Heavy Metal Residue (As, Cd, Hg, and Pb) in Hen Eggs after Applying Different Cooking Methods

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Introduction
Food chain pathways and dietary consumption are the primary causes of toxic heavy metal exposure in humans [1].Food pollution with heavy metals is the main threat to human health, as they cause many diseases and complications.High concentrations of toxic heavy metals (As, Cd, Pb, Cr, Zn, Cu, Co, Fe, and Ni) in the environment, contaminate the water, soil, fauna, and flora [2,3].These metals mostly accumulate in agricultural soils, later showing up in food and ultimately causing health problems [4,5].Heavy metals are present in the environment due to both natural processes and pollutants from human activity [6].
Hen eggs are a valuable supply of high-quality protein and vital nutrients [7][8][9][10].Compared to other sources of animal proteins, hen eggs are a necessary part of the food pattern and are more affordable worldwide [11,12].In addition, hen eggs contain a staggering number of biologically active components to provide nutrients suitable for practically any age [13][14][15].Worldwide, the average annual per capita consumption of hen eggs was 250.The annual number per capita egg consumption in the USA, Japan, Turkey, and Iran was 280, 335, 135, and 192, respectively [12,16].
Eggs may become contaminated with heavy metals through drinking water and chicken feed, both of which are highly affected by the environment [17].Eggs are capable of accumulating heavy metals and are a good indicator of environmental contamination, too [18][19][20].
According to the Environmental Protection Agency (EPA), a group of four heavy metals that includes arsenic (As), mercury (Hg), cadmium (Cd), and lead (Pb) is the most prevalent in the environment [21].These metals gradually spread in water and subsurface soil because they are not degradable and have a long persistence, allowing them to accumulate and amplify along the food chain [22].They can cause both carcinogenic and noncarcinogenic consequences, as well as a variety of detrimental health issues with long-lasting impacts [23].Negative effects can occur through skin contact, inhalation, and ingestion via water, air, and soil [21,24].When heavy metals enter the human food chain after consumption, they react with proteins and enzymes and can become stable oxidants in the acidic environment of the stomach.This causes the body to accumulate heavy metals [25].
Heat (boiling, baking, roasting, frying, and grilling) is applied to food in different ways to improve its hygienic quality by inactivating pathogenic microorganisms to enhance flavor and taste, increase shelf life [17], increase food safety, enhance nutrient quality, and digest proteins better; so, it is recommended to heat hen eggs before consumption [26,27].
Many studies have been conducted to investigate the negative and positive effects of processing in reducing hazardous components in food.Some studies suggest that heating food greatly reduces the amount of heavy metals.A wide variety of food products have been assessed, including fish and seafood, seaweed, fruits, vegetables, rice, milk, and bread.Based on their findings, they have developed protocols and proposed chemical agents to minimize the amount of toxic elements in final food products [2,28].
The different methods of cooking have been reported to significantly affect the amounts of contaminants such as heavy metals present in the final product [29].Cooking methods such as boiling and dry-frying can alter the quantity of hazardous elements through the loss of water and volatile ingredients, the solubilization of the metal, and to some extent, metal binding to macronutrients, including proteins, lipids, and carbohydrates.Harmful substances are eliminated from food during dry-frying, boiling, or canning operations and can be transferred to the frying pan, boiling water, or cooking stocks because they are not evaporated or converted into safer compounds.However, some cooking techniques, including herbal marinating, can lower the bioaccessibility of hazardous substances by combining them with other substances to produce inaccessible complexes [28].
This study was conducted in the framework of a heavy metal monitoring plan and risk assessment in the food chain.Although numerous studies have been conducted on the amount of heavy metals in hen eggs, to the best of our knowledge, this is the first study in which two methods of hen egg cooking, boiling (with and without shell), and dry-frying are compared on the residues of heavy metals (As, Cd, Hg, and Pb).

Material and Methods
2.1.Samples.In the experimental design, forty-four hen egg samples (from each pack of 12, 2 eggs) were randomly chosen from 22 best-selling brands of supermarkets in Tehran, Iran, in February (2023).All samples were initially checked with candles, and only undamaged and fresh eggs (eggs without pinholes, clearly clean eggs, and no cracks) were chosen.All the hen eggs were cleaned with deionized water and categorized into one of four groups (raw, dry-frying, boiling with and without eggshells).Before usage, deionized water was utilized to wash all the glassware (even ceramic nonstick pots and pans) (Figure 1).
Boiling group with eggshells: the deionized water was used to boil hen eggs with shells in ceramic nonstick pots for 10 minutes, and then the shells were removed (n = 11).
Boiling group without eggshells: the deionized water was used to boil.After that, unshelled hen eggs were put in ceramic nonstick pots and boiled for 8 minutes, and then filter papers were used to remove water from hen eggs (poached) (n = 11).
Dry-fried group (without using any oil or butter): whole edible hen eggs (yolk and white) mixed with a glass stirring rod in a beaker) to avoid metal contamination( and then dry-fried for 5 minutes in ceramic nonstick pans which is similar to grilling (n = 11).
Raw group: raw hen eggs, whole (yolk and white), mixed with a glass rod in a glass beaker (without any cooking) (n = 11).

Samples Preparing for
Laboratory.All samples were first cooled at room temperature in Pyrex plates before being put in zipped-lock bags and tagged with a code.Each sample was thoroughly mixed and homogenized before being transported to the laboratory for analysis of heavy metal residues (As, Cd, Pb, and Hg).The samples were stored in a refrigerator at 4 °C immediately before the preparation steps.To prevent chemical contamination, sterile and chemical-resistant flask containers were used.Initial analytical work began immediately after cleaning all equipment with dilute nitric acid (HNO 3 10%) and then rinsing with deionized water.
The whole egg ingredients (yolk and white) were put in a petri dish to dry in the 70 °C oven for 24 hours until it turns into a fine powder [30].For the digestion of .05grams of dried egg samples, 10 ml of 65% nitric acid and 37% hydrogen peroxide (v/v) were purchased (Merk KGaA, Darmstadt, Germany).
The solution was left at room temperature for one night until it was clear.The digestion was stopped at 150 °C after 4 hours after bringing the solution to room temperature (22-23 °C).The solution was then diluted to 50 ml with deionized water, filtered with 0.45 l of acid-resistant filter paper, and stored for later examination [31].Using the heat-blockassisted acid digestion method and the inductively coupled plasma mass spectrometry (ICP-MS) methodology following the FDA (Food and Drug Administration) Elemental Analysis Manual [32], the total amount of As, Pb, Cd, and Hg was analyzed.The blank solution was made similarly but without the use of hen egg.The criteria for detection in hen eggs using ICP-MS are listed in Table 1.The method employed in ICP-MS included ionizing the sample with inductively coupled plasma.ICP-MS is well-known for its application in detecting exceedingly low quantities of different nonmetals and metals in liquid samples.For determining the level of heavy metals in the food industry, this method is thought to be the fastest and most reliable.Estimation was done using ICP-MS (ULTIMA2, 6100 DRC-e Perkin Elmer Elan) [31].
The calculation of the limit of detection (LOD μg kg -1 ) , based on the measurement results obtained with blank filters in the present study was 0.0003.The results were given in micrograms per kilogram of the sample's moist weight.The limit of quantification (LOQ) was estimated to be 0.001 for both metals [33].

Statistical Analysis.
The statistical analysis was performed with the SPSS software version 20.The means with standard errors of values were estimated by one-way analysis of variance (ANOVA).Differences between means were compared by Tukey's method, and p < 0 05 was considered significant.

Results and Discussion
3.1.Heavy Metal Residue in Raw Hen Eggs.According to the results of heavy metals in raw samples in the present study, the average levels of As, Cd, Hg, and Pb were 0.307, 1.654, 0.121, and 6.5 (μg.kg -1 ), respectively.Compared to the provisional tolerated weekly intake (PTWI) of As, Cd, Hg, and Pb of 15, 7, 4, and 25 (μg kg -1 BW/week), all heavy metal residues (As: 0.75, Cd: 4.12, Hg: 0.30, and Pb: 16.25) were determined to be below acceptable permitted values ( [34][35][36][37][38][39][40].On another hand, comparing the results of this study with Iranian assessments [30,[41][42][43] indicated that during almost a decade, food authorities have paid more attention to this issue.2 displays the ICP-MS results on the effect of different cooking methods in terms of heavy metal residues (μg.kg -1 ).The remaining arsenic in boiling with eggshell (0 457 ± 0 918) was more than boiled without eggshell (0 228 ± 0 197) and dry fried eggs (0 222 ± 0 109).This difference was significant by using the ANOVA test (p < 0 05).Also, residual cadmium in hen eggs was shown between boiling (with shell 2 118 ± 0 223; without shell 1 985 ± 0 037) and dryfrying (1 548 ± 0 223).Dry-frying also lowered arsenic and cadmium concentrations to a significant degree compared to raw eggs (p < 0 05).Probably, the eggshell prevents the transfer of As or Cd to the boiling water.The extent of this reduction varies depending on the severity of the element.Heavy metals may be bonded to soluble amino acids and uncoagulated proteins.The mechanism is believed to be the leaching of heavy metals into boiling water due to protein denaturation and solubilization from cooking temperatures.Metals bound more loosely to egg components are released [44].
In general, the results of the present study showed a higher residual of heavy metals including arsenic and cadmium in boiled egg with shell in comparison to raw samples; despite the lack of statistical significance, this increase can be related to the increased permeability of the eggshell during heating [45].According to the results of Hincke et al. [46], an eggshell has many pores in its structure [46].So, it is permeable, and these little alterations might be put on by the surroundings of the chicken, human contamination from handling eggs, the container used to keep the eggs, recycled materials, or anything else that contaminates the eggshell's surface that plays a slight role in these changes, boiled with eggshell compared to the results of raw hen eggs.Therefore, the eggshell was suitable as an indicator of heavy metal pollution [47].
In one study, Canadian researchers investigated the effect of cooking on residual arsenic levels.The results showed that the decrease in arsenic in cooked fish was due to weight reduction in fish which led to an increase in the concentration of arsenic [48].In another study [49], it was claimed that reductions in arsenic levels during cooking were due to the dissolving or evaporating of the metalloid.In contrast, the increase in total arsenic levels was caused by the concentration of arsenic resulting from the loss of water and other soluble compounds during cooking.Arsenic species can be classified as either lipid-soluble or watersoluble arsenic based on their chemical characteristics.Water-soluble As includes both inorganic and organic chemicals, and they can dissolve in water during cooking.However, lipid-soluble arsenic can be dissolved in oil or other fats during cooking.Therefore, understanding the characteristics of different arsenic species can help reduce exposure to this toxic metalloid [50].
Although the level of Pb residue decreased (with eggshell 6 285 ± 1 672 > without eggshell 5 714 ± 1 889 > dry − frying 5 457 ± 1 672), it was not significant.The amount of mercury residue level in hen eggs after boiling decreased lightly (with shell 0 100 ± 0 166 and without shell 0 120 ± 0 005), despite a slight increase in the dry-frying method (0 122 ± 0 012), and, it was also not significant.Mercury and lead showed little change or inconsistent effects from cooking methods.They may bind more strongly within eggs and resist removal through cooking.The mineralization of the shell is characterized by the precipitation of calcium carbonate on the egg membrane [51].As the eggshell is mainly inorganic, Hg is rarely deposited in the eggshell [52].It seems that mercury has a preference for forming bonds with sulfur (-SH) groups in addition to the water loss due to heating, which can explain the slight increase in concentration during grilling [25].
When hen eggs are dry-fried, water molecules inside the hen egg are heated and gain enough energy to evaporate, and the reduction of some heavy metal concentrations, such as As, Cd, and Pb, might be due to the loss of volatile substances during evaporation.The extent of evaporation depends on several factors, such as the cooking temperature, duration, and the amount of egg surface exposed to the air [28].The fact indicates that the concentration of crude protein (amino acids containing sulfur) in food is inversely correlated with the quantity of heavy metals (As, Cd, Hg, Pb), indicating that heavy metals (As, Cd, Hg, Pb) are binding to the SH groups of proteins [49].
Several investigations claimed that the amount of heavy metals in food had significantly decreased after cooking.Cooking parameters, such as duration, temperature, and cooking medium, were factors in studies that showed a reduction of hazardous components [28,53].Boiling and grilling are both widely used cooking methods that can alter the amount of harmful elements present in food.These changes are brought about by the loss of water and volatile compounds during cooking, as well as the solubilization of metals and, to a lesser extent, metal binding to macronutrients like proteins, lipids, and carbohydrates.Harmful elements that are removed from food during frying, boiling, or canning are not converted to safer compounds or evaporated during the cooking process.However, these materials can migrate to the frying oil, the boiling water, or the cooking stocks [28].Particular attention was placed on the chemical binding of both the organic and inorganic forms of each element in diverse diets when discussing the reduction of hazardous elements.Additionally, the types of these reactions as well as the chemical groups and ligands by which food items bond with metals are discussed [28].Since heating methods in different organs of the body have not shown the same results such as in cows' meat, the grilling method reduced Pb concentrations, while in the chicken liver, the level of Pb was above the maximum allowable level after boiling and grilling [54].By selecting an appropriate preparation procedure, it is possible to reduce the amount of harmful ingredients in a food product.

Based on the Findings, Two Hypotheses Were Proposed
(1) Eggshell Has Many Pores in Its Structure [46].So, the eggshell is permeable; therefore, these little alterations might be put on by the surroundings of the chicken, human contamination from handling eggs, the container used to keep the eggs, recycled materials, or anything else that contaminates the eggshell's surface (2) Water Loss and Evaporation.It was suggested that a heating process could reduce the levels of heavy metals by causing the loss of water and volatile materials, causing the metals to dissolve, and causing partial metal binding to macronutrients such as proteins, lipids, and carbohydrates, as well as the interaction of heavy metals with the sulfur group (-SH).Mijin et al. were found to bind heavy metal ions to different sites in egg ovalbumin before thermal treatment.Such binding could not cause a change in the heavy metal level of the hen egg after heating [44].
Tehran is one of the world's most polluted cities [55].This pollution can affect human health directly or indirectly through contamination of the food chain.Chickens play an essential role in the food chain, and their contamination can have irreparable consequences.Hen eggs may become tainted with heavy metals through chicken feed and drinking water, both of which are primarily influenced by the environment.Consistently consuming heavy metals in food at hazardous levels may have negative impacts on humans by impairing a variety of biological and metabolic systems [11].
3.2.2.Limitation.The loss of metals by evaporation, particularly for mercury, cannot be ruled out since we were unable to measure the temperature, which was identified during the cooking operations.Another limitation was that we did not have access to drinking water or chicken feed from the chicken farms from which we obtained the hen eggs for examination.Also, the detection of heavy metals from eggshells and boiled water is suggested for future research.

Conclusions
Hen eggs are a valuable supply of high-quality protein and vital nutrients and are more affordable worldwide.Also, hen eggs are capable of accumulating heavy metals and are a good indicator of environmental contamination.The result of different cooking methods showed that the residue of dryfried samples had the lowest level of the two heavy metals (As and Cd).All heavy metal residues were found to be within acceptable permissible thresholds.Therefore, policymakers should protect the health of the community and prioritize continuous national monitoring of food as well as education and research in the field of reducing heavy metals in the food chain.It can also design programs on how to reduce these heavy metals and improve guidelines to protect consumer health and increase food safety.

Figure 1 :
Figure 1: Flow chart of the experimental design.

Table 1 :
Conditions of ICP-MS apparatus for determining As, Pb, Cd, and Hg in hen eggs.