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Protocol(s) for size-distribution analysis of primary MNM objects in powders and
liquids for compliance with the EU definition (D2.10)
Jan Mast & Pieter-Jan De Temmerman CODA-CERVA
Service Electron microscopy
BE mid-term NANoREG meeting February 20, 2015, Brussels, BE
•24/03/2011 2
« Nanomaterial » means an incidental, natural or manufactured material containing particles, in an unbound state or as an aggregate or as an agglomerate and where, for 50 % or more of the particles in the number size distribution, one or more external dimensions is in the size range 1 nm – 100 nm
There are many “sizes” Few methods can detect 1 nm
>50 %
EC RECOMMENDATION on the definition of nanomaterial
a number based size distribution of aggregated synthetic amorphous silica measured by TEM (this product is used as anti-caking agent in powdered food)
multi-walled carbonnanotubes (their dimensions are up to 2 µm long, 60 nm wide; their width qualifies them as a NM)
In specific cases, 50 % may be replaced by a threshold between 1 and 50%
TEM remains indispensible
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Simultaneous evaluation of size, shape and morphology
•2D, takes in account the morphology (‘dimensionality’)
•The EC definition explicitly states ‘in one dimension’
•Techniques based on scattering and hydrodynamic radius reduce 3D information to 1D
Technical advantages
•< 1 nm resolution
•Can yield quantitative, number-based results
•Distinction between primary particles and aggregates/agglomerates.
OECD end points
•Micrographs, aggregation/agglomeration status, crystal structure, contaminants and number-based size
•Essential for risk analysis and toxicological and toxicokinetical research
Comparison with alternative methods
• Particle tracking analysis (PTA)
• SP-ICP-MS
• Scanning electron microscopy (SEM)
• Dynamic light scattering (DLS)
• Centrifugal liquid segmentation (CLS)
• Asymmetrical flow field flow Fractionation coupled to A4F/ICP-MS
• Analytical TEM including STEM-EDX
• Atomic force microscopy (AFP)
• Direct link to D2.11 (VSSA)
Partners
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Partner Staff Method Contacts RR-EM Comparison
CODA-CERVA (B)
57 TEM, NTA, DLS, SP-ICP-MS
Jan Mast <[email protected]> Pieter-Jan De Temmerman <[email protected]>
NTA, DLS, SP-ICP-MS
VN (I) 19 SP- ICP-MS, A4F, TEM
<[email protected]> Federico Benetti <[email protected]>;
AFFFF, sp-ICP-MS
LTH (S) TEM, DDCLA, DMA, FMPS
UMB (No) 4 FFF, TEM <[email protected]> <'[email protected]>
FFF
IIT (I) 4 TEM [email protected]; <[email protected]>; <[email protected]>
NCRWE (DK)
6 TEM Rambabu Atluri (RBA) <[email protected]>; <[email protected]>
DLS
INL (I) 8 TEM <[email protected]> <[email protected]>
Analytical TEM
Brazil many SEM & TEM Carlos A Achete <[email protected]>; '
South Korea
many SEM & TEM Nam Woong Song <[email protected]>; <young.h@[email protected]>; <[email protected]>;
Planning and milestones – D2.10
1. Guidelines to report the qualitative characteristics of a NM based on a TEM analysis
2. SOPs for the quantitative size and shape analysis of manufactured nanomaterials based on TEM for application for regulatory use
3. Validation of SOPs on reference and representative test nanomaterials based on estimation of the uncertainty budgets
4. Validation of SOPs based on inter-laboratory round-robin testing
•25/03/2015 •6
M A M J J A S O N D J F M A M J J A S O N D J F M A M J J A S O N D J F M A M J J A
6-monthly scientific reporting
Milestones
M 1 M 2 M 3 M 4
D2.10 meeting
Development and implementation of (draft)
SOPs for measurement of primary particles
Round Robin of TEM measurement of primary
particles of selected types of MNAggregates & complex
Inter-method comparison
Final reporting
Colloids Mix tures and rods
2015 201620142013
1. Guidelines for qualitative TEM analysis
o Task 1.3 Interaction with WP 2-6 on the scientific answers to the issues/questions related to regulatory needs for nanomaterials safety assessment and management.
• SOP for preparation of TEM-grids supporting WP2, WP5 and WP6 partners.
• OECD Guidance on descriptive TEM analysis TEM based on CODA-CERVA approach.
o Collaboration with WP5 partners to solve issues associated with the pre-test characterisation.
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2. SOPs for quantitative TEM analysis: colloidal MNM
o Developed procedures
• Preparation of TEM specimens
• TEM imaging
• TEM image analysis of colloidal NM
o Formulated in a platform-independent manner
o Reviewed, tested and accepted by D2.10 partners
o In CODA-CERVA: high degree of automation
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Specimen
•Representative transfer to EM-grid
•Even distribution
•Sufficient amount
TEM Imaging
• Representative
• # particles
• Resolution
• Mode
Image Analysis
•Detection
•Measurement
•Primary particles
Data Analysis
•Particle classification
•Artifact identification
2. SOPs for quantitative TEM analysis: Primary particels in aggregated NM
Proof of principle
o Model: NM-100
o Combines TEM imaging and semi-automatic image analysis
o Measures the minimal dimensions of the primary particles of an aggregated MNM from TEM micrographs and the overlap
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Approach
o The primary particles are detected based on watershed segmentation
o Their minimal size and overlap coefficient are measured based on a Euclidean distance map.
o Euclidean distance (in grey values) corresponds to its straight line distance to the nearest background pixel
o Platform independent:
• Developed in iTEM
• Tested in ImageJ
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Detailled TEM characterisation of fractal-like NM
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Comparative analyses
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A the central limit theorem is invoked to calculate the 95% confidence interval around this ratio B From BET, assuming a material density of 3.9 g/cm³
Testing on other NM
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NM-105 NM-103 Printex 90
NM-200 NM-201 NM-203
3. Validation of SOPs
o Guidelines for validation still have to be developed For sizing of NM by TEM and single particle ICP-MS
o Each method has to be validated for each NM chemical composition, size (distribution), structure, aggregation status, coating,…
o Few (certified) reference materials are available April 2012: one spherical, monodisperse silica NM (20 nm) certified for ECD measurement by TEM
o Implementation of legislation increases pressure
24/03/2011 14
Validation of SOPs: estimation of the uncertainty budgets
•25/03/2015 • 15 23 measurands
Measurands
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Aspect ratio
Convexity
Shape factor Sphericity
Elongation
ECD
Maximal Diameter
Minimal DIameter
Mean Diameter
Convex Perimeter
Perimeter
Convex Area
Area
Rectangle Min
Feret Mean Rectangle Mean
Feret Max
Feret Min
Rectangle Max
Maximal enscribed circle
Shape Surface topology 1D size 2D size
Intra-laboratory validation of other size, shape & surface topology measurands
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1D size
2D size
Validation of the TEM based method to define a colloidal material as a nanomaterial
o A top-down approach for a panel of 9 colloidal NM
• spanning a range of 9 to 200 nm
• Various compositions
o No certified values for TEM size measurement u(t)
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Size (nm) Name Composition Source Type
9 RM8011 Au NIST RM
19 NM-300K Ag JRC, Ispra, Italy RTM
19,4 ERM-FD100 Silica IRMM, Geel, Belgium CRM
20 / 80 ERM-FD102 Silica IRMM CRM
27,8 ERM-FD304 Silica IRMM CRM
30 RM8012 Au NIST RM
60 RM8013 Au NIST RM
100 Latex beads P Latex Nanosight RTM
200 Latex beads H Latex Nanosight RTM
Points of attention
o The trueness (between labs) uncertainty is the largest factor
o Standardization of the TEM methodology required
o Relatively few particles need to be measured: 5 % ~ 10-100 PP
o Overlapping particles can give problems
•25/03/2015 • 19
Intra-lab PTA measurement uncertainties
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PTA Hydrodynamic diameter (nm)
RM-8012 RM-8013 P1 H1 AverageA
Mean measured value, Cm
30.3 nm 53.7 nm 104.6 nm 199.6 nm
Reference value, Cref
27.6 nm B 56.0 nm B 102.0 nm 202.0 nm
Repeatability uncertainty, u(r)
6.86% 2.86% 3.63% 1.56% 3.73%
Intermediate precision uncertainty u(ip)
1.60%C 1.82% 2.40% 3.27% 2.27%
Measurement uncertainty u(m)
1.91% 1.10% 1.42% 1.52% 0.97%
Combined uncertainty uc(x)
7.30% 3.56% 4.58% 3.93% 4.47%
Expanded uncertainty U(x)
14.59% 7.12% 9.15% 7.85% 8.94%
Δm 2.7 nm 2.3 nm 2.6 nm 2.4 nm
U(x)- Δm 1.7 nm 1.5 nm 7.0 nm 13.3 nm
Validation of TEM analysis:
more complex models
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TEM PTA
Feret Min ± U(x) Aspect Ratio ± U(x) Hydrodynamic diameter ± U(x)
NM-100 Aggregates 96 nm ± 10.35% 1.3 ± 5.52%* 123 nm ± 25.41%
PP 98 nm ± 7.16%
NM-300K PP 15.1 nm ± 1,64 % 36.8 nm ± 47.20%
Nanorods 22 x 68 nm 25.1 nm ± 5.14% 2.5 ± 4.57% 51.8 nm ± 29.68%
15 x 54 nm 15.4 nm ± 7.23% 2.8 ± 10.24% 55.1 nm ± 18.26%
4. Validation of SOPs based on ILC
o Distribution of 3 draft SOPs to involved D2.10 partners for review and testing
• Preparation of TEM specimens
• TEM imaging
• TEM image analysis of colloidal NM
o Detailled instructions regarding the ILC
o Phase I of ILC: Testing and determination of measurement uncertainties of the colloidal NM (from Dec. 1)
• CRM-FD100 (silica)
• NM-300K (Ag)
o Data analysis is in process
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Information related to the methods
SOP colloidal NM + measurement uncertainties o De Temmerman et al. (2014) Measurement uncertainties of size, shape, and surface
measurements using transmission electron microscopy of near-monodisperse, near-spherical nanoparticles. DOI 10.1007/s11051-013-2177-1 J Nanopart Res 16:2177
o De Temmerman et al. (2014) Size measurement uncertainties of near-monodisperse, near-spherical nanoparticles using transmission electron microscopy and particle tracking analysis. Journal of Nanoparticle Research. 16:2628
o Braun, A., K. Franks, et al. (2011). Certified Reference Material ERM®- FD100: Certification of Equivalent Spherical Diameters of Silica Nanoparticles in Water. Luxembourg, European Union.
TEM image analysis of the primary particles in aggregates/agglomerated NM
De Temmerman et al. (2014) Semi-automatic size measurement of primary particles in aggregated nanomaterials by transmission electron microscopy. Powder Technology 261: 191–200
Reviews o Mast et al. Physical characterization of nanomaterials in dispersion by transmission
electron microscopy in a regulatory framework. In: Electron Microscopy of Materials, ed. L. D. Francis, A. Mayoral, R. Arenal, Springer (2014)
o Quantitative analysis of the physical characteristics of NM in suspensions by advanced TEM. PhD Pieter-Jan De Temmerman. [email protected]
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Acknowledments
o Pieter-Jan De Temmerman, Eveline Verleysen, Jan Mast Marina Ledecq, Michel Abi Daoud Francisco, Elke Van Doren, Nadine Dubois (CODA-CERVA)
o Jeroen Lammertyn, Bart De Ketelaere (KULeuven)
o Vikram Kestens, Gert Roebben, Thomas Linsinger (IRMM)
o nanokara project (CODA-CERVA)
o NANoREG (FP7/2007-2013, 310584)
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