Effect of Charcoal Grilling on Polycyclic Aromatic Hydrocarbons (Pahs): Content, Composition, and Health Risk in Edible Fish in Japan
Analytical Sciences Advance Publication by J-STAGE Received June 24, 2021; Accepted August 14, 2021; Published online on August 20, 2021 DOI: 10.2116/analsci.21P197
Original Papers
Effect of charcoal grilling on polycyclic aromatic hydrocarbons (PAHs): content, composition, and health risk in edible fish in Japan
Zijun GAO,* Zhen CHEN,* and Shu-Ping HUI*†
* Faculty of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Kita-Ku, Sapporo
060-0812, Japan
† To whom correspondence should be addressed.
E-mail: [email protected]
1 Abstract
The content and composition of polycyclic aromatic hydrocarbons (PAHs) induced by charcoal grilling of fish were determined in this study. By using HPLC-DAD, the simultaneously quantitative method for 16 priority PAHs was developed and applied to three common Japanese fish: mackerel, pacific saury, and sardine. Charcoal grilling largely increased the content of both total and representative PAHs. Moreover, all the three fish showed a similar PAH composition under the effect of charcoal grilling: in the raw samples, naphthalene was observed as the dominant PAH, while in the charcoal-grilled samples, phenanthrene, fluoranthene, and pyrene served as the major
PAHs. Furthermore, health risk assessment showed that charcoal grilling resulted in high levels of the toxic equivalence quotients, the daily dietary intake exposure to PAHs, and the incremental lifetime cancer risk in three fish, which outclassed the raw samples and exceeded the recommended limitations. The results suggested that charcoal grilling-induced accumulation of PAHs and their potential health risk on human health should be greatly concerned, which might contribute to the dietary guidance and risk management of food-contained PAHs.
Keywords: Polycyclic aromatic hydrocarbons (PAHs); charcoal grilling; health risk;
HPLC-DAD; fish; food composition.
2 Introduction
Polycyclic aromatic hydrocarbons (PAHs) are a class of chemicals that include two or more fused aromatic rings.1 They have been known as toxic, mutagenic, and carcinogenic for long.2 And the main causes of these concerns associated with human exposure to PAHs are considered carcinogenicity and mutagenicity.3 It is to be noticed that the mixture of PAHs is supposed to be more dangerous than individual PAH, which is what human exposure to in daily life.4,5 According to the occurrence frequency, the toxicity, and the potential for human exposure, sixteen PAHs were claimed by the
United States Environmental Protection Agency (USEPA) as priority PAHs (PAH16), including naphthalene (Nap), acenaphthylene (AcPy), acenaphthene (Acp), fluorene
(Flu), phenanthrene (PA), anthracene (Ant), fluoranthene (FL), pyrene (Pyr), chrysene
(CHR), benzo[a]anthracene (BaA), benzo[b]fluoranthene (BbF), benzo[k]fluoranthene
(BkF), benzo[a]pyrene (BaP), dibenz[a,h]anthracene (DBA), indeno[1,2,3-cd]pyrene
(IND), and benzo[ghi]perylene (BghiP) (Fig. S1).6 Meanwhile, the European Food
Safety Authority (EFSA) suggests focusing on the eight representative PAHs (PAH8, including BaA, CHR, BaP, BbF, Acp, BkF, BghiP, and DBA), because they represent
80% of all the PAHs.7 Furthermore, EFSA recommends the four most frequent and life-concerning PAHs (PAH4, including BaA, CHR, BaP, and BbF) to be the most worthy-noting targets with regard to their occurrence and content in foods, adverse biological effects, and health risks to human.7 Therefore, not only the content but also the composition for the determination of PAHs is of great importance.
Food processing, cooking, and eating are among the major sources of PAHs in the daily life of humans, especially for non-smokers.8 The burning process of fossil fuels and the high temperature during the cooking result in the organic components been
3 partly cracked to small unstable molecules (pyrolysis) and recombined to relatively stable aromatic hydrocarbons (pyrosynthesis), leading to the release of PAHs.9,10 Fish is a generally consumed food in Japan, especially for outdoor parties, in which grilling using convenient commercial kits is a popular cooking method. At the same time, raw fish meat is also commonly accepted for eating (i.e., Sushi and Sashimi in Japan).
Previous studies have elucidated the composition and concentrations of PAHs during fish cooking processes, such as frying, smoking, and sun-drying.11–13 In terms of grilling, there have been several reports in China and Malaysia,14,15 even though studies on charcoal grilling-induced PAHs variations, especially for fish in Japan, are quite limited.
Furthermore, the PAH-associated potential risks regarding human health are rarely evaluated.
Hence, the current study aimed to determine the content and composition of PAHs and their comparison between raw and charcoal-grilled fish in Japan. The sensitive and accurate method for simultaneous determination of PAH16 was firstly developed and validated by using high-performance liquid chromatography (HPLC) with a diode-array detector (DAD). Moreover, three common Japanese fishes (pacific saury, mackerel, and sardines) before and after grilling were used to compare the content and composition of
PAHs. Moreover, the health risk indexes, including daily dietary intake (DDI), carcinogenic toxic equivalents (TEQ), and incremental lifetime cancer risk (ILCR), were calculated in these raw and charcoal-grilled samples. The impact of charcoal grilling on the profile variation of PAHs from fish in Japan and the association with health risks were thereafter discussed.
4 Experimental
Reagents and chemicals
Standards of all the sixteen PAHs investigated in this study, including Nap, AcPy, Acp,
Flu, PA, Ant, FL, Pyr, CHR, BaA, BbF, BkF, BaP, DBA, IND, and BghiP, were purchased from Accu Standard, Inc. (New Haven, CT, USA). All the chemicals and reagents were of HPLC grade and purchased from Wako Pure Chemical (Osaka, Japan) unless specified.
Sampling and charcoal grilling
Three commonly consumed fish in Japan were selected in this study: mackerel, pacific saury, and sardine. All the fish were produced in Japan from June to July 2019 and were purchased from the local markets (n = 10 for each fish). The fresh fish sample was cleaned with water and then eviscerated. The grilling was performed using the commercial grilling kit purchased from a local supermarket, including the charcoal, stand, and net. The grilling process was accorded to the recommended protocol of the grilling kit, of which each side of the fish sample (approximately 100 g per piece) with skin was grilled for fifteen minutes.
Extraction procedure
The extraction procedure followed the method described by Ikenaka et al.16 Briefly, the fish samples after grilling were homogenized by an Abitelax® kitchen blender (YOSHII
ELECTRIC Co., Ltd., Gunma, Japan). Then, 2.5 grams of the mashed sample, weighed on an ultrasensitive electro-balance (Cubis® ultra micro balance, Sartorius Inc.,
Göttingen, Germany), was saponified using 15 mL 1M KOH dissolved in ethanol for 10
5 hours at 60 °C. Next, 5 mL of hexane and 5 mL of water were added, followed by vortex for 5 min at 25 ℃. Partition was conducted by centrifugation at 3000 rpm for
30 min at 25℃, and the PAHs-contained hexane layer was collected. The obtained
PAHs were dried under vacuum, re-dissolved into 500 μL of methanol, filtered to remove any insoluble matter prior to HPLC injection.
HPLC conditions
HPLC analysis was performed with a Shimadzu LC30 pump system (Shimadzu, Kyoto,
Japan) equipped with an SPD-M20A photodiode array detector as the DAD. The 16 investigated PAHs were separated on a Kinetex Phenyl-Hexyl column (250 mm ×
4.6 mm, 5 µm, Phenomenex Inc. Torrance, CA, USA) maintained at 40 ℃. The tray temperature was set at 4 ℃, and the injection amount was set at 10 μL. The mobile phase consisted of water (A) and acetonitrile (B) with a flow rate of 1.3 mL/min, and the gradient elution was as follows: 0.0–21.5 min, 30%→80% B; 21.5–22.0 min,
80%→100% B; 22.0–35.0 min, kept at 100% B; 35.0–35.5 min, 100%→30% B; this ratio was kept until 38.0 min for rebalancing.
Method validation
Method validation was according to European Union Commission Regulations,17 including linearity, limit of detection (LOD) and quantification (LOQ), reproducibility, and recovery. For linearity determination, a series of PAHs standard mixture solutions were prepared by diluting with methanol. The method of external standards was used, and the coefficient of correlation of six standard points, calculated through the ratio of peak area versus concentration of each PAH standard, was used to evaluate the linearity of the calibration curve. The signal-to-noise ratios of 3:1 and 10:1 were used to measure
6 LOD and LOQ, respectively. Reproducibility and recovery were carried out by 6 independent replications of fish samples spiked with a certain concentration of PAHs, and the spiked amount of each PAH was decided as around 10 folds of its content in the unspiked samples. Method repeatability was described as the percentage coefficient of variation (CV). Precision as CV was calculated from the measured standard deviation
(SD) and mean values. The calculation formula of recovery is shown as below:
Recovery (%) = (Amount found – Amount original)/Amount spiked × 100%
Health risk assessment
(1) Toxic equivalence quotients (TEQ): The TEQ was calculated to express the carcinogenic risk of individual PAH by the toxicity equivalency factor (TEF), which was determined by the toxic PAH to a reference compound BaP according to USEPA
Integrated Risk Information System.18 The TEF of individual PAH is listed in Table S1, and the TEQ of PAHs was calculated using the following equation:19
TEQ = Ci × TEFi
where Ci represents the measured individual PAH content, and TEFi is compound with the assigned toxicity equivalency factor.
(2) Dietary daily intake (DDI): For a Japanese adult of average weight (70 kg), the daily intake of PAHs of charcoal-grilled fish was calculated by multiplying the respective
PAH content in the fish sample by the fish ingestion rate (IR).20 The DDI of PAHs occurred in fish was calculated using the following equation:
DDI = Ci × IR
where IR represents the ingestion rate of fish in Japan, which was estimated at about 45.3 kg/year/capita by the Food Agriculture Organization.21
(3) Incremental lifetime cancer risk (ILCR): The ILCR was identified as the increase in
7 the possibility for human beings causing any type of cancer all their life due to 24 h/day exposure to a given daily amount of a carcinogenic element for seventy years.22 ILCR was calculated using the following equation:23
ILCR = (DDI × TEF × EF × ED × SF × CF)/(BW × AT)
where EF represents the exposure frequency (365 days/year), ED is the exposure duration in years (43 years for adults), SF is the oral cancer slope factor of BaP (7.3),
CF is the conversion factor (10−6), BW is the average adult body weight (70 kg), and AT is the average lifespan of carcinogens (25550 days).24
Statistics
All the data are presented as the means ± SD. Statistical analyses were performed by two-way ANOVA with Sidak's multiple comparisons test as the post hoc test using
Prism 7.0 (GraphPad Software, Inc., La Jolla, CA, USA), and P < 0.05 was considered statistically significant.
Results and Discussion
Optimization and validation of the HPLC method
The detection UV wavelength for each PAH was optimized individually, and consequently, 6 wavelengths were selected as the working wavelength for detecting
PAH16. The optimal wavelength and retention time for each PAH under the present
HPLC parameters are listed in Table 1. In the current study, all the PAH16 were separated within 10 minutes (12–22 min), though the gradient elution from 0 to 12 min was necessary to remove the interferents. While the elution for 22–38 min was for
8 thoroughly wash the column and rebalancing, thus, extending the life of the column and ensuring the data reliability when analyzing multiple batches of samples.
The peak area ratio of each PAH standard against its content was carried out to gain the six-point calibration curve, of which all the analytes showed good linearity with correlation coefficients above 0.999 within their dynamic ranges (Fig. S2 and
Table 2). For sensitivity, the LODs ranged from 0.03 ng/mL to 0.30 ng/mL, while the
LOQs were from 0.10 ng/mL to 0.64 ng/mL (Table 2). The results of reproducibility and recovery are listed in Table 3, in which the CVs of reproducibility for all the PAHs were less than 8.2%, and the recoveries were from 86.5% ± 4.9% to 112.8% ± 3.7%
(with all the CVs below 9.6%). Therefore, the currently established HPLC-DAD quantitative method was considered reliable to investigate the PAHs content and composition in the following experiments.
It should be mentioned that in recent years, most of the analyses on PAHs were based on mass spectrometric (MS) technologies, such as LC/MS and GC/MS.
Specifically, the vast majority of these studies used atmospheric pressure ionization
(API), including atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI), due to the poor ionization efficiency of electrospray ionization (ESI). However, these MS instruments are not as common/economical as
HPLC-DAD. Moreover, for PAHs analysis, HPLC-DAD showed similar or even better performance of LOD and LOQ compared with LC/APCI-MS.25 Therefore, this easy-accessible and well-performing approach was chosen in our experiment to evaluate the grilling-induced PAHs changes and the subsequent health risks.
Comparison of PAHs content in raw and charcoal-grilled fish
Based on this developed method, the contents of the 16 PAHs in all the fish samples
9 were determined (listed in Table 4). In raw meat of mackerel, pacific saury, and sardine,
13, 8, and 8 kinds of PAHs were detected in this study, respectively. While after charcoal grilling, all the 16 PAHs were observed in all the fish samples. These results indicated that charcoal grilling leads to produce the other different kinds of PAHs. The contents of total PAHs investigated (i.e., PAH16) were 5.70 ± 0.65 ng/g,
2.43 ± 0.49 ng/g, 2.34 ± 0.25 ng/g in raw mackerel, pacific saury, and sardine, respectively, which agreed with Masuda et al.26 who determined PAH contents in the raw fish products from Japanese markets. After charcoal grilling, the total PAHs content in the three kinds of fish samples significantly elevated to 178.22 ± 12.63 ng/g,
92.60 ± 5.05 ng/g, and 100.60 ± 6.08 ng/g (P < 0.001 vs. raw for all fish), reaching to
31.3, 38.1, and 43.0 folds of raw samples, respectively.
Furthermore, in terms of the carcinogenic indicator PAH4, the raw fish samples showed relatively low levels (in mackerel: 0.38 ± 0.11 ng/g; in pacific saury and sardine: not detected). Whereas the charcoal-grilled samples exhibited the considerably and significantly high amount in mackerel (24.47 ± 1.64 ng/g), pacific saury
(10.43 ± 0.79 ng/g), and sardine (12.30 ± 0.50 ng/g) (P < 0.001, grilled vs. raw for all fish), of which the mackerel and the sardine exceeded the permittable level (12 ng/g) set by European Union for fish and meat products.17 This indicated that the consumption of charcoal-grilled mackerel and sardine could pose a potential health concern to human health. Previous studies reported similar results: PAH4 ranged from 8.14 ng/g to
11.00 ng/g in grilled Nile perch (Lates niloticus) from Uganda,27 and much higher content of BaA (7.4–39.4 ng/g), CHR (16.3–55.2 ng/g), BbF (5.7–23.2 ng/g), and BaP
(3.0–38.0 ng/g) were observed in smoked or grilled fish (mudfish, jackfish, and mackerel) in Nigerian.28
The elevation of total PAHs indicated that charcoal grilling resulted in the
10 formation of PAHs, particularly the carcinogenic PAH4. Especially, charcoal grilling was proved even more inclined to produce PAHs than gas grilling due to the direct contact between foodstuff and charcoal flame.29 For instance, much more PAHs were formed during charcoal grilling than gas roasting in beef and pork.30 Another report on meat products claimed that PAHs generated from the disposable grilling device were
1.6-fold higher than the traditional wood-burning griller.31 This evidence supposed that the contents of PAHs during grilling were involved with the fuel materials. Besides, the components of foodstuffs also influence the PAHs formation. For example, PAHs were proved to generate through the intramolecular cyclization of unsaturated fatty acids.32 In our present study, raw mackerel exhibited the highest PAHs among the three kinds of fishes, which also contained the highest content of lipids and unsaturated fatty acids
(Data from the standard tables of food composition given by the Japanese government,33 listed in Table S2). These results also supported the view that the higher fat level resulted in the more generated PAHs.34,35 It should be noted that the amount of the produced PAHs might be related to the time of grilling, and the longer grilling time might lead to more PAHs. However, in the present study, only 15 minutes (as the recommendation of the grilling kit use guidance) was investigated. The detailed time-amount relationship should be comparatively investigated in the future.
Comparison of PAHs composition in raw and charcoal-grilled fish
It was noted that the most abundant PAH compound in raw fish samples was Nap, which contributed 23%–41% of all the investigated PAHs. These results were in agreement with Essumang et al.36, who reported that Nap was the dominant PAHs in the fresh fish samples. While after charcoal grilling, PA, FL, and Pyr were found to be the predominant PAHs in these fish samples, accounting for 19%–25% (PA), 16%–21%
11 (FL), and 16%–17% (Pyr) (Fig. 1). The alteration of PAH composition before and after charcoal grilling could be explained as their origins in foods: Nap in raw fish mainly originated from the environment, and due to its lowest polarity among PAHs, Nap was more like to cross the fish biomembrane and accumulates in tissue.37 Moreover, the similar PAH composition in these three charcoal-grilled fish indicated that charcoal might be the key factor for PAH formation. It was reported that PAHs could be produced by pyrolysis of the fish drippings and incomplete combustion of charcoal, which thereafter accumulated on the surface and penetrated the meat.15,38 A similar composition pattern of PAHs residuals was also found by a previous study on grilled meats.39 Nevertheless, whether this characteristic varies along with other factors, such as the fuel kind, the cooking method, and the specific parameters, are to be investigated.
Health risk assessment of charcoal grilling for fish
Since not only raw but also charcoal-grilled fishes are very popular in Japan, the exposure risk to the carcinogenic (or suspect carcinogenic) PAHs is considered a public health concern. Therefore, the health risk estimations based on the consumption of raw and charcoal-grilled fish among the Japanese population were assessed, and the summarized results are listed in Table 5.
The TEQ of the PAHs in the raw mackerel, pacific saury, and sardine was 0.50,
0.07, and 0.26 ng/g, respectively. While these values were elevated to 14.06, 6.38, and
6.91 ng/g after charcoal grilling, which exceeded the screening value of 0.67 ng/g proposed by USEPA.23 For DDI of the total PAHs, the raw fish exhibited relatively low levels (707.74 ng/day for mackerel, 301.71 ng/day for pacific saury, and 290.50 ng/day for sardine). However, after charcoal grilling, mackerel, pacific saury, and sardine expressed the DDI as high as 22117.50, 11492.11, and 12484.03 ng/day, respectively.
12 Moreover, the DDI based on PAH4 was 47.22 ng/day in mackerel and 0 ng/day in both pacific saury and sardine, while in charcoal-grilled fish, they were 3036.22, 1294.23, and 1526.76 ng/g, respectively. It is to be mentioned that, in the year 2008, ESFA reported the dietary exposure of 1168 ng/day of PAH4 on a basis in the average consumption.7 The ILCR resulting from PAHs exposure over the all life via fish consumption was also estimated, of which the value below 1 × 10−6 is the acceptable risk level and over 1 × 10−4 is considered serious for a 70-year lifespan according to
USEPA.40 In the present study, raw fish showed the ILCR values of 4.00 × 10−6 for mackerel, 5.93 × 10−7 for pacific saury, and 2.04 × 10−6 for sardine. Whereas the charcoal-grilled fish, the ILCR values were 1.12 × 10−4 (mackerel), 5.07 × 10−5 (pacific saury), and 5.49 × 10−5 (sardine), reaching 27.93, 85.59, and 26.93 folds of the corresponded raw samples, respectively. Importantly, the grilled mackerel showed the most concerning values among the three fishes, accounting for approximately 2–3 folds of pacific saury and sardine for all these risk indexes, which is directly connected to its highest PAH content. All these results indicated that charcoal grilling could strongly increase the potential cancer risk to humans regarding the exposure to PAHs. It is noted that ILCR might vary due to the variations of foodstuffs, the consumption amount, the contamination degree of raw food, and the processing methods. Therefore, our study also suggested that adequate management process steps should be taken in Japan to control and reduce the health risks associated with dietary PAHs exposure.
Conclusions
In this study, a sensitive, accurate, and reliable HPLC method for the simultaneous
13 quantitation of 16 PAHs in foods has been developed, validated, and applied to three kinds of fish samples. The PAHs content in the charcoal-grilled fish samples largely increased compared with the raw fish. Moreover, a similar PAH composition was found in all the charcoal-grilled fish, indicating that the process of charcoal grilling, rather than fish species, was the primary factor for PAH formation. Three health risk assessment indexes TEQ, DDI, and ILCR were used to compare the health risk of exposure to PAHs between the consumption of raw and charcoal-grilled fish, which indicated the increased probability of human exposure to PAHs through diet as well as the additional health risk induced by charcoal grilling. Our current work not only provided a practical approach to investigate the content and composition of PAHs in foodstuffs and processed foods, but also revealed a non-negligible problem involved in health risk and the consumption of charcoal grilling foods.
Acknowledgements
This study was supported by the Ministry of Education, Culture, Sports, Sciences and
Technology, Japan.
14 References
1. S. Danyi, F. Brose, C. Brasseur, Y.-J. Schneider, Y. Larondelle, L. Pussemier, J.
Robbens, S. De Saeger, G. Maghuin-Rogister, and M.-L. Scippo, Anal. Chim.
Acta, 2009, 633, 293.
2. S. K. Samanta, O. V. Singh, and R. K. Jain, Trends Biotechnol., 2002, 20, 243.
3. G. FALCÓ, J. L. DOMINGO, J. M. LLOBET, A. TEIXIDÓ, C. CASAS, and L.
MÜLLER, J. Food Prot., 2003, 66, 2325.
4. E. H. Pfeiffer, IARC (International Agency Res. Cancer) Sci. Publ., 1977, No. 16,
69.
5. C. Barata, A. Calbet, E. Saiz, L. Ortiz, and J. M. Bayona, Environ. Toxicol. Chem.,
2005, 24, 2992.
6. United States Environmental Protection Agency, Proposed Guidelines for
Carcinogen Risk Assessment, Washington, DC, 1996.
7. EFSA, EFSA J., 2008, 6, 724.
8. X. Duan, G. Shen, H. Yang, J. Tian, F. Wei, J. Gong, and J. Zhang, Chemosphere,
2016, 144, 2469.
9. K. Dost and C. İdeli, Food Chem., 2012, 133, 193.
10. G. Purcaro, P. Morrison, S. Moret, L. S. Conte, and P. J. Marriott, J. Chromatogr.
A, 2007, 1161, 284.
11. J. A. M. Mahugija and E. Njale, J. Food Compos. Anal., 2018, 73, 39.
12. S. Wretling, A. Eriksson, G. A. Eskhult, and B. Larsson, J. Food Compos. Anal.,
2010, 23, 264.
13. C. M. A. Iwegbue, K. O. Osijaye, U. A. Igbuku, F. E. Egobueze, G. O. Tesi, F. I.
Bassey, and B. S. Martincigh, J. Food Compos. Anal., 2020, 94, 103633.
15 14. J. Cheng, X. Zhang, Y. Ma, J. Zhao, and Z. Tang, Sci. Total Environ., 2019, 690,
965.
15. A. Farhadian, S. Jinap, F. Abas, and Z. I. Sakar, Food Control, 2010, 21, 606.
16. Y. Ikenaka, Y. Ito, H. Eun, E. Watanabe, and Y. Miyabara, J. Environ. Chem.,
2008, 18, 341.
17. The European Parliament And The Council of The European Union, Off. J. Eur.
Union, 2011, L 215, 4.
18. United States Environmental Protection Agency, Integrated Risk Information
System., 2011.
19. I. Tongo, O. Ogbeide, and L. Ezemonye, Toxicol. Reports, 2017, 4, 55.
20. United States Environmental Protection Agency, Provisional Guidance for
quantitative risk assessment of PAH.pdf, 1993.
21. FAO, Fishery and Aquaculture Country Profiles Japan, 2019.
22. I. Grzetic and A. Ghariani, J. Serbian Chem. Soc., 2008, 73, 923.
23. United States Environmental Protection Agency, United States Environ. Prot.
Agency, Washington, DC, 2000, 1, 823.
24. W. S. Darwish, H. Chiba, W. R. El-Ghareeb, A. E. Elhelaly, and S.-P. Hui, Food
Chem., 2019, 290, 114.
25. G. M. Titato and F. M. Lancas, J. Chromatogr. Sci., 2006, 44, 35.
26. M. Masuda, Q. Wang, M. Tokumura, Y. Miyake, and T. Amagai, Ecotoxicol.
Environ. Saf., 2019, 178, 188.
27. O. Acaye, W. N. George, M. Jolocam, K. Justus, and M. Margaret, African J.
Pure Appl. Chem., 2013, 7, 164.
28. V. O. E. Akpambang, G. Purcaro, L. Lajide, I. A. Amoo, L. S. Conte, and S.
Moret, Food Addit. Contam. Part A, 2009, 26, 1096.
16 29. A. Farhadian, S. Jinap, H. N. Hanifah, and I. S. Zaidul, Food Chem., 2011, 124,
141.
30. S. Y. Chung, R. R. Yettella, J. S. Kim, K. Kwon, M. C. Kim, and D. B. Min, Food
Chem., 2011, 129, 1420.
31. M. Reinik, T. Tamme, M. Roasto, K. Juhkam, T. Tenno, and A. Kiis, Food Addit.
Contam., 2007, 24, 429.
32. B. H. Chen and Y. C. Chen, J. Agric. Food Chem., 2001, 49, 5238.
33. H. Taira, Nippon Shokuhin Kagaku Kogaku Kaishi, 2001, 48, 549.
34. B. K. Larsson, G. P. Sahlberg, A. T. Eriksson, and L. A. Busk, J. Agric. Food
Chem., 1983, 31, 867.
35. T. H. Kao, S. Chen, C. W. Huang, C. J. Chen, and B. H. Chen, Food Chem.
Toxicol., 2014, 71, 149.
36. D. K. Essumang, D. K. Dodoo, and J. K. Adjei, Food Control, 2014, 35, 85.
37. A. Mohammadi, V. Ghasemzadeh-Mohammadi, P. Haratian, R. Khaksar, and M.
Chaichi, Food Chem., 2013, 141, 2459.
38. W. Lijinsky and P. Shubik, Science, 1964, 145, 53.
39. H. Alomirah, S. Al-Zenki, S. Al-Hooti, S. Zaghloul, W. Sawaya, N. Ahmed, and
K. Kannan, Food Control, 2011, 22, 2028.
40. G. Li, S. Wu, L. Wang, and C. C. Akoh, Food Control, 2016, 59, 328.
17 Table 1 UV detection wavelength and retention times of PAHs
Compound Abbreviation UV detection Retention times (min)
wavelength (nm) In standard In fish
solution sample
Naphthalene Nap 220 12.20 12.26
Acenaphthylene AcPy 230 13.46 13.54
Acenaphthene Acp 230 14.74 14.86
Fluorene Flu 264 14.93 15.03
Phenanthrene PA 251 15.66 15.77
Anthracene Ant 251 16.03 16.14
Fluoranthene FL 230 16.99 17.10
Pyrene Pyr 240 17.25 17.37
Chrysene CHR 264 18.70 18.83
Benzo[a]anthracene BaA 290 18.86 18.99
Benzo[b]fluoranthene BbF 251 19.98 20.13
Benzo[k]fluoranthene BkF 230 20.13 20.22
Benzo[a]pyrene BaP 264 20.33 20.47
Dibenz[a,h]anthracene DBA 290 21.28 21.41
Indeno[1,2,3-cd]pyrene IND 251 21.49 21.63
Benzo[ghi]perylene BghiP 290 21.62 21.78
18 Table 2 Linearity, LOD, and LOQ for the investigated PAHs
PAH Calibration equation R² Dynamic range LOQ LOD
(ng/mL) (ng/mL) (ng/mL)
Nap y = 13144.0x + 3621.5 0.9999 0.20 – 250.00 0.20 0.05
AcPy y = 5613.4x + 781.7 1.0000 0.10 – 125.00 0.10 0.03
Acp y = 6057.5x + 764.1 0.9999 0.20 – 125.00 0.20 0.10
Flu y = 2410.7x + 452.0 0.9999 0.40 – 200.00 0.40 0.16
PA y = 6355.0x + 951.5 0.9999 0.10 – 300.00 0.10 0.04
Ant y = 14626.0x + 951.5 0.9999 0.10 – 125.00 0.10 0.03
FL y = 3770.1x + 1394.5 1.0000 0.64 – 400.00 0.50 0.16
Pyr y = 5769.8x + 629.5 1.0000 0.30 – 200.00 0.16 0.04
CHR y = 8870.0x + 90.7 1.0000 0.32 – 100.00 0.20 0.08
BaA y = 4791.6x – 49.9 1.0000 0.50 – 50.00 0.50 0.20
BbF y = 3571.0x – 244.4 0.9999 0.40 – 125.00 0.40 0.10
BkF y = 3682.0x + 126.3 0.9999 0.20 – 50.00 0.20 0.07
BaP y = 3635.5x – 500.3 0.9991 0.50 – 100.00 0.50 0.30
DBA y = 6180.0x – 1432.1 0.9999 0.50 – 200.00 0.50 0.15
IND y = 3392.2x – 1232.0 0.9998 0.50 – 125.00 0.50 0.13
BghiP y = 2771.3x – 679.0 0.9992 0.64 – 125.00 0.64 0.16
19 Table 3 Repeatability and recovery for the investigated PAHs
PAH Repeatability Recovery
(n = 6) (n = 6) Concentration CV Expected Tested CV Recovery Recovery (ng/mL) (%) concentration concentration (%) (%) Range (%)
(ng/mL) (ng/mL)
Nap 0.34 ± 0.03 8.0 1.5 1.33 ± 0.10 7.7 89.0 ± 6.9 77.0 – 98.3
AcPy 5.49 ± 0.35 6.3 40 41.78 ± 3.31 7.9 104.5 ± 8.3 91.0 – 112.7
Acp 1.22 ± 0.03 2.2 10 10.15 ± 0.55 5.4 101.5 ± 5.5 95.2 – 108.3
Flu 2.36 ± 0.06 2.3 20 18.39 ± 1.09 5.9 91.9 ± 5.5 83.8 – 98.8
PA 23.29 ± 0.79 3.4 200 173.16 ± 16.65 9.6 86.6 ± 8.3 79.1 – 101.8
Ant 5.59 ± 0.33 6.0 100 100.20 ± 4.43 4.4 100.2 ± 4.4 93.6 – 105.6
FL 21.57 ± 1.11 5.1 150 145.72 ± 7.12 4.9 97.1 ± 4.7 88.6 – 101.9
Pyr 15.99 ± 0.46 2.9 100 112.84 ± 3.73 3.3 112.8 ± 3.7 108.3 – 117.2
CHR 1.79 ± 0.06 3.5 15 14.91 ± 0.34 2.3 99.4 ± 2.3 97.7 – 103.0
BaA 3.93 ± 0.18 4.5 30 30.36 ± 0.58 1.9 101.2 ± 1.9 97.7 – 102.9
BbF 2.62 ± 0.14 5.4 20 20.58 ± 0.56 2.7 102.9 ± 2.8 100.2 – 108.2
BkF 4.63 ± 0.18 3.9 40 37.45 ± 3.06 8.2 93.6 ± 7.7 82.8 – 101.9
BaP 4.39 ± 0.24 5.5 40 39.79 ± 2.71 6.8 99.5 ± 6.8 91.7 – 105.7
DBA 0.99 ± 0.07 7.2 10 8.65 ± 0.49 5.7 86.5 ± 4.9 83.3 – 96.3
IND 2.78 ± 0.23 8.2 20 20.68 ± 0.64 3.1 103.3 ± 3.2 98.5 – 106.7
BghiP 2.93 ± 0.13 4.4 20 20.01 ± 1.26 6.3 100.0 ± 6.3 93.6 – 108.7
20 Table 4 PAHs content (ng/g) in raw and charcoal-grilled fishes (Means ± SD)
PAH Mackerel Pacific saury Sardine
(n = 10) (n = 10) (n = 10)
Raw Grilled Raw Grilled Raw Grilled
Nap 1.68 ± 0.56 3.46 ± 0.32 1.00 ± 0.42 3.30 ± 0.23*** 0.54 ± 0.10 0.82 ± 0.20
AcPy 0.51 ± 0.20 10.29 ± 1.85*** 0.10 ± 0.04 6.84 ± 0.57*** 0.22 ± 0.10 5.26 ± 0.60***
Acp 0.30 ± 0.05 3.65 ± 0.84 0.14 ± 0.03 2.06 ± 0.33** N/D 1.30 ± 0.12
Flu 0.16 ± 0.06 5.61 ± 0.59*** N/D 2.92 ± 0.28*** N/D 2.27 ± 0.18**
PA 0.06 ± 0.03 33.10 ± 3.42*** N/D 19.24 ± 2.57*** N/D 24.98 ± 4.02***
Ant 0.16 ± 0.03 9.26 ± 1.14*** 0.03 ± 0.01 4.95 ± 0.32*** 0.04 ± 0.02 5.35 ± 0.63***
FL 0.71 ± 0.14 35.35 ± 5.87*** 0.25 ± 0.07 15.21 ± 1.67*** 0.46 ± 0.15 20.80 ± 2.74***
Pyr 0.89 ± 0.13 30.08 ± 3.00*** 0.18 ± 0.05 15.76 ± 2.16*** 0.12 ± 0.04 16.11 ± 1.82***
CHR N/D 3.72 ± 0.48* N/D 1.57 ± 0.17* N/D 1.86 ± 0.12*
BaA N/D 6.55 ± 0.68*** N/D 2.55 ± 0.27*** N/D 3.54 ± 0.19***
BbF 0.14 ± 0.04 4.70 ± 0.40** N/D 2.13 ± 0.26*** N/D 2.48 ± 0.17***
BkF 0.35 ± 0.06 6.23 ± 1.43*** 0.45 ± 0.10 3.77 ± 0.34*** 0.49 ± 0.08 4.78 ± 0.36***
BaP 0.24 ± 0.08 9.50 ± 1.01*** N/D 4.17 ± 0.30*** N/D 4.42 ± 0.24***
DBA 0.17 ± 0.05 1.81 ± 0.17 N/D 0.83 ± 0.06 0.18 ± 0.04 0.96 ± 0.06
IND 0.34 ± 0.07 6.70 ± 0.59*** 0.27 ± 0.06 3.60 ± 0.21*** 0.29 ± 0.06 2.75 ± 0.23**
BghiP N/D 8.24 ± 1.36*** N/D 3.70 ± 0.27*** N/D 2.91 ± 0.23***
PAH4 0.38 ± 0.11 24.47 ± 1.64*** N/D 10.43 ± 0.79*** N/D 12.30 ± 0.50***
PAH16 5.70 ± 0.65 178.22 ± 12.63*** 2.43 ± 0.49 92.60 ± 5.05*** 2.34 ± 0.25 100.60 ± 6.08***
N/D: not detected. * P < 0.05, ** P < 0.01, *** P < 0.001, compared between raw and grilled samples, calculated by two-way ANOVA with Sidak's multiple comparisons test.
21 Table 5 Health risk estimates due to consumption of raw and charcoal-grilled fish among the Japanese population
Index Mackerel Pacific saury Sardine
Raw Grilled Raw Grilled Raw Grilled
TEQ 0.50 14.06 0.07 6.38 0.26 6.91
DDI of PAH4 47.22 3036.22 N/A 1294.23 N/A 1526.76 (ng/day)
DDI of PAH16 707.74 22117.50 301.71 11492.11 290.50 12484.03 (ng/day)
Cancer Risk 4.00×10-6 1.12×10-4 5.93×10-7 5.07×10-5 2.04×10-6 5.49×10-5 Estimates
N/A: Not available. The DDI value could not be calculated because the PAHs were not detected.
22 Figure Captions
Fig. 1 The representative HPLC-DAD chromatograms of the 16 PAHs (A) in standard mixture solutions and (B) in the grilled fish with 220, 230, 240, 251, 264, and 290 nm as the detecting wavelength. Numbers indicate PAHs: 1, Nap; 2, AcPy; 3, Acp; 4, Flu;
5, PA; 6, Ant; 7, FL; 8, Pyr; 9, CHR; 10, BaA; 11, BbF; 12, BkF; 13, BaP; 14, DBA;
15, IND; 16, BghiP.
Fig. 2 Composition of PAHs in the raw and the charcoal-grilled fish samples. (A) the raw mackerel, (B) the grilled mackerel, (C) the raw pacific saury, (D) the grilled pacific saury, (E) the raw sardine, (F) the grilled sardine.
23
Fig. 1
24
Fig. 2
25 Graphical Index
26