Table of contents

Welcome from the organizers

The rapidly developing field of nanotechnology opens up many exciting opportunities and benefits for new materials with significantly improved properties as well as some revolutionary applications in the fields of energy, environment, medicine, etc. These new materials potentially pave the way to considerable innovations in many industries of the 21st century although associated risks must be perfectly under control for workers, consumers and the environment. So, one can easily understand why Nanosafety is now considered as a specific new scientific area, gaining in importance and maturity every day.

Following the successful outcome of the two past international conferences on the safe production and use of nanomaterials: Nanosafe 2008 and 2010, the organizing committee has the pleasure of welcoming again to Grenoble some of the most famous specialists of the field in the world.

In addition to the standard issues addressed in previous nanosafe conferences such as Toxicology, Eco-toxicology, Expology, Detection, Life Cycle Analysis, Regulation and Standardization, new topics of great interest will be dealt with this year, concerning Governance and practical Risk Management for OSH experts and Societal issues. In order to enhance the exchanges and conviviality three debates werw organized around Nano Governance, Toxicology and Ethics.

We hope that you will appreciate this new Nanosafe edition like the previous ones.

This is your Nanosafe conference, please enjoy.

The Nanosafe conference organisersFrançois Tardif and Frédéric ShusterManagement Vanessa Gaultier

François Tardif	Frédéric Shuster	Vanessa Gaultier

Local Organizing Committee Vanessa GAULTIER (CEA) François TARDIF (CEA) Dominique BAGUET (CEA) Frédédric SHUSTER (CEA) Yves SICARD (CEA) Catherine DURAND (CEA) Didier MOLKO (MINATEC)

International Scientific Committee Chair: Frédéric SCHUSTER (CEA, FR) François TARDIF (CEA, FR) Co-chair: Georgios KATALAGARIANAKIS (EC, BE) Mélanie AUFFAN (CEREGE, FR) Jorge BOCZKOWSKI (INSERM, FR) Jean-Yves BOTTERO (CEREGE, FR) Jacques BOUILLARD (INERIS, FR) Derk BROUWER (TNO, NL) Marie CARRIERE (CEA, FR) Claude EMOND (U. MONTREAL, CA) Cassandra ENGEMAN (UCSB, USA) Eric GAFFET (CNRS, FR) François GENSDARMES (IRSN, FR) Alexei GRINBAUM (CEA, FR) Antje GROBE (U.STUTTGART, DE) Peter HOET (KUL, BE) Eric QUEMENEUR (CEA, FR) Olivier LE BIHAN (INERIS, FR) Robert MUIR (NANEUM, UK) Tinh NGUYEN (NIST, USA) Bernd NOWACK (EMPA, CH) Günter OBERDÖRSTER (U. ROCHESTER, USA) David PUI (U. MINNESOTA, USA) Michael RIEDIKER (IST, CH) Yves SAMSON (CEA, FR) Ken TAKEDA (U. TOKYO, JP) Olivier WITSCHGER (INRS, FR)

The PDF contains a list of sponsor logos, the conference programme and planning documents.

All papers published in this volume of Journal of Physics: Conference Series have been peer reviewed through processes administered by the proceedings Editors. Reviews were conducted by expert referees to the professional and scientific standards expected of a proceedings journal published by IOP Publishing.

Consumer exposure is one of exposures in the life cycle perspective to risk assessment. Many nanoproducts have been commercialized in the fields of health, electronics, home, food etc. Hand dryers are widely used at public rest rooms. In this study, we selected the hand dryer having a filter coated with silver nanoparticles as a popular electronic nanoproduct. A test chamber system was developed to detect nanoaerosols released from the test nanoproduct during the operation. The particle number concentration of the chamber background was confirmed to be <1 particles/cm³ by using a condensation particle counter with a detection limit of 10 nm. When the hand dryer was turn on for 1 min, the particle number concentration in the chamber was almost linearly increased up to the peak concentration (<20 particles/cm³). It was slowly decreased after the hand dryer was turn off. When the operation was extended to 10 min, similar release pattern of nanoaerosols was observed. Therefore, it was concluded that the nanoaerosols was emitted from the hand dryer during the operation. However, the amount of released nanoaerosols was negligible, compared to indoor nanoaerosol concentration (~10,000 particles/cm³). Also, the released nanoaerosols could not be analyzed by filter sampling due to small quantity of mass.

Cascade impactors are widely used to provide particle size distributions for the study of aerosols in workplaces and ambient air. In the frame of exposure assessment to airborne particles, one of their main advantages is the possibility to perform further off-line analysis (e.g. electron microscopy, physical-chemical characterization by XRD, ICP-MS, etc.) on the collected samples according to particle size. However, the large channel width makes the particle size distributions not enough size-resolved. Furthermore, in spite of the sharpness of the collection efficiency curves, the existence of an overlap between stages renders data interpretation difficult. This work aim was to develop a modular program allowing the inversion of data stemming from cascade impactors based on the mass (or any quantity) collected on each impaction stage. Through a precise description of the collection efficiency curves of the different stages, the software provides a continuous curve (from 100 to 1000 points) using the Markowski method, and more particularly the Twomey iterative algorithm, according to several publications about inverse problems in cascade impactors. An additional option consists in determining the experimental error at each point of the inverse curve, performed by realizing several consecutive inversions. The inversion procedure was first tested and optimized for the case of the SIOUTAS personal sampler. Validation of the calculation was performed considering theoretical aerosols. Then, the software was used for two sets of data obtained during field measurement campaigns.

Most of the measurement strategies that are suggested at the international level to assess workplace exposure to nanomaterials rely on devices measuring, in real time, airborne particles concentrations (according different metrics). Since none of the instruments to measure aerosols can distinguish a particle of interest to the background aerosol, the statistical analysis of time resolved data requires special attention. So far, very few approaches have been used for statistical analysis in the literature. This ranges from simple qualitative analysis of graphs to the implementation of more complex statistical models. To date, there is still no consensus on a particular approach and the current period is always looking for an appropriate and robust method. In this context, this exploratory study investigates a statistical method to analyse time resolved data based on a Bayesian probabilistic approach. To investigate and illustrate the use of the this statistical method, particle number concentration data from a workplace study that investigated the potential for exposure via inhalation from cleanout operations by sandpapering of a reactor producing nanocomposite thin films have been used. In this workplace study, the background issue has been addressed through the near-field and far-field approaches and several size integrated and time resolved devices have been used. The analysis of the results presented here focuses only on data obtained with two handheld condensation particle counters. While one was measuring at the source of the released particles, the other one was measuring in parallel far-field. The Bayesian probabilistic approach allows a probabilistic modelling of data series, and the observed task is modelled in the form of probability distributions. The probability distributions issuing from time resolved data obtained at the source can be compared with the probability distributions issuing from the time resolved data obtained far-field, leading in a quantitative estimation of the airborne particles released at the source when the task is performed. Beyond obtained results, this exploratory study indicates that the analysis of the results requires specific experience in statistics.

Multi-walled carbon nanotubes (MWCNTs) have many beneficial characteristics, but it is concerned that exposure to MWCNTs may pose health risks. As an approach to the quantitative exposure assessment of MWCNTs, we have characterized and determined MWCNTs by elemental carbon (EC) using an aerosol carbon monitor. The EC fractions oxidized at different temperatures correspond sample characteristic such as diameter of MWCNT or origins of particles. As MWCNTs aggregate/agglomerate easily, they are usually observed as micron-size particles. Whereas, EC contained in ambient particulate matter (APM) is mainly observed in fine particles. Therefore, the size of airborne particles is a good parameter to distinguish MWCNT from other carbonaceous particles, especially APM. The size and oxidized temperature of EC suggested the origin of the carbonaceous aerosol samples. Exposure assessment of MWCNT was conducted by utilizing the size distribution of EC in the environment where particulate MWCNT or MWCNT-containing composite was handled. A procedure for exposure assessment of MWCNT-related workplace is proposed.

Air pollution harms human health and the environment. Ostrava's agglomeration and its immediate vicinity suffer regular exceeding of air pollution limits due to its geomorphologic location and present heavy industry. Maximum exceedances of air quality standards and especially PM10 which 24 hour limit value is in EU 50μg.m-3, must not be exceeded more than 35 days per year. This limit is being still often exceeded. In the year 2011 such as situation occurred 126 times. It is very important then for identify sources of air pollution to find out maximum information about air borne dust, like size distribution, chemical composition of individual size fractions, morphology of particulate matter together with other parameters like meteorological conditions, year season etc. Our measurement started two years ago. We focus on the critical situation when there are values of PM10 over a long period above the limit. In winter season it is so called inversion. By default, during the campaign it is measured size distribution of air born dust in range 5.6 nm −560nm by FMPS and using the sampler NanoId are collected samples in range 1nm – 35μm in 12 size fractions for chemical analysis and morphological observations. This contribution deals with results of size distributions only.

Nanoscale particles can be found in the air-borne, liquid-borne and surface-borne dispersed phases. Measurement techniques for nanoscale particles in all three dispersed phases are needed for the environmental, health and safety studies of nanomaterials. We present our studies on connecting the nanoparticle measurements in different phases to enhance the characterization capability. Microscopy analysis for particle morphology can be performed by depositing air-borne or liquid-borne nanoparticles on surfaces. Detection limit and measurement resolution of the liquid-borne nanoparticles can be enhanced by aerosolizing them and taking advantage of the well-developed air-borne particle analyzers. Sampling electrically classified air-borne virus particles with a gelatin filter provides higher collection efficiency than a liquid impinger.

This study investigated the use of metal impurities in single-wall carbon nanotubes (SWCNT) as potential tracers to distinguish engineered nanomaterials from background aerosols. TEM and SEM were used to characterize parent material and aerosolized agglomerates collected on PTFE filters using a cascade impactor. SEM image analysis indicated that the SWCNT agglomerates contained about 45% amorphous carbon and backscatter electron analysis indicated that metal impurities were concentrated within the amorphous carbon component. Two elements present as impurities (Y and Ni) were selected as appropriate tracers in this case as their concentrations were found to be highly elevated in the SWCNT parent material (% range) compared to ambient air particles (μg/g range), and background air concentrations were below detection limits for both elements. Bioaccessibility was also determined using physiologically-based extractions at pH conditions relevant to both ingestion and inhalation pathways. A portable wet electrostatic precipitation system effectively captured airborne Y and Ni released during sieving processes, in proportions similar to the bulk sample. These observations support the potential for catalysts and other metal impurities in carbon nanotubes to serve as tracers that uniquely identify emissions at source, after an initial analysis to select appropriate tracers.

Cellulose nanocrystals (CNCs) exhibit advantageous chemical and mechanical properties that render them attractive for a wide range of applications. During the life-cycle of CNC containing materials the nanocrystals could be released and become airborne, posing a potential inhalatory exposure risk towards humans. Absent reliable and dose-controlled models that mimic this exposure in situ is a central issue in gaining an insight into the CNC-lung interaction. Here, an Air Liquid Interface Cell Exposure system (ALICE), previously designed for studies of spherical nanoparticles, was used for the first time to establish a realistic physiological exposure test method for inhaled fiber shaped nano-objects; in this case, CNCs isolated from cotton. Applying a microscopy based approach the spatially homogenous deposition of CNCs was demonstrated as a prerequisite of the functioning of the ALICE. Furthermore, reliability and controllability of the system to nebulise high aspect ratio nanomaterials (HARN, e.g. CNCs) was shown. This opens the potential to thoroughly investigate the inhalatory risk of CNCs in vitro using a realistic exposure system.

The unique optical properties of Quantum Dots have attracted a great interest to use these nanomaterials in diverse biological applications. The synthesis of QDs by methods from the literature permits one to obtain nanocrystals coated by hydrophobic alkyl coordinating ligands and soluble in most of the cases in organic solvents. The ideal biocompatible QD must be homogeneously dispersed and colloidally stable in aqueous solvents, exhibit pH and salt stability, show low levels of nonspecific binding to biological components, maintain a high quantum yield, and have a small hydrodynamic diameter. Polymer encapsulation represents an excellent scaffold on which to build additional biological function, allowing for a wide range of grafting approaches for biological ligands. As these QD are functionalized with poly(ethylene)glycol (PEG) derivatives on their surface, they show long term stability without any significant change in the optical properties, and they are also highly stable in the most common buffer solutions such as Phosphate Buffer Saline (PBS) or borate. However, as biological studies are normally done in more complex biological media which contain a mixture of amino acids, salts, glucose and vitamins, it is essential to determine the stability of our synthesized QDs under these conditions before tackling biological studies.

Engineered nanomaterials (ENMs) quantification in complex media is an area under development, much demanded by stakeholders due to the introduction of a myriad of consumer products containing ENMs. In this work, Particle-Induced X-ray Emission (PIXE) will be shown as capable of quantifying ENMs in complex media for both in vivo and in vitro assessments using minimal conditioning. ENM quantifications (SiC, TiC, SiO₂) were performed on complex media (rat feces, rat lungs and cell culture), with applied ENM concentrations corresponding to ranges of interest for in vivo or in vitro assessments. The case studies presented in this work show the capability and versatility of PIXE measurements for biopersistence, biodistribution and dose assessment studies.

There is a consensus in the nanoecotoxicology community that better analytical tools i.e. faster and more accurate ones, are needed for the physicochemical characterisation of nanomaterials in environmentally/biologically relevant media. In this study, we introduce the concept of μTAS (Micro Total Analysis Systems), which was a term coined to encapsulate the integration of laboratory processes on a single microchip. Our focus here is on the use of a capillary electrophoresis (CE) with conductivity detection microchip and how this may be used for the measurement of dissolution of metal oxide nanomaterials. Our preliminary results clearly show promise in that the device is able to: a) measure ionic zinc in various ecotox media with high selectivity b) track the dynamic dissolution events of zinc oxide (ZnO) nanomaterial when dispersed in fish medium.

Technological advances in nanomaterials have allowed the development of new applications in industry, increasing the probability of finding airborne manufactured and engineered nano-objects in the workplace, as well as in ambient air. Scientific studies on health and environmental risks have indicated that airborne nano-objects in ambient air have potential adverse effects on the health of exposed workers and the general population. For regulatory purposes, ambient measurements of particulate matter are based on the determination of mass concentrations for PM10 and PM2.5, as regulated in the European Directive 2008/50/EC. However, this legislation is not suitable for airborne manufactured and engineered nano-objects. Parameters characterising ultrafine particles, such as particle number concentration and size distribution, are under consideration for future health-based legislation, to monitor workplaces and to control industrial processes. Currently, there are no existing regulations covering manufactured airborne nano-objects. There is therefore a clear, unaddressed need to focus on the toxicology and exposure assessment of nano-objects such as titanium dioxide (TiO₂), which are manufactured and engineered in large quantities in industry. To perform reliable toxicology studies it is necessary to determine the relevant characteristics of nano-objects, such as morphology, surface area, agglomeration, chemical composition, particle size and concentration, by applying traceable methods. Manufacturing of nanomaterials, and their use in industrial applications, also require traceable characterisation of the nanomaterials, particularly for quality control of the process. The present study arises from the OECD WPMN sponsorship programme, supported by the French Agency for Environmental and Occupational Health Safety (ANSES), in order to develop analytical methods for the characterization of TiO₂ nanoparticles in size and count size distribution, based on different techniques to characterize five different manufactured TiO₂ nanoparticles. In this study, different measurement techniques have been implemented: Transmission Electron Microscopy (TEM), Scanning Mobility Particle Sizer (SMPS) and Aerodynamic Particle Sizer (APS). The TEM results lead to a relatively good agreement between data from the manufacturer and our characterizations of primary particle size. With regard to the dustiness, the results show a strong presence of agglomerates / aggregates of primary particles and a significant presence of emitted airborne nanoparticles with a diameter below 100 nm (composed of isolated primary particles and small aggregates / agglomerates formed from a few primary particles): the number proportion of these particles varies from 0 to 44 % in the measurement range 14-360 nm depending on the types of powders and corrections of measurements.

In this work, the evolution of some key physicochemical parameters of oxide engineered nanomaterial (ENM) dispersions was studied during an in vitro biological assessment. Commercial oxide ENMs, SiO₂ and TiO₂, were dispersed in aqueous solutions (20 μg/mL) to A549 cells and N-hTERT keratinocytes and were assessed at several incubation times: 6, 24, 48, and 72 hours. The ENMs deposited dose and its particle size distribution (PSD) were followed each time. Centrifuge Liquid Sedimentation (CLS) measured the PSD and the ENMs deposited dose from a particle entity perspective, while Particle-Induced X-ray Emission (PIXE) measured the ENMs deposited dose from an elemental mass perspective. No significant variations in PSD were observed for SiO₂ ENMs during incubation in A549 cells and TiO₂ ENMs in both cell lines, while a continuous evolution of the PSD is observed for SiO₂ in N-hTERT keratinocytes. The deposited dose for TiO₂ ENMs remained stable and similar in both cell lines due to a smaller specific surface area and a higher quantity of primary particles present during incubation. It is concluded that the observed differences in the deposited dose are related to an interaction between the proteins present in the media and the ENMs specific surface.

TiO₂ nanoparticles (NPs) are contained in different kinds of industrial products including paints, self-cleaning glasses, sunscreens. TiO₂ is also employed in photocatalysis and it has been proposed for waste water treatment. Micrometric TiO₂ is generally considered a safe material, while there is concern on the possible health effects of nanometric titania. Due to their small size NPs may migrate within the human body possibly entering in the blood stream. Therefore studies on the interaction of NPs with plasma proteins are needed. In fact, the interaction with proteins is believed to ultimately influences the NPs biological fate. Fibrinogen and albumin are two of the most abundant plasma proteins. They are involved in several important physiological functions. Furthermore, fibrinogen is known to trigger platelet adhesion and inflammation. For these reasons the study of the interaction between these protein and nanoparticles is an important step toward the understanding of the behavior of NPs in the body. In this study we investigated the interaction of albumin and fibrinogen with TiO₂ nanoparticles of different crystal phases (rutile and anatase) using an integrated set of techniques. The amount of adsorbed fibrinogen and albumin for each TiO₂ surface was investigated by using the bicinchoninic acid assay (BCA). The variation of the surface charge of the NP-protein conjugates respect to the naked NPs was used to indirectly estimate both surface coverage and reversibility of the adsorption upon dilution. Surface charge was monitored by measuring the ζ potential with a conventional electrophoretic light scattering (ELS) system. The extent of protein deformation was evaluated by Raman Spectroscopy. We found that both proteins adsorb irreversibly against electrostatic repulsion, likely undergoing conformational changes or selective orientation upon adsorption. The size of primary particles and the particles aggregation rather than the crystal phase modulate the affinity of fibrinogen for the TiO₂ surfaces.

Physicochemical properties of nanoparticles (NP) strongly affect their influence on cell behaviour, but can be significantly distorted by interactions with the proteins present in biological solutions. In this study we show how different surface functionalities of zinc oxide (ZnO) NP lead to changes in the size distribution and dissolution of the NP in serum containing cell culture media and how this impacts on NP toxicity. NPs capped with weakly bound large proteins undergo substantial transformations due to the exchange of the original surface ligands to the components of the cell culture media. Conversely, NP capped with a tight monolayer of small organic molecules or with covalently conjugated proteins show significantly higher stability. These differences in ligand exchange also affect the toxicity of the NP to the HepG2 liver cell model, with the NP capped with small organic molecules being more toxic than those capped with large proteins. This study highlights the importance of characterising NPs in biological media and the effect the media has during in-vitro analysis.

Since materials on nanoscale have different characteristics from materials on macro scale their biocompatibility should be precisely and specifically investigated. Fullerenes, the third carbon allotrope, are one of the most used nanomaterials. The least stable and the most common is fullerene C₆₀. One of the main disadvantages of fullerene is its low solubility in water. In order to make it soluble, it must be functionalized with polar groups such as -OH and -COOH. From all the water soluble fullerenes the most important ones are those with -OH groups attached named fullerols. We have developed new materials for contact lenses by adding fullerene (C₆₀) and fullerol (C₆₀(OH)₂₄) into PMMA. The aim of our investigation was to compare the influences of those materials on aqueous solutions similar to tear film. For the analysis of the solutions we used opto-magnetic imaging and IR spectroscopy. The acquired spectrums were commented and compared with the standard contact lens material, which was analyzed by the same methods. The ISO 10993 cytotoxicity test on extract of nanophotonic material with incorporated C₆₀ was done as well. This research contributes to better understanding of the biocompatibility of new rigid gas permeable contact lens materials.

The increasing production volumes and commercialization of engineered nanomaterials (ENM), together with data on their higher biological reactivity when compared to bulk counterpart and ability to cross biological barriers, have caused concerns about their potential impacts on the health and safety of both humans and the environment. A multidisciplinary component of the scientific community has been called to evaluate the real risks associated with the use of products containing ENM, and is today in the process of developing specific definitions and testing strategies for nanomaterials. At ECSIN we are developing an integrated multidisciplinary methodological approach for the evaluation of the biological effects of ENM on the environment and human health. While our testing strategy agrees with the most widely advanced line of work at the European level, the choice of methods and optimization of protocols is made with an extended treatment of details. Our attention to the methodological and technical details is based on the acknowledgment that the innovative characteristics of matter at the nano-size range may influence the existing testing methods in a partially unpredictable manner, an aspect which is frequently recognized at the discussion level but oftentimes disregarded at the laboratory bench level. This work outlines the most important steps of our testing approach. In particular, each step will be briefly discussed in terms of potential technical and methodological pitfalls that we have encountered, and which are often ignored in nanotoxicology research. The final aim is to draw attention to the need of preliminary studies in developing reliable tests, a crucial aspect to confirm the suitability of the chosen analytical and toxicological methods to be used for the specific tested nanoparticle, and to express the idea that in nanotoxicology,"devil is in the detail".

Scanning Mobility Particle Sizer (SMPS) is a high resolution nanoparticle sizing system that has long been hailed as the researcher's choice for airborne nanoparticle size characterization for nano applications including nanotechnology research and development. SMPS is widely used as the standard method to measure airborne particle size distributions below 1 μm. It is composed of two devices: a Differential Mobility Analyzer (DMA) selects particle sizes thanks to their electrical mobility and a Condensation Particle Counter (CPC) enlarges particles to make them detectable by common optical counters. System raw data represent the number of particles counted over several classes of mobility diameters. Then, common inversion procedures lead to the estimation of the aerosol size distribution. In this paper, we develop a methodology to compute the uncertainties associated with the estimation of the size distribution when several experiences have been carried out. The requirement to repeat the measure ensures a realistic variability on the simulated data to be generated. The work we present consists in considering both the uncertainties coming from the experimental dispersion and the uncertainties induced by the lack of knowledge on physical phenomena. Experimental dispersion is quantified with the experimental data while the lack of knowledge is modelled via the existing physical theories and the judgements of experts in the field of aerosol science. Thus, running Monte-Carlo simulations give an estimation of the size distribution and its corresponding confidence region.

The studies on potential health risks possessed by engineered nanoparticles (NPs) have been growing rapidly. However, detailed and systemic knowledge on the uptake and biodistribution of NPs in body is still limited. Moreover, there is a need to characterize the relation between the characteristics of NPs (size, surface modifications, etc.) and their behaviours in the body. The aim of this study is to explore how these characteristics will influence the NPs uptake and biodistribution. We have successfully developed a Physiologically Based Pharmacokinetic (PBPK) model for the biodistribution of polyethylene glycol-coated polyacrylamide NPs in rats, modelling the capture and removal of NPs by phagocytizing cells. Based on this PBPK model, the behaviours of other nanoparticles (polymeric, quantum dot, silver, titanium oxide and cerium oxide NPs) are investigated, based on data from several experiments published in the literature. Size is one of the important properties to consider. Our model parameterization suggests that the uptake rate by phagocytizing cells will decrease as the size of nanoparticles increases when the removal rates for these nanoparticles are similar. This could indicate that the phagocytizing cells are saturated by the number of NPs rather than absolute mass. Nevertheless, surface modification, such as polyethylene glycol coating, may reduce the uptake rate by phagocytizing cells. With phagocytizing cells serving as a deposit of NPs, these influences of different characteristics of NPs to the behavior of phagocytizing cells could affect the fate of NPs in the body not only during the initial uptake within the first hour but also in long-term at the kinetic and dynamic levels.

Biogenic silver nanoparticles with 40.3 ± 3.5 nm size and negative surface charge (− 40 mV) were prepared with Fusarium oxysporum. The cytotoxicity of 3T3 cell and human lymphocyte were studied by a TaliTM image-based cytometer and the genotoxicity through Allium cepa and comet assay. The results of BioAg-w (washed) and BioAg-nw (unwashed) biogenic silver nanoparticles showed cytotoxicity exceeding 50 μg/mL with no significant differences of response in 5 and 10 μg/mL regarding viability. Results of genotoxicity at concentrations 5.0 and 10.0 ug/mL show some response, but at concentrations 0.5 and 1.0 μg/mL the washed and unwashed silver nanoparticles did not present any effect. This in an important result since in tests with different bacteria species and strains, including resistant, MIC (minimal inhibitory concentration) had good answers at concentrations less than 1.9 μg/mL. This work concludes that biogenic silver nanoparticles may be a promising option for antimicrobial use in the range where no cyto or genotoxic effect were observed. Furthermore, human cells were found to have a greater resistance to the toxic effects of silver nanoparticles in comparison with other cells.

This work reports the synthesis and toxicological evaluation of surface modified magnetic iron oxide nanoparticles as vehicles to carry and deliver nitric oxide (NO). The surface of the magnetic nanoparticles (MNPs) was coated with two thiol-containing hydrophilic ligands: mercaptosuccinic acid (MSA) or dimercaptosuccinic acid (DMSA), leading to thiolated MNPs. Free thiols groups on the surface of MSA- or DMSA-MNPs were nitrosated leading to NO-releasing MNPs. The genotoxicity of thiolated-coated MNPs was evaluated towards human lymphocyte cells by the comet assay. No genotoxicity was observed due to exposure of human lymphocytes to MSA- or DMSA-MNPs, indicating that these nanovectors can be used as inert vehicles in drug delivery, in biomedical applications. On the other hand, NO-releasing MPNs showed genotoxicity and apoptotic activities towards human lymphocyte cell cultures. These results indicate that NO-releasing MNPs may result in important biomedical applications, such as the treatment of tumors, in which MNPs can be guided to the target site through the application of an external magnetic field, and release NO directly to the desired site of action.

Safe development and application of nanotechnologies in many fields require better knowledge about their potential adverse effects on human health. Evidence of abilities of nanoparticles (NPs) to cross epithelial barriers and reach secondary organs via the bloodstream led us to investigate the translocation of SiO₂ NPs of 50 nm (50 nm-SiO₂-NPs) across human bronchial epithelial cells that are primary targets after exposure to inhaled NPs. We quantified the translocation of fluorescently labelled SiO₂ NPs at non-cytotoxic concentrations (5 and 10 μg/cm²) across Calu-3 epithelial monolayer. After 14 days in culture Calu-3 cells seeded onto 3 μm-polycarbonate Transwell membranes formed an efficient bronchial barrier assessed by measurement of the transepithelial electric resistance and quantification of the permeability of the monolayer. After 24 hours of exposure, we observed a significant translocation of NPs that was more important when the initial NP concentration decreased. Confocal microscopy observations revealed NP uptake by cells and an important NP retention inside the porous membrane. In conclusion, 50 nm-SiO₂-NPs can cross the human bronchial epithelial barrier without affecting the integrity of the epithelial cell monolayer.

In order to study the various health influencing parameters related to engineered nanoparticles as well as to soot emitted by Diesel engines, there is an urgent need for appropriate sampling devices and methods for cell exposure studies that simulate the respiratory system and facilitate associated biological and toxicological tests. The objective of the present work was the further advancement of a Multiculture Exposure Chamber (MEC) into a dose-controlled system for efficient delivery of nanoparticles to cells. It was validated with various types of nanoparticles (Diesel engine soot aggregates, engineered nanoparticles for various applications) and with state-of-the-art nanoparticle measurement instrumentation to assess the local deposition of nanoparticles on the cell cultures. The dose of nanoparticles to which cell cultures are being exposed was evaluated in the normal operation of the in vitro cell culture exposure chamber based on measurements of the size specific nanoparticle collection efficiency of a cell free device. The average efficiency in delivering nanoparticles in the MEC was approximately 82%. The nanoparticle deposition was demonstrated by Transmission Electron Microscopy (TEM). Analysis and design of the MEC employs Computational Fluid Dynamics (CFD) and true to geometry representations of nanoparticles with the aim to assess the uniformity of nanoparticle deposition among the culture wells. Final testing of the dose-controlled cell exposure system was performed by exposing A549 lung cell cultures to fluorescently labeled nanoparticles. Delivery of aerosolized nanoparticles was demonstrated by visualization of the nanoparticle fluorescence in the cell cultures following exposure. Also monitored was the potential of the aerosolized nanoparticles to generate reactive oxygen species (ROS) (e.g. free radicals and peroxides generation), thus expressing the oxidative stress of the cells which can cause extensive cellular damage or damage on DNA.

Oxidative stress constitutes one of the principal injury mechanisms through which particulate toxicants (asbestos, crystalline silica, hard metals) and engineered nanomaterials can induce adverse health effects. ROS may be generated indirectly by activated cells and/or directly at the surface of the material. The occurrence of these processes depends upon the type of material. Many authors have recently demonstrated that metal oxides and carbon-based nanoparticles may influence (increasing or decreasing) the generation of oxygen radicals in a cell environment. Metal oxide, such as iron oxides, crystalline silica, and titanium dioxide are able to generate free radicals via different mechanisms causing an imbalance within oxidant species. The increase of ROS species may lead to inflammatory responses and in some cases to the development of cancer. On the other hand carbon-based nanomaterials, such as fullerene, carbon nanotubes, carbon black as well as cerium dioxide are able to scavenge the free radicals generated acting as antioxidant. The high numbers of new-engineered nanomaterials, which are introduced in the market, are exponentially increasing. Therefore the definition of toxicological strategies is urgently needed. The development of acellular screening tests will make possible the reduction of the number of in vitro and in vivo tests to be performed. An integrated protocol that may be used to predict the oxidant/antioxidant potential of engineered nanoparticles will be here presented.

This study compares toxic effects of uncoated (20, 40, 60 and 80 nm) and OECD (Organization for Economic Co-operation and Development) standard citrate- and polyvinylpyrrolidone (PVP)-coated (10, 50, and 75 nm) silver nanoparticles (Ag-NPs) in J774A. 1 macrophage and HT29 epithelial cells. The cells were exposed to different concentrations (silver content) of Ag-NPs for 24 h. Analysis showed that uncoated Ag-NPs, at a concentration of 1 μg/ml, decreased cell viability by 20–40% and that 20 and 40 nm particles were 10% more cytotoxic than the 60 and 80 nm particles. In exposures to coated Ag-NPs, cell viability dropped at 25 μg/ml or higher concentrations, and the effects were also size-dependent. PVP-coated particles induced greater cytotoxicity than citrate-coated particles. Changes in sub-cellular architecture were observed in J774A. 1 cells upon exposure to test Ag-NPs. Furthermore, uncoated Ag-NPs (1 μg/mL) decreased the expression of selected cytokines including TNF-α, IL-1β, and IL-12 (p70) in J774A. 1 and IL-8 in HT29 cells. In contrast, both citrate- and PVP-coated Ag-NPs increased the expression of these cytokines at higher concentrations (25 μg/ml), and PVP-coated particles elevated cytokine levels the most. Moreover, while uncoated Ag-NPs resulted in decreased glutathione (GSH) content and increased superoxide dismutase (SOD) activity in test cells in a size-dependent manner at 1 μg/ml, coated Ag-NPs caused non-significant changes in GSH and SOD, even at the highest test concentrations. Lastly, uncoated (20 and 40 nm) at 1 μg/ml and coated Ag-NPs (10 nm PVP) at 50 μg/ml slightly increased the production of reactive oxygen species (ROS). Our data showed that uncoated Ag-NPs are more toxic than coated Ag-NPs. While uncoated Ag-NPs appear to suppress inflammatory responses and enhance oxidative stress in the test cells, coated Ag-NPs induce toxic effects through up-regulation of cytokines. Our findings support the toxicity of Ag-NPs as being size- and coating- dependent while providing additional insight on the health impact of Ag-NPs.

Impedance-based assays can constitute a reliable alternative to the conventional methods used in nanotoxicology due to the important advantages of being label-free and monitoring the cells in real-time. In this study, the suitability of impedance-monitoring for the screening of nanoparticle (NP)-induced cytotoxicity was assessed. The effect of titanium dioxide (TiO₂)-NPs on cellular proliferation, viability, spreading, and detachment from substrate was evaluated by continuous impedance-based measurements made with an xCELLigence system. Fibroblasts seeded in microelectrode-embedded E-plates were exposed to spherical anatase nano-TiO₂ (5, 10, and 40 nm in diameter) for up to 120 h. An alternative excitation signal (20 mV control voltage amplitude) was applied at 10, 25, and 50 kHz to the microelectrodes in the E-plates. Cells attached to the electrode surfaces act as insulators and lead to an increase in impedance. For validating the impedance-method, Trypan Blue exclusion and ultrahigh resolution imaging (URI) were employed. The general trend observed was a decrease in impedance following exposure to TiO₂-NPs. Impedance-based results were in most instances in accordance with those from the Trypan Blue exclusion and URI assays indicating that the impedance-based approach has merit. Further studies are needed to validate it as a high-throughput method for evaluating NPs' cytotoxicity.

Titanium and silicon dioxide nanoparticles (TiO₂ and SiO₂ NPs) are now in daily use in many commercial products of which food, sunscreens, toothpastes or cosmetics. However, their effects on human body, especially on the central nervous system, are still unclear. The aim of this study was to determine whether direct exposition of the brain to TiO₂ and SiO₂ NPs results in alternations in nervous system function. C57Bl6 mice were exposed to 5 and 10 μg doses of TiO₂ and SiO₂ NPs through intracerebroventricular administration using a stereotaxic approach. Then the neurologic effects were investigated using motor performance parameters, measured on a rotarod at 20 rpm or at an accelerating rod (from 4 to 40 rpm). Before and after injection, motor activity is registered individually for each mouse exposed, once a week, for 8 weeks. Besides, a group of 3 mice is culled at 1, 2, 3, 4 and 8 weeks after exposure in order to study the time dependant effect on the histopathology of the brain (gliosis, inflammatory process...). Both rotarod tests (accelerating and at 20 rpm) showed that TiO₂ and SiO₂ NPs exposure could significantly impair the motor performances, even several weeks after initial acute exposure. The first examination of the brain histopathology revealed microglial activation. As it appeared to grow throughout the brain in a time dependant manner this suggests the induction of a long lasting neuroinflammation. These primary findings indicated that exposure to TiO₂ and SiO₂ NPs could possibly impair the locomotor ability and this deficit may be possibly attributed at least to an inflammatory process maintained till 8 weeks after exposure in the mouse brain. To fully investigate the neurotoxicological consequences of TiO₂ and SiO₂ NPs exposure, brain contents in these NPs will be also investigated as well as other alterations like neurotransmitter levels. These preliminary data already underline the necessity of more in vivo studies to better characterize TiO₂ and SiO₂ NPs exposure effects especially on human brain for long-term and low-dose treatment.

In both pharmaceutical and toxicological fields, one of major issues has been the possibility of nanoparticle uptake to central nerve system. For the safe use of nanoparticles, it is integral to evaluate the permeability of nanoparticles through BBB. In our collaborative research group reported that a few nanoparticles accumulated in brain in animal experiment, as an in vitro model, we applied commercially available cell-based BBB model for establishing evaluation method, which is quick, quantitative and equivalent to in vivo assay. We assayed 30–1500 nm silica and surface charge dependent Qdots. Our results showed the size-dependency and the surface modification dependency. We compared our assay to several animal experiments. There are both equivalence and discrepancy with animal experiments. Our BBB model can be useful tools for evaluating size-dependent permeability, but not for surface modification-dependent permeability. Our BBB assay is non-serum assay and we have not adequately reflected the serum-related interaction between nanoparticles and cell surfaces. To clear up the discrepancy of our BBB model, serum-based assay and low-concentration detection will be needed.

Carbon nanotubes (CNTs) are important materials in advanced industries. It is a concern that pulmonary exposure to CNTs may induce carcinogenic responses. It has been recently reported that CNTs scavenge ROS though non-carbon fibers generate ROS. A comprehensive evaluation of ROS scavenging using various kinds of CNTs has not been demonstrated well. The present work specifically investigates ROS scavenging capabilities with a series of CNTs and their derivatives that were physically treated, and with the number of commercially available CNTs. CNT concentrations were controlled at 0.2 through 0.6 wt%. The ROS scavenging rate was measured by ESR with DMPO. Interestingly, the ROS scavenging rate was not only influenced by physical treatments, but was also dependent on individual manufacturing methods. Ratio of CNTs to DMPO/ hydrogen peroxide is a key parameter to obtain appropriate ROS quenching results for comparison of CNTs. The present results suggest that dangling bonds are not a sole factor for scavenging, and electron transfer on the CNT surface is not clearly determined to be the sole mechanism to explain ROS scavenging.

The nanocarrier was synthesized by nanoprecipitation, using poly(-caprolactone) (PCL) as polymer, Tween 80 as surfactant and the biopolymer chitosan (CS) as a charge modification agent. Charge, size and morphology were analyzed by zeta potential, photo correlation spectroscopy (PCS), scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). Bactericidal assays were carried out using a resistant strain of Staphylococcus aureus, and the acute ecotoxicity tests were performed with Daphnia similis. The nanoparticle without CS (PCLnp) exhibited an average size of 200 nm and zeta potential of −4.28 mV, while the nanoparticle with 0.04% (w/v) of CS (CS_PCLnp) had 250 nm and +21.3 mV. Both were stables for at least 30 days. 200 μg mL⁻¹ violacein was encapsulated in CS_PCLnp, which was dissolved in the polymer matrix, a shown by DSC analysis. The minimal inhibitory concentration against S. aureus of CS_PCLnp-vio was 25 μmol L⁻¹, while for free violacein it was > 25 μmol L⁻¹. Nanoparticles exhibited an EC50 between 0.3 – 1.1 μmol L⁻¹ with Daphnia, while free violacein was around 3.3 – 5.0 μmol L⁻¹. Thus, it was possible to control the charge of the nanoparticles, without extreme changes in size and that it is possible also to encapsulate a powerful antibactericidal compound such as violacein in nanoparticle.

Wound healing comprises four distinct phases and involves many cell events and biologic markers. The use of nanoparticles for topical application has gaining attention due to its deeper penetration in the skin and the retention capacity of the drug in the site of application. In this study the effect and toxicity of mucoadhesive polymeric nanoparticles loaded with enoxaparin was evaluated in in vivo model of skin ulcer. Our results showed an interesting formulation based on mucoadhesive nanoparticles with enoxaparin that improved wound healing without cytotoxicity in vitro in all endpoint evaluated. Then, this semi-solid formulation is a promising option for skin ulcer treatment.

The aim of this pilot study was to evaluate perspectives of the assessment of nonspecific biological effects of airborne particulate matter including nanoparticles using appropriate immunological assays. We have selected various in vitro immunological assays to establish an array allowing us to monitor activation of the cell-mediated and humoral response of both the innate and adaptive immunity. To assess comprehensive interactions and effects, the assays were performed in whole blood cultures from healthy volunteers and we used an original airborne particle mixture from high pollution period in Ostrava region representing areas with one of the most polluted air in Europe. Even if certain effects were observed, the results of the immunological assays did not prove significant effects of airborne particles on immune cells' functions of healthy persons. However, obtained data do not exclude health risks of long-term exposure to airborne particles, especially in case of individuals with genetic predisposition to certain diseases or already existing disease. This study emphasizes the in vitro assessment of complex effects of airborne particles in conditions similar to actual ones in an organism exposed to particle mixture present in the polluted air.

Renal morphological parameters were determined in rats intratracheally instilled with model cadmium-containing silica nanoparticles (Cd-SiNPs, 1mg/rat), also exploring whether their potential modifications would be associated with toxicogenomic changes. Cd-SiNP effects, evaluated 7 and 30 days post-exposure, were assessed by (i) histopathology (Haematoxylin/Eosin Staining), (ii) characterization of apoptotic features by TUNEL staining. Data were compared with those obtained by CdCl₂ (400μg/rat), SiNPs (600μg/rat), 0.1 ml saline. Area-specific cell apoptosis was observed in all treatment groups: cortex and inner medulla were the most affected regions. Apoptotic changes were apparent at 7 days post-exposure in both areas, and were still observable in inner medulla 30 days after treatment. Increase in apoptotic frequency was more pronounced in Cd-SiNP-treated animals compared to either CdCl₂ or SiNPs. Histological findings showed comparable alterations in the renal glomerular (cortex) architecture occurring in all treatment groups at both time-points considered. The glomeruli appeared often collapsed, showing condensed, packed mesangial and endothelial cells. Oedematous haemorrhagic glomeruli were also observed in Cd-SiNPs-treated animals. Bare SiNPs caused morphological and apoptotic changes without modifying the renal gene expression profile. These findings support the concept that multiple assays and an integrated testing strategy should be recommended to characterize toxicological responses to nanoparticles in mammalian systems.

Nitric oxide (NO) is involved in several physiological and pathophysiological processes, such as control of vascular tone and immune responses against microbes. Thus, there is great interest in the development of NO-releasing materials to carry and deliver NO for biomedical applications. Magnetic iron oxide nanoparticles have been used in important pharmacological applications, including drug-delivery. In this work, magnetic iron oxide nanoparticles were coated with thiol-containing hydrophilic ligands: mercaptosuccinic acid (MSA) and dimercaptosuccinic acid (DMSA). Free thiol groups on the surface of MSA- or DMSA- coated nanoparticles were nitrosated, leading to the formation of NO-releasing iron oxide nanoparticles. The cytotoxicity of MSA- or DMSA-coated magnetic nanoparticles (MNP) (thiolated nanoparticles) and nitrosated MSA- or nitrosated DMSA- coated MNPs (NO-releasing nanoparticles) were evaluated towards human lymphocytes. The results showed that MNP-MSA and MNP-DMSA have low cytotoxicity effects. On the other hand, NO-releasing MNPs were found to increase apoptosis and cell death compared to free NO-nanoparticles. Therefore, the cytotoxicity effects observed for NO-releasing MNPs may result in important biomedical applications, such as the treatment of tumors cells.

Dibucaine (DBC) is powerful long-lasting local anesthetic, but it is also considered fairly toxic to the CNS. Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) have attracted attention as carriers for drug delivery. The aim of this study was to develop and to evaluate the cytotoxic activity of DBC-loaded SLN and NLC against 3T3 fibroblast and HaCat keratinocyte cells. The SLN and NLC had myristyl myristate and Liponate®GC as their lipid matrices, respectively, plus a surfactant. SLN and NLC were characterized in terms in their diameter, size distribution, surface charge and DBC encapsulation efficiency. The particle size of SLN and NLC were around 234.33 and 166.62 nm, respectively. The polydispersity index was kept below 0.2 for both nanomaterials. Negative surface charges were observed for both nanoparticles, which decreased in the presence of the anesthetic. Encapsulation efficiency reached 76% and 90%, respectively, in SLN and NLC. DBC alone was found to be toxic to 3T3 and HaCat cells in culture. However, NLC and SLN loaded DBC decreased its intrinsic cytotoxic effect against 3T3 and HaCat cells. In conclusion, encapsulation of DBC in SLN and NLC decreased the in vitro toxicity of the local anesthetic, indicating the potential of these nanocarriers for clinical applications.

The paint and lacquer industries consider the use of nanosilver, photocatalytic active nanotitanium dioxide or nanosilica dioxide as additives for the protection of surfaces, against microbial, physical and chemical deterioration, as alternative to conventional organic based additives. Nowadays it is not clear, if nanoparticles in paints will achieve the proposed effects, since there are no long time studies available. Another fact is that the potential risks of nanoparticles for the environment and the human health is still controversial discussed.

The most sensitive entry port for nanomaterials is the lung. However other human organs/systems may also be affected by nanoparticles. Therefore the aim of the study was to assess the potential hazard effects of the three most interesting particles for paints on the gastro-intestinal tract and the immune system in vitro.

In our study we could show that:

i) Nanosilver (TEM size 25 nm) was far less toxic than silver ions of comparable concentrations tested with cells representing the gastro-intestinal tract (CaCo-2) and immune cells (Jurkat, T-lymphocytes). A significant amount of necrotic cells could be observed after exposure of CaCo-2 cells to 27 μg/ml nanosilver for 48 h.

ii) Nanotitanium dioxide can adsorb UV-light and in the presence of water hydroxyl radicals are generated photocatalytically. The exposure of CaCo-2 cells and Jurkat cells to photocatalytically active nanotitanium dioxide (Hombikat UV 100, TEM-size 15 nm) under dark conditions, didn't affected the cells significantly. However, the cells were able to incorporate nanotitanium dioxide, especially when cells were exposed to higher concentrations.

iii) Nanosilica dioxide improves the properties of the paints by increasing the water repellence. When cells were exposed to 243 μg/ml nanosilica dioxide (TEM-size 19 nm) for up to 48 h no cytotoxic effect could be observed.

Zinc oxide (ZnO) nanoparticle have been extensively applied to diverse industrial fields because they possess UV light absorption, catalytic, semi-conducting, and magnetic characteristics as well as antimicrobial property. However, up to date, toxicological effects of ZnO nanoparticles in animal models have not been completely determined. Moreover, little information is available about kinetic behaviors of ZnO nanoparticles in vivo, which will be crucial to predict their potential chronic toxicity after long-term exposure. The aim of this study was, therefore, to evaluate the pharmacokinetics and toxicokinetics of ZnO nanoparticles after single-dose and repeated dose 90-day oral administration in male and female rats, respectively. The blood samples were collected following administration of three different doses (125, 250, and 500 mg/kg) and ZnO concentration was assessed by measuring zinc level with inductively coupled plasma-atomic emission spectroscopy (ICP-AES). The result showed that the plasma ZnO concentration significantly increased in a dose-dependent manner, but decreased within 24 h after single-dose oral administration up to 500 mg/kg, without any significant difference between gender. However, when repeated dose 90-day oral toxicity study was performed, the elevated plasma concentrations did not return to normal control levels in all the cases, indicating their toxicity potential. These findings suggest that repeated oral exposure to ZnO nanoparticles up to the dose of 125 mg/kg could accumulate in the systemic circulation, thereby implying that the NOAEL values could be less than 125 mg/kg via oral intake.

We compared in vitro action of highly toxic anticancer drug doxorubicin under its delivery to the mammalian tumor cells in free form and after encapsulation in novel bio-functionalized nanoscale polymeric carrier. Such encapsulation was found to enhance significantly drug uptake by the targeted cells, as well as its cytotoxic action. 10 times higher cytotoxicity of the carrier-immobilized doxorubicin comparing to its free form was demonstrated by direct cell counting, and 5 times higher cytotoxicity of encapsulated doxorubicin was shown by FACS analysis. The polymeric carrier itself did not possess significant toxicity in vitro or in vivo (laboratory mice). The carrier protected against negative side effects of doxorubicin in mice with experimental NK/Ly lymphoma. The life duration of tumor-bearing animals treated with doxorubicin-carrier complex was significantly longer than life duration in animals treated with free doxorubicin. Besides, the effective treatment dose of the carrier-delivered doxorubicin in tumor-bearing mice was 10 times lower than such dose of free doxorubicin. Thus, novel nanoscale polymers possess high potential as drug carrier.

The influence of water temperature on the aggregation and dissolution kinetics of zinc oxide nanoparticles (ZnO NPs) (mean diameter ~40 nm) was investigated. Samples of 100 mg.L⁻¹ ZnO NPs were incubated at 15, 25 and 35 °C, similar to the surface temperature of cold freshwater, temperate estuarine, and tropical/sub-tropical coastal marine ecosystems, respectively. The natural organic matter (NOM) content, pH, electrolyte type, and ionic strength (IS), were adjusted on the basis of the water chemistries of typical aqueous systems. Specifically, the time-dependent hydrodynamic diameters (HDDs) and sedimentation plots were obtained over the first 3 h and after 24 h using time-resolved dynamic light scattering (TR-DLS) and UV-visible spectroscopy, respectively. The settling distance was further modeled for the aggregates with various HDDs according to the Stokes' sedimentation equation. The dissolution kinetics was studied over the first 12 h and after 48 h in term of percentage of released zinc ions.

The results showed that the HDD increased at elevated temperatures, termed as temperature-induced aggregation, while dissolution was reduced. The aggregation at higher temperatures further hindered the dissolution due to the decrease in the surface area of the NPs. We express this process as "aggregate-suppressed dissolution". The maximum aggregation was reached in the tropical coastal marine environment with the HDD >3 μm, and the released zinc ion of 9.2% was obtained in the cold freshwater. Based on the results, the aggregation rate of 1.57 nm.s⁻¹ was estimated for the former, and the dissolution rate of 7.44 × 10⁻⁵ mol.L⁻¹.h⁻¹ was calculated for the latter, respectively. The predicted values successfully fitted to genuine water samples (<26% deviation). This study provides useful data for environmental risk assessment of exposure of ZnO NPs to water column and benthic organisms.

Nanoparticles are reactive, and their final interactions with the surrounding media are ultimately determined by their reactivity, which in turns depends on the nanoparticles morphology, surface chemistry and environment in which they are embedded. One simple and informative approach for the study of the reactivity of nanoparticles is the determination of their photocatalytic activity. In the present work, we briefly summarize the importance of different parameters such as the size, shape and agglomeration state on the photocatalytic activity of colloidal inorganic nanoparticles. The study of the use of nanoparticles as photocatalyts is relevant not only for its potential applications in environmental remediation issues but also it can provide relevant information about the role of these parameters at the nanoscale.

Carbon nanomaterials (CNM) and molybdenum compound nanostructures are materials with various applications yet little is known regarding the toxicity of these nanoparticles in pristine form in aquatic environment. Daphnia magna standard acute toxicity test (EN ISO 6341:1996; freshwater) and Artemia salina standard acute toxicity test (ArtoxKit standard method; 15 ppt saltwater) were applied to assess the toxicity of non-modified CNM and molybdenum compound nanowires in water. It has been observed that CNM are more toxic in freshwater suspensions and somewhat more toxic than the tested molybdenum compound nanowires.

Research about the fate and behavior of engineered nanoparticles in the environment is despite its wide applications still in the early stages. The fast-growing area of nanoparticle research and the high level of uncertainty create a big challenge for describing clearly the recent state of the current scientific knowledge. Therefore, in this study the certain knowledge, the known uncertainties and the identified knowledge gaps concerning mobility of engineered metal oxide nanoparticles in porous media are analyzed. The mobility of nanoparticles is mainly investigated in model laboratory studies under well-defined conditions, which are often not realistic for natural systems. In these model systems, nanoparticles often retain in the pore system due to aggregation and sedimentation. However, under environmental conditions, the presence of natural organic matter may cause stabilization or disaggregation of nanoparticles and favors therefore higher mobility of nanoparticles. Additionally, potential higher mobility of particles using preferential flow paths is not considered. Knowledge of the long-term behavior of nanoparticles concerning disaggregation, dissolution or remobilization in soils under environmental conditions is scarce. Scientific uncertainty itself is rarely mentioned in the research papers. Seldom known methodically uncertainties in nanoparticle characterization are referred to. The uncertainty about the transferability of the results to environmental conditions is discussed more often. Due to the sparse studies concerning natural material or natural pore systems, certain conclusions concerning the mobility of nanoparticles in the soil environment are not possible to drawn.

Carbon nanotubes are promising nanostructures for many applications in materials industry and biotechnology. However, it is mandatory to evaluate their toxicity and environmental implications. We evaluated nitric acid treated multiwalled carbon nanotubes (HNO₃-MWCNT) toxicity in Nile tilapia (Oreochromis niloticus) and also the lead (Pb) toxicity modulation after the nanotube interaction. Industrial grade multiwalled carbon nanotubes [Ctube 100, CNT Co. Ltd] were treated with 9M HNO₃ for 12h at 150°C to generate oxygenated groups on the nanotube surface, to improve water dispersion and heavy metal interaction. The HNO₃-treated multiwalled carbon nanotubes were physico-chemically characterized by several techniques [e.g. TEM, FE-SEM, TGA, ζ-potential and Raman spectroscopy]. HNO₃-MWCNT did not show toxicity on Nile tilapia when the concentration ranged from 0.1 to 3.0 mg/L, and the maximum exposure time was 96h. After 24, 48, 72 and 96h the LC50 values of Pb were 1.65, 1.32, 1.10 and 0.99 mg/L, respectively. To evaluate the Pb-nanotube interaction influence on the ecotoxicity, we submitted the Nile tilapia to different concentrations of Pb mixed with a non-toxic concentration of HNO₃-MWCNT (1.0 mg/L). After 24, 48, 72, 96 h the LC50 values of Pb plus nanotubes were: 0.32, 0.25, 0.20, 0.18 mg/L, respectively. These values showed a synergistic effect after Pb-nanotube interaction since Pb toxicity increased over five times. X-ray energy dispersive spectroscopy (EDS) was used to confirm lead adsorption on the carbon nanotube oxidized surface. The exposure of Nile tilapia to Pb plus HNO₃-MWCNT caused both oxygen consumption and ammonium excretion decrease, when compared to the control. Finally, our results show that carbon nanotubes interact with classical pollutants drawing attention to the environmental implications.

The incorporation of small amounts of nanoadditives in polymeric compounds can introduce new mechanical, physical, electrical, magnetic, thermal and/or optical properties. The properties of these advanced materials have enabled new applications in several industrial sectors (electronics, automotive, textile...). In particular, for the nanomaterials (NM) described in this work, multi-walled carbon nanotubes (MWCNT) and silicon dioxide nanoparticles (SiO₂ NP), the following properties have been described: MWCNT act as nucleating agents in thermoplastics, and change viscosity, affecting dispersion, orientation, and therefore mechanical, thermal, and electrical properties; and SiO₂ NP act as flame retardant and display improved electrical and mechanical properties.

The work described here is focused on the evaluation of the migration and transformation of NM included in polymer nanocomposites (NC) during accelerated climatic ageing. To this aim, we generated polyamide 6 (PA6) NC with different degree of compatibility between the NM and the polymeric matrix. These NC were submitted to accelerated aging conditions to simulate outdoor conditions (simulation of the use phase of the polymeric NC). The NC contain as nanofillers MWCNT and SiO₂ NP with different surface properties to influence the compatibility with the polymeric matrix. The generated NC were evaluated by scanning electron microscopy (SEM), transmission electron microscopy (TEM) with Energy-dispersive X-ray spectroscopy (EDX), thermogravimetry (TGA) and differential scanning calorimetry (DSC) before and after the aging process, to monitor the compatibility of the NM with the matrix: dispersion within the matrix, migration during aging, and modification of the polymer properties.

The dispersion of SiO₂ NP in the NC depended on their compatibility with the matrix. However, independently of their compatibility with the matrix, SiO₂ NP were aggregated at the end of the accelerated aging process. In addition, degradation of the matrix and migration of nanoparticles to the surface was observed as well in the different types of SiO₂ NP aged NC. Oppositely, compatibilized MWCNT (MWCNT_MB) decreased the degradation of the polymer. Nevertheless, the nanomaterial migrated likewise to the surface during the ageing process.

In order to evaluate the possible changes in the structure of nanomaterials due to the aging process, NM were extracted from the polymer by calcination. The nanomaterials extracted were analyzed by TGA, Fourier transform infrared spectroscopy (FT-IR), BET and TEM and its properties compared with calcinated raw NM. SiO₂ hydrophilic nanoparticles were not affected by the aging process. However, both types of MWCNT were affected by the aging of the NC.

Currently, an increase of published studies regarding nano-object release into the air can be observed. In the absence of a harmonized methodology only a few studies provide quantitative data for further use. In this study, an appropriate methodology was adopted and partly enhanced for the characterization of the nanoparticle release from coatings and composites containing nanoscaled pigments. Systematic analyses at two stages of the life-cycle (non-weathered/arterially weathered) were performed for three simulated treatment processes – sanding, dynamic friction and wind erosion. Results show that the sample material, the sample composition, the sample condition (e.g. weathering state) and the type of mechanical treatment have a strong effect on the particle release. SEM, TEM and EDX analyses revealed that none of the released particles were free nanoscaled pigments.

Nano and micro reinforced glass fibre-polymer composites have been manufactured to investigate different effect such as filler type, filler size and matrix materials on the particle emission during low velocity impact test. Nano and micro- silica, as well as nanoclay reinforced crash cones were prepared with a two step extrusion process and final injection moulding of the structures. The addition of secondary filler into the glass-fibre reinforced polymer composites had significant influence on the mechanical behaviour of the material as well as on the particle emission. In general, nano and ultrafine airborne particles were emitted from all investigated materials. However, composite filled with nanoclay emitted higher amounts of particles than those filled with nano and microsilica. One reason for the increase of particle emission of the nanoclay filled composites was the change of the failure behaviour of the matrix. However, similar results of particle emission were obtained for both nano and microsilica fillers, which in general did not vary significantly from the results obtained from traditionally reinforced glass fibres polymer composites.

The treatment of waste containing nanoparticles (NP) will become a matter of first importance being given the increasing production and use of engineered NP. At present no specific end of life treatment is planned for such waste and most of the time it follows the path of conventional waste in incineration plants. The study of the behavior of NP at high temperature may help to define dedicated procedures and eventually lead to new regulations. This work deals with the set up of an incineration mounting at a laboratory scale. This assembly tested on NP samples shows significant results and interesting trends.

Much concern has been raised about the risks associated with the broad use of polymers containing nanomaterials. Much is known about degradation and aging of polymers and nanomaterials independently, but very few studies have been done in order to understand degradation of polymeric nanocomposites containing nanomaterials and the fate of these nanomaterials, which may occur in suffering many processes such as migration, release and physicochemical modifications. Throughout the UE funded FP7 project NANOPOLYTOX, studies on the migration, release and alteration of mechanical properties of commercial nanocomposites due to ageing and weathering have been performed along with studies on the feasibility of recovery and recycling of the nanomaterials. The project includes the use as model nanocomposites of Polyamide-6 (PA), Polypropylene (PP) and Ethyl Vinyl Acetate (EVA) as polymeric matrix filled with a 3% in mass of a set of selected broadly used nanomaterials; from inorganic metal oxides nanoparticles (SiO2, TiO2 and ZnO) to multi-walled carbon nanotubes (MWCNT) and Nanoclays. These model nanocomposites were then treated under accelerated ageing conditions in climatic chamber. To determine the degree of degradation of the whole nanocomposite and possible processes of migration, release and modification of the nanofillers, nanocomposites were characterized by different techniques. Additionally, recovery of the nanomaterials fro m the polymeric matrix was addressed, being successfully achieved for PA and PP based nanocomposites. In the case of PA, dissolution of the polymeric matrix using formic acid and further centrifugation steps was the chosen approach, while for PP based nanocomposites calcination was performed.

We investigated the particle release caused by the grinding of polystyrene-based composites with and without single-wall carbon nanotubes (CNTs). In the results of real-time aerosol monitoring, considerable increases in the number concentration of nano-sized aerosol particles were observed during the grinding of both CNT-containing and CNT-free polystyrene. When a thermodenuder was used, the number of released nanoparticles was reduced by over 99.9%, indicating that the nanoparticles were presumably volatile particles released by the friction heat produced by grinding the composite. In an electron microscopic analysis of the aerosol particles, micron-sized particles with protruding fibers (probably CNTs) were observed, whereas free-standing CNTs were not observed.

We present the main results demonstrating the feasibility of high surface (> A4 format size) semi-industrial fabrication of composites embedding VACNT in organic matrices. The process of growing VACNT exhibits several advantages regarding safety issues: integrating de facto a safe collecting procedure on the substrate, avoiding additional preparation steps and simplifying handling and protection by impregnation into a matrix. The following steps of the overall process: VACNT carpet functionalization, alignment control and impregnation, can be processed on-line in a closed and safe continuous process and lead to dramatically reduced direct nanotube exposure for workers and users. This project opens the route to a continuous, roll-to-roll, safer, cost-effective and green industrial process to manufacture composites with controlled and aligned greener "black" carbon nanotubes.

Nanoparticles addition is widely studied to improve properties of carbon fiber reinforced composites. Here, hybrid carbon fiber results from grafting of carbon nanotubes (CNT) by Chemical Vapor Deposition (CVD) on the carbon fiber for mechanical reinforcement and conductive properties. Both tows and woven fabrics made of the hybrid fibers are added to the matrix for composite processing. However handling hybrid fibers may induce unwilling health risk due to eventual CNT release and a protective layer is required. A thin coating layer is deposited homogeneously by low pressure plasma polymerization of an organic monomer without modifying the morphology and the organization of grafted CNTs. The polymeric layer effect on the electrical behavior of hybrid fiber is assessed by conductivity measurements. Its influence on the mechanical properties is also studied regarding the interface adhesion between fiber and matrix. The protective role of layer is demonstrated by means of friction constraints applied to the hybrid fiber.

Nanoparticles (NPs) surface functionalization with silica (SiO₂) has attracted high attention due to hydrophilicity, biocompatibility, chemical and thermal stability of silica. The present work is addressed to the production and characterization of SiO₂-coatings on titanium dioxide (TiO₂) and silver (Ag) NPs dispersed in aqueous solutions (commercial nanosols) with the aim to manage the potential risk that such NPs could generate in occupational exposure scenarios. A colloidal approach, based on principles of heterocoagulation in which opposite charged NPs are forced to coagulate in hierarchical structures, imposed by their relative size and weight ratio, was followed. The results were compared with a chemical approach, based on nucleation of silica phase from silica precursor solutions on the surfaces of TiO₂ or Ag NPs seeds. In order to increase the adhesion of silica on TiO₂ and Ag surfaces, heterocoagulated sols were spray-dried and subsequently redispersed in water to check the feasibility of such approach at industrial level. Physicochemical properties such as zeta potential, electrical conductivity, particle-size distribution, specific surface area and morphology of the samples produced with different SiO₂:TiO₂ and SiO₂:Ag weight ratios were collected and compared. Indirect evidences of silica coating were obtained.

Growth of aligned carbon nanotubes (CNT) by a secure injection CVD process on large quartz substrates and carbon fiber (CF) cloths has been reported in this study. Adjustments of CVD setup and synthesis parameters have been achieved to control the CNT growth in terms of homogeneous covering of the substrates and to tailor the length of CNT. Two parameters have been optimized such as the precursor feeding rate per reactor surface unit and the carrier gas flow rate.

Industrial needs in terms of multifunctional components are increasing. Many sectors are concerned, from the integrated direct nanoparticles production to the emerging combinations which include the metal matrix composites (MMC), ductile ceramics and ceramic matrix composites, polymer matrix composites (PMC) for bulk application and advanced surface coatings in the fields of automotive, aerospace, energy production and building applications. Moreover, domains with a planetary impact such as environmental issues, as well as aspects for instance health (toxicity) and hazard assessment (ignition and explosion severity) were also taken into account. Nanotechnologies play an important role in promoting innovation in design and realization of multifunctional products for the future, either by improving usual products or creating new functions and/or new products. Nevertheless, this huge evolution in terms of materials could only be promoted by increasing the social acceptance and by acting on the different main technological and economic challenges and developing safe oriented processes. Nowadays, a huge number of developments of nanoparticles are potentially industrial up-scalable. However, some doubts exist about the handling's safety of the current technologies. For these reasons, the main purpose was to develop a self-monitored automation in the production line coupling different techniques in order to simplify processes such as in-situ growth nanoparticles into a nanostructured matrix, over different substrates and/or the nanopowders synthesis, functionalization, dry or wet safe recovery system, granulation, consolidation in single-step, by monitoring at real time the processing parameters such as powder stoichiometry. With the aim of assuring the traceability of the product during the whole life, starting from the conception and including the R&D, the distribution and the use were also considered. The optimization in terms of processing, recovery and conditioning, permits to increase its versatility, thus leading to the increase of the added value to the production. This review put in evidence -by different examples- the progress achieved since the beginning of Nanosafe integrated initiative developed by CEA and its partners as well as promoting, firstly the safety at work place, and then extending to an integrated and controlled in-situ production by of Nanosafe-by-Design controlled processes.

The implementation in many products of manufactured nanoparticles is in strong growth and raises new questions. For this purpose, the CEA – NanoSafety Platform is developing various research topics for health and safety, environment and nanoparticles exposure in professional activities. The working group Nano-CERT/MTD, driven by INERIS, federates actors of the sector: experts, research organizations, industrial users and manufacturers of collective protection. The main activity of this group is to establish specific guidelines or a voluntary certification of collective protection, at a national scale, but with the possibility of a further extension at an European level. The group aims to establish an experimental protocol of certification to characterize collective protections for workers faced with nanomaterials potential exposure. The NanoSafety Platform provides in this presentation a method of collective protection characterization based on the developments in nanoparticles metrology and on the study of existing standards and practices in related areas (chemicals, dust, microbiological and nuclear). This study presents the results obtained during the experimental characterization of a potential nanoparticles transfer in a prototype laboratory fume hood by the use of a particulate tracer of sodium-fluorescein. The efficiency of the equipment and more specifically the efficiency of dynamical air barrier is evaluated, with the experimental results, by calculating the backward diffusion coefficient.

Manufactured nanoparticles (NPs), including titanium dioxide nanoparticles (nTiO₂), now enter in the formulation of several commercial products. Some studies have been carried out to assess the resistance of protective gloves against NPs, they have generally not considered the conditions prevailing in occupational settings.

This study was designed to evaluate the behavior of protective gloves materials against NPs in solution under conditions simulating occupational use. Mechanical deformations, simulating those produced by flexing the hand, were applied to nitrile rubber glove samples in contact with nTiO₂ in water. The first analysis showed that nTiO₂ solution penetrates some of the materials after prolonged dynamic deformations. These results were partly attributed to modifications in the physical and mechanical properties of protective gloves materials that were induced by repetitive mechanical deformations and/or the presence of the colloidal solution.

We investigated the release characteristics of single-wall carbon nanotubes (CNTs) synthesized by a pilot-scale plant. In addition to on-site aerosol measurements at the pilot-scale plant where the CNTs were synthesized, harvested, and packed, we conducted dustiness tests by vortex shaking and by transferring CNTs from one bowl to another. In the results of the on-site aerosol measurements, slight increases in the concentration were observed by aerosol monitoring instruments in the enclosure where CNTs were harvested and packed. In filter samples collected in this enclosure, micron-sized CNT clusters were observed by electron microscopy analysis. For samples collected outside the enclosure or during other processes, no CNTs were observed. The concentrations of elemental carbon at all locations were lower than the proposed occupational exposure limits of CNTs. The results of the dustiness tests revealed that submicron-sized particles were dominant in the number concentration measured by aerosol monitoring instruments, whereas micron-sized CNT clusters were mainly observed by electron microscopy analysis. The results of dustiness tests indicate that these CNTs have a low release characteristic. The lower drop impact of CNT clusters due to their lower bulk density resulted in lower CNT release from falling CNTs.

Engineered NanoMaterials (NM) offer an opportunity to develop a wide variety of new products with unique properties but many studies have shown potential OHS risks specific to NM. Addressing these risks requires knowledge about release of NM into the workplaces. This research aimed to map the state of nanotechnology OHS practices in Quebec through a questionnaire following a first contact by telephone when possible and by compiling the type and volumes of NM used as well as gathering information related to the working conditions and OHS aspects. This survey was conducted among 1310 Quebec industries and 653 researchers working in different specialties potentially involved in the development/production/distribution/integration of NM and use of NM containing products. Overall, 90 questionnaires, including 51 from the industries, were completed. These showed that NM are mainly used into the powder form, in many different sectors and deserve a wide range of markets. The prevention measures implemented vary widely from a workplace to another but about one third of the participants report that they have implemented NP adapted prevention measures but they remain worried on some specific operations. More than 50% of the participants request more information about the safe laboratory/plant design, toxicity, regulation, good work practices and prevention measures, efficiency of personal protective equipment and environmental impacts.

With an increasing number of consumer's products containing nanomaterials (NMs), Nanotechnology is already a reality on the market despite it may only be the emerging part of an iceberg. The potential exponential increase of nanotechnology uses is starting to alert major public bodies due to the uncertainty inherent of every emerging technology. According to the number of publications on safety/toxicity of NMs, major concern seems to be focused on NMs that may be released as free nano-objects. From a nanotechnologist point of view, major identified aspects on inorganic nano-objects are: i) Corrosion of metallic nano-objects, ii) Aggregation to sizes that trigger immune exacerbation, iii) Presence of toxic bystanders, iv) Biological membrane/barrier perturbation, v) Protein association & protein alteration. While the description of phenomena occurring at the nano-scale has not been classified yet, added to the contradictory studies on "unknown" samples, regulatory agencies have no clear idea on what should be considered "nano" or which NMs might pose a threat for health, safety or the environment. In addition to provide full and accessible information on new nano-product, it would seem advisable to plan research and innovation in a responsible and precautionary manner in order to be pro-active towards (responsive) NMs regulation.

Nanotechnology is considered one of the key technologies of the 21st century. The success of this fascinating technology is based on its versatility. It will bring about fundamental changes of basic research as well as of many sectors of industry and also of daily life from electronics to the health care system. However, consumers often miss reliable and understandable information on nanomaterials and all aspects of this versatile technology. A huge body of data on the potential hazards of nanoobjects towards human and environmental health already exists, but is either not easily accessible for a broad audience or presented unprocessable for nonexperts. But risk communication is an essential and thus integral component of risk management. For that purpose, the DaNa-Project aims at filling this gap by collecting and evaluating scientific results in an interdisciplinary approach with scientists from different research areas, such as human and environmental toxicology, biology, physics, chemistry, and sociology. Research findings from the field of human and environmental nanotoxicology are being prepared and presented together with material properties and possible applications for interested laymen and stakeholders. For the evaluation of literature a "Literature Criteria Checklist" has been developed as well as a Standard Operation Procedure template (SOP) based on careful scientific practice.

A novel instrument, the NanoScan SMPS (TSI 3910), is introduced here that provides the capability of employing well-established Scanning Mobility Particle Sizing (SMPS) technology in a format that can be easily utilized in the field. The use of a non-radioactive unipolar charger, radial Differential Mobility Analyzer (rDMA) and isopropanol-based Condensation Particle Counter (CPC) removes transport and application restrictions on occasion encountered with current SMPS technology. The NanoScan SMPS was compared initially to a TSI component SMPS reference system using several sizes of monodisperse silver particles. A further comparison was completed with polydisperse NaCl and Emery Oil particles. A linearity test for the instrument was performed by comparison to the ultra-fine butanol CPC (TSI 3776) as concentration reference. Subsequently, in-vehicle measurements during a freeway journey between two German cities were made to provide an example of everyday exposure to nanoparticles while driving. Coupling continuous in-vehicle measurements with GPS technology enabled an easy on-route characterization of particle size distributions, allowing source appointment of measurements, identification of evident hotspots as well as a variety of other sources of interest. Finally, the NanoScan SMPS was applied to different processes within a production test facility. Here direct comparisons between ventilation (background) and in the breathing zone were made. These measurements demonstrated the variation between the nature of the nanoparticles in the ventilation system from those within the breathing zone. During all tests the NanoScan SMPS was found to provide data that compares very well with that of the reference SMPS. It also showed a clear linearity with the CPC measurement up to concentrations as high as 10⁶ particles/cm³. In conclusion, the NanoScan SMPS was found to be a reliable tool for nanoparticle sizing and counting in applications where current SMPS instruments would prove to be challenging.

Occupational exposure to engineered nanomaterials (ENMs) is considered a new and challenging occurrence. Preliminary information from laboratory studies indicates that workers exposed to some kinds of ENMs could be at risk of adverse health effects. To protect the nanomaterial workforce, a precautionary risk management approach is warranted and given the newness of ENMs and emergence of nanotechnology, a naturalistic view of risk management is useful. Employers have the primary responsibility for providing a safe and healthy workplace. This is achieved by identifying and managing risks which include recognition of hazards, assessing exposures, characterizing actual risk, and implementing measures to control those risks. Following traditional risk management models for nanomaterials is challenging because of uncertainties about the nature of hazards, issues in exposure assessment, questions about appropriate control methods, and lack of occupational exposure limits (OELs) or nano-specific regulations. In the absence of OELs specific for nanomaterials, a precautionary approach has been recommended in many countries. The precautionary approach entails minimizing exposures by using engineering controls and personal protective equipment (PPE). Generally, risk management utilizes the hierarchy of controls. Ideally, risk management for nanomaterials should be part of an enterprise-wide risk management program or system and this should include both risk control and a medical surveillance program that assesses the frequency of adverse effects among groups of workers exposed to nanomaterials. In some cases, the medical surveillance could include medical screening of individual workers to detect early signs of work-related illnesses. All medical surveillance should be used to assess the effectiveness of risk management; however, medical surveillance should be considered as a second line of defense to ensure that implemented risk management practices are effective.

Different approaches have been adopted for assessing the potential risks of conventional chemicals and products for human health. In general, the traditional paradigm is a toxicological-driven chemical-by-chemical approach, focusing on single toxic endpoints. Scope and responsibilities for the development and implementation of a risk assessment concept vary across sectors and areas and depends on the specific regulatory environment and the specific protection goals. Thus, risk assessment implication is a complex task based not only on science based knowledge but also on the regulatory context involving different parties and stakeholders. Questions have been raised whether standard paradigms for conventional chemicals would be applicable and adequate for new materials, products and applications of nanotechnology. Most scientists and stakeholders assume that current standard methods are in principle applicable to nanomaterials, but specific aspects require further development. The paper presents additional technical improvements like the complementary use of the life cycle methodology and the support of risk-based classification systems. But also aspects improving the utility of risk assessment with regard to societal impacts on risk governance are discussed.

A new approach of assessing the risks inherent in the implementation of powders, including nanomaterials has been developed. This tool is based on the method of the OHB (Occupational Hazard Band) widely spread in the chemical industry. The European classification and CLP scales of toxicity have not been modified; only the control of exposure has been worked at. The method applies essentially to the prevention of the exposures by airborne materials, whatever their particle size. The skin exposure is not treated there specifically for the time being. The method considers exposure based on seven parameters to take into account the characteristics of the materials used, their emission potential, the conditions of use, as well as classic parameters of the characterization of the exposure as the duration and the frequency. The method stresses on a pragmatic exploitation of the current knowledge and of the available data, bearing in mind that a lot of them are not easily accessible to plant operators. The product of the reflection is then positioned on a hazard x exposure matrix from which 3 levels of priority of action are defined, as in the classical OHB method applied to pure chemical risk. This approach fills in a gap in term of risk assessment and avoids jeopardizing all that had been set up for years, while introducing newelements of reflection accessible to all operators.

Since the rise of occupational safety and health research on nanomaterials a lot of progress has been made in generating health effects and exposure data. However, when detailed quantitative risk analysis is in question, more research is needed, especially quantitative measures of workers exposure and standards to categorize toxicity/hazardousness data. In the absence of dose-response relationships and quantitative exposure measurements, control banding (CB) has been widely adopted by OHS community as a pragmatic tool in implementing a risk management strategy based on a precautionary approach. Being in charge of health and safety in a Swiss university, where nanomaterials are largely used and produced, we are also faced with the challenge related to nanomaterials' occupational safety. In this work, we discuss the field application of an in-house risk management methodology similar to CB as well as some other methodologies. The challenges and issues related to the process will be discussed. Since exact data on nanomaterials hazardousness are missing for most of the situations, we deduce that the outcome of the analysis for a particular process is essentially the same with a simple methodology that determines only exposure potential and the one taking into account the hazardousness of ENPs. It is evident that when reliable data on hazardousness factors (as surface chemistry, solubility, carcinogenicity, toxicity etc.) will be available, more differentiation will be possible in determining the risk for different materials. On the protective measures side, all CB methodologies are inclined to overprotection side, only that some of them suggest comprehensive protective/preventive measures and others remain with basic advices. The implementation and control of protective measures in research environment will also be discussed.

Despite the lack of data on the human health potential risks related to the engineered nanomaterials (ENM) exposure, ENM handling spreads in industry. The French government officially charged the InVS to develop an epidemiological surveillance of workers occupationally exposed to ENM. An initial surveillance plan was proposed on the basis of literature review and discussions with national and international ENM and occupational safety and health (OSH) experts. In site investigations and technical visits were then carried out to build an adequate surveillance system and to assess its feasibility. The current plan consists of a multi-step methodology where exposure registry construction is paramount. Workers potentially exposed to carbon nanotubes (CNT) or nanometric titanium dioxide (TiO₂) will be identified using a 3-level approach: 1-identification and selection of companies concerned with ENM exposure (based on compulsory declaration and questionnaires), 2-in site exposure assessment and identification of the jobs/tasks with ENM exposure (based on job-expose matrix, further supplemented with measurements), and 3-identification of workers concerned. Data of interest will be collected by questionnaire. Companies and workers inclusion questionnaires are designed and currently under validation. This registration is at the moment planned for three years but could be extended and include other ENM. A prospective cohort study will be established from this registry, to pursue surveillance objectives and serve as an infrastructure for performing epidemiological and panel studies with specific research objectives.

Nowadays one of the principal innovative spheres in Russia is considered to be a rapid development of nanotechnology and nanomaterials and implementation its products into almost all fields of social and industrial life. Nevertheless, despite the pointed advantages an appearance of nano products has set not only a positive example of the scientific progress development and dynamics, but also a potential threat of possible risks for the environment and people's health in the case of its massive use when there is a lack of research about its impacts. In Russian Federation the problem of nanosafety regulation is extremely actual. A number of international documents have noted a lack of legal regulation in the sphere of nanotechnologies in Russian Federation. Thus, according to the strategic EU program, it's mentioned that in 2009 on an annual meeting of OECD Tour de Table Meeting in Paris (November, 2007) Russia took responsibility to develop a long term program of nanoindustry development by 2015 (Nanotechnology Action Plan for Russia-2015). The key role should be dedicated to the nanosafety aspects and potential risk assessment of nanomaterials for environment and humans' health. Unfortunately, until now Russia did not provide this document for discussion at the international level by the partners of nanoconsortcium. Indeed, from 2007 until nowadays in Russian Federation there are no a single federal act of legislation (federal'nyi zakon), establishing the state foundations of regulation of nanosafety. The only one Federeal Legal Act in the described sphere is the Federalniy Zakon from 19 July 2007 "About the Russian corporation of nanotechnologies", which has established the principles of organization, activites, functions and termination of Rosnanotech. However, there are no articles dedicated to the environmental safety provision in regards of nano products applications. Also there is no complex federal act consolidating legal status of action in the field of development and application of nanoproducts, inspite of fact, that these relationships have a significant financial segment. Thus, according to the Federal Act from December 13, 2010 "About federal budget for 2011 year and the period of 2012 and 2013 years" the expenses for realisation of program "The infrastructure development of nano industry in Russian Federation for 2008-2011" were 250 milliard of Russian rubbles. The profound analyses of the current legal systems has demonstrated that nowadays the main legal regulation in nano sphere consist of some range of frame documents (accentuated by the author). Thereby, one of the most dynamic and developed field of innovative activities in Russian Federation – nanotechnology – is left out of environmental and legal area of protection and that is might lead to the raising level of ecological risks at the stage of creation and application nano products to the environment and humans' health. During the analyses of annual norms of the Report OECD "Nano technologies: the Environment, health and safety" the conclusion is following – 2010 Russia has an extremely low degree of conceptual realization of the program in the sphere of forming the policy connected with nanotechnology and its impact on the environment and also a lack of implementations of norms into the national legal systems in terms of the assessment criteria of nano safety EHS (Environmental, Health and Safety) and ELSI (Ethical, Legal and Social Issues). To the great regret, there is no independent and precise legal act about the ways to creat and apply nano products with the certain definitions and principles and, more importantly, with the level of legal obligations and responsibility. This gap is not possible to fill by just altering and editing the existed legal acts due to the lack of the state Russian regulation. Thus, one of the most dynamic fields of innovative activities – nano technologies – is practically out of the regulation. It might lead to an increase of ecological risks' level in the process of creation and application of nano products to the environment and health of people. As a result, implementation of international legal recommendations in the field of safety regulation of nanotechnology is quite crucial for Russian Federation.

As result of the gradually increasing nanotechnology sector there is the necessity of a contemporary analysis of the present regulations used for nanomaterials, to outline the current situation of the nanotechnology sector, to promote international cooperation and research's coordination to overcome disciplinary boundaries, to fill the gap between more and less experienced regions and to turn investments in R&D in industrial innovations.

The general objective of the Central Europe project NANOFORCE, which is developed by national and regional chemistry associations and R&D Centres of the Central Europe area, is to foster the innovative nanotechnology-sector networks across Central Europe regions by bringing together public and private organizations to carry out collaborative and interdisciplinary researches on nanomaterials (in the frame of REACH Regulation) and to turn the most promising laboratory results into innovative industrial applications.

To build up a legal advisory board for chemical enterprises starting in nanotechnology, a state of the art report on existing safety procedures and nanotech related regulations was produced to give an overview on currently available regulations used by chemical industries and manufacturing companies within the European region to secure their products. The main emphasis was placed on REACH regulation to search for relevant sections concentrating on nanomaterials which are applicable for nanotechnology. In addition, all relevant directives and amendments of REACH were screened with regard to identify gaps where action is still needed and give possible recommendations for the European Commission. Beyond literature research a questionnaire for producers, users, researchers and financiers was developed with the goal to collect information about the nanotechnology sector in the CE region concerning development, financial status, and international cooperation within joint ventures, safety and nanotoxicology.

By 1 December 2010 substances manufactured or imported in the EU ≥ 1000 t (as well as certain other substances) had to be registered under the REACH Regulation 1907/2006. The Joint Research Centre (JRC) in close cooperation with the European Chemicals Agency (ECHA) carried out an analysis and assessment of what type of information on nanomaterials was provided in the received registrations. The aim of the assessment was to develop options for an adaptation of the REACH regulation to ensure proper information generation and reporting and an appropriate risk/safety assessment of nanomaterials (Nano Support project). It should be noted that this analysis and assessment was not a compliance check of the dossiers. From 26000 submitted registration dossiers covering 4700 substances finally 25 dossiers (19 substances) were identified to cover nanomaterials or nanoforms of a substance. It is possible that other dossiers are considered to cover nanomaterials or nanoforms by the registrants, however such dossiers could not be identified to address nanoforms given the information contained in those dossiers. The identified 25 dossiers were subject to a detailed analysis and assessment of information provided for all endpoints including substance identity, physico-chemical properties, human health, environmental fate & behaviour, ecotoxicity, PBT⁶ assessment, Classification and Labelling as well as the attached Chemical Safety Report documenting the Chemical Risk/Safety Assessment. In order to evaluate how the safety of workers and consumers was ensured, it was appropriate to check how the "Derived No (Minimum) Effect Levels" (DN(M)ELs) were established for substances, covering nanomaterials or nanoforms. DNELs were established mainly for long term inhalation exposure of workers. Half of the assessed dossiers included an oral long term DNEL for the general population. DNELs were usually not specific for nanosized forms and, in the few cases where they were calculated for nanosized materials, they were not derived from hazard data for the nanoform. Different methods for deriving the DNELs were applied and few dossiers derived DNELs by applying the default assessment factors in the REACH guidance. Several DNELs were based on available Occupational Exposure Limits (OELs) for inhalable and respirable dust or the nuisance dust levels, which have not been established for nanosized materials. In general lower (i.e. less strict) assessment factors were applied with different types of justification. All DNELs were expressed in the mass metrics. It is important to note that submission, identification and selection of the dossiers addressed in this study was done before the adoption of the EC recommendation (2011/696/EU) on a definition of nanomaterial and before the publication of the revised ECHA guidance documents that include recommendations for nanomaterials.

Nanomaterials are widely present in many industrial sectors (e.g., chemical, biomedical, environment), and their application is expected to significantly expand in the coming years. However, nanomaterial use raises many questions about the potential risks to human health and the environment and, more specifically, to occupational health. The available literature supports the ability of the lung, gastrointestinal tract, and skin to act as significant barriers against systemic exposure to many nanomaterials. However, because a potential risk issue exists about the toxicity of nanomaterials to the biological material, tools need to be developed for improving the risk management of the regulators. The goal is to develop a tool that examines the current knowledge base regarding the health risks posed by engineered nanoparticles to improve nanotechnology safety prior to the marketing phase. The approach proposed during this work was to establish a safety assessment constructed on a decision-control pathway regarding nanomaterial production and consumer's product to integrate different aspects. These aspects include: (1) primarily research and identification of the nanomaterial base of physicochemical properties, toxicity, and application; (2) the occupational exposure risk during the manufacturing process; (3) and the engineered nanomaterial upon the consumer product. This approach provides important parameters to reduce the uncertainty related to the production of nanomaterials prior their commercialization, reduce the reluctance from the industry, and provide a certification tool of sanitary control for the regulators. This work provides a better understanding of a critical issue of nanomaterials and consumer safety.

In recent years, large distributed collaborative projects have become very prominent in scientific research, allowing exchanges between laboratories located in different institutions and countries and between various domains of competence. Particularly the work on nanotoxicity – a field which has only been under investigation for a few years and is still lacking regulatory framework – highlighted the need for well-controlled methods, as well as rules for the handling and disposal of used materials. To obtain comparable and reproducible results of experiments conducted in a distributed context, the standardisation and proper documentation of the applied methods is crucial. The European project NanoDiaRA, whose aim is to develop nanoparticles and biomarkers for the early diagnosis of inflammatory disease, faces this situation as it involves 15 European partners and brings together different scientific cultures and professional backgrounds. Protocols especially developed for Superparamagnetic Iron Oxide Nanoparticles and a management system were designed and implemented within the NanoDiaRA project to fulfil those needs. The main goals were the establishment of standardised Standard Operating Procedures assuring transparency and reproducibility and the provision of access to these protocols to every project partner, as well as their clear allocation to carry out precise measurements and production steps.