فاطمة عباس -رسالة ماجستير

Republic of Iraq
Ministry of Higher Education

and Scientific Research
HC
Thi-Qar University H
3
College of Sciences

Department of Biology

Study of Some Heavy Metals Concentration

and blood Parameters of Workers in Fuel
Stations in the Center of Thi-Qar Province
Southern of Iraq
A Thesis Submitted to the Council of

College of Sciences -Thi-Qar University in Partial Fulfillment of
the Requirements for the Degree of Master of sciences
In Biology
By:
Fatima Abbas Moshchil

B.Sc. 2012
( Biology )
Supervised by:
Prof. Dr. Basim Y. Al-Khafaji
April Rajab
2016 1437

Acknowledgements
First, I would like to thank God for His care and support throughout
my life especially through the accomplishment of this research. I would
like also to express my profound thanks and sincere gratitude to my
supervisor Prof. Dr. Basim Y. Al-Khafaji for his invaluable scientific
guidance, encouragement and support, that made it possible for me to
accomplish this study. For his scientific advice and support. I would like
to express my deepest gratitude to dean of science college Prof. Dr.
Mohammed A. Aouda and head of Biological department Prof. Dr. Manal
B. AL-Tamimi for their kindness and help. My special thanks also go to
Prof. Dr. Khalid Al Fartosi. Thanks go to all the staff in the department of
Biology, college of sciences, university of Thi-Qar, with my special
thanks to Assist. Prof. Dr. Afrah A. Magtoof.
I would like to thank all the staff of fuel stations in Al-Nssiriyah city.
With my deep regards and special gratitude to Msc. Karrar K. Abd, Msc.
Moslim Mohsin and my both friends for cooperation and thanks are also
to my family for their support and couragement. whose without them, this
work would not have been accomplished.
Fatima
Dedication
I Dedicate this humble
effort to my Big teacher
My father, to my
compassionate mother,
dear brothers, sisters, my
dear son, and my
husband.
fatima
Abstract
This study was designed during the period from October 2014 to
April 2015 to measure the concentration of some heavy metals
(Cadmium, Cobalt, Copper, Lead & Nickel) in the blood serum and
its effect upon some blood parameters of workers at four fuel stations
have chosen (Alballam station, Nasiriyah station, Alrayat station and
Almohandis station) in Al-Nasiriyah city (Center of Thi-Qar
province southern of Iraq).
100 blood samples have collected randomly, 80 of it were taken
from a group of workers at four stations. They aged from 20 to 55
years (them duration work was 1-12 years) and 20 samples of non-
working as a control group.
Each sample of blood divided into two parts, the first, to measure
blood parameters directly, second part the separate the serum for the
purpose of measuring the concentration of heavy metals.
The results and statistical analysis showed that the concentration
of these heavy metals in the samples were higher than the
concentration in the control group, the concentration of metals were,
Cd (0.024 ppm), Co (0.027 ppm), Cu (0.02 ppm), Ni (0.078 ppm)
and Pb (0.15 ppm), while the control group recorded low values, Cd,
Co, Cu were less than (0.004 ppm), while Ni (0.03 ppm) and Pb
(0.03 ppm).
Blood parameters of this study were (RBCs.= 5.1*106/L , Hb.=
14 g/dL, PCV.= 43.2%, WBCs.= 7.4*103/L & PLTs.=
204.9*103/L). Some of these parameters (RBCs., WBCs. & PLTs.)
I
have been found decreased with the increasing of heavy metals
concentration in the blood serum of workers comparison with control
sample, there were some significant differences in some as well. All
results within the normal range and did not point any existence
critical values.
II
List Contents
Index Subjects Page No.
Summary
List of contents III
List of tables VIII
List of figures IX
Abbreviations XI
Chapter One : Introduction& Literature Review
1.1 Introduction 1
1.2 Aim of study 4
1.3 Literature review 5
1.3.1 Heavy metals 5
1.3.2 Using and Recovery of heavy metals 6
1.3.3 Cadmium (Cd) 7
1.3.4 Cadmium Toxicity 8
1.3.5 Cobalt ( Co) 9
1.3.6 Cobalt Toxicity 10
1.3.7 Copper (Cu) 11
1.3.8 Copper Toxicity 11
1.3.9 Lead (Pb) 12
1.3.10 Lead Toxicity 13
III
1.3.11 Nickel (Ni) 15
1.3.12 Nickel Toxicity 16
1.3.13 Classifications of heavy metal exposure 17
1.3.14 Access pathways of heavy metal to the body 18
1.3.15 Mechanism of heavy metals activity 19
1.3.16 Laboratory testing and diagnosis for the presence of 19

heavy metals
1.3.17 Assessment of exposure to heavy metals 20
1.3.18 Management of Heavy Metal poisoning 23
1.3.19 Heavy metals analytical methods (Quantitative 24

determination)
1.3.20 Vehicle emissions and their sources 26
Chapter Two: Materials and Methods
2.1 Materials 28
2.1.1 Instruments and equipment 28
2.1.2 Chemical materials 29
2.2 Methods 29
2.2.1 The Study area 29
2.2.2 Method of sample collection 29
2.2.2.1 With regard to data of workers at fuel stations 29
2.2.2.2 With regard to collection of blood samples 30
2.2.3 Measurement of blood parameters 30
IV
2.2.4 Preparation of serum 30
2.2.5 Digestion of serum 30
2.2.6 Estimation of heavy metals 31
2.2.7 Study design 31
2.2.8 Statistical analysis 32
Chapter three : Results
3.1 Heavy metal concentrations in serum of study samples 33
3.1.1 Cadmium (Cd) 34
3.1.2 Cobalt (Co) 34
3.1.3 Copper (Cu) 35
3.1.4 Nickel (Ni) 36
3.1.5 Lead (Pb) 37
3.2 Estimation of some blood parameters 38
3.2.1 Hemoglobin (Hb.) 39
3.2.2 Packed Cell Volume (PCV.) 40
3.2.3 Platelets (PLT.) 41
3.2.4 Red blood corpuscles (RBCs.) 42
3.2.5 White blood cells (WBCs.) 43
3.3 The relationship between the values of heavy metals 44

concentrations and periods of work
V
3.4 The relationship between Heavy metals concentration 45
and smoking
3.5 The relationship between Heavy metals concentration 48

and age
3.6 Concentrations of heavy metals in collected samples 51
Chapter four: Discussion
4.1 Heavy metals concentration in blood serum of 52

workers
4.1.1 Cadmium 52
4.1.2 Cobalt 53
4.1.3 Copper 54
4.1.4 Lead 55
4.1.5 Nickel 56
4.2 Heavy metals concentration in blood serum of 57

workers and study stations
4.3 Blood parameters 59
4.4 The relationship between the values of heavy metals 60

4.5 Relationship between smoking and heavy metals 61

concentration in serum of workers
VI
4.6 Relationship between age and heavy metals
concentration in serum of workers 61
Conclusions and Recommendations
I Conclusions 64
II Recommendations 65
References 66
Appendices
Arabic Summary
VII
List of Tables
Table No. Table title Page No.
Chapter One : Introduction& Literature Review
1-1 The most usual methods applied for heavy metals 25

determination
Chapter Two : Materials and Methods
2-1 Instruments and equipment used and their 28

manufacturers
2-2 Chemical materials that used in this study 29
3-1 The mean values of the heavy metals in the serum 33

of workers at each station and control
3-2 The means of some blood parameters for workers 39

and control
3-3 concentration of heavy metals in the serum of 45

workers according to the work period
3-4 Heavy metals concentration in the smoker, non- 46

smoker and control groups
3-5 Relationship between heavy metals concentration 49

(ppm) in workers and their age
Appendices
Appendices-1 The information of study samples
VIII
List of Figures
Figure Figure title Page No.
No.
Chapter Two : Materials and Methods
2-1 Study design 31
3-1 Mean values of Cadmium (Cd)concentration ppm. in 34

study samples
3-2 Mean values of cobalt (Co) concentration ppm. in study 35

samples
3-3 Mean values of copper (Cu) concentration ppm. in 36

study samples
3-4 Mean values of Nickel (Ni) concentration ppm. in 37

study samples
3-5 Mean values of Lead (Pb) concentration ppm. in study 38

samples
3-6 Mean value of Hb. (g/dL) for study samples 40
3-7 Mean value of PCV. (%) for study samples 41
3-8 Mean value of PLT.(*103/L) for study samples 42
3-9 Mean value of RBC.(*106/L) for study samples 43
IX
3-10 Mean value of WBC.(*103/ L) for study samples 44
3-11 Heavy metals concentration for smoker, non-smoker 47

and control groups
3-12 Heavy metal concentration ppm and age of workers 50
3-13 The comparison among means of heavy metals 51

concentrations values for workers in all stations and
control
Appendices
Appen.2 Location of study stations
X
List of Abbreviations
Abbreviations Symbol
UNEP United Nations Environmental Protection
GPA Global Program Of Action
USEPA US Environmental Protection Agency
UNDP United Nations Development Programme
DNA Deoxy Ribose Nucliec Acid
PM particulate matter
USDOL United States Department Of Labor
IARC International Agency for Research on Cancer
ATSDR Agency of Toxic Substances and Disease Register
HPA Health Protection Agency
WHO World Health Organization
EPA Environment Protection Agency
PAHs Polycyclic aromatic hydrocarbons
NIH National Institute Of Health
EDTA Ethylenediaminetetraacetic
AAS Atomic absorption

Spectrometry
XI
ICP-AES Inductively coupled plasma with atomic
emission spectrometry
AFS Atomic fluorescence spectrometry
XRF X-ray fluorescence
NAA Neutron activation analysis
CO carbon monoxide
NO2 nitrogen dioxide
VOCs volatile organic compounds
FAAS Flame Atomic Absorption Spectrophotometer
ppm Part per million
ND None Detected
PCV Packed Cell Volume
Hb Hemoglobin
RBCs Red blood corpuscles
WBCs White blood cells
PLT Platelets
XII
Chapter One
Introduction
and
Literatures Review
Chapter one.Introduction and Literatures review
Chapter one
Introduction and Literatures review
1.1: Introduction
The environmental pollution became a major problem in the world
especially the third world countries with the rapid development of industry,
particularly those dependent on the hydrocarbon products industries as a fuel
and its contaminated products as heavy metals, that considered as significant
threat to the environment and human because of its impact on human life and
other organisms. Heavy metals are group of metallic elements that have certain
chemical and electrical characters and its density is greater than 5g/cm
(Forstner and Wittmann, 1979). Heavy metals are entered the Ecosystem by
many ways such as manufacturers, the using of materials that containing heavy
metals (Alloway, 1990). Cadmium (Cd), Copper (Cu), Lead (Pb), Cobalt (Co)
and Nickel (Ni) are group of elements constitute heavy metals, these heavy
metals considered as natural constituents of the earths crust and are non-
biodegradable and so affect in the living things in the environment. So it can
enter the body through the respiratory system through air and digestive system
through ingestion and bio accumulate for long period of time (Hutton and
Symon, 1986 and UNEP/GPA, 2004).
The pollution by heavy metals can occur by many different ways, directly
or indirectly, may contaminate soils, water and plants by material from the air
or by direct deposition of pollutants "generally humans are exposed to these
metals by ingestion (drinking or eating) or inhalation (breathing)" (Alloway,
1990). Because of their contact with human life and other living organisms,
these dangerous and important pollutants are interest to many researchers and
have been a high number of mortality because of their toxicity (UNEP/GPA,
2004). Some of heavy metals are beneficial to humans and other living
1
organisms where intervention in the physiological and chemical processes

(Margesin & Schinner, 2005).
Because of their solubility in water and can be absorbed by plant (as ions
or its compounds), most of heavy metals are toxic for all biota and after
absorption, these metals bind with biomolecules such as proteins and nucleic
acids and defect their functions (Ming-Ho, 2005). After entered and absorbed
by biota, heavy metals are transport with bloodstream to the liver for
transformation (Dimari et al., 2008). The intake of heavy metals by inhalation
and ingestion may cause different diseases such as anemia, neuropsychological
effects, liver diseases, gastrointestinal pathologies, teratogenic implications
(Needleman & Bellinger, 1991). Such toxic metals can cause DNA-damaging
in humans that can lead to cancer and a decrease of fertility (Snow, 1992).
Heavy metals pollution is widespread due to many sources such as traffic
and fuel stations (Margesin & Schinner 2005). Traffic emission (especially
from cars exhaust) is an important source which affect human health near from
or workers in fuel stations, that estimated in some of European countries
France, Switzerland and Austria, 3% of total mortality per year (i.e. 20 000
deaths) are due to traffic emissions alone (Kunzli et al., 2000). Some of heavy
metal emission are factors for cars, vans, trucks, buses and motorcycles for
each of the emission sources fuel consumption, engine oil, tire wear, brake
wear and road abrasion, the emission components contain Cadmium (Cd),
Chromium (Cr), Copper (Cu), Mercury (Hg), Nickel (Ni), Lead (Pb), Selenium
(Se), Cobalt (Co) and Zinc (Zn), all of them are relevant for emission reporting
to the United Nations Economic Commission for Europe Convention on Long
Range Trans boundary Pollutants (Winther and Slento, 2010). Other studies
indicate that living near roads with heavy traffic may considerably increase the
risks of adverse health effects (Janssen et al., 2001; Heinrich et al., 2005 ;
Beelen et al., 2009).Some of those studies indicate to evidence of effects of
2
such metals which are related to the distance from major roads and traffic
density (Hoek et al., 2002). Brunekreef et al. (2009) reported results from
comprehensive European academic study on the effects of long-term exposure
to traffic pollutants and cause-specific mortalities.
Likewise the fuel stations have been considered as an important source of
pollution by heavy metals which are a group that mostly originate from traffic
and fuel stations emission (Chen et al., 2005; Kong et al., 2012). Heavy metals
are part of content in gasoline and diesel available from fuel stations (Denier
van der Gon, 2009). Pb added to fuels and lubricants to enhance its properties
(Huber and Prokop, 2012).
Working in or living near a fuel stations and industrial sites which inhale
these metals and their compounds which increase ones risk of exposure and the
air in the same environments usually contains a number of chemicals which
inhaled and absorbed by the body, pose a potential risk for workers health
(Gutteridge, 1995). Therefore the workers in the fuel stations are the most
persons that exposure to such pollutants. The presence of workers near the car
exhaust and fuel vapors for long periods leads to the accumulation of heavy
metals, which mostly be harmful to humans.
3
1.2: Aim of study

Heavy metals are group of most important toxic metals for human , and its
presence at high levels in the human body considered as significant threat for
human health. Because of the lack previous studies in this fields, so the present
study was complemented for the following aims:
1. Determine the concentration of selected heavy metals (Cadmium,

Cobalt, Copper, Lead and Nickel) in the blood serum of workers at fuel
stations in Al-Nasiriyah city ( center of Thi-Qar province )
2. Measurement of some blood parameters ( Hb., PCV., PLT., RBC. &
WBC. ) of workers and show the relationship between their values and
metals concentration.
3. The variation between the stations according to the differences in the
heavy metals concentration in their serum.
4. Highlight upon the source of this type of pollutants in the study place.
4
1.3: Literature review

1.3.1: Heavy metals
There is no clear definition of what is heavy metal, density is in most
cases taken to be the defining factor, the term heavy metals refers to metallic
metals that has a relatively high density and even at low concentration is
considered toxic or xenobiotic metals (Lenntech, 2004). Heavy metals is a
general term, which refers to group of metals and metalloids with atomic
density more than 5g/cm or 5 times or more than water density (Huton and
Symon, 1986; Battarbee et al., 1988; Nriagu and Pacyna 1988; Nriagu, 1989;
Garbarino et al., 1995; Hawkes, 1997). Heavy metals a group of elements have
important chemical properties that include Lead (Pb), Cadmium (Cd), Zinc
(Zn), Mercury (Hg), Arsenic (As), Silver (Ag) Chromium (Cr), Nickel (Ni),
Cobalt (Co), Copper (Cu) Iron (Fe), and the Platinum group metals (Farlex,
2005).
Some of these metals are accumulative pollutants, cause environmental
hazards and are reported to be exceptionally toxic (Yargholi and Azimi, 2008).
Such metals are play an important role in oxidative stress in the cell and in the
etiology of diverse human diseases such as carcinogenesis (Frenkel, 1992; Hei
and Filipic, 2004; Leonard et al., 2004; Wang et al., 2004).
There are scientific studies proved that if the concentrations of heavy
metals such as Cu, Cd, Pb, Ni and Co exceed critical thresholds, may pose
ecosystem to potential toxic effects and have a risk impact to human health
(Janko et al., 2000 ; WHO, 2007). A mount of inhaled metals by human are
transmitted to several organs through circulatory system and load in some
certain organs especially lung (Descotes, 1992).
Several diseases may occur by exposure to heavy metal toxicity such as
brain damage, cerebral palsy, mental retardation, gastrointestinal
abnormalities, and dermatitis, lung cancer, and death of the unborn fetus
5
(USEPA, 2002; Rotich et al., 2006 ; UNDP, 2006). Many metals may be form
DNA adducts that can cause directly modify and/or damage DNA (Chakrabarti
et al., 2001).
1.3.2: Using and Recovery of heavy metals

Heavy metal exists in rocks as ores in the different chemical forms, from
which they are recovered as minerals (Habashi, 1992). For thousands years ago
started using of heavy metals by human in different areas, since 5000 years
lead was used in many applications such as pigments of glazing ceramics,
building materials and water pipes. Lead acetate was used to sweeten old wine
in wide areas in ancient Rome and thought Mercury was used as a ointment to
sedative teething pain in infants by the old Romans, and later (from the 1300s
to the late 1800s) used for syphilis treatment. In the mid of 1800s, Claude
Monet used pigments of cadmium extensively (Jarup, 2001).
Heavy metals adverse health effects have been known for a long time and
exposure to it increasing and continues since the 19th century and its
production take to increase for more than 100 years, with continuous emissions
to the environment. From 1990 and 2000 the using of heavy metals decrease
especially in the developed countries such as the UK due to fell down
emissions of heavy metals by over 50% (Jarup, 2001). There are many
chemical forms for heavy metals, as ores include sulphides, as arsenic, iron,
lead, cobalt, lead-zinc, gold, silver and nickel sulphides, oxides as manganese,
antimony, gold, aluminum, and selenium some of it was recovered as both
oxide and sulphide such as copper, iron and cobalt. Naturally, sulphides of
cadmium, lead, arsenic and mercury would be found together with sulphides of
copper (chalcopyrite, CuFeS2) and iron (pyrite, FeS2) as minors, which a
results to processes of various hydrometallurgical or as part of exhaust fumes
in pyro metallurgical and other processes that follow the mining (Habashi,
6
1992). Heavy metals are generally recovered through a lot of ores by many
processing as mineral operations (Peplow, 1999; Lenntech, 2004; UNEP/GPA,
2004 ; USDOL, 2004).
1.3.3: Cadmium (Cd)

Cadmium was used by human relatively recent concomitant to
technological development and in for some decades ago that give cadmium
serious consideration as a possible pollutant. Naturally Cd found in the
environment: in ambient air, soils, sediments and seawater (Harrison, 2001).
Mines, metal smelters and industries are sources for Cadmium emission to air
by using cadmium compounds for alloys, pigments ,batteries and in plastics
(Harrison, 2001). Tobacco smoke in all of its forms contains an amounts of the
metals especially Cd, therefore, the smoking was considered as one of the
important sources of cadmium exposure in humans because of Cd absorption
through the lung is much greater than absorption by gastrointestinal tract
(Figueroa, 2008 ; Ming-Ho, 2005). In general, non-smokers and non-
occupationally exposed workers, food products account for most of the human
exposure burden to cadmium (Extoxnet, 2003).
There many several stable isotopes Cd (106Cd, 108Cd, 120Cd, 111Cd,
112Cd, 113Cd, 114Cd and 116 Cd), There is an abundance of isotopes of
cadmium, starting from the top and is 112Cd, which is transition metal in
group-II B abundance in the periodic table, Cd seems mostly in free Cd+2 and
CdHCO3+ it is found in the soil, However, pH is a great factor for controlling
the solubility of Cd (Bradl et al., 2005). Cd tends to be more available in free
Cd+2 ions, especially in low pH level while the formation of different carbonate
species (CdCO3 and CdHCO3) which is favored in high levels of pH.
Adsorption of Cd. depends largely on the surrounding ionic exchange such as
7
Ca+2 and Zn+2 and this leads to excessive competition for absorption site in the
soil (Bradl et al., 2005).
The production of Cd comes mainly as a by-product from the smelting
and refining of Zn, because the metals often appear together. Therefore,
production of cadmium as the product which comes mainly from the mining
and refining of zinc and some other metals, where most of them look together
and usually Cd pollution accompanied the zinc, lead and copper mining
operation. It is usually used in the electrodes of the batteries and paints and
coatings industry, which protects the iron and other metals from oxidization
(Bradl et al., 2005). There are many ways for the introduction of cadmium into
the environment, one of the main sources is the burning of waste containing
cadmium as one of the heavy metals are relatively volatile, it is easily
introduced into the atmosphere when they are burnt other sources of cadmium
is the application of phosphate fertilizers and sewage sludge in agriculture and
mining and smelting operations (Extoxnet, 2003).
1.3.4: Cadmium Toxicity

Cd is non-essential heavy metal, it can be toxic to humans and some
biological systems even at very low levels of concentration, and is considered
a risk factor as associated with premature arteriosclerosis and high blood
pressure, which can lead to cardiovascular disease (Messner et al., 2009). Cd
has serious effects for humans such as bone degenerations and interferences
with the metabolism of Ca, vitamin D and collagen. The continuously
exposure to Cd. can defect the kidney and lung and causing protein urea and
emphysema (Nordberg, 2004).
Cd and its compounds are considered carcinogenic to humans by the
IARC (International Agency for Research on Cancer, 2010). It and its
compounds are also classified as known human carcinogens by the United
8
States Department of Health and Human Services (Agency of Toxic

Substances and Disease Registry, 2008). Cd breathing has been associated with
lung cancer in humans who exposed in rats (ATSDR., 2008). There is evidence
of increased mortality due to lung and prostate cancer from Cd inhalation over
a long period of time in various occupational settings , exposure for Cd at long
time even at low levels in the workplace or other environment can also result
in kidney dysfunction and cute exposure for Cd by ingestion and inhalation can
lead to skin and eye irritation (Environment Canada & Health Canada, 1994).
In women Cd has also been shown to exert significant effects on ovarian and
reproductive tract morphology even with extremely low doses and exposure
during pregnancy is being associated with decreased birth weights and
premature birth (Henson & Chedrese, 2004).
1.3.5: Cobalt ( Co)

At present the use of cobalt increased significantly because of its
resistance to corrosion and its magnetic qualities and durability of alloys
(Hamilton, 1994). These qualities make cobalt suitable for many fields such as
ceramics, glass, catalysts, and the magnet alloy and also in various steel. Co is
also used in paints, varnishes, inks, foam stabilizers in beverages, it is also
used in paints, varnishes, inks, foam stabilizers in malt beverages,
agrochemicals and many other applications (Hamilton, 1994). There are many
sources that product elevated concentrations of Co which are both natural and
anthropogenic, where natural sources include erosion of rocks and human
sources, mostly resulting from mining operations, various industries and
remnants of cars and uses of oil and its derivatives that are most likely to be
found in all parts of the mining industry and processing (WHO, 2006). Co can
also enters the air through burning of oil which adversely affect the health of
people who breathe the fumes and residues element (ATSDR, 2004).
9
Co is located in the first transition series of group 9 of the periodical table,

where oxidation states of Co are +2 and +3 and there is only one stable isotope,
59Co. Co+3 which is a powerful oxidizer, that can oxidize water and release
oxygen (Bradl et al., 2005). Co has a high affinity towards different organic
matter, Fe and Mn oxides (WHO, 2006).
1.3.6: Cobalt Toxicity

Cobalt is one part of components vitamin B12, which is essential for all
animals, including humans (Bradl et al., 2005). Co involved in many
biological processes such and Co deficiency can affect the gastric secretions,
blood sugar levels and enzyme activities, in human, excessive intake of Co can
cause disorder in the hematologic, neurologic, cardiac and thyroid activities
(Hamilton, 1994).
Exposure to high levels of Co from polluted air where workers breathing
air containing 100,000 times than concentration of Co in normal ambient air
that may result in health problems associated with exposure to high levels of
Co, for example, severe lung effects such as wheezing, asthma, and pneumonia
and other health effects through ingestion may include: vomiting and nausea,
vision problems, thyroid damage, dermatitis, severe damage to the heart, and
even death (Texas Department of State Health Services, 2012).
There are many sever effects due to excessive exposure to Co such as
severe sickness, bleeding, decreased white blood cell count, diarrhea, sterility,
coma, hair loss and even death depending on the amount of the Co source, the
parts of the body that are closest to the source, and how long the exposure lasts
and exposure to high levels of Co can cause genetic materials damage within
cells and may result in the development of some types of cancers, therefore, it
considered as main reason of cancer in human (Texas Department of State
Health Services, 2012).
11
1.3.7: Copper (Cu)

Copper is reddish brown element which is found naturally in soil, water,
air and even food, which is essential for living organisms, including the
human, but the human body needs in small amounts. Cu released from many
sources, including volcanoes and erosion processes, industrial processes,
mining, waste incineration and fuel with little vegetation. Exposure to Cu is
through eating polluted foods with this element and the most important source
is breathing air laden with particles of Cu (HPA., 2010).
Copper is usually heat and electric conductive metal. In the periodic table,
copper is listed in the group I-B and has several isotopes but 63Cu and 65Cu
are the most abundance with 60,9% and 30,1% respectively, the oxidation
states for copper are I and II. Cu has the same properties as Zn+2, Mg+2 and
Fe+2 in the oxidation state II, (Bradl et al., 2005).
Copper is known for use in many industries such as electric wires, cook-
ware, piping systems, bactericides, fungicides, and the fertilizers, feed
additives, bactericides and fungicides are the main source of the introduction
of copper to into the environment (Calvino et al., 2008).
1.3.8: Copper Toxicity

Copper can enter the human body and his bloodstream directly by eating
foods and drinks containing copper, there are a great source for the
introduction of copper to the human body and his bloodstream is breathing air
laden with particles of this element so workers are more exposure to increase
the copper content in their bodies, (ATSDR., 2004). Sometime Cu may also
considered a toxic for human, but its toxicity is incredibly unusual but toxic at
high concentrations (Adams and Keen, 2005). The harmful impact of copper
on human health is depending on the a way and amount of exposure to this
metal, breathing of fumes containing Cu can causes metal fume fever, which is
11
characterized by symptoms such as fever, headache and tiredness, as well as

sore throat, cough, tightening of the chest, nausea, vomiting, sometimes a
metallic taste in the mouth and blurred vision can occur by eating foods or
drinking water containing high levels of copper can cause stomach pain and
diarrhea (HPA., 2010).
Copper is distributed in various tissues of the body and present at highest
concentration in the liver and brain and high concentration generates a
Wilson's disease, which affects the nervous system and causes Liver disease
and breast cancer (Salman and Fadhil, 1990 ; Kruseo, 1994 ; Nicala and
Pauline, 1998 ; Saad, 2002). while decrease level of Copper affect on
mechanism of the heart and lead to an increase low-density cholesterol and
fatty proteins (Retsky et al., 1999).
Exposure of skin to Cu may cause inflammation, itching and burns.
Exposure to high levels of copper will result in the same effects in both adults
and children, but is not clear if effects occur at the same level. There are no
data regarding the effects of copper upon pregnancy, and whether it causes
harm to the unborn child (HPA., 2010). A few studies proved that the
hemolytic anemia has been occurred in a severely burned and debilitated in
which copper sulfate crystals were being applied to granulation tissue and
increased serum and urine copper levels were observed too (Holtzman et al.
1966).
1.3.9: Lead (Pb.)

Lead one of metals which found naturally in the earths crust (Health
Canada, 2009). A little amount of it can enter the environment from many
sources such as natural processes as erosion and anthropogenic source as
industrial activities for example metal smelters or refineries (Health Canada,
2010). Lead is a weak electrical conductivity but it is anti-oxidant metal, and it
12
be found in group IV-A in the periodic table and exists in two oxidation state,
II and IV. It has four isotopes, Pb 204, Pb 206, Pb 207 and Pb 208 and the last
isotope is the most abundant with 52.3% of the total Pb isotopes. Lead is found
in many forms, but it is become toxic in ionic forms only and it is sensitive
against low pH due to its solubility (Bradl et al., 2005).
Because of its physical and chemical properties, it is easy to extract with
other metals and it extensively used in many applications and products such as
gasoline additives, automobile batteries, ammunitions, pigments, pesticides
and different alloys. Since long time ago lead has been used extensively
particularly industry of lead-acid batteries (Environment Canada, 2010),
however it is also used to make fishing nets weights, lead shot, solder, sheet
lead, some brass and bronze products, pipes and professional paints (other than
paints for use by children). Because of Pb toxicity, the use of it in some
applications and products has been banned in many countries, for example, Pb
as a gasoline additives, pesticides and ammunitions are banned in the U.S. and
most of the countries in Europe but still widely used in other countries such as
China, Russia and India. Lead is introduced and accumulated in the
environment by different ways, most of it by Pb dust from pigments, mining of
its ores, combustion of leaded gasoline and use of ammunitions in hunting
(Bradl et al., 2005). There are several ways to be exposed of lead, usually by
inhalation, digestion of food and water and skin contact and due to it is use as
gasoline additives, the workers of fuel station are most exposed to pollution
with Pb (Environment Canada, 2010).
1.3.10: Lead Toxicity

Inhalation, is the main way for lead exposure by workers in the refining,
mining, use and reclamation of lead industries. In countries that still use
gasoline containing lead, fuel stations and remnants of cars and proximity to
crowded roads are major sources of exposure to lead pollution (WHO., 1995).
13
In humans, lead can result in a wide range of biological effects depending

upon the level and duration of exposure. Effects may range from inhibition of
enzymes to the production of marked morphological changes and even death,
overall effect of Pb is usually confined to the nervous system of humans, it
also has an impact on the composition of hemoglobin which is associated with
anemia and a slight rise in blood pressure, as well as affect the kidney, thus
damaging the kidney and high levels of Pb may cause kidney failure (Nordic
Council of Ministers, 2003).
High levels of Pb may cause nerve joint mood swings, memory loss, and
muscle disorders, cardiovascular, skeletal (Environmental Working Group,
2010). Pb exposure can cause a convulsions, coma and intelligence disorder at
the low end of exposures (US. ATSDR., 1999). A studies showed that even
small amounts are have being hazardous to human health (Environment
Canada, 2010). Pb and its inorganic compounds may be carcinogenic for
humans (International Agency for Research on Cancer, 2010). Acute
exposures for high-level characterized by vomiting, diarrhea, convulsion,
coma, and death (Health Canada, 2007). Pb poisoning tends to restrain
important enzymatic functions in human and animals. Ingested lead can be
found in the blood and the surplus often accumulates in the bones and soft
tissues (liver, kidney, brain), and the reproductive effects of lead in the male
are limited to sperm morphology and count. In the female, some adverse
pregnancy outcomes have been attributed to Pb (Nordic Council of Ministers,
2003).
Pregnant women and young children are particularly vulnerable because
Pb can cross the placenta with rest and enter the fetal brain (Campaign for Safe
Cosmetics, 2007). Pb can also be transferred to infants via breastfeeding
(ATSDR., 2007), and Pb store in bones of infants that exposed to Pb by their
mothers (Rothenberg et al., 2000). Infants, toddlers, children, fetuses and
14
pregnant women are most susceptible to its chronic low-dose effects (Health
Canada, 2009 ; Health Canada, 2010). Intelligent quality deficits have been
associated with high blood lead levels (ATSDR., 2007), including those of
low-levels (Sprinkle, 1995). Lead exposure has also been linked to
miscarriage, hormonal changes, reduced fertility in men and women, menstrual
irregularities, delays in puberty onset in girls (Campaign for Safe Cosmetics,
2007).
1.3.11: Nickel (Ni.)

The fifth most abundant metal in the biosphere is Nickel, which is a white
yellowish metal, it was discovered through mining of other metals. Ni and its
compounds are one of the components of Earth's crust, and it releases to the
atmosphere through natural processes as erosion and volcanic eruptions, and
anthropogenic activities. There are 8.5 million kg of Ni are emitted into the
atmosphere each year from natural sources such as windblown dust, vegetation
and volcanoes (Schmidt and Andren 1980 ; Bennett and john, 1984), and five
times of this quantity is estimated which come from anthropogenic sources
(Nriagu and Pacyna, 1988). The incineration of wastes and fuel oil is
responsible for 62% of anthropogenic emissions, followed by metal refining,
municipal incineration, steel production, other Ni alloy production, and coal
combustion (Schmidt and Andren, 1980 ; Bennett and john, 1984).
Nickel is included in the group VIII in the periodic table and exists in five
stable isotopes, 58Ni, 60Ni, 61Ni, 62Ni and 64Ni, whereas the former is the
most abundant with 68.27 % of total Ni isotopes (Bradl et al., 2005).
Historically, focused the many researchers attention on this metal, how to
increase the prevention of this metal risks, and now paying more to examining
nickels role in the health of ecosystems (Ming-Ho, 2005).
15
Nickel carbonyl gas, Ni(CO)4, it is the most toxic form, and there are
other forms of this metal but however remains the most toxic and play an
uncertain role in the safety of workers and the community. Overall demand for
Ni has been increasing over time, mostly due to increasing stainless-steel
production, Ni is used in approximately 250,000 industrial applications, and is
used in the forms of nickel carbonate, nickel carbonyl, nickel chloride, nickel
nitrate, nickel oxide, nickel sulfate, and nickel sulfide (Chau and Kulikovsky-
Cordeiro, 1995). Some applications include the use in iron processing, nickel
plating, and nickelcadmium batteries. Nickel iron is used for electrical
equipment, copper nickel is used as an anticorrosive for marine vessels and
equipment, and nickel titanate is used as a pigment in paints. As Ni refineries
increase production, the concern for this heavy, mobile metal and its effects on
the environment also increases (Ming-Ho, 2005).
Nickel is present in fuel and as trace element in steel. It may also be
included in virgin engine oil in very small amounts - about 1 ppm (mg / litre)
according to Castrol, (2006) and Oil Analysers (2006) operates with a limit
value at 5 ppm for both gasoline and diesel engines while Shell.
1.3.12: Nickel Toxicity

Nickel plays an important and vital role for most living organisms,
including human as well as their role in plant where the excessive uptake of Ni
by plants causes phytotoxicity and leads to chlorisis, growth retardation and
necrosis (Poulik, 1999). As in humans Ni a negative role is also has negative
role in an excessive levels which may cause disorder in their nervous system,
many researchers have also found carcinogenic activities in both animals and
human (Denkhaus and Salnikow, 2002). The main pathway of Ni exposure by
humans is inhalation and ingestion which may cause a chronic Ni allergy in the
form of dermatitis. It can also cause discord in heart, skeleton, muscle, kidney,
and liver (Anke et al, 1983 ; Denkhaus and Salnikow, 2002). Several types of
16
Ni (oxidic, "sulphidic", and "soluble" nickel) considered as toxic because

of concern carcinogenicity to health, and in some cases effect on the
environment (IARC, 2010).
Long-term of humans exposure to Ni via food chain or work place can
lead to chronic diseases or further to mutagenic changes. Relatively, common
form of Ni toxicity in human is Ni dermatitis (Para keratosis-like damage). The
direct contact to Ni can cause as eczema as containing jewelry, bracelets, etc..
Currently, 10-15% of human are Ni sensitive and can suffer from Ni-hand
eczema. The incorporation of Ni into the food chain in soil, plant, animal, and
human is particularly important (Anke et al, 1983).
1.3.13: Classifications of heavy metal exposure

Exposure to toxic heavy metals is generally classified as acute, 14 days or
less, intermediate, 15-354 days, and chronic is more than 365 days. Because
heavy metals are not easily biodegradable, it can accumulate in vital human
organs, which cause varying degrees of sickness based on period and type of
exposures (Demirezen and Aksoy, 2006). Chronic exposure to low levels of
heavy metals have negative effects on human beings and other animals due to
the fact of mechanism for their elimination from the body is not easily
(Bahemuka and Mubofu, 1999). Some of heavy metals are cumulative poisons,
and these metals considered as an environmental hazards and to be
exceptionally toxic (Ellen et al., 1990). The acute toxicity is usually from a
sudden or unexpected exposure to a high levels for short time of the heavy
metal and a chronic toxicity results from a repeated or continuous exposure,
leading to an accumulation of the toxic substance in the body. Chronic
exposure may result from contaminated food, air, water, or dust. Living near a
hazardous waste site, spending time in areas with deteriorating lead paint;
maternal transfer in the womb; or from participating in hobbies that use lead
17
paint or solder. Chronic exposure may occur in either home or workplace.

Symptoms of chronic toxicity are often similar to many common conditions
and may not be readily recognized (WHO, 1998; Dupler, 2001; Ferner, 2001;
Sharma et al., 2008).
An accumulation of heavy metals in humans body can cause disruption of
numerous biochemical processes, such as cardiovascular, nervous, kidney and
bone diseases. Furthermore, the consumption of heavy metal-contaminated
food can seriously deplete some essential nutrients in the body causing a
decrease in immunological defenses, impaired psycho- social behavior,
intrauterine growth retardation, disabilities associated with malnutrition and a
high prevalence of upper gastrointestinal cancer (Jarup, 2003; Arora et al.,
2008).
1.3.14: Access pathways of heavy metal to the body

Upper respiratory system consists of a nasal cavity and tracheas, and
bronchial tubes and lung constitute lower respiratory system, which consider
the passage of the pollutants entry to the body because of the lung contain
hundred millions several small sacs called (alveoli) provide a surface area
equivalent to 50 m for exchange of gases. An average adult breathes
approximately 30 pounds of air per day. Although our respiratory system is the
main gate to enter most of the particles, the suspended particulate of different
size, range, and compositions enter the body by inhalation through nasal
passage, which collects and entraps most of the particles, before they could
reach the alveoli of the lung ( EPA 2002). Heavy metal particles with diameter
0.1 to 2 m can penetrate deeply into the lung or bend to other particles and
adhere with bronchial tree, these may lead to respiratory system defect and
these metals can irreversibly penetrate into the lung (Airnow, 2006 ; USEPA.,
2006). The severity of this defect depends on the amounts which release and
period of exposure. Breathing polluted air even at low concentrations of some
18
heavy metals may be harmful if a person is expose to its sources for long time
(Demirbas, 2003).
The particles that contain toxic and un degradable metals and stay along
leads to absorption and accumulation of these metals in different parts of the
body which can cause disturbing the normal physiological functions (EPA
2002). The most common effect of metal toxicity is anemia, kidney and liver
damage and nervous and gastrointestinal disturbances (Roosli, 2001).
1.3.15: Mechanism of heavy metals activity

Heavy metals ions form complexes with proteins, in which carboxylic
acid (COOH), amine (NH2), and thiol (SH) groups are involved. These due
to lose of the function ability, properly modify biological molecules and result
in malfunction or death of the cells. When metals bind to these groups, they
inhibition the activity of important enzymes, or affect structure of some protein
which link to the catalytic properties of enzymes. This type of toxin can cause
formation of free radicals, dangerous chemicals that cause oxidation of
biological molecules. There are many other mechanisms of heavy metal
toxicity but are rare incidence (Dhar, 1973 ; Neal and Guilarte, 2012).
1.3.16: Laboratory testing and diagnosis for the presence of

heavy metals
Observation of history for potential exposure obtaining thorough
presenting symptoms are necessary to laboratory tests and diagnosis of the
poisoning by heavy metals. A general way for routine laboratory tests include
blood tests, liver and renal function tests, fecal tests, urinalysis, x-rays, and
hair and fingernail analysis. Many of these tests are not routinely performed in
a health services. However, physicians can only take blood samples and send
them to the appropriate laboratory for testing. For persons which Suffer
19
respiratory and abdominal symptoms, chest X-rays are recommended for

detect ingested heavy metals (ATSDR, 1999; ATSDR, 2005; ATSDR, 2007 ;
ATSDR, 2009).
1.3.17: Assessment of exposure to heavy metals

WHO defined Human exposure as the amount of a substance in contact
with the outer boundary of the body for over time and space (WHO, 2004).
Human exposure assessment to such substances can be measured by two major
methods based on different data profiles, thus permitting the verification and
validation of the information. One approach is the environmental monitoring
i.e., determining the chemical concentration in the body. Second methodology
based on estimation of exposure through the use of biomarkers (Peterson,
2007).
Biomarkers are relevant indicators in studies with human health and are
defined by National Institute of Health (NIH) as the characteristic which is
objectively measured and evaluated as an indicator of normal biological
processes, pathogenic processes or pharmacologic responses to a therapeutic
intervention" (NIH, 2001). They may be used with any level within biological
organization (eg. molecular, cellular or organ levels). These tools can be used
to identify exposed individuals or groups, assess the health risks, quantify the
exposure or assist in diagnosis of environmental or occupational disease (Aitio
et al., 2007).
A decisive and conclusive measure for assessment of exposure to
hazardous chemicals is evaluation of potentially exposed populations. This
step also includes the degree, incidence and routes of potential exposure. A
significant of direct approach to assess exposure to hazardous substances
within potentially exposed populations is the determination of chemicals or
their metabolic products in some biological fluids such as blood (blood and
21
serum) and urine, with certain defined levels being a reliable indicator of metal
exposure (Fischer et al., 2009).
Long term storage of some toxic metals take place in hard tissues such as
teeth and bones. In addition to samples of keratinous tissue components such
as nails and hair are commonly used for routine clinical screening and
diagnosis of longer-term exposure of heavy metals. For example, levels of lead
in hair, bones and teeth increase with age, due to a gradual accumulation of
lead in the body. Therefore, pollution of food with lead and may be lead
intoxication through the diet is a chronic, this need to constant monitoring
(Janssen, 1997). In addition, during mineralization of teeth depend on
cadmium and lead within the matrix components (Fischer et al., 2009). In
human urine, recent studies found monomethylarsonous acid and
dimethylarsinous acid in trace amounts (Mandal et al., 2003; Bhattacharya et
al., 2007). DNA and protein adducts, chromosomal aberrations, mutations,
genes that have undergone induction and a host of other early cellular or sub
cellular events all of them considered as potential biomarkers which thought to
link exposure and effect. Biomarkers of exposure include measurements of
parent compound, DNA or metabolites or protein adducts and reflect internal
doses, the biologically effective dose or target dose. Effects biomarkers could
be changes on a cellular level, such as altered expression of metabolic
enzymes, and may also include markers for early pathological changes in
complex disease developments, such as mutations and preneoplastic lesions
(Bhattacharya et al., 2007).
Biomarkers of susceptibility are often constitutive which indicate to an
ability of individual responses to specific exposure. These three categories of
biomarkers cited above were mentioned by Nordberg (2010), in studies of
health effects after exposure to certain heavy metal. Progress in genomics and
proteomics fields has also reported, and more recent attention is focused on
21
proteomics technologies seek to find a new and relevant biomarkers for metal
assessments. For example, preclinical changes in people exposed to heavy
metals were recently monitored by proteomics biomarkers. In addition to urine
and blood analysis proteomic profiling of serum samples, one representing the
metal-exposed group and the other a control group, revealed three potential
protein markers of preclinical changes in humans chronically exposed to a
mixture of heavy metals (Kossowska et al., 2011).
Other symptoms associated with heavy metal exposure may also be
evaluated such as effects on human skin damage, namely stress signals.
Middendorf & Williams (2000) have critically reviewed early indicators of
cadmium damage in kidney, such as a low-molecular-weight protein (2-
microglobulin), usually reabsorbed by the proximal tubules. Glycosuria,
aminoaciduria, and the reduced ability of the kidney to secrete PAH are also
indicators of nephrons damage by cadmium. An increase in urinary excretion
of low- and high molecular-weight proteins occurs as damage increases,
reflecting the decline in glomerular filtration rate. This review also underlines
that cadmium renal damage may occur after many years in workers removed
from exposure in factories where nickel/cadmium was excessive (Middendorf
& Williams, 2000).
More recently, some cellular functions have been used as biomarkers. For
example, the autophagy pathway was proposed as a new sensitive biomarker
for renal injury induced by cadmium (Chargui et al., 2011).
Non-invasive or a minimally invasive monitoring techniques are
nowadays preferred, although these assays may require further improvement
and validation. For example, the use of the buccal micronucleus assay as a
biomarker of DNA damage is a contribution for epidemiological studies
(Ceppi et al., 2010). Previously, children hand rinsing was used as a biomarker
of short term exposure to As (Shalat et al., 2006). The description of the
22
adverse reactions in animal bioassays may provide relevant information for a

better understanding of human health risk. Moreover, the relevance of animal
testing data to humans is well established. However, the differences in
metabolism between species, added to some intra-specific differences (e.g.
gender, nutritional status, age, genetic predisposition, and frequency of
exposure) are some limitations. In order to overlap these differences, a safety
margin must be considered (Ceppi et al., 2010).
Finally, the complexity and number of available potential biomarkers for
heavy metals exposure may be lead to the development of improved prognostic
and diagnostic tools.
1.3.18: Management of Heavy Metal poisoning

Heavy metal toxicity characterized by that the agent poisoning cannot be
metabolized, despite the fact that sometimes the body can be metabolize toxic
metal to facilitate the process of its excretion out of the body, but the body can
not convert the toxic metal to non-toxic by metabolism. One mechanism that
may reduce the toxicity of metals is a metal carrier protein, metallothionein,
which may complex with the metal, preventing it from exerting a toxic effect,
and transport it to the kidney where it may be filtered and excreted (Dhar, 1973
; Neal and Guilarte, 2012).
If someone injured by heavy metal poisoning ( especially the fuel
workers), the use of chelators is common treatment for metal intoxication. A
chelator is a flexible molecule with two or more electronegative groups that
can form stable complexes with cationic metal atoms. The complexes are then
eliminated from the body. The most widely used chelator is
ethylenediaminetetraacetic acid (EDTA). It has four binding positions (two
nitrogen atoms and two oxygen atoms) that focus on the metal ion. It works
very well on many metals, the most notable being calcium, magnesium, and
23
lead (Howland, 2011 ; Neal and Guilarte, 2012). Chelating with drugs is
indicated primarily for acute poisonings by some metals, although the drugs
may have dangerous side effects, but are considered worthwhile in the face of
toxicity which may be fatal or cause serious, even permanent injury (Kosnett,
2010).
Approved chelating drugs include succimer, dimercaprol (BAL), edetate
calcium disodium, deferoxamine, and penicillamine. They are given only for
diagnosed metal toxicity because they may have serious side effects, even
when their use is needed; and they are non-specific and can bind even essential
trace metals in the body, for example copper and zinc. They can sometimes
bind calcium, too. Chelation of these substances can cause symptoms related to
their deficiency (Howland, 2011; Nelson, 2011).
1.3.19: Heavy metals analytical methods (Quantitative

determination)
There is a tremendous challenge to develop sensitive and selective
analytical methods that can quantitatively characterize trace levels of heavy
metals in several types of samples (Rao et al., 2005). Table (1) summarizes the
optical and the electrochemical methods applied for heavy metals
determination (Karadjova et al., 2007 ; Draghici et al., 2010).
24
Table (1-1): The most usual methods applied for heavy metals
determination ( Draghici et al., 2010)
Technique Principle type of analysis Applications
Atomic absorption Absorption of -single element; widely used

spectrometry radiant energy -multielement
(AAS) produced, by a analysis(2-6
special radiation elements)
source, by atoms in
their electronic
ground stat
Inductively measures the optical Simultaneous widely used method
coupled plasma emissionfrom multielement for environmental
with atomic excited analysis analysis
emission atoms
spectrometry
(ICP-AES)
Inductively - argon plasma used Simultaneous -widely used;
coupled plasma as multielement -isotope determination
with mass ion source; analysis
spectrometry used for separating
(ICP-MS) ions based on their
mass-to charge ratio
Atomic measures the light single element -mercury, arsenic, and
fluorescence that selenium;
spectrometry is reemitted after -complementary
(AFS) absorption technique to AAS
X-ray fluorescence -X-rays primar simultaneous -non-destructive

(XRF) excitation source; determination of analysis;
-elements emit most elements -less suitable for
secondary X-rays of analysis of minor and
a trace elements
characteristic
wavelength
Neutron activation -conversion of stable simultaneous -most elements can be
analysis nuclei of atoms into multielement determined;
(NAA) radioactive ones; analysis - highly sensitive
-measurement of the Procedure
characteristic
nuclear
25
radiation emitted by
the radioactive nuclei
Electrochemical -controlled voltage consecutive -analysis for transition

Methods or analysis of Metals and metalloids
current; different metal (total content or
-polarography; ions speciation analysis)
-potentiometry;
-stripping
voltammetry
1.3.20: Vehicle emissions and their sources

The pollution originating from road traffic can be divided into two major
types: exhaust and nonexhaust emissions. Exhaust emissions are caused by
incomplete combustion of vehicle fuel which is a mixture of hydrocarbons and
compounds improving combustion properties. During this process several
types of pollutants are generated such as carbon monoxide (CO), nitrogen
dioxide (NO2), volatile organic compounds (VOCs), polycyclic aromatic
hydrocarbons (PAHs) and particulate matter (PM). Incomplete burn of fossil
fuels as well as traffic-related can be cause of road suspension, soil and
mineral dust leads to direct emission of various liquids and solids into the air
(primary particles). Moreover, the gaseous substances released from exhaust
systems, undergo gas-to-particle conversion (secondary particles) in the
atmosphere (new particle formation by nucleation and condensation of gaseous
precursors) (Pschl, 2005). Secondary particles are mainly composed of
inorganic compounds, including sulphates, ammonium and nitrates. Non-
exhaust emissions are generated through mechanical (e.g. braking, clutch
usage, tyre wear, road abrasion) and chemical processes (e.g. corrosion of
vehicle elements).
26
Among various heavy metals emission sources, vehicle emission has been
known to be one of the main contributors in urban areas (Vassilakos et al.,
2007; Zheng et al., 2010). On a global basis, anthropogenic inputs of Pb
predominate over natural sources, accounting for 96% of the total emissions
(Nriagu, 1989). Among these inputs, vehicle emissions of particles are often
found to be the most significant source. Although Pb-free petrol has become a
popular choice for most transport facilities, Pb is still found to be an important
component of airborne particles throughout the world. The association of Pb
with vehicle emissions can be explained in terms of Pb contamination in crude
oil, which may be of the order of 1015 mg/L (Vassilakos et al., 2007).
Elements that have often been associated with vehicular emissions include
Ba, Br, Ca, Cd, Co, Cr, Cu, Fe, Mg, Mn, Pb, Ni and Zn (Morawska and Zhang,
2002; Sternbeck et al., 2002; Lin et al., 2005 ; Lough et al., 2005). Birmili et
al. (2006) concluded that materials rich in Cu, Ba and Fe serve as an indication
of abrasive vehicular wear, in particular brake linings.
Exhaust and non-exhaust emissions can significantly contribute to the
total mass of urban particulate matter (PM2.5, PM10). As reported by Ketzel et
al. (2007) a large part (from 50 up to 85%) of the total PM10 emissions
originates from non-exhaust emissions. In northern European countries, road
sanding and the use of studded tyres are considered as major sources of the
non-exhaust fraction of PM10, which can account for up to 90% of airborne
particulate matter (Forsberg et al., 2005 ; Omstedt et al., 2005).
27
Chapter two
Materials
and
methods
Chapter Two Materials and Methods
Chapter two
Materials and Methods
2.1: Materials
2.1.1: Instruments and Equipment
The instruments and equipment used in the present study are listed in Table
(2-1).
Table (2-1): Instruments and equipment used and their manufacturers
No Instrument or equipment Source
1- Centrifuge Human / Germany

2- Distillatory Alab Tech / Korea
3- EDTA tube AFCO, Jordan
3- Electric Hood chamber Labnet / USA.
4- Flame Atomic Absorption spectrophotometer United kingdom (UK.)
Phoenix 986 AA.
5- Glass Wear Pyrex /England
6- Hematology analyzer SFRI. H18 Light (France)
7- Hot plate Heidolph /Germany
8- Micropipettes Cyan / Belgium
9- Oven Memmert /Germany
10- Refrigerator Ashtar / Iraq
11- Roll mixer china
12- Supper freezer Ariston / Australia
15- Syringe Ultra health, China
14- Test tube AFCO, Jordan
82
2.1.2: Chemical Materials

The Chemical materials used in the present study are listed in Table (2-2).
Table (2-2): Chemical materials that used in this study
No Chemical materials Manufacture / Origin
1- Alcohol (70%) Nisc, Jordan

2- Distilled water
3- Hydrochloric acid (0.3) GCC ,England
4- Nitric acid (70%) Merck ,Germany
5- Perochloric acid(70%) Merck, Germany
2.2: Methods
2.2.1: The Study Area
During the period from October 2014 to April 2015, the workers of four
fuel ( 1-Al-Ballam, 2- Al-Nasiriyah , 3-Al-Rayat and 4-Al-Mohandis stations)
in the center of Al-Nasiriyah city, These stations are distributed randomly in
Al-Nasiriyah city, as shown in the appendices (Appendices-2).
2.2.2: Method of Sample Collection

2.2.2.1: With regard to data of workers at fuel stations
The current study included the collection of 100 samples (aged from 20
years to 55 years), 80 samples from workers (20 workers from each station)
and 20 samples from healthy people away from direct exposure to the sources
of those elements as a control group. After getting some important information
recorded all in a special form include age, region, smoking, the duration of
working, previous work and illnesses suffered by (as shown in appendices) .
Most of workers were smokers (68%) and have no history of heart disease
as Wilson disease and the history of the work they have not recorded a work in
82
the environment high concentrations of the components of the study metals,

such as a car mechanic, traffic police, workers electric, batteries plants or paint
workers.
2.2.2.2: With regard to collection of blood samples

After obtaining the necessary information from the workers, withdrawal
region was disinfected with a mild antiseptic ,then blood was withdrawn from
radial vein, it was obtained 10 mL of blood, 1ml of blood put it in the EDTA
tube for the purpose of estimating some blood parameters by hematological
analyzer and the rest of the blood 9 ml put in a test tube. All tubes transported
in cool box until separation of serum for purpose of estimating of study metals.
2.2.3: Measurement of blood parameters

Blood parameters (RBCs. , Hb., PCV., PLTs. & WBCs.) were measured by
hematological analyzer according to manufacturers procedure.
2.2.4: Preparation of serum

A 9 ml of blood was centrifuged (3000 rpm/minute) for 20 minutes, then
serum kept at -20 c until the digestion for metals values estimation.
2.2.5: Digestion of serum

The digestion of serum was done according to Xueping and Reny, (2002)
which included an adding of 2 ml. of Nitric acid (70%) and one ml. of
Perochloric acid (70%) to (0.5)ml of serum in a Pyrex tube then heating of this
compound with a water path on a hot plate at 160C for one hour, then cooled
and finally completed to 10 ml with (30%) Hydrochloric acid. We can make
some modifications as the soaking of the serum in Nitric acid for thirty
minutes followed by the addition of Perochloric acid ,and the increment of
heating degree and period to 200C and 2.5 hours respectively in order to
obtain an absolute digestion of serum and vaporization of acids.
03
2.2.6: Estimation of heavy metals

The estimation of heavy metals in the blood serum by Flame Atomic
Absorption Spectrophotometer (FAAS.- Phoenix 986 AA. United kingdom-
UK.) was done according to procedure of manufacturer.
2.2.7: Study design
The current study was briefly in the Figure 2-1 below
Worker's data
Collection of blood sample
9 ml of blood in test tube 1 ml of blood in EDTA

tube
centrifugation Hematological analyzer

to measure of blood
parameters
Digestion of serum
Estimation of heavy metals

by FAAS.
Figure (2-1): Study design
03
2.2.8: Statistical analysis

By using of ANOVA testing from SPSS. Program for investigate the
significant differences among the metal values at level (P 0.05).
08
chapter three
Results
Chapter ThreeThe results
Chapter three
The results
3.1: Heavy metal concentrations in serum of study samples

Table (3-1) shows the mean values of heavy metals concentration for
workers at each station separately, as well as in the control group. It is found in
the present study that the mean value of heavy metals concentrations varies from
one station to the other. All stations were recorded mean values of heavy metal
concentrations higher than the control group and there are some significant
differences among the stations and among stations and control group at level
(P0.05).
Table (3-1): The mean values of the heavy metals in the serum of workers at
each station and control
Cd Co Cu Ni Pb
Stations Concentrations of Metals, ppm / Mean SE.
b a ab ab a
1 0.006 0.028 0.01 0.07 0.19
Al-Ballam 0.0007 0.002 0.0003 0.02 0.05
a a a a ab
2 0.057 0.025 0.03 0.1 0.11
Al-Nasiriyah 0.003 0.006 0.001 0.02 0.006
ab a ab a a
3 0.015 0.028 0.01 0.1 0.19
Al-Rayat 0.004 0.006 0.0006 0.02 0.05
ab a a ab ab
4 0.016 0.027 0.03 0.04 0.11
Al-Mohandis 0.006 0.004 0.009 0.008 0.011
b b
b b b 0.03 0.03
Control ND. ND. ND.
0.003 0.003
LSD. 0.0434 0.0138 0.0233 0.062 0.150
The different letters refer to a significant differences, The same letters refer to no significant
differences, at level (P 0.05)/ND. (non-detected 0.004 ppm), all ND considered 0.004 ppm.
33
3.1.1: Cadmium (Cd)

The mean values of Cd for workers and control group are shown in table (3-
1). All stations recorded Cd values higher than its values in the control group as
shown in the figure (3-1), there is significant difference between the station 2
and each of station 1 and the control group (0.057 0.003 ppm, 0.006 0.0007
ppm and ND. respectively), at level ( P 0.05).
0.06
0.05
Mean concentration of Cd ppm
0.04
0.03
0.02
0.01
0
station 1 station 2 station 3 staion 4 control
Figure (3-1): Mean values of Cadmium (Cd)concentration ppm. in study

samples
3.1.2: Cobalt (Co)
Co concentration in worker samples recorded high values more than its
concentration in the control group for all the study stations, as shown in figure
(3-2), where there are significant differences among the control group with all
the stations at level (P 0.05). As shown in the table (3-1).
33
0.03
0.025
Mean concentration of Co ppm
0.02
0.015
0.01
0.005
0
station 1 station 2 station 3 station 4 control
Figure (3-2): Mean values of cobalt (Co) concentration ppm. in study

samples
3.1.3: Copper (Cu)

Serum samples obtained from workers in all stations record copper mean
values higher than the control group, station 2 and station 4 recorded the highest
values for copper (0.03 0.001 ppm and 0.03 0.009 ppm, respectively) while
station 1 and station 3 record the lowest values (0.01 0.0003 ppm and 0.01
0.0006 ppm, respectively), as shown in figure (3-3), this results were showed a
significant differences between control and both of station 2 and station 4, at
level ( P 0.05) as shown in the table (3-1).
33
0.035
0.03
Mean concentration of Cu ppm
0.025
0.02
0.015
0.01
0.005
0
Figure (3-3): Mean values of copper (Cu) concentration ppm. in in study

samples
3.1.4: Nickel (Ni)
Both station 2 and station 3 were recorded the highest values of nickel
concentration as shown in figure (3-4), in there were a significant differences
among control and both of station 2 and station 3 (0.03 0.003 ppm, 0.1 0.02
ppm and 0.1 0.02 ppm, respectively), at level ( P 0.05), While station 4
record lower values (0.04 0.008 ppm) as shown in table (3-1).
33
0.11
0.1
Mean concentration of Ni ppm
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
Figure (3-4): Mean values of Nickel (Ni)concentration ppm. in study

samples
3.1.5: Lead (Pb)

The lead values of worker samples in all stations are higher than its
concentration in the control as shown in figure (3-5), there are a significant
differences among control and both of station 1 and station 3 (0.03 0.003 ppm,
0.19 0.05 ppm and 0.19 0.05 ppm, respectively), at level ( P 0.05), as
shown in the table (3-1).
33
0.2
0.18
Mean concentration of Pb ppm
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
Figure (3-5): Mean values of Lead (Pb)concentration ppm. in study samples
3.2: Estimation of some blood parameters

Table (3-2) shows the mean values of some blood parameter for workers at
each station and control samples. The mean values of Hb., was recorded a slight
increase compared with control. While the other blood parameters record a low
values compared to control in each of PCV., PLT., RBCs. & WBCs.. The station
4 record approximately the lowest mean values with all blood parameters
compared with the other stations, as shown in table (3-2) bellow.
33
Table (3-2): The means of some blood parameters for workers and control
Blood parameters
Mean SE.
Stations PLT. RBC. WBC.
Hb. g/dL PCV.%
*103/L *106/L *103/L
14.1 a 43.9 b 208.7 a 5.1 a b 7.8 b a
1
0.43 0.65 14.2 0.13 0.59
14.3 a 43.8 b 203.8 a 5.2 a 7.8 b a

2
0.31 0.76 14.6 0.12 0.47
14 a 43.5 b 204.6 a 5.3 a 7.7 b a

3
0.34 0.96 16.6 0.17 0.57
13.4 a 41.7 b 202.6 a 4.7 b 6.3 b

4
0.41 1.15 12.6 0.19 0.33
13.4 a 46.6 a 244.2 a 5.4 a 9.3 a

Control
0.18 1.01 15.4 0.09 0.35
LSD. 1.002 2.64 42 0.42 1.37
differences, at level (P 0.05)
3.2.1: Hemoglobin (Hb.)

Station 2 was recorded the highest value with Hb. (14.3 0.31) g/dL than
other stations, while the lowest was station 4 (13.4 0.41) g/dL, the differences
among these stations showed non-significant as well as with control at level
(P0.05), as shown in figure (3-6) below.
33
14
Mean value of Hb. g/dL
12
10
0
Figure (3-6):Mean value of Hb. (g/dL) for study samples
3.2.2: Packed Cell Volume (PCV.)

The highest value of PCV. was station 1 (43.9 0.65 %) and the lowest value
was station 4 (41.7 1.15 %), there are no significant differences among stations
at level (P0.05).
All stations values have recorded PCV. values lower than the control, where
there are significant differences among these stations and the control at level
(P0.05), as shown in figure (3-7) below.
34
47
46
Mean value of PCV. %
45
44
43
42
41
40
Figure (3-7):Mean value of PCV. (%) for study samples

3.2.3: Platelets (PLT.)
Station 1 was the highest value of PLT. (208.714.2)*103/L compared with
the other stations and the lowest value (202.6 12.6)*103/L was station 4. All
stations were record values less than control, but these differences are non-
significant at level (P0.05), as shown in figure (3-8) below.
34
250
245
Mean value of PLT.*103/L
240
235
230
225
PLT.*103/L
220
215
210
205
200
Figure (3-8):Mean value of PLT.(*103/L) for study samples
3.2.4: Red blood corpuscles (RBC.s)

The highest value of RBC.s was in station 3 (5.3 0.17)*106/L compared
with the other stations, the lowest was station 4 (4.70.19)*106/L. The
statistical analysis shows that station 4 recorded a significant difference
compared with each of station 2,3 and the control group at level (P0.05), as
shown in figure (3-9) below.
34
5.5
5.4
5.3
Mean value of RBC.*106/L
5.2
5.1
4.9
4.8
4.7
4.6
4.5
Figure (3-9):Mean value of RBC.(*106/L) for study samples
3.2.5: White blood cells (WBC.s)

Each of station 1, 2 and 3 record approximate values of WBC.s (7.8
0.59*103/L, 7.8 0.47*103/L and 7.7 0.57*103/L, respectively), while the
station 4 was the lowest (6.3 0.33)*103/L.
Statistical analysis was showed that station 4 recorded significant difference
with other stations and control, and the control group records high values (9.3
0.35) *103/L with significant difference at level (P0.05), as shown in figure
(3-10) below.
33
9.3
9
8.7
Mean value ofWBC.* 103/L
8.4
8.1
7.8
7.5
7.2
6.9
6.6
6.3
6
Figure (3-10):Mean value of WBC.(*103/L)for study samples
3.3: The relationship between the values of heavy metal

The concentration values of heavy metals were divided into groups
according to the period of work (1-4, 5-8 & 9-12) years. Did not show
increasing in the values of the heavy metals according to the period of the work,
except Cu. There were variations in the values of the heavy metals for the same
periods.
Cd within the period 1-4 records the highest value while the period 9-12 the
lowest value. Co in period 9-12 was the highest compared with period 5-8 that
was the lowest value. Cu at period 9-12 was highest value while period 1-4 was
lowest value.
33
Ni at period 5-8 was highest value compared with period 1-4 that was lowest
value, where there are significant differences at level (P0.05). Pb in period 9-12
recorded highest value compared with period 5-8 that recorded lowest value, as
Table (3-3): concentration of heavy metals in the serum of workers
according to the work period
Means of metals ppm SE.

Groups
Cd Co Cu Ni Pb
0.044 a 0.027 a 0.01 a 0.065b 0.175 a

1-4
0.0024 0.0042 0.0005 0.0067 0.0417
0.008 a 0.023 a 0.013 a 0.189a 0.119 a

5-8
0.0017 0.0033 0.0038 0.0403 0.0089
0.006 a 0.028 a 0.021 a 0.134 a b 0.196 a

9-12
0.0007 0.0071 0.0011 0.0383 0.0882
LSD. 0.044 0.0134 0.0153 0.096 0.174
3.4: The relationship between Heavy metals concentration and

smoking
To make sure of the impact of smoking on the concentrations of study metals
in the workers serum so to raise or lower the values of these metals, decide
investigate the values of these metals in the smoker workers and non-smoker
comparing with control. Concentrations of all these metals in smoker workers
and non-smokers are higher than the control.
Cd was recorded highest value in the smokers, without significant differences
at level (P0.05).
33
Co, Cu & Ni were recorded highest values in non-smokers compared with

smoker and control, both of smoker and non-smoker recorded a significant
differences with control at level (P 0.05).
Pb recorded highest value in non-smoker compared with smoker and control
groups. Where there were significant differences between non-smoker and both
of smoker and control, so that between smoker and control at level (P 0.05), as
Table (3-4): Heavy metals concentration in the smoker, non-smoker and

control groups
Metals Mean (ppm) SE.
Groups Cd Co Cu Ni Pb
Smokers 0.028 a 0.028 a 0.009 a 0.109 a 0.111 b a

0.0013 0.0036 0.0003 0.0191 0.0076
Non-smokers 0.007 a 0.029 a 0.010 a 0.113 a 0.134 a

0.0015 0.0043 0.0004 0.0225 0.0102
Control ND. a ND. b ND. b 0.03 b 0.03 b

0.0011 0.0035
LSD. 0.031 0.011 0.0011 0.054 0.022
Figure (3-11) shows the levels of study heavy metals with smokers and non-
smokers workers and control group.
33
0.03 0.03
0.025 0.025
0.02 0.02
0.015 0.015
0.01 0.01
0.005 0.005
0 0
smokers non-smokers cotrol smokers non-smokers cotrol
Mean of Cd Mean of Co
0.12
0.01
0.1
0.008
0.08
0.006
0.06
0.004
0.002 0.04
0 0.02
smokers non-smokers cotrol smokers non-smokers cotrol
Mean of Cu Mean of Ni
0.14
0.12
0.1
0.08
0.06
0.04
0.02
smokers non-smokers cotrol
Mean of Pb
Figure (3-11): Heavy metals concentration for smoker, non-smoker and

control groups
33
3.5: The relationship between Heavy metals concentration and age

The study samples are divide into groups according to age of workers. The
ages of workers were ranged from 20 to 55 years, therefor it was divided into
three groups, each group includes 12 years (20-31, 32-43 & 44-55 years), as
shown in table (3-5) bellow.
Cd showed its lowest concentration in group (20-31), while the highest
concentration was in group (44-55), where there was significant difference
between the first group and each of second and third groups at level (P 0.05).
Co recorded its lowest value in group (20-31), while the highest value was in
group (32-43), where group (20-31) recorded significant difference with the
other groups at level (P 0.05). Cu recorded its lowest value in group (20-31),
while the highest value in group (44-55), where group (44-55) recorded
significant difference with the other groups at level (P 0.05).
Ni and Pb recorded their lowest values in group (44-55), while the highest
value was in group (20-31), where group (20-31) with Ni recorded significant
difference with the other groups at level (P 0.05). As shown in table (3-5)
bellow.
33
Table (3-5): Relationship between heavy metals concentration (ppm) in

workers and their age
Metals Mean (ppm) SE.
Groups Cd Co Cu Ni Pb
20-31 0.018 b 0.026 b 0.009 b 0.163 a 0.121 a

0.001 0.0034 0.0004 0.036 0.0086
32-43 0.117 a 0.110 a 0.010 b 0.028 b 0.056 b

0.0101 0.0240 0.0006 0.0061 0.0027
44-55 0.199 a 0.106 a 0.025 a 0.023 b 0.014 b

0.0639 0.0293 0.0095 0.0036 0.0071
LSD. 0.083 0.051 0.011 0.090 0.049
Figure (3-12) bellow shows relationship between heavy metals concentration in
workers and their age.
33
0.22
0.116
0.19 0.106
0.096
0.16
0.086
0.13
0.076
0.1 0.066
0.056
0.07
0.046
0.04
0.036
0.01 0.026
20-31 32-43 44-55 20-31 32-43 44-55
Mean of Cd Mean of Co
0.03 0.18
0.16
0.025
0.14
0.02
0.12
0.015 0.1
0.08
0.01
0.06
0.005
0.04
0 0.02
20-31 32-43 44-55 20-31 32-43 44-55
Mean of Cu Mean of Ni
0.13
0.11
0.09
0.07
0.05
0.03
0.01
20-31 32-43 44-55
Mean of Pbi
Figure (3-12): Heavy metal concentration ppm and age of workers
34
3.6 : Concentrations of heavy metals in collected samples

It is found from obtained results during the present study that all the means
of worker`s samples record concentration values of heavy metals higher than the
control samples, as shown in figure (3-13) where the mean values of each Cd
(0.024) ppm, Co (0.027) ppm, Cu( 0.02) ppm, Ni (0.078) ppm and Pb (0.15)
ppm which were higher than the control values that recorded non detected
values ( ND. < 0.004) ppm for each Cd, Co and Cu while Ni (0.03) ppm and Pb
(0.03) ppm.
0.15
Concentrations of heavy metals with ppm.
0.16
0.14
0.12
0.1 0.078
0.08
0.06
0.024 0.027 0.03 0.03
0.04 0.02
0.02 ND. ND. ND.
0
Cd. Co. Cu. Ni. Pb.
Workers Control
Figure (3-13): The comparison among means of heavy metals concentrations

values for workers in all stations and control
34
Chapter four
Discussion
Chapter ThreeDiscussion
Chapter four
Discussion
4.1: Heavy metals concentration in blood serum of workers
4.1.1: Cadmium
Cd is restricted to the surface of RBCs (it has a high affinity for the linkage
with RBCs) it must be has low concentrations in serum, the present study
record high concentration of Cd. There were not found a local studies for
estimation of Cd in human blood serum, therefor the present study compared
with few global studies that were deal with Cd concentration in blood serum of
human. It was found that the concentration of blood serum Cd in the present
study is higher than its concentration in the previous studies, Lyengar and
Wolttlez (1988) were found Cd value was (0.001) ppm in blood serum, in an
Italian study by Minoia et al., (1990), in an attempt to found a reference values
for heavy metals in serum of healthy people living in one area in Italy,
compared with Lyengar and Wolttlez(1988), they found concentration of Cd
value was (0.0002) ppm.
Speaking of reference values of Cd concentrations in human serum, there
were several studies which tried to record the reference values for the
concentration of Cd in the blood serum and tissues of the human body where
there is a studies in Italy on non-smokers find Cd concentration in there serum
was (0.0001) ppm (Alimonti et al., 2005). Brazilian study by Gine et al.,
(2008), for the same purpose they found that concentration of Cd in serum of
healthy was (0.1)ppm.
Study by Schultze et al., (2013), for the healthy serum sample from non-
smokers in Swedish society, it was found that concentration of serum Cd was
very low not get a worthwhile. The search about reference for normal values
of Cd in the serum is to investigation the extent pollution of serum workers.
25
There are many reasons for the disparity which requires a comparison only
with control samples of the same study, which may be affected by a person
housing area. For example, the nearby areas of the plants used for Cd or
subtracted Cd with increasing Cd concentration in population bodies of these
areas with varying degrees depend on exposure way (Oral, dermal and
respiratory) is there (Cornelis et al., 1995). Because Cd uptake in the lung is
much greater than its absorption in the gut (Ming-Ho, 2005;Figueroa, 2008)
The smoking may be is one of the sources of pollution by Cd, so Cd in
smokers is higher than non- smokers. The age may be one reason which effect
upon the Cd concentration in serum, where it increases with age cumulatively
but not necessarily have to be the sample, it is taken after acute exposure to
high concentrations of Cd, which leads directly to the high value temporarily.
Therefor the present study studied these two objectives, relationship of heavy
metals concentration with both smoking and age separately.
4.1.2: Cobalt
Co is one of the necessary metal for humans and its role as an essential
ingredient in Vitamin B12,its deficiency makes it an essential metal which may
cause damage to the body ,The inhalation of Co and its compounds fumes can
cause defect to the respiratory system and other organs, such as heart disease
and asthma (Cornelis et al., 1995).
One of the studies that designed to measure concentration of Co in human
serum, it was found Co concentration ranging between (0.0001 and 0. 00045)
ppm (Lyengar, 1985),similarly what was got in study of Gent University
was(0.0036) ppm (Cornelis et al., 1995).
The samples from healthy non-exposed and non-smokers ,it was found that
Co concentration in their serum is (0.00019) ppm (Alimonti et al., 2005). A
study on people with leukemia, the control group in this study from healthy,
non-exposed and non-smokers where Co value in their serum was (0.114) ppm
25
(Elradi, 2010). The reference values in Sweden Co value was(0.00014) ppm

(Schultzeet al., 2013).Co is one of the vehicles and cars fuel components and
their emission (Morawska and Zhang, 2002; Sternbeck et al., 2002; Lin et al.,
2005 ; Lough et al., 2005). Axiomatic the concentration of Co is high in blood
serum of fuel station workers in addition to another reasons such as the
physiology of their bodies, housing place ( may be living near from mining
plants of Co, Ni or Cu and other plants which release high amounts of Co) and
the abuse of alcohol, other chemical materials and drugs that may contain Co.
There are some types of cigarettes may contain Co in its ingredients (Ishiguro
and Sugawara, 1980 ; Morris, 2008).
4.1.3: Copper
The present study was recorded high values of Cu in blood serum of
workers in compared with the its concentration in the control group, but these
results are different from many global studies which have wide range of
variation. Gent University records the natural value of Cu in the serum (0.011)
ppm(Cornelis et al., 1995).Milde et al., (2004) in a study on patients with
colon cancer found Cu concentration in the serum of healthy control group,
(1.05) ppm. Another study in Italy tries to find the natural values of Cu in the
serum found in healthy is 0.947 ppm (Alimonti et al., 2005). Brazil Gine et al.,
(2008) records the natural values of Cu in serum 1.1ppm.
Selvaraju et al., (2009) in a study measure some of the heavy metals in the
serum of patients with viral hepatitis B, where Cu concentration of the control
group was found 1.003 ppm. Another study in Sweden determines the
concentration of Cu in the serum and compares it with its concentration in the
blood and it found that Cu concentration is 0.009 ppm (Schultze et al., 2013).
Cu is an essential for human, everyone must absorb small amounts of Cu
every day. High levels of Cu can be harmful, breathing high levels of Cu can
cause irritation of nose and throat and ingesting high levels of it can cause
25
nausea, diarrhea and vomiting. Over doses of Cu can cause damage to liver
and kidneys, and can even cause death (ASTDR, 2004).There is a great
disparity between Cu values obtained from the workers and this disparity is
followed by personal differences. This metal is one of the essential metals for
the body, so it is constantly consumed every day and non-cumulative that is
supposed to find it in a low concentration values. These values may have to be
not real values. Nonetheless Cu values obtained in the present study, almost
higher than control values, then acute and immediate exposure to Cu may be
harmful for such workers.
4.1.4: Lead
Lead is a non-essential and toxic metal for human even at low
concentrations and can cause several biological effects (Environment Canada,
2010). Because of Pb is a serious metal for human and the fuel consumption is
the main source of Pb emission and release to environment (Pacyna &Pacyna,
2001), the present study investigate the levels of this metal in the blood serum
of fuel station workers. The workers Pb concentration has high levels in
compared with the Pb levels in control group. The present study record
different values of Pb with same global studies which recorded high range of
variation according to the time and place of sample collection.
A study by Lyengar and Wolttlez (1988) the concentration of Pb in the
serum of healthy people is less than 0.001ppm. Alimonti et al., (2005)find that
the Pb concentration in the serum of healthy non- exposed people to those
elements directly in Italy it was (0.00054 )ppm. In Brazil in the study with
serum of non-exposed healthy people are to those metals directly and non-
smokers found that the value of Pb concentration was(0.012) ppm (Gine et al.,
2008). While in Sweden, Schultze et al., (2013) found human Pb serum
concentrations(0. 0006) ppm.
22
The disparity in values of Pb in different studies and among the samples of

the present study is may be subject to several reasons, one of them is the
diversity of lead sources which effect on the Pb concentration in the workers
blood serum, such as using of fossil fuels, living near a traffic, non-ferrous
metal production, iron, steel and cement production and waste. Another reason
may be depend on the level and duration of exposure for Pb.
4.1.5: Nickel
The present study record high values of Ni concentration compared with
control group and there are high disparity between the current study and other
global studies, which studied with different samples of human. Ni
concentration in the serum of people who were not directly exposed to Ni
was(0.0002) ppm (Templetonet al., 1994). In Italy Alimonti et al., (2005), the
concentration of Ni in serum of healthy non- exposure for Ni was(0.00046)
ppm. According to ASTDR, (2005) the normal values in healthy exposed and
non-smokers people and (0.0002) ppm and when the concentration of Ni in
serum increase upper (0.1) ppm considered Hypernickelaemia. In another
study in India to examine the Ni concentration in the serum of people who
were putting teeth fillings metal concentrations as the control group
was(0.0006) (Rai et al., 2011).
Sometimes, cannot compare present study with global studies and even
local if there was a long period of time. There were many reasons to make the
comparison impossible. They give great disparities, including environmental
pollution, which is constantly increasing due to speed industrial development
where increasing pollutants such as Pb, Cd and others over time where the
industrial development the main reason of the pollution of environment as
water, air and soil (Akesson et al., 2008).An industrial waste containing
several types of toxic compounds, including heavy metals. This development
varies with time and space around the world, meaning that the area where the
25
individual lives can affect the concentration of metals in the body, as well as
nearby plants residential areas or factories that cadmium raises the level of
certain metals in the tissues and body fluids of the inhabitants of those areas
somewhat cause pollution of environment by such metals this is proved by
Cornelis et al., (1995).
There are no reference values for those metals. So each study must to be
concerned with concentrations of these metals in study sample to be compared
with control samples to the same study at the same time and geographic area.
In fact, it is expected to express reference values affected by several factors
such as age, sex nature of the individual (smoker, deal alcohol and drugs),
living environment, work environment and some of the diseases that affect the
individual as well as nutrition type (such as feeding the fish) and the use of
dental fillings and its type and number (Alimonti et al., 2005).
The concentration values of metals in the present study is much higher than
in most global studies both in serum of workers and even in the control group
and this may be a risk index on the rise of concentrations of these metals and
their harmful role. And this may be an evidence of environmental pollution in
Al-Nassiriya city with those metals. This may be due to diversity of those
metals sources of such as the remnants of war, weapons and the large number
of factories and cars, and a lack of vegetation in most area of Al-Nasiriyah
city.
4.2: Heavy metals concentration in blood serum of workers at

study stations
The present study include four stations with different geographical
location. The location of station has certain effect on concentration of studied
metals in the blood serum of workers.
Station 1 (Al-Ballam station ) located approximately on north part of city
at a crowded street (Al-Neel street) is longer street in the city, where soldered
25
shops and sell pieces of iron. In addition the passage high number of cars
frequently especially old cars that discharge high amounts of pollutants
through exhaust and crowdedness of population around of this station, all these
reasons that raise the level of pollution in the work environment, so it observed
increasing concentrations of these metals in the blood serum of workers in this
particular station.
Station 2 (Al-Nasiriyah station ) located in the center part of the city, the
geographic location of this station is the most crowding and pollution among
other stations, where there are many sources of pollution, such as the repair
and sale of automotive shops and stores selling automotive oils tires shops as
well as very heavy traffic.
High values of Cd and Pb an indicator of pollution of area around this
station. All of these reasons are additional factors of pollution in addition to the
pollution of this station itself, which would raise the level of contamination
such metals.
Station 3 ( Al-Rayat station ) in the south of the city, and located on sub
highway at the intersection of two important major streets. The location of this
station may be considered as important factor which rise level of pollution with
the study metals. But the population crowdedness around of this station
approximately low than other stations.
Station 4 ( Al-Mohandis station ) east of the city approximately, on high
way road with passage of high number of vehicles and presence of trucks and
all type of cars that across through this road. In addition, this station use a poor
quality of fuel which may be lead to increase level of study metals.
There are no consistent differences between stations in the values of
metals, each station where a particular metal rises, while the other declines,
this indicates that there are many reasons lead to this disparity, for example,
the quality and the components of the used fuel differs from one station to
25
another and from one time to another. In addition to the location of each
station and different of pollution sources. As well as personal differences,
where the worker who housing in rural areas is different than in urban areas
and his previous work, smoking, health status, physiological, age, smoking and
duration of the work of all these reasons lead to the disparity between workers
from one station to another.
4.3: Blood parameters

Some of blood parameters (Hb., PCV., PLT., RBC. & WBC.) were studied
to investigate the impact of have metals concentration on these parameters.
Although, the values of both workers samples and control group were fall
within the normal range.
The accumulation of Cd in the body adversely affects some organs such as the
liver, bones, brain tissue and CNS. (ATSDR, 2008) and other damage were
observed, including damage to reproductive, toxicity, kidney inflammation,
and affect as well as on most of the blood parameters and the immune system
(ATSDR, 2008; Apostoli and Catalani, 2011), and the rise Cu may cause blood
iron deficiency anemia, or perhaps cause iron storage disease. Some types of
anemia, which do not respond to treatment with iron no common with copper
vital Cu are required to convert the ferric form to the ferrous which is
consumable, copper is also required to integrate iron hemoglobin, as well as
the high copper concentration in the tissues of the body can cause a lack of
manganese, which is necessary to stimulate the formation of hemoglobin this
type of anemia resulting from stimulation of copper to manganese (Eck and
Wilson, 1989).
While Pb is a toxic for humans even at low levels it can cause a chronic
effects on heme synthesis by inhibition of the enzyme -aminolevulinic acid
dehydratase in blood, reduction of concentrations of porphyrinsin blood
(especially zinc protoporphyrin), -aminolevulinic acid in urine, coproporhyrin
25
in urine, and, in increased dose hemoglobin itself. Ni and Co effect synthesis

of hemoglobin and the increase of these metals can decline the blood
parameters(Ewers and Schlipkoter, 1991).
The incidence of all values within the normal range make it seem at first
glance that the decline in those values is not important or may indicate that it is
not affected. And is well known that high concentrations of study heavy metals
helps to reduce blood parameters values of the current study. There are several
reasons may be make this increasing and closely values to the control group
values may be air pollution in Al-Nasiriyah city environment is an important
factor in raising the values of those parameters. As well as smoking with
different kinds.
4.4:The relationship between the values of heavy metals

The study samples are divided into groups according to the work period to
investigate the relationship between heavy metals concentration and the work
period. It is not possible to say that the period of work has no effect on the
concentration of the metals despite the verification in the results that have
obtain. They are not relevant to the concentration groups increasingly upward,
the period of work has a major effect on the heavy metals concentration, but
there are many other reasons that act as a helping factor to increase or decrease
the heavy metal values such as smoking may increase the heavy metal values
for lower period workers. The area where the worker lives, and the
physiological differences between the workers in terms of health and safety of
members have a significant effect on the concentrations of heavy metals in the
serum of workers and therefore did not find regularity in the concentrations of
metals values is not found in ascending where they can make sure that the
work period has an effect in the study of equal workers in the way and area of
life and physical and the physiological integrity.
56
4.5: Relationship between smoking and heavy metals

concentration in serum of workers
Compartment results of heavy metals concentrations values in blood serum
of smoker workers and non-smokers showed higher values of most metals (Co,
Cu, Ni & Pb) in blood serum of non-smoker except Cd that was higher in the
serum of smokers. It is known that tobacco plant absorbs high concentrations
of heavy metals from the soil and more of these metals is Cd (Bernhard et al.,
2005), therefore, cigarette smoke contains most of these metals and most Cd as
the focus on tobacco in general (0.5-3.5) at a rate of 1.7 g/g. (Schenker,1984).
This explains the high concentration of Cd in smoker workers in addition
to being exposed to cars fuel residues and vapors understand also exposed to
concentrations of Cd extra derived from cigarette smoke. But the effect of
smoking does not result in smokers only, but extends to non-smoker so that the
smoke produced by cigarettes burning, containing many minerals, including
metals study are inhaled by non-smokers and smokers who was close to the
smoker so, it called (side stream) in this case it is called passive smoking
(Chiba & Masironi, 1992). Other metals was focus in non-smokers which was
higher than smokers and this confirms that the main source of heavy metals
contamination is a motor fuel and its waste, where it was the main reason for
the rise in those metals among workers, smoking cannot be said that was not
influential in the rise in the values of these metals but its contribution was
minimal compared to the constant daily exposure.
4.6: Relationship between age and heavy metals concentration in

serum of workers
Heavy metals were accumulated because they were un -degradable which
make it stored over time in different body tissues such as bones, liver, spleen,
kidney, hair, nails, teeth and central nervous system.
56
Cd and Pb are two metal which have long half time in men from 10 to 30
years (Cornelis et al., 1995). This explain the rising of Cd with age in collected
sample of present study. While Co un regulatory with age. Where the first age
group (20-31) the lowest group, while second and third groups are closed
together. Unnecessary accumulation of cobalt with age is variable periodically
because of the entry in the synthesis of vitamin B-12. And its levels are high in
the liver, kidneys, heart, spleen, brain, pancreas except serum shall be very low
concentrations. And sometimes higher Co be the result of abuse of alcohol
containing salts of cobalt, it raises its levels in the body to produce ten times
more than concentration in non-abuse (Bernhard et al., 2005).
Cu value is rise with age increasing in third group(44-55) recorded
significant increase compared with the each of groups 1 and 2. Cu is associated
with ceruloplasmine protein (blood protein responsible for linking and
transport Cu in the body of the human) level is influenced by circumstance
physiology of the body, but the metabolized Cu controls the balance process
(Eck and Wilson, 1989). And a height with age may explain the low activity of
the body or because of the effect of lead that decrease with age increasing in
table (3-6).
The nickel is low in the results of the current study with age and this is the
natural state of nickel, which is present in cigarette smoke, but the focus will
be in reverse with increasing age (Ikeda et al., 2011).
Pb rising was reversed with increasing in age. It must accumulates in the
tissues of the body when exposure repeated over time. There are several
reasons, for example, high lead concentration in the tissue does not necessarily
reflect accumulate with age may be a higher concentration as a result of too
much exposure before the examination (Cornelis et al., 1995).
55
To study the accumulation of heavy metals in the tissues of human body

with age must be take samples with a long half-life for metals, such as bones,
nails and teeth.
There was no fixed criteria or conditions to see the natural concentrations
of the components of the study in the blood serum of employees. There are
several factors should be taken into consideration, such as the period of work,
smoking, age and geographical location. The sum of these factors led to the
disparity between the concentrations of metals in the blood serum of
employees has therefore examine each of these factors separately.
55
Conclusions
and
Recommendations
.........Conclusions and Recommendations............
...
I. Conclusions
1- High concentrations of heavy metals in blood serum of workers at fuel
stations were more than in blood serum of the control group.
2- Although the control samples record concentrations of heavy metals values
lesser than the study samples much higher than results of global studies, this
may be a risk index of environment province contamination.
3- No clear effect is observed for high concentrations of these metals on the
changing values of the studied blood parameters in this study.
4- No clear effect is observed for a period of work on serum concentrations of
heavy metals in the workers blood serum and this may be due to the
individual differences of the physiological or physical or suspected.
5- The obtained results show that smoking does not have a big impact on the
concentration of heavy metals except Cd.
6- Heavy metal concentration (Cd ,Cu ,Co,)were increased proportional with
them ages ,except Ni& Pb
46
.........Conclusions and Recommendations............
...
II. Recommendations
According to the results of the present study, the following points are
recommended:-
1. The study of heavy metals effects separately on blood parameters was
required .
2. Recommended a study on how these heavy metals affect the blood
parameters and other physiological aspects.
3. suggest that there be a study on the relationship between smoking and
concentration of these metals in the serum.
4. There should be a study on the types of fuel for the purpose of knowing the
species least harm to the environment.
5. There are should be monitor of the type of fuel used at each station by the
officials to ensure the safety of workers and the environment.
6. Periodic checks must be done for workers and monitor the concentrations of
these metals in biological samples to determine if they pose a threat or not.
7. Must be turn off the vehicle engine before starting input fuel.
46
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Appendices
..................................Appendices...................................
Appendices (1): The information of study samples
Other Past work Smoking Work Resident Age No.

notes period area
..................................Appendices...................................
Appendices(2): Location of study stations by

https://www.google.com/maps/@31.05496,46.26,12z/data=!3m1!1e3
Station -1
Al-Ballam
Station-4
Al-Mohandis
Al-husain
hospital
Station-2
Al-Nasiriyah
Al-habboby
statue Highway leading
to Al-basrah city
Al-nser
bridge
Station-3
Al-Rayat
.

HC -
H
3

:

0220
( )

0222 2341
:
1024 1025

.
200 00
. 10 55 21-2 10
.
-

Cd. 02014
Cu. 0201 Co. 02010
Ni. 02000 Pb. 0225

Cu. 02004 Ni. Cd. Co.
0200 Pb. 0200 .
Hb., PCV., RBCs., WBCs. & PLTs.
=(RBCs.= 5.1*106/L , Hb.= 14 g/dL, PCV.= 43.2%, WBCs.
7.4*103/L & =PLTs. )204.9*103/L
RBCs., WBCs. & PLTs.

.

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Republic of Iraq

Ministry of Higher Education

A Thesis Submitted to the Council of

Fatima Abbas Moshchil

I Dedicate this humble

effort to my Big teacher

dear brothers, sisters, my

dear son, and my

List of contents III

List of tables VIII

Chapter One : Introduction& Literature Review

1.2 Aim of study 4

1.3 Literature review 5

1.3.1 Heavy metals 5

1.3.2 Using and Recovery of heavy metals 6

1.3.3 Cadmium (Cd) 7

1.3.4 Cadmium Toxicity 8

1.3.5 Cobalt ( Co) 9

1.3.6 Cobalt Toxicity 10

1.3.7 Copper (Cu) 11

1.3.8 Copper Toxicity 11

1.3.9 Lead (Pb) 12

1.3.10 Lead Toxicity 13

1.3.12 Nickel Toxicity 16

1.3.13 Classifications of heavy metal exposure 17

1.3.14 Access pathways of heavy metal to the body 18

1.3.15 Mechanism of heavy metals activity 19

1.3.16 Laboratory testing and diagnosis for the presence of 19

1.3.17 Assessment of exposure to heavy metals 20

1.3.18 Management of Heavy Metal poisoning 23

1.3.19 Heavy metals analytical methods (Quantitative 24

1.3.20 Vehicle emissions and their sources 26

Chapter Two: Materials and Methods

2.1.1 Instruments and equipment 28

2.1.2 Chemical materials 29

2.2.1 The Study area 29

2.2.2 Method of sample collection 29

2.2.2.1 With regard to data of workers at fuel stations 29

2.2.2.2 With regard to collection of blood samples 30

2.2.3 Measurement of blood parameters 30

2.2.5 Digestion of serum 30

2.2.6 Estimation of heavy metals 31

2.2.7 Study design 31

2.2.8 Statistical analysis 32

Chapter three : Results

3.1 Heavy metal concentrations in serum of study samples 33

3.1.1 Cadmium (Cd) 34

3.1.2 Cobalt (Co) 34

3.1.3 Copper (Cu) 35

3.1.4 Nickel (Ni) 36

3.1.5 Lead (Pb) 37

3.2 Estimation of some blood parameters 38

3.2.1 Hemoglobin (Hb.) 39

3.2.2 Packed Cell Volume (PCV.) 40

3.2.3 Platelets (PLT.) 41

3.2.4 Red blood corpuscles (RBCs.) 42

3.2.5 White blood cells (WBCs.) 43

3.3 The relationship between the values of heavy metals 44

3.5 The relationship between Heavy metals concentration 48

3.6 Concentrations of heavy metals in collected samples 51

Chapter four: Discussion

4.1 Heavy metals concentration in blood serum of 52

4.2 Heavy metals concentration in blood serum of 57

4.3 Blood parameters 59