Characterisation dossier for reference materials · The EU FP7 NanoDefine Project Development of an...

The EU FP7 NanoDefine ProjectDevelopment of an integrated approach based on validated and

standardized methods to support the implementation of the EC rec-

ommendation for a definition of nanomaterial

Characterisation dossier for referencematerials

NanoDefine Technical Report D1.6

Robert Koeber, Vikram Kestens,Ralf Bienert and Philipp Müller

The NanoDefine Consortium 2017

NanoDefine Technical Report D1.6 Characterisation dossier for reference materials

© 2017 The NanoDefine Consortium of 41

NanoDefine in a nutshell

The EU FP7 NanoDefine project was launched in November 2013 and will run until October 2017. Theproject is dedicated to support the implementation of the EU Recommendation on the Definition of Na-nomaterial by the provision of the required analytical tools and respective guidance. Main goal is to de-velop a novel tiered approach consisting of (i) rapid and cost-efficient screening methods and (ii) con-firmatory measurement methods. The "NanoDefiner" eTool will guide potential end-users, such as con-cerned industries and regulatory bodies as well as enforcement and contract laboratories, to reliablyclassify if a material is nano or not. To achieve this objective, a comprehensive inter-laboratory evalua-tion of the performance of current characterisation techniques, instruments and software is performed.Instruments, software and methods are further developed. Their capacity to reliably measure the size ofparticulates in the size range 1-100 nm and above (according to the EU definition) is validated. Tech-nical reports on project results are published to reach out to relevant stakeholders, such as policy mak-ers, regulators, industries and the wider scientific community, to present and discuss our goals and re-sults, to ensure a continuous exchange of views, needs and experiences obtained from different fieldsof expertise and application, and to finally integrate the resulting feedback into our ongoing work on thesize-related classification of nanomaterials.

Bibliographic data

NanoDefine Technical Report D1.6

Report title: Characterization dossier for reference materials

Author): Robert Koeber (JRC Geel), Vikram Kestens (JRC Geel), Ralf Bienert (BAM), Philipp

Müller1 (BASF)

Affiliation: JRC – Joint Research Centre European Commission IRMM, Retieseweg 111, 2440

Geel, BAM – Bundesanstalt für Materialforschung und Prüfung, Unter den Eichen 87, 12205

Berlin, BASF SE – Material Physics and Analytics, Carl-Bosch-Str. 38, 67056 Ludwigshafen

am Rhein

Publication date: 31/10/2017

Publisher: The NanoDefine Consortium

© Copyright 2017: The NanoDefine Consortium

Place of publication: Wageningen, The Netherlands

Citation: Koeber R., Kestens V., Bienert R., Müller P: Characterization dossier for referencematerials, NanoDefine Technical Report D1.6, NanoDefine Consortium, Wageningen, 2017

URL: http://www.nanodefine.eu/index.php/downloads/public-deliverables

Contact: [email protected], www.nanodefine.eu

The NanoDefine project has received funding from the European Union’s Seventh Programmefor research, technological development and demonstration under grant agreement No604347

________________________1 Corresponding author: [email protected]



Table of Contents1 Glossary...............................................................................................................................................6

2 Abbreviations and acronyms ...............................................................................................................7

3 Summary and material characterization report ...................................................................................8

4 Description of materials .......................................................................................................................9

4.1 Suspensions .................................................................................................................................9

4.2 Substances...................................................................................................................................9

4.3 Products .................................................................................................................................... 10

5 Processing of the test materials ....................................................................................................... 11

5.1 Suspensions .............................................................................................................................. 11

5.1.1 Monomodal silica (ID-17) ................................................................................................... 11

5.1.2 Monomodal polystyrene (ID-19)......................................................................................... 11

5.1.3 Trimodal silica (ID-18) ........................................................................................................ 11

5.1.4 Trimodal polystyrene (ID-20).............................................................................................. 11

5.2 Substances................................................................................................................................ 12

5.3 Products .................................................................................................................................... 12

5.3.1 Food with SiO2, Pancake dry mix – BAM-12b.................................................................... 12

5.3.2 Sunscreen, complex formulation – BAM-13a..................................................................... 12

5.3.3 Fe2O3 particles in HDPE – BAM-14 ................................................................................... 12

5.3.4 Toothpaste – BAM-15 ........................................................................................................ 12

6 Assessment of homogeneity and stability ........................................................................................ 13

6.1 Optimisation of DLS and CLS measurement procedures ......................................................... 13

6.1.1 Pigment Yellow 83 (IRMM-380, nano grade)..................................................................... 13

6.1.2 IRMM-381 (Barium sulfate, fine grade) .............................................................................. 16

6.1.3 IRMM-384 (Calcium carbonate, fine grade)....................................................................... 17

6.1.4 IRMM-386 (Pigment Yellow 83, coarse grade) .................................................................. 18

6.1.5 IRMM-387 (BaSO4, ultrafine grade) ................................................................................... 24

6.1.6 IRMM-388 (Coated TiO2) ................................................................................................... 25

6.1.7 BAM-14 (iron oxide in HDPE) ............................................................................................ 26

6.2 spICP-MS method for sample preparation and measurement NPs.......................................... 31

6.3 BET for determination of specific surface area ......................................................................... 33

6.4 Evaluation of homogeneity study samples ................................................................................ 34

6.4.1 Substances......................................................................................................................... 34

6.4.2 Products ............................................................................................................................. 36

6.5 Evaluation of stability study samples......................................................................................... 38

6.5.1 Substances......................................................................................................................... 38

6.5.2 Products ............................................................................................................................. 40

7 Conclusions ...................................................................................................................................... 41



Index of figures

Figure 1: Overlay of light extinction-weighted PSDs and their corresponding cumulative distributions forIRMM-380 (dispersed in purified water after applying 15 min (pink curve) and 30 min (blue curve) ofprobe sonication), obtained by CLS. ................................................................................................ 14

Figure 2: Overlay of number-weighted PSDs and their corresponding cumulative distributions for IRMM-380 (dispersed in purified water after applying 15 min (pink curve) and 30 min (blue curve) of probesonication), obtained by CLS............................................................................................................ 14

Figure 3: Light extinction-weighted PSD and its corresponding cumulative distribution for IRMM-380(dispersed in 0.5 g/L Nekal BX), obtained by CLS. .......................................................................... 15

Figure 4 : Number-weighted PSD and its corresponding cumulative distribution for IRMM-380(dispersed in 0.5 g/L Nekal BX), obtained by CLS. .......................................................................... 15

Figure 5: Overlay of scattered light intensity-weighted PSDs of IRMM-381 (dispersed in 2 g/kg SHMP),obtained by DLS. .............................................................................................................................. 16

Figure 6: Overlay of mass-weighted PSDs of IRMM-381 (dispersed in 2 g/kg SHMP and probesonicated for 10 min and 20 min), obtained by CLS. ....................................................................... 17

Figure 7: Overlay of CLS mass-weighted PSDs from 50 g/kg (pink curve) and 5 g/kg (blue curve) testsamples prepared from IRMM-384................................................................................................... 17

Figure 8: Overlay of transformed density functions of light extinction-weighted PSDs obtained by CLSfor 5 g/kg test samples subjected to different sonication times (1 min, 2 min, 7 min, 10 min and 25min) ................................................................................................................................................... 18

Figure 9: Overlay of density functions of light extinction-weighted PSDs obtained by CLS for 5 g/kg testsamples subjected to different sonication times (1 min, 2 min, 7 min, 10 min and 25 min) ............. 18

Figure 10: Overlay of three DLS intensity-weighted PSDs, obtained on IRMM-386 dispersed in purifiedwater without application of ultrasounds. The inset represents the normalised intensity ACFs. ..... 20

Figure 11: Overlay of transformed density functions of two light extinction-weighted PSDs from CLS,obtained on IRMM-386 dispersed in purified water without application of ultrasounds. .................. 20

Figure 12: Overlay of two representative scattered light intensity-weighted PSDs (left) and normalisedintensity ACFs (right) from DLS, obtained on IRMM-386 dispersed in purified water withoutapplication of ultrasounds (solid curves) and with application of ultrasounds during 1 min (dashedcurves). ............................................................................................................................................. 21

Figure 13: Overlay of light extinction-weighted PSDs from CLS, obtained on IRMM-386 dispersed inpurified water without application of ultrasounds (pink curve) and with application of ultrasoundsduring 1 min (blue curve). ................................................................................................................. 21

Figure 14: Overlay of representative scattered light intensity-weighted PSDs (left) and correspondingnormalised intensity ACFs (right) from DLS, obtained for IRMM-386 dispersed in purified water andafter applying different probe sonication periods.............................................................................. 22

Figure 15: Overlay of light extinction-weighted PSDs from CLS, obtained for IRMM-386 dispersed inpurified water after applying different probe sonication periods. ...................................................... 22

Figure 16: Mean and modal particle size results of IRMM-386 dispersed in purified water as a functionof sonication time, as obtained by DLS (left) and CLS (right). ......................................................... 22

Figure 17: Overlay of transformed density functions of number-weighted PSDs and their correspondingequivalent cumulative distributions from DLS (right) and CLS (left), obtained for IRMM-386dispersed in purified water after applying 10 min and 15 min of probe sonication........................... 23

Figure 18: Overlay of representative scattered light intensity-weighted PSDs (left) and correspondingnormalised intensity ACFs (right) from DLS, obtained for IRMM-386 dispersed in purified water withno sonication (solid curves) and after applying cup/horn sonication during 15 min (dashed curves)and 30 min (dotted curves). .............................................................................................................. 23



Figure 19: Overlay of scattered light intensity-weighted PSDs from DLS, obtained for IRMM-387dispersed in SHMP and after applying probe sonication at different times...................................... 24

Figure 20: Overlay of mass-weighted PSDs from CLS, obtained for IRMM-387 dispersed in SHMP andafter applying probe sonication at different times. ............................................................................ 24

Figure 21: CLS mass-weighted modal Stokes' particle diameter results and DLS (cumulants) scatteredlight intensity-weighted harmonic mean results obtained for IRMM-387 dispersed in SHMP andafter applying probe sonication at different times............................................................................. 25

Figure 22: Overlay of representative scattered light intensity-weighted PSDs from DLS, obtained forIRMM-388 dispersed in SHMP and after applying probe sonication at different times. ................... 26

Figure 23: Overlay of representative scattered light intensity-weighted PSDs from DLS, obtained forIRMM-388 dispersed in SHMP and after applying probe sonication at different times. ................... 26

Figure 24: Influence of the sonication time on the size distribution of BAM-14-2. The number-weightedsum function is shown. To obtain more stable results, the sample was filtered after sonication. .... 27

Figure 25: Effect of filtration on the size distribution of BAM-14-2 using 1 µm GF syringe filter ............. 28

Figure 26: Number-weighted sum function of six individual measurements of BAM-14-2 under sameconditions (procedure 4; filtration: 1 µm GF) .................................................................................... 28

Figure 27: Number-weighted mean sizes derived from NNLS algorithm from six individualmeasurements of BAM-14-2 under same conditions (Procedure 4; filtration: 1 µm GF) ................. 29

Figure 28: Number-weighted sum function of six individual measurements of BAM-14-2 under sameconditions (procedure 5; centrifugation: 2’ 150 x g) ......................................................................... 30

Figure 29: Number-weighted mean sizes derived from NNLS algorithm from six individualmeasurements of BAM-14-2 under same conditions (Procedure 5; centrifugation: 2’ 150 x g)....... 30

Figure 30: Effect of thermal degradation on the size distribution of BAM-14-2 and BAM-14 representedas number-weighted sum function.................................................................................................... 31

Index of Tables

Table 1: Overview on the selected test substances ...................................................................................9

Table 2: Overview on the selected products............................................................................................ 10

Table 3: Direct sonication conditions applied to IRMM-386 dispersed in purified water ......................... 19

Table 4: Direct sonication conditions applied to IRMM-388 dispersed in SHMP .................................... 25

Table 5: Summary of the results derived from measurements using procedure 4 and 5........................ 31

Table 6: Results of the statistical evaluation of the homogeneity studies at 95 % (trends) and 99 %confidence level (outliers) ................................................................................................................. 35

Table 7: Results of the homogeneity studies........................................................................................... 36

Table 8: Results of the statistical evaluation of the homogeneity studies at 95 % (trends) and 99 %confidence level (outliers) ................................................................................................................. 37

Table 9: Results of the homogeneity studies........................................................................................... 37

Table 10: Results of the short-term stability tests.................................................................................... 39

Table 11: Uncertainties of stability during dispatch and storage. ............................................................ 40

Table 12: Uncertainties of stability during dispatch and storage ............................................................. 41



1 GlossaryAmplitude (sonication)

The change of electrical into mechanical energy causes the probe to move up and down. The dis-tance of one movement up and down is called its amplitude. For example, if the maximum ampli-tude for a probe with a diameter of 12.7 mm is 120 m, then the actual amplitude will be 48 mwhen using the probe at 40 % of its maximum amplitude.

Cycle time; pulsed mode (sonication)

Ultrasonic intervals alternated with intervals of no sonication (E.g., specific for vial tweeter, a cycletime of 0.2 means 0.2 s of sonication immediately followed 0.8 s of no sonication). The use of a cy-cle time reduces warming up of the test sample.

FL70 detergent mix (also known as NovaChem100 (Postnova Analytics GmbH))

Composition: 88.8 % water, 3.8 % triethanolamine oleate, 2.7 % sodium carbonate, 1.8 % alcoholethoxylates, 1.4 % tetrasodium ethylenediaminetetraacetate, 0.9 % polyethylene glycol, 0.5 % so-dium oleate and 0.1 % sodium bicarbonate

Power density (sonication)

The amount of power (P), expressed in W/cm3, dissipated to a sample.

Purified water

Tap water, purified by reverse osmosis and collected into a tank that contains an automatic saniti-zation module (e.g., based on chlorine tablets) to prevent against growth and proliferation of bacte-ria. The water is additionally UV-irradiated using a Hg-lamp. The system additionally consists of a0.2 µm pore size built-in filter. According to the technical specifications, the produced purified waterhas a resistivity of 18.2 Mcm at 25 °C and contains < 0.1 CFU/mL, < 0.001 EU/mL and < 1 parti-cle/mL with size > 0.2 m. (CFU = colony forming units; EU = endotoxin units).

(Milli-Q is commonly used jargon referring to deionised water)

Representative test material (RTM) [1]

Material from a single batch, which is sufficiently homogeneous and stable with respect to one ormore specified properties, and which implicitly is assumed to be fit for its intended use in the de-velopment of test methods which target properties other than the properties for which homogeneityand stability have been demonstrated.

Streaming (CLS)

Streaming or bulk settling of a sample occurs if the density of the suspension is much higher thanthe density of the (initial part) of the density gradient. Streaming affects the sedimentation speed,and thus also the measured size, of the separated particles.



2 Abbreviations and acronymsACF : Autocorrelation function

CLS : Centrifugal liquid sedimentation

DLS : Dynamic light scattering

ELS : Electrophoretic light scattering

EtOH : Ethanol

FWHM : Full-width at half peak maximum

HDPE : High-density polyethylene

MeOH : Methanol

NNLS : Non-negative least squares

NP : Nano particle

PSD : Particle size distribution

QCM : Quality Control Material

RM : Reference material

RTM : Representative test material

SEM : Scanning electron microscopy

SHMP : Sodium hexametaphosphate

SI : International System of Units

spICP-MS : single particle Inductively Coupled Plasma Mass Spectrometry (ICP-MS)

TEM : Transmission electron microscopy

TSEM : Scanning electron microscopy in transmission mode

XRF : X-Ray Fluorescence



3 Summary and material characterization reportThis deliverable mainly reports on the homogeneity and stability studies conducted for the sub-stances and products used within the NanoDefine project.

All materials studied can be regarded as sufficiently homogeneous and stable with regardto their use in the NanoDefine project.

Furthermore, the characterization results available at M40 of the substances and products aresummarized in an overview table (Supplement 1). TEM triplicate measurements on materials notincluded in the miniTEM evaluation were conducted by BASF and included in the table. The tablewill be updated with further validated results towards the end of the project.

Some of the test materials may turn out not to be suitable as reference material, e.g. due to unex-pected inhomogeneity, impurities or instability over time. For all materials, the results of homoge-neity and stability tests and the results of the method validation studies in WP6 are compiled in thisdossier: This information will give the basis on which materials can be considered as reference ma-terials. The reference materials will be made available at cost price after the end of the project toother interested partners, hence creating a repository of reference materials (JRC) with demon-strated homogeneity and stability of physicochemical properties by different methods with regard tothe EC ‘nano’-definition recommendation. After the end of the project, JRC Geel has committed tofurther develop three of these Reference Materials into Certified Reference Materials at its own ex-pense.


4 Description of materials

4.1 SuspensionsAccording to the DOW monodisperse and trimodal quality control Materials (QCMs) were sourced, pre-pared and distributed to a list of interested labs within the consortium. The labs received the followingsamples from IRMM: 0.25% suspension ~40 nm silica (the existing certified reference material ERM-FD304) (ID-17) 1 % suspension ~46 nm polystyrene (ID-19) suspension of 0.25% ~50nm, ~90 nm, ~120 nm silica (ID-18) suspension of 0.5% ~46nm; 1% ~100 nm; 1% ~350 nm polystyrene (ID-20)

4.2 SubstancesFor a detailed description of the selection of the test substances for NanoDefine please refer to the re-port D1.2. Table 1 gives an overview on the selected substances.

Table 1: Overview on the selected test substances

Mat

eria

l ID

Material

nano

(n) /

coa

rse

(c)

Che

m. c

ompo

sitio

n

Part

icle

shap

e

Pres

ence

of d

iffer

ent

size

d pa

rtic

les

IRMM-380 Organic pigment n organic particle nano+(non-nano)

IRMM-381 BaSO4 (fine grade) c inorganic particle non-nano

IRMM-382 MWCNT n carbon fibre nano

IRMM-383 Nano Steel n metal platelets nano+(non-nano)

IRMM-384 CaCO3 (fine grade) c inorganic rod / cigar (nano)+non-nano

IRMM-385 Kaolin c inorganic multiscaleplatelets (nano)+non-nano

IRMM-386 Organic pigment c organic particle (nano)+non-nano

IRMM-387 BaSO4 (ultrafine grade) n inorganic particle nano+(non-nano)

IRMM-388 Coated TiO2 c inorganic/composite core-shell non-nano

IRMM-389 Basic methacrylate copolymerparticles (BMA) c organic particle non-nano

BAM-11 Zeolite powder c inorganic particle (nano)+non-nano



4.3 Products

For a detailed description of the selection of the test substances for NanoDefine please refer to the re-port D1.2. Table 1 gives an overview on the selected substances.

Table 2: Overview on the selected products

Mat

eria

l ID

Material

Che

m. c

ompo

sitio

n

Shap

e

Pres

ence

of d

iffer

ent

size

dpa

rtic

les

Trad

e fo

rm

Type

of m

atrix

BAM-12a Food with SiO2, NanoLyse10(tomato soup) inorganic irregular

particles nano suspen-sion organic water

BAM-12a-1 NanoLyse02 (Silica suspen-sion) inorganic particle nano powder organic

BAM-12b *) Food with SiO2, (commercialdry mix; pancake) inorganic irregular

particles nano powder organic

BAM-13a

TiO2 + surface treatment withaluminium compound formulat-

ed in sunscreen (w/ Fe2O3,“micro”-TiO2)

inorganic/composite

Fe2O3:sphericalTiO2:

elongated

nano+bigger lotion emulsion

BAM-13bTiO2 + surface treatment with

aluminium compound formulat-ed in simplified sunscreen

inorganic/composite elongated nano lotion emulsion

BAM-14 Fe2O3 in HDPE inorganic irregularparticles

nano+bigger granulate polymer

BAM-14-1 Raw HDPE (component) inorganic irregularparticles


BAM-14-2 Raw Fe2O3 (component) inorganic irregularparticles


BAM-15 Al2O3 in toothpaste inorganic irregularparticles

nano+bigger paste mixture

*) Material was officially removed from the projects list of products



5 Processing of the test materials

5.1 Suspensions

5.1.1 Monomodal silica (ID-17)

Project partners were provided with 3 units of ERM-FD304, a certified reference material (CRM) of amonomodal silica suspension sold by JRC Geel. It contains monodisperse silica particles of a nominaldiameter of 40 nm. More information can be obtained on the website of JRC Geel: copies of the certifi-cate and the certification report are available.

5.1.2 Monomodal polystyrene (ID-19)

The raw material (monodisperse polystyrene particles with a nominal diameter of 50 nm) was obtainedfrom Thermo Scientific, batch 3050-002. The assigned value by the manufacturer was 46 nm ± 2 nm (k= 2; as determined by TEM; traceable to the SI). The microsphere density was given as 1.05 g/cm3 andthe refractive index as 1.59 at 589 nm. The concentration of the particles was approximately 1 %.

150 ampoules (5 mL amber ampoules) were filled completely with MilliQ water, emptied and air dried inan oven for two days at 60 °C. The bottle with the raw material was shaken and 255.15 g of the sus-pension was poured into a glass beaker where it was stirred with a Teflon coated cross-shaped stirrer.About 2 mL of the suspension were carefully transferred in the ampoule, one by one using a 2 mL(nominal volume) syringe with a stainless steel needle. With this set up, the risk of having a drop fallingon the neck of the ampoule was close to zero. Once all ampoules of a tray were filled with the 2 mL ofsuspension, the tray was transferred to the ampouling machine. The ampoules were flushed with Argonjust prior to flame closure. The ampoules were manually collected and stored in a carton box in the fillorder. In total 125 ampoules were filled.

5.1.3 Trimodal silica (ID-18)

The raw material was obtained from ACE Nanochem, South Korea (batch WP4) with a particle concen-tration of 20.5 %. Analysis by SEM and TEM revealed two large (about 30 nm and 120 nm) and onesmall-fraction mode (about 80 nm).

425 ampoules (5 mL amber ampoules) were filled completely with MilliQ water, emptied and air dried inan oven for 1.5 days at 60 °C. 10 ml of the silica suspension was transferred into a 1000 ml glass flaskthat was topped up with Milli-Q water until the 1000 ml mark was reached. The suspension was stirredwith a Teflon coated cross-shaped stirrer. About 2 mL of the suspension were carefully transferred inthe ampoule, one by one using a 2 mL (nominal volume) syringe with a stainless steel needle. With thisset up, the risk of having a drop falling on the neck of the ampoule was close to zero. Once all am-poules of a tray were filled with the 2 mL of suspension, the tray was transferred to the ampouling ma-chine. The ampoules were flushed with Argon just prior to flame closure. The ampoules were manuallycollected and stored in a carton box in the fill order. In total 425 ampoules were filled.

5.1.4 Trimodal polystyrene (ID-20)

The raw materials (monodisperse polystyrene particles with a nominal diameter of 50 nm, 100 nm and350 nm) were obtained from Thermo Scientific, batches 3050-002, 3100-006, 3350-02. The assignedvalues by the manufacturer were 46 nm ± 2 nm, 100 nm ± 3 nm, and 350 nm ± 6 nm (k = 2; as deter-mined by TEM; traceable to the SI). The microsphere density was given as 1.05 g/cm3 and the refrac-tive index as 1.59 at 589 nm. The concentration of the particles was approximately 1 %.

150 ampoules (5 mL amber ampoules) were filled completely with MilliQ water, emptied and air dried inan oven for two days at 60 °C. The bottles with the raw materials were shaken and 13.699 g, 26.258 g,and 25.948 g of the suspension were poured into a glass beaker and 394.820 g of water was added.The resulting suspension was stirred with a Teflon coated cross-shaped stirrer. About 2 mL of the sus-pension were carefully transferred in the ampoule, one by one using a 2 mL (nominal volume) syringewith a stainless steel needle. With this set up, the risk of having a drop falling on the neck of the am-poule was close to zero. Once all ampoules of a tray were filled with the 2 mL of suspension, the traywas transferred to the ampouling machine. The ampoules were flushed with Argon just prior to flameclosure. The ampoules were manually collected and stored in a carton box in the fill order. In total 133ampoules were filled.



5.2 SubstancesThe solid substances were mixed individually for 0.5 h to 1 h in a Dyna-Mix 200 mixer. Apart fromIRMM-380 were an All Fill machine was used, all substances were filled in 5 ml amber glass vials withthe help of a MCPI Filling Machine that was placed in a glove box. About 250 mg were filled in each vialand closed with a rubber insert and a coloured metal cap. Depending on the substance, about 1000 to2000 units were filled per material.

Zeolite powder (BAM-11) was purchased from Alfa Aesar with an assigned value for the specific sur-face area of 425 m2/g. The substance was mixed for 6 h at 16 rpm in an over-head shaker (HeidolphRFAX 20). For further minimization of between-sample variation a cross-riffling scheme according tovan der Veen and Naeter [5] was applied using a rotation sample divider (PT100, Retsch, 10 divisions).The substance was packed into 1000 amber glass screw neck bottles @ 8 ml with Teflon-lined caps(average weight: 0.5 g). Before use the bottles were cleaned and dried for 1 h at 150 °C. Project part-ners were provided with 5 units of BAM-11.

5.3 Products

5.3.1 Food with SiO2, Pancake dry mix – BAM-12b

The pancake dry mix was purchased on the Dutch market. The substance was mixed for 6 h with adrum hoop mixer made of stainless-steel. For further minimization of between-sample variation a cross-riffling scheme according to van der Veen and Naeter [5] was applied using a rotation sample divider(PT100, Retsch, 10 divisions). The substance was packed into 1000 amber glass screw neck bottles @12 ml with Teflon-lined caps (average weight: 5.1 g). Before use the bottles were cleaned and dried for1 h at 150 °C. Project partners were provided with 5 units of BAM-12b.

5.3.2 Sunscreen, complex formulation – BAM-13a

Sunscreen, complex formulation, (BAM-13a) is a simple formulation containing next to aluminium com-pound coated titanium oxide particles (Rutil, Anatase) iron oxide particles (goethite). The sample wasfully homogenized within the production process. About 4.3 g of the cream were carefully transferred in8 ml amber glass screw neck bottles, one by one using a syringe pump with 20 ml plastic syringes andclosed by Teflon-lined caps. Before use the bottles were cleaned and dried for 1 h at 150 °C. 200 bot-tles were filled. Project partners were provided with 3 units of BAM-13a.

5.3.3 Fe2O3 particles in HDPE – BAM-14

The substance was mixed for 2 h at 16 rpm in an over-head shaker (Heidolph RFAX 20). For furtherminimization of between-sample variation a cross-riffling scheme according to van der Veen and Naeter[5] was applied using a rotation sample divider (PT100, Retsch, 8 divisions). The substance waspacked into 256 amber glass screw neck bottles @ 8 ml with Teflon-lined caps (average weight: 3.9 g).Before use the bottles were cleaned and dried for 1 h at 150 °C. Project partners were provided with 3units of BAM-14.

5.3.4 Toothpaste – BAM-15

The toothpaste was purchased on the Dutch market. The content of 81 tubes (from 2 batches) wereunified and homogenised by vigorous mixing for 30 minutes using Teflon coated sticks and stirrer madeof stainless-steel. About 4.8 g of the cream were manually transferred in each of 1000 amber glassscrew neck bottles @ 8 ml, one by one using 20 ml plastic syringes, and closed with Teflon-lined caps.Before use the bottles were cleaned and dried for 1 h at 150 °C. Project partners were provided with 5units of BAM-15.



6 Assessment of homogeneity and stability

6.1 Optimisation of DLS and CLS measurement procedures

During a homogeneity and stability study (see later), a number of units are traditionally tested under repeat-ability conditions. This approach can allow the distinction of trends that are due to the filling sequence or sta-bility of the candidate RM from trends that may be due to the analytical method. In order to increase thethroughput of samples that can be analysed within one day, and to investigate the precision of the dynamiclight scattering (DLS) and centrifugal liquid sedimentation (CLS) methods, the suitability of the establisheddispersion protocols was tested and protocols were further optimised where possible.

The findings of these feasibility tests are discussed in the following sections.

6.1.1 Pigment Yellow 83 (IRMM-380, nano grade)

The originally established NanoDefine dispersion protocol requires the powder to be dispersed in a mixtureof MeOH and Nekal BX. After evaporation of the MeOH, the surfactant coated particles are re-dispersed inpurified water and ultrasonicated for 15 min. This sample preparation strategy is time consuming and wouldlimit the number of measurements that can be performed under repeatability conditions. Also, at this stage,the recommended anionic surfactant Nekal BX was not yet available. Therefore, alternative dispersion pro-cedures were investigated:

Procedure 1

Step 1: preparation of a 1 g/kg stock suspension in purified water

Step 2: 3 min of bath sonication to improve the dispersibility and homogeneity of the suspension

Step 3: Preparation of a 0.1 g/kg dilution in purified water

Step 4: probe sonication at 40 W/cm3 in pulsed mode (2 s of sonication alternated with 1 s of no sonication)for 15 min and 30 min

After ultrasonication, the obtained suspension had an intense yellow appearance which could cause un-wanted light absorption/scattering interferences during DLS and CLS measurements. Also, to reduce theamount of yellow pigment that could stick to the wall of the DLS measurement cell and to the inner wall ofthe CLS disc, it was decided to further dilute the suspension until a more yellowish opaque appearance wasobtained. Such appearance was obtained at particle mass fractions of 1 mg/kg and 10 mg/kg. The dilutedtest sample was then analysed by CLS and DLS.

The density functions of the normalised CLS light extinction- (Figure 1) and mass-weighted PSDs of the testsamples (in purified water) that were sonicated for 15 min and 30 min appeared to be monomodal with theright tails be drawn to sizes of about 1.2 m. The modal values of the light extinction- and mass-weightedPSDs were about 100 nm. The median values were about 340 nm (15 min sonication) and 280 nm (30 minsonication) for the light extinction-weighted PSDs and about 260 nm (15 min sonication) and 188 nm (30 minsonication) for the mass-weighted PSDs.

While the light extinction- and mass-weighted PSDs were strongly right skewed, the tail of the number-weighted PSDs was much less pronounced (Figure 2). However, the left edge of the PSDs showed to benoisy which is probably due to the presence of a significant fraction of (constituent) nanoparticles with sizesbelow 50 nm. The modal and median values were respectively 74 nm and 73 nm (15 min sonication) and67 nm and 75 nm (30 min sonication). For the applied dispersion protocol, 15 min and 30 min of probe soni-cation did not result in different modal and median particle size results.



Figure 1: Overlay of light extinction-weighted PSDs and their corresponding cumulative distributions forIRMM-380 (dispersed in purified water after applying 15 min (pink curve) and 30 min (blue curve) of probesonication), obtained by CLS.

Figure 2: Overlay of number-weighted PSDs and their corresponding cumulative distributions for IRMM-380(dispersed in purified water after applying 15 min (pink curve) and 30 min (blue curve) of probe sonication),obtained by CLS.

When comparing these results with the CLS results that are presented in the NanoDefine report on disper-sion protocols, it can be clearly concluded that the applied dispersion protocol, i.e. without using MeOH andNekal BX as (pre-)wetting and dispersing agents, was either insufficient to effectively disrupt the agglomer-ates and/or aggregates into smaller constituent particles as those previously detected and quantified(NanoDefine report on dispersion protocols), or to avoid instantaneous re-agglomeration of separated con-stituent particles. A fraction of the constituent particles did appear as a noisy peak at the lower end (30 nm to50 nm) in the number-weighted PSD. The poor signal-to-noise ratio, which is due to the fact that the constit-uent particles are much smaller than the interfering agglomerates/aggregates, prevents quantitative assess-ments.

While repeatable results could be obtained by CLS, very different and non-repeatable results (results notshown) were obtained from the DLS experiments. These results are, however, not surprising knowing (fromthe CLS results) that after probe sonication large particles are still present in the suspension.

Once Nekal BX was made available, a second dispersion protocol was tested. To reduce the time neededfor sample preparation, test samples were directly prepared in 0.5 g/L Nekal BX in purified water.



Procedure 2

Step 1: preparation of a 10 g/kg stock suspension in 0.5 g/L Nekal BX (in ultrapure water); powder pre- wet-ted with 100 L EtOH

Step 2: 10 s vortexing (max. speed) immediately followed by 2 min of bath sonication

Step 3: preparation of a 10 mg/kg dilution in 0.5 g/L Nekal BX

Step 4: probe sonication at 24 W/cm3 in pulsed mode (2 s of sonication alternated with 1 s of no soni- cat-ion) during 15 min

Despite identical probe sonication conditions applied in both dispersion procedures, a lower power densitywas calculated for procedure 2. The reduced power density is because the Nekal BX surfactant caused thesuspension to foam during sonication. The foam interferes with the probe surface thereby delivering less ul-trasonic energy to the suspension. Under the given conditions, foaming could not be avoided.

Applying the second dispersion procedure yielded again right skewed monomodal light extinction- (Figure 3)and mass-weighted PSDs. However, the fraction of particles that was present in the right tail was considera-bly larger than for the test samples that were prepared in purified water. The modal values of the light extinc-tion- and mass-weighted PSD were about 100 nm and 80 nm, respectively. The median values of the lightextinction- and mass-weighted PSDs were about 550 nm and 450 nm.

Figure 3: Light extinction-weighted PSD and its corresponding cumulative distribution for IRMM-380 (dis-persed in 0.5 g/L Nekal BX), obtained by CLS.

Figure 4 : Number-weighted PSD and its corresponding cumulative distribution for IRMM-380 (dispersed in



0.5 g/L Nekal BX), obtained by CLS.

The obtained number-weighted PSDs (Figure 4) had a similar shape to the number-weighted PSDs of thetest samples that were prepared in purified water. However, the modal value had shifted from about 70 nm toabout 40 nm. As the latter result agrees with the results described in the NanoDefine report on dispersionprotocols, protocol 2 was used during the homogeneity and stability study of IRMM-380.

6.1.2 IRMM-381 (Barium sulfate, fine grade)

As instructed by the NanoDefine report on dispersion protocols, test samples of IRMM-381 with mass frac-tions of approximately 2.6 g/kg were prepared in 2 g/kg sodium hexametaphosphate (SHMP). The prepareddispersions were then homogenised in a bath sonicator for 3 min. After homogenisation, the suspension hada turbid almost milky-white appearance and a deposition layer was formed within few minutes. The observedparticle deposition indicated the presence of larger particles, possibly aggregates. In order to break theseaggregates down to the level of their constituent particles, the suspension was probe sonicated (44.2 W/cm2)according to the conditions (i.e. continuous mode using an amplitude of 100 % during 20 min) described inthe NanoDefine report on dispersion protocols. The prepared test samples were analysed by means of CLSand DLS.

As can be seen from Figure 5, the DLS method is unable to provide repeatable results. These non-repeatable results, however, indicate that the prepared test samples contain highly polydisperse particlepopulations.

From the CLS results, it can be seen that probe sonication significantly shifts the peak of the light extinction-and mass-weighted PSDs towards smaller sizes. Also, the modal values of the PSDs (Figure 6) of duplicateresults from test samples which were subjected to 10 min and 20 min of probe sonication agreed well. Basedon these results, it was decided to limit the sonication time to 10 min during the homogeneity and stabilitystudy of IRMM-381.

Figure 5: Overlay of scattered light intensity-weighted PSDs of IRMM-381 (dispersed in 2 g/kg SHMP), ob-tained by DLS.

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Figure 6: Overlay of mass-weighted PSDs of IRMM-381 (dispersed in 2 g/kg SHMP and probe sonicated for10 min and 20 min), obtained by CLS.

6.1.3 IRMM-384 (Calcium carbonate, fine grade)

Test samples of 50 g/kg were prepared in 2 g/L SHMP. As prescribed by the NanoDefine report on disper-sion protocols, the test samples were homogenised by applying 5 min of bath sonication. The relatively highparticle mass fraction was recommended to reduce the impact of dissolved ultra-fine particles. However, theobtained suspension had a milky-white appearance which was too intense for allowing DLS measurementsand it also caused streaming during CLS analyses (Figure 7). Therefore, it was decided to dilute the testsample 10x (i.e. 5 g/kg). The diluted concentration showed to be appropriate as streaming effects were notobserved.

The 5 g/kg test sample was subjected to cup/horn sonication (75 % amplitude and pulsed node 1s on and 1s off) during 1 min, 2 min, 7 min, 10 min and 25 min. The NanoDefine dispersion protocol recommended 10min of sonication using a vial tweeter or probe sonication device.

Figure 7: Overlay of CLS mass-weighted PSDs from 50 g/kg (pink curve) and 5 g/kg (blue curve) test sam-ples prepared from IRMM-384.

Despite the applied dilution, no repeatable DLS results (neither for the cumulants method, nor for the non-negative least squares (NNLS) method), could be obtained for the 5 g/kg test sample. The large scatter be-tween the data (results not shown) is most likely due to the presence of significant fractions of large particles(e.g., agglomerates/aggregates).

10 min and 20 min probesonication

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More repeatable results were obtained from CLS. As can be observed from the transformed density functionof the light extinction-weighted PSDs (Figure 8), two different particle populations with modal values of about480 nm and 1100 nm could be distinguished. When presenting the data with a linear abscissa (Figure 9), thesecond particle population becomes less pronounced and the modal value of the first populated shifted toabout 430 nm. The median value of the number-weighted PSD was determined to be about 240 nm.

Figure 8: Overlay of transformed density functions of light extinction-weighted PSDs obtained by CLS for 5g/kg test samples subjected to different sonication times (1 min, 2 min, 7 min, 10 min and 25 min)

Figure 9: Overlay of density functions of light extinction-weighted PSDs obtained by CLS for 5 g/kg test sam-ples subjected to different sonication times (1 min, 2 min, 7 min, 10 min and 25 min)

Based on the obtained CLS results, it was decided to slightly adapt the originally proposed dispersion proto-col; i.e. the bath sonication process was reduced from 5 min to 3 min and the probe sonication time was re-duced from 10 min to 7 min.

6.1.4 IRMM-386 (Pigment Yellow 83, coarse grade)

According to the established dispersion protocol, a stock suspension of IRMM-386 is to be prepared in puri-fied water at a concentration of 1 g/kg. The stock suspension must be homogenised by means of vortexingor bath sonication and then diluted to a final concentration of 0.1 g/kg. The diluted suspension is must finallybe sonicated (probe of vial tweeter) at an amplitude of 75 % for 15 min.



The recommended sample preparation strategy was followed: a 1 g/kg stock suspension was prepared bydispersing a representative portion of the pigment powder in purified water. After homogenisation (shakingby hand), the obtained dispersion had a non-transparent intense yellow appearance and instantaneous sed-imentation of particles was observed. The suspension was placed in an ultrasonic bath and subjected to ul-trasounds for 3 minutes. The applied moderate dispersion procedure was insufficient to disperse (and/or de-aggregate) the settled particles that could be optically distinguished near the bottom of the sample bottle.The 1 g/kg stock suspension was then diluted in purified water to a concentration of 0.1 g/kg. The dilute sus-pension was treated with a probe and cup/horn style sonicator for 1, 5, 10 and 15 minutes. An overview ofthe sonication conditions and correspondingly calculated energy densities is given in Table 3.

The use of direct sonication, i.e. by immersing a probe in the sample suspension, limits the sample through-put and the suspension may moreover be contaminated by wear particles that can be released by the titani-um probe. This problem can be avoided by using a cup/horn approach which allows multiple samples to besimultaneously and indirectly sonicated. Indirect sonication has as main disadvantage that the ultrasonicwaves that are generated by the horn need first to cross the (cooling) liquid inside the cup and then to crossthe wall of the sample vial. As a result, the ultrasonic intensity inside the sample vial is much lower than inthe case a probe is directly placed inside the sample vial.

Table 3: Direct sonication conditions applied to IRMM-386 dispersed in purified water

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Specifications

1 4.5 Standard titanium-basedprobe of 1/2", operated inpulsed mode (on/off 10 s/5s) at 75 % amplitude with apower density of about54 W/cm2

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15 219.0 Cup/horn setup, operatedin pulsed mode (on/off 10s/5 s) at 75 % amplitudewith a power density ofabout 1 W/cm2

30 433.3

The efficiency of the applied sonication conditions were examined from the intensity-weighted particle sizeresults that were obtained by DLS and from the light extinction-weighted size results from CLS.

Three transformed density functions of scattered light intensity-weighted PSDs that were obtained by DLS(Malvern General Purpose NNLS algorithm) on a single aliquot that was not treated by ultrasounds areshown in Figure 10. These PSDs all had a monomodal shape with maxima in the range of 460 nm to 615 nmand with a typical full-width at half peak maximum (FWHM) of about 335 nm. The corresponding normalisedintensity autocorrelation functions (ACF), which are shown as inset of Figure 10, have noisy, even stronglydeviating, baselines. ACF baseline artefacts of that kind (i.e. increase in correlation at high delay times) aretypically caused by (few) particles that are significantly larger in size than those belonging to the main parti-cle population and which do not move in a random (Brownian motion) manner (e.g. sedimentation). Sincethe PSDs are monodisperse without any indication of such larger particles or agglomerates/aggregates be-ing present, while detectable from the ACF, it can be concluded that the fraction of such large particles isnegligibly small.



Figure 10: Overlay of three DLS intensity-weighted PSDs, obtained on IRMM-386 dispersed in purified wa-ter without application of ultrasounds. The inset represents the normalised intensity ACFs.

The monomodal shape of the DLS PSDs was confirmed by CLS (Figure 11). The small fraction of the largerparticles that was seen in the intensity ACF is likely causing the baseline noise. The modal values of thetransformed (logarithmic scale) density functions of the light extinction-weighted PSDs correspond to 469 nmand 478 nm. The modal values of the density functions (linear scale) are 399 nm and 408 nm.

Figure 11: Overlay of transformed density functions of two light extinction-weighted PSDs from CLS, ob-tained on IRMM-386 dispersed in purified water without application of ultrasounds.

Treating the 0.1 g/kg suspension by probe sonication for 1 minute already shifted the peak of the intensity-weighted PSDs towards significantly smaller particle sizes (Figure 12). The FWHM increased to about 400nm. In particular the deviating baseline of the normalised intensity ACF became less distinct, although thebaseline still contains some degree of noise. From this data, it can be concluded that the few large particlesthat were seen by the ACF, are large agglomerates of constituent particles which can be relatively easilybroken down into constituent particles of smaller sizes.

In contrast to the intensity-weighted PSDs of the non-sonicated suspension, small artefacts (see arrow inFig. 12) could now be persistently observed in the intensity-weighted PSDs at sizes in the range of 4 m to 5m. Particles of such sizes are more likely subject to sedimentation rather than to Brownian motion. Theseartefacts can therefore be most likely attributed due to the ill-posed nature of the DLS NNLS algorithms.

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Figure 12: Overlay of two representative scattered light intensity-weighted PSDs (left) and normalised inten-sity ACFs (right) from DLS, obtained on IRMM-386 dispersed in purified water without application of ultra-sounds (solid curves) and with application of ultrasounds during 1 min (dashed curves).

Similar to the intensity-weighted PSDs from DLS, the effect of 1 min of probe sonication was also clearlyseen from the shifted CLS PSD (blue curve in Figure 13). The CLS peak of the curve from the aliquot thatwas sonicated for 1 min has a light extinction that is about twice of that of the curve from the aliquot with nosonication. For both aliquots, a sample intake of 0.3 mL was used. The increased light extinction, or de-creased light transmission, must therefore be caused by a fraction of constituent particles which were initiallypresent as agglomerates. The modal values of the transformed density functions of the peaks of both PSDscorrespond to 377 nm (1 min probe sonication) and 478 nm (no sonication). When presenting the PSDs asdensity functions, the modal values reduced to 408 nm (no sonication) and 346 nm (1 min of probe soni-cation).

Figure 13: Overlay of light extinction-weighted PSDs from CLS, obtained on IRMM-386 dispersed in purifiedwater without application of ultrasounds (pink curve) and with application of ultrasounds during 1 min (bluecurve).

Longer sonication periods (e.g., 10 min and 15 min) were shown to be effective for shifting the modes of themain peaks of the DLS intensity-weighted PSDs from approximately 400 nm (1 min and 5 min sonication) toapproximately 300 nm (Figure 14). A similar observation was also made from the CLS results (Figure 15).The modal values of the CLS PSDs are 290 nm (for 10 min and 15 min of sonication) and about 350 nm (for1 min and 5 min of probe sonication)

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Figure 14: Overlay of representative scattered light intensity-weighted PSDs (left) and corresponding nor-malised intensity ACFs (right) from DLS, obtained for IRMM-386 dispersed in purified water and after apply-ing different probe sonication periods.

Figure 15: Overlay of light extinction-weighted PSDs from CLS, obtained for IRMM-386 dispersed in purifiedwater after applying different probe sonication periods.

A brief evaluation of the obtained DLS and CLS particle size results as a function of the different applied son-ication times is presented in Figure 16. Each DLS result has been calculated as the mean of the arithmeticmean hydrodynamic diameter values that were computed by the cumulants method and by the MalvernGeneral Purpose NNLS algorithm. In total, two independent aliquots were analysed in triplicate. The CLS re-sults originate from two replicate results that were calculated from the transformed density functions of thePSDs. Error bars correspond to the standard deviation calculated from the replicate results.

Figure 16: Mean and modal particle size results of IRMM-386 dispersed in purified water as a function ofsonication time, as obtained by DLS (left) and CLS (right).

During the development of the preliminary dispersion procedure, one project partner applied vial tweeter ul-

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trasonication (0.5 cycle time and 75 % amplitude) to a 0.1 g/kg water-based suspension (without dispersingagent) for a period of 15 min. Under these conditions, a mean particle size value of 241 nm was experimen-tally determined for the transformed density functions of mass-weighted PSDs by CLS.

During our experiments, vial tweeter sonication was replaced by the more commonly used probe sonication(75 % amplitude and pulsed operation mode). When sonicating the test sample during 10 min and 15 min atthese modified dispersion conditions, mean values of respectively 325 nm and 322 nm were calculated forthe transformed density functions of the mass-weighted PSDs that were determined by CLS.

The nanomaterial definition, as currently given by the EC Recommendation, relates a 100 nm particle sizethreshold to the median, or d50 value, of a number-weighted PSD. The transformed density functions of thenumber-weighted PSDs and their accompanying cumulative distributions that were obtained by CLS on the0.1 g/kg test sample (10 min and 15 min probe sonication) are depicted in Figure 17. The median values ofthese number-weighted results, as determined by DLS and CLS, are about 165 nm and 175 nm, respective-ly. In contrast to the light extinction- and mass-weighted PSDs, a distinct population of particles centredaround sizes of 60 nm is seen in the CLS number-weighted PSD. Due to their weak light scattering behav-iour, these small nanoparticles were not detected by DLS.

Figure 17: Overlay of transformed density functions of number-weighted PSDs and their correspondingequivalent cumulative distributions from DLS (right) and CLS (left), obtained for IRMM-386 dispersed in puri-fied water after applying 10 min and 15 min of probe sonication.

The suitability of indirect sonication was tested for the cup/horn setup. Because of the much lower sonicationintensity, only extended sonication periods of 15 min and 30 min were considered.

Due to the low sonication intensity, the efficiency of a sonication period of 15 min was found to be highly in-adequate as the obtained DLS results were almost identical to the DLS results that were obtained for thenon-sonicated suspension (Figure 18). A sonication time of 30 min did reduce the amount of large agglom-erates, but the mean intensity-weighted particle size of 420 nm ± 80 nm (standard deviation) was still con-siderable larger than the particle size of 320 nm ± 12 nm (standard deviation) that was obtained after 15 minof probe sonication.

Figure 18: Overlay of representative scattered light intensity-weighted PSDs (left) and corresponding nor-malised intensity ACFs (right) from DLS, obtained for IRMM-386 dispersed in purified water with no soni-cation (solid curves) and after applying cup/horn sonication during 15 min (dashed curves) and 30 min (dot-ted curves).

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6.1.5 IRMM-387 (BaSO4, ultrafine grade)

Test samples of 2.6 g/kg were prepared by dispersing 260 mg of powder in about 100 mL of 2 g/L SHMP.The dispersion was then homogenised (3 min) by means of bath sonication. After sonication, the suspensionhad a turbid appearance and a deposition layer was formed within few minutes. The suspension was finallyprobe sonicated (48 W/cm2, continuous mode, amplitude of 66%) for 1, 3 and 5 min. The applied dispersionstrategy was in line with the established NanoDefine dispersion protocol.

As can be seen from Figure 19 and Figure 20, sonication does not significantly alter the size range andshape of the DLS and CLS PSDs (see also Figure 21). The arithmetic mean values of the scattered light in-tensity/light extinction-, mass- and number-weighted PSDs were about 140 nm, 105 nm and 70 nm for DLSand about 100 nm, 45 nm and 20 nm for CLS.

For the homogeneity tests, the dispersion strategy described above was used. The 5 min of probe sonicationwas, however, reduced to 3 min.

Figure 19: Overlay of scattered light intensity-weighted PSDs from DLS, obtained for IRMM-387 dispersed inSHMP and after applying probe sonication at different times.

Figure 20: Overlay of mass-weighted PSDs from CLS, obtained for IRMM-387 dispersed in SHMP and afterapplying probe sonication at different times.

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Figure 21: CLS mass-weighted modal Stokes' particle diameter results and DLS (cumulants) scattered lightintensity-weighted harmonic mean results obtained for IRMM-387 dispersed in SHMP and after applyingprobe sonication at different times.

6.1.6 IRMM-388 (Coated TiO2)

As prescribed by the NanoDefine dispersion protocol, a stock suspension was first prepared at a concentra-tion of 1 g/kg (in 2 g/L SHMP). Before adding the SHMP, it was recommended to pre-wet the powder by us-ing a small volume of ethanol. Since the exact ethanol volume was not provided, we followed the guidelines(0.5 % v/v EtOH in final suspension) as described in reports from other studies (e.g., PROSPECT andNanogenotox). The prepared stock suspension was then moderately sonicated (bath type) during 10minutes. After sonication, a bottom layer of deposited particles was observed. The stock suspension had amilky-white appearance which would not easily allow light scattering/transmission based measurements. Thestock suspension was then diluted in 2 g/L SHMP, thus producing a 0.1 g/kg test sample. The diluted sus-pension was finally treated with ultrasounds for 1 min, 3 min, 10 min and 15 min using a probe sonicator op-erated in pulsed mode (2 s on and 1 s off) at an amplitude of 75 %. The amount of energy that correspondsto the applied sonication times is given in Table 4. The sonicated test sample had a semi-transparentopaque appearance with no large settled or dispersed particles being optically present.

Table 4: Direct sonication conditions applied to IRMM-388 dispersed in SHMP

Time [min] Total amount of energy [kJ] Specifications

1 4.2 Standard titanium-based probeof 1/2", operated in pulsed mode(on/off 2 s/1 s) at 75 % ampli-tude with a power density of53.7 W/cm2

3 11.9

10 35.3

15 Not recorded

The PSDs obtained by DLS and CLS are shown in Figure 22 and Figure 23. From the CLS results it was ob-served that sonication times longer than 1 min do not lead to any further significant decrease in mean/modalparticle size. A deviating PSD was obtained by DLS for the test sample that was sonicated for 3 min. Thisresult was, however, not confirmed by CLS indicating that the deviating PSD is most likely the result of fewlarge aggregates which have erroneously affected the performance of the DLS PSD algorithm. In General,the obtained particle size values are comparable to results obtained by another partner during the develop-

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Figure 22: Overlay of representative scattered light intensity-weighted PSDs from DLS, obtained for IRMM-388 dispersed in SHMP and after applying probe sonication at different times.

Figure 23: Overlay of representative scattered light intensity-weighted PSDs from DLS, obtained for IRMM-388 dispersed in SHMP and after applying probe sonication at different times.

6.1.7 BAM-14 (iron oxide in HDPE)

This section focuses on sample preparation methods for the iron oxide nanoparticles embedded in polyeth-ylene matrix for DLS characterization (and associate needs).

In the course of the development of protocols for preparation of products for microscopy methods (D2.4) thepure iron oxide particles were analysed by TSEM yielding to particle size of 34.6 nm and 35.5 nm for themedian and mean values, respectively. The TSEM images also show the highly agglomerated nanoparticles.TSEM analysis of the hematite particles after thermal degradation at 500 °C for 1 hour show comparable re-sults; 33.2 nm and 34.7 nm for the median and mean values, respectively.

Since TSEM is very time consuming and probing very small amounts of sample this method is not suitablefor assessing the homogeneity of BAM-14. Therefore, a dispersion protocol for the analysis with DLS had tobe developed.

First protocols were developed for BAM-14-2, known as Pigment red 101, to test the suitability of DLS forthis substance.

Procedure 1

1. preparation of a 4 g/L stock suspension in absolute ethanol2. 3 min of bath sonication to improve the dispersibility and homogeneity of the suspension3. preparation of a 0.08 g/L dilution in 2 g/L hexametaphosphate (SHMP)4. bath sonication for 5, 10, 20 and 30 min

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The efficiency of the applied sonication conditions were examined from transformed density functions ofscattered light number-weighted PSDs that were obtained by DLS (Malvern General Purpose NNLS algo-rithm). The obtained mean diameters decrease with increasing sonication times. However, with increasingmeasurement times the mean size decreased as well, indicating the presence of large sedimenting particlesthat were also visible by eye.

Therefore, the solution was treated in procedure 2 by probe sonication (2 mm probe, 460 W/cm2, 75% of 210µm amplitude, cycle time 0.5) for 5, 10, 20 or 30 minutes. The mean particle size decreased even more withincreasing sonication times compared to the bath sonication. However, the repeatability was very limited.The reason for that could be the particles emitted from the sonotrode that may influence the DLS measure-ment result. To minimize the abrasion from a sonotrode the power has to be reduced significantly. Thatwould lead to an insufficient disruption of the aggregates or agglomerates. Therefore, a sonotrode with alarger diameter was used in Procedure 3 (7 mm probe, 300 W/cm2, 75% of 175 µm amplitude, cycle time0.5). The influence of the sonication time on the size distribution of BAM-14-2 is depicted in Figure 24.

Figure 24: Influence of the sonication time on the size distribution of BAM-14-2. The number-weightedsum function is shown. To obtain more stable results, the sample was filtered after sonication.

To obtain a higher reproducibility the suspension was filtered with 1 µm glass fibre syringe filters after soni-cation (see Procedure 4 below). This improves the overall quality of the correlogram mainly by reduction ofthe baseline noise. Removing the very large particles by filtration, also leads to reduction of the width of theintensity-weighted size distribution, which in turn results in smaller number-weighted mean sizes of the parti-cles (see Figure 25).

Procedure 4:1. 5 g/L BAM-14-2 in abs. EtOH (5 g/L PR sol.)2. 5’ vortexing3. 10’ US bath4. 10” vortexing5. 19.9 ml 2 g/L SHMP in MilliQ + 100 µl 5 g/L PR sol6. 15’ probe sonication (sonotrode S7 300 W/cm2; cycle time 0.5; 85% of 175 µm amplitude)7. immediatel filtration using 1 µm GF syringe filter, 1st ml for rinsing the filter, 2nd ml for DLS measure-

ment8. start DLS measurement immediately with a equilibration time of 120 s

The average number-weighted mean size of 6 independent measurements of BAM-14-2 is 95.7 nm with astandard deviation of 4.4 nm.



Figure 25: Effect of filtration on the size distribution of BAM-14-2 using 1 µm GF syringe filter

Figure 26: Number-weighted sum function of six individual measurements of BAM-14-2 under sameconditions (procedure 4; filtration: 1 µm GF)



Figure 27: Number-weighted mean sizes derived from NNLS algorithm from six individual measure-ments of BAM-14-2 under same conditions (Procedure 4; filtration: 1 µm GF)

Another way of increasing the reproducibility is a short centrifugation step directly after sonication.

The corresponding protocol is described in procedure 5:

Procedure 5:1. 5 g/L BAM-14-2 in abs. EtOH (5 g/L PR sol.)2. 5’ vortexing3. 10’ US bath4. 10” vortexing5. 19.9 ml 2 g/L SHMP in MilliQ + 100 µl 5 g/L PR sol in glass jar6. 15’ probe sonication (sonotrode S7 300 W/cm2; cycle time 0.5; 85% of 175 µm amplitude)7. fill immediately 7 ml in 15 ml centrifuge tube and start centrifugation8. 2’ 150 x g (1000rpm rotor #7591); use shortest break period (break 9)9. transfer slowly 1 ml into cuvette, take care that the pipette tip is 5 cm below the surface10. start DLS measurement immediately with an equilibration time of 120 s

The average number-weighted mean size of 6 independent measurements of BAM-14-2 is 96.9 nm with astandard deviation of 3.8 nm.

1 2 3 4 5 660

70

80

90

100

110

120

130

140 Number weighted mean sizeMean valueStandard deviation with a coverage factor k of 2

parti

cle

size

(nm

)

Record #



Figure 28: Number-weighted sum function of six individual measurements of BAM-14-2 under sameconditions (procedure 5; centrifugation: 2’ 150 x g)

Figure 29: Number-weighted mean sizes derived from NNLS algorithm from six individual measure-ments of BAM-14-2 under same conditions (Procedure 5; centrifugation: 2’ 150 x g)

Both protocols, procedure 4 and 5, give comparable results for BAM-14-2 (see Table 5).

10 100 10000

102030405060708090

100 123456

no. w

eigh

ted

sum

fct.

(%)

particle size (nm)

1 2 3 4 5 660

80

100

120

140 Number weighted mean sizeMean valueStandard deviation with a coverage factor k of 2

parti

cle

size

(nm

)

Record #



Table 5: Summary of the results derived from measurements using procedure 4 and 5

Sample BAM-14-2 BAM-14-2

Procedure 4 5

Preparation filtration centrifugation

weighting number number

Number of measurements 6 6

average mean value /nm 95.7 96.9

standard deviation /nm 4.4 3.8

2 * standard deviation /nm 8.7 7.5

To adapt the procedure 5 to BAM-14 the Fe2O3/HDPE rods were burned in a muffle furnace. The tempera-ture was gradually increased from room temperature to 450 °C at 5 K/min and held for one hour. The darkred product was dispersed according to procedure 5 and analysed using DLS. The number-weighted meansize was 429 nm, thus, much higher than expected. To be sure that all HDPE is burned, a new sample washeated up to 650 °C. The number-weighted mean size was even higher. The temperature treatment of BAM-14-2 leads also to elevated mean size values compared to the neat iron oxide powder, although the differ-ence is not as drastic as seen for BAM-14 (see Figure 30).

Unfortunately, it turned out that the ashing procedure leads to the formation of aggregates which could notbe disrupted by probe sonication. Hence, the determined mean size of the particles after ashing is significanthigher than without ashing and does not reflect the real particle size of the particles embedded in HDPE.Thus, procedure 5 is not suitable for BAM-14 to evaluate the homogeneity. Therefore, it was decided to relayon data derived from XRF measurements to assess the homogeneity.

Figure 30: Effect of thermal degradation on the size distribution of BAM-14-2 and BAM-14 representedas number-weighted sum function.

6.2 spICP-MS method for sample preparation and measurement NPsExtraction and single-particle ICP-MS method for sample preparation and measurement of nanoparticles insuspension and in consumer products has been detailed in D4.5 and in D2.6. Brief details follow below.spICP-MS was used to determine (based on the principles of spICP-MS) the particle size (based on particlemass) and particle mass concentration of TiO2 in BAM13a and Al2O3 in BAM15.

10 100 10000

102030405060708090

100 BAM-14-2BAM-14-2 450°CBAM-14-2 650°CBAM-14 450°CBAM-14 650°C

no. w

eigh

ted

sum

fct.

(%)

particle size (nm)



Sample Preparation Toothpaste BAM15

The median particle size of Al2O3 in toothpaste was analysed by means of spICP-MS. In order to determinethe aluminium background a toothpaste which does not contain Al2O3 nanoparticles was measured as blank.

The dispersion of toothpaste BAM-15 and blank toothpaste is performed according to the protocol de-scribed in the NanoDefine deliverable D2.6 and is briefly explained below.

- Weigh 1 ± 0.1 g of the product into a 50 mL plastic tube (Greiner centrifuge tubes, Sigma-Aldrich)add 50 mL of UPW to get a concentration of ~20 mg mL-1

- Homogenize the dispersion by vortexing (180 seconds)

- Dilute the sample for spICP-MS analysis with UPW by applying a dilution factor of 100.000 to a finalconcentration of ~200 µg L-1.

NOTE: This dilution protocol has been optimized in relation to expected transport efficiency on the ICP-MSused in this study, the particle size and initial concentration of alumina in the toothpaste. For new sampletypes the final dilution has to be explored experimentally until the dilution is suitable to i) avoid the occur-rence of double events ii) achieve sufficient statistics.

Sample Preparation Sunscreen BAM13a

The median particle size of TiO2 in BAM13a was analysed by means of spICP-MS. In order to determine thealuminium background a blank-formula which does not contain TiO2 nanoparticles was measured as blank.

The dispersion of BAM-13a and blank sunscreen is performed according to the protocol described in theNanoDefine deliverable D2.6 and is briefly explained below. The sample is prepared by using a matrix dilu-tion approach and it has been adapted from literature 3 .

To prepare samples, 50 mg of cream (BAM13a or Blank cream) was weighted on a 4-digit scale, into a 50mL plastic tube (Greiner centrifuge tubes, Sigma-Aldrich). 50 g of diluting agent was added. Diluting agentwas prepared by weighting 5 g of MelPers® 2450 (BASF, Germany) and 5 g of Triton X- 100 (Nonionic sur-factant, laboratory grade, Sigma Aldrich) in a clean plastic bottle, add 1 L of UPW. Samples were vortexeduntil the sunscreen until was completely detached from the tube walls and dispersion looked visually homog-enous; samples were then sonicated in a water bath for 10 minutes at room temperature; just after soni-cation a first dilution step was performed (1 000x dilution in UPW);samples were further sonicated in a waterbath for 10 minutes at room temperature; final dilution ( 2 000x ) was performed in UPW before spICP-MSanalysis.

Both ionic standards and standard for transport efficiency determination were prepared in the same matrix assamples, and therefore containing an amount of diluting agent as for final dilution step prior to spICP-MSmeasurement.

spICP-MS methodSingle Particle ICP-MS is based on the measurement of highly diluted nanoparticle dispersions by ICP-MSoperated in time resolved mode for a pre-selected mass-to-charge ratio (m/z) value. Ideally, individual parti-cles enter the ion source and are atomised and ionised in the plasma torch to produce a plume of elementions that is transferred to the mass spectrometric detector. The discrete measurement intervals (dwell times)of the MS are set to a value ≤10 ms that allows the registration of the signal of the ion plume from a singleparticle.

A prerequisite to operate in the single particle modus is (besides the short dwell times) that the concentrationof particles is small enough to avoid simultaneous ionisation of more than one particle and the generation ofoverlapping ion plumes per dwell time. When these requirements are met the signal intensity is proportionalto the mass of the respective element in the particle. A typical run time is 60 seconds and is called a timescan. The mass spectrometer can be tuned to measure any specific element, but due to the high time reso-lution only one m/z value can be monitored during a run. In order to calculate concentrations, the nebuliza-tion efficiency has to be determined first using a reference particle. Mass calibration is performed using ionicstandard solutions of the measured element analysed under the same conditions. The detailed characteris-tics of the method are reported in the NanoDefine deliverables D7.2 and D7.6.

The data are exported as a CSV file and imported in Excel to calculate the number and mass concentration,and the size and size distribution of the nanoparticles. The procedure is applicable for the determination ofinorganic nanoparticles, metal and metal oxides (e.g. Ag, Au, Al2O3, TiO2, SiO2, etc.) in particle suspensions



or consumer products after an adequate sample preparation. Depending on the material type, particle sizesin a range of 10 to 1000 nm and mass concentrations in the range of 1 to 1000 ng L-1 can be determined.

In this study we used a quadrupole ICP-MS, ICAP-Q, (Thermo Fisher Scientific); typical tune settings usedon iCAP-Q are reported below:

Forward power : 1550 W

Nebulizer : PFA

Spray chamber : cyclonic, quartz

Gas flows : plasma, 13 L min-1

nebulizer, 1.1 L min-1

Rinsing liquid flow rate : 1 mL min-1

Sample flow rate : 0.35 mL min-1

Data acquisition : time resolved analysis (TRA) mode (npQuant)

Dwell time : 3 ms

Total acquisition time : 60 s

Monitored Isotopes : Au (m/z 197), Al (m/z 27), Ti (m/z 48)

NOTE: For the measurement of elements with potentially occurring interferences the application of anothermeasurement mode or reaction gases can be necessary such us in the case of sunscreen BAM13a andBAM13b, the collision gas nature and flow has to be evaluated in balancing effect on sensitivity and back-ground removal, in this study He at 3 mL min-1 was used as collision gas

6.3 BET for determination of specific surface areaThe Brunauer-Emmett-Teller (BET) theory was derived 1938 to explain the physical adsorption of gasmolecules on a solid surface (Brunauer et al., 1938). BET serves as the most often applied techniquefor the measurement of the specific surface of a material – typically porous materials. BET explainsmultilayer adsorption of gas molecules on a solid and dry material. Nitrogen and argon gas are widelyused for measurements. BET is based on three hypotheses:

1.) gas molecules physically adsorb in infinite layers,

2.) no interaction exist between adsorbed layers, and

3.) the Langmuir theory is applicable for each layer of gas molecules.

The resulting BET equation is applied for fitting experimental gas adsorption isotherms and gives theadsorbed monolayer gas quantity. Knowledge of gas quantity, adsorption cross section of the adsorbinggas and the molar gas volume allows calculation of the specific surface area of the material [1].

The sample is heated in order to desorb the surface and cooled down to 77K. The adsorption of e.g. N2is proportional to the sample surface in a distinct pressure range which is determined by measuring thesaturation under equilibrium pressures. The volume of the adsorbed N2 is determined for 5 points inthat linear region and the surface is derived by the BET plot method.

The volume specific surface area can be used as a proxy to determine if a material is in the nanorange. However, this is only valid for particles which do not feature an accessible internal surface likee.g. sponge-like materials. For materials with a high specific internal area like Zeolite (BAM-11) the totalsurface area is dominated by micropores. Using the t-method micropore analysis according to de Boer,the surface area of micropores and the outer surface can be estimated. For the zeolite material it wasfound that the pressure region of the adsorption isotherm used for the evaluation has a significant im-pact on the results. The optimal working point of the system can be found and reproduces using theTest proposed by Rouquerol. The test is implemented in the software package provided by the manu-facturer.



The BET technique was used for materials that are not measurable by the DLS, CLS or spICP-MStechnique.

6.4 Evaluation of homogeneity study samples

6.4.1 Substances

The material IRMM-389 (methacrylate copolymer) was analysed with BET, but only few meaningful re-sults were obtained. As the samples that were selected by a stratified sampling scheme were used forthe homogeneity and the short-term stability testing, all of them had been exposed to the study temper-ature of 60 °C. An investigation revealed that the surface of the polymer was molten for most sampleswhich makes a BET measurement futile. For this reason, no data were obtained for IRMM-389. Thereare samples from the long-term stability study available that can be used for a possible future stabilitycheck.

A key requirement for any reference material is the equivalence between the various units. In this re-spect, it is relevant whether the variation between units is significant compared to the uncertainty of thecertified value. In contrast to that it is not relevant if this variation between units is significant comparedto the analytical variation. Consequently, ISO Guide 34 requires RM producers to quantify the between-unit variation. This aspect is covered in between-unit homogeneity studies.

The between-unit homogeneity was evaluated to ensure that the reference materials can be consideredsufficiently homogeneous.

The number of selected units corresponds to approximately the cubic root of the total number of theproduced units. The number units were selected using a random stratified sampling scheme coveringthe whole batch for the between-unit homogeneity test. For this, the batch was divided into numbergroups (with a similar number of units) and one unit was selected randomly from each group. However,in order to reduce the number of samples to be analysed for the homogeneity and stability studies, theshort-term stability samples were selected in such a way that they are as much as possible distributedover the batch size. Their results, in relation to their sequence in the filling order, were used for theevaluation of homogeneity.

Two independent samples were taken from each selected unit, and analysed by DLS, CLS or BET. Themeasurements were performed as much as possible under repeatability conditions although the meas-urements had to be performed over two days, and in a randomised manner to be able to separate a po-tential analytical drift from a trend in the filling sequence.

Regression analyses were performed to evaluate potential trends in the analytical sequence as well astrends in the filling sequence. No trends in the filling sequence or the analytical sequence were visible.Some significant (95 % confidence level) trends in the analytical sequence were visible, pointing at asignal drift in the analytical system. The correction of biases, even if they are statistically not significant,was found to combine the smallest uncertainty with the highest probability to cover the true value [2].Correction of trends is therefore expected to improve the sensitivity of the subsequent statistical analy-sis through a reduction in analytical variation without masking potential between-unit heterogeneities.As the analytical sequence and the unit numbers were not correlated, trends significant on at least a 95% confidence level were corrected as shown below:

ib resultmeasuredresultcorrected Equation 1

b = slope of the linear regression

i = position of the result in the analytical sequence

The datasets were tested for consistency using Grubbs outlier tests on a confidence level of 99 % onthe individual results and the unit means.

Quantification of between-unit inhomogeneity was accomplished by analysis of variance (ANOVA),which can separate the between-unit variation (sbb) from the within-unit variation (swb). The latter isequivalent to the method repeatability if the individual samples are representative for the whole unit.



Evaluation by ANOVA requires unit means which follow at least a unimodal distribution and results foreach unit that follow unimodal distributions with approximately the same standard deviations. Distribu-tion of the unit means was visually tested using histograms and normal probability plots.

Minor deviations from unimodality of the individual values do not significantly affect the estimate of be-tween-unit standard deviations. The results of all statistical evaluations are given in Table 6.

Table 6: Results of the statistical evaluation of the homogeneity studies at 95 % (trends) and 99 % con-fidence level (outliers)

Material Measurand Trends(before correction)

Outliers

Analyticalsequence

Fillingsequence

Individualresults

Unitmeans

IRMM-380 Number-based mode (CLS) no no none noneIRMM-381 Mass-based mode (CLS) no no none noneIRMM-381 Intensity-based mode (CLS) no no none noneIRMM-382 Brunauer-Emmett-Teller (BET) no no yes yesIRMM-383 Brunauer-Emmett-Teller (BET) no yes none noneIRMM-384 Mass-based mode (CLS) yes no none noneIRMM-385 Brunauer-Emmett-Teller (BET) no no none noneIRMM-386 Number-based mode (CLS) no no none noneIRMM-387 Number-based mode (CLS) no no none noneIRMM-387 Intensity-based mode (CLS) no no none noneIRMM-388 Number-based mode (CLS) yes no none noneIRMM-389 Brunauer-Emmett-Teller (BET) n.a. n.a. n.a. n.a.BAM-11 Brunauer-Emmett-Teller (BET) no no none none

One has to bear in mind that sbb,rel and swb,rel are estimates of the true standard deviations and thereforesubject to random fluctuations. Therefore, the mean square between groups (MSbetween) can be smallerthan the mean squares within groups (MSwithin), resulting in negative arguments under the square rootused for the estimation of the between-unit variation, whereas the true variation cannot be lower thanzero. In this case, u*bb, the maximum inhomogeneity that could be hidden by method repeatability, wascalculated as described by Linsinger et al. [3]. u*bb is comparable to the limit of detection of an analyti-cal method, yielding the maximum inhomogeneity that might be undetected by the given study setup.

The relative standard deviation (swb,rel) reflecting the method repeatability, the relative between–unitstandard deviation (sbb,rel) and the relative standard uncertainty related to a possible between-unit(u*bb,rel) inhomogeneity were calculated as:

ywithin

rel,wb

MSs Equation 2

yn

MSMS

s

withinbetween

rel,bb

Equation 3

y

νn

MS

u MSwithin

within

*rel,bb

42

Equation 4

MSwithin mean square within a unit from an ANOVA

MSbetween mean squares between-unit from an ANOVA

y mean of all results of the homogeneity study

n mean number of replicates per unit

MSwithinν degrees of freedom of MSwithin



The results of the evaluation of the between-unit variation are summarised in Table 7. The resultingvalues from the above equations were converted into relative uncertainties. In most cases, the uncer-tainty contribution for homogeneity was determined by the method repeatability.

Table 7: Results of the homogeneity studies.

Material Measurand Average swb,rel

[%]

sbb,rel

[%]

u*bb,rel

[%]

ubb,rel

[%]

IRMM-380 Number-based mode (CLS) 34.3 nm 10.9 7.3 4.9 7.3IRMM-381 Mass-based mode (CLS) 539 nm 21.5 n.c. 9.7 9.7IRMM-381 Intensity-based mode (CLS) 641 nm 13.0 n.c. 5.9 5.9IRMM-382 Brunauer-Emmett-Teller (BET) 239 m2/g 4.4 1.0 2.0 2.0IRMM-383 Brunauer-Emmett-Teller (BET) 10.3 m2/g 6.8 n.c. 3.1 3.1IRMM-384 Mass-based mode (CLS) 276 nm 7.0 n.c. 3.2 3.2IRMM-385 Brunauer-Emmett-Teller (BET) 16.3 m2/g 3.2 1.1 1.4 1.4IRMM-386 Number-based mode (CLS) 182 nm 1.55 0.82 0.76 0.82IRMM-387 Number-based mode (CLS) 20.5 nm 14.1 3.7 6.7 6.7IRMM-387 Intensity-based mode (CLS) 102 nm 1.15 n.c. 0.54 0.54IRMM-388 Number-based mode (CLS) 184 nm 0.08 n.c. 0.04 0.04IRMM-389 Brunauer-Emmett-Teller (BET) n.a. n.a. n.a. n.a.BAM-11 Brunauer-Emmett-Teller (BET*) 70.7 m2/g 6.43 n.c. 2.9 2.9

n.c. : cannot be calculated as MSbetween < MSwithin

* : external surface

It can be concluded that there are no issues with the homogeneity of the materials. Only one material(IRMM-383) showed a significant trend in the filling sequence, but this can be explained with a ratherhigh precision; the corresponding ubb value is still acceptable with regard to possible uncertainty contri-butions for a material coming from other sources (stability, material characterisation).

The uncertainty contributions for inhomogeneity (ubb) are very much dominated by the method perfor-mance per material and are mostly in a low range (<3 %). Higher values are due to the high repeatabili-ties of the method for that material, but are still moderate.

6.4.2 Products

The between-unit homogeneity was evaluated as described in the previous chapter (6.4.1). Two inde-pendent samples were taken from each selected unit, and analysed by spICP-MS or XRF.

The datasets were tested for consistency using Grubbs outlier tests on a confidence level of 99 % onthe individual results and the unit means (see Table 6).

Regression analyses were performed to evaluate potential trends in the analytical sequence as well astrends in the filling sequence. No trends in the filling sequence were visible. Some significant (95 % con-fidence level) trends in the analytical sequence were visible for BAM-13a and BAM14 that were causedby individual outliers (see Table 6).

The Si content of BAM-12b (Pancake dry mix) is 0.09 % (w/w) as determined by atomic spectroscopy.



This value is far too small for any reliable particle sizing technique. Furthermore, XRF analysis showsthat other elements like phosphor, potassium, sulphur, calcium and chlorine are present in even higheramounts. This makes the extraction of silica particles more challenging. A possible method could be atwo-step acid extraction, followed by FFF fractionation and ICP-MS analysis. This was shown by Grom-be et al. for spiked tomato soup with a nominal silica concentration of 2 % (w/w) [6]. However, thismethod is very expensive, labour intensive, has security concerns and up to now, there is no validatedprotocol available. Attempts of thermal degradation of the pancake dry mix for 1 h at 500 °C leads toblack speckled white powder. Thus it is not suitable for DLS measurements. In consultation with the ECproject officers, the work on the pancake mix was stopped within the whole project due to additional is-sues in other tasks.

The homogeneity of BAM-13a (sunscreen, complete formulation) was assessed based on data derivedfrom spICP-MS measurement after extraction of titanium oxide. The average median particle size is 53nm, see Table 9.

The material BAM-14 (Fe2O3 in HDPE) was analysed by XRF using a Magix PRO spectrometer system(Panalytical) equipped with a 4 kVA Rh tube. For measurement 1.8 g of each sample were placed into asample holder with a Kapton polyimide window of 27 mm in diameter. To shorten the measurementtime, only the spectral line of Fe-K at 6.4 keV was used and evaluated. The relative iron content wasdetermined and extrapolated to the relative iron oxide content, see Table 9. We remark that XRF onlyprobes the total Fe concentration. The study therefore analysed the homogeneity of the Fe-distributionNOT the particle size of the Fe particles. There was no tier 1 method identified which is able to analysethe homogeneity of the Fe particles in the matrix. TEM is possible but very expensive as additionalsample preparation by Ultramicrotomy is necessary. Furthermore, it only probes tiny sample regionsfrom one single polymer bead.

The homogeneity of BAM-15 (toothpaste) was assessed based on data derived from spICP-MS meas-urement after extraction of aluminium oxide. The average median particle size is 133 nm, see Table 9.

Table 8: Results of the statistical evaluation of the homogeneity studies at 95 % (trends) and 99 % con-fidence level (outliers)

Material Measurand Trends(before correction)

Outliers

Analyticalsequence

Fillingsequence

Individual re-sults

Unit means

BAM-12b Work was stopped n.a. n.a. n.a. n.a.

BAM-13a Inductively coupled plasmamass spectrometry (ICP-MS)

yes no yes none

BAM-14 X-Ray Fluorescence (XRF) yes no yes none

BAM-15 Inductively coupled plasmamass spectrometry (ICP-MS)

no no none none

Table 9: Results of the homogeneity studies

Material Analytical Method Average swb,rel

[%]

sbb,rel

[%]

u*bb,rel

[%]

ubb,rel

[%]

BAM-12b Work was stopped n.a. n.a. n.a. n.a. n.a.

BAM-13a Inductively coupled plasma massspectrometry (ICP-MS) 53.3 nm 2.81 2.40 1.27 2.40

BAM-14 X-Ray Fluorescence (XRF) 3.54 rel. %(w/w)

0.49 0.45 0.22 0.45

BAM-15 Inductively coupled plasma massspectrometry (ICP-MS) 133.3 nm 4.75 3.57 2.15 3.57



6.5 Evaluation of stability study samples

6.5.1 Substances

The material IRMM-389 (methacrylate copolymer) was analysed with BET, but only few meaningful re-sults were obtained. As the samples that were selected by a stratified sampling scheme were used forthe homogeneity and the short-term stability testing, all of them had been exposed to the study temper-ature of 60 °C. An investigation revealed that the surface of the polymer was molten for most sampleswhich makes a BET measurement futile. For this reason, no data were obtained for IRMM-389. Thereare samples from the long-term stability study available that can be used for a possible future stabilitycheck.

Time, temperature, radiation and water content were regarded as the most relevant influences on sta-bility of the materials. The influence of ultraviolet or visible radiation was minimised by the choice of thecontainment which eliminates most of the incoming light. In addition, materials are stored and dis-patched in the dark, thus eliminating practically the possibility of degradation by radiation. For the drypowders (substances), the water content can be considered sufficiently low to avoid microbial growth.Therefore, only the influences of time and temperature needed to be investigated.

Stability testing is necessary to establish conditions for storage (long-term stability) as well as condi-tions for dispatch to the customers (short-term stability). During transport, especially in summer time,temperatures up to 60 °C could be reached and stability under these conditions must be demonstratedif transport at ambient temperature will be applied.

The stability studies were carried out using an isochronous design [4]. In that approach, samples arestored for a certain time at different temperature conditions. Afterwards, the samples are moved to con-ditions where further degradation can be assumed to be negligible (reference conditions). At the end ofthe isochronous storage, the samples are analysed simultaneously under repeatability conditions. Anal-ysis of the material (after various exposure times and temperatures) under repeatability conditionsgreatly improves the sensitivity of the stability tests.

6.5.1.1 Short-term stability study

For the short-term stability study, samples were stored at 60 °C for 0, 1, 2 and 4 weeks. The referencetemperature was set to 18 °C. Three units per storage time were selected using a random stratifiedsampling scheme. From each unit, two samples were measured by DLS, CLS or BET. The measure-ments were performed as much as possible under repeatability conditions although the measurementshad to be performed over two days, and in a randomised manner to be able to separate a potential ana-lytical drift from a trend in the filling sequence.

The results were screened for outliers using the single and double Grubbs test. Some outlying individu-al results were found (

Table 10). As no technical reason for the outliers could be found all data were retained for statisticalanalysis.

Furthermore, the data were evaluated against storage time and regression lines of the particle size ver-sus time were calculated. The slopes of the regression lines were tested for statistical significance. Forall materials, the slopes of the regression lines were not significantly different from zero (on 95 % confi-dence level).

The results of the statistical evaluation of the short-term stability are summarised in



Table 10.

Table 10: Results of the short-term stability tests

Material Measurand Number of individualoutlying results

Significance of thetrend on a 95 % con-fidence level

IRMM-380 Number-based mode (CLS) none noIRMM-381 Mass-based mode (CLS) none noIRMM-381 Intensity-based mode (CLS) none noIRMM-382 Brunauer-Emmett-Teller (BET) 1 noIRMM-383 Brunauer-Emmett-Teller (BET) none noIRMM-384 Mass-based mode (CLS) 1 noIRMM-385 Brunauer-Emmett-Teller (BET) none yesIRMM-386 Number-based mode (CLS) none noIRMM-387 Number-based mode (CLS) none noIRMM-387 Intensity-based mode (CLS) none noIRMM-388 Number-based mode (CLS) none noIRMM-389 Brunauer-Emmett-Teller (BET) n.a. n.a.BAM-11 Brunauer-Emmett-Teller (BET) none no

Two statistical outliers were detected for IRMM-382 and IRMM-384, and were retained for the estima-tion of usts. None of the trends was statistically significant on a 95 % confidence level.

It can be concluded that there are no issues with the short-term stability of the materials. Only one ma-terial (IRMM-385) showed a significant trend in the filling sequence, but this can be explained with a ra-ther high precision; the corresponding usts value is low and acceptable.

The uncertainty contributions for inhomogeneity (usts) are very much dominated by the method perfor-mance per material and in a low range (<3 %). Higher values are due to the high repeatability of themethod for that material, but are still moderate.



6.5.1.2 Long-term stability study

For the long-term stability study, samples are stored at 18 °C for 12, 18, and 24 months. The referencetemperature was set to 4 °C. Three samples per storage time were selected using a random stratifiedsampling scheme.

It is envisaged to measure these stability samples after a decision on the methods and the materials(milestone MS37, M41).

6.5.1.3 Estimation of uncertainties

Due to the intrinsic variation of measurement results, no study can rule out degradation of materialscompletely, even in the absence of statistically significant trends. It is therefore necessary to quantifythe potential degradation that could be hidden by the method repeatability, i.e. to estimate the uncer-tainty of stability. This means, even under ideal conditions, the outcome of a stability study can only be"degradation is 0 ± x % per time".

Uncertainties of stability during dispatch and storage were estimated as described in [3] for each mate-rial. For this approach, the uncertainty of the linear regression line with a slope of zero is calculated.The uncertainty contributions usts are calculated as the product of the chosen transport time/shelf lifeand the uncertainty of the regression lines as:

tt2

i

rel,sts txx

RSDu

Equation 5

RSD relative standard deviation of all results of the stability study

xi result at time point i

x mean results for all time points

ttt chosen transport time (1 week at 60 ºC)

The following uncertainties were estimated:- usts,rel, the uncertainty of degradation during dispatch. This was estimated from the 60 °C studies.

The uncertainty describes the possible change during a dispatch at 60 °C lasting for one week.

The results of these evaluations are summarised in Table 11.

Table 11: Uncertainties of stability during dispatch and storage.

Material Technique/Measurand usts ,rel

[%]IRMM-380 Number-based mode (CLS) 1.79IRMM-381 Mass-based mode (CLS) 2.66IRMM-381 Intensity-based mode (CLS) 1.60IRMM-382 Brunauer-Emmett-Teller (BET) 0.63IRMM-383 Brunauer-Emmett-Teller (BET) 0.84IRMM-384 Mass-based mode (CLS) 1.25IRMM-385 Brunauer-Emmett-Teller (BET) 0.43IRMM-386 Number-based mode (CLS) 0.25IRMM-387 Number-based mode (CLS) 2.09IRMM-387 Intensity-based mode (CLS) 0.14IRMM-388 Number-based mode (CLS) 0.07IRMM-389 Brunauer-Emmett-Teller (BET) n.a.BAM-11 Brunauer-Emmett-Teller (BET) 0.54



6.5.2 Products

6.5.2.1 Short-term stability study

For the short-term stability study, samples were stored at 60 °C for 1, 2 and 4 weeks. The referencetemperature was set to 22 °C. Three units per storage time were selected using a random stratifiedsampling scheme. From each unit, two samples were measured by spICP-MS.

For BAM-14 no short-term stability study was performed since from XRF data no conclusions concern-ing the particle size can be drawn. Instabilities are not expected. The particles are embedded in a solidmatrix.

The calculation of the uncertainty of degradation during dispatch is described in chapter 8.5.1.3. Theuncertainty describes the possible change during a dispatch at 60 °C lasting for one week. The resultsof these evaluations are summarised in Table 12.

Table 12: Uncertainties of stability during dispatch and storage

Material Measurand usts ,rel

[%]BAM-12b n.a. n.a.BAM-13b spICP-MS 2.58BAM-14 n.a. n.a.BAM-15 spICP-MS 0.74

6.5.2.2 Long-term stability study

For the long-term stability study, samples are stored at 22 °C for 12, 18, and 24 months. The referencetemperature was set to 4 °C. Three samples per storage time were selected using a random stratifiedsampling scheme.

For the time being, the stability study is still in progress.

7 ConclusionsThe currently available data demonstrate that there are no indications that the tested materials do ex-hibit a significant inhomogeneity or instability. The quantitative results are often determined by the re-peatability of the respective method that masks the true effect studied. As is can be assumed that theuncertainty of a characterisation of the materials would be clearly above 10 %, the contributions fromhomogeneity and stability would be mostly negligible.

All materials studied can be regarded as sufficiently homogeneous and stable with regard to their use inthe NanoDefine project.

NanoDefine Material Characterisation Data

Genaral informationParameter Details e-tool parameterSample ID official NanoDefine ID reference_signifier BAM-11 IRMM-380 IRMM-381 IRMM-382 IRMM-383 IRMM-384 IRMM-385 IRMM-386 IRMM-387 IRMM-388 IRMM-389 BAM-12a BAM-12b 4) BAM-13a BAM-13b BAM-14 BAM-15Sample ID used for some publications RTM7 RTM2 RTM4 RTM5 RTM8 RTM1 RTM3 RTM9

Description reference_name zeolite pigment yellow 83 nano BaSO4, fine grade MWCNT Nano steel CaCO3, fine grade Kaolin pigment yellow 83 coarse BaSO4, ultrafine grade coated TiO2 Basic methacrylate copolymer food with SiO2-tomato soup food with SiO2-pancake doughsunscreen formulation containing TiO2

simplified sunscreen formulation containing TiO2 HDPE containing Fe2O3 pigment toothpaste containing Al2O3

EM image12)

trade form trade_form powder powder powder powder powder powder powder powder powder powder powdershape SEM shape ~spherical elongated ~spherical elongated flat elongated flat ~spherical ~spherical ~spherical ~spherical

aggregation SEMaggregation/agglomeration yes yes yes yes yes yes yes yes yes yes yes

small dimensions SEM dimensions 3 2 3 2 1 2 1 3 3 3 3polydispersity polydispersity {high_poly} {high_poly} {high_poly} {moderate_poly} {high_poly} {high_poly} {high_poly} {high_poly} {high_poly} {high_poly} {high_poly}multimodality multimodalityhomogeneity ok ok ok ok ok ok ok ok ok ok okshort-term stability 3) ok ok ok ok ok ok ok ok ok ok oklong-term stability 3)

Physical DataSample ID reference_signifier BAM-11 IRMM-380 IRMM-381 IRMM-382 IRMM-383 IRMM-384 IRMM-385 IRMM-386 IRMM-387 IRMM-388 IRMM-389 BAM-12a BAM-12b 4) BAM-13a BAM-13b BAM-14 BAM-15skeleton density He-pycnometry 1) in g/cm3 2.07 ± 0.0006 1.484 4.28 ± 0.0013 2.05 ± 0.0056 5.12 ± 0.0024 2.657 2.61 1.5 4.01 ± 0.0010 3.99 ± 0.0015 1.13 2) 3.607 ± 0.015 3.13 ± 0.015specific surface VSSA 1) in m²/cm³ 875 93 13 480 47 15 41 25 149 58 1specific surface MSSA 1) in m²/g 423 63 3 234 9 6 16 17 37 15 1 23 56crystal structure

stable temperature range [min,max] in °C analysis_temperature [-273,500] [-273,100] [-273,500] [-273,500] [-273,500] [-273,500] [-273,500] [-273,100] [-273,500] [-273,500] [-273,50]sensitive to e-beam electron_beam yes yes no no no no no yes no no yesconductivity conductivity {insulator} {insulator} {insulator} {conductive} {conductive} {insulator} {insulator} {insulator} {insulator} {insulator} {insulator}magnetism magnetism ? ? ? ? {ferromagnetic} ? ? ? ? ? ?light absorption light absorption no no no no no no no no no yes nofluorescsnce fluorescsnce ? ? ? yes ? ? ? ? ? ? ?unstable at vacuum conditions os_vacuum no no no no no no no no no no norefractive index for λ=589 nm 5) 1.78 – 0.024i 1.641 2.76-3.35i 1.529 1.78 – 0.024i 1.641 2.8 1.3825

Chemical DataSample ID reference_signifier BAM-11 IRMM-380 IRMM-381 IRMM-382 IRMM-383 IRMM-384 IRMM-385 IRMM-386 IRMM-387 IRMM-388 IRMM-389 BAM-12a BAM-12b 4) BAM-13a BAM-13b BAM-14 BAM-15material system inorganic organic inorganic carbon inorganic inorganic inorganic organic inorganic inorganic organic

chemical composition sum formula SiO2:Al2O3 = 23:1 C36H32Cl4N6O8 BaSO4 CFe 67-72%, Cr 16-26%, Ni 10-14%, Mo 2-4% CaCO3 Al2O3•2SiO2•2H2O C36H32Cl4N6O8 BaSO4

>92% TiO2 (rutile);aluminium + silicon compound

H31C22N1O6

Inorganic Oxides: TiO2 and Fe2O3 Inorganic Oxides: TiO2 Fe2O3

Aqua, Hydrated Silica, Sorbitol, Alumina, Olaflur, Hydroxyethylcellulose, Aroma, PEG-40 Hydrogenated Castor Oil, Stearic Acid, Sodium Saccharin, Cocamidopropyl Betaine, Citric Acid, Limonene, CI 77891, 3-(N-hexadecyl-N-2-hydroxiethylammonio)propyl-bis(2-hydroxiethyl)ammoniumdifluorid (1400 ppm F-)

chemical composition EDXS

dispersability dispersability{aqueous,nonpolar,polar,specific,aerosol,substrate}

{aqueous,nonpolar,polar,specific,aerosol,substrate}










functionalisation functionalisation no no no no no no no no no yes norelease_iam release_iam

Size MeasurementSample ID reference_signifier BAM-11 IRMM-380 IRMM-381 IRMM-382 IRMM-383 IRMM-384 IRMM-385 IRMM-386 IRMM-387 IRMM-388 IRMM-389 BAM-12a BAM-12b 4) BAM-13a BAM-13b BAM-14 BAM-15expected size range [min,max] in nm wr_size_range [50,750] [20,90] [50,800] [5,20] [10,200] [50,500] [1,1000] [50,500] [10,150] [75,400] [500,10000]

D509) TEM [nm] three-fold determination 6) 33 12 plates - standard TEM not useful 181 250015)

disc AC-tu [nm] validatedautomated TEM11) [nm] validatedFFF [nm] validatedsize according to BET method further details 7) internal porosity nano non-nano nano nano non-nano nano non-nano nano internal porosity (coating) non-nano

miniTEM [nm] three-fold determination 10) 48 191 9 13) 74 13,14) 218 31D50 manual SEM [nm] 1), 8) 133 40 214 12 63 158 128 157 35 185;213 2000 25 23 18.4 33.2D50 TEM [nm] 8) 39 253;281 153;161 221 22;21 180;185D50 spICP-MS [nm] 8) 182D50 PTA [nm] 8) 281 274 210 114 254D50 DEMA spray [nm] 8) 293 148 252 53 253D50 disc AC-tu [nm] 8) 52 223 255 186 66 243D50 cuv AC-tu [nm] 8) 34 258 248 132 153 43 277 413D50 cuv AC-RI [nm] 8) 203 232 98 24 201D50 AF4-LS [nm] 8) 177D50 DLS [nm] 8) 68;81 285 294 290 292;269 76;72 195;215crystallite size SAX/XRD [nm] 1) 8/13.3 108.6 10.2 17.8 65.3 187/43.1 103 rutile 29 (anatase); 21 (rutile); 15 31 (hematite) 26 (AlOx); 36 (anatase) D50 ALS [nm] 8) 635 160 152 168;133 76 483;237 1837D50 USSP [nm] 8) 410 415 315

Remarks1 BAM; D1.32 large error; sample was not dried3 D1.64 work on material was stopped5 reported in D3.3 p. 60; values for additional wavelengths available. Public paper: (https://static-content.springer.com/esm/art%3A10.1007%2Fs11051-016-3461-7/MediaObjects/11051_2016_3461_MOESM1_ESM.pdf). 6 As discussed in the FCM in Wageningen Dec. 2016; Measured by BASF for those substances with no miniTEM data available. Evaluation using ParticleSizer7 D3.5 and Wohlleben et al. (DOI)8 taken from D3.3 (real world performance tests; unvalidated)9 D50 = x50,0

10 joker method reported in D4.611 Benchmark value; taken from WP5 validation12 Images of substances provided by Coda-Cerva; products BASF SE (expect BAM 12a: imaged by BAM)13 2-fold determination only14 issues with sample preparation SOP - fracturing of particles detected; value therefore too small15 only on TEM-grid out of 5 preparations useful. 1-fold determination including only 190 particles

substances products

200 nm

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