Polycyclic Aromatic Hydrocarbon (PAH) Contents of Four Species of Smoked Fish from Different Sites in Senegal

Polycyclic aromatic hydrocarbons (PAHs) are compounds resulting from any incomplete combustion process. These are pollutants that have proven toxicity due to their carcinogenic nature and can contaminate food during traditional smoking methods. Their highly toxic effect on human health requires monitoring of their levels in food products and the development of appropriate analytical methods for their determination. Thus, this study was conducted to assess the level of PAHs contamination of four (4) species of smoked fish (Arius heudelotii, Sardinella aurita, Ethmalosa fimbriata, and Sardinella maderensis) which were sampled in seventeen (17) localities in Senegal. The compounds targeted in this study were benzo(a)pyrene (B(a)P), benzo(a)anthracene (B(a)A), benzo(b)fluoranthene (B(b)F), and chrysene (Chr). The QuEChERS method was used for the extraction of PAHs, and their contents were quantified by gas chromatography (GC) coupled with mass spectroscopy (MS). The validation method was performed in accordance with the French standard NF V03-110 (2010). Satisfactory linearity (R2 > 0.999), LOD (0.05–0.09 μg/kg), LOQ (0.19–0.24 μg/kg), and precision (1.33–3.13%) of the four PAHs were obtained. The results of analysis in the 17 localities showed that all samples are contaminated by the four (4) PAHs with great variability of the contents between the different species and their origin. The B(a)P and ∑4PAHS contents in the samples ranged from 1.7 to 33 µg/kg and from 4.8 to 1082.3 µg/kg, respectively. Twelve (12) samples showed high levels of B(a)P, ranging from 2.2 to 33 µg/kg, thus exceeding the maximum authorized level (2 µg/kg). Fourteen (14) samples showed an overall ∑4PAHS content varying from 14.8 to 1082.3 µg/kg, which is above the maximum authorized limit (12 µg/kg). The principal component analysis showed that sardinella (Sardinella aurita and Sardinella maderensis) have very low levels of B(a)P, B(b)F, B(a)A, and Chr contents. However, high ∑4PAHS contents characterize smoked fish of the Kong species (Arius heudelotii), from Cap Skiring, Diogne, Boudody, and Diaobé, and of the Cobo species (Ethmalosa fimbriata) from Djiffer. Thus, based on the authorized limits for PAHs in smoked fish, it appears that smoked fish of the sardinella species are less carcinogenic for human consumption.


Introduction
Fishing is one of the most dynamic sectors in Senegal as it has experienced strong growth over the past three decades. Catches have increased considerably eight (8) times in thirty-two (32) years [1]. A source of jobs, income, and foreign currency for stakeholders and governments, fshing is a real center for economic and social development. In Senegal, the average of sea fshing landings over the period 2009-2018 was estimated at 421,000 tons, 89% of which was carried out by artisanal fshing. In 2018, the total national fsh production reached more than 461,000 tons [2]. Most of the catches come from small-scale maritime fshing (94.7%) [3]. It should also be noted that fsh is one of the main sources of animal protein for populations, thus contributing to more than 75% of protein intake [4][5][6]. However, the fsh is known for its highly perishable nature [7]. Indeed, it can deteriorate very quickly and become unft for consumption and even dangerous for health due to microbial proliferation, chemical modifcations, and degradation by endogenous enzymes [4]. Tus, due to the large quantities produced, the perishable nature of the fsh, and the lack of appropriate conservation equipment, large quantities of fsh are processed using traditional methods.
Among these artisanal processing methods, smoking occupies a prominent place in developing countries. Tis technique is the main and sometimes the only means of supplying populations with fsh, especially when they are far from fshing sites. Smoke not only gives fsh a special taste and aroma but also improves preservation due to its dehydrating and bactericidal properties [8]. However, smoke, in particular that of wood, is a vector of compounds called polycyclic aromatic hydrocarbons (PAHs), known for several decades for their carcinogenic power in humans [9]. Tis artisanal processing technique is generally known to generate and increase the level of PAHs in food. Moreover, many studies have shown the presence of PAHs in smoked fsh [10,11]. Today, the presence of these PAHs in food products, especially smoked fsh, is a subject of major concern.
It is in this context that this present study occurs, which had as its objective the determination of polycyclic aromatic hydrocarbons (PAHs) in four (4) species of smoked fsh from diferent areas in Senegal. Tus, the contents of benzo(a)pyrene (B(a)P), benzo(a)anthracene (B(a)A), benzo(b) fuoranthene (B(b)F), and chrysene (Chr), enacted by European Commission (EC) Regulation No. 835/2011, were estimated. A principal component analysis (PCA) was done to assess the efect of fsh species, source, and technology of processing on the quality of smoked fsh.

Biological Material.
Te biological material consists of four (4) species of smoked fsh that were chosen from the processors. Tese were Kong (Arius heudelotii), Round sardinella (Sardinella aurita), Cobo (Ethmalosa fmbriata), and Tass or Flat sardinella (Sardinella maderensis). Te choice of these four species was justifed by the fact that they are more consumed in Senegal.

Sampling and Preservation.
Smoked fsh samples were collected between February and June 2021 in seventeen (17) fsh-processing sites in Senegal. Figure 1 presents the geographical plan of the 17 smoked fsh sampling sites. Tese sites are located on the Senegalese coast near the landing centers for artisanal fshing. Tus, for each sample, the fsh were packed in bags and then stored at −4°C. Once in the laboratory, they were crushed, and then introduced into QuECherS (quick, easy, cheap, rugged, and safe) tubes and stored at −20°C in the freezer until the start of the analyses.

Chemicals.
Chemicals and solvents used are of analytical reagent grade. Te acetonitrile (99.9%) and the acetone (99.8%) were, respectively, obtained from Sigma-Aldrich and VWR Chemicals. QuEChERS extraction tubes were purchased from Termo Fisher Scientifc Inc. (USA) and consisted of 50 mL centrifuge tubes containing 4 g anhydrous magnesium sulfate and 1 g sodium acetate.

Methods for the Extraction and Analysis of PAHs in
Smoked Fish. Te QuEChERS method was used for the extraction of PAHs [12]. Te proposed method is fast and efective and can be successfully applied for PAHs determination in difcult matrices such as heat-treated food of animals [13]. For this, a test portion of 5 g of fsh fesh was ground, homogenized, and introduced into a 50 mL QuECherS tube. Ten, 20 mL of an acetonitrile-acetone mixture (60 : 40 v/v) is added thereto. Te whole was stirred vigorously at 2000 rpm using a DLAB MX-S vortex (DLAB Scientifc Inc.) for 30 seconds and then with an ultrasound bath for 5 minutes. After this step, centrifugation for 7 minutes at 3000 revolutions per minute was carried out, using a Medibas + centrifuge mod. 2741 (Auxilab). Te supernatant was transferred to a 250 mL fask and then evaporated to dryness at 35°C using a rotary evaporator. Ten, the concentrated residue was extracted with 10 mL of the acetonitrile-acetone mixture (60 : 40 v/v), and then the whole was transferred into another 50 mL QuEChERS tube and centrifuged at 2000 revolutions per minute for 1 minute. Te supernatant was recovered and placed in a 100 mL fask and then evaporated at 35°C. Te concentrated residue contained in the 100 mL fask was again recovered with 2 mL of ACN using a 1000 μL automatic pipette. Everything was transferred using a Pasteur pipette into a vial and then frozen for 24 hours to freeze the fat. Finally, the supernatant was collected using a Pasteur pipette into another vial. Te latter was placed in a gas chromatograph (GC) of the Agilent 7890A type coupled to a mass spectrometer (MS) (Agilent 5975C). Te apparatus was equipped with an automatic sampler. Separation of PAHs was conducted using a 5% phenyl-methylsilicone (HB-5MS) bondedphase fused-silica capillary column (Hewlett-Packard, 30 m * 0.25 mm I.D., and flm thickness 0.25 µm). Helium (99.999% purity) was used as the carrier gas with a fow rate of 1.2 mL/min. Te injection port was adjusted in splitless mode, and the injection volume was 1.5 µL. Te GC was programed as follows: initial temperature 70°C for 5 min and ramped at 25°C/min to 310°C for 13 min and allowed to stay for 10 min giving a total of run time of 28 min. Te mass spectrometer (MS) was operated in the electron ionization mode, with an electron energy of 70 eV. Te MS transfer line and ion source temperatures were adjusted at 310°C and 290°C, respectively.
In order to increase sensitivity, all the GC-MS measurements of the diferent samples were carried out in triplicate and the results recorded in the various tables represented the average. Tis procedure included successive steps and the determination of diferent parameters such as the linearity of the calibration interval, the detection and quantifcation limits, coefcients of variation for the repeatability, and intermediate precision tests.
Te identifcation of PAHs was verifed by comparing the retention times of each standard solution between each test. For the linearity tests, the standard stock solutions were dissolved in acetonitrile and fve working solutions (0.005, 0.1, 0.2, 0.5, and 1 ppm) were prepared to distinctly construct the calibration curves. Tese curves made it possible to establish an adequate correlation between the areas of the peaks and the concentrations of PAHs found in the samples tested. Te limits of detection (LOD) and quantifcation (LOQ) were calculated from the standard PAH solution by separate 3 × 10 analysis according to the repository (NF-V03-110 2010). Te values of these limits expressed in (µg/ kg) are given by the following formulas: where mb is the average of each marker PAHs content in the standard solution and σ is the value of standard deviation for the norm for each PAH marker. Besides, the analytical stability was assigned by the analysis of a standard solution of 1 ppm by calculating the relative standard deviation of the measurements. By repeating the analysis three times per day (intraday) for three consecutive days (interday), the precision (%) expressed as a coefcient of variation (CV) was obtained.
2.6. Statistical Analyses. All analyses were injected in triplicate to obtain a good precision. GC-MS data were acquired and analysed by Agilent Chemstation software for GC (G2070BA) and a Microsoft Excel version 2016 spreadsheet. A principal component analysis (PCA), which is the most widespread of the factorial methods [14], and a numerical classifcation were carried out on the results of analyses of

Validation of the PAHs Determination Method.
Te method validation results as the limit of detection (LOD), limit of quantifcation (LOQ), linearity, and retention times are shown in Table 1

PAH Content in Smoked Fish Samples.
Te results from the analyses are presented in Table 3. Te results showed a great variability of the contents between the various species and according to their origin. Te B(a)P molecule, representing 40% of the total carcinogenic risk attributed to PAHs [16], is present in all the samples analysed with sometimes levels that exceed the authorized limit (2 µg/kg). Tus, it appears from these analyses that the samples taken in the areas of Dionewar Te highest concentration of this molecule was observed in Djifer with a value of 33 µg/ kg. However, these values are lower than those obtained by [8] on smoked fsh species from south Nigeria, the levels of which were estimated at 204 µg/kg for Clarias gariepinus and 288 µg/kg for the species Ethmalosa fmbriata. Te mean value (8.0 µg/kg) of B(a)P observed in the samples is much higher than that found in [11] whose concentration was below the detection limit (0.01 µg/kg) but lower than that (52.7 µg/kg) obtained in [17] on smoked fsh species.
Te analysis of samples of smoked fsh from various sites also revealed the presence of chrysene at variable concentration levels between 1.3 and 142.6 µg/kg. Tese were observed in samples from Cayar and Boudody, respectively. Te average chrysene content is 35.1 µg/kg. Te latter is higher than the mean values of B(a)A and B(a)P. Compared to the results in [18], a content of 69.7 µg/kg of chrysene was obtained in a species of smoked fsh called Tunnus albacares.
Regarding B(b)F and B(a)A, the levels in the various samples analysed vary, respectively, from 1.7 to 822.3 µg/kg and from 1.2 to 121.6 µg/kg. Te average contents of B(b)F and B(a)A, in the samples, were respectively evaluated at 120.1 and 26.4 µg/kg. Much lower levels were observed in [19] on several species of smoked fsh, in the Beninese Coast. Te average concentrations of B(a)A and B(b)F in these species were estimated, respectively, at 9.99 µg/kg and 5.79 µg/kg.
Te total concentration of the four (4) molecules varies between 4.8 and 1082.3 µg/kg, respectively, observed in the samples from Cayar and Diaobé. Higher concentrations of the sum of PAHs, between 1295 and 2020 µg/kg, were obtained in [20]. Te average content of 4PAHSs in the diferent samples studied is much higher than that obtained in [11] which was estimated at 12.47 µg/kg. Te total PAH content recommended by the European Commission is 12 µg/kg. Tus, only the samples taken in the sites of Cayar It is clear that also samples from sites, such as Boudody, Dioabé, Diogue, Djifer, Sedhiou, and Cap Skiring, have the highest PAH contents. Te latter has been observed in species such as Kong (Arius heudelotii) and Cobo (Ethmalosa fmbriata).

Principal Component Analysis.
Principal component analysis (PCA) was carried out to assess the efects of smoking, the nature of the fsh species, and the place of processing on PAH levels. Te frst two dimensions (Dim 1 and Dim 2) express 98.90% of the total inertia (Table 4).
Te frst dimension (Dim 1) contributes to 81.08% and the second (Dim 2) at 17.82%. Tey thus present the highest eigenvalues, respectively, equal to 3. 24 (Figures 2 and 3). Te third class, which represents smoked fsh of the Kong species from

Discussion
Te analysis of the results of this study shows that the B(a)P content and the sum of the 4PAHS contents in diferent zones and according to the species present a signifcant diference compared to the standard. Tis trend could be attributed, on the one hand, to the diferences in fat and moisture composition of each species as well as to the nature of the skin cover [21]. Indeed, according to [20], smoked fatty fsh are more exposed to the development of PAHs than lean fsh. Fat is associated with the pyro synthesis of PAHs. It would also be linked to the traditional methods of smoking carried out in these areas [22,23]. Indeed, smoking is done exclusively by women, and it is clear that they very often use wood lint or coconut shell as their main fuels. Te pyrolysis of cellulose and lignin contained in the wood gives rise to the production of PAHs [24,25]. Furthermore, the type of wood has a signifcant infuence on the increase of PAH contents in smoked foods. Similarly, the use of cartons during the smoking process could modify the photosensitive properties of PAHs and cause their accumulation in foods [26]. In addition, this is the fact that the PAH content in smoked products varies and depends on environmental factors such as the presence or absence of light or oxygen. In addition, the long smoking times as in the species (Arius heudelotii), the temperature used and multiple smoking could lead to the high numbers obtained. Moreover, studies have shown that a smoking temperature above 450°C would allow the pyrolysis of the wood to generate more (PAH) [17,27].
PAHs would also be present in fsh products before smoking. Indeed, apart from spatiotemporal parameters, it has been revealed that the levels of PAHs would also be affected by several ecological factors. It has been demonstrated that vehicles, especially diesel engines, could also contribute signifcantly to the generation and release of PAHs into the environment [28]. In addition, studies have shown the presence of PAHs in marine sediments which could contaminate the fsh before smoking [29]. Te combined infuence of all these variability parameters associated with possible environmental contamination would certainly constitute the source of the increase in PAH levels in these smoked fsh. Tereby, the high levels of PAHs observed in certain areas of this study would probably be linked to ignorance of the existence of the production of PAHs during technological treatment and ignorance of the processes minimizing the production of PAHs. So, the adoption and use of appropriate facilities as studied in [30,31] are required to considerably reduce the PAH content in smoked fsh and to limit the contamination of fnished products.

Conclusion
Tis study consisted of highlighting the presence of polycyclic aromatic hydrocarbons (PAHs) in traditionally smoked fsh. Indeed, smoking, while helping to preserve fsh, could also induce certain carcinogenic substances such as PAHs in processed fsh. Sampling, carried out in seventeen (17) production areas in Senegal, revealed the presence of benzo(a)pyrene (B(a)P), benzo(a)anthracene (B(a)A), benzo(b)fuoranthene (B(b)F), and chrysene molecules (Chr). Te results showed that twelve (12) or 71% and fourteen (14) sites or 82% present contents higher than the European Commission standards, respectively, for B(a)P and the sum of 4PAH. Te highest PAH concentrations were observed in the Kong species (Arius heudelotii). Tese results, therefore, indicate that it is necessary to improve the smoking process in order to best limit the rate of contamination of the fnished products with PAHs and ensure consumer safety. Moreover, it would be very important to carry out an analysis to assess the possible risks to human health associated with the consumption of these smoked fsh in Senegal.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.