Online-coupling of AF4 and single particle-ICP-MS as an analytical approach for the selective detection of nanosilver release from model food packaging films into food simulants

Accepted Manuscript 4 Online-coupling of AF and single particle-ICP-MS as an analytical approach for the selective detection of nanosilver release from model food packaging films into food simulants Birgit Hetzer, Anna Burcza, Volker Gräf, Elke Walz, Ralf Greiner PII:

S0956-7135(17)30205-0

DOI:

10.1016/j.foodcont.2017.04.040

Reference:

JFCO 5596

To appear in:

Food Control

Received Date: 24 October 2016 Revised Date:

6 April 2017

Accepted Date: 9 April 2017

Please cite this article as: Hetzer B., Burcza A., Gräf V., Walz E. & Greiner R., Online-coupling of 4 AF and single particle-ICP-MS as an analytical approach for the selective detection of nanosilver release from model food packaging films into food simulants, Food Control (2017), doi: 10.1016/ j.foodcont.2017.04.040. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Online-coupling of AF4 and single particle-ICP-MS as an analytical

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approach for the selective detection of nanosilver release from

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model food packaging films into food simulants

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Birgit Hetzer*, Anna Burcza, Volker Gräf, Elke Walz, Ralf Greiner

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Max Rubner-Institut, Department of Food Technology and Bioprocess Engineering,

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Haid-und-Neu-Str. 9, 76131 Karlsruhe, Germany

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*Corresponding author contact: [email protected]

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Keywords

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Migration study; food packaging; nanosilver; single particle ICP-MS; AF4-sp-ICP-MS

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ACCEPTED MANUSCRIPT Abstract

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It is of utmost importance, that reliable exposure data about the migration behavior of

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nanoparticles are collected in order to provide adequate information for the safety

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assessment of engineered nanoparticles (ENPs) used in the food packaging sector. The

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objective of this study was to evaluate the migration behavior of silver nanoparticles (AgNPs)

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from model food packaging films with varying nanosilver content into three different food

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simulants (water, 10 % ethanol and 3 % acetic acid). The overall silver migration determined

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by classical inductively coupled plasma mass spectrometry (ICP-MS) analysis was dependent

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on the silver content of the films, the food simulant used and the contact time and

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temperature applied. Furthermore, single particle ICP-MS analysis was applied in order to

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detect and quantify migrated AgNPs selectively. As coexistent silver ions in the migration

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samples had an impedient effect (decreased signal-to-noise ratio), an optimized analytical

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approach was developed by online-coupling of asymmetric flow field flow fractionation (AF4)

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to single particle-ICP-MS. The enrichment of the nanoparticle fraction and simultaneous

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reduction of the ionic background via AF4 resulted in a clearly improved ICP-MS detection

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sensitivity, which enabled a more refined identification and size characterization of the

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migrated silver species. Scanning transmission electron microscopy (STEM) and energy-

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dispersive x-ray spectroscopy (EDS) confirmed independently the presence of AgNPs and

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silver/polymer heteroaggregates in the migration samples.

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ACCEPTED MANUSCRIPT 1.

Introduction

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Nanosilver is the most incorporated material in nanoparticle containing consumer products

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(Vance et al., 2015) and dominates the market outside Europe as an antimicrobial additive,

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e.g. in food contact materials (FCM).

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Although this new generation of nanocomposite food packaging systems offers significant

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benefits for enhancing the freshness and shelf life of food products, the market growth is

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hindered, because there is still a high degree of public skepticism and concern whether these

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benefits really outweigh the risks arising from the exposure to the used nanomaterials

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(Bumbudsanpharoke & Ko, 2015; Giles et al., 2015).

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Potential risks arising from nanotechnology-based FCMs are mainly dependent on the

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migration and dissolution behavior of the engineered nanoparticles (ENPs) which are

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incorporated in the polymer matrix. The most likely relevant human exposure to

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nanoparticles incorporated in food packaging occurs through oral uptake and ingestion of

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ENPs migrating from the food contact material into the packed food (Cushen et al., 2012;

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Loeschner et al., 2011).

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Despite significant progresses in the elucidation of the toxicological and antimicrobial effects

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of nanosilver the underlying mechanisms are still not completely understood, but it is

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suggested in literature, that toxic effects are related to both AgNPs and their release of

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silver ions (Beer et al., 2012; Reidy et al., 2013). The fate of AgNPs during gastrointestinal

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digestion was investigated by in vitro digestion studies and it was found, that during the

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different digestion steps AgNPs can reach the intestine wall in their nanoparticulate form to

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some extent – despite pH changes and the complex environment with variable

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concentrations of salts, enzymes, proteins etc. (Böhmert et al., 2014; Lichtenstein et al.,

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ACCEPTED MANUSCRIPT 2015; Walczak et al., 2013). Although the overall bioavailability of silver after digestion can

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be assumed as very low, several research groups found out, that both intact AgNPs and

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dissolved Ag+ pass the intestinal wall and can reach other organs (Juling et al., 2016;

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Loeschner et al., 2011; Park, 2013; van der Zande et al., 2012). Therefore silver speciation

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analysis is essential in order to provide reliable exposure data about the release of ionic and

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nanoparticulate silver from FCMs, which are a prerequisite for the safety assessment.

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In the EU safety assessment for food packaging is mostly based on experimental migration

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studies. In these empirical investigations packaging materials are individually tested under

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controlled conditions concerning the ability of containing nanoparticulate compounds to

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migrate into food simulants.

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Because of the lacking information about the incorporated nanosilver in commercially

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available food package material, it is not an easy task to carry out migration studies and

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derive information about the potential risk of the released AgNPs. Especially the low

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concentration of silver being released from the plastic films complicates speciation analysis.

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Nevertheless, the assessment of AgNP migration was already investigated by several studies

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with commercially available FCMs. In summary, because of the low initial silver content of

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the used FCMs (< 40 µg Ag/g plastic) only very small total silver concentrations could be

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determined in the resulting migration samples (mostly by using classical ICP-MS). However,

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in some cases a small fraction of the migrated silver species could be found in

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nanoparticulate form: One of the first research groups engaged in the investigation of the

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silver release from FCMs carried out migration studies with polyethylene bags and confirmed

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the presence of AgNPs in the size range of up to 300 nm with SEM/EDS, whereupon no

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further quantification of the found AgNPs was achieved (Huang et al., 2011). Approaches in

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quantifying the AgNP release from various commercial plastic containers and bags by

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ACCEPTED MANUSCRIPT single particle-ICP-MS were carried out by Echegoyen & Nerín (2013), von Goetz et al. (2013)

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and Mackevica et al. (2016). Although all groups investigated among others two identical

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FCM products, the results regarding the size and/or concentration of released AgNPs differ

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and are difficult to compare, as both the conditions for the migration studies and sample

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analysis varied. Accompanying SEM/TEM analysis showed in all studies that not only small

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primary nanoparticles can be found but also aggregates and even polymer containing

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species (Echegoyen & Nerín, 2013; Mackevica et al., 2016).

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Few further studies exist performing the direct coupling of different analysis methods, e.g.

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asymmetric flow field flow fractionation (AF4) hyphenated to varying techniques such as

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MALLS (multi-angle laser light scattering) (Bott et al., 2014) or (classical) ICP-MS as silver

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detectors for the characterization of migration samples (Artiaga et al., 2015). In these studies

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the hydrodynamic nanoparticle diameter of eventually migrating nanosilver particles could

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be estimated by determining the AF4 retention time and comparing it with the retention

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times of AgNP standards.

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The objective of this application-oriented study was to establish coupling of AF4 with

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single particle-ICP-MS for the characterization of AgNP containing samples derived from

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migrations studies with model FCMs. The combination of these two state-of-the-art

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nanoparticle characterization techniques enabled a highly sensitive detection and

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quantification of nanoparticulate silver species migrating from model films into food

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simulants, which offline sp-ICP-MS or classical AF4-ICP-MS coupling could not provide. By

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interfacing AF4 with sp-ICP-MS the AF4 step can be utilized for two purposes:

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Firstly, during the AF4 step ionic silver is washed out of the system, so that only AgNPs reach

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selectively the ICP-MS detector and can be quantified with increased sensitivity because the

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ACCEPTED MANUSCRIPT intended loss of silver ions leads to an improved signal-to-noise-ratio. Secondly, via

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fractionation with AF4 the heterogeneity/polydispersity of the migration samples is reduced

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because the different occurring silver-containing species are separated by size and thus

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introduced into the online-coupled sp-ICP-MS system successively and not simultaneously as

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in offline sp-ICP-MS analysis.

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To the best of our knowledge, this is the first time that nanosilver detection was carried out

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by the online coupling of AF4 and single particle-ICP-MS in migration assays. This represents

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a new approach for the selective investigation of nanosilver release from FCMs, which helps

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to provide more comprehensive and reliable data for evaluating the relevance of AgNPs in

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FCM safety assessment. So far only one study with AF4-sp-ICP-MS coupling could be found in

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the scientific literature describing the characterization of citrate-coated standard nanosilver

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and Ag-SiO2 samples in ultrapure water (Huynh et al., 2016).

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2.

Materials and methods

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2.1.

Reagents and model films

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Ultrapure water (18 MΩcm) for the preparation of all samples was provided by a water

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purification system unit (Milli-Q Advantage A10, Millipore, Darmstadt, Germany). Nitric acid

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(65 %, suprapur) and sulfuric acid (97 %, suprapur) were purchased from Merck (Darmstadt,

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Germany), acetic acid (100 %, suprapur) and ethanol were obtained from Roth (Karlsruhe,

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Germany). Working concentrations of 3 % (w/v) acetic acid and 10 % (v/v) ethanol were

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freshly prepared via dilution with ultrapure water. Ionic silver solutions, internal ICP-MS

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standards and tuning solutions were obtained from Elemental Scientific (Mainz, Germany)

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and Thermo Fisher Scientific (Bremen, Germany), respectively.

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ACCEPTED MANUSCRIPT AgPURE W10 was a generous gift from RAS AG (Regensburg, Germany). AgPURE is an

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industrially used, well characterized nanosilver product (primary size distribution

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D99 < 20 nm, particle stabilization with polyoxyethylene fatty acid ester) and corresponds to

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the OECD reference material NM-300 (Klein et al., 2011; RAS AG, 2015) and the certified

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reference material BAM-N001 (Menzel et al., 2013). The trade name is further abbreviated

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to AgPURE and used throughout the text.

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The model films containing AgPURE were also obtained from RAS AG. The polymer

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masterbatch was produced with low-density polyethylene (ExxonMobil™ LDPE LD 100.BW,

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Exxon Mobil Chemical Europe, Belgium) as polymer host material and AgPURE nanosilver as

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antimicrobial additive. To obtain tailor-made model polymer films (film 1 – 5) with varying

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nanosilver contents (500 – 11000 ppm) the AgPURE containing polymer master batch was

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mixed with varying portions of pure polymer and extruded to a film thickness of 50 µm.

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Additionally, a film made of pure LDPE without nanosilver was provided as blank test

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material (blank film).

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2.2.

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2.2.1. Determination of the silver content in the model films

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The total silver content of all films was determined at least in quadruplicate by ICP-MS after

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microwave assisted digestion of the films based on Song et al. (2011). In brief, each model

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film was cut into small pieces and about 10 mg of these film fragments were weighed out in

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15 ml PTFE digestion vessels and digested in a microwave oven (MWS-1, Berghof

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Products + Instruments GmbH, Eningen, Germany) by adding 3.75 ml 65 % nitic acid and

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1.25 ml 96 % sulfuric acid using the program shown in Table 1. After cooling the vessels,

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each digested sample was diluted with ultrapure water and analyzed by classical ICP-MS

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(iCAP Qc, Thermo Fisher Scientific, Bremen, Germany) for the total silver content.

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2.2.2. Migration studies

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The migration studies were carried out according to the European Regulation (European

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Commission, 10/2011). For determining the overall silver migration rate self-made test

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pouches (10 cm x 15 cm) of all model films were filled with 100 ml of three different food

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simulants (ultrapure water, 3 % acetic acid and 10 % ethanol) at least in triplicate and sealed

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with a heat sealing device (Krups Vacupack Plus F380, Rowenta, Offenbach/Main, Germany).

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The test pouches were then stored in an incubator (UT-12, Heraeus, Hanau, Germany) for

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10 days at 40 °C or 2 hours at 70 °C in the dark. After completion of the experiments 10 ml of

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each migration extract were transferred into metal-free 15 ml PP tubes (Roth, Karlsruhe,

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Germany) and acidified with 0.2 ml 69 % nitric acid for determining the total silver content

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by classical ICP-MS measurements. The obtained data were analyzed using the software JMP

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(JMP®13.1.0, SAS Institute Inc.) and expressed as means +/- standard deviation. Because of

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the small sample size Gaussian distribution of the data could not be ensured. Thus,

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significance of the differences between the three migration simulants or the two time-

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temperature protocols has been tested using non-parametric one-way analysis (Dunn all

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pairs test) for the highest level of the initial silver amount in films. Statistical significance has

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been considered for p-values <0.05.

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For determining the AgNP fraction via AF4-ICP-MS coupling further short-term migration

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experiments were carried out in which cut film fragments with a side length of 3.5 cm were

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transferred into 50 ml PP tubes (Roth, Karlsruhe, Germany) and immersed in 25 ml of

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simulant (ultrapure water 3 % acetic acid and 10 % ethanol) at least in triplicate. After

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ACCEPTED MANUSCRIPT incubating the samples for 2 hours at 70 °C the film fragments were removed and the

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resulting samples prepared for further analysis.

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Additionally, for the AF4-single particle-ICP-MS method development control samples with

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pristine AgPURE (concentration range 0.05 – 1 µg/L) were prepared in two simulants

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(ultrapure water and 3 % acetic acid) and treated the same way as the migration samples.

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2.2.3. Analysis by Scanning (Transmission) Electron Microscopy

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Images were collected with a field emission scanning electron microscope (Quanta 250 FEG,

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FEI, Eindhoven, Netherlands) in two operation modes, namely SEM under low vacuum

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conditions for the analysis of the model films and STEM for characterizing nanoparticles

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from liquid samples. The settings for both modes are shown in Table 2. Furthermore, the

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presence of nanosilver was confirmed by qualitative energy-dispersive x-ray spectroscopy

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(EDS) (EDAX Genesis Apex SM 2i, EDAX Inc., Mahwah, USA). ImageJ was used for obtaining

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particle size distributions from chosen images.

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For the characterization of the model films in low vacuum mode (LoVac-SEM) a small piece

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of about 1 cm2 of each model film was prepared with a scalpel, rinsed with ultrapure water

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and fixed on an aluminum specimen stub (Agar Scientific, UK) with special adhesive support

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tabs (Leit-Tabs, Plano, Wetzlar, Germany) (Artiaga et al., 2015).

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For imaging pristine AgPURE nanoparticles (dispersed in ultrapure water, working

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concentration of 10 - 100 mg/l) and migration samples, the SEM was operating in STEM

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mode (Scanning Transmission Electron Microscopy). In order to provide migration samples

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with higher silver concentrations, migration experiments with a higher polymer/food

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simulant ratio (ca. 10 cm2/ml) were carried out. Once the migration experiment was

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completed, the film fragments were removed from the tubes and an adequate sample

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volume (6 – 30 µl) was directly taken and prepared by means of drop deposition on carbon

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coated TEM grids (Hassellöv et al., 2008). Afterwards each grid was air-dried for about

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30 minutes and finally transferred to the STEM stage for analysis.

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2.2.4. Analysis by classical and single particle ICP-MS

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The quantification of nanoparticles via sp-ICP-MS is based on the fundamentals which are

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exemplarily described by Laborda et al. (2013). For single particle ICP-MS analysis samples

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containing metal or metal-oxide nanoparticles are nebulized directly, resulting in a non-

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homogeneous distribution of the analyte in the aerosol droplets. In the plasma each single

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particle generates an ion plume which can be detected as transient peak signal (in the range

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of about 300 – 500 µs) when appropriate low dwell times for the time resolved data

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acquisition are chosen. Furthermore, the concentration of the sample has to be very low

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(ng/l-range), so that only one particle may reach the detector per chosen dwell time interval.

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In order to guarantee these basic conditions, sp-ICP-MS measurements were carried out

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with highly diluted samples (approx. 1-5 ng/l) with a chosen dwell time of 5 ms.

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In practice, for this type of measurement 1 ml of a migration sample was diluted with

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ultrapure water to obtain the target concentration of approx. 5 ng/l. The dilution factor

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varied from 10 to 500 depending on the initial silver content in the migration sample. For

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calibration of the instrument, solutions of ionic silver at concentrations ranging from 0 -

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5 µg/l were analyzed. Nebulization efficiency was derived by measurement of 60 nm gold

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nanoparticles (RM8013, NIST). Additionally, a silver standard (1 µg/l) was measured after

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every 10 samples as control.

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The AgNP quantification was performed utilizing the single particle calculation (SPC)

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spreadsheet developed by RIKILT (Peters et al., 2015). In short, by obtaining the signal

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ACCEPTED MANUSCRIPT distribution of the raw transient signal (hence referred to as spikes) from the time scan a

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particle detection limit can be determined, which in turn determines the number of

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detected nanoparticles. Based on the ionic calibration, particle mass and in turn particle

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diameter may be calculated from the signal intensity of the detected spikes. The particle

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number concentration is calculated from the number of detected particles in the measured

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time interval, the transport efficiency and the sample input flow.

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For this study, the number of 500 particle events was specified as minimal particle number

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for evaluating single particle measurements.

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The baseline of the raw signal in the original time scan corresponds directly with the amount

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of silver ions, which also migrated from the model films or which have been released from

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migrated AgNPs during the migration study experiments.

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In the coupling experiments the data acquisition time for the sp-ICP-MS signal was 58 min

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(duration of the AF4 fractionation run). The resulting huge datasets (ca. 3,600,000 data

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points per measurement) could not be evaluated on the whole because the current ICP-MS

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instrument software (Qtegra “npQuant” plug-in) does not support long-term measurements

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yet. Therefore the collected data of the AF4 fractograms were exported, re-imported to the

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RIKILT spreadsheet, manually split into sections of constant time intervals of 300 s and

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evaluated individually in succession.

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2.2.5. Analysis by Asymmetric Flow Field Flow Fractionation (AF4)

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For the coupling experiments the ICP-MS was hyphenated to an AF4 system (AF2000 MT,

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Postnova Analytics, Landsberg am Lech, Germany), connected via polyetheretherketone

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ACCEPTED MANUSCRIPT The method used for the fractionation of samples containing AgPURE nanosilver was

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established in our research group previously (Burcza et al., 2015) and further optimized for

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the current application by increasing the injection volume and correspondingly also the

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focusing time. The mobile phase was ultrapure water. For the chosen model films and the

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control sample with pristine AgPURE 0.5 ml aliquots of each sample were directly injected

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into the system without further sample treatment and as promptly as possible after finishing

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the migration studies. For each sample a coupling experiment set composed of an AF4-ICP-

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MS run and an AF4-single particle-ICP-MS run was carried out at least in triplicate.

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At the start of each experiment set and intermittently between the sample runs the system

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was rinsed with ultrapure water. Table 3 summarizes the instrumental parameters of both

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ICP-MS and AF4.

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Results and discussion

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3.1.

Characterization of AgPURE and model films with STEM, LoVac-SEM and ICP-MS

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In order to obtain information about the size and shape of the used nanosilver, STEM

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analysis was performed. A representative dark field STEM image of pristine AgPURE and a

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corresponding size distribution histogram is presented in Figure 1. The histogram is based on

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the ImageJ analysis of 3361 particles found in 6 images (Figure S1) collected with the same

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high magnification factor (200,000x). A mean diameter of 16.7 ± 3.7 nm was obtained for the

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pristine AgPURE.

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The particles are virtually spherical and more than 80 % of all detected particles exhibited a

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diameter between 14 and 22 nm. Very rarely, somewhat larger particles with polygon or

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triangle-like, flat shapes and needle-like structures were visualized. However, those particles

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were not considered in the determination of the particle size distribution.

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ACCEPTED MANUSCRIPT SEM images in LoVac mode were taken of all model films. However, in Figure 2 images of the

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surface of film 2 and film 5 including a representative EDS spectrum (Fig. 2D) are shown. At

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lower magnifications (Fig. 2, A/B, both 5,000x) nanosilver and nanosilver agglomerates

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and/or aggregates which are directly detached on the film surface or incorporated inside the

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film very close to the surface can be clearly visualized. The size of these species ranges from

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about 300 nm up to about 2 µm for film 2 and up to 5 µm for film 5 (Fig. 2C). At higher

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magnification (11,000x) also single AgPURE nanoparticles and smaller agglomerates and/or

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aggregates are visible.

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The results of the ICP-MS measurements of the digested samples of all films representing

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the total silver content are shown in Table 4. As the values show only a low relative standard

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deviation of 3 - 6 %, it can be assumed, that despite the varying size of the incorporated and

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surface bound AgNP agglomerates/aggregates the distribution all over the films was

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relatively homogeneous, which is an important prerequisite for conducting reproducible

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migration experiments with these model films.

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3.2.

Migration studies

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3.2.1. STEM imaging of migration samples

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The microscopy results in Figure 3 show the release of nanosilver in aqueous migration

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samples with film 2 and film 5. The migrated particles can be found freely dispersed (Fig. 3C)

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in the migration extract, agglomerated (Fig. 3C/D), attached to polymer pieces (Fig. 3A/B) or

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even still embedded in the matrix of these polymer pieces (Fig. 3E/F) which obviously have

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also been released from the model films. The presence of silver was verified by EDS (data not

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shown). Due to the very low concentration of released silver a quantitative evaluation of the

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detected nanosilver particle sizes, shapes or the agglomeration state was not possible.

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In the first set of the migration studies the effect of the initial amount of nanosilver in the

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model films on the migration of both AgNPs and Ag+ into the three food simulants was

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evaluated. According to the EU regulation (European Commission, 10/2011) the chosen

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conditions represent the following storage conditions:

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(A) 10 days at 40 °C: long storage of food in FCMs at room temperature or lower

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(B) 2 hours at 70 °C: long periods of usage

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Figure 4 shows the overall silver migration rate from all films under the different storage

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conditions. The amount of migrated silver was strongly dependent on the initial nanosilver

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content in the films. For the overall migration at 70 °C for 2 h similar values in water, ethanol

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and acetic acid can be determined (Fig. 4A) with no statistically significant differences. In

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comparison, the amount of total silver found after 10 days storage at 40 °C (Fig.4B) was also

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clearly dependent on the particular simulant and showed for film 5 (highest initial silver

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content) a more than 3-fold higher migration under acidic conditions (7.7 +/- 1.1 µg/dm2)

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compared to water (2.2 +/- 0.5 µg/dm2) or ethanol (1.4 +/- 0.2 µg/dm2). In this case

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statistical significance could be found for the migration of silver in acetic acid versus the

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migration in ethanol.

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In nonacidic media the most probable release mechanism is based on desorption of weakly

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bound AgNPs and (hetero)aggregates from the film surface (Störmer et al., 2017). In the case

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of the examined model films the surficial silver release in water and ethanol does not

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increase by applying longer exposure times, because there is no considerable degradation of

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the polymer matrix.

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ACCEPTED MANUSCRIPT Under acidic conditions the release of nanosilver and silver ions is promoted by oxidative

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dissolution and/or polymer degradation processes. In the conducted migration experiments

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the time dependency of these processes can be seen in the increasing migration of total

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silver from short to long-term exposure of the model films in 3 % acetic acid (statistically

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significant differences in the total silver migration values).

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In other recent studies the temperature and storage time has already been shown to affect

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the migration levels of silver (Artiaga et al., 2015; Echegoyen & Nerín, 2013; Song et al.,

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2011). These results are in good agreement with our findings, in so far as the highest

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migration rates occur in acetic acid at longer storage times. Some discrepancy occurs for the

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experiments with short incubation time. However, it has to be taken into account, that

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different commercial FCMs with other types of nanosilver and much lower silver content

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than the model films in this study have been used, so that the results are not directly

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comparable.

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As the released silver concentrations in the short-term migration experiments were

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sufficient for the applied analytical methods, these samples were also used for the method

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development of AF4-sp-ICP-MS coupling.

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3.3.

Characterization of migrated nanosilver

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3.3.1. Offline single particle ICP-MS analysis

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In Figure 5 the raw time scans of pristine AgPURE and of migration samples of film 5 in water

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and 3 % acetic acid are shown. Because of the very low ionic silver background a sufficient

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number of single particle events can be detected for pristine AgPURE (Fig. 5, top left), which

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enables a straight forward data evaluation even though the size of AgPURE lies in the range

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ACCEPTED MANUSCRIPT of the minimal particle size detection limit for sp-ICP-MS, which was reported between

340

13 - 20 nm by Lee et al. (2014).

341

A representative particle size distribution for AgPURE with a mean diameter of 20 nm is

342

shown in Figure 5 (top right). By averaging eight independent sp-ICP-MS measurements a

343

mean particle size of 21.6 +/- 2.3 nm can be derived (data not shown), which is slightly

344

higher than the value determined by STEM analysis. However, as the results are based on

345

different analytical techniques, the values are not directly comparable but still in good

346

agreement.

347

In comparison, the background signal in a characteristic time scan for a highly diluted water

348

migration sample was approx. 10-fold higher compared to the pristine AgPURE samples

349

(Fig. 5, bottom left). Therefore, the particle detection limit has to be chosen much higher

350

and as a consequence the particle number found was too low for further evaluation. In the

351

time scan of a migration sample in acetic acid (Fig. 5, bottom right) the background signal is

352

also very high (4-fold higher in comparison to the water migration sample). Thus, it is

353

impossible to derive information about the presence of AgNPs in the sample.

354

Finally, it has to be taken into account that regardless of the chosen simulant the fraction of

355

smaller AgPURE nanoparticles are completely hidden by the high background signal and

356

therefore not considered in the determination of the particle number.

357

One approach for obtaining an adequate amount of particles for the quantification would be

358

further dilution in combination with a simultaneous increase in measurement time.

359

However, the particle number would decrease dramatically and especially with acidic

360

samples there is the risk that a significant dissolution of nanoparticles during the

361

measurement occurs.

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ACCEPTED MANUSCRIPT 3.3.2. Coupling of AF4 and ICP-MS (classical and single particle mode)

364

As ionic silver is a substantial disruptive factor in confirming the presence of AgNPs in the

365

migration samples, AF4 was used to significantly reduce the Ag+ fraction in the migration

366

samples resulting also in a pre-concentration of the AgNP fraction. Thus, an essential

367

improvement of the signal-to-noise ratio for the following element-specific (online) single

368

particle-ICP-MS analysis of AgNPs was expected to occur.

369

The fundamentals of AF4 and its applications for nanoparticle separation have been

370

thoroughly described before (Dubascoux et al., 2008; Hagendorfer et al., 2012; Loeschner et

371

al., 2013; Mudalige et al., 2015). Nevertheless, in order to explain the occurring process of

372

Ag+ elimination and AgNP pre-concentration, the AF4 fundamentals will be outlined briefly in

373

the following paragraph.

374

Basically, particles having hydrodynamic diameters below 1 µm can be fractionated with AF4

375

in the so-called normal or Brownian elution mode. A typical AF4 run consists of a

376

focusing/relaxation step and an elution/separation step. The focusing occurs, when a cross

377

flow is applied to the injected sample while the solvent is pumped into the channel from

378

both ends simultaneously. The mobile phase and any ionic species can pass through the

379

membrane on the semi-permeable accumulation wall and are thus removed from the

380

system. The remaining undissolved sample components, which are small enough to undergo

381

significant Brownian motion, distribute in the focusing zone according to their sizes and find

382

as diffusional cloud a steady-state equilibrium position where the forces of crossflow and

383

diffusion are counterbalanced (relaxation). In the elution/separation step the retained

384

nanoparticles can be successively eluted in the laminar parabolic main channel flow; smaller

385

particles elute earlier than the larger ones and are directed to the ICP-MS detector. By

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ACCEPTED MANUSCRIPT determining the retention time an equivalent hydrodynamic particle size can be obtained, if

387

a suitable calibration with size calibration standards was carried out.

388

As the focus was on the method development itself, only selected migration samples from

389

incubation experiment with film 2 and film 5 in water and acetic acid were used.

390

In Figure 6 typical fractograms of migration samples with film 2 and 5 after 2 h storage at

391

70 °C in water (B) are presented in comparison to pristine AgPURE (A). In both fractograms

392

the presence of AgNPs can be clearly observed as peak signals, whereas the retention time

393

of the peak maximum in the migration samples is with 15 min distinctly lower than for

394

pristine AgPURE with a retention time of 20 min. This implies that the hydrodynamic

395

diameter of the migrating nanosilver fraction is smaller than the size of the original

396

nanosilver particles. One reason might be, that only AgPURE nanoparticles with sizes far

397

below 20 nm are able to migrate at all from the model films. Secondly, it can be asumed that

398

the the surface of the migrating nanosilver has been changed as a result of the interaction

399

with the polymer matrix of the films resulting in an altered interaction with the AF4

400

membrane. Another reason can be the oxidative dissolution of the nanoparticles themselves

401

and the associated release of ionic silver, which results in the detection of slightly smaller

402

AgNPs at lower retention times. This dissolution process can be clearly observed in samples

403

with acetic acid as used food simulant. In these samples - even with the short incubation

404

time of 2 h at 70 °C - no noteworthy amount of AgNPs could be detected with AF4-ICP-MS

405

(see Fig. 7).

406

In the time range from 25 - 30 (A) and 25 - 40 minutes (B), respectively, peak singals are

407

visible in both fractograms. This shows, that silver containing nanoparticulate species with

408

an obviously higher hydrodynamic diameter are eluted. It can be excluded that the size of

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ACCEPTED MANUSCRIPT the nanosilver particles itself has increased, so the higher retention times are rather an

410

indication for the presence of nanoparticle agglomerates and/or aggregates. In the case of

411

pristine AgPURE (Fig. 6A) these agglomerates and aggregates might be formed in the

412

focussing step during the AF4 run, when the particles are pressed to the membrane of the

413

lower channel wall because of the applied cross-flow. In the case of the migration samples

414

(Fig. 6B) the particle peaks, which occur in a larger time span (in comparison to the AgPURE

415

samples), indicate the elution of polymer-bound AgNP. These type of heteroaggregates

416

could already be verified independently by STEM imaging (see Fig. 3).

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For a possibly more refined analysis of the occuring particle peaks in the migration samples,

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the AF4 was coupled to ICP-MS in single particle mode (5 ms dwell time). In Figure 7 a typical

420

AF4-sp-ICP-MS fractogram for a migration sample of film 5 in acetic acid and water is shown.

421

Since most of the ionic silver has already been eliminated by the focusing step the

422

background signal lies in the range of 200 - 600 cps and the signal-to-noise ratio is highly

423

improved in comparison to the equivalent offline sp-ICP-MS measurements in both

424

simulants, in which the background signal was in the range of 12,000 cps and higher

425

(depending on the used simulant, see Fig. 5). This leads to an increased sensitivity and

426

enables a much better detection of AgNPs (without further dilution) even near the detection

427

limit.

428

Nevertheless, for the migration sample in 3 % acetic acid only 21 particle peaks could be

429

detected in total, which shows, that either no noteworthy AgNP release occurred or that all

430

migrated AgNPs have been dissolved during the storage time of 2 hours in the acidic

431

simulant. By direct comparison to the time scan of the corresponding offline sp-ICP-MS

432

measurement of the same sample (see Fig. 5, bottom right), it is obvious, that the reduction

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ACCEPTED MANUSCRIPT of the ionic background did not “excavate” a significant amount of hidden particles, which

434

proves that in acetic acid there is no significant AgNP migration.

435

The resulting water migration AF4-sp-ICP-MS fractogram is presented in the middle row of

436

Figure 7. As already mentioned in the method section (2.2.4), the AF4-sp-ICP-MS fractograms

437

were analysed in sections: For sub section A of the water migration fractogram no data

438

evaluation is possible because the peaks signals are inseparably close together (too high

439

particle concentration) and prevent the determination of the crucial detection limit for the

440

size characterization. In all following sub sections (B - F) a total of 3588 peaks with varying

441

signal height ranging from approx. 10,000 cps up to more than 500,000 cps occur. Based on

442

the assumption that all particles which have been injected in the AF4 (500 µl injection

443

volume) have reached the ICP-MS, a particle number concentration of 3.4·106 particles/l can

444

be derived for this migration sample. However, this assumption should be concidered with

445

caution, as for sp-ICP-MS analysis there exists both a lower and upper detection limit: The

446

lower detection limit is dependent on the minimal mass of the analyte, which is large

447

enough to generate a ion plume in the ionization step. The upper detection limit for the

448

particle characterization is limited by the behaviour of larger particles, as they may be

449

already removed in the spray chamber or are only partial ionizied in the argon plasma. So

450

far, maximum sizes for sp-ICP-MS analysis are expected to be in the range of 1 - 5 µm

451

(Laborda et al., 2013), which is actually in the range of the found silver/polymer

452

heteroaggregates in this study.

453

The obtained particle size distribution histograms indicate that small nanosilver particles

454

with a size of around 20 nm, which corresponds with the size of the native AgPURE particles,

455

dominate in every section. However, with increasing retention time peaks with significantly

456

higher signal intensities show the prensence of other silver containing species, which are

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ACCEPTED MANUSCRIPT larger than the pristine AgNPs and are probably a mixture of silver agglomerates/aggregates

458

and silver/polymer heteroaggretates. The large variety in the peak intensities demonstrates,

459

that the silver amount of these species is very inhomogeneous. This is also in good

460

agreement with STEM images of these migration samples as presented in Figure 3

461

(unfractionated migration sample) and Figure 8, where two fractions of an AF4 run were

462

collected and analyzed independently. The first fraction correlates here to the peak signal in

463

section A from Figure 7 (Fig. 8 A/B), the second fraction correlates to section B – F

464

(Fig. 8 C/D). Again, all types of nanosilver species – whether isolated and agglomerated or

465

bound as heteroaggregates – can be identified.

466

It should be taken into account, that the AF4 can capture the complete particle (polymer part

467

included), wheras the ICP-MS as element-specific detector for silver completely ignores the

468

polymer part of eventually occuring heteroaggregates. Therefore the size information

469

obtained from the RIKILT SPC spreadsheet evaluation of the signal intensity of

470

heteroaggregate peaks is at least partial rather fictive, as the size is calculated with the

471

assumption, that the signal comes from one single spherical pure silver nanoparticle and not

472

from an AgNP agglomerate/aggregate or even another heterogeneous, probably not

473

spherical particle.

474

In order to overcome this problem, the larger heteroaggregates could be separated before

475

AF4 fractionation (e.g. filtration) in oder to guarantee a meaningful characterization at least

476

for the smaller unbound or only loosely agglomerated AgNP species. With focus on the

477

heteroaggregates any further adaption of the applied AF4 method, which was actually

478

optimized for the fractionation of single AgNPs, could only be carried out with a proper size

479

calibration. However, finding adequate size calibration standards is impossible, because the

480

composition of the silver/polymer heteroaggregates is very inhomogeneous with regard to

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ACCEPTED MANUSCRIPT 481

the shape and the amount of incorporated silver and their distribution in the polymer matrix

482

itself. The application of AF4 in this study was focusing on the isolation of AgNPs from the

483

ionic silver background. Further opimization steps regarding sample preparation and/or the

484

AF4 method parameters in order to improve the fractionation itself are still pending.

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4.

Conclusions

487

In this study the migration of nanosilver from model food packaging films into food

488

simulants was investigated by carrying out migration assays based on the application of the

489

EU regulation. For analyzing the migration samples the coupling of AF4 and sp-ICP-MS was

490

introduced as new application-oriented approach focusing on the selective detection of

491

AgNP migration at very low concentrations in the ng/l-range. This way it could be clearly

492

verified, that no noteworthy AgNP migration occurred in acetic acid. In water as simulant,

493

besides isolated nanosilver particles also nanosilver/polymer heteroaggregates have been

494

released, which could also be confirmed by STEM imaging. By eliminating the ionic silver

495

background and fractionating the released heterogeneous aggregates and agglomerates by

496

AF4, the polydispersity of the samples could be reduced significantly and it was possible to

497

derive information about the AgNP fraction in the heteroaggregates by evaluating the

498

collected online-sp-ICP-MS data.

499

All in all, the coupling of AF4 and sp-ICP-MS with its increased sensitivity represents a

500

promising analytical approach for monitoring the release of nanosilver also in commercial

501

FCMs, which have a much lower silver content than the model films in this study.

502

The finding, that also heteroaggregates and polymer fragments are present, has to be taken

503

into account when analyzing the hydrodynamic diameter of released nanoparticle species

504

and should be considered in nanosilver-related safety assessment studies.

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Acknowledgements

506

The authors would like to thank their teams and colleagues for their technical support and

507

scientific contribution, especially Anja Lauckner-Tessin, Fabian Mohr, Lola Hogekamp,

508

Simone Brümmer and Judith Steinbacher.

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ACCEPTED MANUSCRIPT References

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Artiaga, G., Ramos, K., Ramos, L., Cámara, C. & Gómez-Gómez, M. (2015). Migration and characterisation of nanosilver from food containers by AF4-ICP-MS. Food Chemistry, 166, 7685. Beer, C., Foldbjerg, R., Hayashi, Y., Sutherland, D. S. & Autrup, H. (2012). Toxicity of silver nanoparticles - nanoparticle or silver ion? Toxicology Letters, 208(3), 286-292. Böhmert, L., Girod, M., Hansen, U., Maul, R., Knappe, P., Niemann, B., Weidner, S. M., Thünemann, A. F. & Lampen, A. (2014). Analytically monitored digestion of silver nanoparticles and their toxicity on human intestinal cells. Nanotoxicology, 8(6), 631-642. Bott, J., Störmer, A. & Franz, R. (2014). A Comprehensive Study into the Migration Potential of Nano Silver Particles from Food Contact Polyolefins. In Chemistry of Food, Food Supplements, and Food Contact Materials: From Production to Plate (Vol. 1159, pp. 51-70): American Chemical Society. Bumbudsanpharoke, N. & Ko, S. (2015). Nano-Food Packaging: An Overview of Market, Migration Research, and Safety Regulations. Journal of Food Science, 80(5), R910-R923. Burcza, A., Gräf, V., Walz, E. & Greiner, R. (2015). Impact of surface coating and food-mimicking media on nanosilver-protein interaction. Journal of Nanoparticle Research, 17(11), 1-15. Cushen, M., Kerry, J., Morris, M., Cruz-Romero, M. & Cummins, E. (2012). Nanotechnologies in the food industry – Recent developments, risks and regulation. Trends in Food Science & Technology, 24(1), 30-46. Dubascoux, S., von Der Kammer, F., Le Hecho, I., Gautier, M. P. & Lespes, G. (2008). Optimisation of asymmetrical flow field flow fractionation for environmental nanoparticles separation. Journal of Chromatography A, 1206(2), 160-165. Echegoyen, Y. & Nerín, C. (2013). Nanoparticle release from nano-silver antimicrobial food containers. Food and Chemical Toxicology, 62, 16-22. European Commission. (10/2011). Commission Regulation (EU) No 10/2011 of 14 January 2011 on plastic materials and articles intended to come into contact with food. In Offical Journal of the European Union (Vol. 15.1.2011). Giles, E., Kuznesof, S., Clark, B., Hubbard, C. & Frewer, L. (2015). Consumer acceptance of and willingness to pay for food nanotechnology: a systematic review. Journal of Nanoparticle Research, 17(12), 1-26. Hagendorfer, H., Kaegi, R., Parlinska, M., Sinnet, B., Ludwig, C. & Ulrich, A. (2012). Characterization of silver nanoparticle products using asymmetric flow field flow fractionation with a multidetector approach – a comparison to transmission electron microscopy and batch dynamic light scattering. Analytical Chemistry, 84(6), 2678-2685. Hassellöv, M., Readman, J. W., Ranville, J. F. & Tiede, K. (2008). Nanoparticle analysis and characterization methodologies in environmental risk assessment of engineered nanoparticles. Ecotoxicology, 17(5), 344-361. Huang, Y., Chen, S., Bing, X., Gao, C., Wang, T. & Yuan, B. (2011). Nanosilver migrated into foodsimulating solutions from commercially available food fresh containers. Packaging Technology and Science, 24(5), 291-297. Huynh, K. A., Siska, E., Heithmar, E., Tadjiki, S. & Pergantis, S. A. (2016). Detection and Quantification of Silver Nanoparticles at Environmentally Relevant Concentrations Using Asymmetric Flow Field–Flow Fractionation Online with Single Particle Inductively Coupled Plasma Mass Spectrometry. Analytical Chemistry, 88(9), 4909-4916. Juling, S., Bachler, G., von Götz, N., Lichtenstein, D., Böhmert, L., Niedzwiecka, A., Selve, S., Braeuning, A. & Lampen, A. (2016). In vivo distribution of nanosilver in the rat: The role of ions and de novo-formed secondary particles. Food and Chemical Toxicology, 97, 327-335. Klein, C. L., Comero, S., Stahlmecke, B., Romazanov, J., Kuhlbusch, T. A. J., Van Doren, E., Mast, J., Wick, P., Krug, H., Locoro, G., Hund-Rinke, K., Kördel, W., Friedrichs, S., Maier, G., Werner, J.,

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Linsinger, T. & Gawlik, B. M. (2011). NM-300 Silver Characterisation, Stability, Homogeneity. Publications Office of the European Union, EUR 24693 EN, 1-84. Laborda, F., Bolea, E. & Jiménez-Lamana, J. (2013). Single Particle Inductively Coupled Plasma Mass Spectrometry: A Powerful Tool for Nanoanalysis. Analytical Chemistry, 86(5), 2270-2278. Lee, S., Bi, X., Reed, R. B., Ranville, J. F., Herckes, P. & Westerhoff, P. (2014). Nanoparticle size detection limits by single particle ICP-MS for 40 elements. Environmental Science & Technology, 48(17), 10291-10300. Lichtenstein, D., Ebmeyer, J., Knappe, P., Juling, S., Böhmert, L., Selve, S., Niemann, B., Braeuning, A., Thünemann, A. F. & Lampen, A. (2015). Impact of food components during in vitro digestion of silver nanoparticles on cellular uptake and cytotoxicity in intestinal cells. Biological chemistry, 396(11), 1255-1264. Loeschner, K., Hadrup, N., Qvortrup, K., Larsen, A., Gao, X., Vogel, U., Mortensen, A., Lam, H. R. & Larsen, E. H. (2011). Distribution of silver in rats following 28 days of repeated oral exposure to silver nanoparticles or silver acetate. Particle and Fibre Toxicology, 8(1), 18. Loeschner, K., Navratilova, J., Købler, C., Mølhave, K., Wagner, S., Kammer, F. v. d. & Larsen, E. H. (2013). Detection and characterization of silver nanoparticles in chicken meat by asymmetric flow field flow fractionation with detection by conventional or single particle ICP-MS. Analytical and Bioanalytical Chemistry, 405(25), 8185-8195. Mackevica, A., Olsson, M. E. & Hansen, S. F. (2016). Silver nanoparticle release from commercially available plastic food containers into food simulants. Journal of Nanoparticle Research, 18(1), 1-11. Menzel, M. c., Bienert, R., Bremser, W., Girod, M., Rolf, S., Thünemann, A. F. & Emmerling, F. (2013). Certification Report - Certified Reference Material BAM-N001 - Particle Size Parameters of Nano Silver. In. Berlin: BAM - Federal Institute for Materials Research and Testing. Mudalige, T. K., Qu, H. & Linder, S. W. (2015). Asymmetric Flow-Field Flow Fractionation Hyphenated ICP-MS as an Alternative to Cloud Point Extraction for Quantification of Silver Nanoparticles and Silver Speciation: Application for Nanoparticles with a Protein Corona. Analytical Chemistry, 87(14), 7395-7401. Park, K. (2013). Toxicokinetic differences and toxicities of silver nanoparticles and silver ions in rats after single oral administration. Journal of Toxicology and Environmental Health, Part A, 76(22), 1246-1260. Peters, R., Herrera-Rivera, Z., Undas, A., van der Lee, M., Marvin, H. J. P., Bouwmeester, H. & Weigel, S. (2015). Single particle ICP-MS combined with a data evaluation tool as a routine technique for the analysis of nanoparticles in complex matrices. Journal of Analytical Atomic Spectrometry, 30(6), 1274-1285. RAS AG. (2015). AgPURE - Technical Information. Reidy, B., Haase, A., Luch, A., Dawson, K. & Lynch, I. (2013). Mechanisms of Silver Nanoparticle Release, Transformation and Toxicity: A Critical Review of Current Knowledge and Recommendations for Future Studies and Applications. Materials, 6(6), 2295-2350. Song, H., Li, B., Lin, Q. B., Wu, H. J. & Chen, Y. (2011). Migration of silver from nanosilver– polyethylene composite packaging into food simulants. Food Additives & Contaminants: Part A, 28(12), 1758-1762. Störmer, A., Bott, J., Kemmer, D. & Franz, R. (2017). Critical review of the migration potential of nanoparticles in food contact plastics. Trends in Food Science & Technology, 63, 39-50. van der Zande, M., Vandebriel, R. J., Van Doren, E., Kramer, E., Herrera Rivera, Z. E., Serrano-Rojero, C. S., Gremmer, E. R., Mast, J., Peters, R. J. B., Hollman, P. C. H., Hendriksen, P. J. M., Marvin, H. J. P., Peijnenburg, A. A. C. M. & Bouwmeester, H. (2012). Distribution, Elimination, and Toxicity of Silver Nanoparticles and Silver Ions in Rats after 28-Day Oral Exposure. ACS Nano, 6(8), 7427-7442. Vance, M. E., Kuiken, T., Vejerano, E. P., McGinnis, S. P., Hochella, M. F., Rejeski, D. & Hull, M. S. (2015). Nanotechnology in the real world: Redeveloping the nanomaterial consumer products inventory. Beilstein Journal of Nanotechnology, 6, 1769-1780.

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von Goetz, N., Fabricius, L., Glaus, R., Weitbrecht, V., Günther, D. & Hungerbühler, K. (2013). Migration of silver from commercial plastic food containers and implications for consumer exposure assessment. Food Additives & Contaminants: Part A, 30(3), 612-620. Walczak, A. P., Fokkink, R., Peters, R., Tromp, P., Herrera Rivera, Z. E., Rietjens, I. M. C. M., Hendriksen, P. J. M. & Bouwmeester, H. (2013). Behaviour of silver nanoparticles and silver ions in an in vitro human gastrointestinal digestion model. Nanotoxicology, 7(7), 1198-1210.

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Figure captions

2

Fig. 1. Representative STEM image of AgPURE (left) with particle size distribution histogram

3

(right).

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Fig. 2. LoVac-SEM images of film 2 (A, 5,000x) and film 5 at two magnification factors (B:

6

5,000x/C: 11,000x) with a characteristic EDS spectrum (D) of nanosilver aggregation on/in

7

film 5 at higher magnification (C).

9 10

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Fig.3. STEM analysis of aqueous migration samples of film 2 (A-C) and film 5 (D-F) showing single nanosilver particles and silver/polymer heteroaggregates.

11

Fig.4. Total silver migration into food simulants in dependency on the silver amount in all

13

model films after 2 hours storage at 70 °C (A) and after 10 days storage at 40°C (B). The ratio

14

of polymer contact surface area to food simulant was 1.3 +/- 0.1 cm2/ml. The statistical

15

significant difference between the simulants is marked with a bracket.

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Fig.5. sp-ICP-MS time scan of pristine AgPURE (A) and resulting particle size distribution (B);

18

sp-ICP-MS time scans of migration samples of film 5 in water (C) and 3 % acetic acid (D).

19

20

Fig.6. Comparison of a typical AF4-ICP-MS fractograms obtained for pristine AgPURE (A) and

21

migration samples of film 2 and film 5 in water (B). The total silver concentrations of the

ACCEPTED MANUSCRIPT samples were determined by classical ICP-MS as 0.2 µg/l (AgPURE), 0.42 µg/l (film 2) and

23

22 µg/l (film 5). The ratio of polymer contact surface area to food simulant was 1 cm2/ml.

24

Fig.7. Typical AF4-sp-ICP-MS fractograms showing the nanosilver release of film 5 in acetic

25

acid (top row) and water (middle row). The total silver concentration was determined by

26

classical ICP-MS as 22.3 µg/l (acetic acid migration sample) and 22 µg/l (water migration

27

sample). For sp-ICP-MS data evaluation the fractogram of the water migration samples was

28

sectioned into six time intervals (bottom row); as there was an insufficient number of

29

particle peaks in section 6, section 5 and 6 were merged for evaluation. The number of

30

detected particles and the derived mean particle diameter for the evaluated subsections are

31

as follows: 748 particles/21 nm (section B), 1118 particles/24 nm (section C), 982

32

particles/25 nm (section D) and 740 particles/29 nm (section E/F).

33

Fig.8. STEM images of two collected AF4 fractions (same migration experiment with film 5 in

34

water as shown in Fig. 7) showing the presence of single AgNPs and nanosilver/polymer

35

heteroaggregates. Image A and B are taken from a collected AF4 sample, which correlates

36

with subsection A from Figure 7, image C and D are derived from a collected AF4 sample,

37

which corresponds with subsections B-F from Figure 7.

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Figure Caption for Figure S1 in Supplementary Information

40

Fig. S1. Dark field STEM images of pristine AgPURE for the size distribution analysis with

41

ImageJ.

42

ACCEPTED MANUSCRIPT Table 1 Instrumental parameters for the microwave-assisted digestion of nanosilver model films. Power (W) 680 680 680

Temperature (°C) 110 150 180

Time (min) 10 10 20

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ACCEPTED MANUSCRIPT Table 2 SEM settings for LoVac and STEM mode SEM settings LoVac mode Accelerating voltage Chamber pressure Working distance (WD) Detector Specimen holder STEM mode Accelerating Voltage Chamber pressure Working distance (WD) Detector

Details

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7-10 kV 130 Pa (adjusted with water vapor) 10 mm GAD (Gaseous Analytical Detector) Aluminium stubs (Ø = 12 mm) with adhesive support tabs

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30 kV ∼ 0.0003 Pa (high vacuum) 5 mm STEM detector (integrated in sample stage holder, dark field and bright field observation possible) TEM grids (Ø = 3 mm, copper, 300 mesh) with holey carbon support film

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ACCEPTED MANUSCRIPT Table 3 ICP-MS and AF4 settings ICP-MS settings RF power Flow rates: Cool gas Nebulization gas Auxiliary gas Sample

Details 1550 W

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14 l/min 1 l/min 0.8 l/min 0.4 ml/min (offline measurements) 0.5 ml/min (online measurements) PFA , concentric Cyclonic, quartz, Peltier-cooled

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Nebulizer Spray chamber Additional parameters for single particle analysis: Acquisition time 60 s (for offline measurements) / ca. 58 min (AF4-sp-ICP-MS coupling) Dwell time 200 ms (classical mode) / 5 ms (single particle mode) 107 Monitored mass Ag External calibration Ag ion standard solution in 2% HNO3, concentration range: 0 – 5 µg/l 4 AF settings Membrane 10 kDa regenerated cellulose Spacer 350 µm Injection volume 500 µl Eluent ultrapure water Detector flow 0.5 ml/min Focus step: Focusing time 12 min Injection flow 0.2 ml/min Focus flow 1.5 ml/min 0.5 min Transition time Elution step: Cross flow From 1.2 ml/min to 0.1 ml/min Gradient Linear decrease within 35 min 10 min constant 0.1 ml/min

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nanosilver content (mg/kg)

Blank film film 1 film 2 film 3 film 4 film 5

0.16 ± 0.10 710 ± 49 1279 ± 35 2850 ± 213 5801 ± 419 12281 ± 346

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Sample

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Silver content of the model LDPE films as determined by ICP-MS analysis after microwaveassisted digestion (n = 4).

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ACCEPTED MANUSCRIPT Highlights Model polymer films with varying AgNP content were used for migration studies

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Migrated silver (Ag+ ions and AgNP) correlates with silver content in films

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Sp-ICP-MS was used as key analytical method for the detection of migrated AgNP

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Coupling of AF4 to sp-ICP-MS resulted in improved sp-ICP-MS sensitivity

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Release of AgNP/polymer heteroaggregates was detected by sp-ICP-MS and STEM

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imaging