Risk Assessment of Polycyclic Aromatic Hydrocarbons Concentration in Cold Smoked Mullet Fish ( Mugil cephalus )

The purpose of this work was to determine the concentration and risk assessment of Polycyclic Aromatic Hydrocarbons (PAHs) in cold-smoked mullet fish samples which were pre-frozen at -18°C for 6 months. Fish samples were obtained from two fish farms; A (Al-Batts drain) and B (El-Wadi drain), El-Fayoum governorate, Egypt. 16 components of PAHs concentration were determined by GC-MS. Results showed that the total concentration of PAHs recorded16.2 and 7.4 ppb sample of Aand B-smoked mullet fish products, respectively. Also, levels of Benzo [a] Pyrene (B {a} P) equivalent were 0.0378 and 0.029 ppb in A and B-products, respectively. Besides, content of Low molecular weight (LMW) components was higher in A-smoked mullet product than medium MW and vice versa in case B-smoked product however, high MW was not detected in products. In conclusion, PAHs concentration in smoked products processed from pre-frozen mullet samples for 6 months at -18°C are considered a minimally contaminated (16.2 ppb) for A-smoked product and not contaminated (7.4 ppb) for B-smoked product compared with recommended levels. In addition to the component of Benzo [a] Pyrene (B {a} P) was not detectable in all smoked fish products.


Introduction
Smoking is one of the oldest methods used to process and preserve fish.It is a process of treating fish by exposing it to smoke from smoldering wood or plant materials.This process is usually characterized by an integrated combination of salting, drying, heating and smoking operations in a smoking chamber.The preservation properties of smoking treatment are mainly due to the partial drying and the precipitation of aliphatic and aromatic vapors on fish surface [1][2][3][4][5][6].Food cooking and processing methods at high temperatures such as smoking, drying, roasting, baking or frying are recognized as a major source of food contamination by PAHs [7][8][9][10].
Wood smoke contains a hundreds (a least 100) of PAHs and their derivatives which have carcinogenic compounds such as Benzo [a] pyrene (BaP).BaP consider a marker for carcinogenic PAHs in smoked fish and the maximum level is 2µg/kg.After metabolic activation in mammalian cells to diol-epoxides, PAHs bind covalently to cellular macromolecules, including DNA, thereby causing errors in DNA replication and mutations that initiate the carcinogenic process.This mechanism of activation, with some modifications, occurs with all carcinogenic PAHs [14].The classification of the International Agency of Research on Cancer for benzo [a] pyrene (BaP) was changed from group 2A (probably carcinogenic to humans) to group 1 (carcinogenic to humans), chrysene was changed from group 3 (not classifiable for humans) to group 2B (possibly carcinogenic to humans), and benzo [a] anthracene was regrouped from 2A to 2B [15][16].
Therefore, the main purpose of this work was to determine the concentration and risk assessment of Polycyclic Aromatic Hydrocarbons (PAHs) in cold-smoked mullet fish samples which were obtained from two fish farms, El-Fayoum governorate, Egypt and frozen storage at -18°C for 6 months.

Fish Samples
Mullet fish (Mugil cephalus) samples were obtained after directly catch from two fish farms (A and B).The main resources of irrigation water were agricultural discharge for A (Al-Batts drain) and B (El Wadi drain) during August, 2015 at El-Fayoum governorate, Egypt.They were transported immediately to Fish Processing and Technology Lab, Shakshouk Station for Water Resource, National Institute of Oceanography and Fisheries (NIOF), Egypt.Average of weight 525±25gm and length 36±1cm for raw samples from Farm A (Al-Batts drain) while, the average weight of raw mullet samples from Farm B (Agricultural discharge) was 545±5gm and length was 37.75±0.25cm,respectively.After that, fish samples were carefully washed with tap water, glazed, packed in polyethylene bags and stored at -18ºC for 6 months.

Smoking Process
After 6 months of raw mullet fish frozen storage, Fish samples (from A and B farm) were thawed at 4°C then soaked in 10% brined solution (Sodium chloride) for two hrs., rinsed with tap water for 1 min and semi-dried at 25ºC for two hrs.The smokehouse had inside dimensions of 1.20×1.0×3.5 m with pours-metal plates localized above the smoke source by 75cm. the Semi-dried fish samples were hooked at distance about 250cm in smoking house.Traditional cold smoking was carried out at 28-32ºC for 8-10 hrs.using sawdust as smoke source.After smoking the fish samples were cooled under ambient temperature.

Analytical Methods
The edible of smoked mullet fish products was manually separated, homogenized, packed in polyethylene bags and then stored in a freezer at -20°C till analysis.PAHs were determined at Central Laboratory of Residue Analysis of Pesticides and Heavy Metals in Food (QCAP), Agricultural Research Centre.Cairo, Egypt as described by [17].Chemicals and Reagents; acetone (Riedel-de Hӓen, purity 99.8%), acetonitrile (Sigma-Aldrich, purity> 99.9%), toluene (Merck), dichlorom-ethane chromatography grade, and nhexane (purity>99.0%)were used.
B{a}P equivalent The BaP eqi was calculated as the sum of BaP eqi value for individual PAHs.The BaP eqi value was calculated for each PAH from its concentration in the sample (C PAHi ) multiplied by its toxic equivalency factor (TEF PAHi ) [18].
CPAHi Concentration of each PAH in the sample; TEFPAHi: Toxic equivalency factor for each individual PAH.

Statistical Analysis
The results obtained were analyzed statistically using the least significant difference test (LSD) at (P ≤ 0.05) and were expressed as Mean ± SD using SPSS 16 for windows.

Polycyclic Aromatic Hydrocarbons (PAHs)
Table 1 shows the PAHs concentration of cold smoked mullet fish flesh.16

Toxic Equivalent Factors (TEFs) and B {a} P Equivalent of PAHs Found in Smoked Mullet Fish Samples
Toxic equivalency factor (TEF) is an estimate of the relative toxicity of individual PAH fraction compared to benzo (a) pyrene.TEFs have been applied as a useful tool for the regulation of compounds with a common mechanism of actions (e.g PAHs) [19].Even if this presentation of PAH content is empirical because the effects of PAHs in a mixture are insufficiently understood, with this approach it is possible to express PAH contamination of food by a single value as reported [20][21].Benzo [a] Pyrene (BaP) has been well characterized as the most potent carcinogenic PAH after dibenz [a, h] anthracene.Therefore, the total PAH concentration is expressed as Benzo [a] Pyrene Equivalents (BaP eq ) to illustrate the toxic potency [22].
The toxic equivalent factors (TEFs) and B [a] P Equivalent of PAHs in smoked mullet fish are presented in Table 2.The B [a] P Equivalent of Fluorene; Phenanthrene; Anthracene; Fluoranthene and Pyrene were 0.0038; 0.0049; 0.024; 0.0026 and 0.0025 respectively and the ∑ (BaP eqi ) was 0.0378 for farm A smoked samples.In the other farm samples (B) the B [a] P Equivalent of Anthracene; Fluoranthene and Pyrene were 0.024; 0.0026 and 0.0024 respectively and the sum of B [a] P Equivalent (∑ (BaP eqi ) were 0.029.

Molecular Weight of PAHs in Smoked Fish
Table 3 illustrates the molecular weights (MW) of PAHs in smoked mullet fish.The total concentration of the low molecular weights (LWM) of PAHs was higher than the medium molecular weights (MMW) in smoked fish farm (A).The concentration of LWM in smoked fish farm (A) was 11.1 ppb while MMW was 5.1 ppb.On the other side, for samples from B farm the total concentration of medium molecular weights of PAHs 5 µg / kg, and LMW was 2.4 µg / kg.The high molecular weight compounds not detected in samples from both farms A and B, Most of the carcinogenic PAHs fall within the group of the HMW [23].This can be suggested to have been influenced by low fat and pyrolysis resulted from melted dropping onto the heat source.This is due to the average temperature of the smoking processes does not favor the production of HMW PAHs.The temperature range of 500-900°C is known to favor the production of HMW PAHs from thermal breakdown of lignin in lignocelluloses during wood combustion and also from pyrolysis of fats in fish [24][25][26][27][28].
Category of PAH concentration (ppb) in the studied smoked samples is shown in Table 4. Concentrations of PAHs were 16.2 and 7.4 ppb in smoked fish from farms (A) and (B), respectively.Based on these results, categories of concentration of PAH are considered a minimally contaminated (10-99 ppb) for A-smoked product, may be due to pollutants presented in Al-Bats drain and not contaminated (˂10 ppb) for B-smoked product compared with recommended levels as set by [29].

Conclusion
It could be concluded PAHs concentration in smoked mullet products processed from pre-frozen mullet samples for 6 months at -18°C are considered a minimally contaminated (16.2 ppb) for A-smoked mullet product and not contaminated (7.4 ppb) for B-smoked product compared with recommended levels.In addition to the component of Benzo [a] Pyrene (B {a} P) was not detectable in all smoked fish products.

Table 1 .
Concentration of polycyclic aromatic hydrocarbons (PAHs) in cold-smoked mullet fish samples pre-frozen for 6 months at -18°C.

Table 3 .
Total mean concentration (ppb) of PAHs in cold smoked fish, according to their molecular weights.

Table 4 .
Category of PAH concentration (ppb) in the studied cold smoked mullet samples.