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FP7

NANOPOLYTOX Résumé de rapport

Project ID: 247899
Financé au titre de: FP7-NMP
Pays: Spain

Final Report Summary - NANOPOLYTOX (Toxicological impact of nanomaterials derived from processing, weathering and recycling of polymer nanocomposites used in various industrial applications)

Executive Summary:

Nanopolytox produced 18 nanomaterials (NM) by using existing industrial techniques with tailored functionalizations when necessary, and 21 polymeric compounds or nanocomposites (NC) with a final content of 3% of nanofiller using commercial grade PA6, PP or EVA resins. NC were moulded to obtain homogeneous standard specimen tests by using current extrusion and injection industrial techniques. However, optimal dispersion could not be achieved in some nanoclay-based NC.
NM (9 metal oxide nanoparticles (MOx), 6 nanoclays and 3 MWCNT) and NC were submitted to accelerated aging simulating sunlight and climate. These NM and nanofillers extracted from non-aged and aged NC by non-destructive/mild techniques (filtration, centrifugation, calcination) were studied and compared among them in powder form. In general NM hydrated or oxidized with aging and, if present, lost partially their functional groups. These changes in their chemical structure modified their behavior in aqueous solutions, generally reducing their toxicological effect. When extracted from their matrices, even using the mildest conditions possible, nanofillers showed the same trend. The only exception were NM extracted chemically from PA6. This polymer was not completely eliminated and its residual content exerted a protective effect, avoiding NM or functional groups alteration. However, it prevented to assess on whether the NC aging process resulted in modifications of the functionalizations.
Mechanical properties of NC indicated a clear stabilizing effect of MOx and MWCNT in PP and EVA, which disappeared in almost all aged EVA NC. Contrarily, PA6 NC degraded earlier and faster than plain PA6. Studies of NC surface degradation went in the same direction. The release of nanofillers depended on polymer structure and on the shape of the nanofillers, being the fibrous (MWCNT) and platelet (nanoclays) structures the less released. For a more realistic approach NC were aged outdoors as well during an equivalent period. In general trends, only slight differences were observed in the evolution of their mechanical properties.
The different stages of the life cycle of the 18 NM were evaluated to determine the relationship between physicochemical characteristics and its (eco)toxicological impact: mechanistics, absorption, biodistribution, bioaccumulation, transformation and distribution in water/soil. In summary, ZnO was the most toxic NM tested (related to an intracellular ionization). Nanoclays were also considerably toxic, its effect mainly attributed to the organomodifier. MWCNT caused little impact regardless of their functionalization. Direct climatic aging of the raw NM only changed slightly toxicity of three MOx. It was related to chemical surface changes, which affected NM surface reactivity and interactions with the cells. All the NM were found in cytoplasmic vesicles internalized in cells excepting ZnO, and no free NM could be observed in the cytoplasm or in the nucleus.
In vivo and ecotoxicological studies centered in the most interesting NM. Oral studies indicated a very low systemic absorption, and MWCNT and MOx accumulated mainly liver and spleen. Positively charged and hydrophilic NM tended to remain longer in blood, accumulate less in those tissues and have a quicker renal excretion. Ionization of ZnO altered this biokinetic pattern. Aquatic toxicity studies showed a quick excretion of MOx partly modulated by functionalization, and a very low terrestrial toxicity. These NM did not leachate through the soil, though were affected at least by pH, salinity and the concentration of dissolved organic carbon. In most cases changes in physicochemical properties were not predicted to result on major differences at the biokinetic level and did neither result in relevant changes in cytotoxicity. However, in some cases, particularly at the recycling steps, a physicochemical change of the NM reflected on different toxic profiles and sometimes on slight differences on their predicted biokinetic fate.
Considering all this, as a proof of concept two aged NC were recycled mechanically and one chemically. Disposal of the most toxic/dangerous NM was simulated as well in one case. Although optimization of the recycling methods proposed would be needed, all the strategies proved to be valid for its industrial application. Furthermore, all the NM or NC could be reused for many different purposes. All the data were compiled in specific technical cards (publicly available) for each NM/NC and stage of its life cycle, and used to derive general trends. The complete analysis of the four NM mentioned following a comprehensive LCA approach allowed to quantify the potential environmental impact of NM during its entire life cycle. In production and transformation electricity was the parameter with greatest impact in all cases, with also an important impact of waste generation in toxicity. In the climate change potential NM impact would be mainly caused by the waste treatment of disposal/mechanical recycling in all NM, more pronounced by the use of chemical recycling. All NM had similar contribution to the overall (eco)toxicological impact, and environmental impact was associated to the energy consumption. Thus, similar strategies could be implemented to reduce the impact of the NM studied in Nanopolytox project.

Project Context and Objectives:
The main objective of NANOPOLYTOX will consist of monitoring the evolution of three families of nanomaterials (carbon nanotubes, nanoclays and metal oxide nanoparticles) during their life cycle as nanofillers in selected polymeric hosts. The synthesis of highly pure nanomaterials and the subsequent generation of nanocomposites is not per se the goal of the project. However, the control over the synthesis of these materials will ensure a correct monitoring of their evolution during the whole life cycle. Moreover, working with very pure nanomaterials will allow identifying the properties of the released nanomaterials
analyzing their analogues included in polymeric hosts (released nanomaterials are considered the same as the ones included into the polymeric matrices).
The project will include monitoring of the chemical and physical properties of the nanomaterials and their toxicity from the synthesis, processing, aging and recycling to their end of life (disposal) covering their migration and/or release during their life cycle. The theoretical analysis of the data obtained during the project will lead to the development of predictive models to assess the biological and environmental fate of the studied nanomaterials. Moreover, the overall human health and environmental impact will be assessed by LCA analysis specifically designed for nanomaterials. Additionally, three recycling strategies will be followed in order to give solutions for the disposal of both toxic and innocuous nanomaterials. For this purpose, exhaustive evaluations including the selection of adequate digestion and extraction methods to separate the nanomaterials from the polymeric matrix will be developed. The strategies proposed for the recycling process will be the following: The direct mechanical recycling of nanocomposites, the recycling of nanomaterials and polymers obtained by novel chemical separation techniques, and the recycling of polymers and immobilization of toxic nanomaterials in inert matrices.

Specific objectives

1. The preparation of eighteen highly pure and monodisperse nanomaterials from three different families (carbon nanotubes, nanoclays and metal oxide nanoparticles) including adecuate tailoring for their inclusion in three selected polymeric hosts widely used in several industrial sectors.
2. Generation of eighteen samples of nanocomposites by processing in double screw extruders and further injection in test specimens.
3. Weathering of the raw nanomaterials and the eighteen nanocomposite test specimens in climatic chambers.
4. Fully characterization (physical and chemical properties) of all the samples (raw nanomaterials and nanocomposites) during their life cycle (1000 hours) to obtain an exhaustive overview of the evolution (physical and chemical degradation) of nanomaterial’s properties along their life cycle.
5. Collection of toxicological data (in vitro and in vivo) for selected samples (nanomaterials at the different stages of their life cycle, see Figure 4, p 27) in order to evaluate the risks associated with their manufacturing, use and disposal.
6. Development of predictive models based on the data obtained for the evolution of the properties and toxicity of the nanomaterials along their life cycle, in order to contribute to nanomaterials risk assessments.
7. Detection and quantification of possible migrations and/or releases of the nanofillers from the polymeric matrices, establishing a relationship between weathering cycles and migration/release of nanomaterials.
8. Mechanical and chemical recycling for innocuous and toxic nanomaterials including the development of a new, efficient and cost effective chemical recycling technology based on specific metal oxide nanofiber filters.
9. Development of new solutions for the disposal of toxic nanomaterials as complement for recycling processes based on the inclusion of specific metal oxide nanofibers filters (containing the toxic nanomaterials) in xerogel matrices by sol-gel processes and sintering.
10. Evaluation of the human health and environmental impact of nanomaterials that are highly used in many industrial sectors during their life cycle by LCIA analysis specifically amplified by the data obtained during this and other European projects related to nanosafety.

NANOPOLYTOX will provide important information on a general concern regarding the degradability of polymer nanocomposites and their direct impact on human health and environment. It is expected that these results can prevent or minimize the exposure of workers and consumers, and releases to environment of hazardous manufactured nanomaterials.

Project Results:

SYNTHESIS OF NANOMATERIALS AND PRODUCTION OF NANOCOMPOSITES (WP1 & 2)
The first objective for the development of the NANOPOLYTOX project was the production of 18 different nanocomposites (3kg each) with industrial relevance, moulded as standard specimen tests. For this, 500g of each nanomaterial used as nanofillers were produced as follows:
The three metal oxide (SiO2, TiO2 and ZnO) nanoparticles were synthesized by L'Urederra using the Flame Spray Pyrolisys method and functionalized by Polyrise. Surface modification was reached introducing organic coatings covalently bonded onto the nanoparticles surface, using as precursors triethoxy(3-isocyanatopropyl)silane (SiO2-OH) 3-glycidoxypropyltrimethoxysilane (GPTS; TiO2-OH and ZnO-OH), propyltrimethoxysilane (propylTMS; SiO2-propyl) and octyltrimethoxysilane (octylTMS; TiO2-octyl and ZnO-octyl).
MWCNT (97.8% pure, average diameter of 25-27 nm) were synthesized by Glonatech by fluidized bed chemical vapor deposition. Afterwards, its surface was oxidized to hydroxyl groups (MWCNT-OH); to obtain amine groups (MWCNT-NH2) it was necessary an additional chemical treatment with nitric acid and ethylenediamine.
Nanoclays used were Dellite® montmorillonites (MMTdell), hydrated sodium calcium aluminium magnesium silicate hydroxides (Na,Ca)0.33(Al,Mg)2(Si4O10)(OH)2•nH2O that typically form microscopic or at least very small platy micaceous crystals (90% smectite, 0.5% cristoballite). MMTdell were modified by Laviosa, with the quaternary ammoniums dimethyl dihydrogenated tallow ammonium (34% content in type 43B) or dimethyl benzyl hydrogenated tallow ammonium (42% and 34% in 67G and 72T respectively). The cation exchange capacity of the clays used was 100 meq/100 g. The modifiers were introduced stoichiometrically, excepting in MMT67G clays, in which a 10-15% excess was used. After modification, organoclays were washed several times with warm water under pressure to eliminate the excess of modifier and other impurities. Two different size ranges of clays were used: large and small (obtained by milling the large ones), with Sympatec sizes ranging from 7-9µm and 5-7µm.
An scheme of the tailored functionalizations chosen for each NM type used as nanofiller in the project are detailed in figure 1.
L’Urederra, Leitat and Lati optimized the conditions and processed nanocomposites with metal oxide nanoparticles, MWCNT and organoclays respectively. All the nanomaterials were processed in a double screw extruder, to produce PA6 (Novamid® B24), PP (MOPLEN HP500N) or EVA (ELVAX® 550A) nanocomposites with a final content of 3% of nanofiller. All the polymers selected were of commercial grade, and the use of the selected nanomaterials had a previously demonstrated benefit over the plain polymer formulation. Final samples were injected as test specimens by injection moulding of the nanocomposites processed previously, to obtain standard specimen tests as described by the UNE-EN ISO 527 norm.
The properties of the 18 tailor functionalized nanomaterials and the 21 different types of test specimens (3 plain polymers and 18 composites) were studied using physicochemical and mechanical techniques for its posterior comparison after treatment.

ACCELERATED AGING OF NANOMATERIALS AND NANOCOMPOSITES, AND REAL WEATHERING OF NANOCOMPOSITES (WP3)
The effect of use and weathering on the nanomaterials and nanocomposites was simulated by accelerated aging in an aging chamber where both, rain and sunlight radiation, were combined. Conditions chosen based on the normative ISO 4892/06 (a standard method of exposure to laboratory light sources) were controlled during the entire process. It was used distilled water as artificial rain. To avoid any loss of material due to the formation of aerosols, each sample was aged in an independent tray with drainage for water collection. During the aging process, waters proceeding from the artificial rain collected independently for each nanocomposite were dried completely and the material collected was characterized thorough different techniques.
Simultaneously, a real weathering outdoors of the 21 types of composites was used to determine the acceleration rate achieved with artificial aging. Conditions are comparable to 11 months of exposure in a Mediterranean climate with 100 KLy of radiation and ~625 L/m2/year of rain.

PROPERTIES OF EACH NANOCOMPOSITE TYPE AND ALTERATIONS DURING ITS AGING. MIGRATION/RELEASE OF NANOMATERIALS FROM THE POLYMERIC MATRICES DUE TO AGING. MAIN CONCLUSIONS BY POLYMER/NANOMATERIAL (WP3)
The weathered test specimens were characterized using several techniques and compared with the initial materials. Their thermal properties were determined by DSC and TGA, whilst degradation of the polymeric chains was evaluated by FT-IR. ICP-MS provided information about compatibility between nanofiller-matrix. Migration and release of NM was evaluated by SEM/TEM and by the analysis of the aging waters collected, respectively. Finally, the mechanical properties of the NC were determined following the normative UNE-EN ISO 527.
Generally PA6 composites presented the smallest chemical transformation when exposed to aging. EVA and PP composites suffered radicalary degradation caused by light and the presence of oxygen, and degraded forming new carbonyl groups. Oxidation, chain breakage and cross-linking reactions occurred in PP as well.
Nor SiO2 neither the other metal oxide nanoparticles had significant protective effect in PP or in PA6 matrices, but increased resistance of PA6. With aging PA6 surface showed chemical degradation only with ZnO, though PP composites degraded slightly more intensively, and TiO2 nanoparticles were more exposed after aging. Mechanically, the addition of ZnO (and at a lower degree TiO2), increased the ductility of the composites after aging. The three metal oxides delayed EVA degradation but, in the surface, TiO2 and ZnO induced a strong degradation in EVA and PA6 which favoured the exposure of these nanofillers. It was due to the photocatalytic activity associated to both nanoparticle types; in PA6 it caused a heterogeneous degradation with greyish regions containing mainly nanoparticles that had lost its functionalization. In spite of this, TiO2 nanoparticles increased flexibility in EVA.
The release pattern changed depending on the matrix and the metal oxide used. Compared with the plain polymer, SiO2 favoured slightly the release of material in EVA and intensely in PP (~10 fold higher). When analyzing the nanofiller content released, however, in the three matrices was ~30%, and the particles could be clearly identified. Thus, SiO2 nanoparticles seemed to migrate equally independently of the polymer where were embedded, and its rate of release was more related to the resistance of the matrix itself. With TiO2 nanoparticles, the intense surface degradation of EVA and PA6 allowed migration and release of the nanofiller up to 20% of the total material. ZnO nanoparticles showed a completely different pattern when referring to release: in PA6, the hygroscopic nature of the polymer favoured the leaching/dissolution of the nanofiller to cationic Zn (Zn2+). The more hydrophobic nature of EVA and PP prevented the contact between nanofillers and water, reducing the amount of ZnO/Zn2+ released ZnO. However, the material released contained a <10 times the amount of inorganic content expected, presumably proceeding entirely from ZnO. As it has been extensively demonstrated that the toxicity of these nanoparticles is essentially due to its ionization, special attention must be put in reducing the degradation rate of the nanocomposites produced using ZnO as nanofiller, particularly when using hygroscopic/hydrophilic matrices.
With NWCNT, as it had been described before, our results confirmed the reinforcing and protective effect in PP, allowing the retention of the tensile properties, giving extra heat resistance to the polymer and avoiding photooxidation, even after aging. In PA6, the positive nucleating effect of MWCNT was masked by a bad compatibilization nanofiller-matrix that caused a significant release of nanomaterial (<1g/m2) not observed in the other MWCNT formulations. However, the reinforcement of the composite was evident, especially after aging. Finally, nanotubes in EVA promoted photo-oxidation during aging absorbing UV light, but causing a local warming that favoured the oxidation of the polymer around nanotubes and reorganizing the polymer chains. As a result, EVA with MWCNT became more flexible after aging.
The good performance in the different matrices studied makes this nanomaterial a suitable option to improve mechanical and thermal properties in several polymer types. In addition, resistance to aging conditions would indicate that these composites could probably be recycled at its end of life, to produce new materials and reduce the environmental impact. Indeed, comparing with the other nanofillers used, the positive effect of MWCNT in the three polymer types must be highlighted.
The effect of the accelerated aging on EVA+MMTdell72T/small was globally positive, with improvement in some values: initially a slight hardening and stiffening after aging. In terms of degradation, both EVA+MMTdell formulations showed a significant increase in the retained thermal properties (slight acetate degradation, as occurred in EVA+MWCNT). In PA6+67G/small and PP+43B/small ductility increased respecting the plain polymer, but the degradation was essentially the same as in the neat polymer and nanoclays were more exposed in the surface after aging. A reduction of crystallinity in PP was attributed to a bad exfoliation of both organoclays (normal sized and small). Waters collected from nanoclay formulations contained the smallest amount of material of the nanocomposites studied (essentially organic residue), proceeding from the degradation of the matrix and/or the release of organomodifier. With a release of 50 mg/m2 in what would equate approximately one year of use, PP+MMTdell43B/small NC clearly released the smallest amount of material. This was the result of a reduced degradation of polymer and the shape and size of nanoclays, which made more difficult its migration within the polymer and to its surface. Nevertheless, we demonstrated that the toxicity of this organoclays is mainly driven by its organomodifier, and is the latter which migrates and releases from the matrix. Thus, apparently, the release of those organic groups from NC to the environment should not be despised, as could cause toxic effects to living organisms. On the other side, nanoclays do not migrate within the polymer, leaving recycling of these NC as a suitable option.
In summary, the release of nanofillers from the polymeric matrices depends on polymer structure and on the shape of the nanofillers, being the fibrous (MWCNT) and platelet (nanoclays) structures those that are less released. It has been proven that it is necessary to determine the risk for human health and environment, as there is potential exposure to toxic materials in all the cases studied excepting in EVA+MWCNT and PP+MWCNT.
These results provide important information on a general concern regarding the new properties provided by nanomaterials in three of the most widely used thermoplastics, degradability of polymer nanocomposites and their direct impact on human health and environment. It is expected that this information will be useful to minimize the exposure of workers and consumers, and reduce release to environment of hazardous manufactured nanomaterials.

CHANGES IN THE PHYSICAL AND CHEMICAL PROPERTIES OF NANOMATERIALS ALONG THE PHASES OF THEIR LIFE CYCLE STUDIED (WP4, WP6)
Exhaustive evaluations including the selection of adequate digestion and extraction methods to separate the nanomaterials from the polymeric matrix were developed with three main objectives: i) determine the degree of affectation of the nanofillers when aged embedded in polymeric matrices, ii) study their characteristics (structure, chemical composition and toxicity) at different stages of their life cycle, and iii) determine de feasibility of its recycling as industrial products.
Nanomaterials in powder form (before and after aging) and materials recovered from non-aged and aged nanocomposites were characterized anew using the same physicochemical techniques: ICP-MS, TGA, DLS, Z-pot, UV-Vis, FTIR, BET (surface area adsorption analysis and BJH Porosity) and TEM imaging. Results obtained were compared to determine the degree of modification due to the accelerated aging and extraction processes.
When aged in powder form, generally metal oxide nanoparticles hydrated and lost partially their functional groups. These changes in their chemical structure lead to modifications in their porosity and aggregation, changing their behavior in aqueous solutions: only the 3 dispersions of ZnO nanoparticles and TiO2-octyl reduced their stability after aging; the firsts essentially due to partial ionization and changes in porosity, and the latter for changes in the functionalization, in porosity and in surface area. The oxidation of MWCNT structures due to aging accelerated their thermal degradation at high temperatures (<600°C) and altered their porosity. The behavior in aqueous medium (aggregation and stability) was altered as well, as the increase of –OH groups in the surface of carbon nanotubes facilitated the formation of H bonds, causing aggregation. Finally, in nanoclays surface area and porosity increased significantly due to a displacement of functional groups. In MMTdell43B/small, however, aging altered the chemical structure of the organomodifier, reducing surface area and facilitating aggregation in aqueous solution.
In summary, processing or aging of raw nanomaterials affected them less intensely than extraction from polymeric matrices. Processing essentially was found to damage partially the organomodifiers of nanoclays. After extraction from polymeric composites changes in the physicochemical characteristics of nanomaterials depended more on the method of extraction used, but a common factor was the loss of the functionalization groups and an increase in aggregation (except in MWCNT) of nanoparticles.
Depending on the matrix, the nanofillers were extracted using a different strategy: PP composites were incinerated and PA6 was depolymerised chemically to recover the nanomaterials by centrifugation. This second strategy allowed to repolymerize the polymeric chains and reuse PA6 as well. The extraction processes selected are similar to some of the end-of-life processes that can be applied to nanomaterials: calcination as a surrogate of the thermal waste treatments, and chemical dissolution as a surrogate of chemical recycling. Thus, they provide valuable information on the impact that these end-of-life processes can have on nanomaterials and its (eco)toxicity. Finally, nanomaterials could not be recovered successfully from EVA composites without using very aggressive strategies and in reasonable yields. Thus, unfortunately these nanomaterials could not be characterized. For future studies, mechanical recycling is suggested as the most suitable technique.
After extraction, PA6 composites contained residues of polymer, complicating the analysis of the main physicochemical parameters of nanomaterials and, consequently, the determination of changes caused strictly by the aging processes. However, the incomplete elimination of the polymer protected the functional groups. Thus, nanomaterials extracted from PA6 could be reused again (e.g. as a masterbatch) without any extra treatment. The only exceptions were ZnO nanoparticles (which could not be extracted due to its high reactivity in acid pH) and nanoclays, particularly MMTdell67Gsmall (highly desegregated and with a surface potential dramatically affected).
Respecting PP composites, generally calcination allowed the recovery of almost the totality of the nanomaterials, though these had lost almost completely their functional groups; to be reused would need an extra functionalization step. In addition, metal oxide nanoparticles suffered changes in surface charge, surface degradation or sinterization that derived in more hydrated and aggregated nanomaterials that those extracted from the non-aged nanocomposites. Consequently, metal oxide nanoparticles extracted from aged PP were hydrated and smaller. MWCNT both aged and non-aged gained hydroxyl groups in the surface, but its high hydrophobicity did not avoid aggregation. Finally, extracted nanoclays were clearly affected by aging, as were much more degraded and showed different morphology (fewer layers); its hydration during aging also increased surface area and pore volume.
Regarding release studies, considering the initial 3% of nanofiller, the material collected from nanocomposites degradation during aging contained a bigger amount of nanomaterial (13-42%) in all the metal oxides (excepting PP + TiO2-octyl) and PA6 + MWCNT-NH2, which was a very significant increase. In addition, the amount of NM released was <1.0 g/m2 of nanocomposite surface excepting in three composites: PA6+ZnO-OH, PA6+MWCNT-NH2 and PP+SiO2-propyl, where the tailored functionalization could not avoid NM migration. These results clearly evidence that release of nanomaterials to the environment due to the use of nanocompounds cannot be dismissed. To determine its consequences further toxicological studies will be required.

RECYCLING/DISPOSAL STRATEGIES. RESULTS, RECOMMENDATIONS AND IMPROVEMENTS FOR THEIR INDUSTRIAL APPLICATION (WP7)
Nanopolytox proposed different methodologies to recycle production surpluses or used nanocomposites, and evaluate its efficiency on the recovery of nanomaterials, as well as the quality of the recycled products. As a proof of concept, recycling/disposal strategies were defined in selected representative nanocomposites, considering their mechanical and physicochemical properties as well as their toxicological results.
Composites were classified in two main groups, considering disposable materials those with the highest toxicity. Included in this last group was EVA+ZnO, which was disposed by calcination and immobilization in an inert ceramic matrix. Recycled ZnO nanoparticles were successfully incorporated into a ceramic matrix. The resulting bulk material even at high nanoparticles loading performed well under aging. The small amount of matter released after aging contained both Zn ions and nanoparticles surrounded by the matrix, and the cytotoxicity of ZnO nanoparticles did not change. Therefore, the method employed can be suggested as promising for the disposal of nanoparticles recovered from polymer composites.
As representative of recyclable nanocomposites, aged PA6+TiO2, EVA+MMTdell72T and PP+MWCNT were chosen. The first was recycled chemically with the strategy previously defined and used with PA6, and the recovered material extruded again. The other two composites were recycled mechanically using a grinder. The tree nanocomposites were reinjected anew and exposed to aging conditions to test their properties compared with the previous samples.
The recycling process gave place to a PA6+TiO2 nanocomposite with slight differences with the new one, essentially caused by an important loss of the nanoparticles functionalization during its extraction with formic acid, as those groups are known to be sensitive to acid media. Despite this, the extraction process allowed to recover a good percentage of the nanomaterial that could be reused. After aging, all the properties worsened similarly in the new and recycled nanocomposites, cracked and more brittle, with brown-yellow spots in the surface. In both regions carbonyl and amide groups degraded, and appeared new peroxides, carboxylic acids and/or alcohols. The heterogeneous degradation was photocatalized by TiO2 nanoparticles, more exposed after aging, and the more grayish regions contained mainly nanoparticles which had lost its functionalization. Waters collected from the recycled samples contained a much bigger amount of material considering the total surface exposed of the specimens. Nevertheless the recycled composite (before or after aging) was more ductile than the non-recycled one, even than pure PA6. In summary, the presence of remaining PA6 around TiO2 nanoparticles modified the properties of the nanocomposite, mainly during aging, reducing the stability of the recycled samples.
To improve the recycling process, the complete elimination of this remaining PA6 should enable the reuse of the extracted TiO2 nanoparticles. But at large scale centrifugation is not considered a viable process. During the project it was evaluated the possibility of filtering the solution, and discarded because the filters were easily blocked due to the presence of polymer in the solution. However, at industrial level this process seems viable. To solve the identified problems we propose i) modulate the high viscosity of the nanocomposite by diluting the solution or by breaking the polymer chains with mineral acids, ii) to use flocculation of nanoparticles instead of filtration, or iii) to use a capping agent with affinity for nanoparticles or for the polymeric chains, to extract one of these components with a non-polar solvent. These proposed alternatives should be tested to evaluate its effectiveness and achieve a more optimal recycling of this nanocomposite. The use of UV-absorbers or the increase of the initial functionalization would reduce the photodegradation of the matrix. From an industrial point of view, PA6+TiO2 could be used for sporting goods and for flame-retardant cables. By calcining the composite TiO2 could also be reused as “pure” non functionalized nanoparticles for heavy metal removal from aqueous systems, paints to sunscreen or for their photocatalytic properties. Another interesting option would be the reuse of the polymeric matrix after the dissolution process, as theoretically it would result in useful PA6 polymer.
Recycled EVA+MMTdell72 suffered some degradation of the matrix with aging, with the formation of peroxides and carbonyls comparable to the observed in the first aging process. Waters proceeding from the accelerated aging process indicated a much higher amount of material released from the recycled specimens when compared with the new ones, almost the half of it corresponding to nanoclays. Apart from this, the nanocomposite did not show other observable alterations, neither before nor after undergoing aging. Properties of recycled and aged samples were not far from those of the aged pure EVA, but tensile strength of the recycled material increased. Mechanically, recycling led to a more resistant nanocomposite, though after aging the mechanical properties highly decreased and came back to the level of the “new” nanocomposite after aging. Thus, recycled EVA+MMTdell72 underwent deeper modifications under aging than the new one, but at the end both were close. Recycling did not alter neither the organomodifier nor the clays itself. The only negative effect observed was an easier degradation of the nanocomposite surface after recycling. Thus, the mechanical recycling suggested by the NANOPOLYTOX consortium for the NC EVA+MMTdell72T would be suitable for its application in industry.
Considering these results, the mechanical recycling of EVA+MMTdell72T is a feasible and easy process to implement at industrial scale that gave place to materials with the characteristics and composition comparable to the original nanocomposite. For this type of recycling the separation process is an important activity. Some technologies can be introduced at this point to sort plastics automatically using techniques such as X-ray fluorescence, electrostatics, IR spectroscopy and flotation. The last two can be easily used in the separation of EVA nanocomposite. For the mechanical grinding of this soft sticky nanocomposite, a cryogenic process would produce best fracture surfaces. With this process little or no heat is generated in the process resulting in less degradation of the material and resulting in a high yield of usable product. The final step of the process, extrusion, needs no optimization; at this step the nanocomposite can be modified by adding new components and new types of nanocomposites could be produced. The properties obtained after recycling and aging are compatible with the main target applications of EVA+MMTdell72T: fire-resistant or flame-retardant compounds for cables/electrical wiring or to improve abrasion in footwear. Nevertheless if industrial users considered the recycled composite as not reusable, it would be possible to enhance their properties by adding UV-absorbers… to stabilize the aging of the product.
The last nanocomposite recycled, PP+MWCNT was the most resistant to the superficial degradation of all the 18 studied. After its mechanical recycling, this property was quite maintained, confirming the protective effect exerted by the nanotubes. Mechanical and thermal properties were comparable to the first one as well, no important reductions were showed. After aging both nanocomposites (initial and recycled) kept good properties and showed a similar behavior. Eventual losses were much less important that in the neat resin, thanks to the protective effect of MWCNT in the resin, even after recycling. Only the surface was more prone to degradation with aging, allowing nanotubes to migrate to the surface and reducing its protection of PP.
Consequently, the mechanical recycling of PP+MWCNT seemed realistic and applicable in industry. The techniques to be employed to obtain an optimal recycling process could be implanted easily, as milling and extrusion are standardized and widely used in the industry. Only an optimization of the parameters used in both processes for recycling would be required. The use of UV protection components in the recycled nanocomposite would reduce its degradation. The proposed reuses for PP+MWCNT are in the electrical and electronics industry, to develop rearview housings for the automotive industry with intelligent paintings/electromagnetic shielding, for structural components (injected/blow molded pieces, film extrusion or high-purity piping systems), antistatic materials (the inside of cars, protection clothes, packaging or microelectronics, a very huge market), flame retardancy (wiring) and for electroconductive materials.

(ECO)TOXICOLOGY IN ALL THE STAGES STUDIED OF THE LIFE CYCLE OF NANOMATERIALS (WP5)
At the toxicological level, results obtained in NANOPOLYTOX indicated that all TiO2 and functionalized SiO2 nanoparticles showed no toxic effects nor before neither after aging. Toxicity of raw SiO2 and ZnO was associated with their surface reactivity (or ionization in ZnO) and interactions with the cells, and was reduced with the hydration of the nanomaterials. Only ZnO-octyl nanoparticles increased their toxicity with aging, a phenomenon associated to the loss of octyl groups. No cytotoxicity was observed up to the highest concentration tested (100µg/mL) for any MWCNT type, and no changes in toxicity among raw, aged MWCNT and the nanotubes extracted from PA6 or PP (before or after aging) were observed. With organoclays the toxic effect of was mainly due to the organomodifiers used; aging had no statistically significant effects on their toxicity, only a trend towards lower toxicity in all the types of clays associated to a reorganization of the organomodifier within the clay structure.
Exposure to SiO2 nanoparticles reduced cell viability at the highest concentration tested (500 µg/mL), though aging reduced their cytotoxicity. SiO2-OH and SiO2-propyl nanoparticles either raw or aged were not cytotoxic up to 500 µg/mL; the extraction processes from PP (calcination) or PA6 (chemical dissolution) did not change toxicity in those NP types. Similarly, no significant effects in cell viability were observed when cells were exposed (up to 500 µg/mL) to raw/aged TiO2, to the functionalized raw/aged TiO2 nanoparticles, and to the material extracted from the raw/aged nanocomposites. In the Fish Embryo Toxicity (FET) test (OECD Draft Guideline), SiO2 series were more toxic than TiO2 series as well, and only TiO2-octyl nanoparticles extracted from aged nanocomposites by incineration increased its toxicity significantly. As SiO2, SiO2-OH and SiO2-propyl nanoparticles showed some toxicity and internalization in cells, it was decided to study affectation of these nanoparticles at collembolan reproduction, according to the OECD Guideline 232. In all three tests the Lowest Observed Concentration (LOEC) was estimated to ≥1000 mg/kg DW, indicating that their toxicity was very low.
Among raw, aged MWCNT and the nanotubes extracted from the PA6 or PP (before and after the aging process), no changes in cytotoxicity were observed: no cytotoxicity was observed in any case up to the highest concentration tested. In non-adherent cell lines, concentrations above 100 µg/mL of this nanomaterial could not be evaluated, as the intense absorbance of MWCNT interferes in the readouts of the methods. Any effect on cell viability would not be due to the induction of apoptosis. Other authors had reported higher toxicity, though the level of impurities in our nanotubes is much lower. In FET tests, results went in the same direction, showing low toxicity or no toxicity at all; just MWCNT extracted from PP by an incineration process increased slightly its toxicity respecting to the initial nanotubes.
Oppositely, nanoclays were considerably toxic to cells, and cell viability was affected by the six nanoclays in all cell lines evaluated. A noticeable difference in potency was observed between the 43B/43Bsmall nanoclays and the 67G/67Gsmall/72T/72Tsmall nanoclays, being the first group more toxic than the second. The fact that these two groups differed only in the organomodifier suggested that these might be major players in determining the cytotoxicity of nanoclays. Indeed, we proved that the organomodifiers were the main responsible for the cytotoxicity observed in the incubations with the nanoclays. Due to the fact that for the nanoclays the toxicity is not based on their nano properties but on the presence of an organic compound, no further work was performed with the modified nanoclays and mechanistic studies focused on the pristine nanoclays. Nanoclays reduced cell proliferation and induced apoptosis even at the lowest concentration tested (33µg/mL). Our results and literature suggest that different factors affect toxicity of nanoclays: the proportion of serum in the cell media (which influences dispersion), absorption/ion exchange of components of the cell culture media or the formation of a “protein corona” among others. These results were published in the indexed journal Nanotoxicology (http://informahealthcare.com/doi/abs/10.3109/17435390.2013.776123). The aging process had no statistically significant effects on the cytotoxicity of nanoclays, only a trend towards lower toxicity in all the types of clays associated to a reorganization of the organomodifier within the clay structure. The extraction of nanoclays from the matrices where were embedded degraded the organomodifier, resulting in a reduction of its toxicity.
Finally, ZnO nanoparticles (pristine and functionalized were the most toxic nanomaterials tested, causing significant effects on cell viability at considerably low concentrations among the cell lines evaluated (IC50 from 9 to 22 µg/mL), with results comparable to those available in literature. Also in FET tests ZnO series were the most toxic. In all cases, ZnO-octyl showed the less toxicity, around one order of magnitude under pristine or ZnO-OH nanoparticles. However, such differences in toxicity were lost when concentrations were expressed in terms of surface area instead of mass. Another explanation for the differences in cytotoxicity among these nanoparticles is that the release of Zn2+ differs among them, being more important in non-functionalized nanoparticles. Zn2+ did become free during the incubation period. These ions caused rapid alterations in cell morphology only after a few hours of exposure, but no induction of apoptosis was recorded for ZnO nanoparticles (pristine, hydroxyl- or octyl- modified), and the release of Zn2+ to the cell media does not fully explain the toxicity of ZnO. These conclusions are in agreement with those from other studies. Two different arguments can explain these findings: i) the fact that NP tend to be in close contact with cells, which may lead to local ion concentrations at the cell surface higher than those in the cell media; and ii) an intracellular release of Zn2+ after internalization of the nanoparticles. Consequently, cytotoxicity of ZnO was related not only to its surface area, but also to the degree of intracellular release of Zn2+; its level is controlled by regulatory mechanisms, but when they are overwhelmed, the levels of intracellular zinc ions rapidly increase and toxicity occurs. This explains the sharp dose-response curves for viability observed in the experiments. With aging, ZnO nanoparticles reduced its toxic effect, both in vitro and in the FET tests, oppositely to what happened with ZnO-octyl. It is attributed to a reduction in the surface reactivity in the first case, and a loss of functionalization in the latter.
In summary, direct climatic aging of the initial nanomaterials only changed toxicity of raw SiO2, raw ZnO, and ZnO-octyl nanoparticles. In the first two cases, toxicity decreased, whereas in the last case increased. These differences were, however, relatively small. The decrease in toxicity was possibly related to the hydration of the nanomaterials, which reduces their surface reactivity and interactions with the cells. The increase in the cytotoxicity of ZnO-octyl nanoparticles was associated to the loss of octyl groups. The extraction processes did affect the functionalization but had no impact in the core nanomaterials. Calcination was very efficient to remove the polymeric matrix, but did also remove all the organic functionalizations. Therefore, the results obtained cannot inform on whether the nanocomposites aging process resulted in modifications of the functionalizations. Chemical dissolution of PA6 was less aggressive but did also alter the functionalizations and was not applicable for ZnO nanoparticles because these were dissolved during extraction.
To our knowledge, our studies in NANOPOLYTOX are the first that attempted to evaluate the changes in the toxicity of nanomaterials following the different stages of their life cycle. Difficulties have been encountered in this process that will have to be reconsidered in the future. Nevertheless, overall: i) direct climatic weathering of nanomaterials may result in surface changes that can modulate their toxicity, and ii) calcination results in the degradation of the organic functionalizations. Thus, the hazard modulating effect of the organic modifiers is lost and nanomaterials acquire similar hazard properties as the non functionalized ones.

BIOLOGICAL FATE AND BIOTRANSFORMATION OF NANOMATERIALS (WP5)
In addition to (eco)toxicity, one important factor to understand the possible effects and the biological fate of nanomaterials is the degree of cell internalization and its subcellular location. We evaluated these parameters by TEM and confocal microscopy. After 72h of exposure nanomaterials were often surrounded by membranes within cells, suggesting that they had been internalized by endocytosis. In order to further confirm this hypothesis, we used specific subcellular markers to localize early endosomes and lysosomes in the confocal microscope, to test whether there was co-localization of these subcellular vesicles and nanomaterials.
MWCNT, SiO2, TiO2, raw ZnO and raw/organomodified nanoclays were found internalized in cells. Except ZnO, these NP were present in cytoplasmic vesicles, and no free nanoparticles could be observed in the cytoplasm or in the nucleus of the cells. Four mechanisms of endocytosis have been described: clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis, and phagocytosis. Considering data obtained in NANOPOLYTOC, it seems that the nanomaterials evaluated were endocyted by macropinocytosis or by phagocytosis. The fate of these cytoplasmic vesicles containing nanomaterials could not be derived from these experiments. The cells might try to digest these particles in lysosomes; however, after unsuccessful digestion, the cells might just expulse the vesicles containing nanomaterials (exocytosis). Further studies will be needed to better understand these processes.
To determine bioavailability we selected a series of nanomaterials (TiO2, ZnO) and evaluated two approaches: i) a Caco-2 cells monolayer in vitro model morphologically and functionally resembling the enterocytes lining the small intestine, and an in vivo rat model, due to the uncertainties that still exist on the predictability of in vitro models of absorption for nanomaterials.
In vitro, the results showed that TiO2 nanoparticles could almost completely move through the insert membrane when cells were absent, but a very small proportion (below detection limit, i.e., 0.1 ppm or 0.4% of the applied concentration) was able to cross the cellular membrane, suggesting a low potential for translocation through the intestinal epithelial wall. These results are consistent with existing literature data showing very low absorption after oral absorption of nanomaterials or in ex-vivo intestinal absorption experiments.
The tissues selected for the in vivo analysis comprised those that are known to accumulate nanomaterials that reach systemic circulation and those that due to the administration route could locally accumulate nanomaterials. In summary, our results showed that oral absorption of TiO2 and ZnO nanoparticles is very low. When compared with previous studies, differences in the degree of intestinal absorption seem to be mainly associated to the pattern of administration (prior or after feeding). Thus, in both nanomaterials studied there was a very low degree of oral absorption after gavage administration. These results are consistent with those obtained in the in vitro intestinal barrier model for TiO2 nanoparticles. Nevertheless, for both types of nanomaterials a very low degree of absorption does not imply zero absorption, as demonstrated by a slight statistically significant increase in zinc levels in small intestine and Peyer’s Patches, and by the presence of TiO2 nanoparticles in sporadic cells in the TEM evaluations.
For the evaluation of biotransformation mediated toxicity, an exhaustive literature research led to consider that the nanomaterials included in NANOPOLYTOX were too large to interact with the catalytic sites of the CYP450 enzymes (smallest dimension >15 nm). Thus, as a way to evaluate their capacity to up-/down-regulate or to alter the catalytic activity of CYP450 enzymes (consequence of unspecific interactions) we evaluated the potential of different nanomaterials to increase or decrease liver ethoxycoumarin-O-deethylase (ECOD) activity. ECOD activity was measured in liver microsomes obtained from mice intravenously exposed to ZnO, SiO2 and TiO2 nanoparticles (the same used in biodistribution and clearance experiments). No statistically significant differences in ECOD activity were obtained among experimental groups. Thus, ZnO, SiO2 and TiO2 nanoparticles did not seem to affect CYP450 enzymatic activity. After 24h about half of the injected nanoparticles were located in the liver. Indeed, the liver has been described as one of the major nanomaterials accumulating tissues in the literature. However, their presence in the liver does not necessarily imply a potential interaction with P450 enzymes, and depending on their size and surface properties, nanomaterials have been found on both hepatocytes and Kupffer cells, usually located in cytoplasmic vesicles rather than freely circulating in the cytoplasm.
The limited information available together with the high likelihood of physicochemical changes due to interactions with the gastrointestinal system, lead us to study the biotransformation of nanomaterials in artificial gastric fluids. For these studies, we selected the ZnO series. These showed a relatively high toxicity in the in vitro systems, and an easy dissolution/release of Zn2+ ions that started as soon as during the preparation of the dispersions. This process was more evident with non-functionalized ZnO nanoparticles. ZnO-OH and ZnO-octyl dispersions were surrounded with proteins and bile salts as well, resulting in the partial dissolution of some nanoparticles (nanorods to nanospheres).

BIODISTRIBUTION, BIOACCUMULATION AND ENVIRONMENTAL FATE OF NANOMATERIALS (WP5)
Particulate material is known to be rapidly cleared from blood, due to the action of mononuclear phagocytic cells. Then, nanomaterials are usually retained in tissues of the reticulo endothelial system, from which they are slowly eliminated, though kinetics differ from one nanomaterial to another, due to physicochemical characteristics such as dissolution rate or size. We evaluated biodistribution and elimination kinetics of selected nanomaterials (non-functionalized SiO2, TiO2, ZnO and MWCNT) considering traceability, background levels and aggregation.
At sacrifice, no relevant weight changes, hematological parameters or macroscopic abnormalities were observed in any of the animals, except those exposed to MWCNT, which showed homogeneously dark-coloured liver and spleen. This change in coloration was probably due to the accumulation of MWCNT in these tissues. Apart from size and shape, two more factors may contribute to this fact: a higher recognition by the reticulo endothelial system which results in a lower residence time in blood, accumulation in liver and spleen and lower clearance rates from these tissues. A similar biodistribution pattern was observed for SiO2, TiO2, ZnO and MWCNT, which accumulated in tissues of the reticular endothelial system, mainly liver and spleen. Microscopically however, ZnO nanoparticles could not be observed, supporting the hypothesis of its rapid dissolution in the organism. Thus, broadly speaking, this distribution pattern is independent of the type of nanomaterial. The data generated is also consistent with the information available in literature. In contrast, the elimination pattern did differ between TiO2 and ZnO nanoparticles: A long residence time in tissues was observed for TiO2 (half-life >21 days), whereas no differences with controls were detected after three weeks in animals exposed to ZnO nanoparticles. The differences in the biokinetic pattern are likely to be due to the dissolution of ZnO into Zn2+ ions, a process not expected to occur for the other nanomaterials studied.
In trouts (Oncorhynchuss mykiss), bioaccumulation tests were performed with TiO2 series to determine the difference, if any, depending on the functionalization. Results showed that titania accumulated in fishes, though was also rapidly excreted again after transition to “clean” feed. In Daphnia magna, uptake was observed for ZnO and ZnO-octyl (higher in the latter) while for ZnO-OH nanoparticles no uptake was detected, suggesting that some effect of functionalization occurred. Only ZnO nanoparticles a steady state was reached within the 14 days of exposure. A trophic transfer study showed uptake of both ZnO and ZnO-octyl nanoparticles, reaching values >10-fold the level obtained through aqueous exposure even with 10 times higher concentration than the exposed to the feed stock.
The adsorption/desorption and leaching of nanomaterials in soil and sediment was studied according to the principle of OECD 106. However, the experiments showed that the use of this guideline for testing nanomaterials was not directly applicable due to their precipitation caused by the presence of salts, and the need of a centrifugation step to separate soil and solution for the study. Thus, it was decided to apply the OECD 312 guideline to investigate the leaching of selected nanomaterials (TiO2 series) in soil and sediment. All the soils used had a high background concentration of titania, but TiO2 nanoparticles did not leachate through the columns during the 48h test period. The use of higher concentrations of nanomaterial in this test would by far exceed the expected realistic concentrations in sewage treatment plants and, hence, would not be environmentally relevant. Thus, the principle of OECD 312 is feasible for testing nanomaterials, though of limited use unless labeled nanoparticles are used and/or more specific chemical analyses can be applied to differentiate between naturally occurring substances and added nanomaterials.
Transformation of selected nanomaterials (TiO2 and ZnO series) was studied in water at laboratory scale, as an alternative to a traditional transformation study according to the principles in the OECD 307 and 308. Multiple regression analysis of the data set showed that the magnitude of the Z-pot in general was determined by the pH, the salinity and the concentration of dissolved organic carbon. The factors governing the hydrodynamic particle size were less obvious, especially for the functionalized nanomaterials. Transferring these results to the situation in the real environment we may probably expect that nanoparticles in the aquatic environment might alternately agglomerate and deagglomerate depending on the conditions such as pH, salinity and concentration of dissolved organic matter. The physical-chemical parameters may also influence the behaviour such as the porosity and the surface area.
On the basis of the compiled data, together with the remaining information generated within NANOPOLYTOX (physical, chemical and toxicological data), we derived general trends for predictive models. These models, allowed the prediction of the environmental and biological fate of the nanomaterials studied along their life cycle.

TECHNICAL CARDS (ALL WP)
All the information collected during the execution of the NANOPOLYTOX project regarding physicochemical data of nanomaterials and nanocomposites in all the stages of their life cycle studied, as well as the (eco)toxicological main results, were summarized in specific technical cards for each nanomaterial/nanocomposite pair. The general chart of NANOPOLYTOX with all the stages of the nanomaterials studied is summarized in figure 2.
These cards are complete and uploaded in the website of the project. In the future, the technical cards will be available under request in a digital format.

LCIA, ENVIRONMENTAL AND HUMAN IMPACTS. FATE MODEL: USETOX (WP6)
The data collected in the whole project on physical and chemical properties of nanomaterials, biological and environmental fate during their life cycle were used to generate a new methodology for the Life Cycle Impact Assessment (LCIA) of nanomaterials. We obtained a comprehensive framework describing the impact and risks associated with nanomaterials included in polymeric matrices, highly used in consumer products.
A complete LCIA for four selected nanocomposites (PP+MWCNT, EVA+MMTdell72T, PA6+TiO2 and EVA+ZnO) was done following the international standards ISO 14040 and 14044. Current methodologies, databases and impact methods were used, adapting them to nanomaterials and creating a new methodology for nanotechnology-based products. The goal of LCA studies was to have a global knowledge of nanomaterials’ potential impacts on environment and human health during all their life. This information would cover current data gaps and allow performing future LCAs on nanomaterials and products containing these nanomaterials.
Life Cycle Inventory (LCI) was carried out modelling all life cycle stages. All relevant inputs and outputs were quantified including the release of nanomaterials. The main results at inventory level are:
* Processes modelled in all life stages: synthesis of nanoparticles, functionalization, nanocomposite manufacturing, external use simulation and waste treatment (mechanical and chemical recycling, disposal through immobilisation). All processes were modelled with quantified primary data from processes carried out by NANOPOLYTOX partners.
* Data estimation on nanomaterials released in different Life stages, including use simulations.
A complete methodology was developed for the LCIA, which allowed adapting the existing impact assessment methods (in this case ReCiPe) in order to include specific and new data of nanomaterials potential impacts. The ReCiPe Impact Assessment method has 18 midpoint indicators, and three endpoint indicators: i) damage to human health, ii) damage to ecosystem diversity and iii) damage to resource availability.
Impact factors for Human Toxicity and Freshwater Ecotoxicity categories were derived for nanomaterials. The characterization factors for these categories were estimated following the principles of the USEtox model, which has been approved by UNEP-SETAC as the preferred model for characterization modelling of human and ecotoxicity impacts in LCIA. Exposure, fate and (eco)toxicity of nanomaterials were assessed in order to derive characterisation factors. However, the general equations to generate fate factors in the USEtox model are not directly applicable to nanomaterials. Thus, to predict the environmental distribution of nanomaterials in the NANOPOLYTOX approach we considered additional characteristics governing their fate: size, shape, porosity, agglomeration state, surface area, surface charge, composition, density, reactivity, etc.
With this new methodology developed, environmental impacts were assessed at midpoint and endpoint level in order to see the relative contribution of the different life stages and the main impacting parameters. General distribution of all studied nanomaterials followed a similar scheme; in all production and transformation processes, electricity was the most impacting parameter. Due to energy consumption, climate change appeared to be the most relevant impact category at endpoint level, both on human health damage (84% - 80%) and ecosystem diversity damage (97%-95%).
At endpoint level, characterization factors of freshwater ecotoxicity and human toxicity due to released NM were added to final results. In use stage, only the impacts from released nanomaterials were considered, with no impacts coming from other sources during the application of composites. For each life stage, the relative contribution of released nanomaterials and the other processes was calculated.
For almost all composites, the nanocomposite manufacturing through extrusion and injection had the highest contribution to overall environmental impacts. In the case of MWCNT mechanical recycling had also significant impacts, whereas the synthesis of this nanomaterial (by fluidized bed deposition) had lower impacts associated. For the EVA+ZnO formulation, nanoparticles’ synthesis and composite manufacturing have similar impacts. For the nanocomposite PA6+TiO2, chemical recycling was the stage with higher impacts.
Although direct comparisons among different nanocomposite could not be done, PA6+TiO2 had higher potential damage values in the three levels than the rest of nanocomposites, since TiO2-OH particles had been functionalized before its incorporation into the composite (stage with relevant impacts), and waste treatment by chemical recycling had higher impacts than mechanical recycling.
The relative contribution of released nanomaterials to environmental impacts was included at damage on human health and ecosystem diversity for all nanocomposites except for nanoclays, where it was considered that released clays did not cause any adverse effects since they are natural materials present in the environment. For the worst case scenario, released MWCNT along life cycle contributed only to 1% on human health and 0.003% on ecosystem diversity. The toxic effect of MWCNT on workers was also assessed, being its synthesis and nanocomposite production the stages higher values, which corroborated the convenience of performing Risk Assessment in the different stages of the life cycle of MWCNT together to LCA assessment. In the case of PA6+TiO2 nanocomposites, the relative contribution of the released nanoparticles was higher, being a 24% for human health damage and 0.03% in ecosystem damage indicator. For ZnO contribution in human health damage was 7%, whereas for damage on ecosystem was 0.04%.

MAIN GENERAL CONCLUSIONS
Results are not only interesting for the scientific community but also for a great number of SMEs that work with nanotechnology and material recycling/recovery solutions.
Finally, the consortium has been interacting with relevant technology (e.g. nanotechnological and advanced materials) an environmental management platforms which allowed the consortium to widen the potential applications and the dissemination of the results. On a scientific level, the dissemination activities has been carried out through publications in specialized journal of toxicology, materials chemistry, and nanotechnology related journals, as well as in journals devoted to material science.


Potential Impact:

NANOPOLYTOX project has achieved significant results and reached a number of important conclusions. The project has addressed the toxicological impacts of nanomaterials that are present in polymeric nanocomposites. The project characterized the effects of these nanomaterials during their life cycle including processing, weathering and recycling of these nanocomposites. NANOPOLYTOX results will have a significant impact on various industries including the automotive, food packaging, fire retardant insulations and other consumer products manufacturers. Moreover, the project will have significant social impact mainly with respect to the education and information that it will provide the consumers about the safety if the products they use containing nanomaterials. The results of the project will have a significant return and impact on the SMEs and large industries involved in the project that mainly produced the nanomaterials. The identification of the hazards and toxicological effect of nanomaterials in nanocomposites will allow the industries to evaluate their products that are released to the market and how safe they are. This will also motivate the industries to seek new, safe and more effective nanomaterials and nanocomposites. Determining the potential toxicity of its nanomaterials and their long-term consequences on important ecosystems potentially exposed to multiple stressors (e.g. climate change, pollution). This type of characterization is required by the company’s customers and collaborators in order for them to feel secure when using and handling nanomaterials and nanocomposites during the various stages of their life cycle. By building trust between manufacturers and end users, the market share of the former will rise through increased market penetration, and this will trigger economic benefits for them. The methodologies and results of NANOPOLYTOX can contribute to the development of testing standards and guidelines for nanotoxicity characterization and nanomaterials recycling. They can also lead to strategies for improved product design for nanomaterials manufacturers that aim at reducing the release of nanoparticles from polymeric or other matrices during their life cycle, and thus resulting in safer nano-based products. The project has promised and delivered data on the following i) study of physico-chemical properties of the nanomaterial, ii) environmental fate, iii) environmental toxicology, iv) mammalian toxicology, v) emission and exposure thresholds, vi) nanomaterial safety datasheets and safety labeling.
In depth study of composites made of broadly used polymers (PP, PA and EVA) as polymeric matrices and some of the highest tonnage nanomaterials (SiO2, TiO2, ZnO , MWCNT and some types of nanostructured clays) yielded in obtaining new and useful data that will certainly have an impact on several fields and aspects of our society.
Environmental fate and effect of non-functionalized and functionalized nanoparticles were investigated using in vivo and in vitro studies. A decision three starting with in vitro test for selection of nanomaterials for further investigation was tested and found useful as a first step for identifying the nanomaterials expecting to pose most risk for the environment (and human). Through more thorough investigation of the most toxic nanomaterials, the project has provided new results applicable to industry, academia and authorities on the environmental fate of nanomaterials. This includes results on bioaccumulation, biomagnification, adsorption/desorption to soil particles and initial transformation of nanoparticles in term of stability and tendency to agglomerate/aggregate in the environment. These are areas with only few published results until now. Especially interesting is the new knowledge provided on the influence of the functionalization of a nanoparticle on the toxicity and behaviour in the environment. The project showed that there might be differences in toxicity and fate of nanoparticles depending on their functionalization.
In most of the cases low or no toxicity was observed, and in the cases were significant toxicity of any kind was found (mostly ZnO) that does not implies a significant risk on using composites containing these materials. This is because in order to represent a risk, there must be exposure of them, they must be available for organisms, and once incorporated to the studied polymeric matrices almost no release of them due to aging was found, and when found, it was in low amount that it insignificant to pose any health risks. Nevertheless, this is true regarding the intrigued risks of the studied nanomaterials after they are introduced into the polymeric matrix, which we consider as trapped and inert in terms of health risk, but health risk of some of the studied nanomaterials is a serious issue to deal with when they are manipulated in huge amounts in a powder forms. This situation may occur in composites production industries, were nanomaterials in powder form will be manipulated by operators in order to incorporate them into the polymer/composite during extrusion and injection phases, is there then when safety protocols ensuring no breath, skin or eye contact with the nanomaterials found to be toxic (it would be advisable to do so with any kind of nanomaterial) have to be developed and applied.
NanoPolyTox has also contributed to complex issues of repeated exposures and disposal of nanomaterials. This is vital for industry, which is currently trapped by over-regulated measures due to existing rules for industrial discharge consents, based on continuous exposure data. This would stifle production since industry cannot expand/innovate if it does not have capacity to deal with its waste. Most industrial processes produce effluents intermittently that change in volume and composition with the weekly production cycle
In addition to the hazard identification, the Lifecycle Analysis (LCA) work carried out in Nanopolytox is of significant relevance for both SMEs and large industries since it will help them to gain a broader and more comprehensive perspective of their products’ footprints. LCA has helped the industrial partners to benchmark the environmental performance of their nanoproducts against alternative conventional products with similar functionality – an exercise that may reveal that properly developed nanotechnologies are environmentally preferable in many cases. Moreover, this activity may form the basis for defining proper measures and technological alternatives to reduce the environmental burden of certain nanoproducts. Understanding where they stand in terms of environmental performance of their products would not only encourage companies to push forward with innovative solutions, but also to provide valuable information to regulators regarding the net benefits of nanotechnologies versus their identified risks/impacts.
Recycling procedures developed for some of the studied composites, could also have an impact from an industrial production point of view. Mechanical recycling following a milling and reinjecting strategy has shown maintaining, and in some cases even improving, on the mechanical properties of the recycled composites obtained for used (and aged) old nanocomposites. In all cases recycled nanocomposites showed better mechanical performance than brand-new polymeric matrices with no nanofilling. These both results will certainly be of high interest for the polymer sector, and give economically positive and environmentally friendly solutions to the composites upon the end of their life-cycle. The impact of recycling, waste management and recovery will be seen in the following areas:
(i) Generation of new effective ways of waste management processes, which has been developed in NANOPOLYTOX
(ii) Cost reduction of waste processing by expanding and implementing the new methods and knowledge from the project
(iii) The successful recycling strategies will reduce the cost of environmental remediation
(iv) Reduction of manufacturing cost by the demonstrated ability to reuse and recycled nanocomposite

Characterization of the composites themselves for other properties, including mechanical can shed a light on the potential of improving the properties of polymeric nanocomposites. Significant enhancement and improvement of various mechanical properties has been proven for almost all the chosen combinations of polymer/nanomaterial studied. Few of these composite compositions are already been used in some industrial fields for their prime mechanical properties, but most of the composites generated are new and all the date on their mechanical characteristics can be really valuable and have an impact from a polymer and composite industrial point of view. Better mechanical properties could imply the use of less amount of composite to accomplish the same mechanical requirements or performance, and can thus contribute to lighter or smaller (maybe miniaturizated) products (mechanical equipment, automobiles…). This, depending on the case, can help to reduce the production costs.
Other properties discovered about the nanomaterials developed in the project itself can be of benefit and impact on the industries and consumer. Understanding the risks associated with these newly developed nanomaterials. For example, the developed functionalized nanoparticles are detrimental for their incorporation in some coating formulation due to change of solvent blend from the nanoparticle dispersion to the sol gel based formulations. Currently, Polyrise is developing a new range a high performance anti-reflective coating. For the production of these sol gel formulations, the functionalization of nanoparticles is required for the stability of the solution (and by the way its application in industrial fields such as PV Panels). For this eco-oriented products, the question of their life cycle impact is more and more important as well as their recycling. Since these panels are exposed to severe weather aging, it is important to anticipate the release of aged nanoparticles from the coating surface with possible ecotoxicity impact or not. In that sense the nanopolytox project showed no pronounced ecotoxicity of functionalized metal oxide nanoparticles either (silicon, zinc or titanium).
One of the unique and innovative approaches was to prepare “Technical Cards”. This is essentially a collection of the data gathered for each nanocomposite which include physio-chemical properties of the nanomaterialsas well as toxicity data. The technical cards of the projects can provide a comprehensive toolbox that industry can use for toxicity & risk analysis and management. This tool can be communicated to the EU NanoSafety Cluster with which the project consortium has actively interacted and taken advantage of results stemming from other relevant projects.

NANOPOLYTOX results were expected to have a major impact on industries, mainly automotives and packaging industries. However, in reality any industry that utilizes and exploits nanocomposite superior properties will benefit from the results of the project. nanocomposites have attracted considerable attention in the automotive industry for their interesting properties including mechanical, thermal, electrical and fire retardancy. However, the market for NM-enabled products in the sector is limited to a few applications including external body parts, under-bonnet parts, coatings, etc. Nevertheless, the market potential is expected to grow. According to “Nanocomposites- A Global Business Report”, the market of nanocomposites will reach 1.3bn lbs by 2015. The automotive industry share is projected to be the third-largest market for polymer nanocomposites with over 15% of the market. According to Frost and Sullivan, carbon nanotubes (CNT) are expected to represent 3.6% of the automotive composites. This represents a market in the automotive composites of approximately $36m (for 1% loading of CNTs in these materials). This Packaging by far represents the largest end-use market. Global bioplastic packaging demand is forecast to reach 884,000 tonnes by 2020. A 24.9 % CAGR is expected from 2010-2015, slowing to 18.3 percent in the five years to 2020. By 2020, electric/electronics and others applications are expected to gain a more prominent position demand for bioplastics. Hence, the use of nanocomposite materials is very attractive for improvement of the properties of the polymers at an affordable cost. Therefore, the potential future for market exploitation can only be possible if the issue of safety, among others, is thoroughly evaluated.
The EU needs to shift towards a resource-efficient and low-carbon economy, a perspective reflected in the flagship initiative under the Europe 2020 Strategy ‘A resource-efficient Europe’, where the European Commission calls to create a framework that aims to (1) ‘boost economic performance while reducing resource use’; (2) ‘identify and create new opportunities for economic growth and greater innovation’; (3) ensure the supply of essential resources; and (4) fight against climate change and limit the environmental impacts of resource use (European Commission, 2011). This is also envisioned in NANOfutures Vision where by 2025 Europe will aim to play a leading role in an ever increasing nanotechnology market and create competitiveness in all sectors where nanotechnology has added values.. This will not only increase the market share of existing nano-products, but it will pave the way towards the commercialization of novel nano- products and facilitate the eventual insurability of nanotechnology industrial activities. This in turn will create a competitive market and generate jobs in this multidisciplinary sector. Investigation of waste management, recycling of nano-enabled products will have a significant economic impact in terms of (i) reduction in handling and processing of waste, (ii) reduction of cost of environmental remediation by improvement of NM recovery (iii) reduction of production costs by improving the recycling and reuse methods. Clearly NANOPOLYTOX was in line with these objectives
The social perception of nanotechnology is still greatly divided. A recent survey across a wide socio-demographic distribution (Eurobarometer 2010) was taken to evaluate the European general public’s awareness, opinion and attitude towards nanotechnology. The survey revealed that 46% had heard of nanotechnology and 56% had never heard of it. Following up with perception about the safety of nanotechnology, the survey revealed that one third believed that nanotechnology may cause harm to the environment and is not safe for humans and future generations. One third believed the opposite and one third did not know. The lack of sufficient information about the safety and dangers associated with NMs and products affects the public’s trust in such technologies. NANOPOLYTOX results and dissemination will inform the general public about the nature and safety of the nanomaterials used in consumer goods. The project is also in line with the actions listed in the European Strategy for Nanotechnology regarding the integration of the societal dimension by (i) ensuring public awareness and confidence in nanotechnology, (ii) encouraging a dialogue with EU citizens/consumers to promote informed judgement on nanotechnology, and (iii) committing to ethical principles in order to ensure that R&D in nanotechnology is carried out in a responsible and transparent manner.
NanoPolyTox findings can significantly contribute to the objectives of the NanoSafety Cluster Strategic Research Agenda (Nanosafety 2015-2025: A Strategic Research Agenda towards Safe and Sustainable Nanomaterial and Nanotechnology Innovations) and to EU 2020 Strategy; to provide essential contributions to green technologies and environmental protection, and also to contribute to the inclusive growth by providing new employment and keeping jobs in the EU. The regulatory environment (e.g. nanosafety) affects time to market, marginal cost structure and allocation of resources, especially for SMEs. The innovative strategies, methods and technologies developed will significantly facilitate regulatory compliance and improve industrial safety. The creation of new job opportunities will arise from the new methodologies that will be developed in the project, i.e. experienced personnel for large-scale recycling in the plastics and composites companies and design & synthesis of safer nanomaterials. Increasing safety without compromising the successful scale-up of nanotechnologies will lead to rapid technological development and economic growth.

1 Oral Presentation (GLONATECH) DEFENSYS’10 International Defence and Security Fair 28/10/2010 Thessaloniki, Greece
2 Oral Presentation (GLONATECH) 6th International Congress for Composites: Composites in Automotive & Aerospace (Materialica) 18/10/2010 Munich, Germany
3 Posters (LEITAT) Conference 02/11/2010 Gijon, Spain
4 Oral Presentation (LEITAT) Nanosafety Cluster Meeting 09/11/2010 Prague, Czech Republic
5 Oral Presentation (LEITAT) NEPHH - 1st International Workshop “Industry and nanomaterials: Benefits and risks” 18/11/2010 Milan, Italy
6 Oral Presentation (GLONATECH) Seminar “Activities of Greek Nanotechnology SMEs” 16/12/2010 Thessaloniki, Greece
7 Oral Presentation (LEITAT) Nanosafety Cluster meeting 17/02/2011 Laussane, Swizerland
8 Oral Presentation (LEITAT) ImagineNano Conferece, Life cycle analysis of nanomaterials: An overview 11/04/2011 Bilbao, Spain
9 Posters (GLONATECH) Symposium on Safety issues of Nanomaterials along their Life Cycle 04/05/2011 Barcelona, Spain
10 Posters (LEITAT) Symposium on “Safety issues of nanomaterials along their life cycle” 04/05/2011 Barcelona, Spain
11 Organisation of Workshops (LEITAT) Symposium on “Safety issues of nanomaterials along their life cycle” 04/05/2011 Barcelona, Spain
12 Posters (DHI) Symposium on “Safety issues of nanomaterials along their life cycle” 04/05/2011 Barcelona, Spain
13 Oral Presentation (LEITAT) ENPRA Workshop: Challenges of regulation and risk assessment of nanomaterials 10/05/2011 Ispra, Italy
14 Oral Presentation (LEITAT) Nanosafety Cluster, Outcomes of the Nanosafety Cluster meeting 30/05/2011 Budapest, Hungary
15 Oral Presentation (LEITAT) NanoSustain LCA training Workshop 26/09/2011 London, UK
16 Oral Presentation (LEITAT) In vitro toxicology society annual meeting 08/10/2011 Liverpool, UK
17 Posters (LEITAT) In vitro toxicology society annual meeting 09/10/2011 Liverpool, UK
18 Posters (LEITAT) 6th international conference on the Environmental Effects of Nanoparticles and Nanomaterials 19/11/2011 London, UK
19 Posters (DHI) 6th international conference on the Environmental Effects of Nanoparticles and Nanomaterials 19/11/2011 London, UK
20 Oral Presentation (LEITAT) Metrology and exposure assessment of carbon nanotubes Workshop 29/11/2011 Cologne, Germany
21 Exhibitions (LATI) 21st Fakuma International Trade Fair Hall B2 - Stand 2205 18/10/2011 Friedrichshafen, Germany
22 Exhibitions (LATI) International Expodental Forum, Hall 9 – Stand F15 06/10/2011 Rome, Italy
23 Exhibitions (LATI) Plastics Materials Forum 06/10/2011 Milan, Italy
24 Oral Presentation (DHI) NanoSustain-ability CNBSS Workshop 28/10/2011 Barcelona
25 Oral Presentation (LEITAT) NanoImpactNet – QNano Conference 27/02/2012 Dublin, Ireland
26 Oral Presentation (LATI) Symposium on Safety issues and Regulatory Challenges of Nanomaterials 03/04/2012 San Sebastian, Spain
27 Oral Presentation (LEITAT) IVTIP Spring Meeting 19/04/2012 Bilbao, Spain
28 Oral Presentation (LEITAT) Safe implementation of nanotechnologies: common challenges 29/05/2012 Grenoble, France
29 Oral Presentation (LEITAT) International congress on safety of engineered nanoparticles and nanotechnologies, SENN 2012 28/10/2012 Helsinki, Finland

List of Websites:

www.nanopolytox.ie

Scientific and Technical Coordinator
Dr Socorro Vázquez-Campos
Coordinator of Nanohealth & Safety Group
LEITAT Technological Centre
C/ de la Innovació, 2 · 08225 Terrassa (Barcelona), Spain
Tel. (+34) 93 788 23 00 · Fax (+34) 93 789 19 06
email: svazquez@leitat.org

Administrative Manager
Dr. Amro Satti
Project Manager – International Project Office
LEITAT Technological Centre
C/ de la Innovació, 2 · 08225 Terrassa (Barcelona), Spain
Tel. (+34) 93 788 23 00 · Fax (+34) 93 789 19 06
email: asatti@leitat.org

Informations connexes

Contact

Dirk Saseta, (International Project Office Manager)
Tél.: +34937882300
Fax: +34937891906
E-mail