Assessment of heavy metal pollution transfer and h

RESEARCH ARTICLE
Assessment of heavy metal pollution transfer and human exposure
risks from the consumption of chicken grown
in mining-surrounding areas
Sameh Elkribi-Boukhris
1,2
&Naceur M’hamdi
3
&Iteb Boughattas
1
&Sondes Helaoui
1
&Cecile Coriou
4
&Sylvie Bussiere
4
&
Valerie Sappin-Didier
4
&Mohamed Banni
1
Received: 4 May 2021 / Accepted: 12 August 2021
#The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2021
Abstract
The purpose of this study was to assess heavy metal contamination in soil, plants, earthworms, and chicken in farmlands adjacent
to an old mining site and to evaluate the potential exposure risks to humans through the consumption of chicken. For this purpose,
soil, earthworms, plant, chickens, and eggs were sampled from 5 sites following a gradient of contamination. All samples were
analyzed for heavy metals (Pb, Cd, Cu, and Zn). A food chain model was used in order to characterize heavy metal transfer
between soil-plant-earthworm and chicken organs. Furthermore, target hazard quotient (THQ), estimated daily intake (EDI), and
hazard index (HI) were employed to assess human health risks posed by heavy metal contamination. Despite the higher level of
Pb, our data related to the calculation of EDI and THQ suggested that local consumers are more at risk of Cd contamination. The
calculated HI showed values ranging from 2.58 to 4.74 for adults, and up to 12.34 for children, indicating a considerable risk to
the health of local inhabitants, especially children. This study highlighted the crucial role of diets based on chickens grown in
contaminated areas, on health risks especially for children.
Keywords Estimated daily intake (EDI) .Hazard index (HI) .Heavy metals .Old mine .Target hazard quotient (THQ)
Introduction
Heavy metals are characterized by their non-biodegradability and
long biological half-life, (Tlili et al. 2010; Sobhan 2017).
Consequently, they can be accumulated within soil-plant-food
chains in a considerable amount, which presents a potential
health risk for humans (Rezaei Raja et al. 2016; Boughattas
et al. 2017;Rezvanetal.2019). For example, deficits in intelli-
gence quotient development of physiological abnormalities, and
neurotoxicity effects in infants, and anemia are the primary con-
sequences of exposure to lead (Duran et al. 2009; Iwegbue 2015;
Hariri et al. 2017). In this context, environmental disease out-
breaks of Minamata disease were caused by oral intake of MeHg
in fish and itai-itai disease from cadmium in rice (Gunnar et al.
2019). Cadmium has an extremely long biological half-life
(Javed and Usmani 2016). Thus, it can be damaging to humans,
potentially through its concentrations in kidneys (Bernard 2008;
Nookabkaew et al. 2013; Kim et al. 2016). Decreased rate of
glomerular filtration and significant protein urea along with an
important frequency of kidney stone development are the main
effects of oral exposure to Pb (Zhuang et al., 2017). Copper (Cu)
and Zinc (Zn) are essential trace elements with numerous biolog-
ical functions such as hemoglobin synthesis and enzyme func-
tions (United States Environmental Protection Agency (USEPA)
2012). However, both excess and deficiency of these elements
can result in adverse effects in the human body such as Menkes
and Wilson’s diseases (Tapiero and Kenneth 2003; Sobhan
2017).
Heavy metal contamination occurs generally due to indus-
trial activities and especially with metal manufacture. Indeed,
mining is considered the primary source of long-term soil
Responsible Editor: Lotfi Aleya
*Iteb Boughattas
[e-mail]
1
Laboratory of Agrobiodiversity and Ecotoxicology, Higher Institute
of Agronomy, Chott-Mariem, 4040 Chott-Mariem, Tunisia
2
Department of Biological Sciences, Faculty of Science of Tunis,
University of Tunis El Manar, Tunis, Tunisia
3
Department of Animal Sciences, National Agronomic Institute of
Tunisia, University of Carthage, 1082 Tunis, Tunisia
4
UMR ISPA, INRAE, 33140, Villenave-d’Ornon, Bordeaux, France
Environmental Science and Pollution Research
https://doi.org/10.1007/s11356-021-15995-9
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pollution (Lourenço et al. 2011; Iwegbue 2015; Kim et al.
2016; Boughattas et al. 2018; Mkhinini et al. 2020). Mine sites
are generally located adjacent to farmlands and rural areas.
Consequently, these are exposed to pollution by heavy metals,
through air and soil deposits, and additionally, through direct
exposure of the villagers to these pollutants via different path-
ways (Qing et al. 2015; Giri and Kumar 2017; Rezvan et al.
2019).
In this regard, the Pb-Zn mining site of Jebel Ressas and its
surrounding agricultural area, located in northeastern Tunisia
have been abandoned, and huge quantities of mining waste
have been left in the open. The site is considered a source of
heavy metal contamination of farmland surrounding the
mines, especially since no specific management was recorded
up to now to minimize heavy metals contamination (Ghorbel
et al. 2014; Boughattas et al. 2017). Consequently, a health
risk may exist for the habitants due to their exposure to heavy
metal contamination via ingestion, inhalation, and dermal
contact (Ghorbel et al. 2014).
Conversely, poultry raised in the Jebel Ressas mine area
could uptake heavy metals from different origins, mainly
through food. Metal residues may be accumulated on their
tissues and eggs (Chowdhury et al. 2011; Abdulkhaliq et al.
2012). Therefore, trace heavy metals can be transferred to
humans through food consumption (De Vries et al. 2007;
Rodrigues et al. 2012). Béjaoui et al. (2016) affirmed the
health risk for Jebal Ressas inhabitants being exposed to Pb
and Cd contamination through spontaneous plants (Malva
sylvestris) consumption and soil direct dust inhalation.
Considering this, food chain models are crucial tools to
evaluate soil contamination risks. Thus, it is essential to
promote models to describe the pathways related to soil
pollution, dietary transfer of hazardous to animals, and hu-
man exposure from dietary intake of animal products and
plants. The transfer of chemicals in food chains is in gen-
eral detailed by bioaccumulation factors (BAFs) and
bioconcentration factors (BCFs). These key factors are
based on the hypothesis of the existence of linear relation-
ships between heavy metal loads in plants (Helaoui et al.
2020), earthworms (Boughattas et al. 2016), birds (Indrajit
et al. 2018), mammals (Syr-Song et al. 2013;Jebalietal.
2014; Banni et al. 2017), and the amounts existing in soil.
However, the use of constant factors, BCF and BAF, is
controversial since some authors retain it not suitable to
describe the transfer of contaminants from soil to plants
in a particular context (Römkens et al. 2009a,2009b,
Vries et al., 2007). Therefore, more efficient empirical
models have been suggested to depict the soil-plant-
animal transfer. To realistically consider differences in the
bioavailability of trace elements in corresponding to several
soil types, important soil properties (pH, organic matter:
OM, clay) have been considered in such models (Krauss
et al. 2002; Efroymson et al. 2001; Römkens et al. 2009b).
Despite the available literature about the effects of hazard-
ous on ecosystem components, a gap of knowledge still exists
on pollutants transfer modeling from the ecosystem to
humans. This should be underlined in a heavily contaminated
context such as old mining sites.
The objective of the present work is the evaluation of hu-
man risks after exposure to Pb, Cd, Cu, and Zn from the direct
ingestion of Gallus gallus domesticus, which is the most avail-
able and low coast protein source considering the socio-
economic state of the citizens of the Jebel Ressas region.
This evaluation was performed using a predictive modeling
approach considering the supply chain from soil to the final
consumer.
Materials and methods
Study area and sample collection
Jebel Ressas (JR) mine site was considered in this investiga-
tion (Figure S1). JR is located south of the Tunisian capital
“Tunis City”(30 km) and is under a semi-arid climate. The
site includes fields impacted by old mining activities (Pb-Zn
extraction zone).
For the current research, 5 sites were considered:
–Four sites (S1, S2, S3, and S4) with a decreasing
polymetallic contamination gradient in the surrounding
area of the old mine.
–Site 5: Selected as a reference site and located in Chott-
Mariem, in the center of Tunisia (110 km away from the
mining site). We considered this site because it is used for
organic farming with no source of heavy metals pollution
(Figure S1).
Soil samples were collected from the top 0–30 cm of soil in
the different sites (10 samples from each site) after removing
litter and surface vegetation. Earthworms and plants were also
sampled from each site (n=10 from each site).
A total of 100 chickens (20 from each site) were raised for 6
months (March and July 2017). Soil macro-flora including
earthworms and spontaneous vegetation were their main food
diet. Before their sacrifice, chickens were weighed, and then
they were dissected. All the experimental protocols were car-
ried out in accordance with the principles and recommenda-
tions of Directives 86/609/EEC regulating the welfare of lab-
oratory animals (Louhimies 2002; Elkribi-Boukhris et al.
2020). The liver, kidney, lung, and muscle were carefully
removed. Additionally, the blood from the scarified chicken
was sampled. All the samples were then conserved at −80°C
for further analysis. Fresh eggs were collected (20 eggs from
each site) from the five farms that were used for chicken sam-
pling. The eggs were collected and transferred to the
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laboratory in small plastic bags and were kept in a cool, dark
place (+4) until analysis.
Chemical analysis of soils, plants, worms, and chicken
tissues
Soil samples were homogenized and sieved (2-mm mesh) and
then dried twice: first air-dried until the weights were constant
and then re-dried at 105°C for 24 h. Soils were then ground
into agate balls for 10 min. All the mineral elements in the soil
were quantified after solubilization by aqua regia (HCl/HNO
3
)
(NF X 31-415 method).
Plant samples were brushed to eliminate traces of adhering
soil and dust (Válega et al. 2008). Then, they were dried at
60°C until constant weight (Válega et al. 2008). The dried
plant material was ground into zirconium balls for 10 min.
Then, they were mineralized with acid attack digestion with
a mixture of 4:1 (v/v) H
2
O
2
30% and HNO
3
69% (Aristar® for
trace analysis, VWR Chemicals).
Chicken tissues (muscle, liver, kidney, feather, lung, and
blood) and earthworms were dried to constant weight at
105°C for 48 h and then ground (< 1 μm) using a Retch
Planetary Ball Mill PM 400.
For the eggs, each one was cut into the end of the air cell
using sharp tweezers and dissecting scissors. The shell was
rinsed with distilled water for each egg separately. The content
of each sample was placed in a chemically cleaned glass jar
and the egg white was separated from the yolk. They were
dried at 75°C to obtain constant weight and then, they were
ground into powder.
Aliquots of plant and animal powdered samples were min-
eralized using a mixture of HNO
3
69% and H
2
O
2
30% and
furtherer filtrated and diluted with deionized water.
All extracts were quantified by atomic absorption spectro-
photometer (AAS). The solutions were injected into atomic
spectrometry and the elements analyzed were Cd, Pb, Zn, and
Cu (detection limit was 0.02 μgL
−1
). Samples were tested and
analyzed in triplicates.
A standard reference material (SRM) was used for valida-
tion of the analytical procedure, “Tomato leaves 1573a”cer-
tified by the National Institute of Standards and Technology.
Model concept and data analysis
The SAS software version 9.4 (Statistical Analysis System
2012) was used to perform all statistical analyses. The data
were subjected to basic statistics, tests for normality and cor-
relation significance, and an analysis of variance. Statistical
comparisons between the different metal loads in the various
matrixes were first controlled using the “Levene”test of the
homogeneity of variance, then by one-way analysis of vari-
ance (ANOVA). A forward stepwise multiple linear regres-
sion was employed to obtain the models of the organ’sheavy
metals concentration as functions of the total heavy metals
from soils and plants. The pvalues for the Pearson correlation
coefficients in the regression models were achieved by corre-
lating the metal loads in chicken organs with the total metal
concentrations from the plants and the soil. The pvalues of the
regression model and the Rsquare of the linear regression
were achieved by correlating the expected and measured
metals loads in chicken’stissues.
Model overview description
In the present investigation and in order to assess to what
extend heavy metals in soils may present risks for human
health through food consumption, we developed a human ex-
posure chain model. This integrates soil-heavy metal levels,
soil properties, soil-plant transfer, animal intake, transfer to
animal tissues, and human consumption patterns (to evaluate
human hazardous exposure from daily intake). A schematic
overview of the model is provided in Figure S2
(Supplementary data).
Derivation of soil-plant transfer models
Usually, the transfer of metals in food chains is characterized
by bioconcentration factors (BCFs) and bioaccumulation fac-
tors (BAFs). The BCF is defined as the ratio of the test metal
concentration in an organism (e.g., plant, earthworm) to the
concentration in soil at a steady state. The BAF is defined as
the ratio of the test metal concentration in an organism to the
concentration in its food at steady state (Jongbloed Ben and
Westerheijden 1994).
BCFsp ¼M½p
M½sð1Þ
where,
[M]p metal loads in plant (mg kg
−1
)
[M]s total metal loads in soil (mg kg
−1
)
BAF ¼M½organ
M½fð2Þ
where:
[M]organ metal loads in an organism (mg kg
−1
)
[M]f metal loads in feed (mg kg
−1
)
These factors are calculated assuming that metals in soil
and those in plants, earthworms, mammals, and other target
organisms are linearly correlated (De Vries et al. 2007).
However, better predictions of the hazardous loads in
plants can be retrieved by a nonlinear relationship that con-
siders the contribution of soil properties that controls the
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bioavailability of heavy metals in soils (Adams et al. 2004;
Brus et al. 2002).
Tsp ¼M½p
M½
−n
s
ð3Þ
where,
Tsp transfer parameter from soil to plant (mg/kg
1−n
)
nconstant rendering the nonlinear relationship
Tsp varies with respect to soil clay content, organic matter,
and pH (Brus et al. 2002; Adams et al. 2004; Rodrigues et al.
2012). Using log
10
transformation, Equation (3) can therefore
be presented as:
Log Tsp ¼Iþα:log OM½þβ:log clay½þγ:log
þM½
sþδ:pH ð4Þ
where,
OM is the organic matter content (in percentage, fixed on
3% for all soils) (De Vries et al. 2007; Römkens et al. 2009b;
Boughattas et al. 2016).
Clay is the fraction of clay particles (in percentage, fixed on
25% for all soils) (De Vries et al. 2007).
[M]
s
is the heavy metal level in the soil (in mg kg
−1
of dry
weight).
The regression parameters I(intercept) were obtained by
regression analysis: stepwise multiple regression (Römkens
et al. 2009b; De Vries et al. 2007).
Modeling of heavy metal transfer in animal organs
The following food chain model: soil ➔plant ➔earthworm ➔
chicken. Logarithmic transformation was used based on
Equation (2) which can be therefore presented as:
Log Tf−c¼Iþα:log Ip
þβ:log Is
½þδ:log Iv ½ ð5Þ
where,
–T
f−c
: transfer parameter from food to chickens
–The regression parameters I(intercept), α,β,andδwere
estimated by stepwise regression analysis (Römkens et al.
2009b; De Vries et al. 2007).
–I
p
: daily intake of plants (intake plant) in kg day
−1
of dry weight
–I
s
: daily intake of soil in kg day
−1
of dry weight
–I
v
: daily intake of earthworm in kg day
−1
of dry weight
The daily intake (DI) of heavy metals in animal organs
relates to feed consumption (plant-leaf and worm) and soil
ingestion according to Smith (2009) and Rodrigues et al.
(2012). Based on Equation 2,DIisasfollows:
DIchicken−organ ¼AþBþCðÞ*BAFfeed−chicken ð6Þ
where,
A¼Cplant−leaf *I
plant−leaf
=Iplant−leaf þIsoil ð7Þ
B¼Csoil*I
soil
ðÞ=Iworm þIsoil ðÞ ð8Þ
C¼Cworm*I
worm
ðÞ=Isoil þIworm ðÞ ð9Þ
where,
C
plant-leaf
,C
soil
,andC
worm
concentration of heavy metal in
leaves of plants, soil, and earthworm, respectively, in mg kg
−1
of dry weight.
I
plant-leaf
,I
soil
,andI
worm
are the daily intake of plant leaves,
soil, and earthworm, respectively, by chicken in kg day
−1
of dry
weight.
A combination of Equations (6), (7), (8), and (9) is used to
calculate the concentration of target heavy metal in chicken
organs (Sobhan 2017):
M½
chicken−organ ¼Cplant−leaf *I
plant−leaf
=Iplant−leaf þIsoil þCsoil *I
soi
ðÞ
l=Iworm þIsoil
ðÞþCworm*I
worm
ðÞ=Isoil þIworm ðÞBAFchicken
  ð10Þ
Equation 10 is based on the following assumptions:
–We measured the metal loads in plant leaves, soils, and
earthworms.
–The calculations were realized on a field-by-field basis
considering that chicken feed at the field all the time
and always at the same sites.
–The transfer coefficient of metals from soil to animal or-
gan is equal to the BAF
chicken
;
–Heavy metal uptake from air and water is negligible com-
pared to that of soil and feed (De Vries et al. 2007).
Exposure assessment: human estimated daily intake
(EDI), target hazard quotients (THQ), and hazard
index (HI)
Human intake of toxic metals in this study consists of intake
by soil ingestion, consumption of plant leaves, chicken tis-
sues, and eggs consumption.
The EDI of heavy metals for humans was calculated for
three elements (Pb, Cd, and Zn) according to the following
equation, (United States Environmental Protection Agency
(USEPA) 1997):
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EDI ¼M½IR EF ED
BW AT ð11Þ
where,
[M] the heavy metal concentration (mg kg
−1
)
IR ingestion rate (kg day
−1
)
EF exposure frequency (365 days/year)
ED exposure duration
BW body weight (kg)
AT averaging time for non-carcinogens (365 days/years)
In our case study, we consider that the Jebel Ressas’inhab-
itants are exposed to heavy metal contamination all day. The
above equation changes as follows:
EDI ¼M½хIR
BW mg kg−1day−1ðÞ ð12Þ
According to the National Institutes of Health database
(INS, 2015), the following information about the Tunisian
consumer are available:
–The average weekly consumption of eggs is 9.3kg/year,
therefore 27.48g/day.
–The average daily consumption of chicken is 16.8 kg/
year, therefore 46.03g/day.
–The exposure frequency (EF) has been set at 144 days/
year for people who eat chicken and eggs three times a
week.
–The duration of exposure (ED) is 70 years (equivalent to
the average lifespan) for adults and 7 years for children.
–The average value for adult body weight was considered
65 kg and 25 kg for children for the Tunisian population
(www.ins.tn).
Human health risks posed by heavy metal contamination
are generally characterized by the target hazard quotient
(THQ). The following equation determines the THQ based
on the non-cancerous toxic risk (Equation 13):
THQ ¼EDI=RfDð13Þ
R
f
Dis the reference dose of individual metal (mg kg
−1
day
−1
). It is applied for Cd, Pb, Cu, and Zn, and the values
are 0.001, 0.0035, 0.04, and 0.3 respectively, as established by
US Environmental Protection Agency (United States
Environmental ProtectionAgency(USEPA)2012; Javed
and Usmani 2016).
If the THQ value is under 1, the carcinogenic toxic effects
are considered to be low. When it exceeds 1, the potential
health risks associated with overexposure become concerning.
The HI (hazard index) is used to estimate the total non-
carcinogenic health risks (Wilbur Sharon et al. 2004;Javed
and Usmani 2016). To assess the potential health risks posed
by numerous metals, THQs can be added to create a hazard
index (HI) aiming to evaluate the risk of a mixture of toxic
compounds. HI refers to the sum of many THQ for many
hazardous compounds (Equation 14):
HI ¼THQ1þTHQ2þ…… þTHQnð14Þ
THQi the THQ of an individual metal element;
HI the hazard index for all investigated metals
Results
Chemical study for determination of heavy metals
(Pb, Cd, Zn, and Cu)
Heavy metal content in soils
Trace element concentrations in the investigated soils are re-
ported in Table 1. Results revealed that S3 is most contami-
nated with values reaching 214.58 ± 33.53 mg kg
−1
,48884±
7447.35 mg kg
−1
, 22913.37 ± 3274.13 mg kg
−
1, and 5.07 ±
0.45 mg kg
−1
, respectively, for Cd, Zn, Pb, and Cu. The soil
from S2 recorded heavy metal concentrations with values
77.33 ± 5.67mg kg
−1
of Cd, 17399.54 ± 1298mg kg
−1
of
Zn, and 8306.25 ± 817.8mg kg
−1
of Pb. Regarding S1, the
observed values are within 17.43 ± 0.08mg kg
−1
of Cd,
3530.52 ± 33.44mg kg
−1
of Zn, 1481.28 ± 13.86mg kg
−1
of
Pb, and 2.34± 0.05 mg kg
−1
of Cu.
Heavy metal concentration in plant Malva sylvestris
and earthworms Lumbricus sp.
Heavy metal concentrations in Malva sylvestris from the dif-
ferent sites of Jebel Ressas’mine are presented in Table 2.
Results first demonstrated that the highest levels of Pb were
observed in plants from S3 andS2 with means 425.95 ±
5.01 mg kg
−1
and 63.15 ± 3.34 mg kg
−1
respectively. Cd
and Zn are accumulated mostly in plants from S3 soil com-
pared to other soils with values 8.96 ± 0.28 mg kg
−1
and 1080
±30mgkg
−1
respectively.
Concerning earthworms Lumbricus sp., Pb content was
below the detection limit in animals from the four JR’ssoils
and also in the reference soil. On the other hand, Zn had
recorded the highest values in earthworms from S3 and
S1with means of 4930 ± 1330 mg kg
−1
and 1670 ± 60 mg
kg
−1
, respectively. In addition, earthworms from S3 and S2
accumulated higher Cd concentrations and the values reached
were 33.65 ± 0.82 mg kg
−1
and 13.08 ± 0.69 mg kg
−1
respectively.
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