Occupational Exposure during the Production and the Spray Deposition of Graphene Nanoplatelets-Based Polymeric Coatings.

Graphene-based polymer composites are innovative materials which have recently found wide application in many industrial sectors thanks to the combination of their enhanced properties. The production of such materials at the nanoscale and their handling in combination with other materials introduce growing concerns regarding workers' exposure to nano-sized materials. The present study aims to evaluate the nanomaterials emissions during the work phases required to produce an innovative graphene-based polymer coating made of a water-based polyurethane paint filled with graphene nanoplatelets (GNPs) and deposited via the spray casting technique. For this purpose, a multi-metric exposure measurement strategy was adopted in accordance with the harmonized tiered approach published by the Organization for Economic Co-operation and Development (OECD). As a result, potential GNPs release has been indicated near the operator in a restricted area not involving other workers. The ventilated hood inside the production laboratory guarantees a rapid reduction of particle number concentration levels, limiting the exposure time. Such findings allowed us to identify the work phases of the production process with a high risk of exposure by inhalation to GNPs and to define proper risk mitigation strategies.

Size characteristics of asbestiform fibers in lung tissue of animals that grazed in areas rich in ophiolitic outcrops in Central Calabria (Southern Italy).

In Calabria (Southern Italy) naturally occurring asbestos (NOA) mainly occurs in the ophiolitic sequences cropping in the Mount Reventino area. The most common type of asbestos detected was the amphibole tremolite; fibrous antigorite and minor chrysotile were also found. The development of asbestos-related diseases depends on, among other things, the morphological characteristics of fibers, length and width, affecting the durability of asbestos fibers in the lung. In this work fifteen lung samples of sheep, goats and wild boars, grazing around the Mount Reventino area were collected and asbestos fibers analysed. Observed fibers (357), of which 97 % were tremolite and 3 % antigorite fibers, were grouped according to species, grazing area and age of the animals. The aim of this work was to highlight any differences among the groupings and to compare our size results with data in literature related to exposed populations. Principal Component Analysis (PCA) highlighted a positive correlation between tremolite fiber length and width and revealed groupings in terms of animal age. The Kruskal-Wallis test showed statistically significant differences between fiber mean widths in young and old animals. 63 % observed asbestiform fibers were longer than 5 µm and 7 % of the fibers were longer than 20 µm (critical fiber length connected to the frustrated phagocytosis by the macrophage). Fibers conforming to the Stanton Hypothesis size (predictor of the carcinogenic potency of fibers) were 1 %. Our size parameters of fibers detected in the animal lungs were in fairly good agreement with literature data for human asbestos exposure to tremolite. These results confirmed that an animal-sentinel system could be used to monitor the natural background of the airborne breathable fibers exposure. In addition, the size correlation of animal-human breathed fibers could be useful to study their potential toxicity. Additional data are necessary for improving the agreement with human exposure data.

A follow-up study on workers involved in the graphene production process after the introduction of exposure mitigation measures: evaluation of genotoxic and oxidative effects.

During nanomaterial (NM) production, workers could be exposed, particularly by inhalation, to NMs and other chemicals used in the synthesis process, so it is important to have suitable biomarkers to monitor potential toxic effects. Aim of this study was to evaluate the effectiveness of the introduction of exposure mitigation measures on workers unintentionally exposed to graphene co-pollutants during production process monitoring the presumable reduction of workplace NM contamination and of early genotoxic and oxidative effects previously found on these workers. We used Buccal Micronucleus Cytome (BMCyt) assay and Fpg-comet test, resulted the most sensitive biomarkers on our first biomonitoring work, to measure the genotoxic effects. We also detected urinary oxidized nucleic acid bases 8-oxoGua, 8-oxoGuo and 8-oxodGuo to evaluate oxidative damage. The genotoxic and oxidative effects were assessed on the same graphene workers (N = 6) previously studied, comparing the results with those found in the first biomonitoring and with the control group (N = 11). This was achieved 6 months after the installation of a special filter hood (where to perform the phases at higher risk of NM emission) and the improvement of environmental and personal protective equipment. Particle number concentration decreased after the mitigation measures. We observed reduction of Micronucleus (MN) frequency and oxidative DNA damage and increase of 8-oxodGuo excretion compared to the first biomonitoring. These results, although limited by the small subject number, showed the efficacy of adopted exposure mitigation measures and the suitability of used sensitive and noninvasive biomarkers to bio-monitor over time workers involved in graphene production process.

Airborne LTA Nanozeolites Characterization during the Manufacturing Process and External Sources Interaction with the Workplace Background.

Engineered nanoscale amorphous silica nanomaterials are widespread and used in many industrial sectors. Currently, some types of silicon-based nanozeolites (NZs) have been synthesized, showing potential advantages compared to the analogous micro-forms; otherwise, few studies are yet available regarding their potential toxicity. In this respect, the aim of the present work is to investigate the potential exposure to airborne Linde Type A (LTA) NZs on which toxicological effects have been already assessed. Moreover, the contributions to the background related to the main emission sources coming from the outdoor environment (i.e., vehicular traffic and anthropogenic activities) were investigated as possible confounding factors. For this purpose, an LTA NZ production line in an industrial factory has been studied, according to the Organisation for Economic Cooperation and Development (OECD) guidelines on multi-metric approach to investigate airborne nanoparticles at the workplace. The main emission sources of nanoparticulate matter within the working environment have been identified by real-time measurements (particle number concentration, size distribution, average diameter, and lung-deposited surface area). Events due to LTA NZ spillage in the air during the cleaning phases have been chemically and morphologically characterized by ICP-MS and SEM analysis, respectively.

An integrated and multi-technique approach to characterize airborne graphene flakes in the workplace during production phases.

Graphene is a one-atom-thick sheet of carbon atoms arranged in a honeycomb pattern and its unique and amazing properties make it suitable for a wide range of applications ranging from electronic devices to food packaging. However, the biocompatibility of graphene is dependent on the complex interplay of its several physical and chemical properties. The main aim of the present study is to highlight the importance of integrating different characterization techniques to describe the potential release of airborne graphene flakes in a graphene processing and production research laboratory. Specifically, the production and processing (i.e., drying) of few-layer graphene (FLG) through liquid-phase exfoliation of graphite are analysed by integrated characterization techniques. For this purpose, the exposure measurement strategy was based on the multi-metric tiered approach proposed by the Organization for Economic Cooperation and Development (OECD) via integrating high-frequency real-time measurements and personal sampling. Particle number concentration, average diameter and lung deposition surface area time series acquired in the worker's personal breathing zone (PBZ) were compared simultaneously to background measurements, showing the potential release of FLG. Then, electron microscopy techniques and Raman spectroscopy were applied to characterize particles collected by personal inertial impactors to investigate the morphology, chemical composition and crystal structure of rare airborne graphene flakes. The gathered information provides a valuable basis for improving risk management strategies in research and industrial laboratories.

Occupational exposure to graphene and silica nanoparticles. Part I: workplace measurements and samplings.

Few-Layers Graphene (FLG) are able to improve the performance of materials, due to their chemical-physical properties. Engineered amorphous silica nanoparticles (SiO2NPs) are among the most widespread nanomaterials (NMs) in the world. Such nanomaterials are two case studies of the research project 'NanoKey' that integrated the exposure assessment through personal measurements and sampling in the workplace, as described in the present work (part I), with the biomonitoring of exposed workers (reported in part II). Measurement campaigns were conducted according to OECD and WHO harmonized approach in two production sites. The set of instruments included real-time devices for high-resolution measurements at the nanoscale and time-integrated samplers for the off-line gravimetric analysis and chemical and morphological (SEM-EDS) characterization of exposure in order to identify the contribution of production compared to the background. Values of particle number concentration (PNC) and lung deposited surface area (LDSA) within the FLG production resulted higher than the background far field (FF), even if they are always similar to the near field (NF) ones: the average diameter (Davg) during the production was higher than the NF background but always lower than the FF values. SEM-EDS analysis highlighted the presence of structures comparable to those produced. During the SiO2NPs production, the PBZ values showed PNC and LDSA levels higher than the background, with a decrease in the Davg probably due to NPs emission. SEM-EDS confirms the presence of rare silica nanoparticles. Since the exposure to airborne NMs cannot be excluded in both production sites, a prevention-through-design approach to mitigate the potential risk for workers has been recommended.

Workers' Exposure Assessment during the Production of Graphene Nanoplatelets in R&D Laboratory.

Widespread production and use of engineered nanomaterials in industrial and research settings raise concerns about their health impact in the workplace. In the last years, graphene-based nanomaterials have gained particular interest in many application fields. Among them, graphene nanoplatelets (GNPs) showed superior electrical, optical and thermal properties, low-cost and availability. Few and conflicting results have been reported about toxicity and potential effects on workers' health, during the production and handling of these nanostructures. Due to this lack of knowledge, systematic approaches are needed to assess risks and quantify workers' exposure to GNPs. This work applies a multi-metric approach to assess workers' exposure during the production of GNPs, based on the Organization for Economic Cooperation and Development (OECD) methodology by integrating real-time measurements and personal sampling. In particular, we analyzed the particle number concentration, the average diameter and the lung deposited surface area of airborne nanoparticles during the production process conducted by thermal exfoliation in two different ways, compared to the background. These results have been integrated by electron microscopic and spectroscopic analysis on the filters sampled by personal impactors. The study identifies the process phases potentially at risk for workers and reports quantitative information about the parameters that may influence the exposure in order to propose recommendations for a safer design of GNPs production process.

Data acquired by wearable sensors for the evaluation of the flexion-relaxation phenomenon.

The relationship between flexibility and the pattern formed by the surface electromyography activity of the back muscles while performing a dynamic trunk flexion-extension task is not yet thoroughly understood, although many previous studies have adopted it as their focus in the literature. Additionally, several studies have proposed technologies and algorithms to analyse the flexion-relaxation phenomenon, which is defined by myoelectric silence that occurs when the subject's torso exceeds a certain flexion angle. Before participating in the flexion-relaxation test, subjects involved in the data collection underwent medical examinations, in which their physical condition, perceived pain, and level of disability were reported in their anamnesis. During the flexion-relaxation test, which was conducted with 25 subjects with and without low back pain, subjects wore four surface electromyography electrodes positioned over the back muscles, as well as an inertial sensor to estimate trunk inclination.

Levels of urinary metabolites of four PAHs and cotinine determined in 1016 volunteers living in Central Italy.

Polycyclic aromatic hydrocarbons (PAH) are products of the incomplete combustion of organic materials, and exposure of the general population to PAH is ubiquitous. They are also present in tobacco smoke. Some PAH are classified as carcinogens. Urine samples from 747 non-smoking and 269 smoking subjects living in the same area of Central Italy were analyzed in order to determine reference values for PAHs exposure of a general population. The concentration of cotinine, urinary metabolite of nicotine was also measured in these samples in order to classify the subjects as smokers or not. The median concentration and 50th percentile in females was higher than in males for all metabolites; 1- and 2-hydroxynaphtalene (1-OHNAP and 2-OHNAP) and 1-hydroxypyrene (1-OHPy), are significantly higher in smokers; on the other side 3-hydroxybenzo[a]pyrene (3-OHBaPy) and 6-hydroxynitropyrene (6-OHNPy) do not correlate with the cotinine concentration.

Workers' Exposure to Nano-Objects with Different Dimensionalities in R&D Laboratories: Measurement Strategy and Field Studies.

With the increasing interest in the potential benefits of nanotechnologies, concern is still growing that they may present emerging risks for workers. Various strategies have been developed to assess the exposure to nano-objects and their agglomerates and aggregates (NOAA) in the workplace, integrating different aerosol measurement instruments and taking into account multiple parameters that may influence NOAA toxicity. The present study proposes a multi-metric approach for measuring and sampling NOAA in the workplace, applied to three case studies in laboratories each dedicated to materials with different shapes and dimensionalities: graphene, nanowires, and nanoparticles. The study is part of a larger project with the aim of improving risk management tools in nanomaterials research laboratories. The harmonized methodology proposed by the Organization for Economic Cooperation and Development (OECD) has been applied, including information gathering about materials and processes, measurements with easy-to-use and hand-held real-time devices, air sampling with personal samplers, and off-line analysis using scanning electron microscopy. Significant values beyond which an emission can be attributed to the NOAA production process were identified by comparison of the particle number concentration (PNC) time series and the corresponding background levels in the three laboratories. We explored the relations between background PNC and microclimatic parameters. Morphological and elemental analysis of sampled filters was done to identify possible emission sources of NOAA during the production processes: rare particles, spherical, with average diameter similar to the produced NOAA were identified in the nanoparticles laboratory, so further investigation is recommended to confirm the potential for worker exposure. In conclusion, the information obtained should provide a valuable basis for improving risk management strategies in the laboratory at work.

Thermophysiological response of Newton manikin equipped with power-assisted filtering device incorporating a full-face mask in hot environment.

One of the most common reasons inducing discomfort in wearing a respirator is an unpleasant local thermal perception. In this study, the effect of a power-assisted filtering device incorporating a full-face mask on the thermophysiological response was investigated by performing tests on a Newton manikin in a climatic chamber, where hot conditions were simulated setting the ambient temperature at 34 °C and the relative humidity at 32%. Two different levels of metabolic rate (1 MET and 3 MET) were considered. Results show that the variation of metabolic rate plays an important role in the heat exchanges with the environment causing different values of the local and global temperatures. At 1 MET the manikin is in thermal balance with the external environment with or without respirator. At 3 MET although the thermoregulation system is very stressed (the rectal temperature Tre increases continuously with or without respirator), the effect of the respirator produces a small decrease of the local and global temperatures and also of the amount of sweat generated by active thermoregulation Swa (reaching its maximum value of 30 g/min when the respirator is not worn). Furthermore, a little amelioration of the face and body sensation and comfort was observed. In any case, wearing the respirator seems not to provide additional thermal load.

Evaluation of performance loss of paraffin oil loaded filtering facepieces.

Penetration measurements through commercially available filtering facepieces were performed with monodisperse DEHS aerosols ranging from 0.03 µm to 0.40 µm (either singly charged or neutralized), before and after 500 mg of paraffin oil loading. The distinct behavior of Coulomb and polarization capture efficiency is studied: as in the case of non loading also in the case of loading 500 mg of paraffin oil, the electrostatic capture mechanisms are mainly due to the Coulomb contribution up to aerosol particle diameter of about 0.10 µm, just when the polarization contribution becomes substantial. Both Coulomb and polarization capture mechanisms are influenced by the presence of 500 mg of paraffin oil, resulting less effective than the oil unloaded case of about 12% and 11%, respectively. By the occupational hygiene point of view, there is a degradation in the filter performance due to oil loading that the user does not realize because there is no remarkable variation in the breathing resistance.

Variation in penetration of submicrometric particles through electrostatic filtering facepieces during exposure to paraffin oil aerosol.

Several studies show the increase of penetration through electrostatic filters during exposure to an aerosol flow, because of particle deposition on filter fibers. We studied the effect of increasing loads of paraffin oil aerosol on the penetration of selected particle sizes through an electrostatic filtering facepiece. FFP2 facepieces were exposed for 8 hr to a flow rate of 95.0 ± 0.5 L/min of polydisperse paraffin aerosol at 20.0 ± 0.5 mg/m(3). The penetration of bis(2-ethylhexyl)sebacate (DEHS) monodisperse neutralized aerosols, with selected particle size in the 0.03-0.40 µm range, was measured immediately prior to the start of the paraffin aerosol loading and at 1, 4, and 8 hr after the start of paraffin aerosol loading. Penetration through isopropanol-treated facepieces not oil paraffin loaded was also measured to evaluate facepiece behavior when electrostatic capture mechanisms are practically absent. During exposure to paraffin aerosol, DEHS penetration gradually increased for all aerosol sizes, and the most penetrating particle size (0.05 µm at the beginning of exposure) shifted slightly to larger diameters. After the isopropanol treatment, the higher penetration value was 0.30 µm. In addition to an increased penetration during paraffin loading at a given particle size, the relative degree of increase was greater as the particle size increased. Penetration value measured after 8 hr for 0.03-µm particles was on average 1.6 times the initial value, whereas it was about 8 times for 0.40-µm particles. This behavior, as well evidenced in the measurements of isopropanol-treated facepieces, can be attributed to the increasing action in particle capture of the electrostatic forces (Coulomb and polarization), which depend strictly on the diameter and electrical charge of neutralized aerosol particles. With reference to electrostatic filtering facepieces as personal protective equipment, results suggest the importance of complying with the manufacturer instructions when it is specified that their use has to be restricted to a single shift.

Si nanotubes and nanospheres with two-dimensional polycrystalline walls.

We report on the characteristics of a new class of Si-based nanotubes and spherical nanoparticles synthesized by the dc-arc plasma method in a mixture of argon and hydrogen. These two nanostructures share common properties: they are hollow and possess very thin, highly polycrystalline and mainly oxidized walls. In particular, we get several hints indicating that their walls could constitute only one single Si oxidized layer. Moreover, we find that only the less oxidized nanotubes exhibit locally atomic ordered, snakeskin-like areas which possess a hexagonal arrangement which can be interpreted either as an sp(2) or sp(3) hybridized Si or Si-H layer. Their ability to not react with oxygen seems to suggest the presence of sp(2) configuration or the formation of silicon-hydrogen bonding.

Comparative cyto-genotoxicity assessment of functionalized and pristine multiwalled carbon nanotubes on human lung epithelial cells.

Chemical functionalization extends CNT applications conferring them new functions, but could modify their toxicity. We compared cytotoxicity and genotoxic/oxidative effects of -OH functionalized and pristine MWCNTs to evaluated the influence of the functionalization exposing A549 cells to 1-40µg/ml of both MWCNTs for 2, 4 and 24h. Cytotoxicity was evaluated by MTT and LDH tests and apoptosis induction, direct/oxidative DNA damage by Fpg-modified comet assay. After 24h we found viability reduction significant at 20 and 40µg/ml for both the MWCNTs with a detectable viability reduction already at lower concentrations for MWCNTs. A significant LDH release was found only for MWCNTs. Significant apoptosis induction was found from 10µg/ml of MWCNT-OH. A concentration-dependent increase of direct DNA damage, significant at 40µg/ml of MWCNTs and beginning from 5µg/ml of MWCNT-OH was detected at all exposure times. Oxidative DNA damage was not observed for both CNTs. The results indicate a different cytotoxic mechanism, by membrane damage for MWCNTs and apoptosis for MWCNT-OH, that could be explained by a different cellular uptake. Moreover, we found an earlier genotoxic effect for MWCNT-OH. The findings suggest that further studies on functionalized CNTs are necessary before using them in several applications particularly in biomedical field.

Multi-walled carbon nanotubes induce cytotoxicity and genotoxicity in human lung epithelial cells.

The increasing use of nanomaterials in consumer products highlights the importance of understanding their potential toxic effects. We evaluated cytotoxic and genotoxic/oxidative effects induced by commercial multi-walled carbon nanotubes (MWCNTs) on human lung epithelial (A549) cells treated with 5, 10, 40 and 100 µg ml⁻¹ for different exposure times. Scanning electron microscopy (SEM) analysis, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and lactate dehydrogenase (LDH) assays were performed to evaluate cytotoxicity. Fpg-modified comet assay was used to evaluate direct-oxidative DNA damage. LDH leakage was detected after 2, 4 and 24 h of exposure and viability reduction was revealed after 24 h. SEM analysis, performed after 4 and 24 h exposure, showed cell surface changes such as lower microvilli density, microvilli structure modifications and the presence of holes in plasma membrane. We found an induction of direct DNA damage after each exposure time and at all concentrations, statistically significant at 10 and 40 µg ml⁻¹ after 2 h, at 5, 10, 100 µg ml⁻¹ after 4 h and at 10 µg ml⁻¹ after 24 h exposure. However, oxidative DNA damage was not found. The results showed an induction of early cytotoxic effects such as loss of membrane integrity, surface morphological changes and MWCNT agglomerate entrance at all concentrations. We also demonstrated the ability of MWCNTs to induce early genotoxicity. This study emphasizes the suitability of our approach to evaluating simultaneously the early response of the cell membrane and DNA to different MWCNT concentrations and exposure times in cells of target organ. The findings contribute to elucidation of the mechanism by which MWCNTs cause toxic effects in an in vitro experimental model.