Human health risk assessment of some important trace elements in boneless whole chicken meat [version 1; peer review: 2 approved with reservations]

Background: Excessive trace element exposure has been linked to a variety of harmful health impacts. The goal of this study was to track the amounts of several trace elements in boneless whole chicken samples collected from various shops in Amman, Jordan. Methods: Using inductively coupled plasma-optical emission spectroscopy (ICP-OES), 30 samples were collected and examined for aluminum (Al), arsenic (As), cadmium (Cd), chrome (Cr), copper (Cu), iron (Fe), lead (Pb), manganese (Mn), mercury (Hg), iron (Fe), lead (Pb), and zinc (Zn). The calculated health risk in Jordanians was based on the measured concentrations of these elements. Results: The element concentrations were such that Fe > Zn > Al > Mn > Cu > Cr > As > Pb > Cd > Hg, with As, Cu, Mn, and Zn exceeding the maximum allowable levels. The combined THQs for these trace elements, on the other hand, were 25.22, a value > 1, indicating a non-carcinogenic effects in adult population. As, Cd, and Cr had cancer risk factors that were 46.80, 8.81, and 41.42 times higher than the acceptable lifetime carcinogenic risk (10 -5 ), respectively. Conclusions: Current research shows that Jordanian consumers are at risk of eating boneless whole chicken.


Introduction
Poultry chicken meat is a major source of animal protein and is distinguished by the presence of numerous essential amino acids that are necessary for human health and growth.Despite its high biological value, the quality of poultry meat may be compromised by trace element contamination caused by emissions from various anthropogenic activities (Mottalib et al., 2018).Bioaccumulation of trace elements in meat poses a risk to human health and can result in a variety of adverse health effects due to its persistent and non-biodegradable nature (Al-Subeihi, 2021).Without pollution prevention measures, industrial activities cause increased concentrations of trace elements to enter the environment (Akan et al., 2010).Consumption of contaminated food may establish a significant source of non-occupational exposure for the general population (Filippini et al., 2019).Trace elements in poultry meat may pose potential health risks to humans (Mottalib et al., 2018).Few studies have been conducted to investigate the accumulation and contamination of trace elements in chicken diets in order to assess the potential risk of human consumption (Mottalib et al., 2016).
As a result, it is very important to determine the trace elements content of chicken meat so that it does not harm health and keeps its levels within acceptable ranges (Uysal et al., 2008;Palaniappan and Karthikeyan, 2009).Regulatory bodies in several countries, including the World Health Organization (WHO), Food and Agriculture Organization (FAO) and the European Union (EU), have established maximum permissible concentrations of trace elements in foods (Xue et al., 2012;Sridhara Chary et al., 2008).The maximum acceptable limits for Lead (Pb), Cadmium (Cd), Copper (Cu), and Zinc (Zn) in poultry meat, for example, are 0.1, 0.05, 1, and 20 mg/kg (EU, 2006;FAO/WHO, 2002).Chronic ingestion of large amounts of toxic trace elements from food has been associated with nervous system disorders, increased mortality, cardiovascular disease, morphological abnormalities, kidney disease, growth retardation, various forms of cancer, and genetic effects in humans (Shariatifar et al., 2020).Aluminum (Al), for example, has been shown to cause neurotoxicity in humans.It has been linked to the cause of sporadic Alzheimer's disease (AD) and other neurodegenerative diseases (Yokel, 2012).Mercury can also harm the central nervous system and kidneys (Kimáková et al., 2018).Additionally, the International Agency for Research on Cancer (IARC) has identified several trace elements, including Cd, Nickel (Ni), and Arsenic (As), as carcinogens to humans.(Mulware, 2013).Exposure to inorganic As, for example, has been related to lung and skin cancer (Pershagen, 1981).Cd is a lung carcinogen that also causes prostatic and renal cancers (Rahimzadeh et al., 2017).Long-term exposure to chemical contaminants is most likely associated with potential health risks, which can be estimated using both the cancer risk measure (CR) and the target hazard quotient (THQ) (USEPA, 2011).CR assesses the risk of cancer after exposure to trace elements during one's lifetime (Bello et al., 2017).THQ, on the other hand, is defined as the proportion of exposure to the measured concentration to the oral reference dose, which is the highest concentration at which no adverse health effects are expected (Kortei et al., 2020).Despite Jordan's food and drug administration (JFDA) inspects food for some unwanted chemicals, it is unfortunate that trace elements are not inspected in boneless chicken (Shawarma) in Jordan.The main goal of this study is to use an appropriate spectroscopic method to quantify trace element concentrations in chicken samples (Shawarma) collected from various shops in Amman, Jordan.Another goal is to compare the findings of this study to previous studies published in the literature, as well as the international maximum acceptable levels.Finally, the potential health risk associated with trace element consumption will be estimated, and recommendations for both Jordan's official administrators and consumers will be established.

Sample collection
The sampling period was completed in June 2021.A total of 30 random samples of boneless whole chicken were purchased from various shops in Amman, Jordan, and immediately dried in an oven at the selected temperature.The trace element content of each chicken sample was expressed in milligrams per kilogram of dry weight.

Materials
A purification water system from Millipore was used to deionize water.Nitric acid 70% was bought from Biosolve chemical company (Valkenswaard, Netherlands), and multielement standard calibration solution 1000 mg/L was obtained from Accustandard supplies (New Haven, USA).All glassware used in this study was decontaminated overnight with 10% HNO 3 and rinsed with purified water.

Sample preparation
Whole boneless chicken samples were dried in oven at 105 °C until a consistent weight was achieved.Following that, one gram of each dry sample was placed in a pressure-resistant Teflon (PTFE-TFM) flask and digested with Milestone ETHOS 1 microwave oven (Sorisole, Italy) in accordance with the food safety and standards authority of India (FSSAI) (FSSAI, 2016) with modifications on the digestion protocol which includes the temperature, the time, and the microwave power.For each chicken sample, 7 ml of HNO 3 and 1 ml of H 2 O 2 were introduced into separate flask.The flask was capped tightly then heated following the microwave digestion protocol: 1800 W at 180°C (ramp time 15 minutes, holding time 15 minutes, and cooling time 15 minutes).After cooling, all digested combinations were filtered and diluted to 100 mL with distilled water.

Analysis of digested chicken samples
A Thermo Fisher Scientific iCAP 7400 ICPOES Due was used in combination with a Burgener mira mist atomizer and a glass cyclone chamber to detect trace elements in digested chicken samples.For optimal operating conditions, the ICPO-ES was tuned to a radio frequency (RF) power of 1150 W, nebulizer gas flow at 0.5 L/min, cooling gas flow at 14.5 L/min, and auxiliary gas flow at 0.5 L/min.Each digested sample was tested three times.
Validation of analytical method Percent recovery, linearity, limit of quantitation (LOQ), limit of detection (LOD), and precision are validation parameters for the analytical method, as shown in Table 1.The validation of the method was carried out in accordance with the International Conference on Harmonization (ICH) guidelines (ICH, 1996).Furthermore, a certified reference material (CRM) was used to validate the current study's analytical approach (Animal Origin Reference Material).After being treated to a microwave acid digestion, the CRM was tested with ICP-OES in the same way as actual chicken samples.

Risk assessment
Risk scores for the current study were calculated by examining chicken samples to determine daily intake (EDI), risk ratio (THQ), and carcinogenic risk (CR).

Estimated daily intake (EDI)
Ingestion is a common way humans are exposed to trace elements.The daily intake (EDI) of trace elements for adults eating boneless chicken was calculated using the following equation (Alipour and Banagar, 2018): where Cm is the metal concentration in mg/kg dry weight in boneless chickens.EF -frequency of exposure (365 days/ year).FIR -Chicken consumption rate (for adults, 0.085 kg/(person Â day); ED is the duration of exposure (60 years).Assume an average adult body weight (BW) of 79 kg and TA is the mean time to non-carcinogenic substances (365 days/ year Â ED) (Saha et al., 2016).When calculating health risks, it was assumed that the swallowed pollutant quantity is equivalent to the absorbed contaminant amount (USEPA, 1989) and that cooking had no influence on contaminant concentrations (Cooper et al., 1991).The exposure parameters considered in risk estimations are listed in Table 2 (USEAP, 2012).

Target hazard quotient (THQ)
The non-carcinogenic risk to the population from consuming contaminated chicken was calculated using the target hazard quotient (THQ).THQ was calculated by dividing the estimated daily intake (EDI) of trace elements in polluted boneless chicken by the reference oral dose (RfD) for each element (Mortazavi and Fard, 2017).When THQ is less than 1, trace elements is less likely to have serious health effects.A THQ greater than 1 increases the likelihood that this trace element is hazardous to health (USEAP, 2012).THQ was calculated using the following formula: The CR is a measure of the likelihood of developing any type of cancer as a result of lifetime exposure to carcinogenic elements.Using the cancer slope factor, the CR over a lifetime of exposure was predicted using the following equation (Peng et al., 2016;Shaheen et al., 2016): where CSF denotes the cancer slope factor (mg/kg/day), and the rest of parameters have already been described.An acceptable lifetime carcinogenic risk of 10 À5 has been established by USEPA (Saha et al., 2016).

Statistical analysis
Microsoft Excel software was used in performing all statistical analysis.Descriptive statistics include the arithmetic mean (AM), percentile (P), standard deviation (SD), and range (min-max).Pearson's correlation coefficient analysis (PCA) can be applied to the measurement data to determine the source of trace elements quantified by ICP-OES.PCA calculates the strength of association between two variables on a scale of -1 (perfect inverse relation) to +1 (perfect sympathetic relation) (Adebiyi et al., 2020).If two variables have a strong relationship, they may be derived from the same parent.

Concentrations of trace elements in boneless chicken meat
The central limit theorem (CLT) was used to determine the sample size (n = 30) (Mascha and Vetter, 2018;Brussolo, 2018;Kwak and Kim, 2017).ICP-OES was used to test boneless chicken samples for Al, As, Cd, Cr, Cu, Fe, Hg, Mn, Pb, and Zn.Table 3 displays some basic statistics for trace element concentrations in boneless chicken samples.The mean   5).The mean Cu content in this study surpassed the WHO, EC, and FAO/WHO standards of 0.4, 1, and 1 mg/kg, respectively, as shown in Table 4.
Hg is a harmful trace element that enters the body through the intake of fish (Jaishankar et al., 2014).The type, amount, and frequency of exposure to Hg all contribute to human toxicity.Hg salts mostly harm the renal and intestinal walls, whereas elemental mercury vapour causes severe pneumonitis (Kalay, 1999).Hg is toxic to the human nervous system, and there is evidence of a link between fetal Hg exposure and neurodevelopment in children (Kimáková et al., 2018).In this study, the Hg content (0.0005 mg/kg) did not exceed the maximum EC limit for chickens of approximately 1.0 mg/kg (Mathaiyan et al. 2021).According to Mathaiyan et al. ( 2021), Hg concentrations ranged from 0.0477 to 0.5457 mg/kg (Table 5).
Zn promotes wound healing, lipid and glucose metabolism, hormonal function and hair growth (Topcuoglu et al., 2002).
As shown in Table 4, the average Zn concentration (28.22 mg/kg) in this study exceeded the maximum permissible limit for Zn in chicken meat stipulated by FAO/WHO and EU, but did not exceed the maximum limit stipulated by the People's Republic of China's Ministry of Health of China (MHPRC).According to  Cr has shown to produce skin irritation and ulceration in humans after acute exposure, as well as kidney and liver malfunctions, circulatory and nervous system damage (Wuana and Okieimen, 2011).According to Table 4, the mean Cr concentration was less than the USEPA's concentration threshold (1.0 mg/kg) (Mottalib et al. 2018).The mean Cr concentration in this study was 0.77 mg/kg, which was lower than the value found by Mottalib et al. (2018) in broiler chicken (3.98 mg/kg).
The mean Mn concentration in this study was 3.74 mg/kg, which was lower than the value reported by Korish and Attia (2020).
Pearson's correlation coefficient analysis (PCA) Table 6 shows the results of PCA analysis of trace elements tested in boneless chicken samples.A positive correlation was found between As/Pb, Pb/Fe, Cd/Cr and Cu/Cr in PCA analysis.A positive relationship indicates that the elements in the sample have a similar genetic origin, chemical affinity, and/or a common background level, while a negative relationship indicates that the elements are not chemically similar or originate from different sources.

Health risk assessment
Estimated daily intake (EDI) and target hazard quotient (THQ) Table 7 summarizes the oral reference doses (RfD) of the trace elements studied in this study.The estimated daily intake (EDI) of Al, As, Cd, Cr, Cu, Fe, Mn, Hg, Pb, and Zn in adults is 9.4 Â 10 À3 , 3.12 Â 10 À4 , 1.40 Â 10 À5 , 8.3 Â 10 À4 , 1.5 Â 10 À3 , 4.59 Â 10 À2 , 4.0 Â 10 À3 , 5.38 Â 10 À7 , 6.78 Â 10 À5 , and 3.0 Â 10 À2 , respectively.Except for Al and As, the EDI values of the investigated elements were lower than their corresponding RfD values.Table 7 shows that the calculated THQ of the trace elements (Al and As) was greater than 1, indicating that Jordanians have a potential health risk if they eat boneless chicken containing these elements.

Cancer risk (CR)
The current study found that the CR factors for As, Cd, Cr, and Pb during a lifetime consumption of contaminated fish were 4.68 Â 10 À4 , 8.81 Â 10 À5 , 4.14 Â 10 À4 , and 5.76 Â 10 À7 , respectively (Table 7).The United States Environmental Protection Agency has set an acceptable limit for a lifetime cancer risk of 10 À5 (Saha et al., 2016).The CR of As, Cd, and Cr in this study were higher than the USEPA acceptable value, indicating that people who eat boneless chicken are at a higher risk of developing cancer.

Conclusion and recommendation
Concentrations of ten trace elements (Zn > Fe > Al > Mn > Cu > Cr > As > Pb > Cd > Hg) in boneless whole chicken samples collected from various Jordanian markets were determined using ICP-OES in the present study.Zn has the highest average concentration of trace elements in chicken samples, while Hg has the lowest.Health risk assessment of ten trace elements was investigated for a Jordanian population.As a result, toxic elements were ranked in the order of Al > As > Cr > Zn > Cu > Fe > Mn > Pb > Cd > Hg based on the THQ index, highlighting that the total THQ of all trace elements was greater than one.Furthermore, the CRs of As, Cd, Cr, and Pb were 4.68 Â 10 À4 , 8.81 Â 10 À5 , 4.14 Â 10 À4 , and 5.76 Â 10 À7 , respectively.It can be concluded that exposure to As, Cd and Cr may pose a threat to human health.These metals can accumulate to harmful levels, so JFDA is required to monitor trace elements levels in chicken products commonly consumed in Jordan.In addition, the results of this research shed the light that ICP-OES is a suitable and rapid technique for the determination of trace element concentrations when applied in food industry.Nevertheless, the exposure of these trace elements from many other food sources has not been taken into account in this assessment, which could contribute to the exposure from chicken consumption.
This project contains the following underlying data: -  Reviewer Expertise: Food hygiene I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
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Table 1 .
Validation of the ICP-OES analytical method for determining the selected boneless whole chicken samples.

Table 2 .
Exposure parameters used for the assessment of health risk from food consumption (USEPA, 2012).

Table 3 .
Concentrations of trace elements with various confidence intervals a .
aThe statistical parameters of Min, Max, Mean, SD, and P were used to show the variations in data.

Table 4 .
International guidelines levels.ofAl,As,Cd,Cr,Cu,Fe,Hg,Mn,Pb,andZnwere8.77,0.29,0.013,0.77,1.37,23.44,0.0005,3.74,0.063, and 28.22mg/kg, as shown in Table3.Concentrations of trace elements in boneless chicken samples decreased in the following order: Zn > Fe > Al > Mn > Cu > Cr > As > Pb > Cd > Hg.The concentrations of Al, As, Cd, Cr, Cu, Fe, Mn, Pb, and Zn in boneless chicken samples ranged from 2.25 to 25.52 mg/kg, from 0.142 to 0.419 mg/kg, from 0.006 to 0.024 mg/kg, from 0.524 to 1.089 mg/kg, from 0.943 to 2.316 mg/kg, from 15.629 to 44.48 mg/kg, from 1.532 to 6.473 mg/kg, from 0.003 to 1.69 mg/kg, from 19.51 to 43.33 mg/kg, respectively.On the other hand, the concentration of Hg in all investigated chicken samples was below the limit of detection (<0.0005 mg/kg).According to Table4, the mean As concentration in this study (0.29 mg/kg) was approximately three times higher than the maximum allowable As limits in chicken reported by FAO/WHO and WHO guidelines.This concentration was lower than that reported byDionísio et al. (2010), who found that the concentration range of As determined in chicken production-related samples ranged from 1.30 to 29.8 mg/kg.Cd is a non-essential element that is not present at birth(Thabetelsharawy et al., 2019).The mean Cd content in the current study was lower than that reported by Thabetelsharawy et al. (2019), which was 0.056 mg/kg (Table5).The mean Cd content in this study was below the MHPRC, EC, and FAO/WHO standards of 0.1, 0.05, and 0.05 mg/kg, respectively, as shown in Table4.Cu is involved in blood formation as well as glucose and lipid metabolism(Kwon and Lee, 2001).The mean Cu content in this study was seven times higher than the values published byThabetelsharawy et al. (2019), but equivalent to the concentration reported by Zhuang et al. (2014) (Table a MHPRC (2013).bEl-moselhy et al. (2014).cHosseini et al. (2015).dMottalib et al. (2018).eZhuang et al. (2014).fJayanthi (2019).gMathaiyan et al. (2021).concentrations

Table 5
, the average Zn concentration in this study (28.22 mg/kg) is lower than the corresponding average concentration reported byZhuang et al. (2014).

Table 5 .
Comparison of the total elemental concentration in this study with similar studies.Pb has been linked to delayed development, lower IQ, decreased attention span, hyperactivity, and mental deterioration in children.Memory loss, nausea, insomnia, anorexia, joint weakness, brain, nervous system, red blood cells, and kidney damage in adults(Wuana and Okieimen, 2011).As shown in Table4, the mean Pb concentration in this study (0.063 mg/kg) was lower than the maximum allowable Pb limits in chicken reported by FAO/WHO, the European Commission, and the People's Republic of China's Ministry of Health (MHPRC).Mean Pb concentrations were found to be lower in the current study than the data of 0.30 mg/kg and 0.52 mg/kg reported byThabetelsharawy et al. (2019)andZhuang et al. (2014), respectively.

Table 6 .
Correlation coefficient values between investigated trace elements.

Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate
Table Calculated Cancer Risks (CA) and Target Hazard Quotients (THQ) of trace elements in chicken samples.

Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Yes Competing Interests:
No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Page 5:
All abbreviations used in this equation (Carcinogenic risk (CR)) should be mentioned below under it.Please add them.The author described the equation parameters in the text before in EDI equation but in my opinion all parameters in the equation should be described under each equation.You mentioned in table 1 that LOD of Hg is 0.001.and here you mention that it is 0.0005 (limit of detection (<0.0005 mg/kg).),please revise it.Please transfer such information to the introduction section.What about the other trace elements such as AL, Fe, and others?You have to mention them too.Please discuss all results for all trace elements.The reasons behind these variations in your results compared to the other studies.
○ 5. Results & Discussion: Page 5, table 3: Which form of Hg you have measured?Please indicate organic or inorganic mercury.○ Page 7, the first paragraph: ○ Page 7, the fifth paragraph: ○ Page 8, the third paragraph: ○ Page 9,

table 7 :
Please check this c source under the table I cannot find the superscript letter (c) in this table.Please revise. ○Is

the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No
competing interests were disclosed.