SC IEN

CE

United States

Environmental Protection

Agency

EPA 600/R-09/054 | October 2009 | www.epa.gov/ord

Highly Pathogenic Avian Influenza 

H5N1 Virus Persistence Testing 

and Evaluation of Liquid 

Decontamination Technologies 

INVESTIGATION AND TECHNOLOGY 

EVALUATION REPORT

Office of Research and Development

National Homeland Security Research Center
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EPA/600/R-09/054 | October 2009 | www.epa.gov/ord

INVESTIGATION AND TECHNOLOGY 

EVALUATION REPORT

Highly Pathogenic Avian Influenza 

H5N1 Virus Persistence Testing and 

Evaluation of Liquid Decontamination 

Technologies 

By

Young W. Choi, James V. Rogers, Daniel J. Chappie, 

Michael L. Taylor, Karen B. Riggs, and Zachary J. Willenberg

Battelle

505 King Avenue

Columbus, Ohio 43201

Joseph P. Wood

Task Order Project Officer

National Homeland Security Research Center

Office of Research and Development

U.S. Environmental Protection Agency

Mail Code E343-06

Research Triangle Park, NC 27711

Office of Research and Development

National Homeland Security Research Center, Decontamination and Consequence Management Division
iv

Disclaimer

The U.S. Environmental Protection Agency, through its Office of Research and 

Development’s National Homeland Security Research Center, funded, directed, and 

managed this investigation and technology evaluation through a Blanket Purchase 

Agreement under General Services Administration contract number GS23F0011L-3 

with Battelle. All tests were conducted at Battelle facilities located in West Jefferson, 

Ohio. It has been reviewed by U.S. Environmental Protection Agency but does not 

necessarily reflect the Agency’s views. No official endorsement should be inferred. 

U.S. Environmental Protection Agency does not endorse the purchase or sale of any 

commercial products or services.
v

Foreword

The U.S. Environmental Protection Agency is charged by 

Congress with protecting the nation’s air, water, and land 

resources. Under a mandate of national environmental 

laws, the U.S. Environmental Protection 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 

Agency’s Office of Research and Development provides data 

and scientific 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.

In September 2002, the Agency announced the formation 

of the National Homeland Security Research Center. The 

Center is part of the Office of Research and Development; 

it manages, coordinates, and supports a variety of research 

and technical assistance efforts. These efforts are designed 

to provide appropriate, affordable, effective, and validated 

technologies and methods for addressing risks posed by 

chemical, biological, and radiological terrorist attacks. 

Research focuses on enhancing our ability to detect, contain, 

and clean up in the event of such attacks.

The Center has developed the Technology Testing and 

Evaluation Program in an effort to provide reliable 

information regarding the performance of homeland security 

related technologies. The Technology Testing and Evaluation 

Program provides independent, quality assured performance 

information that is useful to decision makers in purchasing or 

applying the tested technologies. It provides potential users 

with unbiased, third-party information that can supplement 

vendor-provided information. Stakeholder involvement 

ensures that user needs and perspectives are incorporated 

into the test design so that useful performance information is 

produced for each of the tested technologies. The technology 

categories of interest include detection and monitoring, water 

treatment, air purification, decontamination, and computer 

modeling tools for use by those responsible for protecting 

buildings, drinking water supplies and infrastructure, and for 

decontaminating structures and the outdoor environment.

The evaluation reported herein was conducted by Battelle 

as part of Technology Testing and Evaluation Program. 

Information on National Homeland Security Research Center 

and Technology Testing and Evaluation Program can be 

found at http://www.epa.gov/nhsrc.
vi

Acknowledgments

The authors wish to acknowledge the support of all those who helped plan and conduct 

the evaluation, analyze the data, and prepare this report. We also would like to thank 

the following U.S. Environmental Protection Agency’s personnel for their reviews Dr. 

Worth Calfee (National Homeland Security Research Center); Dr. Wendy Davis-Hoover 

(National Risk Management Research Laboratory); and Mr. Jeff Kempter (Office of 

Pesticide Programs).
vii

Contents

Disclaimer......................................................................................................................................................................iv

Foreword.........................................................................................................................................................................v

Acknowledgments..........................................................................................................................................................vi

Abbreviations/Acronyms...............................................................................................................................................ix

Unit Abbreviations..........................................................................................................................................................x

Definitions.......................................................................................................................................................................x

Executive Summary.......................................................................................................................................................xi

1.0 Introduction............................................................................................................................................................1

2.0 Persistence Testing.................................................................................................................................................3

2.1 Test Materials...............................................................................................................................................3

2.2 Cytotoxicity of Material Extracts.................................................................................................................4

2.3 H5N1 Virus Recovery From Test Materials.................................................................................................4

2.3.1 Spiking the Coupons............................................................................................................................4

2.3.2 H5N1 Virus Extraction and Quantification .........................................................................................5

2.3.3 H5N1 Virus Recoveries Based on Results of Virus Spiking Studies ..................................................6

2.4 Persistence Testing Approach.....................................................................................................................10

2.4.1 Temperature .......................................................................................................................................10

2.4.2 Relative Humidity .............................................................................................................................11

2.4.3 Ultraviolet-A/B Radiation .................................................................................................................12

2.5 Test Results.................................................................................................................................................13

2.5.1 Chicken Feces....................................................................................................................................13

2.5.2 Galvanized Metal...............................................................................................................................16

2.5.3 Glass..................................................................................................................................................18

2.5.4 Soil.....................................................................................................................................................20

2.5.5 Comparison of Chicken Feces, Galvanized Metal, Glass, and Soil Results .....................................22

3.0 Decontamination Technology Evaluation............................................................................................................25

3.1 Technology Descriptions............................................................................................................................25

3.2 Cytotoxicity of Neutralized Decontamination Liquids.............................................................................25

3.3 H5N1 Virus Recovery From Extraction Buffer, Neutralized Decontamination Liquid, 

and Trough Decontamination Liquid ................................................................................................................26

3.3.1 H5N1 Virus Recovery From Coupons Using Extraction Buffer.......................................................26

3.3.2 H5N1 Virus Recovery From Neutralized Decontamination Liquid..................................................27

3.3.3 H5N1 Virus Recovery From Trough Decontamination Liquid.........................................................27

3.4 Evaluation of Liquid Decontamination Technologies................................................................................28

3.4.1 Test Matrix.........................................................................................................................................28

3.4.2 Test Results .......................................................................................................................................28

4.0 Quality Assurance/Quality Control......................................................................................................................33

4.1 Equipment Calibration ...............................................................................................................................33

4.2 Audits .........................................................................................................................................................33

4.2.1 Performance Evaluation Audit ..........................................................................................................33

4.2.2 Technical Systems Audit ...................................................................................................................33

4.2.3 Data Quality Audit.............................................................................................................................33

4.3 Quality Assurance/Quality Control Reporting ...........................................................................................33

4.4 Data Review ...............................................................................................................................................33

4.5 Deviations...................................................................................................................................................33

5.0 Summary..............................................................................................................................................................35

5.1 H5N1 Virus Persistence..............................................................................................................................35

5.2 H5N1 Virus Liquid Decontamination ........................................................................................................35

6.0 References............................................................................................................................................................37

Appendix A: H7N2 Virus Testing.................................................................................................................................39
viii

Figures

Figure 2-1. Petri Dishes With Soil ..................................................................................................................................5

Figure 2-2. Soil and Parafilm® in a Conical Vial ............................................................................................................6

Figure 2-3. Mini-Refrigerator Configured With Ultraviolet A/B Lamps......................................................................12

Figure 2-4. Schematic (Top View) of Ultraviolet Sampling Locations........................................................................13

Tables

Table ES-1. Summary of Persistence Test Conditions and H5N1 Virus Recoveries....................................................xii

Table 2-1. Test Materials.................................................................................................................................................3

Table 2-2. Material Extract Cytotoxicity Test Results....................................................................................................4

Table 2-3. H5N1 Virus Recovery From Basswood........................................................................................................7

Table 2-4. H5N1 Virus Recovery From Chicken Feces.................................................................................................7

Table 2-5. H5N1 Virus Recovery From Concrete (Unpainted) ......................................................................................8

Table 2-6. H5N1 Virus Recovery From Galvanized Metal ............................................................................................8

Table 2-7. H5N1 Virus Recovery From Glass................................................................................................................9

Table 2-8. H5N1 Virus Recovery From Pine Wood........................................................................................................9

Table 2-9. H5N1 Virus Recovery From Soil...................................................................................................................9

Table 2-10. Persistence Test Matrix..............................................................................................................................11

Table 2-11. Ultraviolet Measurements..........................................................................................................................13

Table 2-12. H5N1 Virus Persistence on Chicken Feces................................................................................................14

Table 2-12. H5N1 Virus Persistence on Chicken Feces (continued)............................................................................15

Table 2-13. H5N1 Virus Persistence on Galvanized Metal ..........................................................................................16

Table 2-13. H5N1 Virus Persistence on Galvanized Metal (continued).......................................................................17

Table 2-14. H5N1 Virus Persistence on Glass..............................................................................................................18

Table 2-14. H5N1 Virus Persistence on Glass (continued)...........................................................................................19

Table 2-15. H5N1 Virus Persistence on Soil.................................................................................................................20

Table 2-15. H5N1 Virus Persistence on Soil (continued).............................................................................................21

Table 2-16. Summary of H5N1 Virus Persistence ........................................................................................................22

Table 3-1. Neutralized Decontamination Liquid Cytotoxicity Test Results.................................................................25

Table 3-2. H5N1 Virus Recovery From Coupons Using Extraction Buffer .................................................................26

Table 3-3. H5N1 Virus Recovery From Neutralized Decontamination Liquid ............................................................27

Table 3-4. H5N1 Virus Recovery From Galvanized Metal Trough Decontamination Liquid......................................28

Table 3-5. Decontamination Technology Evaluation Matrix........................................................................................28

Table 3-6. Decontamination Efficacy Against H5N1 Virus on Galvanized Metal .......................................................29

Table 3-7. Decontamination Efficacy Against H5N1 Virus on Soil .............................................................................30

Table 3-8. Summary of Decontamination Efficacy Against H5N1 Virus.....................................................................31

Table A-1. H7N2 Virus Titers Obtained Using Chicken Embryo Kidney Cells...........................................................39
ix

Abbreviations/Acronyms

AOAC AOAC International (formerly the Association of Analytical Chemists)

°C degrees Celsius

CaCO3 calcium carbonate

CEK chicken embryo kidney

CO2 carbon dioxide

CPE cytopathic effects

D/E Dey and Engley

EPA U.S. Environmental Protection Agency

H5N1 highly pathogenic avian influenza virus

H7N2 low pathogenic avian influenza virus

MDCK Madin-Darby canine kidney

MTT 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide

Na2CO3 sodium carbonate

NHSRC National Homeland Security Research Center 

PBS phosphate-buffered saline

QA quality assurance

QC quality control

QMP quality management plan

RH relative humidity

TCID50 50% tissue culture infectious dose 

TTEP Technology Testing and Evaluation Program 

UV ultraviolet radiation
x

Unit Abbreviations

°C degrees Celsius

cm centimeter

g gram

hr hour

min minutes

mL milliliter

mm millimeter

ppm parts per million

rpm revolutions per minute

μL microliter

μm micrometer

μW microwatt

Definitions

TCID50 is a quantitative measure of infectivity and growth and is the number of 

organisms that must be present to infect 50% of the cell culture wells tested.

UV-A/B ultraviolet radiation within the wavelength range of 280–400 nanometers

 UV-A 320–400 nanometers

 UV-B 280–320 nanometers

UV-C ultraviolet radiation within the wavelength range of 200–280 nanometers
xi

Executive Summary

The U.S. Environmental Protection Agency’s (EPA) 

National Homeland Security Research Center (NHSRC) 

Technology Testing and Evaluation Program (TTEP) helps 

to protect human health and the environment by carrying 

out performance tests on homeland security technologies. 

Under TTEP, the persistence of the highly pathogenic avian 

influenza H5N1 virus on test coupons prepared from chicken 

feces, galvanized metal, glass, and soil was investigated. 

The performance of liquid technologies to decontaminate the 

H5N1 virus from test coupons of galvanized metal and soil 

was also investigated at room and low temperatures.

For persistence testing, test coupons were contaminated 

by spiking each coupon with a quantity of H5N1 virus 

amounting to a 50% tissue culture infectious dose (TCID50) 

of H5N1 virus (A/Vietnam/1203/04) of at least 1 x 106. The 

persistence of the H5N1 virus was investigated for up to 

three test durations at five different environmental conditions 

[each environmental condition is described by a specific 

temperature, relative humidity (RH) value, and presence/

absence of exposure to ultraviolet (UV-A/B) radiation to 

simulate sunlight]. Please note that all references to and 

quantifications of the H5N1 virus in this document refer to 

viable H5N1 virus. The environmental conditions were:

•	Room temperature, low RH, no UV 

◦ Test durations: 1-, 2-, and 4-day 

• Room temperature, high RH, no UV 

◦ Test durations: 1-, 2-, and 4-day

• Low temperature, low RH, no UV 

◦ Test durations: 4-, 8-, and 13-day

• Low temperature, high RH, no UV 

◦ Test durations: 4-, 9-, and 13-day

• Low temperature, low RH, UV-A/B

◦ Test durations: 24-, 48-, and 96-hours

(continuous UV-A/B exposure)

Note that for all experiments, the target room temperature 

was 22 degrees Celsius (°C), target low temperature was 

4 °C, target high RH was 80%, and target low RH was 40%.

The persistence testing yielded the following results. At room 

temperature (under low RH and high RH, with no UV), the 

H5N1 virus did not persist on galvanized metal and glass at 

time periods of one day or greater while generally persisting 

on chicken feces and soil for less than two days. 

At low temperature (under low RH and high RH, with no 

UV) the H5N1 virus persisted for at least four days on all 

materials. Following exposure to the low temperature, low 

RH, no UV environmental condition, the H5N1 virus was 

detected after 13 days on galvanized metal, glass, and soil. 

The H5N1 was also viable following exposure to the low 

temperature, high RH, no UV environmental condition, 

after nine days on chicken feces, glass, and soil. Although 

testing was not conducted for durations longer than 13 days 

for any of the environmental conditions, the H5N1 virus 

persistence may exceed 13 days, especially on galvanized 

metal and glass under the low temperature, low RH, no UV 

environmental condition. 

With continuous UV-A/B exposure (and under a low 

temperature and low RH environmental condition), the H5N1 

virus persisted less than 48 hours on galvanized metal and 

glass but persisted at least 48 hours on chicken feces and soil. 

A summary of the actual test conditions and the quantities 

of the H5N1 virus (expressed as individual TCID50 values) 

recovered from chicken feces, galvanized metal, glass, and 

soil is provided in Table ES-1.
xii

Table ES-1. Summary of Persistence Test Conditions and H5N1 Virus Recoveriesa

Environmental 

Condition / Test 

Duration

Temperature 

(°C)b RH (%)b

Mean Recovered H5N1 Virus (TCID50)

Chicken feces

Galvanized 

metal Glass Soil

Room Temperature, 

Low RH, No UV

1-Day 23 35 6.37 x 102 c ND ND 8.82 x 103 d

2-Day 22 36 ND ND ND ND

4-Day 23 49 ND ND ND ND

Room Temperature, 

High RH, No UV

1-Day 22 89 7.70 x 104 ND ND 8.26 x 104

2-Day 23 90 3.16 x 103 c ND ND ND

4-Day 24 91 ND ND ND ND

Low Temperature, 

Low RH, No UV

4-Day 4 28 7.11 x 104 3.78 x 105 4.98 x 105 2.47 x 104

8-Day 7 15 3.16 x 103 c 7.33 x 105 1.34 x 106 1.16 x 103

13-Day 7 46 ND 3.44 x 105 5.91 x 105 1.05 x 103 d

Low Temperature, 

High RH, No UV

4-Day 8 89 5.42 x 104 4.69 x 103 1.37 x 105 2.21 x 105

9-Day 7 97 5.90 x 104 ND 3.16 x 103 5.93 x 105

13-Day 7 79 7.86 x 102 d ND ND ND

Low Temperature, 

Low RH, UV-A/Be

24-Hourse 4 25 1.06 x 106 6.42 x 104 2.62 x 102 d 5.93 x 105

48-Hourse -1 30 9.56 x 104 ND ND 3.90 x 104

96-Hourse 0.1 28 3.16 x 103 c Not Tested Not Tested 3.11 x 103 d

a

 Spike amount ranged from 5.01 x 106 to 5.01 x 107 TCID50. b Mean temperature and RH values were based on continuous monitoring at 1-minute intervals, with the exception of the room temperature, low 

RH, no UV environmental condition, which were derived from the mean of the temperature or RH at the start and end of the tests.

c TCID50 was detected but at a level ≤ the procedural blank TCID50 for chicken feces such that the cytopathic effects observed (and used in the 

calculation of the TCID50) may be attributed to the test material rather than H5N1 virus. d H5N1 virus was not detected on some of the replicate test coupons; a value of 1 TCID50 was used for non-detects in the calculation of the mean 

H5N1 virus recovery.

e

 UV-A/B exposures were continuous (i.e., the UV-A/B lamps did not shut off every 12 hours).

ND = No cytopathic effects detected; the detection limit was 1.31 x 103 TCID50.

The liquid decontamination technologies evaluated 

included 1% citric acid, pH-amended bleach, a hospital 

grade 732 parts per million (ppm) quaternary ammonium 

disinfectant, and 8% sodium carbonate (Na2

CO3) (all were 

prepared in water with a hardness of 400 ppm as calcium 

carbonate [CaCO3]). The H5N1 virus was inoculated onto 

galvanized metal and soil test coupons and exposed to the 

decontamination liquid for a 10-minute contact time. The 

decontamination technology evaluation was implemented at 

room temperature and at a low temperature; UV-A/B lamps 

were not used.

The decontamination technology evaluation results indicated 

that only pH-amended bleach was completely effective 

at inactivating the H5N1 virus (i.e., H5N1 virus was not 

detected), and this inactivation only occurred on galvanized 

metal (at both room and low temperatures). On soil, pHamended bleach induced mean H5N1 virus log reductions 

in TCID50 of 2.7 at room temperature and 2.9 at low 

temperature. The mean H5N1 virus log reductions in TCID50

associated with 1% citric acid ranged from 1.5 (with soil 

at room temperature) to 2.1 (with galvanized metal at low 

temperature). The mean H5N1 virus log reductions in TCID50

associated with 732 ppm quaternary ammonium and 8% 

Na2CO3 were less than 1.0. 
1

1.0

Introduction

National Homeland Security Research Center’s (NHSRC’s) 

Technology Testing and Evaluation Program (TTEP) 

works in partnership with recognized testing organizations; 

with stakeholder groups consisting of buyers, vendor 

organizations, scientists, and permitters; and with 

participation of individual technology developers in carrying 

out performance tests on homeland security technologies. 

In response to the needs of stakeholders, TTEP investigates 

the natural persistence of biological and chemical agents 

and evaluates the performance of innovative homeland 

security technologies by developing test plans, conducting 

evaluations, collecting and analyzing data, and preparing 

peer-reviewed reports. All evaluations are conducted in 

accordance with rigorous quality assurance (QA) protocols 

to ensure the generation of high quality data and defensible 

results. TTEP provides unbiased, third-party information 

supplementary to vendor-provided information that is useful 

to decision makers in purchasing or applying the evaluated 

technologies. Stakeholder involvement ensures that user 

needs and perspectives are incorporated into the evaluation 

design to produce useful performance information for each 

evaluated technology. 

Under TTEP, the persistence of viable, highly pathogenic 

avian influenza H5N1 virus was tested and the performance 

of liquid decontamination technologies to inactivate the 

H5N1 virus was evaluated. The primary objectives were 

to determine how long and under what environmental 

conditions the H5N1 virus remains viable; and to evaluate the 

efficacy of four generic liquid decontamination technologies.

Persistence testing investigated the amount of the H5N1 virus 

remaining on chicken feces, galvanized metal, glass, and soil 

for one to three different durations under the following five 

environmental conditions:

■ Room temperature, low relative humidity (RH), no 

ultraviolet radiation (UV) 

■ Room temperature, high RH, no UV

■ Low temperature, low RH, no UV

■ Low temperature, high RH, no UV

■ Low temperature, low RH, UV-A/B

Note that the target room temperature was 22 degrees Celsius 

(°C), the target low temperature was 4 °C, the target high RH 

was 80%, and target low RH was 40%.

Four liquid decontamination technologies (1% citric acid, 

pH-amended bleach, 732 parts per million (ppm) quaternary 

ammonium and 8% sodium carbonate [Na2CO3]) prepared 

in hard water were evaluated for their effectiveness in 

inactivating the H5N1 virus. For this technology evaluation, 

the H5N1 virus was spiked onto two materials (galvanized 

metal and soil) and the spiked test coupons were treated 

with the liquid decontaminant for a 10-minute contact 

time at room temperature and a low temperature (UV-A/B 

lamps were not used for the decontamination technology 

evaluation).

Efforts were conducted according to a peer-reviewed test/

QA plan1 that was developed according to the requirements 

of the TTEP quality management plan (QMP)2. The 

persistence testing and the technology evaluation both used 

a TCID50 (50% tissue culture infectious dose) assay to 

quantify the H5N1 virus extracted from test coupons. This 

report documents the log reductions in TCID50 associated 

with a natural reduction in the H5N1 virus under various 

environmental conditions and the decrease in the H5N1 virus 

exposed to various liquid decontamination technologies.

Please note that the test/QA plan1 also includes testing for 

the low pathogenic avian influenza virus H7N2. Although 

persistence testing and evaluation of liquid decontamination 

technologies were not conducted for the H7N2 virus, 

preliminary research results associated with the H7N2 virus 

are included in Appendix A. 
2

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3

2.0

Persistence Testing

2.1 Test Materials

Materials considered for H5N1 virus persistence testing 

are described in Table 2-1. Basswood, concrete, and pine 

wood were eventually rejected for H5N1 virus persistence 

testing because of the poor recovery of the H5N1 virus 

(i.e., <5% of the applied inoculum of approximately 1 x 106

TCID50 of H5N1 virus per coupon) from these materials 

(see Section 2.3). Persistence testing was performed using 

test coupons of uniform size. As indicated in Table 2-1 test 

coupons for wood, galvanized metal, and glass were each 

1.9 centimeter (cm) x 7.5 cm; concrete test coupons were 

each 1.0 cm x 3.5 cm. Chicken feces and soil test coupons 

consisted of 3.5 cm diameter Petri dishes, 1 cm high, lined 

with Parafilm®, and filled with uncompacted material. Straw 

and feathers were removed from the chicken feces before 

being placed in the Petri dishes.

Table 2-1. Test Materials

Material

Lot, Batch, or 

Observation

Manufacturer/ 

Supplier Name

Approximate 

Coupon Size, 

width x length

Approximate 

Coupon 

Thickness

Material 

Preparation

Basswood (a hardwood) Not applicable Michaels – Arts 

and Crafts Store

1.9 cm x 7.5 cm 0.2 cm and 0.7 cm Autoclaved

Chicken Feces Not applicable Veterinariana --b --b Nonec

Concrete (unpainted) 5 parts sand: 2 parts 

cement

Wysong Concrete 1.0 cm x 3.5 cm 1 cm Autoclaved

Galvanized Metal Heating, ventilation, and 

air conditioning industry 

standard 24 gauge 

galvanized steel

Accurate 

Fabrication

1.9 cm x 7.5 cm 0.06 cm Cleaned with 

acetone; 

autoclaved

Glass ASTM C1036 Brooks Brothers 1.9 cm x 7.5 cm 0.3 cm Autoclaved

Pine Wood (a softwood) Untreated pine lumber 

(i.e., no preservative 

treatment)

Kingswood Lumber 1.9 cm x 7.5 cm 0.2 cm and 1 cm Gamma 

irradiation

Soil (topsoil) Batch No. Py1A0597 GardenScape 

Brand

--b --b Nonec

a

 Feces was collected from chickens on the day of testing by a veterinarian who raises hens for egg production.

b Chicken feces and soil coupons consisted of a 3.5 cm diameter Petri dish with a height of 1 cm lined with Parafilm® and filled with 

uncompacted material. 

c Chicken feces and soil were not autoclaved or gamma irradiated given the uncertainties associated with potential side-effects (e.g., potential 

hydrolysis of humic acids, release of metals, etc.). Instead these materials were filtered (0.2 micrometer [μm]) during the extraction process, 

which involved agitation and centrifugation of the chicken feces or soil/extraction buffer mixture. The filter was capable of removing bacteria (not 

viruses) from the supernatant that could interfere with virus quantification. Blanks (non-inoculated coupons) did not indicate any interference 

with the assay.
4

2.2 Cytotoxicity of Material Extracts

Prior to persistence testing and the decontamination 

technology evaluations, the potential for extracts of test 

coupons (with no virus present) to cause cytotoxicity 

was determined. Cytotoxic effects caused by test coupon 

extracts could interfere with the TCID50 determination. 

The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium 

bromide (MTT) assay was used to determine the cytotoxic 

effects of test coupon extracts. For the MTT assay, MadinDarby canine kidney (MDCK) cells (the cells used to 

quantitate the TCID50) were seeded into each well of a 

96-well microtiter plate and exposed to serial dilutions of 

test material extracts. MDCK cells exposed only to serial 

dilutions of sterile cell culture medium (the medium in which 

the virus particles were suspended) were used as controls. 

The MDCK cells were then incubated in the presence of 

MTT where mitochondrial succinate dehydrogenase (present 

in living cells) converts the yellow MTT to a purple formazan 

salt. The absorbance (optical density) of this purple reaction 

product is determined using a microplate reader and is 

proportional to mitochondrial function (i.e., the number of 

living cells) and is expressed as a percentage of the control 

cell viability.

The cytotoxicity results showing the lowest dilution able to 

achieve ≥90% of the control cell viability are summarized in 

Table 2-2. The cell viability performance criterion (per the 

test/QA plan1) of ≥90% of unexposed control viability with 

dilution of 1 to 8 (1:8) or less was attained for all materials 

except soil. However >90% of unexposed soil control 

viability was obtained with a dilution of 1:16, which was 

approved as an acceptable level of dilution by the EPA Task 

Order Project Officer. Although an increased level of dilution 

was needed for the MTT assay with soil, the TCID50 assay 

was not believed to be affected. This is because cytopathic 

effects were not generally observed in the MDCK cells 

exposed to any dilution of non-inoculated soil extract; the 

detection limit of the TCID50 assay was not generally affected 

by soil. Further, any potential negative bias in the TCID50

results due to cytotoxicity of the soil extract would have been 

indicated through the inadequate positive control recovery, 

which was not the case.

2.3 H5N1 Virus Recovery From Test Materials

2.3.1 Spiking the Coupons

In order to assess the quantity of virus that could be 

recovered, following the addition of a known quantity of 

virus to each of several test coupons, each coupon was 

spiked with a quantity of virus ranging from 1.98 x 106 to 

7.93 x 106 TCID50 of H5N1 virus per coupon. One hundred 

microliters [μL] of stock virus suspension was applied per 

coupon; the target spike level was 1 x 106 TCID50 of H5N1 

virus per coupon. The H5N1 virus was a Battelle stock 

propagated from a parent stock provided by the Centers for 

Disease Control and Prevention. This H5N1 influenza A 

virus is the 1,203rd strain isolated from a human in Vietnam 

in 2004 (A/Vietnam/1203/04). Genetic sequencing was used 

to verify that propagated stocks matched the parent stock. 

Spiking of the 1.9 cm x 7.5 cm coupons was conducted using 

a multichannel micropipette as two rows of five droplets 

(10 μL per droplet) across the surface of the test coupon. A 

single channel pipette was used to apply ten 10 μL droplets 

at separate locations on the surface of each chicken feces and 

soil test coupon. For concrete, which readily absorbs liquid, 

the spiking was conducted using a single channel pipette and 

100 μL of the inoculum was applied as a single line or streak 

across the surface of the test coupon.

After spiking, all coupons were allowed to dry undisturbed at 

22 ± 2 °C and 40–70% RH for one hour prior to H5N1 virus 

extraction. Test coupons were placed in an acrylic Compact 

Glove Box Model 830-ABC (Plas Labs, Inc., Lansing, 

Mich.), with a volume of 317 liters. Feces collected from 

chickens on the morning of testing remained moist during 

the one-hour drying time. The soil, stored in an air-tight 

container prior to use, also remained moist during the onehour drying time. Following the one-hour drying time, the 

H5N1 virus inoculum appeared to absorb into chicken feces 

and soil and was not visible; however, spots indicative of the 

dried H5N1 virus inoculum were visible on galvanized metal 

and glass.

Table 2-2. Material Extract Cytotoxicity Test Results

Material

Mean Control Cell 

Optical Densitya Dilution

Mean Material Extract 

Optical Densitya % Cell Viability

Basswood 0.3612 1:2 0.4604 127b

Chicken Feces 0.3108 1:4 0.4114 132b

Concrete 0.5557 1:8 0.5075 91.3

Glassc

Pine Woodd Not Tested Not Tested Not Tested Not Tested

Galvanized Metal 0.2389 1:8 0.2216 92.8

Soil 0.5657 1:16 0.5255 92.9

a

 The optical density (absorbance) is directly proportional to the number of viable cells present in the sample well.

b

 Viability above 100% may reflect non-specific reduction of MTT to formazan from unknown coupon extract components.

c

 The cytotoxicity associated with glass was not evaluated as previous testing indicated there is no interference with MDCK cell viability. d

 Pine wood was not tested due to insufficient recovery of the H5N1 virus; see section 2.3.3.
5

2.3.2 H5N1 Virus Extraction and Quantification

For basswood, concrete, galvanized metal, glass, and 

pine wood, extraction of the H5N1 virus from individual 

test coupons entailed, following drying, placing a single 

spiked test coupon into a sterile 50 milliliter (mL) conical 

vial containing 10 mL of sterile extraction buffer (i.e., 

phosphate-buffered saline [PBS]). The vials were agitated on 

an orbital shaker for at room temperature for 15 minutes at 

approximately 200 revolutions per minute (rpm). The extract 

was removed from the vial using a pipette and was then 

serially diluted and plated onto MDCK cells. Quantitation 

of the H5N1 virus in the extract was based on 10-fold serial 

dilutions of the material extracts applied to MDCK cells and 

the subsequent observation of cytopathic effects (CPE) in 

the MDCK cells to determine the viral concentration. The 

quantity of virus present was expressed as the TCID50 for the 

H5N1 virus. 

The TCID50 for H5N1 virus in test coupon extracts was 

determined by first preparing 10-fold serial dilutions of each 

extract and then transferring 0.1 mL aliquots of each dilution 

to five wells (of a 96-well microtiter plate) containing 

monolayers of MDCK cells. Following transfer of aliquots 

of the diluted extracts, the 96-well microtiter plate was 

incubated at 37 ± 2 °C under 5% carbon dioxide (CO2) in a 

humidified incubator for 72 to 96 hours or until CPE were 

visually detected by comparing to the control (cell culture 

medium only) wells. Using light microscopy each well of the 

96-well plate was evaluated and observations documented 

(+ = positive CPE; 0 = no observed effect) for CPE. The 

TCID50 was calculated using the Spearman-Karber3

 method 

as follows:

Equation 2-1.

For the titer, take the inverse log of TCID50 per unit volume. 

For an inoculating volume of 0.1 mL, the calculated TCID50

titer is multiplied by a factor of 10 (10 x 0.1 mL = 1.0 mL) to 

achieve TCID50/mL.

The TCID50 assay’s limit of quantitation (i.e., detection limit) 

for all materials using the Spearman-Karber method3,4 is 131 

TCID50/mL (i.e., 1.31 x 103 TCID50 per coupon). 

For quantitating the H5N1 virus in chicken feces and soil, 

each test coupon plus the Parafilm®, which lined each Petri 

dish (Figure 2-1), were placed in the conical vials containing 

the PBS extraction buffer. (Figure 2-2). Each vial was shaken 

on the orbital shaker as described for the other test materials 

(shaken on an orbital shaker at 200 rpm for 15 minutes), 

then each vial was centrifuged at 1000 x g for 10 minutes. 

A syringe was used to withdraw a 0.5 to 1 mL aliquot of the 

extract in each vial, which was then filtered using a 0.2 μm 

(polyether sulfone, low protein-binding) syringe filter, 

enabling the H5N1 virus to pass through while excluding 

larger particles and organisms. The filtered sample was then 

serially diluted and plated onto the MDCK cells for TCID50

determination.

Figure 2-1. Petri Dishes With Soil
6

Figure 2-2. Soil and Parafilm® in a Conical Vial

2.3.3 H5N1 Virus Recoveries Based on Results of Virus 

Spiking Studies

Materials considered for persistence testing were studied to 

determine if an acceptable level of H5N1 virus (an acceptable 

level is defined in the test/QA plan1 as ≥5% of the applied 

inoculum) could be recovered from the coupons. The 

coupons were spiked and allowed to dry for one hour; then 

the H5N1 virus was extracted from the coupons and analyzed 

for TCID50 determinations. Numerous extraction approaches 

and materials (e.g., the use of surfactants) were considered 

for use in an attempt to achieve acceptable H5N1 virus 

recovery. Initial determinations of H5N1 virus recoveries 

were conducted with three replicate coupons per material; 

if acceptable recoveries were obtained final demonstrations 

using five replicate coupons were used to obtain mean 

recovery and variance data. The H5N1 virus recovery results 

and associated extraction approaches for each of the materials 

tested are provided in Tables 2-3 through 2-9. Materials 

studied include: basswood (a hardwood), chicken feces, 

concrete, galvanized metal, glass, pine wood (a softwood), 

and soil. For concrete, special tests were also conducted 

to investigate the impact of the highly alkaline concrete 

extraction solution on the viability of the H5N1 virus and the 

adsorption/deposition of the H5N1 virus into the concrete 

(Table 2-5). The tabulated data indicate that an acceptable 

H5N1 virus recovery (≥5% of the applied inoculum) was 

only obtained from chicken feces, galvanized metal, glass, 

and soil. Hence, these four materials were used in subsequent 

persistence testing.
7

Table 2-3. H5N1 Virus Recovery From Basswood

Material Extraction Approach

Replicate 

Coupons

Inoculum 

(TCID50)

Recovered Virus 

(TCID50)

a

% Virus 

Recoverya

Basswood; 0.7 cm thickness Placed in PBS, agitated on an 

orbital shaker for 15 minutes (min) 

at 200 rpm.

3 2.00 x 106 5.01 ± 0.00 x 103 0.25 ± 0.00

Basswood; 0.2 cm thickness Placed in PBS, agitated on an 

orbital shaker for 15 min at 200 

rpm.

3 2.00 x 106 3.00 ± 1.74 x 103 0.15 ± 0.09

Basswood; 0.2 cm thickness Basswood soaked in 2% skim milk 

(dry milk reconstituted in deionized 

water) for 10 min, allowed to dry 

to the touch, and autoclaved for 

15 min at 121 °C. Once cooled, 

inoculated and allowed to dry for 

1 hour (hr), then placed in PBS, 

agitated on an orbital shaker for 

15 min at 200 rpm, coupons were 

also scraped with a pipette tip, 

and extraction fluid jetted onto the 

inoculated surface in an attempt to 

aid H5N1 virus recovery.

3 2.00 x 106 4.75 ± 2.76 x 103 0.24 ± 0.14

Basswood; 0.2 cm thickness Placed in PBS, agitated on a plate 

shaker for 5 min at 1,100 rpm.

3 5.01 x 106 3.00 ± 1.73 x 103 0.06 ± 0.03

Basswood; 0.2 cm thickness Placed in PBS, agitated on a 

vortexer for 2 min at 2,500 rpm.

3 5.01 x 106 4.00 ± 1.73 x 103 0.08 ± 0.03

a Data are expressed as mean ± standard deviation.

The drying time or elapsed time between inoculation and recovery was 1 hr.

Table 2-4. H5N1 Virus Recovery From Chicken Feces

Material Extraction Approach

Replicate 

Coupons

Inoculum 

(TCID50)

Recovered Virus 

(TCID50)

a

% Virus 

Recoverya

Chicken Feces Placed in PBS, agitated on an orbital 

shaker for 15 min at 200 rpm, then 

centrifuged with the supernatant filtered 

(0.2 μm) for analysis.

3 2.00 x 106 1.74 ± 1.25 x 105 8.7 ± 6.3

Chicken Feces Placed in PBS, agitated on an orbital 

shaker for 15 min at 200 rpm, then 

centrifuged with the supernatant filtered 

(0.2 μm) for analysis.

5 7.92 x 106 5.23 ± 1.72 x 105 6.6 ± 2.2

a Data are expressed as mean ± standard deviation.

The drying time or elapsed time between inoculation and recovery was 1 hr.
8

Table 2-5. H5N1 Virus Recovery From Concrete (Unpainted)

Material Extraction Approach

Replicate 

Coupons

Inoculum 

(TCID50)

Recovered Virus 

(TCID50)

a

% Virus 

Recoverya

Concrete Placed in PBS, agitated on an orbital shaker 

for 15 min at 200 rpm.

3 1.98 x 106 ND 0.00 ± 0.00 

Concrete; 

extract 

solution

The PBS extraction of concrete resulted in a 

highly alkaline solution (i.e., pH > 11). A test 

was conducted to determine if this solution 

inactivates the H5N1 virus. Concrete was 

placed in PBS and shaken to obtain a high 

alkaline solution, the coupon was removed 

and the solution was spiked with H5N1 virus 

then agitated on an orbital shaker for 15 min 

at 200 rpm. 

3 5.01 x 106 5.34 ± 4.51 x 105 11 ± 9.0

Concrete Placed in PBS and H5N1 virus was spiked 

into the tube (not onto the coupon), which 

was then agitated on an orbital shaker for 

15 min at 200 rpm. The purpose of this 

test was to determine if the H5N1 virus was 

depositing or absorbing into the concrete 

compounded by the highly alkaline extraction 

solution. 

3 5.01 x 106 3.00 ± 1.74 x 105 6.0 ± 3.5

Concrete Inoculated with the H5N1 virus and 

immediately extracted (placed in PBS, 

agitated on an orbital shaker for 15 min at 

200 rpm) to prevent the H5N1 virus from 

drying out 

3 5.01 x 106 2.44 ± 2.38 x 104 0.49 ± 0.47

Concrete Placed in sodium phosphate dibasic 

heptahydrate + Tween® 80, agitated on an 

orbital shaker for 15 min at 200 rpm.

3 2.00 x 106 ND 0.00 ± 0.00 

Concrete Pulverized immediately after inoculation with 

the H5N1 virus, placed in PBS, agitated on 

an orbital shaker for 15 min at 200 rpm.

3 2.00 x 106 ND 0.00 ± 0.00 

Concrete Pulverized immediately after inoculation with 

the H5N1 virus, placed in sodium phosphate 

dibasic heptahydrate + Tween® 80 agitated 

on an orbital shaker for 15 min at 200 rpm.

3 2.00 x 106 ND 0.00 ± 0.00

a Data are expressed as mean ± standard deviation.

The drying time or elapsed time between inoculation and recovery was 1 hr, except where immediate extraction is noted.

ND = Not detected; the detection limit is 1.31 x 103 TCID50.

Table 2-6. H5N1 Virus Recovery From Galvanized Metal

Material Extraction Approach

Replicate 

Coupons

Inoculum 

(TCID50)

Recovered Virus 

(TCID50)

a

% Virus 

Recoverya

Galvanized Metal Placed in PBS, agitated on an orbital 

shaker for 15 min at 200 rpm.

3 7.92 x 106 6.96 ± 1.69 x 105 8.8 ± 2.1

Galvanized Metal Placed in PBS, agitated on an orbital 

shaker for 15 min at 200 rpm.

5 5.01 x 106 12.7 ± 7.42 x 105 25 ± 15

a Data are expressed as mean ± standard deviation.

The drying time or elapsed time between inoculation and recovery was 1 hr.
9

Table 2-7. H5N1 Virus Recovery From Glass

Material Extraction Approach

Replicate 

Coupons

Inoculum 

(TCID50)

Recovered Virus 

(TCID50)

a

% Virus 

Recoverya

Glass Placed in PBS, agitated on an orbital shaker 

for 15 min at 200 rpm.

3 1.98 x 106 0.75 ± 1.11 x 106 38 ± 56

Glass Placed in PBS + 0.01% Tween® 20 (a nondenaturing surfactant), agitated on an orbital 

shaker for 15 min at 200 rpm.

3 5.01 x 106 1.42 ± 1.52 x 106 28 ± 30

Glass Placed in PBS, agitated on an orbital shaker 

for 15 min at 200 rpm.

5 2.00 x 106 1.86 ± 1.33 x 105 9.3 ± 6.7

a

 Data are expressed as mean ± standard deviation.

 The drying time or elapsed time between inoculation and recovery was 1 hr.

Table 2-8. H5N1 Virus Recovery From Pine Wood

Material Extraction Approach

Replicate 

Coupons

Inoculum 

(TCID50)

Recovered 

Virus (TCID50)

% Virus 

Recoverya

Pine Wood;

1 cm thickness

Placed in PBS, agitated on an orbital shaker 

for 15 min at 200 rpm.

3 1.98 x 106 ND 0.00 ± 0.00 

Pine Wood;

1 cm thickness

Pine wood soaked in 2% skim milk (dry milk 

reconstituted in deionized water) for 10 min, 

allowed to dry to the touch, and autoclaved for 

15 min at 121 °C. Once cooled, inoculated 

and allowed to dry for 1 hr, then placed in 

PBS, agitated on an orbital shaker for 15 min 

at 200 rpm. 

3 5.01 x 106 ND 0.00 ± 0.00 

Pine Wood;

0.2 cm thickness

Placed in PBS, agitated on an orbital shaker 

for 15 min at 200 rpm.

3 2.00 x 106 ND 0.00 ± 0.00 

Pine Wood;

0.2 cm thickness

Pine wood soaked in 2% skim milk (dry milk 

reconstituted in deionized water) for 10 min, 

allowed to dry to the touch, and autoclaved for 

15 min at 121 °C. Once cooled, inoculated 

and allowed to dry for 1 hr, then placed in 

PBS, agitated on an orbital shaker for 15 min 

at 200 rpm.

3 2.00 x 106 ND 0.00 ± 0.00 

a Data are expressed as mean ± standard deviation.

The drying time or elapsed time between inoculation and recovery was 1 hr.

ND = Not detected; the detection limit was 1.31 x 103 TCID50.

Table 2-9. H5N1 Virus Recovery From Soil

Material Extraction Approach

Replicate 

Coupons

Inoculum 

(TCID50)

Recovered Virus 

(TCID50)

a

% Virus 

Recoverya

Soil Placed in PBS, agitated on an orbital shaker for 

15 min at 200 rpm, then centrifuged with the 

supernatant filtered (0.2 μm) for analysis.

3 1.98 x 106 11.0 ± 8.13 x 105 56 ± 41

Soil Placed in PBS, agitated on an orbital shaker for 

15 min at 200 rpm, then centrifuged with the 

supernatant filtered (0.2 μm) for analysis.

5 7.92 x 106 4.11 ± 2.52 x 105 5.2 ± 3.2

a Data are expressed as mean ± standard deviation.

The drying time or elapsed time between inoculation and recovery was 1 hr.
10

2.4 Persistence Testing Approach

The TCID50 of the H5N1 virus was measured from the liquid 

extracts obtained from test coupons (spiked coupons placed 

in the exposure chamber with temperature, RH, UV-A/B 

radiation, and contact time treatments) and positive controls 

(spiked coupons extracted after the one hour drying time 

[time-zero]). Coupon spiking and H5N1 virus extraction 

and quantification followed the approach described in 

Section 2.3.

The log reduction in TCID50 was calculated as N/N′ where 

N is the mean TCID50 from five positive controls of a given 

material and N′ is the TCID50 from each test coupon replicate 

of a given material, environmental condition, and contact 

time. The log reduction in TCID50 for each individual test 

coupon (R) was calculated for each of the five replicate test 

coupons of each material type, environmental condition, and 

contact time as:

Equation 2-2.

Where:

= log reduction in TCID50 for the ith replicate 

test coupon, jth test material, kth environmental 

condition, and lth contact time

= arithmetic mean TCID50 from the five positive 

controls (which are measured at time-zero) for the 

jth test material

= TCID50 recovered on the ith replicate test coupon, 

jth test material, kth environmental condition, and 

lth contact time.

If no TCID50 (i.e., no CPE is detected) is measured from a 

test coupon (N′), the value one was substituted for N′. Since 

the value one is greater than the observed value of zero, the 

estimate with this substitution becomes a lower bound for the 

true log reduction. Next, the mean log reduction in TCID50

(R) for the five replicate test coupons of a given material/

environmental condition/contact time was calculated as:

Equation 2-3.

Where:

= mean log reduction in TCID50 for the jth test 

material, kth environmental condition, and lth 

contact time

= sum of the log reductions in TCID50 for each 

individual test coupon for the jth test material, 

kth environmental condition, and lth contact 

time n is the number of test coupon replicates 

(five).

The test matrix and various test conditions that were utilized 

for H5N1 virus persistence testing are summarized in 

Table 2-10. The environmental conditions included various 

combinations of temperature, RH, and UV-A/B radiation. 

Persistence was measured on four types of test coupons: 

chicken feces, galvanized metal, glass, and soil, and the test 

durations ranged from one to 13 days. Initial time points

were selected based on comparable data available in the 

literature, but subsequent time points were adaptively chosen 

(i.e., shorter or longer durations) based on the initial test 

results, and tests were not necessarily conducted sequentially 

from the shortest to longest test duration. 

2.4.1 Temperature

Avian influenza viruses, in general, can survive in the 

environment depending on temperature and humidity 

conditions, but the various strains of avian influenza may 

survive longer in cooler and moister conditions5

. The 

persistence tests were conducted at room temperature and 

a lower temperature to better evaluate the influence of 

temperature on the persistence of the H5N1 virus (e.g., the 

ability to detect the presence of H5N1 virus). The actual 

temperatures associated with each test are provided in 

Table 2-10.

The persistence of the H5N1 virus was tested at the room 

temperature, low RH, no UV environmental condition by 

placing test coupons spiked with the virus into a Plas Labs’ 

compact glove box under ambient laboratory conditions; 

temperature was recorded manually with a digital calibrated 

thermometer (FisherbrandTM Traceable Radio-Signal 

Hygrometer/Thermometer, Fisher Scientific, Pittsburgh, 

Penn.) at the start and end of the test but not continuously 

monitored. The temperature for the other environmental 

conditions was recorded continuously at one-minute intervals 

with a HOBO® U10 Temperature Data Logger, (Onset 

Computer Corporation, Bourne, Mass.). 

Low temperature persistence tests (with no UV) were 

conducted by placing the coupons inside a sealed 

Lock&LockTM container and the container was then placed 

inside a refrigerator. For testing at a low temperature with 

UV-A/B, coupons were placed directly beneath the UV-A/B 

lamps mounted inside a mini-refrigerator (Marvel® Scientific, 

Model No. 6CAR, Greenville, MI) modified to include glove 

ports. Some of the temperatures associated with these low 

temperature environmental conditions exceeded 20 oC; this 

generally occurred during the first two hours of a test as 

the temperatures inside the Lock&LockTM container were 

equilibrating (cooling down) relative to the temperature in the 

refrigerator. As noted in Section 4.5 and shown in Table 2-10, 

the range of temperatures often exceeded the allowable test 

measurement tolerance of ± 2 oC (as specified in the test/QA 

plan1) for the target temperatures of 22 oC and 4 oC. Although 

some of the mean test temperatures deviated from the target 

temperatures by a few oC, the associated TCID50 data remain 

valid and useful. 
11

Table 2-10. Persistence Test Matrix

Target Environmental 

Condition / Test Duration Material Temperature (°C) RH (%)

Room Temperature (22 oC), Low 

RH (40%), No UVa

Chicken feces, galvanized metal, 

glass, and soil

Start End Start End

1-Day 22.7 23.4 32 38

2-Day 22.0 22.9 30 42

4-Day 23.2 22.8 36 61

Room Temperature (22 oC), High 

RH (80%), No UV

Chicken feces, galvanized metal, 

glass, and soil

Mean Range Mean Range

1-Day 22.4 19.0 – 23.2 89.1 51.9 – 93.5

2-Day 23.4 22.6 – 24.4 90.4 80.3 – 93.8

4-Day 23.7 22.7 – 24.9 91.2 76.5 – 94.4

Low Temperature (4 oC),Low RH 

(40%), No UV

Chicken feces, galvanized metal, 

glass, and soil

Mean Range Mean Range

4-Day 4.0 3.41 – 21.9 27.7 17.5 – 33.3

8-Day 6.7 6.46 – 22.2 15.2 1.00 – 71.3

13-Day 6.7 6.36 – 22.6 46.3 27.4 – 54.6

Low Temperature (4 oC), High RH 

(80%), No UV

Chicken feces, galvanized metal, 

glass, and soil

Mean Range Mean Range

4-Day 7.8 7.22 – 23.1 89.5 63.6 – 92.8

9-Day 6.7 6.38 – 22.9 96.9 66.4 – 104

13-Day 6.8 6.48 – 22.6 79.0 24.9 – 100 

Low Temperature (4 oC), Low RH 

(40%), UV-A/B

Chicken feces, galvanized metal, 

glass, and soil

Mean Range Mean Range

24-Hours 4.1b 2.26 – 20.8c 24.5b 23.1 – 68.3c

48-Hours -0.91 -2.04 – 21.2 29.7 28.0 – 86.2

96-Hoursb 0.12 -1.76 – 21.7 28.2 25.2 – 65.2

a For the room temperature, low RH, no UV test condition, temperature was recorded manually and RH was measured with a digital calibrated 

hygrometer. Both temperature and RH were recorded at the start and end of the test; neither was continuously monitored.

b

 Galvanized metal and glass were not tested at the 4-day test duration.

c

 Temperature and RH were not recorded during the initial seven hours of the test.

2.4.2 Relative Humidity

Persistence testing was conducted using low and high 

RH levels to better understand the influence of RH on 

the persistence of the H5N1 virus. The actual RH levels 

associated with each test are provided in Table 2-10; the 

range of RH levels often exceeded the allowable test 

measurement tolerance of ±10% specified in the test/QA 

plan1

, as noted in Section 4.5. Deviations from the target RH 

levels do not invalidate the associated TCID50 data. 

For the room temperature, low RH, no UV environmental 

condition, persistence of the H5N1 virus was tested at 

ambient laboratory conditions (no active measures were 

taken to increase or reduce the RH) inside a glove box; RH 

was measured at the start and end of the test with a digital 

calibrated hygrometer but not continuously monitored. 

RH for the persistence testing performed under other 

environmental conditions was recorded continuously at 

one-minute intervals with a device that measures and logs 

temperature and RH data (HOBO® U10, Onset Computer 

Corporation). For the room temperature, low RH, no UV 

environmental condition, the RH level appeared to increase 

with the duration of the test. The moisture content associated 

with the materials being tested (e.g., soil and chicken feces) 

likely contributed to the increasing RH inside the sealed 

glove box as time elapsed. 

Persistence testing at the room temperature, high RH, no UV 

environmental condition was accomplished by placing the 

coupons directly in a pre-humidified (by using an ultrasonic 

fogger) glove box. The RH tended to increase with the 

placement of the coupons into the glove box; consequently 

during the 1-day test, fresh air was introduced into the glove 

box to manually lower the RH at the start of the 1-day test. 

During the 2- and 4-day tests, there were no attempts to 

adjust the RH, which increased during testing. 

Persistence testing at low temperature (with no UV) 

was conducted by placing the coupons inside a sealed 

Lock&LockTM container inside a refrigerator. When testing 

at a low RH, DRIERITE (W. A. Hammond DRIERITE 

Co., Xenia, Ohio) was also added to the container. In an 

attempt to prevent the RH from increasing as the DRIERITE 

became saturated, fresh DRIERITE was exchanged with 

the used DRIERITE on the fourth day of the 8-day test 

and this reduced the RH from 20–30% to <10% (the RH 

briefly spiked to 71.30% during the exchange). For high RH 
12

persistence testing (target RH 80% per the test/QA plan1) 

inside the Lock&LockTM container at low temperature, 

mean RH levels ranging from 79% to 97% were achieved by 

sealing the inherently moist materials (i.e., soil and chicken 

feces) in the container.

For testing with UV-A/B (low temperature, low RH), the RH 

inside the modified mini-refrigerator remained relatively low 

throughout the testing such that the use of DRIERITE was 

not required. 

2.4.3 Ultraviolet-A/B Radiation

Influenza viruses may be inactivated by UV-B wavelengths 

in sunlight6. UV-A/B radiation was implemented in the tests 

in an effort to mimic UV radiation levels associated with 

natural sunlight. The spectrum and intensity of terrestrial 

UV radiation is highly variable and is affected by time of 

day, day of year, geographical location, altitude, atmospheric 

pollution, and clouds. Naturally occurring UV-B levels, 

observed around noon, range from 19.5 to 150 microwatt 

(μW)/cm2, 7–10, and therefore a target exposure chamber 

condition of 70 μW/cm2 UV-B was selected. The amount of 

UV-A generated during testing was quantitated during the 

method development phase and actual testing and was kept 

within the range of UV-A observed in natural sunlight (0 to 

4,500 μW/cm2)11. The target UV-C level to be generated 

during testing was 0 μW/cm2, since UV-C generally does 

not reach the earth’s surface. UV-B, UV-C, and total UV 

were monitored during the persistence testing (UV-A was 

calculated as total UV minus the UV-B and UV-C levels).

The level of UV-B radiation generated and the potential 

of diminishing UV-B output over time was evaluated 

with data obtained from the lamp manufacturer and data 

generated during the method development phase of testing.

A ReptiSunTM 10.0 Linear Fluorescent UV-B Lamp 

was used in the study. The manufacturer, Zoo 

Med Laboratories, Inc. (San Luis Obispo, Calif.) 

indicated that after an initial burn in, UV-B levels of 

the lamp would diminish somewhat over time.

Using a single ReptiSunTM 10.0 Linear Fluorescent 

UV-B Lamp with a baseline UV-B level of 70 μW/cm2, 

and a Solarmeter® Model 6.2 (Solartech, Inc., Harrison 

Township, Mich.) the following UV-B levels were measured 

sequentially over time during preliminary tests: 

■ 69 μW/cm2, 99% of baseline remaining after 1 hour

■ 68 μW/cm2, 97% of baseline remaining after 2 hours

■ 66 μW/cm2, 94% of baseline remaining after 17 hours

■ 64 μW/cm2, 91% of baseline remaining after 64 hours

■ 64 μW/cm2, 91% of baseline remaining after 89 hours

The UV-A/B lamps were placed inside a scientific-grade 

mini-refrigerator, which was customized with a faceplate and 

glove ports (see Figure 2-3). All UV-A/B exposures were 

continuous; the lamps were not turned off during testing. 

In addition to measuring UV-B, UV-C was measured with 

the Solarmeter® Model 8.0 and total UV was measured 

with Solarmeter® Model 5.0. UV data measured during the 

persistence testing are summarized in Table 2-11. During 

the 24-hour test, UV measurements were recorded from the 

center of the testing area at the start and completion of the 

test. During the 48- and 96-hour tests, UV measurements 

were periodically made from five positions beneath the lamps 

(Figure 2-4). UV measurements were taken at approximately 

14 cm below the lamps, which corresponded to the distance 

of the test coupons from the lamps. All UV-B levels 

coincided with the target UV level (70 ± 7 μW/cm2 UV-B as 

specified in the test/QA plan).

Figure 2-3. Mini-Refrigerator Configured With Ultraviolet A/B Lamps

UV Lamps 

UV Monitor 

(what type> 
13

Table 2-11. Ultraviolet Measurements

Low Temperature, 

Low RH, UV-A/B 

Test Durationsa UV-A (μW/cm2) UV-B (μW/cm2) UV-C (μW/cm2)

24-Hourb Start

125

End

130

Start

74

End

75

Start

0

End

0

48-Hourc Mean

109

Range

100 – 122

Mean

68

Range

63 – 74

Mean

0

 Range 

0 – 0

96-Hourc Mean

106

Range

97 – 120

Mean

69

Range

63 – 75

Mean

0

 Range

0 – 0

a

 UV-A/B exposures for the 24-, 48-, and 96-hour tests were continuous.

b

 During the 24-hour test, UV measurements were recorded from the center of the testing area at the start and completion of the test.

c

 During the 48- and 96-hour tests, UV measurements were periodically made from five positions beneath the lamps (see Figure 2-4). 

Figure 2-4. Schematic (Top View) of Ultraviolet Sampling Locations

Mini-fridge work 

surface (top view) 

All coupons located within dashed line 

Five points measured for UV-B, UV-C and 

total UV intensity during persistence trial 

2.5 Test Results

Persistence results for each material/environmental condition 

combination are summarized in Tables 2-12 through 2-15. 

A summary of all persistence data obtained is provided in 

Table 2-16.

2.5.1 Chicken Feces

The results obtained for persistence of the H5N1 virus on 

chicken feces are summarized in Table 2-12.

The H5N1 virus was least persistent (6.3 mean log reduction 

in TCID50 during the 2-day test) on chicken feces for the 

room temperature, low RH, no UV environmental condition, 

and the H5N1 virus was not detected above the TCID50 on 

the laboratory and procedural blanks at any test duration.

Extracts from chicken feces samples not inoculated with 

the H5N1 virus often induced CPE when testing the 

most concentrated serial dilution (no CPE observed with 

subsequent dilutions). This CPE often resulted in a TCID50

of 3.16 x 103

, and was attributed to the inherent properties of 

the chicken feces rather than the H5N1 virus. Test coupons 

with mean recovered TCID50 values less than or equal to the 

associated laboratory and procedural blanks were still used in 

the calculation of the mean log reductions.

At higher RH (room temperature, high RH, no UV 

environmental condition), the mean TCID50 level recovered 

from the test coupons after the 1-day test was greater than the 

TCID50 levels associated with the laboratory and procedural 

blanks. Mold was observed growing on the chicken feces 

during the 4-day test at the room temperature, high RH, no 

UV environmental condition. 
14

Table 2-12. H5N1 Virus Persistence on Chicken Feces

Test Duration / 

Sample

Inoculum 

(TCID50) CPE Detectionsa

Mean Recovered 

Virus (TCID50)

b, c

% Virus 

Recoveryb

Mean Log 

Reductionb

Room Temperature, Low RH, No UV

1-Day

Positive Controld 7.94 x 106 5/5 8.05 ± 4.16 x 105 10.1 ± 5.2 -

Test Coupone 7.94 x 106 5/5 6.37 ± 3.99 x 102 h 0.01 ± 0.01h 3.2 ± 0.26h

Laboratory Blankf 0 1/1 3.16 x 103 - -

Procedural Blankg 0 1/1 3.16 x 103 - -

2-Day

Positive Control 7.94 x 106 5/5 2.11 ± 1.95 x 106 26.6 ± 24.5 -

Test Coupon 7.94 x 106 0/5 NDi 0.00 ± 0.00i 6.3 ± 0.00i

Laboratory Blank 0 1/1 3.16 x 103 - -

Procedural Blank 0 1/1 3.16 x 103 - -

4-Day

Positive Control 5.01 x 106 5/5 3.66 ± 1.87 x 105 7.30 ± 3.73 -

Test Coupon 5.01 x 106 0/5 NDi 0.00 ± 0.00i 5.6 ± 0.00i

Laboratory Blank 0 1/1 3.16 x 103 - -

Procedural Blank 0 1/1 3.16 x 103 - -

Room Temperature, High RH, No UV

1-Day

Positive Control 5.01 x 107 5/5 6.96 ± 3.96 x 106 13.9 ± 7.90 -

Test Coupon 5.01 x 107 5/5 7.70 ± 3.11 x 104 0.15 ± 0.06 2.0 ± 0.2

Laboratory Blank 0 1/1 3.16 x 103 - -

Procedural Blank 0 1/1 3.16 x 103 - -

2-Day

Positive Control 3.16 x 107 5/5 2.54 ± 1.59 x 105 0.80 ± 0.50 -

Test Coupon 3.16 x 107 5/5 3.16 ± 0.00 x 103 h 0.01 ± 0.00h 1.9 ± 0.0h

Laboratory Blank 0 1/1 3.16 x 103 - -

Procedural Blank 0 1/1 3.16 x 103 - -

4-Day

Positive Control 2.00 x 107 5/5 1.39 ± 1.17 x 106 6.96 ± 5.83 -

Test Coupon 2.00 x 107 0/5 NDi 0.00 ± 0.00i 6.1 ± 0.0i

Laboratory Blank 0 1/1 3.16 x 103 - -

Procedural Blank 0 1/1 3.16 x 103 - -

Low Temperature, Low RH, No UV

4-Day

Positive Control 2.00 x 107 5/5 2.02 ± 1.83 x 106 10.1 ± 9.13 -

Test Coupon 2.00 x 107 5/5 7.11 ± 3.32 x 104 0.36 ± 0.17 1.5 ± 0.2

Laboratory Blank 0 1/1 3.16 x 103 - -

Procedural Blank 0 1/1 3.16 x 103 - -

8-Day

Positive Control 5.01 x 107 5/5 8.05 ± 4.16 x 105 1.61 ± 0.83 -

Test Coupon 5.01 x 107 5/5 3.16 ± 0.00 x 103 h 0.01 ± 0.00h 2.4 ± 0.0h

Laboratory Blank 0 1/1 3.16 x 103 - -

Procedural Blank 0 1/1 3.16 x 103 - -

13-Day

Positive Control 2.00 x 107 5/5 7.10 ± 3.94 x 105 3.55 ± 1.97 -

Test Coupon 2.00 x 107 0/5 NDi 0.00 ± 0.00i 5.9 ± 0.0i

Laboratory Blank 0 1/1 3.16 x 103 - -

Procedural Blank 0 0/1 ND - -
15

Table 2-12. H5N1 Virus Persistence on Chicken Feces (continued)

Test Duration / 

Sample

Inoculum 

(TCID50) CPE Detectionsa

Mean Recovered 

Virus (TCID50)

b, c

% Virus 

Recoveryb

Mean Log 

Reductionb

Low Temperature, High RH, No UV

4-Day

Positive Control 5.01 x 107 5/5 8.05 ± 4.16 x 105 1.61 ± 0.83 -

Test Coupon 5.01 x 107 5/5 5.42 ± 4.09 x 104 0.11 ± 0.08 1.3 ± 0.3

Laboratory Blank 0 1/1 3.16 x 103 - -

Procedural Blank 0 1/1 3.16 x 103 - -

9-Day

Positive Control 7.94 x 106 5/5 4.42 ± 4.86 x 105 5.57 ± 6.12 -

Test Coupon 7.94 x 106 5/5 5.90 ± 4.57 x 104 0.74 ± 0.58 1.0 ± .05 

Laboratory Blank 0 1/1 3.16 x 103 - -

Procedural Blank 0 1/1 3.16 x 103 - -

13-Day

Positive Control 2.00 x 107 5/5 7.10 ± 3.94 x 105 3.55 ± 1.97 -

Test Coupon 2.00 x 107 3/5 7.86 ± 7.17 x 102 i 0.00 ± 0.00i 4.0 ± 2.0i

Laboratory Blank 0 1/1 3.16 x 103 - -

Procedural Blank 0 0/1 ND - -

Low Temperature, Low RH, UV-A/B

24-Hourj

Positive Control 2.00 x 107 5/5 7.10 ± 3.94 x 105 3.55 ± 1.47

Test Coupon 2.00 x 107 5/5 1.06 ± 0.86 x 106 5.32 ± 4.29 -0.05 ± 0.37

Laboratory Blank 0 1/1 3.16 x 103 - -

Procedural Blank 0 1/1 3.16 x 103 - -

48-Hourj

Positive Control 5.01 x 107 5/5 1.07 ± 0.26 x 106 2.14 ± 0.51 -

Test Coupon 5.01 x 107 5/5 9.58 ± 4.16 x 104 0.19 ± 0.08 1.1 ± 0.22

Laboratory Blank 0 1/1 3.16 x 103 - -

Procedural Blank 0 1/1 3.16 x 103 - -

96-Hourj

Positive Control 2.00 x 107 5/5 2.07 ± 1.08 x 106 10.4 ± 5.40 -

Test Coupon 2.00 x 107 5/5 3.16 ± 0.00 x 103 h 0.02 ± 0.00h 2.8 ± 0.0h

Laboratory Blank 0 1/1 3.16 x 103 - -

Procedural Blank 0 1/1 3.16 x 103 - -

a CPE detections: the numerator is the number of coupons with CPE detected and the denominator is the total number of replicate coupons. 

b

 Data are expressed as mean ± standard deviation as applicable.

c

 TCID50 values for laboratory and procedural blanks are attributed to CPE observed from the test material rather than H5N1 virus. d

 Positive controls were inoculated, extracted at time-zero (1-hour drying time).

e

 Test coupons were inoculated, exposed to the environmental condition for the test duration.

f

 Laboratory blanks were not inoculated with any virus, and extracted at time-zero.

g

 Procedural blanks were not inoculated, but exposed to the environmental condition for the test duration.

h The test coupon TCID50 was ≤ the procedural blank TCID50 such that the CPE observed may be attributed to the test material rather than

H5N1 virus. 

i A value of 1 TCID50 was used for each test coupon replicate with no CPE observed, in the calculation of % virus recovery and mean log reduction. j UV-A/B exposures for 24-, 48-, and 96-hour tests were continuous.

ND = Not detected; the detection limit was 1.31 x 103 TCID50.

“-” Not applicable.

Lowering the temperature resulted in longer persistence times 

(1.5 mean log reduction in the TCID50 during the 4-day test). 

In fact, the H5N1 virus was recovered (detected above the 

laboratory and procedural blank level) from chicken feces 

after the 4-day test at the low temperature, low RH, no UV 

environmental condition and after the 9-day test at the low 

temperature, high RH, no UV environmental condition. 

Even with exposure to UV-A/B (at a low temperature and 

low RH), the H5N1 virus was detected above the laboratory 

and procedural blank level after 48 hours of continuous UVA/B exposure test on chicken feces.
16

2.5.2 Galvanized Metal

The test results obtained for persistence of the H5N1 virus 

on galvanized metal are summarized in Table 2-13. The 

H5N1 virus was not detected in any of the tests conducted 

at room temperature. When the tests were conducted at the 

low temperature, low RH, no UV environmental condition, 

the H5N1 virus was detected after the 13-day test with only a 

0.69 mean log reduction in TCID50. At the low temperature, 

high RH, no UV environmental condition, the H5N1 virus 

was only detected after a 4-day test; during the 9-day test, 

the galvanized metal appeared to exhibit oxidation (black 

spots formed where the H5N1 virus was inoculated), but the 

H5N1 virus was not detected. When testing under high RH 

conditions, it was common for the dried inoculum drops to 

reform (rehydrate) into liquid drops on non-porous surfaces 

(e.g., galvanized metal). When exposed to UV-A/B (at a low 

temperature and low RH), the H5N1 virus persisted only for 

the 24-hour test.

Table 2-13. H5N1 Virus Persistence on Galvanized Metal

Test Duration / 

Sample Inoculum (TCID50) CPE Detectionsa

Mean Recovered 

Virus (TCID50)

b

% Virus 

Recoveryb

Mean Log 

Reductionb

Room Temperature, Low RH, No UV

1-Day

Positive Controlc 7.94 x 106 5/5 1.56 ± 0.41 x 106 19.6 ± 5.10 -

Test Coupond 7.94 x 106 0/5 NDg 0.00 ± 0.00g 6.2 ± 0.0g

Laboratory Blanke 0 0/1 ND - -

Procedural Blankf 0 0/1 ND - -

2-Day

Positive Control 7.94 x 106 5/5 2.25 ± 1.28 x 106 28.3 ± 16.1 -

Test Coupon 7.94 x 106 0/5 NDg 0.00 ± 0.00g 6.4 ± 0.0g

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND -

4-Day

Positive Control 5.01 x 106 5/5 1.36 ± 1.03 x 106 27.2 ± 20.5 -

Test Coupon 5.01 x 106 0/5 NDg 0.00 ± 0.00g 6.1 ± 0.0g

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND -

Room Temperature, High RH, No UV

1-Day

Positive Control 5.01 x 107 5/5 6.16 ± 3.69 x 106 12.3 ± 7.36 -

Test Coupon 5.01 x 107 0/5 NDg 0.00 ± 0.00g 6.8 ± 0.0g

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND -

2-Day

Positive Control 3.16 x 107 5/5 1.13 ± 0.63 x 106 3.56 ± 1.98 -

Test Coupon 3.16 x 107 0/5 NDg 0.00 ± 0.00g 6.1 ± 0.0g

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND -

4-Day

Positive Control 2.00 x 107 5/5 2.76 ± 2.05 x 106 13.8 ± 10.3 -

Test Coupon 2.00 x 107 0/5 NDg 0.00 ± 0.00g 6.4 ± 0.0g

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -
17

Table 2-13. H5N1 Virus Persistence on Galvanized Metal (continued)

Test Duration /

Sample Inoculum (TCID50) CPE Detectionsa

Mean Recovered 

Virus (TCID50)b % Virus Recoveryb

Mean Log 

Reductionb

Low Temperature, Low RH, No UV

4-Day

Positive Control 2.00 x 107 5/5 2.92 ± 1.42 x 106 14.6 ± 7.11 -

Test Coupon 2.00 x 107 5/5 3.78 ± 2.80 x 105 1.89 ± 1.40 1.0 ± 0.38

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

8-Day

Positive Control 5.01 x 107 5/5 2.08 ± 0.68 x 106 4.16 ± 1.36 -

Test Coupon 5.01 x 107 5/5 7.33 ± 3.58 x 105 1.46 ± 0.71 0.50 ± 0.23

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

13-Day

Positive Control 2.00 x 107 5/5 1.56 ± 0.41 x 106 7.78 ± 2.03 -

Test Coupon 2.00 x 107 5/5 3.44 ± 1.51 x 105 1.72 ± 0.76 0.69 ± 0.20

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

Low Temperature, High RH, No UV

4-Day

Positive Control 5.01 x 107 5/5 2.08 ± 0.68 x 106 4.16 ± 1.36 -

Test Coupon 5.01 x 107 5/5 4.69 ± 3.06 x 103 0.01 ± 0.01 2.7 ± 0.3

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

9-Day

Positive Control 7.94 x 106 5/5 8.81 ± 6.58 x 105 11.1 ± 8.28 -

Test Coupon 7.94 x 106 0/5 NDg 0.00 ± 0.00g 5.9 ± 0.0g

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

13-Day

Positive Control 2.00 x 107 5/5 1.56 ± 0.41 x 106 7.78 ± 2.03 -

Test Coupon 2.00 x 107 0/5 NDg 0.00 ± 0.00g 6.2 ± 0.0g

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

Low Temperature, Low RH, UV-A/B

24-Hoursh

Positive Control 2.00 x 107 5/5 1.56 ± 0.41 x 106 7.78 ± 2.03 -

Test Coupon 2.00 x 107 5/5 0.64 ± 1.41 x 105 0.32 ± 0.70 2.6 ± 1.1

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

48-Hoursh

Positive Control 5.01 x 107 5/5 1.56 ± 0.41 x 106 3.10 ± 0.81 -

Test Coupon 5.01 x 107 0/5 NDg 0.00 ± 0.00g 6.2 ± 0.0g

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

a CPE detections: the numerator is the number of coupons with CPE detected and the denominator is the total number of replicate coupons. 

b Data are expressed as mean ± standard deviation as applicable. 

c Positive controls were inoculated, extracted at time-zero (1-hour drying time).

d

 Test coupons were inoculated, exposed to the environmental condition for the test duration.

e

 Laboratory blanks were not inoculated with any virus, and extracted at time-zero.

f

 Procedural blanks were not inoculated, but exposed to the environmental condition for the test duration.

g

 A value of 1 TCID50 was used when no CPE are observed, in the calculation of % virus recovery and mean log reduction. h

 UV-A/B exposures for the 24- and 48-hour tests were continuous.

ND = Not detected; the detection limit was 1.31 x 103 TCID50.

“-” Not applicable.
18

2.5.3 Glass

The H5N1 virus persistence results on glass are summarized 

in Table 2-14. The H5N1 virus was not detected on any of 

the coupons tested at room temperature. When the tests were 

conducted at the low temperature, low RH, no UV environmental 

condition, the H5N1 virus was detected after a 13-day test with 

only a 0.59 mean log reduction in TCID50. When the tests were 

conducted at the low temperature, high RH, no UV environmental 

condition, the H5N1 virus was detected on glass after the 9-day 

test with a 2.7 mean log reduction in TCID50. As noted previously 

for galvanized metal, when testing under high RH conditions, it 

was common for the dried inoculums to reform (rehydrate) into 

liquid drops on non-porous surfaces (e.g., glass). When exposed 

to UV-A/B (at a low temperature and low RH), the H5N1 virus was 

detected on only one out of five replicate coupons following the 

24-hour test.

Table 2-14. H5N1 Virus Persistence on Glass

Test Duration / 

Sample Inoculum (TCID50) CPE Detectionsa

Mean Recovered 

Virus (TCID50)

b % Virus Recoveryb

Mean Log 

Reductionb

Room Temperature, Low RH, No UV

1-Day

Positive Controlc 7.94 x 106 5/5 6.18 ± 1.60 x 105 7.79 ± 2.02 -

Test Coupond 7.94 x 106 0/5 NDg 0.00 ± 0.00g 5.8 ± 0.0g

Laboratory Blanke 0 0/1 ND - -

Procedural Blankf 0 0/1 ND - -

2-Day

Positive Control 7.94 x 106 5/5 3.29 ± 1.71 x 106 41.4 ± 21.6 -

Test Coupon 7.94 x 106 0/5 NDg 0.00 ± 0.00g 6.5 ± 0.0g

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

4-Day

Positive Control 5.01 x 106 5/5 1.01 ± 0.63 x 106 20.2 ± 12.7 -

Test Coupon 5.01 x 106 0/5 NDg 0.00 ± 0.00g 6.0 ± 0.0g

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

Room Temperature, High RH, No UV

1-Day

Positive Control 5.01 x 107 5/5 1.07 ± 0.26 x 107 21.4 ± 5.09 -

Test Coupon 5.01 x 107 0/5 NDg 0.00 ± 0.00g 7.0 ± 0.0g

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

2-Day

Positive Control 3.16 x 107 5/5 2.66 ± 2.21 x 106 8.43 ± 7.01 -

Test Coupon 3.16 x 107 0/5 NDg 0.00 ± 0.00g 6.4 ± 0.0g

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

4-Day

Positive Control 2.00 x 107 5/5 2.32 ± 0.83 x 106 11.6 ± 4.14 -

Test Coupon 2.00 x 107 0/5 NDg 0.00 ± 0.00g 6.4 ± 0.0g

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -
19

Table 2-14. H5N1 Virus Persistence on Glass (continued)

Test Duration / 

Sample Inoculum (TCID50) CPE Detectionsa

Mean Recovered 

Virus (TCID50)

b % Virus Recoveryb

Mean Log 

Reductionb

Low Temperature, Low RH, No UV

4-Day

Positive Control

2.00 x 107 5/5 1.07 ± 0.59 x 106 5.35 ± 2.93 -

Test Coupon 2.00 x 107 5/5 4.98 ± 2.97 x 105 2.49 ± 1.49 0.41 ± 0.30

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

8-Day

Positive Control

5.01 x 107 5/5 1.10 ± 0.63 x 106 2.20 ± 1.25 -

Test Coupon 5.01 x 107 5/5 1.34 ± 2.07 x 106 2.68 ± 4.12 0.26 ± 0.58

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

13-Day

Positive Control

2.00 x 107 5/5 1.79 ± 0.83 x 106 8.94 ± 4.16 -

Test Coupon 2.00 x 107 5/5 5.91 ± 4.48 x 105 2.96 ± 2.24 0.59 ± 0.36

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

Low Temperature, High RH, No UV

4-Day

Positive Control

5.01 x 107 5/5 1.34 ± 1.09 x 106 2.67 ± 2.17 -

Test Coupon 5.01 x 107 5/5 1.37 ± 0.61 x 105 0.27 ± 0.12 0.63 ± 0.37

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

9-Day

Positive Control

7.94 x 106 5/5 1.58 ± 1.09 x 106 19.9 ± 13.8 -

Test Coupon 7.94 x 106 5/5 3.16 ± 0.00 x 103 0.04 ± 0.00 2.7 ± 0.0

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

13-Day

Positive Control

2.00 x 107 5/5 1.79 ± 0.83 x 106 8.94 ± 4.16 -

Test Coupon 2.00 x 107 0/5 NDg 0.00 ± 0.00g 6.3 ± 0.0g

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

Low Temperature, Low RH, UV-A/B

24-Hourh

Positive Control

2.00 x 107 5/5 1.79 ± 0.83 x 106 1.83 ± 0.66 -

Test Coupon 2.00 x 107 1/5 2.62 ± 5.85 x 102 g 0.00 ± 0.00g 5.6 ± 1.4g

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

48-Hourh

Positive Control

5.01 x 107 5/5 1.31 ± 0.43 x 106 2.62 ± 0.86 -

Test Coupon 5.01 x 107 0/5 NDg 0.00 ± 0.00g 6.1 ± 0.0g

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

a CPE detections: the numerator is the number of coupons with CPE detected and the denominator is the total number of replicate coupons. 

b

 Data are expressed as mean ± standard deviation as applicable. 

c

 Positive controls were inoculated, extracted at time-zero (1-hour drying time).

d

 Test coupons were inoculated, exposed to the environmental condition for the test duration.

e

 Laboratory blanks were not inoculated with any virus, and extracted at time-zero.

f

 Procedural blanks were not inoculated, but exposed to the environmental condition for the test duration.

g

 A value of 1 TCID50 was used when no CPE are observed, in the calculation of % virus recovery and mean log reduction. h

 UV-A/B exposures for the 24- and 48-hour tests were continuous.

ND = Not detected; the detection limit was 1.31 x 103 TCID50.

“-” Not applicable.
20

2.5.4 Soil

The H5N1 virus persistence results in soil are summarized 

in Table 2-15. When the tests were conducted at room 

temperature, the H5N1 virus was detected after the 

1-day test, but not at the longer durations. When the tests 

were conducted at the low temperature, low RH, no UV 

environmental condition, the H5N1 virus was detected 

from soil after a 13-day test with a 3.1 mean log reduction 

in TCID50. When the tests were conducted at the low 

temperature, high RH, no UV environmental condition, the 

H5N1 virus was detected from soil after the 9-day test with 

no mean log reduction in TCID50. When exposed to UV-A/B 

(at a low temperature and low RH), the H5N1 virus persisted 

during the 96-hour test with a 3.3 mean log reduction in 

TCID50.

Table 2-15. H5N1 Virus Persistence on Soil

Test Duration / 

Sample Inoculum (TCID50) CPE Detectionsa

Mean Recovered 

Virus (TCID50)

b, c % Virus Recoveryb

Mean Log 

Reductionb

Room Temperature, Low RH, No UV

1-Day

Positive Controld 7.94 x 106 5/5 7.33 ± 3.58 x 105 9.23 ± 4.51 -

Test Coupone 7.94 x 106 2/5 0.88 ± 1.38 x 104 h 0.11 ± 0.17h 4.2 ± 2.4h

Laboratory Blankf 0 0/1 ND - -

Procedural Blankg 0 0/1 ND - -

2-Day

Positive Control 7.94 x 106 5/5 6.40 ± 2.21 x 105 8.06 ± 2.78 -

Test Coupon 7.94 x 106 0/5 NDh 0.00 ± 0.00h 5.8 ± 0.0h

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

4-Day

Positive Control 5.01 x 106 5/5 2.55 ± 0.88 x 105 5.09 ± 1.75 -

Test Coupon 5.01 x 106 0/5 NDh 0.00 ± 0.00h 5.4 ± 0.0h

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

Room Temperature, High RH, No UV

1-Day

Positive Control 5.01 x 107 5/5 1.48 ± 1.12 x 106 2.96 ± 2.24 -

Test Coupon 5.01 x 107 5/5 8.26 ± 4.31 x 104 0.16 ± 0.09 1.3 ± 0.26

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

2-Day

Positive Control 3.16 x 107 5/5 3.64 ± 2.80 x 105 1.15 ± 0.89 -

Test Coupon 3.16 x 107 0/5 NDf 0.00 ± 0.00f 5.6 ± 0.0f

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

4-Day

Positive Control 2.00 x 107 5/5 1.79 ± 0.83 x 105 0.89 ± 0.42 -

Test Coupon 2.00 x 107 0/5 NDf 0.00 ± 0.00f 5.3 ± 0.0f

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

Low Temperature, Low RH, No UV

4-Day

Positive Control 2.00 x 107 5/5 2.82 ± 1.57 x 105 1.41 ± 0.78 -

Test Coupon 2.00 x 107 5/5 2.47 ± 3.29 x 104 0.12 ± 0.16 1.5 ± 0.73

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -
21

Table 2-15. H5N1 Virus Persistence on Soil (continued)

Test Duration / 

Sample Inoculum (TCID50) CPE Detectionsa

Mean Recovered 

Virus (TCID50)

b, c % Virus Recoveryb

Mean Log 

Reductionb

Low Temperature, Low RH, No UV

8-Day

Positive Control 5.01 x 107 5/5 5.23 ± 1.72 x 105 1.04 ± 0.34 -

Test Coupon 5.01 x 107 5/5 1.16 ± 0.14 x 103 0.00 ± 0.00 2.7 ± 0.1

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

13-Day

Positive Control 2.00 x 107 5/5 3.67 ± 1.31 x 105 1.83 ± 0.66 -

Test Coupon 2.00 x 107 4/5 1.05 ± 0.59 x 103 h 0.01 ± 0.00h 3.1 ± 1.4h

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

Low Temperature, High RH, No UV

4-Day

Positive Control 5.01 x 107 5/5 5.23 ± 1.72 x 105 1.04 ± 0.34 -

Test Coupon 5.01 x 107 5/5 2.21 ± 1.65 x 105 0.44 ± 0.33 0.46 ± 0.30

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

9-Day

Positive Control 7.94 x 106 5/5 2.82 ± 3.02 x 105 3.56 ± 3.81

-

Test Coupon 7.94 x 106 5/5 5.93 ± 3.95 x 105 7.46 ± 4.98 -0.25 ± 0.28

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 1/1 2.00 x 104 - -

13-Day

Positive Control 2.00 x 107 5/5 3.67 ± 1.31 x 105 1.83 ± 0.66 -

Test Coupon 2.00 x 107 0/5 NDh 0.00 ± 0.00h 5.6 ± 0.0h

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

Low Temperature, Low RH, UV-A/B

24-Hoursi

Positive Control 2.00 x 107 5/5 3.67 ± 1.31 x 105 3.55 ± 1.97 -

Test Coupon 2.00 x 107 5/5 5.93 ± 3.95 x 105 2.96 ± 1.98 -0.14 ± 0.28

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

48-Hoursi

Positive Control 5.01 x 107 5/5 5.23 ± 1.72 x 105 1.04 ± 0.34 -

Test Coupon 5.01 x 107 5/5 3.90 ± 1.01 x 104 0.08 ± 0.02 1.1 ± 0.1

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

96-Hoursi

Positive Control 2.00 x 107 5/5 1.07 ± 0.59 x 106 5.35 ± 2.93 -

Test Coupon 2.00 x 107 4/5 3.11 ± 3.28 x 103 h 0.02 ± 0.02h 3.3 ± 1.6h

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

a

 CPE detections: the numerator is the number of coupons with CPE detected and the denominator is the total number of replicate coupons. 

b

 Data are expressed as mean ± standard deviation as applicable.

c

 TCID50 values for laboratory and procedural blanks are attributed to CPE observed from the test material rather than H5N1 virus. d

 Positive controls were inoculated, extracted at time-zero (1-hour drying time).

e

 Test coupons were inoculated, exposed to the environmental condition for the test duration.

f

 Laboratory blanks were not inoculated with any virus, and extracted at time-zero.

g

 Procedural blanks were not inoculated, but exposed to the environmental condition for the test duration.

h

 A value of 1 TCID50 was used when no CPE are observed, in the calculation of % virus recovery and mean log reduction. i

 UV-A/B exposures for the 24-, 48-, and 96-hour test were continuous.

ND = Not detected; the detection limit was 1.31 x 103 TCID50.

“-” Not applicable.
22

2.5.5 Comparison of Chicken Feces, Galvanized Metal, 

Glass, and Soil Results

The H5N1 virus persistence results are summarized in 

Table 2-16. These data denote the longest duration (days) that 

the virus was detected and the shortest duration that the virus 

was not detected to bracket the length of time that the H5N1 

virus remained viable for each material and environmental 

condition. The H5N1 virus persisted for relatively short 

durations on all materials at room temperature, low RH, no 

UV and at room temperature, high RH, no UV. The H5N1 

virus was not detected after the 1-day test on galvanized 

metal and glass and was only detected after the 1-day test on 

soil. For chicken feces, at the room temperature, high RH, 

no UV environmental condition, the TCID50 level measured 

after the 2-day test was less than the TCID50 associated with 

the procedural blank, possibly indicating an effect of the test 

material, rather than the H5N1 virus. Similarly, for the room 

temperature, low RH, no UV environmental condition, the 

TCID50 level measured from chicken feces after the 1-day 

test was less than the associated procedural blank TCID50, 

indicating a possible test material interference rather than the 

H5N1 virus.

The H5N1 virus persisted longer on all materials at both 

low temperature conditions (i.e., low temperature, low RH, 

no UV and low temperature, high RH, no UV) compared to 

the room temperature conditions. For the low temperature, 

low RH, no UV environmental condition, the H5N1 virus 

persisted on galvanized metal, glass, and soil for 13 days (the 

longest duration tested). At the low temperature, high RH, no 

UV environmental condition, the H5N1 virus persisted for 

shorter durations on galvanized metal (four days), glass (nine 

days), and soil (nine days). Interestingly, the H5N1 virus 

persisted on chicken feces longer at the low temperature, high 

RH, no UV environmental condition (13 days) than at the 

low temperature, low RH, no UV environmental condition 

(possibly eight days where the TCID50 level detected was 

less than the TCID50 associated with the procedural blank, 

possibly indicating an effect of the test material).

Table 2-16. Summary of H5N1 Virus Persistence

Material and Environmental Condition

Longest Duration (Days)

H5N1 Virus Detected

Shortest Duration (Days)

H5N1 Virus Not Detected

Chicken feces

Room temperature, low RH, no UV 1a 2

Room temperature, high RH, no UV 2a 4

Low temperature, low RH, no UV 8a 13

Low temperature, high RH, no UV 13 NA

Low temperature, low RH, UV-A/B 4a NA

Galvanized metal

Room temperature, low RH, no UV NA 1

Room temperature, high RH, no UV NA 1

Low temperature, low RH, no UV 13 NA

Low temperature, high RH, no UV 4 9

Low temperature, low RH, UV-A/B 1 2

Glass

Room temperature, low RH, no UV NA 1

Room temperature, high RH, no UV NA 1

Low temperature, low RH, no UV 13 NA

Low temperature, high RH, no UV 9 13

Low temperature, low RH, UV-A/B 1 2

Soil

Room temperature, low RH, no UV 1 2

Room temperature, high RH, no UV 1 2

Low temperature, low RH, no UV 13 NA

Low temperature, high RH, no UV 9 13

Low temperature, low RH, UV-A/B 4 NA

a The TCID50 measured was at a level ≤ the procedural blank TCID50 for chicken feces such that the CPEs observed (and used in the calculation 

of the TCID50) may be attributed to the test material rather than H5N1 virus.

NA = Not available; the H5N1 virus was either detected at all durations or not detected from any duration. 
23

For the low temperature, low RH, UV-A/B environmental 

condition, the H5N1 virus persisted longer on chicken feces 

and soil than galvanized metal and glass. The H5N1 virus 

was detected after only one day (24 hours of continuous 

exposure to UV-A/B) from galvanized metal and glass. 

The H5N1 virus was detected after two to four days of 

continuous exposure to UV-A/B on chicken feces and soil; 

on chicken feces the H5N1 virus was detected after the 2-day 

test at a level above the associated procedural blank and 

potentially detected after the 4-day test but at a level below 

the associated procedural blank, possibly indicating an effect 

of the test material rather than the H5N1 virus. In comparing 

these results to the low temperature, low RH environmental 

condition (no simulated sunlight), the UV-A/B clearly 

diminishes the H5N1 persistence.
24

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25

3.0

Decontamination Technology Evaluation

3.1 Technology Descriptions

The liquid decontamination technologies evaluated consisted 

of:

• 1% Citric Acid

◦ 1% citric acid was prepared by adding 1 gram (g) 

citric acid, anhydrous, (≥ 99.5% purity) to 99 mL 

hard water until completely dissolved. 

• pH-Amended Bleach

◦ pH-amended bleach was prepared by adding 5% 

acetic acid to household bleach (Clorox®; 5–6% 

sodium hypochlorite) to obtain a pH-amended bleach 

solution. The solution was prepared using 9.4 parts 

hard water, 1 part bleach, and 1 part 5% glacial acetic 

acid to obtain a solution having a mean pH of 6.81 

± 0.15 and a mean total chlorine content of 6,215 ± 

212 ppm. 

• 732 ppm Quaternary Ammonium

◦ Hospital grade quaternary ammonium disinfectant 

[n-alkyl dimethyl benzyl ammonium chloride 

(6.25%), n-alkyl dimethyl ethylbenzyl ammonium 

chloride (6.25%), inert ingredients (87.5%)] was 

purchased from a local vendor and prepared per 

the vendor’s guidance (3/4 ounces of disinfectant 

added to 1 gallon of hard water) to obtain a solution 

containing 732 ppm of the quaternary ammonium 

active ingredient. This concentration was not 

independently verified in this evaluation.

• 8% Na2CO3

◦ 8% Na2CO3 was prepared by adding 8 g Na2CO3

(SigmaUltra (Sigma-Aldrich Inc.,St. Louis, Mo.); 

≥99.0% purity) to 92 mL hard water until completely 

dissolved.

All preparations and dilutions were made using AOAC 

International hard water prepared at 400 ppm hardness as 

calcium carbonate (CaCO3) (AOAC Official Method 960.09, 

Germicidal and Detergent Sanitizing Action of Disinfectants, 

Section E. Synthetic Hard-Water, p. 11). All decontamination 

contact times were 10 minutes.

3.2 Cytotoxicity of Neutralized Decontamination 

Liquids

During the decontamination technology evaluation, 

neutralization of each decontamination liquid was required 

in order to terminate activity at the end of the 10-minute 

decontamination contact time. For neutralization to be 

acceptable, the chemicals used could not interfere with the 

methods used for extraction and quantification of the H5N1 

virus. The potential for neutralized decontamination liquids 

to cause cytotoxicity to the MDCK cells, which could 

subsequently interfere with the TCID50 determination, was 

assessed with the MTT assay using the approach described 

in Section 3.2. Neutralization of each decontamination 

liquid was attempted by dilution with hard water (400 ppm 

as CaCO3) alone and by the addition of Dey and Engley 

(D/E) neutralizing broth (solutions ranging from 50-95% 

D/E broth) followed by dilution with hard water. Fifty 

percent D/E broth was used for pH-amended bleach (3.0 mL 

decontamination liquid + 3.0 mL neutralizer), 75% D/E 

broth was used for 1% citric acid (1.5 mL decontamination 

liquid + 4.5 mL neutralizer), and 75% D/E broth was used 

for 732 ppm quaternary ammonium (1.5 mL decontamination 

liquid + 4.5 mL neutralizer). Sodium carbonate only required 

a 1:2 dilution of the decontamination liquid itself, with no 

need for the neutralizer (3.0 mL decontamination liquid 

+ 3.0 mL hard water). The neutralization method for each 

decontamination liquid was generally selected based on the 

lowest amount of D/E broth that needed to be added (if any) 

in order to maintain ≥90% of the mean unexposed control 

MDCK cell viability after diluting the neutralization solution 

(with hard water) at a ratio of 1:2 (i.e., equal amounts of 

neutralization solution and hard water were used). The 

cytotoxicity test results are provided in Table 3-1 for the 

decontamination liquids and the selected neutralization 

approaches.

Table 3-1. Neutralized Decontamination Liquid Cytotoxicity Test Results

Decontamination 

Liquid Neutralization Solution

Dilution with 

Hard Water

Mean Control Cell 

Optical Density

Mean Neutralized 

Decontamination 

Liquid Optical Density

% Cell 

Viability

1% Citric Acid 75% D/E brotha 1:8 0.3336 0.3240 97.1e

pH-Amended Bleach 50% D/E brothb 1:2 1.0299 1.0795 104.8e

732 ppm Quaternary 

Ammonium

75% D/E brothc 1:2 1.0610 1.0216 96.3

8% Na2CO3 No neutralizer (D/E broth)d 1:2 0.8761 0.8354 95.4

a

 1.5 mL citric acid + 4.5 mL D/E broth.

b

 3.0 mL pH-amended bleach + 3.0 mL D/E broth.

c

 1.5 mL quaternary ammonium + 4.5 mL D/E broth.

d

 3.0 mL Na2CO3 + 3.0 mL hard water. e

 Viability above 100% may reflect non-specific reduction of MTT to formazan from unknown coupon extract components.
26

3.3 H5N1 Virus Recovery From Extraction Buffer, 

Neutralized Decontamination Liquid, and Trough 

Decontamination Liquid

The decontamination technology evaluation utilized test 

coupons that were each spiked and dried as described 

in Section 2.3 for persistence testing. For the evaluation 

conducted at the lower temperature, the coupons were first 

dried for one hour at room temperature, then placed in the 

refrigerator for one hour before adding the decontamination 

liquids (also placed in the refrigerator one hour prior to 

testing). In the decontamination technology evaluations, 

spiked test coupons and procedural blank coupons were 

inverted (spiked surface down) and placed into separate 

troughs for galvanized metal and separate vials for soil 

each holding enough decontamination liquid to cover the 

spiked surface of the coupon. At the end of the 10-minute 

decontamination contact time, the coupons were removed, 

neutralized in separate vials, extracted to recover H5N1 virus, 

and extracts assayed as described in Section 2.3.

For neutralization to be acceptable, it should not interfere 

with the methods used for extraction and quantification of the 

virus, and the neutralization must be effective at inhibiting 

the virucidal activity of the decontamination liquid. Three 

method demonstration tests were conducted to determine the 

applicability of each neutralization approach: 

■ H5N1 virus recovery from coupons using extraction 

buffer

■ H5N1 virus recovery from neutralized 

decontamination liquid

■ H5N1 virus recovery from trough decontamination 

liquid.

3.3.1 H5N1 Virus Recovery From Coupons Using 

Extraction Buffer

The intent of these tests was to demonstrate the ability 

to recover the H5N1 virus from coupons undergoing the 

test procedures of decontamination and neutralization by 

using only extraction buffer (PBS), essentially serving 

as a recovery control. Spiked coupons (galvanized metal 

and soil) were added to troughs/dishes containing PBS. 

After a 10-minute contact time, the test coupons were 

then removed and extracted and quantified as described in 

Section 2.3.2. For galvanized metal, the trough solution was 

also quantified to capture any virus washing into the trough 

liquid; soil test coupons became a mixture with the PBS 

in the vial and separate quantification of the trough liquid 

was not applicable. These tests were conducted three times 

(Trials A, B and C; each with three replicates) and the mean 

H5N1 virus recoveries are presented in Table 3-2. Although 

the recoveries were generally less than the performance 

criterion of ≥25% of the spiked level, as discussed in 

the test/QA plan1, adequate quantities of the H5N1 virus 

remained and therefore decontamination efficacy (reported 

as log reduction) could be accurately assessed. Note: EPA’s 

virucidal test guidance does not require a minimum percent 

recovery, but rather a minimum virus recovery (TCID50) of 

≥104, 12.

Table 3-2. H5N1 Virus Recovery From Coupons Using Extraction Buffer

Trial / Material (sample) Inoculum (TCID50)

Mean Recovered Virus 

(TCID50) Mean % Virus Recoverya

Trail A

Galvanized Metal (total; coupon + trough) 5.01 x 106 8.17 x 105 13

Galvanized Metal (from coupon) - 4.39 x 105 8.77

Galvanized Metal (from trough) - 3.78 x 105 4.52

Soil 5.01 x 106 2.99 x 105 3.58

Trial B

Galvanized Metal (total; coupon + trough) 2.00 x 106 5.40 x 105 27

Galvanized Metal (from coupon) - 5.37 x 105 26.9

Galvanized Metal (from trough) - 3.16 x 103 0.09

Soil 2.00 x 106 3.16 x 105 9.48

Trial C

Galvanized Metal (total; coupon + trough) 5.01 x 107 1.52 x 107 30

Galvanized Metal (from coupon) - 9.49 x 106 19.0

Galvanized Metal (from trough) - 5.70 x 106 11.4

Soil 5.01 x 107 1.75 x 106 3.50

a The mean % virus recovery is determined by calculating the % virus recovery for each coupon (recovered virus/inoculum) and then computing the

mean of these percentages.
27

3.3.2 H5N1 Virus Recovery From Neutralized 

Decontamination Liquid

The intent of this test was to determine the ability of the 

neutralization approach (as summarized in table 3-1) to 

inhibit the virucidal activity of the decontamination liquid. 

Briefly, spiked coupons (three for each material) were added 

to the troughs (for galvanized metal) or vials (for soil) of 

neutralized decontamination liquid (i.e., the neutralization 

solutions of Table 3-1; subsequent dilution with hard water at 

1:2 was only required for the MTT assay) for the 10-minute 

contact time. The coupons were then removed and extracted 

and the H5N1 virus quantified as described in Section 2.3.2. 

For galvanized metal the H5N1 virus in the trough solution 

was also quantified to account for any virus washing into 

the trough liquid; soil test coupons became a mixture in 

the vials with the addition of neutralized decontamination 

liquid; since no troughs were used, a separate quantitation 

of the trough liquid was not applicable. The mean TCID50 of 

H5N1 virus recovered from the neutralized decontamination 

liquids are presented in Table 3-3. Although the recoveries 

were generally less than the performance criterion of ≥25% 

of the spiked level (per the test/QA plan), an appreciable 

amount of the H5N1 virus remained (6.92 x 104 to 5.96 x 

106 mean TCID50) and therefore decontamination efficacy 

could be accurately assessed. Note: EPA’s virucidal test 

guidance does not require a minimum percent recovery, but 

rather a minimum virus recovery (TCID50) of ≥104

 (so that an 

adequate log reduction could be demonstrated).

3.3.3 H5N1 Virus Recovery From Trough 

Decontamination Liquid

The intent of these tests was to determine if the H5N1 virus 

remained in the decontamination liquid after the coupons 

(galvanized metal only) were removed from the troughs. 

Spiked galvanized metal coupons were placed in troughs of 

decontamination liquid for the 10-minute contact time and 

then removed. Given the possibility of the H5N1 virus being 

dislodged from the coupon during decontamination, the 

decontamination liquid in the trough was neutralized, and the 

H5N1 virus in the trough liquid was quantified as described 

in Section 2.3.2. The H5N1 virus recovered from the trough 

decontamination liquid is presented in Table 3-4. In view 

of the finding that the H5N1 virus was sometimes detected 

in the trough decontamination solution, the quantification 

of this residual liquid was incorporated into the actual 

decontamination technology evaluation approach to ensure 

the recovery of all of the H5N1 virus.

Table 3-3. H5N1 Virus Recovery From Neutralized Decontamination Liquid

Neutralized Decontamination Liquid / 

Material (sample) Inoculum (TCID50)

Mean Recovered Virus 

(TCID50)

Mean % Virus 

Recoverya

1% Citric Acid + 75% D/E brothb

Galvanized Metal (total; coupon + trough) 5.01 x 107 5.96 x 106 12

Galvanized Metal (from coupon) - 5.96 x 106 11.9

Galvanized Metal (from trough) - 0 0

Soil 5.01 x 107 2.60 x 105 0.52

pH-Amended Bleach + 50% D/E brothb

Galvanized Metal (total; coupon + trough) 2.00 x 106 1.54 x 105 7.6

Galvanized Metal (from coupon) - 1.51 x 105 7.53

Galvanized Metal (from trough) - 3.16 x 103 0.09

Soil 2.00 x 106 6.96 x 105 20.9

8% Na2

CO3 + No D/E brothb

Galvanized Metal (total; coupon + trough) 5.01 x 106 6.96 x 104 1.4

Galvanized Metal (from coupon) - 6.96 x 104 1.39

Galvanized Metal (from trough) - 0 0

Soil 5.01 x 106 6.92 x 104 0.83

732 ppm Quaternary Ammonium + 75% D/E brothb

Galvanized Metal (total; coupon + trough) 5.01 x 106 6.66 x 105 13

Galvanized Metal (from coupon) - 5.96 x 105 11.9

Galvanized Metal (from trough) - 6.96 x 104 0.83

Soil 5.01 x 106 1.60 x 106 19.1

a The mean % virus recovery is determined by calculating the % virus recovery for each coupon (recovered virus/inoculum) and then computing the

mean of these percentages.

b Unlike the cytoxicity test results of neutralized decontamination liquid shown in Table 3-1, subsequent dilution with hard water was not required 

for this assay.
28

Table 3-4. H5N1 Virus Recovery From Galvanized Metal Trough Decontamination Liquid

Decontamination Liquid / 

Material (sample) Inoculum (TCID50)

Mean Recovered Virus 

(TCID50) Mean % Virus Recoverya

1% Citric Acid 5.01 x 107 0 0

pH-Amended Bleach 2.00 x 106 3.16 x 103 0.09

8% Na2CO3

b Not tested Not tested Not tested

732 ppm Quaternary Ammonium 5.01 x 106 0 0

a The mean % virus recovery is determined by calculating the % virus recovery for each coupon (recovered virus/inoculum) and then computing the

mean of these percentages.

b

 8% Na2CO3 (which did not require D/E broth) for trough neutralization, was not tested.

3.4 Evaluation of Liquid Decontamination Technologies

3.4.1 Test Matrix

Log reductions in the H5N1 virus persistence were measured 

on two materials: galvanized metal and soil. Based on 

the relatively longer persistence times for the H5N1 virus 

under cold temperatures and low RH (especially in the 

absence of UV-A/B), the efficacy of decontamination liquids 

was assessed at both room temperature (22 °C) and low 

temperature (5 °C) for the 10 minute contact times. The 

experimental treatments performed are shown in Table 3-5.

3.4.2 Test Results 

The H5N1 virus recovery and log reduction in TCID50

for each decontamination liquid/environmental condition 

combination are summarized in Tables 3-6 for galvanized 

metal and Table 3-7 for soil. Table 3-6 and 3-7 also include 

the number of replicates for positive controls (5), test 

coupons (5), laboratory blank (1), and procedural blank 

(1). A summary of the log reductions obtained in all 

decontamination liquid tests is provided in Table 3-8. For the 

decontamination evaluations, the mean room temperature 

was 23.31 °C with a range of 23.11 °C to 23.33 °C and the 

mean low temperature was 5.34 °C with a range of 4.20 °C

to 6.10 °C.

Following a 10-minute contact time, 8% Na2CO3 and 

732 ppm quaternary ammonium, reduced the H5N1 virus 

on galvanized metal by less than 1.0 log in TCID50 at room 

temperature and low temperature test conditions (Table 3-6). 

When 1% citric acid was applied, mean log reductions in 

H5N1 virus were 2.04 at room temperature and 2.13 at low 

temperature. Only pH-amended bleach with a 10-minute 

contact time, completely inactivated the H5N1 virus from 

galvanized metal (no detectable H5N1 virus), with mean 

log reductions of 7.1 at room temperature and 6.9 at the low 

temperature.

Table 3-5. Decontamination Technology Evaluation Matrix

Decontamination Liquid Material Environmental Condition

1% Citric Acid Galvanized metal Room temperature

Galvanized metal Low temperature

Soil Room temperature

Soil Low temperature

pH-Amended Bleach Galvanized metal Room temperature

Galvanized metal Low temperature

Soil Room temperature

Soil Low temperature

8% Na2CO3 Galvanized metal Room temperature

Galvanized metal Low temperature

Soil Room temperature

Soil Low temperature

732 ppm Quaternary Ammonium Galvanized metal Room temperature

Galvanized metal Low temperature

Soil Room temperature

Soil Low temperature
29

Table 3-6. Decontamination Efficacy Against H5N1 Virus on Galvanized Metal

Decontamination Liquid / 

Sample

Inoculum 

(TCID50) CPE Detectionsa

Mean Recovered 

Virus (TCID50)

b

% Virus 

Recoveryb

Mean Log 

Reductionb

Room Temperature

1% Citric Acid

Positive Controlc 2.00 x 107 5/5 9.86 ± 0.98 x 106 49.3 ± 4.87 -

Test Coupond 2.00 x 107 5/5 1.00 ± 0.58 x 105 0.50 ± 0.29 2.0 ± 0.2

Laboratory Blanke 0 0/1 ND - -

Procedural Blankf 0 0/1 ND - -

pH-Amended Bleach

Positive Control 7.94 x 107 5/5 2.76 ± 1.18 x 107 34.8 ± 14.9 -

Test Coupon 7.94 x 107 0/5 ND 0.00 ± 0.00g 7.1 ± 0.0g

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

8% Na2CO3

Positive Control 7.94 x 107 5/5 2.76 ± 1.18 x 107 34.8 ± 14.9 -

Test Coupon 7.94 x 107 5/5 3.71 ± 0.96 x 106 4.67 ± 1.21h, i 0.88 ± 0.11h, i

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

732 ppm Quaternary Ammonium

Positive Control 7.94 x 107 5/5 2.76 ± 1.18 x 107 34.8 ± 14.9 -

Test Coupon 7.94 x 107 5/5 5.64 ± 1.58 x 106 7.10 ± 1.99 0.70 ± 0.12

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

Low Temperature

1% Citric Acid

Positive Control 1.28 x 108 5/5 5.83 ± 2.95 x 107 45.5 ± 23.1 -

Test Coupon 1.28 x 108 5/5 5.49 ± 4.27 x 105 0.43 ± 0.33 2.1 ± 0.4

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

pH-Amended Bleach

Positive Control 5.01 x 107 5/5 1.43 ± 0.53 x 107 28.5 ± 10.5 -

Test Coupon 5.01 x 107 0/5 ND 0.00 ± 0.00g 6.9 ± 0.0g

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

8% Na2CO3

Positive Control 5.01 x 107 5/5 1.43 ± 0.53 x 107 28.5 ± 10.5 -

Test Coupon 5.01 x 107 5/5 2.19 ± 1.86 x 106 4.37 ± 3.71 0.93 ± 0.35

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

732 ppm Quaternary Ammonium

Positive Control 5.01 x 107 5/5 1.43 ± 0.53 x 107 28.5 ± 10.5 -

Test Coupon 5.01 x 107 5/5 6.05 ± 2.70 x 106 12.1 ± 5.39 0.43 ± 0.29

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

a

 CPE detections: the numerator is the number of coupons with CPE detected and the denominator is the total number of replicates.

b Data are expressed as mean ± standard deviation as applicable. 

c Positive controls were inoculated, placed in PBS and exposed to the environmental condition, extracted after the 10-minute contact time. Only 

one set of positive controls was used for each environmental condition and all four decontamination liquids.

d

 Test coupons were inoculated, exposed to the environmental condition and decontamination liquid for the 10-minute contact time.

e

 Laboratory blanks were not inoculated, extracted at time-zero.

f

 Procedural blanks were not inoculated, placed in PBS, exposed to the environmental condition, extracted after the 10-minute contact time.

g A value of 1 TCID50 was used for non-detects (from the coupon and the trough liquid, which are summed to determine the total amount of virus) 

in the calculation of % recovery and mean log reduction.

h Although H5N1 virus was recovered from all five replicate galvanized metal coupons, H5N1 virus was not detected from the associated trough 

liquid (quantification of virus from the coupon and trough liquid is summed to determine the total amount of virus).

i A value of 1 TCID50 was used for non-detects in the trough liquid for calculation of % virus recovery and mean log reduction.

ND = Not detected; the detection limit was 1.31 x 103 TCID50.

“-” Not applicable.
30

Table 3-7. Decontamination Efficacy Against H5N1 Virus on Soil

Decontamination Liquid / 

Sample

Inoculum 

(TCID50)

CPE 

Detectionsa

Mean Recovered 

Virus (TCID50)

b

% Virus 

Recoveryb

Mean Log 

Reductionb

Room Temperature

1% Citric Acid

Positive Controlc 2.00 x 107 5/5 3.12 ± 2.80 x 106 15.62 ± 14.0 -

Test Coupond 2.00 x 107 5/5 1.25 ± 0.78 x 105 0.63 ± 0.39 1.5 ± 0.4

Laboratory Blanke 0 0/1 ND - -

Procedural Blankf 0 0/1 ND - -

pH-Amended Bleach

Positive Control 7.94 x 107 5/5 4.27 ± 1.01 x 107 53.8 ± 12.8 -

Test Coupon 7.94 x 107 5/5 9.97 ± 3.05 x 104 0.13 ± 0.04 2.7 ± 0.2

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

8% Na2CO3

Positive Control 7.94 x 107 5/5 4.27 ± 1.01 x 107 53.8 ± 12.8 -

Test Coupon 7.94 x 107 5/5 6.86 ± 4.97 x 106 8.64 ± 6.26 0.95 ± 0.49

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

732 ppm Quaternary Ammonium

Positive Control 7.94 x 107 5/5 4.27 ± 1.01 x 107 53.8 ± 12.8 -

Test Coupon 7.94 x 107 5/5 1.63 ± 0.43 x 107 20.6 ± 5.36 0.43 ± 0.11

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

Low Temperature

1% Citric Acid

Positive Control 1.28 x 108 5/5 2.83 ± 1.32 x 107 22.14 ± 10.28 -

Test Coupon 1.28 x 108 5/5 7.10 ± 3.94 x 105 0.55 ± 0.31 1.7 ± 0.3

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

pH-Amended Bleach

Positive Control 5.01 x 107 5/5 2.16 ± 1.63 x 107 43.1 ± 32.5 -

Test Coupon 5.01 x 107 5/5 3.29 ± 1.68 x 104 0.07 ± 0.03 2.9 ± 0.3

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

8% Na2CO3

Positive Control 5.01 x 107 5/5 2.16 ± 1.63 x 107 43.1 ± 32.5 -

Test Coupon 5.01 x 107 5/5 1.15 ± 1.09 x 107 22.9 ± 21.8 0.42 ± 0.38

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

732 ppm Quaternary Ammonium

Positive Control 5.01 x 107 5/5 2.16 ± 1.63 x 107 43.1 ± 32.5 -

Test Coupon 5.01 x 107 5/5 8.09 ± 3.26 x 106 16.1 ± 6.51 0.45 ± 0.17

Laboratory Blank 0 0/1 ND - -

Procedural Blank 0 0/1 ND - -

a

 CPE detections: the numerator is the number of coupons with CPE detected and the denominator is the total number of replicate coupons. 

b

 Data are expressed as mean ± standard deviation as applicable. 

c Positive controls were inoculated, placed in PBS and exposed to the environmental condition, extracted after the 10-minute contact time.

d

 Test coupons were inoculated, exposed to the environmental condition and decontamination liquid for the 10-minute contact time.

e

 Laboratory blanks were not inoculated, extracted at time-zero.

f

 Procedural blanks were not inoculated, placed in PBS and exposed to the environmental condition, extracted after 10-minute contact time.

ND = Not detected; the detection limit was 1.31 x 103 TCID50.

“-” Not applicable.
31

None of the evaluated decontamination technologies 

completely inactivated the H5N1 virus on soil (Table 3-7). 

For 8% Na2CO3 and 732 ppm quaternary ammonium (at 

room and the cold temperature), mean H5N1 log reductions 

on soil were less than 1.0. When 1% citric acid was applied, 

mean log reductions in H5N1 virus were 1.5 at room 

temperature and 1.7 at low temperature. When pH-amended 

bleach was used the mean H5N1 virus log reductions on soil 

were 2.7 at room temperature and 2.9 at the low temperature.

Table 3-8. Summary of Decontamination Efficacy Against H5N1 Virus

Material and 

Environmental 

Condition

H5N1 Virus Mean Log Reduction in TCID50a

1% Citric Acid pH-Amended Bleach 8% Na2CO3 732 ppm Quaternary Ammonium

Galvanized Metal

Room Temperature 2.0 ± 0.2 7.1 ± 0.0b 0.88 ± 0.11c 0.70 ± 0.12

Low Temperature 2.1 ± 0.4 6.9 ± 0.0b 0.93 ± 0.35 0.43 ± 0.29

Soil

Room Temperature 1.5 ± 0.4 2.7 ± 0.2 0.95 ± 0.49 0.43 ± 0.11

Low Temperature 1.7 ± 0.3 2.9 ± 0.3 0.42 ± 0.38 0.45 ± 0.17

a Data are expressed as mean ± standard deviation.

b H5N1 virus was not detected from any of the replicate test coupons or associated trough liquid; a value of 1 TCID50 was used for these nondetects in the calculation of the H5N1 virus mean log reduction in TCID50. c H5N1 virus was not detected from the associated trough liquid; a value of 1 TCID50 is used for non-detects in the calculation of H5N1 virus 

mean log reduction in TCID50.
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33

4.0

Quality Assurance/Quality Control

Quality assurance/quality control (QC) procedures were 

performed in accordance with the test/QA plan1 and the 

TTEP QMP2 for the persistence testing and decontamination 

technology evaluation. QA/QC procedures are summarized 

below. Some experimental procedures could not be 

conducted in accordance with the test/QA plan; those 

deviations are summarized in Section 4.5.

4.1 Equipment Calibration

All equipment (e.g., pipettes, incubators, biological safety 

cabinets) used at the time of use was verified as being 

certified, calibrated, or validated.

4.2 Audits

4.2.1 Performance Evaluation Audit

No performance evaluation audit was performed for 

biological agents and organisms because quantitative 

standards for these biological materials do not exist. 

Performance evaluation audits for analytical measurements 

(e.g., spectrophotometric absorbance, temperature, RH, and 

contact time), will be conducted and reported in conjunction 

with the virus persistence/decontamination testing associated 

with freeze-dried vaccinia virus.

4.2.2 Technical Systems Audit 

A technical systems audit was conducted on April 16, 2009. 

The assessment addressed all steps from removal from the 

cold storage (low temperature) followed by application of 

the decontamination technologies, neutralization, extraction, 

preparation of serial dilutions, and seeding onto cells. Only 

steps missed were staining of plates and reading of final 

plaques. All equipment used was within calibration and no 

other items were noted. In addition, all known deviations 

from the test procedures specified in the test/QA plan have 

been documented in this listing of deviations in Section 4.5. 

4.2.3 Data Quality Audit

At least 10% of the data acquired during the persistence 

testing and decontamination technology evaluation were 

audited. A Battelle QA 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.

4.3 Quality Assurance/Quality Control Reporting

The audit described in Section 4.2.3 was documented 

in accordance with the QMP2. Quality control samples 

including laboratory blanks, procedural blanks, and positive 

controls are reported along with the test coupon results in 

Sections 2 and 3 for each persistence and decontamination 

test conducted. 

4.4 Data Review

Records and data generated from the persistence testing 

and decontamination technology evaluation received a QC/

technical review. All data were recorded by Battelle staff. The 

person performing the QC/technical review was not involved 

in the experiments and added his/her initials and the date to a 

hard copy of the record being reviewed. This hard copy was 

returned to the Battelle staff member who stored the record.

4.5 Deviations

Deviations from the test/QA plan1 occasionally arose once 

work in the laboratory was initiated; these deviations were 

not expected to adversely affect data quality and included: 

1. Soil and chicken feces were not autoclaved (avoiding 

potential effects on the soil matrix, such as hydrolysis 

of humic acids), but instead filtered through a 

0.2 μm filter during the extraction process to remove 

bacteria (not viruses) from the supernatant that could 

potentially interfere with virus quantification. Blanks 

(non-inoculated coupons) confirmed that any naturally 

occurring viruses did not interfere with the TCID50

assay (i.e., the TCID50 was generally not detected). 

For soil, there was only one occasion (the 9-day test at 

low temperature, high RH, no UV) where TCID50 was 

detected in the procedural blank.

2. Unpainted concrete and soil coupons were too small to 

spike with a multichannel pipette, so a single channel 

pipette was used for these coupons. There were no 

adverse impacts on the testing.

3. Regarding the assessment of potential cytotoxic effects 

of materials extracts, that could potentially interfere 

with the TCID50 assay, the cell viability performance 

criterion of ≥90% of unexposed control cell viability 

with dilution of 1:8 or less was not attained for soil; 

however >90% of unexposed control cell viability 

was obtained with a dilution of 1:16, which was 

approved as an acceptable level of dilution by the EPA 

Task Order Project Officer. As noted in Section 2.2, 

although an increased level of dilution was needed 

for the MTT assay with soil, the TCID50 assay was 

not affected as cytopathic effects were generally not 

observed in the MDCK cells exposed to any dilution 

of soil extract. There were no adverse impacts on the 

testing. 

4. Alternative approaches were used for generating high 

RH and low temperature environmental conditions 

than proposed in the test/QA plan1. Specifically, 

instead of using a nebulizer to generate a high RH at 

the room temperature condition, the coupons were 

placed in a glove box pre-humidified by using an 

ultrasonic-fogger. The moist test material (i.e., soil 
34

and chicken feces) maintained a high RH level. For 

conducting low temperature test, rather than running 

tubing from a re-circulating chiller through the glove 

box, testing was conducted inside a refrigerator. When 

testing at a low temperature with no UV the coupons 

were placed inside a Lock&LockTM plastic storage 

container which was placed in the refrigerator. When 

testing at a low temperature with UV-A/B the coupons 

were placed directly into a refrigerator modified to 

include glove ports. There were no adverse impacts on 

the testing.

5. Continuous monitoring of temperature and RH was 

inadvertently not conducted during the H5N1 virus 

persistence testing at the room temperature, low RH, 

no UV environmental condition and during the initial 

seven hours of the 1-day test at the low temperature, 

low RH, UV-A/B environmental condition. No unusual 

events occurred that would lead one to expect that the 

temperature and RH of these tests deviated from those 

of comparable tests.

6. Temperature and RH inside the test chamber were 

found to be difficult to control (especially when trying 

to test non-ambient conditions over relatively long 

periods of time, i.e. days) and were often outside of the 

target environmental conditions (±2 °C for temperature 

and ±10% for RH), as documented in Section 2.4. 

However, these deviations from target environmental 

conditions do not invalidate the TCID50 data. The 

actual (measured) temperature and RH data are all 

reported. 

7. The performance criteria of no observed CPE in 

MDCK cells for laboratory and procedural blanks 

often failed for chicken feces; however laboratory 

and procedural blanks with CPE were not rejected 

because they were attributed to the test material itself. 

Relatively low detections of TCID50 (i.e., ≤3.16 x 

103 TCID50) in chicken feces test coupons were often 

flagged as not being detected above the associated 

blank of 3.16 x 103 TCID50 (i.e., it could not be 

determined if the CPE were attributed to the H5N1 

virus or simply the chicken feces). 

8. Contrary to the method development tests, most of 

the positive control virus recoveries were below 

the performance criterion (>20% and <120% virus 

recovery). However, even the lowest percent virus 

recovery (0.8%) had a mean of 2.54 x 105 TCID50

recovered, which is an appreciable amount of virus 

to assess persistence. EPA’s virucidal test guidance 

does not require a minimum percent recovery, but 

rather a minimum virus recovery (TCID50) of ≥104. It 

is possible that having a lower virus titer initially may 

make the virus seem less persistent than it would have 

been with a higher initial titer. 

9. Regarding the decontamination technology evaluation, 

the positive controls (at the 10-minute contact time) 

were exposed to PBS rather than being exposed only to 

air, which better mimicked the actual application of the 

decontamination liquid.

10. Although the H5N1 virus recoveries from neutralized 

decontamination liquid and extraction buffer were 

generally <25%, the H5N1 virus recoveries were 

>6 x 104 TCID50, which is an appreciable amount of 

virus to assess decontamination efficacy. Note: EPA’s 

virucidal test guidance does not require a minimum 

percent recovery, but rather a minimum virus recovery 

(TCID50) of ≥104. The H5N1 virus was detected 

in the trough decontamination liquid, such that the 

H5N1 virus recovery in the actual decontamination 

technology evaluation was quantified from the coupon 

and the liquid in the trough (for galvanized metal 

only) to ensure that all of the viable H5N1 virus was 

quantified.
35

5.0

Summary

5.1 H5N1 Virus Persistence

The persistence of the H5N1 virus (A/Vietnam/1203/04) was 

investigated on four materials (chicken feces, galvanized 

metal, glass, and soil) under five environmental conditions 

(each condition consisted of various combinations of 

temperature, RH, and exposure to UV-A/B radiation [to 

simulate sunlight]). Under room temperatures at both low 

and high RH (no UV), the H5N1 virus persisted for less 

than two days on chicken feces, galvanized metal, glass, and 

soil. At low temperatures under both low and high RH (no 

UV) the H5N1 virus persisted for at least four days on all 

materials. The H5N1 virus was detected following exposure 

to the low temperature, low RH, no UV environmental 

condition after 13 days on galvanized metal, glass, and soil. 

The H5N1 virus was detected on chicken feces, glass, and 

soil following exposure to the low temperature, high RH, 

no UV environmental condition for nine days. Testing was 

not conducted for durations longer than 13 days for any of 

the environmental conditions; the duration of H5N1 virus 

persistence may exceed 13 days, especially on galvanized 

metal and glass under the low temperature, low RH, no UV 

environmental condition. 

With continuous UV-A/B exposure to simulate sunlight 

and under a low temperature and low RH environmental 

condition, the H5N1 virus persisted less than 48 hours 

on galvanized metal and glass. H5N1 persisted 96 hours 

on soil and chicken feces, although for chicken feces, the 

H5N1 level recovered from the test coupons could not be 

distinguished from the interference of the test material itself.

5.2 H5N1 Virus Liquid Decontamination

Four decontamination liquids (1% citric acid, pH-amended 

bleach, 732 ppm quaternary ammonium, and 8% Na2CO3) 

prepared with hard water were applied to galvanized metal 

and soil coupons inoculated with the H5N1 virus, and tested 

at room and low temperature conditions for 10-minute 

contact times. The decontamination technology evaluation 

indicated that only pH-amended bleach was effective at 

completely inactivating the H5N1 virus (i.e., the H5N1 

virus was not detected), and this result was only obtained 

for galvanized metal (at both room and low temperatures). 

For soil, pH-amended bleach induced a mean H5N1 virus 

log reductions in TCID50 of 2.7 at room temperature and 2.9 

at low temperature. The mean H5N1 virus log reductions 

in TCID50 associated with 1% citric acid ranged from 1.5 

(with soil at room temperature) to 2.1 (with galvanized metal 

at low temperature). The mean log reductions in the H5N1 

virus, determined for 732 ppm quaternary ammonium and 

8% Na2CO3, were all less than 1.0 for both temperature 

conditions tested.
36

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37

6.0

References

1. Technology Testing and Evaluation Program Test/QA Plan for Persistence Testing of Avian Influenza 

Viruses on Outdoor Materials, Version 1, Prepared for EPA by Battelle, Columbus, Ohio,

March 2007.

2. Quality Management Plan (QMP) for the Technology Testing and Evaluation Program (TTEP), 

Version 1, Prepared for EPA by Battelle, Columbus, Ohio, January 2005.

3. Finney, D.J. Assays Based on Quantal Responses. In: Statistical Methods in Biological Assays, 3rd 

ed. Macmillan Publishing Co., Inc., New York, p. 394–398. 1978.

4. Battelle, BBRC Method No. 125/Microbiology, Method for the Determination of the Median Tissue 

Culture Infectious Dose (TCID50 ) for a Viral Sample. Battelle Proprietary.

5. Centers for Disease Control and Prevention (CDC). Guidance & Recommendations, Interim 

Guidance for Protection of Persons Involved in U.S. Avian Influenza Outbreak Disease Control and 

Eradication Activities. http://www.cdc.gov/flu/avian/professional/protect-guid.htm. 

6. Mims, F.M., III. Avian Influenza and UV-B Blocked by Biomass Smoke. Environmental Health 

Perspectives, 2005 113(12) A806–A807.

7. Jeanmougin, M. and J. Civatte. Dosimetry of Solar Ultraviolet Radiation. Daily and Monthly 

Changes in Paris. [Article in French] Ann Dermatol Venereol, 1987 (114):671–676.

8. Kolari, P.J., J. Lauharanta, and M. Hoikkala. Midsummer Solar UV-Radiation in Finland Compared 

with the UV-Radiation from Phototherapeutic Devices Measured by Different Techniques. 

Photodermatol, 1986 (3):340–345.

9. Balasaraswathy, P., U. Kumar, C.R. Srinivas, and S. Nair. UVA and UVB in Sunlight, Optimal 

Utilization of UV Rays in Sunlight for Phototherapy. Indian J Dermatol Venereol Leprol, 2002 

(68):198–201.

10. McNamara, A.E. and W.R. Hill. UV-B Irradiance Gradient Affects Photosynthesis and Pigments but 

Not Food Quality of Periphyton. Freshwater Biology, 2000 (43):649–662.

11. Diffey, B.L. Sources and Measurement of Ultraviolet Radiation. Methods, 2002 (28):4–13.

12. EPA, Antimicrobial Science Policies, Disinfectant Technical Science Section, available online at: 

http://www.epa.gov/oppad001/sciencepolicy.htm.
38

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39

Appendix A:

H7N2 Virus Testing

In an effort to generate paired persistence and 

decontamination efficacy data for highly pathogenic avian 

influenza virus (i.e., H5N1 virus, strain A/Vietnam/1203/04) 

and low pathogenic avian influenza virus (i.e., H7N2 virus 

strain A/chicken/Maryland/Minh Ma/04), preliminary 

research was also conducted with the H7N2 virus. 

To improve comparability of the test results for both viruses, 

it was desired to quantify both viruses using the same 

approach (i.e., the TCID50 assay). However, initial efforts at 

propagating the H7N2 virus resulted in virus concentrations 

that were less than the target titer of 1 x 107 TCID50 (using 

MDCK cells for virus quantitation, as with the H5N1 

virus). Re-propagation of the H7N2 virus continued to yield 

apparently low titers; however, this was eventually attributed 

to the use of a mammalian cell line (MDCK) to quantify 

the H7N2 virus. Attempts were thus made to quantify the 

H7N2 virus using chicken embryo kidney (CEK) cells to 

conduct the TCID50 assay. Initial results indicated that CEK 

cells may be used to quantify the H7N2 virus and yielded 

results indicating that the propagated H7N2 virus stock had 

a titer of 1.26 x 107 TCID50/mL. However unacceptably high 

variability of this assay was obtained with repeated assays. 

Research was conducted to evaluate the effect of different 

aged CEK cells with the TCID50 assay to reduce the assay 

variability, but the titer results remained too variable to draw 

conclusions (see Table A-1). It was determined that additional 

research was needed to develop a reliable CEK assay for 

H7N2 virus, which was beyond the scope of the current 

project. 

Table A-1. H7N2 Virus Titers Obtained Using Chicken Embryo Kidney Cellsa

H7N2 Virus Stock

7/16/07 Titer:

Cells Plated 1 Day After 

Receiving (TCID50/mL)

7/31/07 Titer:

Cells Plated 1 Day After 

Receiving (TCID50/mL)

8/1/07 Titer:

Cells Plated 2 Days After 

Receiving (TCID50/mL)

4/13/07 H7N2 Stock (1:500)b 1.26 x 106 5.01 x 105 1.26 x 106

4/13/07 H7N2 Stock (1:1000)c 2.00 x 104 2.00 x 105 3.16 x 105

4/13/07 H7N2 Stock (1:5000)d 5.01 x 103 3.16 x 105 5.01 x 105

5/4/07 H7N2 Stock 2.00 x 103 1.26 x 105 2.00 x 106

5/25/07 H7N2 Stocke 2.00 x 103 2.00 x 105 2.00 x 106

a The results of using the fresh (plated within 48 hours of receipt from vendor) CEK cells (7/31/07 titer) and the aged (plated after 48 hours after 

receipt from vendor) CEK cells (8/1/07 titer) were compared with initial titer results; all results were based on the fifth day of post-infection. 

b 1:500 = initial 1:10 dilution of H7N2 virus stock (0.01 mL virus stock + 0.09 mL diluent), final 1:50 dilution of initial dilution (0.03 mL 1:10 

virus dilution + 1.47 mL diluent) for a 1:500 quantity of virus needed to infect eggs.

c 1:1000 = initial 1:100 dilution of H7N2 virus stock (0.01 mL virus stock + 0.99 mL diluent), final 1:10 dilution of initial dilution (0.15 mL 

1:10 virus dilution + 1.35 mL diluent) for a 1:1000 quantity of virus needed to infect eggs.

d 1:5000 = initial 1:100 dilution of H7N2 virus stock (0.01 mL virus stock + 0.99 mL diluent), final 1:50 dilution of initial dilution (0.03 mL 

1:10 virus dilution + 1.47 mL diluent) for a 1:5000 quantity of virus needed to infect eggs.

e

 Titered at 1.26 x 107 TCID50/mL on 6/28/07; cells plated two days after receiving.
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