EPA/600/A-95/001
EVALUATION OF FUNGAL GROWTH (PEN1CILUUM G LAB RUM) ON A CEILING TILE
J. C. S. Chang, U. S. Environmental Protection Agency, Research Triangle Park, NC 27711, USA
K. IC Foarde and D. W. VanOsdeU, Research Triangle Institute, Research Triangle Park, NC 27709, USA
ABSTRACT
Laboratory studies employing static chambers have been undertaken to study the impact of different
equilibrium relative humidities (RHs) and moisture conditions on the ability of a new ceiling tile to
support fungal growth. Amplification of the mold, Penicillium glabrum, occurred at RHs above 85 to
90%. Conversely, at lower RHs, decreases were detected. The issue of survival vs. die-off may be
important in the control of fungal contamination in building materials.
KEYWORDS
biocontaminant, fungi, building materials, chambers, moisture, relative humidity, control
INTRODUCTION
With individuals spending as much as 90% of their day indoors (Ott, 1988), exposure of building occu-
pants to biological contamination of the indoor environment is a major health concern (Miller, 1990).
Although some biocontaminants are transported indoors from outdoors, many are produced or amplified
indoors. Building materials that have become contaminated and sustain a population of fungi are a
significant source of indoor air contamination, and a number of investigators have studied fungal growth,
water retention, and their interactions in buildings and building materials (West and Hansen, 1989;
Coppock and Cookson, 1951). Contaminated ceiling tiles (Morey, 1988), as well as contaminated
accumulated ceiling tile dust (Striefel, 1988), may pose a serious health threat.
Laboratory static chamber studies were undertaken to study the impact of different environmental factors
on the ability of building materials to support fungal growth and amplification. This method has been
demonstrated to be useful for the evaluation of microbial growth on materials (Foarde et al.t 1992).
Previous results have demonstrated both organism and substrate differences. For instance, at high RH
(97%), Penicillium glabrum was able to grow and amplify on both used and most new tiles, while
Aspergillus versicolor was not (Foarde et al., 1993). New experiments have been undertaken at a range
of equilibrium RHs from 54 to 97% and the corresponding material moisture contents. The objective of
the experiment described in this paper was to determine the impact of a range of RHs on the ability of
the test organism to grow on new ceiling tiles.
MATERIALS AND METHODS
Modified acrylic-walled desiccators (32 x 39 x 51 cm) were used to provide controlled environments for
the fungal growth tests. Saturated salt solutions maintained specific RHs of 54, 70, 85,90, 94, and 97%
within each chamber (ASTM E104-85), which were in a dark, temperature-controlled (21 ± 3°C) room

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during the experiments. Each static chamber was equipped with a hygrometer and three shelves on which
building mawrial samples were placed.
Samples of one type of new, Class A (light-commercial or residential use) ceiling tile were evaluated.
It was fire-resistant, acoustical, washable, and composed of 20 - 60% mineral wool fiber and 4 - 10%
hydrous aluminum silicate. The ceiling tiles were obtained as 30.5 x 61 cm boards and cut into 3.8 cm2
blocks that served as the actual test samples.
The moisture content (MC) of the ceiling tile blocks was determined gravimetrically and reported as
percent water on a dry basis. An alternative measure of moisture is water activity (a^). Corry (1987)
stated that aw is the proportion of "available water for biological reactions." a^ is a useful laboratory
measurement with limited field use. The ability of microorganisms to grow on foods has been related
to water content through a,, (Pitt, 1981). Because the ceiling tile blocks were at equilibrium with the
chamber RHs, aw was, by definition, equal to RH/100%. For porous materials, MC and a^ are related
through the water adsorption isotherm, and different relationships are obtained for different materials.
Because nutrient content varies widely for various building materials (and between clean and dirty
materials), no single moisture measurement will unequivocally indicate whether microorganisms will
grow in a particular situation or on a material. We have chosen to use MC in the present study because
it is more common in the building industry, although aw is easily calculated from the chamber RHs.
P, glabrum, purchased from the American Type Culture Collection (ATCC) as P. aragonense (ATCC
#4228) and re-identified by R.A. Samson of the Centraalbureau voor Schimmelcultures, Baarn, The
Netherlands, was employed as the test organism. P. glabrum has been isolated from the indoor
environment (Samson and van Reenen-Hoekstra, 1992), and has also been proposed as a causative agent
of asthma in a saw mill (Comptois and Malo, 1990).
To study fungal growth at a single RH, chamber-conditioned (for 72 h), sterile ceiling tile blocks were
inoculated (on the non-white, unfinished side) with approximately 1.0 x 105 colony forming units (CFUs)
suspended in 10 of sterile water and placed in the static chambers. Uninoculated control blocks were
also placed in each chamber. Triplicate blocks were removed for quantitation on days 1,7, 14,21, and
28. To quantify the growth, the blocks were removed from the chambers, weighed, and placed in sterile
receptacles containing phosphate-buffered saline with 0.1% Tween 80. The filled receptacles were
shaken on a wrist-action shaker for 30 minutes to facilitate extraction, then the extract was diluted and
plated on Sabouraud dextrose
agar. Plates were incubated at
room temperature for at least 1
week. CFUs were quantitated
shortly after visible growth was
first observed and again as
moderate growth became
apparent.
RESULTS AND DISCUSSION
The water adsorption isotherm
data for this ceiling tile over the
test RH range of 54 to 97% are
presented in Fig. 1. The MC was
constant at 2% at the lower RHs
of 54 and 70%, demonstrated a
7.5




-


i

6.5
-




h
-


I

j4"5
-




i s
I35
-


i
i

2.5
-




1



i

1.5

, , i . .
, , i , ,






64
69
64
68 74 79 84 88 94 89




Relative Humidity, percent
Figure 1. Water adsorption data for new Class A ceiling tile

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slight increase at 85 and 90%, followed by a much steeper increase to 94 and 97% RH. At 90% RH the
moisture content was 3.8%, while at 94 and 97% RH, the MC was 5.1 and 7.2%, respectively.
Table 1 shows the static chamber test results for new Class A tiles inoculated with P. glabrum. No
growth or a decrease in CFUs below the detection limit (2.5 log CFUs) was obtained within the test
period in chambers with RH s 85%. Fungal amplification (an increase in CFUs by at least 1 order of
magnitude in the test period) was measured in chambers with RHs of 90% and greater. The minimum
RH for growth was therefore between 85 and 90%.
Table 1. Mean log CFUs (± 1 standard deviation) for P. glabrum
DAY
54% RH
70% RH
85% RH
90% RH
94% RH
97% RH
1
4.1 ±0.1
3.9 ±0.1
4.0 ±0.1
4.1 ± 0.2
4.0 ±0.3
4.0 ±0.3
7
3.7 ±0.1
3.2 ±0.3
4.3 ± 0.7
4.0 ± 0.2
4.3 ±0.2
5.1 ±0.1
14
3.1 ±0.5
^2.5 ± na
4.1 ±0.5
4.9 ± 0.2
5.5 ±0.1
5.7 ±0.1
21
3.1 ±0.2
<,2.5 ± na
<2.5 ± na
5.5 ±0.2
6.0 ±0.2
6.2 ±0.1
28
*2.5 ± na
<,2.5 ± na
*2.5 ± na
5.4 ±0.2
6.0 ±0.1
6.2 ±0.2
A comparison of the data in Fig. 1 and Table 1 shows that the minimum MC required for the organisms
to grow on this type of ceiling tile should be between 3.6 and 3.8%, corresponding to an aw of between
0.85 and 0.90, or RH between 85 and 90%. The minimum a,, for germination of P. glabrum has been
shown to be 0.81 at 23 °C (20 days). However, sporulation occurred only when the a,, was at or above
0.86 (10 days, 23°C) (Mislivec and Tuite, 1970). The results of this experiment on ceiling tile agree with
that result.
In the building and building materials industries, MC is the common measurement of a material's
moisture. It is a practical and rapid measurement. It has been reported that a minimum MC of 10% was
required for fungal growth on substrates such as leather, wool, cotton, wood, and cheese, and that a lower
MC would limit fungal growth (Block, 1953). However, the data from this experiment indicate that the
minimum MC for fungal growth on the ceiling tiles tested was considerably less than 10%. The
differences in minimum MC required for fungal growth on ceiling tiles and other substrates reflect the
fact that substrate materials can have significant impacts on the conditions required for fungal
amplification.
CONCLUSIONS
Previous results demonstrated that new ceiling tiles were able to support fungal growth at high RHs
(97%) (Foarde et ai, 1993). That work also showed that used ceiling tile samples were more susceptible
to fungal growth than the new ones. The data in the current experiment indicate that fungal growth (as
measured by sporulation) can occur in ceiling tiles at equilibrium with RHs between 85 and 90%.
Block (1953) and Pasanen et al„ (1991) examined the influence of RH and MC on fungal growth and
suggested that the RH of air did not directly influence the growth of fungi. Previous static chamber work
by Foarde et ai, (1992) at non-equilibrium conditions showed that fungal growth appeared to correlate
with material MC better than it did with chamber RH. The current data, generated under equilibrium
conditions (in which case the MC and RH relationship is fixed by the adsorption isotherm such as the one
in Fig. 1), showed that fungal growth correlated well with both MC and RH. In summary, under
3

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equilibrium conditions, RH (or aj and material MC are both effective measurements of the availability
of water for fungal growth. However, under non-equilibrium conditions fungal amplification may be
more closely correlated with material MC than R.H.
In addition, and perhaps even more importantly, when conditions were unfavorable for growth (insuf-
ficient water in tiles at equilibrium with RHs s 85%), culturable CFUs decreased more than 1 order of
magnitude. Similar results were reported previously for wetted tiles that were dried rapidly (Foarde et
al., 1992). When the MC was lowered to below 3% within 3 days, culturable CFUs decreased almost 2
orders of magnitude within 10 days. This result is presumably a consequence of spore germination and
subsequent inhibition of the resultant vegetative growth. Survival vs. die-off would appear to be an
important element in the control of fungal contamination of building materials.
REFERENCES
ASTM E 104-85 (1991). Standard practice for maintaining constant relative humidity by means of
aqueous solutions. ASTM Annual Book of Standards, Section 11. 11.03. 496-498.
Block, S.S. (1953). Humidity requirements for mold growth. Envir. Micro., pp. 287-293.
Comptois, P. and J.L. Malo (1990). The air spora of East-Canadian sawmills. In: Indoor Air '90
Proceedings, 2:109-114.
Coppock, J.B.M. and E.D. Cookson (1951). The effect of humidity on mould growth on construction
materials. J. Sci. FoodAgri., 2, 534-537.
Cony, J. (1987). Relationships of water activity to fungal growth. Food and Beverage Mycology, pp.
51-99.
Foarde, K., E. Cole, D. VanOsdell, D. Bush, D. Franke, and J. Chang (1992). Characterization of
environmental chambers for evaluating microbial growth on building materials. In: Proceedings of
ASHRAE/ACGIH/AIHA Conf. IAQ '92: Environments for People, San Francisco, CA, pp. 185-189.
Foarde, K., P. Dulaney, E. Cole, D. VanOsdell, D. Ensor, and J. Chang (1993). Assessment of fungal
growth on ceiling tiles under environmentally characterized conditions. In: Proceedings of Indoor
Air '93, 6* Int'i. Conf. on Indoor Air Quality and Climate, Helsinki, Finland, 4:357-362.
Miller, D.J. (1990). Fungi as contaminants in indoor air. In: Proceedings of Indoor Air '90: Indoor Air
Quality and Climate, 5* Int'l. Conf., Toronto, Canada, 5:51-64.
Mislivec, P.B. and J. Tuite (1970). Temperature and relative humidity requirements of species of
Penicillium isolated from yellow dent corn. Mycologia, 32:75-88.
Morey, P.R. (1988). Microorganisms in buildings and HVAC systems: a summary of 21 environmental
studies. In: Proceedings of ASHRAE Conference IAQ '88: Engineering Solutions to Indoor Air
Problems, Atlanta, GA, pp. 10-21.
Ott, W.R. (1988). APCA tech. paper. Human Exposure to Environmental Pollutants, 1,16:88-115
Pasanen, A.L., P. Kalliokoski, and P. Pasanen (1991). Laboratory studies on the relationship between
fungal growth and atmospheric temperature and humidity. Envir. Int'l., 12, 225.
Pitt, J. (1981). Food spoilage and biodeterioration. In: Biology of Conidial Fungi (G.G. Cole and B.
Kendrick, Eds.). Academic Press, London, England, 2, 18:111-142.
Samson, R.A. and E.S. van Reenen-Hoekstra (1992). A compilation of the fungal species in the indoor
environment. In: Proceedings of Int'l Workshop: Health Implications of Fungi in Indoor
Environments, Baarn, The Netherlands, p. 27.
Striefel, A. (1988). Aspergillosis and Construction. Architectural Design and Indoor Microbial
Pollution (Ruth B. Kundsin, Ed.), Oxford Univ. Press, pp. 198-217.
West, M.K. and E.C. Hansen (1989). Determination of material hygroscopic properties that affect indoor
air quality. In: Proceedings of ASHRAE Conf., IAQ '89 ¦ The Human Equation: Health and Comfort,
San Diego, CA, pp. 60-63.	4

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,pPDt n ion TECHNICAL REPORT DATA
A£iXlirC.[_i~ 1 ~ LZl ( (I'lcasc read f/isinicliom on the reverse before compiel

1 RtPORT NO.
EPA/600/A-95/001
2.
3.
4. TITLE AKO SUBTITLE
Evaluation of Fungal Growth (Penicillium Glabrum)
5- REPORT OATE
on a Ceiling Tile


6. PERFORMING ORGANIZATION CODE
7. author(S!j_ Chang (EPA) and K. Foarde and
(RTI)
D. VanCsdell
8 PERFORMING organization report NO.
9. PERFORMING OBOAMZATION NAME AND ADDRESS
Research Triangle Institute
P. C, Box 12194
Research Triangle Park, North Carolina

10, PROGRAM ELEMENT NO.
27709
n. contract/grant no,
CR822642-01
12. SPONSORING AGENCY NAME AND ADDRESS
EPA, Office of Research and Development
Air and Energy Engineering Research Laboratory
Research Triangle Park, NC 27711
13. TYPE OF REPORT AND PERIOD COVEREO
Published paper; 6— 9/94
14. SPONSORING AGENCY CODE
EPA/600/13
15. supplementary notes AEERL project officer is John C. S. Chang, Mail Drop 54, 919/541-
3747. Presented at Indoor Air International Workshop, Indoor Air, an Integrated
Approach, 11/2 7-12 /1/94. Milton. Queensland. Australia.
¦ The paper gives results of a study employing static chambers to study the
impact of different equilibrium relative humidities (RHs) and moisture conditions on
the ability of a new ceiling tile to support fungal growth. Amplification of the mold,
Penicillium glabrum, occurred at RHs above 85-90%. Conversely, at lower RHs,
decreases were detected. The issue of survival vs. die-off may be important in the
control of fungal contamination in building materials.
17.
KEY WORDS AND DOCUMENT ANALYSIS


a. DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
c. COSaTI Field/Group
Pollution
Fungi
Construction Materials
Test Chambers
Moisture
Humidity
Pollution Control
Stationary Sources
Ceiling Tile
Biocontaminants
13 B
06 C
13	C
14	B
07D
04B
13. DISTRIBUTION statement

19. security CLASS (This Report/
Unclassified
21. NO. OF PAGES
Release to Public

20. SECURITY CLASS (This page)
Unclassified
22. PRICE
EPA Form 2220 1 (9-73)

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