EPA/600/AT93/184
ASSESSMENT OF FUNGAL GROWTH ON CEILING TILES UNDER
ENVIRONMENTALLY CHARACTERIZED CONDITIONS
K. Foarde1, P. Dulaney1, E. Cole1, D. VanOsdell1, D. Ensor1, and J. Chang2
Research Triangle Institute, Research Triangle Park, NC, USA
2U.S. Environmental Protection Agency, Research Triangle Park, NC, USA
ABSTRACT
The impact of the building environment on the ability of building materials to support
microbial growth is being investigated using static chambers with defined relative humidity,
temperature, and light conditions. The ability of fungi to grow on materials, given sufficient
moisture and nutrition, is well established. Four different types of ceiling tile were investi-
gated -- one aged, two mineral fiber/crystalline silica, and one fire-resistant. Ceiling tile
pieces were placed in the chambers, allowed to chamber-equilibrate, and inoculated with either
Penicillium glabrum or Aspergillus versicolor. The critical bulk moisture content for organism
growth varied depending upon tile type as well as test mold. P. glabrum counts increased
three orders of magnitude in 28 days. A. versicolor did not grow as well. Organism related
differences are apparent.
INTRODUCTION
A building is not a sterile environment, nor should it be expected to be. In fact, a building
is frequently a reservoir for biocontaminants. The ability of fungi to grow on materials, given
sufficient moisture and nutrients, is well established; however, not all organisms grow under
the same conditions. Each microorganism has a specific environmental tolerance range.
Outside this range, the microorganism may survive in a dormant spore state or may die.
Viable spores can result in significant biocontamination should environmental conditions
become favorable.
Availability of moisture is key for microbial growth in the indoor environment. The
relative humidity (RH) requirement for germination varies for different species (1,2). It has
been suggested that water, more than nutrients, is the primary factor in spore germination, but
nutrients are more important for development (1).
Pasanen, et al. (3) showed that the moisture content of a substrate is a major determinant
of fungal growth and that RH exerts only an indirect influence. However, RH has been shown
to be the most important factor in determining the moisture content of organic materials (4).
Hygroscopic furnishings can change the indoor RH by as much as 15 to 20% for as long as
1 month (5). Materials that are the most hygroscopic are the most susceptible to fungal
growth (6).
Dirt has been shown to have a significant impact on the moisture content of materials.
West and Hansen (5) found that used or aged glass fiber had a moisture content that was 10
to 40 times higher than the new sample. Ceiling tile demonstrated the same tendency.
Yoshida, et al. (7) found that, under normal conditions, tatami mats contained about 10%
-------
moisture, but with the accumulation of din, the moisture content rose to about 20%.
The influence of the building environment on indoor materials is being investigated using
static chambers with defined RH, temperature, and light conditions. The objective of this
research program is to: 1) develop a standardized method for evaluating the capability of
building materials to support microbial growth; and 2) assess the effect of building micro-
environments on materials' ability to sustain the growth of microorganisms. This cooperative
research effort utilizes these chambers to investigate the impact of possible building microen-
vironments on the ability of ceiling tile to support fungal growth.
MATERIALS AND METHODS
Static Environmental Chambers. Static chambers (32 x 39 x 51 cm) were made by modifying
acrylic-walled desiccators (Fisher Sci. 08-647-24). Saturated salt solutions were used to
maintain specific RHs (8) within each chamber. The chambers were placed in a dark,
temperature-controlled (21+3°C), HEPA-filtered (High Efficiency Paniculate Air) room.
The five RH values and the saturated salt solutions used to attain them were:
54% RH - magnesium nitrate 70% RH - potassium iodide
85% RH - potassium chloride 90% RH - barium chloride
97% RH - potassium sulfate
Building Materials. All experiments reported in this paper utilized ceiling tile as the test
building material.
Aged ceiling tile, type "A", (approximately 10 years old, standard white, textured-face, sus-
pended-ceiling tile) was removed from offices. According to manufacturer's specifications,
the ceiling tile was mineral fiber with a vinyl surface. It contained varying proportions of
mineral fiber and/or glass fiber (0 - 90%), gypsum (10 - 15%), starch (10 - 15%), paper fiber
(10 - 15%), clay (0 - 25%), perlite (0 - 30%), silica (0 - 12%), styrene acrylic polymer (0 -
12%), and phenolic resin (0 - 8%). This tile was identified as type "D" of the new tile de-
scribed below. Three types of commercially available die were purchased new from local
building supply companies. The first and second types, tiles "B" and "C," are fire retardant,
acoustical, and washable. They are composed of 20 - 60% mineral wool fiber and 4 - 10%
hydrous aluminum silicate. Although the only discernable difference is that the visible
surfaces differ in texture, they have two different order numbers. The third tile, type "D," was
a fire-resistant acoustical tile.
All ceiling tiles were purchased as 30.5 x 61 cm boards and cut into 3.8 cm squares with
a band saw. The pieces of tile were sterilized by autoclaving before inoculation (on the dark-
colored, unfinished side) with microorganisms.
Bulk Moisture Content. The bulk moisture content of the ceiling tile blocks was determined
gravimetrically. Ceiling tile blocks were chamber-equilibrated at the test RH for at least 3
days before being weighed to determine the equilibrium bulk moisture. Dry weights were ob-
tained after drying for 4 hours at 105°C. The bulk moisture content was computed using the
formula:
M = [Wb-Wd]/Wd x 100%
where: M = bulk moisture content (%), Wb= weight of the block (g), and Wd= the block
2
-------
weight after drying (g).
Test Microorganism. One of the predominant genera of aeroallergenic indoor air molds
isolated from problem buildings is Penicillium. Frequently, the species have not been
identified. For the first set of experiments in this study, Penicillium glabrum was utilized.
The organism was purchased from the American Type Culture Collection (ATCC) as
Penicillium aragonense (ATCC #42228, reported as isolated from air); it has subsequently
been re-identified by R.A. Samson of the Centraalbureau voor Schimmelcultures,
Baarn, the Netherlands, as Penicillium glabrum.
Another mold of concern in indoor environments is Aspergillus. For the final set of
experiments in this study, A. versicolor (ATCC #9577) was used.
Procedure. To study microbial growth at a single RH, chamber-equilibrated ceiling tile blocks
were inoculated with approximately 1 X 105 colony forming units (CPUs) of either P. glabrum
or A. versicolor suspended in 10 uJ of water and placed in the static chambers. The spores
were suspended in sterile water so that no nutrients were introduced into the material.
Triplicate blocks were removed for quantitation on days 3, 7, 14, 21, and 28. Day'44 was
included for A. versicolor only. Uninoculated blocks, treated with sterile water and placed in
the same chamber, were processed with the inoculated blocks.
In order to quantify fungal growth, the ceiling tile blocks were removed from the chambers,
weighed, and placed in sterile receptacles containing phosphate-buffered saline with 0.1%
Tween 80. The blocks in buffer were agitated on a wrist-action shaker for 30 min to ensure
thorough extraction. Aliquots of block/buffer suspension were diluted and plated on
Sabouraud Dextrose Agar. Plates were incubated at room temperature for at least 1 week.
Individual colonies (CPUs) were manually counted shortly after visible growth was first
observed and again as moderate growth became apparent Prolonged incubation was necessary
to confirm the identification of the mold as P. glabrum or A. versicolor.
RESULTS
Growth of Penicillium glabrum on Aged and New Ceiling Tile. The bulk moisture content
of the aged ("A") and new ceiling tiles "B," "C," and "D" are shown in Table 1.
Table 1. Percent bulk moisture
D
A
Y
0
3
7
14
21
28
Aged
A Tiles
54%
RH
1.8
2.0
2.0
2.0
2.0
2.0
97%
RH
5.0
5.4
5.9
6.4
6.7
7.3
New
B Tiles
54%
RH
2.2
1.9
1.9
1.9
1.8
2.0
97%
RH
4.6
5.3
5.9
5.9
6.1
6.3
New
C Tiles
54%
RH
2.8
3.1
3.0
3.1
3.0
3.1
97%
RH
5.8
6.3
6.9
7.1
7.5
7.6
New
D Tiles
54%
RH
1.5
1.0
1.1
1.0
1.1
1.1
97%
RH
3.9
4.3
4.7
5.0
5.2
5.5
-------
The data presented here are from blocks maintained in the 54% RH or the 97% RH
chambers. The bulk moisture varied depending upon the type of tile. All of the moisture
contents were lower at 54% RH than at 97% RH, with the "D" tiles the lowest at about 1%
bulk moisture. The highest levels were found in the "C" tiles and the aged ("A") dies from
the 97% RH chamber. The "B" tiles attained approximately 6% bulk moisture and the "C"
tiles over 7%. The aged tile reached approximately 7.3% bulk moisture by day 28.
Table 2 presents the corresponding CPUs for the above bulk moisture data. The CPUs are
expressed as CPUs in thousands per block. All of the tiles in the 54% RH chamber
demonstrated a decrease in organisms over the 4 weeks with the new tile decreasing more
rapidly than the aged. However, in the 97% chambers the "A," "B," and "C" tiles all demon-
strated varying amounts of growth. The counts increased approximately two orders of magni-
tude on the "B" and "C" tiles in 28 days. Previous experiments demonstrated an increase of
two orders of magnitude in 14 days in 97% RH chambers for P. glabrum on aged tile (9).
These experiments confirm those results and indicate that the counts continue to increase until
day 21 when they plateau at about 10s CPUs/block (an increase of over three orders of
magnitude). It is interesting to note that, in a second experiment (data not presented here),
the CPUs/block attained the same plateau of 10s. The CPUs on the"D" tiles decreased below
the detection limit by the third day.
Table 2. Penicillium glabrum CPUs in thousands/block
D
A
Y
0
3
7
14
21
28
Aged
A Tiles
54%
RH
470
180
210
270
96
91
97%
RH
180
85
1100
28000
390000
360000
New
B Tiles
54%
RH
160
10
10
5
10
5
97%
RH
290
10
30
90
2900
9700
New
C Tiles
54%
RH
150
10
20
10
10
5
97%
RH
90
10
310
1100
1500
15400
New
D Tiles
54%
RH
40
5
1
10
<1
<1
97%
RH
40
<1
<1
<1
<1
<1
Table 3 presents the observed ranges of percent bulk moisture content of the 4 ceiling tile
types where P. glabrum growth begins (critical moisture content). For the "B" tiles, P.
glabrum CPUs begin to increase once the bulk moisture content reaches approximately 6%,
while for the "C" tiles, that point appears to be just over 6.5%. For this aged tile, a bulk
moisture content of around 5.5% seems to be critical for growth of P. glabrum.
Growth of Aspergillus versicolor on New Ceiling Tile. The results of the experiments with
A. versicolor inoculated onto new ceiling tile are presented in Table 4. The "B" tile data are
not presented because the results are very similar to those for the "C" tiles. Bulk moisture
-------
contents were determined. The result for the 54 and 97% RH chambers are essentially the
same as those presented in Table 1 for the P. glabrum. The organisms on the "D" dies are
below the detection limit by day 14 or 21 for all chambers. For the "C" tiles, the organisms
decrease slowly in all the chambers, but by day 44, in the 97% RH chamber, seem to have
begun a slight recovery.
Table 3. Observed critical bulk moisture content range for
growth of P. glabrum on four different ceiling tiles
Tile
A
B
C
D
Description
Naturally Aged Type "D"
New mineral fiber
New mineral fiber
New fire resistant
Critical bulk moisture, %
5.4 - 5.9
5.9 - 6.1
6.3 - 6.9
No Growth
Table 4. Aspergillus versicolor CPUs in thousands/block
D
Y
0
3
7
14
21
28
44
CTile
54%
RH
190
80
140
40
40
20
ND
70%
RH
190
40
70
5
5
1
ND
85%
RH
190
40
70
5
5
1
ND
90%
RH
190
40
60
70
30
10
ND
97%
RH
190
80
140
20
10
10
200
DTile
54%
RH
20
10
10
1
<0.4
<0.4
ND
70%
RH
20
10
10
<0.4
<0.4
<0.4
ND
85%
RH
20
0.4
1
<0.4
<0.4
<0.4
ND
90%
RH
20
0.4
1
<0.4
<0.4
<0.4
ND
97%
RH
20
10
10
<0.4
<0.4
<0.4
ND
ND = Not Detectable
DISCUSSION AND CONCLUSION
These data indicate that it is possible for P. glabrum to grow on new, as well as aged,
ceiling tile. The composition as well as the bulk moisture content is critical to the growth of
the organism. The "D" tiles were identified as the same ceiling tile as the aged tile collected
from office buildings. However, not only did the organism fail to grow, but the counts actu-
ally decreased rapidly and disappeared below the detection limit. It is not known whether the
formulation changed over the last 10 years and an inhibitory substance was added, the com-
-------
position altered with time, or the dirt provided the actual growth media. It should be noted
that the bulk moisture content of the tiles, even after 28 days, never exceeded 5.5% (see Table
1 for "D" tiles). The bulk moisture in the aged tiles reached 5.9% by day 7. These results
correspond to those found by West and Hansen (5) — that used or aged materials were capable
of retaining more moisture than new materials under the same conditions.
The critical bulk moisture content for P. glabrum to grow varied depending upon the actual
tile being tested. For the two different new tiles, "B" and "C," as well as the aged tiles,
between 5.5 and 6.5% bulk moisture is critical. Growth appears to be directly related to the
moisture uptake of the tile.
The fact that A. versicolor did not grow as well as P. glabrum on any of the new tiles de-
monstrates organisms' differences in moisture and substrate requirements.
These results confirm earlier preliminary findings and illustrate that it may be possible to
limit or slow biocontaminant growth on indoor materials if the bulk moisture content can be
controlled (9). The critical bulk moisture content is undoubtedly material as well as organism
specific. The critical bulk moisture content should be one of the factors taken into account
when selecting building materials.
ACKNOWLEDGEMENTS
This combined research effort was performed under U.S EPA cooperative agreement
CR-817083-01-0.
REFERENCES
1. Snow D. The germination of mould spores at controlled humidities. Annals of Appl
Biol., 1949;36(1):1-13.
2. Block SS, Rodriquez-Torrent R, Cole MB, Prince AE. Humidity and temperature
requirements of selected fungi. Dev in Ind Microbiology, 1962;31:204-216.
3. Pasanen A-L, Kalliokoski P, Pasanen P, Jantunen MJ, Nevalainen A. Laboratory studies
on the relationship between fungal growth and atmospheric temperature and humidity.
Environmental International, 1991;Vol 17,pp 225-228.
4. BRE. Surface condensation and mould growth in traditionally-built dwellings. Building
Research Establishment Digest; No. 297; May 1985.
5. West MK and Hansen EC. Effect of hygroscopic materials on indoor relative humidity
and air quality. The Human Equation: Health & Comfort, 1989;pp 56-59.
6. Block SS. Humidity requirements for mold growth. Applied Microbiology, 1953;Vol 1,
pp 287-293.
7. Yoshida K, Ando M, Sakata T, Araki S. Prevention of summer-type hypersensitivity
pneumonitis: effect of elimination on Trichosporon cutaneum from patients' homes.
Archives of Environmental Health, 1989;44(5):317-322.
8. ASTM Designation E 104-85 (reapproved 1991). Standard Practice for Maintaining
Constant Relative Humidity by Means of Aqueous Solutions. ASTM Annual Book of
Standards, Section 11. Water and Environmental Technology, 1991;Vol 11.03.
Atmospheric Analysis: Occupational Health and Safety.
9. Foarde K, Cole E, VanOsdell D, Bush D, Franke D, Chang J. Characterization of
Environmental Chambers for Evaluating Microbial Growth on Building Materials.
Presented at ASHRAE/ACGIH/AIHA's conference - IAQ'92: Environments for People,
OcL 1992, San Francisco, CA, USA.
-------
AEERL-P-1032
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before comp
1. REPORT NO
EPA/600/A-93/184
2.
1. TITLE AND SUBTITLE
Assessment of Fungal Growth on Ceiling Tiles Under
Environmentally Characterized Conditions
5. REPORT DATE
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S) R _ Foarde, P. Dulaney, E. Cole, D.• VanOsdell,
D. Ensor (RTI); and J. Chang (EPA)
S. PERFORMING ORGANIZATION REPORT NO
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Research Triangle Institute
P.O. Box 12194
Research Triangle Park, North Carolina 27709
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
CR817083-01-0
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 COVERED
Published paper; 11/92-2/93
14. SPONSORING AGENCY CODE
EPA/600/13
15. SUPPLEMENTARY NOTES A Tm-iT> T • j. cc- • T u /-i o /^i T\ IT •-i T\ c A m n /
AEERL project officer is John C-S Chang, Mail Drop 54, 919'
541-3747. Presented at Indoor Air '93, Helsinki, Finland, 774-8/93.
16. ABSTRACT
The paper discusses investigation of the impact of the building environment
on the ability of building materials to support microbial growth, using static cham-
bers with defined relative humidity, temperature, and light conditions. The ability
of fungi to grow on materials is well established, given sufficient moisture and nu-
trition. Four types of ceiling tile were investigated: one aged, two mineral fiber/
crystalline silica, and one fire-resistant. Ceiling tile pieces were placed in the
chambers, allowed to chamber-equilibrate, and inoculated with either Penicillium
glabrum or Aspergillus versicolor. The critical bulk moisture content for organism
growth varied depending on both tile type and test mold. P. glabrum counts increa-
sed three orders of magnitude in 28 days. A versicolor did not grow as well. Organ-
ism related differences are apparent.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Pollution
Construction Materials
Tiles
Fungi
Growth
Pollution Control
Stationary Sources
Ceiling Tiles
13B
13 C
06C
06P
B. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (This Report)
Unclassified
21. NO. OF PAGES
20. SECURITY CLASS (This page)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
-------
------- |