Environ Monit Assess (2018) 190:540
https://doi.org/10.1007/s10661-018-6811-3
The impact of strain and feed intake on egg toxic trace
elements deposition in laying hens and its health risk
assessment
Mohammad Hashemi & Abbas Sadeghi & Mahmoud Dankob & Majid Aminzare &
Mojtaba Raeisi & Hamid Heidarian Miri & Masoumeh Saghi
Received: 7 September 2017 / Accepted: 18 June 2018
# Springer International Publishing AG, part of Springer Nature 2018
Abstract The impact of strain or feed intake on food
trace elements and its health risk assessment is still
ambiguous, and therefore, available facts are rare.
Therefore, this study aimed to assess the effects of both
strains and feed intake on trace elements depositions to
egg, toxic heavy metals concentration, and health risk
assessment of egg consumption. In the current cross-
sectional study, the selected strains, including Shaver
White, Hy-Line W36, Bovanse White, Lohman LSL-
Lite, and Native laying hens of Khorasan Razavi prov-
ince, were examined. A total number of 50 samples of
eggs and 15 samples of their feed was purchased from
poultry farms. Yolk and white were separately analyzed.
M. Hashemi
Department of Nutrition, Faculty of Medicine, Mashhad
University of Medical Sciences, Mashhad, Iran
e-mail: Hashemimd@mums.ac.ir
A. Sadeghi
Food and Drug Office, Mashhad University of Medical Sciences,
Mashhad, Iran
e-mail: SadeghiA@mums.ac.ir
M. Dankob : M. Saghi (*)
Department of Environmental Health Engineering, School of
Health, Mashhad University of Medical Sciences, Daneshgah
Ave., Mashhad, Iran
e-mail: Saghim931@mums.ac.ir
M. Dankob
e-mail: DankoobM1@mums.ac.ir
Sample preparation was performed by wet digestion
followed by Inductively Coupled Plasma Mass Spec-
trometry. Trace metals Pb, As, Cd, Hg, Cr, and Ni were
detected. Statistical analysis was performed in Stata11.2
portable software. Although there was a significant dif-
ference in strains and feed, no significant difference was
observed in trace elements in egg contents (weight of
egg white and egg yolk). Mercury concentration in all
the samples was below the instrument detection limit. In
this study, the target hazardous quotients were below
one for all trace elements. Therefore, Iranian does not
experience the adverse health effects due to the con-
sumption of egg.
M. Dankob
Students Research Committee, Mashhad University of Medical
Sciences, Mashhad, Iran
M. Aminzare
Department of Food Safety and Hygiene, School of Public Health,
Zanjan University of Medical Sciences, Zanjan, Iran
e-mail: M.aminzare@zums.ac.ir
M. Raeisi
Department of Nutrition, Faculty of Health, Golestan University of
Medical Sciences, Gorgan, Iran
e-mail: Raeisi.mojtaba@yahoo.com
H. Heidarian Miri
Departments of Epidemiology and Biostatistics, School of Health,
Mashhad University of Medical Sciences, Mashhad, Iran
e-mail: Heidarianh@mums.ac.ir
H. Heidarian Miri
Social Determinants of Health Research Center, Mashhad
University of Medical Sciences, Mashhad, Iran
540 Page 2 of 13
Keywords Spiking recovery . Hazard quotient .
Estimated daily intake . ICP-OES
Introduction
There has been a rising global concern regarding the
environmental pollution in particular trace elements
and their adverse effect on food safety (Kaplan et al.
2011; Gebrekidan et al. 2013). Micropollutants can
be easily found in the air, soil, fertilizer, water, and
industrial processes (Tuzen et al. 2009). Having
positive and negative effects on human health and
the environment (Uluozlu et al. 2009), trace ele-
ments can be classified into three groups; essential
elements, non-essential, and probably essential. Es-
sential elements include iron, manganese, copper,
zinc, and selenium, while non-essential ones include
toxic elements such as arsenic, cadmium, lead, mer-
cury, and aluminum; and finally, probably essential
metals are cobalt, nickel, and vanadium (Uluozlu
et al. 2009). According to the U.S. Environmental
Protection Agency, Cd, Hg, and Pb were introduced
as the top hazardous materials (Salar-Amoli and Ali-
Esfahani 2015).
The egg is among the most inexpensive and highly
nutritious food and it is also a good source of protein,
lipids, vitamins (B12, D, etc.), and minerals (Applegate
2000; Herron and Fernandez 2004; Kiliç et al. 2002).
Therefore, it should be included in everybody’s daily
diet (Kiliç et al. 2002). Strains and genetic greatly affect
the egg’s quality, specifically the egg’s lipids and com-
position (Scheideler et al. 1998), yolk and albumen
proportion, albumen height, etc. (Singh et al. 2009;
Scott and Silversides 2000). Besides, egg quality has a
great impact on consumer’s demand and marketing
(Singh et al. 2009). Polluted feed like fish by-products
or drinking waters (Abbas et al. 2008) are the most
important sources of heavy metals being fed to laying
hens (Vincevica-Gaile et al. 2013; Shah et al. 2010).
Toxic heavy metals can change the species population
because they affect reproductive organs, biochemical
response, egg-shell thickness, etc. (Dauwe et al. 2004).
The organic compounds of an egg like proteins and
lipids provide a suitable substrate for the accumulation
of heavy metals. During chronic exposures, toxic metals
are accumulated in different body organs causing chron-
ic disorders (Iwegbue et al. 2008; Saei-Dehkordi et al.
2010; Fallah et al. 2011).
Environ Monit Assess (2018) 190:540
Hence, continuing research on eggs appears fully
justified. Accordingly, many researchers have fo-
cused on either the trace element assessment of the
egg (Vincevica-Gaile et al. 2013; Van Overmeire
et al. 2009; Uluozlu et al. 2009; Domingo 2014) or
the productive performance of hens based on the
strains (Singh et al. 2009; Scheideler et al. 1998;
Ershad 2005; Ali et al. 2016). However, there has
been no paper focusing on the relationship between
laying hens and toxic heavy metals transition into
the egg. Therefore, this paper was conducted to
assess (1) the effects of both strain and feed intake
on trace elements depositions to egg and (2) health
risk assessment of egg consumption.
Materials and methods
Study area and sampling
In the current cross-sectional study, more common
strains, including Shaver (Shaver White), Hy-Line
(Hy-line W36), Bovanse (Bovanse White), LSL
(Lohman LSL-Lite), and native laying hens of
Khorasan Razavi province (Gallus gallus
domesticus), were examined. A total number of 50
egg samples (20 samples per strain) were collected;
both yolk and white were separately analyzed. In
addition, 15 samples of the feed intake (three rep-
licates from each strain) were obtained from poultry
farms near Mashhad (Latitude/longitude: 36°18′56″
N59°34′04″E), Razavi Khorasan province, Iran
(Fig. 1), during February to April 2016. The eggs
of native hen were purchased from Modifying Cen-
ter of Native Hens of Khorasan Razavi province,
Mashhad, Iran.
The egg and feed samples were taken at the same
time. Samples were immediately brought to the cen-
tral laboratory of the Ferdowsi University of Mashhad
and the analytical preparation was carried out imme-
diately. When there were delays in the preparation
process, the samples were refrigerated at 4 °C and
covered with aluminum foil before preparation pro-
cess began. In order to avoid possible chemical con-
tamination, chemically stable glass tools were used
instead of metal tools. Prior to use, all the glassware
and plastics were rinsed with diluted HNO3 (1:9)
followed by distilled water (Vincevica-Gaile et al.
2013; Scott and Silversides 2000).
Environ Monit Assess (2018) 190:540 Page 3 of 13 540

Fig. 1 The study area (Mashhad

city) located in Razavi Khorasan
province, North-East of Iran

Sample preparation and trace element analysis curves supplied from Carlo Ebra reagents. Each batch of
10 samples had one additional procedure blank. The
Egg samples were kept in ambient air temperature until operating limits for trace element determination by
reaching a stable condition. Then 5.0000 g (± 0.0005 g) ICP-OES were shown in Table 1. The Limit of Detec-
of the fresh weight yolk, albumen, and feed were care- tion (LOD) and the Limit of Quantification (LOQ) and
fully weighed on an analytical balance (MFD BY 8&D
Co LTD-JAPAN) in a glass beaker using powder-free
nitrile gloves. First, 10 ml of H2O2 (Merck, Darmstadt, Table 1 The operation parameters for trace element determination
Germany) and 10 ml of HNO3 65% (Merck, Darmstadt, by ICP-OES
Germany) were added; then the solution was held over- Instrument SPECTRO ARCOS
night followed by being heated at 160 °C on a heater
(IKA C-MAG HS10-GERMANY) until the pure liquid Replicates 3
was obtained. The round clock glass was used to avoid Plasma power (W) 1400
vaporization of some volatile metals, like Hg, As, etc., Spray chamber Scott
during heating process (Vincevica-Gaile et al. 2013). Nebulizer modified Cross flow
The pure liquid was filtered by ashless Whatman filter Nebulizer flow (L per min) 1
papers, then poured into the labeled polypropylene Plasma Torch Fixed, 2.5 mm
containers. Measure time parameter (s) 28
The toxic trace elements: lead (Pb), chromium (Cr), Auxiliary flow (L per min) 0.060
arsenic (As), cadmium (Cd), nickel (Ni), and mercury Coolant flow (L per min 12
(Hg) in feeds and eggs were determined using Induc- Global preflush Fast speed 63, normal
tively Coupled Plasma Mass Spectrometry ICP-OES Parameter (rpm) Speed 30
(SPECTRO ARCOS-GERMANY). Multi-element Pump speed (rpm) 25
standard solutions were used for establishing calibration
540 Page 4 of 13 Environ Monit Assess (2018) 190:540

the results for spiking recoveries for each trace element

were calculated as shown in Table 2. Recoveries are Estimated Daily Intake ðEDIÞ
nearly quantitative in the range of 89–103% for all the ¼ Mean metal Concentration ðmg per LÞ
sample types.
 ½Daily egg consumption per body weight

The health risk assessment of egg In the current study, determining the metals concen-
tration (based on the fresh weight, mg per kg) was
The estimated daily intake (EDI, μg per kgBW.day) was carried out individually for white and yolk.
calculated using the following formula: Mean weights formula is as follows:

Mean weights ¼ Total selected metal concentration in egg white or egg yolk

¼ ðthe weight of white  metal content in white or yolk ðmg per kgÞÞ per the weight of the egg white or egg yolk:

People in Iran consume one egg per day per capita
but in this study, we have used the weight of each white
and yolk samples, not the standard weight of an egg.
The average body weight of a normal adult and child is
60 and 27 kg respectively, which was assumed based on
Shaheen et al. literature (Shaheen et al. 2016; Ki et al.
2017).
The U.S. EPA had described the following method
for the Target Hazard Quotient (THQ) for the noncarci-
nogenic disease (Wang et al. 2005):
study). The standard RfD values are as follows: Cd =
0.001, Pb = 0.004, As = 0.0003, Hg = 0.0005, Cr = 1.5,
and Ni = 0.02 mg per kgbw.day. Below, THQ value
shows an insignificant health risk because of egg con-
sumption (Wang et al. 2005).
The total THQ (TTHQ) of the heavy elements in food
was calculated by the mathematical sum of each indi-
vidual metal THQ value (Hallenbeck 1993):
TTHQ individual foodstuff

¼ THQtoxicant1 þ THQtoxicant2 þ … þ THQtoxicant

THQ ¼ ½ðE F  ED  FIR  CÞ=ðRfD  WAB  TA Þ

 10−3 :
where EF is the exposure frequency (356 days per
year), ED is the exposure duration (70 years) which
described the average lifetime, FIR is the food ingestion
rate (g/person/day), C is the metal concentration of
product, RfD shows oral reference dose, WAB is the
average body weight in adults and children as reported
by Shaheen et al (2016), and TA is the average exposure
time for noncarcinogens (365 days per year × number of
exposure, which was assumed as 70 years in the current
Statistical analysis
The sample size was calculated using a confidence
level of 95%. The mean concentration of the metals
was provided according to the mean frequency
weights of the white and yolk. This study has used
the bootstrap statistical method to place the confi-
dence intervals on phylogenies. When the trace ele-
ment concentration was below the instrument detec-
tion limit, mean LOD was inputted in the analysis

Table 2 LOD, LOQ, and recoveries for white, yolk, and feed samples

Metal Ni Cr Cd As Hg Pb

LOD ppb 2 1 1 4 2 2
LOQ ppb 6 3 3 12 6 6
White spike recovery per cent 102 103 98 89 101 98
Yolk spike recovery per cent 103 93 100 92 96 97
Feed spike recovery per cent 95 94 90 92 98 98
Environ Monit Assess (2018) 190:540 Page 5 of 13 540

(Waegeneers et al. 2009). Pure data was entered the Results and discussion
regression analysis to take the regression coefficient
of strain, feed, weight, and white/yolk on the heavy Metals content of egg samples
metal transition to egg samples. In this study, regres-
sion analysis was used because it shows the negative In the current study, mean concentration of the metals in
and positive effect of each stain on each metal egg white and egg yolk and the total concentration of
concentration, not just a total effect of strain as a different five strains are shown in Fig. 2b–e. Hy-line had
number. The one-sample mean-comparison test was the lowest total egg concentration in the selected trace
used to compare the standard values. Also, the elements. Next place belongs to Shaver except for its Ni
pairwise correlation was performed to study the concentration. Regarding other strains, Bovans, LSL,
association in metals in white, yolk, and feed sepa- and Native were sometimes first, second, and third.
rately. All statistical calculations were performed Among the top hazardous toxic trace elements (Pb,
using Stata11.2 portable software (Stata Corp, Col- Cd, and As), Pb concentration was the highest. The
lege Station, TX, USA) in which the statistically results of the current study were compared to other
significant difference was considered: P < 0.05 literature and are presented in Table 3. In the present
(two-sided). study, the values were higher than other studies. Study

0.040 0.080
a As Mean Con. mg/kg as Fresh Weght ab b
Cd Mean Con. mg/kg as Fresh Weght

0.035 c 0.070
b
0.030 0.060
b
ab a ac
0.025 0.050
a a
0.020 White Con 0.040 a White Egg Con.
a ab
ab Yolk Con Yolk Egg Con.
0.015 a 0.030 ab
c
a Total Con Total Egg Con.
0.010 b ac 0.020
ab b bc
ab ab bc
0.005 c a 0.010
b c c
0.000 0.000
Native Bovans LSL Shaver Hy-line Native Bovans LSL Shaver Hy-line
Strains Strains

0.800 3.000
c c d
Pb Mean Con. mg/kg as Fresh Weght

a
Cr Mean Con. mg/kg as Fresh Weght

c 2.500
0.600 c
2.000 a
a
a a
0.400 bc c White Egg Con 1.500 White Egg Con

ab Yolk Egg Con a Yolk Egg Con

a
Total Egg Con. 1.000 a Total Egg Con
a a ab
0.200 a a
ad 0.500
b b b b
d b b b b b
0.000 0.000
Native Bovans LSL Shaver Hy-line Native Bovans LSL Shaver Hy-line
Strains Strains

2.000
e
Ni Mean Con. mg/kg as Fresh Weght

1.800 a
1.600
1.400
1.200 a
1.000 White Egg Con
0.800 a a Yolk Egg Con
a
0.600 ab ab Total Egg Con
0.400 a b
a bc
0.200 b
c b b
0.000
Native Bovans LSL Shaver Hy-line
Strains

Fig. 2 a–e Mean-weights concentration (mg/kg of fresh weight) of Cd (a), Pb (b), As (c), Cr (d), and Ni (e) in egg samples. Different letters
show a significant difference (P < 0.05) among strains
540 Page 6 of 13 Environ Monit Assess (2018) 190:540

Table 3 Comparison of toxic trace element concentrations (mg/kg FW) in egg samples with the reported values of the other literature

Part of egg Country Strain Cr Ni Cd Reference

White egg Iran Gallus gallus domesticus (Native) 0.985 0.581 0.009 This study
Bovans White 0.887 0.425 0.017
Lohman LSL-Lite 0.680 0.364 0.006
Shaver White 0.031 0.109 0.010
Hy-line white 0.003 0.004 0.002
Total of 5 different strains 0.517 0.297 0.009
Greece Gallus gallus 0.048 0.064 0.001 Giannenas et al. (2009)
Turkey – 0.06 0.09 0.002 Uluozlu et al. (2009)
Poland Lohmann Brown 0.024 – – Dobrzański et al. (2007)
Yolk egg Iran Gallus gallus domesticus (Native) 0.635 0.380 0.005 This study
Bovans White 0.494 0.243 0.008
Lohman LSL-Lite 0.569 0.297 0.005
Shaver White 0.077 0.059 0.005
Hy-line white 0.018 0.014 0.003
Total of 5 different strains 0.359 0.575 0.018
Greece Gallus gallus 0.066 0.063 0.001 Giannenas et al. (2009)
Turkey – 0.01 0.09 0.003 Uluozlu et al. (2009)
Poland Lohmann Brown 0.227 – – Dobrzański et al. (2007)
Total egg Iran Gallus gallus domesticus (Native) 1.621 0.961 0.013 This study
Bovans White 1.381 0.668 0.025
Lohman LSL-Lite 1.249 0.662 0.012
Shaver White 0.107 0.168 0.015
Hy-line white 0.021 0.017 0.005
Total of 5 different strains 1.825 1.029 0.014
Bangladesh Gallus gallus domesticus 1.4 0.279 0.361 Shaheen et al. (2016)
Iran – 0.24 0.45 0.13 Salar-Amoli et al. (2015)
Part of egg Country Strain Pb As Hg Reference
White egg Iran Gallus gallus domesticus (Native) 0.162 0.020 0.01 This study
Bovans White 0.364 0.026 0.01
Lohman LSL-Lite 0.277 0.031 0.01
Shaver White 0.101 0.018 0.01
Hy-line white 0.009 0.001 0.01
Total of 5 different 0073trains 0.183 0.029 0.01
Greece Gallus gallus – 0.005 – Giannenas et al. (2009)
Turkey – 0.01 0.10 – Uluozlu et al. (2009)
Yolk egg Iran Gallus gallus domesticus (Native) 0.130 0.007 0.01 This study
Bovans White 0.166 0.014 0.01
Lohman LSL-Lite 0.249 0.021 0.01
Shaver White 0.056 0.007 0.01
Hy-line white 0.029 0.000 0.01
Total of 5 different strains 0.126 0.029 0.01
Greece Gallus gallus – 0.014 – Giannenas et al. (2009)
Turkey – 0.10 0.08 – Uluozlu et al. (2009)
Total egg Iran Gallus gallus domesticus (Native) 0.634 0.028 0.01 This study
Bovans White 1.028 0.039 0.01
Lohman LSL-Lite 1.100 0.053 0.01
Environ Monit Assess (2018) 190:540 Page 7 of 13 540

Table 3 (continued)

Part of egg Country Strain Cr Ni Cd Reference

Shaver White 0.341 0.025 0.01

Hy-line white 0.098 0.001 0.01

on different strains is one of the possible reasons for this areas, use of fertilizers, and solid-waste disposal activi-
difference between this study and former studies. The ties, in different (developed, developing, or undevel-
mean total of 5 different strains of Pb concentration was oped) countries (Bilo et al. 2015; Shaheen et al. 2016).
0.309 mg/kg FW egg that was more than the value of the Then it is probable that we find these contaminations in
study of Uluozlu et al. equal with 0.01 mg/kg FW but our food and finally in our body, leading to health
they did not mention the strain of chickens studied hazards (Shaheen et al. 2016). However, the most im-
(Uluozlu et al. 2009). In the case of As, the values of portant sources of pollution in poultry products espe-
the current study were 0.029 mg/kg FW less than the cially egg are feed and water (oral intake) (Iwegbue et al.
amount of the study of Uluozlu (0.1 mg/kg FW) but 2008; McEvoy 2002). In the current study, feed, as well,
more than the amount of the study of Giannenas had a significant impact on the concentration of heavy
(0.005 mg/kg FW). Nowadays, there is inevitable accu- metals (Pb, Cd, and Cr) (Table 7).
mulations of heavy metals in living organism through The impact of the strains of laying hens on heavy
the food chain or due to the industrialization of urban metals concentration based on regression coefficient is
shown in Table 4. A significant difference was detected
in trace elements content in most of the five strains with
Table 4 The regression coefficients of strain for heavy metals
concentration in yolk and white according to the type of heavy
high significance values for Cr and Ni (R2 = 54.2 and
metals 67.3%, respectively). There has been no similar study
on the relationship between the strains and trace ele-
Metal Strain β* P value β P value
White Yolk
Table 5 The mean weight (g) of samples in white, yolk, and total
egg according to different strains
Cd Bovans 0.013 0.00 0.008 0.026
LSL − 0.003 0.351 − 0.001 0.789 Strain Mean (g) Std. Err. [95% conf. interval]
Shaver 0.001 0.713 0.003 0.390
White weight 28.776 1.017 26.731 30.820
Hy-line − 0.010 0.001 − 0.007 0.054
Native 35.874 1.499 32.862 38.886
Pb Bovans 0.293 0.069 0.101 0.194
Bovans 30.351 1.175 27.989 2.713
LSL 0.210 0.188 0.257 0.002
LSL 38.193 1.262 35.657 40.729
Shaver − 0.105 0.505 − 0.187 0.019
Shaver 36.150 0.591 34.962 37.338
Hy-line − 0.232 0.145 − 0.304 0.00
Hy-line
As Bovans 0.006 0.640 0.022 0.123
Yolk weight 14.786 0.618 13.544 16.027
LSL 0.020 0.153 0.033 0.021
Native 18.246 0.503 17.235 19.256
Shaver − 0.006 0.637 0.003 0.813
Bovans 19.392 0.285 18.820 9.964
Hy-line − 0.031 0.029 − 0.021 0.136
LSL 15.110 0.550 14.003 16.216
Ni Bovans − 0.226 0.203 − 0.427 0.013
Shaver 18.721 0.391 17.935 19.507
LSL − 0.271 0.129 − 0.392 0.022
Hy-line
Shaver − 0.716 0.00 − 0.945 0.00
Total weight 43.561 1.163 41.224 45.898
Hy-line − 0.863 0.00 − 1.109 0.00
Native 54.120 1.540 51.025 57.214
Cr Bovans − 0.134 0.631 − 0.458 0.065
Bovans 49.743 1.102 47.528 1.958
LSL − 0.355 0.206 − 0.464 0.062
LSL 53.303 1.304 50.682 55.924
Shaver − 1.428 0.00 − 1.665 0.00
Shaver 54.871 0.686 53.493 56.249
Hy-line − 1.466 0.00 − 1.871 0.00
Hy-line 28.776 1.017 26.731 30.820
*β: regression coefficient
540 Page 8 of 13 Environ Monit Assess (2018) 190:540

Table 6 The regression Table 7 The regression coefficients of feed samples for heavy
coefficients of white and Metal β* P value metals concentration in yolk and white according to type of heavy
yolk samples for heavy metals
metals concentration ac- Cd 0.619 0.000
cording to type of heavy Pb 0.638 0.002 Metal β* P value β P value
metals White Yolk
As 0.838 0.000
Ni 0.779 0.000 Cd 0.132 0.000 0.080 0.001
Cr 0.816 0.000 Pb − 0.256 0.007 − 0.246 0.000
*β: regression coefficient
As 0.009 0.088 0.012 0.043
Ni 0.025 0.854 0.019 0.900
ments concentration; however, Canli et al. had studied
Cr 0.433 0.016 0.381 0.043
the impact of trace elements in the tissues of different
types of fish and they discovered that the impact of *β: regression coefficient
strain was significant (P < 0.01) for Cd, Cr, Pb, Cu,
Fe, and Zn (Canli and Atli 2003). The main reasons housing system) might lead to different results
for the observed difference here might be due to differ- (Küçükyılmaz et al. 2012; Iwegbue et al. 2008).
ent metabolic activities and environmental needs of each Mean weight of the samples obtained from white,
strain (Canli and Atli 2003; Fallah et al. 2011). It is also yolk, and whole egg of each strain is shown in Table 5.
probable that genetically different strains have different Our results suggested higher trace element concentra-
ways for use of space or resources in the field (Ali et al. tion in egg white compared to the yolk; however, in a
2016). Silversides had discussed the genetic basis of the study by Giannenas et al., mean concentration of the egg
egg quality (albumen height, egg weight, and albumen/ yolk was above egg white (Giannenas et al. 2009). That
yolk/shell weight) (Silversides et al. 2006). Factors such is because of the mean weight of yolk and white. In fact,
as the investigated strains difference, environmental when the weight of each egg had entered the calculation,
needs like diet, water, air conditioning, temperature, the heavy metals concentration of egg white was detect-
and rearing system (commercial or conventional ed to be more than egg yolk, unlike other studies.

3
b d

2.5 d
Feed Metals Concentraon mg/kg

b
2
Cd
1.5 c c Pb
bc bc
As
a ab
ab a Cr
1 c
a c a
a Ni

0.5 a
c a d
a b b a
0
Native Bovans LSL Shaver Hy-line
Strains

Fig. 3 Metals concentration in feed samples (mg/kg) among different strains. Different letters show a significant difference (P < 0.05)
among strains
Environ Monit Assess (2018) 190:540 Page 9 of 13 540

Table 8 Correlations between metal’s concentrations in white, yolk and feed samples

Metal Cd Ni Pb As Cr

Part of egg R value P value R value P value R value P value R value P value R value

White
Cd 1.000
Ni 0.442 0.001 1.000
Pb 0.713 0.000 0.359 0.011 1.000
As 0.533 0.000 0.219 0.126 0.724 0.000 1.000
Cr 0.505 0.000 0.966 0.000 0.458 0.001 0.261 0.067 1.000
Yolk
Cd 1.000
Ni 0.491 0.000 1.000
Pb 0.420 0.002 0.658 0.000 1.000
As 0.208 0.147 0.218 0.128 0.369 0.008 1.000
Cr 0.452 0.001 0.972 0.000 0.719 0.000 0.274 0.054 1.000
Feed
Cd 1.000
Ni 0.086 0.551 1.000
Pb − 0.474 0.001 0.118 0.417 1.000
As 0.842 0.000 − 0.144 0.318 − 0.710 0.000 1.000
Cr 0.971 0.000 0.028 0.848 − 0.580 0.000 0.944 0.000 1.000

Mercury concentration in all the samples was below the
instrument detection limit. A significant difference was
observed in the trace element concentrations of egg yolk
and egg white (see Table 6). As stated before, these are
the organic compounds chelating the minerals especial-
ly the toxic elements in a structure (Iwegbue et al. 2008;
Fallah et al. 2011). Therefore, the natural difference in
metal-binding capacity of compositions like proteins
resulted in the difference of metal concentration in both
yolk and white (Canli and Atli 2003). Weight had
caused a significant difference in heavy metals concen-
tration of each strain (p < 0.05). In this study, the egg
white assumed to contain an average of 66% of the total
weight, while the egg yolk contains 34%. Demirulus
reported an equal weight for yolk and albumen
(Demirulus 2013). There was no significant difference
between the weight and metal concentration in different
strains.
Metals content of feed samples
Trace elements of feed samples are presented in Fig. 3.
Not enough data is available on the heavy metals as-
sessment of feed intake in hens; however, Dobrzanski
reported a 1.68 mg per kg of Cr concentration in the feed
of Lohmann Brown strain which was closed to the value
obtained in our study, 1.825 mg per kg (Dobrzański
et al. 2007). In the current study, feed had changed along
with the strain; each strain was fed with standard ingre-
dients; therefore, there was little strain-feed interference.
However, in other studies, the role of strain was simul-
taneously investigated with other factors. It is worth
mentioning that the strain’s role can also be studied
completely in isolation (Novak et al. 2006). Table 7
shows the regression coefficients and P values for heavy
metals of feed samples in egg yolk and egg white
according to the type of heavy metals. The statistical
analysis has significantly revealed a difference in feed,
egg white, and egg yolk for all the trace element con-
tents except for Ni. Iwegbue considered feed and water
as the main sources of Pb contamination (Iwegbue et al.
2008). There was a significant negative effect of feed on
Pb concentration of white and yolk samples. Although
the feed given to Hy-line strain had the highest Pb
concentration (1.78 mg per kg), their eggs had the
lowest and even near zero Pb concentration. Regarding
Cd, the highest concentration was observed in Shaver
diet and consequently in Shaver eggs. The regression
540 Page 10 of 13 Environ Monit Assess (2018) 190:540

analysis showed a significant difference in Cd diet in
egg yolk and white (Table 7). Table 8 shows the corre-
lation between metals in egg yolk, egg white, and feed.
There was a reverse strong correlation, 97% (above
70%), between Ni and Cr in egg white and egg yolk of
the egg samples; the correlation coefficients were as
follows: between Cr and Cd concentrations = 0.971, Cr
and As concentrations = 0.944, and an interesting nega-
tive strong correlation between As and Pb concentra-
tions = − 0.710 in feed samples.
The health risk assessment of egg
Table 9 shows the estimated daily intake (μg per
kgBW.day) of Iranian adults and children. The
maximum provisional tolerance daily intake of As and
Pb is based on the provisional tolerable weekly intake
(PTWI), and the maximum provisional tolerance daily
intake of Cd is based on the provisional tolerable month-
ly intake (PTMI) of JECFA(Commission 2013).PTMI
for other metals was obtained from Shaheen et al. liter-
ature (Shaheen et al. 2016). The results were significant-
ly much lower than the standard values. Although for As
and Pb, JECFA withdrew PTWI and mentioned that
PTWI could no longer be considered health protective
because of the adverse effects of inorganic As on lung
cancer, Pb on intelligent quotients of children and sys-
tolic pressure in adults (Commission 2013). However,
the estimated daily intake as shown in Table 9 was
higher in children compared to adults, because of their

Table 9 The estimated daily intake (EDI, μg/kgBW.day) according to mean weight of toxic heavy metals each trace element in total, white,
and yolk of the egg in Iran

Laying strain/part of egg Estimated daily intake (EDI) (μg/kgBW.day)

Cd Pb As Ni Cr

Adult Child Adult Child Adult Child Adult Child Adult Child

Native
White egg 0.004 0.009 0.077 0.171 0.009 0.021 0.274 0.608 0.467 1.037
Yolk egg 0.001 0.003 0.031 0.07 0.002 0.004 0.089 0.199 0.151 0.334
Total egg 0.005 0.012 0.108 0.241 0.011 0.025 0.363 0.807 0.618 1.371
Bovans
White egg 0.011 0.024 0.239 0.531 0.017 0.037 0.257 0.57 0.538 1.196
Yolk egg 0.002 0.005 0.051 0.113 0.004 0.009 0.074 0.164 0.151 0.335
Total egg 0.013 0.029 0.290 0.644 0.021 0.046 0.331 0.734 0.689 1.531
LSL
White egg 0.003 0.007 0.14 0.312 0.016 0.035 0.186 0.413 0.347 0.772
Yolk egg 0.002 0.004 0.08 0.179 0.007 0.015 0.096 0.214 0.184 0.409
Total egg 0.005 0.011 0.22 0.491 0.023 0.05 0.282 0.627 0.531 1.181
Shaver
White egg 0.006 0.014 0.064 0.141 0.012 0.027 0.069 0.153 0.02 0.044
Yolk egg 0.001 0.003 0.014 0.032 0.002 0.004 0.015 0.034 0.02 0.045
Total egg 0.007 0.017 0.078 0.173 0.014 0.031 0.084 0.187 0.04 0.089
Hy-line
White egg 0.001 0.003 0.005 0.012 0.00 0.001 0.002 0.005 0.002 0.004
Yolk egg 0.001 0.002 0.009 0.021 0.00 0.00 0.004 0.009 0.006 0.013
Total egg 0.002 0.005 0.014 0.033 0.00 0.001 0.006 0.014 0.008 0.017
Totally in this study 0.006 0.015 0.142 0.316 0.039 0.031 0.213 0.473 0.377 0.838
Total egg (Shaheen et al. 2016) 0.06 0.07 0.05 0.07 0.06 0.07 0.25 0.33 0.25 0.32
Maximum tolerable daily intake (MTDI) 0.083a 3.57a 2.1a 4.3 2.8

aCODEX ALIMENTARIUS (1993)

Environ Monit Assess (2018) 190:540 Page 11 of 13 540

Table 10 The Target Hazard Quotient (THQ, dimensionless) for each metal caused by the consumption of total, white, and yolk of different
strains’ egg in Iran

Laying strain/part of egg Target Hazard Quotient (THQ, dimensionless)

Cd Pb As* Ni Cr
Adult Child Adult Child Adult Child Adult Child Adult Child

Native
White egg 0.000 0.009 0.019 0.043 0.015 0.035 0.014 0.030 0.000 0.001
Yolk egg 0.001 0.003 0.008 0.018 0.004 0.007 0.004 0.010 0.000 0.000
Total egg 0.005 0.012 0.027 0.060 0.019 0.042 0.018 0.040 0.000 0.001
Bovans
White egg 0.011 0.024 0.060 0.133 0.029 0.062 0.013 0.029 0.000 0.001
Yolk egg 0.002 0.005 0.013 0.028 0.007 0.015 0.004 0.008 0.000 0.000
Total egg 0.013 0.029 0.073 0.161 0.035 0.077 0.017 0.037 0.000 0.001
LSL
White egg 0.003 0.007 0.035 0.078 0.027 0.059 0.009 0.021 0.000 0.001
Yolk egg 0.002 0.004 0.020 0.045 0.011 0.025 0.005 0.011 0.000 0.000
Total egg 0.005 0.011 0.055 0.123 0.039 0.084 0.014 0.031 0.000 0.001
Shaver
White egg 0.006 0.014 0.016 0.035 0.020 0.045 0.003 0.008 0.000 0.000
Yolk egg 0.001 0.003 0.004 0.008 0.004 0.007 0.001 0.002 0.000 0.000
Total egg 0.007 0.017 0.020 0.043 0.024 0.052 0.004 0.009 0.000 0.000
Hy-line
White egg 0.001 0.003 0.001 0.003 0.000 0.002 0.000 0.000 0.000 0.000
Yolk egg 0.001 0.002 0.002 0.005 0.000 0.000 0.000 0.000 0.000 0.000
Total egg 0.002 0.005 0.004 0.008 0.000 0.002 0.000 0.001 0.000 0.000
Totally in this study 0.006 0.015 0.036 0.079 0.023 0.051 0.011 0.024 0.000 0.001
Total egg (Shaheen, 2016) 0.06 0.1 0.01 0.02 0.19 0.33 0.01 0.02 0.08 0.15

*Calculating 50% inorganic arsenic in foods (Islam et al. 2015)

much less weight. Hy-line strain showed a minor health metals as seen in Table 9. EDIs in this study are close to
risk, while Pb presented stronger health risk than other Shaheen et al. values, except for Pb, that is, much higher

0.450
0.400
0.350
0.300 Nave
Total THQ

0.250
Bovans
0.200
LSL
0.150
0.100 Shaver

0.050 Hy-line
0.000
White Egg Yolk Egg Total Egg White Egg Yolk Egg Total Egg
Adults Children

Fig. 4 Total metal Target Hazard Quotient (THQ, dimensionless) for metals caused by the consumption of total, white and yolk of the egg
540 Page 12 of 13
than the recent study (Shaheen et al. 2016). According
to the significant impact of feed on egg’s heavy metals
(except Ni in egg white and yolk and As in white), this
study has also considered feed as an important source of
metal contaminants in egg (Iwegbue et al. 2008). Since
children are more sensitive to contaminants, EDI was
estimated separately for adults and children (Wang et al.
2005).
Table 10 is describing the Target Hazard Quotient
(THQ) for each metal caused by the consumption of the
whole egg, egg white, and yolk in Iran. Obviously, the
results had described the safety range (less than one), but
if the overall potential risk caused by egg plus the other
major products like rice or bread is calculated, the
adverse health effects (THQ > 1) are more probable
(Wang et al. 2005). Hy-line W36 has a near zero non-
carcinogen health risk and after that Shaver has the
lowest health risk. Pb and As showed more THQ than
the other metals. Among all the studied metals, Cr
caused almost zero non-carcinogen health risk. There
was a significant impact of trace elements on EDI and
THQ in strains (P < 0.05).
The total Target Hazard Quotients (sum of individual
metal THQ) for Cd, Pb, As, Ni, and Cr were calculated
and shown in Fig. 4. The exposure of two or more
contaminants may result in additive and/or interactive
properties (Hallenbeck 1993). In this figure, the total
THQ was close to 0.4 in children, but as mentioned
earlier if the other residual metals like Zn and Cu are
combined, more than one THQ is more probable (Wang
et al. 2005; Shaheen et al. 2016). The highest TTHQ
was observed in Bovans and LSL in children. The total
THQ in each strain for adults, as the result of egg
consumption, is about half of that for children. The
mean TTHQ in all the five strains in this study were
0.099 and 0.22 respectively for adults and children, that
is, much less than the values in the study by Shaheen
0.35 and 0.62 (THQ for Cu was omitted from the total
THQ calculations to be able to compare their results
with ours) (Shaheen et al. 2016).
Conclusion
In conclusion, this study discovered the impact of dis-
tinct strain, feed, and weight on heavy metal concentra-
tions in egg white, egg yolk, and the whole egg. The
imposed health risks of Cd, Pb, As, Cr, and Ni in Iranian
population through the consumption of egg white, egg
Environ Monit Assess (2018) 190:540
yolk, and the whole egg were assessed based on the
estimated daily intake (EDI) and target hazardous quo-
tients (THQs). Hy-line W36 strain showed the lowest
toxic metals concentration in egg yolk and egg white
with near zero health risk (THQ). Further genetic studies
and strains’ health risk assessments are recommended.
The target hazardous quotients in this study for all the
trace elements were below one. Therefore, Iran’s popu-
lation does not experience any adverse health effects due
to egg consumption.
Funding information The authors would like to thank the Facul-
ty of Medicine, Mashhad University of Medical Sciences, Mash-
had, Iran, for financially supporting this study (grant no:
940732).References
Abbas, T. E., Elzubeir, E. A., & Arabbi, O. H. (2008). Drinking
water quality and its effects on productive performance and
immune response of layers. International Journal of Poultry
Science, 7(5), 441–444.
Ali, A., Campbell, D., Karcher, D., & Siegford, J. (2016).
Influence of genetic strain and access to litter on spatial
distribution of 4 strains of laying hens in an aviary system.
Poultry Science, pew236, 95, 2489–2502.
Applegate, E. (2000). Introduction: nutritional and functional roles
of eggs in the diet. Journal of the American College of
Nutrition, 19(sup5), 495S–498S.
Bilo, F., Lodolo, M., Borgese, L., Bosio, A., Benassi, L., Depero,
L. E., & Bontempi, E. (2015). Evaluation of heavy metals
contamination from environment to food matrix by TXRF:
the case of rice and rice husk. Journal of Chemistry,
2015(12), 1–12. https://doi.org/10.1155/2015/274340.
Canli, M., & Atli, G. (2003). The relationships between heavy
metal (Cd, Cr, Cu, Fe, Pb, Zn) levels and the size of six
Mediterranean fish species. Environmental Pollution, 121(1),
129–136.
Commission, C. A. (2013). Codex general standard for contami-
nants and toxins in food and feed, CODEX STAN 193–1995.
Dauwe, T., Janssens, E., Kempenaers, B., & Eens, M. (2004). The
effect of heavy metal exposure on egg size, eggshell thick-
ness and the number of spermatozoa in blue tit Parus
caeruleus eggs. Environmental Pollution, 129(1), 125–129.
https://doi.org/10.1016/j.envpol.2003.09.028.
Demirulus, H. (2013). The heavy metal content in chicken eggs
consumed in Van Lake Territory. Ekoloji, 22(86), 19–25.
Dobrzański, Z., Górecki, H., Chojnacka, K., Górecka, H., &
Synowiec, M. (2007). Effect of dietary humic preparations
on the content of trace elements in hens’ eggs. American
Journal of Agricultural and Biological Science, 2(2), 234-
240
Domingo, J. L. (2014). Health risks of human exposure to chem-
ical contaminants through egg consumption: a review. Food
Research International, 56, 159–165.
Ershad, S. (2005). Performance of hybrid layers and native hens
under farmers’ management in a selected area of Bangladesh.
International Journal of Poultry Science, 4(4), 228–232.
Environ Monit Assess (2018) 190:540
Fallah, A. A., Saei-Dehkordi, S. S., Nematollahi, A., & Jafari, T.
(2011). Comparative study of heavy metal and trace element
accumulation in edible tissues of farmed and wild rainbow
trout (Oncorhynchus mykiss) using ICP-OES technique.
Microchemical Journal, 98(2), 275–279.
Gebrekidan, A., Weldegebriel, Y., Hadera, A., & Van der Bruggen,
B. (2013). Toxicological assessment of heavy metals accu-
mulated in vegetables and fruits grown in Ginfel river near
Sheba Tannery, Tigray, Northern Ethiopia. Ecotoxicology
and Environmental Safety, 95, 171–178. https://doi.
org/10.1016/j.ecoenv.2013.05.035.
Giannenas, I., Nisianakis, P., Gavriil, A., Kontopidis, G., &
Kyriazakis, I. (2009). Trace mineral content of conventional,
organic and courtyard eggs analysed by inductively coupled
plasma mass spectrometry (ICP-MS). Food Chemistry,
11 4 ( 2 ) , 7 0 6 – 7 1 1 . h t t p s : / / d o i . o r g / 1 0 . 1 0 1 6 / j .
foodchem.2008.09.079
Hallenbeck, W. H. (1993). Quantitative risk assessment for envi-
ronmental and occupational health. CRC Press.
Herron, K. L., & Fernandez, M. L. (2004). Are the current dietary
guidelines regarding egg consumption appropriate? The
Journal of Nutrition, 134(1), 187–190.
Islam, M. S., Ahmed, M. K., & Habibullah-Al-Mamun, M.
(2015). Determination of heavy metals in fish and vegetables
in Bangladesh and health implications. Human and
Ecological Risk Assessment: An International Journal,
21(4), 986–1006.
Iwegbue, C., Nwajei, G., & Iyoha, E. (2008). Heavy metal resi-
dues of chicken meat and gizzard and Turkey meat consumed
in Southern Nigeria. Bulgarian Journal of Veterinary
Medicine, 11(4), 275–280.
Kaplan, O., Yildirim, N., Yildirim, N., & Cimen, M. (2011). Toxic
elements in animal products and environmental health. Asian
Journal of Animal and Veterinary, 6(3), 228–232.
Ki, S. A., Khaniki, G. J., Shariatifar, N., Nazmara, S., &
Akbarzadeh, A. (2017). Contamination of chicken eggs sup-
plied in Tehran by heavy metals and calculation of their daily
intake. Journal of Health in the Field, 2(4).
Kiliç, Z., Acar, O., Ulaşan, M., & Ilim, M. (2002). Determination
of lead, copper, zinc, magnesium, calcium and iron in fresh
eggs by atomic absorption spectrometry. Food Chemistry,
76(1), 107–116.
Küçükyılmaz, K., Bozkurt, M., Yamaner, C., Çınar, M., Çatlı, A.,
& Konak, R. (2012). Effect of an organic and conventional
rearing system on the mineral content of hen eggs. Food
Chemistry, 132(2), 989–992.
McEvoy, J. (2002). Contamination of animal feedingstuffs as a
cause of residues in food: a review of regulatory aspects,
incidence and control. Analytica Chimica Acta, 473(1–2), 3–
26.
Novak, C., Yakout, H., & Scheideler, S. (2006). The effect of
dietary protein level and total sulfur amino acid: lysine ratio
on egg production parameters and egg yield in Hy-Line W-98
hens. Poultry Science, 85(12), 2195–2206.
Saei-Dehkordi, S. S., Fallah, A. A., & Nematollahi, A. (2010).
Arsenic and mercury in commercially valuable fish species
from the Persian Gulf: influence of season and habitat. Food
and Chemical Toxicology, 48(10), 2945–2950.
Page 13 of 13 540
Salar-Amoli, J., & Ali-Esfahani, T. (2015). Determination of haz-
ardous substances in food basket eggs in Tehran, Iran: a
preliminary study. Veterinary Research Forum, 6(2), 155–
159.
Scheideler, S., Jaroni, D., & Froning, G. (1998). Strain and age
effects on egg composition from hens fed diets rich in n-3
fatty acids. Poultry Science, 77(2), 192–196.
Scott, T., & Silversides, F. (2000). The effect of storage and strain
of hen on egg quality. Poultry Science, 79(12), 1725–1729.
Shah, A., Kazi, T., Baig, J., Afridi, H., Kandhro, G., Khan, S., et al.
(2010). Determination of total mercury in chicken feed, its
translocation to different tissues of chicken and their manure
using cold vapour atomic absorption spectrometer. Food and
Chemical Toxicology, 48(6), 1550–1554.
Shaheen, N., Ahmed, M. K., Islam, M. S., Al-Mamun, H., Tukun,
A. B., Islam, S., Rahim, A.T.M.A. (2016). Health risk as-
sessment of trace elements via dietary intake of ‘non-piscine
protein source’ foodstuffs (meat, milk and egg) in
Bangladesh. Environmental Science and Pollution
Research, 23(8), 7794–7806.
Silversides, F., Korver, D., & Budgell, K. (2006). Effect of strain
of layer and age at photostimulation on egg production, egg
quality, and bone strength. Poultry Science, 85(7), 1136–
1144.
Singh, R., Cheng, K., & Silversides, F. (2009). Production perfor-
mance and egg quality of four strains of laying hens kept in
conventional cages and floor pens. Poultry Science, 88(2),
256–264.
Tuzen, M., Soylak, M., Citak, D., Ferreira, H. S., Korn, M. G., &
Bezerra, M. A. (2009). A preconcentration system for deter-
mination of copper and nickel in water and food samples
employing flame atomic absorption spectrometry. Journal of
Hazardous Materials, 162(2), 1041–1045.
Uluozlu, O. D., Tuzen, M., Mendil, D., & Soylak, M. (2009).
Assessment of trace element contents of chicken products
from Turkey. Journal of Hazardous Materials, 163(2-3),
982–987.
Van Overmeire, I., Pussemier, L., Waegeneers, N., Hanot, V.,
Windal, I., Boxus, L., et al. (2009). Assessment of the chem-
ical contamination in home-produced eggs in Belgium: gen-
eral overview of the CONTEGG study. Science of the Total
Environment, 407(15), 4403–4410. https://doi.org/10.1016/j.
scitotenv.2008.10.066.
Vincevica-Gaile, Z., Gaga, K., & Klavins, M. (2013). Food and
environment: trace element content of hen eggs from differ-
ent housing types. APCBEE Procedia, 5(0), 221–226.
https://doi.org/10.1016/j.apcbee.2013.05.038.
Waegeneers, N., Hoenig, M., Goeyens, L., & De Temmerman, L.
(2009). Trace elements in home-produced eggs in Belgium:
levels and spatiotemporal distribution. Science of the Total
Environment, 407(15), 4397–4402.
Wang, X., Sato, T., Xing, B., & Tao, S. (2005). Health risks of
heavy metals to the general public in Tianjin, China via
consumption of vegetables and fish. Science of the Total
Environment, 350(1–3), 28–37. https://doi.org/10.1016/j.
scitotenv.2004.09.044.