PAHs concentration in heat-treated milk samples

Food Research International 44 (2011) 716–724

Contents lists available at ScienceDirect

Food Research International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f o o d r e s

PAHs concentration in heat-treated milk samples Clara Naccari a,⁎, Mariateresa Cristani a, Francesco Giofrè b, Maria Ferrante c, Laura Siracusa d, Domenico Trombetta a a

Department Farmaco-Biologico, University of Messina, Polo Universitario SS. Annunziata 98168, Messina, Italy ASP 8 Vibo Valentia, Italy Department of Pathology and Animal Health, University of Naples Federico II, Via Delpino 1, 80137, Naples, Italy d Istituto del CNR di Chimica Biomolecolare, Via Paolo Gaifami 18, 95126, Catania, Italy b c

a r t i c l e

i n f o

Article history: Received 20 October 2010 Accepted 16 December 2010 Keywords: Polycyclic aromatic hydrocarbons HPLC Heat-treatment Milk

a b s t r a c t In this study the presence of residual levels of Polycyclic Aromatic Hydrocarbons (PAHs) in milk samples from Calabria was evaluated. A comparative analysis of PAHs concentrations was conducted on raw, pasteurized, UHT semi-skimmed and whole milk. Quantitative determination of PAHs was performed by HPLC using a fluorescence detector and analysis in HPLC–MS was conducted to confirm the presence of these compounds. Residual levels of PAHs were found in all milk samples analyzed, showing higher concentrations in pasteurized and UHT milk than in raw milk samples. The results obtained demonstrate that PAHs presence also in raw milk is dependent from environmental pollution but pasteurization and UHT treatments of milk can influence PAHs formation; the differences found between whole and semi-skimmed samples can be due to different fat content of milk. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are compounds with aromatic structure, considered as ubiquitous environmental contaminants, which derive from the incomplete combustion or pyrolysis of organic materials (Simoneit, 2002), usually found as a mixture containing two or more of these compounds. They are produced by natural combustion (volcanic eruptions and forest fires) and anthropic activities (industries, waste incineration, road traffic and heating systems). PAHs belong to the group of Persistent Organic Pollutants (POPs) because they possess toxic characteristics, are low biodegradable, can bio-accumulate, are prone to long-range atmospheric transport and deposition and are likely to cause significant adverse human health or environmental effects near to and distant from their source (Protocol on Persistant Organic Pollutants, 1999). PAHs are considered carcinogens because the exposition by food ingestion and inhalation is associated with human cancer. The US Environmental Protection Agency (EPA, 2006) has established a list of priority pollutants including 16 PAHs: Naphtalene [NAP], Acenaphtlylene [ACEN], Acenaphthene [ACE], Fluorene [FLU], Phenanthrene [PHEN], Antrhacene [ANT], Fluoranthene [FLR], Pyrene [PYR] Benzo(a) anthracene [B(a)A] Crysene [CHY], Benzo(b)fluoranthene [B(b)F], Benzo(k)fluoranthene [B(k)F], Benzo(a)pyrene [B(a)P], Dibenzo(a,h) ⁎ Corresponding author. Department Farmaco-Biologico, Faculty of Pharmacy, University of Messina, Contrada Annunziata, I-98168 Messina, Italy. E-mail address: [email protected] (C. Naccari). 0963-9969/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2010.12.029

anthracene [DB(a,h)A], Benzo(g,h,i)pyrene [B(g,h,i)P] and Indeno (1,2,3-cd)pyrene [I(c,d)P]. In particular, Benzo(a)pyrene is recently included in group 1A of carcenogen to humans (Kishikawa, Wada, Kuroda, Akiyama, & Nakashima, 2008). PAHs occurred at low level of environmental contamination (water, air or soil) and the human exposure to these compounds occurs mainly by inhalation of airborne particulates and intake of food contaminated (Kishikawa et al., 2003). The occurrence of PAHs in foods is due both to deposition from the air to soil and surface of plants (Killian, Smith, & Jones, 2000; Smith, Thomas, & Jones, 2001) and to pollution resulting from manufacture and alimentary processes such as drying, boiling, cooking, grilling, roasting, toasting and smoking (Codex Alimentarius Commission, 2005; European Commission, 2002; Naccari, Cristani, Giofrè, Licata, & Trombetta, 2008; Perelló, Martí-Cid, Castell, Llobet, & Domingo, 2009; Rey-Salgueiro, Martínez-Carballo, García-Falcón, González-Barreiro, & Simal-Gándara, 2009). The suggested mechanism of PAHs formation in food during alimentary processes (drying, boiling, cooking, grilling, roasting, toasting and smoking) (Codex Alimentarius Commission, 2005; European Commission, 2002; Rey-Salgueiro et al., 2009) is that the combustion of organic matter at high temperatures produces smaller unstable fragments, mostly free radicals, which recombine to form, at first, low molecular mass PAHs (two- or three rings) and then high molecular mass PAHs (four- or six rings) (Simoneit, 2002). Due to their physical and chemical properties, they have a high solubility in lipids, can migrate through the food chain into hydrophobic compartments and be retained by food rich in fats (Feidt, Grova,

C. Naccari et al. / Food Research International 44 (2011) 716–724

Laurent, Rychen, & Laurent, 2000; Moret & Conte, 2000) and the knowledge of transfer pathways through the food chain is a major issue in food safety. PAHs are excreted with urine and feces as hydroxylated metabolites or transferred to milk owing to their lipophilicity (Kishikawa & Kuroda, 2009). Milk is consumed in human diet in its original form and as various dairy products. Several types of milk are known different for the way of production and their fat content (Fidler, Sauerwald, Demmelmair, & Koletzko, 2001). Raw milk is a milk with nothing added or removed, collected from the dairy herd, undergoes various processing techniques before consumption and represents less than 1% of the household milk market. Pasteurized milk is the most consumed milk in Europe, produced with a mild heat treatment to a temperature of no less than 71.7 °C for a minimum of 15 s (max 25 s) according to a process of High Temperature Short Time (HTST), to kill harmful bacteria without significantly affecting the nutritional value of the milk. UHT or ultraheat treated milk is heated to a temperature of at least 135 °C in order to kill off any harmful micro-organisms present in the milk and it has a longer shelf life as a result of the higher temperatures of heating. UHT milk is available in whole, semi skimmed and skimmed varieties: among these semi skimmed milk is the most popular type of milk for a fat content of 1.7%, compared to 4% in whole milk and 0.3% in skimmed milk. PAHs contamination of milk depends on environmental factors related to the rearing system (fodder and potentially contaminated soil, stage of lactation and medical state of the herd) and on lactating ruminant's source of exposure: ingestion during grazing, inhalation of contaminated air or absorption by dermal contact (Fries, 1995). As an excretion of mammary gland, milk can carry various xenobiotics (pesticides, drugs, metals, PAHs and PCBs) (Schaum et al., 2003) which constitute a technological risk factor in dairy products for the health of the consumer (Naccari et al., 2008). Milk can be considerate an interesting biomarker for environmental pollution (Chahin et al., 2008), in fact, the presence of xenobiotics residual levels is considerate a direct indicator of milk and dairy products quality and an indirect indicator of pollution of the environment where the milk is produced (Naccari et al., 2006). Regulation (EC) No 208/2005 of 4 February 2005, amending Regulation (EC) No 466/2001 as regards polycyclic aromatic hydrocarbons, sets maximum levels for certain contaminants in foodstuffs. Regulation (EC) No 1881/2006 of 19 December 2006 confirms that Benzo(a)pyrene can be used as a marker for PAHs contamination and effect of carcinogenic PAH in food and fixes a maximum level of 2 μg/kg wet weight of B(a)P in certain foods containing fats and oils and a lower maximum level of 1 μg/kg wet weight of B(a)P in foods for infants, infant milk and follow-on milk. Recently, the EFSA report on Polycyclic Aromatic Hydrocarbons in Food (EFSA, 2008) established that PAHs with known oral carcinogenicity, in particular BaP and other PAHs such as B(a)A, B(b)F, B(k)F, B(g,h,i)P, CHR, DB(a,h)A, I(c,d)P, individually or in combination (PAH8, PAH4 and PAH2), are the possible indicators of PAHs carcinogenic potency in food to a real analysis on PAHs occurrence and toxicity. There are some data in literature concerning the levels of PAHs in cow's milk (Grova et al., 2000; Ounnas et al., 2009) and same studies admit the transfer of PAHs from feed to milk (Costera et al., 2009; Creäpineau et al., 2003; Grova, Feidt, Creäpineau, et al., 2002; Grova, Feidt, Laurent and Rychen, 2002; Kishikawa et al., 2003; Luitz et al., 2006). The aim of this study was to determine residual levels of 16 polycyclic aromatic hydrocarbons of more toxicological interest, NAP, ACEN, ACE, FLU, PHEN, ANT, FLR, PYR, B(a)A, CRY, B(b)F, B(k)F, B(a)P, DB(a,h)A, B(g,h,i)P and I(c,d)P, in raw, pasteurized and UHT cow's milk samples from Calabria, widely used from consumers, to evaluate the possible role of heating treatment on PAHs concentration in milk and to quantify milk contribution to PAHs dietary exposure.

717

2. Materials and methods 2.1. Sampling This study was conducted on 36 cow's milk samples, 9 whole milk and 9 pasteurized milk, from lactating animals milked by hand, 9 UHT whole and 9 UHT semi-skimmed milk samples, collected at random from farms to Calabria. 2.2. Sample preparation and analysis The milk samples were homogenized with a stainless steel blender and kept at −20 °C until analysis. The lipidic extraction from the samples was done using the method of Naccari et al. (2008). Aliquots of 2 g were taken from each sample and 20 ml of KOH etanolic solution 1 M was added. The mixture was placed in a water bath at 80 °C for 3 h and cooled at room temperature; then, 10 ml of H2O and 20 ml of cyclohexane were added. The mixture was vortexed for 5 min and then centrifuged for 15 min at 4000 ×g; the supernatant layer was recovered and transferred in the glass Erlenmeyer flask, through filtration with a folder filter paper. The samples were reextracted, as previously described, with 20 ml of cyclohexane. The combined extracts were dried over anhydrous Na2SO4 for one night, filtered, reduced to 1 ml by rotavapor and evaporated to dryness under nitrogen. The residue was then recovered with 1 ml of acetonitrile and purged in SPE (solid phase extraction) cartridge pre-treated with 5 ml of acetonitrile. PAHs were eluted with 10 ml of acetonitrile; the eluates were then reduced by rotavapor and evaporated to dryness under nitrogen. The residues were finally recovered with 1 ml of acetonitrile, filtered with filters of 0.22 μm and transferred into special vials for HPLC analyses. 2.3. Reagents and chemicals The solvents used (cyclohexane, acetonitrile and water) were of high performance liquid chromatographic grade (99.9%), obtained from J.T. Backer (Mallinckrodt Backer, Milan, Italy). The other chemicals used, (potassium hydroxide, anhydrous sodium sulphate,) were of analytical reagent grade and commercially available from Sigma-Aldrich (Milan, Italy). PAHs pure reference standard solution (10 μg/ml) was purchased from Sigma-Aldrich (Milan, Italy). 2.4. Apparatus For solid phase extraction (SPE) cleanup, a SPE system (VARIAN 1233-4104, California USA), equipped with EXtrelut NT3 column (Merck, Darmstadt Germany), was used. The HPLC analyses were performed with a SHIMADZU instrument equipped with a system controller (SCL-10A VP), an auto injector (SIL-10AD VP) and a fluorescence detector (HEWLETT PACKARD 1046A). HPLC/UV–Vis-DAD/MS experiments were performed on a Waters instrument equipped with a 1525 Binary HPLC pump, a Micromass ZQ 2000 mass-analyser with a APCI source operating in positive mode, and a 996 Photo Diode Array Detector (PDA). A reverse-phase column SUPELCO SIL LC-PAH HPLC-column (15 cm × 4.6 mm i.d.), 5 μm was used. 2.5. Chromatographic method One hundred microliters of acetonitrile solution were injected into the chromatographic system. PAHs were analyzed at 2 ml/min, at room temperature, with a gradient of elution of mobile phase consisted of 60% H2O and 40% acetonitrile for 2 min, programmed to

718

C. Naccari et al. / Food Research International 44 (2011) 716–724

100% acetonitrile for 16 min, maintained at 100% acetonitrile until to 22 min and to 40% acetonitrile until to 30 min. The fluorescent detection of PAHs was performed by applying the following excitation and emission wavelengths program: λex−em 224–320 nm from 0 to 10.8 min, λex−em 252–400 nm from 10.8 to 14 min, to λex−em 268–398 nm from 14 to 22 min and λex−em 224– 320 nm from 22 to 30 min. 2.6. Calibration method To check the linearity of this method standard mixtures of PAHs at different concentrations (0.1, 0.25, 0.5 and 1 μg/ml) were analyzed. The above mentioned solutions were obtained from a working solution of PAHs at concentration of 10 μg/ml. The calibration lines for each PAHs were constructed using the linear least squares regression procedure (n = 4) of peak area vs standard concentration in μg/ml (R2 = 0.9927 for PA, R2 = 0.9994 for ANT, R2 = 0.9992 for PYR, R2 = 0.9982 for B(a)A, R2 = 0.9989 for CHR, R2 = 0.9991 for B(k)F, R2 = 0.9995 for B(a)P and R2 = 0.9989 for B(g,h,i)P) (Table 1). The accuracy and repeatability of the method was assessed by performing a spike-and-recovery test. Recovery was measured using fortified samples (n = 3 replicates) each at three levels of concentration, corresponding to 85%, 100% and 110%, respectively, of lower concentration of each analyte. Spike recoveries were repeated three times for each concentration and the results were expressed as average percentage of recovery. Recovery values were 91.25% for PA, 92.00% for ANT, 93.12% for PYR, 90.05% for B(a)A, 93.15% for CRY, 93.78% for B(k)F, 93.10% for B(a)P and 90.12% for for B(g,h,i)P and demonstrated the accuracy of analysis (Table 1). The precison of the method (RSD %), expressed as within-day repeatability, was determined by analyzing samples spiked with PAHs standard solutions corresponding to 85%, 100% and 110% of lower concentration of each analyte. For each level three different spiked samples were prepared and each of these samples was analyzed in triplicate. The detection limit (LOD) and the limit of quantification (LOQ) were calculated following AOAC guidelines (1994). LOD and LOQ were defined as the concentration of the analyte that produced a signal-to-noise ratio of three and ten, respectively, and were then tested experimentally by spiking blank samples at such levels. The conversion of LOD and LOQ to μg of PAHs for kilogram of milk results from the injection volume and the mass of sample analyzed. The specificity was confirmed by analysis of blank samples that are un-smoked samples with no detectable levels of PAH. No interferent peaks eluted at the same retention time of PAHs. Good laboratory practice (GLP) was applied throughout and procedural blanks were also analyzed. 2.7. HPLC/UV–Vis-DAD/APCI-MS analysis To further confirm the presence of PAHs in milk samples, a series of HPLC/UV–Vis-DAD/APCI-MS experiments were performed and a

Micromass ZQ 2000 mass-analyser with an APCI interface was used. Chromatographic runs were performed under the same conditions described in section 2.6. DAD analyses were carried out in the range between 400 and 190 nm, and the chromatograms visualised at 254 nm. Total ion current (TIC) chromatograms were acquired in positive mode in the mass range between 50 and 700 m/z units. The parameters used for the acquisition of the TICs were the following: capillary voltage, 2.90 kV; Cone voltage, 30 V; Extractor, 3 V; RF lens, 0,2 V; source temperature,150 °C; and desolvation temperature: 400 °C. Gas (N2) flows were set at 400 L/h for the cone and 500 L/h for the desolvation. Collected data were processed through a MassLynx v. 4.00 software (Waters s.p.a. Milano, Italy). Preliminary direct injections with high purity reference standards allowed us to set up the ideal APCI-MS experimental parameters, in order to obtain the highest ion intensities. A series of blank runs were also carried out to monitor the mobile phase mass spectra throughout the analytical batch. 2.8. Statistical analysis Data, expressed as mean ± SD of at least four determinations, were statistically analyzed by one-way analysis of variance (ANOVA) and the Student-Newman–Keuls test for unpaired data (Tallarida RT and Murray RB, Springer-Verlag, New York, 1986). Statistical significance was accepted when P b 0.01. 3. Results The present results were obtained from quali-quantitative elaboration of samples chromatographic profile acquired by HPLC–FL and confirmed by HPLC–UV–VIS-DAD–MS. 3.1. HPLC–FL chromatographic quali-quantitative analysis High-performance liquid chromatography/fluorescence detection (HPLC–FL) used for PAH detection and quantification offers a good selectivity and separation of the 16 PAHs analyzed. All PAHs were detected by fluorescence, using specific emission and excitation wavelengths for each compound, leading to good detection limits (Windal, Boxus, & Hanot, 2008), except for Acenaphtlylene [ACEN] and Indeno(1,2,3-cd)pyrene [I(c,d)P] since these were not fluorescent. The analytical method in HPLC–FL used in this study to extract and quantify PAHs in milk was shown to be adequate (Fig. 1). In particular the regression analysis of data shows that PAHs levels are linear over the range of analyzed concentrations; the coefficient of correlation exceeded 0.99 demonstrates a good linearity; RSD values (range: 0.800–10.388%) and recoveries (range: 90.12–93.78%) confirm accuracy and precision assessment; the LOQ higher than 0.0240 μg/l and 0.0120 ng/g proves the quality of chromatographic analysis (Table 1).

Table 1 Recovery, precision, linearity of analytical method and limit of quantification of PHEN, ANT, PYR, B(a)A,CHY, B(k)F, B(a)P and B(g,h,i)P. PAHs

ng/g added

Recovery (%)

RSDa (%)

Linearity (R2)

PHE ANT PYR B(a)A CHY B(k)F B(a)P B(ghi)P

1.20–1.42–1.56 1.10–1.30–1.42 1.10–1.30–1.43 0.69–0.81–0.89 0.088–0.104–0.114 0.057–0.067–0.073 0.03–0.035–0.038 0.011–0.01–0.014

91.25 92.00 93.12 89.15 93.15 93.78 93.10 90.12

6.108 7.687 10.388 7.448 6.100 4.154 1.800 0.800

0.9927 0.9994 0.9992 0.9982 0.9989 0.9991 0.9995 0.9989

a b

RSD (%): precision of three independent determinations, expressed as relative standard deviation. LOQ: limit of quantification.

LOQb μg/l of solution

ng/g of milk

0.1813 0.2306 0.4017 0.2234 0.1830 0.1246 0.0540 0.0240

0.0916 0.1153 0.2008 0.1117 0.0915 0.0623 0.0270 0.0120

C. Naccari et al. / Food Research International 44 (2011) 716–724

719

A 0.7

3 6

0.6

0.5

0.4

0.3

9

0.2

13

10

12

4 0.1 1

14

8

5

11

15

7 0.0

0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

22

24

26

28

30

B 35

30

25

20

15

7

10

8 9

5

10 12 13

5

15

0

0

2

4

6

8

10

12

14

16

18

20

Fig. 1. Spectro-fluorimetric chromatograms of (A) PAHs standard solution: NAP (1), ACEN(2), ACE (3), FLU (4), PHEN (5), ANT (6), FLT (7), PYR (8), CHY (9), B(a)A (10), B(k)F (11), B(b)F (12), B(a)P (13), DB(a,h)A (14), B(g,h,i)P (15), I(c,d)P (16) and (B) milk sample. Compounds ACEN(2) and I(c,d)P (16) are no detectable in fluorescence.

3.2. HPLC/MS confirmation analysis Confirmation of the presence of the PAHs identified through the HPLC–FL analyses was needed due to their relatively low abundance

in the matrices; to this purpose, a series of HPLC/MS analyses were performed. Several atmospheric pressure ionization techniques are currently used for the analysis of PAHs in non volatile matrices: electrospray (ES), where addition of suitable cations to improve the

720

C. Naccari et al. / Food Research International 44 (2011) 716–724

ionization of non polar molecules was applied, APPI (atmospheric pressure photo ionization) and APCI (atmospheric pressure chemical ionization) (Gimeno, Altelaar, Mrce', & Borrull, 2002). Among ionization techniques for the analysis of PAHs in non volatile matrices, APCI (atmospheric pressure chemical ionization) coupled with high performance liquid chromatography is undoubtedly the more suitable for the analysis of these molecules (Anacleto, Ramaley, Benoit, Boyd, & Quilliam, 1995). In the APCI interface, a corona discharge from a needle is used as primary ionization source, with the whole ionization process being identical to those occurring in chemical ionization sources (e.g., CI in GC/MS). Several competing reactions take place at the interface, the most common being proton transfers and electron transfers from the solvent molecules to the analytes. The use of an APCI interface in positive mode generally leads to the production of protonated molecules [M + H]+, radical cations [M+]+ as well as analytes-solvent clusters adducts, with the detection limits usually in

the picogram range with linear calibration curves (Anacleto et al., 1995; Galceran & Moyano, 1996). In this work, all PAHs were detected as protonated molecular ions [M + H]+ as water is present in the LC mobile phase, this can be explained with a proton transfer mechanism from protonated water clusters to the PAHs molecules taking place at the APCI interface. Electron transfer to form radical cations is the main competitive reaction which some authors reported to be the dominant process in PAH determination (Gimeno et al., 2002); as a matter of fact, we observed the presence of signals corresponding to radical cation species in the majority of the mass spectra of the analytes, even if not as base peaks. The combined use of APCI-MS and UV–Vis-DAD detectors allowed the acquisition of two independent sets of analytical data, that is, UV– Vis and mass spectra, useful for the qualitative determination of components in complex matrices by giving complementary

Fig. 2. Selected single ion monitoring (SIM) chromatograms of standard mixture of 16 PAHs used as reference: NAP (1), ACEN(2), ACE (3), FLU (4), PHEN (5), ANT (6), FLT (7), PYR (8), CHR (9), B(a)A (10), B(k)F (11), B(b)F (12), B(a)P (13), DB(a,h)A (14), B(g,h,i)P (15), and I(c,d)P (16).

C. Naccari et al. / Food Research International 44 (2011) 716–724

721

Fig. 3. Selected ion chromatograms from pasteurized milk sample of PHEN and ANT (5 + 6), PYR (8), CHY (9), B(a)P (13) and B(g,h,i)P (15).

indications about their chemical nature. Extraction of the protonated ions of PAHs (Figs. 2–3), both in the standard mixture and in the analytical matrices, was needed to the unambiguous confirmation of the peaks previously identified. 3.3. PAHs content in milk Among the 16 PAHs researched only 8 PAHs were detected in milk samples: Phenanthrene [PHEN], Antrhacene [ANT], Pyrene [PYR], Benzo (a)anthracene [B(a)A], Crysene [CHY], Benzo(k)fluoranthene [B(k)F],

PHENANTRENE [PA]

CRYSENE [CHY]

Benzo[a]pyrene [B(a)P] and Benzo(g,h,i)pyrene [B(g,h,i)P] (Fig. 4). PAHs detected were compounds with few aromatic cycles (from three to five), low-medium molecular weight (from 178.23 to 252.29 gmol/L) and high volatility. Except for the Benzo(a)anthracene, only non mutagenic PAHs were found; Benzo(a)pyrene, carcinogen to humans belonging to class 1A (IARC, 2008), was detected in low concentrations in all milk samples. Residual levels of PAHs found in sample of raw, pasteurized, UHT semi-skimmed and UHT whole milk are reported in Table 2, expressed as mean value ± standard deviation (ng/g of milk).

ANTHRACENE [ANT] BENZO(a)ANTHRACENE [B(a)A]

PYRENE [PYR]

BENZO(a)PYRENE [B(a)P]

BENZO(k)FLUORANTENE [B(k)F]

BENZO(g,h,i)PERYLENE [B(g,h,i)P]

Fig. 4. PAHs found in milk samples.

722

C. Naccari et al. / Food Research International 44 (2011) 716–724

Table 2 Residual levels of PAHs (mean values ± DS ng/g of four independent determinations) in raw, pasteurized, UHT whole and UHT semi-skimmed milk samples from Calabria. Samples (n = 9) PAHs

PHE

M ± SD range M ± SD range M ± SD range M ± SD range M ± SD range M ± SD range M ± SD range M ± SD range

ANT PYR B(a)A CHY B(k)F B(a)P B(g,h,i)P a

Σ PAHs

Raw milk (ng/g of milk)

Pasteurized milk (ng/g of milk)

1.425 ± 0.007 1.390–1.510 1.296 ± 0.096 1.134–1.415 1,351 ± 0.223 1.123–1.715 1.072 ± 0.04 0.981–1.140 nd

UHT Semi-skimmed milk (ng/g of milk)

Whole milk (ng/g of milk)

1.636 ± 0.059⁎,⁎⁎ 1.558–1.689 1.749 ± 0.002⁎ 1.745–1.751 1.591 ± 0.056 1.532–1.660 0.813 ± 0.060⁎,⁎⁎ 0.750–0.887 0.104 ± 0.019⁎⁎

1.831 ± 0.073⁎,⁎⁎⁎ 1.724–1.889 2.473 ± 0.083⁎,⁎⁎,⁎⁎⁎ 2.377–2.555 2.132 ± 0.073⁎,⁎⁎,⁎⁎⁎

nd

1.774 ± 0.041⁎ 1.727–1.816 1.835 ± 0.037⁎ 1.795–1.868 1.301 ± 0.233 0.982–1.498 1.060 ± 0.079 0.908–1.137 0.261 ± 0.029 0.226–0.292 nd

0.085–0.129 nd

2.060–2.229 0.830 ± 0.094⁎ 0.751–0.959 0.130 ± 0.022⁎⁎ 0.102–0.158 0.067 ± 0.008⁎⁎

0.270 ± 0.017 0.252–0.300 0.014 ± 0.003 0.011–0.019 5.428

0.268 ± 0.004 0.261–0.271 0.019 ± 0.001 0.019–0.020 6.519

0.035 ± 0.002⁎,⁎⁎ 0.032–0.039 0.013 ± 0.001⁎⁎ 0.013–0.014 5.941

0.055–0.075 0.248 ± 0.021 0.218–0.275 0.039 ± 0.007⁎,⁎⁎,⁎⁎⁎ 0.034–0.049 7.753

⁎ P b 0.01 vs raw milk. ⁎⁎ P b 0.01 vs pasteurized milk. ⁎⁎⁎ P b 0.01 vs UHT semi-skimmed milk.

In all types of milk analyzed, PAHs present in highest levels were PHEN, ANT and PYR, intermediate values were found for B(a)A and lower concentrations for CHY, B(k)F, B(a)P and B(g,h,i)P. Particularly, CHY was detected in pasteurized (0.261 ± 0.029 ng/g of milk), in UHT semi-skimmed (0.104 ± 0.019 ng/g of milk) and UHT whole (0.130 ± 0.022 ng/g of milk) except to raw milk samples; B(k)F only in UHT whole (0.067 ± 0.008 ng/g of milk) milk samples. The analysis of PAH residual levels in different types of milk studied shows that raw milk (Σ PAHs: 5.428 ng/g of milk) presents lower PAH concentrations than pasteurized (Σ PAHs: 6.519 ng/g of milk); on the other hand PAH concentrations in UHT whole samples (Σ PAHs: 7.753 ng/g of milk) are higher than UHT semi-skimmed milk (Σ PAHs: 5.941 ng/g of milk). The statistical analysis of the results and relative significance (P b 0.01) among various groups are reported in Table 2. In addition, residual levels of PAHs found in raw, pasteurized, UHT semi-skimmed and UHT whole milk samples were expressed,

according to guidelines of EFSA report on Polycyclic Aromatic in Food 2008, as PAH8, PAH4, PAH2 (Fig. 5), most suitable indicators of PAHs carcinogenic potency in food and useful to establish the risk characterisation for consumers. From a comparative analysis of PAH2, PAH4 and PAH8 in respect to B(a)P, it is possible observe that PAH2 cannot be an useful alternative marker to B(a)P in raw milk; PAH4 demonstrates to be the higher contributor in PAHs contamination in all type of milk studied; and PAH8 not provide much added value compared to PAHs4. 4. Discussion These results obtained from cow's milk are in accordance with other authors who found PAHs residual levels in goat milk, in particular, fat, half-fat and skimmed milk (Aguinagua, Campillo, Vinas,

B(a)P PAHs 2

1,80

PAHs 4 1,60

PAHs 8

ng/g of milk

1,40 1,20 1,00 0,80 0,60 0,40 0,20

le

T

H

T

w

ho

U H

U

pa

st eu

riz

ra

ed

w

0,00

Fig. 5. Comparison among residual levels of B(a)P and PAH8, PAH4 and PAH2, expressed as ng/g of milk in raw, pasteurized, UHT semi-skimmed and whole milk samples from Calabria.

C. Naccari et al. / Food Research International 44 (2011) 716–724

& Cordoba, 2007), whole, skimmed and condensed milk (Kishikawa et al., 2003). Data present in the literature (Aguinagua et al., 2007; Kishikawa et al., 2003) confirm the presence of eight PAHs, Phenanthrene, Antrhacene, Pyrene, Benzo(a)anthracene, Crysene, Benzo(k)fluoranthene, Benzo(a) pyrene and Benzo(g,h,i)pyrene, detected in our milk samples; however, some authors found also others PAHs in milk, such as Naphthalene, Acenaphtylene, Acenaphtene, Fluorene (Aguinagua et al., 2007; Grova, Feidt, Creäpineau, et al., 2002) and Benzo(b)fluoranthene (Aguinagua et al., 2007; Kishikawa et al., 2003), probably due to the contamination sources in region of milk production. Regarding to PAHs found in this study: Phenanthrene, Anthracene, Pyrene, Benzo(a)anthracene, Benzo(a)Pirene and Benzo(g,h,i,)perylene are ubiquitous in the milk matrices, suggesting that their presence is dependent from environmental pollution; while Crysene and Benzo(k)Fluorene are found only in pasteurized and UHT milk samples, suggesting that the heath treatment used certainly influence the PAHs formation (Dennis et al., 1991). The properties of PAHs (aromatic structure, molecular weight and high volatility) may explain their capacity to be transported from any contaminating sources, to be deposited on fields (Grova, Feidt, Laurent, et al., 2002) and to be transferred from soil to lactating animals and consequently to be excreted through milk (Grova, Feidt, Laurent, et al., 2002; Rychen, Crepineau-Ducoulombier, Grova, Jurjanz, & Feidt, 2005). In addition, for their lipophilic nature PAHs produced during processing techniques and heating treatment (pasteurization, sterilization and ultra-heat treatment) are incorporated in triglycerides, the main components of fats of milk. The differences observed between PAH levels in various types (whole, pasteurized and UHT) of milk analyzed, in fact, can be due to the fat content of milk, to processing techniques and to heattreatments. PAH concentrations were lowest in raw milk (Σ PAHs: 5.428 ng/g of milk) and, considering that this type of milk is not submitted to heating treatment, they are imputable to environmental contamination and to their transferring from soil to lactating animals (Grova, Feidt, Creäpineau, et al., 2002; Rychen et al., 2005). Residual levels of PAHs found in pasteurized (Σ PAHs: 6.519 ng/g of milk) and UHT whole milk (Σ PAHs: 7.753 ng/g of milk) were highest than in raw milk (Σ PAHs: 5.428 ng/g of milk) with differences statistically significant (P b 0.01) and it is possible hypothesize that they are the sum of the PAHs present in milk for environmental contamination and these produced by heath treatments (Simoneit, 2002). Relating to UHT milk samples, residual levels of PAHs in UHT whole milk are higher (Σ PAHs: 7.753 ng/g of milk) than in UHT semiskimmed milk (Σ PAHs: 5.941 ng/g of milk) with differences statistically significant (P b 0.01) probably due to their different fat content (4% in UHT whole and 1.7% in UHT semi-skimmed milk) and this demonstrated that PAH content is reduced by the process of skimming (Aguinagua et al., 2007; Kishikawa et al., 2003). The results obtained confirm that the consumption of milk contributes to the total PAHs intake in the human diet (Lodovici, Dolara, Casalini, Ciappellano, & Testolin, 1995). Because the lactating cows are likely to intake varying quantities of contaminants from soil during grazing (Rychen, Jurjanz, Toussaint, & Feidt, 2008), PAHs can be transferred through the organism and excreted to milk due to their ability to cross blood mammary barrier (Grova, Feidt, Laurent, et al., 2002). The quantity of PAHs secreted in milk would constitute a risk to human consumers (Luitz et al., 2006), especially for children (Ciecierska & Obiedzinski, 2010). In addition, the manufacturing and heating treatment of milk could cause PAHs formation, contributing to their concentration in pasteurized and UHT milk samples. From the results obtained in milk analyzed, PAH residual levels are lower than the maximum levels fixed for B(a)P in infant milk and follow-on milk by Regulation (EC) No 1881/2006 but data on PAH8, PAH4 and PAH2 show that PAH4 could be considered the best indicators of PAHs occurrence in milk samples.

723

Finally, considering the wide use of milk in human diet, it possible to conclude that the consumption of milk from Calabria gives a light contribution to PAHs dietary exposure and constitutes a low risk for consumers.

References Aguinagua, N., Campillo, N., Vinas, P., & Cordoba, M. H. (2007). Determination of 16 polycyclic aromatic hydrocarbons in milk and related products using solid-phase microextraction coupled to gas chromatography–mass spectrometry. Analytica Chimica Acta, 596, 285−290. Anacleto, J. F., Ramaley, L., Benoit, F. M., Boyd, R. K., & Quilliam, M. A. (1995). Comparison of liquid chromatography/mass spectrometry interfaces for the analysis of polycyclic aromatic compounds. Analytical Chemistry, 67(22), 4145−4154. AOAC Peer-Verified Methods Program (1994). Manual on policies and procedures. Gaitherburg, MD: AOAC International. Chahin, A., Guiavarch, Y. P., Dziurla, M. A., Toussanit, H., Feidt, C., & Rychen, G. (2008). 1-Hydroxypyrene in milk and urine as a bioindicator of polycyclic aromatic hydrocarbon exposure of ruminants. Journal of Agricultural and Food Chemistry, 56, 1780−1786. Ciecierska, M., & Obiedzinski, M. W. (2010). Polycyclic aromatic hydrocarbons in infant formulae, follow-on formulae and baby foods available in the Polish market. Food Control, 21, 1166−1172. Codex Alimentarius Commission (CX/FAC 06/38/36) (2005). Joint FAO/WHO food standards programme codex committee on food additives and contaminants. Discussion paper on polycyclic aromatic hydrocarbons (PAH) contamination. Costera, A., Feidt, C., Dziurla, M. A., Monteau, F., Le Bizec, B., & Rychen, G. (2009). Bioavailability of polycyclic aromatic hydrocarbons (PAHs) from soil and hay matrices in lactating goats. Journal of Agricultural and Food Chemistry, 57, 5352−5357. Creäpineau, C., Rychen, G., Feidt, C., Le Roux, Y., Lichtfouse, E., & Laurent, F. (2003). Contamination of Pastures by polycyclic aromatic hydrocarbons (PAHs) in the vicinity of a highway. Journal of Agricultural and Food Chemistry, 51, 4841−4845. Dennis, Mj., Massey, Rc., Cripps, G., Venn, I., Howarth, N., & Lee, G. (1991). Factors affecting the polycyclic aromatic hydrocarbon content of cereals, fats and other food-products. Food Add. Cont., 8(4), 517−530. European Food Safety Autority (EFSA 2008). Polycyclic aromatic hydrocarbons in food. Scientific opinion of the panel on contaminants in the food chain. The ESFA Journal, 2008(724), 1−11. Environmental Protection Agency (EPA 2006). http://www.epa.gov/superfund/programs/clp/svtarget.htm. European Commission, Health and Consumer Protection Directorate-General, Scientific Committee on Food (2002). Opinion of the scientific committee on food on the risk to human health of polycyclic aromatic hydrocarbons –occurrence in food SCF/CS/CNTM/ PAH/29. Feidt, C., Grova, N., Laurent, C., Rychen, G., & Laurent, F. (2000). Le transfert des micropullants organiques dans la chaîne alimentaire. Etat et prospectives de recherche. Oléagineux Corps Gras Lipides, 5, 431−435. Fidler, N., Sauerwald, T. U., Demmelmair, H., & Koletzko, B. (2001). Fat content and fatty acid composition of fresh, pasteurized, or sterilized human milk. Advances in Experimental Medicine and Biology, 501, 485−495. Fries, G. F. (1995). Transport of organic environmental contaminants to animal products. Reviews of Environmental Contamination and Toxicology, 141, 71−109. Galceran, M. T., & Moyano, E. (1996). Determination of hydroxy polycyclic aromatic hydrocarbons by liquid chromatography–mass spectrometry. Comparison of atmospheric pressure chemical ionization and electrospray. Journal of Chromatography A, 731, 75−84. Gimeno, R. A., Altelaar, A. F. M., Mrce', R. M., & Borrull, F. (2002). Determination of polycyclic aromatic hydrocarbons and polycyclic aromatic sulfur heterocycles by high-performance liquid chomatography with fluorescence and atmospheric pressure chemical ionization mass spectrometry detection in seawater and sediment samples. Journal of Chromatography A., 958, 141−148. Grova, N., Feidt, C., Creäpineau, C., Laurent, C., Lafargue, P. E., Hachimi, A., et al. (2002). Detection of polycyclic aromatic hydrocarbon levels in milk collected near potential contamination sources. Journal of Agricultural and Food Chemistry, 50, 4640−4642. 14 Grova, N., Feidt, C., Laurent, C., & Rychen, G. (2002). [ C] Milk, urine and faeces 14 excretion kinetics in lactating goats after an oral administration of [ C] polycyclic aromatic hydrocarbons. International Dairy Journal, 12, 1025−1031. Grova, N., Laurent, C., Feidt, C., Rychen, G., Laurent, F., & Lichtfouse, E. (2000). Gas chromatography–mass spectrometry study of polycyclic aromatic hydrocarbons in grass and milk from urban and rural farms. European Journal of Mass Spectrometry, 6, 457−460. International Agency For Research On Cancer (IARC) (2008). Air Pollution, Part 1. Some Non-heterocyclic Polycyclic Aromatic Hydrocarbons and Some Related Industrial Exposures. Meeting Report, Volume 92, . Killian, E., Smith, C., & Jones, K. C. (2000). Particles and vegetation: Implications for the transfer of particle-bound organic contaminants to vegetation. Science of Total Environment, 246, 207−236. Kishikawa, N., & Kuroda, N. (2009). Evaluation of organic environmental pollutants detected in human milk. Journal of Health Science, 55(1), 1−10. Kishikawa, N., Wada, M., Kuroda, N., Akiyama, S., & Nakashima, K. (2003). Determination of polycyclic aromatic hydrocarbons in milk samples by high-performance liquid chromatography with fluorescence detection. Journal of Chromatography B, 789, 257−264.

724

C. Naccari et al. / Food Research International 44 (2011) 716–724

Lodovici, M., Dolara, P., Casalini, C., Ciappellano, S., & Testolin, G. (1995). Polycyclic aromatic hydrocarbon contamination in the italian diet. Food Add. Contam., 12(5), 703−713. Luitz, S., Feidt, C., Monteau, F., Rychen, G., Le Bize, B., & Jurjanz, S. (2006). Effect of exposure to soil-bound polycyclic aromatic hydrocarbons on milk contaminations of parent compounds and their mono-hydroxylated metabolites. Journal of Agricultural and Food Chemistry, 54, 263−268. Moret, S., & Conte, L. S. (2000). Polycyclic aromatic hydrocarbons in edible fats and oils: Occurrence and analytical methods. Journal of Chromatography B, 882, 245−253. Naccari, C., Cristani, M., Giofrè, F., Licata, P., & Trombetta, D. (2008). Levels of benzo(a) pyrene and benzo(a)anthracene in smoked “provola” cheese from Calabria (Italy). Food Additives and Contaminants B, 1(1), 78−84. Naccari, F., Martino, D., Trombetta, D., Cristani, M., Licata, P., Naccari, C., et al. (2006). Trace elements in bovine milk from dairy farms in Sicily. Italian Journal of Food Science, 18(2), 22−26. Ounnas, F., Jurjanz, S., Dziurla, M. A., Guiavarch, Y., Feidt, C., & Rychen, G. (2009). Relative bioavailability of soil-bound polycyclic aromatic hydrocarbons in goats. Chemosphere, 77, 115−122. Perelló, G., Martí-Cid, R., Castell, V., Llobet, J. M., & Domingo, J. L. (2009). Concentrations of polybrominated diphenyl ethers, hexachlorobenzene and polycyclic aromatic hydrocarbons in various foodstuffs before and after cooking. Food and Chemical Toxicology, 47, 709−715. Protocol on Persistant Organic Pollutants (1999). The 1998 Aahrus protocol to the 1979 convention on long-range transboundary air pollution on persistent organic pollutants (POPs). ReV. Eur. Community International Environmental Law, 8, 224−230. Regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Official Journal of the European Union No L 364/6, 20-12-2006.

Regulation (EC) No 208/2005 of 4 February 2005 amending Regulation (EC) No 466/2001 as regards polycyclic aromatic hydrocarbons. Official Journal of the European Union No L 34/4, 8-2-2005. Regulation (EC) No 466/2001 of 8 March 2001 setting maximum levels for certain contaminants in foodstuffs. Official Journal of the European Union No L 77, 16-3-200. Rey-Salgueiro, L., Martínez-Carballo, E., García-Falcón, M. S., González-Barreiro, C., & Simal-Gándara, J. (2009). Occurrence of polycyclic aromatic hydrocarbons and their hydroxylated metabolites in infant foods. Food Chemistry, 115, 814−819. Rychen, G., Crepineau-Ducoulombier, C., Grova, N., Jurjanz, S., & Feidt, C. (2005). Modalites et risques de transfert des polluants organiques persistants vers le lait. INRA Productions Animales, 18, 355−366. Rychen, G., Jurjanz, S., Toussaint, H., & Feidt, C. (2008). Dairy ruminant exposure to persistent organic pollutants and excretion to milk. Animal, 2(2), 312−323. Schaum, J., Schuda, L., Wu, C., Sears, R., Ferrario, J., & Andrews, K. (2003). A national survey of persistent, bioaccumulative and toxic (PBT) pollutants in the United States milk supply. Journal of Exposure Analysis and Environmental Epidemiology, 13, 177−186. Simoneit, B. R. T. (2002). Biomass burning a review of organic tracers for smoke from incomplete combustion. Applied Geochemistry, 17, 129−162. Smith, K. E. C., Thomas, G. O., & Jones, K. C. (2001). Seasonal and species differences in the air-pasture transfer of PAHs. Environmental Science Technology, 35, 2156−2165. Tallarida, R.T., Murray, R.B., 1986. Springer-Verlag, New York. Windal, I., Boxus, L., & Hanot, V. (2008). Validation of the analysis of the 15 + 1 Europeanpriority polycyclic aromatic hydrocarbons by donor–acceptor complex chromatography and high-performance liquid chromatography–ultraviolet/fluorescence detection. Journal of Chromatography A, 1212, 16−22.