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Estimated daily intake and health risk of heavy metals

by consumption of milk
ab

Amir Ismail , Muhammad Riaz , Saeed Akhtar , Tariq Ismail , Zulfiqar Ahmad & Muhammad
a

Saad Hashmi
a

Department of Food Science & Technology, BahauddinZakariya University, Multan-Pakistan

Department of Food Science, University of Massachusetts Amherst, Amherst-USA

University College of Agriculture and Environmental Sciences, Islamia University

Bahawalpur-Pakistan
Accepted author version posted online: 27 Aug 2015.

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To cite this article: Amir Ismail, Muhammad Riaz, Saeed Akhtar, Tariq Ismail, Zulfiqar Ahmad & Muhammad Saad Hashmi
(2015): Estimated daily intake and health risk of heavy metals by consumption of milk, Food Additives & Contaminants: Part
B: Surveillance, DOI: 10.1080/19393210.2015.1081989
To link to this article: http://dx.doi.org/10.1080/19393210.2015.1081989

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Publisher: Taylor & Francis

Journal: Food Additives & Contaminants: Part B
DOI: 10.1080/19393210.2015.1081989

Estimated daily intake and health risk of heavy metals by consumption of milk

Department of Food Science & Technology, BahauddinZakariya University, Multan-Pakistan

2

Department of Food Science, University of Massachusetts Amherst, Amherst-USA

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University College of Agriculture and Environmental Sciences, Islamia University Bahawalpur-Pakistan

Abstract

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Cd, Co, Pb, Cu and Ni were analyzed in 480 milk samples by flame atomic absorption spectrophotometry. Mean
concentrations were 0.001, 0.061, 0.014, 0.738 and 0.028 mg/kg, respectively. Mean concentration of Cd was very
low, whereas Cu exceeded the maximum limits established by the Codex Alimentarius. In some areas the Pb
concentration exceeded the maximum limit as set by the Codex Alimentarius. The highest estimated daily intake of
Cu calculated for male infants (1-3 year) was 33.534 g/kg bw/day, while the lowest EDI was recorded for Cd
(0.004 g/kg bw/day) in adult females above 16 year. The results suggested that Cu and Pb from milk in the
investigated areas may pose a health risk to the consumers.

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Keywords: Milk; Heavy metal; cadmium; lead; Pakistan.

Introduction

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Amir Ismail1,2, Muhammad Riaz* 1, Saeed Akhtar1, Tariq Ismail1, Zulfiqar Ahmad3 & Muhammad Saad
Hashmi1

The safety of food regarding contaminants such as heavy metals, aflatoxins and pesticide residues is of major
concern for researchers due to their deleterious impacts on human health. More attention is gained when these
contaminants are found in basic food items such as milk, which is most frequently consumed by the most vulnerable
age group. Heavy metals persist naturally in the environment. However elevated concentrations in the food chain are
primarily linked to manmade environmental pollution. Cadmium and lead are reported as the most toxic heavy
*

Corresponding Author
Dr. Muhammad Riaz
riaz@bzu.edu.pk
+923067905770

metals while over consumption of some essential mineral elements also can result in the poisonous effects (Unak et
al. 2007). Heavy metals are a threat for human health due to their potential damage to human cells and the skeletal
system, osteoporosis, kidney failure and lung and blood cancer (Arora et al. 2008; Kumar et al. 2007). The routes of
entry for these heavy metals are ingestion or inhalation (Mendil, 2006). The toxicity of heavy metals is dependent on
a number of factors, the most critical of which are ingestion rate and route, bioavailability, gender, age, excretion
rate and the chemical state of the heavy metal (Enb et al. 2009).

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Pakistan is one of the leading milk producers in the world with a total milk production of 37.86 million tons in 2012.
Milk is mainly obtained from buffalos (62.5%), cows (35.4%) and goats (2.1%), while most of the milk is produced
in rural areas. Per capita availability of milk in Pakistan in 2011 was 286 g/day (FAO, 2015a, b). In Pakistan only
4% of the milk is processed while the remaining 96% is used as fresh milk, mostly supplied by milkmen called
doodhies (Zia et al. 2011), who are responsible for the quality of the milk. Besides other contaminants (Bashir et
al. 2013), milk can also be a major source of heavy metals. However, proper implementation of good quality
practices at dairy farm levels and at each and every step of industrial processing can ensure food safety of milk and
milk products in Pakistan.

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Lactating animals accumulate heavy metals in milk if they are exposed to high concentrations of these metals
through feed or water. The consumption of metal contaminated milk by humans can cause serious health issues.
Increased food safety concerns call upon a study comprising of a complete quantitative evaluation for heavy metal
levels of milk in Pakistan. Therefore, milk samples from various areas of Multan city were analyzed for nickel,
copper, cadmium, lead and cobalt content by using flame atomic absorption spectrophotometry. Estimated daily
intake (EDI) of heavy metals was calculated on the basis of heavy metal content of milk samples, milk consumption
rate and body weight of an individual of various age groups.

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Material and Methods

Sampling Plan

A total of 480 milk samples (cow: 180, buffalo: 160, goat: 140) were collected directly from the farms during
Feburary-August 2014 in Multan city, Punjab Province, Pakistan. Multan city was divided into 4 zones (Fig. 1.),
setting Chungi No 9 as central point, while Zone 1 South west (n = 160), Zone 2 North west (n = 90), Zone 3
South East (n = 110) and Zone 4 North east (n = 120). Zone 1 comprises of the major industrial areas of Multan
city, while all other zones mainly encompas residential and market areas. Milk samples were collected in 250 ml
clean plastic bottles and immediately transported to the labs of the Department of Food Science & Technology,
Bahauddin Zakariya University, Multan in ice boxes and stored at -20C until further analysis. Prior to analysis the
samples were thawed at ambient temperature.

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Since ancient times milk has been regarded as an ideal diet for humans due to the presence of almost all macro and
micronutrients including proteins, fats, carbohydrates, minerals and vitamins (Dobrzanski et al. 2005). Milk and
milk products contribute 15% of the total food intake in European countries (Gonzlez-Montaa et al. 2012) and are
particularly significant not only for infants who completely rely on this food during the first few months of their life
(Ataro et al. 2008) but also for the elderly to prevent them from elevated risks of fractures and osteoporosis (Kira
and Maio, 2004). The presence of toxic elements like heavy metals in milk is therefore of prime concern to ensure
its healthy impacts on human life and is focused on by researchers globally (Aslam et al. 2010; Maas et al. 2011;
Ping et al. 2012; Pilarczyk et al. 2013; Suturovi et al. 2014). Heavy metals are transferred into milk when animals
are fed with metal contaminated fodder or drink water originating from industrial effluents or sewage waste.

Chemicals & Apparatus

The chemicals employed were all of analytical grade and purchased from Merck chemicals (Darmstadt, Germany),
unless otherwise stated. Metals were analyzed by using a flame atomic absorption spectrophotometer (Thermo

Scientific 3000 Series, Waltham, MA, USA). Standard solutions for different elements were purchased from CPA
chemicals (CPA chemicals ltd., Stara Zagora, Bulgaria). For sample digestion and drying, a hot plate (Lab Tech EH
35A plus, Beijing, China) and hot air oven (Memmert UNB 200, Munich, Germany) were used, respectively.
Double de-ionized water was used for dilution of samples and standard solutions.
Analytical Procedure

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Quality Control

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Blanks were analyzed and the recorded concentrations for various elements were below the limits of detection,
calculated as three times the standard deviation of twenty procedural blanks (3.3 SD/b). LODs for Pb, Ni, Cd, Cu
and Co were 0.5, 0.7, 0.1, 3.2 and 0.2 g/kg, respectively. The certified reference material used was NIST 1549
(National Institute of Standards and Technology, Gaithersburg, MD, USA), obtained from the IRMM (Institute for
Reference Materials and Measurements, Geel, Belgium) whose certified values were found very much close to the
observed values (table 2). All analyses were performed in triplicate and repeated when repeatability exceeded the
limit of 1%.
Estimated Daily Intake

The EDI was calculated by the method of Cano-Sancho et al. (2010). A food frequency questionnaire was designed
to calculate the daily intake of milk by the different age groups of Multan city. Five different age groups of both
male and female were surveyed. EDI values were calculated by the formula:

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EDI = Milk intake (kg/day) Heavy metal content in milk (g/kg)

Average individual weight (kg)
Statistical Analysis

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Statistix 8.1 software (Statistix Inc., Florida, USA) was used for statistical analysis of the data obtained for each
parameter. For comparison purposes the data were subjected to one way analysis of variance (ANOVA) followed by
a LSD (Least Significant Difference) test. The differences were considered statistically significant at the probability
level of P < 0.05. Mean values and the measurement uncertainty (standard deviations) were computed with
Microsoft Excel 2007.

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Milk samples were digested by the wet digestion method of Richards (1968). Briefly, 1 ml milk sample and 10 ml
HNO 3 were mixed in a 100 ml flask. The mixture was heated for 20 min at 80 C, cooled at ambient temperature,
followed by addition of 5 ml of HClO 4 . The mixture was again heated, at 180 C, until the solution became clear
and the volume decreased to 2-3 ml. Finally, the solutions were diluted to 50 ml with double distilled water. Metals
were analyzed in an atomic absorption spectrophotometer by employing an air and acetylene flame. The operating
parameters are described in Table 1.

Results and Discussion

The heavy metal content of milk samples collected from different areas of Multan city is presented in Table3.
Statistical analysis showed significant differences in heavy metal concentrations from various zones as well as
among different species. Zone 1 (industrial zone) was the most contaminated zone, while buffalo milk had the
highest concentrations, followed by cow and goat samples. In the current study, milk samples were collected directly
from the farms. Therefore the chances of indirect contamination through utensils, air and milk processing machines
are minimal and most likely contamination sources are fodder and drinking water for the animals.

Cd concentrations were below the limit of detection (<0.0001 mg/kg) in milk samples from zone 2 and zone 3 of all
animal species, while it was highest in goat milk samples from zone 4 (0.004 mg/kg). The maximum tolerance limit
for Cd in milk proposed by the International Dairy Federation (IDF, 1979) is 0.0026 g/g. Comparing our results

with the standard limit, only cow and goat milk samples from zone 1 and zone 4, respectively were found slightly
higher than the maximum limit. The mean concentration of cadmium in the present study (0.001 g/g) is also lower
then earlier reported in Poland (0.0036 mg/kg) by Pilarczyk et al. (2013), in Egypt (0.006-0.018 mg/kg) by Sayed et
al. (2011), in Spain (0.002 mg/kg) by Gonzlez-Montaa et al. (2012) and in Iran (0.002 mg/kg) by Rahimi (2013)
and Najarnezhad et al. 2015 (0.007 mg/kg in bovine milk ). However, all samples from zone 4 were contaminated
with cadmium indicating the presence of possible contamination sources of fertilizer applications, coal production
and burning fossil fuels (ukowska and Biziuk, 2008).

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Nickel is an essential mineral element but is also categorized as a toxin if its level exceeds certain limits. It acts as an
activator or cofactor for a number of enzymes as well as hormones (Belitz et al. 2004), while excessive intake may
lead to cell damage, alteration of enzyme and hormone activities and may also effect bioavailability of other mineral
elements. The average Ni concentration found in this study was 0.027 mg/kg. Maximum nickel concentration (0.069
mg/kg) was found in buffalo milk of zone 4, whereas it could not be detected in goat milk (<0.0007 mg/kg) from the
same zone. Average nickel concentration reported by Enb et al. (2009) in cow and buffalo milk was 0.004 mg/kg
and 0.006 mg/kg, respectively, which were lower then our study. Otherwise, results of the current study are much
lower than those reported earlier by Anetta et al. (2012) in bovine milk (1.01 mg/kg), indicating less chances of
nickel toxicity through milk consumption for people of Multan (Pakistan), when compared to Nitra (Slovakia).
Average nickel concentration in present study is also less then found in Zagreb (0.06 mg/kg) as reported by Vahcic
et al. (2010). The possible reason for the low nickel level in milk in the present study could be the collection of milk
samples directly from the animal farms rather than at markets or lower levels of nickel contamination in feed and
water of the animals as well as in the local environment.

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Copper is an essential element for normal human growth, but becomes a toxin at high levels. Mean concentration of
copper in bovine milk estimated in the present study was 0.738 mg/kg, with a range of 0.244-1.163 mg/kg. All milk
samples analyzed in the present study exceeded the maximum limit of IDF (1979). The short term exposure of
higher doses of copper is harmless to the human body, however elevated and prolonged exposure may disturb the
immune system (Turnlund et al. 2004). Higher doses of copper up to 4-6 mg/kg are reported to cause gastrointestinal
disorders, however all the milk samples were found below this range (Barn et al. 2014). Mean copper concentration
in the milk samples from Multan city is lower than that reported earlier from Egypt (1.45 mg/kg) by Malhat et al.
(2012) and in Slovakia (3.90 mg/kg) by Anetta et al. (2012), but higher than reported in Italy (0.34 mg/kg) by Licata
et al. (2012). Elevated Cu levels in milk samples indicate possible threats of gastrointestinal disorders especially in
infants due to their higher milk intake rates.

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The mean concentration of Pb found in the present study was 0.014 mg/kg, which is below the standard limit of 0.02
mg/kg as set by the Codex Alimentarius Commission (2011). However, mean Pb values of bovine milk samples of
zone 1 and zone 2 and buffalo milk samples from zone 4 were slightly higher than this limit. Increased lead content
in milk is a serious threat for the health of consumers as lead may interrupt the nervous and circulatory systems as
well as several other organs of the body (Malhat et al. 2012). In line with our study Kazi et al. (2009) also reported
lead values higher than the standard limit in milk samples collected from Hyderabad city of Pakistan. In a study
conducted in Nigeria (Jigam et al. 2011) the level of Pb was in the range of 0.16 - 0.62 mg/kg, which is much higher
then in our study. Mean Pb values of the present study are almost in line with earlier bovine milk lead concentrations
of 0.013 mg/kg as reported in Iran by Najarnezhad and Akbarabadi (2013). Elevated Pb levels might be due to the
usage of canal water for irrigation of fodder as well as drinking of canal water by animals, which is common
practice in Pakistan. The canal water has already been reported before to have elevated lead levels in Multan city
(Ismail et al. 2014). Use of waste water and pesticides application on animal fodder is also a potent source of Pb
contamination in animal milk (Hafez and Kishk, 2008; Jan et al. 2009).

Cobalt is a part of vitamin B12 and is essential for nucleic acid formation. Excessive intake may result in reduced
appetite, weight loss, dermatitis and cancer (Andrew, 1965; Basketter et al. 2003). Available data regarding the
prevalence of Co in milk are scarce. Mean cobalt concentration measured in the present study was 0.061 mg/kg,
ranging between 0.015 and 0.137 mg/kg. The maximum level was measured in buffalo milk from zone 2, while the
minimum was found in goat milk from zone 4. Mean values from the present study were almost in line with those
reported earlier by Enb et al. (2009), while mean levels are much lower than those reported earlier from the
unpolluted areas of India (0.19 mg/kg) by Patra et al. (2008).
Estimated Daily Intake

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Conclusion

Acknowledgement

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In this survey most heavy metal levels in milk were within safe limits, except for copper which consistently
exceeded the safety limit. Additionly, lead and cadmium were also excessive in some areas. EDI data of heavy
metals due to milk consumption in the investigated area of Pakistan indicated that mean levels of the studied heavy
metals are within in acceptable RDA ranges. Prevention of excessive lead, cadmium and copper in milk demands
strict regulatory action for control and monitoring of animal feed and water by the health authorities.

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This study is a part of the PhD research of Mr. Amir Ismail, carried out under the supervision of Dr. Muhammad
Riaz, Department of Food Science & Technology, BahauddinZakariya University, Multan, Pakistan. The Higher
Education Commission Islamabad-Pakistan is highly acknowledged for financial aid under project number 20-1932,
titled Safety Status of Street Vended Raw Milk in Southern Punjab.

Conflict of Interest
The authors declare no conflicts of interest.

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EDI data for various heavy metals were in the order Cu > Co > Ni > Pb > Cd. The obtained data (Table 4) show that
children at the age of 1-3 year had the highest daily intake, while above the age of 16 had the lowest risk. Possible
factors contributing to daily intake of heavy metals are the quantity of milk consumption and the mean body weight
(bw). Highest daily intake was calculated for copper (33.534 g/kg bw/day), while the lowest value was recorded
for cadmium (0.004 g/kg bw/day). In a study conducted in Egypt by Hafez and Kishk (2008), EDI values for Pb
and Cd for infants (<1 year) by milk consumption were in the ranges of 0.4-0.82 and 0.004-0.011 g/kg bw/day,
respectively. EDI values for Pb in Egypt were almost in parallel with our data, while those for Cd were slightly
lower. EDI values of Cd and Pb for adults reported by Salah et al. (2013) were 158.5 and 64.4 g/kg bw/day,
respectively, much higher then in our study. EDI values of Pb & Cd and Cu for infants in India reported by Tripathi
et al. (1999) were 1.1 and 0.01 g/kg bw/day, respectively. Compared to our study, EDI values for Pb are higher and
for Cd are lower. Recommended dietary allowance (RDA) values for Cd, Pb and Cu are 52-72, 429 and 2000-3000
g/day, respectively (Farid et al. 2004). Thus, EDI values of Cd, Pb and Cu in the present study are much lower
when compared to RDA values. However, several other dietary and non-dietary factors may also affect heavy metal
daily intake by Pakistan consumers, demanding a separate study from other possible routes.

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Metal

Wavelength (nm)

Fuel flow rate (L/min)

Bandpass (nm)

232
324.8
228.8
217
240.7

0.8-1.0
0.8-1.1
1.0-1.3
0.9-1.2
0.8-1.0

0.2
0.5
0.5
0.5
0.2

ce

pt

Nickel
Copper
Cadmium
Lead
Cobalt

ed

Table 1: Instrumental parameters for the investigated metals

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Tripathi RM, Raghunath R, Sastry VN, Krishnamoorthy TM. 1999. Daily intake of heavy metals by infants through
milk and milk products. Sci Total Environ. 227:229-235.

Table 2. Comparison of measured & certified reference material values (mg/kg)

Metal (CRM)
Cd (NIST 1549)
Pb (NIST 1549)
Co (NIST 1549)
Cu (NIST 1549)
Ni (CPA Chm.)

Certified Value
0.00050.0002
0.0190.003
0.00410.002
0.070.008
0.0030.001

Observed Value
0.000450.0001
0.0180.002
0.00390.001
0.0640.007
0.00260.002

Recovery (%)
90.0
94.7
95.1
91.4
86.7

Table 3: Heavy metal content (mg/kg) in milk samples collected from different areas of Multan city
Animal

Co
c

Pb
a

Cu

Ni

ab

0. 1270.030
0. 0750.004bc
0. 0360.003def

0. 0130.004
0. 0340.004a
0.0090.001cd

1. 0540.05
1. 1630.04a
0. 6930.05fg

0. 0680.007a
0. 0320.002c
0.0120.003e

0. 1370.025a
0. 0420.003cdef
0.0160.004f

0.0120.004c
0. 0220.003b
0.0090.001cd

0. 9650.05bc
0. 6050.04g
0. 2440.03i

0. 0250.003d
0. 0450.004b
0.0140.002e

Bufalo
Cow
Goat

Zone 3

Bufalo
Cow
Goat

<0.0001e
<0.0001e
<0.0001e

0. 0830.003b
0. 0540.005bcde
0. 0250.004ed

0.0060.002d
<0.0005e
0.0130.002c

0. 9370.02cd
0. 7520.04ef
0. 4530.04h

0.0140.002e
0.0040.0005f
0.0140.001e

Zone 4

Bufalo
Cow
Goat

0.00140.0005d
0.00130.0003d
0.00430.0002a

0. 0570.004bcde
0. 0630.003bcd
0.0150.003f

0. 0340.004a
0.0120.002c
<0.0005e

0. 8210.03e
0. 8340.03de
0. 3400.02i

0. 0690.003a
0. 0320.003c
<0.0007f

cr
ip

Zone 2

0.00230.0002
0.00330.0001b
<0.0001e
<0.0001e
<0.0001e
<0.0001e

us

Bufalo
Cow
Goat

Cd

ce

pt

ed

M
an

Total Mean
0.001
0. 061
0.014
0. 738
0. 028
Values expressed as mean standard deviation (=3). Data within a column with different letters are significantly
different (p < 0.05).

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Area
Zone 1

Table 4: Estimated Daily Intake (g/kg bw/day) of Metals through Milk by Different Age Groups
Milk Intake
(kg/day)

30
27
36
38
23
26
37
42
132
104

12.53
10.21
19.89
15.38
29.79
24.66
52.93
43.57
67.32
53.45

0.569
0.395
0.667
0.377
0.344
0.345
0.260
0.240
0.308
0.195

Cd
0.049
0.042
0.036
0.027
0.012
0.015
0.005
0.006
0.005
0.004

Co
2.773
2.362
2.047
1.497
0.705
0.854
0.300
0.336
0.279
0.223

Pb
0.631
0.538
0.466
0.341
0.161
0.195
0.069
0.077
0.064
0.051

Cu
33.534
28.569
24.764
18.101
8.527
10.331
3.627
4.068
3.379
2.694

Ni
1.259
1.073
0.930
0.680
0.320
0.388
0.136
0.153
0.127
0.101

us

Male, 1-3
Female, 1-3
Male, 4-5
Female, 4-5
Male, 6-9
Female, 6-9
Male, 10-15
Female, 10-15
Male, > 16
Female, > 16

g/kg bw/day

Mean
Weight (kg)

cr
ip

No of
Consumers

ce

pt

ed

M
an

Figure1.Map showing the Location of Multan city in Punjab Province of Pakistan and the division of
Multan city in different zones

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Age Group

10