Essential and toxic metals in cow’s whole milk

Volume 1, Issue 1, pp 12-19; December 2012.
Online International Journal of Food Science
©2012 Online Research Journals
Full Length Research
Available Online at http://www.onlineresearchjournals.org/OIJFS
Essential and toxic metals in cow’s whole milk
from selected sub-cities in Addis Ababa, Ethiopia
AG Dawd1, TB Gezmu1, and GD Haki2*
1
Addis Ababa University, Center for Food Science and Nutrition, Ethiopia.
Professor of Food Engineering and Postharvest Technology and Nutrition, Department of Food Science and
Technology, University of Botswana, Botswana College of Agriculture, Private Bag 0027, Gaborone, Botswana.
2
Downloaded 18 November, 2012
Accepted 13 December, 2012
The level of essential (Fe and Zn) and non essential (Cd and Pb) metals in whole cow milk was
determined by Flame Atomic Absorption Spectrophotometer (FA-AAS).Whole cow milk was sampled (n
= 32) from dairy farms of Akaki-kality, Bole, Kolfe- keraniyo, and Yeka subcities in Addis Ababa.
Statistical analysis was performed using SPSS version 17. Significant differences between means were
subjected to one way ANOVA using Duncan’s multiple range test (P < 0.05). The average concentrations
of the elements were Fe (1.213±0.077 mg/kg), Zn (4.923±0.277 mg/kg), Cd (0.100±0.006 mg/kg) and Pb
(0.998±0.251 mg/kg). The levels of toxic metals (Cd and Pb) were beyond the acceptable limit which can
be a potential health concern for consumers.
Keywords: Iron, zinc, cadmium, lead, whole cow milk, FA-AAS.
INTRODUCTION
Milk is considered as a nearly complete food and it is the
main constituent of the daily diet since it is a good source
of protein, fat and major minerals [1,2]. On the other
hand, due to low contents of Fe and Zn, prevalence of
zinc deficiency [3] and iron deficiency anemia [4] were
associated with the intake of whole milk based formula,
and fortification of milk and milk products with these
micronutrients is taken as one preventive strategy.
Many reports indicate the presence of toxic metals in
milk [5-9]. Cd and Pb are amongst the elements that
have caused most concern in terms of adverse effects on
human health [8]. This is because they are readily
transferred through food chains and are not known to
serve any essential biological function. Children have
been shown to be more sensitive to Cd and Pb than
adults and the effects are cumulative [10].
glucose, amino acids, and small peptides are lost in the
urine. Once Cd accumulates in tissues it can not be
removed safely by chelation therapy with out causing kidney
damage [11]. Cadmium affects calcium metabolism and
skeletal changes resulting from calcium loss and ends in
a decrease bone mineral density [12,13].
Cadmium
Iron
Regular absorption of cadmium (Cd) causes damage to
the proximal renal tubules and calcium, phosphorous,
Over 65% of the iron (Fe) content is found in
haemoglobin, whose major function is to transport
oxygen and carbon dioxide. In addition, iron is part of the
composition of the myoglobin molecule of muscle tissue
and acts as an enzyme reaction cofactor in the Krebs
*E-mail: [email protected] or [email protected]; Tel:
+26774925819.
Lead
Lead (Pb) is toxic to the blood, nervous, urinary, gastric
and genital systems [1,11,14]. Furthermore, it is also
implicated in causing carcinogenesis, mutagenesis and
teratogenesis in experimental animals [1]. Lead readily
crosses the placenta and there is evidence that exposure
to high levels increases the risk of spontaneous abortion,
miscarriage and stillbirth [11].
Dawd et al.
13
cycle, and in the synthesis of purines, carnitine, collagen
and brain neurotransmitters. Iron is also present in the
composition of flavoproteins and heme protein catalase
and peroxidase. These enzymes are responsible for the
reduction of the hydrogen peroxide produced in the body
[15]. Fe is present in the brain from very early in life, when it
participates in the neural myelination processes [16].
added in to the 500 ml polyethylene bottle. Samples were
kept in an ice box and transported to Ethiopian Health
and Nutrition Research Institute. On arrival in the
o
laboratory the samples were kept in -20 C until freeze
drying.
Zinc
Lyophilization
Many diverse biochemical roles of zinc (Zn) have been
identified. These include roles in enzyme function, nucleic
acid metabolism and cell signalling. And zinc is essential
for physiological processes including development, lipid
metabolism, brain and immune function [17]. It is also
crucial for normal development and function of cells
mediating nonspecific immunity [16].
Between 2007 and 2050 the world population is
projected to increase from 6.7 to 9.2 billion, and most of
this growth will occur in urban areas of less developed
countries [18]. At the time of urbanization, food insecurity
and environmental pollution are the two problems urban
inhabitants will face. In such phenomenon, most
countries and policy makers use urban and peri urban
agriculture as a food security option including dairy
production. This study assessed the extent of heavy
metals in milk products of Addis Ababa, Ethiopia.
The frozen samples were placed in a freeze-drying unit of
O
Labconco USA working at the temperature of -45 C to O
-3
50 C and vacuum of 324 X 10 millibar until a constant
mass was achieved. The dried milk was crushed with the
tip of a plastic stirrer until a fine powder was obtained and
mixed thoroughly to maintain the homogeneity.
MATERIALS AND METHODS
Reagents
Atomic Absorption Spectroscopic standard solutions for
Fe, Zn, Cd and Pb were prepared by diluting the stock
solution. HClO4 (72%) and HNO3 (68%) UNI-CHEM, all of
analytical reagent quality were used for cleaning glasswares
and digesting milk samples throughout this work. Deionized
water of not more than 2µ Siemens/cm conductivity was
used for dilution and rinsing laboratory glass wares.
Sampling
Purposive sampling was used to select four sub cities
with significant dairy farms and background information
from previous studies on the presence of the heavy
metals of interest in soil, water [19] and vegetables [20]
which increases the likelihood of finding the elements of
interest in cow’s milk. Samples (n = 32) were collected
from dairy farms of four sub-cities namely: Akaki-Kality,
Bole, Kolfe-keraniyo, and Yeka of Addis Ababa as shown
in figure 1. The sampling bottles were soaked in 20%
nitric acid for 24 hours and rinsed with deionized water
before collection of raw milk in order to avoid possible
contamination. The udder of each cow was washed
before milking. Approximately 100 ml of milk samples
were collected from each cow and homogenized and
Sample Preparation
Wet ashing of milk samples
The freeze dried milk samples were digested by wet
ashing method on electric hot plates. A powdered milk
sample aliquot of 0.5g was accurately weighed and
quantitatively transferred to each 100 ml round bottom
flask and treated with 3.5 ml HNO3 and 2.0 ml HClO4
mixed and digested with Gallenkamp Kjeldahl Apparatus
for 4.30 hours. The digest was transferred to 50ml
volumetric flask and diluted with distilled-deionized water
to its mark. Blanks were subjected to similar sample
preparation and analytical procedure. All samples were
digested for triple run. The suitability of this pre-treatment
step was tested by determination of the recovery using
standard addition methods.
Sample analysis
The measurements of Fe, Zn, Cd and Pb were carried
out with Atomic Absorption Spectrophotometer (Specter
AA. 20 Plus) supplied by Varian Pty Ltd Australia at
Ethiopian Health and Nutrition Research Institute. Hallo
cathode lamps of the respected metals were used as a
radiation source. Air acetylene gas mixture was used as
source of flame. Maximum absorbance was obtained by
adjusting the Cathode lamps at specific slit and wave
lengths as indicated in Table 1.
Calibration curve and standard preparation
Standard aqueous solutions of different elements
supplied by SMM INSTRUMENTS (Pty) Ltd were used to
calibrate the Atomic Absorption Spectrophotometer. For
each of the metals; Fe, Zn, Cd and Pb four standards
1ppm, 1.5ppm, 2ppm and 2.5ppm were set for the
calibration. The calibration curves were drawn by using
linear regression analysis of the concentrations of the
standard solutions versus absorbance values. A new
calibration curve was plotted for each element every time
a new batch of milk samples was arranged for analysis.
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Online Int J Food Sci
Figure 1. Map of the study area.
Table 1. Instrumental conditions in the analysis of Cow’s whole milk from Akaki-kality, Bole, Kolfe-keraniyo
and yeka sub cities.
Parameter
Cd
Fe
Pb
Zn
Wave length(nm)
288.8
248.3
217.0
213.9
Slit width(nm)
0.5
0.2
1.0
1.0
The calibration curves obtained were fairly linear. The
calibration curves for Pb and Zn are shown in figures 2
and 3 respectively.
Method Validation
The reliability of the method used was validated by
studying the recovery of the particular metals using
standard addition method. The recovery percentage of
metals in the spiked samples was between 92% and
97%. All of the reported results were corrected taking into
account the recovery percentage. 8 blank samples were
Optimum working range (µg/ml)
0.02-3
0.06-15
0.1-30
0.01-2
analyzed in duplicate and the method detection limit was
calculated as (3.71  blank, n=8). The method detection
limit and the results of recovery percentage are
presented in Table 2.
Statistical analysis
The statistical analysis was conducted using statistical
package of SPSS version 17. Significant differences
between means were subjected to one way ANOVA
using Duncan’s multiple range test. The level of
significance was compared at P < 0.05.
Dawd et al.
15
Figure 2. Calibration curve for lead in the analysis of Cow’s whole milk from Akaki-kality, Bole, Kolfekeraniyo and yeka sub cities.
Figure 3. Calibration curve for Zinc in the analysis of Cow’s whole milk from Akaki-kality, Bole, Kolfekeraniyo and yeka sub cities.
RESULTS AND DISCUSSION
The average micronutrient and toxic metal concentration
in the cow whole milk from the different farms of the subcities are shown in Table 3. All the data obtained during
the analysis are presented on wet weight basis unless
otherwise indicated.
The concentration of both micronutrients and toxic
metals in the present study were found to decrease in the
following
order
Zn
(4.923±0.277mg/kg)
> Fe
(1.213±0.077mg/kg) > Pb (0.998±0.251mg/kg) > Cd
(0.100±0.006mg/kg) (Figures 4 and 5). Lead
concentrations of samples from Kolfe and Yeka were
significantly (p<0.05) higher compared to milk samples
collected from Akaki and Bole subcities. Cd is a metal
that is rightly considered as an industrial risk, however no
significant difference was observed in milk samples
collected from both industrial and non industrial areas.
The levels of iron in the milk did not differ significantly
(P>0.05) between subcities. Milk samples from Bole,
Kolfe and Yeka subcities were not significantly different
with respect to Zn concentration while Akaki was
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Online Int J Food Sci
Table 2. Method detection limit for whole cow’s milk samples (n=8) and recovery percentage of spiked samples.
Analytes
Fe
Zn
Cd
Pb
Amount in
samples
a
(mg/kg)
Amount of
added standard
(mg/kg)
Concentration
after spiking
b
(mg/kg)
Recovery
c
percentage
MDL(mg/kg)
1.16
5.53
0.11
1.153
.300
.500
.030
.300
1.405 ±0.01
5.846 ± 0.06
0.131 ± 0.003
1.366 ± 0.02
96.25 ± 0.92
97.50 ± 1.83
93.35 ± 2.19
94.05 ± 1.20
0.09
0.02
0.08
0.30
a
Mean value of three measurements
Values are mean ± S.D of three measurements
C
Values are mean ± S.D of percent recovery of three measurements
MDL, Method Detection Limit.
b
Table 3. Mean elemental concentration of whole cow’s milk from Akaki-kality, Bole, Kolfe-keraniyo and yeka sub
cities.
Sub city
Akaki
Bole
Kolfe
Yeka
Fe
a
1.285±0.55
a
1.274±0.38
a
1.131±0.28
a
1.165±0.23
Zn
b
4.537±0.39
a
5.124±0.60
ab
4.907±0.90
a
5.127±0.43
Cd
a
0.109±0.053
a
0.095±0.057
a
0.098±0.046
a
0.099±0.047
Pb
b
0.86±0.547
b
0.716±0.55
a
1.169±0.529
a
1.248±0.460
a-b
any two means in the same column not followed by the same letter are significantly different.
Figure 4. Concentration of selected essential elements in cow’s whole milk from dairy farms in Akaki-kality, Bole,
Kolfe-keraniyo and yeka subcities.
significantly different (P<0.05) from the rest but not with
Kolfe. The metal ion concentrations in milk depends on
number of factors influencing its secretion from the
mammary gland such as breed of the animal, season of
the year, feeding and factors related to animal handling
by human. Hence, in the present study the variation in
concentration of the Pb and Zn with respect to subcities
is under investigation in our laboratory.
Dawd et al.
17
Figure 5. Concentration of selected toxic elements in cow’s whole milk from dairy farms in Akaki-kality,
Bole, Kolfe-keraniyo and yeka subcities.
Table 4. Comparison of the elemental concentrations of cow’s whole milk in present study with the values in other studies.
Country
Zn
Fe
Cd
Pb
Reference
Egypt**
Ethiopia***
China**
Saudi Arabia***
Poland***
Italy**
Brazil***
India***
Ethiopia**
3.146±1.081
5.33
2.38±0.50
0.944±2.4
3.163±710.61
2.016
4.59
*
4.923±0.277
0.682±0.406
1.27
1.93±0.96
*
*
*
1.05
*
1.213±0.077
0.086±0.062
0.18
0.04±3.80
0.0047±0.2
*
0.02
*
0.23±0.02
0.101±0.005
0.066±0.056
2.63
0.028±11.23
0.0035±0.2
*
1.32
0.23
0.85±0.11
0.998±0.251
[1]
[23]
[2]
[22]
[21]
[5]
[16]
[8]
This paper
*Not reported **mg/Kg ***mg/L
Comparison of the results of the present study with
reported data
The content of mineral components and trace elements
including toxic ones in milk is determined by a variety of
factors, including mainly the content of a given element in
soil, water, air, veterinary drugs such as antimicrobials,
hormones,
antihelmentic
drugs
and
pesticides,
containers, processing and packaging materials, as well
as phenomena of inter element interactions [21].
Accordingly; Enb et al, [1] reported elemental
concentrations in whole cow milk as Fe: 0.682±0.406
mg/kg, Zn: 3.146±1.081 mg/kg, Cd: 0.086±0.062 mg/kg
and Pb: 0.066±0.056 mg/kg. Farid et al, [22] in his
investigation found Zn: 0.944±2.4 mg/L, Cd: 0.0047±0.2
mg/L, Pb: 0.0035±0.2 mg/L. Admasu et al, [23] collected
samples from farms out of Addis Ababa which are
delivered to the city found the concentration of these
metals as Fe: 1.25 mg/L, Zn: 5.33 mg/L, Cd: 0.18 mg/L,
Pb: 2.63 mg/L and are indicated in Table 4.
In comparison with the average mineral composition of
raw milk obtained by investigations in Italy [5], Egypt [1],
Poland [21], and Saudi Arabia [22] the milk samples in
the present study have higher Zn (4.923±0.277mg/kg),
Pb
(0.998±0.251mg/kg)
and
also
the
Fe
(1.213±0.077mg/kg) concentration is the highest except
milk from china (1.93±0.96mg/kg) as shown in Table 4.
Even though, dairy products are in general low in their
Zn and Fe content[24], results of the present study and
the study by Admassu et al, [23] show better
concentration of these nutrients in milk from Addis Ababa
than reports from other countries. However, considering
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Online Int J Food Sci
daily consumption of 60ml of fresh milk, samples of the
present study only provides 0.295mg and 0.072 mg of Zn
and Fe respectively per day while the recommended
value is 12-15mg/day of Zinc and 10 mg/day for male and
15mg/day for female of Fe respectively and hence
contributes very low amount of these elements per day.
At present, there are no maximum residual levels
(MRLs) for trace elements in milk set by Ethiopian Quality
and Standard Agency. Comparing the results with the
accepted limits both cadmium and lead are beyond the
limit which can be a potential health risk for consumers.
In general; soil fertilisation, vehicle exhaust, aerial
deposition, cattle manure, industrial waste, waste water
irrigation, and geogenic activities such as rock
weathering are major risk factors for the contamination of
soil, water, cattle fodder and then milk with potentially
toxic metals such as lead and cadmium [25-27]. It is,
thus, always important to consider these factors in the
record of toxic metals input to cow’s milk. In addition,
Cadmium contamination of food stuffs in different studies
was associated with application of inorganic fertilizers
[28-30] which is also a common agricultural practice in
Ethiopia and thus the relatively high level of cadmium in
milk of the present study. The level of zinc in the milk
samples of the present study show weak but a positive
significant correlation (R= 0.271) with the corresponding
cadmium concentration; and coexistence of cadmium
with zinc in nature [31] can also be the plausible
explanation for the relatively high Cd level. In addition,
Alemayehu [19] reported a high soil cadmium (0.7mg/kg)
content of Peacock farm which is one of the fodder
(grass) sources for cows in Bole dairy farms. The
persistence of high amount of lead from vehicular
emission of leaded gasoline and geogenic activities in
Addis Ababa soil [32,33] which could mobilize in to cattle
fodder and the very old water distribution pipe lines of the
city can account for the relatively high level of lead in
samples of present study samples.
Level of toxic metals in milk samples associated with
contaminated pasture with industrial influents and mining
area has been of great research interest. However,
concentration of these metals in milk could be high and
become a potential health concern due to geological and
human activities but role of industrial emission is minimal.
We recommend conduction of further studies on the level
of toxic metals taking larger number of milk samples from
other sub-cities
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