Food Chemistry 420 (2023) 136097 Available online 8 April 2023 0308-8146/© 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). Risk assessment of silver and microplastics release from antibacterial food containers under conventional use and microwave heating Estefanía Moreno-Gordaliza , M. Dolores Marazuela , M. Milagros Gómez-Gómez * Department of Analytical Chemistry, Faculty of Chemistry, Universidad Complutense de Madrid, Avda. Complutense s/n, 28040 Madrid, Spain A R T I C L E I N F O Keywords: Silver Antimicrobial containers Nanoparticles Microplastics Risk assessment Single particle-ICPMS A B S T R A C T The evaluation of the migration of ionic silver and nanoparticulated silver (AgNPs) from antimicrobial plastic packaging to food is crucial to ensure its safety. Migration assays were performed on reusable silver-containing polypropylene (PP) food containers and a silicone baby bottle, using food simulants, under conventional or microwave heating and repeated use. The PP containers released significant amounts of silver, increasing with temperature, contact time, acidity and lower crystallinity. Silver migration in the silicone bottle was much lower. Risk assessment of released silver was done considering European authorities safety recommendations, with some containers far exceeding these levels. No significant AgNPs release was detected in the simulants by single particle-ICPMS. Silver-containing microplastics and silicone microparticles were detected by SEM in the food simulants after the migration assays. Consumers may be continuously exposed to the harmful effects of ionic silver and microplastics, which can potentially lead to health issues. 1. Introduction Food packaging has evolved considerably during the last few years to meet the increasing demands of consumers for food quality, safety, and extended preservation. In particular, active packaging systems have been developed (Restuccia et al., 2010), incorporating certain agents, such as antimicrobial compounds, into food contact materials (FCM), with the aim to prevent food spoilage caused by microbial growth and thus, to maintain and extend food shelf-life. Different metallic compounds such as silver (either in the form of silver ions (Ag+) or silver nanoparticles (AgNPs)) (Carbone et al., 2016), zinc and copper (both as oxide nanoparticles, ZnONPs, CuONPs) (Song et al., 2014) have been extensively tested as antimicrobial agents in food packaging materials. It is not yet clear whether the biocide effect of AgNPs is mainly caused by the released particles themselves, by silver ions generated upon particle oxidation, or both. (Hadioui et al., 2013). Features affecting AgNPs environment and biokinetics behavior include dimensions and shape, surface characteristics, disaggregation degree, among others (Mitrano et al., 2012). In vitro and in vivo studies have shown cellular DNA damage, inflammation and oxidative stress that can be caused by AgNPs of different size (Suliman et al., 2015), with their main accumulation taking place in the liver (Juling et al., 2016). Both ionic silver and AgNPs can cause liver toxicity but some recent studies indicate that silver ions produced from AgNPs could be the key in the toxicity exerted by the latter (Smith et al., 2018). Regulations on the use of silver as antimicrobial agent in FCM have been continuously revised due to the difficulty of making definite con- clusions on its toxicity. For this reason, due to the growing concern about potential toxic effects from constant exposure to AgNPs, their use in plastic FCM is currently prohibited in the EU (European Commission, 2011); while the use of silver in its ionic form is permitted. Various silver ion-based agents (including diverse silver zeolites, silver phosphates and silver chloride) employed as biocides are included in the European Commission provisional list of additives temporarily allowed in plastic FCM, but they are still under evaluation for a final decision on their approval. However, in 2021, the biocidal products committee of the European Chemicals Agency (ECHA) proposed not to approve four of them (silver zinc zeolite, silver zeolite, silver copper zeolite and silver sodium hydrogen zirconium phosphate). Therefore, the use of silver in FCM is subject to cautious regulation in the EU and its safety is assessed in terms of ionic silver migrating to food in contact with the material. However, in many countries worldwide, product registration as silver- containing (regardless of its species) is usually sufficient to comply with legislation. Moreover, various plastic food contact products avail- able in the global market claim to include nanosilver as antibacterial, including: food containers, food bags, drinking bottles, or even products * Corresponding author. E-mail addresses: emorenog@ucm.es (E. Moreno-Gordaliza), marazuela@quim.ucm.es (M. Dolores Marazuela), mmgomez@ucm.es (M. Milagros Gómez-Gómez). Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem https://doi.org/10.1016/j.foodchem.2023.136097 Received 1 February 2023; Received in revised form 27 March 2023; Accepted 30 March 2023 mailto:emorenog@ucm.es mailto:marazuela@quim.ucm.es mailto:mmgomez@ucm.es www.sciencedirect.com/science/journal/03088146 https://www.elsevier.com/locate/foodchem https://doi.org/10.1016/j.foodchem.2023.136097 https://doi.org/10.1016/j.foodchem.2023.136097 https://doi.org/10.1016/j.foodchem.2023.136097 http://crossmark.crossref.org/dialog/?doi=10.1016/j.foodchem.2023.136097&domain=pdf http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ Food Chemistry 420 (2023) 136097 2 for babies, such as milk bottles, teethers or pacifiers. Meanwhile, several commercial brands worldwide-available such as Microban Silver- shieldTM, AlphasanTM, SteritouchTM incorporate ionic silver in different chemical forms, such as silver phosphate glass or silver sodium hydrogen zirconium phosphate, in plastic polymers for food contact materials with antimicrobial properties. Commission Regulation 10/2011 (EU) establishes the experimental conditions for migration tests from plastic FCM (Commission Regulation [EU], 2011). Migration testing using food simulants is accepted by EU legislation as a valid and reliable method of verifying compliance of FCM with certain migration limits (Jakubowska et al., 2020). The testing conditions are defined based on the estimated temperature and duration of use of the food containers. Despite the fact that it is not established in the EU law, the assessment of the migration in such FCM under common daily-life use conditions (e.g. microwave heating of stored meals), would be relevant (Bhunia et al., 2013). Recently, the Panel on Food Contact Materials, Enzymes and Pro- cessing Aids (CEP) in the EFSA (EFSA, 2021) issued a scientific opinion on the safety evaluation of the use of AgNPs as biocide in non-polar plastic FCM. The study, considering diverse foods and storing condi- tions, concluded that the AgNPs remain embedded in the polymeric material, with no migration detected, thus neither lead to dietary exposure nor pose toxicological risk, as long as the polymer does not swell upon contact with aqueous foodstuff or simulants. However, contact with the food simulants 3 % acetic acid and 10 % ethanol causes migration of Ag+ up to 6 µg kg− 1 of food from the superficial oxidation of the particles. This is lower than the group restriction of 0.05 mg Ag kg− 1 food proposed by the EFSA Panel on Food Additives, Flavourings, Pro- cessing Aids and Food Contact Materials (AFC) in 2004 (EFSA, 2021), and would represent an exposure from the material lower than the acceptable daily intake (ADI) of 0.9 µg Ag ions kg− 1 body weight (bw) per day set by the ECHA. They concluded that AgNPs do not pose any consumer safety concerns when used as additives up to 0.025 % (w/w) in some polymeric materials, including styrene, polyester or polyolefin. Nevertheless, cumulative exposure to silver including other dietary source may surpass the ECHA-established ADI. In recent years, single particle-inductively coupled plasma mass spectrometry (sp-ICPMS) has been proposed as a useful technique for the characterization and quantification of metallic NPs at the ultratrace level in complex extracts (Laborda et al., 2014). Furthermore, it is appropriate for speciation studies based on the possibility of performing determination of ion species and NPs at once, based on the differential intensities of the time-resolved signals, when analyzing much diluted samples. Previous AgNPs migration studies, following EU Regulation 10/2011, carried out by our group with commercially available food packaging from different worldwide geographical areas, revealed by sp- ICPMS the release of AgNPs into the food simulants and their partial oxidation to Ag+ (Ramos et al., 2016). Size distribution and concen- tration of particles were determined. Additionally, limited sample handling and rapid analysis were involved, minimizing the possibility of particles aggregation and oxidation that could otherwise cause analyt- ical inaccuracy. Exposure to microplastics (MPs) from plastic FCM and its associated risks have been previously reported (Du et al., 2020; Fadare, Wan, Guo, & Zhao, 2020; Kedzierski et al., 2020; Marazuela et al., 2022; Rainieri & Barranco, 2019). Furthermore, triclosan-containing MPs have been detected in the extracts of plastic food packages containing the antimi- crobial triclosan, after microwave migration tests (Marazuela et al., 2022). Therefore, the ingestion of these MPs can cause long term dam- age to certain tissues and organs, and they can serve as carriers for various chemical compounds, such as silver or other food packaging additives. MPs are capable of penetrating tissues and organs, causing inflammation, cell damage, and oxidative stress (Li et al., 2005). On the other hand, the fact that these MPs are associated with metallic biocides can increase their toxicity. In previous literature, no comprehensive study addressing silver migration from FCM and its speciation, polymer characterization, microplastics detection and silver risk assessment has been performed, which is the aim of this work. Consequently, the results obtained in this study can be of great help as a risk assessment of currently commercially available silver-based antimicrobial food packaging and very useful for regulatory authorities and policy makers regarding legislation related to safety in the food chain. 2. Materials and methods 2.1. Reagents and food containers Analytical grade reagents were used in all cases, unless otherwise stated. Nitric acid (65 %) (Merck), hydrogen peroxide (35 %) and hy- drofluoric acid (48 %) (Panreac, Barcelona, Spain) were employed for sample digestion. High purity HNO3 was obtained by distillation of the commercial acid by using an in-house-made polytetrafluoroethylene (PTFE) distiller equipped with an IR-lamp. A silver elemental standard solution of 1000 mg L− 1 in 2 % (v/v) HNO3 (TraceCERT, Merck Life Science, Madrid, Spain) was appropriately diluted and used for ICPMS quantification. An indium standard solution (1000 mg L− 1 in 2 % HNO3, TraceCERT, Merck Life Science, Madrid, Spain) was appropriately diluted and added as an internal standard (1–10 µg L− 1) for all ICPMS quantifications. Citrate-stabilized gold nanoparticles (AuNPs) and AgNPs of 60 and 40 nm, respectively (nanoComposix, San Diego, CA, USA) were used for calibration in sp-ICPMS analysis. Separate stock AuNPs and AgNPs solutions were prepared on a daily basis in Milli-Q water and sonicated for 20 min in an ultrasonic bath to ascertain disaggregation. Solutions were kept in the dark until analysis. Glacial acetic acid (HAc) (Panreac, Barcelona, Spain) and absolute ethanol (HPLC grade, Scharlab, Barcelona, Spain) were used for preparation of food simulants solutions. Ultrapure Milli-Q water (Merck-Millipore, Bedford, MA, USA) was used for preparation of aqueous solutions. Microban antimicrobial polypropylene (PP)-containing FCM from several providers (NeoflamTM CLOC lid, OMADATM Sanaliving drinking cup, and RubbermaidTM EasyFindLids containers with SilvershieldTM) and different geographic origins around the world (South Korea, Italy and USA respectively); in addition to a silicone baby feeding bottle and nipple with nanosilver (ValuederTM, South Korea), were used in this study (Fig. S1). All these world-wide available containers were pur- chased online. These FCM lack clear statements on the specific antimi- crobial agent employed embedded within the polymeric material or its chemical form, and were sold as compatible with microwave heating and both fridge- or freezer-storing. 2.2. Acid digestion of FCM Acid digestion of the FCM was assisted with a microwave (Micro- wave Accelerated Reaction System, MARS 5, CEM, USA) using PTFE Easyprep vessels and a temperature probe. In the case of PP materials, ca. 0.1–0.3 g were acid-digested at 1600 W with 7 mL of HNO3 and 3 mL of H2O2 (Ramos et al., 2016). The temperature program increased from room temperature (RT) to 210 ◦C at 12 ◦C min− 1, with the final tem- perature being kept for 10 min. Silicone baby bottle and nipple (between 0.1 and 0.2 g) were digested in 5 mL of HNO3, 1.5 mL of HF and 2 mL of H2O2 at 1600 W (Wu et al., 1996). Increase from RT to 180 ◦C was performed at 8 ◦C min− 1, and the final temperature was kept for 20 min. Digests were transferred to PTFE beakers with addition of 3 mL HNO3, then evaporated to almost dryness by heating at 200 ◦C and finally 3 mL HNO3 were added for reconstitution. In all cases, digested samples were diluted with Milli-Q water to a final volume of 50 mL for ICPMS analysis. The drinking cup (ca. 1 g) was calcined in a muffle oven (LE 14/11, P300, Naberttherm, Germany) using the following conditions: rise from RT to 250 ◦C at 11 ◦C min− 1 rate, then temperature was increased to 500 ◦C at 12.5 ◦C min− 1, and finally to 600 ◦C at 10 ◦C min− 1, keeping the final temperature for 20 min (Ramos et al., 2016). Reconstitution of E. Moreno-Gordaliza et al. Food Chemistry 420 (2023) 136097 3 ashes was done with 5 mL of HNO3 by heating. The acid was evaporated by heating to ca. 1 mL and further diluted to a final volume of 50 mL with Milli-Q H2O. A total of three replicates per sample and three blanks were prepared. Total silver was determined by ICPMS. 2.3. Migration assays Tests were performed according to Commission Regulation 10/2011 (EU) and those testing recommendations published by the EU Com- mission Joint Research Center (Jakubowska et al., 2020). A total of three replicates per sample and three blanks per experiment were prepared. 2.3.1. Conventional use assays The investigated FCM were sectioned into pieces (typically 1.0 × 1.0 cm, ~1.5–2 mm-thick, total surface area of ~ 2.6–3.0 cm− 2) and weighed. A home-made glass rod was placed on top of the FCM pieces for total submersion in the food simulants within 40-mL borosilicate glass vials (National Scientific, CA, USA), containing between 3 and 5 mL of the solutions, considering a contact of 6 dm2 FCM surface per kg of simulant, namely, ultrapure H2O, 3 % (w/v) HAc, 10 % (v/v) ethanol and 95 % (v/v) ethanol. The latter solutions were intended for simu- lating migration in aqueous, acidic, alcohol-containing and fat- containing food from plastic FCM (Commission Regulation [EU], 2011 & Commission Regulation [EU], 2016). The tubes were screw-capped and kept at either 40 ◦C or 20 ◦C for 10 days, and at 70 ◦C for 2 h in the dark in an incubator (Incudigit – TFT, 36 L, Selecta, Barcelona, Spain). After the incubation, the FCM pieces were removed and the 10 % EtOH, H2O and 3 % HAc extracts were sonicated in an ultrasonic bath (Elma S 60, Elmasonic, Singen, Germany) for 5 min prior to transfer to 5- mL high density polyethylene (HDPE) tubes (Sarstedt AG&Co., Nüm- brecht, Germany). In previous studies we demonstrated that no signifi- cant loss of AgNPs was found in the high density polyethylene (HDPE) tubes (Artiaga, Ramos, Ramos, Cámara, & Gómez-Gómez, 2015). Distilled HNO3 was added to the extracts up to a final acid concentration of 2 % (v/v). Those extracts corresponding to 95 % ethanol were evaporated in a MiniVac duo concentrator (Genevac, Suffolk, United Kingdom) to avoid further unstable spray or plasma during ICPMS analysis. Finally, they were reconstituted in 2 % HNO3, sonicated and transferred to HDPE tubes. All the samples were kept at − 20 ◦C until total silver analysis by ICPMS. 2.3.2. Microwave use assays A microwave oven (Samsung, South Korea) operated at 700 W for 2 min was used to mimic domestic default heating. Pieces of the FCM (typically 1.0 × 1.0 cm, ~1.5–2 mm-thick, surface contact area of ~ 2.6–3.0 cm− 2) were soaked in 5 mL of water or 3 % HAc in 100 mL PTFE vessels (3.2 cm id. × 13.2 cm height, in-house made, mechanical ser- vices facility, UCM), with a tight screw-lid. A total of three replicates per sample and three blanks per experiment were prepared. Afterwards, the vessels were ice-cooled prior to opening, followed by removal of the plastic pieces and transfer of the extracts to 5-mL HDPE tubes. Water extracts were immediately spiked with distilled HNO3 to a final con- centration of 2 % and kept in a freezer at − 20 ◦C until analysis by ICPMS. 2.3.3. Repeated use migration assays With the aim of evaluating silver migration under repeated use of FCM, consecutive extraction experiments were carried out in a micro- wave oven (2 min heating at 700 W) and in a regular oven at 70 ◦C for 2 h, as described in Sections 2.3.1 and 2.3.2, respectively. Three sequen- tial extractions were carried out on the same FCM piece, by using fresh aliquots of food simulant (3 % HAc or Milli-Q H2O) after each extraction, and were stored separately at − 20 ◦C in 5-mL HDPE tubes until analysis by ICPMS. Prior to this, distilled HNO3 was added to water extracts to a final concentration of 2 %. 2.4. Total silver determination by ICPMS Total Ag quantification in the digested FCM and the samples from migration tests was carried out with a quadrupole ICPMS (7700x, Agi- lent Technologies, USA). A quartz Fassel torch (2.5 mm injector), a Meinhard (Conikal) nebulizer and a Scott-type spray chamber kept at 2 ◦C with a Peltier, were used. Monitoring of silver at m/z 107 and 109 was performed, while indium was measured at m/z 115, as internal standard, using a dwell time of 300 ms. ICPMS was operated with a forward power of 1.55 kW and the following Ar flow rates: plasma gas (15 L min− 1); auxiliary gas (0.90 L min− 1); nebulizer gas (1.00 L min− 1). All the samples were sonicated prior to analysis. External calibration was used for quantitation, in the range 0.01–20 µg Ag L− 1 in 2 % HNO3. Matrix effects were discarded by checking the stable signal of an internal standard (In) added to both quantification standards and samples. Determination of limits of detection (LOD) and quantification (LOQ) was done considering the mean signal and the standard deviation of ten procedural blank solutions and the external calibration parameters, resulting in 3.3 ng L− 1 for the LOD and 11 ng L− 1 for the LOQ. 2.5. sp-ICPMS analysis sp-ICPMS analysis was carried out with the Agilent ICPMS, using a MicroMist nebulizer instead, and a quartz torch with an injector diam- eter of 1 mm. A flow rate of 300 µL min− 1 was used for sample intro- duction and 107Ag was monitored for 1 min with a dwell time of 3 ms. Additional optimized parameters are summarized in Table S1. Data were processed with MassHunter software operated in single particle mode, providing a histogram for signal distribution, the AgNPs number con- centration, size distribution and the ionic silver concentration. Trans- port efficiency was calculated using solutions of AuNPs (60 nm, 25 ng L− 1), while AgNPs diluted in water (40 nm, 25 ng L− 1) were employed as reference. Calibration was carried out with Ag+ standards prepared in 2 % HNO3 between 5 and 100 ng L− 1, and was used for method optimi- zation and AgNPs quantification. In-between samples, washing steps were done with H2O for 1 min, then 2 % HNO3 for 1 min and finally, H2O for 1 min, to avoid cross-contamination. The Tygon sample introduction tube used for the sp-ICPMS analysis was daily changed to prevent po- tential NPs adsorption losses. The raw extracts were handled in the dark and immediately analysed after migration assays to minimise the risk of AgNPs instability, after their appropriate dilution with H2O to a final Ag concentration within ng L− 1 range. To promote particle disaggregation, all the samples and solutions were submitted to ultrasonication with a microprobe for 90 s, using a 3-mm titanium tip (SonoPuls HD 2200, Bandelin, Germany), as previously described, prior to the sp-ICPMS analysis (Ramos et al., 2014). 2.6. Scanning electron microscopy (SEM) A SEM instrument (JEOL JSM-6335F; Tokio, Japan), with an energy dispersive X-ray (EDX) analyzer was used to determine the occurrence of Ag and AgNPs in the polymeric FCM and the extracts from the silver migration tests. To increase the sensitivity of the MPs detection, extracts from three consecutive migration experiments were pooled in a glass vial, evaporated at 80 ◦C and finally dissolved in 1 mL EtOH. A sample droplet was deposited on a brass holder, air-dried and, in some cases, a gold-layer was applied for increasing conductivity. An accelerating voltage of 20 kV was used, with an effective distance of 15 mm for analysis. Images were recorded using 2000–15000 X magnification. 2.7. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy An ATR-FTIR spectrophotometer (Nicolet, iS50, Thermo-Fisher Sci- entific, USA) was used for characterizing PP FCM. Absorbance spectra were recorded from 500 to 3250 cm− 1. A resolving power of 4 cm− 1 was E. Moreno-Gordaliza et al. Food Chemistry 420 (2023) 136097 4 employed and averaging of 60 scans was applied. A reference isotactic PP spectrum was used for comparison. 2.8. Differential scanning calorimetry (DSC) DSC analysis was carried out with a DSC-Q200 instrument (TA In- struments, New Castle, Delaware, USA) for the determination of crys- tallinity in the PP FCM samples, using 3–4 mg of the plastic pieces, either unused or after migration assays in water and 3 % HAc at 40 ◦C for 10 days. N2 was employed as purge gas at a 80 mL min− 1 flow rate. Recording of thermograms was done in cycles: step 1: 35 to 300 ◦C at a rate of 10 ◦C min− 1, step 2: 300 to 40 ◦C at − 10 ◦C min− 1 rate, step 3: 40 to 300 ◦C at 10 ◦C min− 1 rate. TA Universal Analysis software (TA In- struments, New Castle, Delaware, USA) was used for data visualization and integration. Calculation of the crystallinity (χ) was done employing equation 1: χc = ΔHf /ΔH0 f ⋅ 100 (1) where: χc is the crystallinity degree calculated as percentage, ΔHf is the fusion enthalpy found by integration of the melting peaks in the ther- mograms, ΔHf 0 is the fusion enthalpy of PP with 100 % crystallinity, in this case, 209 J g− 1 (Tjong & Xu, 1997). 3. Results and discussion 3.1. Determination of silver content in antimicrobial plastic food packaging Considering that the total silver content in the antimicrobial food containers and the baby feeding bottle and nipple is not provided by the manufacturers, this was determined by ICPMS (n = 3), after their acid digestion or calcination. Silver was present in all food containers tested at concentrations of 70 ± 30, 19.5 ± 0.6, 3 ± 1, 0.08 ± 0.02 mg kg− 1 FCM for Rubbermaid container, Neoflam CLOC lid, OMADA cup and the nipple of baby feeding bottle, respectively; these results correspond to Ag surface contents of 320 ± 180, 108 ± 5, 18 ± 9 and 0.7 ± 0.2 µg dm− 2, respectively. The silver found in the baby feeding bottle was lower than the quantification limit of the method (11 ng L− 1), being present only in the nipple in very low amounts. The relative standard deviations of the Ag contents were very high for all containers, except for Neoflam CLOC lid, and this was considered an indication of the heterogeneous distribution of silver in most of these containers. There- fore, according to our results, the tested containers present different silver contents. This has also been observed with other types of anti- microbial food containers using another biocide, such as triclosan (Marazuela et al., 2022). Fig. 1. Migration of silver from antimicrobial PP containers during simulated hot-fill and/or long-term storage. 3 % HAc, 10 % EtOH, 95 % EtOH and water were assayed as food simulants at A) 70 ◦C, 2 h, B) 40 ◦C for 10 days and C) 20 ◦C for 10 days. Mean migration values for 3 replicates are displayed. Error bars represent standard deviations. E. Moreno-Gordaliza et al. Food Chemistry 420 (2023) 136097 5 3.2. Conventional migration experiments The major factors which determine the migration of compounds from FCM are: the characteristics of the packaging material, the nature of food/food simulants and both the contact temperature and time-span (Poças, 2018). According to Commission Regulation 10/2011 (EU), plastic FCM migration testing at 70 ◦C for 2 h, represents hot-fill or heating at 70 < T < 100 ◦C of the containers. On the other hand, migration assays at 20 ◦C for 10 days are representative of storage of food in a freezer or fridge, while at 40 ◦C for 10 days, they represent long-term storing at RT or lower, including a previous hot-fill. Thus, the amount of silver released from the PP containers was evaluated in terms of time and temperature at 70 ◦C for 2 h, 40 ◦C for 10 days or 20 ◦C for 10 days, of contact with the different food simulants (Fig. 1). As can be seen, all packages tested under all conditions of use were able to release significant amounts of silver into the food simulants. However, the amount of silver released to aqueous (10 % EtOH or H2O) or acidic (3 % HAc) simulants is quite high in comparison to the high fat simulant (95 % EtOH). The highest amount of silver release was found in 3 % HAc under 40 ◦C for 10 days, followed by 20 ◦C for 10 days (about 2–5 times lower), being the lowest amount for 70 ◦C for 2 h (about 2–10 times lower). This behaviour could suggest that a longer contact time results in a greater effect than temperature on the diffusion of silver from the inner layers of the material. Therefore, silver migration increases with contact time, temperature, solvent hydrophilicity, and acidity of the medium. This is in agreement with previous studies on AgNPs-containing FCM which used to be available in the market a decade ago (Ramos et al., 2016). Moreover, the degree of silver released also relies on its concentra- tion in the polymeric material. As stated in the previous section, the silver content for the tested plastic materials differs in several orders of magnitude between containers. The Rubbermaid brand provided the highest migration values, probably because this container has a surface silver content around 3 times higher than the Neoflam CLOC lid and 18 times higher than the OMADA cup. Polymer properties may also influence additive migration. Despite the higher silver content in Rubbermaid’s raw material, its high release rate for all temperatures and simulant solutions is still impressive, ac- counting for up to 12 and 8 % of the total Ag in 3 % HAc and H2O, respectively, at 40 ◦C for 10 days. In contrast, in the latter conditions, the release of silver for the OMADA cup and Neoflam CLOC lid was 3 % of their total silver content in 3 % HAc and below 2 % for the rest of conditions and food simulants. This behaviour suggests that silver in the Rubbermaid material diffuses more easily through the polymer back- bone to the food simulant. All the PP materials found in the containers were determined to be of the same type (isotactic PP), considering their FTIR spectra compared with a reference spectrum, showing exactly the same characteristic peaks (Fig. S2). On the other hand, the diffusion of additives through the polymeric matrix is crucially determined by the crystallinity degree of the PP. Therefore, the latter was measured for the original PP FCM and after heating at 40 ◦C for 10 days in water and 3 % HAc. As can be seen in Table S2, the crystallinity degree of the original Rubbermaid container was lower than those calculated for the OMADA cup and the Neoflam CLOC lid. This fact may also contribute to the higher percentage of Ag released from the Rubbermaid container compared to the other two. In fact, it has been reported that the diffusion coefficient of an additive compound in semicrystalline polymers, such as PP, generally decreases with increasing crystallinity (Földes, 1998). Also, the heating in both solvents causes an increase in the crystallinity for the Rubbermaid container, which indicates that some recrystallization took place during heating. The crystallinity values for the OMADA cup and Neoflam CLOC lid were similar, and the heating process in the two simulants did not cause a significant change in their original values, which is reflected in similar percentages of Ag released. As expected, silver migration from the silicone baby bottle nipple was significantly lower compared to PP containers, consistent with its total raw material content being 2 to 3 orders of magnitude lower than those in PP containers. Release of silver from the nipple was detected under water extraction at 70 ◦C for 2 h and 40 ◦C for 10 days, with a migration of 15 ± 9 and 6 ± 5 ng dm− 2, respectively. Unlike PP con- tainers, for this material the migration was lower in 3 % HAc than in water, with a silver release of 5 ± 4 ng dm− 2 for 70 ◦C for 2 h; and was below the detection limit for 40 ◦C, 10-day migration test. All these results may imply a considerable contribution of these FCM to the human daily intake of silver, as will be assessed in section 3.5. 3.3. Migration under repeated use Since the food containers under study are reusable articles, it is important to determine whether the prolonged use of these FCM may lead to a continued migration of Ag into food. Therefore, silver migra- tion in consecutive heating cycles was also investigated for the Rub- bermaid container, the Neoflam CLOC lid and the OMADA cup. Following Regulation 10/2011 (EU) (Commission Regulation 10/2011), silver migration under repeated use of the FCM has been assessed with Fig. 2. Migration of silver from PP antimicrobial containers under simulated repeated use during hot-fill or heating at 70 < T < 100 ◦C. A) 3 % HAc and B) H2O water were assayed as food simulants at 70 ◦C for 2 h, for n = 3 repeated successive extractions. Mean migration values for 3 replicates are displayed. Error bars represent standard deviations. E. Moreno-Gordaliza et al. Food Chemistry 420 (2023) 136097 6 three successive extraction tests at 70 ◦C for 2 h, using the two solvents that provided the highest extraction efficiencies, 3 % HAc (Fig. 2A) and H2O (Fig. 2B). As can be seen, silver migration is quite higher in the first extraction in comparison to the following two cycles. This may be due to the fact that the silver present on the surface of the polymer is leached during the first extraction, while diffusion through the PP structure may limit migration in the following extractions. This effect is more acute for water, since the acid medium could facilitate silver leaching from the polymeric matrix from the very first cycle. However, silver was not fully released for any container after 3 cycles (2.2–2.7 % total release in 3 % HAc). The cumulative silver migration after each cycle can be properly fit to a linear model. Accordingly, up to 200, 240 and 180 cycles heat treatment cycles were estimated to be necessary to release all the silver in the cup, lid and container, respectively, under a 3 % HAc simulant. In water, 400 heating cycles are estimated to release all the silver in the Rubbermaid container, while for the cup and lid, up to 1500 and 1200 cycles, respectively would be required. Considering that in daily-life, meals stored within portable reusable plastic food containers are usually warmed-up in a microwave oven prior to consumption, silver migration has also been evaluated for consecutive microwave heating cycles under conventional conditions of daily use (700 W for 2 min) of the PP containers (Fig. 3). A trend similar to our results for repeated use at 70 ◦C for 2 h, was observed both for 3 % HAc (Fig. 3A) and H2O (Fig. 3B) with a decrease of silver release over time throughout successive microwave heating cycles. Remarkably, migration during microwave heating led to a silver release 2–10 times lower than during heating at 70 ◦C for 2 h, when comparing the first extraction. This difference was also previously observed for food plastic containers incorporating triclosan as antimicrobial agent (Marazuela et al., 2022). As previously demonstrated, the length of contact and higher tem- perature greatly affects the release of silver from PP FCM. For a domestic microwave oven operated at common heating conditions (herein, a measured temperature of 70 ± 3 ◦C was found after every 2-min cycle), the time of use (generally 1–2 min) is much shorter than the heating time involved in the hot-fill simulation at 70 ◦C (2 h), leading to a lower Fig. 3. Migration of silver from PP antimicrobial containers during repeated use under microwave oven heating. A) 3 % HAc and B) H2O water were assayed as food simulants at 70 ◦C for 2 h, for n = 3 repeated successive extractions. Mean migration values for 3 replicates are displayed. Error bars represent standard deviations. Table 1 Risk assessment of antibacterial polypropylene containers under conventional use. Migrated silver to food in contact with the containers was estimated, based on the results from migration tests at 70 ◦C/2h; 40 ◦C/10 d and 20 ◦C/10 d; using 3 % HAc, H2O, 10 % EtOH and 95 % EtOH as simulants. Mean migration values ± their standard deviation are shown for 3 determinations. The silver exposure as a percentage of the acceptable daily intake (ADI) of 0.9 µg Ag kg− 1 body weight (bw) per day set by European Chemicals Agency (ECHA) was calculated using the mean migration values. Conventional use migration tests 3 % HAc H2O 10 % EtOH 95 % EtOH Material Test conditions migrated Ag / µg Ag kg¡1 food % ADI set by ECHA* migrated Ag / µg Ag kg¡1 food % ADI set by ECHA* migrated Ag / µg Ag kg¡1 food % ADI set by ECHA* migrated Ag / µg Ag kg¡1 food % ADI set by ECHA* OMADA cup 40 ◦C, 10 d 3.1 ± 0.4 6 0.9 ± 0.5 2 0.7 ± 0.1 1 0.03 ± 0.01 0.06 Neoflam CLOC lid 22 ± 4 41 3.3 ± 0.1 6 1.6 ± 0.9 3 0.3 ± 0.1 0.6 Rubbermaid container 220 ± 50 410 160 ± 60 296 120 ± 20 222 7 ± 1 13 OMADA cup 20 ◦C, 10 d 0.6 ± 0.1 1 0.02 ± 0.01 0.04 0.13 ± 0.01 0.2 0.07 ± 0.03 0.1 Neoflam CLOC lid 7 ± 1 13 0.4 ± 0.2 0.7 1.4 ± 0.6 3 1.0 ± 0.8 2 Rubbermaid container 40 ± 10 74 40 ± 10 74 21 ± 8 39 1.2 ± 0.5 2 OMADA cup 70 ◦C, 2 h 1.8 ± 0.6 3 0.11 ± 0.03 0.2 0.05 ± 0.03 0.1 0.03 ± 0.01 0.06 Neoflam CLOC lid 9 ± 2 17 1.3 ± 0.4 2 1.1 ± 0.4 2 0.19 ± 0.07 0.4 Rubbermaid container 20 ± 5 37 23 ± 6 43 15 ± 3 28 0.6 ± 0.3 1 * ADI set by ECHA of 0.9 µg Ag kg− 1 bw per day is equivalent to 54 µg Ag kg− 1 food, considering a daily ingestion of 1 kg food in contact with food contact materials and a weight of 60 kg for an adult. E. Moreno-Gordaliza et al. Food Chemistry 420 (2023) 136097 7 amount of released silver. The diffusion of small antimicrobial agents from the polymeric matrix might be enhanced by microwaves and hot spots may appear on the PP surface (Alin & Hakkarainen, 2010) increasing silver migration and leading to a relevant release despite the short warm-up times. Again, by fitting the cumulative silver release after each cycle to a linear model, an estimate of 230 or 470 microwave heating cycles was found to be necessary to remove all silver from the cup or lid/container, respectively, under 3 % HAc. In water, it is esti- mated that around 1,250 microwave heating cycles are required for full silver release from the OMADA cup and the Rubbermaid container, while around 1,680 cycles are required for the lid. Therefore, considering the extensive and repetitive use of these food containers, especially in combination with microwave heating, continued migration of silver into food is expected and consumers may potentially be exposed to the harmful effects of silver after long-term daily use. 3.4. AgNPs identification and speciation of silver by sp-ICPMS The release of AgNPs into the food simulants water and 3 % HAc at 70 ◦C for 2 h was evaluated for all the containers under study. However, no significant AgNPs release could be detected by sp-ICPMS analysis in the extracts for any container (Fig. S3), even when properly diluted. This proved that just ionic silver was released from all the FCM, even for the alleged nanosilver silicone nipple. This could be attributed to a wrap- ping effect caused by the silicone polymeric matrix, which prevents the release of potentially embedded AgNPs, which only after oxidation could leach Ag ions from the silicone. This fact demonstrated compli- ance with EU legislation in terms of lack of AgNPs release from the antimicrobial containers tested, available in the global market. 3.5. Risk assessment of silver release from PP FCM In order to estimate the degree of risk posed by exposure to silver migrating to food in the studied FCM, compliance with the restriction for silver in food proposed by EFSA (50 µg Ag kg− 1 of food) was checked. Migration of silver into food, expressed as µg Ag kg− 1 food, was calcu- lated using the surface migration values, considering a contact of 6 dm2 plastic surface per kg food, according to Commission Regulation 10/ 2011. As can be seen in Table 1, the Rubbermaid container far exceeds the EFSA restriction when subjected to 3 % HAc, H2O or 10 % EtOH at 40 ◦C for 10 days, with a silver release of 220, 160 and 120 µg kg− 1 of Fig. 4. Microplastics are released from PP containers to food simulants upon microwave heating and long-term stor- age. SEM images, along with their respective EDX spectra are shown. Three microwave heating migration cycles at 700 W for 2 min in 3 % HAc were applied for: A) Neoflam CLOC lid; B) OMADA cup and C) Rubbermaid container. A silver-containing MP from the Rubbermaid container released after heating in 3 % HAc at 40 ◦C for 10 days is shown in D). Sample in D) was metallized with gold. Magnification: 2000-15000X. E. Moreno-Gordaliza et al. Food Chemistry 420 (2023) 136097 8 food, respectively. For all other conditions and materials tested, no surpass of EFSA-recommended silver levels was detected under any conventional use condition (Table 1), even during repeated use (Table S3). On the other hand, the ADI of silver established by the ECHA of 0.9 µg silver per kg bw per day, corresponds to 54 µg silver kg− 1 of food when considering a daily intake of 1 kg food in contact with FCM and a 60 kg-weight for an adult (EFSA, 2021). Considering this, the use of the Rubbermaid container represents 410, 296 and 222 % of ADI for Ag when subjected to 3 % HAc, H2O or 10 % EtOH at 40 ◦C for 10 days. This is of special concern for a food container recently launched on the European market. Meanwhile, at 20 ◦C testing for 10 days, in 3 % HAc and 10 % EtOH, the Rubbermaid container reached a 74 and 39 % of the ADI set by ECHA for Ag, respectively; while at 70 ◦C for 2 h, in 3 % HAc and 10 % EtOH, this reached 37 and 28 % of the ADI, respectively. On the other hand, for the Neoflam CLOC lid and OMADA cup, the highest migration found for silver corresponded to 41 % and 6 % of ADI, respectively, for 3 % HAc at 40 ◦C testing for 10 days, as displayed in Table 1. Moreover, the highest silver release found using the 3 % HAc simu- lant in the first microwave heating cycle represents 9, 5 and 0.47 µg Ag kg− 1 food migration for the Rubbermaid container, Neoflam CLOC lid and OMADA cup, respectively, as shown in Table S4. In this case, the amounts of silver migrated during microwave extraction do not exceed the recommended restriction proposed by EFSA, and correspond to 17, 9 and 0.9 % of ADI set by ECHA, respectively (Table S4). However, as hot- fill of the PP portable food containers usually precedes a latter microwave warm-up of daily meals in modern life, an actual higher silver release to ingested food is expected from both contributions, and an increased exposure risk is foreseen. All these results confirm that daily conventional use of this kind of antibacterial plastic food containers represents a source of significant silver human intake and prolonged use and exposure may pose an extended risk to health. 3.6. Microplastics release The characteristics of plastic polymers and their processing largely affect the degree of MPs being released from FCM (Du et al., 2020; Fadare, Wan, Guo, & Zhao, 2020). Thus, H2O and 3 % HAc extracts under conventional and microwave heating of the food containers under study were analized by SEM-EDX, to determine potential MPs release. The presence of micron-sized particles (between 2 and 20 µm) with diverse morphology was detected in all the simulants after microwave or conventional-use migration tests (Fig. 4) for all the PP containers. EDX spectra confirmed that the particles were MPs, considering prominent O and C peaks in the case of PP containers (Rubbermaid, Neoflam CLOC and OMADA), as shown in Fig. 4. The Cu and Zn peaks observed in the EDX spectra belong to the tin coverslip used to fix the extracts. In addition, in the 3 % HAc extract from the Rubbermaid container obtained after a 10-day exposure to 40 ◦C, the presence of silver in the released MPs could be confirmed by SEM-EDX analysis (Fig. 4D). As a conclusion, Ag-containing MPs can be released to food Fig. 5. Silicone microparticles are released from the silicone nipple of the baby bottle during conventional and microwave use. SEM images along with their respective EDX spectra of MPs released after three migration cycles at: A) 70 ◦C for 2 h in 3 % HAc; B) and C) Microwave heating at 700 W for 2 min in 3% HAc and H2O, respectively. Magnification 3000-4000X. Sample in A) was metallized with gold. E. Moreno-Gordaliza et al. Food Chemistry 420 (2023) 136097 9 from these PP FCM during conventional use and microwave oven- heating. In the case of the extracts from the silicone nipple of the feeding bottle, microparticles of approximately 5–30 µm were observed; their EDX spectra showed peaks of C and O, as well as an intense peak of silicon (Si) (Fig. 5), demonstrating the release of silicone microparticles from feeding bottles during contact with food simulants both under microwave heating or conventional use. This would be in agreement with the recent finding that during steam disinfection of silicone-rubber teats used in baby bottles, MPs with a size between 0.6 and 332 μm are released (Su et al, 2022). This poses a risk of exposure to silicone microparticles to infants through ingestion, due to their release from silicone teats both during conventional and microwave heating. However, the potential toxic ef- fect of such microparticles is still unknown. Probably due to the low silver content and inhomogeneity in the nanosilver silicone, SEM could not detect the alleged AgNPs on the surface of the original bottle and nipple (not shown) and no Ag peaks were observed in the EDX spectra of the MPs detected in the food simulants. However, potential silver- containing silicone microparticles would also be released from the silver-containing silicone baby bottles. Therefore, the combination of MPs and chemical additives can intensify the detrimental effects in the long-term. The release of silver containing MPs can be a problem for human health and the environment. 4. Conclusions In summary, the use of certain currently available antimicrobial polymeric food packaging containing silver could be of great concern, as in addition to microplastics, significant amounts of silver have been shown to be released into food simulants, in some cases exceeding the EFSA restriction of 0.05 mg silver kg− 1 food and the ADI of 0.9 µg silver per kg bw per day set by ECHA. Furthermore, considering the extensive use of plastic FCM in modern live, aggravated by daily microwave-use, consumers may be continuously exposed to the harmful effects of released silver and microplastics, which could lead to long-term health problems. The potential toxicological risks of silver-containing MPs released into food from antimicrobial FCM need to be further investigated. CRediT authorship contribution statement Estefanía Moreno-Gordaliza: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing – review & editing, Visualization, Project administration. M. Dolores Marazuela: Concep- tualization, Methodology, Writing – review & editing. M. Milagros Gómez-Gómez: Conceptualization, Methodology, Investigation, Writing – original draft, Supervision, Funding acquisition. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability Data will be made available on request. Acknowledgements This research was granted by the Ministry of Science and Innovation (Spain) (PID2020-116067RB-100) and the Comunidad Autónoma de Madrid (Spain) with funds from FEDER program (EU) (Project PB2018/ BAA-4393, AVANSECAL II). Appendix A. 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