Evaluating Indoor Air Quality: Test Standards for

Report to the Workers’ Compensation Board of British Columbia Research Secretariat Evaluating Indoor Air Quality: Test Standards for Bioaerosols 99FS-64 Karen H. Bartlett, Ph.D. Assistant Professor School of Occupational and Environmental Hygiene University of British Columbia Kit Shan Lee, MSc School of Occupational and Environmental Hygiene Co-investigators: Gwen Stephens, MD Medical Microbiologist William Black, MD Medical Microbiologist UBC-BC Centre for Disease Control Michael Brauer, Sc.D. School of Occupational and Environmental Hygiene University of British Columbia Ray Copes. MD Community Health Specialist Ministry of Health Province of British Columbia November, 2002 Revised July, 2003

Table of Contents List of Tables List of Figures Acknowledgements Abstract Introduction and Background Health effects of mould in the indoor environment Field comparisons of bioaerosol sampling devices Review of available guidance documents for bioaerosol exposures ACGIH Health Canada (1996) New York City Department of Health & Mental Hygiene (2002) Air Sampling for Fungal Particulate Objectives Methods Sampling sites Administrative organizations participating in the study Sampling schedule Bioaerosol Samplers Andersen N6 Single Stage Impactor Surface Air System Super-90 Reuter Centrifugal Air Sampler Standard Air-o-cell Sampler Surrogate measures of fungal mass Comparison of the specifications of the sampling techniques Sampling Media Sampling Protocol Laboratory and Sample Analysis Protocols Results Sampling sites Ventilation Bioaerosol concentrations Descriptive statistics Limits of detection Reproducibility of sequential duplicates Inferential comparisons of geometric means between instruments Correlations Linear regression of relationships between instruments Fungal concentrations and indoor air quality Ergosterol in settled dust Discussion Study Overview Proportion of samples beyond detection limits Reproducibility

ii iii iv 1 2 3 3 4 4 5 5 6 7 8 8 10 10 10 10 11 11 12 12 13 13 16 18 18 19 19 20 21 22 22 24 25 25 26 29

Indoor/ Outdoor Differences Total Yield Comparison of viable samplers Microscopic counting method Limitations of regression equation Analysis of performance characteristics Strengths and Limitations of study Conclusions References Appendix A: ISEE/ISEA abstract August 13, 2002, Vancouver, BC Appendix B: Indoor Air 2002 Abstract pp 455-460 Proceedings, Monterey CA Appendix C: AIHCE abstract June 1-6, 2002 San Diego, CA

31 32 33 33 34 35 35 39 40 43 45 52

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List of Tables Table 1. Summary of particle collection efficiencies Table 2. Comparison of sampling medium, area, and media volume Table 3. Comparison of flow rates and sampling volumes Table 4. Summary of sites by administration organization Table 5. Environmental comfort parameters (June – October 2001) Table 6. Summary of samples analyzed Table 7. Geometric mean concentrations by location Table 8. Indoor geometric means with 95% CI, arithmetic means and ranges Table 9. Outdoor geometric means with 95% CI, arithmetic means and ranges Table 10. Proportion of samples beyond detection limits Table 11. Reproducibility – coefficients of variation (%) Table 12. Comparison of geometric means for Indoor/Outdoor concentration Table 13. Representational proportion of indoor airborne fungal groups Table 14. Pearson r coefficients for linear relationships between sampling results Table 15. Simple linear regression equations between sampling methods Table 16. Rooms with fungal concentrations above Health Canada Guidelines Table 17. Relationship of mechanical ventilation and indoor fungal concentration by sampler type Table 18. Relationship of signs of moisture and indoor fungal concentration by sampler type Table 19. Relationship of presence of carpet and indoor fungal concentrations by sampler type Table 20. Ergosterol in settled dust Table 21. Indoor-Outdoor comparisons by fungal genera and sampler type Table 22. Previous relevant field studies

12 13 14 18 19 19 20 20 21 22 22 23 23 24 25 25 26 26 26 26 27 36

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List of Figures Figure 1. Figure 2. Figure 3. Figure 4. Figure 5.

Geometric means with upper 95% confidence limits Collection efficiency by fungal genera Indoor-outdoor comparisons for N6 sampler Indoor-outdoor comparisons for SAS sampler Indoor-outdoor comparisons for RCS sampler

21 24 27 28 28

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Acknowledgements We would like to thank the following people for their excellent help and involvement in this project: Julie Hsieh, research assistant, School of Occupational and Environmental Hygiene Don Strutt, British Columbia Building Corporation (BCBC) Quinn Danyluk, Simon Fraser Health Region (Burnaby General Hospital) Dan Strand, Vancouver Airport Authority David Bell, Occupational Hygienist, University of British Columbia The staff in the 74 offices that were used as test sites.

Abstract Introduction: No standard method exists for enumerating fungal aerosols, impeding the development of reliable exposure-response data. A field comparison of four bioaerosol samplers, the Reuter Centrifugal Sampler (RCS), the Andersen N6 Single Stage (N6), the Surface Air System Super 90, and the Air-o-Cell sampler (AOC), was conducted in a variety of public buildings for the measurement of fungal aerosols to compare sampling performance efficiencies and to collect baseline data for a pool of buildings Methods: Sampling was conducted at 75 sites in public buildings from June-October 2001 in the greater Vancouver area, British Columbia. Four locations were sampled at each site (1 common area, 2 offices, and 1 outdoor sample). Each location was sampled in parallel, collecting approximately 150 litres of air for each sample. Malt extract agar was used for all growth media. Sequential duplicates were taken at each location. Simple linear regressions were calculated for each method pair to develop betweensampler calibration equations. Results: Data from approximately 592 samples (60 different buildings) were available for analysis from each instrument. Differences were found between samplers for overall yield, detection limits, and reproducibility. The highest spore concentrations were returned by the non-viable method, the AOC. The N6 and RCS were comparable in colony concentrations, but the N6 was more efficient at capturing small particulate such as Penicillium and Aspergillus spores. The SAS-90 returned concentrations that were significantly lower than all other samplers. The surrogate chemicals, ergosterol and (1→3) β D glucan were below the limit of detection of the method for these samples. Conclusions: Concentration data is dependent on the sampling methodology utilized for assessment and should be considered before conducting investigations of bioaerosols in different environments. Exposure guidelines cannot be created until a standard methodology is available. All of the bioaerosol sampling devices tested had unique characteristics which could be seen as beneficial or detrimental depending on the sampling environment and the conclusions drawn from the sample data.

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Introduction and background Workers in the indoor environment of non-industrial buildings make up more than half of the entire workforce of industrialized countries. The number of such workers in BC is increasing as the economy moves into the service sector. Complaints regarding the perceived indoor air quality (IAQ) are pervasive and include all categories of workplaces including office settings, schools, community, and medical facilities. Air quality complaints by workers are associated with significant economic losses including diminished productivity, disability claims, and direct costs for medical assessments and treatment. The spectrum of illness attributed to microbial air pollution has expanded from conditions with known associations such as asthma and allergy to conditions with temporal/spatial associations such as sick building syndrome (SBS) and multiple chemical sensitivity (MCS). SBS may be exacerbated by a number of different pollutants in the indoor environment, many of which are concomitant in the normal office setting. Due to the range of individual susceptibilities and the general lack of definitive doseresponse data for single compounds within the mixture of chemical and biological agents which may be present, adjudications by courts and compensation boards have lacked consistency. Concerns about litigation, worker productivity and illness have resulted in the adoption of IAQ regulations. Many agencies, including the Workers’ Compensation Board of British Columbia (WCB-BC, 1998), have adopted standards developed by professional organizations such as the American Society of Heating and Refrigeration Engineers (ASHRAE). Recommendations for the interpretation of airborne fungal concentrations have been proposed by several agencies. In 1995 a federal-provincial committee struck by Health Canada developed general guidelines for Canadian public buildings, including interpretation of airborne fungal concentrations (Nathanson 1995). Other agencies have been the Occupational Health and Safety Administration (OSHA) (US OSHA 1992), the American Conference of Governmental Hygienists (ACGIH) (Macher 1995), the Central European Committee (CEC 1994) and the World Health Organization (WHO 1988). None of the suggested guidelines for biologic contamination have been adopted as exposure limits, however, primarily due to conflicting exposureresponse data and lack of standardized sampling protocols. Public awareness of mould as

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a hazard is most clearly seen in the United States, where a staggering number of lawsuits are before the courts claiming property and personal damage due to mould colonization in homes and public buildings. The publicity and controversy surrounding litigation has prompted several states to propose “toxic” mold legislation, which would require disclosure of mould damage in buildings prior to sale. In the light of public awareness of the potential for mould to be a health hazard in the indoor environment, standardized methods are required to enable assessment of workplace health for employees and the public, and to allow employers to show compliance. Without scientifically valid standards and guidelines, arbitrary criteria may contribute to inappropriate testing and test interpretations. The human and economic consequences of misleading sampling data are significant. The cost of unsubstantiated interpretation of airborne fungal concentrations may include litigation, labour grievances, disability claims, inappropriate medical treatment, or in extreme cases, the recommendation to demolish public buildings.

Health effects of mould in the indoor environment Many studies have shown a strong and consistent relationship between building dampness and/or the presence of visible mould and respiratory health effects such as wheeze, cough and bronchitis (Bornehag et al 2001). Associations between viable airborne mould concentrations and symptoms have been reported (Husman 1996; Pope 1993). Studies have has evaluated several markers of fungal contamination and mechanisms of action. For example, all fungal cells and spores contain the biochemicals 1 → 3 β-D-glucan and ergosterol. These biochemicals have been used as markers for total biomass, thereby including both viable and non viable cells. Both ergosterol and 1 → 3 βD-glucan have been associated with respiratory or other symptoms in a number of

epidemiological studies (Rylander 1998).

Field comparisons of bioaerosol sampling devices There is no consensus in the literature as to a reliable method to measure fungal exposures that have relevance to health outcomes. This report does not address health outcomes, but focuses on the first requirement, that is, to evaluate methods to measure 3

fungal exposure Four commonly used spore collection devices were evaluated in a field trial conducted in actual work places in BC. Three of the devices depend on the culturability of the organism to enumerate the airborne concentration (Andersen N-6, SAS-90 and Biotest RCS). The fourth device collects fungal spores for microscopic counts and does not require the organisms to be viable (Zeflon Air-O-Cell). Secondly, methods to measure chemical surrogates of exposure (ergosterol and (1→3) β D glucan) were developed. Chemical surrogates may be collected over a longer period of time, thereby integrating the sample over a period of hours. The composition of fungal aerosols indoors is dependent on the abundance and strength of sources, as well as mixing, dilution, and particle removal (Pope et al., 1993). Natural aerosols are typically a mixture of species. Airborne fungal spore concentrations vary over seasons, by diurnal or circadian cycles, and by the presence of source materials such as vegetation or collection surfaces such as carpets, etc. (Gravesen et al., 1986)

Review of available guidance documents for bioaerosol exposures 1. American Congress of Governmental Industrial Hygienists (ACGIH) In the United States, the ACGIH is the scientific organization responsible for the promulgation of occupational exposure limits to chemical, biological and physical hazards (threshold limit values, or TLVs®). The ACGIH does not support any numerical guidelines for the interpretation of bioaerosol data from non-manufacturing environments. The ACGIH Bioaerosl Committee recommendations are to gather the best data possible and use knowledge, experience, expert opinion, logic and common sense to assist in the interpretation of results. As rules of thumb, the ACGIH suggests (1) the comparison of indoor and outdoor concentrations (in office environments, the ratio should be than the d50 are removed from the air stream at increasing efficiency and deposited on the sampling medium. The d50 is generally assumed to be the diameter above which all particles are removed, assuming that the instrument has a sharp cut-off curve. Fungal spores range in aerodynamic diameter from 0.5-20 μm, but are typically larger than 2 μm.

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Objectives Accurate measurement of microbial indoor air pollution is an essential component of workplace safety assessment. New indoor air quality regulations of the British Columbia Workers’ Compensation Board (WCB-BC, 1998) mandate bioaerosol testing when workers have complaints consistent with building related disease. At this time these is no consensus on the part of health, medical, and occupational hygiene experts regarding appropriate test methods for such sampling. This project compares four recommended air sampling methods as the first step toward the development of BC specific field testing and laboratory procedures to measure fungal bioaerosols. Summary of Objectives: 1) To collect baseline bioaerosol measurements from 75 buildings in British Columbia 2) To compare commercially available methods for measuring indoor bioaerosols.

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Methods Sampling Sites The field study was conducted in non-residential buildings in the Greater Vancouver area. A target number of 75 sites were sought to represent a variety of public buildings including traditional offices, waiting rooms, community centers, and educational facilities. Building administrators were approached to obtain contact information and addresses of possible sites. The four administrative organizations that participated in the study were the British Columbia Building Corporation, the University of British Columbia, the Simon Fraser Health Authority, and the Vancouver Airport Authority. Each sampling site was examined over the period of one workday. At each sampling site, four identified areas were studied. 1) Common area (examples: waiting rooms, reception areas, staff lounges, meeting rooms) 2) Private office A (examples: enclosed or semi-enclosed space where employees spent the majority of their work day) 3) Private office B 4) Outdoor control (examples: at the air intake for mechanically ventilated buildings, near windows or doors for naturally ventilated buildings). A building could potentially have more than one sampling site (examples: different floors, area ventilation supplied by different air handling units). Buildings were excluded if they were (a) primarily residential, or (b) were identified as having pre-existing, identified water damage.

Administrative organizations participating in the study: 1. The Building Corporation of British Columbia (BCBC) The BCBC was established in 1977 to provide accommodation and real estate services to the provincial government. Since 1997, BCBC’s mandate was expanded to enable the Corporation to provide its services to the broader public sector. BCBC district managers furnished lists of offices available for study. Sites ranged in size from multistoried downtown office towers to small, portable buildings. Sources of ventilation air

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ranged from fully centralized mechanical air conditioning units to natural ventilation available by opening windows or doors. 2. The University of British Columbia (UBC) The UBC is located at the western tip of the Point Grey peninsula in the city of Vancouver. Buildings on the campus were constructed between 1929 and the present. A general email was sent out to all administrators of departments affiliated with UBC asking for volunteer sampling sites. Departments who wished to participate contacted the study coordinator. Sites included administration offices, research laboratories, performing arts theatre, and gymnasium. Buildings ranged in size from large office buildings to small, portable buildings. Some buildings were ventilated by central air handling units and others were naturally ventilated. 3. The Simon Fraser Health Region (SFHR) The SFHR provides a variety of health services to the residents of Burnaby, New Westminster, Coquitlam, Port Coquitlam, Port Moody, Anmore, Belcarra, Pitt Meadows, and Maple Ridge. An occupational hygienist employed by the SFHR furnished a list of sites available for study. Study sites included hospitals, long term care facilities, and administration offices. Buildings varied in size and sources of ventilation air. 4. The Vancouver Airport Authority (VAA) The VAA is responsible for the management and operation of the Vancouver International Airport (YVR). An occupational hygienist employed by VAA furnished a list of potential sites. Study sites included administrative offices in temporary, mobile buildings that served as offices with either mechanical or natural ventilation

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Sampling schedule A contact person, generally in the management level of the office, was provided for each site. From the pool provided, each site was contacted by telephone. The sites were scheduled for sampling based on the convenience and the availability of the occupants.

Bioaerosol Samplers 1. Andersen N6 Single Stage Impactor (N6) The Andersen N6 Single Stage impactor (Graseby-Andersen, Atlanta, GA, USA) is a sieve type sampler. Air is drawn through 400 holes (diameter of each hole=0.026 cm) at 28.3 litres per minute (lpm). Particulate matter with aerodynamic diameters between approximately 7 – 0.65 μm impact onto agar medium contained in a 100 mm petri dish fitted under the sieve. The N6 can be used to enumerate fungi or bacteria by adjusting the composition of the agar culture medium. In this study, a battery operated, Gilian® AirCon-2 High Flow pump (Sensidyne®, Clearwater, FL) was calibrated to the required airflow of 28.3 lpm through a critical oriface and checked with a calibrated rotameter. 2. Surface Air System Super-90 (SAS) The Surface Air System Super-90 (PBI International, Milan, Italy) is a battery operated (rechargeable 8.4-Volt, 1.2 A/hr, nickel-cadmium battery), single stage, sieve type sampler. Air is drawn through a single sieve plate with 487-holes (diameter of each hole=0.1 cm). Particulate matter is collected by inertial impaction and deposited onto agar medium contained in a 84 mm maxi Replicate Organism Direct Agar Contact (RODAC) plate, (Bioscience International, Rockville, MD). The maximum efficiency of collection is for particulate matter with a d50=2-4μm. The predetermined flow rate is 90 lpm. The SAS is marketed as a device to enumerate fungi or bacteria.

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Field calibration of this instrument is not possible. The instrument is factory calibrated on a regular basis. 3. Reuter Centrifugal Air Sampler Standard (RCS) The Reuter Centrifugal Sampler Standard (Biotest, Frankfurt, Germany) is a portable, battery operated, impaction sampler which draws air into the instrument by a rotating impeller blade (total sampling rate of 280 L/min, effective sampling flow rate of 40 L/min) from a distance of at least 40 cm. Agar medium is distributed into the 34 wells of a flexible strip which is inserted for use around the perimeter of the impeller. Each well is approximately 1 cm2. Particulate matter is collected by centrifugal impaction onto the agar strip (Biotest HYCON, Germany) with an optimum efficiency of d50 = 4μm. The RCS is marketed as a bioaerosol sampling device that can be used to enumerate fungi or bacteria. Although the total sampling flow rate is calculated to be 280 L/min based on the rotation speed of the impeller blade, the effective volume for particulate in the size range of fungal spores is 40 L/min (also called the separation flow rate. Calibration by a primary standard is not possible for this instrument because air enters and exits the instrument through the same opening. The instrument generates 16 electronic pulses per revolution and the number of impulses for each running time is programmed into the unit. Two checks were used to insure the instrument was performing to specifications. 1) The impeller blade angle was checked using a machined mold before use. 2) The impeller rotation frequency was checked by tachometer to be 4,096±82 rpm). 4. Air-o-Cell Sampler (AOC) The Air-o-Cell Sampler (Zefon International, St. Petersburg, FL) consists of a cassette containing a glass slide and an external sampling pump. The battery operated pump draws air through the sampling cassette at 15 L/min. Particulate matter is impacted onto an adhesive coated slide. The maximum efficiency of the device is for particle size of d50=2.6 μm. Spores are counted using a microscope. The Air-o-Cell sampler is

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marketed to enumerate fungal spores, pollen, fibres, and other aerosols (cell fragments, combustion emissions, and insect parts). The pump used in conjunction with the sampling cassette is calibrated using a rotameter designed for this purpose and is provided with the pump. 5. Surrogate measures of fungal mass. Ergosterol is a unique sterol in fungal cell membranes. The chemical quantification of ergosterol is correlated to fungal mass. The measurement of ergosterol has been used in studies conducted by Canada Housing and Mortgage Corporation (CMHC), Health Canada and Agriculture Canada. Fungal particulate is collected on depyrogenated glass fibre filters placed in 37 mm, three-piece cassettes. Battery operated pumps calibrated to 2 lpm are connected to the cassettes and whole day, integrated samples are taken. (1→ 3) β-D glucan (BDG) is a polyglucose, stuctural component of fungal spores. Like ergosterol, BDG is a surrogate marker for airborne fungal biomass. BDG has immunoregulating properties in vivo and has been used as a indicator of fungal exposure in occupational and residential exposures in Dutch and Scandinavian studies. The collection of fungal particulate is the same as described for ergosterol.

Comparison of the specifications of the sampling techniques. Table 1. Summary of particle eollection efficiencies Sampler

Operation

Method

d50 (μm)

N6 SAS RCS AOC

Inertial Inertial Centrifugal Inertial

0.65 2 - 4.0 4.0 2.3 - 2.4

Ergosterol

Filter

BDGa

Filter

Culturable Culturable Culturable Nonviable Nonviable Nonviable

a

Pore size (μm)

Reference Andersen (1958) Lach (1985) Macher and First (1983) Aizenburg et al. (2000)

1.0 1.0

Miller and Young (1997) Rylander (1999)

(1→ 3) β-D glucan

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Sampling Media Culturable sampling methods (N6, SAS, RCS): Malt extract agar (MEA). Formula per litre: maltose, 12.75g, dextrin, 2.75g, glycerol, 2.35g, pancreatic digest of gelatin, 0.78g, agar, 15.0g. Final pH = 4.6±0.2. Non-viable method (AOC): AOC cassettes were supplied with a glass slide coated with an adhesive substance to collect the particulate and were for single use. Surrogate biomass (ergosterol, GDB): Glass fibre filters (37 mm, Gelman type A/E) were baked overnight at 180oC (depyrogenated) to remove any contaminating ergosterol or GDB. Filters were loaded into new, three-piece cassettes (SKC) with a fiberglass supporting pad. Table 2. Comparison of sampling medium, area, and media volume Instrument Sample container Sampling Area Volume of Media cm2 (approximate mL) N6 Petri Dish 78.5 45 SAS Maxi RODAC plate 55 20 RCS Agar Strip 34 10 AOC Cassette with glass slide 0.165 Ergosterol Cassette with filter 10.2 a BDG Cassette with filter 10.2 a (1→ 3) β-D glucan

Sampling Protocol Sampling was conducted on weekdays from Monday to Thursday (June - August 2001) and Monday to Friday (September and October 2001) during normal work hours (8:30am-5pm). Four locations were identified: • • •

One common room Two individual rooms or offices One outdoor location

The locations and times of sampling at each site were determined by consultation with the site contact, and was based on convenience and availability. Occupants were allowed to use the common areas and offices normally during sampling. 13

Indoor sample sites: Sampling was conducted as close to the center of the room as possible. A limit in the battery power of the AOC pump made access to electrical outlets necessary. Each sampler was elevated to a height of 1.5 metres by adjusting tripod bases as necessary. This height was taken to be an average ‘breathing zone’.

Outdoor sites: Sampling was conducted near the air intake for the building, or in proximity to doors or windows that provided natural ventlation.

Instrument Specifications Table 3 summarizes the flow rates, sample times and total volumes collected for each instrument. The volumes represent recommended run/volume times for sampling in an indoor environment. The RCS and the SAS are pre-programmed for operating intervals, and the most appropriate time was chosen for the sampling time. Table 3. Comparison of flow rates and sampling volumes Sampler Flow Rate Sample Time Total Volume 5 min 140 L 1min 20sec 150 L 4 min 160 L 10 min (indoors) 150 L (indoors) AOC 5 min (outdoor) 75 La (outdoor) 2 L/min 360 min 720 L Ergosterol b 2 L/min 360 min 720 L BDG a A lower volume was collected outdoors with the AOC to prevent overloading. b (1→ 3) β-D glucan

N6 SAS RCS

28.3 L/min 90 L/min 40 L/min 15 L/min

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Air Sampling Protocol 1. Culturable methods: (N6, SAS, RCS) Sampling heads were thoroughly wiped with 70% isopropyl alcohol. o N6: a petri dish was placed onto the base of the sampling head. The lid of the petri dish was placed over the inlet of the N6 to prevent contamination. o SAS: a RODAC plate was fitted onto the sampling head. The lid of the RODAC was removed immediately prior to sampling. o RCS: the agar strip was removed from the plastic cover and threaded into the sampling drum. The RCS sampling head was capped with the provided plastic cover until sampling commenced. 2. Non-viable method (AOC, surrogates) o AOC: the sampling cassette was unsealed and fitted onto the pump head immediately prior to each sampling run. Surrogate biomass: o Ergosterol and BDG: inlet and outlet plugs were removed from the cassette and the cassette was attached to a high flow sample pump (SKC) calibrated to 2 lpm. The cassettes were hung from a tripod at approximately 1.5 m height with the inlet facing downwards. For all methods except for surrogate biomass, a sequential duplicate was taken after the first run was complete for all instruments. Cassettes for ergosterol and BDG were run side by side. Upon completion of the test, samples were repackaged into an ice cooler and transported back to the Environmental Bioaerosol Exposure Laboratory at the School of Occupational an Environmental Hygiene, University of British Columbia. Settled dust: Settled dust was collected from a 1 m2 area of the common room at each site. A portable vacuum (Porta-power, 6.8 amp motor,Hoover Canada). The flooring was vacuumed for 2 minutes, sweeping the wand across the area in one direction, then

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repeating the pattern perpendicularly. Dust was collected in sampling socks made of Connaught satin, with an approximate pore size of 10 - 15 μm (Chan-Yeung, 1996)

Laboratory and Sample Analysis Protocols Incubation and Counting of Viable Samples (N6, SAS and RCS) Samples were incubated at room temperature (20oC±4) in a natural light and dark cycle. RCS strips were incubated for 4 days, and the SAS and N6 samples were incubated for 5 days (a shorter incubation period was set for the RCS to prevent overgrowth). Colony forming units (CFU) were counted. Fungal colonies were identified to genus level using microscopy (stereoscope at 30 x and phase contrast at 400 x magnification) and standard mycology texts. Slide preparation (AOC) Cassettes were disassembled and the glass slide removed. Each AOC slide was stained with lactophenol cotton blue and mounted onto a microscope glass slide. Slides were counted using a modified version of the NIOSH Method #7400 (Fibres in air). Spores were counted using light microscopy (Jenamed2 Fluorescence microscope, Carl Zeiss Jena) set at 500x magnification. The field diameter at 500x magnification was determined using a stage micrometer (field diameter at 500x=360μm). Prior to counting, a survey of each slide was conducted to determine the general area of particle impaction. Counting proceeded systematically from the lower edge to top, from the left to right. Spores in the entire field of view were counted. Fungal spores were differentiated from other particulate matter (dust, pollen, etc.) using standard reference guides (Malloch, 1981; Smith, 1990). The general counting rules for the AOC slides were a maximum of 400 spores or 100 fields.

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Surrogate biomass analysis: Ergosterol was measured using a Varian Saturn 2000 Ion Trap instrument operated in the MS/MS mode. The trimethylsilyl derivative of ergosterol yields a unique mass spectrum and with the mass spectrometer operated in the MS/MS mode yields increased specificity. o Calibration standards were spiked with derivatization agents (15 μL of neat pyridine and then 50 μL of BSTFA). o Sample filters were spiked with 50 μL internal standard or surrogate and derivitized. o Standards: The calibration curve of ergosterol-TMS plots the peak ratios of 157/351 m/z versus nanograms injected. The limit of detection of the method was 16 nanograms ergosterol per filter, which was equivalent to 1 x 106 spores (Penicillium brevicompactum). BDG was measured using a commercial kit, Glucatell (Associates of Cape Cod, Inc., Falmouth MA). Briefly, amoebocytes (blood cells) harvested from horseshoe crab degranulate in the presence of fungal BDG. The degranulation releases zymogens which become active serine proteases through the Factor G pathway. A chromogenic peptide substrate permits spectrophotometric quantification of the activated enzyme. o Glass fibre filters were removed from the cassettes and extracted in 0.3M NaOH (in pyrogen free water o A standard glucan (Pachyman) is supplied with the lysate kit. o Measurements of optical density were taken every 10 seconds at 405 nm. The reaction time of the release of the chromatogen was inversely proportional to the amount of glucan in the test well. o The time of onset measures the elapsed time from background optical density (OD405) to an increase of 0.03 OD units. A log-log plot was calculated for the BDG standards. The BDG content of samples is calculated from the standard curve. The limit of detection of the method is 31 picograms BDG per filter.

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Results Sampling Sites A total of 75 sites from 61 different buildings sampled from June-October 2001. These buildings were provided by the BCBC, UBC, SFHR and VAA, and were all located within the greater Vancouver area, British Columbia. One site was excluded from analysis because it did not fit the description of a public building. Table 4 summarizes the total number of sites and buildings by contributing organization. Table 4. Summary of sites by administration organization ORGANIZATION RESULTS BCBC UBC SFHR VAA Total # of sites (# of buildings)

25 (18)

35 (34)

11 (5)

3 (3)

Indoor and Outdoor environments A total of 60 buildings were visited between June and October 2001. The majority of the sites used for sampling were office buildings (n=144, 65%), while health care settings accounted for 39 sites (18%), and a combination of other uses made up the remaining 39 sites (18%) including community buildings, research institutions, and multiple use spaces. The majority of the site buildings were constructed of concrete or concrete and steel (n=120, 85%), only 12 sites were built primarily of wood (9%) or other building materials (n = 9, 6%). The room volumes ranged in size from 6.4 – 1314 m3 (mean 71.8, SD 111 m3). The interiors of the offices were primarily painted drywall (n=142, 71%) or drywall covered with wall paper (n=40, 20%). The remainder of the spaces were finished with a variety of materials including concrete, wood paneling or other (n=18, 9%). The primary material used for ceilings was cellulose acoustic tile (n=166, 81%) followed by painted drywall (n=19, 9%) or a variety of other materials (n = 21, 10%). The majority of offices were carpeted (n = 168, 79%), while 19% had linoleum as the floor treatment. A minority of spaces had wood or ceramic floors (n = 5, 2%).

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Ventilation The majority of office spaces were mechanically ventilated using HVAC systems (75%). Of the naturally ventilated offices, 16% (n=34) had windows open on the day of sampling. The environmental comfort parameters for the test sites are summarized in Table 5. Table 5. Environmental comfort parameters (June – October 2001) Parameter Number Mean (SD) Minimum Maximum Indoor Carbon dioxide (ppm) Temperature (oC) Relative humidity (%) Outdoor Carbon dioxide (ppm) Temperature (oC) Relative humidity (%)

209 212 209

644 (148) 23.8 (1.8) 40.8 (6.6)

430 18.7 25.5

1127 29.1 56.6

36 74 36

458 (43.6) 17.5 (3.0) 46.3 (10.2)

377 8.6 24.5

529 23.3 71.1

Only a minority of test sites had living plants in the offices (73, 33%,) Most of the offices visited were free of signs of moisture or moisture stains (90%).

Bioaerosol concentrations A maximum of 592 samples (296 sequential duplicates) and 74 field blanks were available for analysis. The SAS was sent away for repairs during the study, resulting in a total of 552 samples for this instrument. The distribution of the samples is summarized in Table 6. Table 6. Summary of samples analyzed. Sample type N6, RCS, & AOC SAS Ergosterol & BDG

Field Blanks

Overall Total

74

Indoor Common Room

Room 1

Room 2

Indoor Total

Outdoor

296

74

74

74

222

74

69

276

69

69

69

207

69

74

296

74

74

74

222

74

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The bioaerosol data were lognormally distributed. Counts were transformed to the natural log for analysis using parametric statistics. Table 7 reports the geometric mean concentrations for each room type for each sampling instrument. No significant differences in concentrations between indoor locations were found when analyzed by one-way ANOVA with the Bonferroni post-hoc adjustment for multiple comparisons. Therefore, the three indoor sites were grouped together for all subsequent analyses. Table 7. Geometric mean concentrations by location Room Type – Geometric Mean (GSDa) Sampler Common Room Room 1 Room 2 Outdoor 3 71 (4.3) 64 (3.9) 68 (4.0) 691 (2.3) N6 (CFU/m ) 17 (3.8) 16 (3.0) 17 (2.8) 175 (2.7) SAS (CFU/m3) 3 108 (2.5) 112 (2.7) 126 (2.3) 550 (1.8) RCS (CFU/m ) 906 (3.6) 998 (3.5) 1,042 (3.3) 10,577 (2.4) AOC (Spores/m3) < 22b < 22 Ergosterol (ng/m3) < 0.125 < 0.125 BDG (ng/m3) a Geometric standard deviation b filters from three rooms pooled for analysis

Descriptive Statistics The geometric means, their 95% confidence intervals, standard deviations, arithmetic means and ranges for each method, are shown for indoor samples (Table 8) and for outdoor samples (Table 9) and illustrated by Figure 1. Table 8. Indoor geometric means with 95% CI, arithmetic means and ranges 95% CIc

Meand (SDe)

Range

115 (2.5) 102-130 RCS (CFU/m3) 3 68 (4.1) 56-82 N6 (CFU/m ) 17 (3.2) 14-20 SAS (CFU/m3) 3 980 (2.4) 832-1,155 AOC (Spores/m ) < 22 Ergosterol (ng/m3) < 0.125 BDG (ng/m3) a Geometric Mean b Geometric Standard Deviation c 95% Confidence Interval for the geometric mean d e Arithmetic Mean Standard Deviation

164 (142) 168 (277) 42 (145) 2,118(3,578)

8-984 3.5-2,484 3-1,991 21-29,555

Instrument

GMa (GSDb)

20

Table 9. Outdoor geometric means with 95% CI, arithmetic means and ranges Instrument

GMa (GSDb)

95% CIc

550 (1.8) 478-634 RCS (CFU/m3) 3 691 (2.3) 567-841 N6 (CFU/m ) 175 (2.7) 138-223 SAS (CFU/m3) 3 8,631-12,962 AOC(Spores/m ) 10,577 (2.4) < 22 Ergosterol (ng/m3) BDG (ng/m3) a Geometric Mean b Geometric Standard Deviation c 95% Confidence Interval of the geometric mean d Arithmetic Mean e Standard Deviation

Range

651 (366.9) 986 (1,015) 308 (548) 15,125 (13,759)

141-1,130 60-7,039 18.5-4,394 886-69,286

Indoor Outdoor

Mean Spores/m3

Mean CFU/m3

10000

Meand (SD)e

1000

100

10

RCS

N6

SAS

AOC

Figure 1. Geometric Means with upper 95% confidence intervals

Limits of Detection Table 10 summarizes the upper and lower detection limits and the proportion of samples that were outside detection limits for each instrument.

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Table 10. Proportion of samples beyond detection limits Instrument LOD # of UDL # of samplesUDL (% total samples) (% total samples) 7 CFU/m3 24 (4.1) 18,572 0 (0) N6 3 CFU/m 6 CFU/m3 84 (15.2) 7,471 CFU/m3 0 (0) SAS 3 3 6 CFU/m 7 (1.2) 1,125 CFU/m 25 (8.4) RCS AOCa 11 spores/m3 0 (0) NA 0 (0) ·Indoor 22 spores/m3 0 (0) NA 0 (0) ·Outdoor 22 ng/m3 74 (100) NA Ergosterol 0.125 (ng/m3) 74 (100) NA BDG a Air volume sampled for indoor samples (150 L) different from outdoor samples (75 L)

Reproducibility of Sequential Duplicates The arithmetic mean and median of the coefficients of variation for each sequential duplicate sample for each instrument, stratified into indoor and outdoor values, are presented in Table 11. A significant difference between indoor and outdoor coefficient of variation (CV%) was found for all methods (with indoor>outdoor, pN6=RCS>AOC, for indoor and for outdoor, SAS>N6=RCS=AOC. Table 11. Reproducibility - Coefficient of Variation (%) Instrument Mean CV % (SD) Indoor

Outdoor

p-value*

Range Indoor

32.2 (28.3) 19.1 (22.4)