments [72].
49
Chromium (iii) form complexes with organic and inorganic ligands stable in
aqueous solutions and relatively inert in terms of chemical reactions chromium is
highly toxic, non- essential element for microorganism and plants [73]. The source of
chromium in environment are both natural and anthropogenic, natural source include
burning of oil and coal, petroleum from ferro chromate refractory material, chromium
steels, pigments oxidants, catalyst and fertilizers this element is also used in metal
plating tanneries and oil well drilling[74]. Sewage and fertilizer are also the sources
of chromium [75] Chromium has its effect on certain enzymes such as catastases,
peroxidase, a cytochrome oxidase, which have iron as constituent.
Fifty years ago Walter Mertz) and Klans Schwarz at the US national institute of
health (NIH) discovered that rats fed on pelleted feed developed hyperglycemia
(elevated blood glucose) and hperinsulinemia, association with impaired glucose
tolerance [76]. This effect was reversed by feeding supplement rich in chromium
which led to isolation of “glucose tolerant factor” (G.TF) [77].
Reduction of Cr (vi) is favored by low pH and presence of organic matter, Fe
(II) and oxidation of Cr (iii) is favored by alkaline pH and the presence of Mn-oxide.
Water insoluble chromium (III) compounds and chromium metal are not considered a
health hazard, while the toxicity and carcinogenic properties of chromium (VI) have
been known for a long time [78].
The ratio between Cr (III) and Cr (vi) is the result of the rates of reduction and
oxidation which depends on several factors as pH, presence of Fe and organic matter.
Chromium as hexavalant Cr (vi) is toxic.
50
Chromium is found in all phases, of the environment including air , water and
soil. Naturally occurring in soil, Cr ranges from 10 to 50 mgd kg-1 depending on the
parental material. Cr (VI) is a strong oxidant with a high redox potential in the range
of 1.33-1.38 eV accounting for a rapid and high generation of ROS and its resultant
toxicity [79]. Chromium as an environmental contaminant and its compounds have
multifarious industrial uses. They are extensively employed in leather processing and
finishing [80], in the production of refractory steel, drilling muds, electroplating
cleaning agents, catalytic manufacture and in the production of chromic acid and
specialty chemicals. Hexavalent chromium compounds are used in industry for metal
plating, cooling tower water treatment, hide tanning and, until recently, wood
preservation. These anthropogenic activities have led to the widespread contamination
that Cr shows in the environment and Cr (vi) compounds are widely used in the
chemical industry as ingredients and catalysts in pigments, metal plating and chemical
synthesis. Cr(vi) can also be produced when welding on stainless steel or Cr (vi)-
painted surfaces. The entry routes of chromium into the human body are inhalation,
ingestion, and dermal absorption. Occupational exposure generally occurs through
inhalation and dermal contact, whereas the general population is exposed most often
by ingestion through chromium content in soil, food and water.
Once absorbed into the blood stream, Cr (vi) is rapidly taken up by
erythrocytes after absorption and reduced to Cr (iii) inside the red blood cells. In
contract, Cr(vi) is rapidly taken up by erythrocytes after absorption and reduced to
Cr(iii) inside the red blood cells. In contrast, Cr(iii) does not readily cross red blood
cell membranes, but binds directly to transferring, an iron-transporting protein in the
51
plasma [81].
Reduction of chromium (vi) in the red blood cells occurs by the action of
glutathione. Since the red blood cell membrane is permeable to Cr(vi) but not Cr(iii),
the Cr(iii) forced by reduction of Cr(vi) is essentially trapped within the red blood
cell. Eventually the diffusion of Cr(vi), the reduction to Cr(iii), and complexing to
nucleic acids and proteins within the cell will cause the concentration equilibrium to
change [81]. Chromium toxicity produces chlorosis and necrosis in plants [79].
The major health effects associated with exposure to Cr (vi) include lung
cancer, nasal septum ulcerations and perforations, skin ulceration, and irritant contact
dermatitis.
2.6.2 Iron
Iron is believed to be the tenth most abundant elements in the universe. Iron is
also the most abundant (34.6%) elements making up the earth; the concentration of
iron in the various layers of the earth ranges from high at inner concentration to about
5% in the other crust. Most of this iron is found in various oxides such as the minerals
hematite and taconite. The earth’s crust is believed to consist of a metallic iron-nickel
alloy. Iron has the dominant influence on carbon fixation rates, it play a key role in
controlling carbon and nitrogen cycles, including the biological CO2 pump, which
regulates atmospheric CO2 concentrations and CO2- linked global warming [82].
Over 65% of the iron content is found in hemoglobin, whose major function is
to transport oxygen and carbondioxde. In addition, iron is part of the composition of
52
the myoglobin molecules muscle tissues and as an enzyme reaction cofactor in kreb’s
cycle (responsible for the aerobic metabolism of tissues) and in the synthesis of
purines, carmitine, collagen and brain neurotransmitters [83]. Iron is essential to
almost living things from micro-organism to humans.
Iron can be found in meat, whole meat products, potatoes and vegetables. The
human body absorbs iron in animal products faster than iron in plant products. Iron is
also present in the composition of flavoprotein and heme proteins catalase and
peroxidase (found in erytrocytes and hepatocytes). These enzymes are responsible for
the reduction of the hydrogen peroxide produced in the body [84], it is present in the
brain from very early in life when it participates in the neural myelination processes
[85].
Iron deficiency is seen in the premenopausal woman. In contrast to pre-
menopausal woman, adult men should not use iron supplements because high tissue
level of iron correlate with increased risk of myocardial infection.
Iron may cause conjunctivitis, choroidites, and retinitis if it contact and remains
in the tissues. Chronic inhalation of excessive concentration of iron oxide fumes or
dust may result in development of pneumoconiosis, called siderosis, which is
observable as an X-ray change.
No physical impairment of function has been associated with siderosis.
Inhalation of excessive concentration of iron oxide may enhance the risk of lung
cancer development in workers exposed to pulmonary carcinogens. A more common
problem for human is iron deficiency, which leads to anemia. A man needs average
53
daily intake, of 7mg of iron and a woman 11mg, a normal diet will generally provided
all that is needed.[85].
2.6.3 Lead
Lead is a bluish or silvery grey soft metal with atomic number 82:atomic
weight 207.19; specific gravity 11.34, melting pt. 327.5oC and boiling point 1740oC. It
is the most common industrial metal that has become wide spread in air, water, soil
and food. Lead is slightly soluble in water and is transported mainly through the
atmosphere. Lead is called the horror mineral because it is associated with violence,
lowered 1Q ADD, ADHD and many neurological problems. Recent epidemiological
studies suggest that levels currently found in most industrialized countries,
environmental lead exposure may cause slight deficits in the cognitive development of
children [86]. It behaves like calcium in body and accumulates in bone, liver, kidney
and other tissues. [87].
The main toxic effect of lead is nervous system dysfunction of the foetus and
infants. In adults, it causes: adverse blood effects, reproductive dysfunction; damage
to the gastro intestinal track; nephropathies; damage to the central as well as the
peripheral nervous system and interferences in the enzymatic systems that synthesis
the HEME group [88].
In human exposure to lead can result in a wide range of biological effects
depending on the level and duration of exposure.
Some studies suggest that there may be a loss of up to 21Q points for a rise in
54
blood lead levels from 10 to 2ug.dl in young children.
Although most people receive the bulk of their lead intake from food,
Cosmetics - Believe it or not, lead is commonly found in quite significant levels in
lipstick. This has been verified by testing of a range of brands by the US Food and
Drug Administration (FDA) [89]. In specific populations other source emissions, soil,
and dust, paint flakes in old houses or contaminated land. Lead in the air contributes
to lead levels in food through deposition of dust and rain containing the metal on
crops and the soils.
The main sources of lead pollution in the environment are: industrial
production processes and their emissions, road traffic with leaded petrol, the smoke
and dust emissions of coal and gas- fired power stations, the laying of lead sheets by
roofers as well as the use of paints and anti-rust agents. Problems for foodstuffs were
caused for a long time, and are still cause today on occasion, by the soldered seams of
cans and the soldered closure of condensed milk cans, the metal caps of wine bottles
and, still, by lead pipes in drinking water systems.
In general non-smoking, adult population exposure pathway is from water and
food. Food, air, water, diet/soil are the major potential exposure pathway for infant
and young children. For infants up to 4 or 5 month of age air milk formulated and
water are the significant sources.
Lead is among the most recycled nonferrous metals and its secondary
production has therefore grown steadily in spite of declining lead and prices. Its
physical and chemical properties are applied in the manufacturing construction of
55
chemical industries. It is easily shaped and is malleable and ductile. There are eight
broad categories of uses: batteries rolled and extruded products, alloys, pigments and
compounds, cable sheating shot and ammunition [90].
Thanks to the phasing out of leaded gasoline and lead based paints, lead
poising continues to be a real threat, especially to children living in cities and or
buildings with old lead-based and plumbing.
2.6.4 Zinc
Zinc is one of the most important trace elements in the body for many biological
functions. It is required as a catalytic component for more than 200 enzymes and as a
structural constituents of many proteins, hormones, neuropeptides, homone receptors
and probably polynucleotide [91]
Zinc is essential for normal functioning of cells including protein synthesis,
carbohydrates metabolism cell growth and cell division [92].
The relevance of Zn status to many age-associated diseases and, according to
experimental studies, the aging itself is the major homeostatic mechanisms of the
body i.e the nervous, neuroendocrine and immune system, places Zinc in a pivotal
position in the economy of the aging organisms [93]. Zinc in its ionic form,Zn2+, is
necessary for proper body function, although an excess is toxic.
Many diverse biochemical roles of zinc have been identified. These include
roles in enzyme function, nucleuic acid metabolism, cell signaling. Zn is essential for
physiological processes including development, lipid metabolism, brain and immune
56
function [94]. It is also crucial for development and function of cells mediating non
specific immunity.
Because the diet in developing countries are predominantly base on plants and
often high in phytates, which inhibit zinc absorption strongly, it can be difficult for
children in these countries to obtain their recommended intake of Zn, from their usual
diet [95]. Zn is found in high concentration in most tissues being highest prostate,
liver, kidney, muscle, pancrease, spleen and adrenal [96].
Protein source, phytate concentration and chelating agents, all affect the availability of
Zn to the body [96]. Zinc also interacts with Ca, Cd, Fe, Cu and Mn.
2.6.5 Copper
Copper occurs in the earth crust in several different forms and it is widely used
in industries as a result of its desirable physical and electrical properties [97].
Cu-deficiency as well as cu-abundance may increase the cholesterol content of the
blood serum [98]. Copper is widely distributed throughout the body: and although the
concentration of the metal in tissues and organs varies among species, the liver, brain,
heart and kidney in that order consistently contain the highest concentration Cu.
Cu is an essential trace elements for man and animal, in addition to it’s role in
promoting haematopoiesis. It is also required for normal biological activity of many
enzymes, hemoglobin formation and hair keratin. Copper is an essential substance to
human life, it can cause liver and kidney damage, and stomach and intestinal irritation
[99]. Its requirement in farm animal diets is generally small, being of the order 4 to
57
10ug/g for cattle, sheep, swine and chickens [100].
The distribution of the total body copper among the tissues varies with the
species, age and Cu status. Cu normally occurs in drinking water from copper pipes,
as well as from additives designed to control algal growth.
People with wilson’s disease are at greater risk for health effects from exposure
to Cu. High food accumulation of copper for example, can be the cause of Parkinson’s
disease, anaemia, allergies, hair loss, appetite disturbance hyperactivity, low thyroid
activity, headache, skin conditions, constipation and brain damage, which may follow
hemolytic crisis learning disabilities and/or depression [101].
The deficiency of copper result anemia related to defective iron metabolism,
skeletal defects, affect the central nervous system and the immune and cardiovascular
systems notably in infants, defects in pigmentation and structure of hair or wool,
reproductive failure, and decreased arterial elasticity[102]. Excess intake of copper
can cause vomiting, nervous system disorder and Wilson diseases [103].
2.6.6 Nickel
Nickel is believed to play a role in physiological processes as a co-factor in the
absorption of iron from the intestine Nickel increased the absorption of iron from the
diet in iron deficiency but only when dietary iron was in the unavailable ferric form
[104]. Nickel is released into the air by power plants and trash incinerators. It will
then settle to ground or fall down after reactions with raindrops. It usually takes a long
time for nickel to be removed from air. Nickel can also end up in surface water when
58
it is a part of waste water streams. Although not recognized until the 1970s, nickel
play important roles in the biology of micro organisms and plants [105]. It can
accumulate in aquatic life, but its presence is not magnified along food chains [106].
Nickel is used in many specific and recognizable industrial and consumers products,
including stainless steel, alnico magnet, coinage, recharge batteries, microphone
capsules and special alloy, used for planting and as a green tint in glass, nickel cast
irons and with many other alloys, such as nickel brasses and bronzes, and alloys with
copper, chromium, aluminum, lead cobalt. [107]. It can be found in common metal
products such as jewelry [108]. Food stuffs naturally contain small amount of nickel.
Chocolate and fats are known to contain severely high quantities. Nickel can be found
in detergents.
2.6.7 Cadmium
Pure cadmium is a soft, silver-white metal. The physical property of cadmium is
atomic number 48, atomic weight 122.411, electron negativity 1.5, crystal ions radius
(principal valence state)0.97, ionization potential 8.993, oxidation state +2, electron
configuration kr 4d, 5S2 density 8.64g/cm3, melting point 320.9 oC and boiling pt 765
oC at 100 k pa [109]. It is usually found as a mineral combined with other elements
such as oxygen (cadmium oxide), chlorine (cadmium chloride), or sulphur (cadmium
sulphate, cadmium sulphide)[110] cadmium is a heavy metal that is wildly distributed
in the environment its concentration in the earth’s crust is generally estimated to be
1.5to 0.20 mg/g.
Cadmium is concentrated particularly in the kidney, the liver, the blood
59
forming organs and the lungs. It most frequently results in kidney damage (necrotic
protein precipitation) and metabolic anomalies caused by enzyme inhibitions. It is
now known that the ltai- itai sickness in Japan (with bone damage) is a result of the
regular consumption of highly contaminated rice. Cadmium, like lead, the danger lies
primarily in the regular consumption of foodstuffs with low contamination. However,
in contrast to lead, the definition of an exact toxicity limit is not possible for cadmium.
Cadmium is called the pseudo-macho or the violent element like lead, it is an older
male.
The toxic effects of cadmium are noticeable in various ways. It can interfere
with some of the organisms enzymatic reactions, substituting zinc and other metals,
manifesting its action in several pathological processes such as renal dysfunction,
hypertension, arteriosclerosisi, inhibiting of growth, damages in the nervous system,
bone demineralization and endocrine disruption and stimulate cardio vascular diseases
[111]. High exposure can lead to obstructure lung disease and has been linked to lung
cancer.
Food is one of the principal environmental sources of cadmium as the
cadmium moves through the food chain it becomes more and more concentrated until
it reaches to carnivores where it has increased in concentration by a factor of
approximately 50 to 60 times [112].
Cadmium accumulate in the body, especially in kidney and the liver, over
many years because the body has no homeostatic mechanism to keep cadmium at a
constant safe level such as those that function for zinc. Hence Cd is a cumulative
60
poison, it replace Zn in many enzymes. Therefore, a higher amount of Zn is required
to overcome toxicity effects of Cd [111]. Cd derives it toxicological properties from
it’s chemical similarity to Zn, an essential micro nutrient for plants, animals and
humans. Cd is bioperisistent and once absorbs by an organism, remains resident for
many years (over decades for human) although it is eventually excreted. Cadmium,
found in paints, cigarettes, tires and breaks is toxic in its soluble ionized or salt form
will attempt to participate in the same biochemical reaction as zinc, their presence will
prevent the zinc reacting and performing it’s functions in the body.
Cadmium affects the growth of plants, stomata opening, respiratory and
photosynthesis are affected. Metals are taken up into plants more readily from nutrient
solutions than from soils.
2.6.8 Manganese
Manganese is one of the most abundant metals in soils, where it occurs as
oxides and hydroxides, and it cycles through its various oxidation states. Managanese
occurs principally as pyrolusite (MnO2), braunite, (Mn2+Mn3+6) (SiO12),
[113]psilomelane (Ba,H2O)2Mn5O10, and to a lesser extent as rhodochrosite (MnCO3).
Manganese is an essential element for all species. Some organisms, such as diatoms,
molluscs and sponges, accumulate manganese. Fish can have up to 5 ppm and
mammals up to 3 ppm in their tissue, although normally they have around 1ppm.
Manganese makes up about 1000 ppm (0.1%) of the Earth’s crust, making it
the 12th most abundant element there. Soil contains 7-9000 ppm of manganese with
an average of 440ppm. Seawater has only 10ppm manganese and the atmosphere
61
contains 0.01ug/m3 [114].
Manganese is a very common element that can be found everywhere on earth.
Manganese is one out of three toxic essential trace elements, which means that it is
not only necessary for humans to survive, but it is also toxic where too high
concentrations are present in a human body. When people do not live up to the
recommended daily allowances their health will decrease. But when the uptake is too
high health problems will also occur.
The uptake of manganese by human mainly takes place through food, such as
spinach, tea and herbs. The foodstuffs that contain the highest concentrations are
grains and rice, soya beans, eggs, nuts, olive oil, green beans and oysters. After
absorption in the human body manganese will be transported through the blood to the
liver, the kidneys, the pancreas and the endocrine glands.
Manganese effects occur mainly in the respiratory tract and in the brains.
Symptoms of manganese poisoning are hallucinations, forgetfulness and nerve
damage. Manganese can also cause Parkinson disease, lung embolism and bronchitis.
When men are exposed to manganese for a longer period of time they may become
impotent. A syndrome that is caused by manganese has symptoms such as
schizophrenia, dullness, weak muscles, headaches and insomnia. Because manganese
is an essential element for human health shortages of manganese can also cause health
effects. These are the following effects:
(i) Fatness;
(ii) Glucose intolerance;
62
(iii) Blood clotting;
(iv) Skin problems;
(v) Lowered cholesterol levels;
(vi) Skeleton disorders;
(vii) Birth defects;
(viii) Changes of hair colour; and
(ix) Neurological symptoms
The most common oxidation states of manganese are +2, +3,+4, +6 and +7,
though oxidation states from -3 to +7 are observed. Mn2+ often competes with Mg2+
in biological systems. Manganese compounds where manganese is in oxidation state
+7, which are restricted to unstable oxide Mn2O7 and compounds of the intensely
purple permanganate anion MnO4, are powerful oxidizing agents. [115]. The
derivation of its name from the Greek word for magic remains appropriate, because
scientists are still working to understand the diverse effects of manganese deficiency
and manganese toxicity in living organisms [116].
Manganese (Mn) plays an important role in a number of physiologic processes
as a constituent of multiple enzymes and an activator of other enzymes[117].
Manganese superoxide dismutase (MnSOD) is the principal antoxidant enzyme in the
mitochondria. Because mitochondrian consume over 90% of the oxygen used by cells,
they are especially vulnerable to oxidative stress. The superoxide radical is one of the
reactive oxygen species produced in mitochondria during ATP synthesis. MnSOD
catalyzes the conversion of superoxide radicals to hydrogen peroxide, which can be
63
reduced to water by other antioxidant enzymes [118]. Although the specific
mechanisms for manganese absorption and transport have not been determined, some
evidence suggests that iron and manganese can share common absorption and
transport pathways.
A number of manganese-activated enzymes play important roles in the
metabolism of carbohydrates, amino acid, and cholesterol (4). Pyruvate carboxylase, a
manganese-containing enzyme, and phosphoenolpyruate carboxykanase (PEPCK), a
manganese-activated enzyme, are critical in gluconeogenesis- the production of
glucose from non-carbohydrate precursors. Arginase, another manganese-containing
enzyme, is required amino acid metabolism. In the brain, the manganese-activated
enzyme, glutamine synthetase, converts the amino acid glutamate to glutamine.
Glutamate is an excitotoxic neurotransmitter and a presurseor to an inhibitory
neurotransmitter, gamma-aminobutyric acid (GABA) [119].
2.7 Bioaccumulation and Biomagnifications
Biomagnifications otherwise known as bioamplification or biological magnification is
the increase in concentration of a pollutant from one link in a food chain to another. It
refers to the tendency of pollutants to concentrate as they move from one trophic level to the
next. In order for biomagnification to occur, the pollutant must be:-
(i) long-lived;
(ii) mobile;
(iii) soluble in fats, and
(iv) biologically active
64
It often refers to the process whereby certain substances such as pesticides or
heavy metals move up the food chain, work their way into rivers or lakes, and are
eating by aquatic organisms such as fish, which in turn are eaten by large birds,
animals or human. The substances become concentrated in tissue or internal organs as
they move up the chain. Bioaccumulants are substances that increase in concentration
in living organisms as they take in contaminated air, water or food because the
substance are very slowly metabolized or excreted. [120].
2.7.1 Bioaccumulation
This occurs within a trophic level, and is the increase in concentration of a
substance in certain tissues of organisms’ bodies due to absorption from food and the
environment or increase in concentration of a pollutant from the environment to the first
organism in a food chain. It results in building in the adipose tissue of successive
trophic levels: zooplankton, small neckon, large fish etc.
This is an increase in the concentration of a chemical in a biological organism over
time, compared to the chemical’s concentration in the environment.
Cells have mechanisms for bioaccumulation, the selective absorption and
storage of a great variety of molecules. This allows them to accumulate nutrient and
essential minerals, but at the same time, they also may absorb and store harmful
substances through the same mechanisms. Toxins that are rather dilute in the
environment can reach dangerous level inside cells and tissues through this process
(bioaccumulation). Regulation of metal accumulation by organisms complicates the
interpretation and application of bioaccumulation data for aquatic and terrestrial
65
organisms. Organisms have evolved homeostatic mechanisms that allow metals as
naturally occurring substances, to be stored in non-available forms (sometimes for
later use). These mechanisms regulate the up take and excretion of metals to maintain
tissue concentrations within desirable ranges, as well as to prevent toxicity [121]. For
certain elements and organisms, bioaccumulation is required for organism health and
normal function (e.g for essential trace element such as copper and zinc). In order
situations, bioaccumulation produces residue in plants and animals that cause direct
toxicity to the exposed organism9e.g copper toxicity to aquatic organism) or indirect
toxicity to consumers (as in selenium accumulation by plants). To further complicate
understanding the bioaccumulation and metabolism of an essential element can affect
the metabolism of a non- essential toxic metal, as in the case of calcium and lead in
the central nervous system [122]
Homeostasis should also be considered for metals as it influences or regulates
bioconcentration factors (BCFs), or bioaccumulation factors (BAFs), and exposure
concentration [123]. At low concentrations- where organisms experience nutritional
deficiency - grater uptake and retention of metals occur to meet nutritional
requirements. At concentrations above the nutritional requirement, homeostasis
maintains a concentration limit in the organism. However, beyond that range,
homeostatic mechanism (e.g regulation by excretion) can become overwhelmed,
resulting in toxicity [124]. Organisms also may compensate for exposure to essential
metal concentration beyond their nutritional requirements. The BCFs or BAFs could
decrease with an increase in exposure concentration [125].
66
2.8 Analytical Techniques for Trace Metal Analysis
2.8.1 Preparation of Biological Samples
The effect of sample preparation steps on the quality of the analytical results is
universally recognized. The application of an appropriate digestion procedure and
effective combination with the separation and detection methods are of major
importance in the analysis of trace metals. Complete digestion of the examined
materials and a quantitative transformation of the analytes into stable soluble
complexes that can make the basis of separation, pre concentration and detection steps
are required for ensuring a quantitative recovery of the metals [126].
2.8.2 Drying Ashing
Ashing in analytical chemistry is defined as the heating of a substance to leave
only non-combustible ash, which is analysed for its elemental composition.
2.8.2.1 Ashing Techniques
The sample preparation techniques incorporating some form of ‘ashing’ are as
follows:-
(i) Dry ashing:- this is usually performed by placing the sample in open
inert vessels and destroying the combustible (organic) portion of the sample by
thermal decomposition using a muffle furnace. Typical ashing temperature is 450 to
550 oC. Magnesium nitrate is commonly used as an ashing aid. Charring the sample
prior to muffling is preferred. Charing is accomplished using an open flame [126].
67
(ii) Sulphated ashing:- this involve treatment of the sample charred with
sulphuric acid( the char is wetted using the minimum amount of sulphuric acid and
then brought to dryness before muffling ).
(iii) Wet ashing:- this is the treatment of the sample with a moderate amount
of sulphuric acid before charring. Charring is performed using an open flame. Liquid
samples tend to foam. After the excess sulphuric acid is driven off, the sample is
muffle as above.
(iv) Low temperature ashing:- it involves treatment of the sample at 120oC
using activated ( singlet state) oxygen.
(iv) Closed system ashing:- It involves thermal decomposition in oxygen in a
closed system such as a schnige flask or an oxygen parr bomb.
2.8.2.2 Advantages of ashing
Ashing techniques are understandably used only for samples containing a
significant amount of combustible or organic material as the matrix. The major
advantages of ashing include:-
(i) The ability to decompose large sample sizes
(ii) The need for little or no reagents
(iii)The techniques is relatively safe
(iv) The ability to prepare samples containing volatile combustion elements such
as sulphur, fluorine and chrlorine (the schoniger oxygen flask combustion
68
techniques is very popular) and
(v) The technique tends itself to mass production.
2.8.2.3 Disadvantages.
The trace analyst should be very familiar with their sample type before
performing an ash. Some of the problems that have been encountered are listed as
follows:-
(i) Losses due to retention to the ashing container
(ii) Losses due to volatilization
(iii) Contamination from the muffle furnace
(iv) Physical loss of low density ashes when the muffle door is opened
(v) Difficulty in dissolving certain metal oxides
(vi) Formation of toxic gases in poorly ventilated areas [127].
2.8.3 Wet Digestion
Wet digestion methods for elemental analysis involve the chemical degradation
of sample matrices in solution, usually with a combination of acids to increase
solubility. The various acid and flux treatments are carried out at high temperatures in
specially designed vessel that help to minimize contamination of the sample with
substances in the air, the local environment, and from the vessel wall. Losses from the
sample may occur due to adsorption onto the vessel walls, volatilization, and co
69
extraction, but these can be reduced by procedural modifications. The use of closed
systems, where the digestion reaction is completely isolated from the surroundings,
may help to reduce both contamination and sample loss. The selection of an
appropriate treatment for sample dissolution depends on the nature of the sample, and
different approaches are required for predominantly inorganic and predominantly
organic matrices. Geological, geochemical, and soil samples generally contain silicate,
metal oxides, carbonates, and, in many cases, organic matter. Such samples must be
dried and ground to a fine powder to facilitate dissolution. Minerals and coal often
have a non uniform distribution of elements, while fly ash is very fine and is
composed of metal silicates and oxides. Both these types of samples are difficult to
solubilize. Similarly, alloys can be difficult to dissolve because of the strong bonds
between metal atoms and their brittle nature. Solid and crystalline samples may
possess interstitial water and water of crystallization, so thorough drying of samples is
necessary before and after grinding. Biological samples must be processed with great
care, since the dissolution and total decomposition of all organic matter is required for
the release of trace elements. However, the use of oxidizing acids to decompose
organic matter can produce violent reactions and the alternative procedure of dry
ashing may be more suitable in some cases. Environmental and water samples often
contain mixtures of organic and inorganic substances, so dissolution techniques need
to be modified to take this composition into account. In particular, water samples may
contain dissolved and suspended solid colloids, and microorganisms. Elements
embedded in such samples may be present both in dissolved and solid forms. The
nature of the sample matrix must be given special attention during wet digestion
70
[128].
Naturally occurring inorganic materials, such as ores, must be given special
treatments to facilitate solubilization. The two most common methods employed in
dissolving samples are treatments with hydrochloric, hydrofluoric, nitric, sulfuric, or
perfloric acids (or various combinations thereof) and fusion with an acidic or basic
flux followed by treatment with water or an acid. Organic materials are usually
decomposed by wet digestion with a boiling oxidizing acid or acid mixture, ultimately
producing carbon dioxide, water and other volatile compounds that are driven off to
leave behind salts or acids of the inorganic constituents of the sample. Wet digestions
may be performed in open beakers on hot plates, but kjeldahl flask or specially
designed containment vessels give results that are more satisfactory.[129].
2.8. 3.1 Wet Digestion with Single Acids
The solvent action of an acid depends on several factors:
(i) The reduction of hydrogen ions by metals that are more active than hydrogen, for
example:-
Zn(s) + 2H+ → Zn
2+ + H2(g).. .. .. .. .. (1)
(ii) The combination of hydrogen ions with anions of a weak acid, for example:
CaCO3 (s)+ 2H+ → Ca
2+ + H2O + CO2 (g)… .. .. .(2)
(iii) The oxidizing properties of the acid anion, for example:
3Cu (s) +2NO3 + 8H+ → 3Cu
2+ 3NO(g)+ 4H2O.. .. (3)
71
(iv) The tendency of the acid anion to form soluble complexes with the sample
cation, for example: Fe3+
+ Cl → FeCl2+
. .. .. .. ..(4)
Ideally, the chosen reagent should cause the complete dissolution of the sample.
As a general guide it is useful to classify the more common acid treatments according
to whether they oxidize the sample or not. The non-oxidizing acids include dilute
hydrochloric, hydrofluoric, sulphuric, and perchloric acids, whereas the oxidizing
acids include hot, concentrated nitric, sulphuric, and perchloric acids. Dissolution of
metals by nonoxiding acids is a process of hydrogen replacement. Hydrochloric acid
will dissolve metals above the standard reduction potential of hydrogen, salts of weak
acids, and many oxides. Dilute sulphuric and perchloric acids are useful for metals
above the standard reduction potential of hydrogen. Hot, concentrated sulphuric acid
will often dissolve metals below the standard reduction potential of hydrogen. The
most potent oxidizing conditions are obtained using hot concentrated perchloric acid,
which will dissolve all common metals. Concentrated hydrochloric acid is an excellent
solvent for many metal oxides as well as those metals that are more easily oxidized
than hydrogen. In addition, it is often a better solvent for oxides than the oxidizing
acids. Hot, concentrated nitric acid will dissolve all common metals with the
exception of aluminum and chromium, which are passive to the reagent as a result of
surface oxide formation. Hot nitric acid also readily oxidizes many organic
substances. Hot concentrated sulphuric acid can be use to decompose and dissolve
many substances in part because of its high boiling point (3401oC), and it is
particularly useful for the dehydration and oxidation of organic samples. Most metals
and alloys are also attacked by this hot acid. Perchloric acid is a potent oxidizing agent
72
that leads to the formation of highly soluble perchlorated salts. As with sulphuric acid
perchloric acid dehydrates and oxidizes organic samples very efficiently. It also
attacks iron alloys and stainless steel, which are resistant to other mineral acids. Care
is required when using perchloric acid because it is explosive in contact with certain
organic compounds and easily oxidized inorganic materials. Special chemical hoods
are recommended. Perchloric acid, as a 72-74% solution, boils at 2031oC.
hydrofluoric acid is a weak, nonxidizing acid that is particularly useful for dissolving
silicate samples since it removes the silicon quantitatively as volatile SiF4. In many
cases, hydrofluoric acid dissolution can be achieved by adding sodium fluoride to
samples treated with hydrochloric acid[130].
2.8.3.2 Wet Digestion with Acid Mixtures
Acids in combination are preferred for certain inorganic matrices and are generally
more advantageous for the decomposition of organic compounds. Wet digestion
procedures using acid mixtures can be divided into four types:
(i) Total decomposition, usually with hydrofluoric acid and another mineral acid.
(ii) Strong attacks, for routine analysis but leaving a residue of certain minerals,
particularly silicates. Carried out with various mixture of sulphuric, nitric, and
perchloric acids.
(iii) Moderate attacks, using weaker acid mixtures.
(iv) Partial digestions (acid leaching).
Both (iii) and (iv) are typically employed for environmental analysis where complete
73
dissolution is either hot required or is undesirable and the goal is to determine the
presence of certain trace elements. For geochemical samples containing silicates, the
matrix is decomposed by heating with hydrofluoric acid in combination with either
nitric or perchloric acid, each of which has a higher boiling point than hydrofluoric
acid. The presence of the second acid with a higher boiling point ensures that, once
the hydrofluoric acid has been boiled of and the dry sample re-dissolved, sparingly
soluble metal fluorides are converted to salts that are more soluble. As stated above
however, caution should be exercised with the use of perchloric acid if the sample has
a significant organic component. Perchloric acid is also more expensive than nitric
acid, and can introduce chloride ions as contaminants. For organic samples, a widely
used mixture is aqua regia (1:3) nitric acid –hydrochloric acid. The nitric acid acts as
the oxidizing agent, while the hydrochloric acid provides the complexing properties.
The addition of bromine or hydrogen peroxide can sometimes increase the
solubilizing power of mineral acids. Wet digestion is generally carried out in open
flasks, covered loosely to avoid atmospheric contamination. However, it is becoming
increasingly common to use closed vessels, such as polytetrafluoroethylene (PTFE)
lined containers or ultrapure quartz vessel, especially for small samples. A 1:4 mixture
of sulphuric and nitric acids is also widely employed for organic samples. The nitric
acid decomposes the bulk of the organic matter but does not reach a temperature
sufficient to destroy the last traces. However, as the nitric acid boils off, the sulfuric
acid is left behind. Dense SO3 fumes evolve and begin to reflux in the flask, making
the solution very not and allowing the hot sulphuric acid to decompose the remaining
organic matter. Because of the fumes produce in this method, it must be carried out
74
under a fume hood. More nitric acid may be added to prolong the digestion and
eliminate any stubborn organic material. A very efficient acid mixture is nitric,
sulfuric, and perchloric acid in a volume ratio 3:1:1. For a typical 10.g sample of
tissue of blood, 10 ml of this solution is sufficient for complete dissolution. The
samples are heated until the nitric acid boils off and perchloric acid fumes begin to
appear. Heating continues until the perchloric acid boils off and SO3 fumes appear.
There is little danger of perchloric acid explosions as long as sulphuric acid remains
after the perchloric acid has evaporated to prevent the sample becoming dry.
Perchloric acid should never be added directly to an organic sample. A mixture of
nitric and perchloric acid may also be used [131].
Availability of strong hydrogen peroxide solutions allows a combination of
sulphuric acid and hydrogen peroxide is a vigorous oxidizing agent and is particularly
useful for the degradation of resistant plastics. There is little danger of explosion if
sulfuric acid is present in excess. Most elements can be recovered quantitatively in
this procedure; with the exceptions of ruthenium, osmium, germanium, arsenic, and
selenium. In the case of germanium and arsenic, loss is attributable to volatilization of
chlorides. Additionally, precipitated calcium sulphate may retain lead and silver if not
solubilized. After decomposition, the sulfuric acid solution should be diluted and
boiled gently for 10 min to destroy any remaining hydrogen peroxide.
2.8.4 Microwave Digestion
A microwave sample digestion system consists of a microwave oven, a rotating
carousel holding several sample digestion bombs, and a system for venting these in a
75
controlled fashion. It may also provide monitoring and recording of both temperature
and pressure in the containers. Digesting a sample in a closed container in a
microwave oven has several advantages over open container dissolution methods. The
containers are fabricated of high-temperature polymers, which are less likely to
contain metal contaminants than are glass or ceramic beakers or crucibles. The sealed
containers eliminate the chance of airborne dust contamination, and reduce
evaporation, so that less acid digestion solution is required, reducing blanks. The
sealed container also eliminates losses of more volatile metal species, which can be a
problem in open container sample decomposition, especially in dry ashing [132].
2.9 Detection of Trace Metals
Essentially, the heavy metals have only become a focus of public interest since
analytical techniques have made it possible to detect them even in very trace amounts.
The relatively reckless handling of heavy metals and their compounds in former times
can partly be explained by the fact that their effects were unknown. Today, analytical
detection is possible down to a thousand of a mg/kg (ppb) for certain matrixes.
Analytical methods can be separated into classical and instruct mental [133]. Classical
methods (also known as wet chemistry methods) use separations such as precipitation,
extraction, and distillation and qualitative analysis by color, odor, or melting point.
Quantitative analysis is achieved by measurement of weight (gravimetry) or volume
(volumetric titration).
Instrumental methods use an apparatus to measure physical quantities of the
analyte such as light absorption, fluorescence, or conductivity. The separation of
76
materials when necessary is accomplished using chromatography or electrophoresis
methods.
Although modern analytical chemistry is dominated by sophisticated
instrumentation, the roots of analytical chemistry and some of the principles used in
modern instruments are from traditional techniques many of which are still used
today.
2.9.1 X-Ray Fluorescence (XRF)
XRF is an elemental analysis technique with unique capabilities including
highly accurate determinations for major elements and a broad elemental survey of the
sample composition without standards [134]. X-ray fluorescence (XRF) is a non-
destructive technique that is used to quantify the elemental composition of solid and
liquid samples. X-rays are used to excite atoms in the sample; causing them to emit X-
rays with energies characteristic of each element present. The intensity and energy of
these X-rays are then measures. For example, XRF is used in analysis of rocks and
metals with an accuracy of 0.1% of the major elements.
A material is exposed to X-rays of high energy, and as the X-ray (or photon)
strikes an atom (or a molecule) in the sample, energy is absorbed. If the energy is high
enough, a core electron is ejected out of its atomic orbital. An electron from an outer
shell then drops into the unoccupied orbital, to fill the hole left behind. This transition
gives off an X-ray of fixed or characteristic energy that can be detected by a
fluorescence detector when the energy source is a synchrotron or the X-ray are focus
by anoptic, like a polycappillary, the X- ray beam can be very small and very intense,
77
and atomic information on the sub-micrometer scale can be obtained [134].
There are two types of spectrometer:
(i) Wavelength dispersive spectrometers (WDX or WDS):- the photons are
separated by diffraction on a single crystal before being detected.
(ii) Energy dispersive spectrometers (EDX or EDS):- the detector allows the
determination of the energy of the photon when it is detected
2.9.2 Neutron Activation Analysis (NAA)
Since it was first applied in 1960 for the analysis of tantalum, instrumental
neutron activation analysis (INAA) continues to be one of the most sensitive and
accurate techniques for meeting industries need for the trace element analysis of high-
purity silicon. In keeping with the industry expansion, the demand for INAA of high-
purity silicon has more than doubled over the last three years [135]. The idea of using
neutrons as an analytical probe for elemental analysis was first proposed and
demonstrated by Von Hevesy and Levi for the analysis of trace quantities of rare
earths in geological materials [135]. Since then, the excellent sensitivity, selectivity
and precision of INAA have made it one of the most versatile and widely employed
elemental analysis techniques. Because most materials are “transparent” to both the
probe (neutrons) and the signal (gamma rays), there are few matrix effects associated
with the analysis. Standardization of the measurement is simple and straightforward.
Moreover, because little, if any, sample manipulation is required INAA is a highly
sensitive technique that can be applied to bulk samples and is relatively free of reagent
78
and laboratory contamination.
In INAA, stable nuclei in the sample undergo neutron induced nuclear
reactions when the sample is exposed to a flux of neutrons. The most common neutron
reaction is neutron capture by a stable nucleus that produces a radioactive nucleus.
The “neutron rich” radioactive nucleus then decays, with a unique half-life, by the
emission of a beta particle. In the vast majority of cases, gamma-rays are also emitted
in the beta decay process and a high-resolution gamma-ray spectrometer is used to
detect these “delayed” gamma rays from the artificially induced radioactivity in the
sample for both qualitative and quantitative analysis. The energies of the delayed
gamma rays are used to determine which elements are present in the sample, and the
number of gamma rays of a specific energy is used to determine the amount of an
element in the sample. The physical principles of the analysis are so well understood
that neutron activation analysis is one of the primary techniques used by the National
Institute of Standards and Technology to certify the concentration of elements in
standards reference materials.
The major advantage of INAA is that it provides accurate results for large, bulk
samples (tens of gram) without having to dissolve or digest the sample. Moreover, by
employing an appropriate surface etech procedure, it is possible to ensure that the
trace elements observed in the INAA measurement are coming from the bulk material
and are not a result of surface contamination at the production facility or in the
analytical lab [136]. As with all analytical techniques, there are drawbacks to using
INAA. One major disadvantage is that the technique requires access to a high-flux
neutron source to obtain sensitivities. As a result, the technique cannot be performed
79
“in house” by industry. A second disadvantage of INAA is the time required for the
analysis. The third major disadvantage of INAA is that it cannot provide information
on some of the light elements, particularly B, C, O and Al.
2.9.3 Proton Induced X-Ray Emission (PIXE)
Elemental analysis incorporating proton induced X-ray Emission (PIXE),
provides a non-destructive, simultaneous analysis for the 72 inorganic elements from
sodium through uranium on the Periodic Table for solid, liquid, and thin film (i.e.
aerosol filter) samples. The PIXE technique offers the advantage of analysis, without
the necessity for time consuming digestion, thereby minimizing the potential for error
resulting from sample preparation [136].
Proton Induced X-ray Emission (PIXE) is an X-ray spectrographic technique,
which can be used for the non-destructive, simultaneous elemental analysis of solid,
liquid or aerosol filter samples. The X-ray spectrum is initiated by energetic protons
exciting the inner shell electrons in the target atoms. The expulsion of these inner shell
electrons results in the production of X-rays. The energies of the X-rays, which are
emitted when the created vacancies are filled again, are uniquely characteristic of the
elements from which they originate and the number of X-rays emitted is proportional
to the mass of that corresponding element in the sample being analyzed [137].
In PIXE system, the greater the proton current, the higher the probability for
the production of x-ray. As proton energy changes, so does the probability for X-ray
production. If quantitative analysis is to be assured, then both of these factors must be
accurately known. Since instrument calibration is performed at specific proton energy,
80
knowledge of the proton energy loss is essential for quantitative analysis.
For a PIXE system which bases its calibration on thin film gravimetric
standards, the mass/area may be expressed as a simple ratio of yields. Standard
calibration is carried out under these same conditions, so that each element has a
spectrum in each position. A normalized linear combination of these two positions for
the standards allows for extrapolation to any desired combination of irradiation times
for unknown targets. The calibration is carried out by irradiating each standard with
and without the filter in front of the detector for a preset charge collection [138].
2.9.4 Atomic Spectroscopy
Atomic spectroscopy is the determination of elemental composition by its
electromagnetic or mass spectrum. The study of the electromagnetic spectrum of
elements is called optical atomic spectroscopy. Electrons exist in energy levels within
an atom. These levels have well defined energies and electrons moving between them
must absorb or emit energy equal to the difference between them. In optical
spectroscopy, the energy absorbed to move an electron to a more energetic level and
or the energy emitted as the electron moves to a less energetic energy level is in the
form of a photon. The wavelength of the emitted radiant energy is directly related to
the electronic transition which has occurred. Since every element has a unique
electronic structure, the wavelength of light emitted is a unique property of each
individual element. As the orbital configuration of a large atom may be complex,,
there are many electronic transition which can occur, each transition resulting in the
emission of a characteristic wavelength of light [139].
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2.9.4.1 Atomic (Flame) Emission Spectrometry
Atomic Emission Spectroscopy (AES) is a method of chemical analysis that
uses the intensity of light of particular wavelength emitted by an heated and excited
atom in a flame, plasma, arc, or spark to determine the quantity of the element in a
sample. The wavelength of the atomic spectral line gives the identity of the element
while the intensity of emitted light is proportional to the number of atoms of the
element [139] in the flame and in the sample.
In atomic emission, a sample is subjected to a high energy, thermal
environment in order to produce excited state atoms, capable of emitting light. The
energy source can be an electrical arc, a flame, or more recently, plasma. The
emission spectrum of an element exposed to such an energy source consists of a
collection of the allowable emission wavelengths, commonly called emission lines,
because of the discrete nature of the emitted wavelengths. This emission spectrum can
be used as a unique characteristic for qualitative identification of the element. Atomic
emission using electrical arcs has been widely used in qualitative analysis. Emission
techniques can also be used to determine how much of an element is present in a
sample. For a “quantitative” analysis, the intensity of light emitted at the wavelength
of the element to be determined is measured. The emission intensity as this
wavelength will be greater as the number of atoms of the analyzed element increases.
The technique of flame photometry is an application of atomic emission for
quantitative analysis.
Flame absorption and flame emission techniques usually involve introduction
82
of a sample solution in aerosol form into a flame. Solvent evaporation and
vaporization of the salt occur prior to dissociation of the salt into free gaseous atoms.
At the temperature of an air - acetylene flame, (2300OC) atoms of many elements exist
largely in the ground state. When a beam of radiant energy that consists of the
emission spectrum line for the element that is to be determined is passed through the
flame, some of the ground state atoms absorb energy of characteristic wavelengths
(resonance lines) and are raised to a higher energy state. The radiation not removed by
absorption is isolated by a monochrometer and detected by a photomultiplier. The
amount of radiant energy absorbed as a function of concentration of an element in the
flame is the basis of atomic absorption spectroscopy. The amount of light absorbed is
proportional to the elemental concentration, assuming Beer’s Law holds. For a few
elements such as the alkali metals, sodium and potassium, and air - acetylene flame is
hot enough not only to produce ground state atom, but to raise some of the atoms to an
excited electronic state.
Flame photometry is largely an empirical method and is sensitive to
experimental conditions. The signal intensity from a flame is dependent on the flame
temperature, the rate of flow of liquid into the flame, the pressure and rate of flow of
fuel gases, and any of many other variables which affect the character of the flame or
atomizing of the sample. Thus the viscosity of the solution in which the ion is found
can have a great effect. A consequence of this situation is that reliable results can be
obtained only after painstaking attention to details, with repeated checks of
reproducibility and the effects of altering conditions.
Atomic emission analyses are most commonly and routinely performed on
83
solutions. This is most conveniently done using any of the appropriate digestion
method on the sample, leaving a solution that can then be analyzed. There must be a
sufficient concentration of analyte for the spectrometer to detect. Prior to performing
atomic emission analysis, you will need to determine the minimum detection limit for
the element of interest. The minimum quantifiable limit (the lowest concentration of
analyte which can be quantitatively determined) is generally 3-5 times the minimum
detection limit.
A sample of a material (analyte) is brought into the flame as a gas or sprayed
solution. The heat from the flame evaporates the solvent and breaks chemical bonds to
create free atoms. The thermal energy also excites the atoms into excited electronic
states that subsequently emit light when they return to lower ground electronic state. A
frequent application of the emission measurement with the flame is the regulation of
alkali metals for pharmaceutical analysis [140].
2.9.4.2 Inductively Coupled Plasma Mass Spectrometry (ICP-MS)
It is a type of mass spectrometry that is highly sensitive and capable of the
determination of a range of metals and several non-metals at concentrations below one
part in 1012 (part per trillion). It is based on coupling together an inductively coupled
plasma as a method of producing ions (ionization) with a mass spectrometer as a
method of separating and detecting the ions [141].
In trace elemental analysis, the method has advantage of high, precision and
sensitivity compared to atomic absorption techniques. Analysis of lower
concentrations at the same time is more prone to disruption by trace contaminants in
84
laboratory ware and reagents used. Specific analytes suffer from interference
exclusive to ICP-MS technique. Verification of analysis results requires additional
effort.
The variety of applications exceeds that of ICP-AES and includes isotopic
specification. Inductively coupled plasma is plasma that contains a sufficient
concentration of ions and electrons to make the gas electrically conductive. The
plasmas used in petrochemical analysis are essentially electrically neutral, with each
positive charge on an ion balanced by a free electron. In these plasmas the positive
ions are almost all singly charged and there are few negative ions, so there are nearly
equal amounts of ions and electrons in each unit volume of plasma.
Inductively coupled plasma (ICP) for spectrometry is sustained in a torch that
consists of three concentric tubes, usually made of quartz. The end of this torch is
placed inside an induction coil supplied with a radio-frequency electric current. A
flow of argon gas (usually 14-18 liters per minute) is introduced between the two
outermost tubes of the torch and an electric spark is applied for a short time to
introduce free electrons into the gas stream. The ICP can be retained in the quartz
torch because the flow of gas between the two outermost tubes keeps the plasma away
from the walls of the torch. A second flow of argon (around 1 liter per minute) is
usually introduced between the central tube and the intermediate tube to keep the
plasma away from the end of the central tube. A third flow (again usually around 1
liter per minute) of gas is introduced into the central tube of the torch. This gas flow
passes through the centre of the plasma, where it forms a channel that is cooler than
the surrounding plasma but still much hotter than a chemical flame. Samples to be
85
analysed are introduced into this central channel, usually as a mist of liquid formed by
passing the liquid sample into a nebulizer.
As a droplet of nebulized sample enters the central channel of the ICP, it
evaporates and any solids that were dissolved in the liquid vaporize and then break
down into atoms. At the temperatures prevailing in the plasma a significant proportion
of the atoms of many chemical elements are ionized, each atom losing its most loosely
bound electron to form a singly charged ion.
2.9.4.3 Atomic Absorption Spectrophotometry
2.9.4.3.1 Technique
Atomic absorption spectrophotometry (AAS) is an analytical technique that
measures the concentrations of elements. It is commonly used for determining the
concentration of a particular metal element within a sample. AAS can be used to
analyse for the concentration of over 62 different metals in a solution [142]. AAS
determines the presence of metals in liquid samples. Metals include Fe, Cu, Al, Pb,
Ca, Zn, Cd and many more. Atomic absorption is so sensitive that it can measure
down to parts per billion of a gram (µgdm-3) in a sample. Typical concentrations
range in the low mg/L range. In their element form, metals will absorb ultraviolet light
and get excited. Each metal has a characteristic wavelength that will be absorbed. The
AAS instrument looks for a particular metal by focusing a beam of UV light at a
specific wavelength through a flame and into a detector. The sample of interest is
aspirated into the flame. If that metal is present in the sample, it will absorb some of
the light, thus reducing its intensity. The instrument measures the change in intensity.
86
A computer data system coverts the change in intensity into an absorbance [143].
Atomic absorption spectrometry was first used as an analytical technique. The
underlying principles of atomic absorption spectrometry were established in the
second half of the 19th century by Robert Wilhelm Bunsen and Gustav Robert
Kirchhoff, both professors at the University of Heidelberg, Germany. The modern
form of AAS was largely developed during the 1950s by a team of Australian
Chemists, led by Allan Walsh at the Commonwealth Scientific and Industrial
Research Organization, Division of Chemical physics, in Melbourne, Australia
(CSIRD)[144].
2.9.4.3.2 Principle
Atomic absorption spectrophotometry (AAS) makes use of absorption spectrometry to
assess the concentration of an analyte in a sample. It requires standard solutions to
establish the relation between the measured absorbance and the analyte concentration,
and relies therefore on Beer-Lambert law. In short, the electrons of the atoms in the
atomizer can be promoted to higher orbital (excited state) for a short period of time
(nanosecond) by absorbing a defined quantity of energy (quantized energy or radiation
of a given wavelength). This amount of energy, characterized by the wavelength, is
specific to a particular electron transition in the particular element. In general, each
wavelength corresponds to only one element, while the width of an absorption line is
only of the order of a few picometers (pm), which gives the technique its elemental
specificity. The radiation flux without a sample and with a sample in the atomizer is
measured using a detector, and the ratio between the two values(the absorbance) is
converted to analyte concentration or mass using Beer-Lambert Law[145].
87
2.9.4.3.3 Instrumentation
Fig 2.1 Atomic absorption spectrophotometer block diagram.[145].
In order to analyse a sample for its atomic constituents, it has to be atomized.
The atomizers most commonly used nowadays are flames and electrothermal (graphite
tube) atomizers as shown in fig 2.1 above. The atoms should then be irradiated by
optical radiation, and the radiation source could be an element - specific line radiation
source or a continuum radiation source. The radiation then passes through a
monochromator in order to separate the element-specific radiation from any other
radiation emitted by the radiation source, which is finally measured by a detector.
2.9.4.3.4 Atomizer
Although other atomizers, such as heated quartz tubes, might be used for
special purposes, the atomizers most commonly used nowadays are (spectroscopic)
flames and electrothermal (graphite tube) atomizers.
2.9.4.3.5 Flame Atomizers.
The oldest and most commonly used atomizers in AAS are flames, principally
the air-acetylene flame, with a temperature of about 23000C and the nitrous oxide
(N20) -acetylene flame, with a temperature of about 27000C. The latter flame, in
88
addition, offers a more reducing environment, being ideally suited for analytes with
high affinity to oxygen.
Liquid or dissolved samples are typically used with flame atomizers. The
sample solution is aspirated by a pneumatic nebulizer, transformed into an aerosol,
which is introduced into a spray chamber, where it is mixed with the flame gases and
conditioned in a way that only the finest aerosol droplets (< 10 m) enter the flame.
However, only about 5% of the aspirated sample solution reaches the flame, but it also
guarantees a relatively high freedom from interference with a temperature of about
27000C.
On top of the spray chamber is a burner head that produces a flame that is
laterally long (usually 5-10cm) and only a few mm deep. The radiation beam passes
through this flame at its longest axis, and the flame gas flow-rates may be adjusted to
produce the highest concentration of free atoms. The burner height may also be
adjusted so that the radiation beam passes through the zone of highest atom cloud
density in the flame, resulting in the highest sensitivity.
The processes in a flame include the following stages:-
(i) Desolvation (drying)- the solvent is evaporated and the dry sample nano-
particles remain;
(ii) Vaporization (transfer to the gaseous phase)- the solid particles are
converted into gaseous molecules.
(iii) Atomization-the molecules are dissociated into free atoms;
(iv) Ionization- depending on the ionization potential of the analyte atoms and
89
the energy available in a particular flame, atoms might be in part converted to
gaseous ions.
Each of these stages includes the risk of interference. In case the degree of phase
transfer is different for the analyte in the calibration standard and in the sample.
Ionization is generally undesirable, as it reduces the number of atoms that is available
for measurement, i.e., the sensitivity. In flame AAS a steady-state signal is generated
during the time period when the sample is aspirated. This technique is typically used
for determinations in the mg L-1 range, and may be extended down to a few mg L-1 for
some elements.
If light of just the right wavelength impinges on a free, ground state atom, the
atom may absorb the light and it enters an excited state in a process known as atomic
absorption. Atomic absorption measures the amount of light at the resonant
wavelength which is absorbed as it passes through a cloud of atoms. As the number of
atoms in the light path increases, the amount of light absorbed increases in a
predictable way. By measuring the amount of light absorbed, a quantitative
determination of the amount of analyte element present can be made. The use of
special light sources and careful selection of wavelength allow the specific
quantitative determination of individual elements in the presence of others. The atom
cloud required for atomic absorption measurements is produced by supplying enough
thermal energy to the sample to dissociate the chemical compounds into free atoms.
Aspirating a solution of the sample into a flame aligned in the light beam serves this
purpose. Under the proper flame conditions, most of the atoms will remain in the
ground state form and are capable of absorbing light at the analytical wavelength from
90
a source lamp. The ease and speed at which precise and accurate determinations can
be made with this technique have made atomic absorption one of the most popular
methods for the determination of metals.
2.9.4.4 Inductively Coupled Plasma Atomic Emission Spectroscopy
Inductively coupled plasma atomic emission spectroscopy (ICP-AES), is also
referred to as inductively coupled plasma optical spectrometry (ICP-OES), is an
analytical technique used for the detection of trace metals. It is a type of mission
spectroscopy that uses the inductively coupled plasma to produce exicited atoms and
ions that emit electromagnetic radiation at wavelengths characteristic of a particular
element [146]. The intensity of this emission is indicative of the concentration of the
element within the sample.
The ICP-AES is composed of two parts: the ICP and the optical spectrometer.
The ICP torch consists of three (3) cocentric quartz torch. Argon or helium gas is
typically used to create the plasma as the atomization and excitation source. Plasma is
an electrically neutral, highly ionized gas that consists of ions, and atoms. When the
torch is turned on, an intense electromagnetic field is created within the coil by the
high power radio frequency signal flowing in the coil. The RF signal is created by the
RF generator, which is, effectively a high power radio transmitter driving the “work
coil” the same way a typical radio transmitter drives a transmitting antenna.
The argon gas is ionized in the intense field and flows in a particular
rotationally symmetrical pattern towards the field of the RF coil. Stable temperature
plasma, which is between 600 to 8000k is generated as the result of the inelastic
91
collisions created between the neutral argon atoms and the charged particles.
All three states (solid, liquid, gas) have been successfully introduced into an
ICP. Although both aqueous and non aqueous solvents have been utilized, the most
common analysed sample is cations in solution. For solutions, a nebulizer is used to
convert the liquid stream into an aerosol consisting of particles that are 1-10m in
diameter. Direct injection of liquids into the plasma would either extinguish the
plasma or cause the atoms to be improperly desolvated, making excitation and
emission less efficient shear gas, typically nitrogen or dry compressed air is used to
‘cut’ the plasma at a specific spot. One or two transfer lenses are then used to focus
the emitted light on a diffraction grating where it is separated into its component
wavelengths in the optical spectrometer. More efficient systems have specific
wavelengths at multiple positions simultaneously. The ability of these so-called
polychromators to measure more than one analytical line at a time is a distinct
advantage over monochromators, but polychromators suffer from a lack of flexibility.
The intensity of each line is then compared to previously measured intensities
of known concentrations of the elements, and their concentrations are then computed
by interpolation along the calibration lines. Examples of the application of ICP-AES
include the determination of metals in wine [147], arsenic in food [148], and trace
elements bound to proteins [149] it is also used for motor oil analysis.
Advantages of ICP-AES are excellent limit of detection, can analse multiple
elements at one time and have longer linear ranges compared to AAS and GFAAS.
Low chemical interference, stable and reproducible signal. Disadvantages are spectral
92
interferences (many emission lines), cost and operating expense.
2.9.4.5 Spark and Arc Atomic Emission Spectroscopy
Spark or arc atomic emission spectroscopy is used for the analysis of metallic
elements in solid samples. For non-conductive materials, the sample is ground with
graphite powder to make it conductive. In traditional arc spectroscopy methods, a
sample of the solid was commonly ground up and destroyed during analysis. An
electric arc or spark is passed through the sample, heating it to a high temperature to
excite the atoms within it. The excited analyte atoms emit light at characteristic
wavelengths that can be dispersed with a monochromator and detected. As the spark
or arc conditions are typically not well controlled, the analysis for the elements in the
sample is quantitative. However modern spark sources with controlled discharges
under an argon atmosphere can be considered quantitative. Both qualitative and
quantitative spark analysis are widely used for production quality control in foundries
and steel mills [135].
2.9.4.6 Graphite Furnace Atomic Absorption Spectrometry (GFAAS)
(Also known as Electrothermal Atomic Absorption Spectrometry (ETAAS) is a
type of spectrometry that uses a graphite-coated furnace to vaporize the sample.
Briefly, the technique is based on the fact that free atoms will absorb light at
frequencies or wavelengths characteristic of the element of interest (hence the name
atomic absorption spectrometry). Within certain limits, the amount of light absorbed
can be linearly correlated to the concentration of analyte present. Free atoms of most
elements can be produced from samples by the application of high temperatures. In
93
GFAAS, samples are deposited in small graphite or pyrolytic carbon coasted graphite
tube, which can then be heated to vaporize and atomize the analyte. The atoms absorb
ultraviolet or visible light and make transitions to higher electronic energy levels.
GFAA spectrometry instruments have the following basic features: (i) a source
of light (lamp) that emits resonance line radiation; (ii) an atomization chamber
(graphite tube) in which the sample is vaporized; (iii) a monochromator for selecting
only one of the characteristic wavelengths (visible or ultraviolet) of the element of
interest; (iv) a detector, generally a photomultiplier tube (light detectors that are useful
in low-intensity applications), that measures the amount of absorption; (v) a signal
processor (computer system, strip chart recorder, digital display, meter, or printer).
Most currently available GFAAS are fully controlled from a personal computer
that has windows-compatible software. Aqueous simples should be acidified (typically
with nitric acid, HNO3) to a pH of 2.0 or less. Discoloration in a sample may indicate
that metals are present in the sample. For example, a greenish color may indicate high
nickel content, or a bluish may indicate a high copper content. A good rule to follow
is to analyse clear (relatively dilute) samples first and then analyse colored (relatively
concentrated) samples. It may be necessary to dilute highly colored samples before
they are analysed. They are more sensitive than flame atomic absorption
spectrometers. After the instrument has warmed up and been calibrated, a small
aliquot (usually less than 100 microliters (µL) and typically 20µL) is placed, either
manually or through an automated sampler, into the opening in the graphite tube. The
sample is vaporized in the heated graphite tube; the amount of light energy absorbed
in the vapour is proportional to atomic concentration. Analysis of each sample takes
94
from 1 to 5 minutes, and the results for a sample are the average of triplicate analysis
[150].
95
CHAPTER THREE
3.0 EXPERIMENTAL
3.1 Map and description of study area.
Figure 3.1 Map of Nigeria showing the sheep breeding States.
Kano is the capital city of Kano State. In ancient times a powerful city-state of
the Hausa people, Kano has been an important Islamic city of the West African
savanna for centuries. Kano’s densely populated old city is surrounded by a well-
preserved 22-km- (14-mi-) long wall dating from the 13th century. The old city
contains the 16th century Kurmi Market. Kano is the center of a prosperous, densely
populated agricultural region in which millet, sorghum, peanuts, and beans are
produced. It is an important market center for peanuts, livestock, grains, and
other foodstuffs from the surrounding area. Kano is one of Nigeria’s leading industrial
96
centers. Tanning, oil seed processing, meat packing, and the production of furniture
and enamel ware are long-established industries. Population (2006 estimate)
2,163,225.
Zaria, in Kaduna State. a road and rail hub in a major agricultural area. The city
is a market center for locally produced cotton, peanuts, hides and skins, shea nuts,
corn, sorghum, and vegetables. Industries include cotton ginning, peanut and shea-nut
milling, tanning, cotton seed-oil production, and the manufacture of cigarettes,
bicycles, perfumes, and soap. Population 408,198 (2006 estimate).
Sokoto, is the capital of Sokoto State, near the confluence of the Rima and
Sokoto rivers. With an average annual temperature of 28.3° C (82.9° F), it is one of
the world's hottest cities. Sokoto functions as a trade center for the dry savanna region
of north western Nigeria. Rice and onions are cultivated and livestock is raised in the
area. Industries in Sokoto include tanning and leather crafts, pottery, rice milling, and
cement production.Population (2006 estimate) 427,760.
Gusau, in Zamfara State, is located on the Sokoto River in the savanna region
of Nigeria. The river provides access to water supplies during the dry season. Gusau
serves as a major industrial center of Northern Nigeria. Industries in the city include
textile manufacturing, groundnut and tobacco processing, and cotton ginning. The city
is active in mining of gold and diamonds in the surrounding countryside. Gusau
Population (2006) 383,162.
Katsina, the capital of Katsina State, a historic center of trade and learning.
Peanuts, cotton, and hides are collected at Katsina and sent to Kano, for export.
Sorghum, millet, a variety of vegetables, peanuts, indigo, cotton, goats, sheep, cattle,
97
and poultry are traded in the city’s central market. Traditional crafts in Katsina,
include weaving and dyeing, leather work, and metallurgy. Population (2006
estimate)459,022[151].
3.2 Sample Collection
Forty one (41) sheep were each sampled for intestine, kidney, liver and muscle
in abattoirs in major sheep markets in Zaria, Katsina , Kano, Birnin Kebbi ,
Maiduguri, Sokoto and Gusau, between the months of October and November, 2009,
with the help of veterinary doctors. Altogether, one hundred and sixty four samples
(164 samples) of the four different parts were purchased. The samples were put in
polythene bags pre- washed with 1:1 HCl and stored by freezing. They were later
dried in an oven at 105OC, pulverized, and stored in a dessicator prior to digestion.
More details of sampling are given in Table 3.1 below
Table 3.1: Sampling sites and number of samples collected
State Town No of samples
Kaduna Zaria 24
Kano Kano 24
Katsina Katsina 24
Kebbi Berini-kebbi 24
Borno Maiduguri 24
Sokoto Sokoto 20
Zamfara Gausau 24
Total (n)=164
3.3 Cleaning of Glass wares
98
Washing of the glassware and plastic is an important process to avoid any sort
of contamination especially when trace elements or heavy metals are analysed. The
test tube, polythene bottles (for digestion), watch glass (for drying of samples), and
standard flasks (20mL, 100mL, 500mL) glass wares were soaked in water and soap
for two hours and rinsed several times with water. After that, glass wares has been
rinsed once with de-ionzed water, once with mixture consisting of 520mL of
deionized water, 200mL concentrated HCl and 80 ml H2O2 and once with washing
acid(10% HNO3). Finally they were washed with de-ionised water then air dried in the
incubator away from contamination or dust [152].
3.4 Reagents and Glassware
All the chemicals used were analytical reagent grade for both cleaning of
glass ware and digestion of the meat samples. Deionized water of not more than 2µ
Siemens /cm conductivity was used for dilution and rising of laboratory glass wares.
3.5 Preparation of stock solution for the heavy metals
Cadmium; 1.000 g of cadmium was dissolved in a minimum volume of 1:1 nitric
acid. Dilute to 1 litre to give 1000 µg/mL Cd.
Chromium; 1.000 g of chromium metal was dissolved in 1:1 hydrochloric acid with
gentle heating. Cool and dilute to 1 litre to give 1000 µg/mL Cr.
Copper ; 1.000 g of copper metal was dissolved in a minimum volume of 1:1 nitric
acid and dilute to 1 litre to give 1000 µg/mL Cu.
Iron; 1.000 g of metal was dissolved in 20 mL of 1:1 hydrochloric acid and dilute to 1
litre to give 1000 µg/mL Fe.
99
Manganese; 1.000 g manganese was dissolved in a minimum volume of 1:1 nitric
acid and dilute to 1 litre to give 1000 µg/mL Mn.
Nickel; 1.000 g of nickel was dissolved in 1:1 nitric acid and dilute to 1 litre to give
1000 µg/mL Ni.
Lead; 1.000 g of lead was dissolved in 1:1 nitric acid. Dilute to 1 litre to give 1000
µg/mL Pb.
Zinc. One gram of zinc metal was dissolved in one mL of HCl and volume was made
up to one litre with de-ionized water to make 1000 ppm stock solution of zinc.
3.6 Digestion of Samples-Metal recovery experiment
The dry samples were ground using plastic mortar and pestle. Wet digestion of
the samples was done using 2 gram. of the dried samples in 100 mL polyethylene
bottle and 10 mL of the digestion mixture [3 : 2 HNO3(65%, v/v), and HClO4 (70%,
v/v)] were added. The bottles were tightly closed and the contents were gently swirled
and allowed to stand overnight. The samples were heated for 3hrs in a water bath
adjusted to 70oC with occasional swirling at 30 minutes interval to ensure a complete
digestion of the samples[153].
Finally, the digest was allowed to cool and then transferred into a 20 mL
standard flask, risen with de-ionized water and later made up to mark with de-ionized
water .the solution was transferred into acid – leached polyethylene bottles to avoid
contamination and kept at room temperature until analysis with AAS.
3.7 Preparation of mixed standard solution.
2 mL of 100 ppm solution of Pb, Cd, and Zn, 3 mL of 100 ppm of Cr and 5 mL
100
of 100 ppm of Ni, Cu and Fe were pipetted into a 100mL standard flask and made up
to mark with de-ionized water. This gave 2ppm for Pb, Cd and Zn, 3ppm for Cr and
5ppm for Ni, Cu and Fe in the mixed standard.
Samples were spiked with 1mL, 2mL or 3mL of the mixed standard solution to
give various concentrations in 20mL of the digest as presented in Table 4.2(Co-
efficient of variation). Liver and kidney samples were spiked with various solutions of
the heavy metals under study, recovery repeatability tests and for verifying the
analytical methodology, since no certified reference materials (CRM) was available.
For each metal triplicate sample, spiked sample and blank were carried through
digestion reaction. The % recovery was calculated.
X - Y
% Recovery = x 100.
Z
X = conc. of the spiked sample.
Y = conc. of the unspiked sample.
Z = conc. of the metal ion added.
3.8 Sample Analysis
The determination of the metal concentrations were carried out with GBC
Avanta ver 2.02 atomic absorption spectrophotometer (GBC, Australia). Hallo
cathode lamps of cadmium, chromium, iron, nickel, lead, zinc, manganese and copper
was used as a radiation source. Air acetylene gas mixture was used as source of flame.
Maximum absorbance was obtained by adjusting the cathode lamps at specific slit
101
and wavelengths as shown below:-
Table 3.2 Standard Analytical Conditions for Pb, Cd, Zn, Mn, Ni, Cu, Cr and Fe
using a GBC Avanta ver 2.02 AAS
Metal Wavelenght(
nm)
Lamp current
(mA)
Slit width
(nm)
Slit
height
Sensitivity
(µg/mL)
Detection
limit(µg/mL)
Pb 217.0 5 1.0 Normal 0.06 0.0005
Cd 228.8 3 0.5 Normal 0.009 0.0004
Zn 213.9 5 0.5 Normal 0.008 0.0005
Mn 279 5 0.2 Normal 0.02–5 0.28
Ni 232.0 4 0.2 Normal 0.04 0.009
Cu 324.7 3 0.5 Normal 0.025 0.001
Cr 359.3 6 0.2 Normal 0.09 0.003
Fe 248.3 7 0.2 Normal 0.05 0.005
3.9 Statistical Analysis
The statistical analysis was conducted using statistical package of SPSS version
102
17. Significant differences between means were subjected to one way ANOVA using
Duncan’s multiple range test. The level of significance was compared p<0.05.
CHAPTER FOUR
4.0 RESULTS AND DISCUSSION
103
4.1 RESULTS
4.1.1 Moisture Content
Table 4.1: Moisture content of various organs of sheep from the sheep breeding states in
Northern Nigeria.
The moisture content (Table 4.1) ranged from 70.77 to 82.32% in the organs. There
were no significant differences across the states but the intestine and the kidney have
significantly higher moisture (P≤0.05) compared to the liver and muscle. The kidney
has the highest moisture content on the average.
Table 4.2 % Recovery of trace metals from meat samples
States
Intestine
Organs Kidney
Liver
Muscle
Kaduna 80.97 81.08 73.75 79.05
Kebbi 82.14 81.76 72.85 76.48
Kano 82.32 82.21 72.83 76.77
Katisna 81.52 81.11 75.74 80.44
Bornu 81.24 81.16 71.87 76.88
Sokoto 76.95 78.87 72.05 70.77
Zamfara 70.77 80.59 72.63 75.81
Mean ±
79.41
±
80.96
±
73.10
±
76.60
±
Std.dev
4.2201 1.0633 1.3142 3.0409
104
Metals Added
conc.
(µg/mL)
Conc.of
spiked
(µg/mL)
Conc. of
unspiked
(µg/mL)
Recovered
Conc.
(µg/mL)
%
Recovery
Mean % recovery
± s.d
Pb 0.100 0.209 0.109 0.098 98
100.33 ± 2.08 0.212 0.110 0.102 102
0.211 0.110 0.101 101
Cd 0.100 0.759 0.66 0.099 99
99 ± 1.0 0.768 0.67 0.098 98
0.760 0.66 0.100 100
Zn 0.100 0.757 0.657 0.100 100
98.33 ± 7.64 0.760 0.655 0.105 105
0.744 0.654 0.090 90
Ni 0.100 0.159 0.059 0.100 100
99 ± 1.0 0.166 0.068 0.098 98
0.164 0.065 0.099 99
Cu 0.100 1.051 0.953 0.098 98
100.33± 4.04 1.055 0.950 0.105 105
1.050 0.952 0.098 98
Cr 0.100 0.173 0.072 0.101 101
100 ± 1.73 0.176 0.078 0.098 98
0.163 0.62 0.101 101
Mn 0.100 0.221 0.120 0.101 101
99.33 ± 1.52 0.218 0.119 0.099 99
0.215 0.117 0.098 98
Fe 0.100 1.846 1.748 0.098 98
94.0 ± 7.81 1.885 1.800 0.085 85
1.887 1.788 0.099 99
The table above present an excellent recovery of the various metals, ranging
from 85 to 105%.
105
Table 4.3 Co-efficient of variation for the various metals (%RSD)
Element Sample Concentration (precision)
Pb KdL1 0.109 0.53
KdL2 0.110
KdL3 0.110
Cd Ktk1 0.66 0.87
Ktk2 0.67
Ktk3 0.66
Zn Kdk1 0.657 0.17
Kdk2 0.655 Kdk3 0.654
Ni KnL1 0.059 7.16
KnL2 0.068
KnL3 0.065
Cu KeL1 0.953 0.16
KeL2 0.950
KeL3 0.952
Cr MaL1 0.0072 6.27
MaL2 0.078
MaL3 0.062
Mn SoK1 0.120 3.34
So K2 0.119
SoK3 0.117
Fe ZaK1 1.748 1.53
ZaK2 1.800
ZaK3 1.788
The table above present the co-efficient of variation or precision of the various
metals, which varied from 0.53 to 6.27,these precisions are good being less than 10
[154].
106
Table 4.4 Mean Concentrations of Trace Metals in sheep from Kaduna State
(mgkg-1,n=6)
The concentrations of cadmium and nickel followed the same trend, being
highest in kidney and significantly higher p ≤ 0.05 than muscle and liver. The
concentration of these two elements in muscle and liver is significantly greater than
what was recorded in intestine. The concentration of lead is similar in kidney and liver
(1.085 and 1.190mg/kg). This was significantly higher than the concentration found in
intestine and muscle. The concentration of zinc was highest in the muscle (8.0 mg/kg)
this was significantly higher than concentration of zinc in liver, which was equally
higher than what was the value in kidney and intestine.. Liver had the highest
concentration of copper while muscle and intestine had the least value. The
concentration of chromium in liver was significantly higher than other tissues. The
following trend was observed L>K>M>I. Considering manganese its concentration
ranges between 0.3 – 1.5mg/kg in muscle to 1.5mg/kg in intestine. Liver had the
highest concentration of iron 33.4mg/kg this was significantly higher than iron
concentration found in kidney, intestine and muscle.
Tissue
Pb Cd Ni Zn Cu Cr Mn Fe
Intestine 0.837 ±
0.128
0.085 ±
0.064
0.043 ±
0.120
5.998 ±
1.463
0.463 ±
0.163
0.196 ±
0.246
1.457 ±
1.847
22.505 ±
20.253
Kidney 1.190 ±
0.728
0.227 ±
0.149
0.618 ±
0.365
6.548 ±
1.916
1.550 ±
1.272
0.541 ±
0.519
0.651 ±
0.419
24.843 ±
9.347
Liver 1.085 ±
0.419
0.158 ±
0.716
0.445 ±
0.285
7.196 ±
1.638
4.107 ±
3.529
0.934 ±
0.964
0.969 ±
0.504
34.402 ±
17.547
Muscle 0.817 ±
0.419
0.183 ±
0.172
0.494 ±
0.191
7.995 ±
2.050
0.592 ±
0.474
0.509 ±
0.964
0.328 ±
0.108
18.533 ±
5.251
Total 0.982 ±
0.468
0.162 ±
0.127
0.498 ±
0.251
6.934 ±
1.842
1.678 ±
2.313
0.545 ±
0.708
0.851 ±
1.009
25.072 ±
14.798
107
Table 4.5 Mean Concentrations of Trace Metals in sheep from katsina State
(mgkg-1,n=6)
The concentration of lead in muscle and intestine are almost the same as
shown in Table 4.5 above, but it is significantly higher in kidney and lowest in liver.
The concentration of cadmium and nickel showed same trend with the kidney having
the highest and that of liver and intestine. Liver had the highest concentration of zinc
and copper while muscle had the lowest value. The concentration of manganese and
iron followed the same trend with the intestine having the highest concentration and
liver and kidney showing significantly higher. While intestine showed the least
concentration of chromium but muscle had the highest, followed by the kidney and
liver.
Tissues Pb Cd Ni Zn Cu Cr Mn Fe
Intestine 0.741 ±
0.348
0.216 ±
0.360
0.465 ±
0.193
5.788 ±
1.417
0.508 ±
0.074
0.235 ±
0.192
2.726 ±
2.238
34.380 ±
15.669
Kidney 0.811 ±
0.518
0.661 ±
1.245
0.465 ±
0.193
6.878 ±
2.206
1.293 ±
0.670
0.455 ±
0.646
0.625 ±
0.177
19.761 ±
6.026
Liver 0.688 ±
0.091
0.158 ±
0.088
0.445 ±
0.068
9.105 ±
1.511
1.293 ±
0.670
0.113 ±
0.024
1.268 ±
0.816
30.305 ±
19.725
Muscle 0.716 ±
0.314
0.076 ±
0.032
0.388 ±
0.050
5.390 ±
1.097
0.480 ±
0.765
0.483 ±
0.765
0.551 ±
0.407
15.946 ±
6.153
Total 0.739 ±
0.331
0.278 ±
0.648
0.459 ±
0.196
6.790 ±
2.103
1.279 ±
1.832
0.321 ±
0.504
1.292 ±
1.439
25.223 ±
14.639
108
Table 4.6 Mean Concentrations of Trace Metals in tissues of sheep from Kano State
(mgkg-1,n=6)
In the Kano samples, as shown in Table 4.6 above, the concentration of lead
and nickel is similar in kidney and muscle and closely followed, the liver samples.
The concentrations of other elements in muscle is quite insignificant, in the same vein
the concentration of all the elements in intestine are also insignificant except in
cadmium, were it showed the highest concentration. Copper, zinc, manganese and iron
have highest concentration in liver.
Tissues Pb Cd Ni Zn Cu Cr Mn Fe
Intestine 0.630 ±
0.212
0.558 ±
0.183
0.420 ±
1.125
4.101 ±
0.458
0.545 ±
0.479
0.633 ±
0.712
0.738 ±
0.570
13.633 ±
8.141
Kidney 0.731 ±
0.408
0.136 ±
0.063
0.746 ±
0.112
5.475 ±
1.581
1.125 ±
0.295
0.700 ±
0.171
0.565 ±
0.099
15.183 ±
0.010
Liver 0.678 ±
0.201
0.130 ±
0.076
0.593 ±
0.288
7.678 ±
0.462
6.091 ±
5.831
0.111 ±
0.135
0.931 ±
0.159
26.606 ±
8.516
Muscle 0.701 ±
0.234
0.223 ±
0.370
0.470 ±
0.212
6.870 ±
1.373
0.446 ±
0.935
0.008 ±
0.020
0.636 ±
0.194
9.926 ±
1.976
Total 0.685 ±
0.261
0.137 ±
0.189
0.557 ±
0.225
6.131 ±
1.666
2.052 ±
3.635
0.205 ±
0.431
0.717 ±
0.326
16.330 ±
8.660
109
Table 4.7 Mean Concentrations of Trace Metals in tissues of sheep from Kebbi State
(mgkg-1,n=6)
Table 4.7 above showed the metal concentration in the tissues from Kebbi
State ranging from 0.74 in muscle to 1.38mg/kg in kidney for lead; 0.11 in muscle to
0.44 in kidney for cadmium, 0.49 mg/kg in muscle to 1.21mg/kg in kidney for nickel ,
while for zinc ranges from 6.18mg/kg in intestine to 8.67mg/kg in kidney. Copper
concentration range from 0.75mg/kg in intestine to 9.53mg/kg in liver. In the case of
chromium it is 0.67mg/kg in muscle to 2.80mg/kg in kidney. The variations of
manganese is 0.59 mg/kg to 1.44 mg/kg in kidney and iron concentration is
13.08mg/kg in muscle to 29.82mg/kg in liver. On the whole, the concentration of
these element lead, cadmium, nickel, zinc, chromium and manganese is highest in
kidney and lowest in muscle.
Tissues Pb Cd Ni Zn Cu Cr Mn Fe
Intestine 1.259 ±
1.120
0.133 ±
0.155
0.722 ±
0.513
6.183 ±
2.887
0.750 ±
0.665
1.408 ±
0.128
1.203 ±
0.453
14.461 ±
8.078
Kidney 1.383 ±
0.706
0.448 ±
0.297
1.208 ±
1.265
8.671 ±
2.398
1.738 ±
1.520
2.800 ±
6.526
1.441 ±
1.453
19.343 ±
12.528
Liver 1.111 ±
0.402
0.228 ±
0.037
0.790 ±
0.546
8.560 ±
1.462
9.533 ±
8.779
0.898 ±
0.914
1.321 ±
0.459
29.820 ±
12.763
Muscle 0.740 ±
0.334
0.111 ±
0.049
0.493 ±
0.143
8.560 ±
1.462
0.875 ±
0.228
0.763 ±
1.422
0.586 ±
0.251
13.081 ±
6.458
Total 1.124 ±
0.708
0.230 ±
0.209
0.803 ±
0.734
7.714 ±
2.802
3.224 ±
5.600
1.467 ±
3.561
1.138 ±
0.822
19.176 ±
11.740
110
Table 4.8 Mean Concentrations of Trace Metals in tissues of sheep from Borno State
(mgkg-1,n=6)
Lead concentration is highest in kidney followed by liver and least in intestine,
as shown in Table 4.8 above. The concentration of zinc, copper and iron followed the
same trend though significantly highest in liver and kidney and least in muscle and
intestine. The concentration of nickel, cadmium, manganese is highest in liver and
kidney and least in intestine. On the average the concentration of almost all the
element showed highest value in liver tissues and the least was found in intestine.
Tissue Pb Cd Ni Zn Cu Cr Mn Fe
Intestine 0.576 ±
0.304
0.061 ±
0.019
0.603 ±
0.771
4.477 ±
0.902
0.723 ±
0.912
0.400 ±
0.770
0.425 ±
0.188
8.781 ±
5.757
Kidney 0.818 ±
0.465
0.125 ±
0.049
0.665 ±
0.605
5.541 ±
0.872
1.268 ±
1.029
0.186 ±
0.344
0.473 ±
0.068
14.290 ±
5.560
Liver 0.575 ±
0.287
0.260 ±
0.422
0.413 ±
0.102
7.720 ±
1.238
1.793 ±
1.092
0.071 ±
0.175
0.750 ±
0.183
22.667 ±
12.763
Muscle 0.670 ±
0.102
0.075 ±
0.040
0.358 ±
0.113
7.395 ±
1.294
1.488 ±
1.825
0.236 ±
0.283
0.331 ±
0.178
17.528 ±
11.311
Total 0.660 ±
0.312
0.130 ±
0.214
0.510 ±
0.481
6.283 ±
1.701
1.318 ±
1.229
0.223 ±
0.440
0.526 ±
0.260
15.816 ±
9.849
111
Table 4.9 Mean Concentrations of Trace Metals in tissues of sheep from Sokoto State
(mgkg-1,n=5)
The concentration of lead is significant throughout the tissues, with liver
having the highest and muscle the lowest, as shown in Table 4.9. There is a similar
trend in the concentration of cadmium and copper with liver having the highest value
and muscle the least. Nickel and manganese showed highest concentration in intestine
and least in muscle, though the metals are significant in all the tissues. Chromium has
the highest concentration in intestine and least in kidney.
Tissues Pb Cd Ni Zn Cu Cr Mn Fe
Intestine 0.722 ±
0.081
0.113 ±
0.448
0.700 ±
0.361
5.794 ±
1.145
0.703 ±
0.380
0.198 ±
3.243
2.757 ±
3.408
15.902 ±
9.536
Kidney
0.664 ±
0.179
0.137 ±
0.059
0.400 ±
0.103
5.981 ±
1.097
1.935 ±
1.967
0.134 ±
0.299
1.199 ±
0.906
28.114 ±
17.647
Liver 0.770 ±
0.316
0.166 ±
0.109
0.445 ±
0.047
7.413 ±
1.056
6.102 ±
4.570
0.186 ±
0.209
0.978 ±
0.249
20.030 ±
8.999
Muscle 0.432 ±
0.173
0.056 ±
0.018
0.405 ±
0.146
6.840 ±
1.288
0.743 ±
0.239
0.176 ±
0.394
0.456 ±
0.169
11.942 ±
3.2654
Total 0.647 ±
0.320
0.118 ±
0.074
0.487 ±
0.225
6.507 ±
1.244
2.371 ±
3.224
0.173 ±
0.272
1.348 ±
0.846
18.997 ±
11.901
112
Table 4.10 Mean concentrations of Trace Metals in tissues of sheep from Zamfara
State (mgkg-1,n=6)
Tissues Pb Cd Ni Zn Cu Cr Mn Fe
Intestine 0.830 ±
0.244
0.107 ±
0.035
0.438 ±
0.119
6.352 ±
1.198
0.562 ±
0.207
0.126 ±
0.231
4.601 ±
4.265
15.91 ±
7.372
Kidney
0.814 ± 0.169
0.082 ± 0.017
0.694 ±
0.641
5.288 ±
0.441
1.165 ±
0.248
0.418 ±
0.490
0.598 ±
0.206
17.467 ±
7.841
Liver 0.801 ± 0.108
0.122 ± 0.025
0.662 ±
0.236
7.660 ±
1.212
5.6475 ±
4.0737
1.052 ±
1.636
1.084 ±
0.291
18.542 ±
3.159
Muscle 0.995 ± 0.571
0.122 ± 0.047
0.575 ±
0.196
8.986 ±
2.245
0.898 ±
0.474
1.212 ±
1.322
0.423 ±
0.139
13.619 ±
3.609
Total 0.860 ± 0.314
0.108 ± 0.035
0.592 ±
0.351
7.073 ±
1.945
2.068 ±
2.860
0.702 ±
1.110
1.676 ±
2.650
16.385 ±
5.810
Table 4.10 above; from Zamfara State showed that the muscle has the highest
concentration of lead, cadmium, zinc and chromium and least in intestine. Considering
copper and iron the concentration is highest in the liver and lowest in the muscle and
intestine, and the concentration of copper ranges from 5.6 mg/kg in liver to 0.56
mg/kg in intestine. While the concentration of iron ranges from 18.54mg/kg in liver to
13.62mg/kg found in muscle. Manganese concentration is highest in intestine having a
value of 4.60mg/kg to 0.42mg/kg in muscle.
113
Table 4.10: Overall mean Concentration of Metals in Sheep (mgkg-1
)
Comparing the States together as shown in Table 4.11 above, Kebbi State has
the highest concentration of the metals in their samples these are lead, nickel, zinc,
copper and chromium. While Sokoto State has the lowest, seen in lead, cadmium and
chromium.
States Pb Cd Ni Zn Cu Cr Mn Fe
Kaduna 0.982 ±
0.468
0.162 ±
0.127
0.498 ±
0.251
6.934 ±
1.842
1.678 ±
2.313
0.545 ±
0.708
0.851 ±
1.009
25.072 ±
14.798
Katsina 0.739 ±
0.331
0.278 ±
0.648
0.459 ±
0.196
6.790 ±
2.103
1.279 ±
1.832
0.320 ±
0.504
1.292 ±
1.439
25.223 ±
14.639
Kano 0.685 ±
0.261
0.137 ±
0.189
0.555 ±
0.225
6.131 ±
1.666
2.052 ±
3.634
0.205 ±
0.431
0.717 ±
0.326
16.337 ±
8.660
Kebbi 1.124 ±
0.708
0.230 ±
0.209
0.803 ±
0.734
7.714 ±
2.802
3.224 ±
5.600
1.467 ±
3.561
1.138 ±
0.822
19.176 ±
11.740
Borno 0.660 ±
0.312
0.130 ±
0.214
0.510 ±
0.481
6.283 ±
1.701
1.318 ±
1.229
0.223 ±
0.440
0.526 ±
0.260
15.81 ±
9.849
Sokoto 0.647
± 0.230
0.118
± 0.074
0.487
± 0.225
6.507
± 1.244
2.371
± 3.224
0.173
± 0.272
1.348
± 1.846
18.997
± 11.901
Zamfara 0.860
± 0.314
0.108
± 0.035
0.592
± 0.351
7.073
± 1.945
2.068
± 2.860
0.702
± 1.110
1.676
± 2.650
16.385
± 5.810
114
Table :- 4.12 Comparison of mean elemental concentration of mutton in
present study with values in other studies(mgkg-1
)
Countries Meat Parts
Pb Cd Zn Mn Cu Ni Cr Fe Reference
Pakistan (Lahore )
Liver 4.25 0.41 58.49 - 318.82 - Mariam et al (2004)
Kidney 3.85 0.45 1.38 6.40
Muscle 4.25 0.37 5.82 5.01
Muscle 0.40 0.02 0.27 0.74 0.65 Talib(1991)
Netherland Liver 0.85 0.054 Ger Vos et al (1998)
Kidney 0.36 0.098
Muscle 0.04 0.003
Australia Liver 5.69
Kidney 4.59
Jordan Liver 4.52 Labib A. et al (2006)
Kidney 3.87
Nigeria (Maiduguri
Liver 0.16 0.76 2.34 2.76 0.54 0.09 0.76 3.76 Akan et al (2010)
Kidney 0.08 0.34 1.76 2.04 0.34 0.004 0.65 3.07
Spain Meat 1.35 1.22 Gonzalezweller et al
(2006)
Jordan (North) A
B
C
Liver 0.8 0.17 24.0 107.80 Mutaz Al-alawi
(2008) Kidney 0.96 0.64 18.50 3.80
Liver 0.71 0.32 26.50 52.40
Kidney 0.72 1.23 23.40 3.70
Liver 0.82 0.18 14.40 73.50
Kidney 0.87 0.60 10.30 3.80
Nigeria
Intestine 0.79 0.17 5.52 1.98 0.60 0.48 0.45 17.93 Present study
Kidney 0.91 0.25 6.33 0.79 1.43 0.68 1.00 19.85
Liver 0.81 0.18 7.90 1.04 4.93 0.54 0.52 21.83
Muscle 0.72 0.12 7.43 0.47 0.78 0.45 0.48 14.36
115
According to the result of one way ANOVA in Table 4.4; the metal concentrations in
the organs did not show significant (difference) variation at p < 0.05 in the four
organs except for copper in liver. Among the Kaduna samples, the highest
concentration was for iron (34.403±17.847) in liver and the lowest for cadmium
( 0.80±0.064), in the intestine. In general the mean concentration of the trace metals
from Kaduna samples were found to decrease in the order Fe(25.072±14.798) >
Zn(6.935±1.843) > Cu(1.678±2.313) > Pb(0.983±0.464) > Mn(0.852±1.009) >
Cr(0.546±0.709) > Ni(0.498±0.252) > Cd(0.163±0.128). For the kaduna samples
only Cr was found to be below the instrumental detection limit in about 10
samples(41.07%) as shown in (Appendix 2a).
For samples collected from Katsina State, Duncan multiple range tests showed
that lead, cadmium, nickel, and chromium have concentration that is not significantly
different at p < 0.05 in the four organs as shown in Table 4.5. But the concentrations
of zinc, copper, manganese and iron showed significant difference in the four organs.
Using Duncan multiple range test in the concentrations of zinc, copper, manganese
and iron, significant difference p < 0.05 occur mainly in the liver and intestine. The
mean concentrations of the metals were found to increase in the order
Cd(0.217±0.360) < Cr (0.235±0.192) < Ni (0.465 ±0.193) < Cu (0.508±0.074) < Pb
(0.741±0.348) < Mn (2.726±2.238) < Zn (5.788±1.417) <Fe(34.380±15.669) in the
intestine. While in the kidney Cr (0.455± 0.646) < Ni (0.465 ±0.193) < Mn
(0.625±0.177) < Cd (0.66±1.245) < Pb (0.811± 0.518) < Cu (1.293 ±0.670) < Zn
(6.878±2.206) < Fe (19.761±6.026). In the liver the concentration of Cd (0.158
±0.088) < Ni (0.445±0.068) < Cr (0.455±0.646) < Pb (0.688±0.091) < Mn (1.268
116
± 0.816) < Cu (1.293±0.670) < Zn (9.105±1.511) < Fe (30.805±19.725).
The result of one way ANOVA for Kano samples in Table 4.6 showed that
zinc, nickel, copper, chromium and iron concentrations significantly varied in the
organs (p < 0.05). The metal with highest mean concentration was iron 16.338±8.661
and the lowest was cadmium (Cd) 0.137±0.189. In their mean concentration the metal
varies from Fe (16.330±8.660) > Zn (6.131±1.666) > Cu (2.052 ± 3.635) > Mn
(0.718±0.326) > Pb (0.685±0.261) > Ni (0.557±0.225) > Cr (0.206 ±0.431) > Cd
(0.137±0.189).
Result summarized in Table 4.7 for Kebbi showed; the highest lead levels in
the kidney, intestine and liver, the highest cadmium level in the kidney and liver, the
lowest iron level was observed in the muscle and intestine, a slight decrease in zinc
level in the intestine and muscle. Manganese showed highest concentration in the
kidney and lowest in the muscle. The lowest copper level also in the intestine and
muscle, nickel shows increase in kidney and intestine. Statistically cadmium and
copper, showed significantly high values(p<0.05) in kidney, while iron was in liver.
Samples from Borno (Table 4.8) showed highest level lead, cadmium and
nickel in kidney. Cadmium was also highest in liver. The lowest levels of metals
were copper in intestine, chromium in liver and iron in intestine( p<0.05). The total
mean metal concentration varies, generally with Cd (0.130±0.214) < Cr
(0.223±0.440) < Ni (0.510±0.481) < Mn (0.526±0.260) < Pb (0.660±0.312) < Cu
(1.318±1.229) < Zn (6.283±1.701) < Fe (15.816±9.849).
Samples from Sokoto showed higher lead, nickel and cadmium in intestine as
117
compared with other organs,(Table 4.9). zinc and copper were lower in muscle and
intestine, chromium level was almost same values in all the others samples (tissues).
The lowest manganese level was observed in the muscle and the liver while iron was
in muscle and the intestine.
The result presented for Zamfara State in Table 4.10, showed highest lead level
in muscle, highest Cadmium in liver and muscle, the nicked was highest in kidney
and liver. Lowest zinc level in kidney, copper and Cr levels in the intestine, the
muscle showed a lower level of the iron, compared to other organs (samples).
4.2 Discussion
The mean moisture content shown in Table 4.1 indicates that kidney has the
highest moisture and liver has the lowest. The values favourably compared with
literature data obtained for mutton and other ruminants from different areas of the
world and with international set guideline values. Acid digestion procedures were
employed for the determination of the elements, in order to completely transfer the
analytes into solution and avoid loss of volatile metals e.g zinc and cadmium. The
goal of every digestion process is to acheive complete decomposition of solid (matrix)
while avoiding loss or contamination of the analyte. Digestion in the cold, using
mixture of nitric acid and perchloric acids in a closed vessels present excellent
recovery ranging from 85% to 105% for all the metals(Table 4.2).The good triplicate
recoveries obtained, validates the methodology.
Precision was determined by running selected samples several times and
calculating the co-efficient of variation. Precision is a measure of the spread or
118
dispersion of a set of results from the mean. Precision applies to a set of replicate
measurements and indicate how the individual members of that set are distributed
about the mean value. Thus, indicates the repeatability or reproducibility of
measurements using the tested method as shown in Table 4.3. The values obtained
were all less than 10% showing high precision[155].
Among the eight metals analyzed from seven the States iron has the highest
mean concentration (25.222±14.640),while cadmium was lowest (0.109 ±0.036). In
this study the concentration of the metals were generally in the order Fe (25.222
14.640) > Zn(7.715 ± 2.802) > Cu (3.224 ± 5.600) > Mn (1.677 ± 2.650) > Cr (1.468
± 3.562) > Pb (1.124 ± 0.709) > Ni (0804 ± 0.734) > Cd (0.278 ± 0.648).
Iron concentrations in intestine range from (34.80 ± 15.670) Katsina to
(8.782 ± 5.758) Borno.For Kidney, it ranged between (28.114 ± 17.647) Sokoto to
(14.290 ± 5.561) Borno. Liver concentration lies between 34.402 ± 17.847 to
18.542 ± 3.159 (Zamfara). Muscle was between 18.538 ± 5.351mg-1 (Kaduna) to
9.927 ± 1.976mg kg-1 (Kano).
The zinc concentration for the intestine ranges from 6.352 ± 1.198 (Zamfara) to
4.102 ± 0.459 (Kano). The kidney zinc concentration lies between 8.672 ± 4.398 for
Kebbi to 5.289 ± 0.442 from Kaduna. For liver its 9.105 ± 1.511 (Katsina) to
7.197 ± 1.639 (Kaduna). Muscle’s range is from 8.987 ± 2.246 to 5.390 ± 1.097 from
Zamfara and Katsina respectively.
The least concentration of Copper (Cu) in intestine was 0.463 ± 0.163
(Kaduna) and the highest from Kebbi in 0.750 ± 0.665. The highest copper
119
concentration for kidney was from Sokoto (1.935 ± 1.967) and the lowest from Kano
was 1.125 ± 0.296. The liver from Kebbi has the highest concentration of
9.533 ± 8.780 and 1.293 ± 0.671 for Katsina. Muscle was from 1.488 ± 1.825 (Borno)
to 0.380 ± 0.139 (Katsina).
The Manganese content in intestine was in the range of 4.602 ± 4.602 ± 4.265
(Zamfara) to 0.425 ± 0.189 (Borno). The kidney from Kebbi has the highest of
1.442 ± 1.442 and the lowest for Borno 0.473 ± 0.068. For liver it ranges from
1.322 ± 0.459 (Kebbi) to 0.750 ± 0.183 (Borno),while that of muscle was from
0.637 ± 0.195 (Kano) to 0.328 ± 0.109 (Kaduna).
Almost half of tissues analyzed for chromium were below detection limit of the
instrument, which could be the reasons for observed values and its standard
deviations. The highest mean concentration in the intestine (1.408 ± 0.128) (Kebbi) to
0.126 ± 0.231 (Zamfara). The kidney was 2.800 ± 6.526 (Kebbi) to 0.700 ± 0.171
(Kano). Liver from Zamfara was 1.052 ± 1.536 to 0.071 ± 0.175, in Borno. Muscle
was 1.212 ± 1.022 (Zamfara) to 0.008 ± 0.020 (Kano).
The lead content in the intestine was 1.259 + 1.121 (Kebbi) to 0.577 + 0.304
(Maiduguri) Kidney was from 1.38+0.706 (Kebbi) to Sokoto’s (0.664 + 0.180) Liver’s
concentration ranged 1.112 + 0.403 (Kebbi) to 0.575 + 0.287 (Borno). In Muscle its
from 0.996 + 0.571 (Zamfara) to 0.432 + 0.173 (Sokoto).
Nickel concentration in the intestine was 0.722 + 0.514 (Kebbi) to 0.420 +
0.125 (Kano), liver 0.790 + 0.546 (Kebbi) to 0.413 + 0.103 (Borno), Kidney war from
1.208 + 1.256 (Kebbi) to 0.400 + 0.104 (Sokoto) and Muscle was from 0.575 + 0.196
120
(Zamfara) to 0.358 + 0.113 (Borno).
Cadium being the least varied in the intestine from 0.217 + 0.361 (Katsina) to
0.058 + 0.183 (Kano); Kidney’s was from 0.661 + 1.245 (Katsina) to 0.083 + 0.018
(Zamfara) while liver was 0.260 + or 0.042 from Borno to 0.130 + 0.077 from Kano
and for muscle it was Kano (0.22 + 0.371) to Sokoto (0.056 + 0.018).
The result for trace elements as shown in Table 4.4 – 4.10, indicate high values
of the essential elements; Fe, Mn, Cu and Zn, than the rest especially the toxic ones;
Pb, Cd and Ni. Fe occurs naturally in soil,water, plants and animals. The concentration
of Fe in the sample showed significant variability (P< 0.05) among the animals from
different states.
Nickel being a border line element, is essential at trace levels for human health.
Acute toxicity of the metal arises from it’s competitive interaction with five major
essential elements namely; Ca, Co, Cu, Fe, and Zn [156].
The mean concentration of Ni in intestine from Kaduna is far less than in
samples from other states. These values from Sokoto and Kebbi samples are
significantly high (P<0.05).The Kano, Kastina and Zamfara animals carried relatively
the same burdens of metals .The level of Ni ranged between 0.4133 ±0.102 to 0.7900
± 0.546 for liver and this is in agreement with concentrations obtained of of buffalo
liver reported by Nasser et al [157]. The highest concentration of Ni was found in
kidneys from Kebbi State (1.208 ± 1.25) and the lowest found in the muscle from
Borno (0.3583 ± 0.113), which is within the values earlier reported for sheep in
Borno[159] and for goats, in Bangladesh[158].
121
Of all the samples, 66.66% of the liver from Sokoto, 50% from Kebbi, 33.33%
from Kaduna, Kastina and Kano and 16.67% from Zamfara and Borno contain Ni in
excess of the USSR permissible limit of 0.5 mg/kg in meat and meat products[160].
Nickel showed strong significant correlation (negative) with chromium in the kidney
and muscle (Appendix 4) at p<0.01and with copper at p<0.05 in the liver. These are
essential elements and constitute part of the expected components of the sample.
The results obtained in the present study showed that, the States where the
samples were obtained, the liver samples has the highest mean concentrations of Cu
followed by the kidneys, then muscle and intestines. This is in agreement with other
studies from Poland [161], Jordan [162], Spain [163], Nigeria [159] and Pakistan
[164]. Cu is an essential component of various enzymes and plays key roles in bone
formation, skeletal mineralization and in maintaining the integrity of the connective
tissues. It is essential element but its concentration in the liver of the studied animal is
very low. Though the mean value of Cu (3.22) in the present study is below the
permissible limit of 200ppm (ANZFA) [165], but it is higher than (0.27± 0.12)
reported by Talib Hussain [166] in muscle. Pathological conditions known as
chalcosis may occur due to high concentration of copper , mainly in liver, kidney and
blood occur. These pathological states are caused by either an uptake of an excessive
amount of Cu or by feed containing normal amount of Cu but low amount of Mo or
sulphur radicals [167]. In contrast, lack of Cu causes disturbances which is
characterised by swayback in sheep.
Chromium(iii) is useful in enhances up the action of insulin [167]. But Cr(iv) is
122
carcinogenic for organisms [168]. Concentration of Cr studied in the present work
agreed with Zahurul et al [158], though a little higher than the permissible limit of
0.01mg/kg(WHO,2002) [169] while the highest concentration of Cr is found in the
kidney was(2.800 ± 6.526). The result from the present study is however lower than
those reported in the literature as 47% of the samples all over the States were below
detection limit (BDL), especially in Kano samples, an indication that the animals there
are chromium deficient, since they are free rangers,they may feed more from refuse
dumps. All in all, Cr. frequency in the present work are as follows 60.98 %
(intestine), 48.75% (kidney), 58.5% (liver), and 46.34% (muscle) from Katsina,
Zamfara, Kebbi and Katsina respectively, were found in the liver as reported by
Zahurul et al [158]. Cr is stored in the liver, spleen and soft tissues, the value of the
present study is in agreement with Ihedioha and Okoye[171]. Both human and
laboratory animal studies shows that intestinal chromium absorption is very minute.
The same study on mice showed that Zn administration reduces chromium
absorption.[172], this could also be seen in the Pearson correlations which showed
that Cr correlate negatively with Zn in the liver at p<0.01 . The high concentration
could be as a result of pollution of soil which occurs as a result of the dumping of
chromate waste such as those from tanneries, electroplating manufacturing industries
and textile industries [173].
It is well a known fact, that Manganese activates many enzymes such as
phosphoglucomutase, cholineslerase, oxidative α-Keto-decarboxylase and ATPase in
the muscle. Mn was detectable in the entire sample but the highest concentration
found in the intestine (4.6015 ± 4.26) obtained from Zamfara State is more than any
123
other State. There is a relative higher concentration in the liver across the State more
than in any other organ. Mn occurs in the body principally in the liver, bone and
kidney [166]. The present study also established that concentration of Mn were below
the upper tolerable intake for humans. The major source of Mn in soil is fertilizer,
sewage, sludge and ferrous smelter and thus finds their way into the forage that the
animal feed on. Other sources of other metals have been attributed to vehicles and
industry, such as copper, iron, and manganese from vehicle break pad use and general
engine wear [175].
Relatively, the lead(Pb) concentrations was detectable in all the parts and it
was found that liver and kidney showed highest concentrations of (1.387 ± 0.706 )
from Kebbi State and lowest concentrations of (0.4322 ± 0.173) in the muscle from
Sokoto State. The results showed that the Pb concentrations in kidney and liver from
Kaduna and Kebbi States only were higher than the permissible limit of 1ppm
(ANZFA) [165], and exceeded the codex standard of 0.1mgkg-1 for muscle. The
result of this study were higher than 0.8 ±0.06, 0.71 ± 0.07 and 0.82 ± 0.07 in liver
and 0.9 ± 0.07, 0.72 ± 0.06, and 0.87± 0.04 in the kidney from Northern Jordan[162];
0.85,0.36 in liver and kidney from the work of Ger-Vos et al [176] respectively and
0.16 ± 0.02,0.08 ± 0.05 in liver and kidney obtained from Zahurul et al [158].
However, the values of the present study are lower than reported values from Pakistan
(Lahore) [164], and Jordan [152]. The accumulation of Pb in the muscle, showed that
about 33.33%, from Zamfara exceeded the permissible limit, 50% of the intestine,
66.66% of the kidney from Kebbi State and 50% of the liver from Kaduna State
exceeded the permissible limit. This is an indication that Zamfara and the neighboring
124
States are justifying the current lead poisoning in Zamfara State. The toxicity of lead
(Pb) is attributed to the fact that it interferes with the normal function of number of
enzymes. Bipolar Pb forms strong bonds with enzymes bearing sulphudry groups thus
inhibiting their action [162]. Pb correlate with Cr in the muscle at p<0.05.
The mean concentration of cadmium (Cd) in all the meat samples from the
seven states were lower than values reported by Okoye and Ugwu [177], and Gerber
et al [178]. When the present work was compared to other studies reported from other
countries, it was found that the mean concentrations were higher than that of Jordan
[162], Spain [163], and Switzerland [178]. However, the values in the present work
were lower than those reported from Pakistan [164] and Netherlands [176]. The trend
or variation shown in the present study; is kidney > Liver > Muscle > intestine is
comparable to study reported by Irfana et al [164]. It is also important to note that
only one liver sample from Maiduguri, has Cadmium concentration more than 1ppm,
while a larger percentage has concentration of 0.5ppm; the permissible limit for Cd.
Only one kidney sample from Kaduna contained more than 1ppm and very small
percentage has concentration of between 0.5-1ppm. Generally, the lower level of Cd
in all the tissues from the States is in line with findings of Okunola et al [179] and
Ayodele and Oluyomi [180] on soil from Kano.
Zinc (Zn) is an essential trace element for animals, being one of the most
abundant essential elements in the body, it is a constituent of all cells and several
enzymes depend upon it as a co-factor [181]. Though too little of Zn can cause
problems, however too much of Zn is harmful to human health [182]. Zinc
concentrations was found to be higher in the liver and muscle from Katsina and
125
Zamfara States, while the least value is found in the intestine. The values are relatively
low when compared to the permissible limit of 150ppm (ANZFA, 2001) [165].It has
been equally reported the Zn and copper intoxification by industrial emission in the
livers, kidneys, spleen, musculature and in the ovaries and uterus of some
experimental sheep[183]. Results showed that the highest concentration of Zinc in the
experimental animals, was in the liver and kidneys. The Zinc concentration in the liver
and kidney of the present work is higher than the reported value of 2.3 ± 0.08, and
1.76 ± 0.02 for liver and kidney respectively by Joszef et al [183]. If we assume that
normal concentration of Zn in the livers are 35 – 45 ppm [39]. It is apparent that in all
the seven States, zinc concentration in sheep tissues was low as reported by Mu”taz
Al-Alwi[162],also in Canada[184] and Poland[163] where the concentration of Zn in
kidney and liver of sheep ranged from 23 – 147.2 ppm and 32 – 82.2 ppm.
Considering the other tissues; the intestine and muscle the Zn concentration is low.
The low concentration of Zn may be attributed to Zn deficient soil, consequently the
fodder / cereals available. Though it was also reported by Gerber et al [178], that the
possible explanation to the low level of Zn could be that the tissues concentration of
this element is primarily genetically determined and is only slightly influenced by
feed. Calcium and Phosphorus affect Zn absorption because they form non-
absorbable complexes. Zn strongly correlate with Cr at p<0.05 in the liver, while it
correlate with Cu in the rest of the tissues.
Iron (Fe) is the most abundant transition metal and probably the most well
known metal in biological systems as reported by ATSDR [182].the concentration of
iron in the samples showed significant difference variability (P < 0.05) among the
126
different States. In all the states the concentrations of Fe in liver were significantly
higher (P< 0.05) than in other samples except for kidney. The levels of iron in the
States were higher than those reported in the literature. Among the States the mean
concentration of Fe from Katsina showed the highest in intestine. The higher value of
Fe may be due to the efficiency in accumulating metals during intake of feeds and the
ability of the organ to concentrate the metal in the body from the environment.
The high concentration of Fe in the present work is supported by the work of
Ayodele and Oluyomi [180], who equally reported high concentration of Fe on the
grasses and soil as a direct deposition of metals which is similar to other results earlier
reported by Uwagbue and Hymone [185]. The result of the t-test conducted showed
similar variations among the metals as reported above when compared with the
standard limit.
127
CHAPTER FIVE
5.1 CONCLUSION AND RECOMMENDATION
Due to toxicities, persistence and bioaccumulation problems, heavy metals
become one of the most serious pollutants in our natural environment. Heavy metals
have become a significant figure of concern for scientist in the various fields
associated with the environment, as well as a concern of the general public.
Since heavy metals can be transferred through food chain, there is a potential risk
for ruminant animals grazing in contaminated areas. Increasing industrialization has
been accompanied throughout the world by the extraction and distribution of mineral
substances from their natural deposits. The use of fertilizers and metal based
pesticides in agriculture are also responsible for the contamination with Cu and Zn.
Hence, the need to reduce their usage.
This study, which found important differences in heavy metals levels in the
investigated region, allows a certain generalisation as to the solution of problems
regarding contamination of mutton, with respect to the effects of environmental
factors. If the results of this study are communicated to the populace, it will
contribute to important developments both in reducing and control of diseases directly
related to the lack of heavy metals, and unnecessary elevated levels of heavy metals.
The results of this study supply valuable information about the metal contents in
mutton within the area covered in Nigeria. Moreover, these results can also be used to
test the chemical quality of mutton in order to evaluate the possible risk associated
with their consumption by humans. Moreover, in order to safe consumer from health
risk, further works should be carried out to monitor the sources of the metal more
128
closely in areas where sheep are breed and the meat consumed, in order to reduce the
levels and to provide adequate protection for human health.
The study reveals that mutton is a good source of macro and micro nutrients
and also most of the studied tissues contain the toxic elements within consumable
limits. But some of the tissues bear noticeable amount of toxic metals such as Pb, Cd,
Cr and Ni. The present study suggests avoiding those tissues as much as possible.
Moreover, concerned authority should take necessary steps for reducing the toxic
metal contamination in the food chain. Of all the States, Kebbi samples contain more
of the metals load inferring contamination.
5.2 CONTRIBUTIONS TO KNOWLEDGE
This study has made the following contributions to knowledge with respect to trace
metals in mutton and edible offal of sheep:
(i) Base-line levels of the essential metals (Zn, Cu, Cr, Ni, Mn and Fe) and toxic
metals (Pb and Cd) in sheep have been reported.
(ii) The safety of the consumption of mutton has been evaluated based on the
metals concentration and distribution in the tissues.
(iii) The base- line data of the studied metals in the environment of the major sheep
breeding areas of Nigeria have been reported.
(iv) The distribution in the environment showed that most of the contaminations in
the tissues occur from non-point sources indicating wide spread and occasional
intense contamination.
129
(v) From the study, it is clear that the intestine, liver and kidney contain the highest
metal load and should be avoided as much as possible.
RECOMMENDATIONS
(i) For food safety, it would be advisable to establish maximum residual
limit for the various metals, not only for meat and meat products, but for
all foods.
(ii) Further studies are necessary to evaluate the daily intake of these heavy
metals from sheep sources in various states in Nigeria, since it is the north
which supplies these animals to the south.
(iii) Continuous monitoring and evaluation of levels of toxic metals in soil,
water, air, fodder, and any route of toxic metals into the meat is necessary,
since reducing exposure is the simplest and most cost-effective way to
prevent toxic metal problems.
(iv) Efforts should be made to clean up the water, food (grasses) and air
especially Kebbi State.
(v) The public should be educated on the dangers of toxic metals and how to
avoid them.
130
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APPENDIX 1
1a
CONCENTRATIONS OF TRACE METALS IN SHEEP FROM VARIOUS STATES Mg/kg(Dry weight)
Pb Cd Ni Zn Cu Cr Mn Fe KADUNA
Intestine 5.6 0.45 2.17 33.14 2.02 1.82 2.69 82.83
Kidney 5.604 1.08 1.5 33 7.4 0 1.99 75.4
Liver 2.791 0.547 0.886 29.72 4.99 9.2 2.46 79.35
Muscle 4.88 0.44 3.11 36.57 1.01 0 1.19 118.79
Intestine 4.848 0.329 1.89 37.2 3.342 0 5.763 103.04
Kidney 12.14 2.14 4.93 46.36 6.05 5.01 4.08 161.81
Liver 5.89 0.35 0.77 14.37 5.88 0 0.782 109.42
Muscle 2.45 0.46 1.81 37.22 1.78 2.3 1.035 73.36
Intestine 5.45 1.12 3.309 43.72 3.36 3.35 28.09 344.16
Kidney 3.75 0.54 5.27 36.37 4.77 0 0.611 206.11
Liver 5.61 0.54 2.102 30.77 10.5 5.88 4.57 213.27
Muscle 7.376 0.84 2.13 56.6 3.61 0 2.4 121.1
Intestine 3.84 0.47 2.57 29.15 1.12 0 1.78 89.56
Kidney 3.3 0.47 1.37 20.57 3.64 4.41 1.37 92.25
Liver 3.87 0.51 3.38 30.29 8.81 2.86 3.47 194.13
Muscle 4.2 0.46 3.3 34.97 6.51 0.32 1.33 90.01
Intestine 4.94 0.58 3 34.37 3.6 0 10.12 78.46
Kidney 1.82 2.5 5.56 52.01 2.57 1.32 8.78 167.09
Liver 3.45 1.32 1.33 38.65 26.2 0 7.45 63.43
Muscle 1.52 3.03 1.19 33.73 2.26 0 2.47 85.03
Intestine 3.91 0.35 1.86 25.37 2.56 1.66 1.78 50.25
Kidney 3.38 0.78 1.54 24.12 5.16 5.31 2.18 94.49
Liver 6.1 0.41 1.57 24.68 40.32 4.3 4.39 109.74
Muscle 2.95 0.45 2.4 29.79 1.5 11.26 1.21 48.22
151
1b
KANO Pb Cd Ni Zn Cu Cr Mn Fe
Intestine 3.16 0.23 3.57 22.64 9.06 6.44 1.94 156.74
Kidney 2.54 0.4 4.16 23.81 6.74 2.14 2.82 112.13
Liver 1.96 0.26 3.57 26.69 26.84 1.06 3.14 136.42
Muscle 3.96 0.24 2.7 28.36 1.96 0.21 3.69 54.21
Intestine 6.05 0.6 3.51 27.65 26.83 0 10.92 138.79
Kidney 1.9 0.53 4.22 23.31 5.45 0 2.76 68.58
Liver 3.42 0.43 1.43 28.42 8.1 0 2.71 95.75
Muscle 3.5 0.45 4.15 34.9 1.82 0 1.76 43.61
Intestine 2.07 0.3 2.117 20.06 2.27 0 2.02 46.76
Kidney 5.78 0.67 5.84 34.32 5.9 0 2.89 72.38
Liver 2.36 0.36 1.95 29.15 62.95 2.68 32.84 102.32
Muscle 3.62 0.37 1.03 20.55 2.38 0 2.26 27.05
Intestine 3.76 0.38 1.87 22.99 2.25 0 5.75 67.78
Kidney 4.55 0.91 3.47 43.79 7.9 0 3.88 92.68
Liver 2.11 0.56 1.6 31.88 13.09 0 3.92 82.01
Muscle 1.84 0.21 1.18 28.3 1.99 0 2.95 43.38
Intestine 3.01 0.28 1.88 0.23 1.14 8.68 3.01 40.75
Kidney 2.02 1.38 3.25 33.61 7.11 0 3.44 79
Liver 3.11 0.98 2.6 26.5 19.44 0.57 4.26 83.01
Muscle 3.33 0.29 1.74 33.2 1.27 0 1.79 34.03
Intestine 3.51 0.24 1.62 23.67 1.67 6.01 2.1 34.22
Kidney 9.12 0.76 4.64 41.71 4.52 0 3.24 83.23
Liver 1.82 0.26 1.6 26.4 2.53 0 3.41 75.98
Muscle 1.91 0.51 1.93 33.54 2.19 0 4.39 61.03
152
1c
KATSINA Pb Cd Ni Zn Cu Cr Mn Fe
Intestine 7.29 0.79 4.23 42.5 3.61 0 10.61 203.57
Kidney 1.61 0.78 3.53 30.58 15.61 1.21 4.71 138.54
Liver 2.38 0.62 1.5 33 8.33 0.38 4.97 115.18
Muscle 2.28 0.24 2.38 26.36 1.46 0.89 3.28 55.72
Intestine 2.62 0.18 0.17 5.69 0.85 0.9 0.3 70.88
Kidney 2.67 0.76 0.74 29.53 3.67 0 1.57 48.02
Liver 2.49 0.52 1.42 26.29 2.12 0 1.7 35.29
Muscle 2.38 0.22 1.57 27.17 2.02 0.97 6.31 104.99
Intestine 3.35 0.46 2.19 26.57 2.48 2.02 18.72 211.425
Kidney 4.15 1.18 2.38 39.36 4.14 0 3.3 97.51
Liver 2.63 0.5 1.6 40.46 7.09 0.47 10.8 21.65
Muscle 5.1 0.3 1.91 25.78 1.74 0.79 1.43 99.13
Intestine 6.78 0.537 3.57 46.19 3.06 2.31 6.51 350.28
Kidney 10.77 19.69 7.23 64.46 7.75 9.23 4.28 203.11
Liver 4.88 1.265 3.35 68 17.41 0.86 5 277.49
Muscle 6.12 0.78 2.5 44.59 3.07 2.19 3.72 78.74
Intestine 2.59 0.3 2.25 25.8 2.71 2.23 34.9 147.03
Kidney 2.81 0.51 1.83 31.57 5.26 4.98 3.41 91.46
Liver 2.57 0.35 2.17 43.03 5.52 0 5.83 184.84
Muscle 2.71 0.33 1.87 26.24 1.1 0 1 55.57
Intestine 2.2 0.21 0.71 27 2.89 0.18 15.58 158.18
Kidney 3.73 0.5 2.02 25.06 5.31 0.04 2.09 138.64
Liver 2.76 0.29 0.56 27.36 34.04 1.25 3.44 201.06
Muscle 3.64 0.47 2.11 21.27 2.52 9.26 1.67 89.33
153
1d
KEBBI Pb Cd Ni Zn Cu Cr Mn Fe
Intestine 10.15 0.11 6.75 50.73 9.94 37.89 5.94 126.84
Kidney 15.89 3.26 21.63 99.851 27.48 92.78 6 250.93
Liver 6.97 1.04 7.21 35.62 15.11 0 4.65 166.46
Muscle 3.88 0.5 3.13 31.62 2.09 0 1.11 45.17
Intestine 17.2 2.59 7.9 54.46 4.93 1.68 8.61 118.61
Kidney 6.41 1.16 3.87 38.18 7.04 0 4.23 63.7
Liver 1.86 1.02 0.82 29.01 53.29 3.27 3.19 73.46
Muscle 3.12 0.26 1.33 30.73 2.97 0 1.51 28.6
Intestine 1.53 0.43 2.26 25.52 2.33 0 4.15 62.41
Kidney 4.62 4.09 3.86 39.33 6.84 0 4.19 59.9
Liver 3.98 0.69 2.12 24.75 37.45 2.36 4.5 46.84
Muscle 1.93 0.53 2.5 30.47 4.24 0 2.72 64.51
Intestine 0.68 0.283 1.69 24.93 1.55 1.08 11.3 31.01
Kidney 7.21 0.77 4.26 47.38 3.33 0 24.79 65.49
Liver 3.56 0.81 2.75 31.36 5.77 9.06 6.59 167.44
Muscle 2.49 0.3 1.87 26.41 4.51 4.59 3.1 39.4
Intestine 3.42 0.45 1.69 21.85 2.78 0 6.69 94.5
Kidney 7.19 4.22 3.14 36 7.27 3.69 4.78 111.21
Liver 4.714 0.77 2.16 29.65 9.8 4.67 3.35 113.99
Muscle 5.46 0.4 1.59 30.29 4.6 14.62 3.98 101.92
Intestine 9.34 0.83 3.59 27.88 2.59 1.15 4.07 41.76
Kidney 4.8 1.14 3.85 27.82 6.09 0 4.06 92.25
Liver 3.77 0.74 2.72 38.7 84.25 0.37 6.74 95.63
Muscle 2.11 0.82 2.15 40.16 3.89 0 2.51 46.86
154
1e
BORNO Pb Cd Ni Zn Cu Cr Mn Fe
Intestine 5.52 0.26 11.51 23.41 13.66 10.41 2.46 105.88
Kidney 4.42 0.53 10.22 22.99 17.17 4.8 2.72 136.83
Liver 2.27 0.42 1.13 28.17 9.37 0 3.31 165.32
Muscle 2.96 0.3 1.39 33.42 21.73 0 2.93 159.64
Intestine 2 0.23 2.3 22.22 3.65 1.98 3.16 52.25
Kidney 4.03 0.73 2.28 34.6 6.6 0.65 2.41 74.59
Liver 3.5 0.37 2 24.81 10.55 0 2.14 44.07
Muscle 3.08 0.33 1.16 27.93 4.22 2.75 1.19 51.32
Intestine 3.98 0.5 2.57 29.8 1.95 0.55 3.77 44.88
Kidney 8.38 1.11 3.21 34.85 4.69 0 2.7 66.1
Liver 1.64 0.26 1.58 29.63 7.17 0 3.05 56.49
Muscle 2.25 0.18 1.39 35.54 1.5 1.19 0.93 49.85
Intestine 1.63 0.28 1.61 22.06 1.5 0.02 1.16 24.64
Kidney 2.41 0.47 1.74 30 4.62 0.69 1.91 66.99
Liver 1.26 0.37 1.41 31.73 7.41 1.6 3.11 76.65
Muscle 2.55 0.43 1.59 26.25 5.27 2.19 1 39.32
Intestine 3.9 0.45 1.38 29.65 2.48 0 1.95 28.97
Kidney 1.37 0.8 1.22 25.58 3.61 0 2.57 45.9
Liver 2.54 0.37 1.5 29.51 5.01 0 3.95 79.44
Muscle 3.66 0.64 1.24 42.27 1.02 0 1.36 109.94
Intestine 1.51 0.31 0.41 17.4 1.13 0 1.28 26.99
Kidney 5.35 0.39 2.7 28.77 4.25 0 2.77 67.15
Liver 0.84 0.17 1.08 19.43 3.78 0 2.9 61.71
Muscle 2.91 0.12 2.45 27.5 4.37 0 1.19 45.9
155
1f
SOKOTO Pb Cd Ni Zn Cu Cr Mn Fe
Intestine 3.1 0.35 3.33 28.53 4.1 0 44.76 72.46
Kidney 4.27 1.16 1.59 27.57 5.77 3.44 4.44 86.43
Liver 1.42 0.3 1.54 27.07 21.26 0 3.07 94.4
Muscle 1.35 0.16 1.47 20.38 1.95 2.29 1.69 42.56
Intestine 3.27 0.71 2.74 26.15 2.64 0.45 8.01 49.11
Kidney 1.87 0.52 1.47 22.64 4.85 0 7.43 70.64
Liver 2.11 0.25 1.34 25.89 42.49 0.92 3.86 53.38
Muscle 1.78 0.29 2.01 32.49 3.55 0 1.95 44.92
Intestine 3.89 0.67 6.35 30.65 6.2 1.47 8.4 157.5
Kidney 3.18 0.31 2.18 29.61 21.19 0 10.18 140.98
Liver 2.82 0.62 1.42 25 8.25 1.71 3.75 58.77
Muscle 1.06 0.24 1.46 32.19 4.7 0 2.58 34.19
Intestine 2.73 0.45 1.65 26.33 2.12 0.02 2.73 48.34
Kidney 3.06 0.818 2.013 32.147 5.181 0 2.469 86.36
Liver 4.04 0.48 1.63 28.15 27.57 0 3.14 101.52
Muscle 0.88 0.08 0.57 13.91 1.61 0 0.87 40.86
Intestine 2.68 0.27 1.7 15.04 0.95 2.22 2.43 32.27
Kidney 3.422 0.549 2.154 29.422 0.538 0 2.408 101.7
Liver 3.44 1.158 2.19 26.45 5.22 0.74 2.5 41.7
Muscle 2.51 0.26 1.66 25.78 1.82 0 1.04 40.39
156
1g
ZAMFARA Pb Cd Ni Zn Cu Cr Mn Fe
Intestine 5.07 0.36 1.9 28.42 3.21 0 14.44 65.66
Kidney 3.22 0.41 9.23 23.3 5.71 1.22 2.77 148.46
Liver 3.29 0.62 3.47 28.37 5.79 1.97 3.97 83.52
Muscle 3.34 0.34 2.76 31.47 4.33 0 1.41 49.84
Intestine 3.93 0.65 3.42 36.42 4.19 0 42 117.51
Kidney 4.83 0.5 3.75 30.12 7.15 0 4.65 78.59
Liver 2.45 0.52 2.27 25.88 6.25 0 3.87 53.87
Muscle 2.188 0.59 2.71 37.84 6.866 14.96 2.61 43.15
Intestine 2.16 0.39 2.49 30 4.28 1.04 17.13 68.63
Kidney 3.01 0.25 1.41 24.36 5.99 1.68 2.31 53.05
Liver 2.92 0.34 1.52 27.77 35.38 1.63 3.95 54.14
Muscle 1.5 0.22 1.57 29.13 2.32 5.7 1.22 55.45
Intestine 4.94 0.52 1.94 27.69 1.77 2.911 57.94 135.91
Kidney 3.7 0.42 2.09 24.95 5.8 6.3 3.82 66.13
Liver 2.93 0.42 3.67 35.72 28 16.228 5.68 66.32
Muscle 5.632 0.75 2.29 42.82 2.09 9.13 28.53 75.32
Intestine 4.22 0.75 1.95 38.63 2.73 0 6.3 66.57
Kidney 4.64 0.32 2.32 25.11 5.49 2.19 1.99 91.98
Liver 3.39 0.41 2.31 29.02 36.07 3.64 4.16 72.36
Muscle 7.97 0.68 3.57 53.43 3.15 0 2.7 67.15
Intestine 5.11 0.62 1.88 33.95 1.46 0 4.67 37.76
Kidney 4.46 0.56 1.28 27.13 3.77 0.48 2.04 65.95
Liver 2.59 0.4 1.58 21.47 10.9 0 2.2 77.15
Muscle 4.09 0.49 1.39 29.14 2.29 1.53 1.32 44.91
157
APPENDIX 2
2a
CONCENTRATIONS OF TRACE METALS IN SHEEP
FROM VARIOUS STATES
Mg/kg(wet weight)
KADUNA Pb Cd Ni Zn Cu Cr Mn Fe
Intestine 0.844 0.067 0.32 4.95 0.3 0.27 0.402 12.38
Kidney 0.965 0.185 0.26 5.68 1.28 0 0.34 12.98
Liver 0.648 0.146 0.206 6.9 1.16 2.137 0.57 18.47
Muscle 0.91 0.082 0.58 6.83 0.189 0 0.222 22.18
Intestine 0.956 0.065 0.374 7.35 0.66 0 1.135 20.3
Kidney 2.46 0.433 1 9.4 1.23 1.02 0.83 32.8
Liver 0.886 0.106 0.24 4.45 1.823 0 0.242 33.876
Muscle 0.588 0.11 0.434 8.921 0.427 0.552 0.248 17.6
Intestine 0.994 0.203 0.603 7.97 0.61 0.61 5.12 62.73
Kidney 0.68 0.098 0.96 6.64 0.87 0 0.611 37.61
Liver 1.46 0.141 0.546 8 2.73 1.53 1.19 55.45
Muscle 1.488 0.17 0.43 11.43 0.73 0 0.49 24.4
Intestine 0.85 0.01 0.57 6.44 0.25 0 0.39 19.79
Kidney 0.76 0.109 0.32 4.78 0.85 1.03 0.32 21.45
Liver 1.16 0.15 0.97 8.7 2.53 0.82 0.997 55.78
Muscle 1.01 0.11 0.79 8.45 1.49 0.077 0.32 21.74
Intestine 0.67 0.078 0.41 4.68 0.49 0 1.38 10.69
Kidney 1.66 0.4 0.89 8.34 4.12 0.22 1.41 26.77
Liver 0.78 0.3 0.299 8.71 5.91 0 1.68 14.3
Muscle 0.27 0.53 0.21 5.9 0.4 0 0.43 14.88
Intestine 0.71 0.06 0.34 4.6 0.47 0.3 0.32 9.14
Kidney 0.62 0.14 0.28 4.45 0.95 0.98 0.4 17.45
Liver 1.58 0.11 0.41 6.42 10.49 1.12 1.14 28.54
Muscle 0.64 0.097 0.52 6.44 0.32 2.43 0.26 10.43
158
2b
KANO Pb Cd Ni Zn Cu Cr Mn Fe
Intestine 0.53 0.04 0.59 3.78 1.51 1.08 0.32 25.06
Kidney 0.5 0.08 0.83 4.72 1.34 0.42 0.56 22.23
Liver 0.61 0.08 1.12 8.34 8.38 0.33 0.98 42.63
Muscle 0.84 0.05 0.58 6.05 0.42 0.05 0.79 11.56
Intestine 0.98 0.09 0.57 4.47 0.43 0 1.76 22.42
Kidney 0.36 0.098 0.78 4.38 1.02 0 0.52 12.89
Liver 0.97 0.12 0.4 8.04 2.29 0 0.71 27.09
Muscle 0.7 0.09 0.83 7.03 0.37 0 0.35 8.78
Intestine 0.35 0.05 0.36 3.36 0.38 0 0.34 7.82
Kidney 0.89 0.1 0.9 5.31 0.91 0 0.45 11.2
Liver 0.63 0.1 0.51 7.7 16.65 0.18 0.87 27.04
Muscle 0.91 0.093 0.26 5.14 0.6 0 0.57 6.76
Intestine 0.7 0.07 0.35 4.25 0.42 0 1.06 12.53
Kidney 0.846 0.17 0.65 8.15 1.47 0 0.72 17.25
Liver 0.49 0.13 0.37 7.47 3.07 0 0.92 19.21
Muscle 0.47 0.05 0.3 7.29 0.51 0 0.76 11.18
Intestine 0.54 0.05 0.34 4.16 0.21 1.56 0.54 7.34
Kidney 0.37 0.25 0.6 6.2 1.31 0 0.64 14.58
Liver 0.88 0.28 0.73 7.49 5.49 0.16 1.2 23.45
Muscle 0.92 0.08 0.48 9.2 0.35 0 0.5 9.43
Intestine 0.68 0.05 0.31 4.59 0.32 1.16 0.41 6.63
Kidney 1.42 0.12 0.72 6.49 0.7 0 0.5 12.95
Liver 0.49 0.07 0.43 7.03 0.67 0 0.91 20.22
Muscle 0.37 0.98 0.37 6.51 0.43 0 0.85 11.85
159
2c
KATSINA Pb Cd Ni Zn Cu Cr Mn Fe
Intestine 1.17 0.13 0.68 6.8 0.58 0 1.7 32.57
Kidney 0.27 0.13 0.59 5.15 2.63 0.2 0.79 23.33
Liver 0.54 0.14 0.34 7.46 1.88 0.09 1.12 26.03
Muscle 0.35 0.04 0.36 4.06 0.22 0.14 0.5 8.52
Intestine 0.54 0.04 0.51 5.25 0.39 0.18 0.41 14.49
Kidney 0.61 0.18 0.17 5.69 0.85 0 0.3 9.25
Liver 0.8 0.32 0.46 8.43 0.68 0 0.55 11.31
Muscle 0.5 0.05 0.33 5.71 0.42 0.2 1.33 22.08
Intestine 0.61 0.08 0.4 4.8 0.45 0.36 3.38 38.17
Kidney 0.9 0.26 0.52 8.58 0.9 0 0.72 21.25
Liver 0.68 0.13 0.42 10.52 1.84 0.12 2.81 5.63
Muscle 1.15 0.08 0.43 5.82 0.39 0.18 0.32 22.38
Intestine 1.2 0.95 0.63 8.17 0.54 0.41 1.15 62.01
Kidney 1.78 3.2 1.18 10.48 1.26 1.5 0.69 18.86
Liver 0.72 0.19 0.49 10.09 2.58 0.13 0.74 41.2
Muscle 0.99 0.125 0.4 7.18 0.49 0.35 0.6 12.69
Intestine 0.5 0.06 0.43 4.44 0.52 0.43 6.68 28.14
Kidney 0.576 0.1 0.38 6.47 1.08 1.02 0.7 18.75
Liver 0.64 0.09 0.54 10.69 1.37 0 1.45 45.95
Muscle 0.51 0.062 0.35 4.9 0.21 0 0.19 10.38
Intestine 0.43 0.04 0.14 5.27 0.57 0.03 3.04 30.9
Kidney 0.73 0.098 0.4 4.9 1.04 0.01 0.55 27.13
Liver 0.75 0.08 0.42 7.44 9.26 0.34 0.94 54.71
Muscle 0.8 0.1 0.46 4.67 0.55 2.03 0.37 19.63
160
2d
KEBBI Pb Cd Ni Zn Cu Cr Mn Fe
Intestine 2.086 0.023 1.39 10.43 2.05 7.79 1.22 26.09
Kidney 2.77 0.57 3.77 17.39 4.79 16.11 1.05 43.71
Liver 1.77 0.27 1.83 9.06 3.84 0 1.18 42.34
Muscle 0.9 0.12 0.73 7.34 0.49 0 0.26 10.49
Intestine 2.94 0.44 1.35 9.3 0.84 0.29 1.47 20.26
Kidney 1.18 0.21 0.71 7.01 1.29 0 0.78 11.7
Liver 0.52 0.28 0.23 8.06 14.82 0.91 0.89 20.42
Muscle 0.76 0.063 0.33 7.51 0.73 0 0.37 6.99
Intestine 0.28 0.078 0.42 4.72 0.43 0 0.77 11.53
Kidney 0.86 0.77 0.72 7.36 1.28 0 0.78 11.21
Liver 1.09 0.19 0.58 6.81 10.3 0.65 1.24 12.89
Muscle 0.44 0.12 0.56 6.85 0.95 0 0.61 16.13
Intestine 0.11 0.04 0.284 4.19 0.26 0.18 1.9 5.22
Kidney 1.28 0.14 0.75 8.4 0.59 0 4.4 11.61
Liver 0.99 0.22 0.76 8.7 1.6 2.51 1.83 46.45
Muscle 0.51 0.07 0.43 6 1.03 1.04 0.71 8.96
Intestine 0.61 0.08 0.3 3.89 0.5 0 1.19 16.82
Kidney 1.34 0.79 0.59 6.7 1.35 0.69 0.89 20.7
Liver 1.22 0.2 0.56 7.66 2.53 1.21 0.86 29.46
Muscle 1.32 0.096 0.39 7.34 1.12 3.54 0.97 24.7
Intestine 1.53 0.14 0.59 4.57 0.42 0.19 0.67 6.85
Kidney 0.89 0.21 0.71 5.17 1.13 0 0.75 17.13
Liver 1.08 0.21 0.78 11.07 24.11 0.11 1.93 27.36
Muscle 0.51 0.2 0.52 9.62 0.93 0 0.6 11.22
161
2e
BORNO Pb Cd Ni Zn Cu Cr Mn Fe
Intestine 1.04 0.05 2.16 4.39 2.56 1.95 0.46 19.87
Kidney 0.81 0.09 1.87 4.2 3.14 0.88 0.5 25.02
Liver 0.62 0.11 0.31 7.63 2.54 0 0.9 44.8
Muscle 0.7 0.07 0.33 7.88 5.12 0 0.69 37.63
Intestine 0.36 0.04 0.41 3.97 0.65 0.35 0.57 9.34
Kidney 0.71 0.13 0.4 6.07 1.15 0.11 0.42 13.11
Liver 1.02 0.11 0.58 7.25 3.08 0 0.62 12.87
Muscle 0.7 0.07 0.26 6.3 0.95 0.62 0.27 11.58
Intestine 0.74 0.09 0.4 5.56 0.36 0.1 0.7 8.38
Kidney 1.57 0.21 0.6 6.54 0.88 0 0.51 12.41
Liver 0.47 0.07 0.45 8.43 2.04 0 0.87 16.07
Muscle 0.51 0.04 0.32 8.09 0.34 0.27 0.21 11.34
Intestine 0.32 0.05 0.31 4.266 0.29 0.004 0.22 4.77
Kidney 0.47 0.09 0.34 5.81 0.9 0.13 0.37 12.98
Liver 0.34 0.1 0.38 8.56 2 0.43 0.84 20.69
Muscle 0.62 0.1 0.39 6.39 1.28 0.53 0.24 9.57
Intestine 0.72 0.08 0.26 5.47 0.27 0 0.36 5.35
Kidney 0.26 0.15 0.23 4.79 0.68 0 0.48 8.6
Liver 0.76 1.12 0.45 8.92 0.02 0 1.19 24
Muscle 0.82 0.14 0.28 9.44 0.23 0 0.3 24.55
Intestine 0.28 0.06 0.08 3.21 0.21 0 0.24 4.98
Kidney 1.09 0.08 0.55 5.84 0.86 0 0.56 13.62
Liver 0.24 0.05 0.31 5.53 1.08 0 0.83 17.57
Muscle 0.67 0.03 0.57 6.27 1.01 0 0.28 10.5
162
2f
SOKOTO Pb Cd Ni Zn Cu Cr Mn Fe
Intestine 0.61 0.07 0.65 5.58 0.8 0 8.76 14.17
Kidney 0.83 0.23 0.31 5.36 1.12 0.67 0.86 16.81
Liver 0.4 0.085 0.435 7.647 6.006 0 1.149 26.67
Muscle 0.52 0.062 0.566 7.854 0.751 0.882 0.651 16.4
Intestine 0.801 0.174 0.675 6.409 0.647 0.11 1.96 12.04
Kidney 0.391 0.108 0.307 4.737 1.013 0 1.552 14.76
Liver 0.614 0.072 0.39 7.537 12.369 0.267 1.123 15.54
Muscle 0.436 0.071 0.493 7.473 0.871 0 0.478 11.02
Intestine 0.804 0.138 1.312 6.332 1.28 0.304 1.735 32.54
Kidney 0.818 0.079 0.56 7.62 5.454 0 2.62 36.29
Liver 0.825 0.182 0.416 7.321 2.416 0.501 1.098 17.21
Muscle 0.24 0.054 0.334 7.315 1.068 0 0.586 7.77
Intestine 0.7 0.115 0.423 6.751 0.543 0.005 0.7 12.39
Kidney 0.608 0.163 0.4 6.386 1.029 0 0.49 17.16
Liver 1.256 0.149 0.506 8.755 8.575 0 0.976 31.58
Muscle 0.295 0.026 0.192 4.676 0.541 0 0.292 13.73
Intestine 0.695 0.07 0.441 3.901 0.246 0.575 0.631 8.37
Kidney 0.675 0.108 0.425 5.802 1.061 0 0.475 55.55
Liver 0.755 0.346 0.48 5.806 1.145 0.162 0.548 9.15
Muscle 0.67 0.069 0.443 6.883 0.485 0 0.277 10.79
163
2g
ZAMFARA Pb Cd Ni Zn Cu Cr Mn Fe
Intestine 0.91 0.065 0.34 5.093 0.575 0 2.587 11.767
Kidney 0.682 0.087 1.955 4.936 1.2 0.216 0.586 31.453
Liver 0.884 0.166 0.932 7.626 1.556 0.529 1.067 22.451
Muscle 0.89 0.091 0.736 8.392 1.55 0 0.376 13.291
Intestine 0.775 0.128 0.673 7.183 0.827 0 8.284 23.18
Kidney 0.908 0.094 0.705 5.67 1.345 0 0.877 14.793
Liver 0.658 0.139 0.61 6.956 1.68 0 1.04 14.435
Muscle 0.463 0.126 0.573 8.01 1.453 3.166 0.553 9.133
Intestine 0.381 0.068 0.439 5.295 0.755 0.183 3.023 12.114
Kidney 0.623 0.051 0.292 5.048 1.241 0.348 0.478 10.99
Liver 0.846 0.098 0.439 8.035 10.237 0.47 1.142 15.665
Muscle 0.384 0.056 0.402 7.459 0.594 1.459 0.312 14.199
Intestine 0.975 0.102 0.382 5.47 0.35 0.575 11.442 26.842
Kidney 0.79 0.089 0.446 5.331 1.239 1.346 0.816 14.13
Liver 0.776 0.111 0.972 9.469 7.423 4.302 1.506 17.581
Muscle 1.418 0.188 0.577 10.785 0.525 2.3 0.359 18.97
Intestine 0.857 0.152 0.396 7.857 0.555 0 1.281 13.538
Kidney 1.096 0.075 0.543 5.936 1.297 0.517 0.47 21.744
Liver 0.941 0.113 0.591 8.06 10.019 1.011 1.155 20.1
Muscle 1.88 0.161 0.842 12.595 0.742 0 0.638 15.828
Intestine 1.085 0.132 0.4 7.214 0.31 0 0.992 8.025
Kidney 0.79 0.1 0.226 4.811 0.669 0.085 0.361 11.697
Liver 0.705 0.108 0.43 5.85 2.97 0 0.599 21.021
Muscle 0.938 0.113 0.32 6.68 0.526 0.35 0.301 10.295
164
APPENDIX 3 ONE WAY ANOVA RESULTS
Means (Kaduna)
Report
4.7647 .5498 2.4665 33.8250 2.6670 1.1383 8.3705 124.717
.74708 .29361 .59887 6.37825 .96225 1.37954 10.17699 108.9043
6 6 6 6 6 6 6 6
4.9990 1.2517 3.3617 35.4050 4.9317 2.6750 3.1685 132.858
3.70226 .86219 2.08255 12.24648 1.71275 2.51209 2.98198 52.5327
6 6 6 6 6 6 6 6
4.6185 .6128 1.6730 28.0800 16.1167 3.7067 3.8537 128.223
1.41845 .35511 .96550 8.07538 14.14921 3.56224 2.24762 61.4143
6 6 6 6 6 6 6 6
3.8960 .9467 2.3233 38.1467 2.7783 2.3133 1.6058 89.418
2.08817 1.03235 .79548 9.41597 2.03192 4.47372 .64946 27.7172
6 6 6 6 6 6 6 6
4.5695 .8403 2.4561 33.8642 6.6234 2.4583 4.2496 118.804
2.15949 .72253 1.31892 9.43556 8.80087 3.12632 5.67782 66.8897
24 24 24 24 24 24 24 24
Mean
Std. Deviation
N
Mean
Std. Deviation
N
Mean
Std. Deviation
N
Mean
Std. Deviation
N
Mean
Std. Deviation
N
Tissues
Intestine
Kidney
Liver
Muscle
Total
Pb Cd Ni Zn Cn Cr Mn Fe
One-way
ANOVA
ANOVA
4.071 3 1.357 .263 .851
103.187 20 5.159
107.258 23
1.900 3 .633 1.253 .317
10.107 20 .505
12.007 23
8.706 3 2.902 1.854 .170
31.303 20 1.565
40.009 23
325.033 3 108.344 1.258 .316
1722.653 20 86.133
2047.686 23
740.530 3 246.843 4.743 .012
1040.942 20 52.047
1781.472 23
20.212 3 6.737 .659 .587
204.587 20 10.229
224.800 23
151.781 3 50.594 1.716 .196
589.685 20 29.484
741.466 23
7108.351 3 2369.450 .495 .690
95798.946 20 4789.947
102907.3 23
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Pb
Cd
Ni
Zn
Cn
Cr
Mn
Fe
Sum of
Squares df Mean Square F Sig.
Post Hoc Tests
165
Homogeneous Subsets
Pb
Duncana
6 3.8960
6 4.6185
6 4.7647
6 4.9990
.451
Tissues
Muscle
Liver
Intestine
Kidney
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Cd
Duncana
6 .5498
6 .6128
6 .9467
6 1.2517
.131
Tissues
Intestine
Liver
Muscle
Kidney
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Ni
Duncana
6 1.6730
6 2.3233 2.3233
6 2.4665 2.4665
6 3.3617
.311 .188
Tissues
Liver
Muscle
Intestine
Kidney
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Zn
Duncana
6 28.0800
6 33.8250
6 35.4050
6 38.1467
.099
Tissues
Liver
Intestine
Kidney
Muscle
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Cr
Duncana
6 1.1383
6 2.3133
6 2.6750
6 3.7067
.217
TissuesIntestine
Muscle
Kidney
Liver
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Cn
Duncana
6 2.6670
6 2.7783
6 4.9317
6 16.1167
.614 1.000
Tissues
Intestine
Muscle
Kidney
Liver
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
166
Fe
Duncana
6 89.418
6 124.717
6 128.223
6 132.858
.331
Tissues
Muscle
Intestine
Liver
Kidney
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Mn
Duncana
6 1.6058
6 3.1685
6 3.8537
6 8.3705
.060
Tissues
Muscle
Kidney
Liver
Intestine
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Means (Kano)
Report
3.59333 .33833 2.42783 19.54000 7.20333 3.52167 4.29000 80.84000
1.335555 .138912 .875916 9.772287 10.048963 3.962910 3.555233 53.351017
6 6 6 6 6 6 6 6
4.31833 .77500 4.26333 33.42500 6.27000 .35667 3.17167 84.66667
2.812091 .345297 .926988 8.621730 1.221081 .873651 .436092 15.889407
6 6 6 6 6 6 6 6
2.46333 .47500 2.12500 28.17333 22.15833 .71833 8.38000 95.91500
.653626 .272011 .822016 2.140053 21.718242 1.052130 11.995541 22.086188
6 6 6 6 6 6 6 6
3.02667 .34500 2.12167 29.80833 1.93500 .03500 2.80667 43.88500
.915897 .119290 1.158403 5.318708 .380039 .085732 1.071049 12.501404
6 6 6 6 6 6 6 6
3.35042 .48333 2.73446 27.73667 9.39167 1.15792 4.66208 76.32667
1.695440 .286321 1.273327 8.438141 13.625800 2.413839 6.280064 34.810660
24 24 24 24 24 24 24 24
Mean
Std. Deviation
N
Mean
Std. Deviation
N
Mean
Std. Deviation
N
Mean
Std. Deviation
N
Mean
Std. Deviation
N
Tissues
Intestine
Kidney
Liver
Muscle
Total
Pb Cd Ni Zn Cn Cr Mn Fe
One-way ANOVA
ANOVA
11.326 3 3.775 1.378 .278
54.788 20 2.739
66.114 23
.752 3 .251 4.421 .015
1.134 20 .057
1.886 23
19.071 3 6.357 6.978 .002
18.221 20 .911
37.291 23
624.150 3 208.050 4.106 .020
1013.502 20 50.675
1637.651 23
1398.740 3 466.247 3.247 .044
2871.496 20 143.575
4270.236 23
46.101 3 15.367 3.496 .035
87.911 20 4.396
134.012 23
117.752 3 39.251 .995 .416
789.350 20 39.468
907.102 23
9156.542 3 3052.181 3.262 .043
18714.445 20 935.722
27870.987 23
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Pb
Cd
Ni
Zn
Cn
Cr
Mn
Fe
Sum of
Squares df Mean Square F Sig.
167
Post Hoc Tests
Homogeneous Subsets
Pb
Duncana
6 2.46333
6 3.02667
6 3.59333
6 4.31833
.089
TissuesLiver
Muscle
Intestine
Kidney
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Cd
Duncana
6 .33833
6 .34500
6 .47500
6 .77500
.359 1.000
TissuesIntestine
Muscle
Liver
Kidney
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Ni
Duncana
6 2.12167
6 2.12500
6 2.42783
6 4.26333
.606 1.000
Tissues
Muscle
Liver
Intestine
Kidney
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Zn
Duncana
6 19.54000
6 28.17333
6 29.80833
6 33.42500
1.000 .241
Tissues
Intestine
Liver
Muscle
Kidney
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Cn
Duncana
6 1.93500
6 6.27000
6 7.20333
6 22.15833
.481 1.000
Tissues
Muscle
Kidney
Intestine
Liver
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Cr
Duncana
6 .03500
6 .35667
6 .71833
6 3.52167
.600 1.000
Tissues
Muscle
Kidney
Liver
Intestine
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Mn
Duncana
6 2.80667
6 3.17167
6 4.29000
6 8.38000
.174
Tissues
Muscle
Kidney
Intestine
Liver
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Fe
Duncana
6 43.88500
6 80.84000
6 84.66667
6 95.91500
1.000 .430
Tissues
Muscle
Intestine
Kidney
Liver
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
168
Means (Katsina)
Report
4.13833 .41283 2.18667 28.95833 2.60000 1.27333 14.43667 190.22750
2.280030 .231508 1.571008 14.430777 .938595 1.049146 11.964305 93.146177
6 6 6 6 6 6 6 6
4.29000 3.90333 2.95500 36.76000 6.95667 2.57667 3.22667 119.54667
3.296083 7.737810 2.279603 14.341139 4.468457 3.784805 1.215346 53.101172
6 6 6 6 6 6 6 6
2.95167 .59083 1.76667 39.69000 12.41833 .49333 5.29000 139.25167
.953340 .351318 .932173 15.424329 11.757223 .491352 3.070791 100.2258
6 6 6 6 6 6 6 6
3.70500 .39000 2.05667 28.56833 1.98500 2.35000 2.90167 80.58000
1.584850 .210523 .345582 8.123884 .718825 3.457334 1.986388 21.276364
6 6 6 6 6 6 6 6
3.77125 1.32425 2.24125 33.49417 5.99000 1.67333 6.46375 132.40146
2.125377 3.922254 1.443056 13.436590 7.273315 2.596469 7.571856 80.059240
24 24 24 24 24 24 24 24
Mean
Std. Deviation
N
Mean
Std. Deviation
N
Mean
Std. Deviation
N
Mean
Std. Deviation
N
Mean
Std. Deviation
N
Tissues
Intestine
Kidney
Liver
Muscle
Total
Pb Cd Ni Zn Cn Cr Mn Fe
Oneway
ANOVA
6.480 3 2.160 .443 .725
97.417 20 4.871
103.896 23
53.358 3 17.786 1.184 .341
300.475 20 15.024
353.834 23
4.630 3 1.543 .713 .555
43.265 20 2.163
47.895 23
563.350 3 187.783 1.046 .394
3589.115 20 179.456
4152.465 23
418.740 3 139.580 3.498 .035
797.985 20 39.899
1216.726 23
16.958 3 5.653 .819 .499
138.100 20 6.905
155.058 23
528.673 3 176.224 4.461 .015
789.986 20 39.499
1318.659 23
37448.914 3 12482.971 2.270 .112
109969.2 20 5498.459
147418.1 23
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Pb
Cd
Ni
Zn
Cn
Cr
Mn
Fe
Sum of
Squares df Mean Square F Sig.
169
Post Hoc Tests
Homogeneous Subsets
Pb
Duncana
6 2.95167
6 3.70500
6 4.13833
6 4.29000
.348
Tissues
Liver
Muscle
Intestine
Kidney
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Cd
Duncana
6 .39000
6 .41283
6 .59083
6 3.90333
.165
Tissues
Muscle
Intestine
Liver
Kidney
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Ni
Duncana
6 1.76667
6 2.05667
6 2.18667
6 2.95500
.214
Tissues
Liver
Muscle
Intestine
Kidney
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Zn
Duncana
6 28.56833
6 28.95833
6 36.76000
6 39.69000
.202
Tissues
Muscle
Intestine
Kidney
Liver
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Cn
Duncana
6 1.98500
6 2.60000
6 6.95667 6.95667
6 12.41833
.212 .150
TissuesMuscle
Intestine
Kidney
Liver
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Cr
Duncana
6 .49333
6 1.27333
6 2.35000
6 2.57667
.222
Tissues
Liver
Intestine
Muscle
Kidney
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Mn
Duncana
6 2.90167
6 3.22667
6 5.29000
6 14.43667
.542 1.000
TissuesMuscle
Kidney
Liver
Intestine
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Fe
Duncana
6 80.58000
6 119.54667 119.54667
6 139.25167 139.25167
6 190.22750
.209 .133
Tissues
Muscle
Kidney
Liver
Intestine
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
170
Means ( Kebbi)
Report
7.05333 .78217 3.98000 34.22833 4.02000 6.96667 6.79333 79.18833
6.357684 .917157 2.706954 14.404471 3.111720 15.164147 2.783988 40.156545
6 6 6 6 6 6 6 6
7.68667 2.44000 6.76833 48.09350 9.67500 16.07833 8.00833 107.24667
4.174583 1.592520 7.289734 26.121607 8.841875 37.604967 8.252572 73.167133
6 6 6 6 6 6 6 6
4.14233 .84500 2.96333 31.51500 34.27833 3.28833 4.83667 110.63667
1.675449 .148694 2.194964 4.980316 30.489373 3.329056 1.534062 49.053904
6 6 6 6 6 6 6 6
3.16500 .46833 2.09500 31.61333 3.71667 3.20167 2.48833 54.41000
1.332227 .202427 .652557 4.559801 .991679 5.887419 1.049789 26.050130
6 6 6 6 6 6 6 6
5.51183 1.13388 3.95167 36.36254 12.92250 7.38375 5.53167 87.87042
4.165500 1.167276 4.184382 15.888820 19.644617 19.901210 4.668945 52.248019
24 24 24 24 24 24 24 24
Mean
Std. Deviation
N
Mean
Std. Deviation
N
Mean
Std. Deviation
N
Mean
Std. Deviation
N
Mean
Std. Deviation
N
Tissues
Intestine
Kidney
Liver
Muscle
Total
Pb Cd Ni Zn Cn Cr Mn Fe
Oneway
ANOVA
86.936 3 28.979 1.857 .169
312.146 20 15.607
399.082 23
14.136 3 4.712 5.479 .006
17.202 20 .860
31.338 23
74.151 3 24.717 1.505 .244
328.558 20 16.428
402.708 23
1129.343 3 376.448 1.610 .219
4677.112 20 233.856
5806.456 23
3783.718 3 1261.239 4.954 .010
5092.234 20 254.612
8875.953 23
660.192 3 220.064 .521 .673
8449.146 20 422.457
9109.338 23
104.824 3 34.941 1.762 .187
396.555 20 19.828
501.378 23
12532.316 3 4177.439 1.663 .207
50254.361 20 2512.718
62786.676 23
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Pb
Cd
Ni
Zn
Cn
Cr
Mn
Fe
Sum of
Squares df Mean Square F Sig.
171
Post Hoc Tests
Homogeneous Subsets
Pb
Duncana
6 3.16500
6 4.14233
6 7.05333
6 7.68667
.082
Tissues
Muscle
Liver
Intestine
Kidney
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Cd
Duncana
6 .46833
6 .78217
6 .84500
6 2.44000
.514 1.000
TissuesMuscle
Intestine
Liver
Kidney
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Ni
Duncana
6 2.09500
6 2.96333
6 3.98000
6 6.76833
.080
Tissues
Muscle
Liver
Intestine
Kidney
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Zn
Duncana
6 31.51500
6 31.61333
6 34.22833
6 48.09350
.099
Tissues
Liver
Muscle
Intestine
Kidney
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Cn
Duncana
6 3.71667
6 4.02000
6 9.67500
6 34.27833
.549 1.000
Tissues
Muscle
Intestine
Kidney
Liver
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Cr
Duncana
6 3.20167
6 3.28833
6 6.96667
6 16.07833
.332
Tissues
Muscle
Liver
Intestine
Kidney
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Mn
Duncana
6 2.48833
6 4.83667
6 6.79333
6 8.00833
.061
Tissues
Muscle
Liver
Intestine
Kidney
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Fe
Duncana
6 54.41000
6 79.18833
6 107.24667
6 110.63667
.088
Tissues
Muscle
Intestine
Kidney
Liver
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
172
Means ( Borno )
Report
3.09000 .33833 3.29667 24.09000 4.06167 2.16000 2.29667 47.26833
1.622911 .110167 4.094668 4.823982 4.783620 4.113339 1.038127 30.737371
6 6 6 6 6 6 6 6
4.32667 .67167 3.56167 29.46500 6.82333 1.02333 2.51333 76.26000
2.449038 .265361 3.335988 4.757196 5.166367 1.879113 .322903 31.194211
6 6 6 6 6 6 6 6
2.00833 .32667 1.45000 27.21333 7.21500 .26667 3.07667 80.61333
.963004 .093095 .335500 4.444924 2.548237 .653197 .587798 43.513195
6 6 6 6 6 6 6 6
2.90167 .33333 1.53667 32.15167 6.35167 1.02167 1.43333 75.99500
.481390 .186297 .471155 6.160524 7.721392 1.225715 .748990 48.356862
6 6 6 6 6 6 6 6
3.08167 .41750 2.46125 28.23000 6.11292 1.11792 2.33000 70.03417
1.685758 .223300 2.669198 5.628047 5.168224 2.311324 .904573 38.995658
24 24 24 24 24 24 24 24
Mean
Std. Deviation
N
Mean
Std. Deviation
N
Mean
Std. Deviation
N
Mean
Std. Deviation
N
Mean
Std. Deviation
N
Tissues
Intestine
Kidney
Liver
Muscle
Total
Pb Cd Ni Zn Cn Cr Mn Fe
Oneway
ANOVA
16.407 3 5.469 2.234 .116
48.954 20 2.448
65.361 23
.517 3 .172 5.476 .007
.630 20 .031
1.147 23
22.718 3 7.573 1.073 .383
141.148 20 7.057
163.866 23
210.467 3 70.156 2.708 .073
518.056 20 25.903
728.523 23
35.903 3 11.968 .414 .745
578.439 20 28.922
614.342 23
10.973 3 3.658 .654 .590
111.898 20 5.595
122.871 23
8.377 3 2.792 5.348 .007
10.442 20 .522
18.820 23
4226.967 3 1408.989 .916 .451
30748.245 20 1537.412
34975.212 23
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Pb
Cd
Ni
Zn
Cn
Cr
Mn
Fe
Sum of
Squares df Mean Square F Sig.
173
Post Hoc Tests
Homogeneous Subsets
Pb
Duncana
6 2.00833
6 2.90167 2.90167
6 3.09000 3.09000
6 4.32667
.271 .150
Tissues
Liver
Muscle
Intestine
Kidney
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Cd
Duncana
6 .32667
6 .33333
6 .33833
6 .67167
.916 1.000
Tissues
Liver
Muscle
Intestine
Kidney
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Ni
Duncana
6 1.45000
6 1.53667
6 3.29667
6 3.56167
.221
Tissues
Liver
Muscle
Intestine
Kidney
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Zn
Duncana
6 24.09000
6 27.21333 27.21333
6 29.46500 29.46500
6 32.15167
.098 .126
Tissues
Intestine
Liver
Kidney
Muscle
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Cn
Duncana
6 4.06167
6 6.35167
6 6.82333
6 7.21500
.364
Tissues
Intestine
Muscle
Kidney
Liver
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Cr
Duncana
6 .26667
6 1.02167
6 1.02333
6 2.16000
.218
TissuesLiver
Muscle
Kidney
Intestine
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Mn
Duncana
6 1.43333
6 2.29667 2.29667
6 2.51333
6 3.07667
.052 .091
Tissues
Muscle
Intestine
Kidney
Liver
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Fe
Duncana
6 47.26833
6 75.99500
6 76.26000
6 80.61333
.191
Tissues
Intestine
Muscle
Kidney
Liver
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
174
Means ( Sokoto )
Report
3.13400 .49000 3.15400 25.34000 3.20200 .83200 13.26600 71.93600
.490031 .193907 1.923078 6.043559 2.021910 .978657 17.829762 49.931608
5 5 5 5 5 5 5 5
3.16040 .67140 1.88140 28.27780 7.50580 .68800 5.38540 97.22200
.862445 .327389 .329756 3.547549 7.925884 1.538415 3.370188 26.813560
5 5 5 5 5 5 5 5
2.76600 .56160 1.62400 26.51200 20.95800 .67400 3.26400 69.95400
1.039509 .364449 .335306 1.190722 15.137822 .715318 .554103 26.420102
5 5 5 5 5 5 5 5
1.51600 .20600 1.43400 24.95000 2.72600 .45800 1.62600 40.58400
.651483 .085323 .531818 7.948657 1.345745 1.024119 .695363 3.990693
5 5 5 5 5 5 5 5
2.64410 .48225 2.02335 26.26995 8.59795 .66300 5.88535 69.92400
.998050 .301905 1.166091 5.069622 10.952741 1.023323 9.511445 35.358798
20 20 20 20 20 20 20 20
Mean
Std. Deviation
N
Mean
Std. Deviation
N
Mean
Std. Deviation
N
Mean
Std. Deviation
N
Mean
Std. Deviation
N
TissuesIntestine
Kidney
Liver
Muscle
Total
Pb Cd Ni Zn Cn Cr Mn Fe
Oneway
ANOVA
8.970 3 2.990 4.805 .014
9.956 16 .622
18.926 19
.592 3 .197 2.772 .075
1.140 16 .071
1.732 19
9.027 3 3.009 2.864 .069
16.809 16 1.051
25.836 19
33.486 3 11.162 .393 .760
454.835 16 28.427
488.320 19
1087.799 3 362.600 4.869 .014
1191.490 16 74.468
2279.288 19
.357 3 .119 .097 .960
19.540 16 1.221
19.897 19
398.687 3 132.896 1.611 .226
1320.197 16 82.512
1718.884 19
8050.327 3 2683.442 2.734 .078
15704.319 16 981.520
23754.647 19
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Pb
Cd
Ni
Zn
Cn
Cr
Mn
Fe
Sum of
Squares df Mean Square F Sig.
175
Post Hoc Tests
Homogeneous Subsets
Pb
Duncana
5 1.51600
5 2.76600
5 3.13400
5 3.16040
1.000 .465
Tissues
Muscle
Liver
Intestine
Kidney
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 5.000.a.
Cd
Duncana
5 .20600
5 .49000 .49000
5 .56160 .56160
5 .67140
.062 .324
Tissues
Muscle
Intestine
Liver
Kidney
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 5.000.a.
Ni
Duncana
5 1.43400
5 1.62400
5 1.88140 1.88140
5 3.15400
.523 .067
TissuesMuscle
Liver
Kidney
Intestine
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 5.000.a.
Zn
Duncana
5 24.95000
5 25.34000
5 26.51200
5 28.27780
.378
Tissues
Muscle
Intestine
Liver
Kidney
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 5.000.a.
Cn
Duncana
5 2.72600
5 3.20200
5 7.50580
5 20.95800
.419 1.000
Tissues
Muscle
Intestine
Kidney
Liver
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 5.000.a.
Cr
Duncana
5 .45800
5 .67400
5 .68800
5 .83200
.630
Tissues
Muscle
Liver
Kidney
Intestine
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 5.000.a.
Mn
Duncana
5 1.62600
5 3.26400
5 5.38540
5 13.26600
.079
Tissues
Muscle
Liver
Kidney
Intestine
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 5.000.a.
Fe
Duncana
5 40.58400
5 69.95400 69.95400
5 71.93600 71.93600
5 97.22200
.152 .210
Tissues
Muscle
Liver
Intestine
Kidney
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 5.000.a.
176
Means ( Zamfara )
Report
4.23833 .54833 2.26333 32.51833 2.94000 .65850 23.74667 82.00667
1.127713 .153286 .611708 4.496138 1.186120 1.179304 21.452589 36.900577
6 6 6 6 6 6 6 6
3.97667 .41000 3.34667 25.82833 5.65167 1.97833 2.93000 84.02667
.772701 .113490 3.014343 2.447394 1.091026 2.260287 1.080389 34.208413
6 6 6 6 6 6 6 6
2.92833 .45167 2.47000 28.03833 20.39833 3.91133 3.97167 67.89333
.370751 .100879 .916537 4.646613 14.364079 6.186182 1.104851 12.150005
6 6 6 6 6 6 6 6
4.12000 .51167 2.38167 37.30500 3.50767 5.22000 6.29833 55.97000
2.379705 .203117 .814086 9.573528 1.843128 5.974931 10.911168 12.836964
6 6 6 6 6 6 6 6
3.81583 .48042 2.61542 30.92250 8.12442 2.94204 9.23667 72.47417
1.396479 .148514 1.604654 7.109558 9.981161 4.549118 14.185548 27.447653
24 24 24 24 24 24 24 24
Mean
Std. Deviation
N
Mean
Std. Deviation
N
Mean
Std. Deviation
N
Mean
Std. Deviation
N
Mean
Std. Deviation
N
Tissues
Intestine
Kidney
Liver
Muscle
Total
Pb Cd Ni Zn Cn Cr Mn Fe
Oneway
ANOVA
6.507 3 2.169 1.131 .360
38.346 20 1.917
44.854 23
.068 3 .023 1.036 .398
.439 20 .022
.507 23
4.407 3 1.469 .536 .663
54.816 20 2.741
59.223 23
465.312 3 155.104 4.449 .015
697.242 20 34.862
1162.554 23
1229.736 3 409.912 7.722 .001
1061.606 20 53.080
2291.342 23
73.632 3 24.544 1.220 .328
402.341 20 20.117
475.973 23
1720.009 3 573.336 3.943 .023
2908.276 20 145.414
4628.285 23
3106.202 3 1035.401 1.456 .257
14221.392 20 711.070
17327.594 23
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Pb
Cd
Ni
Zn
Cn
Cr
Mn
Fe
Sum of
Squares df Mean Square F Sig.
177
Post Hoc Tests
Homogeneous Subsets
Pb
Duncana
6 2.92833
6 3.97667
6 4.12000
6 4.23833
.148
Tissues
Liver
Kidney
Muscle
Intestine
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Cd
Duncana
6 .41000
6 .45167
6 .51167
6 .54833
.153
Tissues
Kidney
Liver
Muscle
Intestine
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Ni
Duncana
6 2.26333
6 2.38167
6 2.47000
6 3.34667
.312
Tissues
Intestine
Muscle
Liver
Kidney
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Zn
Duncana
6 25.82833
6 28.03833
6 32.51833 32.51833
6 37.30500
.077 .176
TissuesKidney
Liver
Intestine
Muscle
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Cn
Duncana
6 2.94000
6 3.50767
6 5.65167
6 20.39833
.550 1.000
Tissues
Intestine
Muscle
Kidney
Liver
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Cr
Duncana
6 .65850
6 1.97833
6 3.91133
6 5.22000
.121
Tissues
Intestine
Kidney
Liver
Muscle
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Mn
Duncana
6 2.93000
6 3.97167
6 6.29833
6 23.74667
.653 1.000
Tissues
Kidney
Liver
Muscle
Intestine
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
Fe
Duncana
6 55.97000
6 67.89333
6 82.00667
6 84.02667
.109
Tissues
Muscle
Liver
Intestine
Kidney
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.a.
178
One-way ANOVA
Descriptives
41 4.31556 2.854032
41 4.71673 2.988457
41 3.13427 1.333954
41 3.23088 1.604507
164 3.84936 2.393262
41 .49437 .393542
41 1.46505 3.063156
41 .55171 .287275
41 .46341 .452767
164 .74363 1.608315
41 2.81698 2.067824
41 3.77920 3.531035
41 2.01971 1.141793
41 2.00634 .753431
164 2.65555 2.258330
41 28.43073 10.291230
41 34.03049 13.869082
41 29.97122 8.100469
41 31.95878 8.160794
164 31.09780 10.492703
41 3.82834 4.452221
41 6.81412 4.965650
41 19.03171 18.243960
41 3.29941 3.290923
164 8.24340 11.665875
41 2.40173 6.163116
41 3.69683 14.436707
41 1.89459 3.241605
41 2.12537 3.986669
164 2.52963 8.211889
41 10.38861 13.078570
41 4.02532 3.817941
41 4.70176 4.821193
41 2.76427 4.283012
164 5.46999 8.022355
41 97.19915 75.172555
41 100.33512 46.087298
41 99.63341 55.745647
41 63.52366 29.726457
164 90.17284 55.919139
Intestine
Kidney
Liver
Muscle
Total
Intestine
Kidney
Liver
Muscle
Total
Intestine
Kidney
Liver
Muscle
Total
Intestine
Kidney
Liver
Muscle
Total
Intestine
Kidney
Liver
Muscle
Total
Intestine
Kidney
Liver
Muscle
Total
Intestine
Kidney
Liver
Muscle
Total
Intestine
Kidney
Liver
Muscle
Total
Pb
Cd
Ni
Zn
Cn
Cr
Mn
Fe
N Mean Std. Deviation
ANOVA
76.406 3 25.469 4.754 .003
857.210 160 5.358
933.616 163
28.615 3 9.538 3.883 .010
393.013 160 2.456
421.628 163
86.691 3 28.897 6.209 .001
744.618 160 4.654
831.309 163
726.700 3 242.233 2.251 .085
17219.080 160 107.619
17945.780 163
6657.012 3 2219.004 22.867 .000
15526.087 160 97.038
22183.099 163
79.762 3 26.587 .390 .760
10912.162 160 68.201
10991.925 163
1401.832 3 467.277 8.226 .000
9088.551 160 56.803
10490.382 163
39045.214 3 13015.071 4.425 .005
470647.7 160 2941.548
509692.9 163
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Between Groups
Within Groups
Total
Pb
Cd
Ni
Zn
Cn
Cr
Mn
Fe
Sum of
Squares df Mean Square F Sig.
179
Post Hoc Tests
Homogeneous Subsets
Pb
Duncana
41 3.13427
41 3.23088
41 4.31556
41 4.71673
.850 .434
Tissues
Liver
Muscle
Intestine
Kidney
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 41.000.a.
Cd
Duncana
41 .46341
41 .49437
41 .55171
41 1.46505
.812 1.000
TissuesMuscle
Intestine
Liver
Kidney
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 41.000.a.
Ni
Duncana
41 2.00634
41 2.01971
41 2.81698
41 3.77920
.110 1.000
TissuesMuscle
Liver
Intestine
Kidney
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 41.000.a.
Zn
Duncana
41 28.43073
41 29.97122 29.97122
41 31.95878 31.95878
41 34.03049
.149 .096
Tissues
Intestine
Liver
Muscle
Kidney
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 41.000.a.
Cn
Duncana
41 3.29941
41 3.82834
41 6.81412
41 19.03171
.129 1.000
Tissues
Muscle
Intestine
Kidney
Liver
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 41.000.a.
Cr
Duncana
41 1.89459
41 2.12537
41 2.40173
41 3.69683
.375
TissuesLiver
Muscle
Intestine
Kidney
Sig.
N 1
Subset
for alpha
= .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 41.000.a.
Mn
Duncana
41 2.76427
41 4.02532
41 4.70176
41 10.38861
.276 1.000
TissuesMuscle
Kidney
Liver
Intestine
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 41.000.a.
Fe
Duncana
41 63.52366
41 97.19915
41 99.63341
41 100.33512
1.000 .807
TissuesMuscle
Intestine
Liver
Kidney
Sig.
N 1 2
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 41.000.a.
180
APPENDIX 4
4a Correlations of the various elements in the Intestine
Pb Cd Ni Zn Cu Cr Mn Fe
Pb Pearson Correlation 1 .310 .423 .496 -.316 .742 .080 .044
Cd Pearson Correlation .310 1 .075 .499 -.439 .030 .445 .810*
Ni Pearson Correlation .423 .075 1 .335 -.332 .551 -.203 -.385
Zn Pearson Correlation .496 .499 .335 1 -.963** -.059 .529 .319
Cu Pearson Correlation -.316 -.439 -.332 -.963** 1 .149 -.405 -.288
Cr Pearson Correlation .742 .030 .551 -.059 .149 1 -.471 -.305
Mn Pearson Correlation .080 .445 -.203 .529 -.405 -.471 1 .346
Fe Pearson Correlation .044 .810* -.385 .319 -.288 -.305 .346 1
*. Correlation is significant at the 0.05 level (2-tailed).
**. Correlation is significant at the 0.01 level (2-tailed).
181
Pb Cd Ni Zn Cu Cr Mn Fe
Pb Pearson
Correlation 1 .356 .753 .618 -.268 .744 .464 .088
Cd Pearson
Correlation .356 1 .217 .533 -.250 .363 .241 .069
Ni Pearson
Correlation .753 .217 1 .255 .098 .937
** .440 -.411
Zn Pearson
Correlation .618 .533 .255 1 -.842
* .276 .549 .406
Cu Pearson
Correlation -.268 -.250 .098 -.842
* 1 .201 -.134 -.288
Cr Pearson
Correlation .744 .363 .937
** .276 .201 1 .632 -.176
Mn Pearson
Correlation .464 .241 .440 .549 -.134 .632 1 .504
Fe Pearson
Correlation .088 .069 -.411 .406 -.288 -.176 .504 1
**. Correlation is significant at the 0.01 level (2-
tailed).
182
4 b
Corr
elati
ons
of
vario
us
elem
ents
in the Kidney
. Correlation is significant at the 0.05 level (2-tailed).
183
4c Correlations of various elements in the Liver
Pb Cd Ni Zn Cu Cr Mn Fe
Pb Pearson
Correlation 1 .006 .486 .261 .511 -.141 .432 .529
Cd Pearson
Correlation .006 1 -.054 .409 -.177 -.451 .026 .065
Ni Pearson
Correlation .486 -.054 1 -.089 .834
* .289 .550 -.038
Zn Pearson
Correlation .261 .409 -.089 1 -.340 -.964
** .509 .040
Cu Pearson
Correlation .511 -.177 .834
* -.340 1 .469 .362 .005
Cr Pearson
Correlation -.141 -.451 .289 -.964
** .469 1 -.282 .068
Mn Pearson
Correlation .432 .026 .550 .509 .362 -.282 1 .340
Fe Pearson
Correlation .529 .065 -.038 .040 .005 .068 .340 1
*. Correlation is significant at the 0.05 level (2-
tailed).
**. Correlation is significant at the 0.01 level (2-
tailed).
184
4 d Correlations of various elements in muscle
Pb Cd Ni Zn Cu Cr Mn Fe
Pb Pearson Correlation 1 .407 .744 .290 -.064 .866* -.138 .252
Cd Pearson Correlation .407 1 .518 -.537 .702 .211 .237 -.214
Ni Pearson Correlation .744 .518 1 .202 .060 .871* .080 -.239
Zn Pearson Correlation .290 -.537 .202 1 -.901** .348 -.690 .586
Cu Pearson Correlation -.064 .702 .060 -.901** 1 -.141 .528 -.613
Cr Pearson Correlation .866* .211 .871
* .348 -.141 1 .098 -.102
Mn Pearson Correlation -.138 .237 .080 -.690 .528 .098 1 -.758*
Fe Pearson Correlation .252 -.214 -.239 .586 -.613 -.102 -.758* 1
*. Correlation is significant at the 0.05 level (2-tailed).
**. Correlation is significant at the 0.01 level (2-tailed).
185
