Following the EU definition of nanomaterials as being materials in which more than 50% by number of their primary particles have at least one dimension smaller than 100 nm , the accurate measurement of particle size distributions (PSDs) has become critical. Among the methods used to determine particle size distributions, the evaluation of particle sizes from electron microscopy (EM) images is considered as a confirmatory method for correct classification . In the case of monomodal particle distributions, this method is straightforward . However, when the PSD is broad, the sample preparation plays a crucial role in obtaining reproducible and unbiased results .

The standard approach to measuring particle size by EM is to thoroughly disperse the particles and then to evaluate them using an automatic or semi-automatic procedure. This method is effective for loosely bound monodisperse particles such as polystyrene latex or gold particles. However, it can be challenging when dealing with highly agglomerated and cohesive particles such as TiO2, especially for laboratories without prior experience. The authors have chosen to use “E171” as an abbreviation for the former “European food pigment E171”. The use of TiO2 E171 for food application was banned by the European Commission in January 2022 based on a European Food Safety Authority (EFSA) evaluation that a “concern for genotoxicity of TiO2 particles that may be present in E 171 could not be ruled out” . Recently, papers have been published in which a number of E171 samples were found by transmission electron microscopy (TEM) to be nanomaterials according to the EU definition . For TiO2 producers this is surprising as the former food-grade titanium dioxide E171 is manufactured as a standard white pigment grade, which would be significantly inferior if more than 50% of its particles were below 100 nm in diameter. In other papers, samples of the same materials were measured using scanning electron microscopy (SEM) and found not to meet the EU classification for nanomaterials . In 2018, KRONOS INT. Inc., Precheza a.s and Venator supported EFSA in responding to the questions raised by the EFSA Scientific Panel on the particle size distribution of E171. The results were reported by EFSA in 2019 . The same samples as measured in and were used in a round-robin test in 2023 by three laboratories (KRONOS INT. Inc., Precheza a.s, and Venator). The results are summarised in this publication. Each laboratory followed its own routine procedure to prepare samples, make SEM images, and measure several particle size parameters including the smallest dimension (MinFeret), largest dimension (MaxFeret), and equivalent circular diameter (ECD). Results from the “Regional Centre of Advanced Technologies and Materials”, Olomouc, Czech Republic (RCPTM) were also included.

It is good practice to compare the PSD estimated by EM with other methods for determination and validation. For untreated, non-porous materials, the specific surface area (SSA) serves as a useful independent method. The SSA of the pigment samples was calculated from the particle size distributions, and these values were directly compared with the measured SSA. As with all titanium dioxide materials, the optical properties of E171 are related to the primary particle size, although the degree of dispersion needs to be taken into account .

An overview of particle size measurement methods for nanomaterials is given in . Our paper focuses mainly on number-based particle size measurements for E171 using EM investigations and the comparison with optical properties, SSA, and single-particle inductively coupled mass spectrometry (spICP-MS) to accompany the reported EM results. An overview covering industrially applied measurement methods for pigments and fillers is given in the JRC Technical Report ; EM particle size measurements are covered in OECD guidelines as well as in ISO standards [ISO/TS 19749 (published 07/21) and ISO/TS 21363:2020 (published 11/21)].

The key to any visual EM particle sizing method is to take random images of the sample, to evaluate all measurable particles on each image, and to correctly identify the particle edges. Effective image acquisition and analysis requires training and routine performance checks for both the personnel capturing the images and those interpreting them.

Table 1: Probability that a particle of a given size will fall completely within the 1800 nm × 1250 nm field of view.

As such, any microscopic measurement is biased towards smaller particles because of simple geometric constraints; the higher the resolution and the smaller the field of view, the more pronounced this becomes.

For EM measurements, all five samples were prepared and measured by the participating companies according to their own standard operating procedures as described in the Experimental section. The evaluation procedures and sample preparations used here have been developed over many years and are regularly used in each participating company for quality control and research purposes.

Table 2: Results from three different electron microscopy particle size measurement methods of five anatase E171 samples.

aSE standard error of the median based on .

Figure 1: Comparison of cumulative distribution curves measured by three different manufacturers using three different methods (M1: cross section, M2: dry, and M3: sonicated dispersion) and by the external laboratory RCPTM.

Single-particle inductively coupled plasma mass spectrometry (spICP-MS) measurements are often used to complement EM particle counting results for particulate products with a substantial particle content below 100 nm . Therefore, spICP-MS experiments are also included here for comparison. The experiments were performed on an Agilent 8900 triple quadrupole system at Agilent’s application laboratory in Waldbronn, Germany, and the particle counting threshold was set at 30 nm. The samples were first intensively dispersed by ultrasonication in a polyphosphate solution in water (562 J/mL, 2.5% pigment, 2% polyphosphate) one day before measurement. The energy of 562 J/mL was chosen because aggregates and primary particles start to be destroyed above 250 J/mL, but only at low rates . However, dispersion energies of approximately 600 J/mL or higher may significantly break aggregates into primary particles, causing a shift in the particle size distribution towards a smaller median size (D50n) due to the detection of a higher number of liberated primary particles.

After dispersion, the samples were transported to the Agilent laboratory. The following day, they were re-dispersed in two different ways just before measurement. This was done to evaluate the impact of sample preparation on the results and confirm the method’s suitability for TiO2 pigment analysis. One sample was manually agitated, while the other underwent a 1 min dispersion in an 80 W ultrasonic bath.

Figure 2: spICP-MS of pre-dispersed (562 J/mL ultrasonic treatment prepared one day before measurement) and stabilised (2% polyphosphate) suspension. Left: intensive re-dispersion for 1 min in an ultrasonic bath at 80 W, right: light re-dispersion through shaking by hand. Both particle size distributions show two maxima, one for primary particles and one for aggregates and agglomerates. Re-dispersion destroys agglomerates that formed overnight and increases the number of primary particles.

Table 3: spICP-MS results for samples A–E.

Table 4: Comparison of optical properties, specific surface area, spICP-MS, and particle size measured by EM for additional laboratory samples and E171 pigments A–E.