<i>Post hoc</i> Interlaboratory Comparison of Single Particle ICP-MS Size Measurements of NIST Gold Nanoparticle Reference Materials

Single particle inductively coupled plasma-mass spectrometry (spICP-MS) is an emerging technique that enables simultaneous measurement of nanoparticle size and number quantification of metal-containing nanoparticles at realistic environmental exposure concentrations. Such measurements are needed to understand the potential environmental and human health risks of nanoparticles. Before spICP-MS can be considered a mature methodology, additional work is needed to standardize this technique including an assessment of the reliability and variability of size distribution measurements and the transferability of the technique among laboratories. This paper presents the first <i>post hoc</i> interlaboratory comparison study of the spICP-MS technique. Measurement results provided by six expert laboratories for two National Institute of Standards and Technology (NIST) gold nanoparticle reference materials (RM 8012 and RM 8013) were employed. The general agreement in particle size between spICP-MS measurements and measurements by six reference techniques demonstrates the reliability of spICP-MS and validates its sizing capability. However, the precision of the spICP-MS measurement was better for the larger 60 nm gold nanoparticles and evaluation of spICP-MS precision indicates substantial variability among laboratories, with lower variability between operators within laboratories. Global particle number concentration and Au mass concentration recovery were quantitative for RM 8013 but significantly lower and with a greater variability for RM 8012. Statistical analysis did not suggest an optimal dwell time, because this parameter did not significantly affect either the measured mean particle size or the ability to count nanoparticles. Finally, the spICP-MS data were often best fit with several single non-Gaussian distributions or mixtures of Gaussian distributions, rather than the more frequently used normal or log-normal distributions

Agglomeration of <i>Escherichia coli</i> with Positively Charged Nanoparticles Can Lead to Artifacts in a Standard <i>Caenorhabditis elegans</i> Toxicity Assay

The increased use and incorporation of engineered nanoparticles (ENPs) in consumer products requires a robust assessment of their potential environmental implications. However, a lack of standardized methods for nanotoxicity testing has yielded results that are sometimes contradictory. Standard ecotoxicity assays may work appropriately for some ENPs with minimal modification but produce artifactual results for others. Therefore, understanding the robustness of assays for a range of ENPs is critical. In this study, we evaluated the performance of a standard <i>Caenorhabditis elegans</i> (<i>C. elegans</i>) toxicity assay containing an <i>Escherichia coli</i> (<i>E. coli</i>) food supply with silicon, polystyrene, and gold ENPs with different charged coatings and sizes. Of all the ENPs tested, only those with a positively charged coating caused growth inhibition. However, the positively charged ENPs were observed to heteroagglomerate with <i>E. coli</i> cells, suggesting that the ENPs impacted the ability of nematodes to feed, leading to a false positive toxic effect on <i>C. elegans</i> growth and reproduction. When the ENPs were tested in two alternate <i>C. elegans</i> assays that did not contain <i>E. coli</i>, we found greatly reduced toxicity of ENPs. This study illustrates a key unexpected artifact that may occur during nanotoxicity assays

Agglomeration of <i>Escherichia coli</i> with Positively Charged Nanoparticles Can Lead to Artifacts in a Standard <i>Caenorhabditis elegans</i> Toxicity Assay

The increased use and incorporation of engineered nanoparticles (ENPs) in consumer products requires a robust assessment of their potential environmental implications. However, a lack of standardized methods for nanotoxicity testing has yielded results that are sometimes contradictory. Standard ecotoxicity assays may work appropriately for some ENPs with minimal modification but produce artifactual results for others. Therefore, understanding the robustness of assays for a range of ENPs is critical. In this study, we evaluated the performance of a standard <i>Caenorhabditis elegans</i> (<i>C. elegans</i>) toxicity assay containing an <i>Escherichia coli</i> (<i>E. coli</i>) food supply with silicon, polystyrene, and gold ENPs with different charged coatings and sizes. Of all the ENPs tested, only those with a positively charged coating caused growth inhibition. However, the positively charged ENPs were observed to heteroagglomerate with <i>E. coli</i> cells, suggesting that the ENPs impacted the ability of nematodes to feed, leading to a false positive toxic effect on <i>C. elegans</i> growth and reproduction. When the ENPs were tested in two alternate <i>C. elegans</i> assays that did not contain <i>E. coli</i>, we found greatly reduced toxicity of ENPs. This study illustrates a key unexpected artifact that may occur during nanotoxicity assays

Separation, Sizing, and Quantitation of Engineered Nanoparticles in an Organism Model Using Inductively Coupled Plasma Mass Spectrometry and Image Analysis

For environmental studies assessing uptake of orally ingested engineered nanoparticles (ENPs), a key step in ensuring accurate quantification of ingested ENPs is efficient separation of the organism from ENPs that are either nonspecifically adsorbed to the organism and/or suspended in the dispersion following exposure. Here, we measure the uptake of 30 and 60 nm gold nanoparticles (AuNPs) by the nematode, Caenorhabditis elegans, using a sucrose density gradient centrifugation protocol to remove noningested AuNPs. Both conventional inductively coupled plasma mass spectrometry (ICP-MS) and single particle (sp)­ICP-MS are utilized to measure the total mass and size distribution, respectively, of ingested AuNPs. Scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM/EDS) imaging confirmed that traditional nematode washing procedures were ineffective at removing excess suspended and/or adsorbed AuNPs after exposure. Water rinsing procedures had AuNP removal efficiencies ranging from 57 to 97% and 22 to 83%, while the sucrose density gradient procedure had removal efficiencies of 100 and 93 to 98%, respectively, for the 30 and 60 nm AuNP exposure conditions. Quantification of total Au uptake was performed following acidic digestion of nonexposed and Au-exposed nematodes, whereas an alkaline digestion procedure was optimized for the liberation of ingested AuNPs for spICP-MS characterization. Size distributions and particle number concentrations were determined for AuNPs ingested by nematodes with corresponding confirmation of nematode uptake <i>via</i> high-pressure freezing/freeze substitution resin preparation and large-area SEM imaging. Methods for the separation and <i>in vivo</i> quantification of ENPs in multicellular organisms will facilitate robust studies of ENP uptake, biotransformation, and hazard assessment in the environment