EPA/600/R-13/229 | November 2013 | www.epa.gov/ord
United States
Environmental Protection
Agency
Laboratory Evaluation of the
Efficacy of Chlorine Dioxide
Fumigation for Remediation
of Building Materials
Contaminated with Molds,
Mycotoxins or Allergens
•
Office of Research and Development
National Homeland Security Research Center
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x-xEPA
EPA/600/R-13-229
November, 2013
Laboratory Evaluation of the Efficacy of Chlorine Dioxide Fumigation
for Remediation of Building Materials Contaminated with Molds,
Mycotoxins or Allergens
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
RESEARCH TRIANGLE PARK, NC 27711
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DISCLAIMER
This report has been peer and administratively reviewed and has been approved for publication
as an Environmental Protection Agency document. It does not necessarily reflect the views of
the Environmental Protection Agency. No official endorsement should be inferred. The
Environmental Protection Agency does not endorse the purchase or sale of any commercial
products or services.
Questions concerning this documentor its application should be addressed to:
Doris Betancourt
Office of Research and Development
U.S. Environmental Protection Agency
109 T.W.Alexander Drive, E305-03
Research Triangle Park, NC 27711
919-541-9446
betancourt.doris@epa.gov
Shannon D. Serre
Office of Research and Development
U.S. Environmental Protection Agency
109 T.W.Alexander Drive, E343-06
Research Triangle Park, NC 27711
919-541-3817
serre.shannon@epa.gov
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ACKNOWLEDGMENTS
Sabre Technical Services, LLC, and the EPA Office of Pesticides provided financial support of
this project through a Cooperative Research and Development Agreement (CRADA) that
supported this study.
Contributions of the following individuals and organizations to the development of this document
are gratefully acknowledged.
United States Environmental Protection Agency (EPA)
Worth Calfee
Tim Dean
Carlton Kempter (EPA Retired)
Laura Kolb
Blair Martin (EPA Retired)
Dave Mickunas
Shawn Ryan
Sabre Technical Services (CRADA# 0400-A-07)
RTI International (Contract number EP-C-05-060; Project number0209847.075)
ARCADIS US, Inc. (Contract num ber EP-C-09-027)
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TABLE OF CONTENTS
Disclaimer ii
Acknowledgments iii
Table of Contents iv
List of Tables v
Acronyms and Abbreviations vii
Executive Sum many viii
1. Introduction 1
2. Materials and Methods 4
3. Results and Discussion 14
4. Conclusions and Recommendations 30
5. Quality Assurance and Quality Control 33
6. References 37
APPENDICES
Appendix A: Tables of Results for Allergens and Mycotoxins in Alternate Format
IV
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LIST OF TABLES
Table 1 Test Matrix for Chlorine Dioxide Fumigation 12
Table 2 Summary of Results for Latex Painted Gypsum Wallboard and Wallpapered
Gypsum Wallboard (all vegetative coupons) 15
Table 3 Summary of Results for Ceiling Tile (all vegetative coupons) 17
Table 4 Summary of Results for Unpainted Pine Wood 18
Table 5 Summary of Results for Carpet (all nonvegetative coupons) 20
Table 6 Summary of Results for Glass (all nonvegetative coupons) 20
Table 7 Summary of Results for Pure Allergen rAlt a1 on All Materials (nonvegetative
coupons) 24
Tables Summary of Results for Pure Aflatoxin on All Materials (nonvegetative
coupons) 25
Table 9 Summary of Results for Asp f 1 Allergen from Aspergillus fumigatus CPU
Inoculated Positive Controls and Exposed Coupons in All Materials
(vegetative coupons) 25
Table 10 Summary of Results for Alt a 1 Allergen from Alternaria alternata CPU
Inoculated Positive Controls and Exposed Coupons on All Materials
(nonvegetative coupons)3 27
Table 11 Percent Inactivation Efficacy of CPU on all materials 28
Table 12 DQIs for Critical Measurements 33
Table 13 DQIs for the Assay Critical Measurements 34
Table 14 DQIs for the Allergen Analysis Critical Measurements 34
Table 15 DQIs for the Mycotoxin Analysis Measurements 35
Table A 1 Summary of Results for Pure Antigen rAlt a1 on All Materials (nonvegetative
coupons) 40
Table A 2 Summary of Results for Pure Aflatoxin on All Materials (nonvegetative
coupons) 35
Table A 3 Summary of Results for Asp f 1 Allergen from Aspergillus fumigatus CPU
Inoculated Positive Controls and Exposed Coupons in All Materials
(vegetative coupons) 42
Table A 4 Summary of Results for Alt a 1 Allergen from Alternaria alternata CPU
Inoculated Positive Controls and Exposed Coupons on All Materials
(nonvegetative coupons) 43
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LIST OF FIGURES
Figure 1 Laboratory Fumigation Schematic 10
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ACRONYMS AND ABBREVIATIONS
Ag
Aspfl
ASTM
ATCC
B.a.
CPU
CIO2
CRADA
CM
CT
DAS
DQI
DQO
ELISA
EMS
ERH
FIFRA
FTTA
hr
HVAC
MSM
ng
OPP
ORD
ORIA
PBS
PCR
P&DC
ppmv
QAPP
R&D
T
rAlt a 1
RH
RTI
SJRMC
US EPA
antigen
Aspergillus fumigatus antigen
American Society for Testing and Materials
American Type Culture Collection
Bacillus anthracis
colony form ing unit(s)
chlorine dioxide
Cooperative Research and Development Agreement
Critical measurement
concentration of chlorine dioxide Xtime of exposure
digital acquisition system
Data quality indicator
Data quality objective
enzyme-linked immunosorbentassay
environmental monitoring system
Equilibrium Relative Humidity
Federal Insecticide, Fungicide, and Rodenticide Act
Federal Technology Transfer Act
hour(s)
heating, ventilation and air conditioning
Modified Standard Method
nanogram(s)
Office of Pesticide Programs
Office of Research and Development
Office of Radiation and Indoor Air
phosphate buffered saline
polymerase chain reaction
Processing and Distribution Center
part(s) per million by volume
Quality Assurance Project Plan
research and development
temperature
Alternaria antigen
relative humidity
RTI International
Saint Johns Regional Medical Center
United States Environmental Protection Agency
VII
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EXECUTIVE SUM MARY
The purpose of this project was to determine the efficacy of chlorine dioxide (CIO2) fumigation to
inactivate viable mold, mycotoxins, and allergens on building materials. Alternaria alternata,
Aspergillus versicolor, Aspergillus fumigatus, Chaetomium globosum, and Stachybotrys
chartarum were individually inoculated onto the surface of latex-painted and wallpapered
gypsum wallboard, unpainted pine wood, ceiling tile, carpet, and glass. In addition, neat
preparations of one allergen (rAlt a1 extract from Alt. alternata) and one mycotoxin (aflatoxin)
were spiked onto surfaces in pure form. Spiked building materials were fumigated with CIO2gas
at low and high concentrations for several time periods. The analytical techniques included
counting colony forming units (CPU) for molds and use of enzyme-linked inmunosorbent assays
(ELISA) for quantification of the remaining antigen and mycotoxin. A > 4 Iog10 reduction in CPU
was achieved after exposure at 9000 parts per million by volume (ppmv)-hour(hr) at 75 °F and
a relative humidty of 75% for S.chartarum, C.globosum, A. versicolor, A. fumigatus and A.
alternata on latex-painted wallboard, unpainted pine wood, carpet and glass. A > 4 Iog10
reduction in CPU was achieved for S. chartarum on all materials and all CIO2 concentration x
exposure time (CTs)from 3000 to 9000 ppmv-hr.
Allergens and mycotoxins were partially inactivated under several of the conditions tested.
This research has demonstrated the potential of CIO2 gas to achieve a large percentage
reduction in viable spore counts on all materials as well as a significant percentage reduction of
the concentration of allergens and mycotoxins. Observations of fumigation of actual buildings
with heavy mold contamination are consistent with these results.
VIM
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1. INTRODUCTION
Mold contamination is frequently cited as the causative agent in a variety of health problems
(IOM, 2004; Andersen et al., 2011; Dearborn et al., 1999). In instances of extensive or heavy
contamination, mold contamination can also lead to expensive and time-consuming efforts to
remediate the problem using conventional approaches. This report summarizes the results of
research under a Cooperative Research and Development Agreement (CRADA) to quantify the
efficacy of fumigation using chlorine dioxide (CIO2) for inactivation of a variety of common mold
species on typical building materials.
The objective of this study was to determine the biocidal efficacy of CIO2 fumigation against a
variety of fungi (molds) and their nonviable components, e.g., allergens and mycotoxins. The
organisms chosen for this study are representative of mold species commonly associated with
damp indoor environments (Vesper et al., 2007). In particular, C. globosum was tested at
multiple exposure concentrations and exposure times because this organism is highly resistant
to adverse environmental conditions. This mold produces a multilayered spore that confers
protection against UV light and desiccation (Millner et al., 1977). Likewise, the mold has been
demonstrated to offer protection against oxidation by CIO2 (Wilson et al., 2005).
Mold contamination of structures
Mold spores are ubiquitous in natural and built environments and, under normal circumstances,
are relatively innocuous (Frankel etal., 2012). However, under wet/high moisture conditions
favorable to the formation of the vegetative form, mold has been associated with human health
issues and damage to the impacted structures (DM, 2004; Vesper et al., 2006; Gravesen et al.,
1999). The effects on human health may result from the proliferation of spores, contact with the
vegetative organism and/or production of allergens and mycotoxins. Mold spore contamination
may occur as the result of water dam age associated with normal day-to-day occurrences or with
widespread damage from natural disasters.
Conventional approach to mold remediation
The industry standard for mold remediation is described in the United States Environmental
Protection Agency (USEPA) report entitled "Mold Remediation in Schools and Commercial
Buildings" (USEPA, 2001). Because that approach is beyond the scope of this report, only the
broad outline will be provided as follows:
- Assess the extent and cause of the problem.
- Repair structural damage causing water leaks.
- Remediate other sources of moisture.
- Remove waterlogged contents (e.g., carpet, bedding), if appropriate.
- Determine if full or partial containment and ventilation are necessary to prevent
spread of mold during remediation activities.
- Remediation may entail selective removal of water-damaged finish materials (e.g.,
wallboard, ceiling tile). However, in the case of pervasive water damage, the structure
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may be gutted to the structural elements (e.g., studs and joists) and the electrical and
heating, ventilation and air conditioning (HVAC) systems removed.
- Dry out the affected area(s) of the structure.
- Sample to determine the effectiveness of the remediation.
In cases of extensive water damage, the approach can be time consuming and expensive,
especially in a material and labor constrained market (such as after a natural disaster).
Alternative approaches
In the aftermath of Hurricane Katrina, several vendors offered alternative approaches to mold
remediation that were claimed to be more effective or less disrupting than the conventional
approach. Sabre Technical Services, LLC, offered the CIO2 fumigation technique, which was
used successfully in response to the 2001 "anthrax" incidents (Martin, 2005) and will be
discussed in greater detail in the following section. Sabre received state level registration for
mold remediation under Section S4(C) of the Federal Insecticide, Fungicide, and Rodenticide
Act (FIFRA) in Louisiana, Texas and Mississippi. The approach was applied to structures with
residual issues that had been remediated conventionally and to structures with extensive to
pervasive mold growth that had not previously been remediated. In summary, the approach was
as follows:
- Apply the technique after structural and other sources of water were remediated.
- Remove carpet and other water-saturated materials.
- Tent the structure and fumigate with CIO2; remediating structure and contents in situ.
- Ensure fumigant penetration into all parts of the structure (e.g., attic, wall cavities).
- Vacuum to remove residue once the fumigation is complete.
- While some contents showed material effects (e.g., discoloration of materials or
oxidation of mild steel) and required disposal, many items were suitable for
reclamation/reuse.
Federal Technology Transfer Act
The Federal Technology Transfer Act (FTTA) grants the USEPA the authority to make its
experimental facilities and expertise available to non-Federal organizations to perform research
and development (R&D) of mutual interest. The FTTA allows the non-federal cooperatorto
provide both funding and in-kind resources to the Federal organization. The Federal
organization can provide a variety of in-kind resources for performance of the R&D but may not
provide resources to the cooperator. The CRADA is the mechanism forformalizing the scope of
the R&D to be conducted collaboratively between the Federal organization and the cooperator.
The R&D described in this report was completed under a CRADA between Sabre Technical
Services, LLC (henceforth Sabre) and the USEPAs Office of Research and Development
(ORD), with additional resources from the USEPAs Office of Pesticide Programs (OPP). The
goals of the CRADA were:
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- Evaluate a range of CIO2 fumigation conditions to determine if a 4 log™ reduction of
viable spores could be achieved.
- Evaluate the effects of CIO2 fumigation on mycotoxins and allergens.
As a part of the CRADA, USEPA performed the fumigation using its in-house fumigation
research facility in Research Triangle Park, NC.
Background
A number of techniques for disinfection or sterilization of microbes have been developed for a
variety of applications. When the 2001 "anthrax" incidents occurred, many approaches were
used to remediate the numerous buildings that were affected. One such approach was
fumigation with CIO2, which has been suggested as a method for remediation of buildings
contaminated with mold. One method of CIO2 generation that uses a wet method to generate
CIO2 from the reaction of bleach, hydrochloric acid and sodium chlorite to generate CIO2 was
developed by John Y. Mason et a/. (1993). This approach was used at the Hart Senate Office
Building and the Brentwood Processing and Distribution Center (P&DC), both in Washington,
D.C., and the Hamilton P&DC in NJ (Canter, 2005; Martin, 2003). The conditions that were used
required a minimum CT of 9000 ppmv-hr (750 ppmv CIO2for 12 hours) at a minimum 75%
relative humidity (RH) and 75 °F. This same approach has been used at two fumigations for
mold in contaminated structures: an abandoned Victorian era house in Utica, NY, and an
abandoned department store building in Hudson Falls, NY. The conditions that were used were
9000 ppmv-hr CT (3000 ppm for 3 hours). This approach was also used to remediate the Saint
Johns Regional Medical Center (SJRMC) for mold at a lower CT of 2000 ppmv-hr to minimize
material impacts (Martin et al., 2008). The conditions developed in these previous tests were a
starting point for the work contained in this report.
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2. MATERIALSAND METHODS
Overall technical approach. The laboratory evaluation was designed to simulate some of the
conditions that could be found in mold-contaminated structures. The materials were selected to
represent the most common materials used in building and home construction. The mold
species selected are usually identified in contaminated structures. The overall approach consists
of four components, as follows:
- Fumigation experiments to evaluate the efficacy of CIO2 gas as a function of
concentration, time, overall CT and RH.
- Culturability Assay. The inactivation of culturable fungi was quantified by comparing
the number of colony forming units (CPU) pre- and post-treatment on the material
coupons.
- Mycotoxin Analysis. The inactivation of aflatoxin was measured by an enzyme linked
immunosorbent assay (ELISA).
- Allergen Analysis. The inactivation of purified Alternaria antigen (rAlt a1) was
quantified by direct ELISA. In addition, the inactivation of rAlt a1 from the Alternaria
alternata CPU inoculated coupons and Asp f1 from Aspergillus fumigatus CPU
inoculated coupons was quantified.
Coupon Preparation. The building material coupons were prepared using standard procedures
as summarized below. All coupon preparations and analyses were performed at RTI
International, Research Triangle Park(RTP), NC.
- Coupon Materials. The coupon materials were selected to represent commonly used building
materials known to be subject to mold growth. These materials were bare structural pine wood
(Home Depot, Raleigh, NC), latex-painted gypsum wallboard (Home Depot, Raleigh, NC),
wallpapered latex-painted gypsum wallboard (Home Depot, Raleigh, NC), carpet (Kraus,
Brentwood Carpets, Raleigh, NC), ceiling tiles (Home Depot, Raleigh, NC), and glass (Prism
Research, Raleigh, NC). Wood and ceiling tile are porous materials that support mold growth
not only on the surface but also penetrating into the coupon. Painted and wallpapered gypsum
wallboard has a relatively nonporous surface yet is quite porous internally. If water com promises
the surface (i.e., makes the surface porous), then painted and wallpapered gypsum is a perfect
substrate for mold growth. Mold growth on carpet is dependent on the age and the composition
of the carpet and is subject to spore contamination. Smooth glass was selected as a control
material for biological studies because mold growth was not expected. Most building materials
exhibit properties that might interfere with the CIO2 inactivation of biological contaminants.
However, glass, being a smooth hard surface, eliminates any chemical interference related to
material composition.
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The coupons were 1 inch by 1 inch squares cut from the interior portion of the source sample to
ensure that all coupons had uniform characteristics. The coupons were individually autoclaved
prior to inoculation with the target organism. Contaminants (mold spores, mycotoxins or
allergens) were inoculated onto the surface of the six building materials. Test coupons were
exposed to CIO2 at different concentrations. Positive control coupons were prepared like the test
coupons but were not exposed to the CIO2. The negative control coupons were not inoculated
but were exposed to CIO2 with the test coupons. Field blanks (not inoculated and not exposed)
were also included.
Two types of coupons were prepared - vegetative coupons and nonvegetative coupons. The
mold spores were inoculated on the surface of the vegetative coupons, and the coupons were
placed in a static chamber for at least six weeks to allow active growth to occur. The inactivation
of growing fungi was determined for the latex-painted gypsum wallboard, the wallpapered
gypsum wallboard, the ceiling tile and the wood. Inoculation was performed in a Class II
Biosafety Cabinet (BSCII). The cabinet was decontaminated prior to each use. The fungi
challenge suspensions were prepared by inoculating the test organism onto solid agar media
(MMBD Standard Operating Procedure (SOP) #001), incubating the culture at room temperature
until mature, wiping organisms from the surface of the pure culture, and eluting the organisms
into sterile deionized water (MMBD SOP #002) to a known concentration to serve as a stock
solution (MMBD SOP #012). The organism preparation was viewed microscopically to verify the
purity of spores (absence of hyphae). The suspension was diluted in sterile deionized water
when needed. The coupons were inoculated (usually with five 10 to 100-uL spots in an X
configuration) by pipet onto the surface of the coupon and allowed to dry in the biosafety cabinet
(MMBD SOP #58).
Clear plastic desiccators served as the static environmental chambers. The desiccators have
gasket-sealed doors, which eliminate air exchange and maintain the humidity within the
chamber through the use of sterile water. Temperature was externally controlled and maintained
at room temperature. Prior to use, the chambers were decontaminated (MMBD SOP #005) and
then characterized as per MMBD SOP #017. The chambers were set to 100% Equilibrium
Relative Humidity (ERH) for all tests. The ERH in each chamberwas monitored with a
hygrometer (Vaisala, Woburn, MA) .
Prior to fumigation, the challenge level of CPU on vegetative coupons was determined by
assaying coupons to determine the level of growth. CPU eluted were compared to the baseline
inoculum at Day 0. Growth was defined by an increase of approximately 1 log™ and confirmed
visually by microscopy. The goal was to load each of the individual coupons with 106 to 107
CPU/coupon. However, the nature of C. globosum growth (forms perithecia) and Alt. alternata
growth (produces few very large spores) made achieving 106to 107 CPU/coupon difficult.
Modifications to the level of the inoculum were necessary. By lowering the minimum detection
limit, the log reduction was able to be measured with an appropriate dynamic range without
achieving the high level of inoculum.
Fungal spores were inoculated on the surface of the nonvegetative mold coupons (without an
incubation period) and the coupons were fumigated. Nonvegetative coupons included all of the
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carpet and glass coupons, the wood Stachybotrys and Alternaria coupons, and all of the
allergen and mycotoxin inocula. Carpet and glass did not support growth of the test organisms
because the surfaces were not soiled prior to inoculation; carpet and glass were the only
nonvegetative coupons. Also, in several instances, an organism did not grow on the test
material; the test was performed using nonvegetative coupons. For nonvegetative coupons, the
test fungus was inoculated to the surface of the materials following the same procedure as
previously described for vegetative coupons. The goal was to load each of the individual
coupons with 106to 107 CPU/coupon. The number of fungal spores on the coupons was
enumerated by replicate plating of the positive control coupons (MMBD SOP #009)..
The allergen and mycotoxin preparations were also nonvegetative coupons. For allergen and
mycotoxin, pure Alt. alternata allergen rAlt a1 (INDOOR Biotechnologies, Ltd., Charlottesville,
VA) and Aflatoxin B1 (RomerLabs, Union, MO) were used. The coupons were inoculated by
pipet onto the surface of the coupon and allowed to dry in the biosafety cabinet. The amount of
allergen and mycotoxin inoculated onto the coupons was determined by replicate analysis of the
positive control coupons. Also, rAlt a1 from A.alternata vegetative coupons and Asp f1 from
A. fumigatus vegetative coupons were used for the allergen tests.
- Organisms. The organisms selected for this study are usually identified among the mycoflora
of mold-contaminated indoor environments. The following mold spores are maintained in the RTI
International (Research Triangle Park, NC) (RTI) collection: Aspergillus versicolor(RJ\ 3843),
Aspergillus fumigatus (RTI 3749), and Alternaria alternata (RTI 3211). Stachybotrys chartarum
(EPA 63-07) was obtained from USEPA; Chaetomium globosum (ATCC 34507) was purchased
from the American Type Culture Collection (ATCC, Manassas, VA). Spores were inoculated
onto each coupon at a concentration of 106 - 107 spores/coupon. The coupon preparation and
the inoculation procedure were performed in accordance with the American Society for Testing
and Materials (ASTM) guidelines D 6329-98: "Standard Guide for developing methodology for
evaluating the ability of indoor materials to support microbial growth using static environmental
chambers." (ASTM, 1998)
Transportation of coupons. The CIO2 fumigation of all coupons was performed at the USEPA
laboratory in Research Triangle Park, NC. When a set of coupons was ready for the fumigation
experiment, the coupons were packaged in Tyvek® envelopes under sterile conditions and
transported to the USEPA. Following the fumigation experiment, the set of coupons was
returned to RTI for analysis. Chain of custody forms were completed for each transfer step. The
coupons were trans ported under ambient conditions over a distance of about three miles.
Coupon analysis. For CFU determinations, each material coupon was placed in a separate
container, suspended in sterile phosphate buffered saline (PBS) containing Tween 80 and
shaken for at least thirty minutes. All necessary dilutions were made using the same buffer.
Aliquots of the suspension were plated on fungal culture media and incubated for the optimal
time and temperature for the test organism. CFU were counted and calculated for each coupon
per contractor laboratory standard operating procedures.
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Mycotoxin analysis was performed using AgraQuant Aflatoxin (4-40 ppb) test kit from Romer
Labs (Union, MO). The coupons were extracted using 70% methanol. The extract or aflatoxin
test kit standard was mixed with conjugate in individual dilution wells, and then 100 uL from
each dilution well was transferred to an antibody-coated microwell. After incubation at room
temperature, the plate was washed, enzyme substrate added, and the plate was reincubated.
Stop solution was added, and the intensity of the resulting yellow color was measured optically
with a microplate reader (Molecular Devices Emax, Sunnyvale, CA) at a wavelength of 450/650
nm. The chromogenic color change is inversely proportional to the concentration of the toxin in
the sample.
Coupons for allergen analysis were extracted into phosphate buffer containing Tween 20. A
commercially available ELISAkit by INDOOR Biotechnologies (Charlottesville, VA) was used for
analysis of rAlt a1 (Alt. a., allergen 1). Optical density values of both treated and control coupons
were plotted against the optical density values of the allergen standard to determine
concentration. In addition to the purified rAlt a1 allergen coupons, the coupons prepared for the
CPU analyses of Alternaria alternata and Aspergillus fumigatus were also analyzed for allergen.
A portion of the extract from the CPU coupons was removed and bead beaten to break up the
spores and/or hyphae present. The antigen present on the Alternaria alternata CPU coupons
was also quantified with the rAlt a1 ELISA kit. The antigen present on the Aspergillus fumigatus
CPU coupons was quantified with the Asp f1 ELISA kit.
Calculation of Efficacy. The viability of the organisms was quantified using the ASTM guidelines
D 6329-98. The effectiveness of the CIO2 in inactivation of the culturable test organisms
(culturability assay) was quantified by calculating the log change in CPU. First, the log™ CPU per
coupon was determined. Next, the average and standard deviation of either the replicate
unexposed positive control coupons or the replicate inoculated exposed coupons were
calculated. Finally, the log change was calculated as follows:
Iog10 change = Iog10 CFUC - Iog10 CFUE (Eq. 1)
where:
logio CFUc = mean log™ CFU of positive control coupons
logic CFUE= mean logic CFU of exposed coupons.
The uncertainty of the efficacy was calculated using the standard deviations from both the
exposed and positive control coupons to determine the combined standard error of the
difference for each test.
To quantify the effectiveness of the CIO2 mycotoxins and allergens (mycotoxin and allergen
assays), the log change in ng of mycotoxin or allergen was calculated. The ng per coupon of
mycotoxin or allergen of either the replicate inoculated unexposed positive control coupons or
the replicate inoculated exposed coupons was averaged, and the standard deviation was
calculated. The log change was calculated as follows:
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Iog10 ng change = Iog10 ngc - Iog10 ngE (Eq. 2)
where:
log™ ngc = mean ng of mycotoxin or allergen of positive control coupons
log™ ngE = mean ng of mycotoxin or allergen on exposed coupons
The uncertainty of the efficacy was calculated using the standard deviations from both the
exposed and positive control coupons to determine the combined standard error of the
difference for each test.
Laboratory fumigation procedures: The fumigations were performed at the US EPA laboratory
in Research Triangle Park, NC. The fumigation procedure was designed to simulate the
conditions used in the field mold remediation process and to provide additional information to
assess the efficacy at different CTs. At the time when the application for FIFRA 24 (c)
registration was submitted, there were limited data on the efficacy of CIO2 for mold and related
compounds (USEPA, 2006). As a result, the registration label conditions are based on the same
9000 ppmv-hr CT levels used for remediation of B. anthracis (B.a.) spores. In the response to
the 2001 anthrax remediation, the required CT was achieved by operating at a minimum of 750
ppm for 12 hr. However, for mold remediation, the CTwas achieved by operating at a minimum
of 3000 ppm for three hr (USEPA, 2012). In addition, the label requires a minimum temperature
of 75 °F and 75% RH for the duration of the fumigation. The laboratory fumigation procedure is
described below.
Fumigation conditions. The experimental conditions were chosen to span the range of the field
conditions used in previous applications. In addition, samples could periodically be removed
from the fumigation chamber to allow evaluation of the efficacy of lower CT levels. The baseline
condition was 750 ppm for 12 hr. However, intermediate time points were 1.5, 3, 6 and/or 9
hours, corresponding to CT values of 1125, 2250, 4500, 6750 ppmv-hr. The alternative condition
was 3000 ppmvfor 1 or 3 hours, corresponding to CT values of 3000 and 9000 ppmv-hr. As the
experiments progressed, some conditions were adjusted or eliminated fora given organism
and/or material to provide better resolution of intermediate CT values. Every organism/material
combination was tested at least at 9000 ppmv-hrs CT. Some continuously wetted coupons for
pine wood and wallboard were treated at limited CTs (1125 and 2250 ppm-hr). An additional
treatment level of 12,000, 18,000, and 24,000 ppmv-hr at 45% RH was used in sometests as
included in the test matrix (see Table 1, discussed below).
- Fumigation chamber: The fumigation system was initially designed and constructed to allow
flexible operation for a variety of fumigation systems. The configuration of the system used in
these experiments is shown in Figure 1. The fumigation enclosure is a 300 L (liter) (11 cubic foot
(ft3)) opaque glovebox with provision for periodic removal of a subset of coupons without
affecting the fumigation conditions. The coupons, wrapped in Tyvek® envelopes, were placed on
racks in the chamber. The system was equipped with fans to ensure that the CIO2 concentration
and humidity were well distributed throughout the chamber. The temperature and RH in the
chamberwere monitored by a Vaisala RH/Temperature sensor(Vaisala Model HMD40Y,
8
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Vaisala, Helsinki, Finland) that provided feedback to a digital acquisition system (DAS)
(LabView, National Instruments, Austin, Texas). When the RH dropped below the set point, the
DAS activated a humidity bottle which provided a saturated warm air stream to maintain the
process conditions. The DAS also controlled the heat lamps to maintain the process
temperature based on feedback from the Vaisala sensor. ACIorDiSys Solutions, Inc. EMS
(ClorDiSys EMS, Lebanon, NJ) stand-alone photometric monitor provided continuous monitoring
of the CIO2 concentration in the chamber and provided feedback to the DAS to maintain the
required conditions. Modified Standard Method (MSM) 4500-E samples were taken every 20
minutes during the decontamination phase to confirm the concentration of CIO2 in the chamber.
CIO2 was provided by a Sabre S07-012 system (Sabre, Albany, NY), which supplies a gaseous
CIO2/air stream to the chamber as needed to maintain the required concentration. The aqueous
CIO2 solution was produced externally by mixing hydrochloric acid (FisherScientific, Pittsburgh,
PA) and sodium chlorite (SabreChlor, Odessa, TX) in the presence of aqueous sodium
hypochlorite (12.5%, UnivarUSA, Raleigh, NC). The Sabre system had an emitter, which
stripped the CIO2from the liquid into an air stream and delivered the CIO2to the chamber. The
DAS also provided automatic control of the CIO2 concentration in the chamber. A modified
version of the American Waterworks Association (AWWA) standard method SM 4500-E
(AWWA, 2005) was used to determine CIO2 concentrations every 20 minutes to compare with
the EMS system and to confirm the concentration of CIO2 in the test chamber.
-------
EMS CIO2
concentration
Makeup air
and
pressure
Sabre Generator
Heat Lamp
RH/Temp Sensor
Digital signal line
Digital control line
Heated tubing for gas flow
Cooling water line
Figure 1 Laboratory Fumigation Schematic
*MSM-4500-E= Modified Standard Method 4500-E
- Laboratory-scale Fumigation procedure'. The coupons were placed in the chamber and the
chamber airlock was sealed. The target temperature and RH for the trial were established and
set in the control system. Once the temperature and RH were established, the chamber was
charged with CIO2 to achieve the target concentration. The control system maintained the target
CIO2 concentration, temperature, and RH for the specified time. Time zero is defined as the time
at which the target concentration, temperature and RH was achieved in the chamber. Once the
desired conditions and time had been achieved, the chamber was aerated until a safe CIO2
concentration was achieved in the chamber. To achieve the shorter exposure times, the
coupons were removed through the airlock (without affecting the chamber conditions) at
intermittent time points in the fumigation cycle. The chamber was then opened, and the coupons
were removed and placed in the appropriate sample packaging containers. The fumigated
coupons were then transferred to RTI for subsequent analysis.
10
-------
Test Matrix. Table 1 shows the test matrix for CIO2 fumigation with information on the materials
exposed, the contaminant used (mold spores, allergen or mycotoxin), the CIO2 concentration
(ppmv), exposure time, analyses, and the total number of coupons per run. A total of 20 CIO2
fumigation runs were performed in this study.
11
-------
Table 1. Test Matrix for Chlorine Dioxide Fumigation1
Test ID
Run 1
Run 2
Run 3
Run 4
Run 5
Run 6
Run 7
Run 8
Run 9
Run 10
Run 11
Materials
G, L, W, C
G, L, W, C
G, L, W, C
W
W
W
G, L,C
G, LC
G, L, C
G, L, W, C
G, L, W, C
G, L, W, C
G, L, W, C
G, L, W, C
G, L, W, C
G, L, W, C
G, L, W, C
G, L, W, C
G, L, W, C
G, L, W, C
G, L, W, C
G, L, W, C
G, L, W, C
G, L, W, C
G, L, W, C
G, L, W, C
G, L, W, C
G, L, W, C
G, L, W, C
G, L, W, C
G, L, W, C
Contamination
Stachyb otrys
none (blank)
Stachyb otrys
Chaetomium
none (blank)
Chaetomium
Chaetomium
none (blank)
Chaetomium
mycotoxin
(C1)
none (blank)
mycotoxin
(C1)
rAlt a 1 extract
none (blank)
rAlt a 1 extract
Aspergillus
versicolor
none (blank)
A.versicolor
Alternaria
none (blank)
Alternaria
mycotoxin
(C2)
none (blank)
mycotoxin
(C2)
rAlt a 1 extract
(C2)
none (blank)
rAlt a 1 extract
(C2)
Chaetomium
none (blank)
Chaetomium
Aspergillus
fumigatus
Treatment
none (pos. control)
CIO2(neg. control)
750 ppmvCIO2
none (pos. control)
CIC>2(neg. control)
750 ppmvCIO2
none (pos. control)
CIO2(neg. control)
750 ppmvCIO2
none (pos. control)
CIO2(neg. control)
750 ppmvCIO2
none (pos. control)
CIO2(neg. control)
750 ppmvCIO2
none (pos. control)
CIO2(neg. control)
750 ppmvCIO2
none (pos. control)
CIO2(neg. control)
750 ppmvCIO2
none (pos. control)
3000 ppmvCIO2
3000 ppmvCIO2
none (pos. control)
3000 ppmvCIO2
3000 ppmvCIO2
none (pos. control)
CIO2(neg. control)
3000 ppmvCIO2
none (pos. control)
Time, hours
0
6, 12
6, 12
0
3,6, 9,12
3,6, 9,12
0
3, 12
3, 12
0
6,9, 12
6,9, 12
0
6,9, 12
6,9, 12
0
6, 12
6, 12
0
6, 12
6, 12
0
1 (L&W), 3
(all)
1 (L&W), 3
(all)
0
1 (L&W), 3
(all)
1 (L&W), 3
(all)
0
1 (L&W), 3
(all)
1 (L&W), 3
(all)
0
Analyses
V(6), M(6)
V(1), M(1)
V(6), M(6)
V(6)
V(1)
V(6)
V(6)
V(1)
V(6)
M(6)
M(1)
M(6)
A(6)
A(6)
A(6)
V(6), M(6),
A(6)
V(1), M(1),
A(1)
V(6), M(6),
A(6)
V(6), A(6)
V(1), A(1)
V(6), A(6)
M(6)
M(1)
M(6)
A(6)
A(6)
A(6)
V(6)
V(1)
V(6)
V(6), M(6),
A(6)
Total
Coupons
72
24
144
6
4
24
24
8
48
24
12
72
24
12
72
72
24
144
48
16
96
24
6
36
24
6
36
24
6
36
72
12
-------
Test ID
Run12
Run 13
Run 14
Run 15
Run 16
Run 17
Run 18
Run 19
Run 20
Materials
G, L, W, C
G, L, W, C
G, L, W, C
G, L, W, C
G, L, W, C
G, L, W, C
G, L, W, C
G, L, W, C
L, W, VWV, C
L, W, VWV, C
L, W, VWV, C
Lw, Ww, WWw,
Cw
Lw, Ww, WWw,
Cw
Lw, Ww, WWw,
Cw
Lw, Ww, WWw,
Cw
Lw, Ww, WWw,
Cw
Lw, Ww, WWw,
Cw
L, W, WW, C
L, W, WW, C
L, W, WW, C
L, W, WW, C
L, W, WW, C
L, W, WW, C
L, W, WW, C
L, W, WW, C
L, W, WW, C
L, W, WW, C
L, W, WW, C
L, W, WW, C
Contamination
none (blank)
A. fumigatus
Stachyb otrys
none (blank)
Stachyb otrys
Alternaria
none (blank)
Alternaria
Chaetomium
none (blank)
Chaetomium
Chaetomium
none (blank)
Chaetomium
A. fumigatus
none (blank)
A. fumigatus
Aflatoxin (C1)
none (blank)
Aflatoxin (C1)
A. fumigatus
none (blank)
A. fumigatus
Chaetomium
none (blank)
Chaetomium
Chaetomium
none (blank)
Chaetomium
Treatment
CIO2(neg. control)
3000 ppmvCIO2
none (pos. control)
CIO2(neg. control)
3000 ppmvCIO2
none (pos. control)
CIO2(neg. control)
3000 ppmvCIO2
none (pos. control)
CIO2(neg. control)
750 ppmvCIO2
none (pos. control)
CIO2(neg. control)
750 ppmvCIO2
none (pos. control)
CIO2(neg. control)
750 ppmvCIO2
none (pos. control)
CIO2(neg. control)
750 ppmvCIO2
none (pos. control)
CIO2(neg. control)
750 ppmvCIO2
none (pos. control)
CIO2(neg. control)
3000 ppmvCIO2
45% RH
none (pos. control)
CIO2(neg. control)
4000 ppmvCIO2
45% RH
Time, hours
3
3
0
1 (L&W), 3
(all)
1 (L&W), 3
(all)
0
3
3
0
1.5,3
1.5,3
0
1.5,3
1.5,3
0
1.5,3
1.5,3
0
1.5,3
1.5,3
0
1.5,3
1.5,3
0
3,6
3,6
0
3,6
3,6
Analyses
V(1), M(1),
A(1)
V(6), M(6),
A(6)
V(6), M(6)
V(1), M(1)
V(6), M(6)
V(6), A(6)
V(1),A(1)
V(6), A(6)
V(6)
V(1)
V(6)
V(6)
V(1)
V(6)
V(6), A(6)
V(1), A(1)
V(6), A(6)
M(6)
M(1)
M(6)
V(6), A(6)
V(1), A(1)
V(6), A(6)
V(6)
V(1)
V(6)
V(6)
V(1)
V(6)
Total
Coupons
12
72
48
12
72
48
8
48
24
8
48
24
8
48
48
16
96
24
8
48
48
16
96
24
8
48
24
8
48
1 Yellow highlight indicates runs w here mycotoxin data were not reported. Components of the building materials interfered w ith
mycotoxin assays.
Key: Materials: G = glass; L = latex-painted wallboard; W= unpainted pine wood; C= industrial carpet; WW =wallpapered latex-
painted wallboard; C= ceiling tile (grown at 100% RH, but dry by the time they were treated).
Lw.Ww.WWw, Cw (Same materials as above but grown at 100% RH with wetting prior to inoculation, w etting maintained during
treatment and return for analysis).
Analysis: V= viability; A = antigen; M = mycotoxin. Numbers in parentheses indicate the number of coupons used for that particular
test.
13
-------
3. RESULTSAND DISCUSSION
The objective of this study was to determine the biocidal efficacy of CIO2 fumigation against a
variety of fungi (molds) and their nonviable components. A series of tests was performed using
either glass slides or one of five building materials as the test coupons. These building materials
were latex-painted gypsum wallboard, wallpapered gypsum wallboard, bare structural pine
wood, industrial-grade carpet, and ceiling tile representing real-life structural building materials
(the painted walls, the flooring, and ceiling). These materials permitted the evaluation of the
efficacy of using CIO2 gas as a decontamination approach in the built environment as
realistically as possible in a controlled laboratory setting. Glass was chosen as a control material
that would give the best-case inactivation results with the least amount of variability caused by
confounding factors. The building materials have the potential to impede the inactivation of
biological contaminants by CIO2, whereas glass is a smooth hard surface and is, thus, less likely
to interfere.
At present, there are no standard procedures for the evaluation of biocides used for remediation
of mold-contaminated buildings. The methodology used in this study was developed according
to ASTM D 6329-98. To evaluate the effectiveness of CIO2 gas to inactivate the test organisms,
the CPU of exposed test control coupons were compared to CPU of unexposed positive control
coupons. For the calculations, CPU values were converted to their logarithmic (base 10) form. A
key issue in evaluating the efficacy of CIO2 fumigation was to determine the acceptable number
of CPU remaining after treatment. A panel of EPA ORD researchers and Program Office experts
from OPP and the Office of Radiation and Indoor Air (ORIA) agreed that the target reduction for
mold was 4 log™ (99.99%). Therefore, the challenge level (CPU concentration per coupon prior
to CIO2 exposure) was based on being able to quantify a 4 log™ reduction (change) in CPU after
CIO2 exposure. The USEPA previously reported a similar methodology for the evaluation of the
antimicrobial efficacy of ozone (Menetrez et al., 2009).
Tables 2 through 6 show the results for each of the materials tested. All of the tables have the
same format and include results from both vegetative and nonvegetative coupons. The first
column represents the CIO2 concentration (ppmv). The second column represents the exposure
time (hr). The third column represents the total exposure CT (ppmv-hr). The fourth column
represents the averaged log™ CPU results with standard deviations of unexposed positive
controls, and the fifth column represents the averaged log™ CPU results with standard
deviations of exposed test coupons. The sixth column represents the CPU log™ change as
shown in equation 1 (materials and methods). The seventh column represents the % of
inactivation efficiency, calculated with Equation 3. Table 2 shows the results for the latex-painted
and the wallpapered gypsum wallboard. Table 3 shows the results for ceiling tile. Table 4 shows
the results for unpainted pine wood. Table 5 shows the results for carpet. Table 6 shows the
results for glass. When 0 (zero) CPU were detected on all the replicate plates from one coupon,
0.5 was used as the number of CPU detected and used to calculate of the number of CPU on a
coupon. When that number of CPU on a coupon was transformed to CPU log™, the < symbol
was used in the tables to indicate that no actual CPU were detected. Consequently, when the <
14
-------
CPU log™ was used in the calculation of the log™ change, the > symbol is used to express the
log-io change.
% inactivation efficiency = Cone. Control - Cone. Exposed x 100
Cone. Control
(Eq. 3)
Table 2. Summary of Results for Latex-Painted Wai I board and Wallpapered Latex-Painted
Wallboard (all vegetative coupons)
CIO2
Concentration
(ppmv)
Exposure
Time
(hr)
Total
Exposure -
CT (ppmv-hr)
Control
Coupons
(CPU logio)
Mean ±
St. Dev.
Exposed
Coupons
(CPU logio)
Mean ±
St. Dev.
Log Change
(CPU logio)
Mean ±
St. Dev.
%
Inactivation
Efficacy
S. chartarum
Latex-Painted Wallboard Coupons; 75% RH CIO2 Exposure
3000
750
750
3000
1
6
12
3
3000
4500
9000
9000
6.75 ±0.17
6.78 ±0.05
6.78 ±0.05
6.75 ±0.17
< 2.00 ±0.00
< 2.00 ±0.00
< 2.00 ±0.00
< 2.00 ±0.00
>4.75±0.12
> 4.78 ±0.03
> 4.78 ±0.03
>4.75±0.12
>99.99
>99.99
>99.99
>99.99
C. globosum
Latex-Painted Wallboard Coupons; 75% RH CIO2 Exposure
750
750
750
3000
750
3000
1.5
3
3
1
12
3
1125
2250
2250
3000
9000
9000
5.82 ±0.21
5.82 ±0.21
5.67 ±0.62
5.90 ±0.22
5.67 ±0.62
5.80 ±0.19
2.55 ±0.64
1.58 ±0.31
3.42 ±0.74
2.46 ±0.91
1.73 ±0.67
2.99 ±0.59
3.27 ±0.48
4.24 ±0.28
2.25 ±0.68
3.44 ±0.67
3.94 ±0.64
4.55 ±0.18
81.74
99.99
56.24
85.99
98.49
99.99
Latex-Painted Wallboard Coupons; 45 % RH CIO2 Exposure
3000
4000
3000
4000
3
3
6
6
9000
12000
18000
24000
Latex-Painted Wallboard V
750
750
1.5
3
1125
2250
5.80 ±0.19
5.78 ±0.16
5.80 ±0.19
5.78 ±0.16
2.99 ±0.59
2.91 ±0.56
2.65 ±0.48
1.78 ±0.79
2.81 ±0.44
2.87 ±0.41
3. 14 ±0.36
3.99 ±0.57
70.24
71.74
78.42
99.74
Vetted Coupons* ; 75% RH CIO2 Exposure
5.66 ±0.17
5.66 ±0.17
2. 16 ±0.34
2.23 ±0.65
3.50 ±0.27
3.43 ±0.47
87.49
85.66
Wallpapered Latex-Painted Wallboard Coupons; 75% RH CIO2 Exposure
750
750
1.5
3
1125
2250
5.53 ±0.40
5.53 ±0.40
3.46 ±0.87
3.39 ±0.72
2.07 ±0.68
2. 14 ±0.58
51.74
53.49
Wallpapered Latex-Painted Wallboard Coupons; 45 % RH CIO2 Exposure
3000
4000
3
3
9000
12000
5.71 ±0.35
5.97 ±0.18
2.63 ±0.41
1.73 ±0.44
3.08 ±0.38
4.25 ±0.34
76.99
99.99
15
-------
CI02
Concentration
(ppmv)
3000
4000
Exposure
Time
(hr)
6
6
Total
Exposure -
CT (ppmv-hr)
18000
24000
Control
Coupons
(CPU logio)
Mean ±
St. Dev.
5.71 ±0.35
5.97 ±0.18
Exposed
Coupons
(CPU logio)
Mean ±
St. Dev.
2.03 ±0.16
1.61 ±0.50
Log Change
(CPU logio)
Mean ±
St. Dev.
3.69 ±0.27
4.36 ±0.36
%
Inactivation
Efficacy
92.24
99.99
Wallpapered Latex-Painted Wallboard Wetted Coupons ; 75% RH CICb Exposure
750
750
1.5
3
1125
2250
6.28 ±0.18
6.28 ±0.18
4.84 ±0.31
4.21 ± 0.84
1.44 ±0.25
2.07 ±0.61
A. versicolor
35.99
51.74
Latex-Painted Wallboard Coupons; 75% RH CIO2 Exposure
750
750
6
12
4500
9000
6.67 ±0.02
6.67 ±0.02
1.93±0.12
< 1.88 ±0.00
4.75 ±0.09
> 4.80 ±0.02
A. fumigatus
99.99
>99.99
Latex-Painted Wallboard Coupons; 75% RH CIO2 Exposure
750
750
3000
1.5
3
3
1125
2250
9000
7.58 ±0.17
7.58 ±0.17
7.46 ±0.15
3.91 ± 1.41
2.09 ±0.40
2. 15 ±0.25
3.67 ± 1.01
5.49 ±0.31
5.31 ±0.21
91.74
99.99
99.99
Latex-Painted Wallboard Wetted Coupons ; 75% RH ClOs Exposure
750
750
1.5
3
1125
2250
7. 17 ±0.48
7. 17 ±0.48
7.06 ±0.09
7.05 ±0.19
0.11 ±0.35
0.12 ±0.36
2.75
2.99
Wallpapered Latex-Painted Wallboard Coupons; 75% RH CICb Exposure
750
750
1.5
3
1125
2250
7.52 ±0.22
7.52 ±0.22
6.83 ±0.53
6.69 ±0.68
0.69 ±0.41
0.83 ±0.51
17.24
20.74
Wallpapered Latex-Painted Wallboard Wetted Coupons ; 75% RH CIO2 Exposure
750
750
1.5
3
1125
2250
7.79 ±0.29
7.79 ±0.29
7.12±0.18
7.06 ±0.05
0.67 ±0.24
0.73 ±0.21
A. alternate
16.74
18.24
Latex-Painted Wallboard Coupons; 75% RH CIO2 Exposure
750
750
3000
6
12
3
4500
9000
9000
3.90 ±0.84
3.90 ±0.84
4.23 ±0.30
< 0.88 ±0.00
< 0.88 ±0.00
< 0.88 ±0.00
> 3.03 ±0.60
> 3.03 ±0.60
> 3.36 ±0.21
>99.99
>99.99
>99.99
*Wetted-vegetative coupons were coupons wetted prior to inoculation and maintained w etted during treatment.
Brow n-shaded area - wetted-vegetative coupons.
Pink-shaded area- 45% RH CIO2 exposure.
16
-------
Table 3. Summary of Results for Ceiling Tile (all vegetative coupons)
CIO2
Concentration
(ppmv)
Exposure
Time
(hr)
Total
Exposure-
CT (ppmv-hr)
Control
Coupons
(CPU logio)
Mean ± St. Dev.
Exposed
Coupons
(CPU logio)
Mean ± St.
Dev.
Log Change
(CPU Iog10)
Mean ± St.
Dev.
%
Inactivation
Efficacy
C. globosum
Ceiling Tile Coupons; 75% RH CIO2 Exposure
750
750
1.5
3
1125
2250
7.32 ±0.21
7.32 ±0.21
5.72 ±0.55
3.3 ±0.29
1.59 ±0.41
3.99 ±0.26
39.74
99.74
Ceiling Tile Coupons; 45 % RH CICb Exposure
3000
4000
3000
4000
3
3
6
6
9000
12000
18000
24000
7.29 ±0.18
6.85 ±0.09
7.29 ±0.18
6.85 ±0.09
4.67 ±0.26
3.99 ±0.71
4.14±0.16
3.26 ±0.18
2.62 ±0.22
2.86 ±0.51
3.15±0.17
3.60 ±0.14
65.49
71.49
78.74
89.99
Ceiling Tile Wetted Coupons* ; 75% RH CIO2 Exposure
750
750
1.5
3
1125
2250
7. 13 ±0.23
7. 13 ±0.23
6. 15 ±0.39
5.76 ±0.46
0.98 ±0.32
1.36 ±0.37
24.49
33.99
A. fumigatus
Ceiling Tile Coupons; 75% RH CIC>2 Exposure
750
750
1.5
3
1125
2250
8.00 ±0.40
8.00 ±0.40
2.03 ±0.53
< 1.88 ±0.00
5.97 ±0.47
>6. 12 ±0.28
99.99
> 99.99
Ceiling Tile Wetted Coupons* ; 75% RH CIO2 Exposure
750
750
1.5
3
1125
2250
9.19± 0.12
9.19± 0.12
8.34 ±0.23
8.02 ±0.23
0.85 ±0.18
1.17±0.19
21.24
29.24
*Wetted-vegetative coupons were coupons wetted prior to inoculation and maintained w etted during treatment.
Brow n-shaded area - wetted-vegetative coupons.
Pink-shaded area- 45% RH CIO2 exposure.
17
-------
Table 4. Summary of Results for Unpainted Pine Wood
CI02
Concentration
(ppmv)
Exposure
Time
(hr)
Total
Exposure -
CT (ppmv-
hr)
Control
Coupons
(CPU logio)
Mean ± St.
Dev.
Exposed
Coupons
(CPU logio)
Mean ± St.
Dev.
Log Change
(CPU Iog10)
Mean ± St.
Dev.
%
Inactivation
Efficacy
S. chartarum
Unpainted Pine Wood non-Vegetative Coupons; 75% RH CIC-2 Exposure
3000
750
750
3000
1
6
12
3
3000
4500
9000
9000
7.63 ±0.13
7.24 ±0.17
7.24 ±0.17
7.63 ±0.13
2.05 ±0.12
<2.00±
0.00
<2.00±
0.00
<2.00±
0.00
5.58 ±0.13
> 5.24 ±0.12
> 5.24 ±0.12
> 5.63 ±0.09
C. globosum
99.99
> 99.99
> 99.99
> 99.99
Unpainted Pine Wood Vegetative Coupons; 75% RH CIO2 Exposure
750
750
750
3000
750
750
750
3000
1.5
3
3
1
6
9
12
3
Unpainted
3000
4000
3000
4000
3
3
6
6
1125
2250
2250
3000
4500
6750
9000
9000
4.53 ±0.23
4.53 ±0.23
5.39 ±0.26
5.20 ±0.14
5.39 ±0.26
5. 39 ±0.26
5.39 ±0.26
5.20 ±0.14
1.07 ±0.68
1.02 ±0.62
0.93 ±0.12
0.98 ±0.25
1.03 ±0.35
<0.88±
0.00
<0.88±
0.00
<0.88±
0.00
3ine Wood Vegetative Coupons; 45 % RH CIO2
9000
12000
18000
24000
4.72 ±0.18
4.57 ±0.44
4.72 ±0.18
4.57 ±0.44
<1.57±
0.00
0.61 ±0.98
<1.57±
0.00
0.61 ±0.98
3.46 ±0.51
3.52 ±0.47
4.46 ±0.20
4.22 ±0.20
4.36 ±0.31
£4.51 ±0.18
£4.51 ±0.18
> 4.33 ±0.10
86.49
87.99
99.99
99.99
99.99
> 99.99
> 99.99
> 99.99
Exposure
> 3.15 ±0.76
3. 96 ±0.76
> 3.15 ±0.76
3. 96 ±0.76
> 99.99
98.99
> 99.99
98.99
Unpainted Pine Wood Vegetative Wetted Coupons* ; 75% RH CIO2 Exposure
750
750
1.5
3
1125
2250
5.34 ±0.29
5.34 ±0.29
1.76 ±0.29
<1.18±
0.00
3. 58 ±0.75
4. 16 ±0.20
A. versicolor
89.49
99.99
Unpainted Pine Wood Vegetative Coupons; 75% RH CIO2 Exposure
750
750
6
12
4500
9000
6.23 ±0.12
6.23 ±0.12
<1.88±
0.00
<1.88±
0.00
> 4.35 ±0.08
> 4.35 ±0.08
A. fumigatus
> 99.99
> 99.99
Unpainted Pine Wood Vegetative Coupons; 75% RH CIO2 Exposure
750
750
3000
1.5
3
3
1125
2250
9000
4.71 ±0.45
4.71 ±0.45
6. 56 ±0.32
2.00 ±0.32
2.31 ±0.92
<2.00±
0.00
2.71 ±0.39
2.40 ±0.73
> 4.56 ±0.23
67.74
59.99
> 99.99
18
-------
rcio2
Concentration
(ppmv)
Exposure
Time
(hr)
Total
Exposure -
CT (ppmv-
hr)
Control
Coupons
(CPU logio)
Mean ± St.
Dev.
Exposed
Coupons
(CPU logio)
Mean ± St.
Dev.
Log Change
(CPU logio)
Mean ± St.
Dev.
%
Inactivation
Efficacy
Unpainted Pine Wood Vegetative Wetted Coupons ; 75% RH CIO2 Exposure
750
750
1.5
3
1125
2250
7.58 ±0.22
7.58 ±0.22
2.30 ± 1.53
1.56 ±0.66
5.28± 1.10
6. 02 ±0.49
A. alternate
99.99
99.99
Unpainted Pine Wood non-Vegetative coupons; 75% RH CIC-2 Exposure
750
750
3000
6
12
3
4500
9000
9000
6.29 ±0.08
6.29 ±0.08
7.01 ±0.06
<1.88±
0.00
<1.88±
0.00
<1.88±
0.00
> 4.42 ±0.06
> 4.42 ±0.06
> 5.13 ±0.04
> 99.99
> 99.99
> 99.99
*Wetted vegetative coupons w ere coupons wetted prior to inoculation and maintained w etted during treatment.
Brow n-shaded area - wetted vegetative coupons.
Pink-shaded area- 45% RH CIO2 exposure.
19
-------
Table 5. Summary of Results for Carpet (all non-vegetative coupons)
LCI02
Concentration
(ppmv)
Exposure
Time
(hr)
Anticipated Total
Exposure -CT
(ppmv-hr)
Control
Coupons
(CPU logio)
Mean ± St.
Dev.
Exposed
Coupons
(CPU logio)
Mean ± St.
Dev.
Log Change
(CPU Iog10)
Mean ± St.
Dev
%
Inactivation
Efficacy
75% RH CIO2 Exposure
S. chartarum
750
750
3000
6
12
3
4500
9000
9000
7.15±0.12
7.15±0.12
7.46 ±0.14
2.32 ±0.41
2.04 ±0.11
<2.00±
0.00
4.83 ±0.30
5.11 ± 0.12
> 5.46 ±0.10
99.99
99.99
> 99.99
C. globosum
750
750
3000
3
12
3
2250
9000
9000
7. 19 ±0.07
7. 19 ±0.07
7.64 ±0.08
3.70 ±0.20
2.18 ±0.27
2.62 ±0.55
3.48 ±0.15
>5.01 ±0.20
5. 03 ±0.39
86.99
> 99.99
99.99
A. versicolor
750
750
6
12
4500
9000
6. 94 ±0.20
6. 94 ±0.20
<1.88±
0.00
<1.88±
0.00
> 5.07 ±0.14
> 5.07 ±0.14
> 99.99
> 99.99
A. fumigatus
3000
3
9000
7.16±0.17
<2.00±
0.00
>5.16±0.12
> 99.99
A. alternata
750
750
3000
6
12
3
4500
9000
9000
5. 93 ±0.20
5. 93 ±0.20
6.91 ±0.44
<1.88±
0.00
<1.88±
0.00
<1.88±
0.00
>4.05±0.14
>4.05±0.14
> 5.04 ±0.31
> 99.99
> 99.99
> 99.99
20
-------
Table 6. Summary of Results for Glass (all non-vegetative coupons)
CIO2
Concentration
(ppmv)
Exposure
Time
(hr)
Anticipated
Total
Exposure -
CT (ppmv-
hr)
Control
Coupons
(CPU logio)
Mean ± St.
Dev.
Exposed
Coupons
(CPU logio)
Mean ± St.
Dev.
Log
Change
(CPU logio)
Mean ± St.
Dev
75% RH CIO2 Exposure
%
Inactivation
Efficacy
S. chartarum
750
750
3000
6
12
3
4500
9000
9000
7.46 ±0.01
7.46 ±0.01
7.53 ±0.17
< 2.00 ±0.00
< 2.00 ±0.00
< 2.00 ±0.00
> 5.46 ±0.01
> 5.46 ±0.01
> 5.53 ±0.12
> 99.99
> 99.99
> 99.99
C. globosum
750
750
3000
3
12
3
2250
9000
9000
7.33 ±0.07
7.33 ±0.07
7.75 ±0.07
4.55 ±0.36
3. 10 ±0.87
2.91 ±0.79
2.78 ±0.26
4.24 ±0.62
4.83 ±0.56
69.49
99.99
99.99
A. versicolor
750
750
3
12
4500
9000
6.08 ±0.13
6.08 ±0.13
< 1.88 ±0.00
< 1.88 ±0.00
>4.21 ±0.09
>4.21 ±0.09
> 99.99
> 99.99
A. fumigatus
3000
3
9000
6.91 ±0.22
< 2.00 ±0.00
>4.91 ±0.15
> 99.99
A. alternata
750
750
3000
6
12
3
4500
9000
9000
5.49 ±0.57
5.49 ±0.57
6.98 ±0.11
< 1.88 ±0.00
< 1.88 ±0.00
< 1.88 ±0.00
>3.61 ±0.40
>3.61 ±0.40
>5.10±0.08
> 99.99
> 99.99
> 99.99
A >4 log reduction in CPU was seen on latex-painted gypsum wallboard, unpainted pine wood,
industrial carpet, and glass for A. alternata and S. chartarum at CTs of 3,000 or 4,500 ppm-hr.
A. versicolorwas tested only at very high CTs and showed a >4 log reduction. However, A.
fumigatus was tested at much lower CTs, and the level of kill was highly variable and dependent
upon building material type. The results of the C. globosum testing also show a material
dependence for the efficacy of kill.
Most of the CIO2 fumigations at 75% RH were performed using standard dry materials (coupons)
prior to inoculation with mold spores. However, someCIO2 fumigations at 75% RH using wetted
coupons prior to inoculation were tested with C. globosum and A. fumigatus. The level of kill for
C. globosum on the wetted coupons was similar to that attained for the dry coupons except for
ceiling tile. A >4 log^ reduction was observed for dry ceiling tile at a CT of 2250 ppmv-hr. No
significant inactivation was observed on the wetted ceiling tile coupons exposed to the same CT.
Results were highly variable for A. fumigatus. For wetted wood coupons, a >4 LR was observed
at a CT of 1125 ppmv-hr. All the other materials tested showed a lower level of kill on the wetted
coupons.
A series of CIO2 fumigations was performed at 45% RH at high CTs. C. globosum was the only
organism exposed under these conditions. A >4 Iog10 reduction was observed for latex-painted
21
-------
wallboard, wallpapered latex-painted wallboard and unpainted pine wood at 12000 or 24000
ppmv-hr. A lower kill was observed on ceiling tile exposed to the same conditions.
In some cases, although a >4 log™ reduction in CPU was attained, there were still viable spores
on the coupons (data not shown). These spores may have the ability to germinate should the
environmental conditions become or remain favorable. The germination potential was not
evaluated.
To evaluate the effectiveness of CIO2gas to inactivate mold components, the mass (nanograms,
ng) of allergen or mycotoxin on the exposed test coupons was compared to ng of allergen or
mycotoxin of unexposed positive control coupons. The percent change was calculated using
Equation 4:
% inactivation allergen (mycotoxin) = Cone. Control - Cone. Exposed x 100 (Eq. 4)
Cone. Control
Using this formula, the results were analyzed using the averaged ng concentration of allergens
(mycotoxins) for both unexposed and exposed coupons and excluding negative log™ values. All
the tables have the same format. The first column represents the CIO2 concentration (ppmv).
The second column represents the exposure time (hr). The third column represents the total
exposure CT, in ppmv-hours. The fourth column represents the averaged ng concentrations of
unexposed positive controls and the fifth column represents the averaged ng concentrations of
exposed coupons. The sixth column represents the % inactivation of allergen (mycotoxin)
calculated using equation 4. The log™ change for the allergens and mycotoxins on each of the
materials tested was calculated using equation 2 and is shown inTables A1-A4 (Appendix A).
Table 7 shows the results for pure allergen rAlt a1 on each of the materials tested, except for
latex-painted wallboard. CIO2 exposures > 4500 showed a 99% reduction in recovered allergen
in all the materials tested. For latex-painted wallboard, results were variable; at CTs of 3000 and
9000 ppmv-hr, a 99% inactivation was attained, and all the other CTs tested showed a lower
degree of inactivation. Table 10 shows the results for allergen rAlt a1 on A. alternata non-
vegetative coupons. All materials tested showed a 97 - 99% reduction in Alt a1 allergen. Table
9 shows the results for allergen Asp f1 on vegetative coupons. Results showed that the highest
% of allergen Asp f1 inactivation was attained at 9000 ppmv-hr on latex-painted wallboard
followed by Aspergillus fumigatus antigen (Aspfl) inactivation of > 85.55 % on unpainted pine
wood. Results for all the other materials tested were highly variable, and there was no clear
distinction on which CT effectively reduced the allergen concentrations.
Table 8 shows the results for pure aflatoxin. On latex-painted wallboard, the highest %
inactivation was observed at a CIO2 concentration of 750 ppmv for 12 hours (9000 ppmv-hr) of
exposure. On carpet, the highest % inactivation was observed at a CIO2 concentration of 3000
ppmv for three hours (9000 ppmv-hr). On unpainted pine wood, at CTs > 3000 ppmv, a 93 - 94%
inactivation was attained. Wallpapered wallboard and ceiling tiles were tested only at CTs of
22
-------
1125 and 2250 ppmv-hr. On ceiling tiles, a 96% inactivation was attained for both CTs.
However, for latex-painted wallpapered wallboard, these CTs were ineffective. On glass, all CTs
tested showed an aflatoxin inactivation > 90%. The most effective condition for glass was a CIO2
concentration of 3000 ppmvfor 3 hrs (9000 ppmv-hr) of exposure.
Table 11 shows a summary of the % of inactivation efficacy of CPU on all materials. Only tests
with 99.99% of inactivation efficacy are presented in this table.
23
-------
Table 7. Summary of Results for Pure Allergen rAlt a1 on All Materials (non-vegetative
coupons)
CIO2
Concentration
(ppmv)
Exposure
Time
(hr)
Anticipated Total
Exposure - CT
(ppmv-hr)
Latex-painted Wai
3000
750
750
750
3000
1
6
9
12
3
3000
4500
6750
9000
9000
Control
Coupons
(ng)
Exposed
Coupons
(ng)
% Allergen
nactivation3
board
912.01
831.76
831.76
831.76
912.01
4.57
70.79
79.43
81.28
10.72
99.50
91.49
90.45
90.23
98.83
Unpainted Pine Wood
3000
750
750
750
3000
1
6
9
12
3
3000
4500
6750
9000
9000
602.56
707.95
707.95
707.95
602.56
74.13
<1.00
<1.00
<1.00
4.57
87.70
> 99.86
> 99.86
> 99.86
99.24
Carpet
750
750
750
3000
6
9
12
3
4500
6750
9000
9000
831.76
831.76
831.76
933.25
2.04
1.32
1.41
3.09
99.75
99.84
99.83
99.67
Glass
750
750
750
3000
6
9
12
3
4500
6750
9000
9000
776.25
776.25
776.25
575.44
<1.00
<1.00
<1.00
2.04
> 99.87
> 99.87
> 99.87
99.65
, (concentration control coupons-concentration of exposed coupons /concentration of control coupons) * 100.
24
-------
Table 8. Summary of Results for Pure Aflatoxin on All Materials (non-vegetative coupons)
CI02
Concentration
(ppmv)
Exposure
Time
(hr)
Total
Exposure
-CT
(ppmv-
hr)
Control
Coupons
(ng)
Exposed
Coupons
(ng)
% aflatoxin
inactivatiorf
Latex-painted Wallboard
750
750
3000
750
750
750
3000
1.5
3
1
6
9
12
3
1125
2250
3000
4500
6750
9000
9000
89.13
89.13
66.07
104.71
104.71
104.71
66.07
24.55
11.75
14.79
6.92
4.47
<3.98
4.27
72.46
86.82
77.61
93.39
95.73
> 96.20
93.54
Wallpapered Latex-painted Wallboard
750
750
1.5
3
1125
2250
70.79
70.79
32.36
23.99
54.29
66.12
Ceiling tile
750
750
1.5
3
1125
2250
309.03
309.03
10.96
10.47
96.45
96.61
Unpainted Pine Wood
750
750
3000
750
750
750
3000
1.5
3
1
6
9
12
3
1125
2250
3000
4500
6750
9000
9000
46.77
46.77
67.61
69.18
69.18
69.18
67.61
<7.94
8.13
<3.98
4.57
4.47
4.27
<3.98
> 83.02
82.62
> 94.11
93.39
93.54
93.83
> 94.11
Carpet
750
750
750
3000
6
9
12
3
4500
6750
9000
9000
51.29
51.29
51.29
100.00
26.30
9.33
6.61
7.76
48.71
81.80
87.12
92.24
Glass
750
750
750
3000
6
9
12
3
4500
6750
9000
9000
47.86
47.86
47.86
154.88
<3.98
<3.98
<3.98
5.50
£91.68
£91.68
£91.68
96.45
, (concentration control coupons-concentration of exposed coupons /concentration of control coupons) * 100
25
-------
Table 9. Summary of Results for Asp f 1 Allergen from Aspergillus fumigatusCF\J
Inoculated Positive Controls and Exposed Coupons in All Materials (vegetative coupons)
LCI02
Concentration
(ppmv)
Exposure
Time
(hr)
Anticipated
Total
Exposure -
CT (ppmv-hr)
Control
Coupons3
(ng)
Exposed
Coupons
(ng)
% allergen
inactivationb
Latex-painted Wallboard
750
750
3000
1.5
3
3
Wa
750
750
1.5
3
1125
2250
9000
0.26
0.26
8.32
0.30
<0.40
<0.40
-14.32
£-51.36
> 95.21
Ipapered Latex-painted Wallboard
1125
2250
0.19
0.19
0.22
0.30
-17.49
- 58.49
Ceiling Tile
750
750
1.5
3
1125
2250
0.13
0.13
0.12
0.32
8.80
-139.88
Unpainted Pine Wood
750
750
3000
1.5
3
3
1125
2250
9000
0.17
0.17
2.75
0.18
0.18
<0.40
-2.33
-2.33
> 85.55
a, Aspf 1 alergen data are not shown for carpet and glass because the allergen is a hyphal allergen. Aspf 1is not shownforlatex-
painted w allboard wetted coupons, wallpapered latex-painted wallboard wetted coupons, ceiling tile w etted coupons and unpahted
pine w ood wetted coupons because the allergen concentrations in the controls w ere below the detection limits.
b, (concentration control coupons-concentration of exposed coupons /concentration of control coupons) * 100.
26
-------
Table 10. Summary of Results for Alt a 1 Allergenfrom^/ternar/a alternata CPU
Inoculated Positive Controls and Exposed Coupons on All Materials (non-vegetative
coupons)3
LCI02
Concentration
(ppmv)
Exposure
Time
(hr)
Anticipated
Total
Exposure -
CT (ppmv-hr)
Control
Coupons
(ng)
Exposed
Coupons
(ng)
% allergen
inactivationb
Unpainted Pine Wood
750
750
3000
6
12
3
4500
9000
9000
158.49
158.49
177.83
3.55
2.24
1.12
97.76
98.59
99.37
Carpet
750
750
3000
6
12
3
4500
9000
9000
891.25
891.25
74.13
3.63
1.55
1.15
99.59
99.83
98.45
Glass
750
750
3000
6
12
3
4500
9000
9000
363.08
363.08
89.13
3.55
3.72
<1.00
99.02
98.98
> 98.88
a, Alt a 1 allergen data are not shown for latex-painted gypsum wallboard due to material assay interference or insufficient spore
production on the grow ing sample.
b, (concentration control coupons-concentration of exposed coupons /concentration of control coupons) * 100.
27
-------
Table 11. Percent Inactivation Efficacy of CPU on All Materials
CI02
Concentration
(ppmv)
Exposure
Time
(hr)
Anticipated
Total
Exposure -
CT (ppmv-
hr)
Stachybotrys
chartarum
Chaetomium
globosum
Aspergillus
versicolor
fumigatus
Alternaria
alternata
Latex-painted Gypsum Wallboard
(all vegetative coupons)
75% RH CIO2 Exposure
750
750
750
3000
750
750
3000
1.5
3
3
1
6
12
3
1125
2250
2250
3000
4500
9000
9000
—
—
—
> 99.99
> 99.99
> 99.99
> 99.99
99.99
—
99.99
—
—
—
—
99.99
> 99.99
—
99.99
—
—
—
—
99.99
—
—
—
—
> 99.99
> 99.99
> 99.99
45% RH CIO2 Exposure
4000
6
24,000
—
99.74
—
—
—
Wallpapered Latex-painted Wallboard
(all vegetative coupons)
45% RH CIO2 Exposure
4000
4000
3
6
12,000
24,000
—
—
99.99
99.99
—
—
—
—
—
—
Ceiling Tile
(all vegetative coupons)
75% RH CIO2 Exposure
750
750
1.5
3
1125
2250
—
—
99.74
—
—
99.99
> 99.99
—
—
Unpainted Pine Wood
(A.versicolor, C.globosum, and A.fumigatus\ vegetative coupons;
S. chartarum and A.alternata: non-vegetative coupons)
75% RH CIO2 Exposure
750
750
750
3000
1.5
3
3
1
1125
2250
2250
3000
—
—
—
99.99
99.99
99.99
—
—
—
—
—
—
—
—
—
—
28
-------
CIO2
Concentration
(ppmv)
750
750
750
3000
Exposure
Time
(hr)
6
9
12
3
Anticipated
Total
Exposure -
CT (ppmv-
hr)
4500
6750
9000
9000
Stachybotrys
chartarum
> 99.99
—
> 99.99
> 99.99
Chaetomium
globosum
99.99
> 99.99
> 99.99
> 99.99
Aspergillus
versicolor
> 99.99
—
> 99.99
—
fumigatus
—
—
—
> 99.99
Alternaria
alternata
> 99.99
—
> 99.99
> 99.99
45% RH CIO2 Exposure
3000
4000
3000
4000
3
3
6
6
9000
12000
18,000
24000
—
—
> 99.99
98.99
> 99.99
98.99
—
—
—
—
—
—
75% RH CIO2 Exposure - Wetted Coupons
750
750
1.5
3
1125
2250
—
—
99.99
—
—
99.99
99.99
—
—
Carpet
(all non-vegetative coupons)
75% RH CIO2 Exposure
750
750
750
3000
3
6
12
3
2250
4500
9000
9000
—
99.99
99.99
> 99.99
—
> 99.99
99.99
—
> 99.99
> 99.99
—
—
—
—
> 99.99
—
> 99.99
> 99.99
> 99.99
Glass
(all non-vegetative coupons)
75% RH CIO2 Exposure
750
750
750
3000
3
6
12
3
2250
4500
9000
9000
—
> 99.99
> 99.99
> 99.99
—
99.99
99.99
—
> 99.99
> 99.99
—
—
—
—
> 99.99
—
> 99.99
> 99.99
> 99.99
*, Only tests w ith 99.99% of inactivation efficacy are presented in this table.
29
-------
4. CONCLUSIONS AND RECOMMENDATIONS
The efficacy of CIO2 fumigation for remediation of mold and related allergens and mycotoxins
was evaluated for five commonly found mold species on five typical building materials and glass
as a standard material for biological testing. The different interactions between the various mold
species and the materials resulted in two types of coupons: coupons where the coupons were
incubated and the mold grew (vegetative coupons) and coupons where there was no growth and
the original spores remained (nonvegetative coupons). The reduction in viable spores was
determined by subtracting the average log values of CPU recovered from the treated coupons
from the CPU recovered from the positive controls. In the case of Alternaria, the pre- and post-
inoculated coupons were extracted to quantify the allergen concentration reduction. Coupons
were also prepared by inoculation with either a pure allergen and/or mycotoxin. The results
obtained lead to the following conclusions:
Mold viability. The hypothesis of the experiment was that CIO2 fumigation had the potential to
achieve a 4 log™ reduction in viable spores on typical building materials. The target
concentration to achieve this result was a CT of 9000 ppmv-hr. This CT was achieved by
treating either at 750 ppm for 12 hours or at 3000 ppm for 3 hours. However, additional CT
values (2250, 4500, 6750 ppmv-hr) were also evaluated to determine efficacy at intermediate
time points. A limited set of tests was performed at an alternative fumigation condition of 18,000
or 24,000 ppm-hr at 45% RH. Another limited series of tests was performed on wetted coupons
at low CT, a condition not likely to be encountered in an actual fumigation (but provided for
reference). The results indicate the following:
1. Table 11 shows that a greater than 4 log™ reduction was achieved at the 9000 ppmv-
hr for S. chartarum, A. versicolor, and A. alternata on latex-painted wallboard, unpainted
pine wood, carpet and glass. Likewise, a greater than 4 log™ reduction was achieved at
the 9000 ppmv-hr for C. globosum and A. fumigatus on unpainted pine wood and carpet.
A minimum 4 log™ (99.998% CPU count) reduction was achieved at the 9000 ppmv-hr
for C. globosum and A. fumigatus on latex-painted wallboard and glass. A greater than 4
log™ reduction was achieved for Stachybotrys on all materials and all CTs from 3000 to
9000 ppmv-hr.
2. In most cases, there did not appear to be a significant difference between the
efficacies of the two approaches to achieving the CT of 9000 ppmv-hr for most
organisms. The possible exception is the Alternaria where the 3000 ppm for 3 hours
appeared to be as much as one log™ more effective.
3. The fumigation appears to be somewhat more effective for a given organism on the
nonvegetative coupons as com pared to the same organism on a vegetative coupon.
However, this determination may be an artifact of the higher CPU on the nonvegetative
coupons for some organisms.
30
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4. In virtually all cases where the coupons were treated at the target CT of 9000 ppmv-
hr, no viable mold cells were detected, so the value was reported as <_ the detection limit
of the method, which ranged from log-|0 0.88 to 2.00. This value represents a maximum
number of remaining viable mold cells between 8 and 100.
5. If good mold remediation practices have been followed in the field, the source of water
will have been removed, and the RH will be controlled. Under these circumstances,
regrowth of the residual mold spores would not be expected. In some cases, although a
>4 log™ reduction in CPU was attained, there were still viable spores on the coupons
(data not shown). These spores may have the ability to germinate should the
environmental conditions become or remain favorable. The germination potential of the
spores was not evaluated.
6. The conclusion is therefore that CIO2 fumigation at a CT of 9000 ppmv-hr may be an
effective technique for reducing commonly occurring mold on typical building materials
based upon laboratory testing. In many cases, the target reduction was achieved at
lower CTs.
Allergen reduction: The hypothesis of the experiment was that chlorine dioxide fumigation had
the potential to achieve significant reductions in the allergens and mycotoxins associated with all
species. The reduction as a function of CT was evaluated on two types of samples: coupons of
all four materials inoculated with pure Alternaria allergen Alt a 1 and coupons of the three non-
vegetative materials inoculated with spores of Alternaria. The coupons were fumigated at CT
values between 3000 and 9000 ppmv-hr. The results indicate the following:
1. For the pure allergen Alt a1, the concentration was reduced between 99.2 and 100 %
at CT values of 4500 or greater on pine wood, carpet and glass coupons.
2. For the pure allergen Alt a1 on wallboard, the reduction was between 90.5 and 98.8%
for CT values between 3000 and 9000 ppmv-hrs.
3. The allergens from the Alternaria CFU coupons were reduced between 98.6 to 100%
on the three materials for CT values of 4500 and 9000 ppm-hrs.
4. There is no significant difference between the results for treatment at 750 ppm
compared to the 3000 ppm treatment on the Alternaria CFU coupons.
5. The allergen Asp f 1 from the A. fumigatus latex-painted wallboard coupons showed a
% inactivation > 95 % and on unpainted pine wood showed a % inactivation > 85.55 at a
CT of 9000 ppmv-hr. These results suggest that the efficacy of allergen inactivation is
material-dependent. The other two materials, wallpapered latex-painted wallboard and
ceiling tiles, were tested only at CTs of 1125 and 2250, and the Asp f1 inactivation was
very low.
31
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6. In conclusion, allergen inactivation is dependent on the building material and the type
of allergen.
Mycotoxin reduction: The coupons were fumigated at CT values between 3000 and 9000 ppmv-
hr. The results indicate the following
1. The mycotoxin was reduced by 91.5 to 96.3 % for wallboard, pine and glass for CT
values of 4500 ppm-hrand above.
2. The carpet appears to be the most difficult to fumigate with 92.3% reduction achieved
only with the treatment at 3000 ppm for 3 hours.
3. If similar results were obtained on mycotoxins derived from vegetative mold species,
high reductions of spore loadings by fumigation may provide effective remediation of
mycotoxins and significantly reduce the likelihood of regeneration.
Overall conclusion: In any mold remediation, the EPA guidelines call for repairing the integrity of
the structure and/or removing all other sources of water intrusion, as well as controlling RH. This
research has demonstrated the potential to achieve large percentage reductions in viable spore
counts on all materials as well as significant percentage reductions of the concentration of
allergens and mycotoxins. This laboratory research showed that the results of allergen and
mycotoxin inactivation are very promising. The building materials and most of the contents can
be fumigated in place and effectively reused after the fumigation.
32
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5. QUALITY ASSURANCE AND QUALITY CONTROL
The objective of this study was to determine the efficacy of chlorine dioxide fumigation for mold
remediation. The Data Quality Objectives (DQOs) define the critical measurements (CMs)
needed to address the stated objectives and specify tolerable levels of potential error associated
with simulating the prescribed decontamination environments. The following measurements
were deemed to be critical to accomplish part or all of the project objectives:
• Real-time fumigant concentrations;
• Temperature;
• Relative humidity;
• Fum igation tim e sequence; and
• Mold, allergen, ormycotoxin counts.
Data Quality
The Quality Assurance Project Plan (QAPP) in place for this testing was followed with few
deviations. Deviations included:
• In some of the runs, mycotoxin data were not reported because certain compounds in
the building materials interfered with the mycotoxin assay. Water-soluble components in
the materials may have inactivated the reagents. For this same reason, the polymerase
chain reaction (PCR) method could not be used for analysis.
• Certain coupon materials, like glass, would not sustain mold growth, and in these cases
the spores were inoculated directly onto the materials.
Data Quality Indicator Goals for Critical Measurements
The Data Quality Indicators (DQIs) listed in Table 12 are specific criteria used to quantify how
well the collected data meet the DQOs for the fumigations.
Table 12. DQIs for Critical Measurements
Measurement
Parameter
Real-time CIO2
concentration inside
the test chamber
Extracted CIO2, high
concentration
Analysis Method
ClorDiSys
Environmental
Monitoring System
(EMS) monitor
(0.1-30mg/L)
Modified SM 4500-
CIO2E
Accuracy
15% of
modified
SM-4500-E
10% of
standard
Detection
Limit
0.1 mg/L
36 ppm
0.1 mg/L
(solution)
Completeness1
%
100
90
33
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Relative humidity
Differential time
Temperature(T)
inside the isolation
chamber
RH probes (0-1 00
%)
Computer clock
Thermocouple
± 5 % of 83%
standard salt
solution
1 % of reading
+ 2°C
1%
0.5 sec
NA
95
95
90
1 Completeness goals of 100% are used for those parameters that a re performed manually and infrequently. A
completeness goal of 95% is used forthose data streams thatare automaticallylogged.
The measurements listed in Table 12 are specific criteria used to quantify how well the collected
data met the DQOs. The accuracy of the real-time CIO2 monitor was assessed with respect to
the Modified SM 4500-CIO2 E Method. Corrections to the real time concentration set-point were
made so that the target concentration was attained according to the titration measurement. The
accuracy of the extractive titration was assessed with respect to a standard solution.
The DQIs for the biological assays are found in Table 13.
Table 13. DQIs for the Assay Critical Measurements
Measurement
Parameter
Minimum counts from
coupons
Maximum counts
from coupons
Duplicate CPU
Frequency
Each extraction
Each extraction
Each test,
statistical check
of data quality
Acceptable Criteria
Minimum of 1 CFU/plate. If
there are 0 CPU, 0.5 CPU
will be used in the
spreadsheet as the
minimum detected.
Maximum of 500 CFU/plate
Coefficient of variance
<0.25
Completeness
%
95
95
100
The DQIs for the allergen analysis are found in Table 14.
Table 14. DQIs for the Allergen Analysis Critical Measurements
Measurement
Parameter
Minimum acceptable
allergen
Frequency
Each ELISA
analysis
Acceptable Criteria
Optical density at 405 nm
must be equal to or greater
than 1 5 ng and have a
detection value within
Completeness
%
95
34
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Maximum acceptable
allergen
Duplicate samples
Each ELISA
analysis
Each test,
statistical check
of data quality
standard curve
Optical density at 405 nm
must be equal to or less
1 00 ng and have a
detection value within
standard curve
Coefficient of variance
<0.25
95
100
The DQIs for the mycotoxin analysis are found in Table 15.
Table 15. DQIsforthe Mycotoxin Analysis Measurements
Measurement
Parameter
Minimum acceptable
mycotoxin
Maximum acceptable
mycotoxin
Duplicate Samples
Frequency
Each ELISA
analysis
Each extraction
Each test,
statistical check
of data quality
Acceptable Criteria
Optical density detection
value at 450 nm greater
than or equal to 10 ng per
reaction.
Optical density detection
value at 450 nm less than
or equal to 100 ng per
reaction
Coefficient of variance
<0.25
Completeness
%
95
95
100
The accuracy of the differential time was not assessed but error is expected to be negligible
through the use of a computer clock. The accuracy of the thermocouple was determined through
the measurement of the uncertainty following calibration.
All data from the CIO2fumigations satisfied the precision requirements.
Equipment Calibration
All equipment (e.g., pipets, incubators, biological safety cabinets) used at the time of evaluation
was verified as being certified, calibrated, or validated. All equipment (e.g., pipets,
thermocouples, RH probe, clocks) at US EPA was certified as being calibrated or having the
calibration validated by EPAs on-site (RTP, NC) Metrology Laboratory at the time of use. Any
35
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deficiencies were noted in the laboratory notebooks. Instruments were adjusted to meet
calibration tolerances and recalibrated. If tolerances were not met after recalibration, additional
corrective action was taken, possibly including, recalibration and/or replacement of the
equipment. The MSM 4500-E was used to confirm the real-time EMS CIO2 measurements.
Audits
This project was assigned QA Category III and did not require technical systems or performance
evaluation audits.
36
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6. REFERENCES
American Waterworks Association (AWWA), Standard Method 4500-CIO2-E. Amperometric
Method II. In: Eaton, A. D.; Clesceri, L. S.; Rice, E. W.; Greenberg, A. E., Eds. Standard
Methods for the Examination of Water and Wastewater, 21st ed. American Public Health
Association, American Waterworks Association, and Water Environment Federation.
Washington, DC, 2005.
Andersen, B., Frisvad, J.C., Sondergaard, I., Rasmussen, I.S., and L. S. Larsen. "Associations
between fungal species and w ater-damaged building materials", Applied and E nvironmental
Microbiology 77:4180-4188, 2011.
ASTM, ASTM D 6329-98: Standard guide for developing methodology for evaluating the ability
of indoor materials to support microbial growth using static environmental chambers, West
Conshohocken, PA, 1998.
Canter, D. A, "Remediating anthrax-contaminated sites: learning from the past to protect the
future", Chem. Health Safety, 12:13-19, 2005.
Dearborn, D.G., Yike, I., Sorenson, W.G., Miller, M.J., and R.A Etzel, "Overview of
investigations into pulmonary hemorrhage among infants in Cleveland, Ohio", Environmental
Health Perspectives, 107Suppl 3:495-499, 1999.
Frankel, M., Beko, G., Timm, M., Gustavsen, S., Hansen, E.W., and A.M. Madsen, "Seasonal
variations and indoor microbial exposures and their relation to temperature, relative humidity,
and air exchange rate", Applied and Environmental Microbiology, 78 (23):8289-8297, 2012.
Gravesen, S., Nielsen, P.A, Iversen, R., and K.F. Nielsen, "Microfungal contamination of damp
buildings- examples of risk constructions and risk materials", Environmental Health
Perspectives, 107 Suppl 3:505-508, 1999.
Institute of Medicine (IOM), "Damp Indoor Spaces and Health", National Academies Press, ISBN
0-309-09193-4, Washington, D.C.2004.
Martin, G.B., and FT. Princiotta, "Observations on Engineering Aspects of the Brentwood Postal
Facility Fumigation", AWMA/EPA Indoor Air Quality Problems and Engineering Solutions
Specialty Conference and Exhibition, Research Triangle Park, NC, July 21-23, 2003.
Martin, G.B., "Practical experiences with technologies for decontamination of B. anthracis in
large buildings", AWMA Annual Meeting, Minneapolis, MN, June 2005.
Martin, G.B., Mickunas, D., Ryan, S.P., Fredericks, S., Mattorano, D.,Zmmer, A, Zuroski, D.,
Mason, J.Y., Cavanagh, K., Dechant, D., Brooks, L.R., Intrepido, T., Kowalski, J., T. Leighton.
"Field experience with chlorine dioxide fumigation of a large hospital: Timeline and lessons
learned", Proceedings of the Thirty-first AMOP Technical Seminar on Environmental
Contamination and Response, June 3-5, 2008.
37
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Mason, J.Y., Bechberger, E.J., Matchim, D.N., and D.L Milliard, "Process for the generation of
chlorine dioxide", US Patent # 5204081, Apr 20, 1993.
Menetrez, M.Y., Foarde, K.K., Schwartz, T.D., Dean, T.R., and D.A. Betancourt, "An evaluation
of the antimicrobial effects of gas-phase ozone", Ozone-Science & Engineering, 31:316-325,
2009.
Millner, P.O., Motta, J.J., and P.L. Lentz, "Ascospores, germ pores, ultrastucture, and
thermophilism of Chaetomium", Mycologia, 69:720-733. 1977.
U.S. EPA, "Mold Remediation in Schools and Commercial Buildings", Office of Air and
Radiation, EPA report 402-K-01-001, Washington, DC, March 2001.
U.S. EPA, "Reregistration Eligibility Decision (RED) for Chlorine Dioxide and Sodium Chlorite
(Case 4023))," U.S. Environmental Protection Agency, Washington, DC, EPA/738-R-06-007,
August 2006.
U.S. EPA, http://www.epa.gov/pesticides/factsheets/chemicals/chlorinedioxidefactsheet.htm,
accessed Nov. 27, 2012.
Vesper, S.J., McKinstry, C.,Yang, C., Haugland, R.A, Kercsmar, C.M., Yike, I., Schluchter,
M.D., Kirchner, H.L, Sobolewski, J., Allan, T.M., and Dearborn, D.G., "Specific molds
associated with asthma in water-damaged homes", Journal of Occupational Environmental
Medicine, 48, 852-858, 2006.
Vesper, S.J., McKinstry, C., Haugland, R.A, Wymer, L, Bradham, K., Ashley, P., Cox, D.,
Dewalt, G., and W. Friedman, "Development of an environmental relative moldiness index for
US homes", Journal of Occupational Environmental Medicine, 49:829-833. 2007.
Wilson, S.C., Wu, C., Andriychuk, LA, Martin J.M., Brasel, T.L, Jumper, C.A, and D. C.
Straus, "Effect of chlorine dioxide gas on fungi and mycotoxins associated with sick building
syndrome", Applied and Environmental Microbiology, 71:5399-5403, 2005.
RTI International Operating Procedures
MMBD SOP #001 Standard Operating Procedure for the Preparation of Media -
Dehydrated.
MMBD SOP #002 Standard Operating Procedure for the Preparation of Sterile Water
MMBD SOP #003 Standard Operating Procedure for the Preparation of Sterile Buffer.
MMBD SOP #005 Standard Operating Procedure for the Characterization of Relative
Humidity Chambers.
MMBD SOP #009 Standard Operating Procedure for Quantitative Evaluation of
Microorganisms.
38
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MMBD SOP #012 Standard Operating Procedure for Quantitation of Viable Microorganisms
in Suspension.
MMBD SOP#017 Standard Operating Procedure for Decontamination of Humidity
Chambers.
MMBD SOP #058 Standard Operating Procedure for Direct Inoculation of Materials with a
Spore Suspension
All SOPs are maintained on file at RTI International and access to these files is permitted on-site
at RTI International.
39
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Appendix A: Tables of Results for Allergens and Mycotoxins in
Alternate Format
Table A1. Summary of Results for Pure Antigen rAlt a1 on All Materials (nonvegetative
coupons)
ICIO2 concentration
(ppmv)
Exposure
time
(hr)
Anticipated Total
Exposure -CT
(ppmv-hr)
Control
Coupons
(ng logio)
Mean ± St.
Dev.
Exposed
Coupons
(ng logio)
Mean ± St.
Dev.
Log
Change
(ng logio)
Mean ± St.
Dev.
Latex-painted Gypsum Wallboard
3000
750
750
750
3000
1
6
9
12
3
3000
4500
6750
9000
9000
2.96 ±0.04
2.92 ±0.01
2.92 ±0.01
2.92 ±0.01
2.96 ±0.04
0.66 ±0.61
1.85 ±0.02
1.90 ±0.06
1.91 ±0.05
1.03 ±0.48
-2.30 ±0.43
-1.07 ±0.02
-1.02 ±0.04
-1.01 ±0.04
-1.93 ±0.34
Unpainted Pine Wood
3000
750
750
750
3000
1
6
9
12
3
3000
4500
6750
9000
9000
2.78 ±0.08
2.85 ±0.14
2.85 ±0.14
2. 85 ±0.14
2.78 ±0.08
1.87 ±0.34
< 0.00 ±0.00
< 0.00 ±0.00
< 0.00 ±0.00
0.66 ±0.51
-0.91 ±0.24
>-2.85±
0.10
>-2.85±
0.10
>-2.85±
0.10
-2.12±0.37
Carpet
750
750
750
3000
6
9
12
3
4500
6750
9000
9000
2.92 ±0.01
2.92 ±0.01
2.92 ±0.01
2.97 ±0.08
0.31 ±0.56
0.12 ±0.76
0.15 ±0.24
0.49 ±0.46
-2.61 ±0.39
-2.80 ±0.54
-2.77±0.17
-2.48 ±0.33
Gass
750
750
750
3000
6
9
12
3
4500
6750
9000
9000
2.89 ±0.05
2.89 ±0.05
2. 89 ±0.05
2.76 ±0.09
< 0.00 ±0.00
< 0.00 ±0.00
< 0.00 ±0.00
0.31 ±0.54
>-2.89±
0.04
>-2.89±
0.04
>-2.89±
0.04
-2.45 ±0.38
40
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Table A 2. Summary of Results for Pure Aflatoxin on All Materials (nonvegetative
coupons)
CIO2 concentration
(ppmv)
Exposure
time
(hr)
Anticipated Total
Exposure -CT
(ppmv-hr)
Control
Coupons
(ng logio)
Mean ± St.
Dev.
Exposed
Coupons
(ng logio)
Mean ± St. Dev.
Log Change
(ng logio)
Mean ± St. Dev.
Latex-painted Gypsum Wallboard
750
750
3000
750
750
750
3000
1.5
3
1
6
9
12
3
1125
2250
3000
4500
6750
9000
9000
1.95 ±0.03
1.95 ±0.03
1.82±0.15
2.02 ±0.04
2.02 ±0.04
2.02 ±0.04
1.82±0.15
1.39 ±0.20
1.07 ±0.06
1.17±0.12
0.84 ±0.13
0.65 ±0.08
< 0.60 ±0.00
0.63 ±0.04
-0.56±0.12
-0.88 ±0.05
-0.64 ±0.14
-1.18±0.10
-1.37 ±0.06
>- 1.42 ±0.03
-1.19±0.11
Wallpapered Latex-painted wallboard
750
750
1.5
3
1125
2250
1.85 ±0.32
1.85 ±0.32
1.51 ±0.40
1.38 ±0.46
-0.34 ±0.36
-0.47 ±0.39
Ceiling Tile
750
750
1.5
3
1125
2250
2.49 ±0.14
2.49 ±0.14
1.04 ±0.02
1.02 ±0.03
-1.45 ±0.08
-1.47 ±0.09
Unpainted Pine Wood
750
750
3000
750
750
750
3000
1.5
3
1
6
9
12
3
1125
2250
3000
4500
6750
9000
9000
1.67 ±0.09
1.67 ±0.09
1.83±0.15
1.84±0.17
1.84±0.17
1.84±0.17
1.83±0.15
< 0.90 ±0.00
0.91 ±0.01
< 0.60 ±0.00
0.66 ±0.04
0.65 ±0.02
0.63 ±0.03
< 0.60 ±0.00
>- 0.77 ±0.05
-0.76 ±0.05
>-1.23±0.11
-1.17±0.12
-1.19±0.12
-1.20±0.12
>-1.23±0.11
Carpet
750
750
750
3000
6
9
12
3
4500
6750
9000
9000
1.71 ±0.08
1.71 ±0.08
1.71 ±0.08
2.00 ±0.06
1.42±0.10
0.97 ±0.16
0.82 ±0.19
0.89 ±0.09
-0.29 ±0.09
-0.74±0.13
-0.89±0.15
-1.10±0.08
Gass
750
750
750
3000
6
9
12
3
4500
6750
9000
9000
1.68 ±0.06
1.68 ±0.06
1.68 ±0.06
2.19±0.19
< 0.60 ±0.00
< 0.60 ±0.00
< 0.60 ±0.00
0.74 ±0.29
>- 1.08 ±0.04
>- 1.08 ±0.04
>- 1.08 ±0.04
-1.45 ±0.24
41
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Table A 3. Summary of Results for Asp f 1 Allergen fromAspergillus fumigatus CPU
Inoculated Positive Controls and Exposed Coupons in All Materials (vegetative coupons)
CIO2
concentration
(ppmv)
Exposure
time
(hr)
Anticipated
Total
Exposure -
CT (ppmv-
hr)
Control Coupons*
(ng logio)
Mean ± St. Dev.
Exposed Coupons*
(ng logio)
Mean ± St. Dev.
Log Change
(ng logio)
Mean ± St. Dev.
Latex-painted Gypsum Wallboard
750
1.5
1125
-0.58 ±0.14
-0.52 ±0.31
0.05 ±0.24
750
2250
-0.58 ±0.14
<- 0.40 ±0.00
0.18±0.10
3000
9000
0.92 ±0.51
<- 0.40 ±0.00
£-1.31 ±0.36
Wallpapered Latex-painted wallboard
750
1.5
1125
-0.72 ±0.24
-0.65 ±0.32
0.06 ±0.28
750
2250
-0.72 ±0.24
-0.52 ±0.30
0.20 ±0.27
Ceiling Tile
750
1.5
1125
-0.88 ±0.14
-0.92 ±0.26
-0.04 ±0.21
750
2250
-0.88 ±0.14
-0.50 ±0.26
0.38 ±0.21
Unpainted Pine Wood
750
1.5
1125
-0.76 ±0.40
-0.75 ±0.39
0.01 ±0.39
750
2250
-0.76 ±0.40
-0.75 ±0.39
0.01 ±0.39
3000 3 9000 0.44 ±0.49 <-0.40 ±0.00 >-0.84:
Asp f 1 allergen data are not shown for carpetand glass because the allergen is a hyphal allergen.
* Note that because these data are logio, a negative value is a number less than 1 (e.g., 0.8 ng =-0.1 ng log
>- 0.84 ±0.35
42
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Table A 4. Summary of Results for Alt a 1 Allergen from Alternaria alternata CPU
Inoculated Positive Controls and Exposed Coupons on All Mate rials (nonvegetative
coupons)
CI02
concentration
(ppmv)
Exposure
time
(hr)
Anticipated
Total
Exposure -
CT (ppmv-
hr)
Control Coupons
(ng logio)
Mean ± St. Dev.
Exposed Coupons
(ng logio)
Mean ± St. Dev.
Log Change
(ng logio)
Mean ± St. Dev.
Unpainted Pine Wood
750
750
3000
6
12
3
4500
9000
9000
2.20 ±0.33
2.20 ±0.33
2.25 ±0.07
0.55 ±0.29
0.35 ±0.54
0.05 ±0.11
-1.65 ±0.31
-1.86 ±0.45
-2.20 ±0.09
Carpet
750
750
3000
6
12
3
4500
9000
9000
2.95 ±0.06
2.95 ±0.06
1.87 ±0.37
0.56 ±0.31
0.19 ±0.42
0.06 ±0.14
-2.39 ±0.32
-2.77 ±0.38
-1.82 ±0.11
Gass
750
750
3000
6
12
3
4500
9000
9000
2.56 ±0.25
2.56 ±0.25
1.95±0.16
0.55 ±0.35
0.57 ±0.33
< 0.00 ±0.00
-2.00 ±0.34
-1.98 ±0.33
> -1.95 ±0.05
Alt a 1 allergen data are not shown for latex-painted gyps umwallboard due to material assay interference or
insufficient spore production on the growing sample.
43
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United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Offce of Research and Development (8101R)
Washington, DC 20460
Off cial Business
Penalty for Private Use
$300
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