FACTORS RELATING TO THE RELEASE OF STACHYBOTRYS
CHARTARUM SPORES FROM CONTAMINATED SOURCES
K.K. Foarde1 and M.Y. Menetrez2
'Microbiology Department, Center for Engineering Technology, Research Triangle Insti-
tute, Research Triangle Park, NC 27709, USA
2Air Pollution Prevention and Control Division, National Risk Management Research
Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711,
USA
ABSTRACT
Although traditionally, fungi as building contaminants have primarily been viewed as al-
lergens, adverse health effects resulting from inhalation of fungal spores are likely due to
multiple factors. One factor is the mycotoxins produced by some fungi. This paper de-
scribes the preliminary results of a research project to determine the factors that control the
release of S. chartarum spores from a contaminated source and test ways to reduce spore
release and thus exposure. As anticipated, S. chartarum spore emissions from gypsum
board at low flow are directly proportional to airflow and indirectly proportional to relative
humidity and~support our previous observations with Penicillium and Aspergillus. The re-
lationship between the culturable colony-forming units (CFUs) and total spores varied over
time and needs further investigation, but suggests one reason that correlation between air-
borne field measurements (usually only of culturable organisms) and possible exposure is
so difficult.
KEY WORDS
Bioaerosol, chamber study, duct, emissions, microbial contamination
INTRODUCTION
Although traditionally, fungi as building contaminants have primarily been viewed as al-
lergens (and occasionally, pathogens), adverse health effects resulting from inhalation of
fungal spores are likely due to multiple factors (Miller 1992). One factor associated with
certain fungi is low molecular weight toxins (mycotoxins) produced by these fungi. Tradi-
tionally, mycotoxins are important to human and animal health because of their production
by toxigenic fungi associated with food and feed. However, mycotoxins tend to concen-
trate in fungal spores (Sorenson et al. 1987), and thus present a potential hazard to those
inhaling airborne spores. Toxigenic spores strongly affect alveolar macrophage function
and pose a threat to individuals inhaling mycotoxin-contaminated material. A number of
reports have indicated that Stachybotrys chartarum and several toxigenic species of Peni-
cillium and Aspergillus are potentially hazardous. The mycotoxins found in indoor air are
most likely contained in the aerosolized spores or spore fragments of the toxigenic fungi
(Miller 1992).
This paper describes the preliminary results of a research project to determine the factors
that control the release of 5. chartarum spores from a contaminated source and test ways to
reduce spore release and thus exposure. Although there have been numerous reports of S.

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chartarum growing on a variety of building and structural materials that resulted in con-
tamination of buildings and sick individuals, information on what environmental condi-
tions permitted their growth has been limited. Furthermore, S. chartarum can be difficult
to isolate in air samples. A key issue is understanding how exposure occurs.
MATERIALS AND METHODS
The experiments were conducted in the Dynamic Microbial Test Chamber (DMTC)
(VanOsdell et al. 1996). The DMTC is a room-sized test facility designed and constructed
to conduct studies on the conditions and factors that influence biocontaminant emissions
and dissemination. The chamber, a cube with inside dimensions of 2.44 m, was con-
structed with stainless steel walls and floor, and an acrylic drop-in ceiling. Temperature
(18- 32°C) and relative humidity (RH) (55 to 95%) control is provided through an air
handler unit (AHU) with an air circulation rate of 1.4 to 4.8 m3/min. All air in the DMTC
is filtered in a high-efficiency particulate air (HEP A) filter downstream of the cooling coil
and contains essentially no particles larger than 1 |im in aerodynamic diameter.
The DMTC was adapted to contain eight
room-wall simulators (RWS) in which the
S. chartarum was allowed to grow and
then, with minimal physical disturbance,
release spores. Figure 1 is an artist's rendi-
tion of the DMTC containing the RWS and
the sampling apparatus used to measure
spore emissions. For clarity, only three of
the eight RWS are shown. The DMTC is
used for containment, and as a source of
conditioned air for the eight RWS.
The RWS (17.1 cm2 cross section by 161.7
cm long duct sections) were constructed of
16 gauge stainless steel. The front cover was
glass to permit visual monitoring of growth
on the test material. The inlet and discharge
ends of the RWS transition to 2.5 cm tubes.
Each RWS was connected to a pressurized plenum fed by a blower drawing clean condi-
tioned air from the DMTC. A single piece of gypsum board 107 x 42 cm (4494 cm2) was
scored length-wise to permit it to be folded into a three-sided trough that fit into and
formed the interior walls of the RWS. Air from the plenum entered each RWS through the
inlet transition, flowed down along the gypsum board walls, and discharged through the
outlet transition into the DMTC. As shown in Figure 1, bioaerosol samplers periodically
drew samples from the air leaving the RWS.
The gypsum board pieces were loaded into the RWS and the entire assembly autoclaved.
Each 4494 cm2 gypsum board piece was wetted with 200 mL of sterile water three times
for a total of 600 mL. After each wetting, the water was allowed to soak into the gypsum
board. Preliminary experiments showed that the water wicked throughout the entire piece
and that a uniform distribution of water over all surfaces was easily achieved. The goal
was to simulate a catastrophic wetting of the material. The gypsum board was then inocu-
Figure 1. Artist's rendition of DMTC
with room-wall simulators (RWS).
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lated with 12.9 mL of 106 CFU/mL S. chartarum manually pipetted in 15 |iL spots uni-
formly distributed across the surface of the gypsum board.
The RWS were sealed, and growth was allowed to proceed under static conditions for at
least 1 month until relatively heavy growth was visible. The RWS were then mounted on a
rack within the DMTC. The air velocity was set to either < 10 or 35 cm/s, on the order of
velocities encountered in the conditioned indoor space (ASHRAE 55-1992). The RWS
were designed so that the air flowed down to more closely mimic wall contamination.
The temperature in the RWS was maintained at 23.5°C. At the start of an emission rate
determination, the DMTC RH was lowered to the test RH (i.e., 64%) by lowering the RH
setting on the AHU. The RH change was effective within minutes. Isokinetic air samples
were collected using the Mattson-Garvin slit to the agar sampler for culturable fungi and
Air-O-Cells (Zefon Analytical Accessories, Saint Petersburg, FL) for total spore counts.
Surface samples, collected only at the conclusion of all the air sampling, were obtained by
vacuuming 10 cm2 sections of the gypsum board.
The Mattson-Garvin draws air at 28.3 L/min through a metal inlet with a 0.015-cm slit,
allowing the impaction of airborne organisms on the surface of a rotating 150-mm agar
plate. The sampler plates were incubated at room temperature. CFUs were counted shortly
after visible growth was first noted and again as moderate growth became apparent.
Air-O-Cells are preloaded cassettes containing a glass slide coated with a sticky impaction
medium. The base of the cassette was connected to a pump using flexible tubing, and air
was drawn onto the impaction surface at 28.3 L/min through a slit in the top of the cas-
sette. Total airborne spores were quantified by opening the Air-O-Cell and removing the
internal glass slide containing the impaction medium. The slide was placed onto a micro-
scope slide and stained with lacto-glycerol. Total airborne spores were counted micro-
scopically.
Calculation of Emission Rates
To calculate the emission rates for the culturable organisms, the CFUs on the sampler
plates were enumerated, and the CFUs/min were determined. For total spores, the
spores/min were calculated. Both values were adjusted for the total flow rate, and divided
by the area of the emitting surface.
RESULTS AND DISCUSSION
Previous experiments showed that the spore emission rate for Penicillium chrysogenum
and Aspergillus versicolor were directly related to airflow and indirectly related to relative
humidity (Foarde et al. 1999). As the air velocity increased across the surface of the duct,
emissions increased. As the RH was lowered, the emission rate increased for both organ-
isms. The first step was to determine whether the spore emission rate for S. chartarum
would follow a similar pattern.
The earlier work with P. chrysogenum and A. versicolor was performed under duct flow
conditions because these organisms frequently contaminate fiberglass duct liner. Although
there are reports on S. chartarum growing on fiberglass duct liner, for this series of ex-
periments we selected gypsum wallboard as the test material. The duct airflow levels that
n
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we used previously were not appropriate for wall air flow. For this preliminary work, we
selected two air velocities: 10 cm/s, which we consider very low velocity, and 35 cm/s or
low velocity.
Table 1 shows the results of a series of experiments at very low and low air velocity in the
RWS. The emission rates are expressed as CFU/m2/min. The first-hour emission rates at
each RH are shown in the table and confirm that S. chartarum spore emissions increased
with increased airflow and as the humidity was lowered.
Table 1. Culturable CFUs in CFlJ/niVmin emitted at two airflows at four relative
humidities.
Airflow,
cm/s
Relative Humidity, %
95
85
75
65
< 10
0
0.4
25
50
35
4
29
65
200
The second set of experiments were designed to quantify emissions at 65% RH over an
extended period. The previous study, with P. chrysogenum and A. versicolor, showed that
emissions continued for at least 17 hours. For S. chartarum, emissions were measured until
they were below the detection limit (< 5 CFU/m2/hr).
10000
1000
100
10
1
10
15
0
5
Day
Figure 2. S. chartarum CFU emissions from four RWS at 65% RH and 10 cm/s
airflow.
Figure 2 shows the results from four RWS. Note that the surface concentrations of the
wallboard in the four RWS were essentially the same, ranging from 5 x 104 to 5 x 105
CFU/102 cm2. What may be more important is the age of the growth on the RWS. RWS 1
through 3 were 2 months old when the emission experiments were initiated, whereas RWS
4

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4 was 3 months old. We are continuing to investigate the surface-to-airbome concentration
relationship.
As can be seen in Figure 2 spore emissions from RWS 1 through 3 started at levels be-
tween 500 and 1,000 CFU/m2/hr on day 0 and tapered off by day 2 or 3. RWS 4 started at
the same initial level as RWS 1, 2, and 3 but continued to emit that same level of spores
for the entire first week before the level started to decrease.
The next set of experiments was designed to investigate the emission of spores at RH be-
low 65%. The low air velocity of 35 cm/s was used for this experiment. The RH was al-
lowed to range from 25 to 64%, the normal range of room humidities in controlled envi-
ronments.
Figure 3 shows the hourly emission rates of both culturable CFUs and total spores/m2 of
gypsum board from one RWS (3-month age of growth). Culturable CFUs, designated by
the diamonds, are plotted on the left y-axis. Total spores are plotted on the right y-axis
with squares. Figure 3 shows fairly high levels of emissions, continuing for at least 80
days.
1,000,000
100,000
a 10,000 f
,000
100
10
1
• -S
1 p
1 to
I O
1

oo

0
-A>-
- Culturable CFUs
¦ Total Spores
1,000,000 i
100,000 |
100
10
1
10
20
30
40 50
Day
60
70
H !
Figure 3. S. chartarum spore emissions at RHs between 25 and 64% at low air flow (35
cm/s).
The data show an interesting relationship emerging between the numbers of culturable
CFUs and the numbers of total spores enumerated. For the first 30 days, the CFUs ap-
peared to be about 10% of the number of total spores. Then the levels of culturable CFUs
and total spores seem to converge. Figure 4 shows the percentage of culturable CFUs to
total spores over the nearly 3-month experiment.
Figure 4 shows that, for the first 30 days, the culturable CFUs are approximately 10% of
the total spores. For the next 30 days (days 30-60), the percentage of culturable spores in-
5

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creased notably compared to the first 30 days. For the final 25 days, the percentage of cul-
tivable CFUs was lower than the second 30 days, but higher than the first 30 days. We are
continuing to investigate.
Figure 4. Percent of the total spores that are culturable CFUs.
CONCLUSIONS AND IMPLICATIONS
Significant progress has been made on elucidating release factors and the potential impli-
cation for exposure. As anticipated, S. chartarum spore emissions from gypsum board at
low flow are directly proportional to airflow and indirectly proportional to RH and support
our previous observations with Penicillium and Aspergillus. The relationship between the
culturable CFUs and total spores needs further investigation, but suggests one reason that
correlation between airborne field measurements and possible exposure is so difficult.
Generally, field measurements consist of collecting culturable CFUs. Additional measure-
ments, such as quantifying total spores, are needed.
ACKNOWLEDGMENTS
The authors greatly appreciate the technical assistance of Tricia Webber in collecting the
data, and Douglas VanOsdell for designing the room wall simulators, both of the Research
Triangle Institute. This project was funded under cooperative agreement CR-827977-01
between EPA and RTI.
REFERENCES
ASHRAE. 1992. ANSI/ASHRAE Standard 55-1992, Thermal Environmental Conditions
for Human Occupancy, Atlanta: ASHRAE.
Foarde, K. K., D. W. VanOsdell, M.Y. Menetrez, and J.C. S. Chang. 1999. Investigating
the Influence of Relative Humidity, Air Velocity, and Amplification of the Emission
Rates of Fungal Spores. Indoor Air 99. 2:507-512.
Miller, J. D. (1992). Fungi as contaminants in indoor air. Atm Environment 26: 2163-2172.
Sorenson, W.G., D.G. Frazer, and B.B. Jarvis. 1987. Trichothecene mycotoxins in airborne
conidia of Stachybotrys atra. App Env Microbiology. 53: 1370-1375.
VanOsdell, D., K. Foarde, and J. Chang. 1996. Design and Operation of a Dynamic Test
Chamber for Measurement of Biocontaminant Pollutant Emission and Control. ASTM
STP 1286 Methods for Characterizing Indoor Sources and Sinks, pp. 44-57.
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TECHNICAL REPORT DATA
NRMRL-RTP-P- Q (Please read Instructions on the reverse before completing)
1, REPORT NO. 2,
F.PA / 60(1 / A—07. / 083
3, RE*
4. TITLE AND SUBTITLE
Factors Relating to the Release of Stachybotrys
Chartarum Spores from Contaminated Sources
5- REPORT DATE
6. PERFORMING ORGANIZATION CODE
7. AUTHORiSI
K.K. Foarde (RTI) and H.Y. Menetrez
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Research Triangle Institute
Center for Engineering Technology
P.O. Box 12194
Research Triangle Park, North Carolina 27709
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
EPA Cooperative Agreement
CR 827977
12. SPONSORING AGENCY NAME AND ADDRESS
EPA, Office of Research and Development
Air Pollution Prevention and Control Division
Research Triangle Park, NC 27711
13. TYPE OF REPORT AND PERIOD COVERED
Published Paper; 10/99-9/02
14. SPONSORING AGENCY CODE
EPA/600/13
ts.supplementary notes APPCD project officer is Marc Y. Menetrez, MD-E305-03, phone 919/
541-7981. Indoor Air 2002, Monterey, CA, 6/30-7/5/2002.
is. abstract ^ descrlb€!S preliminary results of a research project to determine
the factors that control the release of S. chartarum spores from a contaminated source
and test^ways ttTreauce spore release and thus exposure. As anticipated, S. chartarum
spore emissions from gypsum board at low flow are directly proportional to airflow and
indirectly proportional to relative humidity and support our previous observations
with Penicillium and Aspergillus. The relationshiD between the cultural rnlnnv-fnrririno
units and total spores varied over time and needs further investigation, but suggests
one reason that correlation between airborne field measurements (usually only of cul-
turable organisms) and possible exposure is so difficult. (NOTE: Although tradition-
ally, fungi as building contaminants have primarily been viewed as allergens,
adverse health effects resulting from inhalation of fungal spores are likely due to
nulti-gle factors. One factor is the mycotoxins produced by sane fungi.)
17. KEY WORDS AND DOCUMENT ANALYSIS
a. DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
c. COSATi Field/Group
Pollution
Aerosols
Spores
Microorganisms
Chambers
Ducts
Emission
Pollution Control
Stationary Sources
Microbial Contamination
Bioaerosols
13B
07D
06C
06M
14G
13K
18. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (This Report)
Unclassified
21, NO. OF PAGES
6
20. SECURITY CLASS (This page)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)

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