Biodiesel exhaust particle airway toxicity and the role of polycyclic aromatic hydrocarbons.

Renewable alternatives to fossil diesel (FD) including fatty acid methyl ester (FAME) biodiesel have become more prevalent. However, toxicity of exhaust material from their combustion, relative to the fuels they are displacing has not been fully characterised. This study was carried out to examine particle toxicity within the lung epithelium and the role for polycyclic aromatic hydrocarbons (PAHs). Exhaust particles from a 20% (v/v) blend of FAME biodiesel had little impact on primary airway epithelial toxicity compared to FD derived particles but did result in an altered profile of PAHs, including an increase in particle bound carcinogenic B[a]P. Higher blends of biodiesel had significantly increased levels of more carcinogenic PAHs, which was associated with a higher level of stress response gene expression including CYP1A1, NQO1 and IL1B. Removal of semi-volatile material from particulates abolished effects on airway cells. Particle size difference and toxic metals were discounted as causative for biological effects. Finally, combustion of a single component fuel (Methyl decanoate) containing the methyl ester molecular structure found in FAME mixtures, also produced more carcinogenic PAHs at the higher fuel blend levels. These results indicate the use of FAME biodiesel at higher blends may be associated with an increased particle associated carcinogenic and toxicity risk.

Methanol-based extraction protocol for insoluble and moderately water-soluble nanoparticles in plants to enable characterization by single particle ICP-MS.

The detection and characterization of soluble metal nanoparticles in plant tissues are an analytical challenge, though a scientific necessity for regulating nano-enabled agrichemicals. The efficacy of two extraction methods to prepare plant samples for analysis by single particle ICP-MS, an analytical method enabling both size determination and quantification of nanoparticles (NP), was assessed. A standard enzyme-based extraction was compared to a newly developed methanol-based approach. Au, CuO, and ZnO NPs were extracted from three different plant leaf materials (lettuce, corn, and kale) selected for their agricultural relevance and differing characteristics. The enzyme-based approach was found to be unsuitable because of changes in the recovered NP size distribution of CuO NP. The MeOH-based extraction allowed reproducible extraction of the particle size distribution (PSD) without major alteration caused by the extraction. The type of leaf tissue did not significantly affect the recovered PSD. Total metal losses during the extraction process were largely due to the filtration step prior to analysis by spICP-MS, though this did not significantly affect PSD recovery. The methanol extraction worked with the three different NPs and plants tested and is suitable for studying the fate of labile metal-based nano-enabled agrichemicals.

Novel Multi-isotope Tracer Approach To Test ZnO Nanoparticle and Soluble Zn Bioavailability in Joint Soil Exposures.

Here we use two enriched stable isotopes, 68Znen and 64Znen (>99%), to prepare 68ZnO nanoparticles (NPs) and soluble 64ZnCl2. The standard LUFA 2.2 test soil was dosed with 68ZnO NPs and soluble 64ZnCl2 to 5 mg kg-1 each, plus between 0 and 95 mg kg-1 of soluble ZnCl2 with a natural isotope composition. After 0, 1, 3, 6, and 12 months of soil incubation, earthworms (Eisenia andrei) were introduced for 72 h exposures. Analyses of soils, pore waters, and earthworm tissues using multiple collector inductively coupled plasma mass spectrometry allowed the simultaneous measurement of the diagnostic 68Zn/66Zn, 64Zn/66Zn, and 68Zn/64Zn ratios, from which the three different isotopic forms of Zn were quantified. Eisenia andrei was able to regulate Zn body concentrations with no difference observed between the different total dosing concentrations. The accumulation of labeled Zn by the earthworms showed a direct relationship with the proportion of labeled to total Zn in the pore water, which increased with longer soil incubation times and decreasing soil pH. The 68Znen/64Znen ratios determined for earthworms (1.09 ± 0.04), soils (1.09 ± 0.02), and pore waters (1.08 ± 0.02) indicate indistinguishable environmental distribution and uptake of the Zn forms, most likely due to rapid dissolution of the ZnO NPs.

Earthworm Uptake Routes and Rates of Ionic Zn and ZnO Nanoparticles at Realistic Concentrations, Traced Using Stable Isotope Labeling.

The environmental behavior of ZnO nanoparticles (NPs), their availability to, uptake pathways by, and biokinetics in the earthworm Lumbricus rubellus were investigated using stable isotope labeling. Zinc isotopically enriched to 99.5% in (68)Zn ((68)Zn-E) was used to prepare (68)ZnO NPs and a dissolved phase of (68)Zn for comparison. These materials enabled tracing of environmentally relevant (below background) NP additions to soil of only 5 mg (68)Zn-E kg(-1). Uptake routes were isolated by introducing earthworms with sealed and unsealed mouthparts into test soils for up to 72 h. The Zn isotope compositions of the soils, pore waters and earthworms were then determined using multiple collector inductively coupled plasma mass spectrometry. Detection and quantification of (68)Zn-E in earthworm tissue was possible after only 4 h of dermal exposure, when the uptake of (68)Zn-E had increased the total Zn tissue concentration by 0.03‰. The results demonstrate that at these realistic exposure concentrations there is no distinguishable difference between the uptake of the two forms of Zn by the earthworm L. rubellus, with the dietary pathway accounting for ∼95% of total uptake. This stands in contrast to comparable studies where high dosing levels were used and dermal uptake is dominant.

Aerosol exposure at air-liquid-interface (AE-ALI) in vitro toxicity system characterisation: Particle deposition and the importance of air control responses.

Experimental systems allowing aerosol exposure (AE) of cell cultures at the air-liquid-interface (ALI) are increasingly being used to assess the toxicity of inhaled contaminants as they are more biomimetic than standard methods using submerged cultures, however, they require detailed characterisation before use. An AE-ALI system combining aerosol generation with a CULTEX® exposure chamber was characterised with respect to particle deposition and the cellular effects of filtered air (typical control) exposures. The effect of system parameters (electrostatic precipitator voltage, air flowrate to cells and insert size) on deposition efficiency and spatial distribution were investigated using ICP-MS and laser ablation ICP-MS, for an aerosol of CeO2 nanoparticles. Deposition varied with conditions, but appropriate choice of operating parameters produced broadly uniform deposition at suitable levels. The impact of air exposure duration on alveolar cells (A549) and primary small airway epithelial cells (SAECs) was explored with respect to LDH release and expression of selected genes. Results indicated that air exposures could have a significant impact on cells (e.g., cytotoxicity and expression of genes, including CXCL1, HMOX1, and SPP1) at relatively short durations (from 10 mins) and that SAECs were more sensitive. These findings indicate that detailed system characterisation is essential to ensure meaningful results.

Analysis of carbon nanotube levels in organic matter: an inter-laboratory comparison to determine best practice.

Carbon nanotubes (CNTs) are increasingly being used in industrial applications, but their toxicological data in animals and humans are still sparse. To assess the toxicological dose-response of CNTs and to evaluate their pulmonary biopersistence, their quantification in tissues, especially lungs, is crucial. There are currently no reference methods or reference materials for low levels of CNTs in organic matter. Among existing analytical methods, few have been fully and properly validated. To remedy this, we undertook an inter-laboratory comparison on samples of freeze-dried pig lung, ground and doped with CNTs. Eight laboratories were enrolled to analyze 3 types of CNTs at 2 concentration levels each in this organic matrix. Associated with the different analysis techniques used (specific to each laboratory), sample preparation may or may not have involved prior digestion of the matrix, depending on the analysis technique and the material being analyzed. Overall, even challenging, laboratories' ability to quantify CNT levels in organic matter is demonstrated. However, CNT quantification is often overestimated. Trueness analysis identified effective methods, but systematic errors persisted for some. Choosing the assigned value proved complex. Indirect analysis methods, despite added steps, outperform direct methods. The study emphasizes the need for reference materials, enhanced precision, and organized comparisons.

Stable isotope tracer to determine uptake and efflux dynamics of ZnO Nano- and bulk particles and dissolved Zn to an estuarine snail.

Zinc oxide nanoparticles (ZnO NPs) are among the most commercialized engineered nanomaterials. Their biological impact in aquatic organisms has been associated with dissolution, but there is also evidence of nanospecific effects. In this study the waterborne uptake and efflux kinetics of isotopically labeled (68)ZnO NPs (7.8 ± 1.2 nm), in comparison to aqueous (68)Zn and (68)ZnO bulk particles (up to 2 µm), were determined for the estuarine snail Peringia ulvae following a 7 d exposure (nominally 20 µg (68)Zn L(-1)) and 28 d depuration. Detection of the (68)Zn label was achieved by high precision multiple-collector ICP-MS (MC-ICP-MS). Previous characterization in artificial estuarine water revealed that the NPs underwent initial aggregation and solubilized up to 60% within 1-2 days. Bulk and aqueous forms were significantly more bioavailable than (68)ZnO NPs (p < 0.05), but after correcting for dissolution, aqueous (0.074 L(-1) g(-1) d(-1)) and NP (0.070 L(-1) g(-1) d(-1)) uptake rate constants were highly comparable. The rate constant of loss for (68)Zn aqueous (0.012 ± 0.005 d(-1)) and (68)ZnO NPs (0.012 ± 0.007 d(-1)) were identical. These results strongly suggest that in this exposure scenario the bioaccumulation of Zn from ZnO NPs is primarily dependent upon solubility.

Identifying Sources of Lead Exposure for Children in the Republic of Georgia, with Lead Isotope Ratios.

In the Republic of Georgia, a 2018 national survey estimated that more than 40% of children aged 2-7 years had a blood lead concentration (BLC) of more than 5 µg/dL. The objective of this study was to document the feasibility of employing lead isotope ratios (LIRs) to identify and rank the Pb (lead) exposure sources most relevant to children across Georgia. A cross-sectional survey between November 2019 and February 2020 of 36 children previously identified as having BLCs > 5 µg/dL from seven regions of Georgia involved the collection of blood and 528 environmental samples, a questionnaire on behaviours and potential exposures. The LIRs in blood and environmental samples were analysed in individual children and across the whole group to ascertain clustering. A fitted statistical mixed-effect model to LIR data first found that the blood samples clustered with spices, tea, and paint, then, further isotopically distinct from blood were sand, dust, and soil, and lastly, milk, toys, pens, flour, and water. Analysis of the LIRs provided an indication and ranking of the importance of Pb environmental sources as explanatory factors of BLCs across the group of children. The findings support the deployment of interventions aimed at managing the priority sources of exposure in this population.

ZnO Nanomaterials and Ionic Zn Partition within Wastewater Sludge Investigated by Isotopic Labeling.

The increasing commercial use of engineered zinc oxide nanomaterials necessitates a thorough understanding of their behavior following their release into wastewater. Herein, the fates of zinc oxide nanoparticles (ZnO NPs) and ionic Zn in a real primary sludge collected from a municipal wastewater system are studied via stable isotope tracing at an environmentally relevant spiking concentration of 15.2 µg g-1. Due to rapid dissolution, nanoparticulate ZnO does not impart particle-specific effects, and the Zn ions from NP dissolution and ionic Zn display indistinguishable behavior as they partition equally between the solid, liquid, and ultrafiltrate phases of the sludge over a 4-h incubation period. This work provides important constraints on the behavior of engineered ZnO nanomaterials in primary sludge-the first barrier in a wastewater treatment plant-at low, realistic concentrations. As the calculated solid-liquid partition coefficients are significantly lower than those reported in prior studies that employ unreasonably high spiking concentrations, this work highlights the importance of using low, environmentally relevant doses of engineered nanomaterials in experiments to obtain accurate risk assessments.

Determination of metallic nanoparticles in biological samples by single particle ICP-MS: a systematic review from sample collection to analysis.

A systematic review of the use of single particle ICP-MS to analyse engineered nanomaterials (ENMs) in biological samples (plants, animals, body fluids) has highlighted that efforts have focused on a select few types of ENMs (e.g., Ag and TiO2) and there is a lack of information for some important tissues (e.g., reproductive organs, skin and fatty endocrine organs). The importance of sample storage is often overlooked but plays a critical role. Careful consideration of the ENM and matrix composition is required to select an appropriate protocol to liberate ENMs from a tissue whilst not promoting the transformation of them, or genesis of new particulates. A 'one size fits all' protocol, applicable to all possible types of ENM and biological matrices, does not seem practical. However, alkaline-based extractions would appear to show greater promise for wide applicability to animal tissues, although enzymatic approaches have a role, especially for plant tissues. There is a lack of consistency in metrics reported and how they are determined (e.g. size limit of detection, and proportions of recovery), making comparison between some studies more difficult. In order to establish standardised protocols for regulatory use, effort is needed to: develop certified reference materials, achieve international agree on nomenclature and the use of control samples, and to create a decision tree to help select the best sample preparation for the type of tissue matrix.

The Use of Pb Isotope Ratios to Determine Environmental Sources of High Blood Pb Concentrations in Children: A Feasibility Study in Georgia.

The incidence of lead (Pb) poisoning in children in Georgia has been identified as a major health concern, with a recent national survey identifying that 41% of children aged 2-7 years had blood lead concentrations (BLCs) greater than the blood lead reference value (BLRV) of ≥5 µg dL-1. This study collected samples of blood, spices, paint, soil, dust, flour, tea, toys, milk, and water from 36 households in Georgia where a child had previously been identified as having a BLC > BLRV. The Pb concentrations of these samples were determined and compared to Georgian reference values. Samples from 3 households were analysed for their Pb isotope composition. The Pb isotope composition of the environmental and blood samples were compared to identify the most likely source(s) of Pb exposure. This approach identified that some spice and dust samples were the likely sources of Pb in the blood in these cases. Importantly, some soil, paint, and dust sources with high Pb concentrations could be discounted as contributing to blood Pb based on their distinct isotope composition. The data presented demonstrate the significant contribution that Pb surveillance and Pb isotope ratio analyses can make to managing Pb exposure in regions where high BLCs are identified.

Biokinetics and Internal Dosimetry of Tritiated Steel Particles.

Decommissioning fission and fusion facilities can result in the production of airborne particles containing tritium that could inadvertently be inhaled by workers directly involved in the operations, and potentially others, resulting in internal exposures to tritium. Of particular interest in this context, given the potentially large masses of material involved, is tritiated steel. The International Commission on Radiological Protection (ICRP) has recommended committed effective dose coefficients for inhalation of some tritiated materials, but not specifically for tritiated steel. The lack of a dose coefficient for tritiated steel is a concern given the potential importance of the material. To address this knowledge gap, a "dissolution" study, in vivo biokinetic study in a rodent model (1 MBq intratracheal instillation, 3-month follow-up) and associated state-of-the-art modelling were undertaken to derive dose coefficients for model tritiated steel particles. A committed effective dose coefficient for the inhalation of 3.3 × 10-12 Sv Bq-1 was evaluated for the particles, reflecting an activity median aerodynamic diameter (AMAD) of 13.3 µm, with the value for a reference AMAD for workers (5 µm) of 5.6 × 10-12 Sv Bq-1 that may be applied to occupational inhalation exposure to tritiated steel particles.

Characterisation of particles within and aerosols produced by nano-containing consumer spray products.

Nanoparticles have been incorporated into a range of consumer spray products, providing the potential for inadvertent inhalation by users and bystanders. The levels and characteristics of nanoparticle inhalation exposures arising from the use of such products are important inputs to risk assessments and informing dose regimes for in vitro and in vivo studies investigating hazard potentials. To date, only a small number of studies have been undertaken to explore both the aerosols generated from such products and the metal nanoparticles within them. The objective of the current study was to add to the limited data in this field by investigating a range of nano-containing spray products available within the UK. Six products were selected and the nanoparticles characterised using a combination of techniques, including: inductively coupled plasma mass spectrometry (ICP-MS), dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), transmission electron microscopy energy-dispersive X-ray spectroscopy (TEM-EDX) and single particle ICP-MS (spICP-MS). The aerosol produced by these products, when sprayed within a glovebox, was characterised by scanning mobility particle sizer (SMPS) and an aerodynamic particle sizer (APS). A cascade impactor with thirteen stages (NanoMOUDI) was used with one product to generate information on the size specific nanoparticle elemental distribution within the aerosol. The results demonstrated the presence of solid nanoparticles (silver, gold or silica) in each of the products at low concentrations (<13 ppm). TEM and (sp)ICP-MS provided reliable information on nanoparticle size, shape, number and mass, while the light scattering methods were less effective due to the complex matrices of the products and their lack of chemical specificity. The aerosols varied significantly across products, with particle and mass concentrations spanning 5 orders of magnitude (10 - 106 cm-3 and 0.3-7600 µg m-3, respectively). The NanoMOUDI results clearly indicated non-uniform distribution of silver within different aerosol particle size ranges.