Summary of Hydrogen Peroxide Vapor Generation using Off-the-Shelf
Items and Equipment for the Disinfection of Spaces and Materials
BACKGROUND
A previous laboratory study found that a 2-h contact time was needed to effectively inactivate both Phi6 and
MS2 bacteriophages on several materials, including those comprising an N95 respirator, when using a
commercial generation system to provide a constant hydrogen peroxide vapor (HPV) concentration of 25
parts per million (ppm).[ 1] The 25-ppm concentration over a two-hour period resulted in a 50-ppm-h dose
of HPV (dose is concentration x time), which was sufficient to inactivate both the Phi6 and MS2
bacteriophages. Phi6 is an enveloped virus that infects Pseudomonas bacteria and MS2 is a non-enveloped
virus that infects Escherichia coli. Non-enveloped viruses are typically more resistant to disinfection than
non-spore forming bacteria and enveloped viruses. Moreover, enveloped viruses are generally considered
the least resistant class of microorganism to germicidal chemicals. [2, 3] For comparison, viruses like H1N1
(influenza), Ebola virus (Ebola virus disease) and SARS-CoV-2 (COVID-19) are in the enveloped virus
class.
One purpose of the experiments described in this Technical Brief was to determine the HPV dosages (in
ppm-h) that could be achieved using commercially available humidifiers and foggers filled with off-the-
shelf hydrogen peroxide aqueous (HPA) solutions. (Humidifiers were shown to be able to generate HPV in
this manner to inactivate B. cmthracis spores and the surrogate. [4, 5]) We also conducted several
experiments to demonstrate the HPV dosages that could be achieved in a small storage bin simply by
allowing the HPA to evaporate from an open tray placed in the bin (with a small fan blowing on the HPA).
These storage bin tests were conducted with and without several N95 respirators, to assess the presence of
the respirators in lowering the HPV concentration. Another purpose of the testing was to determine the
times required to achieve a 100 ppm-h- or 200 ppm-h-dose of HPV, and the quantities of HPA that would
be required. These dosages were chosen as benchmarks and include a 2x and 4x factor of safety beyond
what was needed to inactivate Phi6 and MS2 phages [1], and thus would presumably be effective as well in
inactivating other enveloped viruses such as the Ebola virus and SARS-CoV-2. In addition, the 4x safety
factor dose was selected since it can be practically verified using commercially available chemical indicator
strips (discussed below).
METHODS
HPA sources
A 35% HPA solution (Vaprox®; STERIS Corp., Mentor, OH) was diluted using deionized (DI) water, to
either a 3% or 6% solution of HPA. The HPA concentration of the batches formulated was qualitatively
verified using colorimetric indicator strips (Indigo Instruments; Waterloo, ON; 33815-PClOpc).
Approximately half of the experiments were conducted using 3% HPA since this concentration is
commercially available at grocery stores and drug stores. A 6% solution was also evaluated since this
concentration of HPA is typically available at beauty supply stores. The amount of HPA added to each
generating device before each test and the amounts of HPA recovered after the test were determined
volumetrically and used to assess the amount of HPA transferred to the vapor phase.
U.S. Environmental Protection Agency
Office of Research and Development
EPA/600/S-20/358
September 2020

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Equipment evaluated
Five HPV generating devices were evaluated:
Decontamination test volumes used
Preparation of Humidifier for Testing
Trailer: A small box trailer with a volume of 736 ft3 was used in
these tests. For reference, this size trailer is a little larger than a
small size standard storage container 10 feet long (10x8x8.6 ft = 680 ft3). Tests were conducted with the
trailer closed but empty, using two box fans to help circulate air and HPV. The interior layout of the trailer
includes wood paneling. Tests were initially conducted with the trailer outdoors, but with the sun shining
and the trailer's black color, the interior temperature exceeded 100 °F. The trailer was then tested in a
temperature-controlled building bay where temperatures averaged approximately 70 °F. The fogger was
placed approximately 3 feet off the trailer's floor.
Vehicles: Two vehicles were utilized: 1) A cargo van with an estimated interior volume of 275 ft ', and 2) a
sport utility vehicle with an interior cargo volume estimated at 110 ft3. "Hie vehicles were closed prior to
dissemination of the HPV.
2.
3.
4.
A fogger ( Curtis Dynafogger; Westfield, IN), which generates
droplets of 30 microns or less using pneumatic shear. The capacity
of the tank was 1 gallon, and the fogger was operated on the lowest
setting for all tests to fog at a rate of 1 gallon/h. "Hie fogger was
tested in a 10-foot box trailer, fogging a volume of HP A of either 1
gallon or % gallon, translating to 1.4 or 0.7 gallons per 1000 ft3.
A large evaporative humidifier (Model HCM-6009; Honeywell
International; Charlotte, NC), with a capacity of three gallons, was
used in the humidifier experiments. Evaporative humidifiers work
by passing air through a wicking/sponge material that soaks up
water, or in this case, HPA. The humidifier was operated on the
highest fan setting and highest humidity level. This type of
humidifier will cycle on and off to avoid over- saturation and
condensation once a certain relative humidity (RH) has been
achieved.
A small evaporative humidifier (Vick's Starry Night; Procter &
Gamble; Cincinnati, OH) with a one-gallon capacity was also
used. This humidifier was operated at its highest settings for
humidity and fan speed.
An ultrasonic-based humidifier (Vick's Healthmist) with a one-
gallon capacity was used in some testing. This type of
humidifier generates humidity until the reservoir is emptied and
can produce condensation on surfaces.
A small plastic tray (~ 10-inch diameter, 2-inch depth) of HPA
(~ 8 ounces) was placed in the bottom of an 18-gallon plastic
storage bin with a small fan blowing across it. This approach
utilizes natural evaporation of HPA into the vapor phase, with
equilibrium HPV concentrations proportionate to HPA
concentrations and temperature, as per Henry's Law.
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Storage bin: An 18-gallon plastic storage bin was used. Tests were conducted in the bin with and without
12 N95 respirators to create a demand on the HPV. Tape around the lid of the bin was used to create a seal.
The N95s were placed on a plastic rack inside the bin and spaced out evenly on the rack for five tests. Two
tests were conducted with the N95s stacked tightly together.
Once the experiment was completed, the trailer, vehicle, or storage bin was opened and allowed to aerate
for several hours to allow removal of the HPV.
Location of testing
All tests were conducted at the US EPA's High Bay Facility in Research Triangle Park, NC. Initial tests
were conducted outdoors, but to better control temperature, the trailer and vehicles were brought into a
temperature-controlled bay. When vehicles or the trailer were tested outdoors, interior temperatures
exceeded 100 °F.
HPV measurement
Hydrogen peroxide vapor levels were measured using electrochemical sensors (Model B12-34-1-0010-1,
Analytical Technologies Inc., Collegeville, PA) calibrated by the manufacturer to either 10 ppm or 100
ppm. Data from the sensors were recorded using a data acquisition system.
HPV dose calculation
The times to reach the 100 ppm-h- or 200 ppm-h-benchmark doses of HPV were determined for each
experiment. A ppm-h dose was determined for each one-minute time interval that the HPV data were
logged, and these values were then integrated over the time period of testing.
Temperature and relative humidity
Temperature and RH were measured using relative humidity sensors (HOBO UX100; Onset Computer
Corporation; Bourne, MA). The sensors log their data automatically and were programmed to log a
measurement once each minute. Two sensors were included in the trailer and vehicle tests; an average of
the two sensor values is reported. One sensor was included in the bin tests. Data from these sensors were
downloaded after each test. Data for the temperature and RH are for the time period required to reach the
200 ppm-h dose. If the 200 ppm-h-dose benchmark was not reached, the temperature and RH are reported
for the time required to reach the 100 ppm-h dose.
Hydrogen peroxide vapor dosage check
Two colorimetric strips (Steraffirm PCC060 VH202 Class 4 Indicator, STERIS Corporation) were included in each
test for the trailer, three were used in the vehicles, and one indicator strip was used in each storage bin test. The
colorimetric strips were used to indicate qualitatively whether an HPV dose of 200 ppm-h was achieved, by turning
from a magenta color to yellow. An experiment in which the indicator strips turned orange but not yellow was
determined not to pass.
RESULTS
General overview
Higher HPV concentrations could be achieved with higher air temperatures and higher HPA concentrations, thus
providing shorter contact times to reach dose benchmarks. The minimum ambient air temperature at which we
conducted tests was 66 °F, and the maximum average temperature was 104 °F.
Chemical indicators proved to be useful to ensure the HPV dose was achieved. There were only a few tests in which
the chemical indicators did not sufficiently change color to yellow, indicating that a 200 ppm-h dose was not achieved
during those tests.
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When decontaminating with HPV, materials can react with and/or adsorb the HPV, and thus lower the HPV
concentration, making it more difficult to attain the desired dose. Tests in the trailer and vehicles were conducted
without any cargo or material loaded in them, but in the storage bin tests, the tests were conducted with and without
the presence of N95 respirators.
Fogger
The fogger was only used in the trailer because of its high output and the trailer's larger volume. Refer to Table 1 for
the results of these tests. In the tests, either one gallon or one-half gallon was fogged, translating to either 1.4 or 0.7
gallons per 1000 ft3, based on the volume of the trailer. The 100 ppm-h HPV benchmark dose was reached in every
test conducted, but the 200 ppm-h dose was not reached unless an elevated temperature was used (average
temperatures > 98 °F) or when using 6% HPA. We note that some of the tests in the trailer were terminated after 6-8
h, and that the 200 ppm-h dose most likely could have been reached with additional dwell time. However, in one
experiment, the 200 ppm-h-dose was not reached with a 23-h-contact time.
The fogger rapidly transferred the HPA to HPV. Therefore, high levels of HPA (> 100 ppm) were briefly observed.
Of the seven fogger tests that were conducted, the 100 ppm-h-dose was reached within 1-5 hours. Shorter times to
reach this dose were generally (but not always) associated with the higher temperature, the higher concentration HPA,
or a greater volume of HPA fogged.
Humidifiers in the trailer
In all three of the tests conducted with humidifiers in the trailer, the 200 ppm-h-benchmark was reached. When using
the large evaporative humidifier in the trailer (two tests; refer to Table 1), the times to reach 100 ppm-h were between
approximately 7-8 hours. One test was conducted with 3% HPA at high temperature (average temperature was
estimated to be ~ 100 °F) and produced a result similar to the test using the 6% HPA at the lower temperature
(average temperature was 70 °F).
When the ultrasonic humidifier was tested in the trailer (6% HPA, average T = 66 °F), the time to reach 100 ppm-h
was estimated to be 3.3 h, like the fogger. In this test, one gallon per 1000 ft3 was disseminated. The reason the
ultrasonic humidifier produced a result similar to the fogger may be that both devices generated a constant output of
vapor.
In contrast, the output of the evaporative humidifiers slows as the RH nears 100% (i.e., the air becomes saturated with
moisture), to avoid condensation. In high humidity, the mass transfer rate of HPA to the vapor phase will be reduced
with evaporative-type humidifiers. Hence, for both the fogger and ultrasonic humidifier, excessive condensation may
occur if saturation is reached. Excessive condensation may result in material compatibility issues, a reduction in HPV
concentrations, and longer drying times.
Humidifiers in the vehicles
All three humidifiers (large evaporative humidifier, the small evaporative humidifier, and the ultrasonic humidifier)
were tested in the cargo van, while only the small evaporative humidifier was tested in the SUV. All three
humidifiers demonstrated the ability to generate HPV, with the time to reach the 100 ppm-h-benchmark in the
vehicles ranging from 1.5 to 10.6 hours (refer to Table 2). As with the fogger, generally less time was required to
reach the benchmark dose when testing at a higher temperature. Two of the three tests in which we were able to reach
the 100 ppm-h-dose in less than 2.3 h were at high temperature, and the other case utilized the ultrasonic humidifier.
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Bin/headspace
The HPV concentrations in the empty storage bin agreed with the expected
theoretical concentration based on temperature and HPA concentration. HPV
levels ranged from approximately 9 ppm to mid-30s ppm. With the addition of
twelve N95 masks and then resealing the bin, the new stabilized HPV level had
dropped by approximately a third. In general, a few hours were needed for the
HPV level to stabilize and reach steady state. The times needed to reach the 100
ppm-h-dose when the bin was full of respirators ranged from 5-15 h. The loss
of HPA from a plastic tray over time was measured for a few of the tests and
averaged approximately 1-2 mL/h. For some tests, we reused the same HPA in
the pan for multiple cycles. When we reused the same HPA, a few
measurements showed that the HPA increased in concentration overtime.
SUMMARY
Due to the unlimited number of combinations that could occur due to variations in generation device type and models,
volumes and space configurations, presence of materials, and environmental factors (temperature and RH levels), it is
difficult to predict required contact times and amounts of HPA to use to achieve desired HPV dosages. Our test
matrix only looked at a small subset of available configurations but may provide some indication of the range in
contact times and amounts of HPA per unit volume needed to meet dose benchmarks. From averaging the usage rate
data, the use of 1 gallon of HPA per 1000 ft3, coupled with an average contact time of at least four hours, may be a
good starting point. The use of HPV monitoring or chemical indicators for verifying HPV dose would then confirm if
desired dosages of HPV were met.
Overall, evaporative humidifiers are more widely available, easier to use, and avoid excessive condensation compared
to foggers. The drawback to avoiding condensation is that longer contact times may be needed. Average HPV levels
from the use of humidifiers were in the range of approximately 20-40 ppm, although there were two tests when the
concentration peaked near 100 ppm. Foggers may be more suitable for larger volumes and/or when shorter contact
times are needed. Ultrasonic humidifiers operate like foggers, with a constant output, and may be a more user-
friendly, more commercially available alternative to foggers, although the discharge rate for foggers may be higher.
Small evaporative humidifiers may be a good option for passenger vehicle interiors.
Table 1. Contact Times and HPA Quantities Required to Reach HPV Doses in Trailer
Date
HPV
generator
HPA
(%)
Genera
tion
rate
(GPH)
HPA
gallons
per 1000
ft3
Mean
T
(°F)
Mean
RH
(%)
Time to
reach
100 ppm-
h dose
(h)
Time to
reach 200
ppm-h
dose
(h)
4/6/20
fogger
3
1
1.4
104
79
1
2.4
4/7/20
fogger
3
1
0.7
97
79
5
DNRat 23
h
4/8/20
fogger
3
1
0.7
98
74
3.7
DNR at 8 h
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Date
HPV
generator
HPA
(%)
Genera
tion
rate
(GPH)
HPA
gallons
per 1000
ft3
Mean
T
(°F)
Mean
RH
(%)
Time to
reach
100 ppm-
h dose
(h)
Time to
reach 200
ppm-h
dose
(h)
4/17/20
fogger
3
1
1.4
71
90
4.1
DNRat 7.6
h
4/9/20
fogger
6
1
0.7
90
89
1.9
DNR at 6 h
4/14/20
fogger
6
1
1.4
71
98
1.45
3.4*
4/15/20
fogger
6
1
1.4
66
93
2.0
4.5
4/3/20
Large
evaporative
humidifier
3
UDC
2.8
NAa
NAa
7.0
15.5
4/13/20
Large
evaporative
humidifier
6
UDb
1.4
70
78
7.4
12
4/16/20
Ultrasonic
humidifier
6
0.09
1.0
65
71
3.3**
6.6**
HPV = hydrogen peroxide vapor; HPA = hydrogen peroxide aqueous; GPH = gallons per hour; T = temperature; RH = relative
humidity; ppm = parts per million; h = hour; NAa =T and RH data inadvertently not taken, estimate temp 100 °F during the day;
UC b= undetermined, not sure when 1 gallon was discharged; UDC= Undetermined; discharged 2 gallons over the weekend; times
to reach dosages may have occurred prior to discharging the 2 gallons; DNR=did not reach at the time the test was completed;
* Chem indicator did not pass; "Time to achieve 100 ppm-h and 200 ppm-h based on estimated avg level ~ 30 ppm HPV, from
visual observations of digital readout throughout day.
Table 2. Contact Times and HPA Quantities Required to Reach HPV Doses in Vehicles
Date
HPV
generator
HPA
(%)
Genera
tion
rate
(GPH)
HPA
gallons
per 1000
ft3
Mean
T
(°F)
Mean
RH
(%)
Time to
reach
100 ppm-
h dose
(h)
Time to
reach 200
ppm-h
dose
(h)
Cargo Van
4/6/20
Large evap.
humidifier
3
0.32
2.0
102
63
1.5
DNR at 3 h
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Date
HPV
generator
HPA
(%)
Genera
tion
rate
(GPH)
HPA
gallons
per 1000
ft3
Mean
T
(°F)
Mean
RH
(%)
Time to
reach
100 ppm-
h dose
(h)
Time to
reach 200
ppm-h
dose
(h)
4/14/20
Large evap.
humidifier
3
0.01
1.0
70
80
3
7
4/13/20
Large evap.
humidifier
6
0.03
1.9
73
84
5.4C
9.3°
4/8/20
Small evap.
humidifier
3
0.01
0.7
71
82
10.6
21
4/7/20
Small evap.
humidifier
6
0.07
1.3
100
61
4.3
8.2
4/9/20
Small evap.
humidifier
6
0.01
0.4
71
82
6.9
DNR at 8 h
4/15/20
Ultrasonic
Humidifier
6
0.1
2.3
NAb;
est. 66
NA
2.1
5.1*
Sport Utility Vehicle
4/16/20
Small evap.
humidifier
3
0.01
2.6
65
80
9 i**
15.8**
4/17/20
Small evap.
humidifier
3
0.04
1.6
95
64
2.3
3.5
HPV = hydrogen peroxide vapor; HPA = hydrogen peroxide aqueous; GPH = gallons per hour; T = temperature; RH =
relative humidity; ppm = parts per million; h = hour *Chem indicators did not pass; "Lost recorded data for portion of
cycle (1st ~ 8 h ), estimate avg 10 ppm during that time from visual observations of digital readouts throughout day;
DNR=did not reach at time test completed UDa = undetermined, not sure when 1 gallon was discharged; btemp.
reading not available; estimate temp, was 66 F based on temp of bay next door. CHPV sensor had to be switched during
test, and a few hours into test, we discovered that the humidifier fan was not running.
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Table 3. Contact Times and HPA Quantities Required to Reach HPV Doses in Storage Bin
Date
Bin contents
HPA
(%)
Generation
rate
(mL/h)
Disseminated
HPA mL
(Lost to
evaporation)
Mean
T
(°F)
Mean
RH
(%)
Time
to
reach
100
PPm-
hour
Dose
(h)
Time
to
reach
200
PPm-
hour
dose
(h)
4/3-4/6/20
Empty
3
NM
NM
71
86
8.5
18
4/13/20
Empty
3
NM
NM
74
99
DNR
at 4.5
h
DNR
at 4.5
h
4/13/20
12N95
3
0.9
19 for both
bin tests
combined
73
100
12.3
DNR
at 16 h
4/14/20
Empty
3
NM
NM
74
96
DNR
at 3 h
DNR
at 3 h
4/14 -
4/15/20
12 N95s
stacked
3
1.0
27
73
100
13.7
DNR
at
25.4*
4/16 - 4/17/20
12N95
3
0.75
24 mL in 32 h
72
99
14.3
26.8
4/8/20
Empty
6
NM
NM
75
97
3.9
DNR
at 5.8
h
4/9/20
Empty
6
NM
NM
73
100
3.7
6.8
4/8/20
12 N95s
6
NM
NM
74
98
5.8
10.4
4/9-4/10/20
12 N95s
6
1.5 mL/h
140
74
95
5.3
9.3
4/6/20
12 N95s
6
NM
NM
74.4
98.6
7.4
13.9
HPV = hydrogen peroxide vapor; HPA = hydrogen peroxide aqueous; T = temperature; RH = relative humidity; ppm = parts per
million; h = hour; mL = milliliter; NM = not measured; DNR = Did not reach; *was 197 ppm-h.
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REFERENCES
1.	Wood JP, Richter W, Sunderman M, Calfee MW, Serre S, Mickelsen L. Evaluating the Environmental
Persistence and Inactivation of MS2 Bacteriophage and the Presumed Ebola Virus Surrogate Phi6 Using Low
Concentration Hydrogen Peroxide Vapor. Environ Sci Technol. 2020;54(6):3581-90.
2.	U.S. Department of Health and Human Services. Biosafety in Microbiological and Biomedical Laboratories,
5th ed.; CDC 21-1112, 2009.
3.	McDonnell, G. E. Antisepsis, Disinfection, and Sterilization Types, Action, and Resistance, 2nd ed..
ASM Press: 2017.
4.	Mickelsen RL, Wood J, Calfee MW, Serre S, Ryan S, Touati A, Delafield FR, Aslett LD. Low-concentration
hydrogen peroxide decontamination for Bacillus spore contamination in buildings. Remediation. 2019;30(l):47-56.
5.	Wood JP, Calfee MW, Clayton M, Griffin-Gatchalian N, Touati A, Ryan S, Mickelsen L, Smith L, Rastogi
V. A simple decontamination approach using hydrogen peroxide vapour for Bacillus anthracis spore inactivation. J
Appl Microbiol. 2016:n/a-n/a. doi: 10.1111/jam.13284.
CONTACT INFORMATION
This study was conducted as a joint effort between the EPA's Office of Research and Development/Homeland
Security Research Program, and EPA's Office of Emergency Management, Consequence Management Advisory
Division. The respective websites may be found at https://www.epa.gov/homeland-securitv-research and
https://www.epa.gov/emergencv-response/chemical-biological-radiological-and-nuclear-consequence-management
Technical Contacts: Joseph Wood (wood.ioe@epa.gov)
Leroy Mickelsen (Mickelsen.lerov@epa.gov)
Shannon Serre (serre. shannon@epa. gov)
General Feedback/Questions: Kathy Nickel (nickel.kathv@epa.gov)
U.S. EPA's Homeland Security Research Program (HSRP) develops products based on scientific
research and technology evaluations. Our products and expertise are widely used in preventing, preparing
for and recovering from public health and environmental emergencies that arise from terrorist attacks or
natural disasters. Our research and products address biological, radiological, or chemical contaminants that
could affect indoor areas, outdoor areas, or water infrastructure. HSRP provides these products, technical
assistance, and expertise to support EPA's roles and responsibilities under the National Response
Framework, statutory requirements, and Homeland Security Presidential Directives.
Disclaimer: This report has been peer and administratively reviewed and has been approved for publication as
an EPA document. It does not necessarily reflect the views of the Agency. No official endorsement should be
inferred. EPA does not endorse the purchase or sale of any commercial products or services.
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