Quantitative determination of trace elements in frozen and chilled chicken using ICP OES and related health risk assessment

A graphitic digestion method linked with inductive coupled plasma optical emission spectroscopy (ICP OES) instrument was used to determine the levels of trace elements in frozen and chilled chicken samples collected from markets in Jouf city, Saudi Arabia. The current method is innovative because samples are digested using a graphitic digestion system, which is safer, facile sample handling, short time for assessment, and more environmentally friendly than existing procedures. The results revealed that the chilled chicken samples contained higher concentrations of Na, Fe, Cr, and Mn than the frozen samples, which were below detection limit. Furthermore, the Cu, Al, Zn, and Cr contents in some of the analyzed chicken samples were over the maximum permissible limit of international standards, indicating a serious risk to consumers. The estimation of health-risk indicated that trace metal contamination causes health risks to the consumers, but the effect decreases with decreasing the target hazard quotient value in the rank of Cr > Al > Zn > Cu > Mn > Fe, with all values less than 1 except for Cr in chilled chicken. Moreover, the tolerable limit (1 × 10−6) for the carcinogenic risk for Cr in chilled chicken was observed to be exceeded.


Introduction
Industrial wastes and agricultural activities are the most potent resources of ecosystem deterioration with toxic elements.Once the soil is contaminated, metals can enter the food chain via animals and plants.One of the main sources of animal protein is chicken meat, which is rich in biological vitamins, lipids, minerals, and amino acids essential to human health chicken meat is the most popular kind of meat eaten worldwide, including in Saudi Arabia [1].The chicken meat quality could be influenced by trace elements during processing and environmental contamination caused by human activities [2].Trace elements can also be categorized as toxic metals, including in the case of arsenic, aluminium, mercury, lead, and cadmium, which accumulate in meat and pose a hazard to human health even at low concentrations [3][4][5].Some trace elements are regarded to be necessary, such as zinc, selenium, iron, manganese, and copper which play significant roles in metabolism, physiology, enzyme activities, and human biology [6,7].At higher concentrations, however, these essential trace elements may cause harmful effects.For instance, excessive zinc intake leads to reduced high-density lipoprotein levels among healthy males [8].Excessive Mn intake leads to health issues such as muscle weakness, nausea, and growth retardation [9].Furthermore, long-term exposure to high doses of Cu can cause to the generation of acute gastrointestinal problems and Wilson's disease [10,11].Around 25% of the diseases affecting humans results from long-time exposure to environmental pollution (WHO).From the viewpoint of global health, it is necessary to estimate the important and harmful components in frozen and chilled chicken items in Saudi Arabia marketplaces.
A range of analytical techniques, namely, inductively coupled plasma atomic emission spectroscopy (ICP OES), ICP mass spectroscopy (ICP MS), flame and graphite furnace atomic, absorption spectroscopy (F-AAS and GF-AAS) were used for the assessment of tracemetals in various sample.ICP OES is the most used multi-element analysis technique various fields such as environment [12], food [13], cosmetics [14], medicinal plants and herbal medicines [15], and biological [16] owing to its great sensitivity, high accuracy, and small detection limits [16].
The content of trace elements in various types chicken part samples were studied in different countries in the literature [17][18][19][20][21][22][23].Abdul Mottalib et al. [24] assessed the content and dispersion of metals (Cd, As, Co, Cu, Ni, and Cr) in various kinds of chicken parts after digestion using a blend of HNO 3 and HClO 4 (4:1 vol/vol) at 80°C for 2 h in markets in Bangladesh.They found that the contents of Cu, As, and Cr exceeded the allowable concentrations of international standards, suggesting a health risk [25].Uluozlu et al. assessed the trace element levels of chicken in Turkey by atomic absorption spectroscopy (AAS) after microwave digestion and the results revealed that the lead (Pb) contents in some selected chicken specimens were greater than the permitted limits [26].
Different digestion methods were used for samples [27].To provide information on the total amount of metals in dietary samples, the digesting process chosen must be appropriate.hence, graphitic digestion system that used to digest the frozen and chilled chicken samples in this study has advantages compared with other digestion methods.It is environmentally friendly as there are no gases transmitted to the environment.This system operates in a fume hood under the water suction to dissolve the gases in the water and sink them into the sump to be drained.The digestion system used was safer than direct acidification or microwave systems that did not use pressure and were not exposed to fumes from direct acidification.Also, the advantage of this method is that this system has a corrosion-resistant design with an exhaust gas collection cover attached to it.A crucial tool for assessing potential health impacts in food brought on by various contaminants is health risk assessment [28].This technique has been extensively utilized in the research to assess the potential negative health effects of intake to polluted food [29,30].
As far as we are aware, there are no reports on the trace elements level of frozen and chilled chicken samples in Jouf city.Therefore, the goal of this work was to assess how much of a variety of trace elements, including K, Na, Mg, Ca, Fe, Cu, Se, Mn, Zn, Al, Bi, Cr, Co, and Pb, were present in frozen and chilled chicken samples purchased from markets in Jouf, Saudi Arabia.To ensure the safety of this food by checking whether the comprising elements are within permissible limits.The trace elements in frozen and chilled chicken were assessed by ICP OES following the digestion using a graphite digestion system and the related health risk assessment was estimated.The proposed method showed excellent linearity, accuracy, precision, and recovery as well as adequate LOD and LOQ values.

Instruments
Using a Milli-Q water purification equipment (Millipore, USA), deionized water utilized for dilutions was developed in our lab.All glassware and plastic rinsed by immersing in nitric acid (2%), followed by a washing with purified water, before being used.Under standard working conditions, the digested results of the chicken specimen were measured using an ICP OES Shimadzu ICPE-9000 and graphite digester (model SH220F; Hanon Advanced Technology Group Co., Ltd., Jinan, China) with a temperature range of 5-450°C (Table 1).For sample procedures, additional equipment such an analytical balance and a micropipette were employed.

Sample collection and assessment
Nine frozen and five chilled chicken samples were provided from a local market in Jouf city, Saudi Arabia.Before the assessment of trace elements, the chicken specimens were rinsed with distilled water, divided into smaller pieces, and had the fat and bones removed.The chicken samples were then thoroughly thawed, chopped, hammered with a wooden hammer, and then homogenized.The chicken samples stored in plastic bags until being subject to acid digestion.

Digestion procedure
Graphitic digestion system was utilized to digest the specimen and this approach is eco-friendly due to no gasses passed to the environment which is this system works in the fume hood underwater suction to dissolve the gases into water and sink in the basin for drainage.The digestion system used was safer than direct acidification or microwave systems which was not using pressure and were not exposed to the fumes produced from the direct acidification.Also, the feature of this method is that this system has a corrosionresistant design with a waste gas collection hood linked to it.Typically, each chicken sample was weighed to be around 2 g, then moved to a digestion tube, where 20 mL of 5 M HNO 3 and 5 mL of H 2 SO 4 were added.The digestion tube with the chicken sample was incubated for 10 min at ambient temperature and then sealed.The chicken specimens were then transferred to a digester, where they were digested at 120 °C for 40 min.All the samples were separated after cooling through Whatman paper No.1 a 0.45 μm membrane filter, then 0.1 mL filtrate was transferred to a polypropylene tube comprising 20 mL distilled water.Finally, solutions were stored in a polypropylene tube for metal determination by ICP OES.The ICP OES conditions for element assessment in chicken samples are shown in Table 1.

Quality assurance and quality control
Measures for quality control and assurance were properly followed.Because glassware was thoroughly cleaned with detergent and de-ionized water before use, all chemicals and reagents were of high quality.To ensure the quality of the digestion procedures, QA/QC protocols were employed, which involved the examination of triplicate samples.Element concentrations were measured in triplicate.To ensure quality control of the trace element analysis, certified reference material ERM-CA713 was utilized.Moreover, spiked samples were also prepared.Tables 3 and 4 demonstrate that the replicate analysis of the reference material and spike samples exhibited favourable accuracy, with recoveries obtained from both analyzed samples.Prior to sample analysis, a standard calibration curve with at least seven samples of stock solutions was made, and the correlation coefficients are more than 0.998 as shown in Table 5.The accuracy of the ICP OES analyses was evaluated from results obtained for the reference material as shown in Table 2.The digestion processes were separately performed three times for each sample.Each sample underwent the digestive processes three times in total.Analysis of the reference standard material (ERM-CA713) and addition/recovery calculations, as indicated in Tables 2-4, were used to evaluate the method accuracy.

ICP OES validation
In terms of accuracy, precision, linearity, limits of quantification (LOQ), limits of detection (LOD), and recovery the procedure was verified.The linearity of the ICP OES approach was evaluated by the correlation coefficients  of calibration plots for the standard solution of elements and all calibration plots for element had a regression factor of R 2 ≥ 0.9999.The assessment accuracy of the recipe was determined based on the investigation of reference standard material (ERM-CA713) after acidic digestion using a graphite digestion system.High agreement was obtained between the certified value (μg L −1 ) and measured analyte amounts as given in Table 2. Besides, the RSD% of the three replicated measurements was used to express the method's precision and the results show that the recovery rates were in the range of 96.65%-98.65% with standard deviations (RSDs %) ranging from 0.99 to 0.52% (Table 2).The recovery investigation of the assessment process was conducted using addition/recovery calculations.For this, a chicken specimen was spiked with a known amount of the target element prior to digestion followed by re-measurement of the chicken sample.The recovery of the added element in a chilled sample (CCS3) and frozen sample (FCS6) are shown in Tables 3  and 4, respectively.The rates of recovery were in the range of 95%-100.18% and 95%-102.20%for the studied elements in CCS3 and FCS6, respectively.To test the precision of the ICP OES instrument, a standard solution with a content of 0.5 mg/L was injected during the working run (N = 3), and the standard deviation (RSDs %) ranged from 2% to 5% (Table 5).The limit of detection (LOD) and limit of quantitation (LOQ) were determined as three and ten times the standard deviation on the measured content for ten replicate runs of blank samples, respectively [31] (Table 5).Finally, the proposed method showed excellent linearity, accuracy, precision, and recovery as well as adequate LOD and LOQ values.

Target hazard quotient (THQ), Daily intake (EDI), and Hazard index (HI) assessment
In this study, the consumption of chicken leads to human ingestion of hazardous metals.For three elements for frozen chicken and six elements for chilled chicken, according to the following equation [30], where, ED duration of exposure, EF frequency of exposure (365 days/year), IR rate of ingestion (kg day −1 ), [M] the content of trace metal (mg kg −1 ), AT noncarcinogens averaging time (365 days/years), and BW weight of body (kg).For those who consume chicken three times per week, the exposure frequency (EF) was established at 144 days per year for people who feed chicken three times a week.The average daily chicken intake is 39.9 kilograms per year [32].The exposure duration for adults is 70 years, which is similar to the average lifespan.The mean value for adult body weight was calculated to be 65 kg.
Based on non-cancerous risk, the human health risks are characterized by the THQ.It was determined by the following equation [30].
THQ values less than one indicate low carcinogenic harmful effects.However, if it is greater than one, there are considerable health hazards due to its long-term exposure [21].A blend of toxic substances' overall noncarcinogenic health risks is assessed using the HI (hazard index).As a result, the following equation [23] defines HI as the total of all estimated THQ amounts for various harmful pollutants.HI = THQ 1 + THQ 2 + . . . . . .+ THQ n THQ i is the THQ of each contaminant.

The excess lifetime cancer risk (ELCR) assessment
The following equation is generally utilized to determine the excess life-time cancer risk (ELCR) of the pollutants that have been identified as carcinogenic [33].

ELCR = EDI × SF
Where SF stands for slope factor of the carcinogenic (mg kg −1 d −1 ).The chromium (Cr) CSF value is 5 × 10 −1 [28].The estimated value of ELCR represents the likelihood that cancer will occur during the desired population's lifespan as a result of exposure to carcinogenic pollutants.When the ILCR is less than 1.0 × 10 −6 , the USEPA deems a risk level "acceptable", whereas when the number exceeds 10 −4 , it is deemed "severe" or "high priority".E-4 to E-6 is the tolerable risk value range [28].

Statistical analysis
The mean of three outcomes' standard deviation is used to express the findings of repeated tests conducted in triplicate.The independent student t-test was utilized in the statistical analysis using SPSS (version 13; SPSS Inc.) software.Statistical significance was determined as a probability lower than 0.05.For the correlation between trace metal levels, Pearson correlation coefficients have been used.

Trace element contents of frozen chicken samples
The number of selected frozen chicken products was 9 samples, as presented in Table 6, and the mean content ± standard deviation of selected trace-elements (Na, K, Ca, Mg, Fe, Zn, Cu, Bi, Al, Cr, Co, Ni, Se, and Pb) in frozen chicken specimens obtained from markets in Saudi Arabia were measured by ICP OES, with the results depicted in Table 6.The average trace element contents in frozen chicken samples ranged from 16250 to 29083.3 μg g −1 for potassium, 1005-1872.5μg g −1 for magnesium, BDL-1305 μg g −1 for calcium, 4.75-13.75μg g −1 for copper, BDL-149.75 μg g −1 for zinc, 5.5-242.25 μg g −1 for aluminium, and 21250-49000 μg g −1 for bismuth (Table 6).Comparing the obtained results for all the various frozen chicken samples, the concentrations of Bi were the highest (49000 ± 3441 μg g −1 ), as found in FCS9 (Table 6), while those of Cu were the lowest (4.75 ± 0.29 μg g −1 ), as found in FCS2.Other elements, namely Fe, Cr, Mn, and Na, were not detected in any frozen chicken samples [34].In addition, the metals Co, Pb, Ni, and Se were not observed in any of the chilled and frozen chicken samples.
Cu was present in the range of 4.75-13.75μg g −1 .The smallest and greatest contents were 4.75 ± 0.29 μg g −1 in FCS2 and 13.75 ± 1.94 μg g −1 in FCS6, respectively (Table 6 and Figure 1(f)).All measured concentration of copper in frozen chicken specimens were greater than the allowed limit of 1 ppm set by international standards [35].In addition, the Cu contents  exceed the daily allowance (0.9 mg/adequate intake) recommended by FAO/WHO for humans [36,37].Compared with previous studies, as listed in  7).Although copper is necessary for health, excessive ingestion may result in complications, including kidney and liver damage.Regarding Zn, it was only detected in one sample, FCS6, with 149.75 ± 9.47 μg g −1 (Table 6 and Figure 2(a)).The recommended daily intake of zinc is12 mg for adult females and 15 mg for adult males [26].The obtained value exceeds the permissible level of 20 ppm set by international standards [34,35].Onianwa et al. [26] determined the Zn content in 80 Nigerian food items and found that a Zn concentration of 2.87 mg kg −1 in their chicken sample [38] (Table 7), which is lower than in our results.

Trace element contents in chilled chicken samples
The measured contents ± standard deviation of selected trace-elements (Na, K, Ca, Fe, Mg, Zn, Cu, Bi, Al, and Cr) in chilled chicken specimens were measured by ICP-AES, and the results are shown in Table 6.The Na, K, Ca, Fe, Mg, Zn, Mn, Cu, Bi, Al, and Cr contents were in the range of 22666.7-40083.3μg g −1 , BDL-655 μg g −1 , 1330-2237.5 μg g −1 , BDL-1540 μg g −1 , BDL-2.75 μg g −1 , 5.5-19.5 μg g −1 , BDL-169.5 μg g −1 , BDL-0.5 μg g −1 , BDL-228.25 μg g −1 , 13250-49000 μg g −1 , and 8-20.5 μg g −1 , respectively.Comparatively wide variation of K and Bi was observed among the analyzed chilled chicken samples, wherein CCS3 had the highest K and Bi levels (40083.3± 204.1 and 49000 ± 3629 μg g −1 , respectively), while the CCS1 had the lowest K and Bi levels (22666.7 ± 198.6 and 13250 ± 1289 μg g −1 , respectively) (Table 6).The concentrations of Cr were highest in CCS3 (22 ± 1.23 μg g −1 ) followed by CCS6  (20.5 ± 1.71 μg g −1 ) and lowest in CCS2 (8 ± 2.50 μg g −1 ) (Figure 2(e)).The Cr content of chilled chicken samples exceeds both the recommended daily allowance (0.35 mg/adequate intake) set by FAO/WHO for humans [34] and the highest allowable level of Cr (1 ppm) in collection of poultry products by international standards [34,35].Prolonged exposure to it can result in various biotoxic effects, including damage to the hepatic, renal, and hematological systems [21].Moreover, the Cr concentrations in our samples (CCS) are higher than those cited in the literature [26] in Turkey and by Bratakos et al. 42 [42] in Greece, and Al Brattyet al. 2017 [39] in Saudi Arabia for chicken meat (Table 7).Korish and Attia et al. (2020) reported a mean chromium level of 2.46-8.32ppm in meat products (eggs, meat, burgers, frankfurter liver) from Saudi Arabia during June-July 2017 [35].Sodium was found in all chilled chicken samples except for two, CCS2 and CCS5.CCS4 had the highest levels of Na (655 ± 36.7 μg g −1 ), while CCS3 had the lowest (142.5 ± 26.4 μg/g) (Table 6 and Figure 1(b)).Iron was detected in all samples except one sample (CCS1).The highest content of Fe was in CCS3 (2.75 ± 0.27 μg g −1 ), and the lowest was in CCS5 (2.25 ± 0.03 μg g −1 ) (Table 6 and Figure 1(e)).The Fe content found in all samples is within the acceptable daily limit of 8 mg for adequate intake specified by FAO/WHO for humans [37].The Fe levels found in this investigation are smaller than those cited by Uluozlu et al. 26 [26] and Al Brattyet al. 2017 [39].Regarding Mn content, the levels were below the detectable level in all chilled chicken except for CCS3 (0.5 ± 0.17 μg g −1 ) (Figure 2(b)).This value is smaller than that cited by Uluozlu et al. 26 [26] (Table 7).The Institute of Medicine recommends that intake of Mn from water and food should not exceed the permissible daily upper limit of 11 mg per day [45].The Mn content is lower than the allowable limit of 0.5 mg/Kg set by international standards [46].The role of manganese in neuropsychiatric disorders is documented [47].
Comparing the element levels in chilled chicken with those in frozen chicken, the Mn, Cr, Fe, and Na contents were lower than the detectable limit only in frozen chicken samples.This is due to the different sources of food such as feed, water, and litter which have high concentrations of toxic elements.In addition, the measured levels of K (16250-29083.3μg g −1 ), Ca (BDL-1305 μg g −1 ), Zn (BDL-149.75μg g −1 ), and Cu (4.75-13.75μg g −1 ) contents in chilled chicken samples were greater than the K (22666.7-40083.3μg g −1 ), Ca (BDL-1540 μg g −1 ), Zn (BDL-169.5μg g −1 ), and Cu (5.5-19.5 μg g −1 ) contents in frozen chicken samples.Table 8 shows the significant variation in potassium concentrations between frozen and chilled chicken.This indicates that, among other elements found in chilled chicken.
The results of this study revealed that the chilled chicken samples contained higher concentrations of  Na, Fe, Cr, and Mn than the frozen samples, which were below the detection limit.Furthermore, the Cu, Al, Zn, and Cr contents in some of the analyzed chicken samples were over the maximum permissible limit of international standards, indicating a serious risk to consumers.

Pearson's correlation matrix
Pearson correlation test was conducted to assess the relationship between the obtained element concentrations in this work.Table 9 shows the matrix for Pearson's correlation between metals based on concentration.The results reveal a considerable positive relation of K-Mg (r 2 = 0.874), K-Fe (r 2 = 0.812), K-Cr (r 2 = 0.765), Ca-Zn (r 2 = 0.773), Cu-Mn (r 2 = 0.710), and Zn-Al (r 2 = 0.722).Negative correlations were observed for K-Al (r 2 = −0.104),Na-Bi (r 2 = −0.043),Ca-Al (r 2 = −0.072),Ca-Bi (r 2 = −0.005),and Fe-Al (r 2 = −0.125),Zn-Bi (r 2 = −0.164),Mn-Al (r 2 = −0.185),and Al-Bi (r 2 = −0.072).In cases where there is a positive relation coefficient, it suggests that the element concentrations are probably being controlled by similar sources.Therefore, a positive correlations were observed between the elements which above mentioned indicates that the metal concentration in frozen and chilled chicken samples probably resulted from similar pollution levels and sources such as feeds, water, litter, and the environment [35].  in boneless whole chicken collected from Jordon that includes Cu/Cr, Pb/Fe, As/Pb, and Cd/Cr [48].

Non carcinogenic risk analysis
To assess non-carcinogenic human health risks related to the metal pollution from each type of the tested chickens, THQ, HI and EDI were determined.The findings illustrated in Table 10 and Figure 3 indicate that the contamination with trace metal result in hazards to consumers' health.Still, the effect decreases with decreasing HI value in the rank of Cr > Al > Zn > Cu > Mn > Fe, with all values less than one except for Cr in chilled chicken.Therefore, the exposed adults were unlikely to suffer from apparent detrimental health effects except for Cr in the case of chilled chicken consumption.Consumers of chilled chicken may be subjected to dangers due to the fact that HI is multifaceted.

Carcinogenic risk analysis
Potentially toxic metals can improve the cancer risk in humans, and long-term subject to low concentrations of carcinogenic metals causes various cancers.The intake of trace metals by the consumers was determined using the average ELCR value as listed in Table 11.Cr has a 5.5 × 10 −3 carcinogenicity, which translates to a cancer risk of five per 1000 people.Cr  obtained from chilled chicken carries a very significant risk of cancer.

Conclusion
The current study displayed that frozen and chilled chicken species, provided from the markets in Jouf city, Saudi Arabia comprise various metal contents.The contents of elements were determined using ICP OES after digestion with a graphitic digestion system.The results of selected assessment confirmed that the graphitic digestion system for sample preparation and ICP OES could be used to elements in chicken specimen.The contents of Cu, Al, Zn, and Cr levels in some analyzed chicken samples exceeded the maximum permissible levels of international standards, which may pose a serious threat to consumers.The high contents of Cu, Al, Zn, and Cr levels in some analyzed chicken samples could be attributed to the metals accidentally entering the food chain.Chilled chicken samples exhibited higher levels of Na, Fe, Mn, and Cr than frozen chicken samples, which were below detectable limits.According to the results of the health-risk assessment, trace metal contamination causes carcinogenic and non-carcinogenic health risks to consumers due to Cr present in chilled chicken.Based on the findings, this approach can be utilized as an alternative for assessing various species with a comparable matrix because it is quick and affordable.

Figure 3 .
Figure 3.Estimated target hazard quotient (THQ), and health risk assessment (HI) of metals from chicken samples.

Table 1 .
Operating conditions parameters of ICP OES were used in this study.

Table 2 .
The concentration of elements in the certified ERM-CA713 reference material.

Table 3 .
The relative recovery of metals in the chilled sample (CCS3) with 1 mg/L of elements spiking.
a SD = Standard deviation.

Table 4 .
The recovery of metals in the frozen sample (FCS6) with 1 mg/L of elements spiking.
a SD = Standard deviation.

Table 5 .
Method validation for the metals by ICP OES.

Table 6 .
Concentrations (mean ± SD) of trace elements in Frozen and Chilled Chicken samples.

Table 7 .
Concentrations (mean ± SD) of trace elements in Frozen and Chilled Chicken samples.

Table 8 .
Mean differences of elements concentration between frozen and chilled chicken and their significance level.

Table 9 .
Correlations between metal concentrations of chicken products.

Table 10 .
The reference dose (mg kg −1 d −1 ) and estimated daily intake (EDI in mg kg −1 d −1 ) of trace metals from chicken samples.

Table 11 .
Carcinogenic risk of elements in chilled chicken.