2008 Progress Report: Characterization and Source Apportionment
EPA Grant Number:
Subproject: this is subproject number 001 , established and managed by the Center Director under
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Rochester PM Center
Characterization and Source Apportionment
Hopke, Philip K.
, Prather, Kimberly A.
Hopke, Philip K.
Prather, Kimberly A.
University of California - San Diego
University of Rochester
EPA Project Officer:
October 1, 2005 through
September 30, 2010
(Extended to September 30, 2012)
Project Period Covered by this Report:
October 1, 2007 through September 30, 2008
Particulate Matter Research Centers (2004)
Core 1: Exposure Assessment and PM Source Identification
A central hypothetical mechanism of how particles affect human health involves the generation of reactive oxygen species (ROS) at target sites in the lung. ROS has been defined to include families of oxygen-centered or related free radicals, ions, and molecules. The free radical family includes hydroxyl, hydroperoxyl, and organic peroxy radicals. Ions such as the superoxide, hypochlorite, and peroxynitrite ions, and molecules such as hydrogen peroxide, organic and inorganic peroxides also come under the umbrella of ‘Reactive Oxygen Species'. Much of the attention has focused on the formation of ROS in situ after particle deposition in the respiratory tract generally through the interaction with transition metal ions (Stohs et al. 1997), organic hydrocarbons, such as polycyclic aromatic hydrocarbons and quinones (Squadrito et al. 2001), and ultrafine particle surfaces (Li et al. 2003). However, recent work has shown that ROS is present in the atmosphere on respirable particles to which we are exposed (Hung and Wang 2001, Hasson and Paulson 2003, Venkatachari et al. 2005, 2007). The hypothesis that the ROS present on particles could cause the same kind of systemic dysfunction as endogenously generated ROS has clear merit and represents a fundamental issue for further investigation. Thus, a major function of this core is the study of the concentrations of particle-bound ROS in ambient PM2.5 and studies to better understand their formation so that we can eventually estimate the amounts of particle bound ROS arising from anthropogenic and biogenic sources of the reactive hydrocarbon precursor species.
Development of a Field-Deployable ROS Monitor
We have previously developed a laboratory version of a continuous monitor for particle-bound reactive oxygen species (ROS) (Venkatachari and Hopke, 2008a). This work demonstrated that it is possible to automate the use of dichlorofluorescin (DCFH) as a nonspecific indicator of the oxidative capacity of particle surfaces. To move this system into the field to permit routine monitoring of particle-bound ROS, there are a number of questions to be answered regarding reagent stability and system optimization.
We have conducted a series of experiments to answer these questions. From them, we have concluded that 2 μM DCFH shows a linear response only in 10-7 M H2O2 concentration range, but not in 10-6 M H2O2 concentration range. When we increase DCFH concentration to 5 μM, we extend linear range to 1 x 10-6 M H2O2. With 10 μM DCFH, this range can be extended to 1.5 x 10-6 M H2O2, with 20 μM DCFH to 2 x 10-6 M H2O2 and with 40 μM DCFH we extended that range to 2.5 x 10-6 M H2O2 concentration. We decided to use 5 μM DCFH working solution for future measurement because we do not expect higher ROS ambient concentration than 1 x 10-6 M H2O2.
HRP is catalyst in our reaction, but it can also oxidize DCFH in the absence of any other ROS species and increase fluorescence intensity measured at 535 nm. Thus, we have explored lowering the HRP concentration and make reaction more dependent on the ROS species present in solution. There were no significant differences in results with a range of concentrations so we have decided to use 0.5 units HRP/ml.
In order to test time stability of new working solution (mixture of 5 μM DCFH and 0.5 units/ml HRP), a series of 5 day measurements were conducted. It could be concluded that the solution is stable for at least 5 days (stored in refrigerator). The same working solution (5 μM DCFH) was tested for temperature stability and stored at room temperature for three days. The conclusion from there results is that the 5 μM DCFH working solution is stable for at least three days at room temperature.
The time and temperature stability of H2O2 calibration solutions (10-7M concentration range) were also tested to ensure that those standards could be used during the field measurements when it is not always possible to store them at low temperatures (refrigerator) or prepare them daily. Results for eight days measurements showed that H2O2 calibration solutions are stable for at least one week at room temperature.
Incubation at 37°C using a water bath was one step in ROS continuous monitor system (Venkatachari and Hopke, 2008a). The residence time at this temperature was ~30 sec. Using water bath in field measurements is not always practical because of slow but continuous water evaporation from bath. Thus, we investigated the importance of the incubation step. Two sets of experiments were performed with the same DCFH working solutions: one with incubation and second without the increase in temperature to 37°C. It was found that there was little difference between the two responses. It can be concluded that incubation at increased temperature is not an important step in the ROS continuous monitor system. There are some remaining questions regarding the lower temperature. Considering the standard solution of H2O2 uses a strong oxidant such that the reaction is fast even without incubation. The question is whether this behavior holds for other less potent, but still important ROS species present on the ambient particles. Further studies are in progress.
The result of all of these experiments has led to changes in the ROS continuous system. In the new setup, we have removed the membrane reactor for HRP introduction to system because now we use a premixed solution of DCFH with HRP. The new setup is shown on Figure 1.
Further testing of the system is in progress with the expectation that it will be deployed at the NYS DEC site in Rochester some time in the early fall where it will operate for at least one year. The initial plans were to operate it for a month in each quarter, but if the improvements we have made work as they appear to, it may be possible to operate the system continuously over the entire year.
Figure 1. Scheme of the new ROS continuous monitor system
Chemical Characterization of Particle-Bound ROS
To understand the mechanisms of particle-bound ROS formation, identify its likely sources, and determine the chemical pathways that might be influenced by air quality management strategies, we have pursued the chemical characterization of the constituents in particle-bound ROS. Initially, we used the ROS particle generator described by Venkatachari and Hopke (2008b) to provide samples. These samples were collected on Teflon filters, water leached, and the resulting leachate analyzed by liquid chromatography/mass spectrometry using sequential MS analysis. Using the α-pinene/ozone reaction to produce ROS, several new peroxide species have been separated and identified (Venkatachari and Hopke, 2008c).
These species were those that were sufficiently long lived to permit their collection over extended time periods and then be stable enough for the subsequent LC/MSn analyses. However, more reactive species are not likely to be found in this manner and we have returned to our studies of spin-trap compounds that can react with radicals to stabilize them sufficiently to permit the separation and identification of the resulting adducts. Spin traps have been primarily employed to trap endogenous radical species in biological systems. We are now looking to employ them to stabilize and permit isolation and quantification of reactive radical species associated with airborne particulate matter.
One of the more common spin-trap compounds is 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) that has been widely used for hydroxyl and organic peroxyl radicals. In our studies, particulate matter samples were collected from our 2.4 m3 aerosol chamber in which the α-pinene – ozone reaction was used to produce ROS-containing particles. Samples were collected at 23 LPM during 2 hours on a precleaned, 25 mm quartz fiber filters (PALL, USA) and evenly impregnated with 0.18 mmol of DMPO solution in methanol (Sigma Aldrich, USA).
Samples collected from the aerosol reactor were analyzed first in absence of DMPO. In the MS spectra, monomer compounds with MW up to 250 and dimeric compounds with MW from 300 to 450 could be observed. The MS/MS results for parent ions of m/z 357 and 399 confirmed that the dimers originated from low molecular weight species with m/z 171 and 185. In order to better understand the formation of dimers and higher MW products, we used DMPO to capture reactive species before they can terminate reactions.
Two groups of polymeric species with base monomers m/z 256 and 314 and constant difference among the species of m/z 44 were observed in the DMPO samples, but were not observed in the absence of DMPO. The observed presence of ions with m/z 114, 130 and 146 in the spectra can be attributed to radical adducts with DMPO. An ion with m/z 114 [DMPO+H]+ corresponds to a fragment ion formed from DMPO carbon-centered radical adducts. The ions with m/z 130 [DMPO-O+H]+ and m/z 146 [DMPO-OO+H]+ correspond to fragment ions formed from DMPO oxygen-centered radical adducts (alkoxyl and peroxyl). Other common fragmentations observed in all spectra were the loss of water molecule (-18) and CO2 (-44) group.
These results require more work to explicate the mechanisms of oligomeric compound formation and the role of the spin trap agent in the process. Further work will be done using 5-diethoxy phosphoryl-5-methyl-pyrroline-N-oxide (DEPMPO). DEPMPO is an improvement on DMPO as a spin trap. The spin adduct that it forms is more stable than DMPO, and it is detectable at lower concentrations. Thus, further studies will be made to elucidate the nature of the most reactive particle-bound ROS species.
In summary, prior work has identified the presence of particle-bound reactive oxygen species (ROS) in ambient PM. These initial measurements were made with a manual sampling and analysis system. We have now developed an automated system that would provide continuous measurements of particle-bound ROS. The laboratory system is now being engineered to be a field monitor that will be deployed in Rochester in the fall of 2008. In addition work has continued on the characterization of the important ROS species. Several new peroxide species have been identified in the PM produced by the reaction of α-pinene with ozone. Further studies of more reactive by-products will be studied using spin trapping compounds so that a better understanding of the nature of the particle-bound ROS will be obtained.
Collection of source specific samples of urban aerosol
Research on the characterization of urban ultrafine and accumulation mode particles allows composition and size analysis in real time of single particles using aerosol time-offlight mass spectrometry (ATOFMS). The objective of the research is to greatly expand the understanding of the chemical composition and impact of specific sources of ultrafine particles on human health. New sampling methods coupling ATOFMS with high volume cascade impactors are being developed such that samples will be collected that represent material primarily from specific sources. These samples will permit improved characterization of the PM from specific sources as well as providing material for in vitro and in vivo testing of their toxicity. Also, a new method for collecting size-resolved samples of ultrafine PM from ambient air directly into an aqueous solution has been developed. This method gets around issues required with sample collection on filters, where PM species have been determined to be difficult to extract. These aqueous solutions can be used for in vitro studies and allow 100% of the ultrafine particles to be deposited on cells. Furthermore these are concentrated solutions with small volumes so low concentrations can be used in studies.
Filter samples of ambient ship emissions and wildfires PM were collected by the Prather group and sent to Rochester for in vitro studies. However it was determined that PM could not be effectively extracted from the filters. A sample recovery rate of less than 25% was encountered thus in vitro studies were not performed. Thus it was decided a better method is needed to collect samples for in vitro studies. The Prather group began exploring options and stopped collecting filter samples until this issue was resolved.
Characterization of CAPs
The ATOFMS was moved from the Hopke lab to Rochester to be used to measure the chemistry of ambient PM during exposure studies. A graduate student from the Prather group, Melanie Zauscher, traveled to Rochester to assist in getting the instrument operational.
However, there were problems encountered in measuring particles at the smallest sizes (i.e. <200 nm). A size mismatch existed between the lowest size the ATOFMS could analyze chemically and the sizes of the concentrated particles being used for exposure studies. The next step involves improving the ability of the ATOFMS at Rochester to detect particles at the smaller sizes.
To overcome both issues above, the Prather group worked with Susanne Hering to develop a method for growing small ultrafine particles to larger sizes using a condensational growth tube developed in the Hering lab. This tube is quite compact and can be placed in line with the ATOFMS to grow up size selected particles into larger size so they can be optically detected with the ATOFMS. This approach was recently implemented and tested at UCSD and works very well. It allows the detection of particles as small as 10 nm in the mass spectrometer. This will be implemented in the Clarkson ATOFMS at Rochester in the coming year.
The condensational growth tube also allows one to collect ultrafine particles with close to 100% efficiency in an impinger system. Small spots (300 microns) can also be collected and deposited directly on cells for in vitro studies. This method avoids the filter collection and extraction and allows direct deposition of 100% of the sample to cells. We tested whether the cells remained viable after being exposed to ambient air with these larger particles for different periods of time. The goal of these studies was to be sure the sampling procedure was not lysing the cells. It was shown that no detectable number of cells were destroyed by this sampling procedure. The next steps will involve collecting size resolved source samples for Rochester to use in in vitro studies and helping them set up this sampling method in their laboratories.
on this Report
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|| Venkatachari P, Hopke PK. Development and laboratory testing of an automated monitor for the measurement of atmospheric particle-bound reactive oxygen species (ROS). Aerosol Science and Technology 2008;42(8):629-635.
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|| Yue W, Stolzel M, Cyrys J, Pitz M, Heinrich J, Kreyling WG, Wichmann H-E, Peters A, Wang S, Hopke PK. Source apportionment of ambient fine particle size distribution using positive matrix factorization in Erfurt, Germany. Science of the Total Environment 2008;398(1-3):133-144.
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RFA, Scientific Discipline, Air, particulate matter, Environmental Chemistry, Health Risk Assessment, Biochemistry, cardiopulmonary responses, chemical characteristics, fine particles, atmospheric particles, airway epithelial cells, airborne particulate matter, human exposure, aerosol composition
Progress and Final Reports:
2006 Progress Report
2007 Progress Report
2009 Progress Report
2010 Progress Report
2011 Progress Report
Main Center Abstract and Reports:
Rochester PM Center
Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R832415C001 Characterization and Source Apportionment
R832415C002 Epidemiological Studies on Extra Pulmonary Effects of Fresh and Aged Urban Aerosols from Different Sources
R832415C003 Human Clinical Studies of Concentrated Ambient Ultrafine and Fine Particles
R832415C004 Animal models: Cardiovascular Disease, CNS Injury and Ultrafine Particle Biokinetics
R832415C005 Ultrafine Particle Cell Interactions In Vitro: Molecular Mechanisms Leading To Altered Gene Expression in Relation to Particle Composition