Final Report Summary - NEPHH (Nanomaterials-related environmental pollution and health hazards throughout their life-cycle) The nanomaterial (NM)s and nanotechnology area is an important market for the chemical industry, and the European Union (EU) as a whole. In fact, many sectors are involved ranging from mature high volume markets like automotive applications, high added value parts like space and aeronautic components or even emerging activities like new technologies for energy. Nevertheless, while nanotechnology offers a number of beneficial applications, the potential impact on the environment and human health of certain NMs is not yet fully understood. In the past two decades, polymer nanocomposites (reinforced polymers with low quantities of nanosized organic or inorganic ingredients dispersed into a thermoplastic or thermoset polymer) have emerged as a new class of materials. The use of NMs in composites production offers enormous advantages over traditional macro or microparticles and applications across a wide range of sectors are currently on the market. To date, although NMs are well implemented in some markets, limited know-how exists on the environmental and human health risks these materials pose from a life cycle perspective. The main reason is that most of the research activities related with toxicological evaluation of nanotechnology have been focused on NMs synthesised at laboratory scale or directly supplied from industrial providers, which represent the most relevant form of exposure in production facilities. With the demand growing fast, the consequent exposition of human and environment to these novel materials will undoubtedly increase, not only in the production phase but also throughout all the life cycle of nanocomposites, from NMs manufacturing, to their surface modification for a better dispersion in the polymer matrix, to the production of a variety of consumer products that undergo different end of life processes after usage stage. Therefore, the evaluation of the plausible release of NMs embedded into solid matrixes through the different life cycle stages and the associated toxicological and ecotoxicological impact of these released materials is fundamental for risk assessment. In this sense, NEPHH Project has identified and rated important forms of nanotechnology-related environmental pollution and health hazards that could result from activities involved in nano-structures throughout their life cycle, and it has also suggested means that might reduce or eliminate these impacts. NEPHH project aimed to identify and rate important forms of nanotechnology-related environmental pollution and health hazards that could result from activities involved in silicon-based polymer nanocomposites throughout their life cycle, and also to suggest means that might reduce or eliminate these impacts. Silicon based polymer nanocomposites have been selected because they are a new field of research and development in which great hopes have been placed for significant social and environmental benefits linked to a greater economic development in plastics manufacturing industry. Generated knowledge contributes to better understanding the human health and environmental impacts of the selected NMs. NEPHH's results will set a basis for the establishment of required actions for the efficient management and minimisation of risks, at the time that it also contributes to the acceptance of nanotechnology by the wide public, thus assuring its safe and sustainable introduction into market. Project context and objectives: While nanoscience and nanotechnologies offer a number of beneficial applications, the potential impact on the environment and human health of certain NMs and nanoproducts is not yet fully well understood. Conversely, there exist apprehensive domains with a planetary impact like environment and new products, and functions for health and safety of people. Not only should nanotechnologies be safely applied and produce results in the shape of useful products and services, but there should be also public consensus on their overall impact. In fact, the risk assessment of NMs has become the focus of increasing attention. To date, the widely accepted view is that there are many unanswered questions on the potential environmental and health risks associated with the manufacture, use, distribution and disposal of NMs. At present, the exposure of the general population to nanoparticles originating from dedicated industrial processes is marginal in relation to those produced and released unintentionally e.g. via combustion processes and recycling centres. The exposure to manufactured NPs is mainly concentrated on workers in nanotechnology research and nanotechnology companies. Over the next few years, more and more consumers will be exposed to manufactured NPs, at that time that it is inevitable that in the future manufactured NPs will be released gradually and accidentally into the environment. The biggest concern is that free NPs could be inhaled, absorbed through the skin, dissolved in fluids or ingested. Because of their small size and large surface area, engineered NPs may have chemical, physical, and biological properties distinctly different from larger particles of similar chemical composition. The NEPHH project has the following key motivations: 1. In early studies, engineered NPs have shown potentially toxic properties. They can enter the human body in various ways, reach vital organs via the blood stream, and possibly damage tissue. Due to their small size, the properties of NPs not only differ from bulk material of the same composition but also show different interaction patterns with the human body. The risk assessment for bulk materials is therefore not sufficient to characterise the same materials in nanoparticulate form. Information on the bioaccumulation and potential toxic effects of inhalation and / or ingestion of free engineered NPs and their long-term implications for public health is needed. The environmental consequences associated with the ultimate disposal of these materials also need to be evaluated carefully. There is a dearth of evidence about effects of pollution NPs on environment. Moreover, in common with other chemicals, NPs may reach humans and other organisms by a wide variety of environmental routes. 2. Prioritising and obtaining materials to evaluate are major challenges when studying NMs. Specific NMs with the highest exposure potentials are not well known, making it difficult to identify the most important materials to study. Obtaining materials is also an impediment. In many cases, information about the nanoscale material is proprietary. Consequently, the EU may be unable to study those materials that pose the highest potential exposure to humans. In other cases, the material may be available, but not in sufficient quantities to allow an adequate hazard evaluation, particularly regarding long term, repeated exposure studies. 3. Characterisation of NMs has proven to be more difficult than anticipated for several reasons. First, a standard nomenclature has not been developed. Second, biologists, physicists, and materials scientists working in this area do not always communicate effectively. In addition, an analytical infrastructure to allow characterisation is not consistently available or well-located. The high degree of variability in size and surface chemistry of nanoscale materials and in the coatings, crystal structure, shape, and composition used in preparing these materials increase both their complexity and the multiple permutations that must be considered in their evaluation. 4. Adequate methods to detect NMs in cells and tissues also need further development. Some of these impediments could be addressed by, for example, the development of a repository of well characterised model of NMs for use in both toxicological and biomedical research / reference standards for nanoscale particles targeted for the biomedical and toxicological research. 5. Health, safety and environmental risks that may be associated with products and applications of nanoscience and nanotechnology need to be addressed upfront and throughout their life cycle. Doing complete life cycle analysis on newly developed products, and considering all the ecological as well as the socio-economic components, will help to ensure growth and employment in the European Economic Area. 6. The implications of the special properties of NPs with respect to health and safety have not yet been taken into account by regulators. Size effects are not addressed in the framework of the new European Community regulation on chemicals and their safe use (REACH). Although production volumes for the most commonly used NMs are already approaching the REACH threshold of 1 tonne per year per company. This is why NPs raise a number of safety and regulatory issues that governments are now starting to tackle. Within this context, the NEPHH Project targets to identify and rate important forms of nanotechnology-relate environmental pollution and health hazards that could result from activities involved in nano-structures throughout their life cycle, and suggest means that might reduce or eliminate these impacts. The specific objectives for of the project are listed next: 1. To develop a systematic and continuous practice for selecting and prioritising NMs to assess their safety, environmental and human health implications. 2. To contribute to the standardisation and validation of test methods and schemes for NMs as adaptation of the current physicochemical sampling protocols to present research is envisaged. 3. To collect nanocomposites samples, including laboratory and industrial silicon based materials. These targeted materials represent an innovative selection supplementing ongoing investigations and setting a basis for future ones. 4. To achieve a better understanding of the health impacts of the selected NMs. In vitro methodologies were established for the regulatory demands for the safety assessment of nanotechnology products. 5. To assess the human and environmental exposure throughout the life cycle (synthesis and manufacturing stages) of targeted NMs according to the ISO 14.040:2006 and ISO 14.044:2006 standardised methodologies. 6. To assess the potential of NMs to damage the environment (or human health through the environment). 7. To select and disseminate the best practices (in the fields of manufacture and disposal mainly), and actuation guidelines for exposed workers, to minimise the exposure of workers, in a safe and economic manner. This included the generation and transfer of knowledge, regarding engineered NM safety issues, supporting research and regulation. 8. To contribute to the 'Code of conduct for responsible nanosciences and nanotechnologies research' action to ensure that nanotechnologies are developed in a safe manner. This objective aligned with the European Commission (EC) aims at reinforcing nanotechnology and, at the same time, boosting support for collaborative Research and development (R&D) into the potential impact of nanotechnology on human health and the environment via toxicological and ecotoxicological studies. 9. To contribute to the regulatory frameworks applicable to NMs (chemicals, worker protection, environmental legislation, product specific legislation). Important elements as the test methods and the risk assessment (hazards and exposure) methods serve as a basis for implementing legislation, administrative decisions, manufacturer's obligations or employer's obligations. The NMs selected are silicon based laboratory materials which have been supplemented with NMs from industry. On the one hand, silicon based nanoparticles including nanosilica (SiO2), layered silicates or montmorillonite (MMT), glass(nano)fibres and Foam-glass-crystal (FGCs) materials were selected. On the other hand, a total number of three engineering polymeric matrixes were selected, including polyamide (PA)s and polypropylenes (PP)s as bilsk materials and polyurethane (PU) as foamed polymeric materials. According to this selection, 12 polymer composites were produced on the combination of all NMs and polymeric matrixes. The NEPHH project consortium was made up of ten entities from seven countries, amongst which there are important benchmark references in nanotechnology research such as the University of Cranfield, the University of Technology of Cracow, the Institute of Biochemistry of the Ukrainian National Academy of Sciences, the Polytechnic University of Tomsk, the Tecnalia Research and Innovation Technology Centre and the CNRS. Furthermore, there is an important presence of industrial partners, represented by 3 Small and medium-sized enterprise (SME)s, Ekotek, Grado Sero Espace and Association for the Prevention of Accident and one large company Laviosa Chimica. Regarding the project's work plan, NEPHH project is organised in 8 Work package (WP)s which have covered the following activities: - The development of a technological surveillance system (TSS) during the WP1, resulting in a systematic, continuous practice for the selection of NMs in order to assess their safety, environmental, and human health implications. Within this WP a survey was carried out to assess the occupational health and safety procedures in place. - During the WP2, the selected NMs were synthesised and macroscale structural specimens were manufactured. This enabled the consortium to analyse the implications of such NMs from synthesis to disposal. - The WP3 involved the generation of nanoscale dust particles from the macro-scale fibre reinforced nanostructures fabricated in WP2, to consider the exposure throughout the whole life cycle of NMs in as near 'real life' exposure as possible. - The health implications (WP4) and environmental implications (WP5) of the selected NMs were assessed in parallel. The health effects of NPs on lungs, the structural study of cells and protein expression, and the genotoxicological effects were studied. During WP5 the potential of NMs to damage the environment (or human health through the environment) was assessed, based on persistence, bioaccumulation and ecotoxicity studies. Moreover, the environmental performance of nanocompounds from cradle to grave was also evaluated. - WP6 aimed to make available the understanding of the safety, environmental and health implications of NMs in order to define the appropriate measures and minimise the exposure of workers. Guidelines for responsible management of waste NMs were produced. - Finally, WP7 dealt with the dissemination of the research results and the project management was performed by the WP8. Project results: The aim of NEPHH is to identify and rate important forms of nanotechnology-related environmental pollution and health hazards that could result from activities involved in nano-structures throughout their life cycle, and to suggest means that might reduce or eliminate these impacts. During its execution, the project has considered the safety, environmental and human health implications of nanotechnology-based materials and products. The NMs selected are silicon based NMs which will be supplemented with NMs from industry. On the one hand, Silicon based NPs including (nano)silica (SiO2), layered silicates (MMT), glass (nano)fibres and FGC materials have been selected. On the other hand, a total number of three engineering polymeric matrixes have been selected, including PAs and PPs as bulk materials and PUs as foamed polymeric materials, which will be used to produce nano-induce PU (PU) foams. According to this selection, several polymer composites have been produced on the combination of all NMs and polymeric matrixes. In the following section a description of the main activities and scientific and technological results obtained is presented. WP1 - TSS The first WP of the project dealt with three main activity groups: the design and management of a TSS, the assessment of the occupational health and safety procedures for silicon based NMs at industrial and research organisations within EU, and the evaluation and development of sampling protocols for silicon based NMs. This WP set up the boundaries and scope of the project in terms of current situation of silicon based NMs. 1. Within the TSS development, a complete automated system was developed to capture, evaluate and disseminate information released about a number of topics related to nanotechnologies and, more specifically, materials of interest within NEPHH including: Health and environmental implications, sampling and sample preparation methods, physicochemical and toxicological characterisation, protective systems and working practices when manipulating NMs, regulation and standardisation in nanotechnologies, new uses and applications of NMs and events. The developed vigilance system is a tailormade one, designed and implemented in order to identify all the innovations regarding health and environmental implications of nanotechnologies, but, specifically focusing on silicon based NMs. Around 100 information sources have been tracked continuously, producing a database of 857 documents classified in aforementioned topics. With regards to the information collected, six monthly bulletins were prepared to be disseminated through the consortium and Nanosafety cluster, focused on information relating to silicon based NMs although additional general new information of special interest was also included. Moreover, to provide the consortium with the newest information, relevant information was sent to the partners at the moment it was captured. The TSS has contributed to the research developed in other WPs, as well as in the public dissemination of the project, i.e. gathering information for the newsletters of the project and the 'Guide of actuation for people working on NMs' and 'Guidelines for responsible management of waste NMs' produced in WP6. 2. Within the WP1, a second group of activities targeted to assess the occupational and environmental health and safety procedures in force in the global nanotechnology sector, by means of a survey sent to more than 800 organisations. A structured questionnaire to collect information on Environmental health and safety (EHS) programmes, engineering controls, Personal protective equipment (PPE), waste management, workplace monitoring, risk characterisation and product stewardship, was specifically developed and distributed together with the NEPHH Project information. Most organisations reported having a general EHS programme, but only half of the organisations of these reported having a nano-specific EHS programme. Overall, most organisations communicated having PPE recommendations for their employees while working with NMs, although conventional lab wear was most often reported as the recommended means of protection. Regarding the waste management practices, the majority of respondents dispose of waste containing NMs in the same manner as any other waste (either as hazardous waste or as non-hazardous waste). Finally, the majority of respondents did not perform monitoring of the workplace for NPs, due to the fact that little amounts of NMs were used and only NMs in aqueous solutions were handled. In general, the collected information shows that there is an important lack of information and knowledge about the risks associated to the NMs handling, and that high quality research and legislation development is needed. However, most of organisation surveys are willing to improve their nano-specific practices and are currently planning or implementing new measures or considerations to improve their EHS programmes. The results obtained represented the current situation related to health and safety procedures, providing a baseline for the development of the Guidelines of actuation for people and entities working with NMs in WP6. 3. The last activity within the WP1 drew and evaluated sampling protocols replicating the different life cycle stages of nanocomposites. NEPHH Consortium has defined the envisaged procedure for samples production, collection, storage, labelling and transference amongst partners. Given the importance of the samples and unknown risks associated with these, special procedures were developed covering the samples identification, maintenance and storage, as well as samples transfer to Project partners laboratories for further toxicitity and ecotoxicity investigations. This procedure is a relevant highlight of the project, as it supposes the basis for the standardisation of the testing approaches within the project that can be later replicated at a major scale. It is expected that further activities in the assessment of the toxicological and ecotoxicological potential of NMs from a life cycle perspective will contribute to an international consensus in the area of NMs testing which turns this protocol into a living document that would ideally be updated and completed as new knowledge becomes available beyond NEPHH. For this purposes, series of experiments were conducted to evaluate and optimise the samples collection and handling procedures. Mechanical performance and morphology investigations were conducted to allow successful particles generation and collection to happen (PU Foams). A crash chamber was specifically designed and constructed for the purpose of generating and sampling of NPs through drop weight impact test. For the rest of matrixes - PP and PA - a specific chamber for particles generation and handling while composites and nanocomposites drilling was designed. The generated particles from mechanical loadings were supplied to Project partners in suspension and in dry form. Additionally protocols for macrosamples abrasion followed by accelerated ageing have been designed, representing different approaches for the simulation of real life cycle stages possibly conveying the release of embedded NMs in solid matrixes. WP2 - Working NMs: Supply and preparation NEPHH's WP2 involved the synthesis and/or acquisition of selected NMs and the manufacturing of the macro-scaled structural specimens for the subsequent experiments in WP3 (dust particles generation), WP4 (Heath implications assessment) and WP5 (Environmental implications assessment). With this approach, different nanocomposites were produced, composed of a NM (nanofiller) and a polymer or composite (matrix). The production of macro-scale structural specimens involved a number of steps as hereby listed: 1. Selection and characterisation of Silicon based NPs: (Nano)Silica (SiO2), layered silicates (MMT), glass (nano)fibres and FGC materials to be used as nano-reinforcing agents. 2. Selection and characterisation of engineering polymeric matrixes - PA and PP as bulk materials and PU (PU) as a foamed matrix was carried out. 3. Polymer nanocomposites preparation by using polymers and NPs described in points 1 and 2. Afterwards, developed polymer nanocomposites were used to fabricate macro-scale structural specimens to be physically processed (WP3). Firstly, a systematic and continuous practice for selecting and prioritising NMs was set up, and the selection of the industrial NMs, required for the manufacturing of nanocomposites, was carried out, according next criteria: - the presence of nanostructures available to from uniform dispersion in polymer matrix; - anticipated compatibility with polypropylene, polyamide and PU matrices; - the possibility of using the nanofiller in extrusion technology - material can be quantitatively dosed to the polymer melt or a premix masterbatch of polymer and nanofiller can be prepared; - the detailed knowledge about the technology of production of NMs. The selection of Silicon based NPs acquired from several producers are listed below: - purified sodium montmorillonite and organically modified montmorillonite from Laviosa, Souther Clay and SGM Sebiec; - pyrogenic silica with hydrophilic and hydrophobic surface from Degussa; - glass fibres from Taiwan Glass Ind. Corp; - foamglass from Dennert Poraver GmbH. NMs acquired were subjected to initial testing towards their further application in polymeric nanocomposites. The physicochemical characterisation of the industrial NMs to be applied for preparation of polymer nanocomposites by Wide-Angle X-ray Diffraction (WAXD), Scanning electron microscopy (SEM) and thermogravimetry (TG) methods was carried out. The main goal of the analysis was to ascertain the nanostructure of the supplied nanofillers and investigate their thermal stability, especially in the range of typical processing temperatures of thermoplastic polymer matrices used in this project (PA, PP and PU). Furthermore, this activity was focused on finding possible thermal processes that could influence the structure formation and final properties of polymeric nanocomposites which would be prepared and tested in the forthcoming stages of this WP. The following conclusions were drawn from the structure, morphology and thermal behaviour analysis of these industrial nanofillers: - Structure analysis carried out by WAXD and SEM methods confirmed the presence of NPs (organobentonites, fumed silica) or nanopores (foamglass) in the tested fillers. - Nanofillers and NMs differ in shape and dimensions of elementary particles which, in original state of filler, form bigger porous or branched aggregates and agglomerates of complex morphology, that can be further dispersed in polymer. - As resulted from the TG and Differential scanning calorimetry (DSC) analysis, all of the selected nanofillers do not undergo any significant thermal processes, particularly thermal degradation, in the temperature range up to 200 degrees of Celsius, that could influence the miscibility of nanocomposite components and render difficult the formation of nanostructure. According to the physicochemical characterisation a set of representative and well-characterised fillers from industrial origin were selected, due to their structure, morphology and thermal behaviour, for their plausible application in polymer engineering nanocomposites as listed below: - Dellite 72T and Dellite 43B from Laviosa (montmorillonite). - Aerosil 974 and Aerosil 200 from Degusa (nanosilica). - TGFS 202P and TGFS 473H from Taiwan Glass Ind. Corp. (glass fibres). - Poraver2-4 from Dennert Poraver GmbH (foam glass). The following activity within WP2 dealt with laboratory NPs supply and physicochemical modification. Chemical modification of NPs using designed modification procedures can lead to desirable changes in their structure/morphology towards better compatibility with polymer matrix. In a second group of activities within WP2, physicochemical modification of laboratory NPs, specifically montmorillonite (MMT) and foamglass, was carried out. In the case of montmorillonite (sodium MMT - from southern clay deposits) was modified by exchange of inorganic metal cations with ammonium cation as an organic modifier (trimethyloctadecylammonium chloride) in water medium. The parameters of preparation of organically modified montmorillonite were experimentally tested. Lyophilisation was found to be most convenient method of drying producing fine powder and restraining formation of compact agglomerates. The obtained laboratory NPs were characterised by WAXD, SEM and TG techniques. The physicochemical modification procedure was completed by the evaluation of different ammonium modifiers and general optimisation of the procedure inding the drying process. In the foamglass area, well-founded selection of optimum raw materials was done and tested during the controlled experiment. Facile preparation routes were developed implementing chemical and X-Rayphase analysis techniques. A two-stage method for producing foamglass via the intermediate product (quenched cullet) synthesised by thermal treatment of the mixture of the certain composition was developed. Theoretical design and experimental testing of raw mixtures (including selection of chemical components) for producing foamglass and FGCl materials were performed. Initial tests towards development of a new method of low temperature foamglass synthesis were done and be continued in following activities. This approach intended to prepare raw materials for energy saving in the technology of foam-glass production by using natural and anthropogenic waste material. At processing temperatures not exceeding 950 degrees of Celsius, a residual crystal phase is preserved resulting in mechanical enhancement of FGC along with a possible reduced impact on human health and environment. After that, the next activity was focused on the selection of the polymeric matrices for nanocomposite materials PP, PA and PU (PU)) for the manufacturing of macro-scale structural specimens. This selection was based on the melt flow index value and mechanical properties of the polymers (given by the providers), and it also took into account suitable processing methods and possible application areas of different polymers grades. The next characteristics were considered: - current potential application of nanocomposites (especially engineering applications) that make engineering polymers more competitive; - ability to disperse the filler in nanometric scale, associated with polymer molecular weight and melt flow index as a relative measure of molecular weight; - polymer grade suitable for extrusion and compounding. Based on those criteria, the polymeric matrixes selected were the following ones: 1. PP Moplen HP500J from Basell Polyolefins (melt volume flow rate 4.3 g / 10 min (230 degrees of Celsius / 2.16 kg), tensile modulus 1 500 MPa, tensile stress at yield 34 MPa and Charpy notched impact strength 4 kJ / m2 (23 degrees of Celsius )). 2. Polyamide 6 (PA-6) Tarnamid T-30 from Sak?ady Asotowe w Tarnowie-Mo?cicach (melt volume flow rate 25.0 g / 10 min (275 degrees of Celsius / 5.0 kg), tensile modulus 1 100 MPa, tensile stress at yield 28 MPa Charpy notched impact strength 5 kJ / m2 (23 degrees of Celsius)). 3. Rigid PU foams (PU) were considered for their study since all the prepared materials are designed for crash test and the ability of shock absorption is not desirable. PU foam was synthesised (after a set of optimisation experiments) in a three-step process - first, to polyol (polyether RF-551) catalyst (N,N-dimethyl cyclohexylamine), water and surfactant (SR-321, Union Carbide, Marietta, GA) were added in order to prepare the polyol premix (component A). In the next step, n-pentane as a physical blowing agent was added to component A. In the third step component B (polymeric 4,4'-diphenylmethane diisocyanate (PM 200)) was added to component A and the mixture was stirred for 10 seconds with an overhead stirrer. Finally, the prepared mixtures were dropped into a mould. All the experiments were performed at ambient temperature of approximately 20 degrees of Celsius. The degree of dispersion of lamellar nanofillers in PA-6 melt was reported to be higher for polymers having higher molecular mass. The selected grades of thermoplastic polymers should be characterised by the highest value of mass flow rate (MFR) at which an injection moulding of thick-walled details for e.g. cars is possible. Moplen HP500J is a PP homopolymer with good stiffness in addition to good processability, suitable for compounding, whereby Tarnamid T-30 is PA-6 high quality engineering thermoplastic polymer fabricated in the process of ?-aminocaprolactam polycondensation. It shows high mechanical strength and high chemical/thermal resistance. The PP, PA-6 and PU polymer matrices were characterised by WAXD, SEM, TG, and DSC techniques to confirm their structure and morphology. The next activity dealt with the supplying of mineral charges and their chemical modification of the following NPs - montmorillonite, nanosilica and glass fibres - for the production of laboratory NPs to characterised latter, as well as Foamglass (laboratory material). Following, the modifications of these 4 Si-based industrial NPs are detailed: 1. Water dispersion of purified montmorillonite (MMT - from Southern Clay deposits) was modified by cation exchange of hydrated metal cations located in gallery space with the alkyl- and arylammonium salts with various chemical constitution (dimethyldioctadecyl ammonium chloride, N-p-hydroxybensyl-N-octadecyl-N,N-hydroxyethylammonium chloride, N,N,N-trimethyl-N-alkylammonium chloride, where alkyl group incorporated amide group). 2. Nanosilica and glass fibres were modified by condensation of dichlorodimethyl silane with silanol groups present on the surface of silicon dioxide-based fillers. The obtained laboratory NPs were characterised for what refers to structure, morphology and thermal analysis by WAXD, SEM, Fourier transform infrared (FTIR), DSC and TG techniques. 3. A two-stage method for producing foamglass via the intermediate product (glass granulate) synthesised by thermal treatment of the mixture of a certain composition was developed at Tomsk Polytechnic University. This product acts as the raw material for the further sponging and obtaining FGC products with the pre-set characteristics. It has been found that the temperature of charges treatment calculated according to the base glass composition increases with the rise of SiO2 quantity and with the decrease of impurities content in silica component: from 800 degrees of Celsius for charges on the basis of seolite (SiO2-63 %) to 885 degrees of Celsius for charges with marshallite (SiO2 - 95.7 %). Within the next activity, the preparation of polymer nanocomposites was accomplished. The nanocomposites of PP and PA-6 with four types of fillers (organically modified montmorillonite, nanosilica, FGC materials and glass fibres) were obtained by direct melt mixing in twin-screw extruders. In order to maintain high dispersibility of nanofillers in both apolar (polypropylene) and polar (polyamide) matrix selected nanofillers were used with proper surface functionalisation and macromolecular compatibiliser addition providing good compatibility with both types of polymers - nonpolar polypropylene and polar PA-6. Injection moulding bars of nanocomposites prepared within this task have been examined in terms of structure, morphology and thermal properties by WAXD, Small-angle X-ray diffraction (SAXD), TG, DSC, SEM, Polarised light optic microscope (POLM) and FTIR methods. On the basis of literature review and laboratory results eight different compositions (PP / MMT, PP / nanosilica, PP / FGCM, PP / glass fibres, PA6 / MMT, PA6 / nanosilica, PA6 / FGCN and PA6 / glass fibres) containing 5wt % of nanofiller were selected as a suitable material for preparation of macrosamples that would be further examined in the scheduled physical processes replicating different stages of the life cycle of nanocomposites in WP3. Selected filler concentration provided the highest content of additive in the form of well distributed and not agglomerated NPs. No other additives, e.g. thermal stabiliser, processing aids were used in order to avoid their influence on materials toxicity. Macrosamples for physical processing (crash tests) were prepared by compression moulding technique. The next task within WP2 included the preparation, through injection moulding process, of glass fibre reinforced composite panels. Two types of material were utilised to prepare the glass fibre reinforced panels: PP and PA-6 with / out 5 % glass fibres. The glass reinforced PP and PA-6 composites were based on PP homopolymer (Moplen HP500J, Basell Orlen -Basell Polyolefins Group) and PA-6 (Tarnamid T30, Sak?ady Asotowe w Tarnowie-Mo?cicach, Poland). Additionally, commercial PP and PA-6 with glass fibres (MM-PP BI 24 and MM-PA I 1F30, 30 % glass fibres, MACOMASS Verkaufs AG, Germany) were also used to manufacture injection moulded thermoplastic panels. From the manufactured glass fibre reinforced panels sandwich structure were manufactured using PU / MMT foams. Finally, PU foam was synthesised (after a set of optimisation experiments) in a three-step process comprising (i) preparation of polyol premix with auxiliary components and nanoaditives, (ii) introduction of blowing agent into polyol premix and (iii) addition of isocyanate component. High speed mechanical mixer was applied in order to enhance dispersion of nanoadditives and ensure proper mixing of reagents in the course of synthesis of PU foam. Four types of nanoinduced PU foams were prepared: PU foam / montmorillonite, PU foam / nanosilica, PU foam / FGCM and PU foam / glass fibres with 5wt % of nanofiller. The foams were analysed in terms of structure, morphology and thermal properties by WAXD, SAXD, SEM-Energy Dispersive X-Ray Analysis (EDX), POLM, TG, DSC and FTIR methods. Macroscale samples for physical processing were cut from the large foam bar and their dimensions were adjusted to 10x10x1 cm, concluding that in the frame of this WP, a set of perfectly characterised materials to be processed for nanosize dust generation (WP3) were obtained. WP3 - Dust Particles from Macro-Scale Nanostructures The WP3 focused on generating nanoscale dust particles from the macro-scale nanoreinforced nanostructures fabricated previously, in order to analyse the exposure throughout the whole life cycle of NMs in near 'real life' exposure as possible. Mainly due to the fact that the potential exposure in high performance structures (aerospace, automotive) is deemed to increase when material fracture occurs, WP3 focused on potential exposures in the transport vehicles accidents, recycling centres (especially composites ones), milling, sawing, machining, manufacture and testing operations of nanoreinforced composites. Ageing protocols were also performed to evaluate the effects of Silicon based NMs on recycling and reclamation at the end of the final product lifecycle. A preliminary study comprised the microscopic evaluation of generated samples via mechanical processing as established in the sampling protocol defined in WP1, using electron microscopy along with EDX, X-ray photoelectron spectroscopy (XPS), laser-based particle sise analysis and surface seta potential. This study shown that the incorporation of different nanofillers into a PU foam matrix enhances the mechanical properties as compared with the neat polymer. Furthermore, fracture studies of macrostructural systems using the standard system of FGC blocks fracture were accomplished. These mechanical tests were conducted to define mechanical properties and specifically to determine the tensile ultimate strength of foamglass. Foamglass samples underwent the test for compression down to their complete fracture with registering strain diagram in the automatic mode. The universal machine 'Instron 1185' with the load range between 0-100 N and 0-100 kN was used. For a comparative analysis three kinds of samples were chosen, namely FGC, industrial foamglass and laboratory foamglass obtained from cullet. According to the research findings in relation to the fracture process of foamglass crystral samples obtained in vitro it can be concluded that: - foam glass sample have higher strength as compared to the foamglass obtained from cullet (i.e. traditional foamglass); - fracture mechanisms for FGC samples is well described by the synergetic models of deformation of a quasi-viscous (amorphous) solid; - relation between the interpore partitions of FCS and the ultimate strength is similar to Hall-Ptech relation; - fracture stress is in direct proportion to the pore sise whose diminishing raises the strength of cellular materials. In addition, a literature review on impact behaviour and end of life processes for relevant nanocomposites was conducted. The results showed that there is a strong dependency between energy absorption capacity of nanocomposites and the physicochemical properties of the integrated nanostructures. Numerous studies demonstrated that nanofillers have potential in improving both stiffness and energy absorption of polymer and/or conventional fibre-reinforced polymer composites. Regarding the end of life processes (recycling, combustion and disposal to landfill), several studies were reviewed but few data is available on the degradation of polymer nanocomposites, the state of the embedded NPs during environmental exposures, or how they will be released during their life cycles. From the high performance nano and fibre polymer panels manufactured, NPs were generated by impacting PU nanocomposites with different nanofillers (montmorillonite, nanosilica, glass fibres, FGC) via low velocity impact testing. Also, NPs from polypropylenes and polyamide nano and fibre reinforced panels were generated by mechanical drilling, as impacting of these materials did not generate dust. The released particles were sampled and extracted by suspending them in solution. The solution has been filtrated in several steps and the physical and chemical properties were characterised by means of SEM, Transmission electron microscopy (TEM) measurements, Dynamic light scattering (DLS) and nanosight (SN). Additionally, the materials were investigated by WAXD, TG in inert and oxidative atmosphere DSC. Additionally, dry NPs extraction was performed, as estimated necessary for the avoidance of the occurrence of changes in solutions due to storage time required during the accomplishment of the (eco)toxicological assessment: these changes include the occurrence of biological contamination and variations in the concentrations originally reported. The results showed that NPs can be generated by different mechanical processes. Moreover the characterisation revealed that the physicochemical properties of the generated particles significantly vary depending on filler and matrix material. It is worth to highlight that by integration of MMT into the PA-6 and PP matrices particle quantity and particle size can be reduced. In the particular case of PP panels the drilling of FGC, glass fibres and MMT reinforced panels generates a similar amount of NPs, being maximum for SIO2. However, PP unreinforced generates nearly no particles. Thermal analyses were focused on the effect of mechanical degradation on the structure of polymer matrix and NPs. The presence of NPs in the dust particles was indirectly confirmed by changes in polymer melting and crystallisation behaviour comparing to the polymer dust without NPs. The aim of this study was to investigate the effect of different fillers (nanofillers and fibres) during machining process and low energy impact into reinforced composites panels. Total airborne particle concentration and particle geometric mean sise were measured by means of a Scanning mobility particle sizer (SMPS+C) and particles were sampled with an Electrostatic precipitator (ESP). In contrast with previous task, characterisation data obtained are those of airborne NPs, as collected by means of the SMPS. SEM Micrographs complete measurements on particle size distribution. The results clearly showed that by impacting of nanoreinforced PU, NPs are generated especially in the size range of less than 100 nm. Regarding the PP nanocomposites, the results showed the potential impact of machining of nano and fibre reinforced panels, as for unreinforced, just a slight increase of NPs could be measured, but for the nano-reinforced panels a huge increase could be measured. Aligned with outcomes obtained previously the integration of nanoclay into the PA matrix leaded to a decrease of particles generation. The geometric mean size vs. time distribution showed also a clear difference between the PA-6 / MMT panels. While the PA-6/MMT soon after termination of drilling showed sizes of more than 200 nm, all the others needed longer to reach larger sizes. Within WP3, a series of industrial scale dispersion tests were defined and performed by the use of equipment and techniques as close as possible to existing and future industrial production ones: on this purpose, a specifically dedicated (and set-up) pilot-plant of an Italian masterbatch producer was used (VIBA). The aim of these tests was to provide subsequent WP (WP4 and WP5) with both relevant information and materials that will be useful for the evaluation of possible environmental pollutions and health hazards related to the manufacture, use and disposal of NMs. Such tests involved the dispersion of three classes of charges and two polymeric matrices, obtaining six composite materials to be subsequently processed and analysed: SiO2 / PP, MMT / PP, glass fibre / PP, SiO2 / PA, MMT / PA and glass fibre / PA. All the industrial dispersion tests were accomplished until the production of 2-3 kg of each sample. Subsequently, a second series of industrial scale dispersion tests was performed, by extrusion of four further composite materials: ATH / EVA, ATH / MMT / EVA, MDH / EVA, MDH / MMT / EVA. These materials were selected for the LCA development in WP5. Apart from these tests, the effect of matrix and reinforcement material on the energy absorption capabilities of polymer composite structures was evaluated. The axial crash experiments of glass-reinforced polymeric cones were conducted. The low velocity impact tests were carried out using a high energy drop tower at velocities not exceeding 8 m / s. Furthermore, quasi static compression tests of the conical structures and quasi-static tensile tests were conducted using Instron electromechanical machine. The impact event was recorded using a high-speed camera with maximum speed of 1 000 frames / sec. The experiments showed that by changing the matrix and the reinforcement material it is possible to change the micro-mechanism of the crash and therefore control the energy absorption characteristics of the composite. In this sense, a significant increase in tensile properties (stiffness, strength or elongation to brake), of PA composites was shown. Furthermore, SiO2 and glass-sphere (GS) reinforcements were found to increase the energy absorption capabilities of PA materials, whereas MMT reinforcement caused a decrease in that property. On the other hand, all mechanical properties of PP were decreased after the addition of secondary reinforcement. Bad dispersion of the particles and weak filler-matrix interaction were indicated as a possible reason of that problem. The last activity of WP3 was focused on the development of NMs persistence studies. The aim was to assess the persistence of dust generated during mechanical abrasion reproducing wear and tear of polymeric product incorporating NMs. From all experiments developed, it came out that alteration / aging of dust from polymer incorporating NPs can vary not only as function of the polymer matrix but also the type of incorporated NPs. In the case of PP aging / alteration experiments indicated that Si ions were released from solid materials. Even if Si is initially present in polymers without NMs, it appeared that adding nano-SiO2 and MMT lead to higher level of Si dissolution. In all cases, aggregation of dust from mechanical abrasion occurred. Even that, the fractal dimension of the aggregates were low (1.8 to 2.1). It was shown that in the case of PP-MMT the structure of the nanoclay was modified with the release of the ammonium cations from the interlayer space. In the case of PA aging / alteration had very low effects on both properties and structures of the polymer materials. A partial dissolution of incorporated Si NMs occurred and to a larger extent in the case of Nano-SiO2 than MMT. As for PP materials, aging under light lead to the increase of particle size due to aggregation. However in the dark aggregation remained very limited. In the case of PU aging / alteration experiments revealed a slight oxidation of the PU matrix. Aggregation was also observed with mean particle size increasing from 20 to 40-60 microns. But as opposed to PA and PP incorporating Si NMs, PU - SiO2 and PU-MMT were not affected by Si dissolution indicating that NM were not at the surface of fragment produced by mechanical abrasion. This was further evidenced by SEM analysis. WP4 - Health Implications of NMs NPs are potentially more hazardous than their larger counterparts to human health. Very little information is currently available on the release of NPs throughout product life cycle and related health effect. The development of WP4 aimed to assess the toxicological mechanisms and health impacts of the selected NMs, collaborating in establishing reliable and useful in vitro methodologies for the regulatory demands of the safety assessment of nanotechnological products. To accomplish these evaluations, in vitro experiments were performed to determine effect of nanosized particles of PU, PA and PP nanocomposites containing glass fibres, glass particles, montmorillonite, silica gel and polymers alone (reference materials) generated within WP3 and collected both in liquid suspensions and in dry status. The results of these evaluations are detailed in following sections: Considering the raw NPs, Aerosil 200 as well as Aerosil 974 were analysed for blood serum lipid as well as protein oxidation. A method for blood serum lipid oxidation determination in vitro in presence of Silica NPs by measuring contents of the products of the lipid peroxidation - aldehydes reacting with thiobarbituric acid has been used. A method for blood serum proteins oxidation determination in vitro in presence of Silica NPs by measuring contents of reduced SH-groups in proteins by reaction with fluorescence probe ThioGlo has been used. Aerosil 200 as well as Aerosil 974 at concentrations 0,1; 1,0; 10; 100 mg/L do not affect (increase or decrease) blood serum lipid and proteins oxidation determined in vitro. In contrast, iron containing nanosized particles (used as a control) at concentration 25; 50; 100 mg / L statistically significant increase blood serum lipid oxidation as determined in vitro. The same control particles under identical conditions statistically significant decrease contents of reduced SH-groups in proteins which confirms increase of blood serum proteins oxidation in vitro. Aerosil 200 as well as Aerosil 974 at concentrations 0,1; 1,0; 10; 100 mg/L do not affect (increase or decrease) gluthatione content in blood serum in vitro. In contrast, iron containing nanosized particles (as a control) at concentration 10; 25; 50; 75; 100 mg / L statistically significant decrease gluthatione content in blood serum in vitro. Therefore, it can be concluded that evaluated samples do not influence blood serum lipid oxidation either protein oxidation and are inert towards the non-enzymatic antioxidant gluthatione content in blood serum as determined in vitro. In relation to the results obtained with regards to montmorillonite allow to confirm that Dellite 43B (D43B), Dellite 72T (D72T) as well as Dellite LVF (DLVF) at the investigated concentrations of 100; 200 and 600 mg / L do not effect (increase or decrease) lung bronchoalveolar lavage fluid or blood serum lipids as well as proteins oxidation as determined in vitro by Thiobarbituric acid reactive substances (TBARS) test and SH-group consumption, respectively. In contrast, iron containing nanosized particles (selected positive control) at concentration 100; 200 and 600 mg / L statistically significant increase lipid and protein oxidation as determined in vitro by TBARS test and SH-group consumption, respectively. In relation to the samples collected in liquid suspensions; tests carried out include TBARS test for blood serum lipid oxidation; fluorescence test for blood serum protein oxidation and Fluorescent Test for the evaluation of gluthatione as non-enzymatic antioxidant system. Additionally, lung bronchoalveolar lavage fluid (LBALF) was prepared and the oxidation of LBALF was determined. NPs of PU, PA and PP nanocomposites containing glass fibres, glass particles, montmorillonite, silica gel and polymers alone (reference materials) generated by mechanical means (drilling, crashing) and collected in DDW at the investigated concentrations - 50 % V / V - do not effect (increase or decrease) lung bronchoalveolar lavage fluid or blood serum lipids as well as proteins oxidation as determined in vitro by TBARS test, lipid hydroperoxide-test and SH-group consumption, respectively. In contrast, iron containing nanosized particles (used as a positive control) at concentration 10, 25 and 100 mg / L causes an statistically significant increase in lipid and protein oxidation as determined in vitro by TBARS test, lipid hydroperoxide-test and SH-group consumption, respectively. Concerning the samples delivered as dry powders, after the extraction of the nanosized fraction out, the presence of NPs in the supernatants in several samples was confirmed by TEM. However, this was not confirmed in all of them and the distribution patterns were not uniform (big agglomerates). Dosimetry should always report mass concentration but the aforementioned protocol does not allow quantifying the quantity of nanosized particles assays are being performed with. For this reason, results obtained even if positive effects are reported- should be considered cautiously. According to results obtained it can be summarised: 1. PA nanocomposites containing nanosilica gel (LO_SiO2_PA) generated by drilling and collecting as a dry dust effecting blood serum lipids oxidation in vitro. 2. PP nanocomposites containing glass fibres (LO_GNF_PP), silica gel (LO_SiO2_PP), polymers alone (LO_PP) generated by drilling and collected as a dry dust is effecting blood serum non-enzymatic antioxidant - glutathione in vitro. 3. No effect was observed for the rest of samples. The second group of investigations under WP4 were devoted to investigate toxic effect of NPs on potential cellular targets. Within this task toxic effects of different NPs have been investigated: silica and MMT NPs, dust NPs from crash and drilling tests, and dust suspension produced by mechanical abrasion and aging procedure. This work developed can be divided into three different research lines: 1. the study of different cytotoxicity endpoints including cell viability, membrane damage, oxidative stress, and inflammatory effect induced by dust NPs and raw silica NPs in vitro; 2. cytotoxicity evaluation of the selected NPs (raw NPs, samples from mechanical processing and samples from accelerated aging) by American Society for Testing and Materials (ASTM) ASTM E2526-08, including MTT ((3-4,5-Dimethylthiasol-2yetl)-2,5-diphenyltetrasolium bromide)) and Lactate Dehydrogenase (LDH) assays in two cell lines; 3. study of cellular effect of NP by toxicogenomics. Regarding the assessment of silica and MMT NPs it is worth to highlight that in lung cells (A549), skin keratocytes (HaCAT) and foetalfibroblast (MRC5) cells, all the silica NPs tested at 10-100 microgram / ml induced time dependent loss of cell viability as assessed by MTT test, and early increase in intracellular Reactive oxygen species (ROS) level and membrane damage. The effects appeared not to be concentration dependent and can be detected for the lowest concentration applied. The bioluminiscense (ATP) and biodreduction capacity (XTT) assays as well as transcriptomic studies also show that silica NPs are toxic in A549 cells in a dose-dependent manner, starting from the concentration 7.5 microgram / ml. DLVF, Aerosil 200 and dimethylbensyl hydrogenated tallow ammonium chloride DMBHT are all cytotoxic in a dose-dependent manner. On the contrary, toxicity of Dellite 43B (D43B - a commercial nanoclay deriving from MMT) and Dellite 72T (D72T - a commercial nanoclay deriving from MMT) is not dose dependent attesting a different mode of action. In the case of MMT and compared to SiO2 NPs, CI50 (determined by ATP tests) was the following D43B®: 15 µg/ml < Aerosil 200 (a commercial nanosilica): 60 microgram / ml < D72T:100 microgram / ml > DLVF:500 microgram / ml. The toxicity of Dellite mainly depends on the nature of the cationic organic molecule intercalated in the interlayers of montmorillonite. Particularly CI50 of DMBHT (embedded in MMT 43B) is 2.5 microgram / ml. Transcriptomic study allowed to obtain information about modes of action of these compounds. Study of mode of action showed that silica NPs enter cells through an endocytosis process clathrin-dependent and induce overexpression of many proinflammatory interleukins. On the other hand, MMT and DMBHT probably activate and saturate some external receptors resulting in disturbance of G-protein Coupled Receptor signalling pathway and they induce a more massive inflammation. The toxicity of raw NPs was also assessed in HepG2 and LLCPK1 cell lines. It seems that LLCPK1 cell line is more sensitive to these NPs. In addition, Dellite samples trigger higher level of toxic effects than Aerosil samples at concentrations up to 100ug/ml. Dellite 43B is the sample which triggered more toxic effects. With regards to the assessment of dust NPs from crashing and drilling tests, it is worth mentioning that the dust NPs released from different polymer-composites and after crash and drilling tests exhibit no difference in terms of cytotoxicity as compared with the NPs released from their respective neat polymers in the chosen in vitro models. At 25-100 microgram / ml, the PA based dust NPs showed toxic potency as determined by MTT assay in A549 cells. In comparison, the PU and PP based NPs showed no effect. Concerning the assessment of dust suspension produced by mechanical abrasion and aging procedure, PU suspension incorporating NPs or not did not exhibit any toxic effects while PP-MMT and PA incorporating NPs revealed some cytotoxic effect. In the case of PP-MMT the release of ammonium used as spacer in the montmorillonite is like to be at the origin of the toxicity. The analysis of transcriptome of PA nanocomposites showed that: - PA / SiO2 does not induce any inflammation response contrary to Aerosil 200 Silica NPs, according to their respective expression levels of interleukins. Moreover polymers uptake is based on macropinocytosis when Aerosil 200 silica NPs penetrate through clathrin-dependant endocytosis. - PA / MMT nanocomposite inherits from part of D43B's toxicity. The high toxicity of nanoclay D43B comes from the cocktail of toxicities of its linker DMBHT and its inorganic structure DLVF, but with a cumulative and synergistic effect, likely due to the stiffness brought by the presence of the linker. This organoclay triggers a drastic response of signal transduction, via interconnected signalling pathways and activates the molecular mechanisms of cancer, especially DNA damage through BRCA1 pathway. The nanocomposite PA/MMT displays the same toxicity through the activation of the same pathways. - The organo clay D72T seems also to induce a very drastic cellular response, likely for the same reason. Therefore, it would make sense to analyse the transcriptome of cells exposed to PP/MMT (D72T). However this way was not selected because the linker DMDHT displayed less toxicity than DMBHT, and hypothesising that the final toxicity would be due to the release of the sole linker from the nanocomposite. However our last results let us think that toxicity is due to a release of the organoclay. - Another question would be to find the minimal concentration of PA/MMT (UV) which does not induce any transcriptomic toxicity, since with tested concentrations (20 microgram / mL to 0.075 microgram / mL), toxicity was not dose-dependent. The study of the evolution of the surface properties of NPs and their relation with the cell absorption and cytotoxicity potential was also carried out within WP4. The work accomplished within this activity included the following analysis: - chemical composites of silica NPs in water and in culture medium were obtained by infrared (IR) assay; - analysis of the effects of storage temperature and duration on the chemistry of silica NPs absorption of the selected NPs by the cell; - evolution of surface properties of NPs during biological tests. In order to explore the relationship of physiochemical characteristics of silica NPs with their toxicity potential, the chemistry of silica NPs were analysed by IR. Storage conditions were also investigated for possible effect of storage on silica NPs chemistry. It was demonstrated that both Aerosil 200 and Aerosil 974 shared same IR spectra when dispersed in water stored either at 4oC or at RT, suggesting that silica NPs is stable under the two storage temperatures for 1 week. The IR spectra of the two silica NPs in culture medium were slightly different from that in water. The peak at approximately 1 410 cm-1, corresponding to the C-H bond, appeared to overlap with the peak 1384 cm-1, a hydrogen bond, which was present in the IR spectra of silica NP dispersed in culture medium but not in water. This change could be due to the adsorption of protein on the surface of silica. It appears that the hydrophobic Aerosil 974 was more reactive with proteins than the hydrophilic silica NP Aerosil 200, as indicated by the peak at ~1410-1384 cm-1 being more prevalent in the IR spectra of Aerosil 974 sample, which could imply the difference in their reactivity with cells. During the analysis of the reactivity of raw NPs within nutritive media, it was detected that their structure is strongly affected in nutritive media. Indeed, it was proved that the cationic ammonium organic compound inserted in the interlayer space of montmorillonite was partly released. This structural information combined with results obtained in WP3 strongly suggest that the toxicity of MMT is not related to the size of NP, but much more due to the chemical modification of the clay. For all raw NPs, strong aggregation occurred in nutritive media and then during biological tests. An internalisation of the three studied NPs by the HepG2 cells was observed. Nanosilica and nanoclays were mainly distributed inside the cytoplasm and on the cell membrane, but no NPs were observed in the cell nuclei. These results for silica NPs coincide with the ones reported by Ling Hu et al relating their work on the cellular endocytosis and exocytosis of silica NPs of diverse sizes in the same cell line. Although it is known that there are several possible uptake pathways for the NPs internalisation (endocytosis -fluid-phase endocytosis, adsorptive endocytosis and receptor- mediated endocytosis- phagocytosis and micropinocytosis) according to Ling Hu et al, the mechanism of clathrin mediated endocytosis can be applied universally to the silica NPs with the diameter ranging from 60nm to 600nm. Nevertheless, bigger aggregates of nanosilica have been found in the development of the project (bigger even for nanoclays). Nowadays, the maximum size for internalisation of NPs is still in debate. Although some authors indicate a maximum sise for internalisation by non-phagocyte cells (e.g. 1 um latex beads are not taken by mouse melanoma), gold nanowires that have a length of several micrometers and a 200 nm diameter have been observed inside fibroblasts and HeLa cells. Moreover, some authors have reported that some NPs aggregate during the uptake process, forming clusters inside the cells. Related documents Final Report - NEPHH (Nanomaterials-related environmental pollution and health hazards throughout their life cycle)
