Synthesis and characterization of titanium dioxide ... Special Session 7/All... · Synthesis and characterization of titanium dioxide particles for toxicity tests ... J. J., Flagan,

Synthesis and characterization of titanium dioxide particles for toxicity tests

A. J. Koivisto1, H. Alenius1, J. Joutsensaari2, H. Norppa1, M. Miettinen2, P. Pasanen2, L. Pylkkänen1, E. Rossi1, T. Tuomi1, M. Vippola1, J. Jokiniemi2,3 and K. Hämeri4

1Finnish Institute of Occupational Health, Topeliuksenkatu 41 a A, 00250, Helsinki, Finland

2Department of Environmental Science, University of Kuopio, Yliopistonranta 1 E, 70210, Kuopio, Finland 3VTT Technical Research Centre of Finland, Vuorimiehentie 3, 02044, Espoo, Finland

4Department of physics, University of Helsinki, Gustaf Hällströmin katu 2, 00014, Helsinki, Finland

Keywords: TiO2 nanoparticles, Nanoparticle production, Nanoparticles, characterization, Health effects of aerosols, Morphology.

Nanotechnology is controlling matter by adjusting dimensions of particles at roughly from 1 to 100 nanometers. In this size range the bulk material properties start to change, because number of surface atoms is increasing and quantum mechanical properties are changing. This change in properties may also affect to the matter toxicity and thus nanoparticles should not be treated as a bulk material. Here we introduce an inhalation exposure setup with laminar flow reactor, and present the results of titanium dioxide (TiO2) particle production. The experimental setup is shown in Figure 1. It consists of particle generator, humidifier and diluter, inhalation chamber, fabric filter and aerosol characterization instruments. The setup operates in atmospheric pressure where the produced aerosol is diluted and humidified to be suitable for mice experiments.

Figure 1. Scheme of the experimental setup with typical operation parameters. Nano-sized TiO2 particles were produced by thermal decomposition of titanium tetraisopropoxide (TTIP) (Ti(OC3H7)4 vapour in the tubular flow reactor (Okuyama et al. 1986; Ahonen et al. 2001). Nitrogen carrier gas, with flow rate of Qb, was saturated with the TTIP vapour. Inside the reactor TTIP is thermally decomposed: Ti(OC3H7)4 → TiO2 + 4C3H6 + 2H2O.

TiO2 molecules start to nucleate, and condensate to the seed particles, forming primary particles. Primary particles are further agglomerated by Brownian coagulation. The experiments were done with aerosol mass concentrations of 0.8, 7.2, 10.0 and 28.5 mg/m3. The aerosol size distributions were measured with scanning mobility particle sizer and electronic low pressure impactor. Aerosol particle concentration was measured with condensation particle counter. Composition and shape of the particles were defined from grid samples with transmission electron microscopy (TEM). The fabric filter was used to collect particles for further analysis and experiments. Specific surface area of produced titanium dioxide was defined with Brunauer-Emmet-Teller method to be 61m2/g. The crystalline structure of the particles was defined with X-ray diffraction method to be 74% anatase and 26% of brookite with crystalline size of 41 and 6 nm. In figure 2 is presented aerosol size distribution and figure of collected particles for aerosol with mass concentration of 10 mg/m3.

Figure 2. On the left is particle mobility size distribution. Fit is done for averaged values (○) of experiments. Symbols are median (x), standard deviation (box), and 5% and 95% percentiles (bars). On the right is TEM figure of collected particles. This work was supported by the Academy of Finland. Okuyama, K., Kousaka, Y., Tohge, N., Yamamoto,

S., Wu, J. J., Flagan, R. C. and Seinfeld, J. H. (1986). AICHE Journal, 32, 2010-2019.

Ahonen, P. P., Moisala, A., Tapper, U., Brown, D. P., Jokiniemi, J. K., Kauppinen, E. I. (2001). Journal of Nanoparticle Research, 4, 43-52.

Cite abstract as Author(s) (2009), Title, European Aerosol Conference 2009, Karlsruhe, Abstract T190A01

Characterization of polychlorinated dibenzo-p-dioxin/dibenzofuran emissions from joss paper burned in a furnace

M.T. Hu1, S.J. Chen1*, K.L. Huang1, Y.C. Lin2, G.P. Chang-Chien3,4 and J.H. Tsai1

1 Department of Environmental Engineering and Science, National Pingtung University of Science and Technology, 91201, Taiwan.

2 Institute of Environmental Engineering, National Sun Yat-Sen University, Kaohsiung, 804, Taiwan. 3 Department of Chemical and Materials Engineering, Cheng Shiu University, Kaohsiung County, 833, Taiwan. 4 Super Micro Mass Research & Technology Center, Cheng Shiu University, Kaohsiung County, 833, Taiwan.

Keywords: joss paper burning, PCDD/Fs, emissions.

Burning joss paper in temples is popular in some Asian countries (e.g., China and Taiwan) where Buddhism and Taoism are practiced. During temple activities, those praying and visiting are usually exposed to toxic compounds emitted by burning joss paper. Several studies have measured PCDD/F emissions from burning wood (Bumb et al., 1980; Choudhry & Hutzinger, 1983; Lemieux. et al., 2004). The chlorine ions (Cl-) inside wood enhance formation of PCDD/Fs (Bumb et al., 1980; Choudhry & Hutzinger, 1983). Joss paper is primarily made of recycled paper, bamboo, furniture manufacturing waste, or architectural waste. Hence, PCDD/Fs may form when joss paper is burned in temple furnaces. The joss paper in this study is widely used in Taiwanese temples—Taiwan has over ten kinds of joss paper in different shapes/sizes. The chlorine content (Cl−) in unburned and burned joss paper (B-ash) was 0.093 and 1.71 mg g−1, respectively (Table 1). The theoretically calculated Cl− content in burned joss paper was 1.98 (= 0.093×100/4.69) mg g−1. In other words, about 10% of Cl− content in joss paper was possibly associated with the formation of PCDD/Fs. The ash content in burned joss paper (B-ash) was high (84.6%). Hence, we infer that incomplete combustion and Cl− release are related to PCDD/F emissions from combustion of joss paper in the furnace.

Table 1. Compositions of the selected joss paper and bottom ash (B-ash) of burned joss paper.

Proximate Analysis (%) Joss papers B-ash Analytical MethodCombustible 87.2 15.1 NIEA R205.01CAsh 4.69 84.6 NIEA R205.01CMoisture 8.08 0.32 NIEA R203.01TUltimate Analysis(mg/g)

Chlorine (Cl−) 0.093 1.711 1.522 1.543

NIEA R205.01C /NIEA W415.52B

1 B-ash of burned joss paper obtained from the temple furnace 2 ash of re-burned B-ash obtained in lab 3 ash of burned joss paper obtained in lab

Table 2 lists the mean PCDD/F content and relative standard deviation (RSD) in unburned joss paper and ash from burned joss paper. Notably, ratios >1.0 for PCDDs/PCDFs and their I-TEQ (11.4 and 1.24, respectively) indicate that the predominant

PCDD/Fs in unburned joss paper were PCDDs, not PCDFs, and their toxic contribution was PCDDs > PCDFs. Gullett and Touati (2003) reported that PCDD/F content in ash from burning wheat and rice straw stubble was 0.5 ng I-TEQ kg-1, significantly lower than that from burning joss paper. Additives such as dyes and paint in joss paper are likely responsible for differences in PCDD/F content. The TEQ of total PCDD/Fs in ash in this study was in the range of 1.62–5.23 ng I-TEQ kg-1 (mean = 3.42 ng I-TEQ kg-1) higher than that for bottom ashes obtained from municipal solid waste incinerators in Taiwan (Chen et al., 2006). Therefore, joss paper combustion is a significant source of PCDD/F emissions.

Table 2. Mean PCDD/F content in the joss paper and B-ash of burned joss paper (n = 6)

Joss paper B-Ash PCDD/Fs species Mean

(ng kg-1) RSD (%)

Mean (ng kg-1)

RSD(%)

Total PCDDs 178 13.2 7.79 7.01 Total PCDFs 15.6 5.67 10.7 6.59

PCDDs/PCDFs 11.4 14.6 0.726 2.84 Total PCDD/Fs 193 12.1 18.5 6.62

ng I-TEQ kg-1 % ng I-TEQ kg-1 % Total PCDDs 0.355 16.0 0.449 7.01 Total PCDFs 0.290 14.1 1.47 6.01

PCDDs/PCDFs 1.24 19.5 0.305 2.87 Total PCDD/Fs 0.645 11.9 1.92 6.14

Bumb, R.R., Crummett, W.B., Cutie, S.S., Gledhill,

J.R., Hummel, R.H., Kagel, R.O., Lamparski, L.L., Luoma, E.V., Miller, D.L., Nestrick, T.J., ShadoD, L.A., Stehl, R.H., & Woods, J.S. (1980). Science, 210, 385–90.

Chen, C.K., Lin, C., Wang, L.C., & Chang-Chien, G.P. (2006). Chemosphere, 65, 514–520.

Choudhry, G.G., & Hutzinger, O. (1983). Mechanistic aspects of the thermal formation of halogenated organic compounds including polychlorinated dibenzo-p-dioxins. New York: Gordon and Breach.

Gullett, B., & Touati, A. (2003). Atmospheric Environment, 37, 4893–4899.

Lemieux, P.M., Lutes, C.C., & Santoianni, D.A. (2004). Progress in energy and combustion science, 30, 1-32.


Human type B synoviocytes, as a cellular model for a better knowledge of the pro-inflammatory effects of environmental PM

F. Cetta1, F. Laghi Pasini2, E. Selvi2, A. Dhamo1, M. Natale2, R. Zangari1, P. Laviano1, L. Cantarini2,

E. Bolzacchini3, M. Camatini3, M. Galeazzi2.

1 Department of Surgery, Research Doctorate in Oncology and Genetics and 2 Department of Clinical Medicine and Immunology, University of Siena, Nuovo Policlinico “Le Scotte”, viale Bracci,16, I-53100 Siena, Italy

3 Department of Environmental Sciences, University of Milano-Bicocca, Piazza dell'Ateneo Nuovo, 1,20126 Milan, Italy

Keywords: air-pollution, traffic emissions, field measurements , health effects of aereosols, lung - particle

interaction. Human type B synoviocytes are involved in cartilage damage in chronic inflammatory joint diseases, particularly in rheumatic diseases, by producing inflammatory mediators such as interleukin 6 (IL-6). The increased level of purine and pyrimidine nucleotides in the synovial fluid of rheumatoid arthritis (RA) patients could activate the large family of purinergic P2 receptors. An interesting member of the P2X subfamily is the P2X7 receptor (P 2X7R), - expressed on inflammatory cells and human primary fibroblasts, - coupled to ion fluxes, microvescicle formation, and IL release (Caporali, 2008). Health effects from environmental particulate material (PM), in particular clinically evident diseases related to long-term exposure (asthma, COPD, pulmonary fibrosis etc), show striking similarities with autoinflammatory and autoimmune rheumatic diseases. In particular, epidemiological studies show that only a minority of the general population, namely predisposed people, are those mainly affected by adverse effects, whereas in the vast majority of the general population, clinically evident diseases usually do not occur, even after exposure to high peaks of environmental pollution (Cetta, 2007, 2009). The final clinical outcome is usually determined by common mechanisms such as ROS generation, oxidative stress, activation of pathways, such as NFKB, increased production of proinflammatory cytochines, such as interleukin (IL) 6, IL8, and tumor necrosis factor α (TNF α), similarly to what occurs in chronic inflammatory joint diseases. The aim of the present study has been to assess the “in vitro” effects of variable concentration (1-10-50 µg/cm2) of various types of PM (PM 10,2.5,0.1) when incubated with human synoviocytes, to evaluate the occurrence of proinflammatory effects. Daily levels of PM10 and PM2.5 and PM1

(diameter <10 µm, 2.5 µm, 1 µm, respectively) were measured by PM detection units, both outside and inside 2 schools for children and 2 hospices for retired people in Milan, Italy. Both OPC detectors (GRIMM) and low volume gravimetric detectors (Tecora, Milan, Italy) were used. Usually Teflon filters (47 mmØ, 2 µm, Pall Gilman, USA) were

stored. Samples from these filters were used after sonication in sterile phosphate buffer saline (PBS). Aliquots of these samples at a concentration of 10/50/75µg/ml were incubated for 4 or 6 hours at 37°C with human type B synoviocytes. Preliminary data showed both an increased production of interleukin IL-6, IL-8, and the functional activation of P2X7 receptors. In particular, biological PM - host interaction was mediated by cytosolic Ca2+ increase, triggering the NFKβ pathway, whereas various components of PM (PM10,2.5 and 0.1) interacted different with host cells and tissue, generating a wide variety of reactions involving microvescicle formation, phagocytosis, membrane alterations (cellular membrane holes). Clinical and laboratory evidences show that the occurrence and the severity of evident diseases is not simply related to intrinsic toxicity of the various pollutants, which should be homogeneous in the various hosts, but host-particle interactions generate health end-points, which greatly depend not only on individual susceptibility, but also on the type of the response and on the entity or grade of the reaction. In particular, our working hypothesis is that PM-related diseases are not simply determined by an inflammatory mechanism, mediated by oxidative stress, but more complex responses are generated, namely those typical of autoinflammatory and/autoimmune diseases, which occur only in predisposed subjects. This work was supported by a CARIPLO Foundation Grant to POLARIS Research Center and by the Flagship Project, PROLIFE 2008, City of Milan, Italy. Caporali, F., Natale, M., Selvi, E., Galeazzi, M., Laghi Pasini, F., et al., (2008). J Mol Med., 86,937-949. Cetta, F., Dhamo A., Schiraldi, G., Camatini, M., (2007). Eur Respir J, 10,805-806. Cetta F., Dhamo A., Moltoni L., Bolzacchini E.

(2009). Environ Health Perspect. In press.


Intrinsic toxicity and inflammatory potency and/or health damage of Particulate Material (PM) in physiologic and pathologic conditions

R. Zangari1, F. Cetta1, M. Sala2, A. Dhamo 1, P. Laviano1, F. Cisternino1, G. Malagnino1,

G. Schiraldi3, E. Bolzacchini4

1 Department of Surgery, Research Doctorate in Oncology and Genetics, University of Siena, Nuovo Policlinico

“Le Scotte”, viale Bracci, 16, I-53100 Siena, Italy

2Department. of Pediatrics and 3Toracopulmonary and Cardiovascular Department, University of Milan,Via Festa del Perdono,7,20122,Milan, Italy

4 Department of Environmental Sciences, University of Milano-Bicocca, Piazza dell'Ateneo Nuovo, 1,20126 Milan, Italy

Keywords: air pollution, nanoparticles, health effects of aereosols, lung-paricle interaction

Once Particulate Material (PM) introduced by inhalation has reached pulmonary interstitial sites, uptake into the blood circulation, in addition to lymphatic pathways, can occur, depending on particle size, favouring nanoparticles. In particular, particle size, surface chemistry and possibly charge, govern translocation across epithelial and endothelial cell layers. Depending on particle surface chemistry, nanoparticles have been shown to transcytose across alveolar type I epithelial cells and capillary endothelial cells, but not via cellular tight junctions in the healthy state. However, in a compromised or disease state, translocation across wide and tight junctions occurs as well, because of functional cell membrane damage due to sepsis. Within the frameshift of the Milan Prolife Project, during the last 2 years (2007-2008), a 360° research on health effects of PM has been performed, with particular attention to host particle interactions. In particular, cross sectional and panel longitudinal studies have been performed, both in children and in old patients. Clinical, laboratory (respiratory exhalate) and instrumental data (spirometry), BAL (bronchioloalveolar lavage) and in some cases tissue biopsy, BAL culture and pH analysis, were compared with seasonal PM concentration and speciation and in vitro studies on cell lines, incubated with the same PM (PM 10, PM2,5, PM1 ), that was collected in the same site and period as data collection for clinical study. Namely, cross sectional studies included the comparison of pedriatic acute respiratory admissions to the main pediatric referral center. They were divided into upper airways, lower tract and asthmatic diseases and grouped as seasonal admissions and of old people acute admissions for respiratory or cardiovascular diseases to the entire network of Milan hospitals, with daily and seasonal PM concentration, monitored by fixed monitors. Longitudinal panel studies included: 1) the seasonal comparison of two groups of subjects 100 children

(actual n completing the study=113) attending a school close to busy motorways and 100 (actual = 108) attending school far from vehicle traffic; 2) 100 subjects older than 65 living in hospices for old patients located close (n=89) or far (n= 90) from main motorways. Measures were made during two-week campaigns - both in winter and summer time - with direct measure of PM both outdoor and indoor by optical and gravimetric samplers. Preliminary data showed that the most severe features and complications during PM peaks occurred in patients with previous long-lasting infections asthma, or COPD pulmonary. Overall data suggest that, in addition to individual susceptibility, due to genetic variability, which greatly affects health effects of host particle interactions, in the presence of the same PM concentration and exposure, the occurrence and severity of symptoms greatly varies among subjects with physiologic or pathologic airway conditions. Detection of pathogenetic mechanisms such as oxidative stress, - which could act as a common mechanistic pathway of PM related health damage - is useful, but oversimplification of complex interactions, such as host-particle interaction, could cause understatement or missing of other concomitant mechanistic pathways such as concomitant air tract infection or exposure to endotoxin which also play a major role in the occurrence of clinically evident diseases after PM exposure. Previous personal history and pathophysiological conditions of each subject are major determinants of observed adverse effects of air pollutants. The study has been supported by the City of Milan Flagship Project PROLIFE 2008.


Redox balance of Thiols in the exhaled breath condensate (BEC) in two populations with different exposure to traffic related pollutants.

F. Cetta1, R. Accinni2, G. Schiraldi3, M. Sala4, R. Zangari1, P. Laviano1, M. Guinea Montalvo2, G. Giussani2,

C. Dellanoce2, F. Minardi5 , L.Allegra3

1Department of Surgery, University of Siena, Nuovo Policlinico “Le Scotte”, 53100 Siena, Italy 2CNR Institute of Clinical Physiology, Niguarda Cà Granda Hospital, 20162, Milan, Italy

3Toracopulmonary and Cardiovascular Department and 4Department. of Pediatrics and 5Monzino Cardiologic Center, University of Milan,Via Festa del Perdono,7,20122,Milan, Italy

Keywords: air pollution, traffic emissions, field measurements, health effects of aereosols

Thiols such as cysteine (Cys), cysteinilglycine (Cysgly), homocysteine (Hcy) and glutathion (GSH) are widely distributed in humans and play an important role in biological systems. Normal levels of thiols in physiologic fluids are considered as markers of good homeostatic equilibrium (Bloemen, 2007) On the contrary, their alteration has been associated with various diseases (asthma, COPB), usually related with increased production of reactive oxygen species (ROS). Therefore, levels of reduced and oxidized forms of thiols in plasma, in red cells and, more in general in all biological fluids - including exhaled breath condensate (EBC)- is considered a good marker of ROS activation and an early marker of related diseases (Kharitonov, 2006). Two groups of patients (older than 65 years), living in “hospices for retired peoples” have been recruited and compared, (n= 38, age 82 ± 9y) with the aim of evaluating cardiovascular and respiratory adverse-effects of environmental pollution, namely of pollutants related to urban traffic. In particular, one Hospice was located within 200m from high traffic crossroads, whereas the other was sited in a park , far from the main crossroads, and this group of subjects was compared with 35 subjects (age 70±8y) living in Aprica, a remote alpine site (1118 m. a.s.l.). EBC samples were collected and HPLC analysis was performed for the evaluation of thiol levels. In addition, oxidative stress state was also evaluated by isoprostane (8-iso-PGF2alfa), a lipid peroxidation index, by LC-MS/MS method. No differences in 8-iso-PGF2alfa were found between the two groups as the levels were under the detection limits (150µM). In the EBC of 35 subjects from Aprica the presence of various molecules, showing SH groups, was found, in particular in the oxidized form (fig.). Retention time of eluted peaks did not correspond to known thiols. Therefore, an accurate qualitative and quantitative analysis of the various thiols was not possible, only using HPLC. However, there was a striking individual variability of the various chromatographic peaks, which corresponded to fluorescent adducts detected by fluorimetric detector. Since the method is highly specific for S containing groups, it is presumed that observed peaks mainly correspond to low molecular

weight molecules with active SH groups (fig.). Since no signal concerning reduced species was detected, two possible hypotheses can be envisaged : 1) reduced species could be present in small amounts below the lowest treshold for detection; 2) the absence of reduced species could be due to the pro-oxidant environment of the breath exhaled condensate. In the latter evenience, total thiols, obtained by reduction of all thiols by a specific compound, such as tris (2carboxiethil) phosphine, only included oxidized thiols

The number of samples from Milan, due to the very old age of patients living in public hospices, and then the difficulty of obtaining good quality samples, was not sufficient to permit a significant comparison between the 2 groups of subjects. More sensitive and specific detection instruments, such as mass LC, will provide a more precise detection of observed components with SH groups. However, present findings provide for the first time a method to analyze thiols in the exhaled breath condensate and then the possibility of using EBC thiols as an early marker of altered redox balance in the respiratory tract, in order to evaluate initial local damage from environmental pollution. This work was supported by a CARIPLO Foundation Grant to POLARIS Research Center and by the Flagship Project, PROLIFE 2008, City of Milan, Italy Bloemen, K., Lissens, G., Desagerb, K., et al., (2007). Respiratory Medicine,101,1331-1337. Kharitonov, S.A., & Barnes, P.J., (2006). Chest,130,154


The possible impact of “sequential co-exposure” on ozone associated adverse health effects. Preliminary data from cumulative cross sectional and prospective studies in

Milan.

F. Cetta1, M. Sala2, G. Schiraldi3, E. Bolzacchini4, A. Dhamo1, L. Moltoni1, G. Gerosa5, A. Ballarin-Denti5, L. Allegra3

1 Department of Surgery, Research Doctorate in Oncology and Genetics, University of Siena,53100,Siena, Italy 2 Department of Pediatrics, and 3Thoraco pulmonary and Cardiovascular Dept, University of Milan ,Milan, Italy

4 Department of Environmental Sciences, University of Milano-Bicocca,Milan, Italy 5 Department of Mathematics and Physics, University “Cattolica del Sacro Cuore”, Milan,Italy.

Keywords: air pollution, traffic emissions, exposure and health effects of aereosols

Aim of the study: The aim of this study

has been to report on preliminary data concerning the possible impact of ozone as a single pollutant or together with other pollutants in the occurring of clinically relevant diseases.

Methods: Two monitoring campaigns (July-September 2008) (December-January 2009) covering the entire City area were performed in Milan, Italy. In particular, gaseous pollutants, - O3, NO2, CH6H6 - were monitored using passive samples (PASSAM, CH) in 50 different urban sites. Recorded values were processed by a GIS based geostatistical software to give the concentration profiles for the 3 selected pollutants. Fine particulate matter (PM10) concentrations were measured in 5 urban sites representative of different areas of the City by gravimetric samplers. The heavy metal composition was analyzed by XRF techniques, during a 30-day-period. Results: Benzene and NO2, were representative of traffic emission, in particular during the Summer period. Both NO2 and benzene (representative of traffic emission) showed elevated values in sites with intense vehicular traffic, as in the highly congested city centre and along the heavy- traffic roads. Ozone showed an opposite behaviour, being higher where NOx and benzene were lower. In particular, Ozone values were higher inside the city gardens and parks. In fact, in vegetation-rich areas, the absence of traffic scavenging is summed up to the vegetation-released ozone precursors. Ozone average concentrations seldom reached values of more than 90µg/m3 (Fig. 1). The greatest exposure was found in the City park, where ozone precursors were abundant and traffic related scavengers were relatively scarce. Close to City edge, exposures were lower, due to the reduced quantity of precursors and the increased quantity of traffic related scavengers. City center had an intense vehicular traffic, the emission of which contributed to the local ozone consumption. Interestingly, there was a reduction in ozone concentration during the first 3 weeks of August, in concomitance with the population reduction, because of Summer holidays, and a subsequent increase in September (Fig.1).

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Figure 1. Weekly means of ozone concentration recorded by passive samplers at 50 different sites within the city of Milan in the summer 2008. Conclusions: Clinical effects from environmental pollution can be considered as the end result of a complex mixture, including variable concentrations of various pollutants. In the occurrence of the health outcome not only PM 2,5 and/or PM 10, but also gaseous pollutants (ozone, NOx, SO2) and biological components cooperate together through concomitant or sequential co-exposure. A “sound” possibility for ozone to be dangerous is to be responsible for “sequential coexposure”, i.e. to fill the gap, to make damage even during “the summer window”, which could otherwise permit logical and physiological repair of previous damages, the ones that have been accumulated in the various cells, epithelia , because of other pollutants, the effect of which is more prevalent in Winter, or during Spring or Autumn. In this way, Ozone provides a continuous damage to human tissues, that will have no time left for re-epithelization, cell or tissue repair, due to the uninterrupted activity of multiple pollutants, each one having a preferred season, but also all acting together and in ordered sequence to guarantee a continuous damage. In this respect, not only concomitant co-exposure with SO2 and other pollutants from combustion sources, but also sequential exposure, after damage caused by PM10, PM2,5, pollen and bacteria, could offer a “sound” pathophysiological working hypothesis for a causative role of ozone in the occurrence of pollution related adverse effects. This work was supported by the Flagship Project, PROLIFE 2008-Sustainable Mobility, City of Milan, Italy.


Comprehensive characterisation of manufactured nanoscaled powders following soft dispersion

K. Wittmaack1, 2

1Grimm Aerosol Technik GmbH, 83404 Ainring, Germany

2Helmholtz Zentrum München, Institute of Radiation Protection, 85758 Neuherberg, Germany

Keywords: Nanoparticles, Health aspects of aerosols, Optical particle counter, SMPS, SEM. Concern has been raised that exposure to manufactured nanoparticles (NPs) may impose a significant risk to human health. The arguments largely originated from the adverse health effects that have previously been associated with the inhalation of ultrafine aerosol particles. But convincing evidence supporting this association has not yet been presented. A problem with the potential risk of engineered NPs is that commercially available powders are often assumed to be composed of primary particles with sizes < 100 nm. The purpose of this study was to characterise the size distribution and morphology of NP powders produced by a variety of different techniques.

a

b Figure 1. (a) Size distribution of particles dispersed

from an Fe3O4 powder. (b) Calculated mass distribution assuming spherical particles.

Briefly, dispersion was accomplished by directing a jet of dry nitrogen at the powder deposited in a small vessel. Particles transferred into the gas phase were carried to the analyser by a flow of filtered air. Size analysis was achieved using (i) a

differential mobility analyser (DMA) coupled to a condensation particle counter and (ii) an optical counter. Pressure pulses of only 10-100 hPa were required to achieve efficient dispersion. Quite surprisingly, the fraction of dispersed primary particles contained in the spectra of nanopowders was found to be very small, as illustrated in Fig. 1, which constitutes a representative example for the general features observed with all powders studied (Fe2O3, Fe3O4, CeO2, Co, ZnO, TiO2, Si and Printex-90). Particularly important is a shoulder extending into the micrometer size range. The aggregates generating this shoulder, see Fig. 2(a) and (b), comprise more than 99% of the total dispersed mass. Clearly, discussing the toxic potential of nano-structured matter, these features must be considered.

a

b Figure 2. Examples of NP aggregates collected in a

5-stage impactor, (a) Fe2O3, (b) TiO2. This work was supported by the DIPNA project of the European Commission (Contract NMP4-CT-2006-032131).


Abstract für EAC Conference, September 2009

A new exposure system for an efficient and controlled deposition of aerosol particles onto cell cultures

M. Kalberer1, M. Geiser2, M. Savi2, D. Lang2, A. Gaschen2, M Ryser3, J. Ricka3, M. Fierz4

1 Centre for Atmospheric Sciences, Department of Chemistry, University of Cambridge, Cambridge CB2 1EW,

UK 2Institute of Anatomy, University of Bern, Bern, Switzerland

3Institute of Applied Physics, University of Bern, Bern, Switzerland 4Institute for Aerosol and Sensor Technology, University of Applied Sciences, Northwestern Switzerland,

Windisch, Switzerland

Keywords: Aerosol instrumentation, Deposition, Health effects of aerosols, Lung/particle interaction Epidemiologic studies have shown correlations between morbidity and particles smaller than 2.5µm generated from pollution processes. The increased use of nanoparticles in manufacturing processes is also a source of concern. The interaction of particles with the lung, the main pathway of undesired particle uptake, is poorly understood. In most studies investigating these interactions in vitro, particle deposition differs greatly from the in vivo situation, causing controversial results. We present a novel nanoparticle deposition chamber designed to expose lung cells under conditions closely mimicking the particle deposition conditions in the lung (Savi et al., 2008). In this deposition chamber, particles are deposited very efficiently, reproducibly, and uniformly onto the cell culture, a key aspect if cell responses are quantified in respect to the deposited particle number. Particles are deposited onto the cells directly out of a conditioned air-flow, simulating accurately the physiological conditions in the lung, i.e., the aerosol is humidified to about 90% relative humidity, the CO2 concentration is increased to 5% v/v and the aerosol is heated to 37°C. This assures that all cellular reactions are due to particle deposition and not due to other stress factors. The key feature of the chamber is the electrostatic particle deposition. After passing a bipolar charger (Kr-85 source) particles are efficiently deposited by an alternating electrical field. To avoid accumulation of particles with one polarity on the cells, the polarity of the electrical field alternates (Fig. 1). For on-line monitoring of the state of the cell culture, the chamber is equipped with a device detecting the mucociliary activity, whose most conspicuous signature is the ciliary beat frequency. A fiber optic probe detects the vertical movement of the mucus induced by the beating cilia (Fig. 1). These in situ analyses of the lung cells are complemented by off-line biochemical, physiological, and morphological cell analyses. The particle deposition chamber was extensively characterized to assure that particles are evenly and

efficiently deposited on the cell culture. The distribution of the particles on the filter was determined by counting deposited particles off-line with a microscope on randomly selected locations along the filter insert radius and with modeling results. Particle deposition efficiency was tested with monodisperse polystyrene particles. 15–30% of all particles (50-600nm) are deposited, which is 5-45 times more efficient that existing particle deposition systems. In a first series of experiments secondary organic aerosols were deposited in the chamber onto various lung cell culture types (Baltensperger et al., 2008) and moderate inflammation reactions were observed.

Fig. 1. Details of the particle deposition chamber showing the particle deposition on a cell culture filter insert inside the chamber. 12 cell cultures are simultaneously and individually exposed to particles. Particles are deposited in an electrical field between the particle delivery tube and the electrode. A separate electrode is placed directly beneath each filter insert. This work was supported by SBF grants C03.0052 and C06.0075 as part of COST Action 633, SNSF grant K-32K1-120524/1 and the European Commission Project POLYSOA. Baltensperger, U. et al., (2008). J Aerosol Med Pulm

Drug Deliv. 21:145-154. Savi, M. et al., (2008). Environ Sci Technol,

42:5667-5674.


Trace Element Analyses of Spark Discharge Particles

E. Karg, B. Lentner, W.G. Kreyling and O. Schmid

Institute for Inhalation Biology, Helmholtz Zentrum München, D-85758 Neuherberg / Munich, Germany Keywords: Generation of nanoparticles, nanoparticle characterization, elemental composition, spark discharge. Aggregated carbonaceous particles as an experimental surrogate for Diesel exhaust particles are frequently produced by the spark discharge method (Kim et al., 2006) and have been used for numerous exposure studies (Evans et al., 2003; Alessandrini et al., 2008). In our lab, a commercially available generator (model GFG1000, Palas, Karlsruhe, Germany) was used to produce particles in an inert carrier gas stream (Argon, quality better than 99.999 %) by controlled electric spark discharge between pure carbon electrodes. In this study, elemental composition analyses were carried out from particle samples which were taken directly at the generator outlet. They are compared with analyses of the carbon electrodes and the carrier gas, available at the suppliers. Metal trace elements were analyzed by atomic emission spectroscopy (ICP-AES), C, O, H and N by pyrolytic methods. The results of the particle analyses as well as the corresponding electrode and gas analyses of the suppliers are listed in the table.

Table 1. Trace element concentrations. particles1 electrodes2 carrier gas3

ppm % ppm ppmCa 35 C 82.3 Ca 0.2 Cu 30 O 14.4 Cu 0.1 O2 <2 Fe 75 H 1.4 Fe 0.2 H2O <3 Mg 6 N 0.65 Mg 0.05 N2 <5 Si 2200 Si 0.5 HC <0.2

1 the mass concentration of particles in the aerosol output was 4.7 ppm for the standard operating conditions (6.5 lpm Argon flow rate and 220 s-1 spark frequency, 3.2 mg h-1 particle output). Gravimetry of the electrodes before and after operation showed that 42% of the eroded mass appears as particles at the outlet.

2 data from PALAS, Karlsruhe, Germany for the electrode RW0 3 data from Linde, Germany for Argon 5.0 (99.999%) For the trace metals, the particle-to-electrode-ratio ranges from 120 to 4400. A constant ratio for all metals would explain the metal enrichment by oxidation of the carbon matrix. However, as the ratio is not found to be constant for the different metals, sources must be assumed in chamber walls and connectors which are stressed by the high energetic UV radiation produced during the spark discharge. Desorption experiments with the particles (Matuschek et al., 2007) showed a considerable loss of mass (about 14 %) during thermal treatment up to 800 °C. Analyses of the evolved gas phase showed low amounts of hydrocarbons (HC) but a significant

release of the inorganic gases carbon monoxide (CO), carbon dioxide (CO2) and and water (H2O). In this study, an oxygen mass concentration of 14 % is found in the particles. It may result from the adsorption of gaseous components on the particles’ active surface sites from the ambient atmosphere during sample handling and preparation, but also from the impurities in the carrier gas. The carrier gas impurities are of similar mass concentration as the particles in the aerosol (see table foot notes). Even if we assume a 58 % loss of particles during transport in spark chamber and tubing (see table foot notes), the trace gas concentration of oxygen in the carrier gas is sufficient for about 15 % of oxygen in the particles. The water vapour content of the carrier gas is taken as a hydrogen source as no data for the hydrogen trace gas content are available. From hydrolysis of H2O in the spark discharge plasma, < 0.3 ppm H2 and < 4.7 ppm O2 can be expected. Therefrom, a hydrogen content up to 2.9 % is possible in the particles. The additional O2 would be sufficient for up to 30 % of oxygen in the particles. The nitrogen content of the carrier gas is sufficient for at most 31 % N in the particles. The measured content of 0.65 % shows nitrogen to be relatively inert during the discharge process. We conclude that − during the spark discharge process − interaction takes place between the elemental carbon vaporized from the electrodes and trace elements available from the surrounding. The trace gas content in the carrier is sufficient to account for the impurities found. Sources for the trace metals must be assumed in the spark chamber walls. However, the adsorption of ambient gas components during sample preparation and handling as an additional source should also be considered. We thank the “Mikroanalytisches Labor Pascher” Remagen, Germany for performing the AES measurements.


Cellular responses after exposure of lung cell cultures to secondary organic aerosols

M. Geiser1, M. Kalberer2, 3, A. Gaschen1, D. Lang1, M. Savi1, T. Geiser4, A. Gazdhar4, C.M. Lehr5, M. Bur5, J. Dommen2 and U. Baltensperger2

1Institute of Anatomy, University of Bern, 3000 Bern 9, Switzerland 2 Laboratory of Atmospheric Chemistry, Paul Scherrer Institut (PSI), 5232 Villigen, Switzerland

3 Centre for Atmospheric Sciences, Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, UK

4 Division of Pulmonary Medicine, University Hospital, 3010 Bern, Switzerland 5 Department for Biopharmaceutics and Pharmaceutical Technology, University of Saarland, 66123

Saarbrücken, Germany

Keywords: Aerosol instrumentation, Health effects of aerosols, Lung/particle interaction, SOA Ambient fine and ultrafine particles have a variety of adverse health effects. The chemical and physical properties of aerosol particles causing these effects remain unclear. A major fraction of the ambient aerosol particle mass is composed of secondary organic aerosol (SOA). Within the interdisciplinary POLYSOA project (Baltensperger et al. 2008) this work aimed to examine in vitro the response of target lung cells to SOA particles with the goal to eventually identify particle components that are responsible for cell responses. SOA particles were deposited on the air-liquid interface of cultured porcine and human lung epithelial cells (micro-dissected tracheal epithelium, primary cultures and cell lines) and lung surface macrophages in a recently constructed particle deposition chamber (Savi et al. 2008). Particles were applied under realistic ambient air and physiological conditions occurring when particles are inhaled by mammals. Cellular responses were examined within 24 hrs after exposure to SOA. Ultrastructural changes of cells were assessed by transmission electron microscopy. Necrotic cell death was tested by measuring lactate dehydrogenase release. Phagocytic activity of macrophages was tested by post-exposure treatment with 6-μm polystyrene particles. Inflammatory responses were assessed by measuring TNF-α, IL-6 and IL-8 release. In addition, epithelial repair function was tested by measuring the closure of mechanically wounded alveolar epithelial cell monolayers using a computerized imaging technique (Geiser et al., 2000). Analyses of the lung cells indicate that a short time exposure to realistic concentrations of SOA does not induce cytotoxicity but leads to subtle changes in cell function that are essential for lung homoeostasis. We found decreased phagocytic activity in macrophages (Fig. 1) and cell type specific increases in IL-8 release. The alveolar epithelial wound repair was affected mainly due to alterations

of cell spreading and cell migration at the edge of the wound.

Figure 1. The phagocytic activity of human macrophages, i.e. the number of phagocytosed particles per macrophage (mph), was significantly decreased (*p<0.05) after exposure to SOA from α-pinene (2 experiments) as compared to untreated cells (incubator control) and cells exposed to particle-free air. These analyses of cellular responses induced by organic aerosols will greatly help to understand the particle properties as well as the cellular mechanisms responsible for biological effects. This work was supported by SBF grants C03.0052 and C06.0075 as part of COST Action 633, the European Commission Project POLYSOA, contract No 12719, the 3R Research Foundation Switzerland, project No 89-03 and an Eurochamp travel grant. Baltensperger, U. et al., (2008). J Aerosol Med Pulm

Drug Deliv, 21:145-154. Geiser, T. et al., (2000). Am J Physiol Lung Cell Mol

Physiol, 279:L1184-1190. Savi, M. et al., (2008). Environ Sci Technol,

42:5667-5674.


Minimal analytical characterisation of engineered nanomaterials need for hazard assessment in biological matrices

Hans Bouwmeester1, Iseult Lynch2, Michael Riediker3 and Flemming R. Cassee4

1 RIKILT-Institute of Food Safety, Wageningen UR, P.O. Box 230, 6700 AE Wageningen, The Netherlands

2 University College Dublin, Centre for BioNanoInteractions, Belfield, Dublin 4, Ireland 3 Institute for Work and Health, Rue du Bugnon 21, CH-1011 Lausanne, Switzerland

4 National Institute for Public Health and the Environment, Bilthoven, The Netherlands Keywords: engineered nanomaterials, hazard assessment, biological matrices, characterisation.

The safe and responsible development of engineered nanomaterials (ENM), nanotechnology-based materials and products, together with the definition of regulatory measures and implementation of “nano”-legislation in Europe require a widely supported scientific basis and sufficient high quality data upon which to base decisions. At the very core of such a scientific basis is a general agreement on key issues related to risk assessment of ENMs which encompass the key parameters to characterise ENMs, appropriate methods of analysis and best approach to express the effect of ENMs in widely accepted dose response toxicity tests. The following major conclusions were drawn: Due to high batch variability of ENMs characteristics of commercially available and to a lesser degree laboratory made ENMs it is not possible to make general statements regarding the toxicity resulting from exposure to ENMs. Concomitant with using the OECD priority list of

ENMs, other criteria for selection of ENMs like relevance for mechanistic (scientific) studies or risk assessment-based studies, widespread availability (and thus high expected volumes of use) or consumer concern (route of consumer exposure depending on application) could be helpful. The OECD priority list is focussing on validity of OECD tests. Therefore source material will be first in scope for testing. However for risk assessment it is much more relevant to have toxicity data from material as present in products/matrices to which men and environment are be exposed. For most, if not all characteristics of ENMs,

standardized methods analytical methods, though not necessarily validated, are available. Generally these methods are only able to determine one single characteristic and some of them can be rather expensive. Practically, it is currently not feasible to fully characterise ENMs.

Many techniques that are available to measure the same nanomaterial characteristic produce contrasting results (e.g. reported sizes of ENMs). It was recommended that at least two complementary techniques should be employed to determine a metric

of ENMs. The first great challenge is to prioritise metrics which are relevant in the assessment of biological dose response relations and to develop analytical methods for characterising ENMs in biological matrices. It was generally agreed that one metric is not sufficient to describe fully ENMs. Characterisation of ENMs in biological matrices

starts with sample preparation. It was concluded that there currently is no standard approach/protocol for sample preparation to control agglomeration/aggregation and (re)dispersion. It was recommended harmonization should be initiated and that exchange of protocols should take place. The precise methods used to disperse ENMs should be specifically, yet succinctly described within the experimental section of a publication. ENMs need to be characterised in the matrix as it

is presented to the test system (in vitro/ in vivo). Alternative approaches (e.g. biological or in silico

systems) for the characterisation of ENMS are simply not possible with the current knowledge.

Contributors: Iseult Lynch, Hans Marvin, Kenneth Dawson, Markus Berges, Diane Braguer, Hugh J. Byrne, Alan Casey, Gordon Chambers, Martin Clift, Giuliano Elia1, Teresa F. Fernandes, Lise Fjellsbø, Peter Hatto, Lucienne Juillerat, Christoph Klein, Wolfgang Kreyling, Carmen Nickel1, and Vicki Stone.

This abstract presents results created by NanoImpactNet - The European Network on the Health and Environmental Impact of Nanomaterials. NanoImpactNet is a Coordination Action sponsored by the EC's 7th Framework Programme. However, the abstract does not necessarily reflect the opinion of NanoImpactNet or the European Commission.


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