Monitoring of bisphenols in canned tuna from Italian markets

Monitoring of bisphenols in canned tuna from Italian markets

Food and Chemical Toxicology 83 (2015) 68e75 Contents lists available at ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.c...

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Food and Chemical Toxicology 83 (2015) 68e75

Contents lists available at ScienceDirect

Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox

Monitoring of bisphenols in canned tuna from Italian markets Margherita Fattore a, Giacomo Russo b, Francesco Barbato b, Lucia Grumetto a, b, *, Stefania Albrizio a, b a b

Consorzio Interuniversitario INBB, Viale Medaglie d'Oro, 305, I-00136 Rome, Italy  degli Studi di Napoli Federico II, Via D. Montesano, 49, I-80131 Naples, Italy Dipartimento di Farmacia, Universita

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 December 2014 Received in revised form 19 May 2015 Accepted 20 May 2015 Available online 10 June 2015

Monitoring of food contamination from bisphenols is a necessary process for the consumers' risk assessment. A method for the quali-quantitative analysis of Bisphenol A (BPA), Bisphenol B (BPB), Bisphenol A Diglycidyl Ether (BADGE), and Bisphenol F Diglycidyl Ether (BFDGE), by liquid chromatography with fluorescence detection (LC-FD), was performed and validated for their determination in 33 samples of tuna fish, canned in either oil or aqueous medium. Samples were collected in Italian markets. Tuna and the correspondent preservation medium were analyzed separately. Detected levels of bisphenols ranged from 19.1 to 187.0 ng/g in tuna matrix and from 6.3 to 66.9 ng/mL in oil medium. No bisphenols were found in aqueous medium. At least one of the analytes was found in 83% of the tuna samples in oil medium, whereas tuna samples in aqueous medium showed BPA alone in 67% of samples. 21% of the oil medium samples resulted positive for at least one bisphenol. On the basis of measured concentrations and general daily ingestion rate of canned tuna fish, the probable daily intake of BPA for Italian population was calculated. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Bisphenols Endocrine disruptors Canned tuna Migration Italian population

1. Introduction Bisphenol A (BPA) and its various analogs are important chemical starting substances in the production of polycarbonate (PC) plastics and epoxy resins, with multiple industrial applications (Glausiusz, 2014). PC is widely used in manufacturing food containers, whereas epoxy resins are used as interior protective lining for food and beverage cans (Sendon Garcia and Paseiro Losada, 2004). Due to an incomplete polymerization process, residues of bisphenol monomers in PC food containers or epoxy resin coatings can migrate into foods, especially oily food, during storage and processing at high temperatures (Brede et al., 2002; Cao et al., 2011; Geens et al., 2010; Grumetto et al., 2008; EFSA, 2006; Noonan et al., 2011; Rauter et al., 1999; Simoneau et al., 1999; Sungur et al., 2014; Theobald et al., 2000). Furthermore, migration from parts of facilities and/or utensils routinely employed during the food production process may occur under certain conditions (Casajuana and Lacorte,

* Corresponding author. Dipartimento di Farmacia, Via D. Montesano, 49, I-80131 Naples, Italy. E-mail address: [email protected] (L. Grumetto). http://dx.doi.org/10.1016/j.fct.2015.05.010 0278-6915/© 2015 Elsevier Ltd. All rights reserved.

2003, 2004; Goodson et al., 2004; Grumetto et al., 2013; Guart et al., 2011). The migration limits fixed by the European Commission are 0.6 mg/kg of food for BPA, 9.0 mg/kg of food for BADGE and its hydroxyl derivatives and 1.0 mg/kg of food for BADGE and its chlorinated derivatives (European Commission, 2005; European Commission, 2011). Moreover, the use of BFDGE has been forbidden (European Commission, 2005) and no information exists about BPB migration limits. In the January of 2015 EFSA has published a re-evaluation of BPA exposure from diet and other sources (EFSA, 2015). EFSA experts have reduced the safe level of BPA from 50 mg per kilogram of body weight per day (mg/kg of bw/day) to 4 mg/kg of bw/day, although, on the basis of collected data, the exposure risk is clearly under the “tolerable daily intake” (TDI). The presence of bisphenols (BPs) in food represents one of the most serious chemical contamination problems due to their properties as Endocrine Disrupting Chemicals (EDCs). EDCs are substances that influence synthesis, transport, secretion, action, binding or elimination of natural hormones in the body (GarcíaArevalo et al., 2014; Jeng, 2014; Le Corre et al., 2015). Actually, food, and especially canned food, is considered the predominant source of contamination from BPs, mainly from BPA (Le Corre et al., 2015). Due to its large number of applications, BPA is

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ubiquitously present in the environment so that human exposure routes are multiple. For example, it is present in the fragments of PC or other plastics that represent the majority of anthropogenic debris in watersheds and in the marine environment (Crain et al., 2007; Moore, 2008; Engler, 2012; Richard et al., 2009; Teuten et al., 2009a,b). Therefore, plastic fragments ingested by fishes can represent another contamination source from BPA along the food chain. As further consequence, bisphenol presence in canned fish may occur apart from packaging migration (Mita et al., 2011; Wei et al., 2011; Staniszewska et al., 2014). It is widely recognized that canned tuna is one of the most widespread fish commodities in the world. Europe is the world's largest canned tuna market, and Italy is one of the main European Union (EU) country for tuna consumption, with 2.33 kg/year/inhabitant of canned tuna (Market and Industry Dynamics in the Global Tuna Supply Chain). To date no information is available on the exposure of Italian population to bisphenols by this foodstuff. For these reasons the objective of this study was to determine the concentrations of BPA and its analogs Bisphenol B (BPB), Bisphenol A Diglycidyl Ether (BADGE), and Bisphenol F Diglycidyl Ether (BFDGE), in tuna, canned in either oil or aqueous medium, retailed in Italian markets. We applied and validated a simple and effective method for their extraction, from tuna and correspondent preservation media separately, and for their quantitative analysis. The levels of bisphenols were determined by liquid chromatography/fluorescence detection (LC-FD). Fluorescence detection was chosen because it is sensitive and easy to perform; furthermore it is cheaper than other detection techniques, such as mass spectrometry.

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2.2.1. Analyte extraction from tuna The solid content of each can was homogenized by stainless steel hand blender (700 W, Moulinex) for 5 min and an aliquot was taken for analysis. The remaining content of each can was frozen and stored at 20  C. Ten g of tuna paste were added of 40 mL of a mixture n-hexane/acetonitrile 1:1 (v/v) and extracted in an ultrasound bath at room temperature (Branson ultrasonic 2210, frequency 40 kHz). After sonication, the sample was stirred for 15 min, using a polytetrafluoroethylene (PTFE) stir bar, left in contact with the solvent for further 15 min, and centrifuged in polypropylene (PP) tube at 3500 rpm for 15 min. Finally, the acetonitrile phase was filtrated through a 0.45 mm pore size PTFE microfilter and analyzed by LC-FD. 2.2.2. Analyte extraction from oil of canned tuna 500 ml of oil were added of 1.0 mL of n-hexane, sonicated for 15 min and left in contact with the solvent for further 15 min. The mixture was loaded onto a Florisil cartridge (Chromabond, Florisil, MachereyeNagel, Düran, Germany) previously conditioned with 2.0 mL of n-hexane. After cartridge washing with 10.0 mL of nhexane and 20.0 mL of n-hexane/ethylacetate 95:5 (v/v), the sample was eluted with 10.0 mL of n-hexane/ethylacetate 50:50 (v/v). The eluate was evaporated at dryness and the residue reconstituted with 5.0 mL of acetonitrile for the LC-FD analysis. 2.2.3. Analyte extraction from aqueous medium of canned tuna The aqueous medium samples were centrifuged for 20 min at 3500 rpm in order to eliminate particulate and analyzed by LC-FD after filtration through 0.45 mm pore size PTFE microfilters. 2.3. Equipment and chromatographic conditions

2. Materials and methods 2.1. Reagents and chemicals All chemicals and reagents were of either analytical or LC grade and were purchased from SigmaeAldrich (Dorset, UK). BPA and BADGE standards (minimum purity of 99%), BFDGE (minimum purity of, 99% as mixture of three positional isomers orthoeortho, orthoepara, and paraepara) were purchased from SigmaeAldrich (Dorset, UK), while BPB standard (minimum purity of 99%) was purchased from TCI Europe (Zwijndrecht, Belgium). Stock solutions (1 mg/mL) of the four BPs were prepared in acetonitrile as solvent. Standard solutions containing the analytes were prepared just before use by mixing appropriate quantities of individual stock solutions and diluting with acetonitrile. All solutions were stored at 4  C for not more than 3 months.

2.2. Sample preparation The analyses were performed on 33 tuna fish samples, canned in either oil or aqueous medium, all of different brands retailed in Italian markets. Twenty four tuna samples were in oil medium, while nine were in aqueous medium. For each brand the analysis was performed on the content of two cans with the same batch number. The expiration date of samples ranged from two to five years at the moment of the analyses. Samples were stored sealed at room temperature until analysis. Oil or water from the tuna cans were poured off and analyzed separately. Throughout the analyses glassware and plastic equipment was properly treated to avoid any  mez et al., possible BPs background contamination (Ballesteros-Go 2009). Pure water was also verified as bisphenol-free, since it was reported that it may contain detectable levels of BPA (Gallart-Ayala et al., 2010).

The liquid chromatograph was a LC-10AD VP (Shimadzu Corp., Kyoto, Japan) equipped with a 7725 Rheodyne injection valve fitted with a 20 ml loop. The stainless steel column was a reversed-phase Ascentis C18 HPLC column (250 mm  4.60 mm i.d., 5 mm particle size) with a Supelguard Ascentis C18 guard column (both from Supelco, Bellefonte, PA, USA). The mobile phase was acetonitrilewater 60:40 (v/v). Analyses were carried out at room temperature (20 ± 2  C) at a flow rate of 0.5 mL/min in isocratic mode. The fluorescence detector was a model 20A (Shimadzu Corp., Kyoto, Japan) set at 273 nm excitation wavelength and 300 nm emission wavelength. The FD signals were processed with a personal computer software (Chromatoplus 2008, Shimadzu Corp., Kyoto, Japan). Each sample was analyzed in triplicate. 2.4. Mass spectrometry All the samples found positive for at least one of the four bisphenols under investigation were also analyzed by mass spectrometry to confirm the identity of the peaks.

Table 1 Summary of LC-MS/MS optimized conditions. CE: Collision Energy, CXP: Collision Cell Potential. Compound

Precursor ion [M  H] m/z

Product ion m/z

CE

CXP

Bisphenol A

227.2

Bisphenol B

241

BADGE

341.2

BFDGE

313.1

212.2 133.2 212 226 191.2 173.1 189.2 163.1

25 38 15 20 18 22 17 15

13 10 15 15 13 12 10 10

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Chromatographic separation was performed using a LC apparatus equipped with two micropumps Series 200 (Perkin Elmer, Shelton, USA), using the same column and chromatographic conditions described in “Equipment and chromatographic conditions”. Mass spectrometry analyses of bisphenols were performed on an API 3000 triple quadrupole mass spectrometer (Applied Biosystems, Canada) equipped with a Turbo-Ion Spray source. Ionization conditions were optimized infusing standard solutions of bisphenols (10 mg/ml) directly into the mass spectrometer, at a constant flow rate of 8 ml/min, using a model 11 syringe pump (Harvard Apparatus, Holliston, MA, USA). Drying gas (air) was heated to 400  C, the capillary voltage (IS) was set to 4000 V and 5000 V for negative and positive ions, respectively, and the declustering potential (DP) was set to 60 V and 30 V. Analyses were performed in the negative ion mode for BPA and BPB, and in positive mode for BADGE and BFDGE, in MRM (Multiple Reaction Monitoring), using specific combinations of a precursor-product ion transition for each compound (m/z 227.2, 212.2, 133.2, for bisphenol A; m/z 241.0, 226.0, 212.2, for bisphenol B; m/z 313.1, 189.2, 163.1, for bisphenol BFDGE; m/z 341.2, 191.2, 173.1, for bisphenol BADGE) (Table 1). 2.5. Method validation Quality parameters of linearity, accuracy, limit of detection (LOD), limit of quantification (LOQ), precision (repeatability and intermediate precision), and selectivity were evaluated for method validation. Glass tuna samples, in either oil or aqueous medium, analytically found as bisphenol-free (production site: Olbia e Italy, origin: FAO fishing areas 51, 57, and 71), were used as blanks to verify method selectivity and, after spiking, to confirm the assignment of peak identity and to calculate linearity, precision and accuracy of the method. All parameters were calculated for each of the three different matrices, i.e. tuna, oil, and aqueous medium. Bisphenol quantitative analyses were performed on the basis of matrix-matched calibration curves, peak area versus concentration. The external standard procedure was used for calibrations. The calibration curves were obtained from the analyses of blank samples spiked of each bisphenol in order to obtain five different concentrations: 5e120 ng/g for BPA and BPB and 15e90 ng/g for BADGE and BFDGE in tuna matrix; 5e120 ng/mL for BPA and BPB and 15e90 ng/mL for BADGE and BFDGE in preservative liquid matrices. For samples out of the maximum concentration level of linearity range, the analyses were repeated after appropriate dilution. As reported by other authors (Alabi et al., 2014), BFDGE generated three chromatographic signals being the mixture of three

Table 2 Calibration parameters.

positional isomers (orthoeortho, orthoepara, and paraepara). The appearance of three equidistant peaks is strongly indicative of the presence of BFDGE in the sample. On the other hand, the relative abundance of the three isomers in the samples may be different from that of the standard, making the quantitative determination affected by high uncertainty. We estimated the BFDGE content by taking into account the total area of the three signals. Sixty microliters of each calibration sample, i.e. three times the loop volume, were injected and the signal from the fluorescence detector was recorded. The obtained chromatograms were examined to determine the presence of any peak possibly interfering with the analysis of the four bisphenols. The analyte recovery was evaluated at low, medium, and high concentrations (15 ng/mL, 30 ng/mL, 60 ng/mL for liquid matrices, and 15 ng/g, 30 ng/g and 60 ng/g for tuna matrix). The blanks were spiked with appropriate quantities of BPA, BPB, BADGE, and BFDGE standards and three injections were made for each level. LODs and LOQs of the analytical procedure were assumed as the analyte concentrations producing an analytical signal three (LOD) and ten (LOQ) times the ratio between their respective standard deviations and the slope of the calibration line signal vs. concentration. Every spiked sample, after the addition of the suitable quantities of the standard solutions, was stored at 4  C for at least one hour before being examined. The robustness of the method of analysis was established on three different columns in different conditions of reversed-phase HPLC elution: i) Synergi 4m Fusion-RP80 Å 250  4.60 mm i.d. (Phenomenex, Torrance, CA), water/acetonitrile 40:60 (v/v); ii) Jupiter 5m C18 300 Å 250  4.6 mm i.d. (Phenomenex, Torrance, CA), water/acetonitrile 50:50 (v/v); iii) Sphereclone 5m ODS2 250  4.6 mm i.d. (Phenomenex, Torrance, CA), water/acetonitrile 40:60 (v/v). Peak resolution was only negligibly affected by both the different stationary phases and eluent composition. 2.6. Statistical analysis A commercially available statistical package for personal computer (Microsoft Excel 2010) was used. Data is expressed as mean ± standard deviation (mean ± SD). 3. Results and discussion Comparative tests on different kinds of SPE cartridges (data not shown) indicated that the extraction procedure above reported for the oil matrix was crucial to both substantially reduce interferences and obtain good recoveries, so allowing the correct identification and quantification of the analytes. 3.1. Method validation

BPA Tuna Range (ng/g) 5e120 Linearity (r2) 0.9999 Slope 172.35 Intercept 2005.6 Oil preservative medium Range (ng/mL) 5e120 Linearity (r2) 0.9998 Slope 150.49 Intercept 132.43 Aqueous preservative medium Range (ng/mL) 5e120 Linearity (r2) 0.9994 Slope 158.21 Intercept 152.71

BPB

BFDGE

BADGE

5e120 0.9993 89.81 122.57

15e90 0.9989 148.74 935.14

15e90 0.9999 129.96 1061.3

5e120 0.9998 98.93 51.67

15e90 0.9995 156.27 235.43

15e90 0.9999 125.67 301.33

5e120 0.9994 138.23 29.42

15e90 0.9993 173.31 624.29

15e90 0.9997 164.94 376.80

For quantitative analysis, calibration procedures were performed on both liquid and solid blank matrices, spiked with the analytes, and repeated every two weeks. For all four analytes, linearity of the method was verified on the five different concentrations reported in Material and Methods; r2 coefficient values ranged from 0.9989 to 0.9999. Three replicates of each bisphenol concentrations were performed. r2, slope, and intercept values of the calibration curves are summarized in Table 2. No degradation product was observed after injection. Table 3 summarizes LOD, LOQ, recovery, and precision values for the four bisphenols in tuna matrix, oil and aqueous preservative media, respectively. LOD and LOQ values for BPA, BPB, BADGE and BFDGE were in the same range of the values reported in literature for the analyses on

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Table 3 Method validation parameters. LOD

LOQ

Recovery (%)

Precision (RSDs%) Repeatability

ng/g Tuna fish BPA 1.3 BPB 3.0 BFDGE 3.7 BADGE 2.2 Oil medium BPA 1.1 BPB 0.9 BFDGE 2.1 BADGE 2.3 Aqueous medium BPA 1.1 BPB 1.0 BFDGE 2.1 BADGE 2.4

Intermediate-precision

ng/g

15 ng/g

30 ng/g

60 ng/g

15 ng/g

30 ng/g

60 ng/g

15 ng/g

30 ng/g

60 ng/g

4.3 10.0 12.3 7.5

100 105 85 106

91 94 96 80

100 101 104 100

3 4 3 4

5 4 4 5

1 1 5 3

4 5 8 10

5 6 10 9

5 9 10 8

3.7 3.0 6.9 7.8

99 87 60 65

77 72 66 65

100 99 70 75

4 5 5 3

5 4 5 4

3 5 4 4

7 6 10 10

8 10 9 10

10 6 6 8

3.9 3.5 7.0 7.9

89 92 70 75

95 87 75 80

102 100 80 85

3 1 1 1

3 2 0 2

2 1 1 2

8 3 9 8

7 6 8 6

6 3 6 5

the same food matrix by the same detection system (Berger et al., 2001; Braunrath et al., 2005; Cabado et al., 2008). Precision was expressed as both repeatability (intraday precision) and intermediate precision (interday precision) at three different concentration levels (15 ng/mL, 30 ng/mL, 60 ng/mL for liquid matrices and 15 ng/g, 30 ng/g and 60 ng/g for tuna matrix). Intraday and interday precision values were based on 5 and 10 measurements, respectively, and the relative standard deviations (% RSD) did not exceed 5%, for intraday, and 10%, for interday precision. Accuracy of the method was evaluated from the recovery (n ¼ 3) of the tested BPs in spiked blank samples, treated by the sample preparation procedure described, at three concentration levels. Recovery values ranged from 80.0% to 106.0% for tuna matrix, from 60.0% to 100.0% for oil medium and from 70.0% to 102.0% for aqueous medium. All procedures for method validation were performed on an Ascentis C18 HPLC column as reported in Materials and Methods section. Fig. 1 shows the chromatograms of spiked blank samples of tuna (60 ng/g), oil (60 ng/mL), and aqueous (60 ng/mL) matrices in comparison with the corresponding real samples.

3.2. Analysis of real samples In this study we analyzed 33 samples of canned tuna fish, 24 in oil and 9 in aqueous medium, of various brands retailed in Italy. All cans were coated with gold epoxyphenolic lacquer or with a low BADGE enamel, as indicated by manufacturers. The results of the analyses are summarized in Table 4. Peak identity for all bisphenolpositive samples was confirmed by LCe tandem MS (LC-MS/MS). Fluorescence detection has shown a valuable tool to monitor the presence of BPs even though the analysis by FD requires further confirmation by MS in order to reasonably exclude possible occurrence of method artifacts. It is well known that the release of bisphenols is dependent from different conditions such as temperature, storage and acidic or basic features of the foods in direct contact with the can. In particular Munguia et al. demonstrated that heat processing enhanced the rate of migration for BPA and BADGE from epoxy resin in aqueous food stimulants (Munguia-Lopez and Soto-Valdez, 2001). Storage time did not show any effect in BPA migration from cans but it affected BADGE migration due to its susceptibility to hydrolysis in aqueous simulants.

We detected bisphenol levels ranging from 19.1 to 187.0 ng/g in tuna matrix and from 6.3 to 66.9 ng/mL in oil preservative medium. No bisphenols were found in aqueous medium (Table 4). Only four samples of tuna matrix preserved in oil were found bisphenol-free, whereas twenty samples were positive for at least one of the analytes. In contrast, nineteen samples of oil medium were bisphenol free, since BPA and BADGE were found in only four and one samples, respectively. Tuna in aqueous medium showed BPA alone in six samples, while three samples were bisphenol-free (Fig. 2). With regard to BADGE and BFDGE it should be reminded that these two analytes can generate different hydroxy and chlorinated derivatives during food storage by hydrolysis of epoxy groups (e.g. BADGE$2HCl, BADGE$HCl, BADGE$H2O,BADGE$2H2O and BADGE$HCl$H2O) (Sajiki et al., 2007). However BADGE and BFDGE derivative levels have not been investigated in our study. We found that BPA and BADGE concentrations in all positive oil samples were lower than those of the respective solid parts. This result is in agreement with those of Noonan et al., 2011 that quantified BPA concentrations in different highly consumed canned foods, comprising canned tuna, and also investigated the partitioning of BPA between solid and liquid food portions. They found that in foods with separate liquid and solid portions, the BPA concentrations were higher, generally 10-fold, in the solid food (Noonan et al., 2011). BPA levels in our tuna matrix samples were comparable to those reported in the most recent literature (Sungur et al., 2014; Cao et al., 2010; Lim et al., 2009). Instead, BADGE and BFDGE concentrations were, respectively, slightly higher and lower than those detected by other authors (Cabado et al., 2008; Zhang et al., 2010; Theobald et al., 2000). To the best to our knowledge, no authors have analyzed BPB in canned fish. We used a one-way analysis of variance (ANOVA) to study tuna BPA levels depending on the preservation medium (oil or water). We compared samples of tuna in oil and in aqueous medium from the same brands (seven couples of samples). ANOVA results indicated that the differences of tuna BPA concentrations were significant (p < 0.05), depending on the preservation medium. Indeed, BPA levels were higher in tuna from oil containing cans than from aqueous medium containing cans. This observation suggests that oil may promote the bisphenol migration from the can lining to the tuna. In 2008 the European Commission indicated that canned foods contributed for at least 60% of BPA total daily intake among adults in EU, but recent opinions suggest a more realistic percentage as

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Fig. 1. Chromatograms of a spiked (60 ng/g) tuna sample (A), a real tuna sample (B), a spiked (60 ng/mL) oil sample (C), a real oil sample (D), a spiked (60 ng/mL) water sample (E) and a real water sample (F).

high as 90% (European Commission, 2008). In 2008 canned tuna consumption in Italy, the second consumer in EU, was 2.33 kg/year/ inhabitant (Market and Industry Dynamics in the Global Tuna Supply Chain). Taking into account both the Italian consumption data and the results of our study, we calculated the Probable Daily Intake (PDI) of BPA by this commodity and its ratio with TDI.

The PDI was calculated as follows:

PDI ¼ ðC  IÞ=bw where PDI is expressed as mg/kg bw/day, C is the mean concentration of BPA taking into account only the samples resulted

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Table 4 Bisphenol contents in tuna, oil and water medium. N Sample

Expiry date Net weight Drained weight

Origin

Indian Ocean * * * * * Ecuador Thailand Ivory Cost Spain Spain * Spain Atlantic Ocean Italy * * * * * * * * * * FAO 34,47 51,57,71 FAO 67-71-77-87 * * Pacific Ocean * * *

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Tuna in olive oil Tuna in e.v oliva Tuna in olive oil Tuna in olive oil Tuna in olive oil Tuna in olive oil Tuna in olive oil Tuna in olive oil Tuna in olive oil Tuna in olive oil Tuna in olive oil Tuna in olive oil Tuna in olive oil Tuna in olive oil Tuna in olive oil Tuna in olive oil Tuna in olive oil Tuna in olive oil Tuna in olive oil Tuna in vegetal oil Tuna in olive oil Tuna in e.v.olive oil Tuna in olive oil Tuna in olive oil Natural Tuna Natural Tuna

11.2016 03.2017 12.2017 12.2017 09.2017 12.2017 12.2017 11.2014 09.2017 12.2018 12.2016 12.2018 12.2018 12.2019 06.2018 04.2016 08.2018 06.2018 12.2018 12.2018 12.2017 12.2017 12.2018 08.2017 06.2017 09.2016

80 g 80 g 80 g 80 g 111 g 80 g 80 g 80 g 80 g 80 g 80 g 80 g 80 g 80 g 80 g 80 g 80 g 160 g 80 g 80 g 80 g 80 g 80 g 80 g 80 g 111 g

52 g 52 g 52 g 52 g 81 g 52 g 52 g 52 g 52 g 52 g 52 g 52 g 52 g 52 g 52 g 52 g 52 g 104 g 52 g 52 g 52 g 52 g 52 g 52 g 56 g 81 g

27 28 29 30 31 32 33

Natural Natural Natural Natural Natural Natural Natural

07.2016 12.2016 04.2016 03.2016 12.2015 06.2017 12.2016

80 80 80 80 80 80 80

56 56 56 56 56 56 56

Tuna Tuna Tuna Tuna Tuna Tuna Tuna

g g g g g g g

g g g g g g g

Bisphenols ng/mL of mediuma

Bisphenols ng/g of tuna BPA

BPB

BFDGE

BADGE

55.8 ± 4.8 147.5 ± 4.7 109.3 ± 4.3 51.6 ± 3.7 44.9 ± 5.6 104.1 ± 1.8 45.6 ± 4.6 81.1 ± 8.0 nd 38.9 ± 5.5 49.1 ± 4.9 nd nd 25.4 ± 0.4 30.4 ± 1.5 48.2 ± 5.0 37.6 ± 1.8 132.9 ± 2.6 nd 79.4 ± 5.6 94.4 ± 0.8 31.3 ± 2.1 nd 124.1 ± 4.7 50.46 ± 1.6 nd

72.7 ± 4.2 nd 145.9 ± 5.3 nd nd 74.8 ± 8.7 nd nd nd nd nd nd nd nd nd nd nd 19.1 ± 0.1 nd nd nd nd nd nd nd nd

nd nd nd nd nd 49.7 nd nd nd nd nd nd nd 24.6 nd nd nd nd nd nd nd nd nd nd 38.5 ± 4.9 nd nd nd nd nd nd nd nd 91.1 nd 36.1 nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd

38.5 ± 4.8 nd 55.4 ± 2.8 187.0 ± 4.4 49.0 ± 4.0 60.6 ± 3.7 nd

nd nd nd nd nd nd nd

nd nd nd nd nd nd nd

nd nd nd nd nd nd nd

± 3.1

± 0.1

± 3.9 ± 2.5

BPA

BPB BFDGE BADGE

nd nd nd nd nd 6.3 ± 0.9 nd nd nd nd nd nd nd nd nd nd nd 10.2 ± 0.6 nd nd 7.9 ± 1.1 16.2 ± 2.1 nd nd nd nd

nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd

nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd

nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd 66.9 ± 3.6 nd nd nd nd nd nd nd nd nd

nd nd nd nd nd nd nd

nd nd nd nd nd nd nd

nd nd nd nd nd nd nd

nd nd nd nd nd nd nd

Notes: * indication of the origin not present on the label. a Medium is oil in tuna olive oil and water in natural tuna.

Fig. 2. Percentages of BPs in sample of tuna in oil, oil medium, and natural tuna.

positives (72.5 mg/kg), I is the average of daily tuna consumption in Italy, and bw is the average body weight for an adult person (70 kg). Its value is 0.007 mg/kg b.w./day and the ratio PDI/TDI (2015 EFSA,

TDI 4 mg/kg bw/day) is 0.001, corresponding to 0.2%. We underline that the latter value should be considered as a rough estimate of the

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risk, since the general consumption data considered do not include details for subsets of the population. 4. Conclusion The effect of bisphenols on human health is still a matter of debate. Their concentration in food, considered as the main source for human exposure, should be determined through extensive monitoring programs to establish possible cause/effect relationships. In the January of 2015 EFSA experts have reduced the safe level of BPA from 50 mg per kilogram of body weight per day (mg/kg of bw/day) to 4 mg/kg of bw/day. The results of this study are quite encouraging, since the bisphenol levels found are below the legal limits. On the other hand, since the scientific literature reports that a synergistic action of different EDCs is possible, a significant effect may be observed even when each chemical is present at so low doses that individually they would not induce observable effects (Kortenkamp, 2007; Rajapakse et al., 2002). For this reason a constant monitoring of their presence in different foodstuffs should be strongly recommended. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgment This work was supported by the Ministry of Health under Grant code RF-2009-1536185. We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript. References Alabi, A., Caballero-Casero, N., Rubio, S., 2014. Quick and simple sample treatment for multiresidue analysis of bisphenols, bisphenol diglycidyl ethers and their derivatives in canned food prior to liquid chromatography and fluorescence detection. J. Chromatogr. A 1336, 23e33.  mez, A., Rubio, S., Pe rez-Bendito, D., 2009. Analytical methods for the Ballesteros-Go determination of bisphenol A in food. J. Chromatogr. A 1216, 449e469. Berger, U., Oehme, M., Girardine, L., 2001. Quantification of derivatives of bisphenol A diglycidyl ether (BADGE) and novolac glycidyl ether (NOGE) migrated from can coatings into tuna by HPLC/fluorescence and MS detection. Fresenius J. Anal. Chem. 369, 115e123. Braunrath, R., Podlipna, D., Padlesak, S., Cichna-Markl, M., 2005. Determination of bisphenol a in canned foods by immunoaffinity chromatography, HPLC, and fluorescence detection. J. Agric. Food Chem. 53, 8911e8917. Brede, C., Skjevrak, I., Herikstad, H., Anensen, E., Austvoll, R., Hemmingsen, T., 2002. Improved sample extraction and clean-up for the GC-MS determination of BADGE and BFDGE in vegetable oil. Food Addit. Contam. 19, 483e491. Cabado, A.G., Aldea, S., Porro, C., Ojea, G., Lago, J., Sobrado, C., Vieites, J.M., 2008. Migration of BADGE (bisphenol A diglycidyl-ether) and BFDGE (bisphenol F diglycidyl-ether) in canned seafood. Food Chem. Toxicol. 46, 1674e1680. Cao, X.L., Perez-Locas, C., Dufresne, G., Clement, G., Popovica, S., Beraldin, F., Dabeke, R.W., Feeley, M., 2011. Concentrations of bisphenol A in the composite food samples from the 2008 Canadian total diet study in Quebec City and dietary intake estimates. Food Addit. Contam. Part A 28, 791e798. Cao, X.L., Corriveau, J., Popovic, S., 2010. Bisphenol a in canned food products from canadian markets. J. Food Prot. 73, 1085e1089. Casajuana, N., Lacorte, S., 2003. Presence and release of phthalic esters and other endocrine disrupting compounds in drinking water. Chromatographia 57, 649e655. Casajuana, N., Lacorte, S., 2004. New methodology for the determination of phthalate esters, bisphenol A, bisphenol A diglyciyl ether, and nonylphenol in commercial whole milk samples. J. Agric. Food Chem. 52, 3702e3707. Crain, D.A., Eriksen, M., Iguchi, T., Jobling, S., Laufer, H., LeBlanc, G.A., Guillette Jr., L.J., 2007. An ecological assessment of bisphenol-A: evidence from comparative biology. Reprod. Toxicol. 24, 225e239. EFSA, 2015. Scientific opinion on the risks to public health related to the presence of bisphenol A (BPA) in foodstuffs. EFSA J. 13, 3978. EFSA, 2006. European Food Safety Authority. Opinion of the scientific panel on food additives, flavourings, processing aids and materials in contact with food on a

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