DETERMINATION OF LEVELS OF SOME HEAVY METALS (Pb, Cr

AND Cd) IN THREE COMMERCIALLY AVAILABLE BRANDS OF

MILK POWDER FOUND IN HARAR TOWN, EASTERN HARARGE,
ETHIOPIA

M.Sc. Graduate Project Research

ABERA MOGES

October, 2014
Haramaya University
DETERMINATION OF LEVELS OF SOME HEAVY METALS (Pb, Cr
AND Cd) IN THREE COMMERCIALLY AVAILABLE BRANDS OF
MILK POWDER FOUND IN HARAR TOWN, EASTERN HARARGE,
ETHIOPIA

M.Sc. Graduate Project Research Submitted to the Collage of Natural

and Computational Sciences
Department of Chemistry, School of Graduate Studies
HARAMAYA UNIVERSITY

In Partial Fulfillment of the Requirements for the Degree of

MASTER OF SCIENCE IN CHEMISTRY (ANALYTICAL CHEMISTRY)

By

Abera Moges

October, 2014
Haramaya University
 SCHOOL OF GRADUATE STUDIES
HARAMAYA UNIVERSITY

As project based thesis research advisor, I hereby certify that, I have read and evaluated this
project based thesis. Entitled DETERMINATION OF LEVELS OF SOME HEAVY
METALS (Pb, Cr AND Cd) IN THREE COMMERCIALLY AVAILABLE BRANDS
OF MILK POWDER FOUND IN HARAR TOWN, EASTERN HARARGE, ETHIOPIA
and I recommend that it can be submitted as fulfilling the project requirement prepared under

my guidance, by Abera Moges

Prof. O.P.Yadav ___________________ _______________

Major Advisor Signature Date
As members of the Board of Examiners of the Final M.Sc. project research open Defense
Examination, we certify that we have read and evaluated the project research prepared by
Abera Moges and examined the candidate. We recommend that the project based thesis be
accepted as fulfilling the project research requirements for the degree of Master of Science in
Chemistry (Analytical Chemistry).

________________________ ____________________ _________________

Chairperson Signature Date
________________________ ____________________ _________________
Internal Examiner Signature Date
________________________ ____________________ _________________
External Examiner Signature Date

iii
 DEDICATION

I dedicated this project manuscript to my Mother Yewubinesh Tefasse, to my beloved wife

Fatuma Jemel and to my daughters Meron and Yayeshyirad Abera for nourishing me with
affection and love and for their dedicated partnership in the success of my life.

iv
 STATEMENT OF THE AUTHOR

First, I declare that this work is my own work and that all sources of materials used for this
project work have been duly acknowledged. This work has been submitted in partial
fulfillment of the requirements of M.Sc. degree at Haramaya University and is deposited at
the University Library to be made available to borrow under rules of library. I solemnly
declare that this project manuscript is not submitted to any other institution anywhere for the
award of any academic degree, diploma or certificate.

Brief quotations from this project research manuscript are allowable without special
permission provided that accurate and complete acknowledgement of the source is made.
Requests for permission for extended quotations from, or reproduction of, this project
manuscript in whole or in part may be granted by the Head of the Department or the Dean of
the School of Graduate Studies when in his or her judgment the proposed use of the material
is in the interest of scholarship. In all other instances, however, permission must be obtained
from the author of the project based thesis.

Name of the Author: Abera Moges Signature----------------------

Place: Haramaya University, Haramaya
Date of Submission: ________________

v
 BIOGRAPHICAL SKETCH

The author was born from his father Ato Moges Shibeka and his Mother W/ro Yewbnesh
Tafesse in August 1963 E.C in South Nations Nationality and People Region, Gamo Goffa
Zone, Arba Minch. He attended his elementary and junior school studies at Kulfo Elementary
School in Arba Minch; he then attended his secondary education in Arba Minch at Arba
Minch Secondary and Comprehensive High School. After completion of his secondary school
education he joined Arba Minch teachers training institute (TTI) in September 1980 E.C and
graduated as elementary school teacher. After serving as elementary school teacher for about
15 years he then joined distance education program launched by The Ministry of Education
and graduated with diploma in chemistry from Hawasa teachers training college (TTC) in
September 1997 E.C and then, he joined Arba Minch University in July 1998 E.C and
attended his undergraduate studies and received B.Ed. degree in chemistry in September 2002
E.C. After completion of his undergraduate study he served as a secondary school teacher at
Geressie Secondary & Preparatory School in Bonke woreda Gamo Gofa Zone until he joined
the School of Graduate Studies at Haramaya University in July 2003 E.C to pursue his M.Sc.
study in Chemistry.

vi
 ACKNOWLEDGEMENTS

First and foremost, I would like to praise the Almighty God and his mother St.Marry for
bestowing up on me health, strength, patience and protection throughout my study period.

I express my deepest and heartfelt gratitude to my advisor Prof O.P.Yadav for his utmost
cooperation, concrete suggestion valuable comments, supervision, appreciable encouragement
and fatherly consultancy. I also like to appreciate his patience in following up this work from
the very beginning to the end and for the provision of useful reference materials in the
preparation of this project manuscript.

I would like to extend my gratitude to chemistry department, Central soil Laboratory of

Haramaya University, for providing the necessary materials during my Laboratory work. I
would like to extend my gratitude to Ato Gemechu Tafa technical assistant of the Animal
Nutrition laboratory for technical assistance in the digestion process; I would also like to
extend my gratitude to Ato Wubishet Legessie Central Laboratory technical assistant for
majority work assistance. I would also like to thank Ato Fikadu Tsegaye for helping me
during running FAAS of my samples.

I am also indebted to all my family especially to my mother Yewbinesh Tafesse, my wife

Fatuma Jemal, my daughters Meron and Yayeshiyirad Abera for their unreserved
encouragement, love, support and patience. Last but not least, my heartfelt gratitude goes to
my friends and colleagues. They deserve special thanks for their valuable contributions and
encouragement.

vii
 LIST OF ABBRIVIATIONS
AAS Atomic Absorption Spectroscopy
ANOVA Analysis of Variance
CSRM Certified Reference Material
DSS Dietary Supplement Standard
DNA Deoxyribo Nucleic Acid
EPA Environmental Protection Agency
FAO Food and Agricultural Organization
FNBIM Food and Nutrition Board, Institute of Medicine
GEMS Global Environmental Monitoring System
GF-AAS Graphite Furnace Atomic Absorption Spectroscopy
ICOEABS 2nd International Conference on Environment and Bio Science
ICP-OES Inductively Coupled Plasma-Optical Emission Spectroscopy
IDL Instrumental detection Limit
JECFA Joint Expert Committee on Food Additives
LOQ Limit of quantization
MDL Method detection limit
ppm Parts Per Million
RDA Recommended Dietary Allowance
RNA Ribonucleic Acid
SD Standard deviation
US EPA United States Environmental Protection Agency
US FDA United States Food and Drug Authority
WHO World Health Organization

viii
 TABLE OF CONTENTS

STATEMENT OF THE AUTHOR v

BIOGRAPHICAL SKETCH vi
ACKNOWLEDGEMENTS vii
LIST OF ABBRIVIATIONS viii
TABLE OF CONTENTS ix
LIST OF TABLES xii
LIST OF TABLES IN THE APPENDIX xiii
LIST OF FIGURES IN THE APPENDIX xiv
ABSTRACT xv
1. INTRODUCTION 1
2. REVIEW OF LITERATURE 5
2.1. Minerals in the Diet 5
2.2. Classification of Minerals 6
2.3. Heavy Metals 6
2.4. Pollution Problems 7
2.5. Lead as Pollutant 9
2.6. Chromium as Pollutant 11
2.7. Cadmium as Pollutant 11
2.8. Effects of Heavy Metals on Human Health 13
2.9. Preparation of Milk Samples for Analysis 13
2. 9. 1. Sample Decomposition Technique 14
2. 9. 1. 1. Wet-ashing technique 15

2.10. Flame Atomic Absorption Spectrometry 16

3. MATERIALS AND METHODS 18
3.1. Experimental Site 18
3. 2. Materials 18

ix
 TABLE OF CONTENTS(CONT)

3.2.1. Equipment and Apparatus 18

3.2.2. Reagents and Chemicals 19

3.3. Sample Collection 19

3.4. Methods and procedures 19
3.4.1. Preparation of 1000ppm stock AAS standard solution for selected heavy metals
(Pb, Cr and Cd). 19
3.4.2. Preparation of series of standard solutions of the respective metal ion 20
3.4.3. Sample preparation 20
3. 4. 3. 1. Digestion of samples 20

3.4.3.2. Optimization for Digestion Procedure of Powder Milk Samples 20

3.4.3.3. Digestion of Powder Milk Samples 21
3.4.4. Determination of metal content by AAS 21
3.4.4.1. Preparation of calibration curve 21
3.4.4.2. Determination of metal contents of each digested sample 22

3.5. Flame Atomic Absorption Spectrophotometric Analysis 24

3.6. Method Validation 24
3.6.1. Precision and accuracy 24
3.6.2. Method detection limit 25
3.6.3. Limit of quantitation (LOQ) 25
3.6.4. Validation of optimized procedures 25

3.7. Analysis of Metal contents of Digests 26

3.8. Data Analysis 26
4. RESULTS AND DISCUSSION 27
4.1. Analysis of Powder Milk Samples for Metal Level 27

x
 TABLE OF CONTENTS(CONT)

4.2. Instrumental Calibrations 28

4.3. Method of Performance and Method of Validation 28
4.3.1. Precision 29
4.3.2 Validation of the optimized procedure 29

4.4. Determination of Heavy Metals in Different Brands of Powder Milks 30

4.4.1. Lead 30
4.4.2. Chromium 32
4.4.3. Cadmium 32

5. SUMMARY, CONCLUSION AND RECOMMENDATION 34

5.1. Summary and conclusion 34
5.2. Recommendation 35
6. REFERENCES 36
7. APPENDICES 45

xi
 LIST OF TABLES

Page
Tables

Table 1. Ranking of Risks Associated with Various Heavy Metals. 9

Table 2. The Use and Health Effects of Some Heavy Metal on Human Being 13
Table 3 .Standard AAS condition for Cd, Pb and Cr 23
Table4. Metal Concentration (mg/L) in Powder Milk from Harar Super Markets (MeanS.D)
27
Table 5. Concentration Values of Working Intermediate Standard, Working Standard and
Correlation Coefficients of Calibration Graph. 28
Table 6. MeanSd,n=3mg/l and %RSD of Pb,Cd and Crin powder milk samples 29
Table 7. Recovery Tests for the optimized procedure for the Milk Powder Samples 30

xii
 LIST OF TABLES IN THE APPENDIX

Appendix Table page

Table 1.Optimization for Reagent Volume 46

Table 2.Optimization for Temperature 46
Table.3. Optimization for Digestion Time 47
Table 4 Provisional Tolerable Daily Intake of Toxic Heavy Metals 47
Table 5. Range of Metals Concentrations in Powder Milk Samples 48
Table 6 Instrument Detection Limit, Method detection Limit and Method Limit of
Quantitation limits. 48

xiii
 LIST OF FIGURES IN THE APPENDIX

Page
Appendix figures

Fig.1 Plot of Absorbance Versus Concentration for Lead ...................................................... 49

Fig.2 Plot of Absorbance Versus Concentration for Chromium ............................................. 49
Fig.3 Plot of Absorbance Versus Concentration for Cadmium .............................................. 50

xiv
 DETERMINATION OF LEVELS OF SOME HEAVY METALS (Pb, Cr
AND Cd) IN THREE COMMERCIALLY AVAILABLE BRANDS OF
MILK POWDER FOUND IN HARAR TOWN, EASTERN HARARGE.
ETHIOPIA

ABSTRACT

This project was aimed to measure the concentration of Lead (Pb), Chromium (Cr) and
Cadmium (Cd), in brands of full cream powder milk available commercially in Harar using
Atomic Absorption spectrometer. To this effect samples of three different brands of full cream
milk powder were collected from Harar local market, Each of 0.5g of powder milk samples
were wet digested in 5ml of HNO3 (69-72%) and 3ml HClO4 (70%) for 2:30 h at a
temperature of 210 0C. The wet digestion procedure was tested by varying reagent volume,
digestion time and digestion temperature to develop a procedure that consume less reagent
volume, clear solution, that requires minimum digestion time and minimum digestion
temperature applied. The concentrations of heavy metals (Pb, Cr and Cd) in the powder milk
collected from Harar were determined by using flame Atomic Absorption spectrometer.
Concentration of Lead, Chromium and Cadmium in the powder milk brands were found to be
in the range: 0.038-0.1688ppm, 0.0597-0.222ppm, and ND for sample X and Z but 0.068ppm
for sample Y, respectively. The accuracy of the optimized procedure was evaluated by
analyzing the digest of the spiked samples. The recoveries of the metals were in the range of
95.91-99.3% and the standard deviations were less than 10%. These values are compared
with recommended dietary allowance (RDA) values and also with the corresponding values of
different countries available in the literature.

Key Words: Powder milk, Cadmium, Chromium, Lead, Toxic elements

xv
 1. INTRODUCTION

Milk and its products are very common in our food list due to their nutrient value, since it is a
source of vitamins and lot of mineral constituents which are necessary for proper development
and functioning of different tissues and organs. However, overdose of these vitamins or
mineral constituents can be harmful (Farid et al., 2004). Determination of minerals and trace
elements in foodstuffs is an important part of nutritional and toxicological analyses. Copper,
Chromium, Iron and zinc are essential micronutrients for human health. In addition, these
elements play an important role in humans and animals body metabolism. The interest in
these elements is increasing due to the available reports of relationships between heavy metals
status in food and drinking water and the prevalent oxidative diseases in living beings. Lead,
Cadmium, Chromium, Nickel, Arsenic and Mercury are the most common toxic metals of
concern due to the reports of their contamination in various herbal preparations and herbal
ingredients and Chromium is associated with diabetes and cardiovascular diseases (Nathani et
al., 2010).

Cadmium is recognized as a metal posing threat to agricultural food quality due to its mobility
in the soilplant system added to soil from the metal-working industries, waste incinerators,
urban traffic, cement factories, and as a byproduct of phosphate fertilizers. In areas with low
anthropogenic activity, Cadmium can be released as a result of rock mineralization processes.
It enters into plant cells due to its similar chemical and physical characteristics to plant
nutrients. It can cause many toxic symptoms, such as inhibition of growth and photosynthesis,
deactivation or inhibition of enzymes, disturbances in plant water relationships, ion
metabolism, and formation of free radicals (Kumars et al., 2008).

The alarming contaminations of heavy metals in soils and plants have become an important
issue worldwide because of their adverse effects on human health and serious threat to food
safety. Metal pollutants can easily enter the food chain if heavy metal-contaminated soils are
used for the production of food crops.

1
Some elements as essential micro-nutrients have a variety of biochemical functions in all
living organisms and form an integral part of several enzymes (Sommers, 1974). Although
they are essential, they can be toxic when taken in excess; both toxicity and necessity vary
from element to element and from species to species (Tripathi, 1997). Thus, information on
the intake of heavy metals through food chain is important in assessing risk to human health.
These values need to compare with recommended dietary allowance (RDA) values and also
with the corresponding values for milk in different countries available in the literature.

Environmental pollution has been a major area of concern worldwide. Industrial and
agricultural processes have caused an increased concentration of toxicants like heavy metals
in the environment and as a result being taken up by plants or animals into their systems
which cause further distribution of toxicants to the environment (Ahmad, 2002). This is the
case in cows milk that is being consumed by humans in liquid or powder form. Heavy metals
can contaminate milk through exposure of lactating cow to pollution or consumption of
feeding stuffs and water with toxicants (Carl et al., 1991 and Okada et al., 1997). Moreover,
contamination can also be attributed to the manner of manufacturing dairy products where
toxicants have the possibility to be included during the production process (El-Batanouni, and
Abo-El-Ata, 1996 and Okada et al., 1997).

According to Salha Fathy Ahmed Abd-El Aal (2012) the chemical composition of dairy
products depends not only on the concentration of raw milk components and on its treatment,
but also on the mode of packing and quality of the packing materials and on the conditions
and time of storage. With the increasing number of toxicants being present in the environment
the safety of people is at risk. Children are particularly vulnerable to environmental toxicants
because of their possibly greater relative exposure and the effects on their growth and
physiological development (Jamison et al., 2006).

Powdered milk is the essential food needed by growing children. It contains both the basic
and additional requirements needed by children especially during their developmental years.
This milk may be contaminated by heavy metals that at certain levels may cause toxicity to
consumers. This toxicity is attributed to accumulation of heavy metals in the body which are

2
not metabolized to extractable products. Knowing that toxic heavy metals may be present in
powdered milk products, confirmation of their presence and corresponding quantities are
necessary to assess if the commercial products being marketed are safe to use. The acceptable
concentration limit of Cadmium in dietary supplement as proposed by the World Health
Organization ranges from 0.008ppm-0.010ppm. Furthermore United States Environmental
Protection Agency (US EPA) and United States Food and Drug Authority (US FDA)
estimates the daily exposure to total cadmium in an average 9.2 kg infant as 1.3g/ day. In
Chromium, daily intake should not exceed 30g/ day (DSS 173, 2011; 2nd ICOEABS, 2012).

Rapid industrialization, use of agricultural chemicals and automobile exhaust fumes has
increased the toxic heavy metals in the environment affecting all the global air, water and
food. Human population is directly affected through the food chain. In this context, the
increase in Lead and Cadmium concentrations draws the attention of researchers. Lead is
known for its devastating effects on brain development (Bryee-Smith Stephenes, 1982) while
Cadmium causes renal problems, anemia, reproductive failure and other complications
(Murthy and Reha, 1971). The concentration of Lead in milk is a matter of special concern
because it is a major dietary constituent for the infants and childrens.

Determination of trace quantities of Lead, Chromium and Cadmium were carried out all over
the world using various analytical techniques (Narres et al., 1985). The most commonly
employed method for metal analysis is Flame Atomic absorption spectrometry. Here, the
interference by other elements is almost nil or negligible because the estimation is carried out
for an element at a specific emission spectra line. In fact, in Atomic absorption spectrometry,
traces of one element can be determined accurately in the presence of a high concentration of
other elements (Motsara and Roy, 2008).

In this project, the atomic absorption spectrometric technique was employed for the
determination of three toxic heavy metals Lead, Chromium and Cadmium levels in three
brands of full cream powdered milk available in different super markets at Harar town,
Eastern Hararge, Ethiopia. This study was designed with the general objective of determining
the levels of some heavy metals in various commercially available brands of full cream milk

3
powder obtained from different super markets in Harar, Eastern Hararge, Ethiopia and
comparing the results with existing standards for allowable amounts set by WHO, With this
general objective, the specific objectives of this study were:

o To optimize the digestion method used in preparing milk samples for analysis.
o To determine the levels of Pb, Cr and Cd in the digested powdered milk
samples using atomic absorption spectrophotometer.

4
 2. REVIEW OF LITERATURE

2.1. Minerals in the Diet

Varieties of metals are found in a range of foods in the diet, and in this context, are termed
minerals, along with some non-metals, such as iodine and fluorine. These minerals can be
macro minerals those that are needed by the body in relatively large amounts, e.g. sodium,
potassium, chlorine, calcium, phosphorus and magnesium. It presents in virtually all cells of
the body, maintaining general homeostasis and required for normal functioning (Kehoe., et
al., 1994). Acute imbalances of these minerals can be potentially fatal, although nutrition is
rarely the cause of these cases. Diet can affect levels of 12 macronutrients in the body, but
effects are generally chronic, e.g. a high intake of sodium can lead to hypertension (FNBIM,
1997). And micro/trace minerals are those needed in small amounts, e.g. selenium, iron, zinc,
copper, manganese, molybdenum, chromium, arsenic, germanium, lithium, rubidium and tin.
Many of these minerals have been classed as essential elements; necessary for utilization by
the body to ensure good health, but the function of these minerals and their benefits to the
body is still uncertain and has been widely speculated. It contributes to good health if they
originate from an organic source because they have essentially been processed. Plants take up
minerals from the ground, digest them, making them ionic so that when consumed by humans,
assimilation into the body occurs much more easily, and toxicity by accumulation does not
occur. However micro minerals from inorganic sources, such as heavy metals, cannot be used
by the body as they tend to build up in the tissues (FNBIM, 1989).

Much research has been carried out concerning the role of minerals in the body, but in many
cases, difficulties in investigating their individual effects has been expressed because intake is
often in combination with other vitamins and minerals (FNBIM, 2001), e.g. fruit and
vegetables contain several minerals. There is, however, strong evidence that supplementation
of certain minerals would benefit those suffering from deficiency disorders. It is also
important to note though that intake of minerals does not necessarily correlate with absorption
and a balance must be obtained.

5
2.2. Classification of Minerals

Minerals are usually classified in to two main groups on the basis of their relative amounts in
the body. One of the groups is macro elements or macro minerals occurring in relatively
larger amounts and needed in quantities of 100mg or more per day which include calcium,
magnesium, sodium and potassium. Minerals occurring in small amounts and needed in
quantities of a few milligrams or less per day are called microelements or trace elements ,
Which include iron, zinc, copper, manganese, cobalt, nickel, chromium and boron. Other trace
metals like aluminum, lead, cadmium, mercury and arsenic are till now recognized as
potentially harmful. Actually all essential elements may also be toxic in animals and humans
if ingested at sufficiently high level and for a long enough period (WHO, 1996; Sanger et al.,
2005).

2.3. Heavy Metals

The term Heavy Metals refer to any metallic element that has a relatively high density and is
toxic or poisonous at low concentration (Lenntech, 2004). Heavy Metals are a general
collective term, which applies to the group of metals and metalloids with atomic density
greater than 4 g/cm3, or 5 times or more, greater than water (Syman and Huton,1986;
Nriagu,1988; Hawks,1997) and also Heavy Metals are defined as those elements with a
specific density at least five times the specific gravity of water, Heavy Metals include
Cadmium (Cd), Copper (Cu), Lead (Pb), Zinc (Zn), Mercury (Hg), Arsenic (As), Silver (Ag),
Chromium (Cr), Iron (Fe) and Platinum group elements, Copper and Zinc are essential trace
elements for living organisms at low concentration (<10mg/L). However, they become toxic
at high concentration (>10mg/L). Most of these metal ion (Cd, Cu, Zn, Hg, As, Ag, Cr and
Fe) can be released from the industries are in simple cationic forms (Volesky, 1995). The
characteristics of heavy metals are described by Wang (2006). Toxicity that can last for a long
time in nature. Heavy Metals cannot be degraded including bio treatment and are very toxic
even at low concentration (1.0-10.0mg/L).

6
Heavy Metals are dangerous because they tend to bioaccumulate. Bioaccumulation means an
increase in the concentration of a chemical in a biological organism over time, compared to
the chemicals concentration in the environment. Compound accumulate in living things any
time they are taken up and stored faster than they are broken down (Metabolized) excreted
(Tsoumbarist et al.,1994).World Health Organization (WHO) has established levels of metals
in foods above which, they should not be consumed. For this reason the levels of trace metals
in our food should be of much importance and concern to us.

2.4. Pollution Problems

Milk is considered as nearly a complete food in that its a good source of protein, fat, and
major minerals (Enb et al., 2009). Cow milk contains some major elements such as calcium,
potassium, phosphorous, and magnesium in addition to sodium, chlorine and a wide range of
micro elements and even heavy metals. Increase in industrial and agricultural processes have
resulted in increased concentration of metals in the air, water and soil. These metals are taken
in by plants and consequently accumulate in their tissues. Animals that graze on such
contaminated plants and drink from polluted waters also accumulate such metals in their
tissues and milk if lactating (Yahaya et al., 2010). A large amount of these metals taken in by
plants and animals subsequently find their way into the food chain. This ever increasing
pollution has given rise to concern on the intake of harmful metals in humans. Metals enter
the human body through inhalation, ingestion or absorption through the skin (Ogabiela et al.,
2010 and Ahmad, 2002). The intake through ingestion depends on food habit. Cow milk
which is a very important food stuff consumed by man is one of the major sources (Farid et
al., 2004). It has been reported that the content of the main elements in milk are fairly
constant and undergoes slide changes depending on lactation phase, quality of nutrition and
environmental conditions mainly chemical pollutants (Farid et al., 2004 and Dobrzanski et
al., 2005).

In recent times, the amount of metals in cow milk is widely studied, particularly in
industrialized and polluted areas of the developed and the developing countries of the world
since animals grazed freely on open fields are considered as bio-indicators of environmental

7
pollution (Korenekovg et al., 2002 and LI-Quang et al., 2009). Metal level in uncontaminated
milk is generally low, but by inhalation of polluted air, intake of contaminated feeds and
absorption through the skin, many dangerous elements or compound such as metals and
metalloid, accumulate along the food chain (Samaghail Binghila et al., 2008). The toxicity of
metals depends on a number of factors: the particular metal in question, dose absorbed and the
age of the person concerned. For instance, children are vulnerable to the effect of lead
exposure because they absorb several times the percentage ingested compared to adults and
even brief exposure may influence developmental processes (Samara and Richard, 2009).
Lead, cadmium and mercury residue in milk are therefore of great concern because milk is
largely consumed by infants and children. Although some metals are essential nutrients, have
a variety of biochemical functions in all living organisms and important industrial uses, their
potential toxicity to humans and animals is a source of concern. It is, therefore, necessary to
monitor and control their levels in consumed food.

The measurement of metal levels is helpful not only in ascertaining risk to human health but
also in the assessment of environmental quality (Farid et al., 2004; Samaghail Binghila et al.,
2008). Many reports indicated heavy metals in milk and attributed the presence of these heavy
metals in milk and dairy products to exposure of lactating cows to environmental pollution,
consumption of contaminated feed stuffs and water as well as the production process. Cow
milk is a good source of both micro and macro elements in human diet and the location of the
cows determine their concentration (Dobrzanski et al., 2005). The levels of lead and cadmium
in milk from cows grazed on open fields in Kaduna were reported to exceeded the permissible
maximum daily intake (0.05 mg/kg body weight) recommended by WHO (lawal et al., 2006).

The greatest demand for metal sequestration today comes from the need to immobilize the
metals released to the environment or mobilized by and partially lost through human
technological activities. It has been established that dissolved metals (particularly heavy
metals) escaping into the environment pose a serious health hazard. They accumulate in living
tissues throughout the food chain, which has humans at its top, multiplying the danger. Thus,
it is necessary to control emissions of heavy metals into the environment. Due to increase in

8
the world population and development of industrial applications, environmental pollution
problem became important. Communities produce both liquid and solid wastes.

The liquid -wastewater is essentially in industrial effluent and the water thrown out by
community after it has been used in a variety of applications. In recent years, heavy metals,
besides other pollutants, have increased to reach dangerous levels for living environment in
many regions. The presence of toxic and polluting heavy metals in wastewaters from
industrial effluents, water supplies and mine waters and their removal has received much
attention in recent years. The amount of heavy metals that industrial wastewaters often
contain is considerable and would endanger public health and the environment if discharged
without adequate treatment.

Heavy metals are elements such as Cu (Copper), Cd (Cadmium), Ni (Nickel), Pb (Lead), Zn

(zinc), Ag (Silver), Cr (Chromium), Hg (Mercury), Fe (Iron), Co (Cobalt) and As (Arsenic)
which are usually associated with toxicity and are natural components of the earth's crust.
They cannot be degraded or destroyed. To a small extent they enter our bodies via food,
drinking water and air. As trace elements, some heavy metals (e.g. Copper, Selenium, and
Zinc) are essential to maintain the metabolism of the human body. However, at higher
concentrations they can lead to poisoning. Heavy metal poisoning could result, for instance,
from drinking water contamination (e.g. through Lead pipes), high ambient air concentrations
near emission sources, or intake via the food chain.

Table 1. Ranking of risks associated with various heavy metals.

Relative priority High Medium Low

Environmental risk Pb, Cd and Hg Cr, Co, Cu, Ni and Zn Al and Fe

2.5. Lead as Pollutant

Lead is regarded as highly hazardous for plants, animals and particularly for microorganisms.
The main sources of lead pollution in agriculture and plants are lead mines, fuel combustion,

9
sewage sludge applications and farm yard manure. The maximum acceptable concentration
for lead in food stuffs is around 1 mg/kg. Long-term exposure to lead can result in a buildup
of lead in the body and severe symptoms. These include anemia, pale skin, a decrease
handgrip strength, abdominal pain, nausea, vomiting and paralysis of the wrist joint.
Prolonged exposure may also result in kidney damage. If the nervous system is affected,
usually due to very high exposure, the resulting effects include severe headache, coma,
delirium and death. Continued exposure can lead to decreased fertility and/or increased
chance of miscarriage or birth defects (Dobrzanski. et al., 2005).

Lead is rarely found as the free metal in nature, but it is present in several minerals,
principally in Galena (PbS) the main source for lead production. It is also found as Anglesite
(PbSO4) and Cerrusite (PbSO3). Lead is one of the most commonly used non-ferrous metals.
It has many applications; its largest use is in making storage batteries, most of which are
recycled. As a result of its resistance to corrosion and its malleability, it finds use in building
constructions, storage tank lining and corrosive liquid containers. Other uses of the metal are
for radiation shielding, ammunition, solder, cable sheathing and pipe work. Lead compounds
are used as pigments in paints and ceramics, catalysis, antibacterial substances and wood
preservatives.

A major use is the production of anti-knock compounds particularly tetraethyl lead, Pb

(C2H5)4 for addition to petrol. The exhausts from vehicles are a major source of the
environmental contamination by lead. Lead is present in exhaust gases mainly as lead halides
and oxides, but incomplete combustion results in about 10% of alkyl lead compounds also
being present. Other source of lead emissions are copper and nickel smelters, iron and steel
production. Estimates vary as to the importance of vehicle emissions as the sources of the lead
2+ 4+
contamination. Lead exists in the oxidation states Pb and Pb , with the divalent form
being the more stable in most aquatic environments. The speciation of lead compounds in
water is complicated and depends upon a number of factors, principally pH, dissolved oxygen
and the concentration of other organic and inorganic compounds. And concentration of lead in
waters is usually limited by the solubility of PbCO3, and by its adsorption onto particulate
matter (Dojlido and Best 1993).

10
2.6. Chromium as Pollutant

Chromium is one of the known environmental toxic pollutants in the world. The main sources
of chromium contamination are tanneries, steel industries and sewage sludge application and
fly ash. Besides these, chromium plating and alloys in motor vehicles are considered to be a
more probable sources of chromium (McGrath and Smith, 1990 and Shaheen, 1975) at an
elevated concentration it could be toxic for plants and animals. Concentrations between 5-30
mg/kg are considered critical for plants and could cause yield reductions. The problems that
are associated with chromium exposure are skin rashes, upset stomach, ulcers, respiratory
problems, weakened immune systems, kidney and liver damage, alteration of genetic material,
lung cancer and ultimately death (McGrath and Smith 1990 and Pendias et al., 1984).

Among the different heavy metals, Chromium is a common and very toxic pollutant
introduced into natural waters from a variety of industrial wastewaters. The two main forms
of Chromium, (chromate and dichromate) pose significantly higher levels of toxicity than the
other valence states (Krejpcio, 2001; Agaje, 2007; Qaiser et al., 2009). Chromium (Cr) below
toxic limit balances blood sugar levels, regulates hunger, reduces cravings, protect DNA and
RNA improves heart function, helps control fat and cholesterol levels in the blood (Krejpcio,
2001).

2.7. Cadmium as Pollutant

Cadmium is a toxic metal and can cause serious health problems. Recently attention has been
focused on its availability in water, soil, milk, dietary products, medicinal plants, herbal
drugs, etc. The most common sources for cadmium in soil and plants are phosphate fertilizers,
non-ferrous smelters, lead and zinc mines, sewage sludge application and combustion of fossil
fuels (Davies, 1990 and Mc Bride, 1995). Critical levels for cadmium in soil are between 3-5
mg/kg. This level, in most cases, it cannot cause toxic or excessive accumulation
concentration in plants; the lowest level of the element concentration in plants that can cause
crop yield reduction is between 5-30 mg/kg. Surprisingly, a small amount of cadmium was
detected in the stem, leaves, and seeds of the plant sample collected from polluted areas. This
may be due to the polluted air from the surrounding area.

11
The major route of cadmium exposure for the general population is via food. An increase in
soil Cadmium content generally results in an increase of plant uptake of Cadmium although
some soil and plant factors may influence Cadmium accumulation by plants. Crops grown in
Cadmium contaminated areas have been found to contain elevated Cadmium content
compared with normal levels. Therefore, human Cadmium exposure via food in contaminated
areas can be many times above normal intakes and lead to Cadmium toxicity. Cadmium is
used in plating, alloying, pigments, plastics and batteries.

Cadmium is known to be toxic for living organism even if it is present in low levels
(Krejpcio, 2001). Cadmium is obtained from the ore minerals Shalerite (ZnS, CdS) and
Greenockite (CdS) (Tadesu, 2011).

12
2.8. Effects of Heavy Metals on Human Health

Table 2. The Use and Health Effects of Some Heavy Metal on Human Being

Heavy Uses Health effects Reference

metals
Cadmium Electroplating, fertilizers, mineral Cancer, lung insufficiency Sharma,
processing and battery disturbances in liver and 1995
manufacturing kidney damage
Chromium Metal plating electroplating leather, Ulcer, skin irritation liver Kumar et
mining galvanometry, dye and kidney damage al.,2006
production
Lead Metal plating, textile, battery spontaneous abortion, Tunali et
manufacturer, automotive and damage nervous system, a.,2006
petroleum industries kidney and brain damage
Mercury Metallurgy industries, chemical Memory problems, Abia et
manufacturing and metal finishing increased heart rate, al., 2005
tremors, kidney and brain
damage

2.9. Preparation of Milk Samples for Analysis

Elemental analysis of the majority of organic and inorganic matrices requires the partial or
total dissolution of the sample prior to instrumental analysis. Only a few direct methods allow
the introduction of the samples without any preparation. In this case lack of reliable
calibration is the major problem. On the other hand, sample preparation allows the separation
and /or pre-concentration of analytes and makes possible the use of several determination
methods (Kingston and Jassie, 1988; Barnes, 1991; Jarvis et al., 1992).

Sample preparation of milk involves digestion, extraction and preparation of analyte before
analysis. So this step is time limiting, requiring 61% of the total time required to perform the
complete analysis and is responsible for 30% of the total analysis error, Nowadays the goals

13
to be reached are the best results, in the shortest time, with minimal contamination, low
reagent consumption and generation of minimal residue or waste. In order to achieve the real
objectives of the analysis, some aspects of sample preparation should be taken in to account;
Focusing on the chosen procedure, thus, simplification is simple manipulation, use of high
purity water and reagents in suitable amounts, correct cleaning of recipients and blank
preparation in parallel to sample are desirable. Also the validation of methodology is
important. Usually with certified reference materials (Bock, 1979).

2. 9. 1. Sample decomposition technique

Sample decomposition is useful for converting all the species in which a given element is
present in such a way that it becomes present in one defined form eliminating interfering
substances from the matrix and obtaining the element in a homogeneous and easily accessible
matrix. The choice of decomposition techniques should take into account the objective of the
final determination and factors such as the matrix composition, the elemental contents, the
possible interferences, the risk of loses and contaminations, the practicality and possible
safety hazards in the laboratory (Harvey, 2000). Different decomposition methods could be
classified into dry ashing, wet digestion and microwave digestion (Mustafa et al., 2006).

Sample digestion processes prior to quantification of heavy metals includes closed or open
digestion systems and the use of different combinations of acids, such as HNO 3, HCl, HClO4,
HF and others (Melo & Silva, 2008; Nemati et al., 2010; Garca-Delgado et al., 2012), as well
as oxidants such as H2O2. Due to their chemical composition, degree of polymerization and
the presence of molecules resistant to digestion, the recovery of heavy metals in organic
residues is subject to variation (Abreu et al., 1996; Hseu, 2004; Nemati et al., 2010; Garca-
Delgado et al., 2012). Therefore, digestion methods must be chosen considering the residue
and the recovery rate of the heavy metals investigated (Hseu, 2004; Garcia-Delgado et al.,
2012).

Wet digestion in open systems, according to Azcue & Mudrock (1994), is time consuming
and subject to contamination and loss of some chemical elements by volatilization. In the case

14
of muffle furnace digestion, if the analyte measured is volatile (as is the case of As, Cr, Pb
and Cd), losses may occur by volatilization and/or adsorption of the chemical element onto
the walls of the furnace due to the high combustion temperature, usually from 500-550 oC as
well as cross-contamination of samples(Azcue & Mudroch, 1994).

According to Sastre et al., (2002), the use of microwaves in wet digestion has greatly
accelerated the determination of trace elements and reduced the risk of contamination and loss
of volatile chemical elements. However, open systems allow the analysis of a larger number
of samples and facilitate evaporation of acid and drying of the digests, thereby reducing
analytical problems in stages subsequent to digestion. Zheljazkov and Warman (2002)
verified that HNO3 with HClO4 digestion provided higher recovery of Cd and Pb as compared
to breakdown of organic matrices in a muffle furnace (i.e., dry ashing). Hseu, (2004) also
described good results with HNO3 digestion, mainly in regard to the recovery of Cd, Mn and
Ni.

2. 9. 1. 1. Wet-ashing technique

Wet digestion methods involve the use of both heat and mineral acid/s. Acids that have been
used in this procedure include H2SO4, HNO3 and HClO4, either in combination or alone
(Maria, 2002). Hydrogen peroxide is also used to enhance the reaction speed and to ensure
complete digestion. Most laboratories have eliminated the use of HClO 4 due to risk of
explosion. Wet digestion can be carried out in open vessels, in tubes, on a hot plate or in an
aluminum heating block or in closed vessels at elevated pressure (digestion bombs) with
thermal or microwave heating. Microwave-assisted digestion is an attractive method,
especially for small samples. The applicability of this technique strictly depends on the type
of food: carbohydrates are easily mineralized with nitric acid at 1800C, while fats, proteins
and amino acids cause incomplete digestion due to the relatively low oxidation potential of
nitric acid at 2000C; these materials require the addition of sulfuric and/or perchloric acid with
all the problems related to their use at high temperature and pressure. The type of acid/s used
can have important consequences in the measurement step. It is commonly known that in all
atomic spectrometric techniques nitric acid is the most desirable reagent. In spite of

15
occasionally observed signal suppression in its presence (e.g.in ICP-OES), no severe
analytical problems are encountered in practice with nitric acid at concentrations up to 10%,
sometimes higher, in all atomic spectrometric techniques as long as its concentration is
similar in calibration and sample solutions. Hydrogen peroxide added in most mineralization
procedures is also rarely responsible for analytical problems (Arruda, 2007). The presence of
hydrochloric acid is not troublesome in ICP-OES analysis; however, its exclusive use is
prohibited in GFAAS analysis because of the possible formation of volatile and difficult-to-
dissociate analyte chlorides that could cause vapor phase and/or spectral interference (Welz
and Sperling, 1999).

Main features associated with wet digestion methods are: (1) much lower temperatures as
compared to dry ashing procedures, however minimizing volatilization losses or retentions
caused by reactions between analytes and vessel materials, they may lead to incomplete
solubilisation of sample constituents and (2) co-precipitation of analytes with precipitates
formed by main matrix elements within reactive mixtures.

This method was chosen based on its less contamination, lower rate of evaporation, cost and
time and extraction efficiency. In a study developed by Abreu et al, (1996), however, the
highest levels of heavy metals in residues were found when analytical reagent was employed
in the digestion.

2.10. Flame Atomic Absorption Spectrometry

For the determination of essential and toxic trace metals in powder milks different
spectrochemical methods are used. However, FAAS in one of the most extensively used
techniques for discriminating various elements with significant precision and accuracy. The
main advantages of FAAS with atomization in a widespread air acetylene flame are low
operational costs and reasonably good analytical performance (Lopez Garcia et al., 1999;
Loannidou et al., 2005). The possibility of only sequential analysis and the narrow ranges of
linear response can be regarded as disadvantages of this method. Using FAAS for the
determination of metals in powder milks the samples are usually mineralized in order to avoid

16
possible matrix-related interference and organic matter (Abu-Trboush et al., 1993; Downey et
al., 2005). In this case, aqueous external standard solutions can be used for calibration (Soares
et al., 2008). Otherwise, multiple linear regressions can be applied to minimize the effects of
chemical interferences in the flame. This draw back can be alleviated by decreasing the
concentration of organic compounds supplied to the nebulizers by using on line automatic
dilutors or discrete sample introduction manifolds (i.e. flow injection) (Lopez Garcia et at.,
1999).

17
 3. MATERIALS AND METHODS

3.1. Experimental Site

All experimental works were done at Haramaya University, Sample preparation and
digestions were done at the Animal Nutrition Laboratory and the preparations of stock
solutions as well as analysis of metal concentrations with AAS were done at the Soil Science
and Central Laboratories of Haramay University.

3. 2. Materials

The equipments and instruments used in this study were all calibrated to check their status
before and in the middle of the experiments. All Glass ware was cleaned by 10% concentrated
Nitric acid (HNO3) in order to clear out any heavy metal on their surfaces. Apparatus such as
volumetric flasks, measuring cylinder and digestion flasks were thoroughly washed with
detergents and tap water and then rinsed with deionized water. The digestion tubes were
soaked with 1% (w/v) potassium dichromate in 98% (v/v) H 2SO4 and the volumetric flasks in
10% (v/v) HNO3 for 24 hours followed by rinsing with deionized water and then dried in oven
and kept in dust free place until analysis began. Prior to each use the apparatus were soaked
and rinsed in deionized water. Details of the equipment and materials are described as follow:

3.2.1. Equipment and Apparatus

Analytical balance (Mttlerto leadop, model AG 204, Switzerland) with precision of + 0.001g,
100 ml round bottomed flasks, Borosilicate volumetric flasks (25, 50 ml, 100 ml & 1000 ml),
measuring cylinders (Duran, Germany), pipettes (Pyrex. USA), micropipettes (Dragonmed 1-
10 ml, 100-1000 ml, Shangao, Chaina) Muffle furnace, kjeldahy reflux condenser or Gerhardt
hot plate, Desiccators, porcelain crucible, flame atomic absorption spectrophotomers (buck
scientific model 210VGP AAS, USA) equipped with deuterium arc back ground correctors
and lead, cadmium and chromium hollow cathode lamp with air-acetylene flame used to
conduct spectral analysis of the concentration of Pb (II), Cr (VI) and Cd (II) at equilibrium
time. Filter paper whatmann No 1 and other Glass ware were used.

18
3. 2. 2. Reagents and Chemicals

Reagents and chemicals used for the laboratory works were all analytical grade: HNO3 (69-
72%), HClO4 (70%), Pb (NO3)2 (99.5%), Cd (NO3)2 (99.99%), Cr (NO3)2 (99.99%), H2SO4
(98%), Deionized water (chemically pure with conductivity 1.5 s/cm and below) was used
for dilution of sample and intermediate metal standard solutions prior to analysis and rinsing
glassware and sample bottles.

3. 3. Sample Collection

Three different foreign brands of full cream powder milk samples commercially available
were collected at random from different super markets in Harar, Ethiopia. Before analysis,
samples of the same brands were thoroughly mixed to get homogeneous and representative
samples.

3. 4. Methods and procedures

3. 4.1. Preparation of 1000ppm stock AAS standard solution for selected heavy metals
(Pb, Cr and Cd).

The determination of a given metal concentration in the experimental solution was based on
its respective calibration curve. In plotting the calibration curves for lead, chromium and
cadmium a stock solution of each metal ion of (1000ppm) was prepared by dissolving;
1.5980g of Pb (NO3)2, 7.6960g of Cr (NO3)2 .9H2O and 2.1032g of Cd (NO3)2 in deionized
water and then diluting to 1 liter in a volumetric flask with deionized water, respectively.
Blank solutions were prepared for the methods and for the standard working solution. To
prepare 100ppm, 10ml of the standard Pb (NO3)2, Cr (NO3)2 .9H2O and Cd (NO3)2 stock
solutions were pipetted and added in to 100 ml calibrated flasks finally diluted with deionized
water and the solution was mixed thoroughly. The standard working solutions was prepared
from 100ppm by pipetting it in to calibrated flasks and made up to volume with deionized
water.

19
3. 4. 2. Preparation of series of standard solutions of the respective metal ion

The selected heavy metals were Lead, Chromium and Cadmium. Calibration curves for each
of the selected metals were prepared using six standard solutions. The usual procedure in
quantitative analysis method is to prepare a series of standard solutions over a concentration
range suitable for the sample being analyzed i.e. such that the expected sample concentrations
are within the range established by the standard. These standards prepared by dilution from
1000ppm stock solution were as follows: 0.5ppm, 1ppm, 1.5ppm, 2ppm and 2.5ppm for Lead,
0.2ppm, 0.4ppm, 0.6ppm, 0.8ppm,1.0ppm ,1.25 for Chromium and 0.2ppm 0.4ppm, 0.6ppm,
0.8ppm, 1.0ppm and 1.2ppm for Cadmium. Calibration curves were drawn for Pb, Cr and Cd
by plotting absorbance versus metal ion concentration.

3. 4. 3. Sample preparation

3. 4. 3. 1. Digestion of samples

Exactly 0.5g of powdered milk with 5 ml Nitric acid was taken in triplicate in flasks fitted
with a condenser and then heated at 210 0C. After cooling the mixture, 3 ml of Perchloric
acid (70%) was added and heated again at 210 0C with occasional shaking until white fumes
cease to evolve. The solution was cooled down in 25 ml measuring flask and A specified
amount (10ml) of distilled water was added in the digested residue and filtered through
Whatman filer paper No 1.Then the volume of the filtrate was made up to mark with
deionized water.

3. 4. 3. 2. Optimization for digestion procedure of powder milk samples

The basic requirements for sample preparation for analysis are to get an optimum condition
for digestion. The optimum condition is the one which required minimum reagent volume
consumption, minimum digestion time reflection and clear digestion solution, ease of
simplicity and absence of undigested powder milk sample. In this study, to prepare a clear
colorless sample solution that is suitable for the analysis using FAAS different digestions

20
were carried out using HNO3 and HClO4 acid mixtures by varying parameters such as volume
of the acid mixtures, digestion time and digestion temperature. Different conditions tested for
optimization of digestion procedure for 0.5g of powder milk samples are summarized in
appendix table 1, 2 and 3. From the optimization procedure the acid mixture of 5 ml of HNO 3
(69-72%) and 3 ml of HClO4 (70%), digestion time of 2 hours and 30 minutes and digestion
temperature of 2100c were found to be the optimal conditions for digestion of 0.5g powder
milk samples.

3. 4. 3. 3. Digestion of powder milk samples

Applying the optimized conditions, 0.5g of dried, homogenized and representative of each
brand of powder milk samples were transferred in to 100ml round bottom flask and mixed
with 8 ml of a mixture of HNO3 (69-72%) and HClO4 (70%) with a volume ratio of 5:3 (v/v)
and digested at 210 0c for 2 h and 30 minutes on a kjeldahl digestion apparatus fitted with
reflex condenser. The digested sample was allowed to cool at room temperature. A specified
amount of distilled water was added in to the digested residue and filtered through whatman
filter paper No 1. The volume of the filtrate was made up to 100 ml using distilled water and
the solution was further diluted 10 times before determinations of lead (Pb), chromium (Cr)
and cadmium (Cd) using FAAS. The digestion was carried out in triplicate for both blank
samples. Digestion of a reagent blank was also performed in parallel with powder milk
samples keeping all digestion parameters the same.

3. 4. 4. Determination of metal content by AAS

3. 4. 4. 1. Preparation of calibration curve

Stock solutions of (1000mg/l) of each metal ion was prepared by dissolving calculated
amounts of each metal salt in 100ml of distilled water and diluted to 100ml. Six standard
solutions was prepared by serial dilution of each stock solution. Calibration curves were
prepared to determine the concentration of the metals in the sample solution. The instrument
was calibrated using six series of working standards. The working standard solutions of each
metal were prepared from the 10 mg/l intermediate standard solutions of their respective

21
metals. Calibration curve for each metal ion to be analyzed was prepared by plotting the
absorbance as a function of metal ion standard concentration.

3. 4. 4. 2. Determination of metal contents of each digested sample

Concentration of the metal ions present in the sample was determined by reading their
absorbance using AAS and comparing it on the respective standard calibration curve. Then
Pb, Cr & Cd were analyzed with FAAS (Buck scientific model 210GP) equipped with
deuterium arc back ground corrector and standard air acetylene flame system using external
calibration curve after the parameters (burner and lamp alignment, slit width and wave length
adjustment) were optimized for minimum signal intensity of the instrument. Three replicate
determinations were carried out on each sample. Hollow cathode lamps operated at the
manufacturers recommended conditions were used at their respective primary source line.

The acetylene and air flow rates were managed to ensure suitable flame conditions. The
metals were determined by absorption /concentration mode and the instrument readout was
recorded for each solution manually. The same analytical procedure was employed for the
determination of elements in digested blank solutions and for the spiked samples. The
standard Atomic Absorption Condition was recorded in Table 3 below.

22
 Table 3 .Standard AAS Condition for Cd, Pb and Cr

M Wave Slit Relative Characteristic Linear Recommended Operation with high Characteristics
E length noise s concentration range Flame Sensitive concentration
T Nebulizer With a N2O-C2H2
A Impact bead
Flame
L

Cd 228.8 0.7 1.0 0.028mg/l 2.0 Air-acetylene, Will typically provide 2-3 At
nm nm mg/l Oxidizing (lean, blue) sensitivity improvement 228.8nm: 0.11mg/L

Pb 283.3 0.7 0.43 0.45mg/l 20.0 mg/l Air-acetylene, Will typically provide 2-3 At
nm nm Oxidizing (lean, blue) sensitivity improvement 283.3nm: 2.7mg/L

Cr 357.9 0.7 1.0 0.078mg/l 5.0 Air-acetylene, Will typically provide 2-3 At 357.9nm:0.31mg/L
nm nm mg/l reducing (rich, yellow) sensitivity improvement

23
3. 5. Flame Atomic Absorption Spectrophotometric Analysis

Lead, Chromium and Cadmium was determined by a procedure described by Khalid et al

(1987). Before analysis, samples of same brands will be thoroughly mixed to get
homogeneous and representative samples. The digested samples were analyzed for Pb, Cr and
Cd using AAS. Concentration of each metal was obtained by comparing the observed
absorbance on respective standard (calibration curve). Average concentration was taken for
comparing with provisional tolerable weekly intake as stated by the food and agriculture
organization/world health organization with joint expert committee on food additives
(JECFA).

3. 6. Method Validation

3. 6. 1. Precision and accuracy

Accuracy and precision are probably the most often quoted terms to express the extent of
errors in a given analytical results. Analytical results must be evaluated to decide on the best
values to report and to establish the probable limits of errors of these values (Kikuchi et al.,
2002).The analyst will thus be concerned with the question of precision (repeatability of
results), that is, the agreement between a set of results for the same quantity and also with
accuracy, that is the difference between the measured value and the true value of the quantity,
Which is determined (Dean, 1997). In this study, the precision of an analytical procedure is
usually expressed as the variance, relative standard deviation and percentage relative standard
deviation of a series of measurements (Matusiewicz and kopras, 1997). The precision of the
results were evaluated by percentage relative standard deviation of the results of three samples
(N=3) and triplicate readings for each sample giving a total of nine measurements for a given
bulk sample. On the other hand, the accuracy and validity of the measurements were
determined by analyzing spiked samples.

24
3. 6. 2. Method detection limit

Method detection limit is the smallest mass of analyte that can be distinguished from
statistical fluctuations in the blank, which usually corresponds to the standard deviation of the
blank solution times a constant. The limit of detection is most commonly defined as the
amount of analyte that gives a signal equal to three times the standard deviation of a blank
(Butcher and Senddon, 1998). In this study, after digestion of three blank solutions containing
HNO3 and HClO4 three readings was taken for each blank and the standard deviation of these
was calculated. The method detection limit of each element was obtained by multiplying the
standard deviation of the reagent blank by three. As shown in Appendix-Table 7.
MDL=3xblank
Where blank is standard deviation of the blank readings

3. 6. 3. Limit of quantitation (LOQ)

The lowest concentration level at which a measurement is quantitavely meaningful is called

the limit of quantitation (LOQ).The LOQ is most often defined as 10 times the signal/noise
ratio if the noise is approximated as the standard deviation of the blank ,the LOQ is 10xSD of
the blank(Mitra,2003).In this study, LOQ was obtained from triplicate analysis nine reagents
blanks Which were digested in the same digestion procedure for powder milk samples .The
LOQ was calculated by multiplying pooled standard deviation of the reagent blank by 10
(LOQ =10xSDblank ,n=3)and the values for the elements was listed in Appendix table 7.

3. 6. 4. Validation of optimized procedures

Direct determination of the validity of the above optimized procedure towards analysis of the
powder milk samples with respect to each of the selected metals (Pb, Cr and Cd) could not be
made possible because of the absence of certified reference materials (CSRM) for these
metals. Instead, spiking method on the above (same) digestion procedure was adopted for the
same purpose. Accordingly, the efficiency of the optimized procedure was checked by adding
different volumes of standard solutions containing 10 mg/L of each metal in to 0.5g powder
milk. The spiked samples were then digested in the same manner as for original sample. Then

25
the digests were transferred in to 50 ml volumetric flask and diluted to the mark with
deionized water. Finally, the solutions were analyzed for metal concentration with FAAS. As
used for the original samples, triplicate spiked samples were prepared and the readings were
recorded. Recovery was calculated using the equation given below.

Where,

s= concentration equivalent of analyte added to the sample

Cs= metal content of the spiked sample
c = metal content of non-spiked sample
R = percent recover

3.7. Analysis of Metal contents of Digests

Atomic Absorption Spectroscopy was used to determine the concentration of digested

samples after treatment through different conditions of the parameters. The analysis was done
at working conditions of the Atomic Absorption Spectroscopy for Lead, Chromium and
Cadmium analysis as given in Appendix table 4.

3. 8. Data Analysis

Data was analyzed using Microsoft Office Excel. The data were expressed in term of
descriptive statistics while the figures were presented with Mean values as (MeanSD). A p-
value less than 0.05 were considered as Significant.

26
 4. RESULTS AND DISCUSSION

4. 1. Analysis of Powder Milk Samples for Metal Level

The samples were analyzed using flame atomic absorption spectrometer for the determination
of lead, chromium and cadmium at their corresponding wave length 283.3, 228.8 and 357.9
nm, respectively. The concentrations of these metals in powder milk samples were obtained in
mg/l (ppm) after calibration as recorded in Table 4 below.

Table 4. Metal Concentration (mg/L) in brands of Powder Milk from Harar Super Markets
(MeanS.D)

Powder Milk Concentration of Metals (ppm) Values in the Samples (MeanSD)

Brand Type Lead (Pb) Chromium (Cr) Cadmium (Cd)
a a
X 0.164 0.0004 0.222 0.015 ND
Y 0.038b 0.0014 0.206a 0.018 0.0680.004

Z 0.169a 0.0162 0.0597b 0.0055 ND

Mean 0.1234 0.1626 0.023

Note: ND=not detected at p<0.05

: Values with different letters within a column are significantly different at p< 0.05.

Table 4 shows metal concentrations in powder milk sample from three foreign brands. Lead,
Chromium and Cadmium levels showed significant difference between brands. The mean
Lead levels in powder milk sample of brand Y was significantly lower than those from other
brands and the mean chromium levels in powder milk of brand Z was significantly lower than
those from other brands. The mean concentration levels of the metals were 0.1628, 0.1234
and 0.023 mg/l (ppm) for Chromium, Lead and Cadmium, respectively. Levels of metals in
the powder milk were having the following order; Cr>Pb>Cd.

27
4. 2. Instrumental Calibrations

The data qualities obtained from FAAS for metal analysis are highly affected by the
calibration and standard solutions preparation procedures. The instrument was calibrated
using a series of standard working solutions of each of the metals prepared freshly by
appropriate dilution of the intermediate standard solutions. The intermediate standard
solutions were prepared from stock solution of each metal. The concentrations of the
intermediate standards, working standard solutions and values of correlation coefficients of
the calibration graphs for the three metals of interest is presented in Table 5 and their
calibration graphs were depicted in Appendix figure 1,2 and 3.

Table 5. Concentration Values of working Intermediate Standard solutions, Working Standard

solutions and Correlation Coefficients of Calibration Graphs.

NO Metal Concentration of Concentration of working Correlation

intermediate standard(mg/L) Coefficients of
Standard(mg/L) calibration curves (R2)
1 Lead 10 0.5, 1.0, 1.5, 2.0 and 2.5 0.9937

2 Chromium 10 0.2, 0.4, 0.6, 0.8 , 1.25 and1.5 0.9979

3 Cadmium 10 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 0.9994

4. 3. Method of Performance and Method of Validation

The criteria used for evaluating analytical methods are called figures of merit. Based on these
characteristics, one can predict whether a method meets the needs of intended purpose or not.
These figures of merit are accuracy, precision, sensitively, detection limits and the
quantitation limits (Mitra, 2003).

28
4. 3. 1. Precision

In this study, the precision of the results were evaluated by the relative standard deviation of
the results of triplicate digests. Triplicate measurements of each sample (n=9) were used for
the analysis of trace metals in powder milk samples, values of relative standard deviations (%
RSD) are less than 10% for all of the mean concentrations of metals. The precision of an
analytical procedure expresses the closeness or agreement between a replicate measurements
obtained from multiple sampling of the same homogenous sample under the prescribed
conditions (repeatability or reproducibility). The common terms used to measure variability is
the coefficient of variation (CV) or relative standard deviation (RSD). Which may also be
expressed as a percentage and it is a parameter of choice for expressing precision in analytical
sciences (Mitrs; 2003). The results of percent relative standard deviation for the studied
metals were presented in Table 6 below.

Table 6 MeanSD, n=3mg/l and %RSD of Pb, Cd and Cr in powder milk samples

Metal type Sample X Sample Y Sample Z

Lead Mean 0.16350.0004 0.0380.0014 0.16880.0162

%RSD 0.243 3.589 9.5799

Chromium Mean 0.2220.015 0.2060.018 0.0597 0.0055

%RSD 6.76 8.659 9.23

Cadmium Mean ND 0.0680.004 ND

%RSD ------ 5.48 -------

4. 3. 2 Validation of the optimized procedure

Since there was no certified reference material for brands of powder milk in our laboratory, in
order to ascertain the accuracy of optimized wet digestion procedure, recovery tests were
performed using spiked samples with known concentration. Accordingly, known amounts

29
from stock solution (1000mg/L) of each metal element were spiked on 0.5 g samples. The
spiked and non-spiked samples were digested in parallel using the optimized wet digestion
and concentration of each metal was determined in triplicate samples by taking three reading
for each. As shown in Table 7. The results of percentage recoveries for the studied metals
were all ranged between 95.91-99.294 %. The results were obtained through calculation.
Generally good recoveries were obtained for all metals. The results of the recovery tests for
samples were within the acceptable range for most metals 85-103% (Mico et al., 2006).

Table 7. Recovery Tests for the optimized procedure for the milk powder samples

Metal Milk Concentration of Concentration of Amount added %Recovery

sample spiked sample non spiked (ppm)
(ppm) sample (ppm)
Lead X 0.19050.011485 0.16350.000396 0.000250.000138 96.875.9334
Y 0.04190.000434 0.03790.00136 0.000040.000007 98.916.874
Z 0.20010.00434 0.16880.0162 0.0003240.000035 97.516.37
Chromium X 0.5290.019 0.2220.015 0.003130.00018 98.173.3923
Y 0.50.0125 0.2060.018 0.0030.00013 99.2941.47
Z 0.3540.02 0.0600.0055 0.0030.00012 95.912.87
Cadmium X ----------- ---------- ----------- -----------
Y 0.0730.0012 0.0680.004 0.0000420.000011 97.343.09
Z ---------- ----------- ----------- -----------

Note: Since Cd is not detected in all except sample Y the %Recovery is not calculated

4. 4. Determination of Heavy Metals in Different Brands of Powder Milks

4. 4. 1. Lead

Lead contamination of the environment is primarily due to anthropogenic activities, making it

the most ubiquitous toxic metal in the environment. Lead readily accumulates in the humus
rich surface layer of the soils due to its complexity with organic matter and it was reported to

30
be the least mobile heavy metal in soil under reducing and non-reducing conditions. (Abdus-
Salam and Adekola, 2005)

The results of elemental analysis of the three brands of powder milk samples collected from
Eastern Harage, Harar Town super markets, and the range of their average concentrations of
Pb, Cr and Cd in the samples are presented in Appendix Table 5. The concentration of Lead in
this study ranges from 0.038-0.169ppm.The highest concentration of lead was recorded for
milk sample Z (0.169ppm) and the lowest concentration for milk sample Y (0.038ppm). The
difference was statically significant at P<0.05. The mean elemental concentrations of Lead in
this study were higher than those reported in previous studies (Ostapezuk et al., 1987; Cortes,
E et al., 1994; Amal, 2000; Park, 2000; Sharkawy and Manal, 2002; Abdulkhaliq et al.,
2012). However, higher lead levels than the present study were also recorded in other studies
(Madeha et al., 1994; Garcia et al., 1999; Abdou and Emana, 2001; Hamouda, 2002; Jorhem
and Engman, 2000; Mohammod, et al., 2013). The average concentration of lead in milk
samples X, Y and Z was found to exceed the maximum permissible limit of lead for milk and
dairy product taken as 0.02mg/kg (CE Regulation, 2001). European diet provides maximum
potential for weekly intake of lead to be 4.31mg per kg bodyweight (GEMS/Food, 2000). The
presence of lead in brands of powder milk samples could be attributed to the exposure of
lactating cows from which powder milk was made to contaminated fodder, climatic factors
such as wind, use of Agro-chemicals and very importantly drinking water. Furthermore, the
lactating cows graze along rail lines, road sides etc. Moreover, the elevated level may be
related to contaminations during industry processing environmental pollution and storage
time. Lead which is a fuel additive could be emitted from the car exhaust to contaminate the
environment.

Lead is one of the limited classes of element that can be described as purely toxic. Most other
elements thought toxic and high concentration are actually required nutrient at lower levels.
There is no exposure level below which lead appears to be safe. High level of lead is
particularly of great concern especially due to the fact that milk and dairy products are
consumed mostly by infants and children who are uniquely susceptible to the effect of lead. It
induces reduced cognitive development and intellectual performance in children, increased

31
blood pressure, and cardiovascular diseases in adult as well as liver and kidney
dysfunctioning (Abdallah, 2011).

4. 4. 2. Chromium

Chromium is an essential nutrient for plant and animal metabolism, however, the increasing
accumulation of chromium in the environment from industrial outputs has caused great
concern. Chromium exists in III (+) and VI (+) oxidation states as all other oxidation states
are not stable in aqueous solutions. Both valences of chromium are potentially harmful
although the later is more toxic (Dakiky et al, 2002).

The elemental concentration of Chromium in examined powder milk samples ranges from
0.059722ppm - 0.222ppm. The highest concentration was recorded for milk sample X
(0.222ppm) and the lowest for milk sample Z (0.059722ppm). The difference was statistically
significant at p<0.05. The levels of chromium in this work in all samples were within the
maximum acceptable daily intake (0.429mg/day). Lower values of chromium in milk were
reported in previous studies (Bulinki, R, et al 1992 and Samaghul Bingghila et al 2008). In
addition a comparable result to this study has been reported by Binghila et al (2008). In which
the average concentration of chromium in powder milk was obtained as 0.18ppm. It is
gratifying to know that the brands of powder milk from the different super markets, in this
study, are free from Chromium contamination and, therefore, are safe to consume. Chromium
is an important mineral the body must have to function properly. The body stores chromium
in the blood and in the hair. Its responsible for stimulating the activities of insulin in the body
and also help controls blood cholesterol levels. Low level exposure of chromium can cause
skin irritation, and ulceration. Long term exposure can cause kidney and liver damage as well
circulatory and nerve tissue problems.

4. 4. 3. Cadmium

Of all the toxic metals resulted in large quantities in to the environment cadmium is generally
regarded as the most likely to accumulate in the human food chain (Johannesson, 2002).

32
Fortunately, the concentration of the toxic heavy metal cadmium in the studied brands of
powder milk sample was too low to be detected by the analytical technique used in this study.
Except in the brand powder milk Y which has a concentration of 0.068ppm. Comparable
reports revealed that the concentration found in this study to those reported by Mohammod et
al., (2013). A similar result of cadmium concentration in powder milk above the permissible
limit was reported by Abdallh, (2011). Lower levels were reported by Cortes et al., (1994);
Amal ( 2000); Park, (2000); Sharkawy and Manal, (2002); Farid et al.,(2004); Lawal et al.,
(2006); Semaghuil et al., (2008); Abdulkhaliq et al., (2012). The tolerable cadmium intake
established by WHO is 0.06mg/day for adult women and 0.070 mg/day for adult men. CE
Regulation, (2001) put the permissible limit for cadmium in food to 0.075mg/g while the
Egyptian standards put it at 0.05mg/g, Egyptian standard, (2001). However, higher level of
Cadmium above the permissible limit concentrations was reported in previous studies such as
those by Madeha et al., (1994); Garcia et al., (1999); Abdou and Emana,( 2001); Hamouda,(
2002); Jorhem and Engman, (2003).

The non detectable level of cadmium indicates the powder milk brands imported were not
contaminated with cadmium. It is gratifying to know that the brands of powder milk from the
different super markets, in this study, are free from cadmium contamination and, therefore,
are safe to consume except brand powder milk Y.

The presence of Pb Cr and Cd in the imported powder milk samples are certainly due to
industrial pollution as well as the addition of some fertilizers and herbicides on the farm and
farm sides. Although the study does not aim to point out the source of contamination, there
are probable sources. Heavy metals accumulate in the water supply through human activities,
such as industrial and consumer waste, commercial processes like mining, agriculture,
manufacturing and the discarding of wastes in landfills are common sources of heavy metal
contamination. Even rain water, with its acidic pH, can cause these compounds to leach into
the surface and underground water supplies from the surrounding soil and rock (Bock, 2005).

33
 5. SUMMARY, CONCLUSION AND RECOMMENDATION

5. 1. Summary and conclusion

In this study the levels of Pb Cr, and Cd in commercially available powder milk brands in
eastern Hararghe at Harar were determined by flame atomic absorption spectroscopy. The
powder milk brands were digested by using HNO3 and HClO4 as digestion reagents. The main
objective of the study was to determine the concentration of some heavy metals Pb (III), Cr
(VI) and Cd (II) in some selected commercially available brands of powdered milk samples
using atomic absorption spectrometry. All the experiments were done at Haramaya University
Animal Nutrition Laboratory, Soil Science Laboratory and Central Laboratory.

The Concentrations of Lead, Chromium and Cadmium in the studied milk powder brands
were in the ranges: 0.038-0.169ppm, 0.0597-0.222ppm and ND for milk samples X and Z
where as for milk sample Y the concentration was 0.068ppm, respectively. These values were
found to be within the safe limits as recommended for intake of these metals by WHO. Except
for lead metal in all brands of powder milk. The results show the intake of Chromium through
powder milk samples was quiet higher than that of Lead and Cadmium intake Yet all these
values are much lower than the tolerance level set by WHO (1972) except for lead metal. The
optimized wet digestion procedure developed for brands of powder milk was found efficient
for all of the metals. It was evaluated through the recovery experiment. And a good
percentage recovery was obtained for all of the metals identified. Since the studied powder
milk samples were imported types they may be contaminated in their way of transportation as
well as storage. The chemical composition of dairy products depends not only on the
concentration of raw milk components and on its treatment, but also on the mode of packing
and quality of the packing materials and on the conditions and time of storage. Therefore,
regulating agencies should increase the supervision of powder milk products imported to
avoid the risk of their adverse effect on the consumers health. Thus, this study may have
significance in delivering a preliminary data in the levels of heavy metals in the powder milk
brands studied. It might be useful in pointing directions for heavy metals levels of different
brands found.

34
5. 2. Recommendation

In light of the findings and the conclusion drawn above, the following recommendations are
forwarded.
The efficiency of other extractants like NHO3: H2O2, HNO3: H2S04, H2S04: HCLO4
should also be checked.
This study might be repeated with GFAAS and ICP-OES to compare the metal
contents of the selected powder milks.
The concentration of heavy metals in similar powder milk samples from Dire Dawa
and other markets in the eastern part of Ethiopia that are not covered by this research
should be determined.
Monitoring of the levels of heavy metals in commercially available powder milk
should be encouraged.

35
 6. REFERENCES

Abdallah, M. I. M., 2011. Evaluation of some heavy metals residue in whole milk powder
used at confectionary plant regarding the public health significance, Animal research institute,
agricultural research center, Giza Egypt.

Abdou, K. A. and K. Eman, 2001. Lead, Cadmium and Manganese in milk and some milk
products in Upper Egypt, Assiut Veterinary Medical Journal 45(89): 36-348.

Abdulkhaliq, A. Salie, K. M., Hussein, R. M. and M. Matani, 2012. Levels of metals (Cd, Pb,
Cu and Fe) in cows milk, dairy Product and hens eggs from the West Bank, Palestine,
International Food Research Journal. 19(3) :1089-1094.

Abdu-salam, N., FA. Adekola, 2005. The influence of pH and concentration on adsorption of
Pb and Zn on a natural goethite. African Journal of science adsorbent and Technology, 6:55-
66.

Abia, A., J. Igwe, C. Iroh, 2005. Removal of Hg+2,Pb+2andNi+2 ions from waste water using
modified granular activated carbon (GAC); Adsorption and Kinetic studies. Journal
Environmental Management, 654(3): 763-768.

Abreu, M.F., R.S. Berton, J.C. Andrade, 1996. Comparison of methods to evaluate heavy
metals in organic wastes. Commun. Soil Sci. Plant Anal., 27:1125-1135.

Abu-Tarboush, H. M., H. A. Al-Kahtani, M. S. El-Sarrge, 1993. Floral-type identification and

quality Evaluation of some honey types. Food Chemistry, 64: 13-17.

Agaje, B., 2007. Removal of Chromium from Waste Water Using Locally Available
Adsorbent. Graduate project Addis Ababa, Ethiopia .p: 1-52.

Ahmad, W. M. S., 2002. Studies on heavy metal pollution in poultry farms in relation to
production performance Ph.D. Thesis Fac. Of Vet. Med. Zag. University.

Amal, A. G., 2000. Evaluation of aluminum migration from cooking utensils into food, Egypt
Journal of food science.26: 73-80.

Arruda, M.A.Z., 2007. Trends in sample preparation: Nova Science Publishers: New York
aspen garden soils and vegetation. J. Environ.Qual., 21:82-86.

Azcue, J. and A. Mudroch, 1994. Comparison of different washing, ashing and digestion
methods for the analysis of trace elements in vegetation. Intern. J. Anal. Chem., 12:211- 221

Barnes, R. M., 1991. Short course on plasma spectrochemical analysis. Inductively coupled
plasma analysis .department of chemistry, university of Massachusetts: Amherst.

36
Birghila, Simona Dobrinas, Gabriela Stanciu, Alina, Soceana, 2008. Determination of major
and minor elements in milk through ICP-AES Semaghiul. Environmental Engineering and
management Journal.76:805-808.

Bock, R., 1979. A Handbook of determination methods in analytical chemistry, instrumental

text Book Company: London.

Bock, R. A., 2005. Handbook of decomposition methods in analytical chemistry, international

text Book Company: London

Bryee-Smith, O. And R. Stephenes, 1982. Lead and Brian function. Dev. Med. Child neutral,
24:90-91,

Bulinski, R., J. Bloniarz, and B. Libelt, 1992. Trace element content in milk and some milk
products.Bromat.chem.toskykol:327-331

Butcher, D. J. and J. Senddon, 1998. A Practical Guide to Graphite Furnace Atomic

Absorption Spectrometry, John Wiley and Sons, New York, PP: 34-149.

Carl, M., 1991. Heavy metals and other trace elements. Monograph on residues and
contaminants in milk and milk products. International Dairy Federation IDF, Belgium
Special Issue 9101, pp: 112-119.

CE Regulation, 2001. CE Regulation no 2001/466 March 8, 2001, GUEl77/1March 16 2001.

Cortes, E., H. A. Das, D. A. Tarafdar, and B .A. Vasconcellos, 1994. Toxic Heavy metals and
other trace element in food stuff from 12 different countries. Biological trace Element
Research, 20: 415-422.

Dakiky, M. K., M. H. Khamis, A. N. Manassra, and M. R. Mereb, 2002. Selective Adsorption

of Chromium (VI) in Industrial Waste Water using Low-Cost Abundantly available
Adsorbents. Journal of Advance in Environmental Ressearch.6:533-540.

Davies, B. E., 1990. Cadmium in Heavy Metals in Soil; Alloway, B. J. Ed.; Blackie:
Glasgow, pp: 100.

Dean, J.R. 1997. Atomic Absorption and Plasma Spectroscopy. John Wiley and Sons:
Chichester, p: 84.

Dietary supplements standard 173. (Accessed: Nov. 19, 2011). Metal contaminant acceptable
Levels NSF International [online] 2003.http://www.nsf.org/business /newsroom/pdf/DS
_Metal Contaminant_Acceptance_Levels.

Dobrzanski, Z., R. Kalaeu, H. Gorecka, K. Cholnaka, and Barkowalk, 2005. The content of
micro elements and Trace Elements in Raw Milk from cows in Silesian Region. Polish
Journal of Environmental Studies, Volume 14 (5): 685-689.

37
Dojlido, J. R. and G. A. Best, 1993. Chemistry of Water and Water Pollution. (EllisHorwood
Limited, Herts), pp: 65-69, 84-91, 108-114, 200- 202.

Downy, G., K. Hussey, J. D. Kelly, T. F. Walshe, and P. G., Martin, 2005. Preliminary
contribution to the characterization of artisanal honey produced on the island of Ireland by
palynolgical and physic-chemical data. Food Chemistry, 91: 347-354.

Egyptian Standard, 2001. Milk powder. Egyptian Organization for Standardization and
Quality Control. E.S., No. 1648.

El-Batanouni, M. M. and G. Abo-El-Ata, 1996. Metals in food. Conference on Food-borne

Contamination and Egyptians Health, Fac. Of Agri. Mansoura, November. P: 11-25. 26-27.

Enb, A., M. A. Abou Donia, Abd-Rabou, Abou Arab and M. H. El-Senaity, 2009. Chemical
Composition of Raw Milk and Heavy Metals Behavior during Processing of Milk Products,
Global Veterinaria, 3(3): 268-275.

FAO / WHO, 1972. Evaluation of certain Food Additives and contaminants Hg, Pb, and Cd.
Sixteenth Report of the Joint FAO / WHO Expert committee of Food Additives. WHO
Technical Reprt Sr. No. 505: 16 -29.

FAO / WHO, 1978. Evaluation of certain Food Additives and Contaminants. Report of the
joint FAO / WHO Expert Committee of Food Additives. WHO Technical Rep. Ser. No. 631:
11 -17.

Farid, S. M., M. A. Enani, and S. A. Wajid, 2004. Determination of trace elements in cow
milk in Saudi Aribia, JKAU: Egn. Sci. Volume 15(0-2). Pp: 131-140.

Food and Nutrition Board, Institute of Medicine, 1997. Dietary Reference Intakes for calcium,
phosphorus, magnesium, vitamin D and fluoride. National Academy Press, Washington. DC.

Food and Nutrition Board, Institute of Medicine, 1989. Recommended Dietary Allowances,
10th Edition, Food and Nutrition Board, National Academy Press, Washington, D.C.

Food and Nutrition Board, Institute of Medicine, 2001. Dietary Reference Intakes for Vitamin
A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum,
Nickel, Silicon, Vanadium, and Zinc. National Academy Press, Washington, D.C.

Garcia, E. M., M. L. Lorenzo, C. Cabera, M. C. Lopez, and J. Sanchez, 1999. Trace elements
determination in different milk slurries. Journal of Dairy research, 66(4): 569-578.

Garca-Delgado, C.; V. Calab, & E. Eymara, 2012. Inuence of chemical and mineralogical
properties of organic amendments on the selection of an adequate analytical procedure for
trace elements determination. Talanta, 88:375-384,

38
GEMS/Food., 2000. Regional Diets prepared by Global Environmental monitoring system
/food contamination monitoring and Assessment programme (GEMS/FOOD).

Hamouda, A. A. T., 2002. Heavy metal residues and preservatives in some imported dairy
products, Zagazig, Egypt, Zagazing University PhD thesis.

Harvey D. 2000. Modern Analytical Chemistry, McGraw Hill Companies, Inc. PP: 418.

Hawks, J. S., 1997. Heavy Metals. J. chem. Educ, 74: 1374.

Hseu, Z.Y., 2004. Evaluating heavy metal contents in nine composts using four digestion
methods. Bioresour. Technol., 95:53-59,

2nd International Conference on Environment and Bio Science, 2012. IPCBEE vol.44
IACSIT Press, Singapore DOI: 10.7763/IPCBEE. V (44): 626.

Jamison, D. T., J. G. Breman, A. R. Measham, G. Alleyne, M. Claeson, D. B. Evans, P. Jha,

A. Mills, P. Musgrove, 2006. Disease Control Priorities in Developing Countries. 2nd edition.
The International Bank for Reconstruction and Development/ the World Bank Group.

Jarvis, K. E., A. L. Gray, R. S. Houk, 1992. Handbook of inductively coupled plasma mass
spectroscopy, Blackie and son: Glasgow.

Johannesson, M., 2002. A review of risk associated to Cadmium, lead, mercury and zinc.
International Journal of Physics Science.2, p: 62-64.

Jorhem, L. and J. Engman, 2000. Determination of Lead, Cadmium Zinc, Copper and iron in
food by atomic absorption spectrometry after microwave digestion, NMKL collaborative
study, Journal of AOAC International .83(5): 1198-1203.

Kehoe, R. A., J. Chalak, E. J. Largent, 1994. The concentration of certain trace metals in
drinking waters. J. Am. Water works Assoc. 36: 637-643.

Khalid, N., S. Rehman, R. Ahmed, and H. Qureshi, 1987. Determination of lead and cadmium
in milk by electronics atomic Absorption Spectrophotomery. Intern. J .Environ. Anal. Chem.
28: 133-141.

Kikuchi, Y.T.Nomiyama, N.Kumagai, T.Uemura, K.Omae, 2002.Cadmium concentration in

current Japanese food and beverages, Journal of Occupational Health, 44,240-247.

Kingston, H. M., L. B. Jassie, 1988. Introduction to micro oven sample preparation, theory
and Practice, ACS professional reference book: Washington D.C.

Korenekovg, B., M. Skalicka, and P. Nai, 2002. Concentration of some heavy metals in cattle
reared in the vicinity of metallurgic industry Vet. Archive. 72: 254-267.

39
Krejpcio, Z., 2001. Essentiality of Chromium for Human Nutrition and Health. Journal of
Environmental studies, 10(6):399-404.

Kumar, S., U. J. Mehta1, & S. Hazra1, 2008. Accumulation of cadmium in growing peanut
(Arachis hypogaea L.) seedlingsits effect on lipid per oxidation and on the antioxidative
Enzymes catalase and guaiacol peroxidase. J. Plant Nutr. Soil Sci.171: 440447.

Kumar, Y., P. P. King, and V. S. R. K. Prasad, 2006. Removal of copper from aqueous
solution using Ulva fscata sp.A marine green algae, Journal of Hazardous Materials.137,
367-368.

Lawal, A. O., S. S .Muhammed, And D.Damisa, 2006. Assessment of levels of Cu, Cd and Pb
in secretion of mammary gland of cow grazed on open fields. Science World Journal Volume
I. No. I.

Lenntech, 2004. Water Treatment, Published by Lenntech, Rotterdam sewage, Netherlands.

Water Purification, 9: 43-47.

LI-Quang in, Xino Ping Wang, Wei Li., Xing Tong and Wei-jun Tong, 2009. The
minerals and heavy metals in cows milk from China and Japan: Journal of Health Science
555(2): 300- 330.

Loannidou, M. D., G. A. Zachariadis, A. N. Anthemidis, J. A. Stratis, 2005. Direct

determination of toxic trace metals in honey sugars using inductively coupled plasma atomic
emission spectrometry.Talanta, 65: 92-97.

Lopez Garcia, I., P. Vinas, C. Blanco, M. Hemandez Cordba, 1999. Determination of mineral
elements in honey from different floral origins by flow injection analysis coupled to atomic
spectroscopy, Spanish Journal of Food Science and Technology, 34: 441-449.

Madha, A. A., M. A. AbdEl-Kader, and I. Y .Tork, 1994. Lead Cadmium and Mercury in
milk products, Assiut Veterinary Medical Journal, 30(60): 139-146.

Maria B, 2002. Sample digestion methods for determination of traces of precious metals by
Spectrometric techniques, Anal. Sci, 18,737-750.

Matusiewicz, H. and M. Kopras, 1997. Methods for improving the sensitivity in Atom
Trapping FAAS. Journal of Analytical Atomic spectrometry, Vol (12) pp: 1287-1291.

Mc Bride, M.-B. 1995 J. Environ. Qual. (24): 154-158.

McGrath, S. P., S. Smith, 1990. Chromium and Nickel in Heavy Metals in Soils; Alloway, B.
J. Ed; Blackie: Glasgow, pp 125-150.

Melo, L.C.A. & C.A Silva, 2008.Caracterizaofsico-qumica e comparao de mtodos de

digesto de resduosorgnicos. Qum. Nova, 31:556-561.

40
Mico, C., M. Peris, J.Sanchez and L., Recatala, 2006. Heavy metal content of agricultural
soils in a Mediterranean semiarid area: the Segura River Valley (Alicante, Spain). Spanish
Journal of Agricultural Research, 4(4):363-372.

Mitra, S., 2003. Sample preparation Techniques in Analytical Chemistry, John Wiley and
Sons, Inc. Hoboken, 227-244.

Mohammed Abdalbasid, A., Gasmalla, Khadir E.Khadir, Abu-Bakr, Musa, Waleed Aboshora
and Wei Zhao, 2013. Evaluation of Some Physicochemical Parameters of three Commercial
milk products, Pakistan Journal of Food Science, Volume 23, Issue 2, p: 62-65.

Mosara, M.R and R.N.Roy, 2008. Guide to laboratory establishment for plant nutrient
analysis.FAO fertilizer and plant nutrition bulletin, Rome, Italy. 19:1-103.

Murthy, G. M. and U. S. Reha, 1971. Cadmium, Copper, Iron, Lead, Manganese and Zinc in
evaporated milk infant products. J. Dairy Sci, 54:1001-1005.

Mustafa, 2006. Comparison of Digestion Procedures on Commercial powdered Soup Sample

for the Determination of Trace Metal Contents by Atomic Absorption Spectrometry, Journal
of Food and Drug Analysis, 14:62-67.

Naithani, V., N. Pathak, M. Chaudhary, 2010. Evaluation of Heavy Metals in Two Major
Ingredients of Ampucare. International Journal of Pharmaceutical Sciences and Drug
Research. 2: 137-141.

Narre, H. D., M. Carola, and S. Markus, 1985. Metal Analysis in difficult Materials with
Platform furnace Zeeman-Atomic Absorption Unters Forsch, 182.

Nemati, K.; N.K.A. Bakar, M.R.B. Abas, E. Sobhanzadeh, & K.H. Low, 2010. Comparative
study of open system digestion and microwave assisted digestion methods for metal
determination in shrimp sludge compost. J. Hazard. Mater, 182:453-459.

NRC, 1980. Recommended Daily Dietary Allowances, Food National Board, National
Research Council (NRC), Washington, DC, USA.

Nriagu, J. O., 1988. A Silent epidemic of environments metal poisoning. Environmental

Pollution, 50: 139-161.

Ogabiela, E. E., G. G. Yebpella, A. F. Ade-Ajayi, U. J. Mmereole, C. Ezeayanaso, E. M.

Okonkwo, D. O. Ahola, U. U. Udiba, A. Mahmood, and I. Gandu, 2010. Determination of the
level of some elements in edible oils sold in Zaria, Northern Nigeria. Global Journal of Pure
and Applied Sciences vol.6 (3): 325-331.

41
Okada, I. A., A. M. Sakuma, F. D. Maio, S. Dovidauskas, and O. Zenebon, 1997. Evaluation
of lead and cadmium levels in milk due to environmental contamination in Parabia valley
region of south eastern Brazil. Revista-de-Saude-Publica, 31 (2): 140-143.

Ostapezuk, P., P. Valenta, H. Rutzel, and H. W. Nurnberg, 1987. Determination of Heavy

metal in environmental sample, sc. of total environs, 60: 1-16.

Park, Y.W., 2000. Comparison of mineral and cholesterol composition of different

commercial goat milk products manufactured in U.S.A. small Ruminant Research 37(1-2):
115-124.

Pendias, A. K. H., 1984. Trace Elements in Soils and Plants, 1st ed. Boca Raton, Fl: CRC
press, p 365.

Qaiser*, S., A. R. Saleemi, & M. Umar, 2009. Biosorption of lead (II) and chromium (VI) on
Groundnut hull: Equilibrium, kinetics and thermodynamics study. Electronic Journal of
Biotechnology.12 (4): 0717-3458.

Rabinson, C. H., 1978. Fundamentals of Normal Nutrition, Macmillan, UK.

Salah Fathy Ahmed Abd-El Aal, 2012. Assessment of Toxic Heavy Metals in some Dairy
products and the effect of storage on its distribution, Food Control department-Faculty of
Veterinary medicine Zagazig university-Egypt. Journal of American Science.8: 8.

Samara, S. and H. Richard, 2009. Heavy metal toxicity, medicine Specialist > Emergency
Medicine Toxicology. New York.

Sanger, M., J. Hoesch, 2005. Macro and Micro-Element Levels in Cereals Grown in lower
Austria. Central European Agriculture, 6: 461-467.

Sastre, J., A.Sahuquillo, M. Vidal, and G. Rauret, 2002. Determination of Cd, Cu, Pb and Zn
in environmental samples: Microwave assisted total digestion versus aqua regia and nitric
acid extraction. Anal. Chim. Acta, 462:59- 72

Semaghuil Binghila, Simona, D., G .Stanciu, A. Soceanu, 2008. Determination of Major and
Minor Elements in milk through ICP-AES. Environmental Engineering and Management
Journal. No 6: 805-808.

Shaheen, D. G., 1975. Environ. Pot. Agency Report, EPA 600/2-75-004.

Sharkawy, A. A. and S. H. Manal, 2002. Evaluation of some metallic pollutants in milk and
milk powder in Beni-Suef Governorate, Assiut Veterinary Medical Journal 47(84): 211-232.

Sharma, D. C. and C. F. Foster, 1995. Column studies in to the adsorption of chromium (VI)
using sphagnum moss peat, Bioresource Technology.52: 261

42
Soares dos Santos, J., N. Soares dos Santos, S. Pires dos Santos, J. Novaes dos Santos, J. de.
Jesus Lacerda, 2008. Honey classification from Semi-Arid, Atlantic and transitional forest
Zones in Bahia, Braszil.Journal of Brazilial Chemical Society, 51:169-175.

Sommers, E., 1974. The toxic potential of trace elements in foods. A review, Environ. Res.
Technology, 39: 215 - 227, 11-116.

Symon, C., M. Hutton, 1986. The Quantities of Cadmium, Lead, Mercury and Arsenic
entering the U.K. Environment from Human activities. Sci.Total Environ, 57: 129-150

Tadesu, H., 2011. Determination of some physico-chemical characteristics of waste water

from Batu Tannery Industry at Different Batch, Addis Ababa, Ethiopia. Graduate thesis,
Awasa University, p: 169.

Tripathi, R. M., R. Raghunath and T. M. Krishnamoorthy, 1997. Dietary intake of heavy

metals in Bombay city, India, Sci. of Total Environ, 208: 149 - 159.

Tsoumbaris, P. and H. Tsoukali-Papadopoulou, 1994. Heavy Metals in common foodstuff:

Qualitative analysis. Bulletin of Environmental Contamination and Toxicology.53: 267-269.

Tunali, S., A. Cabuk, A., Akar, 2006. Remval of lead and copper ions from aqueous solutions
by bacterial strain isolated from soil. Chemical Engineering Journal.115: 203-211.

Volesky, B. and H. May-Phillips, 1995. Biosorption of heavy metals by Saccharomyces

Cerevisiae. Journal of applied Microbiology Biotechnology.42:797-806.

Wang, J. L., 2006. Biosorption of copper (II) by chemically modified biomass of

saccharomces cerevisiae. Process Biotechemistry.37: 847-850.

Welz B. and M. Sperling, 1999. Atomic Absorption Spectrometry, Wiley-VcH:Weinheim

tomato wastes, Egyptian J. Agri Research, 78(5): 2085-2096.

WHO, 1973. Evaluation of certain food additives and contaminant: Hg, Pb, Cd. 16 th Report of
Expert committee, WHO Technical Report Series. No 51.

WHO, 1983. Recommended Baseline in occupational Exposure to Heavy metals. Report of

WHO study Group. Technical Report. No 647.

World Health Organization/WHO, 1996. Trace elements in human nutrition and health, (A
report of a re-evaluation of the role of trace elements in human health and nutrition).Geneva.

World Health Organization, 1972. Technical report Series. No 505.

Yahaya M. I., G. C. Ezor, Y. F. Musa and S. Y. Muhamad, 2010. Analysis of heavy metals
concentration in road side soils in Yauri, Nigeria. African journ. Of pure and applied chem.
vol 4: 3.

43
Zheljazkov, V.D. and P.R. Warman, 2002. Comparison of three digestion methods for the
recovery of 17 plant essential nutrients and trace elements from six composts. Compost Sci.
Util., 10:197-203,

44
7. APPENDICES

45
7. 1 Appendix of Tables

Table 1. Optimization for Reagent Volume

Trial Reagent Reagent Temperature Digestion Observation

No Used Volume(ml) (0C) Time
(h)
1 HNO3:HClO4 3:1 250 2:00 Deep Yellow
2 HNO3:HClO4 3:2 250 2:00 Deep Yellow
3 HNO3:HClO4 3:3 250 2:00 Yellow
4 HNO3:HClO4 4:1 250 2:00 Light Yellow
5 HNO3:HClO4 4:2 250 2:00 Light Yellow
6 HNO3:HClO4 4:3 250 2:00 Almost Clear
7 HNO3:HClO4 5:3* 250 2:00 Clear and Colorless
8 HNO3:HClO4 5:4 250 2:00 Clear and Colorless
9 HNO3:HClO4 5:5 250 2:00 Clear and Colorless

* The optimized condition for the given parameter

Table 2. Optimization for Temperature

Trial Reagent Reagent Temperature Digestion Observation

No Used Volume(ml) (0C) Time
(h)
1 HNO3:HClO4 5:3 80 2:00 Deep Yellow
2 HNO3:HClO4 5:3 100 2:00 Yellow
3 HNO3:HClO4 5:3 110 2:00 Light Yellow
4 HNO3:HClO4 5:3 140 2:00 Clear Yellow
5 HNO3:HClO4 5:3 180 2:00 Almost Clear
6 HNO3:HClO4 5:3 210* 2:00 Clear and Colorless
7 HNO3:HClO4 5:3 240 2:00 Clear and Colorless
8 HNO3:HClO4 5:3 270 2:00 Clear and Colorless
9 HNO3:HClO4 5:3 300 2:00 Clear and Colorless

* The optimized condition for the given parameter

46
Table 3. Optimization for Digestion Time

Trial Reagent Reagent Temperature Digestion Observation

0
No Used Volume(ml) ( C) Time
(h)
1 HNO3:HClO4 5:3 210 1:00 Deep Yellow
2 HNO3:HClO4 5:3 210 1:30 Light Yellow
3 HNO3:HClO4 5:3 210 1:45 Yellow
4 HNO3:HClO4 5:3 210 2:00 Clear Yellow
5 HNO3:HClO4 5:3 210 2:15 Almost Clear
6 HNO3:HClO4 5:3 210 2:30* Clear and Colorless
7 HNO3:HClO4 5:3 210 2:45 Clear and Colorless
8 HNO3:HClO4 5:3 210 2:50 Clear and Colorless
9 HNO3:HClO4 5:3 210 3:00 Clear and Colorless

* The optimized condition for the given parameter

Table 4. Provisional Tolerable Daily Intake of Toxic Heavy Metals,

Heavy Metal Recommended/Permissible Value

Concentration
Lead 0.050-0.200 mg/day
Cadmium 0.057-0.072 mg/day
Chromium 0.429 mg/day

Source:
Tripathi et al (1997), FAO/WHO (1972), FAO/WHO (1978), NRC (1980) and
Rabinson (1978)

47
Table 5. Range of Metals Concentrations in Powder Milk Samples

Type of Metal Concentration Range in mg/L

Lead 0.038-0.169

Cadmium 0.060-0.222

Chromium ND-0.068(only for sample Y)

Table 6. Instrument detection limit, Method detection limit and Method Limit of Quantitation
limits.

Metal Instrument detection Limit Method detection limit for Method Limit of
(mg/L) powder milk Sample mg/l Quantitation(LOQ)
mg/l
Lead 0.01 0.013172 0.043906

Chromium 0.05 0.064 0.2133

Cadmium 0.005 0.0065 0.022

48
7. 2 Appendix of Figures

Calibration curve for Lead

0.3
y = 0.133x - 0.003
0.25 R = 0.9937
0.2
Absorbance

0.15

0.1

0.05

0
0 0.5 1 1.5 2 2.5
Concentration (mg/L)

Fig.1 Plot of Absorbance versus Concentration for Lead

Calibration curve for Chromium

0.009
0.008 y = 0.008x - 0.001
0.007 R = 0.9979
0.006
Absorbance

0.005
0.004
0.003
0.002
0.001
0
0 0.2 0.4 0.6 0.8 1 1.2
Concentration(mg/L)

49
Fig.2 Plot of Absorbance versus Concentration for Chromium

Calibration curve for Cadmium

0.25

0.2 y = 0.171x - 0.005

R = 0.9994
Absorbance

0.15

0.1

0.05

0
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Concentration (mg/L)

Fig. 3 Plot of Absorbance versus Concentration for Cadmium

50