Evaluation of Endocrine Disrupting Compounds Migration in

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Evaluation of Endocrine Disrupting Compounds Migration in Household Food Containers under Domestic Use Conditions Jorge Sáiz, and Belén Gomara J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02479 • Publication Date (Web): 22 Jul 2017 Downloaded from http://pubs.acs.org on July 23, 2017

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Journal of Agricultural and Food Chemistry

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Evaluation of Endocrine Disrupting Compounds Migration in Household Food Containers under Domestic

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Use Conditions

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Jorge Sáiz, Belen Gómara*

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Institute of General Organic Chemistry, Spanish National Research Council (IQOG-CSIC). Calle Juan de la

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Cierva, 3, 28006 Madrid, Spain.

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*Corresponding author:

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Tel: +34 91 5622900

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Fax: +34 91 5644853

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E-mail address: [email protected] (B. Gómara).

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1 ACS Paragon Plus Environment


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ABSTRACT

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Plasticizers and plastic monomers are commonly used in packaging. Most of them act as endocrine

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disrupters and are susceptible to migrate from the packaging to the food. We evaluated the migration of

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endocrine disrupting compounds from three different household food containers to four food simulants

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under different domestic treatments and for different periods of time, with the aim of reproducing real

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domestic conditions. The results showed that the migration to the simulants increased with the storage

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time, up to more than 50 times in certain cases. The heating power seemed to increase the migration

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processes (up to more than 30 times) and reusing containers produced an increase or decrease of the

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concentrations depending on the container type and the simulant. The concentrations found were lower

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than other concentrations reported (always less than 4000 pg/mL, down to less than 20 pg/mL), which

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might be a consequence of the domestic conditions used.

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Keywords: endocrine disrupting compounds; migration; food containers; food simulants; UHPLC-MS/MS.

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INTRODUCTION

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The presence of contaminants in food due to migration processes from packaging material is a well-known

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issue1,2. These migration processes have been proved to occur in many different packaging of different

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materials, such as recycled paperboard packaging,2 baby bottles,3 canned foods,4,5 or water bottles,6 among

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many others. Nowadays, the majority of food packaging used in food industry and for home-cooked meals

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are made of organic polymers, such as polyethylene (PE) bags, polyethylene terephthalate (PET), or

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polypropylene (PP). In the manufacturing of these plastics, plasticizers are used as additives, which provide

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certain characteristics to the final product, such as plasticity, viscosity, transparency, thickness, or increased

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durability. Most of these plasticizers are phthalates, which can migrate to food and are considered to act as

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endocrine disrupters.1 Besides, other plastic monomers, such as bisphenols and bisphenol derivatives, are

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being used as constituent of some plastics and epoxy resins and could, therefore, also migrate to the food.

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These compounds are also not exempt from controversy and their migration processes have also been

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studied.4,7-11

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Although the migration of plasticizers and monomers to real food samples for human consumption has

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been studied by some authors,12-14 food simulants are commonly used in order to simplify testing and for

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regulatory compliance purposes, since the simulants are less complex than foods.15 Moreover, their use

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allows to easily compare results obtained in different laboratories. According to the European Union,16 food

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simulants mimic the use of foods of different nature, such as oily food, alcoholic drinks, foods with

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hydrophilic character, or foods with specific pH values. Commonly, migration tests are performed with

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samples consisting of small pieces of the plastic packaging of study, which are totally immersed in the food

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simulant. Some authors17 used four different food simulants in order to study the migration of sixteen

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phthalic acid esters. The experimental setup consisted of cutting a piece of plastic from bottles, bags, or

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films and soaking them with the selected simulants for a pre-defined time and temperature. Similarly,

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other authors18 performed a study of plasticizer migration with pieces of bottles and total immersion in

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food simulants. Another work19 studied the migration of phthalates from cork stoppers also by soaking the



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corks in a food simulant. Other groups20-23 cut cling film in pieces, which were added to the selected food

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simulant. The total immersion test was also performed10 for the evaluation of the migration of some

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plasticizers from plastic bottles, caps, and septa to food simulants. Although the total immersion test is

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widely performed for the evaluation of chemical migration to food, it does not faithfully reproduce real

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storing and/or cooking conditions, since both sides of the container are in contact with the simulant and

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because, frequently, the surface-to-volume ratio differs from real storage situations. In these regards, other

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authors have focused their studies in reproducing specific storage conditions using the entire container for

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it. Plastic baby bottles have attracted the interest of several authors, who studied bottles of different

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materials filled with a milk simulant,24-27 while other food containers, such as cans, tetra-packs, or yogurt

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containers have been studied less frequently.9 However, there is still a need for more tests that emulate

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certain uses of specific food containers. Regarding the storage conditions, some authors kept the samples

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at high temperatures, up to 121 ºC, in order to accelerate the experiments.19,21,22,28,29 Other studies also

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used elevated temperatures following the indications stablished in ISO 10106,9,17,19,20,23 which simulate

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storage at room temperature for indefinite time,30 while other authors used a combination of high

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temperatures and long periods of time, such as 1.5 years at 40 ºC.31 Although these situations simulate

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extreme conditions and are of interest in certain cases, they hardly simulate real domestic conditions of

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food storage or cooking, such as freezing or heating.

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While plastic baby bottles have been widely studied9, 24-27 for migration tests, in homes one of the most

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used food packaging are household food containers. They are used to store food in the refrigerator or in

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the freezer and also to transport food, for example to the work place. Household food containers are also

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frequently used to heat the food and they are repeatedly reused over time. While the effects of these

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preservation and cooking actions are relatively unknown in terms of plasticizer and monomer migration,

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the possible adverse effects of these practices on health has become a growing concern for society. There

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exist, in fact, the general idea that food should not be heated in the food container and that quality food

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containers should be used in order to avoid “bad” plastics. Actually, there is a big market for household

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food containers, which offer many types of containers. The user can purchase food containers of different


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materials at very different prices, from cheap disposable containers made of plastic to very expensive glass

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food containers. The decision on which product will be purchased will depend on economic aspects but

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also on health concerns, based on the prejudices of the customer.

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In view of this discussion, it seems important to perform new studies on the endocrine disrupting

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compound (EDC) migration. For this reason, present paper is focused on commercially available household

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food containers, which have scarcely been studied previously, instead of industrial packaging materials

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such as plastic bottles, cans, stoppers, etc. Besides, the migration conditions propose in this work mimic

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conventional domestic storing and cooking conditions instead of using accelerated conditions based on

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high temperatures and long periods of time. Therefore, the aim of this work was to evaluate the plasticizer

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and monomer migration from different types of household food containers to food simulants of varied

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natures under specific conditions that simulate real situations at home, depending on different storage

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type, times, and cooking processes. The EDCs selected for the study were dimethyl phthalate (DMP), 1,

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diethyl phthalate (DEP), 2, dibutyl phthalate (DBP), 3, butyl benzyl phthalate (BBP), 4, diethyl hexyl

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phthalate (DEHP), 5, and di-iso-nonyl phthalate (DiNP), 6, bisphenol A (BPA), 7, bisphenol B (BPB), 8,

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bisphenol F (BPF), 9, bisphenol A diglycidyl ether (BADGE), 10, bisphenol F diglycidyl ether (BFDGE), 11,

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BADGE·HCl, 12, BADGE·H2O, 13, BADGE·HCl·H2O, 14, and BADGE·2H2O, 15. In addition, three different food

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containers (i.e. hermetic semi-disposable containers, BPA-free hermetic containers, and glass hermetic

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containers) were also investigated.

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MATERIAL AND METHODS

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· Reagents and standards

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Native standards of dimethyl phthalate (DMP), 1, diethyl phthalate (DEP), 2, dibutyl phthalate (DBP), 3,

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butyl benzyl phthalate (BBP), 4, diethyl hexyl phthalate (DEHP), 5, di-iso-nonyl phthalate (DiNP), 6,

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bisphenol A (BPA), 7, bisphenol B (BPB), 8, bisphenol F (BPF), 9, bisphenol A diglycidyl ether (BADGE), 10,



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bisphenol F diglycidyl ether (BFDGE), 11, BADGE·HCl, 12, BADGE·H2O, 13, BADGE·HCl·H2O, 14, and

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BADGE·2H2O, 15, were supplied by AccuStandard (New Haven, CT,). Chemical structures are gathered in

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Figure 1.The isotopically labeled standards DMP-D4, DEP-D4, DBP-D4, BBP-D4, DEHP-D4, and BPA-13C12 were

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supplied by Cambridge Isotope Laboratories (Andover, MA).

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Water, acetonitrile, ethanol, and methanol, all of LC–MS Ultra CHROMASOLV® grade, formic acid, acetic

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acid, and ammonium formate (purity ≥ 99.0%) were supplied by Sigma-Aldrich (St. Louis, MO).

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· Apparatus

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The ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS)

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experiments were carried out in a UPLC Acquity system (Waters, Milford, MA) coupled to a Xevo TQ-S triple

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quadrupole mass spectrometer (Waters) using an electrospray ionization (ESI) interface. A 2.1 mm × 50 mm

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i.d., 1.7 µm, Acquity UPLC® BEH Phenyl column (Waters) was used as separation column and a 2.1 mm × 30

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mm i.d., 1.7 µm, Kinetex C18 delay column (Phenomenex, Torrance, CA) was placed between the LC pump

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and the injection valve, in order to retard the phthalates coming from the mobile phase and separate them

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from the analytical peaks. Nitrogen (99.5% purity) was used as desolvation and cone gases for the MS

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experiments, at 16.7 L/min and 2.5 L/min, respectively, and argon (99.999% purity) was used as collision

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gas (0.17 mL/min). The ESI source was operated in the fast polarity switching mode. Phthalates, BFDGE,

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BADGE, and derivatives were detected in the positive mode, while BPA, BPB, and BPF were recorded in the

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negative mode. Optimization and quantitative analysis were carried out in the multiple reaction monitoring

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(MRM) mode, using two precursor-product ion transitions for each compound. The most intense ion of the

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ESI spectra of each analyte was selected as precursor ion. After that, the two most intense product ions

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formed at their optimum collision energies were chosen, obtaining the two most abundant ion transitions.

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The most abundant transition was used for quantitative purposes and the second most abundant one for

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confirmatory purposes. The compounds, along with their retention times, ion transitions, transition ratios,

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optimized collision energies, and polarities are shown in Table 1. The instrumental determination of DMP,


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DEP, DBP, BBP, DEHP, and BPA was already optimized,32 while the instrumental conditions for DiNP, 6, BPB,

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8, BPF, 9, BADGE, 10, BFDGE, 11, BADGE·HCl, 12, BADGE·H2O, 13, BADGE·HCl·H2O, 14, and BADGE·2H2O, 15,

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were optimized and characterized in this work. The first transition indicated in the table corresponds to the

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quantitative transition and the second one to the confirmatory transition. The value of dwell time used

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during the analysis was 0.001 s for all analytes.

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The determinations of pH values were carried out using a pH meter that was calibrated using aqueous

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buffers of pH 4.01, 7.00, and 9.21. Values of pH were taken in aqueous solution.

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The confirmation of the nature of the plastic containers was carried out with a Spectrum One FT-IR

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Spectrometer (Perkin Elmer, Shelton, CT) using a Universal Attenuated Total Reflectance (ATR) sampling

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accessory scanning from 4000 to 650 cm-1 with a resolution of 4 cm-1.

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· Migration tests and samples

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Three different types of household food containers were purchased in a local supermarket (Madrid, Spain)

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and used for the evaluation of EDC migration to food simulants: hermetic semi-disposable containers, BPA-

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free hermetic containers, and glass hermetic containers. The food containers were washed with soap and

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warm water before their first use, according to the manufacturers’ recommendations for their domestic

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use. Then, they were rinsed with ultrapure water three times. Four different food simulants (A, B, C, and

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D1) were employed, according to the Official Journal of the European Union.16 Simulant A consisted of

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ethanol 10% (v/v), simulant B was prepared with acetic acid 3% (w/v), simulant C was ethanol 20% (v/v),

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and simulant D1 consisted of ethanol 50% (v/v), all of them in water. All the simulants were daily prepared

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and a volume of 250 mL of each simulant was introduced in each household food container, which were

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then stored or treated under different experimental conditions, according to Table 2. Different sets of food

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containers were prepared: each set was composed of three different containers (one semi-disposable, one

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BPA-free, and another one made of glass) and each type of container was filled with the four simulants

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described above, making a total of 12 different containers per set. Each set was kept in the refrigerator at 4

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ºC for 1, 3, and 7 d, respectively. Another set of food containers was stored in the freezer at -18 ºC for 1, 4,



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and 12 w, respectively. The experiments of heating in a microwave were as follows. The food containers

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were heated for 2 min at 800 W. Then, each food container was heated for 1 min more at 800 W. This set

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of food containers was reused in the same conditions (2 min + 1 min at 800 W). One more set of household

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food containers was prepared and kept in the freezer for 1 w. Then, the defrosting of their contents was

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carried out in a microwave at 180 W for 5 min, according to the manufacturer’s indications. A total of 132

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experiments were carried out. After the treatments, 1 mL of the simulants were transferred to glass vials

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for UHPLC and a volume 20 µL of a solution of 5 mg/L of the isotopically labeled standards was added to

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the vial. The vials were kept at -18 ºC until their analysis and each sample was injected in the

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chromatographic system without any further pre-treatment. Blank samples were prepared every day of

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sample preparation and the EDC signals (if any) were subtracted from the signal of the samples.

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RESULTS AND DISCUSSION

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· MRM method development and analytical characterization

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As previously mentioned, the instrumental determination of DMP, DEP, DBP, BBP, DEHP, and BPA was

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already optimized and characterized previously,32 so here the same procedure was followed for DiNP, 6,

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BPB, 8, BPF, 9, BADGE, 10, BFDGE, 11, BADGE·HCl, 12, BADGE·H2O, 13, BADGE·HCl·H2O, 14, and

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BADGE·2H2O, 15, optimization. Two different ion transitions were selected for each analyte, in order to use

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the most abundant as the quantitative transition and the other transition as the confirmatory one. In the

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case of bisphenol derivatives (BADGE, 10, BFDGE, 11, BADGE·HCl, 12, BADGE·H2O, 13, BADGE·HCl·H2O, 14,

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and BADGE·2H2O, 15), ammonium adducts were selected as precursor ions for showing higher intensities,

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as experienced previously.33 The energy in the collision cell was optimized for all the transitions

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independently, in order to gain in signal intensity. First, the energies were studied in the range from 5 to 30

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eV (5, 10, 15, 20, 25, and 30 eV). Then a fine optimization was done around the optimal energy in a range of

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10 eV, one by one (Table 1). Two detection windows were programmed for different groups of analytes,

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according to their retention times, in order to enhance the global sensitivity of the analysis. The first


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detection window was from 0 to 2.3 min and included DMP, 1, BPF, 9, and BADGE·2H2O, 15. The second

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window included the compounds eluted between 2.1 and 6.0 min and included DEP, 2, DBP, 3, BBP, 4,

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DEHP, 5, DiNP, 6, BPA, 7, BPB, 8, BADGE, 10, BFDGE, 11, BADGE·HCl, 12, BADGE·H20, 13, and

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BADGE·HCl·H2O, 14.

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As previously reported,32 once the MRM method was developed for DiNP, 6, BPB, 8, BPF, 9, and bisphenol

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derivatives, 10-15, it was characterized in terms of precision, instrumental limits of detection and

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quantitation (iLODs and iLOQs), and linear dynamic ranges. Precision was evaluated at two different levels,

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the repeatability (calculated as the relative standard deviation, RSD (%), of three consecutive injections)

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and the intermediate precision (calculated as the RSD of four injections carried out on different days

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covering two different weeks), at three different concentration levels, i.e. 5, 50, and 500 pg on column for

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all the compounds except DiNP, 6, (50, 500, and 5000 pg on column) and BADGE·HCl·H2O, 14, (10, 100, and

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1000 pg on column). RSD values were lower than 12% and 16% for repeatability and intermediate

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precision, respectively, except in the case of DiNP, 6, BADGE·HCl, 12, and BADGE·HCl·H2O, 14, which RSD

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values were quite higher. iLOD and iLOQ calculations were based on the measure of the standard deviation

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(sb) of the signal of a standard in which the analytes are spiked at a concentration close to iLOQ and the

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slope of the calibration equation for each compound (m) using the formulas 3sb/m and 10sb/m for iLOD and

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iLOQ, respectively.32 iLOD values were between 0.12 and 2.2 pg on column, for all the compounds

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characterized, except for BADGE·HCl·H2O, 14, and DiNP, 6, which presented higher iLOD (6.4 and 12 pg on

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column, respectively). Dynamic ranges were tested using nine calibration points in the interval from 5 to

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2500 pg on column, and different linear ranges were observed for the different compounds. Calibration

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curves adjusted to a linear fit between 5 and 500 pg on column for all the compounds, except BPF (5-2500

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pg on column) and DiNP, 6, and BADGE·HCl·H2O, 14, (50-2500 pg on column). In all cases, correlations

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coefficients were higher than 0.98.

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Characterization parameters for DMP, 1, DEP, 2, DBP, 3, BBP, 4, DEHP, 5, and BPA, 7, can be found in the

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previous paper .32

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· Migration test



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In order to test the migration of EDCs, food simulants were used in accordance with the European Union

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regulations.16 Simulants A, B, and C were used to simulate foods with hydrophilic character. Food simulant

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B simulated the use of foods with pH values below 4.5. Simulant C was for those foods containing a

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relevant amount of organic ingredients, which make the food to show a more lipophilic character and for

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foods with alcohol content of up to 20%. Simulant D1 simulated the use of food that has lipophilic

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character. In particular, this simulant was for oil-in-water emulsions and alcoholic foods with alcohol

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content above 20%. Food simulant A was used to simulate the rest of general foods.

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Three different food containers (semi-disposable plastic, bisphenol A-free plastic, and glass) were selected

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for this work, which form a representative sample of commercially available household containers found in

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supermarkets to be used for food storage and transportation. ATR-IR analyses showed that semi-disposable

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food containers were made of flexible polypropylene (PP), both body and lid, and, according to the

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manufacturer’s specification, they should not be used more than five times. BPA-free food containers were

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made of rigid PP (the body of the container as well as the lid), with a piece of ethylene propylene diene

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monomer (EPDM) rubber in the lid to guarantee the hermetic sealing and, according to the manufacturer,

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BPA is not used in their fabrication. These containers were 15 times more expensive than the semi-

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disposable ones. Glass food containers had a glass body, while the lid was made of rigid PP with an EPDM

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rubber piece to ensure a hermetic closing, similar to the BPA-free food containers. Glass food containers

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were 25 times more expensive than semi-disposable food containers. Figure 2 shows, as an example, the

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ATR-IR spectra of the body of semi-disposable and BPA-Free containers compared to PP library spectra. The

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household food containers were filled with 250 mL of simulant, which is the approximate volume of a meal

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portion.

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For the common population, household food containers are usually employed to store food in the

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refrigerator for short periods of time or in the freezer for longer periods, in order to preserve it. They are

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also used to transport food, the work place being the most common. Then, the food is heated up, most of


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the times inside the food container and using microwave ovens. Food containers are repeatedly used over

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time for the same purposes. In certain cases, microwaves are also employed to defrost the food inside the

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food container, before using it. In order to reproduce, as faithfully as possible, the use that consumers

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make of the food containers, all these situations were covered in the experimental design (Table 2), in

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which all the possible combinations of food containers with food simulants were prepared. Therefore, the

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household food containers were stored in the refrigerator for three different periods of time within a

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normal use, from 1 d to 7 d, and inside the freezer also for three different typical times, from 1 w to 12 w,

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with the aim of studying the effect of the time of storage on the migration of EDCs. The food simulants

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were also heated, firstly, for 2 min and then it was heated for 1 min more in a microwave at maximum

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power (800 W), in order to study the migration of EDCs depending on the temperature reached and the

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same food containers were used again to study the migration depending on the aging of the container. The

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microwave was also used to defrost the simulants stored in the freezer at the power specified by the

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manufacturer (180 W) during 5 min, with the aim of evaluating the effect of the microwave when it is used

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for longer periods of time with lower power.

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Most of the studies reviewed in the introductory part of this manuscript have focused on specific food

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containers under extreme conditions. Unlike those references, the experiments performed in this work

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were designed to consider the most common food containers used daily by regular users and their habitual

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practices. The aim was to reproduce real situations that any user might experience at home every day in

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order to offer valuable information about the EDC migration occurring during these habitual practices.

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· Concentration levels of EDCs

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Tables 3 and 4 show the total concentration of EDCs found in the migration tests, expressed in pg/mL of

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simulant and calculated as the sum of the concentrations of the fifteen compounds included in the study.

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BPB, 8, BFDGE, 11, and BADGE·HCl, 12, were not detected in any of the analyzed samples. DMP, 1, DiNP, 6,

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BBP, 4, BADGE·H2O, 13, and BADGE·HCl·H2O, 14, were found in less than 1% of the experiments. On the



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other hand, DEP, 2, and DBP, 3, were the most frequently detected EDCs, being present in more than 33%

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and 63% of the experiments, respectively. This might be due to the extensive use of these EDCs in the

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formulations of plastics for household food containers. It is also remarkable that BPA-free containers did

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not show the use of BPA or other alternative bisphenols, such as BPB, 8, and BPF, 9, which was in

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agreement with the specifications from the manufacturer.

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The migration of EDCs depended on the type of simulant, the type of food container, as well as the type of

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storage, time, and use in the microwave. However, some general trends can be drawn from the results

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obtained in the present study. Considering the storage experiments (in the refrigerator and in the freezer),

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an increasing tendency along the storage time was observed for all the containers tested. In Figure 3A it

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can be seen this increase in the EDCs concentration, which behaved similarly in the three types of food

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container and that was more intense in simulants B and C after the storage. The total EDC concentrations

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found in each particular experiment and time are quite similar for the three containers investigated (i.e.,

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semi-disposable, BPA-free, and glass) showing a similar behavior independently of the nature of the

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container (plastic of different characteristics or glass). Although it was not expected to find plasticizers in

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glass containers, their presence might be explained by the plastic lids with the EPDM rubber sealing used to

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close the glass body. This could indicate that, although the body is made of glass, the lids also contribute to

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EDC migration. A similar trend can be observed for the different simulants in the storage experiments

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(Figure 3B). The total migration increases with the storage time for all the simulants tested, mainly for

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those with a low percentage of ethanol and acetic acid (simulants A, B, and C). This increase is less

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pronounced in the case of simulant D1, which corresponds to food with highly lipophilic character.

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In the case of the heating experiments carried out in the microwave, a wide variation was observed for

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both, the type of container used and the simulant employed (Figure 4A and 4B, respectively). In the first

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case (Figure 4A), semi-disposable containers showed higher migration after the first use, the total ECD

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concentrations decreasing after the second use. This might indicate that, although the migration of EDCs to

301

food simulants can be prolonged in time, the intensity of this migration is different and might be more


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intense after the first stages of heating than after subsequent uses of the containers. On the other hand,

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although for all types of containers the concentrations of EDCs found decreased after the 2 min of reuse,

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the concentrations of EDCs were increased again after the 2+1-min-reuse experiments. This seems to

305

indicate that the EDC migration will be increased at the high temperatures reached during the experiments

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and longer heating times. In all cases, the lowest migration was observed for the defrosting experiments,

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which used lower heating energies. As it has been stated above, an increase in the microwave power will

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lead to more intense EDC migrations into the food simulants. When the different simulants are considered

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(Figure 4B), the most remarkable finding is that, contrary to that observed in the storage experiments, the

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highest migration was produced when simulant D1 was used. Simulants B and C showed a similar behavior,

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the migration increasing with the heating time in the first use, then decreasing in the experiments of 2-min-

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reuse and increasing again, reaching the maximum concentrations in the experiments of 2+1-min-reuse.

313

Simulant D1 behaved similarly. However, the concentrations of EDCs found in this simulant were much

314

higher in general than in simulants B and C. On the other hand, the migration processes found for simulant

315

A were completely different. The total concentrations found in this case were the lowest among all the

316

food simulants used. Moreover, after the first heating for 2 min, the concentrations of EDCs decreased

317

after the following experiments. This indicates that simulant A is the simulant with least extracting capacity

318

and, therefore, the foods simulated by simulants B, C, and D1 (foods with pH below 4.5 and foods with

319

lipophilic characters) have more extracting capacity of EDCs from household food containers and might be

320

less appropriate for their storage in these types of food containers.

321

In general, the concentration levels for the analytes found in the present work were lower than those

322

reported in previous works where the migration of the same endocrine disruptors were evaluated10, 12, 17, 24-

323

29

324

comparison with the milder but real domestic conditions used in this work.

. This is probably due the strong conditions (temperature, storage time, etc.) used in those works in

325 326

ACKNOWLEDGMENTS

327

Authors thank Mrs. Sagrario Calvarro for instrumental maintenance and control and Enrique Blázquez for

328

ATR-IR analyses.



Page 14 of 28

329 330

ABBREVIATIONS USED

331

ATR, attenuated total reflectance; BADGE, bisphenol A diglycidyl ether; BADGE·2H2O, bisphenol A bis(2,3-

332

dihydroxypropyl) glycidyl ether; BADGE·H2O, bisphenol A (2,3-dihydroxypropyl) glycidyl ether; BADGE·HCl,

333

bisphenol A (3-chloro-2-hydroxypropyl) (2,3-dihydroxypropyl) ether; BADGE·HCl·H2O, bisphenol A (3-

334

chloro-2-hydroxypropyl) (2,3-dihydroxypropyl) ether; BBP, butyl benzyl phthalate; BFDGE, bisphenol F

335

diglycidyl ether; BPA, bisphenol A; BPB, bisphenol B; BPF, bisphenol F; DBP, dibutyl phthalate; DEHP, diethyl

336

hexyl phthalate; DEP, diethyl phthalate; DiNP, di-iso-nonyl phthalate; DMP, dimethyl phthalate; EDCs,

337

endocrine disrupting compounds; EPDM, ethylene propylene diene monomer; iLODs, instrumental limits of

338

detection; iLOQs, instrumental limits of quantitation; PE, polyethylene; PET, polyethylene terephthalate;

339

PP, polypropylene; RSD, relative standard deviation.

340 341

FOUNDING SOURCES

342

Financial support was obtained from MICINN (project AGL2012-37201) and Community of Madrid (Spain),

343

and European funding from FEDER program (project S2013/ABI-3028-AVANSECAL).

344 345

SUPPORTING INFORMATION DESCRIPTION

346

Analytical characteristics of the developed UHPLC-QqQ(MRM) method in terms of instrumental

347

repeatability (relative standard deviation, RSD), instrumental intermediate precision, and instrumental

348

limits of detection (iLOD) and quantitation (iLOQ), for the analytes characterized in the present study (Table

349

S.1.).

350 351

REFERENCES

352

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bisphenol A from polycarbonate glasses. J. Chromatogr. A 2014, 1360, 23–38.

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435

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437


Page 19 of 28


438

FIGURE CAPTIONS

439

Figure 1. Chemical structures of EDC studied.

440

Figure 2. ATR-IR spectra of the body of semi-disposable and BPA-Free containers compared to PP library

441

spectra.

442

Figure 3. Variation of total EDC concentrations (pg/mL of simulant) with time in storage experiments

443

(refrigerator and freezer) considering A, the container and B, the simulant used.

444

Figure 4. Variation of total EDC concentrations (pg/mL of simulant) with time in heating experiments

445

(defrost and heat in the microwave) considering A, the container and B, the simulant used.

446



Page 20 of 28

Table 1. MRM parameters for the studied analytes. Compound DMP, 1

Retention time (min) 1.44

Transitiona

Ion transitionb

162.9-76.9

4.2

162.9-91.9 DEP, 2

2.50

223.1-177.0

1.3

223.1-148.9 DBP, 3

3.60

279.2-205.1

3.5

279.2-149.0 BBP, 4

3.63

313.2-149.0

2.6

313.2-90.9 DEHP, 5

4.04

391.3-279.1

10.3

391.3-149.0 DiNP, 6

4.10

419.6-85.0

1.05

419.6-149.2 BPA, 7

2.33

227.0-211.9

3.1

227.0-132.9 BPB, 8

2.72

241.1-212.1

2.3

241.1-211.1 BPF, 9

1.58

199.1-93.0

1.6

199.1-105.0 BADGE, 10

3.51

358.2-191.1

5.3

358.2-135.1 BADGE·H2O, 13

2.87

376.2-209.1

1.6

376.2-135.1 BADGE·2H2O, 15

1.92

394.2-209.1

1.6

394.2-135.1 BADGE·HCl, 12

3.54

394.2-227.3

1.7

394.2-167.2 BADGE·HCl·H2O, 14

3.09

412.2-167.4

1.2

412.2-131.0 BFDGE, 11

3.34

330.2-163.3

1.1

330.2-133.1

CID voltage (eV) 23

ESI mode

26

POS

17

POS

7

POS

7

POS

13

POS

20

POS

22

POS

7

POS

23

POS

13

POS

28

POS

21

NEG

19

NEG

18

NEG

29

NEG

20

NEG

20

NEG

11

POS

28

POS

12

POS

31

POS

15

POS

33

POS

13

POS

22

POS

26

POS

31

POS

11

POS

16

POS

POS

a

The first transition corresponds to the quantitation transition and the second one to the confirmatory transition. b Calculated as quantitation transition/confirmatory transition intensities.


Page 21 of 28


Table 2. Different food containers, food simulants, treatments, and times used in the 132 experiments carried out. Experimental conditions Containera S-Dis BPA-Free Glass S-Dis BPA-Free Glass S-Dis BPA-Free Glass S-Dis BPA-Free Glass

Simulant

Refrigerator

Refrigerator

Refrigerator

Freezer

Freezer

Freezer

Defrost

Heat (800 W)

Heat (800 W)b

Heat (800 W)

Heat (800 W) b

A

1d

3d

7d

1w

4w

12 w

180 W 5 min

First use 2 min

First use +1 min

Reuse 2 min

Reuse +1 min

B

1d

3d

7d

1w

4w

12 w

180 W 5 min

First use 2 min

First use +1 min

Reuse 2 min

Reuse +1 min

C

1d

3d

7d

1w

4w

12 w

180 W 5 min

First use 2 min

First use +1 min

Reuse 2 min

Reuse +1 min

D1

1d

3d

7d

1w

4w

12 w

180 W 5 min

First use 2 min

First use +1 min

Reuse 2 min

Reuse +1 min

a b

S-Dis, semi-disposable food container; BPA-Free, BPA-free food container; Glass, glass food container. Containers were firstly heated in the microwave for 2 min at 800 W and then they were heated for 1 more min at 800 W.



Page 22 of 28

Table 3. Total EDC content in food simulants stored in refrigerator or freezer in different containers for three different periods of time.

Containera

Refrigerator Refrigerator Refrigerator 1d 3d 7d

Freezer 1w

Freezer 4w

Freezer 12 w

pg/mL of simulant

Simulant A

Simulant B

Simulant C

S-Dis

114

393

250

1025

-

1652

BPA-Free

295

113

34

478

-

1435

Glass

203

-

101

995

-

1268

S-Dis

50

-

-

448

772

2602

BPA-Free

51

146

59

814

816

3140

Glass

94

69

35

611

-

2441

S-Dis

37

-

126

361

469

1981

BPA-Free

112

138

28

396

1057

2600

Glass

77

84

99

38

488

3747

S-Dis

464

90

217

112

37

431

63

107

215

336

348

633

286

160

106

216

-

1412

Simulant D1 BPA-Free Glass a

S-Dis, semi-disposable food container; BPA-Free, BPA-free food container; Glass, glass food container.


Page 23 of 28


Table 4. Total EDC content in food simulants after heating the different containers in the microwave oven.

Containera

Defrost (180 W)

First use (800 W) 2 min

First use (800 W) 3 min

Reuse (800 W) 2 min

Reuse (800 W) 3 min

pg/mL of simulant

Simulant A

Simulant B

Simulant C

Simulant D1

a

S-Dis

118

284

200

124

55

BPA-Free

76

419

106

176

53

Glass

14

242

130

52

33

S-Dis

-

338

207

219

531

BPA-Free

-

29

-

196

182

Glass

-

263

352

278

1309

S-Dis

54

518

649

270

555

BPA-Free

559

200

135

84

361

Glass

54

147

277

147

440

S-Dis

376

412

1374

805

549

BPA-Free

56

483

763

404

1059

Glass

22

739

952

787

818

S-Dis, semi-disposable food container; BPA-Free, BPA-free food container; Glass, glass food container.



Page 24 of 28

TOC Graphic


Page 25 of 28


Figure 1.

Bisphenol B (BPB), 8

Dimethyl phthalate (DMP), 1

Bisphenol F (BPF), 9 Diethyl phthalate (DEP), 2

Bisphenol A diglycidyl ether (BADGE), 10 Dibutyl phthalate (DBP), 3

Bisphenol F diglycidyl ether (BFDGE), 11

Butyl benzyl phthalate (BBP), 4

BADGE·HCl, 12

Diethyl hexyl phthalate (DEHP), 5

BADGE·H2O, 13

Di-iso-nonyl phthalate (DiNP), 6

BADGE·HCl·H2O, 14

Bisphenol A (BPA), 7 BADGE·2H2O, 15

ACS Paragon Plus Environment


Page 26 of 28

Figure 2.

100

80

T (%)

60

40

20

PP (library) S-Dis BPA-Free

0 1000

1500

2000

2500

cm

3000

-1


3500

4000

Page 27 of 28


Figure 3.

A

Total EDC concentration (pg/mL of simulant)

S-Dis

BPA-Free

Glass

2500

2000

1500

1000

500

0 1d Refrigerator

3d Refrigerator

7d Refrigerator

1w Freezer

4w Freezer

12 w Freezer

Storage experiments

B


Simulant A

Simulant B

Simulant C

Simulant D1

3000 2500

2000 1500 1000 500 0

1d Refrigerator

3d Refrigerator

7d Refrigerator

1w Freezer

Storage experiments


4w Freezer

12 w Freezer


Page 28 of 28

Figure 4.

A


S-Dis

BPA-Free

Glass

700 600 500 400 300

200 100 0 (180 W) Defrost

2 min First use (800 W)

3 min (800 W) First use

2 min (800 W) Reuse

3 min (800 W) Reuse

Heating experiments

B


Simulant A

Simulant B

Simulant C

Simulant D1

1200 1000 800 600 400 200 0 (180 W) Defrost

2 min First use (800 W)

3 min (800 W) First use Heating experiments


2 min (800 W) Reuse

3 min (800 W) Reuse

Evaluation of Endocrine Disrupting Compounds Migration in

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