W. Tang, QLAB, Cherry Hill, NJ.
Many approaches have been used to compare indoor and outdoor fungal spores to aid the investigation of possible indoor fungal growth. These approaches include inside to outside concentration ratios (I/O >1 and I/O >10), rank order, complicated statistical analysis, and diversity comparison. A simple and effective way is presented to interpret the results by selecting important groups of fungal spores for data interpretation. The first group of fungal spores is the dominant airborne spores (ascospores, basidiospores, Cladosporium spores, and Aspergillus/Penicillium-like spores). Their sizes are mostly less than 8 µm, and they stay airborne much longer than larger spores. This is the group of spores that can be used for both concentration and percentage comparisons. Studies have shown that indoor spore level in normal commercial buildings is only about 10% (on average) of the outdoor spore level. Spore levels in indoor air in residential buildings are also usually lower than levels outdoors. The airborne spores in an indoor environment are settling and being filtered out constantly. If the indoor fungal growth is hidden, the concentrations of those airborne spores from the hidden source could be low. Therefore, comparing only the concentrations will likely fail to identify the hidden source. However, the percentage of those spores in indoor air should show significant elevation. The second group is water-damage indicators (Stachybotrys, Chaetomium, Ulocladium, etc.). This group of fungi requires high water activity (Aw) to grow (minimal Aw: 0.89-0.90). Since their spores do not easily become airborne, the presence of their spores even in low number is an indication of a possible water-damage history. The rest of the spores usually come from outdoors if no indoor mold growth is disturbed. They do not usually signify an indoor source unless the concentration is more than 100 to 300 spores/m3 and significantly higher than the outdoor count.
H. Burge, D. Gallup, Environmental Microbiology Laboratory Inc., San Bruno, CA.
The MoldRange is a large database of outdoor spore sampling results with more than 100,000 entries. Samples were collected by field investigators and submitted to Environmental Microbiology Laboratory Inc. for analysis using standard quality control procedures. Counts were entered directly into a computer database and then manipulated in Excel. We sorted the files first for Penicillium/Aspergillus spore types, then by date, then by state, then by date within state. Penicillium/Aspergillus spores were present in more than 85% of all outdoor samples. These values were similar when analyzed by month for Arizona, Alabama, and Arkansas. Penicillium/Aspergillus, which are often considered “indoor” molds, usually present in outdoor air, and median concentrations (when present) are above what some consider to be an indoor guideline (200 spores/m3). Maximum concentrations can exceed 100,000 spores/m3. These data affirm the necessity of understanding the outdoor aerosol if one is to interpret indoor data. It is unreasonable to set clearance criteria for naturally ventilated buildings at concentrations below those of the outdoor aerosols.
P. Fallah, Indoor Environmental Hygiene Laboratory, Redmond, WA; C. Robbins, M. Krause, B. Geer, Veritox Inc., Redmond, WA.
The nonculturable air sampling for mold (spore trap) is one of the most common sampling methods used by industrial hygienists (IH) conducting indoor air quality investigations. While this sampling technique allows for capturing a wide variety of spore types from the air, including viable and nonviable spores, it provides little or no information on generic distinction between morphologically similar spores of Aspergillus and Penicillium. As a result, the spores of these genera are grouped as “Aspergillus/Penicillium-like spore.” At present, culturable air sample methods are required to distinguish between the two genera. A method to distinguish Penicillium and Aspergillus spores on spore trap samples would provide additional useful data for the IH. Data were collected to examine the utility of this approach. The spores of 30 species of Penicillium and 28 species of Aspergillus were examined microscopically at 400´ and 1000´ magnification. Spores were evaluated based strictly on surface texture (smoothness or roughness). Fifty spore traps (Zefon) samples and 50 culturable (Andersen) samples were taken side by side in indoor environments with high background mold spore levels. Aspergillus and Penicillium colonies from these cultured air samples were identified to species level and enumerated. Spores on spore trap samples that are typically categorized as Aspergillus/Penicillium-like were counted and separated based on surface texture. The majority of distinctly rough spores belonged to common aspergilli species found indoors. Penicilli species recovered from cultures had distinctly smooth spores and belonged to common species found indoors. While species identification based on spore morphology alone is generally impossible, generic separation of the two genera appears possible when culturable data are not available. However, generic separation based on only a few spores provides inconclusive results.
D. Gallup, D. Bell, H. Burge, Environmental Microbiology Laboratory Inc., San Bruno, CA.
We have developed the MoldScore to aid in interpreting spore trap data from paired indoor/outdoor samples. Previously we compared MoldScore to scoring by three experts in aerobiology and to other published methods for making such comparisons. We used 100 field reports, all with paired indoor/outdoor samples, derived from samples submitted to Environmental Microbiology Laboratory Inc. for analysis. MoldScore correlated strongly with the aerobiologists’ scores. We asked three experienced field investigators to score the 100 reports, and have compared their scores to the aerobiologists’ consensus scores, and to the MoldScore. For 25 of the 100 reports, all investigators agreed within 20 points with both the MoldScore and the consensus score. However, overall correlations with these two measures were only moderate or weak (R2=0.57, 0.37, 0.39 for the three investigators), and the investigators did not agree among themselves. We evaluated each report to discover, if possible, why there were discrepancies. Three general patterns appeared. First, if a few Penicillium/Aspergillus spores were present indoors and none out, one or more of the investigators scored the report high, while the consensus and the MoldScore produced low scores. Second, there was much disagreement on the significance of the presence of one or two Stachybotrys spores. Third, when high levels of Penicillium/Aspergillus were recovered indoors with equal or slightly higher levels outdoors, the consensus and the MoldScore tended to produce moderate or higher scores, while one of the field investigators considered this situation not to be a problem. In this case the consensus was based on the possibility that the indoor spores were different from those outdoors. While we will continue to test and improve the MoldScore, we are confident that it accurately represents the opinion of the aerobiological experts, and should provide a strong tool to be used with expert on-site visual observation.
W. Tang, QLAB, Cherry Hill, NJ.
Cladosporium and Aspergillus/Penicillium-like spores can be commonly found in both indoor and outdoor air. Further identification of those spores based on taxonomy is not possible for direct microscopic examination. When outdoor air has predominantly Cladosporium herbarum-like spores and indoor air has predominantly Cladosporium cladosporioides-like spores in similar concentrations, it is an indication of a possible indoor growth of Cladosporium. However, there is no way to indicate this difference when they are being grouped and reported together under the same name. Same fungal colony produces spores with identical or similar microscopic morphology. Spores with different microscopic morphology are most likely originated from different fungal colonies even further identification can not be made. A new approach is presented to differentiate those two groups of airborne fungal spores based on their microscopic morphology and chemical reactivity. Cladosporium are sorted into 3 groups and Aspergillus/Penicillium-like spores are sorted into 8 to 12 groups. When indoor and outdoor Cladosporium and Aspergillus/Penicillium-like spores are differentiated into several groups, possible differences can be shown in indoor and outdoor distribution. Otherwise, when the concentrations of those common dominant airborne fungal spores are observed both in indoor and outdoor air in similar concentrations, regular spore-counting results cannot indicate any difference.
A. Havics, pH2, LLC, Indianapolis, IN.
Since the retraction of a draft quantitative exposure limit for airborne fungal concentrations originally proposed in 1989 by the ACGIH Bioaerosol Committee, many have struggled to find traditional quantitative exposure limits for fungi. Although numerical standards are available in other countries, a consensus has been lacking in the United States. The classical creation of an occupational exposure limit (OEL) or community risk-based screening level (RBSL) can be used to ballpark an acceptable point estimate for select fungal taxa for specific populations. For example, a numerical limit for Stachybotrys sp. will be presented using the OEL approach. The OEL approach is an end point-based approach that allows for several kinds of limits, depending on the “toxic” end point. Indeed, it also makes use of several methodologies or approaches that flow from a particular end point. These approaches include the RD50, low-dose extrapolation, benchmark dose (BMD), margin of safety (MOS), analogy, correlation, and physiologically based pharmacokinetic (PBPK) modeling. The example limit in this case could be used to aid in the selection of respiratory protection during mold remediation activities as well as to gauge an upper bound for nonoccupational exposures.
D. Kahane, Forensic Analytical, Hayward, CA.
The U.S. Environmental Protection Agency’s development of the EPA relative moldiness index (ERMI) over the past several years is based on its priority of improving indoor air quality. The ERMI was made feasible by the advances in molecular genetic identification of fungal species based on the quantitative polymerase chain reaction (QPCR). QPCR has been used by many industrial hygienists (IHs) for the identification of specific Aspergillus species during hospital construction and renovation, but with the advent of the ERMI, a newly proposed approach for characterizing the residential environment through comprehensive dust sampling is emerging. Commercial kits are expected to be released in 2007. A summary of the analytical method was provided by EPA researchers to IHs in the April 2006 Synergist. In essence, the collection of house dust samples and the analysis of specific mold species form the basis for the determination of whether a home falls into one of the four levels of moldiness. Thirty-six species of mold, broken into two categories, are compared mathematically in obtaining the level of moldiness. The adoption and use of the ERMI raises as many questions as answers for the IH. This session will address the implications of adopting and using ERMI in any routine fashion including: (1) How will IHs incorporate and reconcile the ERMI into data collected from an informed visual inspection? (2) Will the ERMI be used in as index of exposure in addition to relative risk? (3) How will the legal community view a state-of-the-art EPA-derived protocol for mold inspection? IHs must always remain on the leading edge of recognition, evaluation, and control issues, and the EPA-derived ERMI requires that the IH community take a critical look at what might become an adopted strategy for mold inspections.
J. Dobranic, C. Cutler, EMSL Analytical Inc., Westmont, NJ.
There is a critical need to standardize the sampling and analysis of fungal contamination in buildings. The U.S. Environmental Protection Agency (EPA) has been developing and working on a dust-based sampling regiment for determining the moldiness of homes for the past several years. EPA was able to do this using the polymerase chain reaction (PCR) as the analysis method of choice. Using dust and PCR, EPA researchers came up with an indicator of moldiness called the EPA relative moldiness index (ERMI). The ERMI is calculated by using PCR to analyze 36 different fungi. This study looks at the practical usefulness of the ERMI as a predictor of mold problems in homes. Five consultants from five companies with extensive indoor air quality experience were asked to participate in this study. They performed their regular protocols for assessing moldiness in 4 residential properties each (20 total homes assessed). The consultants were not required to take any samples if their in-house protocols did not call for sampling. They were asked to collect a composite dust sample from two primary rooms (living room and bedroom) and to complete a questionnaire based on their visual inspection and sampling results. The questionnaire asked about their conclusions on the moldiness of the homes. After the completion of the questionnaire, the consultants were give the results of the ERMI. Data will be presented on whether the ERMI results corroborated their initial conclusions.
D. Bridge, Rimkus Consulting Group Inc., Houston, TX; M. Krotenberg, Rimkus Consulting Group Inc., Phoenix, AZ; M. Wiseman, Houston Baptist University, Houston, TX.
Total fungi in settled dust on indoor carpeting has been considered as a measure of fungal contamination. The most commonly used metric for expressing the relative amount of fungi in settled dust is colony forming units per collected dust mass (CFU/g). However, collecting a sufficient amount of dust is required. When the mass of dust collected is below the laboratory detection limit, the data cannot be reliably compared to published guidelines/recommendations regarding contamination. As a result, we evaluated the relationship between fungal levels in carpeting as expressed in CFU/g (mass) and CFU/cm2 (area). The relationship between CFU/g and CFU/cm2 was analyzed for two independent sets of carpet dust samples. Samples were collected from non-water-damaged and water-damaged carpeting using open-face and closed-face cassette sampling methods. The results indicate that fungal loading expressed as CFU/g was well correlated with results expressed as CFU/cm2 (R2 = 0.90). The open-face method (R2 = 0.81) correlated similarly to the closed-face method (R2 = 0.93). There was virtually no relationship between collected dust mass and CFU (R2 = 0.05) or sample area and CFU (R2 = 0.17).
H. Neill, 1Source Safety and Health Inc., Exton, PA; W. Tang, QLAB, Cherry Hill, NJ.
The traditional culture method for analyzing culturable fungi requires six to seven days. Sometimes it is not very useful for indoor fungal sampling because of the incubation time required. A new method has been developed to obtain viable fungal count in 24 to 48 hours with limited or no identification. The samples were extracted from collection media and then diluted or concentrated to proper concentrations. The suspensions were then inoculated onto fungal culture media. After 24 to 48 hr, germination of spores or the formation of microcolonies of yeasts was observed under microscope. Viable spores were counted with limited or no identification. The numbers from the rapid method and traditional culture method show good correlation, and the difference can be as low as less than one order of magnitude. Some limitations includes interference from high amounts of debris in surface and bulk samples, desiccation of airborne spores collected on filter membranes, inhibition from biocides, slow growing species, and limited identifications.