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Novel Concepts, Methods, and Technologies for the Production of Portable, Easy-to-Use Devices for the Measurement and Analysis of Airborne Engineered Nanoparticles in Workplace Air

Final Report Summary - NANODEVICE (Novel Concepts, Methods, and Technologies for the ... the Measurement and Analysis of Airborne Engineered Nanoparticles in Workplace Air)

Executive summary:

An executive summary

The main results of the project cover developing innovative concepts and reliable methods for characterizing ENP in workplace air with novel, portable and easy-to-use devices suitable for workplaces.

Identification of relevant physico-chemical properties and metrics of airborne ENP was achieved through designing and building a nano-metal oxide reactor together with engineered nanoparticle (ENP) aerosol synthesis reactors. The materials were produced and characterized. A list of 23 reference materials for the project´s needs was established.

The association between physico-chemical and toxicological properties of ENP was successfully identified and the knowledge on the ENP's biological behaviour and the biologic responses was increased.

Analyzing industrial processes as a source of ENP in workplace air was performed using the exposure scenarios and sampling strategies that were generated during the project.

The device development sub-project progressed during the project life-time excellently and exceeded the high expectations. In the outset, seven pre-prototype devices and measurement instruments were planned to be developed in the project, but in the end, the result was that 17 new pre-prototype devices and measurement instruments in four different device families were developed and also tested in the field.

These four device families are:
1) total or size specific N-S-M concentration in real time;
2) material specific monitors in real-time or quasi-real-time;
3) samplers for off-line particle analysis; and
4) pre-separator modules for size fractions relevant to the human respiratory tract.

Project context and objectives:

Summary of project context

Engineered nanoparticles (ENP), defined as having at least one dimension =100 nm, have attracted a great deal of interest during recent years, due to their many technologically interesting properties. The unique properties of ENP and their applications have given birth to immense technological and economic expectations for industries using ENP. However, some of these properties have given rise to concern that they may be harmful to humans. This has prompted scientists, regulators, and the industrial representatives to investigate the features of ENP in order to be sure of their safe use in nanotechnologies (NT), i.e. technologies utilizing ENP. The European Commission has also explored in-depth the characteristics of ENP and issued a document on ways to assure the safety of ENP.

Progress beyond the state of the art

At the time of planning the project, there were no portable and easy-to-use measurement devices for the characterization and assessing the levels of ENP in workplaces. There are devices that can be used to measure particle size distribution, nano-sized particle number concentrations, or effective surface area of ENP, but these devices are heavy, laborious to use, very expensive, and there are limitations in their sensitivity. Moreover, many of the devices do not allow on-line measurement of given ENP, an important feature for using of these devices in workplaces for assessing the levels of exposure of workers potentially exposed to ENP's. One of the shortcomings of the current devices, even of the expensive and heavy pieces of equipment, has also been the limitations in terms of the particle parameters. Thus, a major challenge at the current situation is to be able to develop devices which provide solutions to these challenges and that allow inexpensive assessment of exposure of workers to ENP (see Maynard et al., 2006).

Project results:

A description of the main Science and Technology(S&T) results/foregrounds (not exceeding 25 pages)

WP1 Physicochemical characterization of reference materials and production of tailored ENP aerosols as well as purchasing and characterization of commercially available nanomaterials

During the first reporting period AALTO has characterised iron catalysts particles from hot wire generator (HWG) and preliminary tests have been performed of SWCNT synthesis with the concept reactor. TUT has been building up the portable metal oxide particle generator, carrying out test measurements with TiO2 and SiO2. Also, negotiations with Partner 23 on participating in the coming experiments in Bochum, July 2010 have been performed. UEF has been building up of metal nanoparticle reactors (CVS, FSP) and running test measurements of CVS and FSP reactors with iron, silicon composite and lithium titanium oxide.

During the second reporting period Deliverable D1.1 'Nanocarbon, metal and metal oxide ENP aerosol synthesis reactions are operational' was achieved. Partner 2, AALTO has designed, built and used a SWCNT floating catalyst synthesis reactor based on iron catalyst nanoparticles and carbon monoxide as carbon precursor to generate fresh SWCNT aerosols. SWCNT parameters (tube diameter distribution, bundle diameter, chiral angle distribution) can be controlled. Partner 17, TUT has finalized the metal oxide flame reactor for nanoparticle generation and the system is operational. Analysis of titanium dioxide and magnetic FexOy-particles generated by flame reactor were carried out and the analyses are continuing. Nanopowder collection and further development of collection by ESP (electrostatic precipitator) were carried out and are continuing. TUT Participated in two campaigns in the Nano Test Facility in Dortmund (July 2010 and July 2011) with portable flame reactor and the data analysis of the two campaigns was performed. Nanoparticle measurements at TUT nanoparticle processing site, as a nanoparticle workplace, during summer 2011, were performed for WP14. Partner 19 UEF designed built and tested CVS, FSP metal nanoparticle reactors and produced and characterised iron, silicon composite and lithium titanium oxide nanoparticles. Sampling system has been build up and tested as well as dilution systems (PRD) for fresh aerosol.

During the third reporting period WP1 'Physicochemical characterization of reference materials and production of tailored ENP aerosols as well as purchasing and characterization of commercially available nanomaterials' is led by AALTO. The objective is to develop and study the syntheses of different kinds of ENP aerosols, including carbon nanotubes and nanobuds, metals and metal oxides.

WP2 Physical and chemical properties by electron microscopy

During the first reporting period the standard methods for generating, analysing, characterising and reporting the electron microscopy analysis of nano-objects and agglomerates were in preparation. There was also a delay in the full selection of powders in order to integrate with other nanoprojects. Some initial characterization of titanium dioxide and carbon nanotube materials, selected in the workplan for WP4 toxicological tests were carried out by FIOH. Characterization included FEG-SEM and TEM analysis of the morphology and EDS analysis for the composition.

A final selection of common nanomaterials were selected and sourced and distributed for use by SP2 (WPs 2, 3 and 4) in September 2010. A total 23 different nanopowders were obtained to link and bridge analysis in SP1 and SP2 as well as choosing a wide range of powders with different characteristics and properties for device challenging. During the second reporting period the standard EN15051 rotating drum dustiness tester (and a smaller drum version) was used to produce airborne dispersion of these nanopowders for both WP2 and WP3 analyses to allow direct comparison between different on-line methods with electron microscopy.

The electron microscopy results showed that that most of the nano-objects generated from a powder were agglomerates of primary nano-objects. This suggests that the energy from the dustiness test, which replicates typical handling situations such as pouring and shovelling, has insufficient energy to completely break up the agglomerates into the primary objects; although rapid re-agglomeration could also be taking place.

Typically the mode of the electron microscopy size distributions was between 100 - 500 nm with some nanopowders showing evidence of a second but smaller size mode, usually above 1000 nm. Functionalised calcium carbonate nanopowders gave much higher releases (two orders of magnitude) than the nano-objects from which they are made but the size distribution was similar. The electron microscopy size distributions are being used to compare the performance of several on-line size measurement instruments that were run simultaneously during the dust generation.

WP3 Physical-chemical properties of ENP evaluated by using non-imaging techniques

During the first reporting period in WP3, there were no significant results obtained as the WP activities have focused in selecting and getting the material and start analysis. WP3 did not have to fulfil any deliverable or milestone in year 1. According to the DOW, there will be one Milestone in the next period and we are on track with analysis of particles in suspension.

During the second reporting period the following parameters were studied: 1. Primary physicochemical characteristics of particles, 2. Particle dustiness, 3. Particle dispersion in liquids, 4 and 5. Hydrochemical reactivity and biodurability.
1. In the physicochemical characteristics X-ray diffraction was used to determine the average crystalline size and the specific surface area was determined by BET method. It was discovered that the particles were larger than the reported by the industry/ vendors. Thermogravimetric analysis (TGA) combined with Gas Chromatography Mass Spectrometry (GC-MS) and/or Liquid Chromatography (LS) MS were important tools for identification of particles with presence of potential surface coatings and their identification. Surprisingly many of the commercial nanoparticles contained surface-coatings or were associated with organic material, even-though it was not reported by the producer/vendor.
2. In the particle dustiness studies 23 nanoparticle powders were tested and a wide range of dustiness from very low to very high was observed. A specific test-campaign was held to perform a multi-instrumental analysis of the surface area of released respirable dust as compared to the specific surface area of test powders. Analyses are now being completed to determine whether there is a link between physicochemical characteristics and dustiness levels of the tested powders. Preliminary results suggest that the specific surface area of powders in general may be used to calculate the surface area of respirable dust.
3. In the particle dispersion studies, particles are dispersed into cell-medium cRPMI (1 mg/ml) in a 37 kHz ultrasound bath for 20 minutes following the test item preparation protocol used by WP4 . It was found that the procedure can be used to disperse oxides and poorly agglomerated particles, but the method is less capable for dispersing entangled carbon nanotubes and not suitable for dispersion of hydrophobic materials. Detailed analysis of dynamic light-scattering and disc-centrifuge data is on-going to quantify the size-distributions in the dispersions.
4 and 5. In the hydrochemical reactivity and biodurability tests a method using an atmosphere-temperature-pH controlled stirred batch reactor has been developed for test of particle dissolution, acidity and redox activity of nanoparticles dispersed in surrogate biological fluids and cell mediums under highly controlled conditions. Proof of concept has been made and the system awaits final modification for optimal control of test atmospheres. An alternative and faster procedure is being established using incubation plates with build-in on-line pH and O2 sensors as a screening method. The system still awaits delivery.

During the third reporting period the following parameters were studied: 1. Primary characteristics of particles,
2. Particle dustiness,
3. Particle dispersion in liquids,
4. Hydrochemical reactivity and
5. ENP biodurability.

1 Primary physicochemical characteristic:
Continuation of the materials- and data analysis from period 2, showed that the specific surface area determined by the BET method did not always indicate the existence of a nanoparticulate powder when particles were partially aggregated, nanostructured or coated. This was highly significant for some carbon nanotubes and Ag nanoparticles with original stabilizers or direct surface-coating. This has implications for the methods available for identifying particulate nanomaterials.

2. Particle Dustiness: Additional tests increased the dustiness tests to comprise a total of 23 powders. The powders were shown to have a wide range in dustiness ranging (very low to high). High similarity was found for respirable dust tested using both the standard EN15051 and the downscaled NRCWE rotating drum. The INRS Vortex shaker generates different dustiness levels and characteristics. No immediate link was observed between primary physicochemical characteristics and dustiness level. Preliminary results suggests that the specific surface area of powders may be used to estimate the surface area of respirable dust based on the mass of respirable dust times the specific surface area, but deviations may occur. For an embedded instrument challenge study between dust surface area measurements using on-line diffusion charge surface area monitors and FMPS surface areas converted from particle-size-distribution analysis. Two surface area monitors showed modest to poor comparability and material dependent linear to non-linear relationships were found between the surface area monitor data and dust surface area recalculated from size-distribution analysis.

3. Particle dispersion in liquids: From continuation of period 2, quantitative analysis of dispersions was completed. We conclude that only 2-3 nanomaterials were well-dispersed in the cRPMI and DMEM+FBS. There was generally good correlation between the size-distributions obtained in cRPMI and DMEM+FBS at higher doses (0.3 mg/ml), but poor at low concentrations (0.03 mg/ml). Interestingly, there was generally poor correlation between primary hydrodynamic size-modes achieved by DLS and DCS. This is partly due to the methodological differences. DLS data may be affected by use of different optical data on the test materials. However, for proper comparison of sizing true viscosities in specific dispersion are needed.

WP4 Toxicological properties of ENPs

During the first reporting period for toxicological analyses tests were performed on: the immunotoxic effects, cell viability, cytotoxicity and DNA strand breaks. The expression of cytokines and chemokines was assessed in mouse macrophages to study the immunotoxic effects of exposure to selected nanomaterials. Cell viability was assessed by counting the proportion of dead cells in treated cultures versus control cultures using Trypan blue dye exclusion or LDH assay. The genotoxicity of selected nanomaterials was assessed in human bronchial epithelial BEAS 2B cells in vitro. The doses tested were chosen on the basis of two cytotoxicity tests - cell count in using Trypan blue dye exclusion and a luminescent assay detecting ATP. The comet assay was applied to measure DNA strand breaks produced by the nanomaterial exposure.

Based on the studies performed during the first period, following conclusions were made:
1. Immunotoxicology: Some nanomaterials induced pro-inflammatory effects by secretion of essential cytokines and chemokines in mouse macrophages. However, these effects seemed to be material-specific.
2. Cytotoxicity: The cell viability test showed that each nanomaterial studied induced cell death at high concentrations but no drastic effects were seen in the assays. All nanomaterials examined decreased the number of cells in cultures of BEAS 2B cells in a dose-dependent fashion, showing a cytotoxic effect.
3. Genotoxicology: Preliminary results on the genotoxicity studies thus far performed indicated that TiO2 (mix) induces a dose-dependent increase in DNA damage. TiO2 (alumina) also produced a significant increase in DNA breaks, but no dose dependency was detected.

During the second reporting period the assessment of the toxicological properties of ENPs was continued and the ENP selection for the toxicological testing was finished. In the immunotoxicological analysis of ENPs cell death and production of pro-inflammatory cytokines were studied using human alveolar epithelial cells, skin cells and primary macrophages; oxidative stress analysis was performed with human alveolar and skin cells. The genotoxicity studies of selected ENPs included studies of cell death and their ability to induce DNA damage and micronuclei (NM) in human bronchial epithelial cells. Also, the template for the data bank of toxicological effects of ENPs was established.

Based on the studies performed during the second period, following conclusions were made:
1. Most of the ENPs studied do not cause cell death or oxidative stress or induce the secretion of pro-inflammatory cytokines in vitro. Only SiOx was able to cause cell death in all cell types studied, and it also induces low levels of oxidative stress in the alveolar epithelial and skin cells.
2. Studying the secretion of IL-1 -family cytokines utilizing LPS-priming of macrophages seems suitable for the recognition of clearly pro-inflammatory ENPs.
3. Most of the studied ENPs induce DNA damage but do not NM in human bronchial epithelial cells.

The last period of project was used to finish the analysis of toxicological properties of ENPs. Human primary macrophages, THP-1 cell line, human alveolar epithelial cells and skin cells were used in the immunotoxicity testing. Different ENP groups clustered according their chemical properties and their ability to induce responses in different cell types were compared using different tools of statistical analysis. Also, the correlation between ENP's catalytic activity and their ability to induce ROS production was investigated in co-operation with WP11. The genotoxicity studies with selected ENPs were finished according to the DoW. The data bank of toxicological characterization of ENPs was finalized and summary sheets of toxicity are published in the http://www.nano-device.eu.

Based on the studies performed during the last period, following conclusions were made:
1. There are similar cellular effects on cytokine response between nanoparticle types in different cell types.
2. There is a strong correlation between the catalytic activity of Pd nanoparticles (generated by WP11) with their ability to induce intracellular ROS production.
3. From the 14 materials studied, metal nanoparticles (TiO2, SiOx) induce the highest level of DNA damage after 24 h exposure indicating possible genotoxic effects. Still, all materials studied failed to induce NM in human bronchial epithelial cells.

WP5 Industrial processes, leaks of ENP, burden at workplaces, and levels and size distribution of ENP for requirements of the concepts, methods and pre-prototype devices

During the first reporting period the following tasks were targeted:
Task 1. Development of a conceptual model of exposure to (engineered) nanoparticles.
Result: Development of a conceptual model for inhalation exposure to ENP
Task 2. Inventory of expected specifications and requirements/desirables of WP 6-8 devices.
Result: A survey was sent to WP6-8 leaders to achieve 'Requirements for the newly developed devices'. The questionnaire was summarized and documented.
Task 3. Description/evaluation of existing and future (if possible) workplace exposure scenarios with emphasis on exposure conditions, concurrent exposures etc.
Result: A preliminary analysis was made using data from the project 'NANOSH'
Task 4. Identification and filling of knowledge gaps by limited and small-scale experiments.

Result: During the 2nd WP meeting (November 2009, Hoofddorp, NL) knowledge gaps were identified, with focus on the conceptual model. It was concluded that the input value of parameters for (both mathematical and CFD) modelling should be as realistic as possible. Consequently, it was concluded that more data were needed with respect to 'background' aerosols, e.g. ranges of size distributions, particle number concentrations etc. An (overarching) outline of the experimental studies was drafted and agreed (M5.2). During the 3rd WP meeting (April 2010, Nancy) further details were discussed. Actual (experimental) experiments will be in close collaboration with WP1 (particle generation) and have been scheduled for August 2010.

During the second reporting period the following tasks were done:
Task 1. Development of a conceptual model of exposure to (engineered) nanoparticles.
Result: Publication of a conceptual model for inhalation exposure to ENP
Task 2. Inventory of expected specifications and requirements/desirables of WP 6-8 devices.
Result: A survey was sent to WP6-8 leaders to achieve 'Requirements for the newly developed devices'. The questionnaire was summarized and documented.

3. Identification and filling of knowledge gaps by limited and small-scale experiments.

Result: An overview of measured and expected concentrations for various exposure scenarios was reported.

4. The description/evaluation of existing and future (if possible) workplace exposure scenarios with emphasis on exposure conditions, concurrent exposures etc. is in progress. During the fall of 2010, actual experiments (measurements) have been conducted in close collaboration with WP1. The dimensions and experimental set-up have been used for meshing the geometry to run CFD modelling. The first preliminary conclusions based on experiments and modelling confirm that coagulation is only relevant at high concentration, obstacles in the room do not relay affect these processes, and the effect of ventilation clearly can be observed. Currently, the first draft report on the experimental study is under review, whereas the CFD modelling will run again including real workplace data for background aerosol simulations.

During the third reporting period the following task was achieved: Identification and filling of knowledge gaps by limited and small-scale experiments. An (overarching) outline of the experimental studies was drafted and agreed (M5.2). Temporal and spatial evolution of particle number concentration and size distribution, affected by initial concentration, background aerosols, and ventilation were considered to be major knowledge gaps. In general, the different aerosol instruments show reasonable agreement with respect the (average) particle size and particle size distribution. In both experiments ELPI showed the highest concentration followed by FMPS. SMPS showed noticeably lower concentrations in both cases.

WP6 Portable active surface area aerosol monitor

During the first reporting period partner 17 TUT has been gathering information about the means to produce ions for diffusion charging of the particles. Base on the literature survey and tests, it was decided that the corona discharge is chosen as a way to charge the particles in the developed portable aerosol monitor. The physical limits of miniaturization of unipolar corona charger have been studied with modelling and experiments. The aim of this study is to find out the physical limits on how small the charger can be and at how low voltage it can be operated. A prototype of a small corona charger has been built, evaluated. The prototype was also modelled and the model was verified against the experimental results. This part of the work has not yet been finished; some of the measurements are still on-going.

Device development is mainly partner 12 Dekati's task. During this period the focus has been in electronics development where major tasks are optimization of high voltage power supply for ECT charger, isolated power supply design and electrometer for current measurement. New concepts for all these parts are built and currently being tested. Integration for a complete system has not started. Mechanical design will start after Milestone M6, decision of the configuration of the pre-prototype (Month 20). Overall development is on time.

During the second reporting period the pre-prototype instrument has been designed and built based on the research findings from the first reporting period. Based on the previous work the corona discharge was chosen for the particle charging. Electrical components (high voltage power supply, ECT unit and electrometer) were integrated to a functional system.

The geometry of the charger was tested by TUT prior to the integration to the measurement electronics using an electrical low pressure impactor (ELPI) as reference and the charging efficiency of the charger for different size particles was measured from 8 to 130 nm. The tests results implied a good sensitivity for the pre-prototype sensor. The prototype charger was also tested with the measurement electronics to test the feasibility of the pre-prototype sensor as a whole. The prototype sensor showed comparable results with the NSAM.

The pre-prototype sensor was then manufactured by Dekati based on the tested geometry. The work will be continued with the verification of the sensor operation both using laboratory test aerosols and monitoring the working environment of different nanoparticle processes. Also the possibility of different weight functions will be evaluated.

During the third reporting period the work continued from the previous reporting period and the focus in the work was turned from device development into testing, calibration and performance evaluation of the pre-prototype sensor. Well-known laboratory aerosols were used for the calibration and the sensor response was compared to reference instruments. Deliverable D6.2 'Test report on pre-prototype instrument performance' summarizes the results of the performance evaluation. To evaluate the real-world performance, the sensor was additionally tested at different facilities both at TUT and other NANODEVICE partners. These tests were performed in co-operation with WP1, WP13, and WP14.

Based on the field tests (WP14 and other test locations) some modifications were made to the built pre-prototype sensors, focusing on improving the reliability and sensitivity of the built pre-prototypes. The main change was adding a possibility for mains (110-230VAC) power use as an alternative for battery. In long-term measurements or in monitoring purposes this was seen as a more practical approach.

WP7 Size-discriminating number and surface area aerosol monitor and sampler

During the first reporting period the main results handled single components of the system developed in WP7.

Conditioner. A suitable commercial conditioner will be chosen after the other components have been developed and the requirements for the conditioner are fixed.

Pre-Separator. A pre-separator was developed that includes a virtual impactor and a cyclone. Optimal settings for the pre-separator were studied and they were achieved.

Charger. A literature survey on existing unipolar and bipolar charger designs was conducted and the results compared. It was decided to use a unipolar charger. The charging efficiency was tested and found to be in very good agreement with literature data.

Classifier. The approach for the classifier was studied. The classifier will use the integral approach. The design is currently underway.

Counter + Size Dependent Detector. Suitable materials for the tailored particle depositions have been identified based on a literature review of the deposition characteristics and were ordered. First experiments revealed that the deposition behaviour does not exactly follow the theoretically predicted one. This may be due to incompleteness of the used theory or –more likely- due to inhomogeneities of the ordered materials.

Thermal Precipitator. Two different versions of a thermal precipitator will be available in WP7. One is an existing TP that needs to be adjusted to fit the overall system. The second TP will be a new development within WP7 and designed to deposit particles directly onto live cells by depositing the particles directly from the airborne state and thus making in-vitro testing more representative for the true hazard in the workplace.

Electrostatic Precipitator. The development of a miniaturized ESP which can be used as stand-alone device or a component in the modular system is therefore considered to be of high priority in WP7. The designing is still on-going. Overall system design and periphery. The design of the overall system and periphery will start as soon as the designs of the single components are finalized.

During the second reporting period the work focused on the design of the single components, their evaluation and the assembly of the single components to the complete system.

Conditioner. The instrument will be kept at an elevated temperature to avoid humidity and particle-bound water to affect the measurement. Since the electrometer itself is heated and the internal pumps also release heat, the device should not need any additional heater, thus reducing battery requirements. Although the elevated temperature may cause semi-volatile particles to evaporate inside the instrument, this can be considered as a first step towards background elimination, since engineered nanoparticles are usually non-volatile. The pre-separator was developed during the first reporting period. Further experiments were conducted to investigate the effect of the pressure drop caused by the pre-separator on the efficiency of a unipolar diffusion charger, which is very similar to the charger that is used in our system.

Charger. A unipolar diffusion charger, based on the proven and established opposed flow charging principle was developed. The charger was subject to intensive investigations of the charging efficiency and the charge distributions. The charge distributions were mathematically described in a way that they can be used in the data acquisition and evaluation software. The Classifier used is of radial type that can be used in integral and differential modes. The transfer functions of the classifier were measured and mathematically describe to include them in the data acquisition and evaluation software.

Counter + Size Dependent Detector. The idea of the counter is to use a tailored deposition of charged particles on a low efficiency filter and measure the current which is then proportional to the particle number concentration. The idea of the second detector is to use a high efficiency filter and deposit all particles that penetrate the low efficiency filter and measure the total current. Five low efficiency filters (screens) have been tested that have different fibre diameters and mesh widths.

Thermal Precipitator. Two different versions of a thermal precipitator are available in WP7. One for sampling on silicon substrates for electron microscopic analysis, the other one for deposition on living cells for cytotoxicity studies. The prior one is part of the aerosol sizer and sampler whereas the latter one is a stand-alone device and referred to as 'Cyto-TP'.

Experiments were conducted to investigate the performance of the TP. A manuscript on the experimental validation of the TP has been drafted and will soon be submitted to a scientific journal. The Cyto-TP has been designed and constructed and is now ready for testing Electrostatic Precipitator. A miniaturized electrostatic precipitator for highly efficient sampling of particles has been developed. The ESP is designed such that particles are homogenously deposited on substrates when used along with the aforementioned charger. The substrates can hence be used for qualitative analyses of the sampled particles. The charger-ESP combination has proven to work reliably and sample even micron particles efficiently and homogenously. Overall system design and periphery. The first pre-prototype ('prototype 0') of the aerosol sizer system has been manufactured and delivered. First measurements were carried out with the new pre-prototype and show good agreement with SMPS measurements.

During the third reporting period the work focused on: improvement of the first pre-prototype towards a clearer design by the use of an internal manifold, implementation of full flow control, improvement of the electronics towards operational stability and user friendliness and safety, testing and calibration of the screen sensor, testing and calibration of the complete system, improvement of the software, including general troubleshooting, improving user friendliness and inclusion of an intelligent decision logic for use in a tiered approach.

In addition, the Cyto-TP has been developed in WP7 that uses thermophoresis to deposit particles directly onto living cells that can be evaluated for cytotoxic effects. Extensive performance tests have been conducted with the device to verify the deposition of nanoparticles on the cells as well as test for the survivability of the cells under zero exposure conditions. These tests were directed at the proof-of-concept of the Cyto-TP.

WP8 Wide-range size-resolving aerosol monitoring and sampling system

During the first reporting period the following results were achieved. Several numerical models for particle deposition in tubes, on plates and on nets for different geometrical and material setups have been developed; Several numerical models for particle deposition in a cyclone as a function of cyclone geometry and operating conditions have been developed; Preliminary calculations with the numerical models for deposition in tubes and on plates showed that such a pre-separator would be too bulky and not suitable for a personal sampler; The models interesting from a design point of view, have been verified and tested; One of the models was employed to calculate geometry and to design a personal nanoparticle size fractionating sampler; The first prototype of the size fractionating nanoparticle personal ENP selective sampler has been manufactured Preliminary tests in Naneum laboratories shows that the performance is in the expected range of parameters; The first draft of operation instruction has been prepared and sent to WP leaders involved in testing; The first tests under SP5 are scheduled with INRS.

During the second reporting period the following results were achieved. Four prototype devices have been developed - 2 by Naneum and 2 by SU and LU to a stage where they are ready to be tested by laboratories within SP5. The devices are: a personal sampler for collecting nano sized particles in the range 2nm-5μm, an on-line monitor for detecting Carbon Nanotubes (CNT), and two modular pre-separators to measure the aerosol fraction deposited in the first extra-thoracic region and the alveolar region of the respiratory tract. Naneum CNT monitor was employed for preliminary tests at working places on the site of a European manufacture of CNT. The pre-separators are still in a process of continuous evaluation and both the design and the measured characteristics may be modified due to recent findings
During the third reporting period the following results were achieved on four devices:

Device 1: Personal nano-sampler

The device has been tested in Naneum laboratories and gives results which are in good agreement with expectations and theory. The device has been tested by partners 7 (DGUV-IFA) and 8 (INRS) as independent testers within WP13. A report has been written and submitted by partner 8 (INRS) confirming the results achieved by the sampler are consistent with expectations and theory.

The sampler has been tested in the field within WP14. Results and comments on usability have been received and form part of the final report.

Device 2: Real time CNT Detector

Naneum CNT monitor was employed for preliminary tests at working places on the site of a European manufacture of CNT. A CNT detector was manufactured and made available to WP14 for field testing. Feedback from initial tests indicated a tendency to blocking. The unit was redesigned and two units built and made available for further field testing. Results from field tests have been reported.

Device 3: Sampler / Pre-separator for aerosol fraction deposited in the Gas-Exchange Region.

The GE sampler/pre-separator has been tested within WP13 at the laboratories of SU, LU (Partners 10 and 26), in collaboration with WP13 at INRS and IGF (Partners 8 and 23) and within the workplace test campaign of WP14. Collaboration with P23 resulted in a redesign of the unit and retesting in the field. The results from these tests are reported in Deliverable Reports D8.3 D13.3 D14.2 and D14.3.

Device 4: Sampler / Pre-separator for aerosol fraction deposited by diffusion in the Anterior Nasal Region (ca. 5 nm – 400 nm)

The 7ET1 sampler/pre-separator has been tested within WP13 at the laboratories of SU, LU (Partners 10 and 26), in collaboration with WP13 at INRS and IGF (Partners 8 and 23) and within the workplace test campaign of WP14. The results from these tests are reported in Deliverable Reports D8.3 D13.3 D14.2 and D14.3.It is expected that most of the comments presented in D14.3 can be addressed either in the next design version of the sampler/pre-separator or the next version of the User Manual.

WP9 High-sensitivity optical sensor for single-particle number and mass

During the first reporting period objectives included the investigation of several methods for the reliable detection of optical signals arising from light scattering at nanoparticles. Until now an optimized laser source and a highly sensitive receiver turned out to be the most promising approach to detect the scattered light originating from small particles in the 100 nm-regime. In order to achieve the project aim of WP9, the optoelectronic components of the measuring cell have to be improved and the opto-mechanical and electronic periphery is to be adjusted and enhanced.

During the second reporting period previously developed hardware and software modules have been assembled into a functional prototype. The GRIMM NANODEVICE prototype consists of an optical and an electrical sensor, whose readings of an integrated electronic system merged directly into the unit and output as a data telegram. Outputs via the interfaces (either RS232 or Bluetooth) are evaluated using special software and are graphically displayed. These evaluations are already available online during measurement. The separation of measuring instrument and control system allows the user great flexibility: The device can also be used in exposed locations and / or health-endangering conditions.

Thus all required properties of the GRIMM NANODEVICE prototype and its metrics are met in the context of NANODEVICE.

During the third reporting period the development on the prototype has made it easy to use; it can be completely operated with a single button at the front site. The combination of an optical and an electrical sensor allows simultaneous measurement of a wide size range of particles:

WP10 High-sensitivity MEMS-based sensor for single-particle number and mass

During the first reporting period the following results were achieved. Development of an algorithm for detection and weighing of several particles with resonant beams. Proofing of sensor principle by measuring micro particles with cantilevers and strings. Fabrication process for micro beams with perfect clamping. Achieving an analytical model for actuation of cantilevers made from arbitrary materials.

During the second reporting period the following results were achieved. Development of an innovative method making use of the Brownian particle motion to efficiently deposit nanoparticles onto a micro resonator. Successfully developed a resonant micro-string based aerosol nanoparticle sensor pre-prototype with a particle number sensitivity competitive to commercial aerosol sensors. Introduction of a novel sensor method for nanoparticle composition measurements based on photothermal resonance spectroscopy.

During the third reporting period the following results were achieved. Airborne nanoparticles were measured using a pre-prototype in a controlled laboratory environment. Micro and nanomechanical string resonators were introduced for the efficient inertial sampling of airborne nanoparticles, facilitating the in-situ gravimetric mass and number concentration detection, and the in-situ chemical analysis with photothermal IR spectroscopy. The sampling technique was studied with a 28 nm silica aerosol and successfully compared to a theoretical model. With the inertial sampling technique, the mass concentration of 25 nm sucrose aerosol was gravimetrically measured. The impaction of single 100 nm Ag particles was detected, which opens the door for real-time nanoparticle mass spectrometry. With the photothermal IR spectroscopy with microstring resonators, a novel sensing technique was established and it allows the chemical analysis of airborne nanoparticles, which enables the background distinction of toxic engineered nanoparticles from the natural background. Chemical fingerprint of organic nanoparticles was measured. The spectroscopy technique is sensitive enough to distinguish TiO2 nanoparticles with different surface coatings. Furthermore, the analysis and distinction of single particles was shown.

WP11 Catalytic and surface-chemical aerosol monitors

During the first reporting period the detection of catalytically active ENP and detection of surface chemical functions of aerosol particles were studied. For the detection of catalytically active ENP several model ENP materials and suitable test reactions had been selected:
- Platinum: Oxidation of H2 - calculated detection limit 2,4 μg*
- Nickel: CO-Methanation - calculated detection limit 4,7 μg*
- Palladium: Hydrogenation of ethylene (C2H4) - calculated detection limit 0,03 μg*
- Iron Oxide (Fe2O3): Oxidation of CO - calculated detection limit 27,7 μg*

Two different approaches were investigated for the detection of catalytically active ENP, considering preliminary laboratory set-ups: catalytic reaction on airborne ENP and catalytic reaction on deposited ENP. An improved design of the laboratory set up for catalysis experiments using deposited nanoparticles is under development. The modifications enable a combination of sampling aerosol nanoparticles and a chemical reaction in one instrument. This offers the possibility to investigate whether the calculated detection limits in the range of nanograms are achievable.

Detection of surface chemical functions of aerosol particles. The proof of principle for a simultaneous determination of particle size and a fluorescence signal on single particles was accomplished on a laboratory setup. A functional relationship between the amounts of a fluorescent dye on the surface of micron sized test particles could be established. Further work is planned using a fluorescent marker molecule specific to redox-active sites.

During the second reporting period the experimental setup for the detection of catalytically active ENP was completed. The results indicate that catalysis is indeed suitable for a substance-specific detection of ENP based on their catalytic activity. The sampling and reaction unit is already portable and allows a particle sampling directly in the breathing zone of a worker. The detection unit currently consists of a mobile infrared spectrometer. Future investigations will be directed toward a further down-sizing of the CAAM, above all with regard to the detection unit. In addition, a combination of the SRU and the heat exchanger was designed and will also be investigated.

Detection of surface chemical functions of aerosol particles. From the tests it could be shown, that the simultaneous detection of scattered light and a fluorescence signal from individual particles can be detected. Further experiments will be conducted to enable a comparison between the two support materials for the dye. Furthermore, first experiments regarding the detection limit and the dependence of the fluorescence signal on the concentration of the fluorescent dye will be conducted.

During the third period the laboratory prototype of the Catalytic Activity Aerosol Monitor (CAAM) was further downsized by the integration of a small IR sensor. In addition, a new functionality-based metric was defined. Its usability for the assessment of workplace air was demonstrated in several experiments concerning the dependence of the catalytic signal on mass, size and support. Besides, the inherent ability of the CAAM to discriminate the target engineered nanoparticles from non-active background aerosols allows the detection of catalytically active nanoparticles even if they are attached to the background. Moreover, first investigations concerning a potential correlation of the activity measured by the CAAM in the gas-phase and a biologic activity in suspension were done. A strong correlation of the CAAM signal with the ability of the particles to produce reactive oxygen species in suspension could be observed which demonstrates the usefulness of the CAAM and its functionality based metric for a quasi-real time detection of airborne catalysts in workplace air.

In the third period of NANODEVICE, the method of detecting surface chemical functions of aerosol particles was investigated into its suitability to detect smaller particles in the range of nm which could be achieved by using TiO2 agglomerates.

WP12 Nanofibre monitor

During the first reporting period the following results were achieved:
- a working setup of a AFM measurement station to measure deposited nanoparticles on surfaces situated in a Cleanroom ISO class 1 (no cross contamination possible due to clean room surroundings)
- A selection of suitable surfaces for the later usage in the fiber monitor
- First successful measurements with nanoparticles on the surfaces
- A CNT generator to produce CNTs for the later usage in the development of the fiber monitor

During the second reporting period following results were achieved:
- Design and construction of a personal sampler Version 1
- Several sampling approaches at the KIT using the CNT-generator on the KIT setting and subsequent measurements using the AFM technique at Fraunhofer IPA without any significant success. It was not possible to detect single CNTs. The agglomeration status and shape of the produced CNT was determined using TEM and SEM analysis showing that a fast scanning device using AFM will not lead to acceptable results.
- It was proposed to use light microscopic inspection instead and a modified sampler regarding some special characteristics of the CNTs.
- First optical inspections after using a modified personal sampler Version 1 showed the high potential of optical inspection. This is now been investigated further.
- Subsequent optical measurements gained different qualitative data between different market-available CNTs. This approach seems to show very good performance even in the discrimination between different CNT manufacturers. These results will be shown at the 4M conference in November 2011 in Stuttgart as talk/poster presentation.
- First testing of the personal sampler Version 1 on a real-scenario industrial setting (in collaboration with IFA). The used parameters and sampling duration showed good deposition of particles and agglomerates. Fibrous agglomerates were visible using light microscopy. But the later preliminary performed spectroscopic analysis detected no significant CNT contamination. Further investigations are in progress.
- Design and Construction of a miniaturized personal sampler Version 2 using CNT-specific characteristics, in progress.
- Submission of a paper for the 4M conference in November in Stuttgart. The paper was accepted after minor amendments. See http://www.4m-association.org/conference/2011

During the third reporting period

- Deliverable D12.3 Optimized nanofibre monitor as pre-prototype is ready and submitted.
- Development has been done on a CNT specific deposition method: magnetic particle collector. The collector uses a specific metric: magnetism. The method very simple to install in a personal sampler. To distinguish between background and CNTs, the particles and agglomerates on the surface are examined via Raman spectroscopy. Raman spectroscopy can detect using database correlation easily CNTs from other magnetic particles, as iron, cobalt or manganese.
- Miniaturization is ready of the system using commercially available components (for a possible fast implementation of the system in larger numbers). A personal sampling pump samples with 100 ml/min air through the magnetic particle deposition system.

WP13 Calibration, testing, and background distinction

During the first year the following results were achieved. The report was completed on the adjustment of the nanoparticle calibration system for NANODEVICE which is in itself ready to use by developers. A ready-to-use version of a primary discontinuous calibration tool for coagulation aerosols and an additional prototype for a continuously working similar system was achieved. The ready-to-use version (for urban aerosol) Nano Test Facility in IGF-BGRCI in Dortmund was achieved.

During the second reporting period the following results were achieved. The nanoparticle calibration system at INRS was operational on time has been used extensively device developers in the project. The calibration tool at Fraunhofer ITEM has been further developed and demonstrated successfully in Dortmund NanoTest Facility in March 2011 and the 1st working mobile device of the calibration tool has been successfully tested. The NanoTest Facility at IGF-BGRCI is fully operational under all requirements of the NANODEVICE project and WP21 human mobility programme has been used travelling of device developers and instruments to the facility and two round robin tests have been performed. The questionnaire to assess the practicability and ease-of-use of the developed devices has been developed according to the requirements of users. It is currently applied by the device developers and measurement personnel to encourage its use with established aerosol measurement techniques and thus provide a comparison dataset for the later novel instruments from the NANODEVICE project.

WP14 Testing of the devices in the field

During the first reporting period the following results were achieved. Current existing instruments for measuring ultrafine and nanoparticles at workplaces were reviewed and checked for parallel application during the planned workplace comparisons in WP 14. Contact with the group of external technicians in Germany has been established and procedures how to distribute devices have been discussed. The draft of the questionnaire will be distributed among them to clarify the meaning of the questions. A document on the sampling strategy (D 14.1) has been drafted in a core group and discussed with external stakeholders in Germany, which also do measurements. The draft will be discussed within the consortium. For milestone 14 it has been agreed that the partners concerned will deliver documents on the justification of their work on the pre-prototypes until summer 2010. At the next subproject 5 meeting in September these documents will be scrutinized.

During the second period the following results were achieved. The group of external technicians in Germany has been contacted and procedures for training and distribution of the new devices are under discussion. The draft of the questionnaire will be distributed among them to clarify the meaning of the questions. Milestone 14 was discussed at the SP 5 meeting in September 2010 and finalized. All instrument developers presented their concepts of feasibility, most including first results of their realised setups. After thorough discussions all reports had been accepted. All concepts were assessed as being feasible. A document detailing the sampling methodology and intercomparison protocols for the workplace measurements (D 14.1) had been discussed and finalised in April 2011. Current existing instruments for measuring ultrafine and nanoparticles at workplaces were reviewed and checked for parallel application during the planned workplace comparisons in WP 14. A comparison of reference instruments (SMPS, ELPI, CPCs, surface area instruments, electrically detecting monitors) was conducted at the NanoTest Facility of IGF in Dortmund in June 2011. The aim was to get information on differences of all reference instruments which will be used in the field tests.

WP15 Educational materials on safety of nanotechnologies

During the third reporting period following results were achieved. Screening was done for the official public website and the internet domain http://www.nano-device.eu was reserved. A template for the creation of a public available internet page together with other EU-projects was developed and the later structure of the homepage was set-up. First templates for presentations and leaflets were prepared and the layout will be mostly delivered from EU-VRi. Possible places for workshops were screened (Reinraum Lounge Karlsruhe, Conferences..)

During the second reporting period the following results were achieved. The internet domain http://www.nano-device.eu was created and fed with all relevant information. The domain is constantly updated. The contents of the domain were commented and the logos provided by the partners. Several poster presentations were held at international conferences on NANODEVICE and a journal paper was achieved.

During the third reporting period the following results were achieved: Set-up of leaflets and posters of each device, Set-up of a questionnaire for the collection of the relevant data for the leaflets and posters from the device developers, sending the questionnaire to all device developers with two reminder loops,

Collecting all relevant data, Design and implementation of a master leaflet, Design optimization together with HSE-HSL, Proof reading of each leaflet/poster form the device developers, Finalizing the leaflets and posters, uploading the files onto http://www.nano-device.eu website.

WP16 Data-base supporting regulations of the safety on ENP

During the first reporting period the goal was to create structure of the databank supporting risk assessment of ENP and the goal was achieved.

During the second reporting period the following results were achieved. The database was opened to users in Mo 20. A project sub domain was created by EU-VRi and access to the website has been limited to partners in the EU-VRi team. The users can access: reference database, NANODEVICE Knowledge base and Survey tool. Reference database contains metadata about the relevant nano databases identified so far. For disseminating the project an official poster and easy-to-use brochures were created in the WP. WP16 has offered to take over the support for standardization activities after the finalization of WP17.

During the third reporting period the following results were achieved. The brochures were prepared and distributed at several occasions in particular at the following events:
- June 2012: Annual Forum for Nanosafety in Copenhagen (1st brochure)
- October 2012: SENN2012 conference in Helsinki
- November 2012: NANOSAFE 2012 in Grenoble
- February 2013: Quality NANO in Prague

D16.3 (brochures) have been submitted.

D16.4 has been submitted, including a survey on standardisation activities.

WP17 Support for standardization of the safety of ENP

During the first reporting period the following results were achieved. WP17 identified in total 30 national, European and international standards, which are of interest for the NANODEVICE consortium. The results were obtained by a literature search and analysis of standards relevant to the NANODEVICE project. Among them 14 standards are also including the nanoscale region and are thus highly relevant for the NANODEVICE project. A scientifically categorized list (4 categories) and a prioritized list (3 prioritized) of standards related to the NANODEVICE project outputs were created. A list of priority 1 standards was scientifically categorized and a list of contacts to various relevant, national and international standardization committees was compiled. A presentation on the standardization issues in general and on the status of work package 17 on the occasion of the Annual Safety Forum 2010 was given.

During the second reporting period the following results were achieved. The report on potential standardization objectives of the NANODEVICE project (D17.1) which summarized the standardization needs of the NANODEVICE consortium and on standards relevant to the project, was completed. The report on a strategy on the standardization of the NANODEVICE technologies (D17.2) summarized the work package´s efforts to develop a concept of the implementation of the standardization potentials of the NANODEVICE project. The results were achieved through a survey on standardization requirements among partners. Major conclusions are that the development of European standards is a major challenge for a single EU co-funded project, even for a large-scale integrating project such as NANODEVICE. Even if adequate resources have been assigned in the planning phase the synchronization of the project life cycle with the standardization process for the development of a Technical Report (TR) or a Technical Specification (TS) will be not possible in most cases, as the project outputs are typically obtained towards the end of Research and Development (R&D) project, but are required prior to the start of a standardization project. WP17 achieved its goals and was finished as planned in the DoW in Mo 24. WP16 offered to take over the support for standardization activities after WP17 is completed.

WP18 Establishment of an Annual Forum for nanosafety

During the first reporting period the Coordinator office held multiple meetings on selection of eligible candidates and created a draft list of names. Careful consideration was used to select the members with interdisciplinary backgrounds and take care that geographically Europe, USA, Asia and Africa were represented.

WP19 Organizing an international congress on safety of engineered nanoparticles and nanotechnologies in 2012

During the first reporting period the following has been decided for the International Congress: the Host, Date, Venue and the PCO. Programme: No final programme has been set yet. Speakers. No confirmed speakers have been invited yet. Practical arrangements: FIOH negotiated the venue of the Congress and has held multiple meetings internally on arranging the preliminary working timetables. All other practical arrangements, such as reserving the hotel rooms and catering, FIOH will give the task to the PCO. FIOH expects the PCO to begin working on this Congress by summer 2010.

During the second reporting period the preliminary programme has been set. The scientific programme consists of invited speakers, free oral and poster communications and a small-scale exhibition. Further education lectures are also being held on the day before the Congress starts. The speakers for the further education sessions and the keynote speakers during the scientific programme have been confirmed. On practical arrangements the Congress website is up and running. The registration fees are set. Early registration is open and it can be accessed through the website.

WP20 Handbook on safety of engineered nanoparticles

During the first reporting period a new book proposal was prepared, which defines the feature and targeted audience of the book. A Draft Outline of the Handbook was proposed and discussed within the WP20. The potential authors of each chapter were suggested and contacted. As a part of preparation for the handbook, we reviewed published books on environmental and human health impacts of ENP and safe production and handling of ENPs.

WP21 Human mobility within NANODEVICE project

During the first reporting period a leaflet inviting applicants for the mobility programme had been prepared and distributed among partners and heavy advertisement aimed to the consortium on the mobility programme was done by the work package members and the management office of the project.

Potential impact:

The potential impact (including the socio-economic impact and the wider societal implications of the project so far) and the main dissemination activities and exploitation of results (not exceeding 10 pages)

The potential impact

The project results will have a marked societal impact especially on workplace safety in small and medium size enterprises and micro-enterprises through bringing into market affordable on-line measuring devices providing reliable exposure information on engineered nanomaterials. This will not only greatly increase our knowledge and understanding on the workplace exposure to these materials. It will also support regulators and decision makers in making evidence-based conclusions and actions on necessary measures to protect the workers from excessive exposure, especially the setting of occupational exposure limits (OEL) for different ENM. One can expect remarkable savings to the industry through reliable exposure information at low or affordable expenses. These outcomes of the project will also enable safe use of these materials, and promote safe production of ENM, and thereby increase the competitive edge of nanotechnologies through emphasizing safety aspects in all areas of nanotechnology applications.

The main dissemination activities

During the course of the project the 26 partners of 21 work packages took part actively in conferences and meeting. Multiple oral presentation, poster presentations and thesis were held during the project. In addition to this list, the project management office presented the project posters and distributed the 3 brochures prepared during the course of the project and the SENN2012 International Congress flyers.

Exploitation of results

The project's successful culmination may have (or should have) on the general acceptance of ENPs and their positive perception by the public and users. It can be assumed that at least some of the nano technology 'scares' of recent years may have been due to inadequate knowledge and/or lack of precise regulatory limits. One issue is the lack of systematic knowledge and the devices have been out of reach for small enterprises regarding the price and regulators have not required to perform the measurements. Project will provide affordable devices and also means for exposure assessment. Regulators have the possibility to expect occupational measurements when there devices are accessible. Availability of reliable and accurate methods and devices is important to measure ENPs (and especially to distinguish between them (e.g. CNT) and naturally occurring NPs). Results of NANODEVICE project are extremely important for the overall success of Nano Technology and its further penetration in the market and public usage.

List of websites:

http://www.nano-device.eu.

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