Development and Evaluation of an Aerosol Generation and Supplying

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Environ. Sci. Technol. 2009, 43, 5529–5534

Development and Evaluation of an Aerosol Generation and Supplying System for Inhalation Experiments of Manufactured Nanoparticles ,†

nm, respectively. By feeding these aerosols into a whole-body exposure chamber for rats, a stable supply of the aerosol nanoparticles could be achieved for long hourly durations (6 h per day) as well as for long terms (5 days per week for 4 weeks).

Introduction †

MANABU SHIMADA,* WEI-NING WANG, KIKUO OKUYAMA,† TOSHIHIKO MYOJO,‡ TAKAKO OYABU,‡ YASUO MORIMOTO,‡ ISAMU TANAKA,‡ SHIGEHISA ENDOH,§ KUNIO UCHIDA,§ KENSEI EHARA,| HIROMU SAKURAI,| KAZUHIRO YAMAMOTO,⊥ AND JUNKO NAKANISHI# Department of Chemical Engineering, Graduate School of Engineering, Hiroshima University, Higashi-Hiroshima, Japan, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Kitakyushu, Japan, Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan, National Metrology Institute of Japan, National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan, Research Institute of Instrumentation Frontier, National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan, and Research Institute of Science for Safety and Sustainability, National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan

Received March 25, 2009. Revised manuscript received May 13, 2009. Accepted May 14, 2009.

Risk assessment of nanoparticles by inhalation experiments is of great importance since inhalation is considered the most significant route of exposure to nanoparticles suspended in air. However, there have been few inhalation experiments using manufactured nanoparticles, mainly because of the difficulty in stably dispersing the nanoparticles in air for a long period of time. In this study, we report for the first time the development of a rational system for stably and continuously dispersing and supplying manufactured nanoparticles for inhalation experiments. The system was developed using a spray-drying technique, in which a nebulizer was used to atomize nickel oxide (NiO) and fullerene (C60) nanoparticle suspensions, and the resulting droplets were dried to generate aerosol nanoparticles. The size, concentration and morphology of the aerosol particles were evaluated by in-line measurements using an aerosol measuring device and off-line measurements based on the collection of the aerosol particles. After examining the effects of the conditions for the suspensions and the aerosol generation, we were able to obtain NiO and C60 aerosol nanoparticles with average diameters of 53-64 and 88-98 * Corresponding author phone: 81-82-424-7717; fax: 81-82-4247851; e-mail: [email protected]. † Hiroshima University. ‡ University of Occupational and Environmental Health. § Research Institute for Environmental Management Technology. | National Metrology Institute of Japan. ⊥ Research Institute of Instrumentation Frontier. # Research Institute of Science for Safety and Sustainability. 10.1021/es9008773 CCC: $40.75

Published on Web 06/05/2009

 2009 American Chemical Society

Nanoparticles, with a representative size of below 100 nm, exhibit unique physical and chemical properties resulting from their small sizes and corresponding large specific surface areas. Consequently, nanotechnology has the potential to make significant contributions to various applications in semiconductors, biotechnology, energy, agriculture, food, consumer products, and so on (1, 2). However, new technologies have attendant risk, and nanotechnology is no exception (3). For most uses, nanomaterials consist of nontoxic matter. The properties of nanoparticles, however, could alter the environmental effect, and has been a recent topic of concern (4). For example, nanoparticles may access regions of the body that may not be accessible by larger particles via different mechanisms. In addition, the very high surface free-energy of nanoparticles may lead them to facilitated adsorption of molecular contaminants, thereby offering a potential route for these contaminants into the body (2, 5). For industrial applications, nanoparticles must be manufactured in large quantities. These manufactured nanoparticles may leak out or be exposed to the environment during manufacture, shipping, handling, or use. Therefore, it is preferable that a thorough risk assessment of nanomaterials be made before marketing. In particular, inhalation assessment is of great importance since inhalation is considered a significant exposure route (6). For inhalation assessment, a suitable aerosol nanoparticle preparation system must be designed to ensure the stability of particle size and concentration over the duration of exposures. However, nanoparticle dispersion and supply techniques are a challenge since nanoparticles tend to form agglomerates during handling. In general, manufactured nanoparticles are usually supplied either as dry powders or as corresponding suspensions. For inhalation experiments using dry powders, conventional dust feed devices and/or air classifiers are used. However, these apparatuses usually produce particles with geometric mean diameters of several micrometers or even larger (7, 8). Aerosol synthesizing methods have recently been used to directly supply nanoparticles for inhalation experiments. For example, Veranth et al. used an evaporation/condensation particle generator to produce hydrocarbon nanoparticles with mean diameters of 30-50 nm for inhalation toxicology studies (9). Gupta et al. prepared fullerene aerosol nanoparticles in high mass concentration by sublimating bulk fullerenes at high temperatures (2). By using the above methods, a controlled size and size distribution of aerosol nanoparticles could be obtained. However, they are difficult to apply to various industrially available nanoparticles for testing. A simple way to supply existing manufactured particles for inhalation assessment is to deliver them from their corresponding suspensions, e.g., a spray-drying technique. However, generation of nanoparticles using a conventional spray-drying process is difficult. The produced particles are usually larger than 100 nm due to the limitation in the generation of small droplets, based on the one-droplet-toone-particle (ODOP) mechanism (10-12). For example, Oyabu et al. reported the generation of nickel oxide (NiO) VOL. 43, NO. 14, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Schematic diagram of the experimental setup. aerosol nanoparticles using an ultrasonic spray-drying technique for inhalation experiments. The geometric mean diameter of the particles was as large as about 140 nm (13). Grassian et al. recently reported an investigation on the generation of titanium dioxide (TiO2) aerosol particles from TiO2 nanoparticle suspensions using a Collison nebulizer for inhalation exposure (14). The resulting aerosol particles had a geometric mean diameter of 128 nm, which were actually aggregates of primary nanoparticles with sizes ranging from 2 to 5 nm. In another example, commercialized spray dryers using a two-fluid nozzle as an atomizer have generally been employed in the pharmaceutical industry for medicine fabrication. The resulting particles, however, were usually hollow in morphology and large in size (i.e., from submicrometer to several micrometers) (15, 16). Although these spray-drying techniques can generate aerosol particles at a mass concentration sufficient for inhalation experiments, conducting an experiment using airborne particles of the nanoparticle size is difficult. An electrospray is capable of generating very fine droplets in a submicrometer size range, leading to the formation of nanoparticles easily (17). However, it will suffer from two major problems, i.e., a low mass concentration output and short duration time. Therefore, it is important to develop a carefully designed system for aerosol nanoparticle dispersion and supply. The purpose of this work was to develop a nanoparticle dispersion and supply system that could continuously feed stable aerosol nanoparticles for in vivo inhalation experiments. In this work, an atomizer with a pressurized two-fluid nozzle, hereafter referred to as a pressurized nebulizer, was used for dispersion of nanoparticle suspensions. The effects of operating conditions and operation for long durations, and over the longterm, were investigated. NiO and fullerene (C60) nanoparticles were selected, since these are frequently used in electronic devices and consumer products, and are now being produced in large quantities (4, 6). NiO particles was reported to be an important candidate as positive control of nanomaterials because agglomerated NiO particles dispersed in water with a size below 100 nm, induced persistent injury and inflammation in animal lungs (18).

Experimental Section Aerosol Generation and Characterization. The entire experimental setup for aerosol generation and characterization is schematically shown in Figure 1. This system mainly consists of a pressurized nebulizer as atomizer, a drying section, a stainless steel whole body exposure chamber (inner volume: 0.52 m3, which consists of a rectangular space with 5530

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three dimensions of 800 (L) × 800 (W) × 500 (H) mm, and flow inlet and outlet spaces) with rat cages, an electrostatic precipitator to collect aerosol particles for off-line analysis, and a particle spectrometer (model 1000XP WPS, MSP Corp., Shoreview, MN), consisting of a differential mobility analyzer and a condensation particle counter (DMA-CPC) system for in-line monitoring. The pressurized nebulizer (Nanomaster, JSR Corp., Tokyo, Japan) was used to atomize suspensions into aerosol particles. This nebulizer was equipped with an ejector to break liquid threads into droplets and resulting aerosol particles, and was proven effective in generating aerosol particles with good monodispersion (a standard geometric deviation of 99.5%) C60 powder (nanom purple, Frontier Carbon Corp., Kitakyushu, Japan) of >100 µm in size was dispersed in deionized and anaerobic water. Since C60 particles have hydrophobic surfaces, polyoxyethylene (20) sorbitan monooleate (Tween 80, Wako Pure Chemical Industries, Ltd., Osaka, Japan) was added to the water as a dispersant to ensure the stability of the suspension. The median diameter of the C60 particles became about 100 nm after the bead milling. The suspension was centrifuged to remove large particles. To avoid the oxidation of the C60 as much as possible, the preparation processes were mostly conducted under anaerobic and darkened conditions. The concentration of C60 was determined using a high performance liquid chromatography (HPLC) technique (Agilent 1100, Agilent Technologies, Inc., Santa Clara, CA), where the C60 nanoparticles were dissolved into toluene. Using DLS measurement, the geometric mean diameter of the particles in the suspension was found to be 20-30 nm. Both NiO and C60 nanoparticle suspensions were found to maintain a good dispersion for at least two months.

FIGURE 2. Size distributions of NiO aerosol nanoparticles as a function of airflow rate in the nebulizer (a) and suspension concentration (b), measured by the particle spectrometer. The suspension concentration for (a) and airflow rate for (b) were 0.5 mg/mL and 35 L/min, respectively.

Results and Discussion Investigation into Aerosol Generation Conditions. An investigation of aerosol generation conditions while using the pressurized nebulizer was first carried out. Figure 2 shows the size distributions of the NiO aerosol nanoparticles measured in-line using the particle spectrometer, before use of the whole-body exposure chamber, as a function of the airflow rate through the nebulizer (Figure 2a) and the suspension concentration (Figure 2b). Figure 2a shows that a rather indistinct, broad peak with low concentration was obtained when the airflow rate was 10 L/min, which suggests that this low flow rate was not enough to break up the liquid thread into droplets. The peak concentration increased significantly when the flow rate increased to 20 L/min and the peak diameter was located at around 60 nm. The peak concentration further increased, while the peak diameter of aerosol particles decreased slightly, when flow rate increased to 40 L/min. This is because a higher flow rate leads to smaller droplets based on the basic droplet formation mechanism in a two-fluid nozzle (20) and, following the ODOP principle, subsequently leads to a smaller particle size (12). The aerosol particles obtained here are smaller compared with Grassian et al. (14), who used a Collison nebulizer. This is considered to be due to the capability of the pressurized nebulizer of generating smaller droplets. To investigate the effects of suspension concentration, four concentrations were used: 0.50 mg/mL, 0.25 mg/mL, 0.125 mg/mL, and 0.0625 mg/mL. As Figure 2b shows, the peak diameter of the aerosol particles decreased with decreasing concentration. This result is explained simply by assuming that the aerosol particle formation follows the ODOP principle, as discussed above. An increase in the peak and total number concentration of the aerosol nanoparticles

FIGURE 3. Size distribution of NiO aerosol nanoparticles measured by the particle spectrometer (open circles) and from SEM images (histogram). The airflow rate and suspension concentration were 35 L/min and 0.50 mg/mL. with increasing suspension concentration is thought to be caused by a change in the physical properties of suspensions. Previous research has shown that the average droplet size decreased with increasing precursor concentration due to the increase in viscosity (12). Since the volume of the suspension sprayed per unit time was found to change little for different suspension concentrations, the number concentration of the droplets, subsequently the aerosol nanoparticles, should increase as suspension concentrations increase. Figure 3 shows a comparison of the in-line aerosol particle size distribution (line with open circles) obtained by the particle spectrometer and the off-line size distribution (histogram) of the aerosol particles as determined by their corresponding FE-SEM image (as inset, can be also found in Supporting Information (SI) Figure S1) under conditions of an air flow rate of 35 L/min and a suspension concentration of 0.50 mg/mL. From the SEM image, the particles were well dispersed, and most of them were nearly spherical and below 100 nm in size, consisting of many primary NiO nanoparticles. VOL. 43, NO. 14, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. Size distributions of NiO (a) and C60 (b) aerosol nanoparticles fed continuously into the whole-body exposure chamber. The geometric mean diameters from in-line and off-line analyses were in good agreement, which likely is due to the particle shape. Since the aerosol particles are nearly spherical, the particle size determined by the particle spectrometer (electrical mobility equivalent diameter) should be very close to the physical diameter (diameter determined with SEM images). In other words, an in-line measurement (that enables real-time monitoring) of aerosol particle size distributions was made possible in this study because nearly spherical particles could be generated. Additional experiments in which water used to prepare the suspensions was sprayed with no particles added showed that the particle number concentration was more than 3 orders of magnitude lower than the results in Figure 3, suggesting that the effect of water contamination was negligible. In addition to the above investigation, aerosol distribution inside a whole-body exposure chamber was also analyzed, which can be found in SI Figure S2. The results show that the peak diameters of the aerosol nanoparticles sampled at the three positions, i.e. A, B, and C (SI Figure S2a), were almost the same, and the difference in the total number concentration was smaller than 10% (SI Figure S2b), indicating that a sufficiently uniform aerosol distribution was achieved across the chamber. In the following experiments, in-line monitoring of aerosol size distribution was performed at position A. The durations for buildup and decay of the aerosol concentration inside the chamber were found to be within 10 min, which are negligibly short compared with the duration of exposure of each day. Long-Time Stability for Whole-body Exposure. Longtime stability (stability for 6 h for each day of the study) of aerosol exposure is required to perform inhalation experiments. NiO nanoparticle suspensions with a concentration of 0.25 mg/mL and C60 nanoparticle suspensions with a C60 concentration of 0.17 mg/mL were used as models. Figure 4a shows the typical size distributions of NiO aerosol nanoparticles in the chamber under a continuous operation of 6 h. The geometric mean diameter was about 55 nm. 5532

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Consistent particle size distribution was obtained for 6 h, indicating that the present system, including the pressurized nebulizer and the particle transport lines, achieved a stable aerosol generation and supply. A similar conclusion was drawn in the case of the C60 suspension as shown in Figure 4b. From this figure, a larger average particle size, 92 nm, than that of the NiO aerosol was found, which was likely due to contents and physicochemical properties of the C60 suspension (0.17 mg-C60 and 0.50 mg-Tween 80 per 1 mLwater) that differed from that of the NiO suspension (0.25 mg-NiO/mL-water). Aerosol Particles from C60 Suspension. The aerosol particles generated from the C60 suspension were spherical (see SI Figure S3), in which the inset suggested that the generated particle was formed not by C60 primary nanoparticles alone. Detailed TEM analysis results of the particles are shown in Figure 5. From Figure 5a the amorphous parts and crystalline phases can be seen coexisting in a single aerosol particle. The crystalline phase shown in a higher magnification (Figure 5b) is considered to be a C60 primary nanoparticle, since the distance between the adjacent lattice fringes was found to be 0.847 nm, which agrees with that of the (111) plane of C60 (PDF No. 44-0558). In addition, the crystal size in Figure 5b is comparable to that of the original C60 nanoparticles in the suspension (Figure 5d). A highmagnification image of the amorphous phase in Figure 5a is shown in Figure 5c. This phase is considered to correspond to solidified Tween 80 since the aerosol particles were heated in the drying section at a much lower temperature (50 °C) than the decomposition temperature of Tween 80 (around 250 °C). A carbon analysis using an ECOC (elemental carbon and organic carbon) monitor (Carbon Aerosol Analysis Lab Instrument, Sunset Laboratory Inc., Tigard, OR) was also conducted for the collected aerosol particles. The analysis revealed that the weight fraction of C60 in the particles was about 1/4. This fraction is almost the same as the weight ratio of C60 to the sum of C60 and Tween 80 in the suspension, which also suggests that the aerosolized particles are actually the composite particles of C60 nanoparticles and dried Tween 80. It is believed that a health effect assessment should detect no dominant influence of coexisting Tween 80 in aerosol nanoparticles, since the toxicity of Tween 80 is reportedly nondetectable in animal experiments (21). Long-Term Stability for Whole-body Exposure. The actual inhalation experiments using male Wister rats were conducted for 6 hours per day and 5 days per week for as long as 4 weeks. The particle size distribution of the aerosols in the whole body exposure chamber was analyzed in-line three times daily, at 0.5, 3.0 and 5.5 h, after the aerosols were fed into the chamber. Based on this thrice-daily analysis, the average particle size and number concentration for each day were calculated. In the case of the NiO aerosol inhalation experiment, NiO suspension with a concentration of 0.50 mg/mL was tried for the first 4 days, and a suspension at 0.25 mg/mL was used for the remaining 15 days. For C60 aerosol generation, suspensions containing 0.085 mg-C60/mL and 0.17 mg-C60/mL were used in the first 3 days and in the following 16 days, respectively. The aerosol concentrations in the chamber and their stability were examined in the earlier days, and after confirming the actual mass concentrations, different suspension concentrations were employed so that the mass concentrations of the two materials were brought close to one another. Both of the C60 suspensions contained Tween 80 at triple the C60 weight concentration. Experimental data of NiO aerosol nanoparticles for the entire experiment period are summarized in Figure 6 and SI Table S1. The average diameters of the NiO aerosol nanoparticles were kept stable for each of the two subperiods with the two suspension concentrations (the first 4 days and

FIGURE 5. TEM image of a typical C60 aerosol nanoparticle (a), magnified image of a crystalline part (b), magnified image of an amorphous part (c), and TEM image of a C60 nanoparticle in the suspension. the remaining days). The average particle diameter for the entire experiment period was approximately 60 nm. The number concentration derived from the corresponding particle size distribution with the size range of 10-500 nm (Figure 6b) also shows that concentration fluctuations were small for each of the two subperiods. As mentioned earlier, the mass concentration of the aerosol nanoparticles was determined gravimetrically using filter collection. The daily change of the mass concentration obtained by this method is plotted with the solid lines in Figure 6c. Besides this direct measurement method, determination of the mass concentration was tried using in-line particle size distribution data. As mentioned earlier, the particle size determined by the particle spectrometer corresponded well with the physical diameter of the aerosol particles. Therefore, if the density of the aerosol particles is known, the mass concentration can be calculated by properly integrating the size distribution with the density. This density should be different from the bulk density of the particle material, i.e., NiO, since the particles are actually agglomerates. This density, referred to as the effective density, of NiO aerosol nanoparticles generated by using the same type of nebulizer was investigated using a DMA-APM (aerosol particle mass analyzer)-CPC system and found to be about 3.0 g/cm3 for particles with an average diameter of approximately 60 nm (22). This value is about half the bulk density of NiO, suggesting that the present NiO aerosol nanoparticles were actually porous. Using this effective density, the mass concentration of the NiO aerosol particles was calculated from the corresponding size distribution datasplotted by dotted lines in Figure 6csand agrees well with that determined using the gravimetrical method. It follows that monitoring of mass concentration using in-line measurement, which is advantageous compared to off-line analyses such as the gravimetrical method, is possible when the effective density is known. Similar to the average diameter and number concentration, the mass concentration of the NiO aerosol nanoparticles was again found to be considerably stable for each of the two

subperiods. The mass concentration (by the gravimetrical method) averaged for the later 15 days was 0.15 mg/m3 with the standard derivation of 0.04 mg/m3, which was considered sufficient for inhalation experiments. This concentration is much higher than the EPA National Ambient Air Quality Standards (NAAQS) primary standards, e.g. 35 µg/m3 for PM2.5. Stability was also obtained in the case of the C60 aerosol particles (see SI Figure S4). SI Figures S4a and b indicate that variations in the average diameter and number concentration were sufficiently small for each of the subperiods. The particle diameter and number concentration averaged for the two subperiods are included in SI Table S1 together with their standard deviations. Estimation of mass concentration from the in-line measurement data was not attempted for the C60 aerosol particles since the effective density of the particles was not available. The mass concentrations plotted in SI Figure S4c were those determined by the gravimetrical method. The mass concentrations were also found to be sufficiently stable. The mass concentration averaged for the later 16 days was 0.50 mg/m3. By considering the weight fraction of C60 in the aerosol particles described earlier, the mass concentration of C60 in the subperiod was estimated to be about 0.12 mg/m3. This concentration was fairly close to that of NiO aerosol particles in the later 15 days. In summary, an aerosol generation and supply system using a pressurized nebulizer was developed in this study for inhalation experiments of manufactured nanoparticles. The size, concentration, and morphology of the resulting aerosol nanoparticles was evaluated using both in-line (i.e., DMA-CPC) and off-line (i.e., SEM/TEM) methods. The results revealed that well-dispersed, almost spherical aerosol particles could be obtained at a sufficient concentration for both NiO and C60 materials. Although the aerosol particles were actually agglomerates, the geometric mean particle diameter was below 100 nm. In the actual inhalation experiments, stable dispersing and supplying of the aerosol nanoparticles was achieved for long periods of time (6 hours per day) and over the long-term (5 days per week for 4 weeks), showing VOL. 43, NO. 14, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 6. Daily change of average diameter (a), number concentration (b), and mass concentration (c) of NiO aerosol nanoparticles during the whole-body exposure experiment. great promise of the currently developed system for future inhalation experiments.

Acknowledgments We thank Dr. Yoshitaka Yonezawa of National Institute of Advanced Industrial Science and Technology for providing valuable information regarding this work. We also thank Mr. Yoshikazu Fukai of JSR Corp. for his cooperation on the nebulizer. This research was funded by the New Energy and Industrial Technology Development Organization of Japan (NEDO) Grant “Evaluating Risks Associated with Manufactured Nanomaterials (P06041).”

Supporting Information Available A summary of aerosol properties during the exposure experiment, SEM images of NiO aerosolized nanoparticles and their size distribution inside the exposure chamber, SEM images and daily change of C60 aerosol nanoparticles in the exposure term. This material is available free of charge via the Internet at http://pubs.acs.org.

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