August 2004
Environmental Technology
Verification Report
CERTEK, INC.
1414RH FORMALDEHYDE
GENERATOR/NEUTRALIZER
Prepared by
Battelle
Baiteiie
The Business oj Innovation
Under a contract with
RrX U.S. Environmental Protection Agency
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August 2004
Environmental Technology Verification
Report
ETV Building Decontamination Technology Center
CERTEK, Inc.
1414RH Formaldehyde
Generator/Neutralizer
by
James V. Rogers
Carol L. Sabourin
Michael L. Taylor
Karen Riggs
Young W. Choi
Darrell W. Joseph
William R. Richter
Denise C. Rudnicki
Battelle
Columbus, Ohio 43201
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Notice
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development, has financially supported and collaborated in the extramural program
described here. This document has been peer reviewed by the Agency and recommended for
public release. Mention of trade names or commercial products does not constitute
endorsement or recommendation by the EPA for use.
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Foreword
The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting
the nation's air, water, and land resources. Under a mandate of national environmental laws,
the Agency strives to formulate and implement actions leading to a compatible balance
between human activities and the ability of natural systems to support and nurture life. To
meet this mandate, the EPA's Office of Research and Development provides data and
science support that can be used to solve environmental problems and to build the scientific
knowledge base needed to manage our ecological resources wisely, to understand how
pollutants affect our health, and to prevent or reduce environmental risks.
The Environmental Technology Verification (ETV) Program has been established by the
EPA to verify the performance characteristics of innovative environmental technologies
across all media and to report this objective information to permitters, buyers, and users of
the technology, thus substantially accelerating the entrance of new environmental
technologies into the marketplace. Verification organizations oversee and report verification
activities based on testing and quality assurance protocols developed with input from major
stakeholders and customer groups associated with the technology area. ETV consists of
seven environmental technology centers. Information about each of these centers can be
found on the Internet at http://www.epa.gov/etv.
Effective verifications of monitoring technologies are needed to assess environmental
quality and to supply cost and performance data to select the most appropriate technology
for that assessment. In 2002, EPA established the Building Decontamination Technology
Center at Battelle. Battelle plans, coordinates, and conducts verification tests of
decontamination technologies and reports the results to the community at large. Information
concerning this specific environmental technology area can be found on the Internet at
http://www.epa.gov/etv/centers/center9.html.
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Acknowledgments
The authors wish to acknowledge the support of all those who helped plan and conduct the
verification test, analyze the data, and prepare this report. In particular we would like to
thank Dr. John Chang, U.S. Environmental Protection Agency (EPA); Ms. Doris
Betancourt, EPA; Ms. Shirley Wasson, EPA; Mr. Jeff Kempter, EPA; Mr. Bruce Henschel,
EPA; Dr. Lloyd Larson, U.S. Army West Desert Test Center, Dugway Proving Ground; and
Dr. Barry Pyle, Montana State University who reviewed the test/quality assurance plan
and/or verification report.
IV
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Contents
Notice ii
Foreword iii
Acknowledgments iv
List of Abbreviations vii
1. Background 1
2. Technology Description 2
3. Test Design and Procedures 4
3.1 Introduction 4
3.2 Test Design 5
3.3 Agents and Surrogates 5
3.4 Test Sequence 6
3.5 Coupon-Scale Testing 6
3.5.1 Preparation of Test Materials 7
3.5.2 Application of Agents to Test Coupons 7
3.5.3 Confirmation of Surface Applications 8
3.5.4 Decontamination 8
3.5.4.1 Verification Testing Apparatus and Parameters 8
3.5.4.2 Formaldehyde Measurement 11
3.5.4.3 Decontamination Efficacy 12
3.5.5 Observation of Surface Damage 13
4. Quality Assurance/Quality Control 14
4.1 Equipment Calibration 14
4.2 Audits 14
4.2.1 Technical Systems Audit 14
4.2.2 Audit of Data Quality 14
4.3 QA/QC Reporting 15
4.4 Data Review 15
5. Statistical Methods 16
5.1 Efficacy Calculations 16
5.2 Statistical Analysis 16
6. Test Results 17
6.1 Efficacy 17
6.1.1 Bacillus anthracis Ames Spores 17
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6.1.2 Bacillus subtilis (ATCC 19659) Spores 21
6.1.3 Geobacillus stearothermophilus (ATCC 12980) Spores 24
6.1.4 Statistical Analysis 26
6.2 Damage to Coupons 27
6.3 Other Factors 27
6.3.1 Operation of the 1414RH Unit 27
6.3.2 Operator Bias 28
7. Performance Summary 30
8. References 32
Figures
Figure 2-1. CERTEK, Inc. #1414RH 2
Figure 3-1. Test Materials 4
Figure 3-2. Overview of Plas-Labs Compact Glove Box 9
Figure 3-3. Nebulizers in the Plas-Labs Compact Glove Box 10
Figure 3-4. Formaldehyde Monitor 11
Figure 6-1. Representative Cycle Parameter Data from a Single Experiment 29
Tables
Table 3-1. Test Sequence and Parameters 6
Table 3-2. Material Characteristics 7
Table 4-1. Summary of Data Recording Process 15
Table 6-1. 1414RH Unit Decontamination of Bacillus anthracis Ames Spores 18
Table 6-2. Liquid Culture Assessment of Bacillus anthracis Ames Spores 19
Table 6-3. Liquid Culture Assessment of Biological Indicators/Spore Strips
(Week 1 B. anthracis Decontamination) 20
Table 6-4. Liquid Culture Assessment of Biological Indicators/Spore Strips
(Week 2 B. anthracis Decontamination) 20
Table 6-5. 1414RH Unit Decontamination of Bacillus subtilis Spores 21
Table 6-6. Liquid Culture Assessment of Bacillus subtilis Spores 22
Table 6-7. Liquid Culture Assessment of Biological Indicators/Spore Strips
(Week 1 B. subtilis Decontamination) 23
Table 6-8. Liquid Culture Assessment of Biological Indicators/Spore Strips
(Week 2 B. subtilis Decontamination) 23
Table 6-9. Liquid Culture Assessment of Biological Indicators/Spore Strips
(Week 3 B. subtilis Decontamination) 23
Table 6-10. 1414RH Unit Decontamination of Geobacillus stearothermophilus Spores.... 24
Table 6-11. Liquid Culture Assessment of Geobacillus stearothermophilus Spores 25
VI
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Table 6-12. Liquid Culture Assessment of Biological Indicators/Spore Strips (Week 1
G. stearothermophilus Decontamination) 26
Table 6-13. Liquid Culture Assessment of Biological Indicators/Spore Strips (Week 2
G. stearothermophilus Decontamination) 26
Table 6-14. Statistical Analysis of Mean Efficacy (Log Reduction) for Spores 27
vn
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List of Abbreviations
ANOV A analysis of variance
BDT Building Decontamination Technology
BSC biological safety cabinet
BWD bare wood (pine lumber)
CPU colony-forming unit
cm centimeter
DL decorative laminate
EPA U.S. Pnvironmental Protection Agency
ETV Environmental Technology Verification
FMI Fluid Metering, Inc.
g gram
GM galvanized metal ductwork
GS glass
HEPA high-efficiency particulate air
1C industrial-grade carpet
in inch
MFC mass flow controller
min minute
mL milliliter
PC painted (latex, semi-gloss) concrete cinder block
ppb part per billion
ppm part per million
psig gauge pressure
PW painted (latex, flat) wallboard paper
QA quality assurance
QC quality control
QMP Quality Management Plan
SD standard deviation
TSA technical systems audit
USAMRIID United States Army Medical Research Institute of Infectious Diseases
Vlll
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Chapter 1
Background
The U.S. Environmental Protection Agency (EPA) supports the Environmental Technology
Verification (ETV) Program to facilitate the deployment of innovative environmental tech-
nologies through performance verification and dissemination of information. The goal of the
ETV Program is to further environmental protection by accelerating the acceptance and use
of improved and cost-effective technologies. ETV seeks to achieve this goal by providing
high-quality, peer-reviewed data on technology performance to those involved in the design,
distribution, financing, permitting, purchase, and use of environmental technologies.
ETV works in partnership with recognized testing organizations; with stakeholder groups
consisting of buyers, vendor organizations, and permitters; and with the full participation of
individual technology developers. The program evaluates the performance of innovative
technologies by developing test plans that are responsive to the needs of stakeholders, con-
ducting field or laboratory tests (as appropriate), collecting and analyzing data, and
preparing peer-reviewed reports. All evaluations are conducted in accordance with rigorous
quality assurance (QA) protocols to ensure that data of known and adequate quality are
generated and that the results are defensible.
The EPA's National Risk Management Research Laboratory and its verification
organization partner, Battelle, operate the Building Decontamination Technology (BDT)
Center under ETV. The BDT Center recently evaluated the performance of the CERTEK,
Inc., Model# 1414RH formaldehyde gas generator/neutralizer for decontaminating
buildings.
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Chapter 2
Technology Description
The objective of the ETV BDT Center is to verify the performance characteristics of
technologies that can be used to decontaminate indoor surfaces in buildings contaminated
with either chemical or biological agents as a result of an intentional attack. This verifica-
tion report provides results for testing the CERTEK, Inc. 1414RH formaldehyde gas
generator/neutralizer. The following is a description of the 1414RH unit, based on
information provided by the vendor.
The information provided below was
not verified in this test.
The 1414RH unit generates
formaldehyde gas for decontam-
inating a sealed area. Formulas in the
operating manual are used to
calculate the appropriate amount of
water, paraformaldehyde, and
neutralizer (ammonium carbonate)
based on the volume of the space
intended to be decontaminated. The
1414RH unit generates the appro-
priate relative humidity (50 to 90%),
then generates formaldehyde gas for
an operator-selected contact time,
and finally it generates neutralizer
into the decontaminated space. The hexamethylenetetramine formed by the reaction of
formaldehyde with the neutralizer is a white powder with a slight "fishy" odor.
Figure 2-1. CERTEK, Inc. # 1414RH
The 1414RH unit has a capacity of 240 grams (g) of paraformaldehyde per canister. Based
upon the recommendation of the U.S. Army Medical Research Institute of Infectious
Diseases (USAMRIID), 0.3 g of paraformaldehyde per cubic foot should be used to
decontaminate Bacillus anthracis. Therefore, 240 g of paraformaldehyde is sufficient to
treat an enclosure of approximately 800 cubic feet (23 cubic meters). The operation of the
1414RH unit can be modified to utilize a second canister filled with 240 g of
paraformaldehyde; therefore, 480 g of paraformaldehyde can treat an enclosure of
approximately 1,600 cubic feet (45 cubic meters). The 1414RH unit weighs 55 pounds
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(25 kilograms), and is 12 inches (in) [30 centimeters (cm)] wide by 20 in (51 cm) in depth
by 12 in (30 cm) in height.
The 1414RH unit was attached to a Plas-Labs Compact Glove Box (Model No. 830-ABC)
modified for this verification test (see Section 3.5.4.1). The connections between the
1414RH unit and the glove box consisted of flexible supply and delivery gassing hoses
connected to high-efficiency particulate air (HEPA) filters. A formaldehyde monitor also
was connected to the glove box to measure the concentration of formaldehyde during each
run of this verification test. A hygrometer was added inside of the glove box to measure
relative humidity.
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Chapter 3
Test Design and Procedures
3.1 Introduction
This verification test was conducted according to procedures specified in the Test/QA Plan
for Verification of Formaldehyde Vapor Technologies for Decontaminating Indoor Surfaces
Contaminated with Biological or Chemical Agents.^ The biological and chemical agents
that pose a threat to buildings include toxic industrial chemicals, chemical warfare agents,
and biological warfare agents (including
biotoxins). The biological agent selected for
this verification test was Bacillus anthracis
(Ames strain). In addition, two biological
surrogates were used: B. subtilis (ATCC
19659) and Geobacillus stearothermophilus
(ATCC 12980). Seven materials
representing indoor surfaces commonly
found in buildings were used for the
verification testing. The indoor surfaces
tested (Figure 3-1) include
Industrial-grade carpet (1C)
Bare wood (pine lumber) (BWD)
Glass (GS)
Decorative laminate (DL)
Galvanized metal ductwork (GM)
Painted (latex, flat) wallboard paper
(PW)
Painted (latex, semi-gloss) concrete
cinder block (PC).
Figure 3-1. Test Materials.
The objective of the verification testing was to evaluate the efficacy of the 1414RH unit to
decontaminate a biological agent or surrogate. Efficacy was tested by applying a biological
agent and surrogates to the surfaces of test coupons and, after using the 1414RH unit,
comparing the number of viable spores on decontaminated and control (non-
decontaminated) samples. Visual inspection of the physical integrity of the test materials
was performed, and observations were recorded before and after using the 1414RH unit in
an effort to detect any degradation or chemical destruction of the material itself.
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3.2 Test Design
Coupons were cut from larger pieces of the representative materials for each of the seven
indoor surfaces (Section 3.1). These coupons measured 3/4 x 3 in (1.9 x 7.5 cm) and varied
in thickness from about 1/32 in (0.079 cm) to 3/8 in (0.95 cm), depending upon the material.
In triplicate, the coupons were placed into a biological agent safety hood, and aliquots of an
aqueous suspension of the biological agent were added to the surface of each coupon. Based
upon the concentration of the spores in the aqueous suspension, the number of spores added
to each coupon was calculated. The coupons were allowed to dry overnight. After drying,
the inoculated coupons intended for decontamination were transferred into a custom-
modified glove box and placed horizontally on a wire rack. Both blank (uncontaminated;
N=2) and control (inoculated with spores, but not decontaminated; N=3) coupons were
prepared, together with the inoculated coupons that were to be decontaminated (N=3).
Efficacy of the 1414RH unit was determined by comparing the number of viable spores on
the control coupons (not decontaminated) to the number present on the decontaminated
coupons, expressed as a log reduction. Following extraction of spores from the test, control,
and blank coupons, efficacy was further evaluated for each biological agent or surrogate by
transferring each coupon into liquid growth medium and assessing bacterial growth after
1 and 7 days.
Physical degradation of the indoor materials used as test surfaces was evaluated informally
in conjunction with the efficacy testing procedure. After decontaminating the test coupons,
the appearance of the decontaminated coupons was observed; and any obvious changes in
the color, reflectivity, and apparent roughness of the coupon surfaces were noted.
3.3 Agents and Surrogates
The following biological agent was used for verification testing:
• Bacillus anthracis spores (Ames strain).
To provide correlations with the biological agent results, two biological surrogates also were
used:
• Bacillus subtilis spores (ATCC 19659)
• Geobacillus stearothermophilus spores (ATCC 12980).
Biological indicators and spore strips that were used to evaluate decontamination efficacy
included:
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Biological indicators (Apex Laboratories, Apex, North Carolina), approximately 1x10
spores each: Bacillus subtilis (ATCC 19659) and Geobacillus stearothermophilus
(ATCC 12980) spores on steel disks and sealed Tyvek pouches
Spore strips (Raven Biological Laboratories, Omaha, Nebraska): with Bacillus
atrophaeus (ATCC 9372) spores, approximately 1 x 106 spores per strip on a filter paper
matrix in sealed glassine envelopes.
3.4 Test Sequence
In Table 3-1, a summary of the verification testing of the 1414RH unit is presented.
Verification testing was performed during a 7-week period that commenced in November
2003 and concluded in January 2004.
Table 3-1. Test Sequence and Parameters
Test
Procedure
Parameters Evaluated
Data Produced
Biological
Efficacy Test
Coupon
Damage
Enumerations
B. anthracis
B. subtilis
G. stearothermophilus
Liquid culture assessment of coupons
B. anthracis
B. subtilis
G. stearothermophilus
Biological indicators/spore strips
B. subtilis
G. stearothermophilus
B. atrophaeus
Damage to test coupons
Log reduction (Efficacy)
Positive/negative bacterial growth (1 and 7 days)
Positive/negative bacterial growth (1 and 7 days)
Visual observation of every test coupon in all
biological efficacy tests before and after
decontamination
3.5 Coupon-Scale Testing
Coupon-scale testing was used to evaluate the decontamination efficacy of the 1414RH unit
by extracting and measuring the viable biological spores on test coupons.
3.5.1 Preparation of Test Materials
Coupons used for biological agent decontamination were cut to about 3/4 x 3 in (1.9 x
7.5 cm) and prepared as shown in Table 3-2 by Battelle staff. Test coupons were visually
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inspected, and the condition of each coupon was recorded. The length, width, and thickness
of the test coupons were measured and recorded. Chain-of-custody forms were used to
ensure that the test coupons were traceable throughout all phases of testing.
Table 3-2. Material Characteristics
Material
Decorative
Laminate
Galvanized
Metal
Ductwork
Glass
Industrial-
grade Carpet
Concrete,
Cinder Block
Wallboard
Paper
Wood
Lot, Batch, or
ASTM No., or
Observation
Laminate/ Formica/
White Matte Finish
Industry HVAC
standard 24 Gauge
Galvanized Steel
C1036
ShawTek,
EcoTek 6
ASTM C90
05-16-03; Set-E-
493; Roll-3
Screen Molding
(Pine Wood)
Manufacturer/
Supplier Name
Solid Surface
Design
Accurate
Fabrication
Brooks Brothers
Shaw Industries,
Inc.
Wellnitz
United States
Gypsum
Company
Kingswood
Lumber
Approximate
Coupon Size,
L x W (inch)
3x3/4
3x3/4
3x3/4
3x3/4
3x3/4
3x3/4
3x3/4
Material Preparation
Wiped with 70% isopropanol
Cleaned with acetone; wiped
with 70% isopropanol
Cleaned with acetone; wiped
with 70% isopropanol
Wiped with 70% isopropanol
Brush and roller painted all
sides. One coat Martin Senour
latex primer (#71-1185) and one
coat Porter Paints latex semi-
gloss finish (#919); wiped with
70% isopropanol
Roller painted on one side using
Martin Senour Paints. One
primer (#71-1185) and two
finish (flat, #70-1001) coats;
wiped with 70% isopropanol
Wiped with 70% isopropanol
3.5.2 Application of Agents to Test Coupons
Biological agent test coupons were laid flat in a Biological Safety Cabinet (BSC) Class III
and contaminated at challenge levels of approximately 1 x 108 spores per coupon. Working
stock suspensions of the spores at the required concentration were transferred to the coupon
using a micropipette by placing the suspension over the surface as small droplets. After
contamination with biological agent or surrogate suspension, the test coupons were allowed
to dry overnight, undisturbed. The next day, the inoculated test materials intended for
decontamination (and one blank) were transferred to the glove box that was attached to the
1414RH unit (see Section 3.5.4.1). The control inoculated test materials (not intended for
decontamination) and one blank were left undisturbed in a BSC Class II.
3.5.3 Confirmation of Surface Applications
To confirm the application density of the biological agent and surrogates, the B. anthracis
and surrogate spore suspensions used to contaminate the coupons were serially diluted and
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plated each day of use and enumerated the following day. The plating and enumerating were
carried out as described in Section 3.5.4.3.
3.5.4 Decontamination
3.5.4.1 Verification Testing Apparatus and Parameters
A Plas-Labs Compact Glove Box (Model 830-ABC) was utilized as the test chamber
(Figure 3-2). The parameters used for this test, as specified by CERTEK, Inc., were
according to the CERTEK Model 1414RH Formaldehyde Generator/Neutralizer Operating
Protocol.^ Specifically, the temperature should be between 60 and 90°F (16-32°C) and the
relative humidity must be held between 50 and 90%. The concentration of paraformalde-
hyde and contact time (0.3 g of paraformaldehyde per cubic foot treated volume with a
10-hour contact time) were recommended by the vendor and based upon the
recommendations of the USAMRIID (as stated in the CERTEK Model 1414RH
Formaldehyde Generator/Neutralizer Operating Protocol).
For this verification test, it was difficult to generate the required relative humidity using the
capability of the RH1414 unit at the operating level as stated above. To solve this problem,
Battelle staff configured a series of six nebulizers (Figure 3-3) inside the glove box to
generate water vapor without using the capability of the 1414RH unit. These nebulizers
were joined to a HEPA filter that was connected to an air pump. Air was pumped through
the nebulizers at 5 to 7 psig (gauge pressure), and a relative humidity of 75% was achieved
within 5 minutes. The 1414RH unit has a Humidify/Bypass switch, enabling initiation of the
Formaldehyde Insert mode once 75% relative humidity was achieved.
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Gas Sampling
Line to Monitor
Figure 3-2. Overview of Plas-Labs Compact Glove Box
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Figure 3-3. Nebulizers in the Plas-Labs Compact Glove Box
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Figure 3-4. Formaldehyde Monitor
3.5.4.2 Formaldehyde Measurement
A previously developed monitor® was used to measure the formaldehyde concentration
within the Plas-Labs Compact Glove Box during each run. This monitor (Figure 3-4) was
developed to measure formaldehyde concentrations within a range of approximately 1 part
per billion (ppb) to 1 part per million (ppm). For this verification test, the concentration of
formaldehyde within the glove box is much higher than 1 ppm (theoretical calculation of
approximately 8,600 ppm); therefore, this monitor had to be modified to dilute the gas
sample from the glove box approximately 1:10,000. This dilution system was designed as
two identical 1:100 systems in series, where each subsystem was made up of a Sierra
Instruments mass flow controller (MFC) and a Fluid Metering, Inc. (FMI), valveless
rotating and reciprocating piston metering pump. In the 1:100 dilution subsystems, the FMI
pumps are set to exactly 10.0 milliliter per minute (mL/min) flow rate. The first FMI
subsystem pulled 10 mL/min from the glove box, which was mixed with 990 mL/min air
gas stream from a gas cylinder controlled by the MFC. From the exhaust stream of the first
FMI, the second FMI subsystem pulled 10 mL/min, which was also mixed with 990 mL/min
air gas stream from the same gas cylinder controlled by the second MFC. The sample from
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the glove box was then diluted 1:10,000. All gas flows were calibrated against a Buck
bubble meter.
For approximately 15 min prior to operating the 1414RH unit, the glove box was monitored
for background formaldehyde concentration (ppm). Once a background baseline had been
established, the 1414RH unit was operated according to the vendor's instructions. The
formaldehyde concentration in the glove box was monitored in real-time throughout the
complete operational cycle of the technology, and the data were recorded on a strip chart.
Using a formaldehyde standard and the known dilution factor, the data from the strip chart
were calculated and expressed as ppm formaldehyde.
3.5.4.3 Decontamination Efficacy
Biological agent or surrogate decontamination efficacy was quantified by measuring the
viable spores on both exposed (test) and unexposed (control) coupons. Each coupon was
placed in a 50 mL test tube containing 10 mL of sterile phosphate-buffered saline to which
0.1% Triton X-100 had been added. The purpose of the Triton X-100 was to minimize
clumping of spores. For spore extraction, the tubes were agitated on an orbital shaker for
15 minutes at room temperature. Each tube was then heat-shocked at 60 to 65°C for one
hour to kill vegetative bacteria. Following the heat-shock, 1.0 mL of each extract was
removed, and a series of dilutions through 10"7 were prepared in sterile water.
Spore viability was determined by dilution plating, using both the undiluted extracts and the
successive dilutions of each extract. One hundred microliters of the undiluted extract and of
each serial dilution were plated onto tryptic soy agar plates in triplicate, allowed to dry, and
incubated overnight at 35 to 37°C for B. anthracis and B. subtilis and at 55 to 60°C for
G. stearothermophilus. Plates were enumerated the next day, and the colony-forming units
(CFU)/mL were determined by multiplying the average number of colonies per plate by the
reciprocal of the dilution. Data were expressed as a mean ± standard deviation (SD) of the
number of CPUs observed. To calculate the efficacy of the decontamination treatment, the
number of spores remaining on the decontaminated test coupons was compared to the
number of spores on the control coupons. Efficacy for biological agents was expressed in
terms of a log reduction.
An additional qualitative assessment of the 1414RH unit efficacy was conducted following
spore extraction. After the extraction process described above, each coupon was transferred
to a sterile 50-mL tube containing 20 mL of tryptic soy broth culture medium. The vials
were sealed and incubated on an orbital shaker at the appropriate temperatures (see above)
for each organism. At 1 and 7 days post-decontamination, the tubes were visually assessed
qualitatively for viability as "growth" or "no growth." The biological indicators and spore
strips were also evaluated at 1 and 7 days post-decontamination for "growth" or "no
growth."
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3.5.5 Observation of Surface Damage
Following decontamination, each test surface was examined visually to establish whether
decontamination using the 1414RH unit caused any obvious damage to the surface. The
coupons were observed immediately after completing the decontamination process, but
before post-decontamination sampling. The surface was inspected by comparing the
decontaminated test surface with control coupons of the same test material. Differences in
color, reflectivity, contrast, and roughness were assessed and recorded.
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Chapter 4
Quality Assurance/Quality Control
Quality assurance (QA)/quality control (QC) procedures were performed in accordance with
the Quality Management Plan (QMP) for the BDT Center(4) and the test/QA plan for this
verification test.(1) QA/QC procedures and results are described below.
4.1 Equipment Calibration
All equipment (e.g., pipettes, incubators, biosafety cabinets) used at the time of testing was
verified as being certified, calibrated, or validated.
4.2 Audits
Two types of audit were performed during the verification test: a technical systems audit
(TS A) of the verification test performance and an audit of data quality. Audit procedures are
described below.
4.2.1 Technical Systems Audit
The Battelle Quality Assurance Unit conducted a TSA on January 21, 2004, to ensure that
the verification test was being conducted in accordance with the test/QA plan(1) and the
BDT Center QMP.(4) As part of the TSA, test procedures were compared to those specified
in the test/QA plan, and data acquisition and handling procedures were reviewed.
Observations and findings from the TSA were documented and submitted to the Battelle
Verification Test Coordinator for response. None of the findings of the TSA required
corrective action. TSA records are permanently stored with the ETV Quality Assurance
Manager.
4.2.2 Audit of Data Quality
At least 10% of the data acquired during the verification test were audited. A Battelle
Quality Assurance Auditor traced the data from the initial acquisition, through reduction and
statistical analysis, to final reporting to ensure the integrity of the reported results. All
calculations performed on the data undergoing the audit were checked.
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4.3 QA/QC Reporting
Each audit was documented in accordance with Section 3.3.4 of the QMP for the ETV BDT
Center.(4) Once the audit reports were prepared, the Battelle Verification Test Coordinator
ensured that a response was provided for each adverse finding or potential problem and
implemented any necessary follow-up corrective action. A Battelle Quality Assurance
Auditor ensured that follow-up corrective action was taken.
4.4 Data Review
Records generated in the verification test received a QC/technical review and a QA review
before they were used to calculate, evaluate, or report verification results. Table 4-1
summarizes the types of data recorded and reviewed. All data were recorded by Battelle
staff. The person performing the QC/technical review added his/her initials and the date to a
hard copy of the record being reviewed.
Table 4-1. Summary of Data Recording Process
Data to Be
Recorded
Dates, times of test
events
Test parameters (agent or
surrogate identities,
concentrations, test
surfaces, test conditions,
etc.)
Sampling data
Biological enumeration
and liquid culture
assessment, chain of
custody, and results
Records and
observations of 1414RH
unit use
Surface damage
Where
Recorded
Data forms
Data forms
Data forms
Data forms
Printout from the
formaldehyde
monitor; data
forms
Data forms
How Often
Recorded
Start/end of test, and at
each change of a test
parameter
When set or changed, or
as needed to document the
sequence of test
At least at start/end of
reference sample, and at
each change of a test
parameter
Throughout sample
handling and analysis
process
Throughout
implementation of the
1414RH unit
Start/end of test
Disposition of
Data
Used to organize/check test
results; manually incorporated
into spreadsheets as necessary
Used to organize/check test
results; manually incorporated
in data spreadsheets as
necessary
Used to organize/check test
results; manually incorporated
into spreadsheets as necessary
Transferred to spreadsheets
Reviewed and summarized to
support data interpretation
Used to assess damage of test
materials following use of the
1414RH unit
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Chapter 5
Statistical Methods
The statistical methods for evaluating the efficacy of the 1414RH unit are presented in this
chapter. Qualitative observations also were used to evaluate verification test data.
5.1 Efficacy Calculations
For biological agents and surrogates, decontamination efficacy was calculated as the log
reduction in viable organisms achieved by the 1414RH unit. The efficacy (E), or log
reduction, for the biological agent, or surrogates was calculated as
E = log (N°/N)
where N° is the mean number of viable organisms recovered from the control coupons (i.e.,
those not subjected to decontamination), and N is the number of viable organisms recovered
from each test coupon after decontamination. For decontaminated samples where viable
organisms were not detected, the efficacy was calculated as the log of the mean number of
viable organisms on the control coupons. Using the calculated log reduction for each test
coupon, the mean log reduction (efficacy) ± SD was calculated.
Percent recovery was calculated for each type of test material inoculated with each
biological agent or surrogate. Percent recovery (mean ± SD) was calculated by dividing the
number of biological organisms in the treated sample by the number of biological organisms
in the controls (non-decontaminated).
5.2 Statistical Analysis
For each material and species combination, log reduction was calculated as described above,
resulting in a total of 63 log reduction values. In cases where no viable colonies remained
after decontamination, one colony was assumed to be present for the purpose of this calcula-
tion. A two-way analysis of variance (ANOVA) model with main effects for Bacillus
species and test material and interactions was fitted to the log reduction data. This model
was used to compare each mean to zero, compare each surrogate to B. anthracis (within
material), and compare each surrogate to B. anthracis for porous and non-porous materials.
T-tests or statistical contrasts were used for the comparisons, with no adjustment for
multiple comparisons. The ANOVA model was fitted using the SAS (Version 8.2) GLM
procedure.
16
-------
Chapter 6
Test Results
The results of the verification test of the 1414RH unit are presented in this section.
6.1 Efficacy
6.1.1 Bacillus anthracis Ames Spores
Exposure of material test coupons contaminated with B. anthracis Ames spores to the
1414RH unit, resulted in decontamination that varied according to the type of the test
material (Table 6-1). The mean log reduction of detectable viable B. anthracis Ames spores
ranged from 5.17 to 7.86 across all seven test materials. Three of these test materials (1C,
BWD, PC) can be considered porous (on the inoculated surface), while the other four test
materials (GS, DL, GM, PW) can be considered non-porous (on the inoculated surface). The
log reduction in viable spores detected on the porous materials was > 7.00, > 7.61, and 7.15
for 1C, BWD, and PC, respectively. The log reduction in viable spores detected on the non-
porous materials was > 7.71, 6.47, > 7.86, and > 5.17 for GS, DL, GM, and PW,
respectively. For the PW, the log reduction in viable spores was calculated to be > 5.17,
although no viable spores were detected during the enumerations. This suggests that the
> 5.17 calculated log reduction may not accurately reflect the decontamination process, but
may be a result of the low recovery rate of 0.16%.
A liquid culture growth assessment at 1 and 7 days post-decontamination was performed to
determine whether viable B. anthracis Ames spores remained on the test materials following
the extraction step (Table 6-2). The extraction efficiency for spores on all seven test
materials was less than 100%; therefore, it was assumed that viable spores could remain on
the test materials. Each test material was wiped with 70% isopropanol prior to inoculation
(or non-inoculated blanks) with B. anthracis Ames spores; however, this isopropanol wash
does not guarantee sterility, especially with the porous materials. The test materials were not
autoclaved due to the risk of the materials being damaged during the autoclaving process.
Therefore, to maintain equivalent treatment and handling of the test materials, a 70%
isopropanol wipe was used. The liquid culture assessment was intended to detect spores that
remained on the test material following the extraction step. However, since the materials
were not sterilized by autoclaving, this type of assessment may not discriminate between the
growth of B. anthracis and/or other microorganisms.
17
-------
Table 6-1.1414RH Unit Decontamination of Bacillus anthracis Ames Spores8
Test Material
Industrial-Grade Carpet (1C)
Control
Decontaminated
Blank (control)
Blank (decontaminated)
Bare Wood (BWD)
Control
Decontaminated
Blank (control)
Blank (decontaminated)
Glass (GS)
Control
Decontaminated
Blank (control)
Blank (decontaminated)
Decorative Laminate (DL)
Control
Decontaminated
Blank (control)
Blank (decontaminated)
Galvanized Metal Ductwork
(GM)
Control
Decontaminated
Blank (control)
Blank (decontaminated)
Painted Wallboard Paper (PW)
Control
Decontaminated
Blank (control)
Blank (decontaminated)
Painted Concrete (PC)
Control
Decontaminated
Blank (control)
Blank (decontaminated)
Inoculum
9.33 x 107
9.33 x 107
0
0
1.02xl08
1.02xl08
0
0
9.33 x 107
9.33 x 107
0
0
1.02xl08
1.02xl08
0
0
1.02xl08
1.02xl08
0
0
9.33 x 107
9.33 x 107
0
0
1.02xl08
1.02xl08
0
0
Total No. Spores
1.01 ± 0.37 xlO7
0
0
0
4.03 ± 0.24 x 107
0
0
0
5.13 ± 1.42 xlO7
0
0
0
4.58 ± 0.60 x 107
4.87 ±5.81x10
0
0
7.24±1.50xl07
0
0
0
1.49±0.43xl05
0
0
0
5.76 ± 0.25 x 107
2.23 ± 3.87 x 10
0
0
% Recovery
10.8 ±3.98
0
0
0
39.5 ±2.37
0
0
0
55.0 ±15.2
0
0
0
44.9 ± 5.89
< 0.0001
0
0
71.0 ±14.7
0
0
0
0.16 ± 0.05
0
0
0
56.4 ± 2.45
< 0.0001
0
0
Efficacy
b
> 7.00 ± 0 (7.00)
> 7.61 ±0(7.61)
> 7.71 ±0(7.71)
6.47 ±1.07 (5.61-7.66)
> 7.86 ± 0 (7.86)
> 5.17 ±0(5.17)
7.15 ±1.05 (5.93-7.76)
aData are expressed as mean (± SD) total number of spores, percent recovery, and efficacy (log reduction). The
efficacy range is shown in parentheses.
bNot Applicable
18
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Table 6-2. Liquid Culture Assessment of Bacillus anthracis Ames Spores
Test Material
Industrial-Grade Carpet (1C)
Bare Wood (BWD)
Glass (GS)
Decorative Laminate (DL)
Galvanized Metal Ductwork (GM)
Painted Wallboard Paper (PW)
Painted Concrete (PC)
Control
Decontaminated
Control
Decontaminated
Control
Decontaminated
Control
Decontaminated
Control
Decontaminated
Control
Decontaminated
Control
Decontaminated
Dayl
SI S2 S3 Bl
.
+ + + +
+ + + -
+ + + -
+ + + -
+ + + -
+ + + -
Day?
SI S2 S3 Bl
.
+ + + +
+ + + -
+ + + -
+ + + -
+ + + -
+ - +
+ + + -
SI = Sample 1
S2 = Sample 2
S3 = Sample 3
Bl = Blank (not inoculated with B. anthracis Ames spores)
"+" = growth; "-" = no growth
Following the extraction step, each test coupon was placed into liquid culture to promote
spore germination, thereby enabling the vegetative bacteria to proliferate. Growth was
determined if the liquid culture medium turned cloudy, while no growth was determined
when the liquid medium remained clear.
None of the liquid culture samples for 1C (both control and decontaminated) exhibited
bacterial growth. The brand of 1C used for this test contains a product known as FlorSept,
which is considered a broad spectrum antimicrobial that is effective against Gram-positive
and Gram-negative bacteria, as well as mold and fungi. It appears that, under the conditions
employed for this verification test, the FlorSept may not be sporicidal since viable
B. anthracis Ames spores were extracted from the 1C and cultured on tryptic soy agar plates.
Therefore, it is possible that, in the liquid cultures, FlorSept may inhibit growth of
vegetative cells derived from germination of the B. anthracis Ames spores. This growth
inhibition was also observed for B. subtilis, with the exception of one control sample at
7 days (Table 6-6). For G. stearothermophilus (Table 6-11), growth was observed in one
control sample at Day 1, and all 3 control samples at Day 7.
19
-------
Qualitative assessments of biological indicators and spore strips are shown in Tables 6-3
and 6-4. For all tests using B. anthracis, the control (not exposed to formaldehyde)
biological indicators and spore strips exhibited growth in the liquid cultures at both 1 and
7 days. Growth in some of the liquid cultures was also observed at 1 and 7 days for the
biological indicators and spore strips subjected to formaldehyde exposure using the 1414RH
unit.
Table 6-3. Liquid Culture Assessment of Biological Indicators/Spore Strips (Week 1
B. anthracis Decontamination)
Indicator (Organism)
Biological Indicator (B. subtilis ATCC 19659) Control
Biological Indicator (G. stearothermophilus ATCC 12980) Control
Spore Strip (B. atrophaeus ATCC 9372) Control
Biological Indicator (B. subtilis ATCC 19659) Decontaminated
Biological Indicator (G. stearothermophilus ATCC 12980) Decontaminated
Spore Strip (B. atrophaeus ATCC 9372) Decontaminated
Dayl
SI S2 S3
-
Day 7
SI S2 S3
+ +
SI = Sample 1
S2 = Sample 2
S3 = Sample 3
"+" = growth;"-"
= no growth
Table 6-4. Liquid Culture Assessment of Biological Indicators/Spore Strips (Week 2
B. anthracis Decontamination)
Indicator (Organism)
Biological Indicator (B. subtilis ATCC 19659)
Biological Indicator (G. stearothermophilus ATCC 12980)
Spore Strip (B. atrophaeus ATCC 9372)
Biological Indicator (B. subtilis ATCC 19659)
Biological Indicator (G. stearothermophilus ATCC 12980)
Spore Strip (B. atrophaeus ATCC 9372)
Control
Control
Control
Decontaminated
Decontaminated
Decontaminated
Dayl
SI S2 S3
+ + +
+ + +
+ + +
+
Day 7
SI S2 S3
+ + +
+ + +
+ + +
+ +
+ - +
+ - +
SI = Sample 1
S2 = Sample 2
S3 = Sample 3
"+" = growth;"-"
= no growth
20
-------
6.1.2 Bacillus subtilis (ATCC19659) Spores
Exposure of test coupons contaminated with B. subtilis spores to the 1414RH unit resulted
in decontamination that varied according to the type of test material. The log reduction of
detectable viable B. subtilis spores ranged from approximately 6.02 to > 8.04 for all seven
test materials (Table 6-5). The log reduction in viable spores detected on the porous
materials was > 8.04, 6.58, and 6.02 for 1C, BWD, and PC, respectively. The log reduction
in viable spores detected on the non-porous materials was > 7.79, 7.29, 6.24, and > 7.68 for
GS, DL, GM, and PW, respectively.
Table 6-5.1414RH Unit Decontamination of Bacillus subtilis Spores3
Test Material
Industrial-Grade Carpet (1C)
Control
Decontaminated
Blank (control)
Blank (decontaminated)
Bare Wood (BWD)
Control
Decontaminated
Blank (control)
Blank (decontaminated)
Glass (GS)
Control
Decontaminated
Blank (control)
Blank (decontaminated)
Decorative Laminate (DL)
Control
Decontaminated
Blank (control)
Blank (decontaminated)
Galvanized Metal Ductwork
(GM)
Control
Decontaminated
Blank (control)
Blank (decontaminated)
Painted Wallboard Paper (PW)
Control
Decontaminated
Blank (control)
Blank (decontaminated)
Painted Concrete (PC)
Control
Decontaminated
Blank (control)
Blank (decontaminated)
Inoculum
1.24 xlO8
1.24 xlO8
0
0
1.05 x 108
1.05 x 108
0
0
1.24 xlO8
1.24 xlO8
0
0
1.04 xlO8
1.04 xlO8
0
0
1.04 xlO8
1.04 xlO8
0
0
1.24 xlO8
1.24 xlO8
0
0
1.05 x 108
1.05 x 108
0
0
Total No. Spores
1.10±0.08xl08
0
0
0
1.21 ± 0.41 xlO7
1.10± 1.91x10
0
0
6.21±2.18xl07
0
0
0
5.52±1.27xl07
2.23 ±3.87x10
0
0
7.42±1.89xl07
1.89±1.65xl02
0
0
4.82±1.22xl07
0
0
0
5.47±0.43xl07
6.63 ±5.77x10
0
0
% Recovery
88.4 ± 6.26
0
0
0
11.6±3.91
< 0.0001
0
0
50.1 ± 17.6
0
0
0
53.1 ±12.2
< 0.0001
0
0
71.4 ±18.2
< 0.001
0
0
38.9 ±9.81
0
0
0
52.1 ±4.10
< 0.0001
0
0
Efficacy
b
> 8.04 ± 0 (8.04)
-
-
-
6.58 ±0.88 (5 .57-7.08)
-
-
-
> 7.79 ±0(7.79)
-
-
-
7.29 ±0.78 (6.38-7.74)
-
-
_
6.24 ±1.42 (5. 39-7.87)
-
-
-
> 7.68 ±0(7.68)
-
-
-
6.02 ±0.35 (5.61-6.22)
-
-
aData are expressed as mean (± SD) total number of spores, percent recovery, and efficacy (log reduction). The
efficacy range is shown in parentheses.
bNot Applicable
21
-------
A liquid culture growth assessment at 1 and 7 days post-decontamination was performed to
determine whether viable B. subtilis spores remained on the test materials following the
extraction step (Table 6-6). As stated above, each test material (or non-inoculated blank)
was wiped with 70% isopropanol prior to inoculation with B. subtilis spores; however, this
isopropanol wash does not guarantee sterility, especially with the porous materials. There-
fore, growth observed in some of the test materials not inoculated with B. subtilis spores
may have resulted from growth of other microorganisms not affected by the 70%
isopropanol wash. This type of assessment may not discriminate between the growth of
B. anthracis and/or other microorganisms.
Table 6-6. Liquid Culture Assessment of Bacillus subtilis Spores
Test Material
Industrial-Grade Carpet (1C)
Bare Wood (BWD)
Glass (GS)
Decorative Laminate (DL)
Galvanized Metal Ductwork (GM)
Painted Wallboard Paper (PW)
Painted Concrete (PC)
Control
Decontaminated
Control
Decontaminated
Control
Decontaminated
Control
Decontaminated
Control
Decontaminated
Control
Decontaminated
Control
Decontaminated
Dayl
SI S2 S3 Bl
.
+ + + +
+ + + -
+ + + -
+ + + -
+ + + -
+ +
Day?
SI S2 S3 Bl
+
+ + + +
+
+ + + -
+ + + +
+
+ + + -
+ + + +
+ - - -
+ + + -
SI = Sample 1
S2 = Sample 2
S3 = Sample 3
Bl = Blank (not inoculated with B. subtilis spores)
"+" = growth; "-" = no growth
Qualitative assessment of biological indicators and spore strips are shown in Tables 6-7,
6-8, and 6-9. For all tests using B. subtilis, the biological indicators and spore strips not
exposed to formaldehyde using the 1414RH unit exhibited growth in the liquid cultures at
both 1 and 7 days. No growth in the liquid cultures was observed at 1 and 7 days for the
biological indicators and spore strips subject to formaldehyde exposure using the 1414RH
unit, with the exception of a single spore strip exhibiting growth at Day 7 for week one of
testing.
22
-------
Table 6-7. Liquid Culture Assessment of Biological Indicators/Spore Strips (Week 1
B. subtilis Decontamination)
Indicator (Organism)
Biological Indicator (B. subtilis ATCC 19659)
Spore Strip (B. atrophaeus ATCC 9372)
Biological Indicator (B. subtilis ATCC 19659)
Spore Strip (B. atrophaeus ATCC 9372)
Control
Control
Decontaminated
Decontaminated
Dayl
SI S2
+ +
+ +
-
Day 7
SI S2
+ +
+ +
+
SI = Sample 1
S2 = Sample 2
"+" = growth; "-" = no growth
Table 6-8. Liquid Culture Assessment of Biological Indicators/Spore Strips (Week 2
B. subtilis Decontamination)
Indicator (Organism)
Biological Indicator (B. subtilis ATCC 19659)
Spore Strip (B. atrophaeus ATCC 9372)
Biological Indicator (B. subtilis ATCC 19659)
Spore Strip (B. atrophaeus ATCC 9372)
Control
Control
Decontaminated
Decontaminated
Dayl
SI S2
+ +
+ +
-
Day 7
SI S2
+ +
+ +
-
SI = Sample 1
S2 = Sample 2
"+" = growth; "-" = no growth
Table 6-9. Liquid Culture Assessment of Biological Indicators/Spore Strips (Week 3
B. subtilis Decontamination)
Indicator (Organism)
Biological Indicator (B. subtilis ATCC 19659)
Spore Strip (B. atrophaeus ATCC 9372)
Biological Indicator (B. subtilis ATCC 19659)
Spore Strip (B. atrophaeus ATCC 9372)
Control
Control
Decontaminated
Decontaminated
Dayl
SI S2 S3
+ + +
+ + +
-
Day 7
SI S2 S3
+ + +
+ + +
-
SI = Sample 1
S2 = Sample 2
S3 = Sample 3
"+" = growth; "-''
= no growth
23
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6.1.3 Geobacillus stearothermophilus (ATCC12980) Spores
Exposure of test coupons contaminated with G. stearothermophilus (ATCC 12980) spores
to the 1414RH unit resulted in variable decontamination. The log reduction of detectable
viable G. stearothermophilus spores (ATCC 12980) ranged from approximately 5.68 to
> 7.64 for all seven test materials (Table 6-10). The log reduction in viable spores detected
on the porous materials was 5.68, > 6.82, and 6.20 for 1C, BWD, and PC, respectively. The
log reduction in viable spores detected on the non-porous materials was > 7.24, > 7.12,
> 7.64, and > 7.19 for GS, DL, GM, and PW, respectively.
Table 6-10.1414RH Unit Decontamination of Geobacillus stearothermophilus Spores3
Test Material
Industrial-Grade Carpet (1C)
Control
Decontaminated
Blank (control)
Blank (decontaminated)
Bare Wood (BWD)
Control
Decontaminated
Blank (control)
Blank (decontaminated)
Glass (GS)
Control
Decontaminated
Blank (control)
Blank (decontaminated)
Decorative Laminate (DL)
Control
Decontaminated
Blank (control)
Blank (decontaminated)
Galvanized Metal Ductwork
(GM)
Control
Decontaminated
Blank (control)
Blank (decontaminated)
Painted Wallboard Paper (PW)
Control
Decontaminated
Blank (control)
Blank (decontaminated)
Painted Concrete (PC)
Control
Decontaminated
Blank (control)
Blank (decontaminated)
Inoculum
8.80 x 107
8.80 x 107
0
0
7.33 x 107
7.33 x 107
0
0
8.80 x 107
8.80 x 107
0
0
7.33 x 107
7.33 x 107
0
0
7.33 x 107
7.33 x 107
0
0
8.80 x 107
8.80 x 107
0
0
7.33 x 107
7.33 x 107
0
0
Total No. Spores
1.50 ± 0.19 xlO7
1.22±1.17xl02
0
0
6.59 ± 1.57 xlO6
0
0
0
1.74±0.05xl07
0
0
0
1.31 ± 0.59 xlO7
0
0
0
4.34 ± 4.80 xlO7
0
0
0
1.53 ± 0.20 xlO7
0
0
0
1.94 ± 0.60 xlO7
0.61 ± 1.06 xlO3
0
0
% Recovery
17.1 ±2.15
< 0.001
0
0
8.99 ±2. 14
0
0
0
19.8 ±0.51
0
0
0
17.9 ±8.08
0
0
0
59.2 ±65.6
0
0
0
17.4 ± 2.30
0
0
0
26.5 ±8. 15
< 0.001
0
0
Efficacy
b
5.68 ±1.30 (4.81-7.18)
-
-
_
> 6.82 ±0(6.82)
_
-
_
> 7.24 ±0(7.24)
_
-
_
> 7.12 ±0(7.12)
_
-
_
> 7.64 ±0(7.64)
_
-
_
> 7.19 ±0(7.19)
_
-
_
6.20 ±1.88 (4.03-7.29)
-
-
aData are expressed as mean (± SD) total number of spores,
efficacy range is shown in parentheses.
bNot Applicable
percent recovery, and efficacy (log reduction). The
24
-------
A liquid culture growth assessment at 1 and 7 days post-decontamination was performed to
determine whether viable G. stearothermophilus spores remained on the test materials
following the extraction step (Table 6-11). As stated previously, each test material (or non-
inoculated blank) was wiped with 70% isopropanol prior to inoculation with
G. stearothermophilus spores; however, this isopropanol wash does not guarantee sterility,
especially with the porous materials. Therefore, growth observed in some of the test
materials not inoculated with G. stearothermophilus spores may have resulted from growth
of other microorganisms not affected by the 70% isopropanol wash. This type of assessment
may not discriminate between the growth of B. anthracis and/or other microorganisms.
Table 6-11. Liquid Culture Assessment of Geobacillus stearothermophilus Spores
Test Material
Industrial-Grade Carpet (1C)
Bare Wood (BWD)
Glass (GS)
Decorative Laminate (DL)
Galvanized Metal Ductwork (GM)
Painted Wallboard Paper (PW)
Painted Concrete (PC)
Control
Decontaminated
Control
Decontaminated
Control
Decontaminated
Control
Decontaminated
Control
Decontaminated
Control
Decontaminated
Control
Decontaminated
Dayl
SI S2 S3 Bl
+
+ + + +
+ + + -
+ + + -
+ + + -
+ + + -
+ + + -
Day?
SI S2 S3 Bl
+ + + -
+ + + +
+ + + -
+ + + -
+ + + -
+ + + +
+ + + -
+ - - -
SI = Sample 1
S2 = Sample 2
S3 = Sample 3
Bl = Blank (not inoculated with G. stearothermophilus spores)
"+" = growth; "-" = no growth
Qualitative assessment of biological indicators and spore strips is shown in Tables 6-12 and
6-13. For all tests using G. stearothermophilus, the biological indicators and spore strips not
exposed to formaldehyde using the 1414RH unit exhibited growth in the liquid cultures at
both 1 and 7 days. Growth in the liquid cultures was observed for one of the biological
indicators at day 1 and three biological indicators at Day 7. No growth was observed for the
spore strips subjected to formaldehyde exposure using the 1414RH unit.
25
-------
Table 6-12. Liquid Culture Assessment of Biological Indicators/Spore Strips (Week 1
G. stearothermophilm Decontamination)
Indicator (Organism)
Biological Indicator (G. stearothermophilm ATCC 12980) Control
Spore Strip (B. atrophaeus ATCC 9372) Control
Biological Indicator (G. stearothermophilus ATCC 12980) Decontaminated
Spore Strip (B. atrophaeus ATCC 9372) Decontaminated
Dayl
SI S2 S3
+ + +
+ + +
-
Day 7
SI S2 S3
+ + +
+ + +
+
SI = Sample 1
S2 = Sample 2
S3 = Sample 3
"+" = growth; "-"
= no growth
Table 6-13. Liquid Culture Assessment of Biological Indicators/Spore Strips (Week 2
G. stearothermophilus Decontamination)
Indicator (Organism)
Biological Indicator (G. stearothermophilus ATCC 12980) Control
Spore Strip (B. atrophaeus ATCC 9372) Control
Biological Indicator (G. stearothermophilus ATCC 12980) Decontaminated
Spore Strip (B. atrophaeus ATCC 9372) Decontaminated
Dayl
SI S2 S3
+ + +
+ + +
+
Day 7
SI S2 S3
+ + +
+ + +
+ - +
SI = Sample 1
S2 = Sample 2
S3 = Sample 3
"+" = growth; "-" = no growth
6.1.4 Statistical Analysis
Table 6-14 presents the mean log reduction in spores sorted by material type. Significant
differences are denoted in the table as well. All means were significantly different from zero
indicating that the technology decontaminated statistically significant numbers of spores on
these materials.
26
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Table 6-14. Statistical Analysis of Mean Efficacy (Log Reduction) for Spores
Material
Porous
Non-
Porous
Industrial-Grade Carpet (1C)
Painted Concrete (PC)
Bare Wood (BWD)
Glass (GS)
Decorative Laminate (DL)
Painted Wallboard Paper (PW)
Galvanized Metal Ductwork (GM)
B. anthracis
> 7.00a
>7.15a
>7.61a
>7.71a
6.47a
>5.17a
7.86a
B. subtilis
> 8.04a
6.02a
6.58a
> 7.79a
7.29a
>7.68a'b
6.24a'b
G. stear other mophilus
5.68a'b
6.20a
> 6.82a
> 7.24a
>7.12a
>7.19a'b
> 7.64a
aMean significantly different from 0 at the (P > 0.05)
bSurrogate significantly different from B. anthracis for specified material (P > 0.05).
While there was no significant overall effect of spore species utilized, a significant
interaction between the spore species utilized and the test coupon materials was noted in the
ANOVA model (P=0.0001). Overall comparisons of the porous and the non-porous
materials were not useful due to this interaction, as opposing interactions appeared to cancel
each other out. That is, it appears that each of the three spore species interacts with certain
test coupons in such a way that the efficacy of the formaldehyde decontamination is
influenced. These spore-coupon interactions differ, depending on the spore type; for
example, the log reductions for B. subtilis and G. stearothermophilis are similar for PC
(6.58 and 6.82, respectively), but B. anthracis was reduced to a greater extent (7.61).
Comparisons within each material indicated that the 1414RH unit decontaminated
significantly more B. subtilis and G. stearothermophilus spores than B. anthracis spores for
PW; significantly fewer B. subtilis spores than B. anthracis spores for GM; and significantly
fewer G. stearothermophilus spores than B. anthracis spores for 1C.
6.2 Damage to Coupons
Subsequent to decontamination, the test coupons were evaluated qualitatively for visible
surface damage. No damage (e.g., change in surface texture, color) and no visible changes
to any of the test materials were observed during this verification test.
6.3 Other Factors
6.3.1 Operation of the 1414RH Unit
The 1414RH unit was operated for approximately 140 hours during this verification test. By
following the user manual, the 1414RH unit was set up for operation within minutes. The
only maintenance that was required for the 1414RH unit during this verification test was the
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addition of new paraformaldehyde and neutralizer at the beginning of each ran. At the end
of each ran, the hexamethylenetetramine formed during the neutralization step had to be
cleaned from all surfaces within the Plas-Labs Glove Box prior to the start of the next ran of
the 1414RH unit. A towel dampened with ethanol was used to remove the
hexamethylenetetramine powder.
The formaldehyde concentration was monitored in real-time, and the data were recorded on
a strip chart. Figure 6-1 is a graphical representation of the real-time formaldehyde
measurement from 0 to 11.5 hours. Paraformaldehyde was added to the 1414RH unit at the
specified concentration, leading to a theoretical concentration of formaldehyde gas in the
test chamber of approximately 8,600 ppm. The observed measured concentration of
formaldehyde gas in the test chamber averaged approximately 1,100 ppm. Therefore, it
appears that the majority of the formaldehyde was deposited on all surfaces within the test
chamber. This deposition seems possible since a film formed on all surfaces within the test
chamber during operation of the 1414RH unit.
As described in Section 3.5.4.1, a nebulizer system had to be utilized to achieve the
appropriate relative humidity (50 to 90%) within the Plas-Labs Compact Glove Box for
each ran of the 1414RH unit.
6.3.2 Operator Bias
Due to the automated capabilities of the 1414RH unit, there is little room for operator error
although operator error was not evaluated in this verification test. Once the appropriate
canisters were filled with their respective components (e.g., paraformaldehyde), the timer
was set for the appropriate contact time (10 hours for this verification test). Next, the "Start"
button was pressed and the 1414RH unit ran through the decontamination cycle. The
decontamination and neutralization steps were run overnight and shut off the next morning;
therefore, a total run time from start to finish was approximately 16 to 18 hours.
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Formaldehyde Concentration
1600 i
01234567
9 10 11 12
Figure 6-1. Representative Cycle Parameter Data from a Single Experiment
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Chapter 7
Performance Summary
For this verification test, the 1414RH unit demonstrated decontamination efficacy for
B. anthracis Ames, B. subtilis (ATCC 19659), and G. stearothermophilus (ATCC 12980) on
all seven test materials. The test showed that, for all three organisms, material type appeared
to influence decontamination. The 1414RH unit promoted a significant decrease in viable
spores for all three organisms on all seven test materials.
The ETV testing to measure the effectiveness of the 1414RH unit for inactivating
B. anthracis Ames strain and surrogate spores on seven indoor surfaces provided a range of
results. A quantitative evaluation of the results indicated that the log reduction values for
detectable viable B. anthracis Ames spores ranged from 5.17 to > 7.86 across all seven test
materials. The log reduction values for detectable viable B. subtilis spores ranged from 6.02
to 8.04 for all seven test materials. The log reduction values for detectable viable
G. stearothermophilus spores (ATCC 12980) ranged from 5.68 to > 7.64 for all seven test
materials. For the porous materials, a significant difference in efficacy was observed only
between B. anthracis and G. stearothermophilus on industrial carpet. For non-porous
materials, significant differences in efficacy between B. anthracis and both surrogates were
observed for painted wallboard paper, and a significant difference was observed between
B. anthracis and B. subtilis on galvanized metal. No damage was observed for any of the
test materials subjected to the 1414RH unit.
A qualitative evaluation of the performance of the 1414RH unit was performed using
biological indicators and spore strips. For all procedures for this verification test, the control
(not exposed to the 1414RH unit) biological indicators and spore strips used in this test
displayed growth in the liquid cultures at both 1 and 7 days. When the biological indicators
and spore strips were subjected to exposure by the 1414RH unit, growth was observed in
some of the liquid cultures at 1 and 7 days. For these samples, the number of samples
exhibiting growth varied among the dates of experimentation, and no clear trend was
observed. The 1414RH unit was partially successful in inactivating both the biological
indicators (containing B. subtilis and G. stearothermophilus) and spore strips (containing
B. atrophaeus}, all of which contain spore loads of approximately 1 x 106 spores per
indicator or spore strip. It is possible that this partial inactivation resulted from the
biological indicators and spore strips remaining in the sealed Tyvek and glassine pouches,
respectively. The Tyvek and glassine may have inhibited the penetration of formaldehyde to
some extent, thereby preventing complete inactivation of the biological indicators and spore
strips.
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The 1414RH unit was set up and ready for operation in the laboratory within minutes. The
1414RH unit is not able to measure parameters such as relative humidity and formaldehyde
concentration. Within the Plas-Labs Compact Glove Box, the relative humidity was
determined by using a traceable hygrometer, and the formaldehyde was measured using a
formaldehyde monitor. The importance of operator skill level to using the 1414RH unit,
while not verified in this test, should be minimal due to the automated capabilities of the
1414RH unit; which left little room for operator error.
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Chapter 8
References
1. Test/QA Plan for Verification of Formaldehyde Vapor Technologies for
Decontaminating Indoor Surfaces Contaminated with Biological or Chemical Agents,
Battelle, Columbus, Ohio, November, 2003.
2. CERTEK Model 1414RH Formaldehyde Generator'/Neutralizer Operating Protocol,
CERTEK, Inc., Raleigh, North Carolina, June 2003.
3. Kelly, T. J., Fortune, C. R. (1993). "Continuous Monitoring of Gaseous Formaldehyde
Using an Improved Fluorescence Approach." International Journal of Environmental
and Analytical Chemistry 54: 249-263, 1993.
4. Quality Management Plan (QMP)for the Technology Verification of Commercially
Available Methods for Decontamination of Indoor Surfaces Contaminated with
Biological or Chemical Agents, Version 1, prepared by Battelle, Columbus, Ohio,
November 22, 2002.
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