Characterizing nanoplastic suspensions of increasing complexity: inter-laboratory comparison of size measurements using dynamic light scattering

Environmental Science Nano PAPER Cite this: DOI: 10.1039/d5en00645g Received 16th July 2025, Accepted 19th September 2025 DOI: 10.1039/d5en00645g rsc.li/es-nano Characterizing nanoplastic suspensions of increasing complexity: inter-laboratory comparison of size measurements using dynamic light scattering Korinna Altmann, * a Raquel Portela, * b Francesco Barbero, c Esther Breuninger, d Laura Maria Azzurra Camassa, e Tanja Cirkovic Velickovic, f Costas Charitidis, g Anna Costa, h Marta Fadda, i Petra Fengler, a Ivana Fenoglio, c Andrea M. Giovannozzi, i Øyvind Pernell Haugen, e Panagiotis Kainourgios, g Frank von der Kammer, d Markus J. Kirchner, jk Madeleine Lomax-Vogt, d Tamara Lujic, f Frank Milczewski, a Mhamad Aly Moussawi, l Simona Ortelli, h Tatjana N. Parac-Vogt, l Annegret Potthoff, m Julian J. Jimenez Reinosa, n Sophie Röschter, m Alessio Sacco, i Lukas Wimmer, o Ilaria Zanoni h and Lea Ann Dailey o Understanding the potential human health risks associated with microand nanoplastic exposure is currently a priority research area. Nanoplastic toxicity studies are complicated by the lack of available, wellcharacterized test and reference materials. Further, many nanoplastic test materials are inherently more polydisperse and heterogenous in shape compared to polystyrene beads, making accurate and representative size distribution measurements particularly challenging. The aim of this study was to conduct an inter-laboratory comparison of dynamic light scattering measurements, the most commonly used particle sizing method for nanomaterials. Using a published standard operating procedure, size measurements in water and a standardized cell culture medium (CCM) were generated for spherical, carboxy-functionalized polystyrene nanoparticles (PS-COOH; 50 nm; benchmark material), and for increasingly complex in-house produced spherical poly(ethylene terephthalate) (nanoPET) and irregularEnviron. Sci.: NanoThis journal is © The Royal Society of Chemistry 2025 a Bundesanstalt für Materialforschung und -prüfung (BAM), Unter den Eichen 87, 12205 Berlin, Germany. E-mail: [email protected] b Instituto de Catalisis y Petroleoquimica (ICP), Materials Science Institute of Madrid (CSIC), –C/Marie Curie 2, Madrid, 28049, Spain. E-mail: [email protected] c Department of Chemistry, University of Torino (UNITO), Torino, Italy d Department of Environmental Geosciences (EDGE), University of Vienna, JosefHolaubek-Platz 2, 1090 Vienna, Austria e Norwegian Institute of Occupational Health (STAMI), 0363 Oslo, Norway f University of Belgrade Faculty of Chemistry (UBFC), Belgrade, Serbia g National and Technical University of Athens (NTUA), Athens, Greece h National Research Council of Italy - Institute of Science, Technology and Sustainability for Ceramics (CNR-ISSMC), Via Granarolo 64, 48018 Faenza, Italy, Via Granarolo 64, 48018 Faenza (RA), Italy i Quantum Metrology and Nano Technologies Division, Istituto Nazionale di Ricerca Metrologica (INRiM), Strada delle Cacce 91, 10135, Torino, Italy j University of Bayreuth, Animal Ecology I, Bayreuth Center of Ecology and Environmental Research (BayCEER), Universitätsstr. 30, 95447 Bayreuth, Germany k Department of Chemical and Product Safety, German Federal Institute for Risk Assessment (BfR), Max-Dohrn-Str. 8-10, 10589 Berlin, Germany l Department of Chemistry, KU Leuven, Celestijnenlaan 200F, 3001 Leuven, Belgium m Fraunhofer Institute for Ceramic Technologies and Systems (IKTS), Dresden, Germany n Materials Science Institute of Madrid (CSIC), Instituto de Ceramica y Vidrio (ICV), C/Kelsen, Madrid, 28049, Spain o Department of Pharmaceutical Sciences, University of Vienna, Josef-HolaubekPlatz 2, 1090 Vienna, Austria. E-mail: [email protected] Environmental significance Nanoplastic test materials with increased complexity regarding shape, surface chemistry and polydispersity are developed to mimic environmental nanoplastics. These materials are used to study eco-corona formation, biodistribution, and toxicity. Particle size is a key parameter and dynamic light scattering (DLS) is widely used for size analysis. Nanoplastic complexity is challenging for DLS, which calculates size based on the assumption of monodisperse, spherical particles. To evaluate how nanoplastic complexity influences DLS measurements, an inter-laboratory comparison was performed. Nanosized PET (spherical, polydisperse) and nanosized PP (irregular, polydisperse) showed a similar variability for measurements in water and cell culture medium compared to spherical monodisperse polystyrene beads. We conclude that nanoplastic complexity does not increase DLS variability if validated protocols are used. Open Access Article. Published on 14 October 2025. Downloaded on 10/27/2025 11:58:05 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online View Journal Environ. Sci.: Nano This journal is © The Royal Society of Chemistry 2025 shaped polypropylene (nanoPP) test materials. The weighted mean of hydrodynamic diameters of PSCOOH dispersed in water (55 ±5 nm) showed moderate variation between labs (coefficient of variation, CV = 8.2%) and were similar to literature reports. Measurements of nanoPET (82 ±6nm)andnanoPP(182 ±12 nm) in water exhibited similar CV values (nanoPET: 7.3% and nanoPP; 6.8%). Dispersion of PS-COOH and nanoPET in CCM increased the CV to 15.1 and 14.2%, respectively, which is lower than literature reports (CV = 30%). We conclude with a series of practical recommendations for robust size measurements of nanoplastics in both water and complex media highlighting that strict adherence to a standard operating procedure is required to prevent particle agglomeration in CCM. 1. Introduction Understanding the potential human health risks associated with microand nanoplastic (NP <1μm) 1 exposure is currently a priority research area. Particularly, exposure to nanosized plastic materials, which may be intentionally produced for industrial/consumer applications (primary nanoplastic, increasingly regulated) 2,3 or occur in the environment/consumer products as a result of microplastic breakdown (secondary nanoplastics) 2,4 is of high concern. Nanoplastics are especially relevant since smaller particulates may be able to access cellular compartments or cross mucosal barriers to a greater extent than larger plastics. 5 Nanoplastic in vitro toxicity studies are complicated, among other reasons, by the lack of available, well-characterized materials. 6–8 Since nanoplastics developed for research purposes should have characteristics reflective of environmentally relevant nanoplastics, the materials under current development are inherently more polydisperse and heterogenous in shape compared to spherical, monodisperse polystyrene (PS) beads. They can exhibit a different surface charge and variable hydrophobicity making their homogenous dispersibility challenging, particularly in complex media. 9 This is especially true for materials developed to mimic nanoplastics which have undergone environmental weathering. 10 A second challenge is that model nanoplastics are required for different intended uses, the two most common being: 1) for the calibration of analytical instruments and 2) for testing the behavior and effects of nanoplastics in both an environmental and physiological context. 11,12 While ideally the nanoplastics themselves should be identical in all applications, the nanoplastic products developed, which includes the choice of packaging (single unit or multi-unit containers), the concentrations provided, the presence of stabilizing additives, as well as the scope of testing and certification, will be very different depending on application. For example, a nanoplastic product developed as a standard for instrument calibration is typically provided at a concentration optimized for the instrument calibration process. Multiple handling steps, such as dilutions, are avoided to reduce sources of error. Additives, such as suspension stabilizers or preservatives, are acceptable if they do not influence the measurement. These products must also be rigorously tested for homogeneity and stability with respect to a specific property, in order to achieve reference material status (Table 1). 11,13–15 In contrast, nanoplastic products used for toxicology studies or to assess environmental disposition are ideally provided in concentrated form to allow for dosing flexibility. This necessitates additional handling steps, such as dilutions. Additives are frequently undesired, as they can cause artefacts in different assays. The suite of characterization methods required for these applications are typically quite different to those required for reference materials, often including detailed characterization of surface chemistry, product sterility, and/or endotoxin content. 11,12 Based on these distinctions, such nanoplastic products are mainly categorized as research grade test materials (Table 1). Since new nanoplastic materials are becoming increasingly available to the research community for a variety of applications, 6,8,10,12,17–19 questions have been raised as to the accuracy and reproducibility of size measurements using dynamic light scattering (DLS) for these more irregular, polydisperse materials. 20 To evaluate the precision and accuracy of DLS measurements on such nanoplastic dispersions, an inter-laboratory comparison (ILC) focused on nanoplastics was conducted. Two types of nanoplastic product formats were examined. The first was a research grade test material comprised of nanosized polyethylene terephthalate (nanoPET), which was provided in concentrated form requiring multiple dilution steps. Since the nanoPET product was designed for use in in vitro toxicity assays, an understanding of colloidal behavior and size stability in cell culture medium (CCM) was also investigated. 9,21–24 The second product comprised a nanosized poly propylene (nanoPP) dispersion. This product was designed for instrument calibration, in particular for size measurements using DLS, with the aim of applying for reference material status following completion of homogeneity and stability testing. Several key reports on the evaluation of DLS measurements for nanoplastics (in particular polystyrene) in both simple and complex media have been published. 21,25–27 Notably, Langevin et al. (2018) conducted an ILC investigating the accuracy and reproducibility of nanoparticle size measurements in biological media for two commonly used particle sizing methods, i.e. DLS and differential centrifugal sedimentation. 26 They recruited 40 labs to participate, although not all labs provided data in all rounds. They measured three types of well-characterized materials: 1) near-spherical silica nanoparticles (reported diameters: 19 nm and 100 nm), 2) spherical, carboxy-modified polystyrene Environmental Science: NanoPaper Open Access Article. Published on 14 October 2025. Downloaded on 10/27/2025 11:58:05 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online Environ. Sci.: NanoThis journal is © The Royal Society of Chemistry 2025 nanoparticles (PS-COOH; reported diameter: 50 nm) and 3) spherical, amine-modified polystyrene nanoparticles (reported diameter: 50 nm). All materials were measured first in water, then in CCM. In the first ILC round, each laboratory used their inhouse established procedures. Following this, a harmonized standard operating procedure (SOP) was developed by four expert laboratories and tested for robustness by eleven independent users in a second round (published in full in the SI). A bespoke SOP was developed for measurements in CCM and was tested by eight participating laboratories. The authors concluded that well-established and fit-for-purpose SOPs are indispensable for obtaining reliable and comparable particle size data, especially when measuring in complex media. Importantly, the SOPs must be optimized with respect to the intended measurement system (e.g. particle size technique, type of dispersant) and must be sufficiently detailed to avoid ambiguity. In subsequent studies, Takahashi et al. (2019) and Coones et al. (2025) addressed the question of how to relate particle sizes measured using a fixed-angle DLS instrument with those measured using a multi-angle DLS. 25,27 The focus of these more technical studies was to establish the functional dependence of the measured particle size on the scattering angle and particle concentration. However, since the aims and scope of the Langevin et al. (2018) study more closely matched the interests of the nanoplastic research community, we chose to adopt their study design and use their SOPs for the current study. To enable direct comparison of our results to published data, we chose to include the same spherical, monodisperse PS-COOH nanoparticles (nominal 50 nm diameter) as a benchmark material. Additionally, we evaluated two nanoplastic test materials (Fig. 1) produced by projects of the CUSP cluster funded by the Horizon2020 program of the European Commission (https://cusp-research.eu/) and the Metrology Partnership project 21GRD07 PlasticTrace (https://plastictrace.eu/). NanoPET test materials were produced via a bottom-up anti-solvent precipitation method with a final concentration of ∼6mgmL −1 . The preparation via precipitation yielded particles with a spherical morphology, moderate polydispersity and an electronegative surface charge with a zeta potential of −42 ± 2 mV (pH 4.75; MilliQ water, conductivity: 0.009 ± 0.0005 mS cm −1 ). NanoPP reference materials were prepared via a top-down approach using milling in chilled acetone to produce submicron-sized fragments at a more dilute concentration (0.04 mg mL −1 ). Since nanoPP materials were produced via mechanical breakdown, 6 the particles exhibited an irregular morphology (Fig. 1). In MilliQ water (pH 4.75; conductivity: 0.009 ± 0.0005 mS cm −1 ), the nanoPP also displayed an electronegative surface charge with a zeta potential of −43 ± 2mV. Since PS-COOH and nanoPET test materials were designed for multiple applications and therefore provided as highly concentrated suspensions, both systems had to be diluted prior to DLS measurement to a concentration of 0.1 mg mL −1 . NanoPP test materials, in contrast, were designed to be used as reference materials for instrument calibration purposes only. As stated above, it is advantageous in such applications to provide the material in a ready-to-use form which negates the need for additional handling steps. Thus, the nanoPP provided in this study had a low concentration of 0.04 mg mL −1 in water. Since it was not possible to dilute this material with CCM and remain in a measurable concentration range, nanoPP was only tested in water during this study. Firstly, we hypothesized that increased nanoplastic material complexity, especially regarding shape and polydispersity, would result in higher measurement variability when measuring in water. Secondly, it is known that dilution into complex media with high ionic strength, such as serum-supplemented CCM, may influence the particle size distribution determined by DLS 9 and increase variability. Therefore, a second aim of this study was to establish simple quality criteria for DLS measurements of more complex nanoplastic samples, which can be easily adopted and understood by user groups without expertlevel knowledge of DLS. Since particle size and size distribution are crucial parameters in toxicity studies, this ILC provides recommendations to harmonize size Table 1 National Institute of Standards and Technology (NIST) and International Organization for Standardization (ISO) categories and definitions of materials 13–16 Category Definition Research grade test material Exploratory materials developed for current research needs, which are subject to continuous stability measurements. The extent of characterization depends on the needs of the user community and is therefore not standardized Reference material A material, homogeneous and stable with respect to one or more specified property values, which has been established to be for its intended use in a measurement process Certified reference material A material characterized by a metrologically valid procedure for one or more specified properties, accompanied by a reference material certificate that provides the value of the specified property, its associated uncertainty, and a statement of metrological traceability NIST standard reference material® A certified reference material issued by NIST that also meets additional NIST-specific certification criteria and is issued with a certificate or certificate of analysis that reports the results of its characterizations and provides information regarding the appropriate use(s) of the material Environmental Science: Nano Paper Open Access Article. Published on 14 October 2025. Downloaded on 10/27/2025 11:58:05 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online Environ. Sci.: Nano This journal is © The Royal Society of Chemistry 2025 characterization measurements across the nanoplastic research community. 2. Materials and methods 2.1. Materials Aqueous dispersions of polystyrene beads with carboxyl surface functionalization (PS-COOH) were purchased from Polysciences, Inc. (catalog number 15913-10). Two lots were used in the study: 1) lot#A844160 used in labs #3, 8, 10, and 11 contains 2.8% solids (w/v) with a reported diameter of 49 nm and a coefficient of variation (CV) of 14%; 2) lot #A844160 used in labs #1, 11 and 15 contains 2.7% solids with a reported diameter of 49 nm and a CV of 14%. Polypropylene granules were kindly provided by PlasticsEurope (Moplen RP320M, LyondellBasell). NanoPET was produced from RAMAPET N180 granules. MilliQ water had electrical resistivity <18.2 MΩcm. Complete cell culture medium (CCM) typically contained modified Eagle medium (MEM), Glutamax provided by different suppliers (Gibco: #41090-028; VWR: #VWRMS024F; Corning: #10-009-CV); 10% fetal bovine serum (FBS;, non-heat inactivated from Gibco: #P220303; Avantor; Biowest: #1860; Sigma: # 1670543; Capricorn Scientific: LOT CP20-3579; Biological Industries: LOT 1348500; ATCC: 80715235) and 100 U mL −1 penicillin/ 100 μgmL −1 streptomycin (Invitrogen:15070063). Labs 8, 12, 13, 14 substituted Dulbecco's modified Eagle medium (DMEM; Gibco: 2858875) for MEM. 2.2. Production of nanoPET In a fume hood, following the method described by, 29 1g PET (pellets) was dissolved in 100 mL hexafluoroisopropanol (HFIP) at room temperature and injected at controlled velocity into 1 L of ice-cooled, sterile MilliQ water. Excess water and HFIP were removed via rotary evaporation (−70 °C, −86 kPa for a minimum of 2 h) until a desired final concentration of ∼6mgmL −1 was achieved. The internal code was PET_c003. 2.3. Production of nanoPP NanoPP test materials were prepared according to a method described by Hildenbrandt el al. 6 Briefly, polypropylene granules (6 25 g) were added to a 400 mL glass beaker containing acetone (250 mL) chilled to 0 °C. The polymer granules were milled with an IKA T 18 digital ULTRATURRAX for 10 min at a rotation speed of 18 000 rpm. The suspension was then filtered using a folded filter to remove the larger aggregates. Acetone in the filtrate was evaporated until ca. 10% of the liquid remained, then MilliQ water (250 mL) was added to the mixture, the remaining acetone was removed with a rotary evaporator and the suspension filtered using a folded filter. The nanoPP is provided as a MilliQ water suspension without any further stabilizing additives and a concentration of 0.04 mg mL −1 . 2.4. Scanning electron microscopy and size analysis NanoPET and nanoPP suspensions were dropcast onto a glass slide, fixed onto a sample holder with double-sided conductive adhesive tape and dried overnight. On the following day, the samples were gold-sputtered and imaged with an EVO MA 10 (Zeiss, Oberkochen, Germany) using secondary electron contrast mode with voltages of 10 kV. Particle size distributions were generated from the SEM images by measuring the Feret diameter of the spherical nanoPET particles (n= 100) and the minimum and maximum Feret diameter for the irregular nanoPP particles (n= 84) following recommendations provided by Bresch et al. 30 Aspect ratios for were calculated as the ratio between the maximum:minimum Feret diameter. The PS-COOH distribution was calculated from the reported diameter and CV% provided by the manufacturer, while the image is provided by the manufacturer. 2.5. Preparation of dispersions in water and CCM PS-COOH and nanoPET were assessed by preparing three separate dilutions (three experimental replicates) and Fig. 1 Description and representative scanning electron micrograph (SEM) images of the three test materials used in the study: PS-COOH, nanoPET and nanoPP. Note: The image of the polystyrene beads is a representative image from the manufacturer website 28 and is not provided at scale. Environmental Science: NanoPaper Open Access Article. Published on 14 October 2025. Downloaded on 10/27/2025 11:58:05 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online Environ. Sci.: NanoThis journal is © The Royal Society of Chemistry 2025 measuring each sample three times (three technical replicates). MilliQ water and CCM were filtered (0.2 μm pore diameter) prior to use to remove ambient particulates. Stock suspensions (1 mg mL −1 ) of PS-COOH and nanoPET were prepared with the filtered MilliQ water and mixed for 30 s using a benchtop vortex at full speed. Immediately, 100 μL stock suspension was added to either 900 μL water or CCM (final sample concentration: 0.1 mg mL −1 ) in separate sterile 1.5 mL polypropylene tubes and further vortexed for a further 30 sec at full speed. Samples were then measured immediately. It should be noted that each lab sourced their own CCM components, resulting in slight variations between the providers of medium and FBS (see section 2.1). Labs 8, 12, 13 and 14 also substituted DMEM for MEM in their experiments. The nanoPP samples were provided as an aqueous dispersion at a concentration of 0.04 mg mL −1 and were therefore measured as received without dilution. Prior to measurement, samples were briefly vortexed for 30 s at full speed and measured immediately (n= 3 separate aliquots with n= 3 technical replicates each). 2.6. DLS measurements Variability due to handling and dilution was assessed by preparing three separate sample dilutions and measuring each sample three times (n= 9 total). Within one measurement sequence, the DLS instrument performs 3–10 measurements for each sample, which correspond to one technical replicate. The DLS instruments listed in Table 2 were used in the study. Quartz or high-quality optical glass cuvettes were recommended, but good quality plastic cuvettes were also included in the study parameters. All cuvettes, but in particular the more scratch-prone plastic cuvettes, were routinely inspected prior to use and discarded if surface scratches or defects were visible. Clean cuvettes were prerinsed with filtered MilliQ water at least three times prior to sample loading (preferably in a high efficiency particulate air-filtered clean bench if available). The required volume of NP dispersion was filled into the DLS cuvette using the minimum volume necessary to ensure that the liquid level was at least 2 mm above the entrance height of the laser beam. Overfilling was avoided to prevent thermal gradients that adversely impact measurement accuracy. Cuvettes were visually inspected to ensure that air bubbles were not present within the optical window area prior to insertion into the instrument. Measurements were conducted at temperatures close to ambient room temperature, ideally between 23–25 °C. Diluent viscosity values and refractive indices for all materials can be found in Table 3. The Z-average (Z-Ave) value of the nine DLS measurements is derived from the cumulants approach for calculating the average size of a distribution of particles based on analysis of the linear form of the measured correlogram (scattered light intensity-weighted harmonic mean hydrodynamic diameter). The analysis assumes that the particles belong to a single population which follows a Gaussian distribution. The polydispersity index (PDI) is the relative variance of the hypothetical Gaussian distribution. 32–34 Representative examples of particle size distribution curves and fitted correlograms are provided in the (SI) Fig. S1 and S2. No evidence of particle sedimentation or flotation was reported during the time course of all measurements. Files with all raw data are available in Zenodo: https://doi.org/10.5281/ zenodo.17105630. 2.7. Statistical analysis The DLS results were analyzed using the arithmetic mean (x i ) and standard deviation (SD i ) for individual datasets from each laboratory. Samples with polymodal size distributions, indicative of particle agglomeration, were excluded from the statistical analysis (example SI Fig. S3). Global means from the ILC were calculated using both a weighted and nonweighted approach. Non-weighted averages represent the Table 2 Instruments used in the ILC study, including information on the detection angle and operator experience level in years Lab Instrument Detection angle°Operator experience (years) 1 NanoZS (Malvern Panalytical) 173°backscatter 2 2 Nano ZSP (Malvern Panalytical) 173°backscatter 3 3 Zetasizer Pro Blue Light Scattering System (ZSU3200; Malvern Panalytical) Not provided 4 4 ZetaSizer Ultra (Malvern Panalytical) 173°backscatter 7 5 NanoZS ZEN 3600 (Malvern Panalytical) 173°backscatter 10 6 NanoZS (Malvern Panalytical) 173°backscatter 2 7 NanoZSP (Malvern Panalytical) 173°backscatter 10 8 NanoZS (Malvern Panalytical) 173°backscatter 4 9 NanoZS ZEN 3600 (Malvern Panalytical) 173°backscatter 2 10 NanoZS (Malvern Panalytical) 173°backscatter >10 11 NanoZS (Malvern Panalytical) 173°backscatter >25 12 NanoZS (Malvern Panalytical) 173°backscatter 12 13 NanoZS (Malvern Panalytical) 173°backscatter 1 14 Litesizer500 (Anton Paar) 175°backscatter 1 15 Litesizer500 (Anton Paar) 90°and 175°backscatter >10 16 NanoPlus-3 (Micromeritics) 160°(automatic) 1 Environmental Science: Nano Paper Open Access Article. Published on 14 October 2025. Downloaded on 10/27/2025 11:58:05 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online Environ. Sci.: Nano This journal is © The Royal Society of Chemistry 2025 arithmetic mean (x nw ) and standard deviation (SD nw ) from the x i values of all partners. The weighted mean (eqn (1)) was defined as: xw¼ P n i¼1 wixi P n i¼1 wi (1) whereby, w i = 1/(SD i ) 2 and x i is the arithmetic mean for individual laboratory measurements. The weighted mean reduces the influence of data sets with high dispersion values on the final average value. It is considered to be more robust compared to the non-weighted arithmetic mean because the contribution of highly scattered data is reduced. The SD and coefficients of variation (CV) were calculated using both non-weighted and weighted means for comparison. The CV is defined as the ratio between the SD and the corresponding mean multiplied by 100. According to ISO/IEC 17043:2010, 35 the measurements of ILC contributor laboratories are acceptable if x i ±SD i falls within the range of x w ± 2SD w . Thus, all black lines in figures represent x w while dashed lines represent ± 2SD w and define the consensus interval in which the results are expected to fall assuming a confidence level of 95%. 3. Results and discussion 3.1. Size analysis from SEM images All analytical techniques for measuring particle size have inherent strengths and weaknesses, depending on the principle underlying the measurement. They can also provide different types of information, such as size metrics (i.e. equivalent diameters) or size and shape metrics. 35,36 It is therefore recommended that a combination of direct sizing methods (image-derived) and indirect methods (including but not limited to small angle X-ray scattering, DLS, laser diffraction, nanoparticle tracking analysis, analytical centrifugation, field flow fractionation combined with light scattering methods, acoustic spectroscopy, tunable resistive pulse sensing) is performed. 35,36 Manufacturers of nanoplastic test materials typically have access to a wider range of these analytical techniques and will employ multiple sizing methods during the material characterization phase. Material users, in contrast, typically only have access to a limited range of sizing equipment and rely heavily on DLS measurements for size characterization. This is why DLS benchmarking studies between material manufacturers and users is highly valuable. SEM images of nanoPET and nanoPP (Fig. 1) were used to assess the Feret diameter distributions and particle aspect ratios of the nanoPET and nanoPP samples (Fig. 2). Size distribution data of the PS-COOH samples was provided in the material data sheet of the commercial product. The analysis verifies the hypothesis that particle complexity increases in terms of polydispersity and shape irregularity over the series of nanoplastics tested (complexity: PS-COOH <nanoPET <nanoPP). 3.2. DLS evaluation of PS-COOH and benchmarking to the literature 3.2.1. Measurement of PS-COOH in water. Eleven labs independently acquired the PS-COOH benchmark material and provided intensity-based Z-average (Z-Ave) measurements in water. Ten of eleven lab datasets (x i ± 1SD i ) were within the consensus range of x w ± 2SD w (Fig. 3). Both the weighted and non-weighted mean hydrodynamic diameters (55 and 57 nm, respectively) were similar to the first round of the ILC reported by Langevin et al. (2018) but larger than the sizes measured in the second round (Table 4), where the same SOP was used. 26 As expected for the PS-COOH, the PDI values were close to 0.05 (Fig. 3) indicating a uniform monodisperse sample in all cases except for two labs. According to guidelines, the PDI is a dimensionless and scaled value, calculated from a two-parameter fit to the correlation data (the cumulants analysis). PDI values can be interpreted as follows: <0.05 = uniform monodisperse, 0.05–0.1 = narrow monodisperse, 0.1–0.4 = moderately polydisperse, 0.4–0.7 = broadly polydisperse, and >0.7 = too polydisperse for DLS measurement. 32,36 3.2.2. Measurement of PS-COOH in CCM. Eight of the eleven labs provided measurements of PS-COOH in CCM; however, three of the eight datasets reported sample agglomeration (example SI Fig. S3) and were excluded from the statistical analysis. Variations in sample handling were identified as the cause of agglomeration and are discussed in detail in sections 3.5 and 3.6. The remaining five monomodal datasets (example SI Fig. S2) were within the consensus range (Fig. 3). The global weighted mean x w increased from 55 nm in water to 60 nm in CCM, with a low CV w of 5.3%, which was substantially lower than the 30% reported by Langevin et al., 2018. This may be influenced by the low sample number in the current study as well as the exclusion of agglomerated samples. The PDI of monomodal PS-COOH dispersions in CCM clustered around ∼0.2 (moderately polydisperse; Fig. 3). Interestingly, the CCM composition (MEM vs. DMEM) did not appear to influence the size and Table 3 Values used for instrument settings. Viscosity values were taken from 26 Viscosity (mPa s) Refractive index Water 23 °C 0.932 1.330 a Water 24 °C 0.910 Water 25 °C 0.890 Cell culture medium 1.090 1.335 a PS-COOH —1.590 b nanoPP —1.490 b nanoPET —1.569 b a Suitable for wavelengths between 488–750 nm within the temperature range of 20 °Cto25°C. b Suitable for wavelengths between 400 nm –2μm within the temperature range of 20 °Cto25 °C. 31 Environmental Science: NanoPaper Open Access Article. Published on 14 October 2025. Downloaded on 10/27/2025 11:58:05 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online Environ. Sci.: NanoThis journal is © The Royal Society of Chemistry 2025 PDI, although the limited sample number prevents conclusive interpretations. Overall, results from this smallscale benchmarking study showed that measurements of uniform, monodisperse PS-COOH test materials in water were in the expected size range with a low variability between labs. Measurements in CCM were generally comparable to the results from Langevin et al., 2018. This observation reinforces previous literature discussions which highlight that the dispersion of nanomaterials in complex media can negatively impact colloidal stability and therefore requires strict adherence to validated SOPs for robust DLS size measurements. 9,26 3.3. DLS evaluation of nanoPET and nanoPP 3.3.1. Measurement of nanoPET in water. All sixteen labs had access to the same batches of nanoPET and nanoPP. As seen in the SEM image in Fig. 1, the nanoPET preparation method produced nearly spherical particles with an expected size of ∼80 nm and a narrow but not uniform distribution. Fig. 2 SEM-derived particle size distributions were generated from the Feret diameters of the spherical nanoPET particles (B; n= 100) and the minimum and maximum Feret diameters for the irregular nanoPP particles (C; n=84). 30 The PS-COOH distribution (A) was calculated from the reported diameter and CV% provided by the manufacturer. Fig. 3 Scattered light intensity-based harmonic mean hydrodynamic diameter (Z-Ave, left) and PDI values (right). PS-COOH measurements in water (top) are compared to CCM (bottom). Values for each lab depict the x i ±1SD i (n= 9). Solid red lines depict the reported diameter provided by the manufacturer, black lines show the weighted global mean x w , and dashed lines correspond to ±2SD w . Lab numbers without asterisks denote Malvern Pananalytic devices, *Anton Paar, **MicroMeritics. Environmental Science: Nano Paper Open Access Article. Published on 14 October 2025. Downloaded on 10/27/2025 11:58:05 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online Environ. Sci.: Nano This journal is © The Royal Society of Chemistry 2025 When diluted in water to the test concentration (0.1 mg mL −1 ), all size distribution curves were monomodal; however, two datasets were outside the consensus range (Fig. 4). The measurement variability was hypothesized to increase for nanoPET, due to the different production method (nanoprecipitation for nanoPET compared to emulsification polymerization for PS-COOH). However, the CV w of nanoPET (7.3%; Table 5) was slightly lower than the corresponding value for PS-COOH (8.2%). The PDI values cluster around 0.1 (Fig. 4), indicating that the material is on the border between narrowly monodisperse and moderately polydisperse. 3.3.2. Measurement of nanoPET in CCM. Nine datasets for measurements of nanoPET in CCM were submitted (Fig. 4). All datasets were monomodal except for labs #10 and 15, which reported particle size distribution curves containing a second minor peak at 5–10 nm (example SI Fig. S4), typical of proteins/protein aggregates in CCM. 37 The presence of this minor peak was sufficient to shift the Z-Ave values for these samples to lower diameters, compared to samples without Table 4 Summary of statistical analysis of PS-COOH hydrodynamic diameter values following dispersion in water and CCM reported in the current study and by Langevin et al. (2018). The reported diameter from the manufacturer was 49 nm. Mean, SD and CV values were calculated from the nonweighted and weighted global means Water CCM Current study b Langevin et al. ILC #1 a Langevin et al. ILC #2 b Current study b Langevin et al. ILC #3 b Nonweighted global mean x nw (nm) 57 n.d. n.d. 62 n.d. 1SD nw (nm) 4 n.d. n.d. 3 n.d. CV nw (%) 6.4 n.d. n.d. 4.7 n.d. Weighted global mean x w (nm) 55 55 46 60 50 1SD w (nm) 5 3 2 3 15 CV w (%) 8.2 5.5 4.4 5.3 30.0 n87 162 199 45 72 a Denotes no common SOP. b Denotes use of the same SOP. n.d. = not determined. Fig. 4 Scattered light intensity-based harmonic mean hydrodynamic diameter (Z-Ave, left) and PDI values (right). nanoPET measurements in water (top) are compared to CCM (bottom). Values for each lab depict the x i ±1SD i (n= 9). Black lines depict the weighted global mean x w , and dashed lines correspond to ±2SD w . Lab numbers without asterisks denote Malvern Pananalytic devices, *Anton Paar, **MicroMeritics. Environmental Science: NanoPaper Open Access Article. Published on 14 October 2025. Downloaded on 10/27/2025 11:58:05 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online Environ. Sci.: NanoThis journal is © The Royal Society of Chemistry 2025 the second peak (Fig. 4). This is a recognized phenomenon explored by Balog et al. (2015). To reduce the background signal arising from a biological matrix, they employed technique known as depolarized DLS, which may be useful for measuring certain types of nanomaterials which exhibit sufficient optical anisotropy. 37 Since this approach has not been explored for nanoplastics, we recommend a simpler solution. For example, in samples where a serum peak is present, it is possible to substitute the Z-Ave value with the mode of the highest intensity peak. This approach (example #2 in Table 5) reduces the differences between the global means measured in water and CCM and decreases the overall data variation. The PDI values range between 0.1–0.4 (Fig. 4), although it should be noted that labs reported serum peaks were included in the figure. Again, the CCM composition (MEM vs. DMEM) did not appear to influence the size and PDI, although the limited sample number requires further investigation. 3.3.3. Measurement of nanoPP in water. As seen in the SEM image in Fig. 1, the nanoPP preparation method produced irregular particles with an aspect ratio of ∼1.5, an expected size centered around 200 nm and a polydisperse distribution (Fig. 2). The top-down method of preparation, i.e., wet-milling of larger plastics to smaller sizes, is often associated with low yields in the submicron size range and is the primary reason why this material is not provided as a concentrated suspension like PS-COOH and nanoPET. Since this material is designed for use as a reference material for instrument calibration, the lower concentration (0.04 mg mL −1 ) can have benefits since the product does not require additional handling steps like dilution. Datasets from two of sixteen labs were outside the consensus range (Fig. 5 and Table 5), but otherwise there was a relatively low variability with a CV of 6.8%. The PDI values of cluster around 0.1 (Fig. 5), indicating that the material is on the border between narrowly monodisperse and moderately polydisperse. This observation is of particular interest, since it demonstrates that nanoplastic dispersions with irregular shapes and a moderate aspect ratio, can still be measured with suitable reliability using DLS. However, it should be highlighted that despite their higher polydispersity and irregular shape, the nanoPP material is still a model nanoplastic and therefore exhibits a greater degree of homogeneity than nanoplastics extracted from the environment. DLS users should be aware that if environmentally derived nanoplastic samples show too high a polydispersity, DLS is likely not a suitable method for size characterization. 3.4. Colloidal stability of nanoplastics in water Nanoplastic suspensions can be classified as colloidal dispersions, where the solid particles are typically in the size Table 5 Statistical analysis of the DLS data from the nanoPET and nanoPP test materials. Two analyses were performed for the nanoPET dispersion in CCM: #1 standard analysis with Z-Ave values and #2 alternative analysis with mode values from labs #10, and 15 nanoPET (water) nanoPET (CCM) #1 nanoPET (CCM) #2 nanoPP (water) Nonweighted global mean x nw (nm) 83 78 80 187 1SD nw (nm) 6 9 8 13 CV nw (%) 7.6 11.2 9.5 7.1 Weighted global mean x w (nm) 82 75 75 182 1SD w (nm) 6 11 12 12 CV w (%) 7.3 14.2 16.5 6.8 n132 81 65 138 Fig. 5 Scattered light intensity-based harmonic mean hydrodynamic diameter (Z-Ave, left) and PDI values (right). nanoPP size measurements were performed with undiluted samples in water. Black lines depict the weighted global mean x w , and dashed lines correspond to ±2SD w . Values for each lab depict the x i ±1SD i (n= 9). Lab numbers without asterisks denote Malvern Pananalytic devices, *Anton Paar, **MicroMeritics. Environmental Science: Nano Paper Open Access Article. Published on 14 October 2025. Downloaded on 10/27/2025 11:58:05 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online