Determination of the Transport Efficiency in spICP-MS Analysis Using Conventional Sample Introduction Systems: An Interlaboratory Comparison Study.

In single particle inductively coupled plasma mass spectrometry (spICP-MS), the transport efficiency is fundamental for the correct determination of both particle number concentration and size. In the present study, transport efficiency was systematically determined on three different days with six carefully characterised gold nanoparticle (AuNP) suspensions and in seven European and US expert laboratories using different ICP-MS instruments and spICP-MS software. Both particle size-(TES)-and particle frequency-(TEF)-methods were applied. The resulting transport efficiencies did not deviate much under ideal conditions. The TEF method however systematically resulted in lower transport efficiencies. The extent of this difference (0-300% rel. difference) depended largely on the choice and storage conditions of the nanoparticle suspensions used for the determination. The TES method is recommended when the principal measurement objective is particle size. If the main aim of the measurement is the determination of the particle number concentration, the TEF approach could be preferred as it might better account for particle losses in the sample introduction system.

Particle size analysis of pristine food-grade titanium dioxide and E 171 in confectionery products: Interlaboratory testing of a single-particle inductively coupled plasma mass spectrometry screening method and confirmation with transmission electron microscopy.

Titanium dioxide is a white colourant authorised as food additive E 171 in the EU, where it is used in a range of alimentary products. As these materials may contain a fraction of particulates with sizes below 100 nm and current EU regulation requires specific labelling of food ingredient to indicate the presence of engineered nanomaterials there is now a need for standardised and validated methods to appropriately size and quantify (nano)particles in food matrices. A single-particle inductively coupled plasma mass spectrometry (spICP-MS) screening method for the determination of the size distribution and concentration of titanium dioxide particles in sugar-coated confectionery and pristine food-grade titanium dioxide was developed. Special emphasis was placed on the sample preparation procedure, crucial to reproducibly disperse the particles before analysis. The transferability of this method was tested in an interlaboratory comparison study among seven experienced European food control and food research laboratories equipped with various ICP-MS instruments and using different software packages. The assessed measurands included the particle mean diameter, the most frequent diameter, the percentage of particles (in number) with a diameter below 100 nm, the particles' number concentration and a number of cumulative particle size distribution parameters (D0, D10, D50, D99.5, D99.8 and D100). The evaluated method's performance characteristics were, the within-laboratory precision, expressed as the relative repeatability standard deviation (RSDr), and the between-laboratory precision, expressed as the relative reproducibility standard deviation (RSDR). Transmission electron microscopy (TEM) was used as a confirmatory technique and served as the basis for bias estimation. The optimisation of the sample preparation step showed that when this protocol was applied to the relatively simple sample food matrices used in this study, bath sonication turned out to be sufficient to reach the highest, achievable degree of dispersed constituent particles. For the pristine material, probe sonication was required. Repeatability and reproducibility were below 10% and 25% respectively for most measurands except for the lower (D0) and the upper (D100) bound of the particle size distribution and the particle number concentration. The broader distribution of the lower and the upper bounds could be attributed to instrument-specific settings/setups (e.g. the timing parameters, the transport efficiency, type of mass-spectrometer) and software-specific data treatment algorithms. Differences in the upper bound were identified as being due to the non-harmonised application of the upper counting limit. Reporting D99.5 or D99.8 instead of the effectively largest particle diameter (D100) excluded isolated large particles and considerably improved the reproducibility. The particle number-concentration was found to be influenced by small differences in the sample preparation procedure. The comparison of these results with those obtained using electron microscopy showed that the mean and median particle diameter was, in all cases, higher when using spICP-MS. The main reason for this was the higher size detection limit for spICP-MS plus the fact that some of the analysed particles remained agglomerated/aggregated after sonication. Single particle ICP-MS is a powerful screening technique, which in many cases provides sufficient evidence to confirm the need to label a food product as containing (engineered) titanium dioxide nanomaterial according to the current EU regulatory requirements. The overall positive outcome of the method performance evaluation and the current lack of alternative standardised procedures, would indicate this method as being a promising candidate for a full validation study.

Insights into the nature of uranium target proteins within zebrafish gills after chronic and acute waterborne exposures.

New data on the nature of the protein targets of uranium (U) within zebrafish gills were collected after waterborne exposure, with the aim of a better understanding of U toxicity mechanisms. Some common characteristics of the U protein target binding properties were found, such as their role in the regulation of other essential metals and their phosphorus content. In total, 21 potential protein targets, including hemoglobin, are identified and discussed in terms of the literature.

Different uranium distribution patterns in cytosolic protein pool of zebrafish gills after chronic and acute waterborne exposures.

The toxicity of uranium (U) to aquatic organisms depends notably on its compartmentalization in organs, tissues, cells as well as on its distribution among biomolecules. In order to contribute to the understanding of U accumulation and associated toxicity mechanisms in case of waterborne exposure, this study focused on U fate in the gills epithelia, uptake pathway, of the fish model Danio rerio (zebrafish). U distribution among cytosolic biomolecules was investigated after no addition (0µgL(-)(1) (c0) for 3 and 30d), chronic (20µgL(-)(1) (c20) for 30d) and acute (20µgL(-)(1) (c20) and 250µgL(-)(1) (c250) for 3d) exposures to depleted U. Cytosolic U accounted for an average of 24-32% of gills burden for c20 and c250, respectively. Size Exclusion Chromatography (SEC) coupled with Inductively Coupled Plasma-Sector Field Mass Spectrometry (ICP-SFMS) allowed identification of ecotoxicologically relevant U-containing fractions among cytosolic biomolecules as a function of exposure conditions. In c0 and c20 samples, most U (ca.80%) was found in the Low Molecular Weight fraction (LMW, <18kDa), often considered as a detoxifying fraction. In c250 exposed fish, U was equally distributed between LMW (40%) and High Molecular Weight (HMW, 150-670kDa; 40%) fractions, the latter including sensitive metalloproteins. Uranium-biomolecules were co-eluted with endogenous essential metal (Fe, Cu and Zn) species, however, no major influence on their cytosolic concentration and distribution pattern among cytosolic proteins was found.

Development of non-denaturing off-gel isoelectric focusing for the separation of uranium-protein complexes in fish.

An off-gel non-denaturing isoelectric focusing (IEF) method was developed to separate uranium-biomolecule complexes from biological samples as a first step in a multidimensional metalloproteomic approach. Analysis of a synthetic uranium-bovine serum albumin complex demonstrated the focusing ability of the liquid-phase IEF method and the preservation of most of the uranium-protein interactions. The developed method was applied to gill cytosol prepared from zebrafish (Danio rerio) exposed to depleted uranium. The results were compared in terms of resolution, recovery, and protein identities with those obtained by in-gel IEF using an immobilized pH gradient gel strip.