EXPOSURE ASSESSMENT TO ESSENTIAL ELEMENTS THROUGH THE CONSUMPTION OF CANNED FISH IN SERBIA

The aim of this study was to provide a quantitative exposure assessment to essential elements through the consumption of canned fish in Serbia. This objective was fulfilled by analyzing content of essential elements in canned fish and by using data from a food consumption survey. Consumption survey of canned fish was designed and performed to general principles and EFSA guidelines on data collection of national food consumption. The questionnaire was performed on 1,000 respondents during 2018. Determination of copper, zinc and iron levels were performed on 454 canned fish and seafood samples divided into four groups (canned tuna, canned sardines, canned other sea fish and canned seafood) during five consecutive years (2014–2018). This study showed significant association between sex, BMI and weight and consumption patterns. Obtained average weekly consumption of canned fish confirms our assumption that consumption of canned fish is significant in Serbia. Zinc and iron were found in all 454 samples (100%), and copper in 222 samples (48.9%). The average obtained concentration in all samples were 1.268 mg kg–1 for Cu, 5.661 mg kg–1 for Zn and 9.556 mg kg–1 for Fe. The highest concentration for all three minerals were found in canned sardines (Cu — 6.49 mg kg–1, Zn — 37.2 mg kg–1 and Fe — 21.8 mg kg–1). Obtained mean exposure to intake of copper, zinc and iron from canned fish was 1.2241 μg/kg bw/day, 5.4634 μg/kg bw/day and 9.2231 μg/kg bw/day, respectively. Exposure of Serbian population to zinc, copper, and iron through consumption of canned fish is less than recommended daily reference intakes and there is no risk of reaching toxic levels by consuming fish. FOR CITATION: Petrovic, J., Jovetic, M., Štulić, M., Redžepović-Đorđević, A., Vujadinović, D., Djekic, I.V., Tomasevic, I.B. (2021). Exposure assessment to essential elements through the consumption of canned fish in Serbia. Theory and practice of meat processing, 6(3), 219225. https://doi.org/10.21323/2414-438X-2021-6-3-219-225 Jelena Petrovic1, Milica Jovetic1, Milica Štulić1, Azra Redžepović-Đorđević1, Dragan Vujadinović2, Ilija V. Djekic3, Igor B. Tomasevic3* 1 Center for Food Analysis, Belgrade, Serbia 2 University of East Sarajevo, Faculty of Technology, Zvornik, Bosnia & Herzegovina 3 University of Belgrade, Faculty of Agriculture, Belgrade, Serbia DOI: https://doi.org/10.21323/2414-438X-2021-6-3-219-225


Introduction
It is considered that eating fish has beneficial impact on human health. It is reach in omega-3 fatty acids, highly digestible good quality proteins containing all the essential amino acids and liposoluble vitamins (A and D), as well as vitamin B 12 [1]. Amounts of essential elements in fish, such as zinc, copper, and iron, are significant [2]. Although copper, zinc and iron have important biochemical functions, their excess can cause adverse health effects, as well as deficit. Copper and zinc are present in numerous metalloenzymes and co-factors [3]. Copper is present in numerous oxidative stress-related enzymes and enzymes involved in redox system [4]. However, copper generates superoxide and hydroxyl radicals, which are toxic and excessive exposure can lead to cellular damage. Copper intoxication could be related to Wilson's disease and development of Alzheimer's disease [5,6]. Zinc is involved in signal conversion and gen expression. If there is excess or deficit of zinc in the organism, it may affect cell function and multiplication, threatening cell survival, which may lead to disease [7]. Iron has an important role in major metabolic processes in the body, such as oxygen transfer, electron transfer and DNA synthesis [8]. Excess levels of iron in human body can lead to liver damage, and can affect pancre-as, heart, and lungs. It can lead to hormonal irregularities, diabetes mellitus, pancreatic hypertrophy and other health disorders [9].
Whether fish can be considered a significant source of zinc, copper, and iron? To obtain such an information it is needed to perform exposure assessment to these elements through fish consumption. Levels of exposure of population depends on fish consumption pattern and levels of these essential elements in fishery products in the local markets. Since Serbia does not have a developed fish production industry, mainly imported fish is consumed. Canned fish is cheaper, and it can be assumed that it is widely consumed. There is publication of essential elements exposure of Serbian population from canned fish consumption, but using SORS (Statistical Office Of Republic Serbia) data [10]. SORS includes only official data of imported fish, which may differ from real consumption. However, to estimate real exposure of the population to essential elements through canned fish consumption, it is needed to perform valid canned fish consumption survey. In order to obtain relevant data on food consumption, EFSA has issued a guide [11] which provides general principles on the collection of data on food consumption at the national level.
The aim of this study was to provide a quantitative exposure assessment to essential elements through the consumption of canned fish in Serbia. This objective was fulfilled by analyzing content of essential elements in canned fish in period of five years (2014-2018) and by using data from a food consumption survey performed during 2018.

Consumption of fish and seafood
Consumption survey of canned fish was designed and performed to general principles and EFSA guidelines on data collection of national food consumption [11]. The questionnaire was performed on 1,000 respondents. The tested population was similar in gender and age to the Serbian population. Average body weight of all interviewees was 71.3 kg which corresponds to recommendation given by EFSA to take 70 kg as average body weight of an adult European citizen, when unknown [12]. Demographic profile of tested population is given in Table 1. The questionnaire was done anonymously during 2018 and all respondents were informed of the purpose of the survey. After excluding the respondents that do not consume canned fish (155), for further analyses left 845 fully answered questionnaires. n -represents the number of respondents; (%) represents their share in the sample.
The questionnaire consisted of three sections. In the first section respondents were asked about general demographic information (sex, age, weight and height, and BMI was calculated later as BMI = weight/height 2 ). The second section included questions about frequency of consumption of canned fish. In the third section the respondents were asked to recall their consumption of canned fish in the last seven days and to state the amount of consumed canned fish (in grams) in four defined categories (canned tuna, canned sardines, other canned sea fish or canned seafood). By choosing 7-day recall instead of 1-day recall, authors tried to avoid bias from dietary habits, according to EFSA recommendations.

Samples
Analyses were performed on 454 canned fish and seafood samples divided into four groups (canned tuna, canned sardines, canned other sea fish and canned seafood) during five consecutive years (2014-2018).

Chemicals and Standards
The chemicals were of analytical grade and supplied by Merck (Darmstadt, Germany): nitric acid 65% and hydrogen peroxide 30% (for analysis EMSURE® ISO). Deionized water (electrical resistivity 18.2 MΩ cm -1 ) was obtained using the Simplicity® water purification system (Merck Millipore, Burlington MA, USA). For the quantification of copper, zinc, and iron certified standards were used (Certipur®, Merck, Darmstadt, Germany) in concentrations of 1000 μg mL -1 . For quality control of the analytical procedure the certified reference material DORM-2 (dogfish muscle, NRC Canada) was used.

Sample preparation
After homogenization, test portion of about 0.5 g were used for further analyses. Into polytetrafluoroethylene (PTFE) vessels with test portions, 7 mL of nitric acid and 2 mL of hydrogen peroxide were added. Samples were mineralized in a microwave closed digestion system (Ethos Touch, Milestone, Italy). The following temperature program was used: heating up to 180 °C for 15 min, followed by heating up to 220 °C for 15 min, and then heating up to 240 °C for 10 min, with a maximum power of 1000 W. After digestion, solutions were quantitatively transferred into 50 mL volumetric flasks and diluted with deionized water.
Instrumentation and analytical procedure Content of copper, zinc and iron was determined according EN14084:2003 [13]. Flame atomic absorption spectrometer (932 plus, GBC, Australia) was used.

Method validation and assurance of the quality of the results
Validation of analytical procedure was performed according ISO/IEC17025:2017 [14] requirements, by determination of the following parameters: linearity, accuracy, precision, limit of detection (LOD) and limit of quantification (LOQ) ( Table 2). Certified reference material (DORM-2, NRC Canada) was used for determination of accuracy and precision. Linearity is expressed as a correlation coefficient (r 2 ), accuracy as recovery, and precision as relative standard deviation in repeatability conditions (RSD r ), and in reproducibility conditions (RSD R ). LOD and LOQ were expressed as the analyte concentration corresponding to 3 times and 10 times, respectively, the standard deviation (SD) of 10 sample blanks. Assurance of the quality of the results were provided by analyzing CRM (DORM-2, NRC Canada) within every sample set. The obtained results were monitored through control charts. If recovery of CRM was within satisfactory range, results of all samples in the sample set were accepted, if it was not, all samples were analyzed again.

Exposure estimation
Obtained results from the analyses of levels of copper, zinc and iron in canned fish and results from the canned fish consumption survey were combined to estimate exposure of Serbian population to these elements through eating canned fish. Estimation was calculated according following equation [15]: To assess exposure, with including the variability of the population, the Monte Carlo simulation, as one of the most common methods [16], was used.

Statistical methods
Canned fish consumption patterns of demographic groups (defined by sex, age, weight, and BMI) were compared by Chi-square test for association. An independent sample t-test was used for analyzing average consumption with statistical significance of 0.05.
For estimation the intake of copper, zinc and iron from canned fish consumption, the Monte Carlo analysis of 100,000 simulations was used. Calculating levels of Cu/Zn/ Fe was based on their mean values (Table 4). According FAO/WHO [17] it can be assumed that an individual exposure over time is equal to this level.
Minitab was used for probability distribution fitting for body weight and weekly intake of canned fish and for Monte Carlo simulation. For fitting of the probability distributions visual analysis was equally considered [18].

Results and discussion
Consumption of canned fish Frequency consumption patterns for canned fish obtained from consumption survey are given in Table 3. Obtained average weekly consumption of canned fish is 228.8 g (Table 1). It confirms our assumption that consumption of canned fish is significant in Serbia.
Relationships between canned fish consumption patterns and demographic characteristics of the sample were tested (Chi-square test for association). On average, depending on the demographic group, consumption on a weekly basis was confirmed by 26.3% to 62.0% of interviewed consumers. This study showed significant association between sex, BMI and weight and consumption patterns (p < 0.05). Male respondents, respondents with weight over 70 kg and with BMI >24.9 eat canned fish more frequently than females, consumers with weight below 70 kg and with "ideal" BMI. There were no

Levels of copper, zinc, and iron in canned fish
Copper, zinc, and iron determination were done in 452 samples of canned fish and 2 samples of canned seafood. Obtained concentrations of copper, zinc and iron in canned fish and seafood are given in Table 4.
Zinc and iron were found in all 454 samples (100%), and copper in 222 samples (48.9%). Values of copper below the LOQ were substituted with a constant value of LOQ/2 for further analyses, as it is recommended [19]. Monte Carlo uncertainty analysis of 95% CI of the mean values shows that such results have little effect on the upper percentile exposures [20]. The average obtained concentration in all samples were 1.268 mg kg -1 for Cu, 5.661 mg kg -1 for Zn, and 9.556 mg kg -1 for Fe. The highest concentration for all of three minerals were found in canned sardines (Cu -6.49 mg kg -1 , Zn -37.2 mg kg -1 , and Fe -21.8 mg kg -1 ), as well as the highest average concentrations (Cu 2.100 mg kg -1 , Zn 7.382 mg kg -1 , and Fe 11.607 mg kg -1 ).
There are two published investigations of levels of Cu, Zn and Fe in canned fish from Serbian local markets. Popovic et al. [10] performed analyses in 207 samples of canned tuna, sardines and mackerel and Novakov et al. [21] in 98 samples of canned tuna, sardines and sprouts. Popovic et al. [10] found the highest average concentration for all the three minerals in canned sardines, which was in line with our results. Found levels (Cu -1.28/1.37 mg kg -1 oil/tomato sauce, Zn -15.1/14.05 mg kg -1 , and Fe -13.8/16.78 mg kg -1 ) were lower than ours for Cu, but significantly higher for Zn and Fe. On the other hand, Novakov et al. [21] reported higher levels of Cu and Zn in canned tuna (2.60 mg kg -1 , 21.96 mg kg -1 , respectively) than in canned sardines (2.49 mg kg -1 , 18.21 mg kg -1 , respectively), but higher level of Fe in canned sardines (21.98 mg kg -1 ) then in canned tuna (20.36 mg kg -1 ). All results are significantly higher than ours, except for Cu, which is in line for canned sardines and little higher in canned tuna.

Exposure assessment
According to our research on the level of minerals in canned fish and eating habits of the Serbian population, mean estimated daily intake of copper, zinc and iron in canned fish were 1.2241 μg/kg bw/day, 5.4634 μg/kg bw/day, and 9.2231 μg/kg bw/day, respectively (Table 5). Estimated total daily intake of the three minerals after a Monte Carlo analysis of 100,000 simulations is shown in Figures 1a-c. Similar investigation of contribution of Cu, Zn and Fe from fish to dietary intake in Serbia was performed by Popovic et al. [10]. Investigation included canned fish (tuna, sardines, and mackerel in oil and in tomato sauce). Results were expressed as%RDA for men and women for each type of fish separately. Reported EDI for Cu were in range 0.28-0.46%RDA, EDI for Zn were in range 0.21-0.57%RDA and EDI for Fe were in range 0.14-0.64%RDA. The authors concluded that levels of Cu, Zn and Fe in canned fish do not represent any health risk, and even do not represent important dietary source of Cu, Zn and Fe. Reported EDI of Cu (1.671 μg/kg bw/day) and Zn (6.629 μg/kg bw/day) in fish from Spain, were slightly higher than our results [30].
Provisional maximum tolerable daily intake (PMTDI) established by JECFA for Cu is 0. 5  Obtained results indicates that there is no risk of exposure to the toxic values of copper, zinc and iron through canned fish consumption and, moreover, canned fish does not represent source of these three essential minerals.

Conclusion
Consumption of canned fish is significant in Serbia (228.8 g/w). On average, depending on the demographic group, consumption on a weekly basis was confirmed by 26.3% to 62.0% of interviewed consumers. Male respondents, respondents with weight over 70 kg and with BMI > 24.9 eat canned fish more frequently than females, consumers with weight below 70 kg and with "ideal" BMI. There were no statistically significant association between age groups.
Copper was detected in 49% of tested samples, where zinc and iron were found in all the tested samples. The highest levels of all three essential elements were found in canned sardines.
Exposure of Serbian population to zinc, copper, and iron through consumption of canned fish is less than recommended daily reference intakes and there is no risk of reaching toxic levels by consuming fish.