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OBJECTIVES/GOALS: In vitro models that mimic the human respiratory system are needed to assess the toxicity of inhaled contaminants. Therefore, our goal is to establish a Dosimetric Aerosol in-Vitro Inhalation Device (DAVID) that delivers aerosols in different patterns to human lung cells cultured at an air-liquid interface (ALI). METHODS/STUDY POPULATION: The collection unit of DAVID was modified in this study to accommodate different deposition patterns (spots, annular ring, rectangle & circle). CuO aerosols of varying concentrations were generated using a 6-jet Collison nebulizer for varying time periods to achieve different doses. To quantify the doses that were delivered to cells, the samples were digested with nitric acid & analyzed by Inductively Coupled Plasma-Optical Emission Spectrometry. Following the exposure of A549 cells to CuO aerosols, cytotoxicity and mRNA expression (i.e., HMOX1 & IL-8) will be assessed via LDH and RT-qPCR to determine the effect of regional (mass deposited/area of the pattern) and global (mass deposited/area of the cell culture insert) doses in cells. RESULTS/ANTICIPATED RESULTS: The deposition areas covered by rectangular, spot, annular ring, and circular patterns are estimated to be 6, 17, 27 and 85% of the insert’s surface area, onto which cells are cultured. Results for the patterns tested (spots and annular ring) show that both the regional and global doses were greater for spots than annular ring. Also, the regional doses were higher than global doses. Irrespective of the patterns, the global doses were the same for nebulizer suspensions of 0.1-1 mg/mL. Statistical analysis by ANOVA revealed there was no significant difference in doses between replicate inserts used in the same trial. We anticipate that regional doses with aerosol deposition to a larger surface area of the cell culture insert will correspond with higher cytotoxicity and mRNA expression of HMOX1 and IL-8 in cells. DISCUSSION/SIGNIFICANCE: There are limited in vitro exposure systems that can efficiently deliver aerosols to lung cells, while also mimicking inhalation by humans. In addition to addressing this knowledge gap, we will show the role of regional & global doses in studying cellular response & the ability of DAVID to deliver aerosols in different deposition patterns.

The B.1.617.2 (Delta) variant of SARS-CoV-2 emerged in India in October of 2020 and spread widely to over 145 countries, comprising over 99% of genome sequence-confirmed virus in COVID-19 cases of the United States (US) by September 2021. The rise in COVID-19 cases due to the Delta variant coincided with a return to in-person school attendance, straining COVID-19 mitigation plans implemented by educational institutions. Some plans required sick students to self-isolate off-campus, resulting in an unintended consequence: exposure of co-inhabitants of dwellings used by the sick person during isolation. We assessed air and surface samples collected from the bedroom of a self-isolating university student with mild COVID-19 for the presence of SARS-CoV-2. That virus' RNA was detected by real-time reverse-transcription quantitative polymerase chain reaction (rRT-qPCR) in air samples from both an isolation bedroom and a distal, non-isolation room of the same dwelling. SARS-CoV-2 was detected and viable virus was isolated in cell cultures from aerosol samples as well as from the surface of a mobile phone. Genomic sequencing revealed that the virus was a Delta variant SARS-CoV-2 strain. Taken together, the results of this work confirm the presence of viable SARS-CoV-2 within a residential living space of a person with COVID-19 and show potential for transportation of virus-laden aerosols beyond a designated isolation suite to other areas of a single-family home.

Fitness centers are considered high risk for SARS-CoV-2 transmission due to their high human occupancy and the type of activity taking place in them, especially when individuals pre-symptomatic or asymptomatic for COVID-19 exercise in the facilities. In this study, air (N = 21) and surface (N = 8) samples were collected at a fitness center through five sampling events from August to November 2020 after the reopening restrictions were lifted in Florida. The total attendance was ~2500 patrons during our environmental sampling work. Air samples were collected using stationary and personal bioaerosol samplers. Moistened flocked nylon swabs were used to collect samples from high-touch surfaces. We did not detect SARS-CoV-2 by rRT-PCR analyses in any air or surface sample. A simplified infection risk model based on the Wells-Riley equation predicts that the probability of infection in this fitness center was 1.77% following its ventilation system upgrades based on CDC guidelines, and that risk was further reduced to 0.89% when patrons used face masks. Our model also predicts that a combination of high ventilation, minimal air recirculation, air filtration, and UV sterilization of recirculated air reduced the infection risk up to 94% compared to poorly ventilated facilities. Amongst these measures, high ventilation with outdoor air is most critical in reducing the airborne transmission of SARS-CoV-2. For buildings that cannot avoid air recirculation due to energy costs, the use of high filtration and/or air disinfection devices are alternatives to reducing the probability of acquiring SARS-CoV-2 through inhalation exposure. In contrast to the perceived ranking of high risk, the infection risk in fitness centers that follow CDC reopening guidance, including implementation of engineering and administrative controls, and use of personal protective equipment, can be low, and these facilities can offer a relatively safe venue for patrons to exercise.

Although exposure to ambient particulate matter (PM) is linked to asthma, the health effects of co-existing vapor-phase organic pollutants (vapor) and their combined effects with PM on this disease are poorly understood. We used a murine asthma model to test the hypothesis that exposure to vapor would enhance allergic sensitization and this effect would be further strengthened by co-existing PM. We found that vapor and PM each individually exerted adjuvant effects on OVA sensitization. Co-exposure to vapor and PM during sensitization further enhanced allergic lung inflammation and OVA-specific antibody production which was accompanied by pulmonary cytokine/chemokine milieu that favored T-helper 2 immunity (i.e. increased IL-4, downregulation of Il12a and Ifng, and upregulation of Ccl11 and Ccl8). TNFa, IL-6, Ccl12, Cxcl1 and detoxification/antioxidant enzyme responses in the lung were pollutant-dependent. Inhibition of lipopolysaccharide-induced IL-12 secretion from primary antigen-presenting dendritic cells correlated positively with vapor’s oxidant potential. In conclusion, concurrent exposure to vapor and PM led to significantly exaggerated adjuvant effects on allergic lung inflammation which were more potent than that of each pollutant type alone. These findings suggest that the effects of multi-component air pollution on asthma may be significantly underestimated if research only focuses on a single air pollutant (e.g., PM).

A self-sustaining, motion-tolerant, water-based condensation particle counter (CPC) has been designed, fabricated, and tested. Referred to as “MAGIC” for moderated aerosol growth with internal wate...

The progression of COVID-19 worldwide can be tracked by identifying mutations within the genomic sequence of SARS-CoV-2 that occur as a function of time. Such efforts currently rely on sequencing the genome of SARS-CoV-2 in patient specimens (direct sequencing) or of virus isolated from patient specimens in cell cultures. A pilot SARS-CoV-2 air sampling study conducted at a clinic within a university student health care center detected the virus vRNA, with an estimated concentration of 0.87 virus genomes L(−1) air. To determine whether the virus detected was viable (‘live’), attempts were made to isolate the virus in cell cultures. Virus-induced cytopathic effects (CPE) were observed within two days post-inoculation of Vero E6 cells with collection media from air samples; however, rtRT-PCR tests for SARS-CoV-2 vRNA from cell culture were negative. Instead, three other fast-growing human respiratory viruses were isolated and subsequently identified, illustrating the challenge in isolating SARS-CoV-2 when multiple viruses are present in a test sample. The complete SAR-CoV-2 genomic sequence was nevertheless determined by Sanger sequencing and most closely resembles SARS-CoV-2 genomes previously described in Georgia, USA. Results of this study illustrate the feasibility of tracking progression of the COVID-19 pandemic using environmental aerosol samples instead of human specimens. Collection of a positive sample from a distance more than 2 m away from the nearest patient traffic implies the virus was in an aerosol.

Since mask use and physical distancing are difficult to maintain when people dine indoors, restaurants are perceived as high risk for acquiring COVID-19. The air and selected environmental surfaces in two restaurants in a mid-scale city located in north central Florida that followed the Centers for Disease Control and Prevention (CDC) reopening guidance were sampled three times from July 2020 to February 2021. Sixteen air samples were collected for 2 hours using air samplers, and 20 surface samples by using moistened swabs. The samples were analyzed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for the presence of SARS-CoV-2 genomic RNA. A total of ~550 patrons dined in the restaurants during our samplings. SARS-CoV-2 genomic RNA was not detected in any of the air samples. One of the 20 surface samples (5%) was positive. That sample had been collected from a plastic tablecloth immediately after guests left the restaurant. Virus was not isolated in cell cultures inoculated with aliquots of the RT-PCR-positive sample. The likelihood that patrons and staff acquire SARS-CoV-2 infections may be low in restaurants in a mid-scale city that adopt CDC restaurant reopening guidelines, such as operation at 50% capacity so that tables can be spaced at least 6 feet apart, establishment of adequate mechanical ventilation, use of a face covering except while eating or drinking, and implementation of disinfection measures.

One limitation in air sampling of airborne microorganisms is their inactivation by forceful impaction and/or dehydration during the collection process. Proper inhalation risk assessments require proof of viability, as non-viable microorganisms cannot cause infectious diseases. In this study, laboratory-generated aerosols of a vegetative bacterium (E. coli) or yeast (S. kudriavzevii) were collected by a laminar-flow water-based condensational “growth tube collector (GTC),” and the GTC’s collection efficiencies were compared with those using an industry standard BioSampler. Collection efficiencies resulting from two types of collection media, phosphate-buffered saline (PBS) and nutrient media (Nutrient Broth, NB, for E. coli, and Yeast Tryptone Glucose Broth, YTGB, for S. kudriavzevii) were also assessed. Both the GTC and the BioSampler performed equally when PBS was used as the collection medium for E. coli, whereas more viable E. coli cells were collected in the GTC than the BioSampler with NB. For S. kudriavzevii, the GTC outperformed the BioSampler using either PBS or YTGB. This is likely because aerosolized E. coli cells can better survive impaction than S. kudriavzevii under the conditions used, and the BioSampler has a much higher collection efficiency for particles in the size range of single-celled E. coli than S. kudriavzevii. Moreover, the GTC had a detection limit one order of magnitude lower for yeast aerosols compared with that of the BioSampler. These results indicate that the GTC is a promising device for sampling viable aerosolized gram-negative bacteria and yeast, as it is less damaging to these types of microorganisms during the collection process.

Individuals with COVID-19 are advised to self-isolate at their residences unless they require hospitalization. Persons sharing a dwelling with someone who has COVID-19 have a substantial risk of being exposed to the virus. However, environmental monitoring for the detection of virus in such settings is limited. We present a pilot study on environmental sampling for SARS-CoV-2 virions in the residential rooms of two volunteers with COVID-19 who self-quarantined. Apart from standard surface swab sampling, based on availability, four air samplers positioned 0.3–2.2 m from the volunteers were used: a VIable Virus Aerosol Sampler (VIVAS), an inline air sampler that traps particles on polytetrafluoroethylene (PTFE) filters, a NIOSH 2-stage cyclone sampler (BC-251), and a Sioutas personal cascade impactor sampler (PCIS). The latter two selectively collect particles of specific size ranges. SARS-CoV-2 RNA was detected by real-time Reverse-Transcription quantitative Polymerase Chain Reaction (rRT-qPCR) analyses of particles in one air sample from the room of volunteer A and in various air and surface samples from that of volunteer B. The one positive sample collected by the NIOSH sampler from volunteer A's room had a quantitation cycle (Cq) of 38.21 for the N-gene, indicating a low amount of airborne virus [5.69E-02 SARS-CoV-2 genome equivalents (GE)/cm3 of air]. In contrast, air samples and surface samples collected off the mobile phone in volunteer B's room yielded Cq values ranging from 14.58 to 24.73 and 21.01 to 24.74, respectively, on the first day of sampling, indicating that this volunteer was actively shedding relatively high amounts of SARS-CoV-2 at that time. The SARS-CoV-2 GE/cm3 of air for the air samples collected by the PCIS was in the range 6.84E+04 to 3.04E+05 using the LED-N primer system, the highest being from the stage 4 filter, and similarly, ranged from 2.54E+03 to 1.68E+05 GE/cm3 in air collected by the NIOSH sampler. Attempts to isolate the virus in cell culture from the samples from volunteer B's room with the aforementioned Cq values were unsuccessful due to out-competition by a co-infecting Human adenovirus B3 (HAdVB3) that killed the Vero E6 cell cultures within 4 days of their inoculation, although Cq values of 34.56–37.32 were measured upon rRT-qPCR analyses of vRNA purified from the cell culture medium. The size distribution of SARS-CoV-2-laden aerosol particles collected from the air of volunteer B's room was >0.25 μm and >0.1 μm as recorded by the PCIS and the NIOSH sampler, respectively, suggesting a risk of aerosol transmission since these particles can remain suspended in air for an extended time and travel over long distances. The detection of virus in surface samples also underscores the potential for fomite transmission of SARS-CoV-2 in indoor settings., Graphical abstract Image 1

Presented is a laminar-flow, water-based condensation particle counter capable of particle detection near 1 nm. This instrument employs a three-stage, laminar-flow growth tube with a “moderator” stage that reduces the temperature and water content of the output flow without reducing the peak supersaturation, and makes feasible operation at the large temperature differences necessary for achieving high supersaturations. The instrument has an aerosol flow of 0.3 L/min, and does not use a filtered sheath flow. It is referred to as a “versatile” water condensation particle counter, or vWCPC, as operating temperatures can be adjusted in accordance with the cut-point desired. When operated with wall temperatures of ∼2°C, >90°C, and ∼22°C for the three stages, respectively, the vWCPC detects particles generated from a heated nichrome wire with a 50% efficiency cut-point near 1.6 nm mobility diameter. At these operating temperatures, it also detects 10–20% of large molecular ions formed from passing filte...

State-of-the-art bioaerosol samplers have poor collection efficiencies for ultrafine virus aerosols. This work evaluated the performance of a novel growth tube collector (GTC), which utilizes laminar-flow water-based condensation to facilitate particle growth, for the collection of airborne MS2 viruses.Fine aerosols (500 nm) containing MS2 coliphage were generated from a Collison nebulizer, conditioned by a dilution dryer and collected by a GTC and a BioSampler. The GTC effectively condensed water vapour onto the virus particles, creating droplets 2-5 μm in diameter, which facilitated collection. Comparison of particle counts upstream and downstream revealed that the GTC collected93% of the inlet virus particles, whereas the BioSampler's efficiency was about 10%. Viable counts of the GTC-collected viruses were also one order of magnitude higher than those of the BioSampler (P = 0·003).The efficiency of the GTC for the viable collection of MS2 viruses exceeds that of industry standard instrument, the BioSampler, by a factor of 10-100.This study reveals that the GTC is an effective collector of viable MS2 aerosols, and concludes the instrument will be an effective tool for studying viable virus aerosols and the inhalation risks posed by airborne viruses.

The Sequential Spot Sampler (S3), a newly developed instrument to collect aerosols for time-resolved chemical composition measurements, was evaluated in the laboratory and field for the measurement of particulate sulfate and nitrate. The S3 uses a multi-temperature condensation growth tube to grow individual aerosols to droplets which are then deposited as a ∼ 1 mm diameter dry spot at the end of the growth tube in a 100 µL well of a multi-well plate. The well plate advances automatically to provide a sequence of time-resolved samples. The collected aerosols are subsequently analyzed in the laboratory. The sample is concentrated during the collection process, and the laboratory extraction and analysis steps can be automated. The well plate, as received from the field, is placed onto a needle-based autosampler that adds liquid for sample extraction and injects sample extract from each well onto an ion chromatograph for analysis. Laboratory evaluation for sulfate and nitrate ions showed that poly ether ether ketone (PEEK) used as well plate material does not contribute any artifacts; a 60 min extraction procedure leads to the recovery of sulfate and nitrate from the dry spots at above 95 % extraction efficiency; and samples stored frozen and analyzed up to 23 months later show less than a 10 % change in sulfate and nitrate concentrations. The limit of detection was 0.5 µg m−3 for sulfate and 0.2 µg m−3 for nitrate for a 1 h sampling period. In a month-long field study conducted in southern California, two S3s were deployed alongside a URG denuder–filter-pack and a Particle-Into-Liquid Sampler combined with an Ion Chromatograph (PILS-IC). Collocated S3 sampler concentrations compared by linear regression show good agreement, with r2 = 0.99 and slope = 0.99 (±0.004) µg m−3 for sulfate and r2 = 0.99 and slope = 1.0 (±0.006) µg m−3 for nitrate. When compared to the URG denuder–filter-pack and the PILS-IC, the S3 sulfate and nitrate concentrations yielded correlations above 0.84 for the square of the correlation coefficient and regression slopes close to 1.

In assessing the biological impact of airborne particles in vitro, air-liquid interface (ALI) exposure chambers are increasingly preferred over classical submerged exposure techniques, albeit historically limited by their inability to deliver sufficient aerosolized dose. A novel ALI system, the Dosimetric Aerosol in Vitro Inhalation Device (DAVID), bioinspired by the human respiratory system, uses water-based condensation for highly efficient aerosol deposition to ALI cell culture. Here, welding fumes (well-studied and inherently toxic ultrafine particles) were used to assess the ability of DAVID to generate toxicological responses between differing welding conditions. After fume exposure, ALI-cultured cells showed reductions in viability that were both distinct between welding conditions and linearly dose-dependent with respect to exposure time; comparatively, submerged cell cultures ran in parallel did not show these trends across exposure levels. DAVID delivers a substantial dose in minutes (> 100 μg/cm2), making it preferable over previous ALI systems, which require hours of exposure to deliver sufficient dose, and over submerged techniques, which lack comparable physiological relevance. DAVID has the potential to provide the most accurate assessment of in vitro toxicity yet from the perspectives of physiological relevance to the human respiratory system and efficiency in collecting ultrafine aerosol common to hazardous exposure conditions.

A water condensation-based ion charging system has been developed to enhance both the charging efficiency and the concentration of sub-20 nm particles. This NanoCharger consists of a bipolar ion source followed by a parallel plate water-based condensation system, an embedded ion scavenger, and an aerodynamic focusing stage. Sufficient numbers of ions are transported through the system to attach to the formed droplets. An ion scavenger removes the ions immediately after the droplet formation to minimize multiple charging. A subsequent cold-walled condensation stage removes most of the water vapor, lowering the dew point to below 16 °C, while a set of focusing nozzles concentrates the droplets into ∼10% of the flow. The flow is then slightly heated to evaporate the droplets. The physical enhancement of electrical charging was evaluated in the laboratory using mobility-selected particles, and found to provide ∼40-fold enhancement over bipolar charging for 6–15 nm particles. Chemical artifacts were evaluated through thermal desorption chemical ionization mass spectrometry. Data comparing ion spectra for flow that passed through the NanoCharger to that obtained without it showed nearly equivalent ion spectra, indicating that no significant artifacts were introduced from the condensation–evaporation process. Copyright © 2018 American Association for Aerosol Research

The capacity of airborne particulate matter to generate reactive oxygen species (ROS) has been correlated with the generation of oxidative stress both in vitro and in vivo. The cellular damage from oxidative stress, and by implication with ROS, is associated with several common diseases, such as asthma and chronic obstructive pulmonary disease (COPD), and some neurological diseases. Yet currently available chemical and in vitro assays to determine the oxidative capacity of ambient particles require large samples, analyses are typically done offline, and the results are not immediate.Here we report the development of an online monitor of the oxidative capacity of aerosols (o-MOCA) to provide online, time-resolved assessment of the capacity of airborne particles to generate ROS. Our approach combines the Liquid Spot Sampler (LSS), which collects particles directly into small volumes of liquid, and a chemical module optimized for online measurement of the oxidative capacity of aerosol using the dithiothreitol (DTT) assay. The LSS uses a three-stage, laminar-flow water condensation approach to enable the collection of particles as small as 5 nm into liquid. The DTT assay has been improved to allow the online, time-resolved analysis of samples collected with the LSS but could be adapted to other collection methods or offline analysis of liquid extracts.The o-MOCA was optimized and its performance evaluated using the 9,10-phenanthraquinone (PQ) as a standard redox-active compound. Laboratory testing shows minimum interferences or carryover between consecutive samples, low blanks, and a reproducible, linear response between the DTT consumption rate (nmol min−1) and PQ concentration (µM). The calculated limit of detection for o-MOCA was 0.15 nmol min−1. The system was validated with a diesel exhaust particle (DEP) extract, previously characterized and used for the development, improvement, and validation of the standard DTT analysis. The DTT consumption rates (nmol min−1) obtained with the o-MOCA were within experimental uncertainties of those previously reported for these DEP samples. In ambient air testing, the fully automated o-MOCA was run unattended for 3 days with 3 h time resolution and showed a diurnal and daily variability in the measured consumption rates (nmol min−1 m−3).

A viable virus aerosol sampler (VIVAS) effectively collected viable influenza A and B viruses from air inside a student health care center during an influenza outbreak. The viruses had “drifted” genes, showcasing the usefulness of the VIVAS for air sampling and noninvasive surveillance of viruses in circulation.

A new sampling approach has been developed to enable affordable, time-resolved monitoring of particulate chemical compositions, and more generally to provide concentrated samples of airborne particles. Using a newly developed, moderated water-based condensational growth technology, individual particle samples are deposited in a 1-mm diameter dry "spot". The moderated condensation technology enables this collection with minimal temperature rise, providing robust collection for volatile constituents. Measured collection efficiencies are above 95% for particles in the size range from 0.010 μm to 2.5 μm. A set of 20 or more time-resolved samples, plus blanks, may be collected onto a multiwell collection plate. For chemical analysis the plate is returned to the laboratory, and placed directly into a modified autosampler, without extraction or preparation. The autosampler handles the addition of eluent, extraction, and sample injection without user manipulation. This paper presents the design and laboratory evaluation of a 1.5 L/min sampling rate version of this system.

Diesel and biodiesel emissions are important sources of ambient air pollution. Therefore, the influence of using different blending percentages of biodiesel to diesel as fuel on redox activity, particulate matter (PM) and polycyclic aromatic hydrocarbons (PAH) emission profiles, and the corresponding carcinogenicity index (BaPE) size distributions are not well known. In this way, the present work aims to investigate the effect of diesel/biodiesel blends on the physical, chemical, and toxicological properties of diesel engine exhaust at low condition, through the study of the changes in size-distribution and emission factors of PAH associated to PM, corresponding carcinogenicity index (BaPE) size distributions and PM redox activity. For that, particle emissions from commercial petroleum-based diesel with 4% of soy biodiesel (B4), a biodiesel blend of 25% and 50% (B25 and B50) and also pure biodiesel (B100) were measured on a steady-state dynamometer in a test using a diesel engine at low load. Size-fractionated PM samples were collected by using the NanoMOUDI impactor and analyzed for the 16 priority PAH by HPLC–fluorescence detector. In addition, PM2.5 samples were collected and analyzed for redox activity by DTT assay. PM was distributed in all sizes, while PAH size distribution was found in higher levels in the accumulation mode (30 nm kg fuel - 1 ) for B4, B25, B50 and B100 were: 237, 111, 182 and 319 ng kg fuel - 1 , respectively. Individual PAH emission factors showed that PAH containing four or more rings (MW > 202) such as BBF, BAA, PYR and BGP were the main PAH emitted by the four studied fuels. The percentage reductions of individual PAH emission factors for the blend fuels in comparison with B4 were 37% and 22% for B25 and B50, respectively, and an increase around 31% for B100. On the other hand it was observed increase in redox activity for B25, B50 and B100 when compared to B4. In general, the results from our study suggest that emissions from pure waste cooking biodiesel may not be the better fuel choice in terms of PM, PAH and BaPE particle size distribution and emission factors as well as redox activity. However, B25 and B50 blends presented some improvements in terms of PM, HPA, BaPE size distribution and redox activity of engine exhaust in comparison to B4. This suggests that addition of low percentages of biodiesel to diesel promotes benefices in both environmental and human health concerns.

The capacity of airborne particulate matter to generate reactive oxygen species (ROS) has been correlated with the generation of oxidative stress both in-vitro and in-vivo. The cellular damage from oxidative stress, and by implication with ROS, is associated with several common diseases such as asthma, chronic obstructive pulmonary disease (COPD), and some neurological diseases. Yet currently available chemical and in-vitro assays to determine the oxidative capacity of ambient particles require large samples, analysis are typically done offline, and the results are not immediate. In this manuscript we report the development of an on-line monitor of the oxidative capacity of aerosols (o-MOCA) to provide on-line, time-resolved assessment of the capacity of airborne particles to generate ROS. Our approach combines the Liquid Spot Sampler (LSS), which collects particles directly into small volumes of liquid, and a chemical module optimized for on-line measurement of the oxidative capacity of aerosol using the dithiothreitol (DTT) assay. The LSS uses a three-stage, laminar-flow water condensation approach to enable the collection of particles as small as 5 nm into liquid, without subjecting the sample to temperature extremes. The DTT assay has been improved to allow the on-line, time-resolved analysis of samples collected with the LSS. The o-MOCA was optimized and its performance evaluated using the 9,10-Phenanthraquinone (PQ) as standard redox-active compound. Laboratory testing shows minimum interferences or carry-over between consecutive samples, low blanks, and a reproducible, linear response between the DTT consumption rate (nmol/min) and PQ concentration (µM). The calculated limit of detection for o-MOCA was 0.15 nmol/min. The system was validated with a Diesel Exhaust Particle (DEP) extract, previously characterized and used for the development, improvement, and validation of the standard DTT analysis. The DTT consumption rates (nmol/min) obtained with the o-MOCA were within experimental uncertainties of those previously reported for these DEP samples. In ambient air testing, the fully automated o-MOCA was run unattended for 3 days with 3-h time resolution, and showed a diurnal and daily variability in the measured consumption rates (nmol/min/m3).

In this study, it is reported the fractal dimension and morphology of ultrafine particles emitted by a diesel engine run with B4, B50, and B100. Transmission electron micrographs evidenced two main groups of particles with characteristic and distinct morphological behaviors: (i) particle agglomerate structures self-organized within a fractal-like geometry, and (ii) amorphous and irregular particles with diffuse edges in a liquid-like particle appearance, mainly associated with some sub-fractions of samples collected from B50 and B100 combustion. Particles from (i) showed typical diesel soot morphology and fractal dimension. Primary particle diameter (Dpp) was 24 ± 4 nm, 23 ± 4 nm, and 20 ± 4 nm for B4, B50, and B100, respectively. Mean aggregate gyration diameters (Dg) ranged from 242 ± 140 nm for B4, 210 ± 135 nm for B50, and to 232 ± 140 nm for B100. In turn, fractal dimensions (Df) were 1.94, 1.89, and 1.99 for B4, B50, and B100, respectively. On the other hand, particle from (ii) had their morphology impaired from the addition of biodiesel to petrodiesel in a way it did not follow a fractal geometry.

Highly efficient collection of infectious pandemic Influenza H1N1 virus (2009) through laminar-flow water based condensation

Motor vehicles are a major source of polycyclic aromatic hydrocarbon (PAH) emissions in urban areas. Motor vehicle emission control strategies have included improvements in engine design, exhaust emission control, and fuel reformulation. Therefore, an updated assessment of the effects of the shifts in fuels and vehicle technologies on PAH vehicular emission factors (EFs) is needed. We have evaluated the effects of ambient temperature on the size-resolved EFs of nine US EPA Priority Pollutant PAH, down to 10 nm diameter, from on-road California gasoline light-duty vehicles with spark ignition (SI) and heavy-duty diesels with compression ignition (CI) in summer 2004 and winter 2005. During the winter, for the target PAH with the lowest subcooled equilibrium vapor pressure --benzo[a]pyrene, benzo[ghi]perylene, and indeno[1,2,3-cd]pyrene-- the mass in the nucleation mode, defined here as particles with dp

A novel sampling system was developed to provide concentrated vapor phase only semi-volatile organic species for in-vivo exposure studies. The system consists of two units: particles (including their semi-volatile component) are first concentrated by means of the Versatile Aerosol Concentration Enrichment System (VACES), and subsequently drawn through a heating section, in which semi-volatile particle-bound components partition to the gas phase, while non-volatile particles are removed by a quartz filter placed after the heater. The vapors are then cooled to ambient temperatures, without producing nano-particles by nucleation, and can be readily used for exposure studies. Laboratory tests were carried out at various heater temperatures using ammonium sulfate, adipic acid and glutaric acid to investigate the occurrence of nucleation in the cooling section. Subsequently the system was tested in the field with concentrated particle and vapor samples taken upstream of the heater, immediately downstream of the filter, and after the cooling section. Chemical analysis of particle and vapor phases upstream and downstream of the system was conducted for selected polycyclic aromatic hydrocarbons (PAHs), and showed very good PAH recovery. These tests indicate that the modified VACES-heater-filter (VHF) system can provide concentrated PM (including their semi-volatile compounds) and PM-bound semi-volatile species purely in the vapor phase for inhalation exposure studies separately, a feature that makes this system an attractive approach for toxicity studies, particularly in light of the increasing interest in health effects of exposures to multi-pollutant atmospheres.

Ambient vapor-phase samples collected in Riverside, California had shown that both redox and electrophilic activity were present, with the vapor phase containing higher levels of electrophiles than the particle phase. In this study, the biochemical effects of the vapor-phase electrophiles were examined using the purified thiol proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), protein tyrosine phosphatase 1B (PTP1B) and KELCH-1 like ECH-associated protein 1 (Keap1). The results demonstrated that the thiol proteins were inactivated by the vapor-phase samples through covalent modifications. Next, two cellular responses, epidermal growth factor receptor (EGFR)/mitogen-activated protein (MAP) kinase and NF-E2-related factor 2 (Nrf2), to the ambient vapor-phase samples were assessed in A549 and RAW 264.7 cell lines, respectively. The vapor-phase samples, at non-oxidative concentrations, increased phosphorylation of EGFR, which is negatively regulated by PTP1B, and its downstream MAP kinase, extracellular signal-regulated kinase (ERK)1/2. Activation of Nrf2, which requires Keap1 alkylation, and expression of its downstream proteins were also observed. The electrophilic compounds present in ambient vapor-phase were shown to modify cellular proteins through covalent modification and to activate diverse cellular responses that can lead to inflammatory and adaptive responses.

Particulate matter (PM) has been the primary focus of studies aiming to understand the relationship between the chemical properties of ambient aerosols and adverse health effects. Size and chemical composition of PM have been linked to their oxidative capacity which has been postulated to promote or exacerbate pulmonary and cardiovascular diseases. But in the last few years, new studies have suggested that volatile and semi-volatile components may also contribute to many adverse health effects. The objectives of this study were: (i) assess for the first time the redox and electrophilic potential of vapor-phase components of ambient aerosols and (ii) evaluate the relative contributions of particle- and vapor-fractions to the hazard of a given aerosol. To achieve these objectives vapor- and particle-phase samples collected in Riverside (CA) were subjected to three chemical assays to determine their redox and electrophilic capacities. The results indicate that redox active components are mainly associated with the particle-phase, while electrophilic compounds are found primarily in the vapor-phase. Vapor-phase organic extracts were also capable of inducing the stress responding protein, heme-oxygenase-1 (HO-1), in RAW264.7 murine macrophages. These results demonstrate the importance of volatile components in the overall oxidative and electrophilic capacity of aerosols, and point out the need for inclusion of vapors in future health and risk assessment studies.

Numerous studies have suggested the association of reactive oxygen species (ROS) with adverse health effects derived from exposure to airborne particulate matter (PM) and diesel exhaust particles (DEP). This redox activity has been attributed to both inorganic and organic species present in these particles, but a clear distinction has not been established between the contribution of each. This article describes an application of an analytical procedure, based on the reaction of salicylic acid with hydroxyl radical to form dihydroxybenzoate (DHBA) isomers, to measure transition metal-based redox activity associated with ambient and diesel exhaust particles. In the procedure, ascorbic acid (AA) is used as electron source for reduction of metal ions and oxygen to generate superoxide, which is further reduced to hydroxyl radical in the presence of transition metal ions. Hydroxyl radical reacts with salicylate to generate DHBA isomers, which are measured by high-performance liquid chromatography (HPLC) with electrochemical detector. Both copper (Cu) and iron (Fe) ions generated DHBA isomers in a concentration-dependent manner but at different rates. The procedure was applied to DEP and ambient particles and the results showed Cu ion to be the major contributor to DHBA formation. The procedure provides a quantitative measure of transition metal-based redox activity associated with ambient samples with different physicochemical properties.

Quinones are reactive organic compounds known to initiate reactions associated with a host of toxicological events. Their presence in different atmospheres has been demonstrated although their sources remain uncertain. As a result of their reactivity and instability during chemical analysis, only a limited number of studies have reported on atmospheric concentrations of quinones in ambient air. Furthermore, besides the limited information on quinones associated with particulate matter, no previous studies have quantified vapor-phase quinones. We report vapor- and particle-phase concentrations of 1,2- and 1,4-naphthoquinones (1,2-NQ, 1,4-NQ), 9,10-phenanthraquinone (9,10-PQ), and 9,10-anthraquinone (9,10-AQ), measured over a 5-year period in Southern California. The results showed that vapor-phase concentrations of the target quinones were in general higher than those in the particle-phase. Vapor-phase concentrations ranged from 80 pg/m 3 for the AQ to 1747 pg/m 3 for the 1,4-NQ, and the particle-phase con...

A mobile exposure and air pollution measurement system was developed and used for on-freeway ultrafine particle health effects studies. A nine-passenger van was modified with a high-efficiency particulate air (HEPA) filtration system that can deliver filtered or unfiltered air to an exposure chamber inside the van. State-of-the-art instruments were used to measure concentration and size distribution of fine and ultrafine particles and the concentration of carbon monoxide (CO), black carbon (BC), particle-bound polycyclic aromatic hydrocarbons (PAHs), fine particulate matter (PM2.5) mass, and oxides of nitrogen (NOx) inside the exposure chamber. This paper presents the construction and technical details of the van and air pollutant concentrations collected in 32 2-hr runs on two major Los Angeles freeways, Interstate 405 (1-405; mostly gasoline traffic) and Interstate 710 (1-710; large proportion of heavy-duty diesel traffic). More than 97% of particles were removed when the flow through the filter box was switched from bypass mode to filter mode while the vehicle was driving on both freeways. The filtration system thus provides a great particulate matter exposure contrast while keeping gas-phase pollutant concentrations the same. Under bypass mode, average total particle number concentration observed inside the exposure chamber was around 8.4 x 10(4) and 1.3 x 10(5) particles cm(-3) on the I-405 and the I-710 freeways, respectively. Bimodal size distributions were consistent and similar for both freeways with the first mode around 16-20 nm and the second mode around 50-55 nm. BC and particle-bound PAH concentrations were more than two times greater on the I-710 than on the I-405 freeway. Very weak correlations were observed between total particle number concentrations and other vehicular pollutants on the freeways.

Ambient concentrations of 15 vapor-and particle-phase (PM 2.5 ) polycyclic aromatic hydrocarbons (PAHs), listed by the US EPA as priority pollutants, were measured between July 2002 and November 2003 in six Southern California communities participating in a multi-year chronic respiratory health study of schoolchildren. The communities were geographically distributed over two hundred kilometers, extending from Long Beach in coastal Los Angeles, to high mountain areas to the north and west of the Los Angeles basin, and south into Eastern San Diego County. Seasonal and spatial variation in the atmospheric concentrations of PAHs is of interest because this class of compounds includes potent mutagens, carcinogens, and species capable of generating reactive oxygen species (ROS) that may lead to oxidative stress. Naphthalene accounted for 95% of the total PAH mass; annual averages ranged from 89 to 142 ng m − 3 . Benzo[ghi]perylene (BGP) and the pro-carcinogen benzo[a]pyrene (BAP), present almost exclusively in ...

Worldwide people are exposed to toxic ultrafine particles (UFP, with diameters (dp) less than 100 nm) and nanoparticles (NP, dp50 nm) under a variety of circumstances. To date, very limited information is available on human exposure to freshly emitted UFP and NP while traveling on major roads and freeways. We report in-cabin and outdoor measurements of particle number concentration and size distributions while driving three vehicles on Los Angeles freeways. Particle number concentrations and size distributions were measured under different vehicle ventilation settings. When the circulation fan was set to on, with substantial external air intake, outside changes in particle counts caused corresponding in-cabin changes approximately 30-60 s later, indicating an maximal air exchange rate of about 120-60 h(-1). Maximum in-cabin protection (approximately 85%) was obtained when both fan and recirculation were on. In-cabin and outdoor particle size distributions in the 7.9-217 nm range were observed to be mostly bimodal, with the primary peak occurring at 10-30 nm and the secondary at 50-70 nm. The vehicle's manufacture-installed particle filter offered an in-cabin protection of about 50% for particles in the 7-40 nm size range and 20-30% for particles in the 40 to approximately 200 nm size range. For an hour daily commute exposure, the in-vehicle microenvironment contributes approximately 10-50% of people's daily exposure to UFP from traffic.

Coarse and fine particles were collected using an ambient particle concentrator (VACES system) in the north, center and south regions of Mexico City during May and November of 2003 with the aim of collecting enough particulate matter (PM) to examine their chemical and physical characteristics, biological content, and toxicity potential. The chemical, morphological and biological composition of PM was determined, together with the redox activity, induction of apoptosis and DNA damage. Carbonaceous species determined by thermal-optical transmittance (TOT) showed that the highest concentrations were found in PM2.5 from the north and in PM10 from the center. When analyzed by inductively coupling plasma (ICP), levels of metals were higher in the coarse fraction, mainly in the north. Morphological analysis by Scanning Electron Microscope & Energy Dispersive X-ray Spectrometer (SEM-EDX) is shown. Bacteria, fungi and endotoxin were present mostly in the coarse samples from the north. Fine PM had higher redox activity, than the coarse PM assessed by the dithiothreitol (DTT) assay. Early apoptotic cell death assessed by annexin V was observed in A549 cells exposed to PM from all regions, particularly with those collected in May. The fine fraction from the south induced higher apoptotic cell death compared to the coarse fraction, in contrast, the coarse fraction from the north induced significantly higher apoptosis than the fine fraction. All PM samples induced DNA damage assessed by the comet assay on THP-1 cells when exposed to a concentration of 10 μg/mL, the highest DNA damage was produced by both particle fractions collected in the north in May and November. In conclusion, PM from the north showed a higher metal and biological content, apoptotic cell death induction and more extensive DNA damage. Also, fine PM fractions from all sampled regions showed more redox activity than the coarse fraction. In summary, location, season and size of PM collection influenced their chemical, morphological and biological composition and thus their toxicity to cells.

The regional distribution of, and human exposure to, naphthalene are investigated for Southern California. A comprehensive approach is taken in which advanced models are linked for the first time to quantify population exposure to the emissions of naphthalene throughout Southern California. Naphthalene is the simplest and most abundant of the polycyclic aromatic hydrocarbons found in polluted urban environments, and has been detected in both outdoor and indoor air samples. Exposure to high concentrations of naphthalene may have adverse health effects, possibly causing cancer in humans. Among the significant emission sources are volatilization from naphthalene-containing products, petroleum refining, and combustion of fossil fuels and wood. Gasoline and diesel engine exhaust, with related vaporization from fuels, are found to contribute roughly half of the daily total naphthalene burden in Southern California. As part of this study, the emission inventory for naphthalene has been verified against new field measurements of the naphthalene-to-benzene ratio in a busy traffic tunnel in Los Angeles, supporting the modeling work carried out here. The Surface Meteorology and Ozone Generation (SMOG) airshed model is used to compute the spatial and temporal distributions of naphthalene and its photooxidation products in Southern California. The present simulations reveal a high degree of spatial variability in the concentrations of naphthalene-related species, with large diurnal and seasonal variations as well. Peak naphthalene concentrations are estimated to occur in the early morning hours in the winter season. The naphthalene concentration estimates obtained from the SMOG model are employed in the Regional Human Exposure (REHEX) model to calculate population exposure statistics. Results show average hourly naphthalene exposures in Southern California under summer and winter conditions of 270 and 430 ng m −3 , respectively. Exposure to significantly higher concentrations may occur for individuals close to local sources, or in naphthalene “hotspots” revealed by simulations and observations. Such levels of naphthalene exposure may be used to gauge the potential health impacts of long-term naphthalene exposure. Results are also given for the distributions of 1,4-naphthoquinone, a naphthalene reaction product that may have significant health effects.

Quinones are reactive organic compounds and are known to initiate reactions associated with many toxicological events. Their presence in air pollution has been demonstrated, but routine quantitative measurements are lacking. A quantitative method for the determination of four quinones was developed using diesel exhaust particles (DEP) and National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 1649a. The method was then used to analyze ambient air samples from different sites in Southern California. After extraction in dichloromethane, the target compounds were converted to their stable diacetyl derivatives and determined by electron impact GC-MS using selected ion monitoring. Calibration plots were obtained with deuterium-labeled internal standards. The four quinones, 1,2-naphthoquinone (1,2-NQ), 1,4-naphthoquinone (1,4-NQ), 9,10-phenanthraquinone (9,10-PQ), and 9,10-anthraquinone (9,10-AQ), were quantified in DEP, in SRM 1649a, and in ambient air samples of PM2.5 collecte...

As part of the Southern California Particle Center and Supersite (SCPCS) activities, we measured, during all seasons, particle size distributions of 12 priority pollutant polycyclic aromatic hydrocarbons (PAHs), concurrently with elemental carbon (EC), organic carbon (OC), sulfate (SO 4 2− ), and nitrate (NO 3 − ) size distributions, from October 2001 to July 2002 in Claremont, CA, a receptor site located about 40 km downwind of central Los Angeles. Samples were collected approximately once every week, for 24-h periods, from midnight to midnight. MOUDI impactors collected samples at 30 LPM which were composited for analysis into monthly periods in three aerodynamic diameter size intervals, defined for the purpose of this work, as: 0–0.18 μm (ultrafine mode), 0.18–2.5 μm (accumulation mode), and 2.5–10 μm (coarse mode). For the monthly composites from October to February, the size distributions of the target PAHs are similar. However, from March to July, notable differences are observed: a significant fraction of the PAH mass is found in the coarse mode, as compared with the previous period. During the entire 1-year period, the form and shape of the EC size distributions did not vary much and are distinguished by prominent mass in the ultrafine and accumulation size mode. For the individual modes of the major species, the highest Pearson's correlation coefficients for the variation of temperature with species concentration were found in the ultrafine mode for both SO 4 2− (0.92) and EC (0.90), and in the coarse mode for both OC (0.85) and NO 3 − (0.54). High SO 4 2− correlations are consistent with increased gas-to-particle formation during the warmer months from (precursor) SO 2 emissions in the Los Angeles and Lon Beach seaport areas and, similarly for EC, increased atmospheric transport to Claremont as the season progresses from winter to summer. Although not statistically significant, the correlations were negative for the less volatile or particle phase group (log [ p L o ]⩽−3.22), consistent with increased partitioning from the vapor phase with decreasing temperature.

Fifteen priority polycyclic aromatic hydrocarbons (PAHs) were measured in two rural communities (Atascadero and Lompoc) located several hundred km northwest of Los Angeles and in four urban communities 40–100 km downwind of Los Angeles (San Dimas, Upland, Mira Loma, and Riverside), during all seasons, from May 2001 to July 2002. PM2.5 and vapor-phase PAHs were collected, on prebaked quartz fiber filters and PUF-XAD-4 resin, respectively, at 113 LPM, during 24 h periods, every eighth day, and quantified by HPLC-Fluorescence. At all sites vapor-phase PAHs contained > 99.9% of the total PAH mass and were dominated by naphthalene (NAP), which varied from about 60 ng m − 3 in Lompoc, a community with light traffic, to ∼580 ng m − 3 in Riverside, a community traversed by ∼200,000 vehicles day− 1. During summer pollution episodes in urban sites, NAP concentrations reached 7–30 times annual averages. Except for summer episodes, concentrations of low MW PAHs showed small seasonal variations (∼2 times higher in win...

The concentrations of 15 "priority pollutant" semivolatile and particulate polycyclic aromatic hydrocarbons (PAHs) were determined in three sets of samples supplied by the National Institute for Standards and Technology (NIST) as part of an interlaboratory analytical exercise. The purpose of the exercise, organized by NIST and the U.S. Environmental Protection Agency, was to determine the comparability of measurements for various organic analytes among the participating laboratories, and to establish consensus values for SRM 1649a and interim materials. The commercially available SRM 1649a Atmospheric Urban Dust and two subsamples of this popular reference material were analyzed: an extract designated as Air Particulate Extract, and a resieved portion labeled Air Particulate I. The method used in our laboratory for the exercise consists of the extraction of the PAHs from the solid samples by ultrasonication, followed by separation and quantification using high-performance liquid chromatographyfluorescence...

A versatile and compact sampling system, the Sequential Spot Sampler (S3) has been developed for pre-concentrated, time-resolved, dry collection of fine and ultrafine particles. Using a temperature-moderated laminar flow water condensation method, ambient particles as small as 6 nm are deposited within a dry, 1-mm diameter spot. Sequential samples are collected on a multiwell plate. Chemical analyses are laboratory-based, but automated. The sample preparation, extraction and chemical analysis steps are all handled through a commercially-available, needle-based autosampler coupled to a liquid chromatography system. This automation is enabled by the small deposition area of the collection. The entire sample is extracted into 50–100μl volume of solvent, providing quantifiable samples with small collected air volumes. A pair of S3 units was deployed in Stockton (CA) from November 2011 to February 2012. PM2.5 samples were collected every 12 hrs, and analyzed for polycyclic aromatic hydrocarbons (PAHs). In parallel, conventional filter samples were collected for 48 hrs and used to assess the new system’s performance. An automated sample preparation and extraction was developed for samples collected using the S3. Collocated data from the two sequential spot samplers were highly correlated for all measured compounds, with a regression slope of 1.1 and r2=0.9 for all measured concentrations. S3/filter ratios for the mean concentration of each individual PAH vary between 0.82 and 1.33, with the larger variability observed for the semivolatile components. Ratio for total PAH concentrations was 1.08. Total PAH concentrations showed similar temporal trend as ambient PM2.5 concentrations. Source apportionment analysis estimated a significant contribution of biomass burning to ambient PAH concentrations during winter.

We apportioned the elemental carbon (EC) component of ambient PM2.5 attributable to emissions from spark ignition (SI) vehicles in samples collected over a three-year period in twelve Southern California communities, including coastal, rural, and urban areas using the chemical mass balance model (CMB8) modified for polycyclic aromatic hydrocarbon (PAH) reactivity. Selected PAH/EC ratios, measured in samples collected in the Caldecott tunnel were evaluated for use as fingerprints. PAH reactivity which occurs during atmospheric transport and affects the source contribution estimates during the summer/fall/spring months was accounted for using experimentally measured decay constants. Results showed that benzo[ghi]perylene and indeno[1,2,3-cd]pyrene can be used successfully as specific tracers of EC contributions from SI vehicles. The average EC portion of PM2.5 attributed by the model to SI emissions at these communities was 39, 58 and 62%, respectively, during the summer, spring/autumn, and winter. For all seasons, coastal community contributions represent about twice those found in the rural and urban inland communities, before December 2003 when MTBE was still in use in California. Repartimos a parcela do componente de carbono elementar (CE) em PM2,5 atmosférico atribuível à emissões de veículos com ignição por faísca (SI) em amostras coletadas durante dois anos em doze comunidades no Sul da Califórnia, incluindo zonas costeiras, rurais e urbanas usando o Modelo de Balanço de Massa Química (CMB8) modificado para levar em conta a reatividade dos hidrocarbonetos policíclicos aromáticos (HPAs). Foram avaliadas as razões HPA/CE em amostras coletadas no túnel Caldecott para utilização como assinaturas das fontes. A reatividade dos HPAs que ocorre durante o transporte de aerossóis atmosféricos que pode afetar as estimativas de contribuição das fontes (ECF) durante o verão/primavera/outono foi considerada com o uso de constantes de decaimento medidas experimentalmente. Nossos resultados mostram que o benzo[ghi]perylene e o indeno[1,2,3-cd]pireno podem ser utilizados com sucesso como marcadores específicos de CE nas emissões por veículos com SI. A estimativa média da porção de CE atribuído pelo modelo à emissões provenientes de SI nessas comunidades foi de 39, 58 e 62%, respectivamente, durante o verão, primavera/outono, e inverno. Para todas as comunidades costeiras, as atribuições a veículos por SI representam cerca do dobro das estimativas para as áreas rurais e urbanas, antes de dezembro 2003 quando MTBE ainda era usado na California.

Carbon nanotubes are difficult to aerosolize in a controlled manner. We present a method for generating aerosols not only of carbon nanotubes, but also of many reference and proprietary materials including quantum dots, diesel particulate matter, urban dust, and their mixtures, using electrospraying. This method can be used as a teaching tool, or as the starting point for advanced research, or to deliver nanomaterials in animal exposure studies. This electrospray system generates 180 microg of nanotubes per m(3) of carrier gas, and thus aerosolizes an occupationally relevant mass concentration of nanotubes. The efficiency achievable for single-walled carbon nanotubes is 9.4%. This system is simple and quick to construct using ordinary lab techniques and affordable materials. Since it is easy to replace soiled parts with clean ones, experiments on different types of nanomaterial can be performed back to back without contamination from previous experiments. In this paper, the design, fabrication, operation and characterization of our versatile electrospray method are presented. Also, the morphological changes that carbon nanotubes undergo as they make the transition from dry powders to aerosol particles are presented.