Environmental Science and Pollution Research https://doi.org/10.1007/s11356-021-15995-9 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; Rezvan et al. 2019). For example, deficits in intelligence quotient development of physiological abnormalities, and Responsible Editor: Lotfi Aleya * Iteb Boughattas [email protected] 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 neurotoxicity effects in infants, and anemia are the primary consequences of exposure to lead (Duran et al. 2009; Iwegbue 2015; Hariri et al. 2017). In this context, environmental disease outbreaks 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 biological functions such as hemoglobin synthesis and enzyme functions (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 industrial activities and especially with metal manufacture. Indeed, mining is considered the primary source of long-term soil Content courtesy of Springer Nature, terms of use apply. Rights reserved. Environ Sci Pollut Res 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 pathways (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 human exposure from dietary intake of animal products and plants. The transfer of chemicals in food chains is in general detailed by bioaccumulation factors (BAFs) and bioconcentration factors (BCFs). These key factors are based on the hypothesis of the existence of linear relationships 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; Jebali et al. 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-plantanimal 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 hazardous 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 human risks after exposure to Pb, Cd, Cu, and Zn from the direct ingestion of Gallus gallus domesticus, which is the most available and low coast protein source considering the socioeconomic 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 investigation (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 ChottMariem, 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 carried out in accordance with the principles and recommendations of Directives 86/609/EEC regulating the welfare of laboratory 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 sampling. The eggs were collected and transferred to the Content courtesy of Springer Nature, terms of use apply. Rights reserved. Environ Sci Pollut Res 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/HNO3) (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) H2O230% and HNO369% (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 mineralized using a mixture of HNO3 69% and H2O2 30% and furtherer filtrated and diluted with deionized water. All extracts were quantified by atomic absorption spectrophotometer (AAS). The solutions were injected into atomic spectrometry and the elements analyzed were Cd, Pb, Zn, and Cu (detection limit was 0.02 μg L−1). Samples were tested and analyzed in triplicates. A standard reference material (SRM) was used for validation of the analytical procedure, “Tomato leaves 1573a” certified by the National Institute of Standards and Technology. metals concentration as functions of the total heavy metals from soils and plants. The p values for the Pearson correlation coefficients in the regression models were achieved by correlating the metal loads in chicken organs with the total metal concentrations from the plants and the soil. The p values of the regression model and the R square of the linear regression were achieved by correlating the expected and measured metals loads in chicken’s tissues. 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 exposure 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 factors (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 ¼ ½M p ½M s ð1Þ where, [M]p [M]s metal loads in plant (mg kg−1) total metal loads in soil (mg kg−1) BAF ¼ ½M organ ½M f Model concept and data analysis ð2Þ where: 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 correlation 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 variance (ANOVA). A forward stepwise multiple linear regression was employed to obtain the models of the organ’s heavy [M]organ [M]f metal loads in an organism (mg kg −1) 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 considers the contribution of soil properties that controls the Content courtesy of Springer Nature, terms of use apply. Rights reserved. Environ Sci Pollut Res bioavailability of heavy metals in soils (Adams et al. 2004; Brus et al. 2002). Tsp ¼ ½M p ½M −n s ð3Þ where, – – where, Tsp n – – – transfer parameter from soil to plant (mg/kg1−n) constant 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 log10 transformation, Equation (3) can therefore be presented as: Log Tsp ¼ I þ α:log ½OM þ β:log ½clay þ γ:log þ ½M s þ δ: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 T f −c ¼ I þ α:log I p þ β:log ½I s þ δ:log ½I v ð5Þ ½M chicken−organ ¼ – – – 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, DI is as follows: DIchicken−organ ¼ ðA þ B þ CÞ*BAFfeed−chicken 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 organ is equal to the BAFchicken; Heavy metal uptake from air and water is negligible compared to that of soil and feed (De Vries et al. 2007). ð6Þ where, A ¼ C plant−leaf * I plant−leaf = I plant−leaf þ I soil ð7Þ B ¼ C soil *ðI soil Þ=ðI worm þ I soil Þ ð8Þ C ¼ C worm *ðI worm Þ=ðI soil þ I worm Þ ð9Þ where, Cplant-leaf, Csoil, and Cworm concentration of heavy metal in leaves of plants, soil, and earthworm, respectively, in mg kg−1 of dry weight. Iplant-leaf, Isoil, and Iworm 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): C plant−leaf * I plant−leaf = I plant−leaf þ I soil þ C soil *ðI soi Þl =ðI worm þ I soil Þ þ C worm *ðI worm Þ=ðI soil þ I worm Þ BAF chicken Equation 10 is based on the following assumptions: – Tf−c: transfer parameter from food to chickens The regression parameters I (intercept), α, β, and δ were estimated by stepwise regression analysis (Römkens et al. 2009b; De Vries et al. 2007). Ip: daily intake of plants (intake plant) in kg day−1 of dry weight Is: daily intake of soil in kg day−1 of dry weight Iv: daily intake of earthworm in kg day−1 of dry weight ð10Þ 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 tissues, 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): Content courtesy of Springer Nature, terms of use apply. Rights reserved. Environ Sci Pollut Res EDI ¼ ½M IR EF ED BW AT ð11Þ where, the heavy metal concentration (mg kg−1) ingestion rate (kg day−1) exposure frequency (365 days/year) exposure duration body weight (kg) averaging time for non-carcinogens (365 days/years) [M] IR EF ED BW AT In our case study, we consider that the Jebel Ressas’ inhabitants are exposed to heavy metal contamination all day. The above equation changes as follows: EDI ¼ ½M х IR ðmg kg−1 day−1Þ BW 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 ¼ EDI=R f D ð13Þ RfD is 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 Protection Agency (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 noncarcinogenic 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 ¼ THQ1 þ THQ2 þ …… þ THQn THQi HI ð14Þ the THQ of an individual metal element; 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 reported in Table 1. Results revealed that S3 is most contaminated 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 different 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 compared to other soils with values 8.96 ± 0.28 mg kg−1 and 1080 ± 30 mg kg−1 respectively. Concerning earthworms Lumbricus sp., Pb content was below the detection limit in animals from the four JR’s soils 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. Content courtesy of Springer Nature, terms of use apply. Rights reserved. Environ Sci Pollut Res Table 1 Heavy metal concentrations (Pb, Cd, Zn, and Cu) in exposed sites (S1, S2, S3, and S4) compared to control site. Data presented as the mean ± SD. *Significant difference in comparison with the reference site (p <0.05). [Hm], heavy metal concentration [Hm] (mg kg−1) Reference site Sites S1 S2 S3 S4 Pb 0.27±5.96 1481.28±13.86 * 8306.25±817.8 22913.37±3274.13* 273.67±57.25* Cd Zn Cu 0.33±0.11 72.61±8.71 0.38±0.06 17.43±0.08 * 3530.52±33.44* 2.34±0.05 * 77.33±5.67* 17399.54±1298* 2.33±0.18 * 214.58±33.53 * 48884.66±7447.35* 5.07±0.45 * 4.21±0.66* 450.56±46.5* 0.88±0.13 * Copper concentrations in plant and earthworm samples were below the limit of detection. Heavy metal content in chickens’ tissues and eggs limit. In addition, the concentration of Zn in lungs and muscles was low in comparison with the other chicken organs. The kidney revealed the highest level of Pb accumulation in chickens from S1 and S2, with means 36.73 ± 0.73 mg kg−1 and 27.06 ± 0.065 mg kg−1 respectively. Conversely, the feathers showed very high Pb values in all exposed sites, and the highest values were 349.10 ± 13.52 mg kg−1 in site S1 and 68.8 ± 4.18 mg kg−1 and 63.73 ± 7.43 mg kg−1 in sites S2 and S3 respectively. However, the concentration of Pb in the liver, muscle, blood, and eggs was below the detection limit. The copper concentrations for all chicken samples and eggs were below the detection limit. The accumulation of heavy metals in chicken’s tissues exposed to polymetallic polluted soils is presented in Table 3. Our data revealed that Cd was most accumulated in the liver (of chickens from S2) and kidneys (of chickens from S1) with rates of 7.80 ± 0.31 mg kg−1 and 62.93 ± 2.08 mg kg−1 respectively. Additionally, the lung showed an important accumulation rate of Cd in all polluted sites. Moreover, the muscle recorded the accumulation of Cd, especially in chicken from S1 (0.15±0.008mg kg−1). For Zn, the rate of heavy metal accumulation in the liver was higher than that in the kidney. Thus, the chicken’s liver from the S1 site showed the highest concentration of Zn with a value of 2083.45 ± 151. 8mg kg−1. In addition, Zn was noted mostly in the kidneys of animals from S1 and S4 with values of 179.08 ± 4. 59mg kg−1 and 140.79 ± 0.25mg kg1, respectively. Interestingly, Zn was the only metal accumulated in eggs and the other heavy metals were all below the detection Table S1 summarizes stepwise multiple regression to estimate regression parameters: I, α, β, γ, and δ. The values of the coefficient of regression (R2) were between 0.87 and 0.91 and the estimated parameters were significant at the p< 0.05 Table 2 Determination of heavy metals (Pb, Cd, Zn, and Cu) concentrations in the plant (Malva sylvestris) and the earthworm (Lumbricus sp.) exposed to the old mine compared to the control site. Data are presented as mean ± standard deviation; *significant difference compared with the reference (p <0.05, ANOVA, Tukey). [Hm], heavy metal concentration [Hm] (mg kg−1) Samples Plant (Malva sylvestris ) Earthworm (Lumbricus) Pb Cd Zn Cu Pb Cd Zn Cu Control 0.18±0.67 0.13±0.007 180±0.07 <LD <LD 0.68±0.03 162±0.9 <LD Heavy metal transfer model and associated human risk assessment Derivation of soil-plant transfer Sites S1 S2 S3 S4 18.99±0.22* 0.4±0.006* 250±0.8* <LD <LD 6.14±0.26* 1670±60* <LD 63.15±3.34* 3.12±0.1* 390±5* <LD <LD 13.08±0.69* 910±110* <LD 425.95±5.01* 8.96±0.28* 1080±30* <LD <LD 33.65±0.82* 4930±1330* <LD 17.17±0.83* 0.17±0.03* 90±0.2* <LD <LD 3.91±0.16* 390±20* <LD Content courtesy of Springer Nature, terms of use apply. Rights reserved. Environ Sci Pollut Res Table 3 Determination of heavy metals (Pb, Cd, Zn, and Cu) concentrations in the different organs of G. domesticus and eggs grown in sites from the Jebel Ressas’ old mine. Data are presented as mean ± Samples Liver Kidney Lung Muscle Blood Feather [Hm] (mg kg−1) Sites S1 S2 S3 S4 <LD 4.70±0.17* 2083.45 ±151.8* <LD 36.73±0.73* 62.93±2.08* 179.08±4.59* <LD 7.80±0.31* 176.99±3.54* <LD 4.09±0.08* 193.89±0.57* <LD 27.06±0.065* 19.84±0.38* 129.31±8.71* <LD 15.45±0.13* 13.52±3.13* 120.89±1.83* <LD 0.66±0.13 221.35 ±4.59* <LD 4.54±0.21* 8.16±0.02* 140.79 ±0.25* <LD <LD 0.042 ±0.002* Pb Cd Zn <LD 0.14±0.01 89.89±1.5 Cu Pb Cd Zn <LD 0.17±0.56 0.15±0.02 93.27±2.86 Cu Pb Cd <LD <LD 0.007 ±0.002 <LD <LD 0.425±0.021* <LD <LD 0.307±0.007* <LD <LD 0.159±0.009* Zn 53.24 ±20.62 <LD <LD 0.009 ±0.001 19.42±0.67 <LD <LD <LD 90.81±4.43 70.32±1.47 64.45±2.7 56.6±12.8 <LD <LD 0.15±0.008* <LD <LD 0.07±0.021* <LD <LD 0.04±0.002* <LD <LD 0.01±0.0001 31.6±2.73* <LD <LD 0.02±0.0009* 21.22±4.26 <LD <LD 0.002 ±0.0004* 5.03±0.45 <LD 68.8±4.18* 0.47±0.29* 22.45±1.46 <LD <LD <LD 22.85±0.19 <LD <LD <LD 4.54±0.12 <LD 63.73±7.43* 0.37±0.022* 225.63 ±20.01* <LD <LD <LD 73.25±2.6* <LD 218.52 ±14.15* <LD <LD <LD 76.22±2.12* <LD 8.8±0.12* <LD 3.54±0.15* 0.05 ±0.0022* 223.79 ±5.07* <LD <LD <LD 69.47±10.56 <LD Cu Pb Cd Zn Cu Pb Cd Zn Cu Pb Cd Zn Eggs Control standard deviation. *Significant difference in comparison with the control site (p <0.05). [Hm], heavy metal concentration; <LD, under the limit of detection Cu Pb Cds Zn Cu 3.95±0.92 <LD 0.24±0.18 0.01 ±0.0011 116.22 ±2.14 <LD <LD <LD 70.54±2.58 <LD 13.35±2.95* <LD 349.1±13.52* 2.15±0.06* 616.63±32.13* <LD <LD <LD 71.53±1.83* <LD level. Our results showed that transfer of Pb and Cd was influenced by the pH with δ=2.68 and δ=0.93, respectively. However, Zn transfer was influenced by his own concentration in the soil (γ= 0.91). Calculation of daily intake (DI) and bioaccumulation factor (BAF) of heavy metals for G. domesticus A summary calculation of chicken daily intake (DI) of heavy metals due to the consumption of plants, earthworms, and soil ingestion is listed in Table 4. The results showed that the most important DI of heavy metals was following soil ingestion, especially for Pb, whose value reached a rate of 2245.51mg day−1 from the S3 site followed by a rate of 814.01 mg day−1 from the S2 site. In addition, the DI of Pb following the consumption of plants was 404.5 E−03 mg day−1 in chicken from soil S3. However, results showed that earthworms do not contribute to the intake of lead for chickens. Content courtesy of Springer Nature, terms of use apply. Rights reserved. Environ Sci Pollut Res Table 4 Calculation of daily intake (DI) of heavy metals by the domestic chickens through the ingestion of plant, soil, and earthworms from Jebel Ressas’ soils, according to Equations 7, 8, and 9 Sites S1 S2 S3 S4 Daily intake (DI) in (mg day−1) Source Zn Cd Pb Plant Soil Earthworm Plant Soil Earthworm 218.65 161.82 0.15 335.85 797.49 0.08 0.3 E−03 0.8 E−03 0.6 E−03 2.7 E−03 3.5 E−03 1.2 E−03 18.0E−03 145.16 60.0 E−03 814.01 - Plant Soil Earthworm Plant Soil Earthworm 931.74 2240.57 0.45 73.69 0.19 0.04 7.7 E−03 9.8 E−03 3.1 E−03 0.1 E−03 0.2 E−03 0.4 E−03 404.5 E−03 2245.51 16.3 E−03 26.82 - Also, the consumption of plants, earthworms, and soil by chicken contributes to the intake of Cd, and the rate reached 9.8 E−03 mg day−1 following soil ingestion in the S3 site. Concerning Zn intake, it was mainly influenced by soil ingestion with a rate of 2240.57mg day−1 in the S3 site. Also, plant consumption impacts Zn intake mostly in chicken from S3, with value coming to 931.74 mg day−1. Values of bioaccumulation factor (BAF) of heavy metals in different internal organs of chickens (liver, muscle, kidney, and lung) are given in Table 5. Our results revealed that Pb bioaccumulation is observed only in kidneys (1.59 E−04 mg kg−1), whereas for Cd, we found significant levels in all analyzed organs. However, they do not exceed limits of food safety and animal health except for Cd in the liver by 1.11 mg kg−1. Derivation of feed-animal transfer A summary of the stepwise multiple regression parameters (I, α, β, and δ) is available in Table 2. An empirical transfer model following food intake explains the heavy metal levels Table 5 Calculation of bioaccumulation (BAF) from feed to chicken organs in comparison with limit concentrations for food safety and animal health limits according to EC (2002) and De Vries et al. (2007) Samples Liver Kidney Muscle Lung variability in chicken organs (0.014<R2< 0.998) between 0.014 and 0.99%. Significant values of determination coefficient were estimated for the transferred concentration of Cd concentration in blood (R2= 0.99). The latter was due, in the first level, to the consumption of earthworms (δ = 0.036). Additionally, for feathers, liver, kidney, and muscles, the transferred concentration of Cd was influenced by the consumption of earthworm with δ = 4.6, δ = 5.39, δ = 2.11, and δ = 0.27 respectively. In the same context, Zn transfer to internal chicken organs was influenced more by earthworm consumption. Moreover, Pb transfer in the kidney was influenced more by plant consumption (α= 0.14). Calculation of the estimated daily intake (EDI), the target hazard quotient (THQ), and the hazard index (HI) through the consumption of the chicken and eggs sampled from Jebel Ressas’ mine site The values of the estimated daily intake (EDI), the target hazard quotient (THQ), and the hazard index (HI) of heavy metals via the consumption of chickens and eggs sampled from Jebel Ressas’ mine site (according to Equations 12, 13, and 14) have been listed in Table 6. The latter data were calculated only for Cd and Zn as the remaining elements were not detected in the chicken muscle and egg samples. The highest EDI for chicken samples was shown for Cd (5.58 mg kg−1 day−1) at the S2 site followed by Zn (1.49 mg kg−1 day−1) at S1 site for adults. Besides, EDI rates for children were up to twice than adult values, reaching 8.93 for Cd in the S1 site. Additionally, results demonstrated that numerous THQ values exceed 1. The highest is for Cd following the consumption of chicken meat with 4.4 at S2 site followed by S1 site with 2.71, for adults. However, THQ values were three more times more pronounced for children and reached 11.44 for Cd in the S2 site. For adults, the HI values (Figure 1) ranged from 0.67 to 4.74 while for children they are up to twice than adult values (children HI values range from 1.74 to 12.34) indicating a BAF (mg kg−1) Food safety limits (mg kg−1) Animal health limits (mg kg−1) Pb Cd Zn Pb Cd Zn Pb Cd Zn 1.59E−04 - 1.11 10.09 0.03 0.079 155 99.1 16.3 41 0.5 0.5 0.1 - 0.5 1.0 0.05 - 150 150 - 2 3 - 1.4 5 0.02 - 600 135 - Content courtesy of Springer Nature, terms of use apply. Rights reserved. Environ Sci Pollut Res Table 6 Estimated daily intake (EDI in mg kg−1 day−1), target hazard quotient (THQ), and health risk assessment (HI) of heavy metals through the consumption of chicken and eggs reared in Jebel Ressas’ old mining site by adults and children Adults Location EDI (Zn) Children EDI (Cd) THQ (Zn) THQ (Cd) HI EDI (Zn) EDI (Cd) THQ (Zn) THQ (Cd) HI Exposed sites S1 1.52 3.44 2.05 1.4 3.4 3.97 8.93 5.29 3.52 8.82 Control site S2 S3 S4 S5 0.17 0.18 0.19 0.10 5.58 2.92 0.48 0.10 0.25 0.27 0.29 0.16 2.2 1.15 0.18 0.04 2.45 1.4 0.5 0.21 0.45 0.48 0.52 0.28 14.50 7.60 1.24 0.27 0.65 0.71 0.76 0.43 5.72 3.00 0.49 0.11 6.37 3.71 1.25 0.54 notable risk to the health of people living near the old mine especially children. Discussion One of the major purposes of ecotoxicology is to assess the transfer of pollutants into the food chain (Hattab et al. 2015; Banni et al. 2017). This becomes of a major interest in agricultural areas adjacent to mine sites (Boughattas et al. 2016). With this regard, the present research is, to the best of our knowledge, the first to use regression and mathematical models to estimate the transfer of heavy metal contamination from mine soils to consumed chicken organs and eggs and consequently, to humans. In the present study, we investigated a food chain model constituted by soil as an interface, with plants, earthworms, and domestic chickens living sedentarily in a mining area. Interestingly, we worked on 5 sites characterized by a decreasing polymetallic gradient ranging from the old mine dumps to the agricultural surrounding areas. The first three sites (S1, S2, and S3) were classified as extremely polluted because of their location next to the highly polluted dump (Figure S1). Site S4 was moderately polluted, located in an agricultural area (Boughattas et al. 2016). Soils from sites S1, S2, and S3 contained Pb, Cd, and Zn concentrations exceeding the prescribed standard limits of the CEE Directive (1986) (ElkribiBoukhris et al. 2020). The results of the present investigation revealed that chickens reared in the polluted area revealed significant heavy metal accumulations in their tissues. This might be due to their direct exposure to heavy metals through the ingestion of contaminated food and/or soil particles, as suggested by Lourenço et al. (2011), Zhuang et al. (2014), and Giri and Kumar (2017). Our results revealed that Pb accumulation occurred only in chicken kidneys and feathers. This can be explained by the chicken’s ability to eliminate this heavy metal (Zamani et al. 2010; Haziri et al. 2019). Indeed, feathers are considered one of the optimum ways to eliminate metals from a chicken’s body (Malik and Zeb 2009; Zamani et al. 2010). For this reason, numerous researchers use avian feathers for environmental pollution biomonitoring programs. In this study, Pb and Zn were found in the chicken feather samples from S3 in the concentrations 68.8±4.18 mg kg −1 and 225.63± 20.01 mg kg−1 respectively. These values were higher than that of Haziri et al. (2019), 15.50 ± 0.45 mg kg−1 and 111.0 ± 2.36 mg kg−1 Pb and Zn respectively were found in the feathers of domestic chickens reared in the Kosovo industrial region. Figure 1 HI due to ingestion of contaminated chicken and eggs for adults and children as a function of site localization of the studied area Content courtesy of Springer Nature, terms of use apply. Rights reserved. Environ Sci Pollut Res Moreover, the concentrations of Cd found in the tissues of chickens were in decreasing pattern as follows: kidney> liver> feather> lung> muscle>blood. According to the maximum permissible levels of trace elements in poultry set by the European Communities (2006) and FAO/WHO (1989), heavy metal distribution in organs can be linked to the physiological functions of each element (Jebali et al. 2014). This is in agreement with Leontopoulos et al. (2015), as well as Indrajit et al. (2018). These latter authors reported that different tissues of backyard chickens reared in an industrial area presented pathological issues due to heavy metal accumulation. Our data may suggest that earthworms have the capacity to accumulate high levels of heavy metal, especially Cd and Zn. This was in agreement with the work of Boughattas et al. (2016), Mkhinini et al. (2020), and Hattab et al. (2015). Additionally, Osioma and Hamilton-Amachree (2019) revealed that heavy metals were accumulated in earthworm Lumbricus sp., making it a good bioindicator for soil pollution as is the case for other annelid species (Bouraoui et al., 2010). Also, plants from Jebel Ressas accumulated high levels of Pb. The work of Hattab et al. (2015) showed that Medicago sativa, plant from Jebel Ressas had accumulated high levels of heavy metals. Béjaoui et al. (2016) has, also, worked on mallow (Malva sylvestris) as locally grown plants in Jebel Ressas and wild food for the local population. Interestingly, our data described a chain model approach to assess the transfer of heavy metal contamination from soil to human beings. This model was divided into two main parts: the first was employed to assess the transfer of soil-plant taking into account the heavy metals loads in soil and its properties (organic matter (OM), pH, clay). The second part used regression models to derive the transfer of heavy metals into the tissues of chicken and to analyze the risks that humans may face from the dietary intake of contaminated chicken. The multiple regression results, for the derivation of soil-plant transfer, reported high values of determination factors (0.87<R2<0.91) from the estimated contribution of pH and heavy metal concentration in the soil. Therefore, it is concluded that the transfer of Pb and Cd from soil to plant was further influenced by the pH of the soil, while Zn transfer was influenced by its concentration in the soil. Accordingly, changes in soil pH cause variability in the availability of Pb and Cd for plants. The results of the regression model of the food transfer (soil, plant, and earthworm) of heavy metals into the tissues of chicken rendered a determination factor R2 from 0.014 to 0.99. A significant R2 value (0.99) was found for the transfer of Cd concentration from food to chicken blood. More precisely, Cd transfer in chicken blood happened from the consumption of (in decreasing order) earthworm>soil>plant. Further, Zn concentration transfer (R2= 0.91) and Cd concentration transfer (R2= 0.5), to chicken kidneys were more influenced by earthworm consumption at the first level, and then by plant consumption. The derivation model results showed that heavy metal transfer to the chicken tissues was influenced mainly by earthworm consumption. Other studies have demonstrated that heavy metals in plants may contribute to the transfer of Pb in different parts of chicken such as the kidney, liver, and feathers. Using regression and mathematical models, Franz et al. (2008) proved that Cd was transferred from vegetalbased food to cattle kidneys, liver, and meat. To assess human health risk associated with heavy metal contamination from chickens and associated eggs in Jebel Ressas mining area, the EDI, THQ, and HI were calculated. Results of the EDI of heavy metals suggest that heavy metal pollution causes health risks to the consumers of Jebel Ressas. According to the classification of New York State Department of Health (NYSDOH (New York State Department of Health) 2007), ratios obtained for analyzing heavy metals were classified as low for Zn in all sites except for S1 (which was classified as moderate risk), whereas Cd was several times higher than the RfD, indicating it to be a potential health hazard to the public. Our findings are in concordance with the results of Javed and Usmani (2016), which proved a potential risk of Cobalt (Co) through the consumption of freshwater fish inhabiting a polluted effluent-loaded canal. Target hazard quotient (THQ) proved that Cd (exceed 1 for S1, S2, and S3 sites) and Zn in S1 may pose potential health risks associated with overexposure. For Zn, in the other sites and Pb and Cu, the risk of non-carcinogenic toxic effects is assumed to be low. The hazard index (HI) was above the unity in the mine sites (S1, S2, and S3) and values were between 2.3 and 4.4, which may indicate a possible synergy between trace elements. Therefore, possible interactions between heavy metals even for the essential metals could present an alarming public health risk. Studies have shown that high intake of Zn induces production of Cu binding proteins (metallothionein) in intestinal cells and prevents systemic absorption (King et al., 2006; FDA (Food and Drug Agency) 2011). Conclusion The present investigation aimed to develop a model that links heavy metal content in soil, earthworm, and plant to chicken dietary exposure in order to assess health risks. Our study provided clues of the transfer of heavy metals suggesting that it contributes to their accumulation in the organs of reared chickens. Also, results revealed a considerable risk to the health of local inhabitants especially children. Indeed, the calculated HI ranged between 2.58 and 4.74 for adults and reached 12.34 for children. Therefore, this study highlighted the crucial role of diets based on chickens grown in the contaminated areas, on health risks for local residents of Jebel Ressas especially for children. Content courtesy of Springer Nature, terms of use apply. Rights reserved. Environ Sci Pollut Res Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/s11356-021-15995-9. Acknowledgements This work was supported by funds from the Ministry of Higher Education, Research Laboratory Agrobiodiversity, and Ecotoxicology (LR21AGR02). The authors thank the University of Tunis Al Manar, Faculty of Mathematical, Physical and Natural Sciences of Tunis, Doctoral School “Sciences et Technologies du Vivant et Sciences de la Terre” for the Research fellowship to the student who developed the research. Author contribution Sameh Elkribi-Boukhris (SEB): data curation; formal analysis; investigation; roles/writing—original draft; writing— review and editing Naceur M’hamdi (NM): data curation; formal analysis; investigation; writing Iteb Boughattas (IB): formal analysis; investigation; supervision Sondes Helaoui (SH): formal analysis Cecile Coriou (CC): formal analysis Sylvie Bussiere (SB): formal analysis Valerie Sappin-Didier (VS): data curation; funding acquisition; methodology; resources; supervision; validation; roles/writing—original draft; writing—review and editing Mohamed Banni (MB): data curation; funding acquisition; methodology; resources; supervision; validation; roles/writing—original draft; writing—review and editing Data availability All data generated or analyzed during this study are included in this published article. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. 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