DSpace Institution

DSpace Repository http://dspace.org

Chemistry Thesis and Dissertations

2019-12-04

Determination of Some Selected

Essential and Non-essential Metals in
Fresh Cow Milk from Wombera Woreda,
Benishangul Gumuz, Ethiopia

Million, Demeki

http://hdl.handle.net/123456789/10025
Downloaded from DSpace Repository, DSpace Institution's institutional repository
 BAHIR DAR UNIVERSITY
COLLEGE OF SCIENCE POST GRADUATE PROGRAM
DEPARTMENT OF CHEMISTRY

MSc Thesis

Determination of Some Selected Essential and Non-essential

Metals in Fresh Cow Milk from Wombera Woreda,
Benishangul Gumuz, Ethiopia

By
Million Demeki

September, 2019

BahirDar
 Determination of Some Selected Essential and Non-essential
Metals in Fresh Cow Milk from Wombera Woreda,
Benishangul Gumuz, Ethiopia

A thesis submitted in partial fulfillment of the requirement for the degree

of masters of Science in chemistry (Analytical chemistry)

By: Million Demeki

Advisor: Meareg Amare(PhD)

September, 2019

Bahir Dar
 Letter of Approval

The thesis entitled “Determination of Some Selected Essential and Non-

essential Metals in Fresh Cow Milk from Wombera Woreda, Benishangul
Gumuz, Ethiopia” Presented by Million Demeki has been accepted as the partial
fulfillment for the degree of Master of Science in Chemistry

Name of student Signature Date

Approved by: Board of Examiners

Name Signature

Advisor ------------------------ --------------

Internal examiner ------------------------ ----------------

External examiner ------------------------ ---------------

Chairperson ------------------------ -------------

 Declaration

I declare that the work described in this thesis is my original work done under the
supervision of my advisor Dr Meareg Amare at the Department of chemistry, Bahir Dar
University for the degree of M.Sc. in Chemistry. I also declare that the work embodied in
this thesis is the results of my own research and where work of any other researcher has
been used, that has been duly acknowledged.

Name: Million Demeki

Signature .......................................

Date of submission ………………………..

i
 Acknowledgements

I am so grateful to my advisor, Dr Meareg Amare for his support from the very
beginning in giving me the opportunity to work on this thesis, facilitating conditions in
chemistry department laboratory in the digestion and analysis of my samples, in making
the lab staff members to cooperate me when things were difficult and for his comments
through every step of my work.

My acknowledgement is also extended to my best friend Gemechu Senbeta for his

material, technical and moral support to accomplish my work.

Finally, I must express my very profound gratitude to my parents for providing me with
unfailing support and continuous encouragement throughout my years study and through
the process of researching and writing the thesis.

ii
 Acronyms and Abbreviations

ANOVA…………………………….. Analysis of Variance

FAAS………………………………... Flame atomic absorption spectrometer

FAO………………………………….Food and Agriculture Organization of United

Nations

FBDs………………………………… food-borne diseases

HDL…………………………………high – density lipoprotein

ICP-OES……………….. …………..Inductively Coupled Plasma Optical Emission

Spectroscopy

IDL………………………………….Instrumental detection limit

MDL…………………………………Method of Detection Limit

NAFDAC…………………………...National Agency for Food and Drugs

Administration Control

R……………………………………The correlation coefficient

RDA………………………………… Recommended Daily Allowance

RF……………………………………radio frequency

SD…………………………………… Standard deviation

WHO………………………………..World Health Organization

iii
Table of content page

Declaration ........................................................................................................................... i
Acknowledgements ............................................................................................................. ii
Acronyms and Abbreviations ............................................................................................ iii
Table of content ................................................................................................................. iv
List of tables....................................................................................................................... vi
List of figures .................................................................................................................... vii
Abstract ............................................................................................................................ viii
1. Introduction ................................................................................................................... 1
1.1 Back Ground of the Study....................................................................................... 1
1.2 Statement of the Problem ........................................................................................ 2
1.3 Objective of the Study ............................................................................................ 3
1.3.1 General objective ......................................................................................... 3
1.3.2 Specific objectives ....................................................................................... 3
1.4 Significance of the Study ........................................................................................ 3
2. Literature Review.......................................................................................................... 4
2.1 Definition of Milk ................................................................................................... 4
2.2 Metal Contamination in Milk.................................................................................. 4
2.3 Composition and Mineral Content in Milk ............................................................. 6
2.4 Effects of High Concentration of Metals in Cow Milk........................................... 8
2.4.1 Manganese (Mn) .......................................................................................... 8
2.4.2 Zinc (Zn) ...................................................................................................... 8
2.4.3 Copper (Cu) ................................................................................................. 9
2.4.4 Iron (Fe) ....................................................................................................... 9
2.4.5 Cadmium (Cd) ............................................................................................. 9
2.4.6 Lead (Pb) ..................................................................................................... 9
2.5 Analytical Techniques used for Metal Analysis ................................................... 10
2.5.1 Flame Atomic Absorption Spectrometer (FAAS) ..................................... 10
2.5.2 Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) 10
3. Materials and Methods................................................................................................ 12
3.1 Description of the Study Area............................................................................... 12
3.2 Chemicals and Reagents ....................................................................................... 12

iv
 3.3 Apparatus / Instrument .......................................................................................... 13
3.4 Sample Collection ................................................................................................. 13
3.5 Milk Sample Digestion and Preparation of the Sample for ICP-OES Analysis ... 14
3.6 Calibration Curve and Measurement of Metal Concentrations ............................ 14
3.7 Method Validation ................................................................................................ 15
3.8 Method and Instrument Detection Limits ............................................................. 16
3.9 Statistical Data Analysis of Methods .................................................................... 17
4. Results and Discussion ............................................................................................... 18
4.1 Calibration of the Instrument ................................................................................ 18
4.2 Percent Recovery Test .......................................................................................... 20
4.3 Mean Concentration of Metals in Cow Milk ........................................................ 20
4.4 Distribution of the Metals in the Study Areas ...................................................... 24
4.5 Comparison of Results of the Present Study with Literature Values.................... 26
4.6 Recommended Daily Allowance of Metals .......................................................... 28
4.7 Analysis of variance (ANOVA)............................................................................ 29
5. Conclusions ................................................................................................................. 30
6. References ................................................................................................................... 31
7. Appendix A: Output for ANOVA............................................................................... 37

v
 List of tables
Table 1:- The elemental concentration in fresh cow milk from different countries. .......... 7
Table 2:- Recommended daily allowance (RDA) for metal concentration in fresh cow
milk..................................................................................................................... 8
Table 3:- Instrumental operating conditions for determination of metals in milk samples
use ICP-OES..................................................................................................... 13
Table 4:- Series of working standards and correlation coefficients of the calibration
curves for the determination of metals in milk using ICP-OES. ...................... 15
Table 5:- Method detection limits for the determination of metals in cow milk samples. 16
Table 6:- Recovery results for the validation of the optimized procedure. ...................... 20
Table 7:- Average concentration (mg/L) of metals in digested solutions of milk samples
collected from different farms of Wombera woreda. ....................................... 21
Table 8:- Average concentration of metals in fresh cow milk samples collected from
different farms of Wombera woreda. ............................................................... 22
Table 9:- Comparison of daily intakes of metals from 53 mL milk by Ethiopian
population with recommended / permissible values. ....................................... 28

vi
 List of figure
Figure 1: Map of the Study Area ..................................................................................... 12
Figure 2: Plot of Intensity as Function of the Concentration for each Studied Metals ... 19
Figure 3: Distribution of Metals Mg, Fe and Zn in Study Areas ..................................... 25
Figure 4: Distribution of Metals Cu, Mn and Pb in Study Areas ................................... 26

vii
 Abstract

Fresh cow milk samples were collected from three different farms of Deberezeit,
Manasibu and Ateshimo site of Wombera woreda, Ethiopia and were placed below 4 ºC
in a refrigerator. The cow milk samples were digested by hot plate using 65 % HNO3 and
30 % H2O2. The concentrations of Mg, Fe, Mn, Cd, Cu, Zn and pb were determined by
Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES). The element, Cd
was not detected at all in three sites of the milk samples under the study. The element Pb
was not detected in Manasibu and Ateshimo sites. But it was detected in Deberezeit site.
The mean concentrations of Pb in Deberezeit was 0.352 ± 0.144 mg/L and the mean
concentrations of Cu, Fe, Mn, Zn, and Mg were 0.742 ± 0.123, 18.700 ± 2.232, 0.575 ±
0.063, 6.645 ± 1.111 and 147.792 ± 9.819 mg/L, respectively in the milk samples of three
sites under study. The validity of the method of the analysis was confirmed by recovery
test and the result was in the range (80.3 to 103.7 %). There was significant difference in
the mean concentrations of Cu, Mn, Zn, Mg, Fe and Pb between the milk samples of three
farms under study. The concentrations of essential metals observed were somewhat below
or above when compared with some of the reported values in literature. Consumption of
beneficial metals through milk were estimated and compared with Recommended Dietary
Allowance set by FAO, WHO and National Agency for Food and Drugs Administration
Control (NAFDAC) standards.

Keywords: Essential metals, Cow milk, Digestion, Deberezeit, Manasibu, Ateshimo and
Inductively Coupled Plasma Optical Emission Spectroscopy

viii
 1. Introduction

1.1 Back Ground of the Study

Milk is the first food that human encounters which serves as a source of essential
nutrients required for the biological functions and growth during early stages of life [1].
Among foods, milk represents an important intake in a typical diet due to its high nutrient
and mineral content. Milk is an excellent source of calcium, vitamin D, riboflavin and
phosphorus and a good source of protein, potassium, vitamin A, vitamin B-12 and niacin
[2, 3]. Milk is a complex, bioactive substance that promotes growth and development of
mammalian infants. It is considered as a nearly complete food since it is a good source of
proteins, fats, sugars vitamins and minerals. Therefore, milk and dairy products are
important components of human diets that are widely consumed by children and adults
around the World [4].

Milk and milk products are an essential source of dietary minerals for consumers. Milk
products are very important human nutrients and their consumption has increased in
recent years. According to several reports, milk is an excellent source of calcium,
magnesium, zinc and provide very small amount of Fe and Cu. In addition milk products
are the main constituents of human daily diet, especially for vulnerable groups such as
infants, school-age children as well as old aged people [5 - 7]. Milk and milk products are
the most diversified of the natural food stuffs in terms of composition contain more than
twenty different trace elements most of them are essential and very important such as
copper, zinc, manganese and iron [8]. Actually, all elements, including essential ones,
may be toxic if their intakes exceed safe levels. Elements such as arsenic (As), cadmium
(Cd), lead (Pb) and mercury (Hg) in foods pose significant risks for human health [9].
Therefore, such non-essential elements need to be determined in milk and dairy products.
Aluminum, arsenic, cadmium and lead may accumulate especially in the lungs, liver,
kidney, thyroid and brain [10].

The entry of pollutants like heavy metals in food such as milk and dairy products is one
of the issues that are very important. By far, various strategies and measures have been
taken to improve milk quality and to lower the amount of pollutants in it [11]. Report
according to [12] indicated that good quality measurements of cow milk are very
1
important in order to control and often play a vital role in maintaining products and
processes quality, in both manufacturing, trading and in research. Consumption of milk
particularly fresh cow milk is associated with beneficial health effect beyond its pure
nutritional value.

Several reports indicated that low-fat milk consumption could reduce risk of arterial
hypertension, coronary heart disease, colorectal cancer and obesity [13, 14]. Report
according to [15] showed that apart from the essential benefits derived from consuming
cow milk, contamination of milk also arises from industrial pollutants in the environment,
modern Agricultural practices, animals feeds and use of sewage sludge and polluted
water in Agriculture which is increasing on daily basis and required urgent attention.
Environmental conditions are responsible for most of the toxic metal content of milk and
milk products. The contamination of soil and water pollution by heavy metals exposes
man and grazing animals to health risks, as such it becomes paramount to determine and
monitor the concentration of toxic metals such as Pb, Cu, Zn., Mn, Fe etc. in milk,
because this can significantly influenced human health [16].

1.2 Statement of the Problem

This study was conducted at Deberezyeit, Menasibu, and Ateshimo site, and the problem
is identified based on milk quality because of dependence of farmers on livestock
production mainly from cattle. Milk represents an important intake in a typical diet in
Wombera due to its high nutrient and mineral content. Milk is an ideal source of essential
elements (Ca, Mg, Cu, Fe, Zn and Se). However in addition amounts of contaminant
metals might enter milk reach level that are harmful to humans [17].

The contamination of milk is considered as one of the main dangerous aspects. The
elements such as Cd, Cu, Fe, Pb, and Zn which are commonly associated with pollution
and toxicity problems [18]. One of the main problems with metals is their ability to bio-
accumulate. Metal residues in milk are of particular concern because milk is largely
consumed by infants and children [19]. However, at high concentrations, these metals can
cause toxicity to living organisms [20]. Residue of Pb and Cd contained in the food can
accumulate in the livestock and human organs, in the long run can disrupt livestock and
humans health [21]. Excessive intake of Cd and Pb can lead to cardiovascular disease,
2
damage to the nervous system and the bones [22, 23]. However, no information is
available about the concentration of Mg, Zn, Fe, Cu, Cd, and Pb in cow milk in Wombera
woreda. To mean that no more analysis was made on determination of essential and non-
essential elements in cow milk in the Wombera woreda related to their beneficial and
negative impacts to humans healthy.

1.3 Objective of the Study

1.3.1 General objective

 The main objective of this study was to determine the level of selected essential and
non-essential metals in fresh cow milk from Wombera woreda, Benishangul Gumz,
Ethiopia.

1.3.2 Specific objectives

 To determine the concentrations of selected essential metals (Mg, Cu, Fe, Zn, and
Mn) in cow milk samples collected from selected kebeles of Wombera woreda.
 To determine the concentrations of selected non-essential metals (Pb and Cd) in Cow
milk sample.
 To compare the results with international/national standards and reported works.

1.4 Significance of the Study

This study may have a significant contribution in recognizing and understanding the
essential and non-essential metals in cow milk samples collected from different sites and
compares the identified result with recommended values. It helps to give information
about the selected metal concentration level in the studied area. It also helps to know the
people, the quality of the milk at the area and used to recommend the people living
around the study area depend on the concentration of metals in cow milk. Finally, the
study is useful to the researchers to serve as starting information for further study.

3
 2. Literature Review

2.1 Definition of Milk

The free dictionary simply defines milk as a whitish liquid containing milk proteins, fats,
lactose, and various vitamins and minerals, produced by the mammary glands of all adult
female mammals after childbirth and serves as food for their young.

Milk is a yellowish-white non-transparent liquid secreted by the mammary glands of all

mammals. It is the primary source of nutrition and Sole food for offspring of mammals
before they are able to eat and digest other types of food. It contains in a balanced form
of all the necessary and digestible elements for building and maintaining the human and
animal body [24]. Milk is an excellent source of calcium, vitamin D, riboflavin and
phosphorus and a good source of protein, potassium, vitamin A, vitamin B-12 and niacin
[2, 25].

Milk is one of the most important food for human nutrition, as it contains all the
macronutrients namely protein, carbohydrates, fat, sugars, vitamins (A, D and B groups)
and trace elements particularly calcium, magnesium, zinc and selenium [26]. Milk is an
important source of nutrients to human and animals and it is meant to be the first and the
only food for the offspring of mammals as is almost complete food [27, 28]. Milk is
addressed as an ideal food. It is also considered as an excellent source of Calcium (Ca),
fewer amount of Zinc (Zn), and a very small content of Copper (Cu) and Iron (Fe) [29].

2.2 Metal Contamination in Milk

The quality of cow's milk is influenced by many things, including genetic, feed and
management. The feed and drinking water used should be free from heavy metal
contamination. Livestock farming management such as sanitation of cages, workers and
equipment should be guaranteed hygiene and meet food safety requirements. Dairy cattle
generally feed grass and concentrate. The quality of milk is determined by its
composition and overall hygiene. However, consumption of contaminated food like milk
may lead to food-borne diseases (FBDs). Specifically, human may be infected with milk-
borne pathogens through consumption of infected raw or unpasteurized milk and milk
products [30, 31]. The main source of contamination of Pb and Cd derives from air and

4
water pollution that contaminates soil. Furthermore, all plants grown on contaminated
soil will accumulate the metals in all parts (roots, stems, leaves and fruit) [32].

The food chain is an important source of Cd and Pb accumulation, especially for plants
grown on polluted soils. Significant amounts of Cd and Pb can be transferred from
contaminated soil to plants and grass, causing accumulation of these potentially toxic
metals in grazing ruminants, particularly in cattle [33, 34]. Accumulation of Cd and Pb in
ruminants causes toxic effects in cattle, but also in humans consuming meat and milk
contaminated with toxic metals [35]. For that reason, the concentration of Cd and Pb in
cow's milk should be monitored to ensure the consumers' health [36].

Milk and dairy products become contaminated with heavy metals either through
contamination of the original cow’s milk, which may be due to exposure of lactating cow
to environmental pollution or consumption of contaminated feed stuffs and water [37,
38]. Heavy metals have been considered as one of the deadly toxins that have
accumulated in plant and animal tissues through the biological cycle and have dispersed
across a wide geographical area. Heavy metals are persistent contaminants in the
environment that can cause serious environmental and health hazards. They are released
into the environment from natural as well as man-made activities. Some heavy metals
(like Mn, Fe, Cu and Fe) are essential to maintain proper metabolic activity in living
organisms; others (like Pb and Cd) are non- essential and have no biological role [39].

Milk and milk products contains trace metals which are co-factors in many enzymes and
play an important role in many physiological function and lack of these metals cause
disturbances and pathological conditions [40]. Milk and dairy products are an important
source of dietary minerals in many European countries, accounting for 10 - 20 % of daily
dietary intake. However, the content of major and trace elements in milk depends upon
the content of these elements in soil and cattle feed, which varies considerably among
and within countries [41, 42]. Essential minerals are vital for the functioning of the
human organism, since they participate in many metabolic processes, and regulate
biochemical reactions. The macro elements (Ca and Mg), are among the essential
minerals required for a correct nutrition and are necessary in large quantities, as they play
an important role in the human organism. For example, magnesium (Mg), a metal of

5
much importance for maintaining electrolytic balances [43]. On the other hand, trace
elements or microelements (Fe, Cu, Zn, and Mn) are required in smaller amounts but
perform numerous physiological functions in the organism. As in the case of iron (Fe),
this forms part of the hemoglobin necessary for the transport of oxygen [44]. However, at
high concentrations, essential metals also cause toxicity to living organisms [45].

Toxicity of metal is closely related to age, sex, route of exposure, daily intake, solubility,
metal oxidation state, retention percentage, duration of exposure, frequency of intake,
absorption rate and mechanisms/efficiency of excretion [46].

2.3 Composition and Mineral Content in Milk

Milk is a considerable resource of products whose composition varies. Four components

are dominant in quantitative terms: water, fat, protein and lactose; while the minor
components are minerals, enzymes, vitamins, and dissolved gases. From these
components, Mineral distribution within the body’s tissues is not uniform, since some
tissues selectively concentrate specific elements. There is no disagreement concerning the
essential nature of major and trace elements for livestock [47]. The composition of
minerals and trace elements in milk can be linked to the breed of the animal from which
it is sourced [48], as well as forage, feed, water and the drugs administered [49]. From the
nutritional point of view, metals contents of milk and dairy products can be grouped into
essential elements (iron, copper and zinc) at low doses and non-essential or toxic ones
(lead and cadmium). The presence of the latter, even in low concentrations, is invaluable
and leads to metabolic disorders with extremely serious consequences [50]. Dairy
animals ingest metals while grazing on the pasture and when fed on contaminated
concentrate feeds. However, in the cow, transfer of minerals to milk is highly variable
[51].

Metal levels 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 [12]. Cow
milk is a good source of both micro and macro element in human diet and the location of
the cows determine their concentration [41].

6
Table 1:- The elemental concentration in fresh cow milk from different countries.

Country Concentration (mg / kg) Reference

Mg Cu Zn Mn Pb Cd

Japan --- 0.1 3 --- 0.001 0.05 45

Poland --- 0.09 3.77 --- 0.015 0.02 45

USA --- 0.019 2.235 --- 0.009 0.034 45

Saudi Arabia --- 0.048 0.944 --- 0.004 0.003 45

Ethiopia --- 0.109 0.559 0.427 --- --- 52

Nigeria 0.560 1.215 0.517 0.092 ----- ---- 53

The concentrations of essential and non-essential metals are different in fresh cow milk
for different researchers. For example, Semaghiul reported in mean (mg/L) as Mn = 0.08,
Cu = 0.17, Fe = 0.72, Zn = 0.98, Cd = 0.004 and Pb = 0.12 [12]. Another researcher
Ogabiela reported at Kano area as Mn = 0.179 ± 0.13, Cu = 0.252 ± 0.123, Fe = 5.987 ±
4.02, Zn = 3.239 ± 7.21, Cd = 0.163 ± 0.89 and Pb = 0.550 ± 0.32 in mg/L [54].

7
Table 2:-Recommended daily allowance (RDA) for metal concentration in fresh cow
milk.

Metals Recommended /Permissible value(mg/day) Reference

Mg Cu Zn Fe Mn Pb Cd

FAO/WHO,1972 -- -- -- - - 0.429 0.057- 45

0.072

FAO/WHO,1978 - 2-3 12–15 - - 0.429 0.057- 45

0.072

NAFDAC 50 2 5 - 0.2 - - 53

Ogabiela Report - - - 15 - - - 54

2.4 Effects of High Concentration of Metals in Cow Milk

2.4.1 Manganese (Mn)

Manganese is an essential nutrient that is important for normal processes in the human
body, though adverse effect have been reported at higher doses. Exposure to high
concentration of manganese is associated with impaired neurological and neuromuscular
control, mental and emotional disturbances (muscle stiffness, and lack of
coordination).Exposure to very high doses result in impaired male fertility, birth defects,
and impaired bone development [54].

2.4.2 Zinc (Zn)

Zinc has numerous functions in the body and it is essential element for human health. At
the same time, zinc function as a cofactor for many enzymes of the body. Excess intake
of zinc in to the body through food, water or dietary supplements can affect health. If
large doses of zinc by mouth even for a short time, stomach cramps, nausea and vomiting
may occur. Ingesting high levels of zinc for several months may cause anemia, damage
the pancreas and decrease levels of high – density lipoprotein (HDL) cholesterol [52].

8
 2.4.3 Copper (Cu)
Copper is a constituent of many enzymes. It is also a constituent of many pigments and is
important for immune functions. Although copper homeostasis plays an important role in
the prevention of copper toxicity, exposure to excessive levels of copper can result in a
number of adverse health effects including liver and kidney damage, anaemia,
immunotoxicity, and developmental toxicity. Many of these effects are consistent with
oxidative damage to membranes or macromolecules [55].

2.4.4 Iron (Fe)

Iron as an essential trace element participates as catalyst in several metabolic reactions.
As a component of hemoglobin, myoglobin, citochrome and other proteins, iron plays an
important role in the transport, storage and utilization of oxygen. It is also a co-factor of
many enzymes [56]. Exposure to excessive levels of iron can result health effects
including in liver, kidney and the cardiovascular system are the target organs for iron
toxicity [54].

2.4.5 Cadmium (Cd)

Cadmium is a cumulative toxic agent with half-life of several years and their burden of
the body increases with age. Cadmium and solutions of its compounds are toxic, chronic
exposure can cause irreversible damage to the lungs and eventually, death. Eating food or
drinking water with high cadmium concentration irritates the stomach causing vomiting
and diarrhea. It accumulates in the kidney and liver causing kidney dysfunctioning and
liver failure, in addition to being a teratogenic and carcinogenic agent [54].

2.4.6 Lead (Pb)

Lead is one of the limited classes of element that can be described as purely toxic. Most
other elements thought toxic at 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. Lead absorption constitutes serious risk to public health.

9
It induces reduced cognitive development and intellectual performance in children,
increased blood pressure, and cardiovascular diseases in adult as well as liver and kidney
dysfunctioning [54].

2.5 Analytical Techniques used for Metal Analysis

The determination of metal concentration can be performed by several instrumental

techniques including indirect photometric chromatography, ion chromatography, flame
atomic absorption spectrometry, furnace atomic absorption spectrometry, inductively
coupled plasma optical emission spectrometry, potentiometric stripping, capillary zone
electrophoresis, differential pulse anodic stripping voltammetry, mid-infrared
spectrometry, particle induced x-ray emission and complexometric titration [52].

2.5.1 Flame Atomic Absorption Spectrometer (FAAS)

FAAS is a suitable technique for determining metals at part per million (ppm)
concentration levels with good precision for many elements. FAAS offers air-acetylene
and/or nitrous oxide flame atomizer. Samples are introduced into the atomizer as an
aerosol by the nebulizer.

FAAS technique provides fast analysis of 10-15 s per sample, with very good precision
(repeatability), moderate interferences that can be easily corrected, and relatively low
cost [57]. FAAS was successfully applied for the determination of heavy metals in
various matrices. Some elements like arsenic (As) and zirconium (Zr) may not be
determined by FAAS because the maximum atomization temperature is not sufficient to
induce complete atomization. In FAAS moderate interferences can be easily corrected
such as background interference due to light scattering by particles in the flame which is
usually controlled by the use of deuterium based lamp [58].

2.5.2 Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-

OES)
Inductively coupled plasma optical emission spectroscopy is a technique for analyzing
the concentration of metallic elements in solid and liquid samples. For ICP OES, solid
samples are dissolved (digested) in an appropriate solvent (typically acid) to produce a
solution for analysis. The resulting sample solution (or an original liquid solution for

10
analysis) is often diluted in water to obtain a final specimen suitable for analysis. The ICP
OES instrument uses argon gas flowing through a torch consisting of three concentric
quartz tubes. A copper coil circumscribing the top of the torch is connected to a radio
frequency (RF) generator. The use of the copper coil with the RF power is called
inductive coupling. When the RF power is applied in the copper coil, an alternating
current occurs within the coil. The oscillation of the alternating current causes electric
and magnetic fields at the end of the torch. A spark applied to the argon gas causes some
electrons to be stripped from the argon atoms. The electrons are caught and accelerated
by the RF generated electric/magnetic field. The high energy free electrons collide with
other atoms, stripping off more electrons in a chain reaction, resulting in plasma of
electrons, ions, and atoms. This is known as an inductively coupled plasma (ICP)
discharge. This ICP discharge is maintained as the RF energy is continually transferred to
the plasma by the copper coil. The liquid samples are nebulized into an aerosol and
introduced into the center of the plasma. The plasma excites the sample atoms, which
subsequently relax to a lower energy state by emitting light at elementally characteristic
wavelengths. The intensities of these characteristic wavelengths are detected, measured,
and compared to intensities for known standards to provide quantitative results [59].

11
 3. Materials and Methods

3.1 Description of the Study Area

The study was conducted at Wombera woreda which is among the 7 woredas in Metekel
zone, Benishangul Gumuz regional state of Ethiopia. Wombera is located at about 614km
away from Addis Ababa, 523 km from Asossa and 165 km from Metekel zone. The study
area was selected based on the dependence of farmers on livestock production mainly
from cattle at Wombera woreda. Fresh cow milk samples were collected from three site
farms of Deberezeit, Manasibu and Ateshimo sites of the Wombera woreda.

Figure 1: Map of the study area

Source: (Gemechu, 2010)

3.2 Chemicals and Reagents

Reagents and chemicals used for the laboratory works were all analytical grade. De-
ionized water was used for dilution of samples and rinsing all glassware. Nitric acid (65
%) (N43725-4K, UNI-CHEM) and hydrogen peroxide (30 %) (Dallul pharmaceuticals
PLC, Addis Abeba) were used for digesting milk samples throughout this work. 1000
mg/L stock standard solutions of Mn, Fe, Mg, Cu, Zn, Cd, and Pb were used to prepare
intermediate standard solutions of concentration 10 mg/L. Working standards were
prepared from intermediate standards of each metals.

12
 3.3 Apparatus / Instrument

The Apparatus / Instrument used in this study were measuring cylinder, 25 mL

volumetric flask, 100 mL beaker, analytical balance (Nimbus, ADAM), hot plate, ice-
box, polyethylene bottle, filter paper and inductively coupled plasma optical emission
spectroscopy (ICP-OES) (Prkin Elmer Optima 8000).

All glassware were washed before use with de-ionized water, soaked in nitric acid (30
%), then rinsed in de-ionized water and air dried. The glassware kept in clean place, to
avoid contamination. Hot plate with temperature adjustment is used in flam hood for the
digestion of milk samples. The ICP-OES was used for the determination of metals in
milk sample.

Table 3:-Instrumental operating conditions for determination of metals in milk

samples use ICP-OES.

RF Power Plasma Gas Auxiliary Pump

Flow Gas Flow Nebulizer Speed
Gas Flow Gas

1500W 8.0 L/min 0.2 L/min 0.7 L/min 1.0 ml/min Argon

3.4 Sample Collection

The polyethylene sampling bottles was washed with distilled water before collection of
raw milk in order to avoid possible contamination. The udder of each cow was washed
with distilled water before milking. Two cows from three farmers were randomly
selected from each of Deberezeit, Manasibu, and Ateshimo sites and one composite
sample representative of each site was prepared. Milk samples of 400 mL were collected
during morning milking time before grazing from each cow and homogenized then
packed immediately with a plastic and were kept in ice box. The samples were
transported to the laboratory of Bahir Dar University and were placed below 4 ºC in a
refrigerator until analysis.

13
 3.5 Milk Sample Digestion and Preparation of the Sample for ICP-OES Analysis

The milk sample needs to be brought into clear solution for analysis by inductively
coupled plasma optical emission spectroscopy (ICP-OES). For this reason the sample was
digested to dissolve the milk and to remove the organic components of the milk. 5 g of
raw cow's milk is treated with 5 mL (65 % nitric acid) and 2 mL (30 % hydrogen
peroxide) and then digested on electric hot plate at 90 ºC and the temperature of this
mixture was gradually increased to 120 ºC until brown fumes appeared, indicating
completion of oxidation of organic matter. The organic matrix of milk was destroyed and
leaving behind the elements in clear solution. After cooling, the clear solution was
filtered in to 25 mL volumetric flask and diluted up to the mark with double distillated
water. A blank digestion solution was made for comparison [60]. Finally the milk
samples were directly analyzed for essential and non-essential metals by ICP-OES. For
those metals above the standard, further dilution was carried out. 0.6 mL of solution was
taken from each analyzed samples to dilute Mg in 25 mL volumetric flask and 11 mL of
solution of Ateshimo site was taken to dilute Fe in 25 mL volumetric flask and the
samples were analyzed again for these metals.

3.6 Calibration Curve and Measurement of Metal Concentrations

The calibration curves were drawn for Mn, Fe, Mg, Cu, Zn, Cd and Pb using linear
regression analysis of the concentrations of the standard solutions versus intensity values.
Six series of working standard solutions (Table - 4) of metals were prepared by diluting
the intermediate standard solution with de-ionized water. A blank and standards were run
in ICP-OES and six points of calibration curve were established. Each standard solution
was measured three times and the mean was plotted. The correlation coefficients of
calibration curves are given in Table - 4. The correlation coefficient of more than 0.99
showed that there is strong linear relationship between concentrations and absorbance.
Each of the sample solutions was aspirated into the ICP-OES instrument then the
concentrations of each metal and emission/intensity values of the metals were recorded.
Triplicate determinations were carried out on each sample.

14
Table 4:-Series of working standards and correlation coefficients of the calibration
curves for the determination of metals in milk using ICP-OES.

No Metals Concentrations of standards (mg/L) Correlation coefficient, R

1 Mg 0.05, 1.05, 2.05, 3.05, 4.05, 5.05 0.999865

2 Cu 0.05, 1.05, 2.05, 3.05, 4.05, 5.05 0.999902

3 Fe 0.05, 1.05, 2.05, 3.05, 4.05, 5.05 0.999975

4 Mn 0.05, 1.05, 2.05, 3.05, 4.05, 5.05 0.999944

5 Cd 0.05, 1.05, 2.05, 3.05, 4.05, 5.05 0.999912

6 Zn 0.05, 1.05, 2.05, 3.05, 4.05, 5.05 0.999889

7 Pb 0.05, 1.05, 2.05, 3.05, 4.05, 5.05 0.999870

3.7 Method Validation

The method was validated using recovery of spiked standard metals. For method
validation a milk sample from Ateshimo site was selected randomly and known amount
(1.00 ppm) of standard solution was added to the sample and digested with the same
procedure of hot plate digestion for milk samples. After diluting the spiked samples to the
required volume with de-ionized water, they were analyzed with the same procedure
followed for the analysis of milk samples. The recoveries of the spiked samples were
calculated using the following formula.

concentration in spiked sample − concentration in sample

Percentage recovery= Amount spiked
× 100 %

15
 3.8 Method and Instrument Detection Limits

Method detection limit is the lowest analyte concentration that can be distinguished from
fluctuations in a blank, which usually corresponds to average of the blank signal plus
three times standard deviation of the blank [52].

In order to determine the method detection limits three blank samples were digested
following the same procedure as the milk samples and each of the blank samples were
analyzed for Mn, Mg, Fe, Cu, Zn, Cd and Pb by using ICP-OES. The standard deviation
for each element was calculated from the three blank measurements to determine method
detection limit of the instrument. Instrument detection limits are directly obtained from
the instrument manual for all the elements under study. It can be observed that the
method detection limit of each element is somewhat above the instrument detection limit.

Table 5:-Method detection limits for the determination of metals in cow milk
samples.

Elements MDL ( mg/L) IDL ( mg/L)

Cu 0.098 0.0097

Mg 3.984 0.0016

Mn 0.063 0.0014

Fe 1.474 0.0046

Zn 0.459 0.0059

Cd 0.001 0.0027

Pb 0.002 0.042

Note: MDL = Method Detection Limit, IDL = Instrument Detection Limit

16
 3.9 Statistical Data Analysis of Methods

In addition to comparison of data with the international standard for milk quality, all data
were statistically analyzed by evaluating the means and standard deviation. For statistical
analysis tools like micro soft Excel, 2007 and IBM SPSS statistics version 20, were used.

17
 4. Results and Discussion

4.1 Calibration of the Instrument

The data qualities obtained from the Inductively Coupled Plasma Optical Emission
Spectroscopy for metals analyses are highly affected by the calibration and standard
solutions. The calibration graphs and determination coefficient of each of the elements
were determined by plotting working standards concentrations versus their intensities.
The determination coefficient (R2) shows that there was a good relationship between
concentration and intensity.

8 Y = 87347.195 + 1.54733E6 *X
2.0 Y = 5956.19648 + 372545.3085 *X
R2 = 0.9997
6 R2 = 0.99993
1.5
Intensity (10 )
6

Intensity (106)
4 Mg 1.0
Fe
2 0.5

0 0.0

0 1 2 3 4 5 0 1 2 3 4 5
Concentration (mg/L) Concentration (mg/L)

20 Y = -73993.8401 + 3.80073E6 *X 2.0 Y = 5956.19648 + 372545.3085 *X

R2 = 0.99983
16 R2 = 0.99993
1.5
Intensity (106)

Intensity (106)

12
Cu 1.0
8 Mn
0.5
4

0 0.0

0 1 2 3 4 5 0 1 2 3 4 5
Concentration (mg/L) Concentration (mg/L)

18
 0.4
Y = 7735.98386 + 73395.75143 *X
1.6 Y = 5032.48 + 294363.01 * X
R2 = 0.9997 R2 = 0.99972
0.3
1.2
Intensity (106)

Intensity (106 )
0.2
Zn 0.8 Cd

0.1 0.4

0.0 0.0

0 1 2 3 4 5
0 1 2 3 4 5
Concentration (mg/L)
Concentration (mg/L)

Y = 732.74 + 19856.27 * X
0.09
R2 = 0.99963
Intensity (106)

0.06
Pb
0.03

0.00

0 1 2 3 4 5
Concentration (mg / L)

Figure 2: Plot of intensity as a function of the concentration for each studied metals

19
 4.2 Percent Recovery Test

In this study the precision of the results was evaluated by the standard deviation of the
triplicate samples (n = 3), analyzed under the same conditions. The accuracy and validity
of procedure used for digestion was evaluated by analyzing the digests of spiked sample.
The recoveries of the detected metals in the spiked milk samples were given in Table - 6.

Table 6:-Recovery results for the validation of the optimized procedure.

Metal Amount spiked Conc. in Conc. in Recovery

sample, spiked (%)
Mg/L Volume mg/L sample, mg/L
(mL)

Cu 1.00 2.5 0.116 1.153 103.7

Fe 1.00 2.5 1.661 2.598 93.7

Mn 1.00 2.5 0.053 0.906 85.3

Pb 1.00 2.5 0.016 0.834 81.8

Zn 1.00 2.5 0.572 1.391 81.9

Mg 1.00 2.5 0.500 1.303 80.3

4.3 Mean Concentration of Metals in Cow Milk

The average metal concentrations in the fresh cow milk from the three farms of Wombera
woreda were presented in Table - 8. Among the seven elements analyzed, six elements
(Fe, Pb, Mg, Mn, Cu and Zn) were detected at Deberezeit site, where as Cd was not
detected. But five elements (Fe, Mg, Mn, Cu and Zn) were detected at Manasibu, and
Ateshimo sites, whereas Cd and Pb were below their corresponding method detection
limit.

20
From the detected metals, the highest mean concentration obtained was for Mg (147.792
± 9.819 mg/L) and lowest for Pb (0.352 ± 0.144 mg/L) among all the studied farms of
Wombera woreda.

The mean concentrations of metals detected in the milk samples in the present study can
be arranged in the order of Mg (147.792 ± 9.819) > Fe (18.700 ± 2.232) > Zn (6.645 ±
1.111) > Cu (0.742 ± 0.123) > Mn (0.575 ± 0.063) > Pb (0.352 ± 0.144) in mg/L (Table-
8).

Table 7:-Average concentration (mg/L) of metals in digested solutions of milk

samples collected from different farms of Wombera woreda.

Study sites

Metals Deberezeit Manasibu Ateshimo Total mean

Cu 0.116 ± 0.030 0.130 ± 0.023 0.190 ± 0.017 0.145 ± 0.024

Fe 2.132 ± 0.391 1.968 ± 0.643 3.033 ± 0.043 2.387 ± 0.435

Mn 0.072 ± 0.003 0.072 ± 0.012 0.194 ± 0.016 0.113 ± 0.012

Zn 0.987 ± 0.030 0.875 ± 0.056 2.045 ± 0.372 1.302 ± 0.218

Pb 0.069 ± 0.028 <MDL <MDL 0.069 ± 0.032

Cd <MDL <MDL <MDL -------

Mg 0.828 ± 0.048 0.875 ± 0.038 0.724 ± 0.066 0.695 ± 0.171

NB: <MDL = below Method Detection Limit

21
Table 8:-Average concentration of metals in fresh cow milk samples collected from
different farms of Wombera woreda.

Level of metals in cow milk by the study sites (mg/L)

Metals Deberezeit Manasibu Ateshimo Mean concentration

(mg/L)
Cu 0.592 ± 0.153 0.663 ± 0.120 0.969 ± 0.087 0.742 ± 0.123

Fe 10.877 ± 1.996 10.049 ± 3.282 35.175 ± 0.436 18.700 ± 2.232

Mn 0.365 ± 0.018 0.367 ± 0.065 0.991 ± 0.085 0.575 ± 0.063

Zn 5.035 ± 0.155 4.464 ± 0.287 10.437 ± 1.897 6.645 ± 1.111

Pb 0.352 ± 0.144 < MDL < MDL 0.352 ± 0.144

Cd < MDL < MDL < MDL -----

Mg 176.089 ± 9.161 113.377 ± 7.050 153.910 ± 12.474 147.792 ± 9.819

In the present study the concentration of Pb and Cd was found to be less than method
detection limit and hence not detected except Deberezeit site in which Pb was detected.
This might be due to less bioavailability of this element to the animal feed and hence to
the Cow milk. Also there are no industries and vehicle emissions which are the basic
sources of these toxic metals around the study area. Since it is rural area, there is no
exposure of these toxic metals. The feeds and the water which the cows use are also free
from these toxic metals. The mean concentration of lead in this study at Deberezeit was
0.352 ± 0.144mg/L. The calculated daily intake of Pb in the present study was 0.019
mg/day which was below the recommended dietary allowance of 0.429 mg/day (Table -
9). The presence of this concentration at this study area may be due to a lot of human
activities were taken part like motor garage situated there, as such there is high
concentration of car exhaust which source of lead thereby polluting the grazing land for
cows and milk in return.

22
The detected metals such as Mg, Fe, Mn, Cu and Zn, can be found naturally in food.
They are essential metals and thus, these metals can be found in cow milk since it is one
of the most important types of food. It is clear that magnesium is detected in all the
samples collected from three different farms and the concentrations varied from 113.377
to 176.089 mg/L. When compared with the other detected elements, the average
concentration of magnesium from all the sites was high (147.792 ± 9.819mg/L). This
shows that, the cow milk in the study area is rich in magnesium. Magnesium activates
100 enzymes and play role in over 300 enzyme reactions in the body, many of which are
directly related to cardiovascular health and helps nerves and muscles function.
Manganese is important in protein metabolism; bone and teeth formation [61]. The
calculated daily intake of Mg in the present study was 7832.976 μg/day which was very
low compared to the recommended daily allowance of Mg (50000 μg/day) which was set
by NAFDAC standard values (Table - 9).

In this study, zinc is present in all the milk samples with concentrations ranging
from4.464 to 10.437 mg/L. The average concentration of zinc from all the sites is 6.645 ±
1.111 mg/L. Zinc has numerous functions in the body and it is essential element for
human health. At the same time, zinc function as a cofactor for many enzymes of the
body. Taking too much of zinc into the body through food, water, or dietary supplements
can affect health [52]. In the present study, the calculated daily intake of zinc was0.352
mg/day which was below the recommended dietary allowance of Zn (12-15 mg/day)
which was set by FAO/WHO standard values (Table - 9).

In the case of copper, the mean concentrations in the present study were found in the
range of 0.592 to 0.969 mg/L. The average concentration of copper from all the sites was
(0.742 ± 0.123 mg/L). This indicates that the cow milk of Wombera woreda is relatively
poor source of copper. Copper is an essential trace element that plays a vital role in the
physiology of animals for fetal growth and early post-natal development. Excess copper
in the body leads to Wilson’s disease which is characterized by deficiency of
ceruloplasmin. In cow milk of Wombera woreda, the daily intake was determined to be
0.039 mg/day. Therefore, the result obtained in this work for copper was below the
recommended dietary allowance of 2-3 mg /day which was set by FAO/WHO standard
values (Table - 9).
23
The concentrations of iron obtained in this research work occur in the range of 10.049 to
35.175 mg/L. The average concentration of iron from all the sites was high (18.700 ±
2.232 mg/L) next to magnesium. This shows that, the cow milk in the study area is rich in
iron. Iron is an essential element participates as catalyst in several metabolic reactions.
Exposure to excessive levels of iron can result health effects. The daily intake of Fe in the
present study was 0.991 mg/day. The result obtained in this work for iron was below the
recommended dietary allowance of 15 mg /day (Table - 9).

In this study, manganese was obtained from the fresh cow milk samples with
concentrations ranging from 0.365 to 0.991 mg/L. The average concentration of
manganese in milk sample was the lowest (0.574 ± 0.063 mg/L) among all the sites of
essential elements. Manganese plays an important role in a number of physiologic
processes as a constituent of multiple enzymes and an activator of other enzymes.
Exposure to high concentration of manganese is associated with impaired neurological
and neuromuscular control, mental and emotional disturbances (muscle stiffness, and lack
of coordination). Exposure to very high doses result in impaired male fertility, birth
defects, and impaired bone development. The calculated daily intake of Mn in the present
study was 0.03 mg/day. Therefore the result obtained in this work for manganese was
below the recommended dietary allowance of 2 - 5 mg/day (Table - 9).

4.4 Distribution of the Metals in the Study Areas

As shown in figure - 3, the concentration level of Mg in fresh cow milk sample was
higher than Fe and Zn throughout the three sample sites. In this study concentration
distribution pattern of Mg determined in fresh cow milk sample by sampling sites
decreased in the order of Deberzyit > Manasibu > Ateshimo.

The level of Fe in cow milk sample in Ateshimo site was higher than Debrezyit and
Manesibu site. The concentration of iron in the milk samples of three sites was found to
follow decreasing order of Ateshimo > Debrezyit > Manesibu. Among the three sites, the
highest concentration of zinc was investigated at Ateshimo. The level of Zn in cow milk
at Debrezyit and Manesibu sites were almost comparable.

24
 200

180

160

140
Concentration (mg/L)

120

100 Deberzit
Manesibu
80
Ateshimo
60

40

20

0
Mg Fe Zn
Metals

Figure 3: Distribution of metals Mg, Fe and Zn in study areas

In figure - 4, among sampling sites high metal concentration of Cu was investigated in

Ateshimo followed by Manesibu and Debrezyit. The highest metal concentration of
manganese was detected in Atheshimo site. The level of Mn in cow milk samples in
Manesibu and Debrezyit sites were almost similar. The concentration level of lead was
determined in Debrezyit among the three sampling site. However, the level of Pb was not
detected in Manesibu and Atheshimo sites at all.

25
 1.2

1
Concentration

0.8

0.6 Debrzeyit
Manasibu
0.4
Ateshimo

0.2

0
Cu Mn Pb
Metals

Figure 4: Distribution of metals Cu, Mn and Pb in study areas

4.5 Comparison of Results of the Present Study with Literature Values

There are a variations in the published data for the elemental concentrations of cow milk
of different countries as shown in Table-1.The observed wide variation in the
concentrations of metals in milk is possibly due to nature and composition of the soil of
the specific region. This results in different level of accumulation in the grass, which in
turn results in the varying concentration of metals in the cow milk. The concentrations of
Cd and Pb were found to be less than method detection limit in present study except
Deberezeit site in which Pb was detected, where as in some of the literature these
concentrations are present in the detectable levels. The result of Pb (0.352 ± 0.144
mg/L) in Deberezeit site was greater than the values of USA (0.009 mg/Kg), Japan (0.001
mg/Kg), Saudi Arabia (0.004 mg/Kg), Poland (0.015 mg/Kg) and Semaghiul report (Pb =
0.12 mg/L) Table 1 and 8. However, Pb had less concentration compared with Ogabiela
report (0.55 ± 0.32 mg/L) in review literature. This showed that the cow milk in
Deberezeit area is rich in lead. This might be due to the presence of vehicle and motor
garage which are the source of lead around Deberezeit site because Deberezeit site is the
city.

26
The concentration of Cu in Deberezeit (0.592 ± 0.153mg/L), Manasibu (0.663 ±
0.120mg/L), and Ateshimo (0.969 ± 0.087 mg/L)sites in the present study were high
compared with the values of Japan (0.1mg/kg), Poland (0.09 mg/kg), USA (0.019
mg/kg), Saudi Arabia (0.048 mg/kg), Ethiopia (0.109 mg/kg), Ogabiela report (0.252 ±
0.125 mg/L) and Semaghiul report (Cu = 0.17 mg/L) in review literature. But Cu had low
concentration in these three sites compared with that of Nigeria (1.215 mg/kg) Table 1
and 8.

The result of Zn in Deberezeit (5.035 ± 0.155mg/L), Manasibu (4.464 ± 0.287mg/L) and

Ateshimo (10.437 ± 1.897 mg/L) sites in this study were greater than the values of USA
(2.235 mg/Kg), Japan (3 mg/Kg), Poland (3.77 mg/Kg), Saudi Arabia (0.944 mg/Kg),
Ethiopia (0.559 mg/Kg) and Nigeria (0.517 mg/Kg) Table-1. Zn also had high
concentration in three sites compared with Semaghiul report (Zn = 0.98 mg/L) and
Ogabiela report (3.239 ± 7.21 mg/L) Table 1 and 8 in review literature.

There are limited reports on the concentration of magnesium in fresh cow milk samples.
The concentration of Mg in the present study in Deberezeit (176.089 ± 9.161mg/L),
Manasibu (113 ± 7.050 mg/L) and Ateshimo (153.910 ± 12.474 mg/L) sites were too
much greater than report of Nigeria (0.56 ± 0.025 mg/kg) Table- 1 and 8. The level of Mg
in Deberezeit site (176.089 ± 9.161mg/L) was greater than Zamberlin report (90 – 160
mg/kg) [62]. But the concentration of Mg in Manasibu (113 ± 7.050 mg/L) and Ateshimo
(153.910 ± 12.474 mg/L) sites were less than Zamberlin report (90 – 160 mg/kg).

Iron concentration from Deberezeit (10.877 ± 1.996mg/L), Manasibu (10.049 ± 3.282

mg/L) and Ateshimo (35.175 ± 0.436 mg/L) sites in the present study were greater than
that of Semaghiul report (Fe = 0.72 mg/L) and Ogabiela report (5.987 ± 4.02 mg/L) in
literature. The result of Mn concentration in Ateshimo site (0.991 ± 0.085 mg/L) was
greater than the value reported for the milk samples of Ethiopia (0.427 mg/Kg) but Mg in
Deberezeit (0.365 mg/L) and Ateshimo (0.367± 0.065 mg/L) sites was less than milk
samples of Ethiopia (0.427 mg/Kg), but greater than that of Nigeria (0.092 ± 0.02 mg/L)
report (Table 1 and 8).

In general, the concentrations of metals detected in the present study were less
comparable with the reported literature values. Hence, relatively higher concentration of
27
Fe, Cu and Zn, were observed in this study in comparison to the reported values, whereas
the concentration of Mg, Mn and Pb in this study were somewhat above and below to the
reported literature values.

4.6 Recommended Daily Allowance of Metals

The daily intake of the metals depends on both the concentration and the amount of food
consumed. The daily dietary intake of milk for an average Mumbai (India) population is
113 g. The reported values of daily milk consumption in USA and Spain are 224 g and
124 g, respectively. However, in Ethiopia the daily consumption of milk is very low. It is
considered to be 53 mL/day which is calculated from 19 Kg per capita per year. The
value given per year is changed to per day to know the approximate daily intake in
Ethiopia. Thus it is possible to assume that 53 g is equal to 53 mL of whole milk [52].
Assuming a value of 53 mL of milk consumption per day, the daily intake of the detected
metals from samples of the present study was determined.

Table 9:- Comparison of daily intakes of metals from 53 mL milk by Ethiopian

population with recommended/ permissible values.

Element in Concentration Daily intake Recommended/ permissible

fresh milk value References
(μg/mL)

(μg /day) mg/day ( μg /day) mg/day

Cu 0.742 39.326 0.039 2000 – 3000 2-3 45

Fe 18.700 991.1 0.991 15000 15 54

Mn 0.575 30.475 0.030 200 2– 5 54

Zn 6.645 352.185 0.352 12000 – 15000 12-15 45

Mg 147.792 7832.976 7.833 50000 50 53

Pb 0.352 18.656 0.019 429.00 0.429 45

28
From the Table- 8, it is observed that the daily intakes of detected metals in the study area
were less than the recommended/permissible levels set by different international
organizations. The daily intakes of these metals were far less than the recommended daily
intake values.

Therefore, it is suggested that some other alternative sources of food rich in this elements
might be taken to compensate the deficiency in milk.

4.7 Analysis of variance (ANOVA)

The statistical analysis was performed using one way analysis of variance (ANOVA).
The P-value of each parameter was calculated using the SPSS version 20 software.
Comparing the means of all the three sites of Wombera woreda for all metal content, at
the 95 % confident level, the means were significantly different (p < 0.05).

The mean values of copper, iron and manganese were significantly different (p < 0.05) in
the three sites of Wombera woreda. The content of Cu, Fe and Mn between Deberzyit
and Manesibu sites was significantly the some (P > 0.05). The mean value of Mg and Pb
were significantly different (p < 0.05) in the three sites of Wombera woreda. In summary,
the calculated P-values of the parameters, Mg (P = 0.00), Fe (P = 0.00), Zn (P = 00), Cu
(P = 0.00), Mn (P = 0.00) and Pb (P = 0.00). Therefore P-value shows that these metals
have significant difference between Deberezeit, Manasibu and Ateshimo site (p < 0.05).

29
 5. Conclusions

The main objective of this study was to assess the levels of essential and non-essential
metals in fresh cow milk of Wombera woreda in Benishagul Gumz regional state of
Ethiopia. Three sampling sites of this woreda were purposely selected using composite
sampling method. Inductively coupled plasma optical emission spectrometer was used to
assess the level of metals in fresh cow milk.

The toxic metals such as Pb, and Cd were not detected in the cow milk of Wombera
woreda even though they were reported in the cow milk in several studies but the
concentration of Pb was detected at Deberezeit site. The essential metals such as Mg, Fe,
Cu, Mn and Zn were detected in milk samples of Wombera woreda. The concentration of
Mg, Fe, Cu, Mn and Zn were 147.792 ± 9.819, 18.700 ± 2.232, 0.742 ± 0.123, 0.575 ±
0.063and 6.645 ± 1.111 mg/L respectively in the milk samples of three sites under study.
From the detected metals, the highest mean concentration obtained was for Mg (147.792
± 9.819mg/L) and lowest for Pb (0.352 ± 0.144mg/L) among all the studied farms of
Wombera woreda. The concentrations of metals observed were compared with some of
the reported values in literature. The results obtained for detected metals in the present
study were also compared with international daily intake guidelines of different
international organizations for food and were found to be below the levels of
Recommended/ permissible value. Therefore, the fresh cow milk in this study area is safe
for human healthy.

30
 6. References

[1] Leotsinidis M, Alexopoulos A, Kostopoulou-Farri E. 2005. Toxic and essential

trace elements in human milk from Greek lactating women: association with
dietary habits and other factors. Chemosphere. 61, 238 - 247.

[2] AjaiA. I, Ochigbo S. S, Ndamitso M. M, Olaoluwajuwon J. 2012. Proximate and

mineral compositions of different raw cow’s milks in Minna, European. J. App.
Eng and Scien. Res. 1, 23 - 29.

[3] Anetta L, Peter M, Agnieszka G, Jozef G. 2012. Concentration of selected elements

in rawand ultra-heat treated cow’s milk. J. Micro. Biotech and Food Sci. 2, 795 -
802.

[4] Arab, A. A. K. and El-Senaity M. H. 2009. Chemical composition of raw milk and
heavy metals behavior during processing of milk products, Glob. Vet. 3, 268 - 27.
[5] Enb A, Abon M. A, Abd-rabou N. S, Arab A. A. K, El-Senaify M.
H.2009.Chemical composition of raw milk and heavy metal behavior during
processing of milk products. Glob.Vet. 3, 268 - 275.
[6] Qiang L, Xiao-ping W, Tong X, Tong W. 2009. The minerals and heavy metals in
cow’s milk from China and Japan. J. Health Science. 55, 300 - 305.
[7] Torrejon C. S, Castillo–Duran C, Hertram E. D, Ruz M. 2004. Zinc and iron
nutrition in Chilean children fed fortified milk provided by the complementary
national food program. Nutri. 209:177 - 180.
[8] Pennington J. A, Schoen S. A, Salmon G. D, Young B, Johnson R. D, Marts, R.W.
1995. Composition of core foods of the US Food Supply. .J. Food Compos. Anal.
8, 171 - 217.
[9] Santos E. E, Lauria D. C, Portoda S, C. L. 2004. Assessment of daily
intake of trace elements due to consumption of foodstuffs by adult inhabitants of
Riode Janeiro city. Sci. Total. Environ. 327, 69 - 79.

[10] Fernandez-Lorenzo J. R., Cocho J. A, Rey-Goldar M. L, Couche M, Fraga J. M.

1999. Aluminum contents of human milk, cows’ milk and infant formulas. J. Pediat
Gastro and Nut.28, 270 - 275.

31
[11] Bonyadian M, Moshtaghi H, SoltaniZ. 2006. Examination of lead of raw milk and
processing milk in Shahrekord. J. Veter. Med. 50 - 59.

[12] Semaghiul B, Simona D, Gabriela S, Alina S. 2008. Determination of major and

minor elements in milk through ICP–AES. Environ Eng. Manage, 7, 805 - 808.
[13] Jigam A, Ali B, Daud E.N, Jimoh T, Hauwa N. 2011. Determination of copper,
zinc, lead and some biochemical parameters in fresh cow milk from different
locations in Niger State,Nigeria. Afri. Jour. Food. Sci. 5, 156 -160.
[14] Agrawal R. P, Swami S. C, Beniwal R. 2003. Effect of camel milk on glycemic
control risk factors and diabete quality of life in type – 1diabetes: A randomized
prospective controlled study. J. Camel Pract.10, 45 - 50.
[15] Abdullahi M. 2011. Evaluation of some heavy metals residue in milk powder
confectionary plant regarding the public health significance. Animal Research
Institute, Agricultural Research Center, Giza, Egypt.
[16] Liobet J, Falco G, Casas C, Teizido A, Domingo J. 2003. Concentration of arsenic,
cadmium, mercury and lead in common foods and estimated daily intake by
children, adolescents and adults of Catalonia, Spain. J. Agric. Food Chem. 5, 838 -
842.
[17] Qin L. Wang X, Long X and Long W. 2009. The Minerals and Heavy Metals in
Cow Milk from China and Japan. J. Health Sci. 55, 300 - 305.
[18] Malhat F, Hagag M, Saber A, Fayz A. 2012. Contamination of cow’s milk by
heavy metal in Egypt.Bullet Environ Cont and Toxic. 88, 611- 613.
[19] Tripathi R, Raghunath R, Sastry V, Krishnamoorthy T.1999. Daily intake of heavy
metals by infants through milk and milk products. Sci. The tot. Environ. 227, 229-
235.
[20] Abdulkhaliq A, Swaileh K, Hussain R, Matani M. 2012. Levels of metals (Cd, Pb,
Cu, and Fe) in Cow’s milk dairy product and Hen’s Eggs from the west bank,
Palestine. J. Int. Food Res. 3, 1089 - 1094.

[21] Harlia E. 2018. Food safety of milk and dairy products of dairy cattle from heavy
metal contamination IOP Conf. Ser. Earth Environ.Sci.10, 12 - 50.

32
[22] Rubio C, González-Iglesias T, Revert C, Reguera J, Gutiérrez A, Hardisson A.
2005. Lead Dietary Intake in a Spanish Population (Canary Islands). J Agric Food
Chem. 53(16), 6543 - 6549.

[23] Rubio C, Hardisson A, Reguera J, Revert C, Lafuente M, González, I. T. 2006.

Cadmium dietary intake in the Canary Islands, Spain. Environ Res. 100, 123 -129.

[24] Pandey G. S, Voskuil G.C. 2011. Manual on milk safety, quality and
hygiene.Golden Valley Agricultural Research Trust, Zambia. 52.

[25] Anetta L, Peter M, Agnieszka G, Josef G. 2012. Concentration of selected

elements in raw and ultra-heat treated cow milk. J. Micro, Biotech and Food Sci.
2, 795-802.

[26] Buldini P. L, Cavalli S, Sharma J. L. 2002. Matrix removal for the ion
chromatographic determination of some trace elements in milk. Microch. J. 72,
277 - 284.

[27] Pandey G.S and Voskuil G.C. 2011. Manual on milk safety, quality and
hygiene.Golden Valley Agricultural Research Trust, Zambia. 52.

[28] Pal M. 2012. Public health hazards due to consumption of raw milk. Eth. Herald.
10.

[29] Pennington J. 1995. Composition of Core Foods of the US Food Supply, 1982 -
1991: I.Sodium, Phosphorus and Potassium. J. Food Compo and Analysis. 8, 91 -
128.

[30] Bertu W. J, Dapar M, Gusi A. M, Ngulukun S. S, Leo S, Jwander L. D. 2010.

Prevalence of Brucella antibodies in marketed milk in Jos and environs. J. Afr. F.
Sc. 4, 62 - 64.
[31] Pal M, Jahdav V. J. 2013. Microbial contamination of various Indian milk
products. Bev. Food World.40, 43 - 44.
[32] Yintao L, Hong Y, Dan S, Yichen J, Shichao Z, Jun Y. 2015. Heavy Metal
Residues in Soil and Accumulation in Maize at Long -Term Wastewater Irrigation
Area in Tongliao, China. Res. Article.J. Chem. 9

33
[33] López A. M, Prieto M. F, Miranda M, Castillo C, Hernández J, Luis B. J. 2004.
Interactions between toxic (As, Cd, Hg and Pb) and nutritional essential (Ca, Co,
Cr, Cu, Fe, Mn, Mo, Ni, Se, Zn) elements in the tissues of cattle from NW Spain.
Bio Metals.17, 389 - 397.
[34] Miranda M, López A. M, Castillo C, Hernández J, Benedito J. 2005. Effects of
moderate pollution on toxic and trace metal levels in calves from a polluted area
of northern Spain. J. Environ. Int. 31, 543 - 548.
[35] González W. D, Karlsson L, Caballero A, Hernández F, Gutiérrez A, González I.
T. 2006. Lead and cadmium in meat and meat products consumed by the
population in Tenerife Island,Spain.Food Additives and Cont. 23, 757 - 763.
[36] Jen S, Lee S, Lin S. 1994. Determination of cadmium and lead in raw milk by
graphite furnace atomic absorption spectrophotometer. Dairy Sci.77, 945 - 949.
[37] Carl M. 1991. Heavy metals and other trace elements. Monograph on residues and
contaminants in milk and milk products. Special Issue 9101. 112 - 119.
[38] Okada I. A, Sakuma A. M., Maio F. D, Dovidauskas S, Zenebon O. 1997.
Evaluation of lead and cadmium levels in milk due to environmental
contamination in Parabia valley region of South- Eastern Barazil. Revista-de-
Saude-Publica. 31, 140 -143.
[39] Ayar A, Akin D. 2009. The Trace Metal Levels in Milk and Dairy Product
ConsuIn Middle Ana.
[40] Schuhmacher M., Bosquem M. A, Doming J. L, Corbella J. 1991. Dietary intake
of Pb and Cd from food in Tarragona province, Spain Bull. Env. Contam.
Toxicol. 46, 320 - 328.
[41] Dobrzański Z, Kołacz R, Górecka H, Chojnacka K, Bartkowiak A. 2005. The
Content of Microelements and Trace Elements in Raw Milk from Cows in the
Silesian Region. Polish J. Environ. Studies 14, 685 - 689.
[42] Malbe M, Otstavel T, Kodis I, Viitak A. 2010. Content of selected micro and
macro elements in dairy cows’.Agronomy Res.8, 323 - 26.
[43] Gonzalez W. D, Rubio C, Gutiérrez A, Pérez B, Hérnandez S. C. 2015. Dietary
Content and Evaluation of Metals in Four Types of Tea (White, Black, Red and

34
 Green) Consumed by the Populations of the Canary Islands. Pharm Anal Acta. 6,
1-10.
[44] Moll R, Davis B. 2017. Iron, vitamin B12 and folate. Medici. 45, 198 - 203.

[45] Farid S. M, Enani M. A, Wajid S. A. 2004. Determination of trace elements in

cow milk in Saudi Aribia. JKAU. Egn. Sci. 15, 131 - 140.

[46] Mertz W.E.D. 1986. Trace elements in human and animal nutrition (Vol. I and II,
5th ed, Academic Press, New York).

[47] MCDOWELL L.R. 1992. Minerals in animal and human nutrition. Acad. Press,
Inc., San Diego, USA.

[48] Linn J. G. 1988. Factors affecting the composition of milk from dairy cows. In:
Designing foods: animal product options in the marketplace. National Research
Council (US) Committee on Technological Options to Improve the Nutritional
Attributes of Animal Products.

[49] Angela G. P, Giuseppa D. B, Vincenzo L. T, Rossana R, Giacomo D. 2013. Non-

toxic and potentially toxic elements in Italian donkey milk by ICP-MS and
multivariate analysis. J. Food Compo and Analysis.31, 161-172.

[50] Khan S, Cao Q, Zheng Y. M, Huang Y. Z, Zhu Y. G. 2008. Health risks of heavy
metals in contaminated soils and food crops irrigated with wastewater in Beijing,
China. Environ. Pollut. 152, 686 - 692.
[51] Maas S Lucot E, Gimbert F, Crini N, Badot P. M. 2011. Trace metals in raw
cow’s milk and assessment of transfer to Comte´ cheese. Food Chem. 129, 7 - 12.
[52] Tassew B, Ahmed H, Rao V. M. 2014. Determination of Concentrations of
Selected Heavy Metals in Cow’s Milk: Borena Zone, Ethiopia. J. Health Sci. 4,
105 - 112.
[53] Alexander P, Ali B. F, Lawrance L. 2018. Determination of Micro and Macro
Minerals and Some Biochemical Parameters in Fresh Cow Milk from Different
Locations in Maiduguri Metropolis, Borno State, Nigeria.Asian Food Sci. J. 3, 1 -
7.

35
[54] Ogabiela E. E, Udiba U. U, Adesina O. B, Hammuel C, Ade A. F. A, Yebpella G.
G, Mmereole U. J, Abdullahi M. 2011. Assessment of metal levels in fresh milk
from cows grazed around Challawa Industrial Estate of Kano, Nigeria. J. Basic
Appl. Sci. Res. 1, 533 - 538.

[55] ATSDR .2004. Agency for Toxic Substance and Disease Registry. Lead Toxicity:
How Are People exposed to lead, Achieved Prom the Original.

[56] Bates C. J, Prentice A. 1996. Vitamins, minerals and essential trace elements.
Drugs and Human Lactation 533 - 607.
[57] Lajunen L. H, Perämäki P. 2004. Spectrochemical analysis by atomic absorption
and emission. Cambridge UK, Royal Society of Chemistry. 6 - 8.
[58] Eka N, Retno S, Rohman A. 2012. Validation and quantitative analysis of
cadmium and lead in snake fruit by flame atomic absorption spectrophotometry.
In .Food Res. J. 19, 937 - 940.
[59] Long X. 2006. Hyphenating multi-syringe flow injection lab-on-valve analysis
with atomic fluorescence spectrometry for on line bead injection pre
concentration and determination of trace levels of hydride forming elements in
environmental samples. Anal. Chem. 8290 - 8298.
[60] Rao M. V. H, Murthy A. C. H. 2017. Determination of Concentrations of
Selected Heavy metals in Cow’s Milk: Dodoma Urban District, Tanzania,Vegi
Maheswara Rao. Int. J. Eng. Res and Appli. 7, 76 - 84.

[61] WHO/FAO report. 1996. Trace elements in Human nutrition and health.

[62] Zamberlin S, Antunac N, Havranek J, Samaržija D. 2012. Mineral elements in

milk and dairy products. Mljekarstvo.62, 111 - 125.

36
 7. Appendix A: Output for ANOVA
ANOVA

Sum of Squares df Mean Square F Sig.

Between Groups .028 2 .014 24.064 .000

Cu Within Groups .014 24 .001

Total .042 26
Between Groups 141.381 2 70.690 155.043 .000
Fe Within Groups 10.943 24 .456
Total 152.323 26
Between Groups .090 2 .045 295.240 .000
Mn Within Groups .004 24 .000
Total .094 26
Between Groups 7.508 2 3.754 79.027 .000
Zn Within Groups 1.140 24 .048
Total 8.648 26
Between Groups .202 2 .101 94.194 .000
Pb Within Groups .026 24 .001
Total .228 26
Between Groups 533.408 2 266.704 114.791 .000

Mg Within Groups 55.761 24 2.323

Total 589.170 26

37
Dependent Variable (I) kebeles (J) kebeles Mean Difference Std. Error Sig. 95% Confidence Interval

(I-J) Lower Bound Upper Bound

Mensibu -.0144444 .0113990 .217 -.037971 .009082

debrezit
*
Ateshimo -.0745556 .0113990 .000 -.098082 -.051029

Debrezit .0144444 .0113990 .217 -.009082 .037971

Cu mensibu
*
Ateshimo -.0601111 .0113990 .000 -.083637 -.036585

Debrezit .0745556* .0113990 .000 .051029 .098082

ateshimo
*
Mensibu .0601111 .0113990 .000 .036585 .083637

Mensibu .1630000 .3183080 .613 -.493955 .819955

debrezit
*
Ateshimo -4.7706667 .3183080 .000 -5.427622 -4.113711

Debrezit -.1630000 .3183080 .613 -.819955 .493955

Fe mensibu
*
Ateshimo -4.9336667 .3183080 .000 -5.590622 -4.276711
*
Debrezit 4.7706667 .3183080 .000 4.113711 5.427622
ateshimo
*
Mensibu 4.9336667 .3183080 .000 4.276711 5.590622

Mensibu -.0002222 .0058237 .970 -.012242 .011797

debrezit
*
Ateshimo -.1226667 .0058237 .000 -.134686 -.110647

Debrezit .0002222 .0058237 .970 -.011797 .012242

Mn mensibu
*
Ateshimo -.1224444 .0058237 .000 -.134464 -.110425
*
Debrezit .1226667 .0058237 .000 .110647 .134686
ateshimo
*
Mensibu .1224444 .0058237 .000 .110425 .134464

Mensibu .1115556 .1027428 .288 -.100495 .323606

debrezit
*
Ateshimo -1.0586667 .1027428 .000 -1.270717 -.846616

Debrezit -.1115556 .1027428 .288 -.323606 .100495

Zn mensibu
*
Ateshimo -1.1702222 .1027428 .000 -1.382273 -.958172
*
Debrezit 1.0586667 .1027428 .000 .846616 1.270717
ateshimo
*
Mensibu 1.1702222 .1027428 .000 .958172 1.382273
*
Mensibu .1050000 .0154541 .000 .073104 .136896
debrezit
*
Ateshimo .2121111 .0154541 .000 .180215 .244007
*
Debrezit -.1050000 .0154541 .000 -.136896 -.073104
Pb mensibu
*
Ateshimo .1071111 .0154541 .000 .075215 .139007
*
Debrezit -.2121111 .0154541 .000 -.244007 -.180215
ateshimo
*
Mensibu -.1071111 .0154541 .000 -.139007 -.075215
*
Mensibu 10.8000000 .7185468 .000 9.316992 12.283008
debrezit
Ateshimo 6.5922222* .7185468 .000 5.109215 8.075230
*
Debrezit -10.8000000 .7185468 .000 -12.283008 -9.316992
Mg mensibu
Ateshimo -4.2077778* .7185468 .000 -5.690785 -2.724770
*
Debrezit -6.5922222 .7185468 .000 -8.075230 -5.109215
ateshimo

38
39