Gert Roebben

Nanoscale reference materials for environmental, health and safety measurements: needs, gaps and opportunities

Abstract The authors critically reviewed published lists of nano-objects and their physico-chemical properties deemed important for risk assessment and discussed metrological challenges associated with the development of nanoscale reference materials (RMs). Five lists were identified that contained 25 (classes of) nano-objects; only four (gold, silicon dioxide, silver, titanium dioxide) appeared on all lists. Twenty-three properties were identified for characterisation; only (specific) surface area appeared on all lists. The key themes that emerged from this review were: 1) various groups have prioritised nano-objects for development as “candidate RMs” with limited consensus; 2) a lack of harmonised terminology hinders accurate description of many nano-object properties; 3) many properties identified for characterisation are ill-defined or qualitative and hence are not metrologically traceable; 4) standardised protocols are critically needed for characterisation of nano-objects as delivered in relevant media and as administered to toxicological models; 5) the measurement processes being used to characterise a nano-object must be understood because instruments may measure a given sample in a different way; 6) appropriate RMs should be used for both accurate instrument calibration and for more general testing purposes (e.g., protocol validation); 7) there is a need to clarify that where RMs are not available, if “(representative) test materials” that lack reference or certified values may be useful for toxicology testing and 8) there is a need for consensus building within the nanotechnology and environmental, health and safety communities to prioritise RM needs and better define the required properties and (physical or chemical) forms of the candidate materials.

Reference materials and representative test materials: the nanotechnology case

An increasing number of chemical, physical and biological tests are performed on manufactured nanomaterials for scientific and regulatory purposes. Existing test guidelines and measurement methods are not always directly applicable to or relevant for nanomaterials. Therefore, it is necessary to verify the use of the existing methods with nanomaterials, thereby identifying where modifications are needed, and where new methods need to be developed and validated. Efforts for verification, development and validation of methods as well as quality assurance of (routine) test results significantly benefit from the availability of suitable test and reference materials. This paper provides an overview of the existing types of reference materials and introduces a new class of test materials for which the term ‘representative test material’ is proposed. The three generic concepts of certified reference material, reference material(non-certified) and representative test material constitute a comprehensive system of benchmarks that can be used by all measurement and testing communities, regardless of their specific discipline. This paper illustrates this system with examples from the field of nanomaterials, including reference materials and representative test materials developed at the European Commission’s Joint Research Centre, in particular at the Institute for Reference Materials and Measurements (IRMM), and at the Institute for Health and Consumer Protection (IHCP).

A new certified reference material for size analysis of nanoparticles

A certified reference material, ERM-FD100, for quality assurance and validation of various nanoparticle sizing methods, was developed by the Institute for Reference Materials and Measurements. The material was prepared from an industrially sourced colloidal silica containing nanoparticles with a nominal equivalent spherical diameter of 20xa0nm. The homogeneity and stability of the candidate reference material was assessed by means of dynamic light scattering and centrifugal liquid sedimentation. Certification of the candidate reference material was based on a global interlaboratory comparison in which 34 laboratories participated with various analytical methods (DLS, CLS, EM, SAXS, ELS). After scrutinising the interlaboratory comparison data, 4 different certified particle size values, specific for the corresponding analytical method, could be assigned. The good comparability of results allowed the certification of the colloidal silica material for nanoparticle size analysis.

Measurement uncertainties of size, shape, and surface measurements using transmission electron microscopy of near-monodisperse, near-spherical nanoparticles

Transmission electron microscopy (TEM) in combination with a systematic selection procedure for unbiased random image collection, semi-automatic image analysis, and data processing has been validated for size, shape, and surface topology measurements of silica nanoparticles. The validation study, assessing the precision and accuracy of the TEM method, consists of series of measurements on two colloidal silica-certified reference materials, with number-based modal area-equivalent circular diameters (ECD) of 19.4xa0nm (ERM-FD100) and 27.8xa0nm (ERM-FD304). The measurement uncertainties are estimated for the modal and median particle size, shape, and surface topology parameters of single primary particles. The single primary particles are distinguished from agglomerates using a linear discriminant analysis approach. After optimization of the binning process, the mode associated with the number-based particle size distribution is obtained by lognormal fitting. The methodology described in this paper relies on a high level of automation of calibration, image acquisition, image analysis, and data analysis and gives robust results for the modal ECD. The expanded uncertainty of the modal ECD is estimated to be about 3xa0%. The largest contribution to the expanded uncertainty stems from the uncertainty associated with the trueness of the TEM method.

Challenges in the size analysis of a silica nanoparticle mixture as candidate certified reference material

A new certified reference material for quality control of nanoparticle size analysis methods has been developed and produced by the Institute for Reference Materials and Measurements of the European Commission’s Joint Research Centre. The material, ERM-FD102, consists of an aqueous suspension of a mixture of silica nanoparticle populations of distinct particle size and origin. The characterisation relied on an interlaboratory comparison study in which 30 laboratories of demonstrated competence participated with a variety of techniques for particle size analysis. After scrutinising the received datasets, certified and indicative values for different method-defined equivalent diameters that are specific for dynamic light scatteringxa0(DLS), centrifugal liquid sedimentationxa0(CLS), scanning and transmission electron microscopy (SEM and TEM), atomic force microscopyxa0(AFM), particle tracking analysis (PTA) and asymmetrical-flow field-flow fractionation (AF4) were assigned. The value assignment was a particular challenge because metrological concepts were not always interpreted uniformly across all participating laboratories. This paper presents the main elements and results of the ERM-FD102 characterisation study and discusses in particular the key issues of measurand definition and the estimation of measurement uncertainty.

Reference materials and representative test materials to develop nanoparticle characterization methods: the NanoChOp project case

This paper describes the production and characteristics of the nanoparticle test materials prepared for common use in the collaborative research project NanoChOp (Chemical and optical characterization of nanomaterials in biological systems), in casu suspensions of silica nanoparticles and CdSe/CdS/ZnS quantum dots (QDs). This paper is the first to illustrate how to assess whether nanoparticle test materials meet the requirements of a “reference material” (ISO Guide 30, 2015) or rather those of the recently defined category of “representative test material (RTM)” (ISO/TS 16195, 2013). The NanoChOp test materials were investigated with small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and centrifugal liquid sedimentation (CLS) to establish whether they complied with the required monomodal particle size distribution. The presence of impurities, aggregates, agglomerates, and viable microorganisms in the suspensions was investigated with DLS, CLS, optical and electron microscopy and via plating on nutrient agar. Suitability of surface functionalization was investigated with attenuated total reflection Fourier transform infrared spectrometry (ATR-FTIR) and via the capacity of the nanoparticles to be fluorescently labeled or to bind antibodies. Between-unit homogeneity and stability were investigated in terms of particle size and zeta potential. This paper shows that only based on the outcome of a detailed characterization process one can raise the status of a test material to RTM or reference material, and how this status depends on its intended use.

Metrological traceability of the measured values of properties of engineering materials

Global comparability of the measured values of material properties is based on some fundamental metrological concepts. These concepts are either already widely implemented in current procedures for materials testing or they are being further developed and increasingly accepted and used. An important aspect of the comparability of measurement results is metrological traceability. This paper aims at illustrating with practical examples how to apply the concept of metrological traceability as defined in ISO/IEC Guide 99:2007, known also as the VIM (International Vocabulary of Metrology), in the field of engineering material properties. VIM distinguishes three different types of references for traceability: either to a system of units, such as the SI, to a measurement procedure or to a physical measurement standard. For each approach, an example is given in the field of engineering material properties, including appropriate traceability statements and means to achieve the traceability. The role of certified reference materials is highlighted, as well as practical consequences of traceability requirements for the design of reference material certification projects.

Improved Metrological Traceability of Particle Size Values Measured with Line-Start Incremental Centrifugal Liquid Sedimentation

Line-start incremental centrifugal liquid sedimentation (disc-CLS) is a powerful method to determine particle size based on the principles of Stokes law. Because several of the input quantities of the Stokes equation cannot be easily determined for this case of a rotating disc, the disc-CLS approach relies on calibrating the sedimentation time scale with reference particles. To use these calibrant particles for establishing metrological traceability, they must fulfill the same requirements as those imposed on a certified reference material, i.e., their certified Stokes diameter and density value must come with a realistic measurement uncertainty and with a traceability statement. As is the case for several other techniques, the calibrants do not always come with uncertainties for the assigned modal diameter and effective particle density. The lack of such information and the absence of a traceability statement make it difficult for the end-user to estimate the uncertainty of the measurement results and to compare them with results obtained by others. We present the results of a collaborative study that aimed at demonstrating the traceability of particle size results obtained with disc-CLS. For this purpose, the particle size and effective particle density of polyvinyl chloride calibrants were measured using different validated methods, and measurement uncertainties were estimated according to the Guide to the Expression of Uncertainty in Measurement. The results indicate that the modal Stokes diameter and effective particle density that are assigned to the calibrants are accurate within 5% and 3.5%, respectively, and that they can be used to establish traceability of particle size results obtained with disc-CLS. This conclusion has a great impact on the traceability statement of certified particle size reference materials, for which the traceability is limited to the size and density values of the calibrant particles.

A comparison of techniques for size measurement of nanoparticles in cell culture medium

Plain and aminated silica nanoparticles dispersed in purified water, in 50 mM Tris–HCl buffer and in cell culture medium were measured using dynamic light scattering (DLS), centrifugal liquid sedimentation (CLS), small-angle X-ray scattering (SAXS), and particle tracking analysis (PTA). The test samples were measured by all methods immediately after dispersion and after incubation at room temperature for 24 h. The effect of the biological dispersion medium on the modal value of the particle size distribution was compared for each method taking into account the estimated uncertainty. For the methods based on light scattering, DLS and PTA, the size distributions obtained were significantly altered due to the formation of a protein corona and induced agglomeration effects. With SAXS and CLS, the measured size of the primary particles was mostly unchanged. While SAXS offers excellent precision and traceability to the SI unit system if the model fitting approach is used for data analysis, CLS provides detailed size distributions from which additional information on the agglomeration state can be deduced.

Comments on the article by A.J. Lecloux (J Nanopart Res (2015) 17:447) regarding the use of volume-specific surface area (VSSA) to classify nanomaterials

In November 2015, an article by A. J. Lecloux was published in this journal (J Nanopart Res, 17:447, 2015). The article focused on the use of volume-specific surface area (VSSA) for the implementation of the European Commission’s recommended definition of “nanomaterial”. In that paper, VSSA values were calculated for polydisperse particulate materials using a particle number-based averaging method which do not agree with earlier results of VSSA simulations of polydisperse materials reported in 2014 by the Joint Research Centre (JRC) of the European Commission (EC). In this contribution, we explain the difference between traditional view of VSSA which was used by the JRC and the proposed model of Lecloux. Through the use of some simple examples for polydisperse materials, it is demonstrated that the latter produces values which neither correspond to the generally accepted definition of VSSA nor relate to the commonly used experimental methods for determining VSSA using gas adsorption. Lecloux’s model therefore does not constitute a basis for practical implementation of the EC’s definition of nanomaterial using gas adsorption techniques.