Determination of the Content of Heavy Metals and Nitrites in Meat Products (Pâtés and Meat Spreads) from the Market of the Republic of Srpska ^†

Abstract

Animal-based food is significant for human nutrition, as it represents an easily digestible source of high-quality proteins, fatty acids, fat-soluble vitamins, and energy. During evolution, humans developed anatomical, metabolic, and biochemical adaptations in the digestive tract, becoming increasingly dependent on nutritionally valuable food, such as animal-based products. Animal-based food can be a source of chemical substances that are harmful to health, such as contaminants (heavy metals, mycotoxins, organochlorine pesticides, etc.), veterinary drug residues, and additives. The subject of this paper is the determination of the content of essential (copper—Cu, iron—Fe, and chromium—Cr) and heavy and toxic metals (lead—Pb, cadmium—Cd, arsenic—As, and tin—Sn), as well as nitrites, in meat products, particularly pâtés and meat spreads available on the market in the Republic of Srpska. Determination of the content of metals was performed using the ICP-OES method, while nitrites were analyzed using the standard SRPS ISO 2918/1999 method. The obtained results indicate that the content of heavy and toxic metals and nitrites is lower than the maximum prescribed by national regulations, namely the Rulebook on Maximum Amounts of Certain Contaminants (Official Gazette of BA, No. 68/14, 79/16, 84/18) and the Rulebook on Additives in Food (Official Gazette of BA, No. 33/18 and 6/21).

1. Introduction

All living beings are exposed daily to a large number of substances from the environment that can negatively affect health. Some of these substances are present in food. In addition to chemicals that have been intentionally added, such as additives, food can also contain contaminants as a result of environmental pollution, improper treatment in agricultural production, and/or the food industry during the production and packaging of the finished product, which cannot be avoided due to physical and chemical features. The assessment of the risk of food to human health is the scientific basis for making decisions about the measures that are needed. In order to ensure impartiality in the risk assessment process, an independent food safety agency—the EFSA—was established in the European Union, whose main task is to provide independent scientific evidence and publish information on existing and new risks associated with the food chain [1]. In accordance with the provisions of Article 53 of the Law on Food [2], the activity of the Food Safety Agency of Bosnia and Herzegovina involves providing scientific advice and scientific and technical assistance for the legislation and policy of the country in all areas that have a direct or indirect impact on food safety [3].

Food of animal origin is important for human nutrition because it is an easily digestible source of quality proteins, fatty acids, fat-soluble vitamins, and energy [4]. Meat occupies an important place in the human diet, because the consumption of meat provides proteins of high biological value, essential amino acids, fats, essential fatty acids, vitamins, and mineral substances. Meat contains B complex vitamins, phosphorus (P), iron (Fe), zinc (Zn), calcium (Ca), potassium (K), magnesium (Mg), manganese (Mn), and many other essential macro- and micronutrients [5]. Meat is a rich source of essential trace elements, but it can accumulate heavy metals. High concentrations of heavy metals in muscles are very rare, but their accumulation often occurs in fatty tissues, offal (liver and kidneys), and, consequently, in meat products [6]. The development of society imposes high requirements regarding the appearance, taste, smell, and shelf life of food. Because of this, manufacturers are increasingly using different additives during food production. Among the most important additives in meat processing technology are nitrites and nitrates [7]. The use of nitrites and nitrates is strictly defined by regulations and directives, and in Bosnia and Herzegovina, the Rulebook on Food Additives is valid [8]. The Rulebook is aligned with EU legislation, i.e., EU Regulation 1333/2008 and EU Regulation 231/2012, when it comes to special cleanliness criteria. The Scientific Committee on Food (SCF) established in 2002, and the European Food Safety Authority, the EFSA, confirmed in 2017 that the Allowed Daily Intake (ADI) for nitrites is from 0 to 0.07 mg/kg of body weight per day, so in practice, there is an increasing effort to reduce the amount of these compounds to preserve the microbiological integrity of food [9]. An adult’s body needs 0.8 g of protein per 1 kg of body weight per day, or an average of 45 to 55 g, which could be met with around 250 to 300 g of meat. However, due to the positive effect of nutritional fibers, it is desirable that 1/3 of protein be of animal origin, and 2/3 of vegetable origin, which means that it is enough for a person to consume 100 to 150 g of meat per day [10].

In the industry of packaging materials, tin cans are mostly used for packing meat products [11]. Cans contains a thin passivating film consisting of tin oxide, chromium oxide, and elemental chromium, and its composition plays an important role in preventing the development of electrochemical corrosion of metals [12,13]. Meat products contain water, acids, nitrites, or other additives that can lead to electrochemical corrosion of metals, and thus increase the concentration of metals and damage cans. Because of all of the above, we saw the importance of product testing in our market.

The aim of this work is to determine the content of nitrite, cadmium, arsenic, lead, iron, copper, tin, and chromium in samples of pâté and meat spreads from the market of the Republic of Srpska, as well as to compare the obtained amounts with the permitted values from the relevant domestic regulations.

2. Materials and Methods

In this paper, three samples of liver pâté made from pork, and three samples of pork spread, purchased at a retail store in the Republic of Srpska during December 2024 and selected using the random sampling method, were examined. The reagents used to prepare the samples for determining their metal content were of analytical-grade purity, p.a.

The following equipment, reagents, and consumables were used during the work:

A Radwag AS220.R2 analytical scale (Radwag Wagi Elektroniczne S.A., Radom, Poland); a Milestone ETHOS microwave oven equipped with HPR1000/10S software (Milestone S.r.l., Bergamo, Italy); an Inductively Plasma Optical Emission Spectrometer—ICP-OES (Shimadzu 9820,Kyoto, Japan); a UV/VIS spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan); a meat grinder, with an opening-plate diameter of less than 4 mm; normal class A vessels of 50 mL and 25 mL; concentrated nitric acid, w ≈ 65%, ρ = 1.4 g/mL, p.a., Lachner; hydrogen peroxide, w ≈ 30%, ρ ≈ 1.11 g/mL, G.R., Lachner; multi standard (Accu standard LPCS-01R, Lot: 216035110, New Haven, CT, USA); Quality Control Standard (Accu Standard, QCS-ASL-19, Lot: 215025098-0, New Haven, CT, USA); HPLC-grade water (High-Performance Liquid Chromatography grade water); argon with a purity of 99.999%; potassium hexacyanoferrate(II)-trihydrate; zinc-acetate-dihydrate; glacial acetic acid; disodium tetraborate decahydrate; sodium nitrate—standard solution; sulfanilamide; concentrated hydrochloric acid, ρ ≈ 1.19 g/mL; N-naphthyl-1-ethyleneamide chloride; automatic pipettes; glass and PTFE/PFA laboratory ware; a water bath; wrinkled filter paper, with a diameter of about 15 cm, free of nitrite; and tested samples of pâté and meat spreads.

2.1. Determination of Metal Content in Canned Meat Samples (Liver Pâté and Meat Spreads)

The contents of the cans were dissolved in a Milestone ETHOS microwave oven equipped with HPR1000/10S software, according to the standard method DG-FO-47, for recording on an ICP-OES spectrometer (Shimadzu 9820,Kyoto, Japan). The contents of the cans were mechanically homogenized and 0.5 g of each thus-prepared sample was transferred to a digestion cuvette. All samples were triplicated. Eight milliliters (8 mL) of a mixture of concentrated nitric acid and hydrogen peroxide was then added to the cuvettes (in a ratio of 8:1–8 mL of 65% HNO₃ and 1 mL of 30% H₂O₂). The time taken to reach the digestion temperature was 30 min. A digestion temperature of 200 °C and a pressure of 400 psi were maintained 10 min. A cooling time of 20 min was taken for the temperature to fall below 40 °C. The solution obtained by digestion was transferred to volumetric bottles—normal vessels with a volume of 50.0 mL—and then they were filled with HPLC water, closed, and shaken for about 5 min for homogenization. After homogenization, 10 mL of the contents was taken from each normal vessel and transferred to a vial. The prepared samples were recorded on an ICP-OES instrument (Shimadzu 9820) with prior preparation of standard solutions, for the formation of calibration curves. All measurements were performed in three repetitions, and the results were presented as the mean value of three repetitions. The obtained measurement results were compared with the maximum allowed concentrations of metals prescribed by the Rulebook on the Maximum Amounts of Certain Contaminants [14].

2.2. Determination of Nitrite Concentration in Canned Meat Samples (Liver Pâté and Meat Spreads)

The method was based on the extraction of part of the test sample with warm water, the precipitation of proteins, and filtration. In the presence of nitrite, a red coloration was obtained by adding sulfanilamide and naphthylethylenediamine chloride to the filtrate. Photometric measurement was performed at a wavelength of 538 nm with a previously prepared series of standard solutions of NaNO₂ at concentrations of 0.05, 1.0, 2.5, 5.0, and 10.0 µg/mL [15]. The nitrite content was evaluated in relation to the values prescribed by the Rulebook on Food Additives [8], which is in accordance with the European Rulebook [16]. According to this regulation, the content of nitrites in meat and meat products differs depending on the type of product and the method of processing and sterilization. The maximum allowed amount of nitrite that can be added during production to sterilized products is 100 mg/kg.

3. Results and Discussion

Two types of sterilized meat products, liver pâté (Samples 1–3) and meat spreads (Samples 4–6), were analyzed for their contents of arsenic, cadmium, copper, iron, lead, tin, chromium, and nitrite.

Table 1. The content of metals and nitrites in the examined samples of meat products.

Sample Number	Metal Contents, mg/kg
	As	Cd	Cu	Fe	Pb	Sn	Cr	Nitrites
1	0.0651 ± 0.001	0.0363 ± 0.002	74.4987 ± 0.005	67.5612 ± 0.001	0.0620 ± 0.005	79.0422 ± 0.005	0.0214 ± 0.003	5.6214 ± 0.003
2	0.0066 ± 0.001	0.0371 ± 0.002	73.4183 ± 0.001	58.9825 ± 0.001	0.0271 ± 0.004	53.2282 ± 0.003	0.0289 ± 0.03	3.1989 ± 0.03
3	0.0713 ± 0.003	0.0291 ± 0.003	70.3514 ± 0.004	72.3521 ± 0.001	0.0515 ± 0.007	22.4825 ± 0.005	0.0688 ± 0.004	4.8687 ± 0.004
4	0.0997 ± 0.003	0.0252 ± 0.001	5.1518 ± 0.001	75.8898 ± 0.003	0.0213 ± 0.008	33.2144 ± 0.003	0.0242 ± 0.005	3.5842 ± 0.005
5	0.0658 ± 0.005	0.0309 ± 0.002	4.5641 ± 0.005	62.4111 ± 0.002	0.0637 ± 0.006	49.7551 ± 0.002	0.0248 ± 0.002	4.3345 ± 0.002
6	0.0532 ± 0.002	0.0322 ± 0.006	4.9921 ± 0.002	65.3026 ± 0.003	0.0841 ± 0.004	31.2623 ± 0.002	0.0284 ± 0.003	4.7584 ± 0.003

gives an overview of the obtained results.

• As

According to the Rulebook on the Maximum Permitted Amounts for Certain Contaminants in Food, the maximum permitted concentration of arsenic in meat products is 0.1 mg/kg. From Table 1, it can be seen that in the considered samples of sterilized canned meat products, the arsenic content ranged from 0.0066 to 0.0713 mg/kg (liver pâté) and from 0.0532 to 0.0997 mg/kg (meat spread), and that not a single sample exceeded the maximum allowed concentration recommended by the national regulations.

• Cd

The Rulebook on the Maximum Permitted Amounts for Certain Contaminants in Food prescribes the maximum permitted concentration of cadmium in meat products and it amounts as 0.05 mg/kg. The cadmium content in all the tested samples was below the maximum allowed concentration prescribed by national regulations. In the liver pâté, the cadmium content ranged from 0.0291 to 0.0371 mg/kg, and in the meat spread samples, it ranged from 0.0252 to 0.0322 mg/kg.

• Cu

According to the Rulebook on the Maximum Permitted Amounts for Certain Contaminants in Food, the maximum permitted concentration of copper in liver products (liver pâté) is 80 mg/kg, while it is not prescribed for other meat products. However, looking at Table 1, it can be concluded that none of the samples of meat spreads or liver pâté exceeded the maximum allowed concentration of 80 mg/kg. Also, as the recommended daily intake of copper is 0.9 mg/day, and the maximum is 10 mg/day. With an average daily intake of 50 g of meat products, there is no risk of exceeding the maximum allowed intake of copper [17]. However, care should be taken, especially with sensitive populations, such as children with autism. Also, it should be kept in mind that there are other sources of copper that are consumed as part of peoples’ daily diet.

• Fe

The content of iron in the liver pâté ranged from 58.9825 to 72.3521 mg/kg, and in the meat spread samples, it ranged from 62.4111 to 75.8898 mg/kg. No iron content recommendations are defined by the Rulebook on Maximum Allowable Amounts for Certain Contaminants in Food.

• Pb

The Rulebook on Maximum Permitted Amounts for Certain Contaminants in Food prescribes a maximum permitted concentration of lead in meat products of 0.10 mg/kg. The content of this metal in the considered samples of liver pâté and meat spreads did not exceed the maximum allowed concentration prescribed by national regulations.

• Sn

For food that is packed in cans, the content of tin must be determined, due to the possibility of transfer from the can into the food when it is damaged. The Rulebook on Maximum Permitted Amounts for Certain Contaminants in Food prescribes a maximum permitted concentration of tin in canned food of 200 mg/kg. It can be seen from Table 1 that not a single sample reached or exceeded the maximum allowed concentration of tin. The tin content in the liver pâté ranged from 22.4825 to 79.0422 mg/kg, and in the meat spreads, it ranged from 31.2623 to 49.7551 mg/kg.

• Cr

From Table 1, it can be concluded that the chromium content was very similar in all the analyzed samples, ranging from 0.0214 to 0.0688 mg/kg. No chromium content recommendations are defined by the Rulebook on Maximum Permitted Amounts for Certain Contaminants in Food.

• Nitrites

Table 1 shows the nitrite content, expressed as mg NaNO₂/kg, in the examined samples of liver pâté and meat spreads. Based on the obtained results, it can be assumed that in all the samples, the amount of nitrite initially added in the production process was in accordance with the legally defined limit (100 mg/kg). As only the residual fraction of nitrite could be determined by the analytical methods, it can be assumed that the amount added during production is greater than that shown in Table 1. Namely, during the processing of meat and meat products, some nitrite (up to 5%) is lost due to the formation of nitric oxide—NO, up to 10% is oxidized to nitrate, 5–10% remains as free nitrites, 5–15% reacts with sulfhydryl compounds and myoglobin, and 20–30% is bound to proteins. As a product of reaction with proteins, the toxic and carcinogenic compounds N-nitrosamines can be formed [4,7,17]. The nitrosation reaction through which N-nitrosamines are formed directly depends on the pH, temperature, redox potential, and amount of added nitrites. In order to accurately determine the content of nitrosamines, which can be potentially dangerous to human health, it is necessary to perform additional qualitative and quantitative tests of N-nitrosamines using the method of gas chromatography coupled with mass spectrometry.

4. Conclusions

Public health monitoring of risks in food is important for the safety of consumers. The control of meat and meat products is particularly important, considering the importance and frequency of meat consumption in all population groups. This paper has shown that the quality of the tested samples of meat products from the Republic of Srpska, selected by the random sampling method, was satisfactory in terms of compliance with legal regulations.

The nitrite content in the tested samples, 3.1989–5.6214 mg/kg, represents only the residual fraction, so it can be assumed that the amount added during production is higher, but within the permissible values prescribed by valid domestic and European Union regulations. It has been shown that meat products can represent significant sources of essential metals, such as iron, copper, and chromium. In the tested samples, the iron content was 58.9825–75.8998 mg/kg, and the copper content was 4.5641–74.4987 mg/kg. The content of the heavy and toxic metals cadmium 0.0252–0.0371 mg/kg, lead 0.0213–0.0841 mg/kg, and arsenic 0.0066–0.0997 mg/kg in all the samples was within the permissible limits prescribed by domestic and European Union regulations. White tin is the most frequently used material for packaging food products. The last operation in the production process involves its chemical protection using chrome passivation, where a thin passivating film coating is formed on the surface of the tin. This film consists of tin oxide, chromium oxide, and elemental chromium, and its composition plays an important role in preventing the development of electrochemical corrosion of metals and the adhesion of lacquers. In the tested samples, the contents of tin—22.4825–79.0422 mg/kg—and chromium—0.0214–0.0688 mg/kg—were very low, confirming that there was no interaction between the contents of the can and the passivation film, that is, that the can packaging was in perfect condition.