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Carbon Nanotubes Released from an Epoxy-Based Nanocomposite: Quantification and Particle Toxicity Lukas Schlagenhauf, Tina Bürki-Thurnherr, Yu-Ying Kuo, Adrian Wichser, Frank Alain Nüesch, Peter Wick, and Jing Wang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b02750 • Publication Date (Web): 06 Aug 2015 Downloaded from http://pubs.acs.org on August 11, 2015

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Environmental Science & Technology

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Carbon nanotubes released from an epoxy-based

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nanocomposite: quantification and particle toxicity

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Lukas Schlagenhaufa,b,c, Tina Buerki-Thurnherrd, , Yu-Ying Kuob,c, Adrian Wichserb, Frank

4

Nüescha, Peter Wickd, and Jing Wang*,b,c.

5

a

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Science and Technology, Dübendorf, Switzerland;

7 8

Laboratory for Functional Polymers, Empa - Swiss Federal Laboratories for Materials

b

Laboratory for Advanced Analytical Technologies, Empa - Swiss Federal Laboratories for

Materials Science and Technology, Dübendorf, Switzerland;

9

c

10

d

Institute of Environmental Engineering, ETH Zurich, Zurich, Switzerland; Laboratory for Materials-Biology Interactions, Empa - Swiss Federal Laboratories for

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Materials Science and Technology, St. Gallen, Switzerland.

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*Corresponding author: Jing Wang; [email protected]; Überlandstrasse 129, CH-

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8600 Dübendorf; Tel: +41 58 765 6115; Fax: +41 58 765 46 14

ACS Paragon Plus Environment


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ABSTRACT

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Studies combining both the quantification of free nanoparticle release and the toxicological

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investigations of the released particles from actual nano-products in a real life exposure

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scenario are urgently needed, yet very rare.

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Here, a new measurement method was established to quantify the amount of free standing

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and protruding multi-walled carbon nanotubes (MWCNTs) in the respirable fraction of

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particles abraded from a MWCNT/epoxy nanocomposite. The quantification approach

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involves the pre-labeling of MWCNTs with lead ions, nanocomposite production, abrasion

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and collection of the inhalable particle fraction and quantification of free standing and

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protruding MWCNTs by measuring the concentration of released lead ions. In vitro toxicity

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studies for genotoxicity, reactive oxygen species formation and cell viability were performed

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using A549 human alveolar epithelial cells and THP-1 monocyte-derived macrophages.

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The quantification experiment revealed that in the respirable fraction of the abraded

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particles, approx. 4000 ppm of the MWCNTs were released as exposed MWCNTs which

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could contact lung cells upon inhalation, and approx. 40 ppm were released as free standing

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MWCNTs in the worst case scenario. The release of exposed MWCNTs was lower for

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nanocomposites containing agglomerated MWCNTs. The toxicity tests revealed that the

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abraded particles did not induce any acute cytotoxic effects.

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1. INTRODUCTION

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Composite materials with carbon nanotubes (CNTs) as filler materials have superior

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properties compared to the neat matrix material. The CNTs can improve the mechanical

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properties 1 and add new features like electrical 2 and thermal conductivity 3. CNT composites

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have numerous applications and are used in different industries, e.g. automotive, aerospace,

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defense, electronics, energy, and sporting goods 4.


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Because of the high aspect ratio and biopersistent nature of CNTs, concerns have been raised

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that CNT composites can pose a risk to producers and consumers if the CNTs are released

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into the environment 5–8. One possible pathway for a release of CNTs from nanocomposites is

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the generation of particles by an abrasion process. The toxicity of abraded particles from

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nanocomposites has been investigated already by several in vitro and in vivo studies

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study found an additional toxic effect caused by the added nanoparticles in comparison with

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the neat matrix materials, however for all tested composites, no release of the nanoparticles

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was detected.

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After abrasion of an epoxy/CNT nanocomposite, three different kinds of CNTs are potentially

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present in the produced dust: CNTs that are completely embedded in the polymer matrix,

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CNTs that are protruding from polymer particles, and free standing CNTs that are released

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from the epoxy matrix. When inhaled, the free standing and protruding CNTs may directly get

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into contact with lung cells and thus can have a toxic impact, thus they are defined as exposed

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CNTs. Completely embedded CNTs are not expected to be toxic. In a recent study, we

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showed that free standing single and agglomerated

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(MWCNTs) as well as protruding MWCNTs can be released when an abrasion process was

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applied on a MWCNT/epoxy nanocomposite

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sufficient for a risk assessment study, which would require information on the released dose

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of each abraded particle type (embedded, protruding, free) and on their hazard assessment.

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Therefore, the purpose of the present study was i) to develop a novel method for the

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quantification of the different types of abraded particles released from a CNT/epoxy

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nanocomposite upon abrasion, ii) to apply the method on samples with different CNT

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dispersion states, and iii) to investigate if these particles induce acute toxic, inflammatory or

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genotoxic effects on lung epithelial cells or macrophages. Although several methods have

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already been published that allow the detection of total CNT levels

15

9–14

. No

multi-walled carbon nanotubes

. However, the provided data were not


16–22

, none of them can


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determine the sub-fraction of free standing CNTs or protruding CNTs, which are most critical

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regarding potential adverse health effects. We developed a novel method, in which MWCNTs

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were pre-labeled with appropriate metal ions by their ability of surface adsorption

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abrasion of a composite containing the marked MWCNTs, the generated particles were

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collected and immersed in an acidic solution in order to desorb the ions from the exposed

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MWCNTs 25,26 while the ions on the embedded MWCNTs remained bound. By determination

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of the ion concentration using inductively coupled plasma mass spectrometry (ICP-MS), the

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quantity of the free standing and protruding MWCNTs was determined. A scheme of the

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quantification experiment is shown in Fig. 1. The same measurement principle can be applied

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on an ion release from the residues of the production catalyst in the CNTs

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approach is not applicable for CNTs with low or no catalyst residues, e.g. functionalized

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CNTs with an acid refluxing method. Nevertheless, quantification by dissolution of the

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intrinsic catalyst ions was used as comparison control of the adsorption method. To explore

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the potential and the limits of this new quantification method, we assessed two sets of 1 wt%

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MWCNT/epoxy composites that mainly differ in the type of curing agent resulting in

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different released amounts of the three abraded particle types. The first nanocomposite

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showed a release of protruding MWCNTs but no free standing MWCNTs when an abrasion

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process was applied. Here we tested three different MWCNT dispersion conditions to

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investigate if the dispersion quality had an impact on the release of MWCNTs and if such

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differences could be detected by our quantification method. The second nanocomposite, for

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which we have detected a release of both protruding and free standing MWCNTs upon

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abrasion 15, was included in this study to see if the presented quantification method was able

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to exclusively measure the free standing MWCNTs.

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Finally, we performed in vitro toxicity studies to investigate the potential hazard of the

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abraded dust containing free standing MWCNTs. Human alveolar epithelial cell line A549


20

23,24

. After

. However, this

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and human monocytic leukemia cell line THP-1 were chosen as they represent crucial cell

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types of the lung, important for the formation of the air-blood barrier and for the removal of

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foreign materials, respectively. For many nanoparticles including CNTs, it has been shown

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that they exert adverse effects through an increase in reactive oxygen species (ROS) leading

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to an oxidative stress that may finally culminate in the severe damage of cell compounds, the

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release of pro-inflammatory factors, genotoxicity and finally cell death

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assessed the effects of abraded particles from MWCNT/epoxy nanocomposites on the

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formation of ROS by the DCF assay, on cytokine release by enzyme linked immunosorbent

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assay (ELISA), on DNA strand breaks by the comet assay and on cell viability by the MTS

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assay, in comparison to pure MWCNTs and abraded particles from the neat epoxy matrix or

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the abrasion wheel.

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2 MATERIALS AND METHODS

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2.1 MATERIAL PREPARATION

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MWCNTs (Baytubes, C150p) were supplied by Bayer Material Science AG. The used epoxy

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resin was Araldite GY 250 (Huntsman, USA), which was based on bisphenol A. The curing

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agents were either the aromatic di-amine curing agent Epikure 3402 (Hexion, USA) or the

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polytheramine Jeffamine D-230 (Huntsman, USA). The resin/hardener ratio was 100:24 for

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the Epikure 3402 curing agent and 100:32 for the D-230 curing agent. The nanocomposite

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preparation was identical for both epoxy systems. The MWCNTs were dispersed in the epoxy

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resin first for 30 min by ultrasonication and then by three-roll milling (SDY200, Bühler AG,

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Switzerland) at 30 °C and at a gap pressure of 1 MPa. The milling process was applied one to

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three times. After mixing with the curing agent, the composite was cured at 80 °C for 12 h,

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followed by post curing at 120 °C for 4 h.

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2.2 INSTRUMENTATION AND METHODS


27,28

. Therefore, we


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2.2.1 ABRASION AND PARTICLE COLLECTION

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The same setup was used for the abrasion experiment as described by Schlagenhauf et al. 15,

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who also presented the treatment of the background particles and particle loss in the sampling

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system. This setup included a Taber Abraser (5135, Taber, USA) with the abrasive wheel H-

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18 (Taber, USA); 0.75 kg of weight was applied at 60 rpm. Particles were sucked with a tube

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through an inlet and collected on Nuclepore track-etched polycarbonate membranes (111106,

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Whatman, UK) with a pore size of 0.2 µm. The air flow was generated with a pump

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(N816.1.2KN.18, KNF, Germany). The particle size distribution and the particle

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microstructure of the abraded particles from the samples that were cured with the curing agent

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3402 were presented in Schlagenhauf et al.

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curing agent D-230 can be found in the supplemental information (SI) in Fig. S1(a)-(f).

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The experiments required to collect particles either with an aerodynamic diameter below

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1 µm (particulate matter PM1) or below 100 nm (ultrafine particles UFPs). For the collection

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of PM1 particles, a combination of two cyclones was added to the sampling system. The first

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cyclone, originally designed for a fast mobility particle sizer spectrometer (FMPS) (3901,

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TSI, USA; part number of cyclone: 1031083R), was used to remove the majority of the coarse

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particles. The second cyclone was used to remove the rest of the micrometer sized particles

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(URG-2000-30EQ, URG, USA). The collection efficiency was measured during an abrasion

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experiment with an aerodynamic particle sizer (APS) (3321, TSI, USA) (see SI, Fig. S2(a)).

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The measured particle diameter for 50 % collection efficiency D50 was approx. 0.9 µm, and

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no particles bigger than 2 µm passed the cyclones. Depending on the abrasion rate, the two

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cyclones were partially clogged within a few minutes and thus could allow bigger particles to

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pass. The size of particles, passing the cyclones, was monitored by an APS connected to the

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sampling system through an accessory loop. The abrasion process was stopped and the

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cyclones were cleaned as soon as large particles were detected.

15

and the same analysis for the samples with the


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For the collection of UFPs, in addition to the cyclones, a neutralizer (Isotope: Kr85, Eckert &

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Ziegler Isotope Products, USA) and a Micro-Orifice Uniform-Deposit Impactor (MOUDI)

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(122-NR, MSP, USA) were added to the sampling system. The MOUDI was operated with a

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flow rate of 13 l/min and the stages 1-11 were used. The calibration of the MOUDI was done

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with NaCl particles and D50 was approx. 126 nm (see SI, Fig. S2(b)). A schematic diagram of

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the particle collection setup is shown in Fig. 2.

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For the toxicity tests, the abraded particles were collected without the separation of large

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particles on Nuclepore track-etched polycarbonate membranes.

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2.2.2 MWCNT DETECTION BY ION UPTAKE AND RELEASE

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For the ion uptake method, lead ions (Pb2+) were used as the marking element. A comparison

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of different elements can be found in the SI, Fig. S3(a). For the preparation of the composite

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samples, a master-batch of Pb2+ marked MWCNTs was produced with 5 g of MWCNTs,

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immersed in 1 l of water with a Pb2+ concentration of 200 mg/l (Pb(II) acetate trihydrate

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(Merck, Germany). The MWCNTs were dispersed in the solution for 30 min in an

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ultrasonication bath and further stirred at 250 rpm for 2.5 h. Afterward, the solution was

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filtered and the collected MWCNTs were rinsed several times. The MWCNTs were dried

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overnight in a vacuum oven at 50 °C. The ion uptake capacity was determined by immersion

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of the MWCNTs into a 0.1 M HNO3 solution (Suprapure® Nitric acid 65 %, Merck KGaA,

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Germany) for 1 h. The MWCNTs then were removed by centrifuge filtration (Amicon Ultra-4

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30 kDa, Merck Millipore, USA) and the concentrations of the marker ions and also of the

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released catalyst ions were determined by ICP-MS (Elan 6000, Perkin Elmer, USA).

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The results of the master-batch characterization are shown in the SI in Fig. S3(b). The Pb2+

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uptake and release for this preparation procedure was 8.7 ± 0.4 µg/mg, and the released



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catalyst ions per unit weight of MWCNTs were 2.82 ± 0.1 µg/mg for manganese (Mn2+) and

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2.24 ± 0.1 µg/mg for cobalt (Co2+).

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Composite samples with ion-loaded MWCNTs were produced as described above. In

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addition, two control samples (A & B) were produced to verify the experimental setup. For

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control sample A, Pb2+-loaded MWCNTs were dispersed in epoxy resin by sonication for

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60 min. Afterwards, 1 g of the resin was dissolved in acetone, filtered to remove the

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MWCNTs and dissolved in 0.1 M HNO3. This sample was produced to test if the loaded Pb2+

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ions might be detached from the MWCNTs and be dissolved in the epoxy resin prior to the

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curing. Control sample B was a neat epoxy sample without the addition of MWCNTs. Before

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curing with D-230, Pb2+ ions were added to the resin and equally dispersed. The ion

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concentration was equal to the loaded ions in a 1 wt% MWCNT sample. This sample was

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used in the same abrasion and quantification procedure.

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2.2.3 QUANTIFICATION OF EXPOSED MWCNTs

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For the quantification experiment, 100 mg of mass was abraded from each sample and

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collected on a filter. The filter then was immersed in 5 ml 0.1 M HNO3 and sonicated for

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30 min. After 1 h, the remaining particles were removed by centrifuge filtration and the ion

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concentration was determined by ICP-MS. For improved accuracy, the ion release of the

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Nuclepore filter and of the neat epoxy was also measured and the value was subtracted from

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the result. The fraction of detected MWCNTs was calculated by Eq. 1,

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To measure the effect of the MWCNT dispersion on the release of exposed MWCNTs in the

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respirable fraction of abraded particles, three 1 wt% MWCNT samples with the curing agent

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D-230 were produced. The first sample was milled only once with the three roll mill, the


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second twice, and the third three times. The more milling steps, the better were the nanotubes

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dispersed and the less MWCNT agglomerates were present in the nanocomposite. For

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nanocomposites that were prepared with the curing agent D-230, no release of free standing

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MWCNTs was detected in preliminary experiments by inspection of transmission electron

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microscopy (TEM) images. To measure the difference between a nanocomposite that released

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free standing MWCNTs and one without release, one 1 wt% MWCNT sample with the curing

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agent Epikure 3402 was produced. The three roll mill was applied three times and for samples

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with this curing agent, a release was detected 15.

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2.3 TOXICITY EXPERIMENTS

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2.3.1 CELL CULTURE AND CELL TREATMENT

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The human alveolar epithelial cell line A549 (CCL-185, ATCC, USA) and human acute

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monocytic leukemia cell line THP-1 (TIP-202, ATCC) were maintained in complete cell

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culture medium (RPMI-1640 medium (Sigma-Aldrich, USA) supplemented with 10 % FCS

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(Lonza, Switzerland), 0.2 mg/ml L-glutamine (Gibco®, Life Technologies (Thermo Fisher

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Scientific), USA), and 1 % penicillin-streptomycin-neomycin (PSN) (Gibco®). Cells were

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grown at 37 °C in a 5 % CO2 atmosphere, and were subcultured twice a week. For

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cytotoxicity testing, abraded particles or MWCNTs were suspended in Millipore water

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containing 0.016 % Pluronic F127 (Sigma-Aldrich, USA) to a final stock solution of

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500 µg/ml. The particle suspensions were sonicated in an ultrasonic bath (Sonorex Super RK

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156 BH, Bandelin, Germany) for 10 min and further diluted with complete cell culture

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medium prior to the experiments. The final water content (16%) was the same for all dilutions

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(0-80 µg/ml) to exclude unspecific effects due to different dilution levels of the cell culture

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medium. To differentiate THP-1 monocytes into macrophages, cells were treated for 3 days

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with 200 nM phorbol 12-myristate 13-acetate (PMA) (Fluka, Sigma-Aldrich, USA) before

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particle exposure. ACS Paragon Plus Environment


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2.3.2 ANALYSIS OF CELL VIABILITY/ACTIVITY (MTS ASSAY)

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Cell viability was determined using the CellTiter96 Aqueous One Solution (Promega, USA)

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containing MTS as the tetrazolium compound according to the manufacturer’s instructions. In

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brief, 8 × 103 A549 cells or 4 × 104 THP-1 cells were seeded in 200 µl of complete cell

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culture medium per well of a 96 well plate and grown for 1 day (A549) or 3 days (THP-1, in

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presence of PMA). Cells were then treated with 200 µl per well of abraded particles,

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MWCNTs or the positive control CdSO4 at the indicated concentrations for 3, 24 or 48 h.

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Thereafter, medium containing stimuli was replaced by 120 µl of MTS working solution,

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incubated for 60 min at 37 °C and 5 % CO2 before optical density was measured at 490 nm in

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an EL800 microplate reader (BioTEK Instruments, USA). Original OD(490) values were

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blank-corrected and normalized to untreated samples.

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2.3.3 DETECTION OF REACTIVE OXYGEN SPECIES (DCF ASSAY)

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The formation of intracellular reactive oxygen species (ROS) was determined using the

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dichlorofluorescein

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dichlodihydrofluorescein, Molecular Probes) to fluorescent DCF by ROS. Briefly, 2 × 104

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A549 cells or 4 × 104 THP-1 cells were seeded per well of a 96 well plate in a volume of

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200 µl and grown for 1 day (A549) or 3 days (THP-1, in presence of PMA). Thereafter, the

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medium was replaced by 100 µl of 50 µM H2DCFDA in Hank’s buffered salt solution

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(HBSS) and cells were incubated for 60 min at 37 °C and 5 % CO2. After washing with

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prewarmed HBSS, cells were exposed to 100 µl of the indicated particle concentrations. The

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peroxinitrite donor 3-morpholinosydnonimine (Sin-1, Sigma-Aldrich) was used as a positive

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control. Sin-1 generates both superoxide anion and nitric oxide that spontaneously form

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peroxynitrite, a potent oxidant. Fluorescent intensities were measured after 2 h using a

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FLX800 fluorescence microplate reader (BioTEK Instruments) at an excitation wavelength of

(DCF)

assay,

measuring

the

conversion


of

H2DCF

(2’,7’-

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485 nm and an emission wavelength of 528 nm. Fluorescence values were blank-corrected

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and normalized to untreated controls.

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2.3.4 DETERMINATION OF DNA DAMAGE (COMET ASSAY)

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A549 and THP-1 cells were seeded in 6-well plates at 2.5 × 105 cells /well one day (A549) or

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3 days (THP-1, in presence of PMA) before treatment. Cells were exposed to the indicated

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concentrations of particles for 30 min and 3 h. Ethylmethanesulfonate (EMS, 5 mM, Sigma-

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Aldrich) was used as a positive control and added 30 min before the end of the incubation.

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The alkaline comet assay was then performed as previously described

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provided in the SI). Comets were analysed using a Nikon Eclipse TS 100 microscope

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equipped with a Stingray F046B IRF Jenofilt camera (Allied Vision Technologies, Germany)

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and the software “Comet IV” (Perceptive Instruments, United Kingdom) respectively. Per

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slide, 100 randomly chosen comets were analysed blinded.

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2.3.5 CYTOKINE ASSAY (ELISA)

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8 × 103 A549 cells or 4 × 104 THP-1 cells were seeded in 200 µl of complete cell culture

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medium per well of a 96 well plate and grown for 1 day (A549) or 3 days (THP-1, in presence

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of PMA). Cells were then treated with 200 µl per well of abraded particles, MWCNTs or

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positive controls (TNFα for A549 cells; lipopolysaccharide (LPS) for THP-1 cells) at the

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indicated concentrations for 3, 8 or 24 h. Supernatants were stored at 20 °C until

249

measurement. IL-8 or TNFα levels were determined using a commercial ELISA kit (IL-8 or

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TNFα ELISA Ready-SET-Go, eBioscience, USA) according to the manufacturer’s

251

instructions.

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2.4 STATISTICAL ANALYSIS


29

(a full description is


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For the quantification experiments, the samples were abraded and measured five times. The

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significance of difference between different quantification datasets was measured with F-tests

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by usage of one-way analysis of variance (ANOVA) (OriginPro 8, OriginLab corporation,

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USA).

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The toxicity data are shown as mean ± standard error of the mean (StEM) from at least three

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independent experiments. Statistical significance was determined using a two-tailed Student's

259

t test (Microsoft Excel, Microsoft Corporation, USA). A p-value below 0.05 was considered

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to be statistically significant.

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3 RESULTS AND DISCUSSION

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3.1 NANOPARTICLE DETECTION BY ION RELEASE

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The results of the quantification experiment for the PM1 particles from the control samples,

264

the samples with different MWCNT dispersion conditions, and the sample that releases free

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standing MWCNTs are shown in Fig. 3(a). Control sample A, the uncured epoxy resin with

266

Pb2+-loaded MWCNTs, showed that no lead or catalyst ions were transferred to the epoxy

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resin during the sonication. This is a prerequisite for the quantification method. For control

268

sample B (Pb2+-loaded neat epoxy D-230), the fraction of the released Pb2+ out of the total

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amount in the epoxy was 790 ± 1000 ppm. Compared to the abrasion samples with Pb2+-

270

loaded MWCNTs (1wt% MWCNT D-230 triply milled), significantly less Pb2+ was emitted

271

(P < 0.025). Even though only particles < 1 µm were collected, the diffusion pathway was too

272

long for all ions to be released. This result shows that the measured ions from the

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nanocomposites origin mainly from exposed MWCNTs. It is not known whether the ions

274

loaded on the totally embedded MWCNTs can migrate to the solution at all. Since diffusion

275

would be the major mechanism in this process, the experiment could be improved by

276

shortening the period between particle dissolution and filtering, at least if only the fast


Page 13 of 25


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desorbing loaded ions are measured 25 and not the catalyst ions, whose dissolution needs more

278

time. This shows the limits of the quantification experiment when catalyst ions are used.

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For the four 1 wt% MWCNT samples, both applied ion detection methods measured a similar

280

percentage of released ions independent of the sample preparation. Therefore, either method

281

can be used to quantify free and protruding MWCNTs in PM1 samples provided that enough

282

MWCNT catalyst is present in the MWCNTs. The three 1 wt% MWCNT samples with the

283

curing agent D-230 revealed a release behavior that was dependent on the dispersion

284

condition of the MWCNTs. If measuring the intrinsic catalyst ions, the sample with the worst

285

dispersion (one milling step) released significantly less exposed MWCNTs in the respirable

286

fraction than the standard sample with three milling steps (P < 0.007). For the measurement

287

with the Pb2+ ions, the result was less clear due to a larger measurement uncertainty of this

288

method (P < 0.14). We did not detect a difference in Pb2+ release between the triply milled D-

289

230 sample and the Epikure 3402, which implies that free standing MWCNTs are a minor

290

fraction of the PM1 particles. Therefore we performed quantification measurement for the

291

UFPs, in which the fraction of free standing MWCNTs was expected to be higher.

292

In a previous publication it was found that an epoxy composite containing poorly dispersed

293

MWCNTs was more likely to release free standing MWCNTs to the environment due to an

294

abrasion process 30. Interestingly, our results indicate the contrary, considerably less exposed

295

MWCNTs were present in once milled composites. These results imply that the low energy

296

abrasion process of the Taber Abraser was not capable of breaking up the MWCNT

297

agglomerates within the composite. Moreover, those agglomerates still bound to the epoxy

298

matrix, either were in the fraction of particles larger than 1 µm or they remained largely

299

covered by the epoxy. Therefore the total quantity of exposed MWCNTs (protruding or free

300

standing) was smaller for samples with poorly dispersed MWCNTs. For other abrasion



Page 14 of 25

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processes that use high energy input to abrade particles such as a sanding machine, the effect

302

of particle agglomeration on particle release has to be verified separately.

303

The results of the UFP measurements are shown in Fig. 3(b). In contrast to the measurements

304

of PM1 samples, the measured average values for ion release in UFP samples were much

305

lower and lay between 10 and 80 ppm. For the three samples with different dispersion states,

306

no different ion release among the samples was detected. Interestingly, when testing for the

307

expected increase of ion release in the Epikure samples, we only observed a significantly

308

increased ion release for Pb2+-loaded samples (approx. 50 ppm, p

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