Carbon Nanotubes Released from an Epoxy-Based Nanocomposite

Aug 6, 2015 - (15) However, the provided data were not sufficient for a risk assessment study, which would require information on the released dose of...
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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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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

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

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a

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Science and Technology, Dübendorf, Switzerland;

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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;

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c

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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

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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

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9–14

. No

multi-walled carbon nanotubes

. However, the provided data were not

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, 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

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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

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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.

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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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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

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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

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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

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instructions.

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2.4 STATISTICAL ANALYSIS

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(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

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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,

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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

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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

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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+-

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

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

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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

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

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

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

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

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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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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