J Nanopart Res (2011) 13:2949–2961 DOI 10.1007/s11051-010-0186-x
RESEARCH PAPER
Aerosol characterization and lung deposition of synthesized TiO2 nanoparticles for murine inhalation studies Antti J. Koivisto • Maija Ma¨kinen • Elina M. Rossi • Hanna K. Lindberg • Mirella Miettinen • Ghita C.- M. Falck • Hannu Norppa • Harri Alenius • Anne Korpi • Joakim Riikonen • Esa Vanhala • Minnamari Vippola • Pertti Pasanen • Vesa-Pekka Lehto • Kai Savolainen • Jorma Jokiniemi • Kaarle Ha¨meri
Received: 21 May 2010 / Accepted: 11 December 2010 / Published online: 7 January 2011 Ó Springer Science+Business Media B.V. 2011
Abstract This study presents a novel exposure protocol for synthesized nanoparticles (NPs). NPs were synthesized in gas phase by thermal decomposition of metal alkoxide vapors in a laminar flow reactor. The exposure protocol was used to estimate the deposition fraction of titanium dioxide (TiO2) NPs to mice lung. The experiments were conducted at aerosol mass concentrations of 0.8, 7.2, 10.0, and 28.5 mg m-3. The means of aerosol geometric mobility diameter and aerodynamic diameter were 80 and 124 nm, and the geometric standard deviations were 1.8 and 1.7, respectively. The effective density of the
particles was approximately from 1.5 to 1.7 g cm-3. Particle concentration varied from 4 9 105 cm-3 at mass concentrations of 0.8 mg m-3 to 12 9 106 cm-3 at 28.5 mg m-3. Particle phase structures were 74% of anatase and 26% of brookite with respective crystallite sized of 41 and 6 nm. The brookite crystallites were approximately 100 times the size of the anatase crystallites. The TiO2 particles were porous and highly agglomerated, with a mean primary particle size of 21 nm. The specific surface area of TiO2 powder was 61 m2 g-1. We defined mice respiratory minute volume (RMV) value during
A. J. Koivisto (&) E. M. Rossi H. K. Lindberg G. C.-M. Falck H. Norppa H. Alenius E. Vanhala M. Vippola K. Savolainen Finnish Institute of Occupational Health, Topeliuksenkatu 41 a A, 00250 Helsinki, Finland e-mail:
[email protected]
J. Riikonen V.-P. Lehto Department of Physics and Mathematics, University of Eastern Finland, P.O. Box 1627, 70211 Kuopio, Finland
M. Ma¨kinen M. Miettinen A. Korpi P. Pasanen J. Jokiniemi Department of Environmental Science, University of Eastern Finland, P.O. Box 1627, 70211 Kuopio, Finland
J. Jokiniemi Technical Research Centre of Finland (VTT), Fine and Nano Particles, P.O. Box 1000, 02044 VTT Espoo, Finland K. Ha¨meri Department of Physics, University of Helsinki, P.O. Box 64, 00014 Helsinki, Finland
J. Riikonen Department of Physics and Astronomy, University of Turku, 20014 Turku, Finland M. Vippola Department of Materials Science, Tampere University of Technology, P.O. Box 589, 33101 Tampere, Finland
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exposure to TiO2 aerosol. Both TiO2 particulate matter and gaseous by-products affected respiratory parameters. The RMV values were used to quantify the deposition fraction of TiO2 matter by using two different methods. According to individual samples, the deposition fraction was 8% on an average, and when defined from aerosol mass concentration series, it was 7%. These results show that the exposure protocol can be used to study toxicological effects of synthesized NPs. Keywords Inhalation exposure Nanoparticle TiO2 Aerosol Lung deposition Health effects EHS
Introduction Nanotechnology is a novel field of science which offers promising new applications for increasing the quality of life (Adlakha-Hutcheon et al. 2009; Kuhlbusch et al. 2009). It is based on controlling matter at an atomic and molecular level where material properties may be adjusted and enhanced. A common property of engineered nanomaterials is that they have at least one dimension less than 100 nm (Borm et al. 2006). Nanoparticles (NPs) form a subset of nanomaterials consisting of manmade uniform particles (Borm et al. 2006). In ambient aerosols, ultrafine (diameter\100 nm) particulate matter is considered to be the most harmful fraction as concerns inhalation uptake (Seaton et al. 1995; Peters et al. 1997; Oberdo¨rster et al. 2001, 2002; Nel 2005). The comparability of NPs to ultrafine particles suggests similar toxicological behavior which may further be enhanced by the unique properties of the NPs (Borm 2002; Oberdo¨rster et al. 2005; Gwinn and Vallyathan 2006) Present knowledge of NPs behavior suggests that they may potentially cause adverse health effects (Hoet et al. 2004; Oberdo¨rster et al. 2005; Gwinn and Vallyathan 2006; Borm et al. 2006; Xia et al. 2009). However, there is currently no evidence that NPs would cause adverse health effects in humans. This may be because mass production of nanomaterials has just begun and there are no epidemiological data on potential exposure to NPs. The concern is that the production rate of nanomaterials is growing exponentially with an
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increasing number of novel applications being based on nanotechnology (Aitken et al. 2006). Several factors that may influence the toxicity of NPs have been identified but up to date the key parameters have remained unknown (Oberdo¨rster et al. 2007). The main metrics that can currently be used for measuring nanoparticles are (1) mass concentration (mg m3 ), (2) number concentration (units m3 ), and (3) surface area concentration (units m2 m-3). Workplace Exposure limits for hazardous materials and chemicals are mostly based on mass concentration, typically expressed as mg m3 of air (Borm 2002). The surface area concentration is indicated to be linked with inflammation caused by nanoparticles (Nel 2006). The number concentration has been associated with respiratory and cardiovascular diseases (Seaton et al. 1995). As no single metrics have been shown to characterize health risk to nanoparticles, it is recommended that all three metrics are determined simultaneously, if possible. Exposure to NPs at the workplace may be separated into process particles (synthesis) and postprocess particles (e.g., packing, storing, use in applications). These two different exposure scenarios can be simulated in inhalation experiments by producing aerosols with online synthesis or aerosolizing commercial nanopowders. One clear difference between these exposure methods concerns the agglomeration state which can be seen as a lower particle concentration of nanopowder dispersions although the mass concentration is the same (Rossi et al. 2010). Exposure to synthesized NPs is a relevant exposure scenario in occupational environments. For example, Balas et al. (2010) showed that 60.5% of research laboratories synthesized their own nanomaterials. Thus, toxicological studies should also be performed with NPs synthesized online. For NP inhalation studies, we built a laminar flow reactor to synthesize titanium dioxide (TiO2) NPs via vaporization of liquid TTIP which thermally decomposes in a hot wall reactor to produce supersaturated TiO2 vapor. The TiO2 vapor immediately nucleates, thus producing TiO2 NPs. The laminar flow reactor was applied to a whole body inhalation exposure setup which enables direct exposure to synthesized NPs in the gas phase. The exposure setup was used to characterize the generated NPs and to study the potential of NPs to remain in the murine airways. The
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study shows that the exposure setup enables NP inhalation studies with murine.
Materials and methods Synthesis of TiO2 particles TiO2 particles were generated by the thermal decomposition of titanium tetraisopropoxide (TTIP 97%, TiðC3 H7 OÞ4 , Sigma-Aldrich, St. Louis, MO) vapor in a laminar flow reactor (Komiyama et al. 1984; Okuyama et al. 1986; Kirkbir and Komiyama 1987; Okuyama et al. 1990; Kobata et al. 1991; Seto et al. 1995, 1997; Moravec et al. 2001; Nakaso et al. 2001, 2003; Backman et al. 2004; Miettinen et al. 2009). The TTIP precursor was in a bubbler thermostatic bath where it was vaporized at constant rate and carried to the reactor with nitrogen gas. Inside the reactor the overall reaction stoichiometry was (Kirkbir and Komiyama 1987): TiðC3 H7 OÞ4 ! TiO2 þ 4C3 H6 þ 2H2 O The primary TiO2 particles were produced by homogenous nucleation and condensation from the supersaturated TiO2 vapor. Secondary particles were formed by the agglomeration of the primary particles and by the condensation of TiO2 vapor on the particle which produced aggregates. The particle formation was catalyzed by TiO2 deposit on the reactor wall. Thus, the reactor was stabilized over 24 h to achieve maximum conversion of TTIP at 500 C (Komiyama et al. 1984). Miettinen et al. (2009) have quantified the concentrations of the TTIP precursor decomposition gaseous byproducts with fourier transform infrared spectrometer (FTIR). At a mass concentration of ½TiO ¼2 m ¼ 30 mg m3 gas concentrations were: propene ½C3 H6 v ¼ 86 ppm, carbon monoxide ½COv ¼ 5 ppm, and nitrogen oxides ½NOxv ¼ 6 ppm. Exposure setup Figure 1 shows the inhalation exposure setup and Table 1 shows the reactor parameters used in experiments A–D. Volume flows were adjusted with mass flow controllers (MFC, Bronkhorst High-Tech B.V., Netherlands). Nitrogen flow Qb to the TTIP precursor was warmed in a tube coil located in the thermostatic
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bath, over 80% of the thermostatic bath temperature (Table 1). Transport lines from the TTIP precursor to the reactor were heated to 80 C to avoid TTIP vapor condensation. Aerosol mass concentration was set by adjusting the precursor flow rate Qb and thermostatic bath temperature Tb (Table 1). Reactor inner temperature was adjusted with two temperature controllers, T1 and T2, to obtain the temperature profile shown in Fig. 1. In the experiments, temperature T1 was set to 230 °C. In one of the experiments (D), complete TTIP conversion was ensured by increasing the reactor temperature T2. Aerosol from the reactor was diluted with dry and humid air with Qdry ¼ 9:2 L min1 and Qhumid ¼ 10:1 L min1 . Aerosol flow through the 69 liters exposure chamber was set with an external pump from 7 to 11 L min1 . Including sampling flow rates, the ventilation rate varied from 14 to 17 h1 . The excess flow went through a particle collector to exhaust. Aerosol characterization Mobility size distribution was measured with an aerosol mobility spectrometer which was similar to the scanning mobility particle sizer (SMPS) designed by Wang and Flagan (1990). It consisted of 63Ni bipolar aerosol neutralizer, 28 cm Vienna type differential mobility analyzer (DMA) and TSI model 3010 condensation particle counter (CPC) (TSI Incorporated, Shoreview, NM 55126 U.S.A.). A positive voltage was applied to the DMA with a FUG HCN 7E 12500 voltage source (FuG Elektronik GmbH, 83024 Rosenheim, Germany). A closed-loop sheath airflow of 5 L min1 was set to the DMA. The aerosol mobility spectrometer control and data acquisition were carried out using a Labview program and NI DAQCard-6024E I/O controller (National Instruments, Austin, Texas). Aerosol mobility spectrometer size range was set from 10 to 1,000 nm with 24 size bins and measurement time was 1 min with 6 s retrace. Size calibration was performed with sucrose particles by selecting 14 monodisperse mobility distributions ranging from 20 to 123 nm. This gave a good correlation on linear fit: Dbg ¼ 0:2 0:4 þ 0:983 0:009 D0bg
ð1Þ
Geometric mobility diameters were: Dbg corrected value and D0bg measured value. This calibration was
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Fig. 1 Schematic figure of experimental setup and operating volume flows. Reactor temperature profile was measured with temperatures T1 ¼ 500 C and T2 ¼ 230 C and reactor flows were turned off
Table 1 Reactor parameters in experiments A–D Exp.
Time (min)
No. of exposures
T2 (°C)
Tb (°C)
Qb ðN2 ) (L min1 )
Qr ðN2 Þ (L min1 )
tra (s)
[O2]va (%)
A
246
10
500
50
0.210
1.50
4.7
19.3
B
246
10
500
50
0.752
1.83
3.1
18.6
C
128
23
500
50
0.745
1.86
3.1
18.5
D
245
10
600
58
1.21
1.77
2.7
18.2
Reactor residence flow temperature T1 was kept at 230 °C. TiO2 mass concentration was adjusted with precursor flow Qb and residence time in reactor tr with residence flow Qr. TTIP precursor was located in a thermostatic bath at temperature Tb. TTIP was decomposed in the reactor at temperature T2 a
Calculated value
used to correct the aerosol mobility spectrometer mobility size. Particle concentration was measured using a TSI model 3776 ultrafine condensation particle counter, operated at high flowrate (TSI Incorporated, Shoreview, NM 55126 U.S.A.). The CPC was calibrated with monodisperse silver particles in respect of the electrometer. The lower detection limit of the CPC followed the efficiency function proposed by Mertes et al. (1995):
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gðDbg Þ ¼ 1 b 1 þ expððDbg D1 Þ=D2 Þ
1
ð2Þ
where Dbg is the particle geometric mobility diameter and parameters were: b ¼ 1:09; D1 ¼ 2:89; D2 ¼ 0:40. The coincidence correction was measured up to 5 105 cm3 where the decline in counting efficiency was less than 7%. In these studies, the maximum concentration was adjusted with a dilution below 3 105 cm3 , so that the CPC coincidence correction worked properly.
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Aerodynamic size distribution was measured from 7 nm to 6 lm with an electrical low pressure impactor with filter stage (ELPI, Dekati Ltd., Tampere, Finland) (Marjama¨ki et al. 2000). Logging time interval was set to 1 s which was averaged at 1 min samples with the ELPIVI 4.0 program. Aerosol was sampled with an ejector diluter (DI1000, Dekati Ltd., Tampere, Finland) with a volume flow of QDD ¼ 5:48 0:04 L min-1, which was further diluted for the aerosol mobility spectrometer and the CPC with a capillary tube diluter (QCD). The dilution flow uncertainties were ±7% for the ejector diluter and ±2% for the capillary tube diluter. The sampling line diffusion losses were corrected on the aerosol mobility spectrometer and the ELPI with a laminar flow penetration theory (Hinds 1999). Particle losses in diluters and tube bends were defined with experimental calibration with a particle concentration of 3 9 105 cm-3, using the CPC 3776 and the aerosol mobility spectrometer. The shape of the mobility size distribution was correct, but particle concentration was corrected for the CPC by a factor of 1.25 and for the aerosol mobility spectrometer by 1.05. With another sampling line, gravimetric samples were collected on nitrocellulose filters (Millipore) with a volume flow of Qs = 3.57 ± 0.05 L min-1. Particle properties were characterized from fabric filter samples and from the powder accumulated and collected from exposure chamber. Specific surface area was defined with the Brunauer–Emmet–Teller (BET) method, using Coulter Omnisorb 100 CX (Florida, U.S.A.) gas adsorption analyzer with nitrogen. The crystallite structure and phases were characterized with Siemens D500 and Philips PW1820 diffractometers and X-ray diffraction (PW1830 generator and PW1710 control unit, Philips, Industrial & Electron-acoustic Systems Division, Almelo, The Netherlands). A full profile fitting was carried out on the diffractograms by using Rietveld analysis (Rietveld 1969; Lutterotti et al. 1999). The structure and primary particle size were determined with a transmission electron microscope (TEM, Jeol Model JEM 2010, Tokyo, Japan). Particles for TEM analysis were sampled from the exposure chamber onto lacy carbon film coated copper grids with 200 mesh (SPI West Chester, U.S.A.) both directly from the aerosol and indirectly from the powder accumulated and collected from the chamber.
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Respiratory parameters Breathing flow rate is described with respiratory minute volume RMV (mL min-1), which is a product of tidal volume VT (mL) and breathing frequency f (min-1) (Wong 2007). RMV ¼ f VT
ð3Þ
Respiratory parameters are expected to change when pollutants are deposited in the respiratory system. The change in respiratory parameter X is described as dX ¼ Xe =Xb , where subscript ‘‘e’’ refers to exposure to pollutants and ‘‘b’’ to baseline. Respiratory minute volume values were determined using a specific mouse bioassay method (ASTM 1984), further developed as described by Vijayaraghavan et al. (1993, 1994), Boylstein et al. (1995, 1996), and Alarie (1998). Groups of four male Crl:OF1 mice (Charles River Laboratories, France, 5–6 weeks old, mean weight 30.4 g) were exposed head-only to 8, 20, and 30 mg m3 of TiO2 for 30 min. A control group was exposed to HEPA (Pall HEPA capsule) filtered air, purified from a TiO2 aerosol stream of 30 mg m3 . The exposure was preceded and followed by 15-min baseline and recovery periods, respectively, when only HEPA filtered room air was led into the exposure chamber. For female BALB/c/Sca mice (8-weeks old, mean weight 19.2 g, Scanbur Ab, Sollentuna, Sweden), only baseline breathing parameters were defined. The measurement of airflow (mL s1 ) of the mice was accomplished via pneumotachographs (A. Fleisch, Switzerland) to which differential pressure transducers (model 8T-2, Gaeltec, Dunvegen, Isle of Skye, Scotland, UK), and a Gould WindoGraf recorder (Gould Instrument Systems Inc., Valley View, Ohio, U.S.A.) were attached. The analogue signal was digitized, and Notocord Hem (Notocord Systems SA, France) data acquisition software was used to collect the RMV values. Inhalation exposure protocols To study the deposition of NPs, eight female BALB/ c/Sca mice (6–8 weeks old and mean weight 18.5 g, Scanbur Ab, Sollentuna, Sweden) were exposed by inhalation to 10 mg m3 of TiO2 for 2 h at a time for either 1 day, for 4 consecutive days, or for 4 days a week for 4 weeks (experiment C in Table 2). Either 4 or 24 h after the last exposure the mice were
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Table 2 Aerosol properties in the experiments Exp.
[TiO2 ]m (mg m-3)
CPC
Aerosol mobility spectrometer 6
-3
6
-3
ELPI
N (910 cm )
Dbg (nm)
N (910 cm )
r
Dag (nm)
N (9106 cm-3)
r
A
0.8 ± 0.2a
0.40
84.5
0.4
1.66
–
–
–
B
7.2 ± 0.7a
5.84
73.9
6.5
1.80
102
5.7
1.71
C
10.0 ± 0.7
a
6.11
80.4
7.7
1.83
125
5.6
1.71
D
28.5 ± 0.9a
–
–
–
–
144
12.1
1.58
–
89.2
23.6
1.67
–
–
–
Aein
28.5
Experiments A–D are measured from the exposure chamber and Aein was measured from the aerosol inlet before the exposure chamber ‘‘-’’ not measured/available a
Standard deviation
sacrificed and samples collected. A piece of the lungs was affixed in formalin and stored in ?4 °C until ICP-MS analysis. Male C57BL/6J mice (6–8 weeks old, weight 17– 31 g and mean weight 24.9 g, Scanbur Ab, Sollentuna, Sweden) were exposed by inhalation to three (0.8, 7.2, and 28:5 mg m3 , experiments A, B, D in Table 2) doses of TiO2 NPs for 5 consecutive days, 4 h per day. After the lavage of lungs with 35 mL protease solution for 30 min, the trachea and main bronchi were removed, and a small piece of each lung was cut and stored at -70 °C for analysis of TiO2 concentration in the lung tissue. The lung pieces of six treated mice per dose and six unexposed controls were pooled for TiO2 concentration analysis. Lung TiO2 concentration analysis The lung tissue samples were dried to constant weight and digested with nitric acid (HNO3 ). Lung dry mass concentration ½TiO2 m;L (mg g1 ) was determined using Inductively Coupled Plasma Mass Spectrometry (ICP-MS, Thermo X Series II, Thermo Electro, Germany). The subscript ‘‘L’’ refers to the lung dry mass concentration. Lung deposition analysis Exposure dose (in mg) by inhalation depends on the respiratory minute volume under exposure RMVe, aerosol mass concentration ½TiO2 m (mg m3 ), exposure time T (min) and the fraction of deposited material DF. Lung TiO2 mass concentration with
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respect to lung dry mass md was quantified with ICPMS analysis. It can be expressed as ½TiO2 m;L ¼
RMVe DF T ½TiO2 m md
ð4Þ
If retention half-life is long compared to inhalation experiment time, the dose is the sum of single exposure doses. Insoluble particles, such as TiO2 particles, have a retention half-life over 90 days for rats (Hsieh et al. 1999) When concentration is the same in each experiment, the summing can be done over single exposure times. The exposure time is T ¼ n t where n is the number of exposures and t is the duration of a single exposure. It is expected that the respiratory minute volume depends on the amount of pollutants entering the respiratory system and thus RMVe ¼ RMVe ð½TiO2 m ) (Snipes 1989). Assuming homogenous TiO2 concentration in the lung tissue, the deposition fraction is DF1 ¼
md ½TiO2 m;L mTiO2 ;L ¼ mTiO2 ;ae RMVe T ½TiO2 m
ð5Þ
where mTiO2 ;L is mass of TiO2 particles in the mouse lung and mTiO2 ;ae is mass of TiO2 aerosol particles in a volume inhaled by the mouse at time T. In lung deposition fraction calculations, the following assumptions were needed: (1) the mouse lung/body weight ratio was 16 (mg g1 ) (Mauad et al. 2008), (2) lung dry weight was 10% of lung wet weight, (3) particles were homogenously deposited in the lung tissue, (4) lung clearance was negligible during the experiment, (5) changes in respiratory parameters were the same in full body exposure as in head only
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9
x 10
8 −3 Particle concentration, N [cm ]
exposure, (6) relative changes in respiratory parameters were the same for different mice populations, (7) aerosol properties were the same between the head only and full body exposure setups (Miettinen et al. 2009), and (8) aerosol particle size distributions were similar in geometric mean diameter and geometric standard deviation in all experiments (Table 2). Assumptions (1), (2), and (3) were used to estimate mTiO2 ;L (Table 4). Because lung weights were not available, the lung weight was assumed from the mouse body weight (Mauad et al. 2008). The lung dry mass had to be estimated, because tissue samples were stored in 70 C or at formalin which probably affected lung tissue density. Uncertainties in the calculation of mTiO2 ;L cause major errors in the deposition fraction calculations. Another error may be caused if the particles are not deposited homogenously in lung tissue. These errors could be avoided by measuring the TiO2 weight of the whole lung, which was not possible in this study, because the mice lungs were primarily used for toxicological studies (Rossi et al. 2010; Lindberg et al. 2010).
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7 6 5 4 3 2 1 0
0
20
40
60
80
100
120
140
Time, t [min]
Fig. 2 Repeatability of the generator in the ½TiO2 m ¼ 10:0 mg m3 experiment: plot of 23 exposures particle concentration time series measured with CPC
approximately 1.5 times Dbg. The difference in mobility and aerodynamic diameters is caused by particles effective density. The effective density, qeff, is defined as (Ristima¨ki et al. 2002): Cca Dag q; Ccb Dbg 0
Results and discussion
qeff ¼
Aerosol properties
where Cca and Ccb were Cunningham slip correction factors for aerodynamic and mobility diameters, respectively, and q0 is unit density (1 g cm3 ). According to Table 2 values, and by assuming the same shape of log-normal particle size distributions, the effective density was in the range of 1.5 to 1:7 g cm3 . Keskinen et al. (2007) measured an effective density of 1:5 g cm3 for TiO2 NPs produced by liquid flame spray. Surface area concentration can be estimated from log-normal distribution by using Hatch–Choate conversion equations and by assuming spherical particles (Hinds 1999). For experiment C, the surface area calculated from mobility size distribution is approximately 0.3 m2 m-3. CPC particle concentration was of the same magnitude in experiments A–C as that measured by the aerosol mobility spectrometer and ELPI. Mobility size distributions Dbg and r were similar to those measured by Miettinen et al. (2009). Deviation in instruments’ size channels was caused by the variation in particle concentration at the beginning and the end of the experiment, and by a ±7% sampling flow
The average temperature and relative humidity in the exposure chamber were 22 1 C and 40 10%. Oxygen concentration ½O2 v calculated from nitrogen and air volume flows was at minimum 18.2% (Table 1). This is 0.8% lower than the guideline for an acute inhalation toxicity test (US EPA 1998). Figure 2 shows the stability and the repeatability of the generator in ½TiO2 m ¼ 10:0 mg m3 exposures executed 23 times. In three exposures, the increase in particle concentration was slower than usual (Fig. 2). We expect that this was due to maintaining the reactor temperatures for 22 h between the exposures. During this time, TiO2 particles were thermally re-suspended from the reactor wall and the reactor was unstabilized. Figure 3 shows the particle size distributions measured during the experiments. Log-normal fits were plotted to the distributions and the fit parameters are listed in Table 2. The mobility geometric mean diameter, Dbg was around 80 nm in experiments A, B, and C. Experiments B and C showed that the aerodynamic geometric mean diameter, Dag, was
ð6Þ
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dN/dLog(Dp) [cm−3 ]
5
10
x 10
3
0.8 mg/m
6
15
x 10
3
7.2 mg/m
(B)
(A) 10 5 5
0 10
100 6
x 10
dN/dLog(Dp) [cm−3 ]
Fig. 3 Statistical values of particle mobility and aerodynamic distributions measured with aerosol mobility spectrometer and ELPI. Diameter, Dp, indicates particle mobility or aerodynamic diameter. Value statistics are average (open circle), median (times) and standard deviation (white box for aerosol mobility spectrometer and gray box for ELPI). Log-normal fits (solid black line) are presented at average dN/ dLog(Dp) values
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1000
0 10
3
10 mg/m
7
3
15
(C)
x 10
1000 3
28.5 mg/m
(D)
10
2
5
1
0 10
100
100
1000
Diameter, Dp [nm]
0 10
100
1000
Diameter, Dp [nm]
uncertainty in the ejector diluter which depended on atmospheric pressure. In experiment A, the large standard deviations were caused by adjustment of Qr and Qb reactor flows after the first exposure. The adjustment was made to obtain similar particle size distribution as in experiments B and D. In Table 2, Aein particle size distribution was measured before the exposure chamber using the same reactor parameters as in experiment D. It can be seen that the particle concentration was approximately twice as high as in experiment D which may have been caused by particle coagulation and particle deposition on the surfaces of the exposure chamber. Particle properties Rietveld analysis of X-ray diffractograms (data not shown) revealed that the TiO2 powder consisted of 74% anatase and 26% brookite in volume concentrations, and crystallite sizes were 41 and 6 nm, respectively. From the electron diffraction pattern in Fig. 4, the brookite phase can be identified from ˚ diffraction ring. According to anatase and 2.9 A brookite crystallite sizes and volume concentration ratio, there should be approximately 100 times more brookite crystallites than anatase crystallites. Miettinen et al. (2009) also found anatase and brookite phases with similar volume concentration ratios
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Fig. 4 TEM micrograph of TiO2 particles collected on copper grid during experiment C. Electron diffraction pattern reveals diffraction rings both for anatase and brookite crystalline structures (rings are marked with dots)
where crystallite sizes were 20 and 7 nm, respectively. This shows that in the particle synthesis anatase crystallite size was not repeatable. The mean size of primary particles was 21 nm when determined with TEM for 450 particles. Figure 4 shows that the TiO2 particles were agglomerated,
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consisting of crystallites in a size range of under 10 nm to approximately 60 nm. The specific surface area of titanium oxide powder was 61 m2 g-1. Miettinen et al. (2009) measured a slightly higher surface area of 69 m2 g-1. According to the BET method, the surface area concentration in experiment C was 0:6 m2 m3 , which is twice as much as defined from mobility size distribution. This is explained by the fact that the mobility measurement quantifies active surface area and the BET method accounts all surface molecules (Ku and Maynard 2005).
volume decrease compared to the control group was caused by lower concentrations of by-product gases. The effect of TiO2 particles on respiratory parameters could be estimated from differences between the control group and the ½TiO2 m ¼ 30 mg m3 exposure group where by-product gas concentrations were the same. According to Table 3, by-product gases decreased tidal volume by 9%, and TiO2 particles at mass concentration of ½TiO2 m ¼ 30 mg m3 further decreased breathing frequency by 4% and tidal volume by 7%. Table 3 shows that RMV decreased from baseline level during the exposure. The decreased level in RMV value depended on TiO2 mass concentration and was mainly caused by the decrease in tidal volume. This shows that respiratory system flow velocities varied little and thus only had a small effect on particle impaction efficiency. The respiratory parameters were defined for Crl:OF1 mice and baseline values for BALB/c/Sca mice (Table 3). For C57BL/6J male mice, RMV values were assumed to be the same as those for Crl:OF1 male mice with body weight correction. The body weight was assumed to be directly proportional to the tidal volume, which meant that tidal volume was, on average, 17% less for the C57BL/6J mice than the Crl:OF1 mice. This lead to RMVe values of 37.3 and 29:1 mL min1 for experiments B and D, respectively. For experiment A, we used the same RMVe value as in experiment B.
Respiratory minute volume Temperature in the head only exposure chamber varied from 23 to 35 C and relative humidity was approximately 30%. Oxygen concentration ½O2 v was a minimum of 17%. According to TTIP reaction stoichiometry, and FTIR measurements by Miettinen et al. (2009), aerosol from the reactor included the following by-product gases: C3 H6 ; CO, and NOx. The concentration levels of the gases depended on the TTIP precursor flow rate and reactor temperature parameters. The control group of mice was exposed to reactor by-product gases, which decreased the tidal volume by 9% from baseline level. When the mice were exposed to 8 mg m3 TiO2 aerosol, the breathing frequency decreased by 8% and tidal volume by 2% compared to the baseline level. The lower tidal
Table 3 Average respiratory parameters for baseline and during TiO2 aerosol exposure [TiO2]m (mg m-3) (No. of mice)
RMVb (mL min-1)
fb (min-1)
RMVe (mL min-1)
fe (min-1)
dRMV (%)
df (%)
dVTc(%)
Control (6)b
47
262
43
262
9
0
9
8 (4)a
52
295
47
272
10
8
2
20 (8)b
61
293
52
274
15
6
9
30 (7)b
46
263
37
252
20
4
16
44
238
–
–
–
–
–
Crl:OF1 male
BALB/c/Sca female 4 groups of 4 mice (16)
Decrease in respiratory parameters is denoted by d and presented in percentages Control: during the exposure period, mice breathed HEPA filtered air from the reactor (by-products included) ‘‘–’’ not measured/available Subscripts: ‘‘b’’ refers to baseline and ‘‘e’’ refers to exposure a
One group of 4 mice
b
Two groups of 4 mice
c
Calculated value
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Table 4 Lung deposition fractions calculated with Eq. 5 and calculation parameters Exp. (no. of samples)
[TiO2]m (mg m-3)
n
t (min)
[TiO2]m,L (mg m-3) (No. of mice)
md (mg)
mTiO2 ;L (lg)
mTiO2 ;ae (lg)
RMVe (mL min-1)
DF (%)
Controla (1)
0
–
–
0.006
43
0.2
–
–
–
Aa (1)
0.8
5
246
0.069
39
2.7
30
37.3
9
Ba (1)
7.2
5
246
0.42
43
18.0
330
37.3
5
a
D (1)
28.5
5
245
38
84.0
1,020
29.1
9
C1b (3)
10
1
128
0.19 (0.09)
30
5.6 (2.6)
50
39.6
11(5)
C2b (2)
10
4
128
0.43 (0.14)
30
12.6 (4.0)
200
39.6
6(4)
2.2
For experiments C1 and C2, mean values of the samples are shown (standard deviations in parenthesis) Control: unexposed, lived in the same room Parameters are: [TiO2 m is aerosol mass concentration, n is number of exposures, t is single exposure time, ½TiO2 m;L is TiO2 lung dry mass concentration, md is lung dry mass, mTiO2 ;L is TiO2 mass in lung tissue, mTiO2 ;ae = RMVe ½TiO2 m n t is TiO2 mass in the breathed volume by the mouse during the experiment, RMVe is respiratory minute volume during the exposure, and DF is deposition fraction a
C57BL/6J male mice, samples pooled from six mice
b
BALB/c/Sca female mice
For the BALB/c/Sca mice in experiment C, RMVe ¼ 39:6 mL min1 value was obtained by correcting the baseline level dRMV value at ½TiO2 m ¼ 8 mg m3 (Table 3). Table 4 shows the derived RMVe values for the C57BL/6 and BALB/c/Sca mice. Respiratory minute volume is often estimated from body weight with a formula by (Guyton 1947). This approximation gives an RMV value that is about half of the RMVb values we measured for the Crl:OF1 and BALB/c/Sca mice. Ryman-Rasmussen et al. (2009) used an RMV ¼ 27:3 mL min1 value for C57BL6 mice of the same age as those in our experiment, which is slightly less than that obtained in the present study at ½TiO2 m ¼ 8 mg m3 . Lung deposition To estimate the uptake of TiO2 particulate matter by the mice, the lung deposition fraction must be quantified. This was done by using two different methods. In both methods, the lung TiO2 mass content was compared to aerosol TiO2 mass content in a volume inhaled by the mouse during the experiment. In the first method, deposition fraction was calculated for each mouse separately using Eq. 5. In the second method, the deposition fraction was estimated from a slope factor of lung concentration as a function of aerosol mass concentration. The slope
123
factor method extrapolates lung deposition fraction for different aerosol mass concentrations. A good correlation (0.98) between mTiO2 ; L and mTiO2 ; ae indicated that assumptions 1, 2, 3, and 4 were reasonable for these experiments (Table 4). Table 4 shows that deposition fractions varied from 5% up to 11% and were on an average 8%. Differences in deposition fractions between BALB/c/Sca mice and C57BL/6J mice could not be seen (Table 4). The control groups showed that the TiO2 lung concentration in unexposed mice was negligible. Any conclusions from lung clearance in experiments C1 and C2 could not be estimated, because of the high standard deviation of the DF values and the lack of statistics. The deposition fractions in all experiments were of the same order of magnitude as expected. This was caused by similar aerosol size distributions and geometric standard deviations in all experiments (Table 2, Fig. 3), and since breathing velocity did not vary significantly, particle deposition by impaction was nearly the same. Experiment C showed a high standard deviation in deposition fraction, which was caused by variation in TiO2 particle uptake in the full body exposure. In the full body exposure chamber some of the mice slept in a bunch and the nose of the mouse may have been located in fur. The fur may have acted as a particle filter and reduced the uptake of the particles.
J Nanopart Res (2011) 13:2949–2961
2959
Linear fit: [TiO2]m,L = 0.076*[TiO2]m
−1
in the lung [mg g ]
2
TiO mass concentration
2
10
0
10
−2
10
−4
10
−1
0
10
10
1
2
10
10 −3
Aerosol TiO2 mass concentration [mg m ]
Fig. 5 Mice TiO2 lung concentration measured from three inhalation experiments with TiO2 aerosol mass concentrations of 0.8, 7.2, and 28:5 mg m3
Figure 5 shows that there was a linear correlation between the TiO2 mass concentration of lung dry weight and aerosol mass concentration. Slope factor k was 0:076 0:004 m3 g1 , and according to Eq. 5, the mathematical formulation is k¼
RMVe DF T md
ð7Þ
where RMVe is constant in first order approximation. According to Eq. 6, and using an average respiratory minute volume of RMV ¼ 35 mL min1 , the average deposition fraction was 7%. This shows that within these accuracies this method may be used to calculate deposition fractions. The lung deposition obtained with these two methods shows that aerosol particles were deposited into mice lungs during the exposure. Because the particles were insoluble, and retention time was long compared to duration of the experiments, uptake of the TiO2 matter increased linearly when the dose was increased. Approximately, 7–8% of aerosol mass was deposited on mice lungs. This is approximately 5% less than reported by Snipes (1989) for particles of less than 1 lm. However, the deposition fraction depends on RMV value, which may vary among different mice populations and exposure conditions.
different aerosol mass concentrations ranging from 0.8 to 28:5 mg m3 with respective particle concentrations varying between 0:4 106 and 12:1 106 cm3 . Particle size distribution’s geometric standard deviation was 1.83 or less and Deff was on average 80 nm. Effective density of particles varied from 1.5 to 1.7 g cm-3. The TiO2 particles consisted of anatase (74 vol%) and brookite (26 vol%) phases, with crystallite sizes of 41 and 6 nm, respectively. The mean primary particle size of the TiO2 agglomerates was 21 nm and specific surface area was 61 m2 g-1. For experiment C this corresponded to surface area concentration of 0.6 m2 m-3. The RMV value changed during exposure and depended on both gaseous by-products and particulate matter. Respiratory minute volume showed a decreasing trend when TiO2 mass concentration was increased. This trend needs to be considered when uptake to pollutants is estimated. The lung dry mass concentration was linearly related to aerosol mass concentration and the slope factor was 0.076. The deposition fraction was, on average, 8% and according to the slope factor method 7%. The deposition fraction was independent of aerosol mass concentration because particle size distributions and breathing parameters were similar in each experiment. The results of the aerosol and particle characterization show that the particle generator is suitable for producing NPs at mass concentrations of up to approximately 30 mg m3 with good repeatability. We have shown that aerosol particles are deposited on the lungs of mice exposed to different concentrations of NPs. These findings indicate that the exposure protocol presented can be used for NP inhalation studies in mice. Acknowledgements The article was supported by the Academy of Finland, FinNano-program, Engineered Nanoparticles: Synthesis, Characterization, Exposure and Health Hazards (NANOHEALTH)-project (project number 117 924).
Conclusions This study presents a novel method for conducting inhalation experiments where NPs were directly synthesized in the gas phase. We exposed mice at TiO2 NPs and estimated deposition fractions of particulate matter at different aerosol mass concentrations. The experiments were carried out with four
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