Load of Polycyclic Aromatic Hydrocarbons in Edible Vegetable Oils: Importance of Alkylated Derivatives

Transcription

1 1904 Journal of Food Protection, Vol. 67, No. 9, 2004, Pages Copyright, International Association for Food Protection Load of Polycyclic Aromatic Hydrocarbons in Edible Vegetable Oils: Importance of Alkylated Derivatives MARÍA D. GUILLÉN* AND PATRICIA SOPELANA Tecnología de Alimentos, Facultad de Farmacia, Universidad del País Vasco, Paseo de la Universidad, no. 7, Vitoria, Spain MS : Received 14 November 2003/Accepted 24 February 2004 ABSTRACT The presence of polycyclic aromatic hydrocarbons (PAHs) has been studied in different samples of olive oil, extra virgin olive oil, and refined seed oils. A high number of PAHs have been found, with a wide range of molecular weights and in concentrations that are high or even very high compared with the data obtained by other authors, especially in the seed oils. Among the PAHs identified, more than half are alkylated compounds, which account for the major part of the total PAH concentration in some of the samples. The total PAH concentrations in olive oils and extra virgin olive oils are similar, but the former present a higher proportion of heavy PAHs than the latter. The seed oils, in general, have much higher concentrations than the different types of olive oil and their PAH profiles are different. One of the olive oil samples exhibited a PAH distribution similar to that observed in olive pomace oil, suggesting possible adulteration. These data reveal that, in some cases, PAH profile provides useful information in relation to the possible origin of the contamination. We also observed large differences in PAH distribution between oils with the same label but from different batches. PAHs with varying degrees of carcinogenicity have been identified in all the samples, including benzo[a]pyrene, although this PAH was identified neither in the extra virgin olive oils nor in two of the seed oil samples. Polycyclic aromatic hydrocarbons (PAHs) constitute a group of contaminants that are widespread in the environment and in foods. Their presence in foods is attributed to both environmental contamination and some processes implicated in food manufacture or cooking, such as smoking or grilling (16). Although much attention has been given to smoked foods because they have been traditionally related to high concentrations of PAHs (16, 19), several studies on the presence of these contaminants in the total diet of different countries (10 12, 26, 31) have revealed that there are other groups of foods that contribute more than smoked products to the total PAHs in the diet. Dennis et al. (12) found that groups of cereals and oils or fats account for the highest dietary PAH load, followed by fruit and sugar and some vegetables, whereas the contribution of the meat group, including smoked products, is much lower. These authors also found that not only was the group of oils or fats one of the major contributors to the total dietary PAH intake but also the highest PAH concentrations arose from this group, especially for the heaviest PAHs. De Vos et al. (10), in their study of the Dutch total diet, also found that the main load of PAHs came from cereals and oils or fats. However, in another diet study performed in Italy (31), the contribution from the oils or fats group was lower than in the previous studies. Therefore, the extent of the contribution of each food group to the total of dietary PAHs may depend on the food preparation and consumption habits of each country. Taking into account both the lipophilic nature of PAHs, * Author for correspondence. Tel: ; Fax: ; knpgulod@vf.ehu.es. which makes oils and fats very prone to PAH contamination, and the importance of this group of foodstuffs in the diet, it is not surprising that there are many studies that deal with the determination of PAHs in this type of food (1, 2, 14, 18, 21, 27, 29, 40, 45, 46, 52). These studies cover a variety of animal fats and vegetable oils, such as olive, sunflower, maize, coconut, grapeseed, rapeseed, linseed, peanut, and safflower, but they usually include analysis of a limited number of PAHs and in some cases only benzo[a]pyrene is determined (42, 43, 50). The presence of PAHs in edible oils is mainly attributed to atmospheric contamination and to contamination during processing. Thus, Corradetti et al. (8) found very high levels of both total PAHs and benzo[a]pyrene in virgin Italian oils from plants exposed to industrial emissions of pitch condensate and somewhat lower levels in oils coming from plants exposed to vehicular exhausts. In relation to oil processing, drying of the plant material before oil extraction with gases that come from direct combustions can lead to high levels of PAHs in the final product, as has been shown with coconut oil (48). The solvents used for oil extraction can also be possible PAH contamination sources (29); however, according to some authors, extraction solvents do not seem to be responsible for the contamination of oils (6, 20, 21). Contamination by rediffusion from recycled polyethylene bottles used for oil packaging and contamination from mineral or lubricating oils is also a possible PAH source (36). A biosynthetic origin of PAHs in plants also cannot be excluded (39). However, PAH contamination of oils can be avoided or at least reduced to a great extent during the refining process, since the deodorization of the oils and their treatment with activated carbon can reduce the initial

2 J. Food Prot., Vol. 67, No. 9 PAHS IN VEGETABLE OILS 1905 PAH contamination to very low levels (29), even though these treatments do not affect all PAHs in the same way (3, 4, 11, 18, 40). Different methods have been described for the determination of PAHs in vegetable oils (21, 27, 35, 38, 39, 49, 51). Most of them begin with a dilution step of the sample in an apolar solvent such as n-pentane, n-hexane, or cyclohexane, followed by extraction of PAHs by liquid-liquid partition with dimethylformamide and water (9:1, vol/vol) (2, 18, 29, 37, 46) or with dimethyl sulfoxide (DMSO) (21, 25, 35); extraction of PAHs can also be performed by complexation with caffeine in an acid formic solution (27, 44). In other procedures, the oil is subjected to a saponification process to extract PAHs from the triacylglycerol matrix before the liquid-liquid partition (1, 14, 47). Whatever extraction technique is used, oil extracts need further purification to isolate PAHs; this has been commonly performed by column chromatography and/or size-exclusion chromatography. Moret et al. (37) studied several extraction and clean-up procedures, and they concluded that the extraction method using liquid-liquid partition of the oil with cyclohexane and dimethylformamide-water and subsequent clean-up by silica gel solid-phase extraction cartridges was best for the study of PAHs in oils. However, new methods, which are faster and less complex, are continuously being developed (36). Among the wide variety of vegetable oils, olive oil deserves special attention because of its healthy properties (28); however, the presence of PAHs in this type of oil is well documented (2, 21, 34, 40, 42, 49, 50). For this reason, in this study, samples of olive and extra virgin olive oils, as well as of other commonly consumed vegetable oils, have been analyzed for PAH contamination and PAH profile. The PAHs present were identified and quantified by gas chromatography mass spectrometry (GC-MS) operating in selective ion monitoring (SIM) mode. MATERIALS AND METHODS Samples. The samples studied were olive oils (blends of virgin olive oil and refined olive oil) from three different commercial brands (O1, O2, and O3), extra virgin olive oils from two brands (EV1 and EV2), two refined sunflower oils (SF1 and SF2), and one refined oil from a mixture of seeds (SM), all of them purchased from local supermarkets. In the case of oil SF1, samples from two different batches were also studied (SF1-A and SF1-B). Each sample was analyzed in duplicate. Reagents and materials. The solvents used were n-hexane for analyses, DMSO for spectroscopy, cyclohexane and methanol, high-pressure liquid chromatography grade (99.9%), and dichloromethane (99.8%). Other reagents and materials used were potassium hydroxide, sodium chloride, anhydrous sodium sulfate, and 3-ml (500-mg) Supelclean LC-Si SPE (solid-phase extraction) tubes. All solvents, reagents, and materials mentioned are commercially available from Riedel-de Haën (Seelze, Germany), Merck (Darmstadt, Germany), Aldrich (Steinheim, Germany), Panreac (Barcelona, Spain), Symta (Madrid, Spain), and Supelco (Bellefonte, Pa.). Standards. Three groups of PAH standards were used for the identification and quantification of the PAHs present in the samples: i. A commercial mix of PAHs dissolved in a mixture of dichloromethane-benzene (75:25), containing naphthalene, acenaphthene, acenaphthylene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[c]phenanthrene, benz[a]anthracene, chrysene, 7,12-dimethylbenz[a]anthracene, benzo[b]fluoranthene, benzo[j]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, 3-methylcholanthrene, indeno[1,2,3-cd]pyrene, dibenz[a,h]anthracene, benzo[ghi]perylene, dibenzo[a,l]pyrene, dibenzo[a,i]pyrene, and dibenzo[a,h]pyrene in concentrations of approximately 500 g/ml. ii. Commercial individual cyclohexane solutions of 1,7-dimethylnaphthalene, 1,4-dimethylnaphthalene, 1,5-dimethylnaphthalene, 1-methylphenanthrene, 2,3-dimethylanthracene, 9,10-dimethylphenanthrene, 2-methylfluoranthene, 1-methylfluoranthene, 11H-benzo[c]fluorene, 1-methylpyrene, 6- methylbenz[a]anthracene, 7-methylbenz[a]anthracene, 3- methylchrysene, 2-methylchrysene, 5-methylchrysene, 4- methylchrysene, 6-methylchrysene, 1-methylchrysene, dibenz[a,j]anthracene, benzo[b]chrysene, picene, anthanthrene, coronene, and dibenzo[a,e]pyrene, in concentrations of approximately 10 g/ml, and benzo[ghi]perylene-d 12 in a concentration of approximately 100 g/ml. iii. Pure standards of 2,6-dimethylnaphthalene, 2,3-dimethylnaphthalene, o-terphenyl, 2-methylanthracene, 9-methylanthracene, 3,6-dimethylphenanthrene, m-terphenyl, p-terphenyl, 11H-benzo[a]fluorene, 11H-benzo[b]fluorene, benzo[e]pyrene and perylene, and of naphthalene-d 8, acenaphthene-d 10, phenanthrene-d 10, pyrene-d 10, p-terphenyl-d 14, chrysene-d 12, and perylene-d 12. From these compounds, whose purity ranged from 97.0 to 99.5%, different solutions containing mixtures were prepared in dichloromethane or cyclohexane. All the standards, both solid and in solution, were obtained from Sigma, Aldrich, Supelco, and Symta. Methods. Analysis began with the addition of a mixture of internal standards constituted by the deuterated PAHs mentioned above. This included the dilution of the oil with n-hexane, extraction of PAHs with DMSO, back extraction of PAHs with cyclohexane, washing of the extract, drying on anhydrous sodium sulfate, clean-up by solid-phase extraction tubes following the procedure described in a previous paper (17), and determination of PAHs by GC-MS operating in SIM mode. Samples O2, O3, EV1, SF1-B, SF2, and SM were also subjected to an alkaline treatment, since their analyses by GC-MS in scan mode showed that they contained fatty acids, which interfere with the determination of PAHs and are detrimental to the equipment. For this purpose, approximately 11.2 g of potassium hydroxide dissolved in 100 ml of a mixture of methanol and distilled water (9:1, vol/ vol) and boiling chips were added to the previous samples and the whole mixture was refluxed for 4 h. The resulting mixture was diluted with 100 ml of methanol-water (8:2, vol/vol), and PAHs were newly extracted with cyclohexane. This extract was again dried on anhydrous sodium sulfate, concentrated to 1 ml, and analyzed by GC-MS in SIM mode. All glassware was cleaned before use with dichloromethane in an ultrasonic bath several times, and the washing solvent was concentrated and analyzed by GC-MS in SIM mode to check the absence of residual contamination. The purity of the solvents was also checked to avoid the incorporation of impurities or even of additional PAHs to the sample subjects of study. A Hewlett-Packard gas chromatograph model HP 6890 series, equipped with a Mass Selective Detector 5973 and a Hewlett-

3 1906 GUILLÉN AND SOPELANA J. Food Prot., Vol. 67, No. 9 Packard Vectra XM Series 4 computer, was used for identification and quantification of PAHs. The column used was a fused-silica capillary column (60-m long by 0.25-mm inner diameter by m film thickness) coated with a nonpolar stationary phase (HP- 5MS, 5% phenyl methyl Siloxane). Operating conditions were as follows: the oven temperature was set initially at 50 C (0.50-min hold), increased to 130 C at 8 C/min, and again increased to 290 C at a rate of 5 C/min (50-min hold); the temperatures of the ion source and the quadrupole mass analyzer were kept at 230 and 150 C, respectively; helium with a purity of % was used as carrier gas at a constant flow of 1.0 ml/min; injector and transference line temperatures were held at 290 and 300 C, respectively; pulsed splitless mode was used for injection with a pressure pulse of 30 psi; and 1 l of each sample was introduced in the gas chromatograph. The data acquisition modes used were scan and SIM. Scan mode was used to know the type of compounds present in the samples, whereas SIM was used to identify and quantify the PAHs present. Quantification was performed by using the deuterated internal standards previously mentioned. Thus, naphthalene-d 8 was used for quantification of naphthalene and its methyl derivatives, acenaphthene-d 10 for acenaphthylene and acenaphthene, phenanthrene-d 10 for phenanthrene, anthracene, and their methyl derivatives, pyrene-d 10 for fluoranthene and pyrene, p-terphenyl-d 14 for m-terphenyl, p-terphenyl, benzofluorenes, methyl derivatives of fluoranthene and pyrene, chrysene-d 12 for benz[a]anthracene, chrysene, and their methyl derivatives, perylene-d 12 for benzofluoranthenes and benzopyrenes, and, lastly, benzo[ghi]perylene-d 12 for PAHs with higher molecular weights. The response factors of each compound relative to the internal standard chosen for its quantification were calculated for each sample. RESULTS AND DISCUSSION Sample composition influences the extraction procedure. We observed that the separation of the phases in the liquid-liquid partition of extra virgin olive oils with DMSO is less clear and slower than for other samples. This is perhaps because extra virgin olive oil is relatively unprocessed and consequently contains more compounds that are not present in refined oils. It is therefore sometimes necessary to introduce extraction modifications for individual samples. The recoveries of the deuterated internal standards added varied from 84.76% for phenanthrene-d 10 to 94.13% for p-terphenyl-d 14 in the samples without alkaline treatment, whereas in those subjected to alkaline treatment they ranged from 74.88% for phenanthrene-d 10 to 83.58% for benzo[ghi]perylene-d 12. The recoveries of naphthalene-d 8 and acenaphthene-d 10 were lower than those of the other internal standards, both in the samples without alkaline treatment (54.39 and 66.10%, respectively) and in the samples subjected to alkaline treatment (38.74 and 51.51%, respectively). A lower recovery of naphthalene (37.6%) was also observed by Moret and Conte in spiked peanut oil (38). The PAHs identified in the samples studied and their concentrations are given in Tables 1 and 2. Table 1 gives the results corresponding to the samples of olive and extra virgin olive oils, and Table 2 gives the results from the seed oils. These results come from duplicate analyses of each sample, and they are expressed as the mean standard deviation. Both tables also show the total PAH concentration of each sample, as well as the total concentrations of parent PAHs and alkyl derivatives separately. A high number of PAHs were identified (33 53), even though this is slightly higher in the olive and extra virgin olive oils (47 53) than in the seed oils (33 44). Compounds with a wide range of molecular weights and with different numbers of aromatic rings were found, such as naphthalene (two rings), phenanthrene (three rings), pyrene and benz[a]anthracene (four rings), benzofluoranthenes (five rings), or indeno[1,2,3-cd]pyrene (six rings). The lack of methyl-fluoranthenes or methyl-pyrenes and benzofluorenes in sample SF1-A could be attributed to difficulties in their identification due to interfering compounds more than to an absence of these PAHs. The presence of a high number (35) of alkylated derivatives should be noted from both lowmolecular-weight PAHs (naphthalene, phenanthrene) and higher-molecular-weight ones (benz[a]anthracene, chrysene). As far as we know, there are no studies of PAHs in vegetable oils where such a high number of compounds have been identified, and, moreover, most of the authors do not refer to alkylated PAHs, even though some of them should be taken into account due to their carcinogenicity, such as some methyl derivatives of benz[a]anthracene, chrysene, and benzo[a]pyrene (5, 32). In relation to the concentrations of the PAHs identified, the seed oil samples (Table 2) have the highest total PAH levels, which range from g/kg in sample SF1-B to 1, g/kg in SF1-A. These values, in general, are far higher than those of olive and extra virgin olive oils, despite the higher PAH number in the latter, as mentioned earlier. These results differ from other studies in which it was observed that the PAH content of refined oils is generally lower than that of crude oils, since the refining process reduces the initial amount of PAHs (6, 11, 18, 29). However, the extent of this reduction is a function of the type of refining operations; thus, deodorization reduces mainly the concentration of light PAHs (6, 11, 18, 29), whereas treatment with activated carbon has an effect on heavy PAHs (3, 4, 29, 30). There is also a significant difference between the total PAH contents of the two batches of refined sunflower oil SF1 (Table 2, SF1-A and SF1-B). With regard to the olive and extra virgin olive oil samples (Table 1), their total PAH concentrations, much lower than in the seed oils, are of a similar order, ranging from g/ kg in sample O3 to g/kg in sample O1. Nevertheless, although the total PAH concentrations in samples O1 and O2 are almost the same ( and g/kg, respectively), the levels of heavy PAHs in sample O2 are far higher than those in sample O1. PAH concentrations did not follow the same pattern in all the samples studied (Tables 1 and 2). Thus, in the seed oil samples (Table 2) the highest concentrations correspond to the lightest PAHs, naphthalene and its alkyl derivatives, and, as the molecular weight of the PAHs increases, in general, the concentrations decrease very sharply. However, in most of the olive and extra virgin olive oil samples (Table 1), in general, the most abundant PAHs are naphthalene, phenanthrene, and their alkyl derivatives and, from phenanthrene onward, PAH concentrations decrease. This is in

4 J. Food Prot., Vol. 67, No. 9 PAHS IN VEGETABLE OILS 1907 agreement with other studies in which it was also observed that phenanthrene was the most abundant PAH and that PAH concentrations went down with the molecular weight (2, 34, 40, 46, 49). Sample O2 is an exception, since the most abundant PAHs are chrysene plus triphenylene and some of their alkyl derivatives, followed by fluoranthene and pyrene. With regard to alkylated PAHs, except for sample O2, their total concentrations are higher than those of parent PAHs to such an extent that in samples O1, EV1, EV2, SF1-B, and SF2 alkylated PAHs account for the major part of the total PAH concentration. In general, the total of both monomethyl and dimethyl derivatives of naphthalene, phenanthrene, and benz[a]anthracene or chrysene are sometimes of the same order and, in most cases, far higher than the concentrations of their respective parent PAHs. Table 3 shows the proportions of the main groups of PAHs identified in the samples, together with the proportions of light and heavy PAHs. In the seed oil samples, in general, the main PAH contribution comes from naphthalene and its alkyl derivatives (68.65 to 83.15%), followed by fluorene and phenanthrene plus anthracene plus alkyl derivatives, whereas the rest of the PAHs account for very small percentages. In the olive and extra virgin olive oils, in general, the highest proportion also corresponds to naphthalene and its alkyl derivatives, but their contribution is lower than in the case of the seed oil samples, ranging from 40.48% in sample O3 to 57.87% in sample EV1. The group of phenanthrene plus anthracene plus alkyl derivatives is the second group, and its proportions are higher than in the seed oils (30.22 to 39.65%); the remaining PAHs contribute only a small proportion to the total. Sample O2 is an exceptional case, having a PAH distribution that is substantially different from the others, since the major PAH contribution for this sample corresponded to the group of benz[a]anthracene plus isomers plus alkyl derivatives, followed by fluoranthene plus pyrene plus alkyl derivatives and phenanthrene plus anthracene plus alkyl derivatives. This difference in the proportions of PAHs could be due to a different contamination source in sample O2. A study on the PAH content of olive pomace oils (unpublished data) revealed that their PAH profile was very similar to that of sample O2. The adulteration of extra virgin olive oil with olive pomace oil has been noted by some authors (33). In relation to the proportions of light and heavy PAHs observed in the different samples, the percentages observed in samples O1 and O3 (97.88 and 96.25%, respectively) match well with those obtained by other authors in olive oils (1, 2, 18, 34, 49). In general, the proportions of both groups of PAHs in olive and extra virgin olive oils are very similar (Table 3), unlike the findings of Moret et al. (40), who observed a lower proportion of light PAHs in olive oils (approximately 34 to 87%) than in virgin olive oils (93 to 100%, except for one sample, which was 73%). In relation to the seed oils, the proportions of light PAHs are also very high (98.92 to 99.93%), whereas in other studies these are variable, ranging from 59 to almost 100% in sunflower refined oils (1, 18, 27, 47, 52) and from 12 to 99% in vegetable oils from different origins and with different degrees of contamination (1, 11, 18, 27, 29, 52). Given that other authors do not usually include alkylated PAHs in their studies, only the total parent PAH concentrations will be used for comparisons with other studies. In relation to extra virgin olive oils (EV1 and EV2), their total PAH concentrations are in the range of the concentrations found by other authors (14, 34, 38, 46), who reported total PAH contents varying between 48 and 80 g/kg, even though Moret et al. (40) found a wider range from numerous samples of extra virgin olive oils from different origins (2.946 to g/kg). Although different authors have reported benzo[a]pyrene levels in virgin olive oils ranging from 0.1 to g/kg (14, 38, 40, 46, 50), this carcinogenic PAH has not been identified in the extra virgin olive oil samples of this study. On the other hand, Menichini et al. (34) found neither benzo[a]pyrene nor any heavy PAHs in seven samples of virgin olive oil. The results we obtained for olive oils agree with the values of some authors (1, 49, 51), but they are higher than the concentrations reported by others (2, 18, 25, 31, 34, 40, 47, 52), who found low levels of PAHs in olive oil samples ranging from 1.0 to 45 g/kg. Menichini et al. (34) did not find heavy PAHs in olive oils. With regard to benzo[a]pyrene, which has been identified in the three olive oils studied, the concentrations in samples O1 and O3 (0.31 and 0.39 g/kg, respectively) are in the range of the values found by other authors in olive oils (1, 2, 31, 49, 50); however, the level of benzo[a]pyrene in sample O2 (5.38 g/ kg) is much higher than the levels usually reported for this type of oil, except for Vreuls et al. (51), who found 19 g/ kg in one sample, and Pupin and Toledo (42), who found benzo[a]pyrene levels ranging from 0.6 to 10.3 g/kg in olive oils from different origins. If we compare the PAH levels in olive and extra virgin olive oils, although the total concentrations are of the same order, the individual amounts from fluoranthene and pyrene onward are higher in olive oils than in virgin olive oils. These results differ from those of other authors (34, 40), who found that virgin olive oils contained higher total PAH concentrations than olive oils, which was explained by a reduction of the amounts of light PAHs in olive oils; however, Moret et al. (40) also observed an increase in the concentrations of some heavy PAHs in olive oils in relation to virgin olive oils. In contrast, Ciusa et al. (7) found higher PAH amounts in refined olive oils than in pressed. In relation to the seed oil samples, the total of parent PAHs in refined sunflower oils SF1-A ( g/kg) and SF2 ( g/kg) are very high compared with the values obtained by other authors in refined sunflower oils (1, 18, 27, 47, 52) and even in native or cold pressed oils (14, 18). The parent PAH content of sample SF1-B, much lower than in the other two sunflower oils of this study, is in the range of the concentrations found by some authors (1, 27, 51), but it is still higher than the levels reported by others (18, 47) in both refined and native sunflower oils. The total parent PAH concentration of sample SM ( g/kg) is also higher than the concentrations found in different vegetable oils (1, 14, 18, 27, 29, 52), except for peanut oil (27) and some samples of grapeseed oil (1). The concentration of benzo[a]pyrene in sample SF1-A (2.13 g/kg) is slightly

5 1908 GUILLÉN AND SOPELANA J. Food Prot., Vol. 67, No. 9 TABLE 1. PAHs identified in the olive oil and extra virgin olive oil samples studied and their concentrations Concentrations ( g/kg) a O1 O2 O3 EV1 EV2 Naphthalene Methylnaphthalenes 2-Methylnaphthalene 1-Methylnaphthalene Dimethylnaphthalenes 2,6-Dimethylnaphthalene 1,7-Dimethylnaphthalene 1,6-Dimethylnaphthalene 1,4-2,3-Dimethylnaphthalene 1,5-Dimethylnaphthalene Dimethyl- or ethyl-napthalene Acenaphthylene Acenaphthene Fluorene Phenanthrene Anthracene Methyl-phenanthrenes and methyl-anthracenes 3-Methylphenanthrene 2-Methylphenanthrene 2-Methylanthracene 9-Methylphenanthrene 1-Methylphenanthrene Dimethyl-phenanthrenes and dimethyl-anthracenes Dimethylphenanthrene or isomer (1) Dimethylphenanthrene or isomer (2) Dimethylphenanthrene or isomer (3) Dimethylphenanthrene or isomer (5) Dimethylphenanthrene or isomer (6) Dimethylphenanthrene or isomer (7) Dimethylphenanthrene or isomer (8) Dimethylphenanthrene or isomer (9) Dimethylphenanthrene or isomer (10) o-terphenyl Fluoranthene Pyrene Methyl-fluoranthenes and methyl-pyrenes 2-Methylfluoranthene Methylfluoranthene or isomer (1) Methylfluoranthene or isomer (2) Methylfluoranthene or isomer (4) Methylfluoranthene or isomer (5) 1-Methylpyrene 1-MFt 11H-B[a]Fl d 11H-Benzo[b]fluorene 11H-Benzo[c]fluorene m-terphenyl p-terphenyl Benz[a]anthracene Chrysene and triphenylene Methyl-benz[a]anthracenes and methyl-chrysenes Methylbenz[a]anthracene or isomer (2) Methylbenz[a]anthracene or isomer (3) 3-Methylchrysene 2-Methylchrysene 4- or 6-Methylchrysene 1-Methylchrysene Dimethylbenz[a]anthracene or isomer (3) Benzo[b]fluoranthene Benzo[j k]fluoranthenes b c

6 J. Food Prot., Vol. 67, No. 9 PAHS IN VEGETABLE OILS 1909 TABLE 1. Continued Concentrations ( g/kg) a O1 O2 O3 EV1 EV2 Benzo[a]fluoranthene Benzo[e]pyrene Benzo[a]pyrene 276 e Indeno[1,2,3-cd]pyrene Dibenz[a,h or a,c]anthracene Benzo[b]chrysene Picene Benzo[ghi]perylene Anthanthrene Total Parent PAHs Alkyl derivatives a Values are means standard deviations. b Not identified. c Identified only in one of the aliquots. d Possibly 1-methylfluoranthene plus 11H-benzo[a]fluorene. e Isomer of indeno[1,2,3-cd]pyrene and benzo[ghi]perylene. higher than the concentrations found by other authors in refined sunflower oil (1, 18, 27, 43), which vary between not detected and 1.59 g/kg, except for Vreuls et al. (51), who found 40 g/kg in one sample of sunflower oil. Finally, the benzo[a]pyrene level of sample SM (0.68 g/kg) is in the range of the values found by other authors in different vegetable oils, even though the range is very wide, depending on the origin and on the processes implicated in their production (1, 14, 18, 27, 29, 43), reaching benzo[a]pyrene concentrations of even g/kg in a sample of peanut oil (27). Among the PAHs identified in the samples of this study, several are considered carcinogenic by different authors (5, 23, 24, 32), such as benz[a]anthracene, benzo- [b]fluoranthene, benzo[j]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, indeno[1,2,3-cd]pyrene, and dibenzo[a,e]pyrene. In relation to the alkyl derivatives of benz[a]anthracene and chrysene identified in these samples, the carcinogenic 6-methylbenz[a]anthracene, 7-methylbenz[a]anthracene, and 5-methylchrysene (5, 32) were not found. However, the possible dimethylbenz[a]anthracene or isomer identified in sample O2 is not the potent carcinogen 7,12-dimethylbenz[a]anthracene, but it could coincide with another carcinogenic isomer (5, 32). None of the dibenzopyrenes identified apart from dibenzo[a,e]pyrene coincides with dibenzo[a,h]pyrene, dibenzo[a,i]pyrene, or dibenzo[a,l]pyrene, which are also carcinogenic (5, 23, 24, 32). As in most foodstuffs, there are no legal limits for PAHs in vegetable oils in the European Union. However, in Spain, a legal disposition was put into effect in 2001 (41) for olive pomace oils, but it was not extended to other types of edible oils. Taking as reference the legislation for olive pomace oil, which permits a maximum of 2 g/kg for individual concentrations of benz[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[e]pyrene, benzo[a]pyrene, dibenz[a,h]anthracene, indeno[1,2,3- cd]pyrene, and benzo[ghi]perylene, and a maximum of 5 g/kg for the sum of all, the concentrations of many of the PAHs mentioned in the disposition exceed the limits in two of the samples of this study, O2 and SF1-A. The limits set for PAHs in olive pomace oil are far higher than that established for benzo[a]pyrene in foods smoked with smoke flavorings (0.03 g/kg) (9), even though vegetable oils contribute to the dietary PAH intake in a much higher proportion than smoked foods (10 12). In conclusion, a high number of PAHs were found in the vegetable oil samples in concentrations that are high relative to those found by previous studies, especially in the seed oils. Among the PAHs identified, more than half were alkylated compounds, which account for the major part of the total PAH concentration in some of the samples. Moreover, the presence of alkyl derivatives should not be overlooked, since many of the most carcinogenic PAHs belong to this group (5, 32), even though they have not been identified. The PAH content of these samples could be attributed to both contamination of the raw material and contamination arising from the oil production process itself. The high proportion of light PAHs in all the samples of this study, either virgin or refined, seems to be in disagreement with the results of some authors (6, 11, 18, 19), who have found that the refining process causes a reduction of the concentrations of light PAHs. Therefore, either the refining process has been deficient or a posterior contamination of the oils has taken place. However, the PAH proportions observed in sample O2, which are very similar to those observed in olive pomace oil, suggest the possibility of adulteration with the latter. Therefore, the PAH profile of the different samples could be an indicator of the PAH contamination source. There are also large differences between oils with the same label but from different batches, revealing the need for more control of the manufacturing process to ob-

7 1910 GUILLÉN AND SOPELANA J. Food Prot., Vol. 67, No. 9 TABLE 2. PAHs identified in the refined seed oil samples studied and their concentrations Concentrations ( g/kg) a SF1-A SF1-B SF2 SM Naphthalene Methylnaphthalenes 2-Methylnaphthalene 1-Methylnaphthalene Dimethylnaphthalenes 2,6-Dimethylnaphthalene 1,7-Dimethylnaphthalene 1,6-Dimethylnaphthalene 1,4-2,3-Dimethylnaphthalene 1,5-Dimethylnaphthalene Dimethyl- or ethyl-naphthalene Acenaphthylene Acenaphthene Fluorene Phenanthrene Anthracene Methyl-phenanthrenes and methyl-anthracenes 3-Methylphenanthrene 2-Methylphenanthrene 9-Methylphenanthrene 1-Methylphenanthrene Dimethyl-phenanthrenes and dimethyl-anthracenes Dimethylphenanthrene or isomer (1) Dimethylphenanthrene or isomer (2) Dimethylphenanthrene or isomer (3) Dimethylphenanthrene or isomer (5) Dimethylphenanthrene or isomer (6) Dimethylphenanthrene or isomer (7) Dimethylphenanthrene or isomer (8) Dimethylphenanthrene or isomer (9) Dimethylphenanthrene or isomer (10) o-terphenyl Fluoranthene Pyrene Methyl-fluoranthenes and methyl-pyrenes 2-Methylfluoranthene Methylfluoranthene or isomer (1) Methylfluoranthene or isomer (2) Methylfluoranthene or isomer (4) Methylfluoranthene or isomer (5) 1-Methylpyrene 1-MFt 11H-B[a]Fl d 11H-Benzo[b]fluorene 11H-Benzo[c]fluorene m-terphenyl p-terphenyl Benz[a]anthracene Chrysene and triphenylene Methyl-benz[a]anthracenes and methyl-chrysenes Methylbenz[a]anthracene or isomer (2) Methylbenz[a]anthracene or isomer (3) 3-Methylchrysene 2-Methylchrysene Benzo[b]fluoranthene Benzo[j k]fluoranthenes Benzo[e]pyrene Benzo[a]pyrene Indeno[1,2,3-cd]pyrene Picene b c c e e e c c c 0.14 c c

8 J. Food Prot., Vol. 67, No. 9 PAHS IN VEGETABLE OILS 1911 TABLE 2. Continued Concentrations ( g/kg) a SF1-A SF1-B SF2 SM Benzo[ghi]perylene Anthanthrene Dibenzopyrene or isomer (1) Dibenzo[a,e]pyrene Dibenzopyrene or isomer (2) Total Parent PAHs Alkyl derivatives , a Values are means standard deviations. b Not identified. c Identified only in one of the aliquots. d Possibly 1-methylfluoranthene plus 11H-benzo[a]fluorene. e Probably these compounds, but it cannot be certain due to the presence of interferences. tain products with the same characteristics and as free of PAHs as possible. The results from this study reveal that the contamination of vegetable oils with PAHs must be better controlled by either avoiding the contamination before and during processing of oils or applying treatments to remove or at least reduce the PAHs present, such as adsorption on activated carbon. Although some carcinogenic PAHs have been identified in the samples of this study, some considerations must be made. First, the carcinogenesis induction mechanism by PAHs, which need metabolic activation in the organism to TABLE 3. Proportions of the main groups of PAHs identified in the vegetable oil samples studied Proportion (% of total) O1 O2 O3 EV1 EV2 SF1-A SF1-B SF2 SM Naphthalene plus alkyl derivatives Naphthalene Methylnaphthalenes Dimethylnaphthalenes Acenaphthylene plus acenaphthene Fluorene Phenanthrene plus anthracene plus alkyl derivatives Phenanthrene plus anthracene Methyl-phenanthrenes and methyl-anthracenes Dimethyl-phenanthrenes and dimethyl-anthracenes Fluoranthene plus pyrene plus alkyl derivatives Fluoranthene plus pyrene Methyl-fluoranthenes and methyl-pyrenes Benzofluorenes Benz[a]anthracene plus isomers plus alkyl derivatives Benz[a]anthracene plus chrysene plus triphenylene Methyl-benz[a]anthracenes and methyl-chrysenes Dimethylbenz[a]anthracene or isomer Benzofluoranthenes plus isomers Benzofluoranthenes Benzopyrenes plus perylene PAHs with five or more aromatic rings Benzo[ghi]perylene plus isomers Dibenzanthracene plus isomers Dibenzopyrenes Light PAHs b Heavy PAHs c a a Compounds included in this group have not been identified. b Sum of PAHs with two, three, and four aromatic rings, except for alkyl-derivatives of benz[a]anthracene and isomers. c Sum of PAHs with more than four aromatic rings and alkyl derivatives of benz[a]anthracene and isomers.

9 1912 GUILLÉN AND SOPELANA J. Food Prot., Vol. 67, No. 9 exert a carcinogenic effect, is very complex and is affected by many factors, both external and endogenous, that determine the formation of the carcinogenic metabolites and, in consequence, their final effect (15). Second, there are some dietary factors that can protect against the induction of carcinogenesis by PAHs by either hindering or avoiding the absorption of these compounds or inhibiting certain reactions necessary for the metabolic activation of PAHs (15). Finally, all vegetable oils, but especially olive oils and most of all extra virgin olive oils (13), have natural antioxidants, which are compounds that can inhibit the toxic effect of PAHs (22). Nevertheless, despite the presence of some protective compounds in vegetable oils, the presence of carcinogenic PAHs should not be overlooked and, in consequence, measures should be taken to avoid the contamination not only of edible oils but also of any foodstuff with PAHs. 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10 J. Food Prot., Vol. 67, No. 9 PAHS IN VEGETABLE OILS Marcos Lorenzo, I., J. L. Pérez Pavón, M. E. Fernández Laespada, C. García Pinto, and B. Moreno Cordero. Detection of adulterants in olive oil by headspace-mass spectrometry. J. Chromatogr. A 945: Menichini, E., A. Bocca, F. Merli, D. Ianni, and F. Monfredini Polycyclic aromatic hydrocarbons in olive oils on the Italian market. Food Addit. Contam. 8: Menichini, E., A. Di Domenico, and L. Bonanni Reliability assessment of a gas chromatographic method for polycyclic aromatic hydrocarbons in olive oil. J. Chromatogr. A 555: Moreda, W., M. C. Pérez-Camino, and A. Cert Gas and liquid chromatography of hydrocarbons in edible vegetable oils. J. Chromatogr. A 936: Moret, S., R. Bortolomeazzi, S. Rebecca, and L. S. Conte HPLC evaluation of polycyclic aromatic hydrocarbons in olive oils: a comparison of some extraction and clean up methods. Riv. Ital. Sost. Grasse 73: Moret, S., and L. S. Conte Off-line LC-LC determination of PAHs in edible oils and lipidic extracts. J. High Resol. Chromatogr. 21: Moret, S., and L. S. Conte Polycyclic aromatic hydrocarbons in edible fats and oils: occurrence and analytical methods. J. Chromatogr. A 882: Moret, S., B. Piani, R. Bortolomeazzi, and L. S. Conte HPLC determination of polycyclic aromatic hydrocarbons in olive oils. Z. Lebensm.-Unters.-Forsch. A 205: Orden de 25 de julio de 2001, por la que se establecen límites de determinados hidrocarburos aromáticos policíclicos en aceite de orujo de oliva (Boletín Oficial del Estado, 178, 26 July 2001). 42. Pupin, A. M., and M. C. F. Toledo Benzo(a)pyrene in olive oils on the Brazilian market. Food Chem. 55: Pupin, A. M., and M. C. F. Toledo Benzo(a)pyrene in Brazilian vegetable oils. Food Addit. Contam. 13: Sagredos, A. N., and D. Sinha-Roy A method for rapid determination of polycyclic aromatic hydrocarbons in fats and oils via coffein complexes. Deut. Lebensm. Rundsch. 75: Speer, K., and A. Montag Polycyclic aromatic hydrocarbons in native vegetable oils. Fat Sci. Technol. 90: Speer, K., E. Steeg, P. Horstman, Th. Kühn, and A. Montag Determination and distribution of polycyclic aromatic hydrocarbons in native vegetable oils, smoked fish products, mussels and oysters, and bream from the river Elbe. J. High Resol. Chromatogr. 13: Stijve, T., and C. Hischenhuber Simplified determination of benzo(a)pyrene and other polycyclic aromatic hydrocarbons in various food materials by HPLC and TLC. Deut. Lebensm. Rundsch. 83: Swetman, T., S. Head, and D. Evans Contamination of coconut oil by PAH. Inform 10: Van Stijn, F., M. A. T. Kerkhoff, and B. G. M. Vandeginste Determination of polycyclic aromatic hydrocarbons in edible oils and fats by on-line donor-acceptor complex chromatography and high-performance liquid chromatography with fluorescence detection. J. Chromatogr. A 750: Vazquez Troche, S., M. S. García Falcón, S. González Amigo, M. A. Lage Yusty, and J. Simal Lozano Enrichment of benzo(a)pyrene in vegetable oils and determination by HPLC-FL. Talanta 51: Vreuls, J. J., G. J. de Jong, and U. A. Th. Brinkman On-line coupling of liquid chromatography, capillary gas chromatography and mass spectrometry for the determination and identification of polycyclic aromatic hydrocarbons in vegetable oils. Chromatographia 31: Welling, P., and B. Kaandorp Determination of polycyclic aromatic hydrocarbons (PAH) in edible vegetable oils by liquid chromatography and programmed fluorescence detection. Z.-Lebensm.- Unters.-Forsch. 183: