WORKSHOP REPORT
Options for Characterizing Organic Particulate Matter New research strategies could help elucidate the mechanisms and causes of aerosol health effects. BARBARA J. T U R P I N cientists and engineers gathered this April at die Electric Power Research Institute (EPRI) in Palo Alto, Calif., to participate in a workshop aimed at developing effective strategies for organic aerosol characterization for use in health and particulate matter (PM) studies. They also sought to identify critical research needed to improve the scope and accuracy of organic characterization over the next five-year period and advised EPRI managers, who are directing a large, 18-month Aerosol Research Inhalation Epidemiology Study (ARIES). The EPRI field study, being carried out in the Atlanta, Ga., area will provide daily data for an epidemiological investigation of me association between heakh endpoints and air quality. ARIES is leveraging work already planned as part of the 3-year PM2 5 Southeastern Aerosol Research and Characterization program, which will investigate PM2 5 compliance and source apportionment issues in the southeastern United States. The primary challenge for workshop participants was to devise strategies for characterization of carbonaceous species in the atmosphere. The selected strategies should provide a comprehensive description of the character of carbonaceous species in no more tiian 6-10 variables, a limiting amount for practical use in epidemiological studies. Although epidemiological studies link atmospheric PM with excess morbidity and mortality, little is known about the composition of organic PM found in the atmosphere, and little effort has been made to study the health effects produced by this complex fraction of atmospheric PM. Solubility in lung fluid, reactivity or potential for free radical formation, concentration of endotoxins, and effect on particle hygroscopicity are properties of organic PM that are considered important in seeking to understand human health impacts. The lack of knowledge regarding causal agents of PM-induced injury
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is hampering development of effective air pollution control strategies. An understanding of the critical chemical and physical properties responsible for the biological activity of PM must be obtained to ensure adequate public health protection. However, the complexity of the organic fraction of atmospheric PM presents a substantial challenge to efforts to characterize biological activity. Traditional toxicological studies using laboratorygenerated aerosols containing known PM constituents have been unable to identify a plausible mechanism or the causal agents responsible for observed effects. Some recent work with concentrated atmospheric aerosols, however, has begun to yield clues. For example, using a Harvard particle concentrator, Godleski and coworkers (1) have observed alterations in canine electrocardiograms that suggest potentially fatal rhythm disorders following inhalation exposure to concentrated atmospheric PM The magnitude of these effects varies considerably from day to dav presumably as a result of changes in particle composition or other particle properties Against this background of unresolved health effects issues and novel research initiatives, the workshop participants ultimately recommended two types of measurement strategies. One approach is to measure the entire organic (gas and particle) mass after categorization of collected material into several sample fractions based on compound class. A second suggested approach uses source tracers or source contributions to describe the aerosol. Associations between adverse health endpoints and pollutant source types would then be examined. Because of recent toxicological interest, workshop participants also recommended analysis of samples of lipopolysaccharides an endotoxin found in the outer membranes of gram-negative bacteria At high expodiese toxins can illnesses such as diarrhea © 1999 American Chemical Society
and hemorragic shock; they are responsible for many of the virulent effects of gram-negative bacteria.
NARSTO (North American Research Strategy for Tropospheric Ozone) ozone transport sites (2). Interest in oxygenated VOCs is growing because of their value in studies of secondary organic aerosol formation and the Scope of current efforts A majority of the organic mass in atmospheric par- environmental fate and effects of organic chemicals on ticles has not been chemically characterized or sub- human and ecosystem health. It should be noted that jected to controlled inhalation studies. To over- accurate analysis of VOCs requires particular care in come this difficulty, inhalation studies are being sample collection and handling because of the reacconducted with atmospheric PM that has been con- tivity of some gassphase organics. centrated 30-100 times through inertial or centrifParticle-phase organic compounds, which acugal separation schemes. The main objectives of these count for 20-60% of the dry PM2 5 mass are not well studies have been to evaluate the plausibility of ep- characterized—only about 10-20% of the organic idemiological findings and to develop insights into PM2 5 mass has been identified on a molecular level the mechanisms of PM-induced injury. (3) (see box below (4)). Moreover, the characterizaThree approaches are now applied in seeking to tion of water-soluble organic PM constituents is much more limited than that of water-insoluble oridentify biologically active PM constituents. • In current toxicological studies, laboratory- ganic PM constituents, because highly polar comgenerated aerosols of known composition and size are pounds do not elute through standard gas chroused to test specific hypotheses regarding causal agents. matographic columns. • Using concentrated atmospheric PM, researchOrganic PM is emitted in particulate form (primaers look for statistical associations during inhala- ry particulate matter) and formed in the atmosphere tion studies between measured PM properties and from products of gas-phase photochemical reactions observed effects in animal models. (secondary particulate matter). Both biogenic and an• In intensive field campaigns, PM species are thropogenic sources contribute to primary and secmeasured for use in "next-generation" epidemiolog- ondary organic particulate matter. Inclusive of contributions from primary sources, as well as precursors and ical analyses. The first approach relies on knowledge about the products of photochemical reactions, compounds such as alkanes, alkenes, aromatic and cyclic hydrocarcomposition of atmospheric PM2 5. Such knowledge, however, is severely limited for the organic frac- bons, polycyclic aromatic hydrocarbons, aldehydes, altion, a situation that might limit any hypotheses that cohols, ketones, esters, carboxylic acids, sulfur- and niare developed and tested. Both other approaches use trogen-containing organics, halogens, terpenoids, and statistical associations between measured aerosol pa- peroxides are found in atmospheric samples. Organrameters and biological indicators or health end- ics of biological origin, such as pollen, spores, and mipoints in animal models or humans. Consequently, crobial products (including endotoxins) also contribthese approaches will require characterization of the ute to atmospheric PM (5). Many of these organic entire organic mass with enough chemical detail to compounds—polycyclic aromatic hydrocarbons carenable identification of biologically active organic boxylic acids and organic peroxides for example constituents should they exist. are found in both gas and particle phases Their phase Carbonaceous compounds constitute a substan- distributions are a function of atmospheric propertial portion of the PM2 5 mass. The gas- and particle- ties such as temperature and relative humidity sorpphase organics include a multitude of compounds from tive,properties of the PM to which they partition and a variety of sources, presumably with differing bioac- properties of the organic compounds themselves tivities. ARIES measurements wiil lnclude S02, CO, NOx, such as vapor pressure and solubility 0 3 , HNO3, NH3, and VOCs in the gas phase; major ions, elements, water-soluble metals, and carbon in the particle phase; pollen and mold; and particle number and size distributions. In support of this undertaking, workParticle-bound organics shop participants considered practical strategies for furSeveral classes of organic compounds have been measured in at ther characterization of carbonaceous species in the atmospheric particles or are predicted to be present in atmospheric mosphere to provide comprehensive chemically useful particles based on thermodynamic arguments and measurements data in few enough variables (6-10) that the data could other media. be used in epidemiological analyses. In developing possible strategies the workshop participants considn-alkanes aliphatic (di)carboxylic acids ered organic speciation knowledge and current health n-alkanoic acids aromatic (di)carboxylic acids hypotheses. Organic speciation knowledge A substantial portion of the atmospheric organic mass is found in the gas phase. Although volatile organic compound (VOC) analysis has largely focused on hydrocarbons and their role as ozone precursors, oxygenated compounds compose a significant fraction of VOC mass. For example, oxygenated compounds are estimated to account for 30-50% of the VOC mass at
aldehydes esters diterpenoid acids aromatic polycarboxylic acids polycyclic aromatic hydrocarbons polycyclic aromatic ketones polycyclic aromatic quinones
glyoxal organic peroxides ketoacids polyols hydroxyamines amino acids nitrophenol
Source: References 3, 4.
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Variables for use in epidemiological studies Workshop participants outlined four possible schemes for developing variables that could be used in performing epidemiological studies. Workshop participants strongly recommended that substantial sample mass should be retained for later analysis, as needed. Level 1 • Gas: Determine aliphatics, aromatics, oxygenates by gas chromatography-flame ionization detection (GC-FID). • Particle: Measure organic and elemental carbon by thermal-optical analysis. Level 2 • Gas: Determine aliphatics, aromatics, diacids, and oxygenates by GC-FID. • Particle: Analyze nonpolar, low-polar, polar, highly polar fractions by solvent extraction in hexane, dichloromethane, acetone, and water, followed by carbon analysis of dried extracts or by liquid chromatographic separation and gravimetric analysis. Level 3 • Gas: Determine aliphatics, aromatics, diacids, and oxygenates by GC-FID. • Particle: Perform nondestructive analyses for functional groups and free radicals by Fourier transform infrared spectroscopy, Raman spectroscopy, 3-D fluorescence, X-ray absorption fine structure spectroscopy, nuclear magnetic resonance spectroscopy, and electron paramagnetic resonance spectroscopy. Level 4 • Source tracers or source contributions measurements: Metals, sulfate, nitrate, organic and elemental carbon, and frequently molecular-level organic measurements are used to determine the contributions of major source types that would then serve as epidemiological variables. Additional desirable measurements • Endotoxins, lung fluid-soluble and -insoluble organic carbon should be measured, as well as the reactivity of various organics.
The presence of multiphase (semivolatile) organic compounds complicates the collection of organic particulate matter because the equilibrium between the gas and particle phases is disturbed during sample collection. Sampling artifacts can result in sizable measurement errors for total particulate organic carbon and individual organic species (6). Thus, sampling methods are needed that measure both the gas and particle phases of multiphase organics. Theories of cause and effect Several active hypotheses seek to explain the toxicity of atmospheric particulate matter. One theory suggests that PM toxicity results from the accumulation of solid matter in the epithelial lining of the lungs and that effects are primarily associated with a certain particle size fraction (ultrafine particles or the ultrafine cores of mostiy soluble fine particles). An alternative theory is that certain chemical constituents are responsible—transition metals might, for example, participate in oxidation-reduction reactions in vivo, inducing injury to the lungs. Anotiier hypothesis assumes that injury results from the ac7 8 A • FEB. 1, 1999 /ENVIRONMENTAL SCIENCE & TECHNOLOGY / NEWS
tivation of lung macrophages by water-soluble oxidants (hydrogen peroxide and organic peroxides) that are transported into the lower lung through association with atmospheric particles. Lipopolysaccharaides—immunoreactive surface antigens of bacteria— have also been suggested as causal agents. Once particles are deposited in the lung, the host response will depend on the solubility of the PM constituents in lung fluid. Insoluble materials are typically internalized by phagocytic cells (alveolar macrophages), which attempt to destroy die material by producing cytotoxic mediators (enzymes and reactive oxygen species). Allhough macrophages are the primary line of defense against respiratory insult, alterations in tiieir activity can also lead to increased host susceptibility to infection, pulmonary injury, and inflammation (7). Lung fluid-soluble PM constituents could alter the lung fluid composition and might be absorbed by tissues or enter the lymphatic or circulatory system directly. Therefore, the solubility of particle-phase material in lung fluid large influence on the mechanism of action Because the organic compounds present in atmospheric particles are largely unknown, the workshop participants concluded that it would be unwise to limit die scope of organic analysis to that suggested by current health hypotheses, witiiout first conducting toxicological studies on realistic organic mixtures. Strategies for organic characterization Effective strategies for organic characterization in support of PM and health studies were defined in the workshop. One suggested approach involves die measurement of the entire organic gas and particle mass after categorization into 4-10 fractions by polarity or compound class and appropriate storage of samples (frozen) for further analysis. If a positive association is observed between an organic fraction and an adverse healtii endpoint, chemical analyses ranging from carbon, hydrogen, oxygen, nitrogen analysis to molecular-level characterization and possibly bioassays could then be conducted on this fraction. The second approach involves the use of source tracers or source contributions to describe the aerosol, followed by an analysis of associations between adverse health endpoints and source types. Three additional types of measurements were recommended by workshop participants. These included analysis of the concentration of endotoxins, mass fraction of lung fluid-soluble and lung fluidinsoluble organic carbon, and a measure of the "reactivity potential" of the organic aerosol. The interest in these types of measurements stems from the current mechanistic hypotheses and questions concerning the role of endotoxins. A limulus amoebocyte lysate endotoxin assay is commercially available as a kit through several environmental laboratories (8). However, workshop participants noted that several common constituents of atmospheric particles, most notably calcium, provide a false positive response in die endotoxin assay. An endotoxin-neutralizing protein, Limulus polyphemus, binds to endotoxin and can be added to identify the signal from otiier PM constituents, en-
abling the true endotoxin concentration to be determined by difference. An alternative approach is to measure specific structural components as biomarkers of die endotoxin (such as 3-hydroxy fatty acid and 2-keto-3-deoxyoctonate). Mass fractions of lung fluid-soluble and -insoluble organic carbon have apparently not been measured in atmospheric aerosols. However, given the complexity of atmospheric organic PM, separate measurements of organic compounds that will be present in the lung as solid matter and those that will become incorporated into the lung fluid(s), tissues and/or circulatory fluids are likely to be useful. Two fluids should be considered: Mucus (95% water, 2-3% glycoproteins, and 2% lipids and minerals) lines the upper airways and surfactant—comprising highly surface-active dipalmitoyl phosphatidycholine—coats the alveoli. A measure of the redox potential or free radical production capacity of the organic compounds could also provide information relevant to current health hypotheses. One candidate technique is electron paramagnetic resonance spectroscopy, which measures steady-state free radical production. Also, the aerosol scientists and chemists participating in the workshop believed that a bioassay, which could serve as an indicator of acute PM toxicity, would be extremely valuable for field use. A major challenge presented in the identification of epidemiological variables is that PM fractionation must be sufficient to separate biologically active constituents from inactive constituents, while keeping die total number of variables small, to prevent loss of statistical power. An additional challenge is that molecularlevel characterization requires substantial particle mass (a few milligrams to a few hundred milligrams, depending on the degree of characterization). The workshop participants suggested four sets of organic variables for use in epidemiological studies of atmospheric PM and health (see box on previous page). Note that level 1 characterization would not permit the identification of a biologically active organic PM constituent that is not correlated with organic PM mass. The approach described in level 2 is similar to die approach that has been used by many researchers to investigate die mutagenicity of organic particulate matter. After identifying the most biologically active fraction, detailed chemical analyses and additional mechanistic studies are tiien focused on die bioactive fraction. The concept proposed in level 3 involves die broad characterization of particle-phase organic compounds based on structural information (such as functional group absorbances). Scientists use techniques like Fourier transform infrared spectroscopy, Raman spectroscopy, three-dimensional fluorescence, X-ray absorption fine structure spectroscopy, and/or nuclear magnetic resonance spectroscopy, and steady state free radical production using electron paramagnetic resonance (EPR) spectroscopy. These techniques are nondestructive and provide a great deal of information about the behavior of the organic fraction with a limited number of variables. The level 3 approach, however is die most experimental of the four suggested. The variables of level 4 are inherently different in character than those of levels 1-3 and involve use of
source tracers or source contributions as variables for epidemiological studies. Source tracers might be selected based on experience or, more rigorously, from factor analysis results, as is done in hybrid factor analysis-multiple linear regression source apportionment. Alternatively, source contributions can be determined using chemical mass balance receptor modeling techniques; for example, chemical mass balance could be used as variables in the epidemiological analysis. Some sources (motor vehicles, wood combustion, and meat cooking) are not easily resolved witii elemental profiles alone but can be resolved witii the addition of organic molecular markers. The major liability to this approach is the cost involved in molecularlevel chemical analysis of daily samples. Samples would be analyzed as for PM source apportionment. Either molecular or elemental tracers of die major source tvoes the contributions themselves would then be used as variables in the epidemiological analysis PM samDles would be analyzed for sulfate nitrate metals nrganic and elemental carhnn and a suite oforganic comt>ounds The workshop participants are confident mat the development of metiiods for die characterization of polar organics can inform toxicologists developing mechanistic hypotheses and guide the design of studies witii laboratory-generated aerosols. Organic measurements tiiat provide some chemical detail about the entire organic fraction can assist die interpretation of toxicological studies utilizing concentrated atmospheric PM and examination of associations between PM constituents and health endpoints in epidemiological studies. Resolution of PM source contributions may lead to more effective air pollution control strategies. Acknowledgments Workshop p r e s e n t a t i o n s were m a d e by Daniel Grosjean, Lara Gundel, Pradeep Saxena, Frank Huggin, Murray Johnston, Jay Odum, Rie Rasmussen, Jamie Schauer, Jian Yu, Barbara Zielinska, Rod Zika, Anita Orendt, Ron Wyzga, and Eric Edgerton. I also would like to acknowledge the assistance of Steven Eisenreich, Debra Laskin, and Zareen Dodwad.
References (1) Godleski, J. J. Plenary Lecture: Health Effects of PM2 5. Paper presented at PM2 5: A Fine Particle Standard; Long Beach, Calif., Jan. 28-30, 1998. (2) Rasmussen, R. Hydrocarbon measurements NARSTONortheast 1995 and 1996; Oregon Graduate Institute: Beaverton, OR, 1998. (3) Rogge, W. E; Mazurek, M. A.; Hildemann, L. M.; Cass, G. R. Atmos. Environ. .193,27, ,309-11330 (4) Saxena, R; Hildemann, L. /. Atmos. Chem. 1996, 24, 57109. (5) Young, R. S.; Jones, A. M.; Nicholls, P. J. /. Pharm. Pharmacol. 1998,50, 11-17. (6) Turpin, B. J.; Saxena, P.; Andrews, E. Atmos. Environ..,i press. (7) Laskin, D. J.; Pendino, K. J. Annu. Rev. Pharmacoll Toxicol. 1995, 35, 655-677. (8) Jacobs, R. R. Int. J. Occup. Environ. Health 1997, 3, SlS48. Barbara J. Turpin is an assistant professor with the Rutgers University Department of Environmental Sciences and Rutgers Cooperative Extension in New Brunswick, N.J. FEB. 1, 1999/ENVIRONMENTAL SCIENCE & TECHNOLOGY/NEWS " 79 A
