A R T I C L E  I N F O		ABSTRACT
ORIGINAL ARTICLE		Introduction: Exposure to mercury (Hg) by consumption of fish is a recent health concern. So, it is important to evaluate the health risks related to canned fish consumption. The purpose of this study was to investigate the potential health risk based on Hg concentration in people who consumed canned fish with a probabilistic approach in Isfahan City, the central province in Iran. Materials and Methods: In this study, 20 popular brands of canned fish prepared in Iran and other countries were selected and analyzed for Hg concentration with atomic absorption spectrometer. The results were compared with the European Communities and JECFA guidelines. Then, a probabilistic method with Monte-Carlo simulation was used to assessment the Provisional Tolerable Daily Intake (PTDI) and the Hazard Quotient (HQ) for consumers in Isfahan City. Results: The average Hg concentrations in samples were 0.251 ± 0.204 and 0.189 ± 0.152 µg/g in canned fish of Iran and other countries, respectively. The Hg level was found below the guideline limit for European Communities and JECFA. The estimated PTDI was 0.037 µg person/day and HQ was 0.074. Conclusion: The results indicated that canned fish available in the markets of Iran did not have a health risk for adults. Moreover, canned fish consumption has a possible influence on the risk estimate and its risk should be assessed  for vulnerable groups.
Article History: Received: 25 May 2019 Accepted: 10 July 2019
*Corresponding Author: Malihe Moazeni Email: malihe_moazeni@hlth.mui.ac.ir Tel: +989133201671
Keywords: Mercury, Fish, Risk Assessment, Monte-Carlo Simulation.

Introduction

Humans usually consume fish in many parts of the world because it contains an appropriate source of protein and is considered as a necessary source of healthy nutrients. Based on the Food and Agriculture Organization of the United Nations (FAO), fish has consist of 16.6% of protein source in the world population's food and 6.5% of the total protein that consumed in the past years ¹. In comparison to the good health benefits, many cases of contamination were reported by fish due to the chemical pollutants in the environment ². Fish may be polluted by heavy metals through the commercial handling and canning process ³. Exposure to toxic metals is important for children and pregnant women. The recent surveys found a correlation between fish consumption and hazardous effects on human health due to increased concentrations of mercury (Hg) and other pollutants ^{4- 7}.
Among the environmental contaminants, Hg is the most harmful to human health ⁸. It is one of the most toxic components and a well-known global contaminant ⁹. Human exposure to Hg leads to slow growth, cerebral palsy, blindness, and other birth defects. In addition, Hg is a neurological toxicant to humans and mainly affects organs such as brain and kidney ¹⁰. The toxic limit of Hg for human fetus, determined by the US-EPA, is 0.1 μg/kg/day ¹¹. In 2003, more attention was paid to the risk attributed to Hg amounts in fish; then, canned fish has been considered as a main supply of Hg ¹².
Adverse health impacts may occur if the fish is consumed too often or in large quantities. As a result, a perfect correlation was observed between Hg levels in humans and the amount of consumed fish ^{12, 13}. The incident of Hg in fish is a common issue for human health risk assessment and is of special concern to the United States Food and Drug Administration (US-FDA) ¹⁰. Therefore, it is essential to know Hg concentrations in fish and ultimately advise the population about the health risks related to fish consumption ¹².
Although some studies were conducted on the health risks of the canned fish consumption around the worlds ^{10, 13-17}, limited data exist on the health risk assessment of Hg in canned fish in Iran. In other words, only a few studies attempted to deal with this in Tehran ¹⁸ and Mashhad ¹⁹, the two crowded cities in Iran. Moreover, all conducted studies focused on the deterministic risk assessment. Therefore, it is necessary to determine the risks of Hg contained in canned fish consumed by the Iranians. Furthermore, the hazard levels of the pollution should be controlled in these sources. Risk assessment is a systematic procedure that provides an assessment of the probability and severity of a specific risk related to consumption of contaminants in food sources ²⁰. Risk assessment studies consist of the deterministic and probabilistic methodologies. The deterministic or “point estimation” approach provides a single assessment of the risk to describe a variable in the model. However, the probabilistic or statistical method uses probability distribution functions (PDFs) to define uncertainty and variability of the model variables ^{21, 22}. Codex Committee on Food Additives and Contaminants (CCFAC) increasingly recommends application of the probabilistic technologies to determine the risk assessment of pollutants in the food sources ²³. Probabilistic methods conducted with Monte-Carlo simulation approach is a suitable computer-based method to reduce irresolvable calculations of the distribution densities by estimating an empirical distribution similar to the distribution of the risk across the population. It is based on statistical sampling methods for estimating a probabilistic approximation to the solution of a mathematical model. For each variable in the model, the probable values are calculated according to a probability distribution, which are determined
by goodness-of-fit tests such as chi-square, Kolmogorov–Smirnov, and Anderson–Darling tests. If a Monte-Carlo simulation is run for 10,000 trials, 10,000 possible outcomes are anticipated and exposure and risk distributions of the population would be estimated using these simulated values ^{24, 25}.
The objective of this study was to determine the concentrations of Hg in canned fish samples prepared in Iran and other countries consumed by Iranian people. In addition, this study represented the potential health risk assessment based on Hg concentrations in canned fish using a probabilistic approach.
Materials and Methods
Laboratory procedures
Twenty popular brands of canned fish samples including 14 samples prepared in Iran and 6 samples prepared in other countries (U.S, U.K and Australia) were purchased directly from markets in the city of Isfahan, the central province in Iran.
In this study, 1 g of each sample was mixed with 5 ml of nitric acid (HNO₃) (1 N), 3 ml H₂O₂, and 15 ml of KMnO₄ 5% in a Teflon vessel. The mixture was heated on a hot plate to 150˚C for 10 min for digestion. Then, the digested samples were diluted with 100 ml of double-deionized water. The Hg concentration in the samples was analyzed using a Perkin Elmer 4100 atomic absorption spectrometer equipped with cold vapor system ²⁶. The limit of detection (LOD) was 0.015 µg/g and the level of quantification (LOQ) was 0.005 μg/g. Analyses of each sample were performed in triplicate and no sample had an Hg concentration of less than LOD. In this study, all used reagents were of analytical grade (Merck, Germany).
Statistical analysis
The descriptive statistics (mean ± standard deviation, and median), Mann-Whitney test, and t-test analysis were directed using SPSS (Version 22, SPSS Inc., Chicago, IL, USA). A P-value of less than 0.05 was considered to indicate the statistical significance.
Human health risk assessment and model implementation
The health risk for human imposed by the intake of Hg caused by consumption of the canned fish was implemented by a probabilistic method in which variables were defined using with probability distributions and the risk estimate was obtained by Monte-Carlo simulation ²⁰.
Exposure assessment
Probability distributions of Hg concentration, body weight, and canned fish consumption were fitted to normal, lognormal, uniform, exponential, logistic, beta, gamma, and Weibull distributions. The goodness of fit was measured using Chi-squared, Kolmogorov–Smirnov, Anderson–Darling  statistics, and graphs (empirical and theoretical distributions plot and, P–P plot and Q–Q plot). Hg levels in canned fish and body weights (mean ( ) and standard deviation ( )) were fitted with the generalized log normal distribution and canned fish consumption were fitted with the Weibull distribution as follows, respectively:
                                        (1)
                                   (2)
Where, k > 0 is the shape parameter and λ > 0 is the scale parameter.
Canned fish consumption and average body weights (kg) were examined based on the questionnaires collected from 4763 adults in Isfahan City ²⁷.
Risk characterization
Exposure has normalized for time and body weight and has typically expressed in units of µg Hg/kg body weight/day. The estimation of health risk was based on the PTDI through the following equations ²⁸:
NTMID = MI × F                              (3)
PTDI = NTMID / WAB        (4)
Here, NTMID is the daily intake of Hg (µg/person/day), MI is concentration of Hg in canned fish (µg/g wet weight), F is the canned fish intake (g/person/day), WAB is the body weight (kg), and PTDI is the daily intake per body weight (µg/kg person body weight/day).
The HQ is calculated using the following equation:
HQ = EF × ED × PTDI / (RfD × TA)                        (5)
where, EF is the exposure frequency (365 days/year), ED is the exposure duration corresponding to the average lifetime (70 years), the Reference oral Dose (RfD) is an estimate of a safe daily oral exposure (0.5 µg/kg/day), and TA is the average exposure time for noncarcinogens (365 days/year x ED) ^{1, 12}.
The Hazard quotient (HQ) indicates the health risk of the Isfahan population to Hg exposure and is obtained from the ratio between the canned fish ingestion rate and RfD. If HQ < 1, there will be no evident risk; however, if HQ = 1, the contamination itself seems to cause no risk; and if HQ > 1, we cannot exclude the possibility of the harmful effects to human health ²⁹.
Sensitivity analysis
The sensitivity analysis was performed using the Spearman rank order correlation (ρ) between the Weibull distribution for canned fish consumption and the log normal distributions for Hg concentration, and body weight. The Monte-Carlo simulation analysis was done in 10,000 iterations by R version 3.4.3 software.
Results
Concentration of Hg in canned fish
The results of the present study showed the mean Hg concentration of samples prepared in Iran and in other countries (Table 1).
Risk assessment for human health
Dietary exposure (PTDI)
In this study, the PTDI values were calculated by taking into consideration the similar Hg concentrations for the canned fish samples prepared in Iran and other countries. Other parameters, used for PTDI calculation, were body weight and canned fish consumption rates based on the completed questionnaires in the study area ²⁷. In this study, mean ± SD of the daily consumption of the canned fish and the human body weight (with log normal and Weibull distributions, respectively) were earned 10.7 ± 11.8 g/person/day and 68.7 ± 12.8, respectively.
As the Hg concentrations were above the LOD, all data were used in the PTDI calculation. The calculated log normal distribution Hg concentration was 0.229 ± 0.1824 µg/g based on mean ± SD.
Finally, the 95% confidence interval (CI) for PTDI using a probabilistic approach by Monte Carlo simulation ranged from 0.0260 to 0.0525 µg/person/day (Mean = 0.037 µg/person/day) (Figure 1,a), which is below the respective guideline value recommended by European Communities and JECFA, 0.5 µg/g body weight ²⁰.
The 95% CI for HQ was 0.0521 to 0.1051 (Mean = 0.074) and showed that ingestion of the canned fish had no risk. Consequently, HQ lower than 1 is considered as a healthy and harmless food item with regard to Hg (Figure 1, b) and has no potential health impacts on consumers.
The results of the sensitivity analysis of the canned fish consumption through Hg concentration and average body weight are shown in Figure 2.

Table 1: Hg concentration (µg/g) in canned fish samples
Samples	No of samples	Median [Min-Max]	Mean ± SD	P-value
Iranian	14	0.138 [0.079-0.668]	0.251 ± 0.204	0.444
Other countries	6	0.102 [0.089-0.449]	0.189 ± 0.152
P-value is based on Mann-Whitney test

Discussion
Concentration of Hg in canned fish
We compared the mean Hg concentration obtained in this study with those of other studied (Table 2). According to our findings, Hg concentrations in the Iranian samples of canned fish were not much different from other areas of the world ^{2, 3, 13, 14, 33-37}. Burger and Gochfeld, Reported that the maximum Hg was 0.997 µg/g, but 25% of white tuna samples exceeded 0.5 µg/g ³⁴.
The differences in accumulation patterns of Hg can be explained by their pathways of absorption and accumulation in the fish body ³⁸. The results of the present study demonstrated no significant difference between the Iranian and foreign canned fish with respect to Hg concentration (P-value = 0.44). In other words, the Hg contents of the canned fish samples prepared all over the worlds are similar.

Table 2: Mean concentration of Hg in Iranian canned fish samples compared to that in other literature
Country	Hg concentration (µg/g)	Reference Number
Iran	0.251	Present study
Iran	0.125	2
Iran	0.117	32
Iran	0.13	18
Turkey	0.14	3
Spain	0.222	16
Canada	0.6	30
India	0.62	30
Italy	0.41	13
Saudi Arabia	0.31	31
U.S	0.623	35
U.S	0.285	14
Australia	0.14	33
U.K	0.055	36
Lebanon	0.075	37
Libya	0.29	37

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