Analytical chemistry of engineered nanomaterials: Part 1. Scope, regulation, legislation, and metrology (IUPAC Technical Report)

2.1 Nanomaterial definitions and classification

2.1.2 Classification

The ISO definitions are accepted by the Uniform Description System (UDS) which was co-developed by CODATA (Committee on Data of the International Science Council) and VAMAS (Versailles Project on Advanced Materials and Standards) and provides a framework that identifies the data and information that are necessary to describe a nanomaterial. The UDS is designed to provide a common method to accurately describe a nanomaterial for use by various communities including researchers, producers, regulators and standard developers and to allow differentiation between materials with similar compositions but different physical properties [19]. It divides nanomaterials and the objects that comprise them into four major types: (1) individual nano-objects, (2) collections of nano-objects that may be identical or different, (3) bulk materials containing individual nano-objects, and (4) bulk materials with nanoscale features. Bulk material is defined as a “solid material that has all external physical dimensions larger than the nanoscale” but that may have nanoscale internal or surface features. Somewhat different information is needed for a complete description of each of these four categories.

A recent report has addressed the need for a systematic approach for describing nanomaterials that is compatible with building datasets for nanoinformatics and regulatory purposes [20]. This approach is an extension of the IUPAC International Chemical Identifier (InChI) machine-readable system for identifying chemicals. The first version of Nano InChI covers an initial set of nanomaterial properties, including composition, size and shape, crystal structure, and surface chemistry, but does not yet include dynamic properties that are affected by the environment, such as agglomeration, dissolution, and formation of protein coronas.

Nanomaterials are frequently classified into groups based on their composition; these include metals and metal oxides, non-metal inorganics, nanocarbon and organic nanomaterials. Metals and metal oxides are by far the most commonly found nanomaterials in consumer products as indicated by recent reviews and databases of nanomaterials [21], [22], [23]. As illustrated in Fig. 1, silver and titanium or titanium dioxide account for more than half of the nanomaterials in products for which the nanomaterial is identified in a database of consumer products available in Europe [22]; two earlier reports covering different geographic areas and time periods reached similar conclusions [21, 23]. Metal and metal oxide ENMs typically have spherical or irregularly shaped particles and relatively broad size distributions, although metal nanoparticles (e.g., gold) can be synthesized in a variety of well-defined shapes and with low polydispersity. Nanocarbon materials are also increasingly used for applications, particularly carbon nanotubes, fullerenes, nanodiamond, and two-dimensional nanoplates such as graphene and graphene oxide. Other (non-metal-containing) inorganic nanomaterials include semiconductor nanocrystals (e.g., CdS or CdSe quantum dots) and aluminosilicates such as zeolites and clays. Organic nanomaterials include synthetic or naturally occurring polymers, dendrimers, and self-assembled structures such as micelles and liposomes, many of which are widely used in biomedical applications. Note that naturally occurring nanomaterials and incidental materials such as nanoplastics are not covered in this report. Both physical characterization techniques and analytical chemistry methods will vary depending on the composition and particle size of the nanomaterial.

Fig. 1:

The most common types of engineered nanomaterials in consumer products (left) and their distribution in different product categories (right). Information is extracted from a web-based database of 5000 consumer products that contain nanomaterials and are available in Europe [22]; the database also contains a large number of products for which the nanomaterial composition is not provided.

2.2 Analytical chemistry of nanomaterials

Analytical Chemistry is defined by IUPAC as a “Scientific discipline that develops and applies strategies, instruments, and procedures to obtain information on the composition and nature of matter in space and time” [24]. The influence of nanotechnology on the development of analytical chemical disciplines has been recognized. To facilitate discussions and define fields, several terms have been proposed to describe the analytical chemistry and the analytical use of nanomaterials. Some of these are noted in this report without endorsing any specific approach. Valcarcel introduced “Analytical Nanoscience and Nanotechnology” [25] which includes a classical theme on the characterization/determination of nanomaterials and a second theme focused on the use of nanomaterials as tools (e.g., sorbents, inert/active supports, and sensors) for improving analytical processes. The second theme enriches the field of analytical chemistry [4].

The term “Nanoanalytics” has been used by the Shtykov, Zolotov, and Glatzel groups to cover similar concepts [26], [27], [28]. As illustrated in Fig. 2, this includes chemical and physical analysis methods for pristine nanomaterials as well as those in complex matrices, and the use of nano-objects as tools to modify sensitivity and selectivity of chemical analyses. In contrast, others have defined nanoanalytics more narrowly as “a scientific discipline that develops and applies methods, instruments and strategies to obtain information on the chemical composition, and physical and chemical nature of matter in the form of nano-objects, in space and time, as well as on the value of these measurements, i.e. their uncertainty, validation and/or traceability according to fundamental standards” [29]. This work recognized that nanoanalytics should include studies of the dynamic behavior of nano-objects, and the behavior of agglomerates and aggregates with sizes above 100 nm. Independent of the various concepts that have been used to define the analytical chemistry of nanomaterials, the use of nano-objects as tools to improve analytical methods is beyond the scope of this report. Part 1 provides information on both physical characterization and analytical methods and Part 2 focuses on descriptions of sampling and analytical methods that are used for detection, identification, and quantification of nanomaterials, with a focus on applications to complex environments.

Fig. 2:

Scheme of the nanoanalytics concept, adapted from Ref. [27].

2.2.1 Nanomaterial properties

Nanomaterials differ from the conventional analytes typically studied using analytical methods for which composition/structure and concentration are generally the relevant properties. Nanomaterials require both chemical (composition, surface characterization, mass and/or particle number concentration, presence of impurities) and physical information (e.g., size, size distribution, shape, surface area, aggregation, crystal structure) to ensure that nanomaterials with similar compositions but different physical properties can be distinguished from each other. Such information is obtained by measurement or examination of the properties. For measurement, the quantity intended to be measured with respect to the nanomaterial as the analyte is the measurand [30].

Chemical information (characterization) should include all expected components as well as undesired impurities [31], which can be responsible for the unexpected behavior of ENMs. Because nano-objects can exhibit complex structures (e.g., core–shell nanoparticles, functionalized nano-objects), the spatial variation of composition should also be considered, although chemical characterization is often restricted to the determination of the bulk and surface compositions. Engineered nanomaterials interact with each other and their environment via surface functional groups, which makes the chemical characterization of the surface and any adsorbed coatings a critical factor for understanding their behavior [3, 32]. Thus, assessment of a significantly more extensive number of parameters is required for nanomaterials than for conventional analytes.

In this sense, the analytical chemistry of nanomaterials can be likened to speciation analysis, particularly to physicochemical speciation. A new approach is represented by nanoparticle imprinted matrices (NAIMs), which is based on an idea similar to conventional molecularly imprinted polymers (MIPs) that are frequently used in analytical chemistry. In NAIMs the nanoparticles are imprinted in a polymer matrix followed by their removal to form highly selective voids that can recognize the original nanoparticles based on differentiation according to size, shape, and surface shell [33].

Table 1 lists the common properties that are typically required for adequate characterization of an ENM. For specific applications or materials it may be necessary to measure additional properties (e.g., mechanical, optical, or electrical properties). For risk assessment purposes, properties such as redox potential, dustiness, and radical formation capability are usually also required.

Table 1:

Characterization of nanomaterial properties.

General category	Properties
Composition	Bulk composition, impurities, spatially resolved composition for complex architectures (e.g., core shell structures)
Content (of material)	Amount of substance concentration, mass concentration, mass fraction, particle number concentration
Structure	Crystal structure, amorphous content, density, porosity
Morphology	3D shape, aspect ratio, particle size, particle size distribution, agglomeration/aggregation state
Surface	Specific surface area, surface charge, surface functional groups, coatings
Other	Dispersibility, solubility, stability

Typically, the necessary physical and chemical properties (Table 1) can be separated into two categories. Intrinsic properties include size, shape, composition (bulk, core, surface shell) and structure (e.g., crystal structure, fraction of crystalline and amorphous content, porosity, and density). They provide a basic description of the material and the minimum set of information needed to differentiate the nanomaterial from other similar materials. These properties are generally independent of the medium or environment in which the nanomaterial is located provided that chemical transformations are avoided. Most properties can be assessed using several different methods and it is important to consider that the methods will differ in principle of operation and will have different method-specific measurands. In other words, the measurands are method-defined and different techniques may give different values for the same property. As an example, one expects the bulk elemental concentration of a specific material to be independent of the measurement method. The same may not apply to methods for measuring particle number concentration which have different principles of operation and different method-specific measurands.

Other properties, generally referred to as extrinsic (or synergistic), depend on the environment and include surface charge, surface coatings, aggregation/agglomeration, and dispersibility. These properties often determine the interaction of the nanomaterial with other components and ultimately its behavior and fate in complex environments. Extrinsic parameters can be modified by pH or ionic strength of the liquid phase, temperature, the presence of redox-active materials, and the presence of surfactants, natural organic matter, or peptides/proteins that can modify the surface coating. As noted above, it is frequently important to correlate several material characteristics to understand the behavior of the material [29]. Fig. 3 illustrates the range of chemical and physical properties needed to characterize ENMs and some of the commonly applied methods; note that fractionation methods and hyphenated techniques that allow multiple measurements on a single instrument platform are not included.

Fig. 3:

Chemical and physical properties needed for characterization of nanomaterials and the most commonly used methods that are applicable to a range of nanomaterial types. Methods for aerosol nanoparticles are not included; adapted from Refs. [4, 34, 35].

2.3 Analytical scenarios

The analytical scenario to be employed for a specific nanomaterial will depend on the composition of the material, the environment in which it is found, and the objective of the investigation. As noted above, there are typically many physical and chemical properties that must be assessed to adequately identify, characterize, and quantify the amount of the material. The overall goal of the study will often determine which parameters must be assessed. As an example, different parameters will be of interest depending on whether the objective is quality control for synthesis of the pristine nanomaterial or determination of the identity and amount of a nanomaterial in a consumer product or an environmental sample. The technique and approach will vary significantly for these two scenarios.

3.1 Nanomaterials and their identification for legislation purposes

One of the first challenges in risk assessment for regulatory purposes is deciding whether a (nano)material requires additional assessment beyond that necessary for conventional chemicals. This will typically involve consideration of the nanomaterial definition adopted for the specific jurisdiction. A variety of regulatory or advisory definitions are used by different regulatory bodies [10]. Although most regulatory bodies have adopted size as one of the metrics, the applicable size range, the manner in which the size distribution is measured (i.e., mass vs particle number concentration) and the inclusion of other properties varies considerably. Furthermore, in some cases the measurement methods that are needed to demonstrate whether a material should be classified as nano are not always available. This is problematic for definitions that require measurement of the particle number concentration. For example, the European Commission (EC) in the updated definition has recommended that a nanomaterial be defined as “natural, incidental or manufactured material consisting of solid particles present, either on their own or as identifiable constituent particles in aggregates or agglomerates where 50 % or more of these particles in the number-based size distribution fulfils at least one of the following conditions: (1) one or more external dimensions of the particle are in the size range 1–100 nm; (2) the particle has an elongated shape, such as a rod, fibre or tube, where two external dimensions are smaller than 1 nm and the other dimension is larger than 100 nm; (3) the particle has a plate-like shape, where one external dimension is smaller than 1 nm and the other dimensions are larger than 100 nm” [49]. Regarding implementation of the nanomaterial definition, the challenges have been highlighted [50]. Two EC Joint Research Council (EC-JRC) reports have attempted to clarify some of the terms used and the availability of methods to comply with the definition [51, 52]. Despite the lack of clarity and the variations across regulatory bodies, it is clear that size and size distribution and number-based concentration are key parameters.

Regulatory agencies in other countries have adopted mass-based criteria, rather than a particle number-based criterion for deciding whether a material is “nano” or not. For example, the US Environmental Protection Agency (EPA) nanospecific guidance applies to chemical substances that are “solids at 25 °C and standard atmospheric pressure, manufactured or processed in a form where any particles, including aggregates and agglomerates, are in the size range of 1–100 nm in at least one dimension and are manufactured or processed to exhibit one or more unique and novel properties.” Materials with less than 1 % by weight of any particles, including aggregates and agglomerates, between 1 and 100 nm are excluded [53]. Health Canada’s working definition is similar in terms of size range and novel properties but notes that either a mass (1 %) or number (10 %) based limit may be applied.

The standard methods required for risk assessment for regulatory purposes are being developed by international standards organizations such as ISO, ASTM (American Society for Testing and Materials, now ASTM International), and CEN (European Committee for Standardization) (see Section 3.2). There are also many large multicenter projects that are tackling the development of methods, both for physico chemical characterization and for ecological and human health toxicology. Notably, most of these large collaborative projects are supported by OECD (Organization for Economic Cooperation and Development) or various EU programs; examples include NanoDefine, Gracious, NanoValid, NanoReg, and several European Metrology Program for Innovation and Research projects (NanoChOP, nPSize, METVES, and InNanoPart) as summarized recently [12, 54].

3.2 Emerging regulations and related analytical drivers

There are several well-established EU regulations in place with regards to the use of nanomaterials in consumer products and medical devices. For example, EU regulation 1169/2011 on food information for consumers indicates that nanomaterials should be clearly indicated in the list of ingredients and names should be followed by (nano) [55]. The Novel Foods Regulation EC 2015/2283 states that foods consisting of ‘engineered nanomaterials’ are in the list of criteria to be considered for novel foods and therefore subjected to pre-market authorization [56]. Regulation EC 1935/2004 includes new active and intelligent food contact materials introduced to maintain/improve or monitor the quality of food, respectively [57]. Medical Devices Regulation (EU) 2017/745 establishes that there is scientific uncertainty about the risks and benefits of nanomaterials used for devices [58]. It also states that in the design and manufacture of devices, manufacturers should take special care when using nanoparticles for which there is a high or medium potential for internal exposure. Such devices should be subject to the most stringent conformity assessment procedures.

The EU Cosmetics Regulation EC 1223/2009 states that all cosmetic products must be notified and the notification must contain, as a minimum, the following information [59]: identification of the nanomaterial, including its chemical name and other descriptors specified in the regulation; specification of the nanomaterial, including its particle size and its physical and chemical properties; an estimation of the quantity of the nanomaterial contained in the cosmetic product to be placed on the market per year; a toxicological profile of the nanomaterial; safety data of the nanomaterial; all information related to the reasonably foreseeable exposure conditions. The EC 1223/2009 and the Scientific Committee on Consumer Safety (SCCS) 1501/12 guidance currently include TiO₂, ZnO, and carbon black as approved additives [59], [60], [61]. Table 2 provides examples of properties that have to be controlled and measured by industry for TiO₂ and ZnO, during the production process and after incorporation in final products. These are two nanomaterials that are used in a relatively high volume in a variety of applications (see Fig. 1), including cosmetics.

Regulation of nanomaterials in the environment is not well established so far but there are emerging regulatory efforts by different countries. These include Danish Statutory Order 644 which provides guidelines for a nanoproduct inventory from the Danish Environmental Protection Agency [62], a Belgian Royal Decree on the mandatory registration of nanomaterials [63] and guidelines on assessing health and environmental risks of nanoparticles from the Dutch National Institute for Public Health and the Environment [64]. The scientific committee on Health, Environmental and Emerging Risks (SCHEER) is active in looking at nanoparticles in the wider context of emerging risks and has issued statements on micro and nano-plastics in the environment and release of nanoparticles from building materials and construction waste to the environment [65].

Regulation of nanomaterials in North America is less well-developed than within the EU. Some nanomaterials are regulated under the EPA Toxic Substances Control Act which has specific guidance for nanomaterials [66]. The EPA reporting and record-keeping guidelines were implemented in 2017 and involved requirements for reporting for existing forms of certain nanoscale materials, and for new discrete forms of certain nanoscale materials before they are manufactured or processed [67]. The use of nanomaterials in food, drugs, and cosmetics is regulated by FDA (US Food and Drug Administration) under their Food, Drug and Cosmetics Act [68]. The situation in Canada is very similar with nanomaterials that are not specifically covered by other acts regulated as a new substance under the Canadian Environmental Protection Act. Currently, nanomaterials with existing CAS numbers that are listed on the approved Domestic Substance List do not require additional consideration as nanomaterials. However, a risk assessment framework is being developed to cover these materials. Health Canada regulates the use of nanomaterials under the Food and Drugs Act [69] with specific regulations that apply to food, drugs, cosmetics, and medical devices [70].

3.3 Recent guidance and scientific opinions

European Food Safety Authority (EFSA) has published several guidance documents and scientific opinions with regard to the safe use of nanomaterials in food and feed and active food contact materials. Key EFSA guidance documents include the 2011 EFSA Guidance on the risk assessment of the application of nanoscience and nanotechnologies in the food and feed chain (parameters such as shape, solubility, surface charge, and surface reactivity for characterization and identification of engineered nanomaterials) [71] and the 2018 Guidance on risk assessment of the application of nanoscience and nanotechnologies in the food and feed chain: Part 1, Human and animal health [17]. A further guidance document on environmental risk assessment was published in fall 2020 [18] and a guidance document on requirements to establish the presence of small particles, including nanoparticles, is currently available for consultation [72]. Key recent scientific opinions from European committees include the following:

1. Scientific Opinion of EFSA on the safety assessment of selenium nanoparticles for use in active food contact materials [73]. This scientific opinion deals with the safety assessment of selenium nanoparticles, which are intended to be used as an antioxidant. The conclusion is that there is no safety concern for the consumer if selenium nanoparticles are used in multilayer films and separated from the food by a polyolefin food contact layer for any type of food and under any food contact conditions.

2. Titanium dioxide (E 171) in food. A May 2021 decision by EFSA stated that E 171, currently approved as a food additive in the EU, can no longer be considered safe for this application [74]. Its safety was previously evaluated in 2016, at which time significant data gaps were identified. The more recent evaluation was the first use of EFSA’s 2018 guidance document on risk assessment for nanomaterials in the food and feed chain [17]. The main finding of this study was that although absorption of the particles is low, they may accumulate and one cannot exclude genotoxicity, potentially leading to carcinogenic effects.

3. Colloidal silver (nano). The SCCS was requested to give its opinion on the safety of the nanomaterial colloidal silver when used in cosmetics including toothpastes and skin-care products with a maximum concentration limit of 1 %, taking into account the reasonably foreseeable exposure conditions [75]. Only a limited amount of data was provided by the applicants to comply with the SCCS Guidance on Safety Assessment of Nanomaterials in Cosmetics (SCCS 1484/12). The provided data were also not in line with the SCCS Memorandum on Relevance, Adequacy and Quality of Data in Safety Dossiers on Nanomaterials (SCCS/1524/13). Due to a number of major data gaps, the SCCS was not in the position to draw a conclusion on the safety of colloidal silver in nano form when used in oral and dermal cosmetic products. The SCCS was requested to address any further scientific concerns with regard to the use of Colloidal Silver in nano form in cosmetic products and recommended that consideration be given to the presence of ionic silver.

4.1 Reference materials

Reference materials are used to calibrate analytical methods and equipment, to develop and validate new methods, to assess whether existing methods are suitable for new materials, and for quality control to ensure that methods are performing reliably. They are necessary for linking measurements to a common point of reference through calibration preferably to the International System of Units (SI). Occasionally, measurements in novel fields of development where there are no documentary standards available so far could also be traceable to an artifact such as a certified reference material (CRM). The CRMs are useful for laboratories that wish to verify that their measurements can be compared with those obtained elsewhere or for manufacturers who wish to benchmark their materials against a well-characterized and widely available material. Although the terminology used for reference materials varies in different communities, the following definitions are recommended by ISO (Guide 30, [79]) and agree with those provided in the VIM and IUPAC recommendations [24, 30].

A reference material is a material, sufficiently homogeneous and stable with respect to one or more specified properties, which has been established to be fit for its intended use in a measurement process [30]. By contrast, a CRM is a reference material characterized by a metrologically valid procedure for one or more specified properties and accompanied by a certificate that provides the value of the specified property, its associated uncertainty, and a statement of metrological traceability.

Certified reference materials can typically be grouped in two major categories, “neat materials” and matrix reference materials. Neat materials, often called high purity materials or calibrators, are typically single-substance formulations containing only the analyte of interest (sometimes in carrier medium). These neat standards are low complexity materials that CRM producers are able to characterize to a very high degree of precision and accuracy and that typically serve as calibration standards. In contrast to single-substance CRMs, matrix reference materials are intended to represent real samples encountered by the analyst where the chemical or physical substance of interest is surrounded by or embedded into another often much more complex material. Consider 10,000 μg/g lead standard solution as a neat standard CRM vis-à-vis 10 μg/g of lead in soil as a matrix CRM. This distinction is especially significant for the nanomaterial analysis community since at the time of writing this report practically all available reference nanomaterials (Fig. 4) fall into the category of neat materials/calibrators such as gold nanoparticle suspension, for example (Fig. 4, right). Producers of CRMs world-wide have been studying nanomaterials in soils, biological tissues, waters, etc.; however, detection of nano-objects in complex, multiphase systems is a great chemical/physical measurement challenge, which is further discussed in Part 2 of this report [14].

Fig. 4:

National Research Council Canada certifed reference material for cellulose nanocrystals (CNCD-1, left); CNCD-1 has a certified value for sulfur content for surface sulfate half ester groups and reference values for size measured by microscopy and dynamic light scattering. Quality control material from LGC – LGCQC5050 (right) has an assessed value for gold nanoparticle number concentration.

Certified reference materials are extensively studied for homogeneity and stability of the measurand of interest. Stability experiments must demonstrate that over the expected lifetime of the material the measurand will not change or will change in a predictable manner under various typical usage and storage conditions. Both intraunit and interunit homogeneity are assessed by measuring multiple replicates for one unit and multiple units, respectively. The extensive requirements for homogeneity, stability, and metrological traceability mean that these materials are generally produced by national metrology institutes or other qualified CRM producers in compliance with the ISO 17034 standard [80], which outlines the competency requirements for reference material producers. Compliance, typically accreditation to ISO 17034, is an indication of the competence of the CRM producer so ideally reference materials should be sourced from such laboratories.

Both reference and certified reference materials (Table 3) are available for nanomaterials and frequently only some of the available property values have been certified. It is also common to provide indicative or informational values for some properties. Reference nanomaterials are supplied as either dry powders or as aqueous suspensions and are frequently spherical and monodisperse nanoparticles. Multiple properties have been characterized for most reference nanomaterials. They can, therefore, be used to develop or calibrate methods for measuring composition, specific surface area, surface charge, surface functional group content, and particle size distributions by microscopy and/or ensemble solution methods such as DLS. Among the earliest and most widely used reference materials are the NIST gold nanoparticles which were produced in three sizes (10, 30, and 60 nm) as aqueous suspensions stabilized with citrate (Table 3). Size measurements using five different methods were provided and showed some variation as expected for method-defined measurands. Although none of the property values were certified, these materials have been extensively used for method development and validation for particle size measurements. Other available reference materials now include silver, silica, metal oxides, and polystyrene nanoparticles. Although initially, most reference materials were spherical with monomodal size distributions, different shapes (e.g., Au nanorods, carbon nanotubes, cellulose nanocrystals), and bimodal mixtures (e.g., silica) are now available (Table 3). Most of these are assumed to be “pure” materials, although in several cases information on metal ion impurities (e.g., carbon nanotubes, cellulose nanocrystals) is available.

The CRMs are accompanied by documents describing key characteristics of the material including use, certified quantity values and associated uncertainities, information about the producer of the material and the validity period of the reference material. Reference to the accreditation status of CRM producers against competency standards ISO 17034 and ISO 17025 is often indicated [80, 81].

An alternate approach that has proven to be very useful, particularly for access to standard samples for nanotoxicology experiments, is the availability of representative test materials (RTM, ISO 16195 [82]). This development was prompted by the wide range of available nanomaterials, the many properties that are necessary for their adequate characterization and the relatively small number of reference materials that are available. A RTM is a material from a single batch that is sufficiently homogeneous and stable with respect to one or more properties and which is assumed to be fit for purpose for its intended use in the development of test methods that target properties other than those for which homogeneity and stability have been demonstrated [82, 83]. A series of nano RTM has been produced and characterized by the EC-JRC for use by the OECD Working Party for Manufactured Nanomaterials program on safety testing. They include a range of metal oxide, gold, clay, and carbon-based nanomaterials and have been extensively characterized with data available in JRC reports; however, these RTMs have not been subjected to the full certification process required for CRMs. The OECD program was designed to test the suitability of existing test guidelines for physical chemical characterization and toxicological testing of a series of 13 commercial ENMs and to provide guidance on whether methods could be adapted for nanomaterials or whether new methods were needed. Note that since these RTMs are primarily materials produced industrially on a relatively large scale, they are often more challenging to characterize than most reference materials. This is due to their range of shapes, broad size distributions, a strong propensity to agglomerate and aggregate and frequently ill-defined surface chemistry and level of impurities. Nevertheless, they have been extensively used to benchmark characterization methods and for nanotoxicology studies, facilitating comparability of results obtained in different laboratories.

Despite the availability of reference materials and representative test materials, there are current gaps in the availability of suitable test materials. The main issue is the lack of availability of nanomaterials in complex matrices such as those encountered in environmental samples, food, consumer products and biological samples. Early studies that generated reference materials for silver nanoparticles in meat and silica in tomato soup found that stability was a significant issue, and the materials are not commercially available, Table 3 [84, 85]. A detailed electron microscopy study of these materials identified sampling, sample preparation for EM (including matrix removal) and image analysis as the main sources of uncertainty [86]. Another gap is the lack of materials for which the particle number concentration has been measured; this is an important metric for the identification of a nanomaterial in some jurisdictions (see Section 2.1). An initial step toward addressing this issue is provided by the recent release of a gold nanoparticle quality control material (LGCQC5050, Table 3 and Fig. 4) for which the particle number concentration has been measured by spICP-MS.

4.2 Advances in standardization

Number of national, regional, and international organizations are involved in developing standards for nanotechnology. Standard methods are important to ensure quality control and allow inter-laboratory comparability, for environmental and occupational health and safety purposes, and for material specifications for use by commercial producers. The ISO Technical Committee (TC) 229, Nanotechnologies has a mandate to pursue standardization that includes (1) understanding and controlling matter and processes at the nanoscale and (2) utilizing the novel properties of nanoscale materials to create improved materials, devices, and systems that exploit these new properties. There are currently five working groups: (1) terminology and nomenclature, (2) measurement and characterization, (3) health, safety, and environment, (4) material specifications, and (5) products and applications. The standards development process is based on reaching a consensus among member countries prior to finalizing a standard method and is meant to focus on areas that are relevant to industry. The ISO TC 229 maintains liaison relationships with number of other organizations including IUPAC, other ISO technical committees, ASTM, and CEN, ensuring that various nano-related standardization activities are complementary rather than duplicative.

The ISO TC 229 has published an extensive collection of standards that deal with the characterization of nanomaterials, mostly from the measurement and characterization working group. Table 4 lists those that provide general introductions to methods and sample preparation for nanomaterial characterization that apply to a broad range of materials. Standards that describe methods for a specific type of nanomaterial are summarized in Table 5. Many of the initial standards in this area focused on the characterization of carbon nanotubes for a range of physical properties as well as structural and compositional measurements using infrared and UV–Vis–IR spectroscopy, thermogravimetric analysis, and ICP-MS (Table 5). More recently published standards target other nanomaterials, including graphene and related 2D materials and cellulose nanomaterials. The graphene standards provide protocols for chemical and structural characterization using several methods and a standard method for analysis of surface groups (sulfate half esters) on cellulose nanocrystals using ICP-OES and conductometric titration has been published.

Standards that focus on specific physical or analytical characterization methods are summarized in Table 6. The latter includes two comprehensive international standards on SEM and TEM and technical specifications on spICP-MS, static multiple light scattering, field-flow fractionation, and ellipsometry. Note that the mandate of ISO TC 229 specifically excludes work on subjects that are covered by other ISO TCs. Therefore, topics directly related to instrumentation (e.g., general principles of a method, calibrations) are generally covered in other ISO TCs. Some nano-relevant methods, such as dynamic light scattering and zeta potential, are covered by ISO TC 24 (particle analysis) and ISO TC 201 provides standards on surface analysis, many of which are relevant for examining nanomaterials, and particularly surface coatings or modifications.

Standards for the physical and chemical characterization of nanomaterials are also developed by ASTM and OECD. Several relevant ASTM standard guides that cover measurement methods for particle size and surface charge are listed in Tables 5 and 6. The OECD Working Party on Manufactured Nanomaterials has carried out an extensive series of studies to assess whether their existing test guidelines and guidance documents are suitable for characterization and toxicological testing of nanomaterials. Since the release of the data approximately 5 years ago, they have embarked on a program to update documents where feasible and to develop new ones when necessary [87]. This includes developing new test guidelines on measuring particle size distribution, solubility and dissolution rate, and specific surface area and dustiness of dry nanomaterials, as well as guidance documents on surface chemistry and biodurability. Two general documents outlining guiding principles for measurement and reporting of physical chemical parameters and a decision framework for physical chemical studies to inform risk assessment have been recently published [88, 89].

The above summary indicates that many available standards focus on the measurement of the physical properties of the materials and are often suitable for pristine nanomaterials as produced, rather than those found in consumer products or in complex environmental or biological matrices. There are fewer standards that deal with compositional analysis and very few that cover the detection and characterization of nanomaterials in complex media. Two notable examples (Table 4) are a recent technical specification (ISO/TS 21361) which provides a method to identify and quantify air concentrations of carbon black and amorphous silica nanoparticles in a manufacturing environment using electron microscopy with energy dispersive spectrometry for elemental analysis after sampling with a cascade impactor and a technical report (ISO/TR 22293) that summarizes current methods for assessing release of nanomaterials from polymers. An ASTM guide with a tiered approach for detection and characterization of Ag nanoparticles in consumer products (textiles) and specific test methods for Ag analysis by ICP-OES and ICP-MS are also available (Table 4).

The second gap in available standards is the lack of standard methods for measuring particle number concentration, which as noted above is important for identifying nanomaterials in some jurisdictions. Note, however, that the release of the first quality control material (LGCQC5050, Table 2) and an ISO technical report (ISO/TR 24672, in progress, Table 3) are beginning to address this gap. Finally, a third gap is the lack of methods to assess aggregation and agglomeration, processes that occur frequently for many ENMs and which vary with the surrounding medium. Methods to assess these dynamic processes remain challenging to implement and are infrequently applied and as yet unstandardized. It should be noted that measurement of particle number concentration and assessment of agglomeration/aggregation changes as a function of time and environment are particularly important for assessment of applied dose for nanotoxicology experiments. A range of dose metrics is used, including mass, surface area, and particle number. Currently, there are no standardized methods to measure routinely the surface area in a relevant environment (e.g., liquid media), to measure particle number concentration or to assess agglomeration and aggregation; all these factors can impact on accurate assessments of the ENM dose delivered.

4.3 Collaborative/interlaboratory studies for assessment of methods and materials

Measurement methods that are deemed ready for standardization are typically assessed and validated by carrying out an interlaboratory comparison (ILC). An ILC is defined as the organization, performance, and evaluation of measurements or tests on the same (or a similar) sample by multiple laboratories in accordance with a predetermined protocol that all participants must follow [90, 91]. Usually, ILCs are designed to test the performance of new or existing measurement methods, but in some cases they may be used to test the performance of laboratories or to determine property values of the test material. In the first case, the ILC is designed to test method reproducibility and should use an appropriate material that is typical of those for which the method is generally used. The material should be homogeneous and stable, which means that a reference material is often used. Participating laboratories should have a demonstrated level of expertise but should not all be considered “experts” as that would result in an overestimation of the method’s reproducibility. The measurement protocol must be detailed and clear, similar to that required for the development of a standard. The ILC data provide the pre-normative demonstration of the suitability of the method for standardization. Note that prior to an ILC the method/protocols to be tested should be carefully optimized in one or more laboratories to provide a preliminary assessment of the necessary calibrations and the sources of uncertainty.

Validation of methods for characterization of physical properties, primarily particle size distributions, has been accomplished in number of ILC and round-robin studies. The earlier studies typically used silica, gold, or polystyrene nanoparticles that were spherical with monodisperse, or occasionally bimodal, size distributions. Combinations of dynamic light scattering, electron microscopy, atomic force microscopy, and nanoparticle tracking provided useful insight on the factors that require optimization to obtain reproducible results across laboratories and to minimize sources of uncertainty [92], [93], [94], [95]. Other ILCs measured zeta potential as a metric for surface charge [92, 93] and specific surface area [NIST SRM 1898]. Large multi-laboratory ILCs were also organized to provide certified and informational particle size data for several EC-JRC reference materials [96] and to compare methods for size measurements by microscopy and scattering methods [97].

More recently several large-scale projects have organized multi-laboratory comparisons to examine more complex nanomaterials with different shapes and a stronger propensity to agglomerate, including some commercial materials. For example, the EU NanoDefine project has published a manual with 23 Standard Operation Procedures that are aimed at facilitating and harmonizing sample preparation and particle size distribution measurements for a number of nanomaterials [98]. This project also compared the particle size distributions obtained using different methods and illustrated the differences that can be expected between methods, particularly for commercial materials; it was concluded that electron microscopy was likely to be the optimal sizing method for demonstrating compliance to a particle number based metric for identifying nanomaterials [47]. A recently published ISO standard (ISO 21363 [99]) for TEM was developed and validated using data from six ILCs for nanomaterials of different sizes and shapes that vary from simple spherical, monomodal gold nanoparticles to heavily aggregated nanoparticles such as carbon black and titanium dioxide. Another recent study compared size distribution measured for rod-shaped cellulose nanocrystals using two microscopy methods, AFM and TEM to support the development of ISO/TS 23151 (Table 4) [100, 101]. The combined ILC studies in support of these standards provide valuable information on particle analysis and data processing, both important factors in measuring reliable particle size distributions. The OECD has recently completed a large international ILC with over 30 participating laboratories that evaluated both microscopy and ensemble methods for particle sizing using 11 different nanofibers and nanoparticles. This study provides validation of a new test guideline for particle sizing for both nanoparticles and nanofibers [87].

Several ILCs have validated chemical analysis methods for nanomaterial characterization. Gold nanoparticles from NIST were used in a post hoc comparison to validate spICP-MS, a method that has the advantage of providing information on particle composition, size and number concentration in a single experiment [102]. The results compared well to the initial characterization of this reference material using other particle sizing methods (Fig. 5). The ability to obtain reliable data from this method is enabled by several careful studies demonstrating appropriate methods for calibration and for assessing the accuracy, precision, and robustness of the method [103, 104]. Such studies are necessary for complete validation of the method and its adoption by the broader research community, as exemplified by a study of commercial gold nanoparticles using spICP-MS [105].

Fig. 5:

Results of an ILC in which two gold nanoparticle reference materials were characterized by spICP-MS and the results compared to data obtained using other methods during the characterization of the reference material [102].

Recent ILCs have validated methods to quantify surface functional groups. A combination of conductometric titration and ICP-OES was used to quantify sulfate half ester groups on the surface of cellulose nanocrystals, achieving agreement between the two methods within the uncertainties of the measurement [106]. A VAMAS ILC to validate protocols for secondary ion mass spectrometry measurement of surface coating on titania nanoparticles is currently underway [107]. Additionally, the new VAMAS Technical Working Area 41 on graphene and related 2D materials has initiated several new projects aimed at testing protocols for elemental analysis using XPS, determination of metal impurities by ICP-MS, structural characterization by Raman spectroscopy, and chemical characterization by TGA [108].

A protocol for analytical ultracentrifugation that was intended to measure particle number concentration (volume and median diameters) to comply with EU regulations was validated in a bilateral comparison using trimodal silica mixture and two barium sulfate samples with different size ranges [109]. Reported uncertainties were below 12 %. Of particular interest are two recent studies, one from the International Bureau of Weights and Measures Consultative Committee on the Quantity of Material (BIPM CCQM-P194) [110] and the other one from VAMAS Project 10 (Technical Working Area 34 [111]). These studies compared for the first time the performance of methods for the determination of particle number concentration and used seven methods (spICP-MS, SEM, differential mobility analysis (DMA), UV–Vis, DLS, PTA, and SAXS) to quantify the number of 30 nm spherical gold nanoparticles in suspension for LGCQC5050 (before the release of the reference material). For CCQM-P194, the results from individual participants have not been made available to the public so far as this requires agreement from all participants but it is important to note that a good agreement between most metrology institutes was obtained within the window defined by the consensus value (1.420 × 10¹¹ g⁻¹) and its associated expanded uncertainty (0.277 × 10¹¹ g⁻¹, k = 2). It is interesting to note that the relative standard deviation of nine datasets obtained by spICP-MS was 13.4 %. For the VAMAS study, broadly, particle-by-particle methods were found to be intrinsically more accurate than the ensemble methods, but the latter had superior reproducibility [111].

Despite the availability of protocols that have been validated in multi-laboratory studies for physical and chemical properties of pristine nanomaterials, there are so far very few validated methods for characterization of nanomaterial properties in consumer products and in environmental or biological matrices. One early report provided a general framework for the validation of methods for detection and quantification of nanoparticles in food samples [112]. This framework outlines some general principles for validating the identity of the material and assessing particle size and mass and particle number concentration, taking into consideration the selectivity, detection limit, and precision of the method(s) employed. A 2015 study used ICP-MS to determine total titanium concentration in digested rat organs following ingestion of titanium dioxide nanoparticles [113]. Results were in reasonable agreement across four laboratories (relative standard deviations < 28 %) for digested samples that contained Ti > 4 μg/g tissue. More recently, an ILC on particle size analysis of pristine food-grade titanium dioxide in confectionary products by seven European food research laboratories has validated methods for number-based particle size distributions and particle number concentration using spICP-MS and TEM [114]. Given the difficulties involved in measuring nanomaterial concentration in complex media, there is interest in developing predictive models particularly for environmental samples; however, current analytical measurements do not yet allow validation of modeled environmental concentrations [115].