Spray Decontamination of Vegetation with Sporicidal Liquids for the Inactivation of Bacillus anthracis Surrogate Spores and Phytotoxicity Assessment


EPA 600/R-22/051 I September 2022 | www.epa.gov/research

Spray Decontamination of Vegetation
with Sporicidal Liquids for the Inactivation
of Bacillus anthracis Surrogate Spores and
Phytotoxicity Assessment

Office of Research and Development Center for
Environmental Solutions and Emergency Response (CESER)

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EPA 600/R-22/051 I September 2022 I www.epa.gov/research

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EPA 600/R-22/051

Spray Decontamination of Vegetation with Sporicidal
Liquids for the Inactivation of Bacillus anthracis
Surrogate Spores and Phytotoxicity Assessment

U.S. EPA Principal Investigator:
Joseph Wood

U.S. Environmental Protection Agency
Research Triangle Park, NC 27711

Prepared by:

Stella McDonald
Timothy Chamberlain
Abderrahmane Touati
Joseph Wood

Jacobs

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DISCLAIMER

The U.S. Environmental Protection Agency (EPA) through its Office of Research and
Development (ORD) directed and managed this work. This study was funded through the
Analysis for Coastal Operational Resiliency (AnCOR) Project by the U.S. Department of
Homeland Security Science and Technology Directorate (DHS S&T) under interagency
agreement IA 070-95937001. This report was prepared by Jacobs Technology Inc. under EPA
Contract Number 68HERC20D0018; Task Order 68HERC21F0023. This report has been
reviewed and approved for public release in accordance with the policies of the EPA. Mention of
trade names or commercial products does not constitute endorsement or recommendation for use
of a specific product. The contents are the sole responsibility of the authors and do not
necessarily represent the official views of EPA, DHS S&T, or the United States Government.

Questions concerning this document, or its application should be addressed to the principal
investigator:

Joseph Wood

U.S. Environmental Protection Agency
Office of Research and development (ORD)

Center for Environmental Solutions and Emergency Response (CESER)

Homeland Security and Material Management Division (HSMMD)

109 T.W. Alexander Drive
MD-E343-06

Research Triangle Park, NC 27711
E-mail Address: wood.ioe@epa.gov

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Foreword

The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting the
Nation's land, air, and water resources. Under a mandate of national environmental laws, the
Agency strives to formulate and implement actions leading to a compatible balance between
human activities and the ability of natural systems to support and nurture life. To meet this
mandate, EPA's research program is providing data and technical support for solving
environmental problems today and building a science knowledge base necessary to manage our
ecological resources wisely, understand how pollutants affect our health, and prevent or reduce
environmental risks in the future.

The Center for Environmental Solutions and Emergency Response (CESER) within the Office of
Research and Development (ORD) conducts applied, stakeholder-driven research and provides
responsive technical support to help solve the Nation's environmental challenges. The Center's
research focuses on innovative approaches to address environmental challenges associated with
the built environment. We develop technologies and decision-support tools to help safeguard
public water systems and groundwater, guide sustainable materials management, remediate sites
from traditional contamination sources and emerging environmental stressors, and address
potential threats from terrorism and natural disasters. CESER collaborates with both public and
private sector partners to foster technologies that improve the effectiveness and reduce the cost
of compliance, while anticipating emerging problems. We provide technical support to EPA
regions and programs, states, tribal nations, and federal partners, and serve as the interagency
liaison for EPA in homeland security research and technology. The Center is a leader in
providing scientific solutions to protect human health and the environment.

This report assesses decontamination options for vegetative materials that may become
contaminated with Bacillus anthracis spores. The findings can be used to better plan and execute
the cleanup of a US Coast Guard port/base, or any large outdoor urban area, following such a
biological contamination incident. This work was coordinated with and managed by the EPA's
Homeland Security Research Program (HSRP) under the Department of Homeland Security
(DHS) Science and Technology (S&T) Directorate funded Analysis for Coastal Operational
Resiliency (AnCOR) project.

Gregory Sayles, Director

Center for Environmental Solutions and Emergency Response

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ACKNOWLEDGMENTS

The principal investigator (PI) from the U.S. Environmental Protection Agency (EPA)
directed this effort with input from the project team consisting of personnel from EPA, the US
Coast Guard, and DHS. The contributions of the individuals listed below have been a valued
asset throughout this effort.

EPA Project Team

Joseph Wood (PI)

Lukas Oudejans
Shannon Serre
Worth Calfee
Timothy Boe
Erin Silvestri
Anne Mikelonis
Katherine Ratliff

Katrina McConkey (ORD Contractor)

Benjamin Franco

Duane Newell

Stephen Wolfe

Christine Tomlinson

Jason Musante

EPA Quality Assurance

Ramona Sherman, CESER/HSMMD

Jacobs Technology, Inc.

Abderrahmane Touati
Stella McDonald
Stacy Cross
Timothy Chamberlain
Eric Morris
Denise Aslett
Ahmed Abdel-Hady
Mariela Monge
Brian Ford
Rachael Baartmans
Sandoval Lesley Mendez

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Coast Guard and DHS Team members

Donald Bansleben
Andrea Wiggins
Kirsten Trego
Omar Borges
Benjamin Perman

EPA Reviewers of report

Michael Pirhalla (Office of Research and Development)
H. Ray Ledbetter (Office of Emergency Management)

External Reviewers of report

Alden Adrion, CBRN Defense Division, Army Evaluation Center
Robert Miknis, USDA APHIS

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EXECUTIVE SUMMARY

The Analysis for Coastal Operational Resiliency (AnCOR) program is an interagency
collaboration between the U.S. Environmental Protection Agency (EPA), the Department of
Homeland Security (DHS), and the United States Coast Guard (USCG). The overall purpose of
this multiagency program is to develop and demonstrate capabilities and strategic guidelines to
prepare the U.S. for a wide-area release of a biological agent, including mitigating effects on
USCG facilities and assets. The study described in this report was conducted under the AnCOR
program, with the goal of assessing decontamination options and their efficacy for vegetative
materials contaminated with Bacillus anthracis spores. Following a wide-area release of B.
anthracis spores, vegetation may become contaminated with the biological agent.
Decontamination of vegetative materials will be a challenge, due to the organic nature of
vegetation and the potentially large and complex surface areas of foliage. The scientific literature
contains minimal documentation related to the decontamination of vegetation contaminated with
B. anthracis spores or other biological agents.

This study included four phases: In Phase 1, bench-scale tests using small "coupons" of
plant leaf material confirmed that Bacillus globigii (B.gsurrogate for B. anthracis) spores could
be readily inoculated and recovered from a variety of different vegetative materials. Efficacy
tests utilizing a variety of decontaminants, active ingredient concentrations, and plant types were
conducted for proof of concept and down selection of decontaminants to be used in the Phase 2
trials.

Phase 2 was conducted at pilot-scale, with the spray application of either peracetic acid-
(PAA) or dichlor-based decontaminants to a variety of small plants and pine bark. Fifteen tests
were conducted to assess the effect of parameters such as plant type, decontaminant, active
ingredient concentration, spray quantity, and sprayer type on decontamination efficacy. Of the 11
experiments conducted with small plants, four experiments resulted in having no samples (of 15
samples for each experiment) positive for B.g., and this absence of B.g. occurred using either
dichlor or PAA. Generally, PAA was somewhat more effective than dichlor if test conditions
were similar. Of the four decontamination experiments conducted with pine bark, none resulted
in having all samples negative for B.g., signifying the difficulty of decontaminating this type of
material. The test with PAA concentration at 0.55% resulted in only 2 samples of 15 testing
positive for B.g. Dichlor performed poorly against the pine bark matrix, returning nearly all 15
samples positive for B.g. spores in both experiments.

Phase 3 of the study was similar to Phase 2, but instead of using small plants, 1 square
foot "coupons" of sod were used. Nine decontamination efficacy experiments were conducted
with three types of sod, using either PAA or dichlor. Within each experiment, there was no
apparent association between increasing the spray volume of the decontaminant and improved
decontamination efficacy. Overall, the zoysia sod was the most amenable to decontamination, in
that it had the least number of samples that were positive (9 positive out of a total of 45)
following treatment. The Bermuda grass had the most positive samples (35 out of 45). The
average number of positive samples per test (out of 15) for the PAA was 5.25, while for dichlor,
the average number of positive samples per test was 9.8.

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Phase 4 of the study examined the phytotoxicity of the decontaminants. The impacts to
plant health were assessed by spraying the small plants and sod under the decontamination
conditions found to be effective in inactivating the B.g. spores (results from Phase 2 and 3 tests)
and comparing effects to plants sprayed with water only. Assessment of any damage to plants
occurred over the course of four weeks following application of the decontaminant. While none
of the small plants died during the month-long observation following exposure to the
decontaminant, there were some mixed results with respect to other phytotoxic effects that varied
by plant type, decontaminant, and the type of phytotoxic effect. No obvious trends in effects
were noted. The phytotoxic effects of the decontaminants on the Indian Hawthorn plants were
generally indistinguishable from water. For the Creeping Jenny plants, there did appear to be
more damage to leaves by both decontaminants, compared to the water controls, with the dichlor
impact likely being more pronounced than the PAA. For the blueberry bushes, which were
assessed in the water-rinsed and unrinsed conditions, the phytotoxic effect results were
inconclusive or at best counterintuitive. For example, the PAA seemed to have more of an effect
on the rinsed blueberry plants compared to the unrinsed plants (in terms of leaf damage and leaf
shedding), although plant growth was greater for the rinsed blueberry plants.

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Table of Contents

List of Figures	x

List of Tables	xi

List of Acronyms and Abbreviations	xiv

1	Introduction	1

2	Experimental Approach	3

2.1	Bench-Scale Efficacy Tests	4

2.1.1	Sample Neutralization	5

2.1.2	Leaf Material Inoculation	5

2.1.3	Sporicidal Solutions Preparation	5

2.1.4	Decontamination Process	6

2.1.5	Sample Retrieval	6

2.2	Small Plant Pilot-Scale Efficacy Tests	7

2.3	Sod Decontamination Tests	9

2.4	Phytotoxicity Tests	10

3	Materials and Methods	12

3.1	Test Facilities	12

3.2	Test Equipment	14

3.2.1	Hand-held Sprayer	14

3.2.2	Electrostatic Sprayer	14

3.2.3	Backpack Sprayer	15

3.3	Plant Material and Coupon Preparation	16

3.3.1	Preparation of 18-mm Leaf Material	16

3.3.2	Full-sized Plant Preparation	17

3.3.3	Tree Bark Preparation	18

3.3.4	Grass (Sod) Preparation	19

3.4	Sterilization, Sanitization, and Cross-contamination Prevention	20

3.5	Bacillus Spore Preparation and Surface Contamination	22

3.6	Decontamination Solutions	25

3.7	Sampling and analysis	27

3.7.1	Bacterial Spores Analysis	27

3.7.2	Extractive Sampling of Plant Material	28

3.7.3	Tree Bark Sampling	29

3.7.4	Sod Sampling	30

4	Quality Assurance/Quality Control	32

4.1 Sampling, Monitoring, and Equipment Calibration	32

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4.2	Acceptance Criteria for Measurements	32

4.3	Data Quality Objectives	34

4.4	Data Quality Indicators	34

5	Results and Discussion	37

5.1	Bench-scale Efficacy Tests	37

5.1.1	Chlorine-based Sporicides	37

5.1.2	PAA-based Sporicidal Solutions	38

5.2	Pilot-Scale Small Plant Decontamination Tests	39

5.3	Sod Decontamination Tests	45

5.4	Phytotoxicity Effects of Sporicidal Solutions	50

6	Summary and Conclusions	56

7	References	58

Appendix A	60

A.1 Preliminary Test Results	61

A.1.1 Recovery Efficiency from Plant Leaves-Test Results	61

A.1.2 Characterization of Spore Deposition onto Plant Material Using the LV-ADA	61

Appendix B	65

Appendix C	74

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List of Figures

Figure 2-1. Bench-scale Test Sequence	5

Figure 2-2. Bench-scale spray apparatus with coupon holders (a) and plant coupons staged

horizontally (b)	6

Figure 2-4. Test setup for spray decontamination for evergreen (a), deciduous (b), tree bark (c),

ground cover (d), grass vegetation categories	9

Figure 3-1. Bench-scale test facilities (a) biosafety cabinet and (b) chemical hood	12

Figure 3-2. Spray chamber (a) exterior and (b) and interior	13

Figure 3-3. Greenhouse (a) exterior and (b) interior	14

Figure 3-4. RL Flo-Master® pressurized hand sprayer	14

Figure 3-5. SC-ET HD electrostatic sprayer	15

Figure 3-6 SHURflo ProPack rechargeable electric backpack sprayer	15

Figure 3-7. Punched leaves (a), plant disks (b), and finished 18-mm coupons (c)	17

Figure 3-8. Shrub branches repositioned to the center of the pot	17

Figure 3-9. Individual ground cover plants with carrying tray	18

Figure 3-10. Pine tree bark in stainless-steel test tray	19

Figure 3-11. Sod material cut into 12-in by 12-in coupons	19

Figure 3-12. Front (a) and top (b) view of a sod coupon prepared for phytotoxicity testing and

evaluation	20

Figure 3-14. Plants and bark trays inside LV-ADA (a and b), sealed LV-ADA (c), and MDI adaptor

(d)	24

Figure 3-15. Diagram of extractive sampling areas - Top view of the plant (a) and the leaf hole

punch tool (b)	29

Figure 3-16. Punching of a leaf (a) and punches collected in a sample tube (b)	29

Figure 3-17. Pine bark nuggets sampling set-up	30

Figure A-1. Sample areas for spore distribution tests	62

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List of Tables

Table 3-1. Detailed Summary of Vegetation	16

Table 3-2. Sterilization Methods for Test Materials and Equipment	21

Table 3-3. Sanitization Methods for Test Materials and Equipment	21

Table 3-4. Surface Contamination Levels for Each Sample Type	22

Table 3-5. Decontaminant Information	26

Table 4-1. Sampling and Monitoring Equipment Calibration Frequency	32

Table 4-2. Analytical Equipment Calibration Frequency	32

Table 4-3. Summary of QA/QC Checks	33

Table 4-4. Assessment of DQI Goals for Dichlor (FAC) Solutions	35

Table 4-5. Assessment of DQI Goals for PAA Solutions	36

Table 5-1. Average Spore Loading and Recovery (CFU/Sample ± SD)	37

Table 5-2. Bench-Scale Decontamination Efficiency Results for Leaf Material Coupon	38

Table 5-3. Average Spore Loading (CFU/Sample ± SD)	38

Table 5-4. Bench-Scale Decontamination Efficiency Results for Leaf Material Tests Using

Peracetic Acid	39

Table 5-5. Small Plant Positive Control Recovery	40

Table 5-6. Summary of Spore Recovery from Bark Sample Controls	41

Table 5-7. Summary of Baseline Contamination and Cross-contamination Results	42

Table 5-8. Decontamination Results for Evergreens (Indian Hawthorn)	43

Table 5-9. Decontamination Results for Ground Cover Plants	43

Table 5-10. Decontamination Results for Deciduous Plants (Blueberry Shrubs)	44

Table 5-11. Decontamination Results for Bark	44

Table 5-13. Decontamination Results forZoysia Sod Coupons	47

Table 5-14. Decontamination Efficacy for Fescue Sod Coupons	48

Table 5-15. Decontamination Efficacy for Bermuda Sod Coupons	49

Table 5-16. Number of Sod Coupons (of 15) Positive for Spores	49

Table 5-17. Average Number of Damaged Leaves for Indian Hawthorne Plant (± SD)	50

Table 5-18. Average Number of Shed Leaves per Indian Hawthorne Plant	50

Table 5-19. Average Height for Indian Hawthorne Plant (Inches)	51

Table 5-20. Average Number of Damaged Leaves for Each Rinsed Creeping Jenny	51

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Table 5-21. Total Shed Leaves for Each Rinsed Creeping Jenny Plant	51

Table 5-22. Average Height of Rinsed Creeping Jenny Plants (lnches±SD)	52

Table 5-23. Average Number of Damaged Leaves for Each Rinsed Blueberry Plant (±SD)	52

Table 5-24. Total Number of Shed Leaves for Each Rinsed Blueberry Plant (±SD)	52

Table 5-25. Height for Rinsed Blueberry Plant (Inches)	53

Table 5-26. Average Number of Damaged Leaves for Each Unrinsed Blueberry Plant (±SD)	53

Table 5-27. Total Number of Shed Leaves for Each Unrinsed Blueberry Plant(±SD)	53

Table 5-28. Height for Unrinsed Blueberry Plant (Inches ± SD)	54

Table 5-29. Percent of Fescue Sod Coupon Measured as Damaged by Photoanalysis (±SD)	54

Table 5-30. Height of Fescue Grass in the Center of the Coupon (inches ± SD)	54

Table 5-31. Average Percent of Zoysia Sod Coupon Measured as Damaged per Photo Test

(±SD)	55

Table 5-32. Height of Zoysia Grass in the Center of the Sod Coupon (inches ±SD)	55

Table A-1. Recovery Efficiency from Leaves	61

Table A-2. Average CFU Recoveries from Horizontal and Vertical Sample Sections	62

Table A-3. Residual pAB and Dichlordecontaminant evaluation	63

Table A-4. Residual Decontamination Evaluation for PAA	64

Table B-1. Bench-scale decontamination efficacy test matrix with Dichlor	66

Table B-2. Bench-scale decontamination efficacy test matrix with Jet-Ag	66

Table B-3. Bench-scale decontamination efficacy test matrix with Oxidate 2.0	66

Table B-4. Bench-scale decontamination efficacy test matrix with pAB	66

Table B-5. Pilot-Scale Control Test with Deionized Water	67

Table B-6. Pilot-scale decontamination efficacy test matrix with Jet-Ag	68

Table B-7. Pilot-scale decontamination efficacy test matrix with Dichlor	70

Table B-8. Phytotoxicity test matrix with deionized water	72

Table B-9. Phytotoxicity test matrix with Dichlor	72

Table B-10. Phytotoxicity Test Matrix with Jet Ag	72

Table C-1. Average Damaged Leaves Per Indian Hawthorne Plant (Unrinsed)	75

Table C-2. Total Shed Leaves per Indian Hawthorne Plant (Unrinsed)	75

Table C-3 Height for Unrinsed Indian Hawthorne Plant (Inches)	75

Table C-4. Average Damaged Leaves for Each Rinsed Creeping Jenny	76

Table C-5. Total Shed Leaves for Each Rinsed Creeping Jenny Plant	76

Table C-6. Height of Rinsed Creeping Jenny Plants (Inches)	76

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Table C-7. Average Number of Damaged Leaves for Each Rinsed Blueberry Plant	77

Table C-8. Total Number of Shed Leaves for Each Rinsed Blueberry Plant	77

Table C-9. Height for Rinsed Blueberry Plant (Inches)	78

Table C-10. Percent of Unrinsed Fescue Sod Coupon Measured as Damaged by Photoanalysis	78

Table C-11. Height of Grass at the Center of the Unrinsed Fescue Sod Coupon (Inches)	79

Table C-12. Average Percent of Zoysia Sod Coupon Measured as Damaged per Photo Test	79

Table C-13. Height of Grass at the Center of the Fescue Sod Coupon (Inches)	79

Table C-14. Decontaminant Effect Substantially Worse than Baseline Plants	80

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List of Acronyms and Abbreviations

AnCOR	Analysis for Coastal Operational Resiliency

B. cmthracis Bacillus cmthracis

B.g.	Bacillus globigii

BP	backpack

CESER	Center for Environmental Solutions and Emergency Response (EPA)

CFU	colony-forming unit(s)

CMAD	Consequence Management Advisory Division (EPA/OLEM/OEM)

COTS	commercial off the shelf

Dichlor	sodium dichloro-s-triazinetrione dihydrate

DE	Dey-Engley broth

DHS S&T	Department of Homeland Security Science and Technology Directorate

DI	deionized water

DQO	data quality objectives

EPA	U.S. Environmental Protection Agency

ES	electrostatic

EtO	ethylene oxide

FAC	free available chlorine

Ft	feet

HDPE	high density polyethylene

HSMMD	Homeland Security and Materials Management Division

HP	hydrogen peroxide

HSRP	Homeland Security Research Program

LR	log reduction

LV-ADA	Large-volume aerosol deposition apparatus

MDI	metered dose inhaler

PI	Principal investigator

psi	pounds per square inch

NIST	National Institute of Standards and Technology

OEM	Office of Emergency Management (EPA/OLEM)

OPP	Office of Pesticides Programs (EPA/OCSPP)

ORD	Office of Research and Development (EPA)

OLEM	Office Land and Emergency Management (EPA)

pAB	pH-adjusted bleach

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PAA

peracetic acid

PBST

phosphate-buffered saline with Tween 20

PVC

polyvinyl chloride

RSD

relative standard deviation

QA

quality assurance

QAPP

quality assurance project plan

QC

quality control

QMP

quality management plan

RMC

reference material coupon

RTP

Research Triangle Park

SD

standard deviation

SEM

scanning electron microscope

SHEM

Safety, Health, and Environmental Management (Program Office, EPA)

STS

sodium thiosulfate

TC

thermocouple

USCG

U.S. Coast Guard

UV

ultraviolet

VHP

vaporized hydrogen peroxide

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1 Introduction

Following a wide-area release of B. cmthracis spores (the environmentally persistent
bacterium causing anthrax disease), vegetation may become contaminated with the biological
agent. Decontamination of vegetative materials such as trees, grass, and crops, especially on a
large scale, will be a challenge, due to the organic nature of vegetative materials (which can
chemically reduce the active ingredient concentration of some decontaminants), and the
potentially large and complicated surface areas of foliage. Other challenges include being able to
deliver the decontaminant to tall trees, and to effectively decontaminate the plants without killing
or damaging them. (Note that the use of large-scale commercial, industrial, and/or agricultural
sprayers to apply decontaminants to outdoor infrastructure and vegetation following a wide-area
release of B. anthracis spores is being evaluated in another AnCOR study.) In discussions with
the USCG, in addition to aesthetic value, maintaining the health and viability of the vegetation
on their bases is needed to mitigate soil and shoreline erosion, which in turn is needed to
maintain the integrity of maritime structures found on their bases.

Since USCG bases are located throughout the U.S., there is no typical vegetation that might
be encountered on such a base. Therefore, small plants were selected for testing based on
whether they were evergreens, deciduous, ground cover, sod/grass, and representative of tree
bark. All tests and analyses described in this report were conducted in the US EPA's Research
Triangle Park (RTP), NC, laboratories, using the well characterized benign surrogate for B.
anthracis, Bacillus globigii (B.g.).

A search of the scientific literature for the decontamination of vegetation contaminated with
biological agents (such as B. anthracis) resulted in a minimal number of articles. Thus, we
looked for the commercial availability of antimicrobial pesticides, such as fungicides, that are
used on plants, and more specifically, used for commercial purposes and contain active
ingredients known to inactivate B. anthracis spores. As a result, we found two commercial off-
the-shelf peracetic acid-based products registered for agricultural uses (as fungicides,
bactericides, and algicides) that we included in our evaluation. The study also evaluated the use
of dichlor (i.e., sodium dichloro-s-triazinetrione dihydrate, also known as sodium
dichloroisocyanurate dihydrate; chemical formula C3ChN3Na03-2H20), a widely available, off
the-shelf dry chemical commonly used for disinfection of swimming pools and spas. In a
previous study, dichlor solutions were shown to be effective in inactivating B.g. spores on
several outdoor materials under certain conditions (U.S. EPA, 2021B).

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The study described in this report was organized into four phases:

•	Phase 1 was conducted at bench-scale, using small "coupons" of plant leaf material,
to confirm that B.g. spores could be readily inoculated on and recovered from a
variety of different vegetative materials. Phase 1 also included scouting efficacy tests
of various decontaminants, active ingredient concentrations, and plant types for
proof of concept and down selection of decontaminants to be used in the Phase 2
trials.

•	Phase 2 was conducted at pilot-scale, with the spray application of decontaminants
(down-selected from Phase 1) to a variety of small plants and pine bark. Fifteen tests
were conducted to assess the effect of parameters such as plant type, decontaminant,
active ingredient concentration, spray quantity, and sprayer type on decontamination
efficacy.

•	Phase 3 of the study was similar to Phase 2, but instead of using small plants, 1
square foot "coupons" of sod/grass were used. Nine decontamination efficacy
experiments were conducted in this phase, to evaluate two decontaminants for three
types of sod.

•	Phase 4 of the study examined the phytotoxicity of the decontaminants. The impacts
to plant health were assessed by spraying the small plants and sod under the
decontamination conditions found to be effective in inactivating the B.g. spores
(results from Phase 2 and 3 tests). Visual assessment of any damage to plants
occurred over the course of four weeks following application of the decontaminant.

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2 Experimental Approach

The general experimental approach used to meet the project objectives is described below.

1. Overview of test matrix:

a. Bench-Scale Proof of Concept Tests: Initial experiments were performed to
assess inoculation and recovery of spores from plant material. Decontamination
tests were conducted using 18-mm-diameter test coupons made of different types
of leaf material. The coupons were assembled in-house by punching 18-mm-
diameter circular discs of plant leaf material and/or galvanized steel (as a
reference material). The coupons were placed in a test stand and sprayed with a
hand-held sprayer.

b. Pilot-Scale Small Plant Decontamination Phase: Application of the
decontamination procedure was completed on small plants (maximum plant
height of approximately 32 inches) that could be accommodated inside a custom-
built spray chamber. The plants were set on electric rotating tables to allow
uniform spraying of the entire plant. Pine bark nuggets covering the bottom of
stainless-steel test trays were also decontaminated in the spray chamber.

c. Sod Decontamination Tests: Application of the decontamination procedure for
sod coupons (1 square foot) was performed in an enclosed, single-access-point
chamber within the current Homeland Security Consequence Management and
Decontamination Evaluation Room (COMMANDER) located on the EPA RTP
campus, in North Carolina.

d. Phototoxicity Evaluation: Phototoxicity evaluations were performed after
exposure of the small living plants and sod to a decontamination spray process in
a fabricated greenhouse enclosure made of ultraviolet (UV)-coated polycarbonate
panels to allow for sunlight diffusion.

2. Inoculation of leaf material surfaces using a standardized inoculum of target
organisms: Target surfaces were inoculated using an aerosol deposition method. A
known quantity of the surrogate organism (105 or 101 B.g. CFUs (colony-forming units))
was deposited onto the surfaces, followed by quantitative assessment of pre-
decontamination spore loading by sampling positive control (non-decontaminated)
surfaces.

3. Preparation of neutralizer - Dey-Engley (DE) broth: For the bench-scale study, a
known volume of neutralizer (DE broth) was used to halt the decontamination activity at
the end of the contact time.

4. Preparation of decontamination formulations: In the Phase 1 bench-scale tests, four
decontamination solutions were evaluated for their sporicidal effectiveness against
bacterial spores. Two PAA-based decontaminant solutions that were evaluated in Phase 1
included prepared solutions of Jet-Ag 15% and Oxidate 2.0. Two chlorine-based
solutions were also evaluated in the Phase 1 tests and included pH-adjusted bleach (pAB)
and sodium dichloro-s-triazinetrione dihydrate (dichlor). Based on the results of the
bench-scale tests, the remainder of the study focused on the use of either PAA or dichlor.

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5. Decontamination of test material:

a. Bench-scale Tests: Coupons of leaf materials were inoculated with 5 x 107 B.g.
spores. A set of 3 replicate leaf coupons were used to assess each test condition.
Additionally, a set of 3 positive controls and 1 material blank per material were
included for each test. Quantitative assessment of residual (background)
contamination of B.g. spores was performed by recovering any spores from
procedural blanks (non-inoculated leaf coupons that went through the same
decontamination process as the test coupons).

b. Pilot-Scale Small Plant Decontamination: The spraying of the plants was
performed using either an electrostatic sprayer (to cover hard-to-reach areas of the
plant leaves and foliage) or a backpack sprayer for a full soaking of the plants.
Each experiment utilized four plants; three were inoculated with B.g. spores and
the fourth served as a procedural blank. The plants were decontaminated inside
the spray chamber in pairs: 2 decontamination events were required to process the
4 plants. The initial spray event included 2 B.g.-inoculated plants and the second
spray event included the third inoculated plant along with a procedural blank
plant.

c. Pilot-Scale Sod/Grass Decontamnation. Each sod decontamination experiment
focused on one type of sod and one decontaminant. Each experiment utilized a
total of 24 sod coupons: four of these coupons served as positive controls, and
were not sprayed with the decontaminant. Of the remaining 20 test coupons, all
were sprayed with the decontaminant, but five served as field blanks and were not
inoculated. The remaining 15 sod coupons were divided into five sets of three
coupons, with each set sprayed a different amount of decontaminant.

6. Decontamination effectiveness:

a. Bench-Scale Decontamination effectiveness: For these tests, decontamination
results are reported as decontamination efficacy in terms of log reduction (LR) in
viable spores. For laboratory assessments of decontamination efficacy, an LR > 6
is considered effective when the CFU recovered from positive control samples is
greater than 7 log.

b. Pilot-scale Decontamination effectiveness. For the small plant and sod tests,
results are reported in terms of the number of samples that were positive for B.g.
for a given experiment, regardless of the number of CFU recovered. (In some
cases, only 1 CFU may have been recovered, but the sample would still be
reported as positive.) The small plant and sod positive control foliage samples
typically ranged from approximately 3-5 log CFU, thus negating the use of
reporting decontamination efficacy in terms of LR.

7. Phytotoxicity evaluations: Visual inspections focused on any changes in leaf or grass
blade color (indicative of disease or stress), leaf loss, and plant growth. Evaluations were
typically performed once per week for 4 weeks following exposure.

2.1 Bench-Scale Efficacy Tests

The bench-scale testing sequence is listed below and shown in Figure 2-1.

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1. Neutralization Volume Determination

2. Inoculation of the test materials

3. Decontamination of the test materials

4. Sampling of the test materials

Figure 2-1. Bench-scale Test Sequence

2.1.1 Sample Neutralization

For the bench-scale testing, initial trials were conducted to identify the optimal volume of
neutralizing agent (DE broth) to use to stop the inactivation of spores at the end of the contact
time. The procedure was performed by inoculating B.g. spores on 5 replicate blank plant and
galvanized steel coupons. Coupons were staged in the spray apparatus and sprayed with the test
decontaminant solution for 3-second intervals every 5 minutes for a total of 10 minutes. After the
15-minute contact time, the sprayed coupons were immediately transferred to sample tubes
preloaded with 10 ml of phosphate-buffered saline with Tween 20 (PBST). The sample tubes
with coupons were vortexed for 10 seconds and then transferred into a 250-ml beaker and
individually titrated using the appropriate titration method to determine FAC (free available
chlorine) or PAA and hydrogen peroxide concentrations. For each sample, the remaining active
ingredient concentration and the volume of DE neutralizing broth required for neutralization
were determined. The volume of DE broth used for neutralization was equal to 1.5 times the
stoichiometric coefficient as determined by average volume required for neutralization. Refer to
Appendix A for further details and results of the neutralization tests.

2.12 Leaf Material Inoculation

Leaf material coupons were inoculated with 5 x 107 B.g. using metered dose inhalers
(MDIs) as described in Section 3.5. (MDI canisters were prefilled to specification by Catalent
Pharma Solutions (Morrisville, NC) with the target organism inside. The canisters were verified
upon receipt by the HSMMD Biodecontaminant Laboratory (or Biolab) to deliver the target
concentration (± 5%) with each actuation.) A set of 3 replicate coupons of plant and galvanized-
steel materials were used to assess each test condition. A set of 3 positive controls and 1 material
blank per material were included for each test. This procedure is further described in Section 3.5.

2.13 Sporiciclal Solutions Preparation

All sporicidal solutions were prepared and analyzed for the active ingredient
concentration and pH. Each prepared solution was transferred to the hand-held sprayer described
in Section 3.2.1. To avoid contamination, each sporicidal solution had its own designated
sprayer.

The sporicidal solutions evaluated included PAA, pAB, and dichlor and are further

-------
described in Section 3.6. Test parameters for each test are detailed in Appendix B. For the PAA-
based sporicidal solutions, the active ingredient concentrations used corresponded to the
concentration used in previous studies, as well as the manufacturer recommended concentrations
for rescue, curative, and preventive treatments of diseased plants.

2.14 Decontamination Process

Coupon stages were removed from the refrigerator at least 1 hour (h) prior to evaluation
and allowed to reach room temperature. A matching set of triplicate dosed plant coupons and a
single blank plant coupon were transferred to a spray test stand. A matching set of triplicate
dosed galvanized-steel coupons and a single blank coupon were transferred to a second spray test
stand. Galvanized steel material was designated as the reference material for this project.

A procedural blank was decontaminated with each material type. For tests that included
multiple concentrations of the same sporicidal solution, the procedural blank coupon was
exposed to the lowest concentration as a worst-case scenario evaluation.

The decontaminant spray was applied using a pressurized hand sprayer, with 3-second
sprays every 5 minutes, for a total contact time of 15 minutes (spray times at 0, 5, and 10
minutes). Figure 2-2 shows the bench-scale spray apparatus with coupon holders (a) and plant
coupons staged horizontally (b).

(a) (b)

Figure 2-2. Bench-scale spray apparatus with coupon holders (a) and plant coupons staged horizontally (b)
2.15 Sample Retrieval

After completing the 10-minute contact with the sporicidal soluti on, leaf coupons were
immediately transferred to sample tubes that were preloaded with 10 ml of PBST and 1.5 times
the previously determined volume of DE broth required for neutralization. For tests that did not
utilize neutralizer (refer to Appendix A), inactivation by the residual active ingredient remaining
on the surface of the coupon was stopped by dilution when introduced to 10 ml of PBST
preloaded in the sample container. Samples were relinquished to the sample processing
laboratory for same-day processing. The runoff collected in the coupon holders was not analyzed
for spores.

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2.2 Small Plant Pilot-Scale Efficacy Tests

For the small plants, decontamination testing was conducted in the large spray chamber
described in Section 3.1. A smaller chamber of similar construction positioned directly beside
the large chamber was used for overnight drying of the small plants. The volume of sporicidal
solutions disseminated, the active ingredient concentration, and contact time were the
independent variables evaluated for decontamination efficacy. The plant categories used for this
investigation include deciduous, evergreen, ground cover, and pine bark. The plants species
used to represent each category are described in Section 3.3.

The test sequence was performed over a period of 3 days as shown in Figure 2-3.

Figure 2-3. Pilot-Scale Test Sequence

Baseline sampling (to assess the presence of B.g. on plants and other materials prior to
spore inoculation) and inoculation were performed on Day 1 of the test sequence. In some cases,
the shape and size of the plants were modified to prevent contact with other surfaces inside the
inoculation and spray chambers. Baseline sampling was performed using a composite sample
approach, i.e., by combining leaf samples from all 4 plants into one sterile 50-ml sample tube
preloaded with 10 mL of PBST (Section 3.7.2). Swab samples were collected from the
inoculation chamber, or large volume aerosol deposition aparatus (LV-ADA) bases and lids prior
to transferring the plants and reference material coupons (RMCs) inside. The LV-ADA
contained 3 plants and 2 sets of triplicate RMCs. Two sets of 3 RMCs were included in each
inoculation. A set of RMCs was positioned in the center of the LV-ADA and the other set was
placed along the side. Galvanized-steel RMCs (18 mm) were used for shrubs, ground cover, and
pine bark. Plants were dosed using a single actuation from the MDI discharged directly into the
LV-ADA. The inoculation procedure for grass was different and is described later in this section.

A singlel8-mm galvanized-steel inoculation control was dosed before and after
discharging the MDI into the LV-ADA. The purpose of the inoculation controls was to assess the
total CFU released per MDI acutation. A single uninoculated plant remained outside the LV-
ADA to serve as the field blank during positive control sampling on Day 2. This plant would be
subjected to the decontamination procedure and sampled as the procedural blank on Day 3.

Field blank and positive control sampling would start Day 2 activities. Samples were
collected in the order of increasing levels of contamination to avoid cross-contamination.
Therefore, the field blank was sampled prior to opening the LV-ADA. The inoculated plants
were removed from the LV-ADA one at a time, sampled as positive controls, then replaced in
LV-ADA until decontaminated.

The decontaminant solution was prepared as described in Section 3.6. The active
ingredient concentration, pH, and temperature were measured prior to use. With the exception of
grass, the plants were decontaminated inside the spray chamber in pairs; two decontamination

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events were required to process all 4 plants. The initial spray event included 2 dosed plants and
the second spray event included the third dosed plant and the procedural blank (formally the field
blank). The flow rate of the sprayer was measured prior to testing, and the time required to
disemininate the prescribed volume of sporicide was determined. Two turntables were used to
rotate the plants during the spray application to distribute the sporicide evenly. The speed of each
turntable was adjusted to complete exactly 2 rotations during the spray time. Figure 2-4 shows
the decontamination setup inside the spray chamber for each plant except grass. Plants used for
this investigation included Gold Dust Aucuba and White Indian Hawthorn.

8

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Figure 2-4. Test setup for spray decontamination for evergreen (a), deciduous (b), tree bark (c), ground cover (d),

grass vegetation categories

After the decontamination procedure was completed and after a 1-h contact time, the
plants were rinsed with deionized (DI) water sourced from the facility's DI water system, then
placed in the drying chamber overnight. The spray chamber was decontaminated with the
sporicidal solution between each decontamination event.

Postdecontamination sampling wrapped up the test sequence on Day 3. Plants were
removed from the drying chamber, one at a time, and sampled in the order of increasing
contamination (i.e., field blank, then dosed plants). With the exception of pine bark, leaf samples
were collected in sterile 50-ml conical sample tubes prefilled with 10 ml of PBST. Water
samples from the DI water system and the backpack sprayer were collected and analyzed for
background contamination.

For one of the small plant decontamination tests, deionized water was used in lieu of a
decontaminant chemical to serve as a control, to provide data on the loss of spores due to
physical removal as opposed to chemical inactivation.

2.3 Sod Decontamination Tests

Sections of grass sod measuring 5-foot length by 2-foot width were purchased from a
local vendor and prepared for decontamination as described in Section 3.3.4.

On Day 1, the sod was cut into 24 12-inch by 12-inch coupons. Inoculations were
performed on sanitized folding tables that were covered with fresh bench liner. Each sod coupon
was placed on the table and covered with 14-inch (in) x 14-in Aerosol Deposition Apparatus

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(ADA). The ADA assembly for sod material was modified from the traditional assembly (Calfee
et al., 2013). AD As were not fastened to the sod, but instead, placed over each sod coupon and
gently pushed down for a close fit around the coupon. The sod coupons were inoculated with
B.g. as described in Section 3.5 and allowed at least an 18-h settling time. Positive control
sampling was completed on Day 2 using the sampling procedure described in Section 3.7.4.

Each sod decontamination experiment (a type of sod decontaminated with a particular
decontaminant) utilized a total of 24 sod coupons. Four of these coupons served as positive
controls and were not sprayed with the decontaminant. Of the remaining 20 coupons, all were
sprayed with the decontaminant, but five served as field blanks and were not inoculated. The
remaining 15 sod coupons were divided into five sets of three, using increasing volumes of
sporicide for each set.

The greenhouse was used as the test facility for the initial test and the COMMANDER
was used thereafter. An 84-in long x 120-in wide polyvinyl chloride (PVC) pond liner was used
to line the floor of both the greenhouse and COMMANDER. A coupon was removed from the
ADA, transferred by hand to the test facility, and positioned on the PVC liner. The prescribed
spray procedure was applied and then the test sample was covered with a sterile plastic bin for
protection from overspray. The procedural steps were repeated for the next test sample, one at a
time, until all were processed.

All samples were relinquished to the Biolab for enumeration. For sampling of the sod, a
total of 10 grass blades were cut from each sod coupon with sterile snips, then aseptically
transferred to a 50-ml sterile conical tube as described in section 3.7.4.

2.4 Phytotoxicity Tests

Simple and expedient phytotoxicity evaluations were performed on plants using spray
conditions that were determined to be efficacious in the pilot-scale tests. (More robust
phytotoxicity evaluations were beyond the scope of the study, due to time and funding
constraints.) The prescribed spray procedures were identical to the pilot-scale spray procedures
previously described in Section 2.2. Test parameters for each test are detailed in Appendix C.

Phytotoxicity evaluations were performed as simple visual inspections and focused on
changes in leaf or grass color, leaf loss, and plant growth. Evaluations were performed every
week for the four weeks following exposure to the sporicidal solution spray procedure. Between
evaluations, the plants were stored and watered twice a week in the greenhouse described in
Section 3.1. (Plants in gallon-size pots or more (shrubs and blueberry trees) received
approximately 1500 ml of tap water. Smaller plants (ground cover and grass) received
approximately 750 ml of tap water. The overall percent changes from baseline conditions (or
week 1 for shed leaves) to week 4 were determined for plants sprayed with water and then
compared to plants sprayed with each decontaminant, to arrive at an assessment of phytotoxic
impacts.

Evaluations included quantifying the total number of leaves showing evidence of
damage, i.e., fading or changes in color, the total number of leaves shed, and the height of the

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plant. To qualify as "showing decay", the affected area of a leaf needed to be approximately
larger than a Vi in diameter (equivalent to a standard hole punch). Digital photographs for each
plant were taken at 2 different angles for comparison.

For the sod phytotoxicity evaluations, each sod coupon was sprayed with a target of 100
ml of the decontaminant, an amount based on the higher end of the spray quantities used for the
efficacy tests. As a quick and "low-tech" approach to assess impacts, the Leaf Doctor mobile
phone application was used to digitally distinguish between diseased and healthy plant tissue.
(Pethybridge, S.J., and Nelson, S.C. 2015). (Note, this "Leaf Doctor" approach for assessing sod
damage is a method under development, and so no data quality indicators are provided.) Briefly,
a digital photo of the sod was uploaded into the application. Through touchscreen input, the user
would provide the app with color samples of healthy sod by touching the photo to specify up to 8
various colors of healthy grass. An algorithm evaluated the color of each pixel, designated them
as healthy or diseased, and quantified the diseased percentage. Physical growth was evaluated by
the maximum height at the center of the sod coupon. The center of the coupon was located, and a
tape measure used to determine the height of the tallest blade of grass.

The plants included in the phytotoxicity testing were as follows: White Indian Hawthorne
(Rhaphiolepsis indica), Goldilocks Creeping Jenny (Lysimachia nummularia), Blueberry Shrubs
(Vaccinium corymbosum), Tall Fescue sod (Festuca), and Zoysia (Zoysia) sod. The small plants
were rinsed with water following the spraying of the decontaminants, and the sod was not rinsed
- consistent with decontamination efficacy procedures. The exception to this was that two sets
of blueberry shrubs were evaluated for phytotoxicity, with and without the water rinse, to
evaluate the effect of rinsing. For each plant tested, four replicates were used for each
decontaminant and four replicates were used as controls (sprayed with DI water only). An effort
was made to purchase each set of plants at the same vendor at the same time, when possible.

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3 Materials and Methods

3.1 Test Facilities

All work described in this report was performed in the laboratory facilities located at the
EPA RTP, NC, campus. Indoor facilities were environmentally controlled and under slight
negative pressure to prevent outflow into the adjoining offices.

Tasks related to bench-scale testing (e.g., coupon inoculation, spray application, and
sampling) were completed in a Class II, Type BI biosafety cabinet (BSC) (NuAire; Plymouth,
MN), and a walk-in chemical hood (Safeaire; Hamilton Laboratory Solutions; Manitowoc, WI ).
The BSC was used for microbiological manipulations (inoculation and sampling) of 18-mm
material coupons.

Bench-scale spray applications were completed under a chemical hood measuring 8 ft
high by 5 ft wide by 3 ft deep. Airflow is filtered and then vented directly into the air handling
system of the facility where it is recirculated throughout the building. Tasks were performed on a
solid stainless-steel bench placed inside the hood.

Prior to use, the BSC and chemical hood surfaces were sanitized using the secondary
work surface sanitation procedure described in Section 3.4. Both hoods were routinely inspected
to ensure proper functionality as required by the EPA's Safety, Health, and Environmental
Management (SHEM) Program Office and used within their certification dates. Figure 3-1
shows the BSC (a) and the walk-in chemical hood (b).

Figure 3-1. Bench-scale test facilities (a) biosafety cabinet and (b) chemical hood

For the small plant decontamination testing, the spray chamber has dimensions of 4 feet
(ft) high by 4 ft wide by 4 ft deep; the spray chamber is of solid stainless-steel construction with
the excepti on of the front and top surfaces, which were fabricated from clear acrylic plastic. The

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front surface acrylic section was a door that allowed full access to the inside of the spray
chamber from outside. The acrylic surfaces on the door contained 3- and 4- in diameter circular
openings evenly positioned horizontally across the top 1/3 to allow various types of sprayer
nozzles and power sources into the spray chamber once the door is sealed. The inverted pyramid-
shaped bottom surface was modified to provide a flat, stable surface for the plants during testing.

The spray chamber was fitted with connections allowing air to exit via a connection to
the facility's air handling system. For the safety of the test personnel, during the application of
the spray decontamination procedure, the spray chamber blast gates were opened to maintain
constant negative pressure inside the spray chamber. The blast gates were maintained in a closed
position at all other times. The small plants were placed on two electric turntables located inside
the spray chamber to promote uniform spraying. Figure 3-2a shows the interior of the spray
chamber and Figure 3-2b shows a plant on a turntable.

Figure 3-2. Spray chamber (a) exterior and (b) and interior

Between phytotoxicity evaluations, plants were stored in a 6 H -ft high by 6 M -ft wide by
8 % -ft long fabricated greenhouse (p/n 47712; Harbor Freight Tools; Calabasas, CA) made of
UV-coated polycarbonate panels for sunlight diffusion. The greenhouse was constructed with an
aluminum frame, two roof vents for air circulation, sliding door for access, and was mounted on
a concrete floor. The greenhouse was located outdoors in full sunlight. Heavy duty steel utility
shelving (Kobalt; New York, NY) was placed inside the greenhouse to increase the storage
capacity. Exterior (a) and interior (b) views of the greenhouse are shown in Figure 3-3.

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Figure 3-3. Greenhouse (a) exterior and (b) interior

3.2 Test Equipment
3.2.1 Hand-held Sprayer

The RL Flo-Master model 56HD (Root-Lowell Manufacturing Co., Lowell, MI)
pressurized hand sprayer was used for bench-scale spray applications, with the leaf coupons
staged in the test stand. The adjustable spray nozzle could provide spray ranging from a fine mist
to a solid stream. For this investigation, the spray was applied as a fine mist. The sprayer had a
capacity of 4 pints (1.9 liters (L)), and the nozzle and tank were made of chemical-resistant high-
density polyethylene (HDPE). A designated sprayer was used for each sporicidal solution to
prevent interactions with another residual chemical. Each sprayer was triple rinsed with
deionized water after each used to maintain functionality. Figure 3-4 shows the RL Flo-Master.

Figure 3-4. RL Flo-Master® pressurized hand sprayer

3.2.2 Electrostatic Sprayer

An air-assisted electrostatic sprayer (SC-ET HD Model Number, ESS, Electrostatic
Spraying Systems ESS, Watkinsville, GA) was used in this investigation for most of the small
plant decontamination tests, and a few of the sod decontamination and phytotoxicity tests.
Towards the end of the study, the ESS malfunctioned and was no longer used.

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The manufacturer's intended use for the SC-ET HD electrostatic sprayer is light-duty
quick disinfection and sanitization applications. The sprayer is compatible with most
conventional chemicals. It is equipped with a patented MaxCharge technology electrostatic spray
gun that delivers droplets with a volume median diameter of 30 to 60 urn. This sprayer has a
nominal flow rate of 1 gallon per hour, and the spray range is up to 8 feet. Figure 3-5 shows the
SC-ET HD electrostatic sprayer.

Figure 3-5. SC-ET HI) electrostatic sprayer

3.2.3 Backpack Sprayer

The backpack sprayer (shown in Figure 3-6) was another spray device used in this
investigation. A SHURilo SRS 600 ProPack is a rechargeable electric backpack sprayer
(SHURflo, Cypress, CA) that features a 4-gallon capacity tank and was maintained at a pressure
of 35 pounds per square inch (psi) and a flow rate of approximately 1 L/min during its use.

Figure 3-6 SHURflo ProPack rechargeable electric backpack sprayer

15

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3.3 Plant Material and Coupon Preparation

The vegetation used for this investigation included deciduous trees (leaves shed at
maturity), evergreen shrubs (foliage remains green and functional for more than one growing
season), ground cover, and grass. Table 3-1 lists all the plants used for this effort.

Table 3-1. Detailed Summary of Vegetation

Plant Name

Vendor

Plant Matrix

Test Matrix

Test Material

Southern Magnolia

(Magnolia grandiflora)

Field Vegetation

Deciduous

Bench-scale

decontamination

efficacy

18-mm leaf coupons

Bradford Pear (Pvrus
calleryana)

Field Vegetation

Deciduous

Bench-scale

18-mm leaf coupons

English Ivy (Hedera helix)

Field Vegetation

Ground cover

Bench-scale

18-mm leaf coupons

Manhattan Euonymus

(Euonymus kiaitschivicus)

Lowe's

Evergreen

Bench-scale

18-mm leaf coupons

Gold Dust Aucuba (A ucuba
japonica)

Lowe's

Evergreen

Bench-scale

18-mm leaf coupons

White Indian Hawthorn

(Rhaphiolepsis indica)

Lowe's

Evergreen

Bench-scale /Pilot-
scale

18-mm leaf coupons /
3.5 gal-potted plant

Blueberry Shrub (Vaccinium
corymbosum)

Lowe's

Deciduous

Pilot-scale /
phytotoxicity

3.5 gal-potted plant

Blueberry Shrub

Home Depot

Deciduous

Pilot-scale /
phytotoxicity

3.5 gal-potted plant

Pine Tree Bark (from Pinus
taeda)

Lowe's

Evergreen

Pilot-scale

Full sized nuggets

Goldilocks Creeping Jenny

(Lvsimachia nummularia)

Lowe's

Ground cover

Pilot-scale

6,1-pint potted plants

Nena Ivy (Hedera helix)

Lowe's

Ground cover

Pilot-scale

6,1-pint potted plants

Japanese Spurge

(Pachysandra terminalis)

Lowe's

Ground cover

Pilot-scale

6,1-pint potted plants

Zenith Zoysia Sod (Zovsia)

Super Sod of Cary

Grass

Pilot-scale /
phytotoxicity

12-inby 12-in coupons

Tall Fescue Sod (Festuca)

Super Sod of Cary

Grass

Pilot-scale /
phytotoxicity

12-inby 12-in coupons

Bermuda Sod (Cvnodon
dactylon)

Super Sod of Cary

Grass

Pilot-scale

12-inby 12-in coupons

Decontamination efficacy testing includes bench-scale and pilot-scale tests

3.3.1 Preparation of 18-mm Leaf Material

Bench-scale tests included 18-mm plant material coupons and reference material coupons

16

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(RMCs). The 18-mm coupons were assembled by punching 18-mm diameter circular discs from
plant leaves and galvanized steel, then affixing each disk to an aluminum 18-mm scanning
electron microscope (SEM) pin mount (Ted Pella; Redding, CA; p/n 16199) using an adhesive
tab (Ted Pella, p/n 16082). Plant leaf coupons were visually inspected to exclude those with
visible damage or decay prior to use. Figure 3-7 shows examples of (a) punched leaves, (b) plant
disks, and (c) finished 18-mm coupons.

Figure 3-7. Punched leaves (a), plant disks (b), and finished 18-mm coupons (c)
3.3.2 Full-sized Plant Preparation

Small full-sized plants used for decontamination efficacy and phytotoxicity testing such
as shrubs, ground covering, grass, and pine bark were modified as needed to fit the size
limitations of the test facilities. While these modifications were unavoidable, every effort was
made to maintain their natural shape and appearance. Shrubs were trimmed not to exceed 32-
inches in height (measured from the surface of the soil) and 24 in in width (measured from the
center of the pot). Occasionally, a shrub would be positioned off-center in the pot causing a large
portion of the foliage to exceed the 24-in width requirement. To avoid trimming an excessive
amount of foliage, the branches were recentered by tying the main branch to a stake placed
against the side of the pot. The shrubs were maintained in the disposable plastic pots (provided at
purchase) for the duration of testing and evaluation. Figure 3-8 shows the use of a stake and tie
to reposition the branches of a shrub over the center of the pot.

Figure 3-8. Shrub branches repositioned to the center of the pot

Ground-covering plants were purchased in 1 -pint disposable pots. Six pots were grouped

17

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together to represent a single test replicate using the store-provided carrying trays. Grouping the
individual pots increased the foliage surface area and improved the representation of the natural
profile of the plant. The final dimensions of ground-covering plants did not exceed 24 in length
and 16 in width. Figure 3-9 provides an example of the ground cover pots and the carrying tray
used to group them together.

Figure 3-9. Individual ground cover plants with carrying tray

3.3.3 Tree Bark Preparation

Timberline pine bark nuggets (Oldcastle Lawn and Garden; Atlanta, GA) were placed in
a single layer covering the bottom of stainless-steel test trays, fabricated in-house with
dimensions for each of 12-in long by 12-in wide. The bottom surface of the trays was made of
stainless-steel wire mesh to allow liquid to drain away from the bark during application of the
spray procedure. The draining trays were part of an overall effort to ensure data integrity, in this
case, by preventing residual spore inactivation from extended contact with pooling liquid
sporicides. Figure 3-10 shows pine bark positioned in the test trays.

18

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Figure 3-10. Pine tree bark in stainless-steel test tray
3.3.4 Grass (Sod) Preparation

Sod was received from a local vendor in rolls measuring 5-ft long by 2-ft wide and were
prepared for testing within 3-4 days of being harvested. Only enough material needed for each
test was ordered beforehand. Sod coupons were prepared by cutting the roll into 12-in long by
12-in wide coupons. Once cut, the sod coupons were ready to use for decontamination efficacy
testing.

For phytotoxicity testing, the sod coupon was placed in a 14-in by 14-in polypropylene
storage container prefilled with approximately 0.11 cubic feet of "Soil Humus Compost" (Super-
Sod; Fort Valley, GA). Figure 3-11 shows 12-in long by 12-in wide sod coupons used for
decontamination efficacy and Figure 3-12 shows sod coupons prepared for phytotoxicity testing.

Figure 3-11. Sod material cut into 12-in by 12-in coupons

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Figure 3-12. Front (a) and top (b) view of a sod coupon prepared for phototoxicity testing and evaluation

3.4 Sterilization, Sanitization, and Cross-containination Prevention

Adequate sterilization of all test materials and equipment was critical in preventing cross-
contamination during the whole testing sequence (inoculation, decontamination, and sampling).
The sterilization method used for materials and equipment was determined by material
compatibility and size.

The LV-ADA, used for inoculation of the target plants and described in Section 5, and
the stainless-steel trays, used for positive controls, were sterilized using vaporous hydrogen
peroxide, VHP® (STERIS VHP® ED1000 generator, STERIS Corp., Mentor, OH, USA) using a
250 ppm 4-h cycle.

Ethylene oxide (EtO) sterilization cycles were performed on small equipment such as
MDIs and 18-mm coupons and stages in the EOGas™ 333 Cabinet (Anderson Sterilizers, Haw
River, NC). Test materials and equipment were prepared for sterilization cycles using the
materials provided in the EOGas kit (AN-1005 and AN-1006) which included a sterilization bag,
a 100% EtO cartridge, a Humidichip for humidification, and a chemical indicator (dosimeter) for
visual verification of cycle efficacy. Upon completion of the 18-h sterilization cycle, the
sterilization bag and its contents were removed and stored until use.

Autoclave sterilization cycles were completed in the Biolab with a STERIS Amsco
Century SV 120 Scientific Pre-vacuum Sterilizer (STERIS, Mentor, OH). Only autoclave-safe
test materials (heat resistant up to at least 121 °C) were sterilized using this method. Approved
materials were processed using 1-h gravity cycles at the sterilization temperature of 121 °C.
Liquid cycles (30-minutes) were used to sterilize small volumes (less than 2 L total) of DE broth.

Table 3-2 lists the sterilization methods used for test materials and equipment.

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Table 3-2. Sterilization Methods for Test Materials and Equipment

Material/ Equipment

Process Used

Sterilization Method(s)

MDI actuator

Bench- and pilot-scale inoculations

EtO

Large-volume ADA

Pilot-scale inoculations

VHP

Stainless-steel test tray

Pilot-scale inoculations and spray
decontamination

VHP

18-mm coupon stages

Bench-scale inoculations

EtO

12-in long by 14-in wide stainless-
steel reference coupons

Pilot-scale inoculations

Autoclave

18-mm galvanized steel RMCs

Pilot-scale inoculations

EtO

DE Broth

Bench-scale sample neutralization

Autoclave

Sanitation procedures were used to prepare secondary work surfaces (i.e., surfaces that
were not in direct contact with samples). Secondary work surfaces were sanitized using portions
of the prepared sporicidal solution intended for testing or using Dispatch bleach wipes (Caltech
Industries, Inc.; Midland, MI). All nonsample surfaces were maintained wet for 15 minutes,
rinsed with DI water, then dried with 70% ethanol solution (the large spray chamber was not
dried with ethanol). Table 3-3 lists the sanitation methods used for test materials and equipment.

Table 3-3. Sanitization Methods for Test Materials and Equipment

Material/ Equipment

Process Used

Sanitization Method(s)

Chemical hood

Bench-scale decontamination spray

Dispatch wipe

BSC

Bench- and pilot-scale spray
decontamination

Dispatch wipe

Turntable

Pilot-scale spray decontamination

pAB, dichlor, PAA

Large spray chamber

Pilot-scale spray decontamination

pAB, dichlor, PAA

Living plant materials were not subjected to sterilization or sanitization procedures prior to
testing. Material blanks were collected from each plant specimen prior to testing to establish
baseline contamination levels of B.g. spores prior to dosing. There were no reports of material
blank samples testing positive for the target organism for this investigation.

-------
3.5 Bacillus Spore Preparation and Surface Contamination

The test organism for coupon inoculation was a powdered spore preparation of B.g.,
mixed with silicon dioxide particles obtained from the U.S. Army Dugway Proving Ground Life
Sciences Division. The preparation procedure is described in Brown et al. (2007). After 80 to
90% sporulation, the suspension was centrifuged to generate a preparation of approximately 20%
solids. A preparation resulting in a powdered matrix containing approximately 1 x 1011 viable
spores per gram was prepared by dry blending and jet milling the dried spores with fumed silica
particles (Degussa, Frankfurt am Main, Germany).

This investigation included several sample types with varying levels of contamination to
serve specific analytical purposes. Table 3-4 details each type of sample analyzed in this study
and the corresponding level of contamination.

Table 3-4. Surface Contamination Levels for Each Sample Type

Sample Material

Purpose

Level of
Contamination

Process

Material Blank

Demonstrated the initial level of
contamination of the material

Uninoculated

Not exposed to laboratory environment
Not exposed to the test treatment

Laboratory Blank

Demonstrated the level of
contamination in the test environment

Uninoculated

Exposed to the laboratory environment
Not exposed to the test treatment

Procedural Blank

Demonstrated the degree of cross-
contamination to which samples were
subjected

Uninoculated

Exposed to the laboratory environment
Exposed to the test treatment

Test Material or
Coupon (TC)

Demonstrated level of remaining
viable spores following application of
the decontamination procedure

Inoculated

Exposed to the laboratory environment
Exposed to the test treatment

Positive Control
(PC)

Demonstrated initial spore loading

Inoculated

Exposed to the laboratory environment
Not exposed to the test treatment

RMC

Demonstrated deposition consistency
for each test set and throughout the
study using a well-established
reference material (stainless steel)

Inoculated

Exposed to the laboratory environment
Not exposed to the test treatment

Test and positive control coupons were inoculated with B.g. spores using an MDI with a
procedure described by Calfee et al. (2013). Briefly, each coupon was contaminated
independently with a separate custom-made, pyramid-shaped ADA designed to overlay and
cover a specific area to be contaminated (18-mm diameter circular coupons, 14-in x 14-in square
surfaces, and full trees or shrubs). The MDI and the actuator were positioned in the opening
located at the ADA's top center. The MDI was discharged a single time to inoculate the target
surface area. The spores were allowed to settle onto the coupon surface for at least 18 hours. The
ADA was removed immediately before sampling.

The accuracy and precision of inoculations for each experimental batch were determined
by analysis of the stainless-steel coupons (18-mm or 14-in x 14-in). Each coupon was placed

-------
inside a chamber and positioned in front of an MDI canister containing B.g. spores suspended in
ethanol solution and HFA-134A propellant gas. The MDI was situated inside an actuator so that
each time the actuator was depressed, a repeatable number of spores was deposited on the
coupon. (Lee et all., 2011). The MDI actuator was a small plastic tube into which the MDI was
inserted (Figure 3-13). Each MDI was charged with a volume of spore preparation plus
propellant sufficient to deliver 200 discharges of 50 microliters (|iL) per discharge. MDI use was
tracked so that the number of discharges did not exceed 200. Additionally, MDIs selected for
testing were required to weigh more than 10.5 grams. MDIs weighing less than 10.5 grams were
retired and no longer used. Test and positive control coupons were inoculated a maximum of 48
h before decontamination.

Figure 3-13. MDI and MDI Actuator

For bench-scale tests, material coupons were inoculated with 5 x 10 ' B.g. MDI canisters
were prefilled with the target organism to specification by Catalent Pharma Solutions
(Morrisville, NC). A set of 3 replicate coupons of plant and galvanized-steel materials were used
to assess each test condition. A set of 3 positive controls and 1 material blank per material was
included for each test. Coupon inoculation was sequenced so that the first sample for each
material was completed first, followed by the second replicate set, then the third replicate set. For
example: Material A - 01, Material B 01, Material A - 02, Material B - 02, Material A - 03,
Material B - 03. Inoculations were completed in this order to minimize possible variances in the
performance of the MDI as inoculations progressed. Coupons were typically inoculated the day
prior to decontamination testing and stored in the refrigerator overnight.

Pilot-scale inoculations utilized a LV-ADA that was fabricated inhouse of high-density
polyethylene material to inoculate full-scale shrubs and small trees from a 200-gallon trilayered
water storage tank (Rotoplas; Edgecliff Village, Texas). The usable space inside the ADA
measured approximately 40-in diameter and 31-in high. Plants and RMCs were transferred into

-------
the ADA through the modified lid, the severed top M section, of the tank. The LV-ADA lid was
retrofitted with an MDI actuator adaptor (fabricated in-house) made of plexiglass, positioned in
the center.

The MDI was placed inside the actuator adaptor and activated to dispense the spores.
Figure 3-14 shows plant materials positioned inside the LV-ADA (a and b), the sealed LV-ADA
(c), and the actuator adaptor on the lid (d).

Figure 3-14. Plants and bark trays inside LV-ADA (a and b), sealed LV-ADA (c), and MDI adaptor (d)

For the sod inoculation, each 12-in by 12-in sod section was positioned under a 14-in by
14-in ADA, then inoculated with a target spore concentration of 1 x 1()7 CFU B.g. by discharging
a single MDI actuation into each ADA. Three 14-in by 14-in stainless-steel coupons were used as
inoculation controls. Inoculation control coupons were strategically inoculated to receive the first,
middle, and last actuation from the MDI. After inoculation, coupons were allowed at least 18
hours for settling of spores prior to testing. After the 18-h settling period, AD As were removed

24

-------
and the coupons were sampled according to the procedure detailed in Section 3.7.4. AD As were
removed immediately prior to sampling each coupon to avoid cross-contamination.

3.6 Decontamination Solutions

Four decontamination solutions were selected to evaluate their sporicidal effectiveness
against contamination by bacterial spores. Two PAA-based decontaminant solutions that were
evaluated for this effort include prepared solutions of Jet-Ag 15% and Oxidate 2.0. Two chlorine-
based solutions that were evaluated include pAB and dichlor. A summary of each of the
decontamination solutions is discussed below.

Jet-Ag 15% (EPA Registration No. 81803-9) is marketed as an agricultural fungicide,
bactericide, and algicide by the manufacturer. The stock solution is 22.0% hydrogen peroxide and
15.0%) peracetic acid. The Jet Ag 15% product label recommends curative and preventive
treatments be applied with prepared dilutions of stock in deionized water at ratios of 1:246 and
1:533, respectively. For the bench-scale scoping tests, PAA concentrations ranged from
approximately 0.04-0.6%. For the small plant decontamination tests, PAA concentrations ranged
from approximately 0.1-0.6 %. For the sod decontamination and phytotoxicity tests, the PAA
concentration was targeted at 0.5%.

Oxidate 2.0 (EPA Registration No. 70299-12), also marketed as a bactericide and
fungicide, is 21.1% hydrogen peroxide and 2.0%> peracetic acid. According to the Oxidate 2.0
product label, rescue, curative, and preventive treatments correspond to dilutions of 1:40, 1:100,
and 1:400, respectively. This brand of PAA was used only in the bench-scale tests (to reduce the
number of test variables), with concentrations of PAA ranging from 0.02 - 0.5%.

The PAA analytical method was applicable for PAA solutions (1% to 35% PAA by weight)
containing H2O2. The H2O2 content was first determined by an oxidation reduction titration with
eerie sulfate reagent. After the endpoint of this titration is reached, an excess of potassium iodide
is added to the sample. The hydroiodic acid formed in acidic media reacts with the PAA to liberate
iodine. A standard sodium thiosulfate (STS) reagent is used to titrate the liberated iodine. The
endpoint of this titration is used to calculate the PAA content.

The pAB decontaminant is a pH amended sodium hypochlorite solution, prepared using household
bleach, water, and 5% acetic acid. This is a FIFRA-registered disinfectant with reported activity
against various biological and chemical agents. PAB was only used in the bench-scale tests, with
FAC levels ranging from approximately 6,000 - 6200 ppm and pH levels of around 6.3 - 7.0.

Dichlor, commonly known as chlorinating granules, is used for daily chlorination of small
pools or spas with high chlorine demand. (Chlorine availability for dichlor solutions typically ranges
between 56 - 62%, depending on the formulation). Dichlor solutions were prepared in a borosilicate
volumetric flask by dissolving the granules in deionized water. The prepared solution was stored at
room temperature and deployed within 4 hours of preparation.

A HACH high-range bleach test kit (CN-HRDT), an iodometric titration method, was used
to determine FAC levels of chlorine-based sporicides per HACH method 10100. Briefly, a 5-mL
sample was transferred to a 250-mL Pyrex flask and diluted to 150 mL with DI water. Acid reagent
(HACH p/n 104299) and one potassium iodide (HACH p/n 2059996) pillow were added to the

-------
sample and allowed to dissolve. Starch indicator (HACH p/n 34932) was added for endpoint
detection during titration. With the solution on the stir plate, the HACH digital titrator was used to
deliver 2.26 N STS to a colorless endpoint.

Table 3-5 lists the information for each decontaminant, including the manufacturer/vendor
and active ingredient(s).

Table 3-5. Decontaminant Information

Decontaminant Manufacturer/Vendor Active Ingredient(s)	Test Series

Jet-Ag 15%

Jet Harvest Solutions
Longwood, FL

Peracetic Acid, Hydrogen
Peroxide (15% and 22%,
respectively, in stock
solution)

•	Decontamination Efficacy
(Bench-Scale and Pilot-
Scale)

•	Phytotoxicity

Oxidate 2.0

Biosafe Systems
East Hartford, CT

Peracetic Acid, Hydrogen
Peroxide (2.0% and 27.1%,
respectively, in stock
solution)

• Decontamination Efficacy
(Bench-scale only)

pH-Adjusted
Bleach

Made with Clorox
Concentrated Germicidal
Bleach p/n 174273
Lowe's, Morrisville, NC

Free available chlorine

• Decontamination Efficacy
(Bench-scale)

Dichlor

Brilliance for Spas
B&G Builders, Durham, NC

Sodium dichloro-s-
triazinetrione dihydrate (99%)

•	Decontamination Efficacy
(Bench-scale and Pilot-scale

•	Phytotoxicity

26

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3.7 Sampling and analysis
3.7.1 Bacterial Spores Analysis

Numerous microbiological samples and assays were used to characterize bacterial spore
presence or absence for each test. Samples or assays were either quantitative (providing a
numerical result) or qualitative (indicating either presence or absence of bacterial growth).
Laboratory blanks of items such as growth media and sampling materials were also employed in
each experiment to check for aseptic conditions.

Following spore extraction procedures, the liquid solution used to extract spores from
each coupon was 10-fold serially diluted and plated. When fewer than 30 CFU were detected on
plates from undiluted extract, a portion of the remaining extraction solution was filter plated
using one or two larger volumes of the extract.

CFU counts per sample were calculated by multiplying the number of counted colonies
by the dilution factor. Efficacy is defined as the extent (by log reduction or LR) to which the
agent extracted from the material surface after the treatment with the decontamination procedure
is reduced below that extracted from a similar material coupon before decontamination. Efficacy
was calculated for each material type within each combination of fogging parameters (i) and test
material (j) as follows:

£ (log 10C,e)

w,jk = "'"i	'"H'VJ (Equation 3_1>

lJc

where:

Cijc is the number of viable organisms recovered from C control coupons for the zth

decontamination procedure and/h test material,

Nijc is the number of control coupons for the zth decontamination procedure and/h test

material, and

Nijk is number of viable organisms recovered on the klh replicate test coupon for the zth

decontamination procedure and/h test material.

If no viable spores were detected, then the detection limit of the sample was used, and the
efficacy reported as greater than or equal to the value calculated by Equation 2-1. The detection
limit of a sample depends on the analysis method and might vary. The detection limit of a plate
was assigned a value of 0.5 CFU, but the fraction of the sample plated varies. For instance, the
detection limit of a 0.1-mL plating of a 20-mL sample suspension is 100 CFU (0.5 CFU/0.1 mL *
20 mL), but if all 20 mL of the sample is filter-plated, the detection limit would be 0.5 CFU.

The standard deviation of LRi is calculated by Equation 2-2:

SDn,r

^S ,	N

2l(v )

N'Jk 1 (Equation 3-2)

27

-------
where:

SDr,i is standard deviation of ?//,

LRij is the average log reduction of spores on a specific material surface, and

Xikj is the average of the LR of each from the surface of a decontaminated coupon (Equation

2-3):

X (Z^CFU^Nc-^CFU^ m

r _ k c=i	_		 (Equation 3-3)

N*

where:

represents the "mean of the logs," the average of the logarithm-
^}og(.CFUr)! Nra transformed number of viable spores (determined by CFU)

recovered on the control coupons:

C is control,

j is coupon number,

Nc is number of coupons (l,j), and

CFU is number of CFU on the surface of the klh
decontaminated material surface.

3.7.2 Extractive Sampling of Plant Material

For purposes of collecting samples from each small plant in a uniform manner, the plant
foliage was divided into four quadrants and a center section: Ql, Q2, Q3, Q4, and CT,
respectively. One sample was collected from each area for a total of 5 samples per plant. Each
sample was a composite, consisting of 3 leaf punches, taken from leaves at the bottom, middle
and top portion of he quadrant of the plant being sampled. (Figure 3-15 a)

To ensure consistent quantities of plant material were collected for each plant sample, the
hole punches from the leaves were performed using the EK Tools 3/4" Circle Punch (EK Success,
Ltd., Clifton, NJ) shown in Figure 3-15 b.

28

-------
(a)	I	(b)

Figure 3-15. Diagram of extractive sampling areas - Top view of the plant (a) and the leaf hole punch tool (b)

For each sample, a sterile hole punch tool was used to cut leaf material and have it drop
directly into the sample container. A single leaf was removed from the plant prior to hole
punching in an effort to minimize unnecessary contact with the plant foliage. With a fresh pair of
sterile gloves, a single leaf was removed by the stem from the predefined sample area. A 3A"
(0.635 cm) circle punch was taken from the center of the leaf and aseptically transferred to a
prelabeled sample tube containing 10 mL of PBST solution. A single hole punch was collected
per leaf. Figure 3-16 shows the punching of a leaf (a) and multiple leaf punch samples collected
in a sample tube (b).

Figure 3-16. Punching of a leaf (a) and punches collected in a sample tube (b)

3.7.3 Tree Bark Sampling

Timberline pine bark nuggets (Oldcastle Lawn and Garden, Atlanta, GA) were placed in
a single layer covering the bottom of five 12-in by 12- stainless-steel trays constructed in-house
with stainless-steel mesh bottoms. Three trays were designated as test coupons (TCs), one as a
positive control coupon (PC), and one as a laboratory/procedural blank (FB/PB). The mass of
bark in each box was determined by measuring the pre- and postweight of the tray and recorded.
The negative control sample was collected directly from the bulk material container by

29

-------
aseptically transferring a single bark nugget to 1-L sterile Nalgene bottle. The 3 TC trays, one PC
tray, and six 18-mm RMCs (3 placed in the center and 3 on the side) were transferred to the LV-
ADA and dosed with 1 X 107 B.g. using the procedure outlined in Section 2.5, then allowed to
settle for a minimum of 18 h.

After the settling period, the PC tray was removed from the ADA and 5 bark nuggets
sampled individually. The natural variability was significant in the shape and size of the bark
nuggets. To promote uniform sample selection, a'%% -in by 2144n rectangular template was
used to assist with sampling comparable sized nuggets. An attempt was made to choose a bark
nugget close to but no larger than the template. A total of 5 bark nuggets (one collected from
each side of the tray and the center) were sampled from each tray by aseptically transferring a
single nugget to a sterile sample container. Post-decontamination TC sampling was performed
using the same procedure. Figure 3-17 shows the sampling setup for bark.

WW,

I \>

Figure 3-17. Pine bark nuggets sampling set-up

3.7.4 Sod Sampling

Sod materials were sampled starting with first test replicate (01) and ending with the third
replicate (03). The procedural blank was sampled between replicates 02 and 03 to monitor the
levels of cross-contamination. Results for procedural blanks are summarized in Section 6.3.

A single composite sample was collected for each sod coupon. The sample was
comprised of 10 blades of grass; two blades were cut from each corner and the center of the
coupon. The 10 blades were aseptically transferred to one 50-ml empty sterile conical tube and
relinquished to the processing laboratory the same day of collection .

In the processing laboratory, 10 ml of phosphate buffer soluti on with 0.05% Tween 20
(PBST) was added to each sample and sonicated for 10 minutes followed by vortexing for 2
minutes continuously. Inoculation control samples were spiral plated at the (-2) dilution in
triplicate until three plates were within countable range for spiral plates (>30 CFU/plate).

Sod positive control samples were spread-plated at either the (-1), (-2), or (-3) dilution
with various aliquots in triplicate to obtain three plates within the countable range for spread

-------
plates (30-300 CFU/plate).

The wipe field blank was filter-plated with 1 ml/remainder aliquots. The sod negative
was filter plated with 500 |il/l ml due to potential background contamination. All filter plating
was performed on Pall filters #4852. All sod samples required heat treatment at 80 °C for 20
minutes due to potential background contamination. Following plating, plates were incubated at
35 ± 2° C overnight and enumerated the following day.

Cross-contamination prevention measures included the use of sterile metal snips and
forceps to cut and transfer the grass blades, fresh bench liner on the sampling surface between
coupons, and donning new clean gloves between coupons.

31

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4 Quality Assurance/Quality Control

Quality Assurance/Quality Control (QA/QC) procedures were performed in accordance
with the Quality Management Plan (QMP) and the Quality Assurance Project Plan (QAPP). The
QA/QC procedures and results are summarized below.

4.1 Sampling, Monitoring, and Equipment Calibration

Approved operating procedures were used for the maintenance and calibration of all
laboratory equipment. All equipment was verified as being certified calibrated or having the
calibration validated by the EPA's Metrology Laboratory at the time of use. Standard laboratory
equipment such as balances, pH meters, biological safety cabinets and incubators were routinely
monitored for proper performance. Calibration of instruments was done at the frequency shown
in Table 4-1 and 4-2. Any deficiencies were noted, and any deficient instrument was adjusted to
meet calibration tolerances and recalibrated within 24 h. If tolerances were not met after
recalibration, additional corrective action was taken, including recalibration or/and replacement
of the equipment.

Table 4-1. Sampling and Monitoring Equipment Calibration Frequency

Equipment Calibration/Certification

Expected

Tolerance

pH and

temperature

sensor

Measurements of pH and temperature of the decontaminant solutions were
performed each day of testing using a calibrated pH meter (Oakton
Acorn™ pH 5, Oakton Instruments, USA). The temperature sensor
included with the pH meter was factory-calibrated and checked monthly by
comparison of the displayed value to a National Institute of Standards and
Technology (NIST)-certified thermometer.

±5%

Stopwatch

Compare against NIST Official U.S. time once every 30 days at
htti)s://www.time.20v/

± 1 min/30
days

Table 4-2. Analytical Equipment Calibration Frequency

Equipment

Calibration
Frequency

Calibration Method

Responsible Party

Acceptance Criteria

Pipettes

Annually

Gravimetric

Carter Calibrations.

Manassas, VA

± 1 % target value

Incubator
Thermometers

Annually

Compared to NIST-
traceable thermometer

Metrology
Laboratory

± 0.2 °C

Scale

Before each
use

Compared to Class S
weights

Laboratory staff

±0.01 % target

4.2 Acceptance Criteria for Measurements

QA/QC checks associated with this project were established in the QAPP. A summary of these
checks is provided in Table 4-3.

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Table 4-3. Summary of QAJQC Checks

Matrix

Measurement

QA/QC Check

Frequency

Acceptance
Criteria

Corrective Action

Negative test coupon
samples (field blank)

CFU/sample

Field blank

One per sample type
per sampling event

0 CFU

(no detection)

Revise handling procedures; investigate
sources of contamination; reject results of
the same order of magnitude

Biolab Materials

CFU/sample

Biocontaminant material
blanks of PBST, dilution
tubes, and plating beads
(check that plating materials
are not contaminated)

3 per each material
used per test

0 CFU

(no detection)

Investigate sterilization procedure;
investigate sources of contamination

Positive test samples

CFU

Positive controls (inoculated
with spores, but not subject to
any treatment)

3 per material per
test

5 x 106to
5 x 107 CFU

Revise deposition or sampling protocol if
mandated by PI

Test coupon samples

CFU

Agreement of triplicate plates
of single coupon at each
dilution

Each sample

Each CFU count
must be within 50
% of the other two
replicates

Replate or filter samples

Sporicidal solutions

pH

Oakton Acorn meter

1 per use

>6.5 and <7.0 for
fresh pAB

Reject solution; replace reagents and
prepare a new solution

Sporicidal solutions
containing bleach or
dichlor

Concentration of FAC

HACH test kit, model
CN-HRDT

Once upon
production

±5%

Reject solution; replace reagents and
prepare a new solution

Sporicidal solutions
containing PAA and
hydrogen peroxide (HP)

Concentration of PAA in
fresh solution

Titration with standardized
eerie sulfate and sodium
thiosulfate

Once upon
production

± 10%

Reject solution; replace reagents and
prepare a new solution

Contact time

Time

NIST-traceable timer,
comparison with official NIST
U.S. time

Once per 1 second

± 0.5 second

Synchronize timer with official NIST U.S.
time

33

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4.3 Data Quality Objectives

The Data Quality Objectives (DQOs) defined the critical measurements (CMs) needed to
address the stated objectives and specify tolerable levels of potential errors associated with
simulating the prescribed decontamination environments. The following measurements were
deemed to be critical to accomplish part or all the project objectives:

•	FAC concentration of the chlorine-based decontaminant solutions

•	PAA concentration of the PAA-based decontaminant solutions

•	temperature of sporicidal solutions

•	pH of the sporicidal solutions

•	flow rate of the decontaminants

•	turntable RPMs

•	CFU counts

4.4 Data Quality Indicators

In general, data quality indicator goals for the prepared dichlor and PAA solutions were
based on either: (1) published specifications, (2) related quantities, or (3) engineering judgment
based on previous experience with similar systems. The concentrations of the active ingredients
were the targeted test parameter while pH and temperature were monitored to ensure
consistency.

Producing batches of sporicidal solutions with consistent pH, temperature, and active
ingredient concentration was a high priority for this investigation. Precision was used to evaluate
the degree to which data quality indicator goals were achieved and was defined as the deviation
from the average measured values over the duration of the test. The best way to represent all of
the replicate responses to an average value is with a relative standard deviation (RSD) for all
measurements of sporicides of a target concentration.

RSD

V

X (Yi Y)	(Equati on 4-1)

*JnTl Y

The accuracy of a measurement was expressed in terms of percent bias. Percent bias
assumes the units of the measurement and, for this effort, was expressed as a percentage of the
average measurement. Percent bias as defined as:

R -C

Percent Bias =	x 100	(Equation 4-2)

where:

R = instrument response or reading
C = calibration standard or audit sample value

34

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Completeness was defined as the total number of data points that satisfy the acceptance
criteria compared to the total number of data points measured. All measured data were recorded
electronically, on data sheets, or in project notebooks. Tables 4-4 and 4-5 detail the assessments
of data quality indicators for the dichlor and PAA decontamination solutions, respectively, for
the pilot-scale tests, i.e., the small plant and sod decontamination tests. For the dichlor tests, the
target concentration was 20,000 ppm FAC, and so accuracy was determined relative to that
benchmark. For the tests with PAA, accuracy was determined relative to the target
concentrations of 0.3% PAA or 0.5% PAA.

Table 4-4. Assessment of DQI Goals for Dichlor (FAC) Solutions

FAC

Test ID

Temperature

(°C)

Measured
(parts per million)

Accuracy
(% Absolute Difference)

6.51

21.2

20,431

2.16

7.08

22.5

20,030

0.15

6.57

22.6

20,431

2.16

6.58

20.8

20,832

4.16

6.65

16.3

19,403

2.99

6.61

24.7

19,830

0.85

6.54

22.9

20,030

0.15

6.37

23.9

21,400

7.00

6.52

23.5

20,151

0.76

6.55

23.8

20,030

0.15

6.22

24.1

19,630

1.85

6.61

20.2

20,609

3.05

Average

6.57

22.2

20,234

RSD (±%)

3.0

10.5%

2.7

With the exception of one instance, all the dichlor batches were determined to have FAC
concentrations that were within 5% of the 20,000-ppm target. RSD values for pH, temperature,
and FAC ranged from approximately 3-10%. There was one test (Test 20) in which the actual
dichlor FAC concentration was beyond the ±5% of 20,000 ppm target goal.

-------
Table 4-5. Assessment of DQI Goals for PAA Solutions

Test ID

Temperature

PAA

(%)

I"1

(°C)

Measured
(%)

Target (%)

Accuracy
(% Absolute Difference)

5

2.39

22.7

0.31

0.30

3.3

6

2.49

19.9

0.31

0.30

3.3

7

NA

20.3

0.31

0.30

3.3

8

2.59

21.9

0.30

0.30

0.0

Average

2.49

21.2

0.31





RSI)

(±%)

4.0

6.2

1.3

















2

2.28

26.1

0.58

0.50

16

4

2.32

23.4

0.53

0.50

6

9

2.38

19.3

0.55

0.50

10

15

NA

NA

0.58

0.50

16

19

2.29

23.7

0.57

0.50

14

21

2.13

24.3

0.57

0.50

14

24

2.23

18.9

0.46

0.50

8

Average

2.27

21.9

0.54





RSI)

(±%)

4.2

12

8.2





NA= not available; data inadvertently not taken.

While there were 4 of 7 tests in which the actual PAA concentration differed by more than
10% of the target value, these data were deemed to be still usable for the purposes of the study.

The "Leaf Doctor" approach for assessing sod damage was an experimental method
under development, and so no data quality indicators are provided. This method was meant to be
an expedient approach. The assessment of phytotoxic effects due to the decontaminants was only
a secondary objective of the study.

36

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5 Results and Discussion

5.1 Bench-scale Efficacy Tests
5.1.1 Chlorine-based Sporicicles

In these tests, leaf material coupons were inoculated with MDIs nominally rated to
deliver 1 x 107 B.g. per actuation, allowing for the minimum 6 log recovery required to assess
decontamination efficacy. Table 5-1 shows the average spore loading for each of the chlorine-
based decontamination tests, which ranged from 6.77-7.24 log CFU. In two of the tests, the
recovery of spores from the leaf coupons was greater than the recovery from the standard
reference material.

Table 5-1. Average Spore Loading and Recovery (CFU/Sample ± SD)

Test II)

Plant Type

Positive Control
Average log CFU
Leaves

Positive Control Average
log CFU RMCs

Percent Recovery1

Gold Dust Aucuba

7.22 ±6.14

Not available

Not applicable

4.2

Gold Dust Aucuba

7.24 ± 6.67

6.91 ±5.68

211

Gold Dust Aucuba

6.77 ±6.07

6.60 ±5.00

148

White Indian Hawthorn

6.78 ±6.32

6.83 ±5.61

Relative to galvanized-steel RMCs; n=3

Note that other plants were evaluated for the inoculation and recovery of B.g. spores from
their leaves in initial proof of concept tests not associated with decontamination, and all
recoveries were greater than the spore recovery from the RMC coupons; refer to Appendix A for
additional details.

As shown in Table 5-2, chlorine-based sporicides were used in the bench-scale scouting
tests 4, 4.2 (repeat of 4 but with neutralizer), 6 and 7, using leaf coupons from the plants Gold
Dust Aucuba and White Indian Hawthorn. Two concentrations of dichlor solution were
evaluated for decontamination efficacy: 10,000 and 20,000 ppm FAC. The pAB solutions were
prepared with FAC concentrations ranging from 6000 ppm to 6700 ppm. Both Test 6 dichlor
spray test results for the Aucuba plant show only a ~ 2 LR despite the increase in FAC from
10,000 ppm to 20,000 ppm. For Test 7, when switched to the White Indian Hawthorn plant with
the same treatment (20,000 ppm FAC dichlor), efficacy improved by nearly 2 LR. The pAB
treatments used for Test 4.2 and Test 4 provided efficacies of nearly a 5 LR for the Gold Dust
Aucuba coupons and nearly achieved a 6 LR for White Indian Hawthorn coupons.

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Table 5-2. Bench-Scale Decontamination Efficiency Results for Leaf Material Coupon
Tests Using Chlorine-Based Decontaminants

Test II)

Decontaminant

Plant Type

ppm

FAC

Average LR
Leaves

Avg. LR
Galvanized Steel
RMCs

pAB

Gold Dust Aucuba

6240

6.92

1.57 ±0.04

N/A

4.2

pAB

Gold Dust Aucuba

6080

6.95

4.8 ±0.7

6.0 ± 1.6

Dichlor

Gold Dust Aucuba

10,516

6.36

2.14 ±0.50

3.27 ±0.44

Dichlor

Gold Dust Aucuba

20,231

6.35

1.99 ±0.79

3.11 ± 1.05

Dichlor

White Indian Hawthorn

20,320

4.03 ±0.35

3.37 ±0.57

pAB

White Indian Hawthorn

6173

5.98 ±0.90

4.89 ±0.21

5.1.2 PAA-based Sporicidal Solutions

As shown in Table 5-3, data from Tests 4, 5, and 8 demonstrate more than 7 log CFU
were recovered from plant and RMC coupons. Test 5 showed 89% of CFU were recovered from
the plant coupons while 67% were recovered for Test 8. Table 5-4 shows the average CFU
recovered from plant and RMCs and the percent recovery of CFU from plant materials relative to
the RMCs.

Table 5-3. Average Spore Loading (CFU/Sample ± SD)

Test II)

Plant Type

Plant Material Positive Control Avg.
log CFU/Sample

Galvanized Steel
Positive Control Avg.
CFU/Sample

Percent
Recovery1

Gold Dust Aucuba

7.22 ±6.14

N/A

Gold Dust Aucuba

7.19 ±6.42

7.25 ±5.93

White Indian Hawthorn

7.10 ±6.06

7.27 ±6.59

Relative to galvanized-steel RMCs

Table 5-4 below shows the average LR for each of the PAA-based decontamination
efficacy tests. Multiple concentrations of PAA from the Jet Ag and Oxidate 2.0 solutions were

-------
evaluated for decontamination efficacy in the bench-scale scoping tests. While efficacy for the
plant material generally improved with increasing PAA concentration, PAA concentrations of
approximately 0.5% were required to obtain nearly a 6 LR or higher. However, none provided
>4 LR for Gold Dust Aucuba coupons. The 0.57% PAA formulation for Test 8 achieved a 7 LR
for the White Indian Hawthorn coupons, the highest efficacy achieved for the PAA-based
decontaminants. With the exception of the Oxidate 2.0 formulation used for Test 8, all tests that
included galvanized-steel RMCs returned >7 LR for the RMC coupons. That the PAA-
decontaminants performed notably better on the RMC material would suggest the inhibition of
sporicidal activity by the leaf material, possibly due to the organic nature of the leaves or
interactions at the surface of the leaf.

Table 5-4. Bench-Scale Decontamination Efficiency Results for Leaf Material Tests Using Peracetic Acid

Test II)

Decontaminant

Plant

%PAA

AvgLR

RMC Avg. LR

Jet Ag 15

Gold Dust Aucuba

0.039

1.88 ± 1.01

N/A

Jet Ag 15

Gold Dust Aucuba

0.072

2.21 ± 1.26

N/A

Jet Ag 15

Gold Dust Aucuba

0.13

3.07± 1.11

N/A

Jet Ag 15

Gold Dust Aucuba

0.28

3.78 ± 1.40

7.49 ±0.01

Jet Ag 15

Gold Dust Aucuba

0.58

3.63 ±0.52

7.36 ±0.17

Jet Ag 15

White Indian Hawthorn

0.57

7.03 ± 0.02

7.20 ±0.01

Oxidate 2.0

Gold Dust Aucuba

0.02

0.35 ±0.19

N/A

Oxidate 2.0

Gold Dust Aucuba

0.079

2.59 ±0.74

N/A

Oxidate 2.0

Gold Dust Aucuba

0.11

3.51 ±0.37

N/A

Oxidate 2.0

Gold Dust Aucuba

0.18

3.93 ±0.14

N/A

Oxidate 2.0

Gold Dust Aucuba

0.30

3.63 ±0.52

7.36 ±0.17

Oxidate 2.0

Gold Dust Aucuba

0.52

4.68 ±0.41

7.46 ± 0.0

Oxidate 2.0

White Indian Hawthorn

0.44

5.76 ±0.51

5.51 ± 1.73

5.2 Pilot-Scale Small Plant Decontamination Tests

Table 5-5 summarizes spore inoculation and recovery data for the small plants, the RMCs
(placed at the bottom of the LV-ADA), and the inoculation controls. As discussed in Section
3.7, positive control samples were included in each test to evaluate the spore loading onto the
leaves while the RMCs defined the level of B.g. deposited onto the surfaces inside the ADA
during inoculation. The inoculation controls verified the levels of B.g. released from the MDI
during actuation. The results are reported in log CFU per square inch, to allow for comparisons
between the three sample types.

An MDI that delivered 5 log CFU per actuation was used to inoculate the plants for Test
1. For the remaining pilot-scale tests, the small plants were inoculated with MDIs that delivered

-------
7 log CFU per actuation. Excluding Test 1, the average log recoveries (CFU/square inch (in2))
for the positive controls ranged from 2.5 log CFU (± 0.77 SD) to 3.9 log CFU (± 0.32 SD). The
recoveries of spores from the RMCs were approximately 1 log per square inch higher than for
the leaf samples.

Table 5-5. Small Plant Positive Control Recovery

Test ID Vegetation Type Avg. Log Recovery (CFU/in2 ±SD)

Test ID

Vegetation
Category

Positive
Control3

RMCb

Inoculation
Control2

1

Evergreen

1.1 ±0.97

1.9 ± 1.1

6.1 ± 0.15

2

Evergreen

3.4 ±0.46

4.3 ± 1.1

7.5 ±0.09

3

Evergreen

3.3 ±0.62

4.2 ±0.5

7.04 ±0.13

4

Evergreen

3.4 ±0.56

3.5 ±0.4

7.6 ±0.12

5

Evergreen

3.1 ± 0.51

3.7 ±0.7

7.5 ±0.1

6

Deciduous

2.5 ±0.77

3.9 ±0.2

7.7 ±0.15

7

Ground cover

3.8 ±0.36

4.6 ±0.3

7.6 ±0.01

11

Evergreen

3.2 ±0.79

5 ±0.5

7.9 ±0.17

13

Ground cover

3.9 ±0.52

4.9 ±0.7

7.9 ±0.02

14

Ground cover

2.8 ±0.69

4 ±0.39

7.7 ±0.07

26

Deciduous

3.9 ±0.32

4.76 ± 0.25

6.9 ±0.04

a Total surface area per leaf sample = 1.3 in2
b Total surface area per RMC or inoculation sample = 0.39 in2

Table 5-6 below summarizes the CFU recovered per bark sample positive control, and their
associated RMCs and inoculation controls. The data for the inoculation controls show that each
MDI actuation dose ranged from 7.3 ± 0.18 to 7.7 ± 0.11 log CFU. The RMC data show the
sample loading ranged from 3.8 (± 0.5 SD) to 4.8 (± 0.4 SD) log CFU. Positive controls show
recoveries of 3.7 (± 1.04 SD) to 5.4 (± 0.37 SD) log CFU.

40

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Table 5-6. Summary of Spore Recovery from Bark Sample Controls



Vegetation

Average Log Recovery (CFU/Sample)

Test II)

Type

Positive Control

RMC

Inoculation Control

8

Bark

4.3 ±0.54

3.8 ±0.5

7.7 ±0.1

9

Bark

5.4 ±0.37

4.4 ± 1.01

7.3 ±0.18

10

Bark

4.8 ±0.34

4.8 ±0.4

7.7 ± 0.11

12

Bark

3.7 ± 1.04

4.3 ±0.78

7.6 ±0.04

Table 5-7 shows the spore recovery results for the small plant material and procedural
blanks. Material and procedural blanks were included in each test to characterize the initial levels
of B.g. prior to any testing and to identify any cross-contamination resulting from the test
procedure. The results show minimal contamination occurred. For example, B.g. was not
detected on any material blank or procedural blank samples for the evergreen or deciduous
plants. There were only three tests that had blank samples positive for the target organism. These
plant samples were typically contaminated with just a few spores, and are indicative of potential
aerosolized spores in the lab space during inoculation, but should not impact evaluation of
decontamination results.

41

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Table 5-7. Summary of Baseline Contamination and Cross-contamination Results

Test II)

Vegetation
Category

Material Blank Positive for
Spores (of 5)

Procedural Blanks Positive for
Spores (of 5)

Evergreen

Deciduous

Ground cover

Bark

Evergreen

Bark

Ground cover

Deciduous

Table 5-8 below summarizes the decontamination results for the evergreen category of
small plants, in terms of the number of samples (of 15) that were positive for B.g. Also shown in
the table is the percentage of samples (out of the 15) in which no spores were detected, i.e., the
percent of samples completely inactivated. The small evergreen plants used in testing were either
the White Indian Hawthorn or Eleanor Tabor Hawthorn varieties, and all but one test (Test 2)
were sprayed with the electrostatic sprayer. A control test using only deionized water was
performed on the evergreen plants; as expected, all 15 test samples were positive for B.g. (We
do note that although all 15 samples from the plants sprayed with deionized water were positive,
there was a small amount of reduction (up to 0.65 LR on one of the plants) that most likely
occurred due to removal from the spraying action alone. This is consistent with other studies
showing water spray may remove up to 90% of spores from surfaces. In the one test utilizing
dichlor (Test 11), 2 of the 15 samples returned positive for B.g. spores. Four tests were
performed with PAA with varying concentration or spray quantities; three of these tests resulted
in no samples positive for B.g, even with the PAA concentration as low as 0.3%. The test
showing incomplete inactivation was sprayed with 0.09% PAA, but it still retained 75% efficacy.
In Test 2, the backpack sprayer was used, which allowed for a much larger volume of
decontaminant to be sprayed and resulted in no samples returning positive.

-------
Table 5-8. Decontamination Results for Evergreens (Indian Hawthorn)

Test
ID

(CFU per
actuation)

Sporicide

Dichlor

FAC
(ppm)

PAA (%)

Sprayer

Volume
sprayed (ml
per plant)

Positive
Samples
(of 15)

Percent of
samples
inactivated

(%)

1

E5

Jet Ag
15%

N/A

0.09

ES

45

3

80

2

E7

Jet Ag
15%

N/A

0.58

BP

1116

0

100%

3

E7

DI Water

N/A

N/A

ES

99

15

0

4

E7

Jet Ag
15%

N/A

0.53

ES

107

0

100

5

E7

Jet Ag
15%

N/A

0.31

ES

118

0

100

11

E7

Dichlor

20030

N/A

ES

130

2

87

ES= electrostatic sprayer; BP=backpack sprayer

Table 5-9 below summarizes the decontamination results for the three ground cover plant
experiments. Tests 13 and 14 both used dichlor with a target FAC of 20,000 ppm but with
different spray volumes. The decontamination procedure for Test 13 disseminated twice the
sporicidal solution volume of Test 14 (260 ml and 131 ml, respectively). A total of 5 samples
were positive for B.g. from Test 14, and all samples were below the detection limit for Test 13.
For Test 7, which used PAA, 3 of 15 samples were positive for spores. When compared to Test
14, it appears at equal spray volumes and spray method, 0.3% PAA was slightly more effective
than the 20,000 ppm FAC dichlor.

Table 5-9. Decontamination Results for Ground Cover Plants

Test
ID

MDI Level
(CFU per
actuation)

Plant

Sporicide

Dichlor

FAC
(ppm)

PAA

(%)

Sprayer

Volume
sprayed
(ml)
per
tray

Positive
Samples
(of 15)

Percent of
samples
inactivated

(%)

7

E7

Japanese
Spurge

Jet Ag
15%

N/A

0.31

ES

60

3

80

14

E7

Nena

Ivy

Dichlor

19403

N/A

ES

65

5

67

13

E7

Creeping
Jenny

Dichlor

20832

N/A

ES

130

0

100

ES= electrostatic sprayer

43

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Two tests were performed with deciduous plants (blueberry shrubs): a test with 0.3%
PAA and a test using dichlor (20,609 ppm FAC). While the electrostatic sprayer was used for
Test 6 and the backpack sprayer for Test 26, equivalent volumes of each sporicidal solution were
disseminated. The PAA spray procedure returned 1 positive sample (93% inactivation) and the
dichlor spray procedure returned 8 positive samples (47% inactivation). Table 5-10 provides
decontamination efficacy for the deciduous plants.

Table 5-10. Decontamination Results for Deciduous Plants (Blueberry Shrubs)

Test
ID

CFU per
actuation)

Sporicide

Dichlor

FAC
(ppm)

PAA

(%)

Sprayer

Volume
sprayed
(ml) per
plant

Positive
Samples
(of 15)

Percent of
samples
inactivated

(%)

6

E7

Jet Ag® 15%

N/A

0.306

ES

121

1

93

26

E7

Dichlor

20609

N/A

BP

120

8

47

ES= electrostatic sprayer; BP=backpack sprayer

Table 5-11 below summarizes the decontamination results for the bark material. Of the
four tests conducted, none resulted in having all 15 samples negative for E.g., signifying the
difficulty in decontaminating this type of plant material. Test 8 and Test 9 used PAA at varying
concentrations: all 15 bark samples exposed to 0.3% PAA were positive for E.g. Increasing the
PAA concentration to 0.55% improved the outcome, resulting in only 2 samples of 15 testing
positive for E.g. In the two tests utilizing dichlor solutions, both performed poorly against the
bark, as indicated by the results for Tests 10 and 12. Increasing the spray quantity by nearly a
factor of 10 (with the use of the backpack sprayer) only minimally improved decontamination
results (reduced the number of positive samples from 15 to 13).

Table 5-11. Decontamination Results for Bark

Test
ID

CFU per
actuation)

Sporicide

Dichlor

FAC
(ppm)

PAA (%)

Sprayer

Volume
sprayed
(ml) per

bark
"coupon"

Positive
Samples
(of 15)

Percent of
samples
inactivated

(%)

8

E7

Jet Ag
15%

N/A

0.3

ES

130

15

0

9

E7

Jet Ag
15%

N/A

0.55

ES

130

2

87

10

E7

Dichlor

20431

N/A

ES

132

15

0

12

E7

Dichlor

20431

N/A

BP

1074

13

13

ES= electrostatic sprayer; BP=backpack sprayer

44

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5.3 Sod Decontamination Tests

Overall, three decontamination tests were conducted for each of the three sod types
(Zoysia, Fescue, and Bermuda), for a total of nine decontamination tests.

Table 5-12 below summarizes the results for the sod inoculation and blank samples. Test
15, which was the first sod decontamination test, used a 1 x 105 CFU MDI. An average of 3.6 log
CFU per gram of grass was recovered from the positive control samples and an average of 5.7
log was recovered from the set of 3 inoculation controls. Due to this lower-than-expected CFU
recovery from the grass, an MDI delivering 7 log CFU was used for the remainder of the sod
decon tests. For these remaining tests, the average log CFU recoveries of the sod positive
controls ranged from 5.4-7.0 per gram of grass (excluding Test 23 positive control recovery
results, which are believed to be in error due to a sampling issue.) For the inoculation controls,
average log recoveries ranged from 6.2 (± 0.05 SD) to 7.61 (± 0.11 SD).

Except for a single spore detected in one of the five blanks collected for Test 21, all other
tests returned 0 CFU for the procedural blanks. All material blank samples were also negative.

We do note that there is the small possibility that the spray application of the
decontaminant may have resulted in physical removal of the spores but without inactivation, and
that some of these spores may have been driven into the soil. Although it is uncertain this
occurred since only grass was sampled in this study, i.e., soil samples were not taken.

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Table 5-12. Summary of Control Coupon Results for Sod Tests

Test
ID

Positive Control
Avg. Log CFU

recoveries
(CFU/g ±SD)

Inoculation Control

Avg. Log CFU Recovery
(CFU/sample ±SD)

Procedural
Blanks Positive
for B.g. (Of 5)

Material Blanks
Positive for B.g.
(Ofl)

15a

3.6 ±0.41

5.7 ±0.06

6.1 ±0.42

6.2 ±0.05

7.02 ±0.85

6.47 ±0.11

5.48 ±0.53

6.57 ±0.08

6.58 ± 1.12

6.54 ±0.14

5.35 ± 1.12

6.44 ±0.04

23°

2.69 ± 1.2

6.58 ±0.03

6.53 ±0.55

6.48 ±0.06

5.91 ±0.79

7.61 ± 0.11

a An MDI verified to deliver 1 x 105 CFU per actuation was used for Test 15. The remaining tests were
inoculated using an MDI verified to deliver 1 x 107 CFU per actuation.
b A single spore was recovered from an 8-mL sample.

c Positive control sample collection was inadvertently sampled incorrectly; Test 25 was a repeat with confirmed
original sampling technique.

The results for the three decontamination tests performed on the zoysia sod coupons are
summarized in Table 5-13. The highly efficacious results for Test 15, in which no spores were
detected on any of the 15 sod test coupons, may be due to the lower quantity of B.g. (log 5 CFU)
than the other sod tests. Additionally, other differences in the procedures for Test 15 that may
have impacted (improved efficacy) results include using the electrostatic sprayer, conducting the
test in the greenhouse, and then coupon sampling out-of-doors, immediately outside the
greenhouse. After Test 15, the electrostatic sprayer malfunctioned, and as a result,
decontamination for all other sod tests was performed using the high flow backpack sprayer.
Additionally, all tests after Test 15 were conducted in the COMMANDER chamber, using a 1 x
107 CFU MDI.

Test 19 was essentially a repeat of Test 15 but used a higher inoculum of spores and the
backpack sprayer to apply the PAA. The 16-ml treatment in Test 19 returned 3 positive coupons,
although samples from each of the three subsequent treatments were below the detection limit.
The 83-ml spray volume returned 2 of 3 coupons that were positive for CFU. In Test 18, dichlor
was applied in quantities similar to the quantities used in the PAA tests and resulted in 4 of 15
sod coupons positive for spores. In general, for a given test, there was no trend in which
increasing spray amounts improved decontamination results.

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Table 5-13. Decontamination Results for Zoysia Sod Coupons

Test
ID

PAA

(%)

FAC (ppm)

Sprayer

Total Volume
Sprayed
(mL)

Total Coupons Positive
for B.g. CFUs

(of 3)

Percent
Inactivation

(%)

15

0.58

NA

ES

26

0

100









52

0

100









78

0

100









105

0

100









131

0

100

18

NA

20,030

BP

18

2

33









54

0

100









72

1

67









108

1

67









126

0

100

19

0.57

NA

BP

16

3

0









33

0

100









50

0

100









66

0

100









83

2

33

ES= electrostatic sprayer; BP=backpack sprayer

Table 5-14 provides a summary of the decontamination test results for the fescue grass.
Tests 20 and 22 both used dichlor as the decontaminant, but with the latter test utilizing higher
spray quantities. Decontamination improved with the greater spray amounts used in Test 22 (7 of
15 sod coupons positive for spores) compared to Test 20 (14 of 15 coupons positive). In Test 21,
which used PAA in spray amounts equal to Test 20, only 4 of 15 sod coupons returned positive
for B.g. spores. As with the Zoysia sod coupon test results, increasing the spray amount for a
given test did not appear to improve efficacy.

47

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Table 5-14. Decontamination Efficacy for Fescue Sod Coupons

Test

PAA

FAC Level

Sprayer

Total Volume

Total Coupons

Percent

ID

(%)

(ppm)



Sprayed

Positive for B.g. CFU

Inactivation







(mL)

(out of 3)

(%)

20

NA

21,400

Back Pack

18

3

0

36

3

0

54

3

0

72

2

33

90

3

0

21

0.57

NA

Back Pack

18

2

33

36

1

67

54

0

100

72

0

100

90

1

67

22

NA

20,151

Back Pack

57

2

33

76

1

67

95

0

100

114

2

33

133

2

33

Table 5-15 below presents the decontamination results for the Bermuda sod coupons.

Test 25 was essentially a repeat of Test 23 (both with dichlor) since the positive control recovery
results for Test 23 were questionably lower than expected (presumably due to a sampling error).
The results for Test 23 showed better decontamination efficacy (9 of 15 coupons positive for
spores) compared to Test 25, in which all 15 coupons were positive for spores, regardless of the
spray volume. Test 24 utilized PAA and resulted in 11 of 15 coupons positive for spores. As with
the other sod decontamination results, there was no trend showing improved decontamination
with increasing spray volume.

48

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Table 5-15. Decontamination Efficacy for Bermuda Sod Coupons

Test
ID

PAA

(%)

FAC Level
(ppm)

Spray Method
(ES/BP)

Total Volume
Sprayed
(mL)

Total Coupons
Positive for B.g.

CFU out of 3

Percent
Inactivation

(%)

23

NA

20,030

BP

19

1

67









38

1

67









57

1

67









76

3

0









95

3

0

24

0.46

NA

BP

18.7

2

33









37.4

3

0









56.1

2

33









74.8

3

0









93.5

1

67

25

NA

20,609

BP

18.1

3

0









36.2

3

0









54.3

3

0









72.4

3

0









90.5

3

0

ES= electrostatic sprayer; BP=backpack sprayer

Table 5-16 is an overall summary of the sod decontamination results, reported as the
number of sod coupons that were positive for a given experiment, for each type of sod and
decontaminant. Three tests were conducted for each sod type. Results are summarized in this
manner since increasing the spray volumes within each test did not appear to improve
decontamination results. Overall, the Zoysia had the least number of total samples that were
positive (9), while Bermuda had the most (35). In addition, the average number of positive
results per test (of 15) for the PAA was 5.25, while for dichlor, the average number of positive
samples per test was 9.8.

Table 5-16. Number of Sod Coupons (of 15) Positive for Spores

Sod Type

Dichlor

PAA

Zoysia

4

0a 5

Fescue

14

7b

5

Bermuda

9C

15

11

a=log 5 MDI used; b=2nd test with dichlor used higher spray volumes; c=low positive control recovery, so test repeated

49

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5.4 Phytotoxicity Effects of Sporicidal Solutions

The phytotoxicity phase of testing included 5 plant types: Indian Hawthorne (evergreen),
Creeping Jenny (ground cover), blueberry shrubs (deciduous), fescue sod, and zoysia sod. To
replicate how the decontamination efficacy tests were conducted, the Indian Hawthorn and
Creeping Jenny plants were rinsed after exposure to the decontamination treatment, while the
Zoysia and Fescue sods were not rinsed after decontaminant exposure. For the blueberry shrubs,
phytotoxic effects were evaluated both with and without rinsing off the decontaminant. For each
plant/decontaminant experiment, three replicate plants were used.

For the Indian Hawthorne plants, both the dichlor and PAA sporicides did not appear to
affect the number of damaged leaves compared to the deionized water controls (Table 5-17). By
week 4, plants exposed to dichlor had an average of 68 damaged leaves (204% increase from the
baseline average) and plants exposed to the PAA had an average of 70 damaged leaves (193%
increase from the baseline). Similarly, the Indian Hawthorne plants exposed to the two sporicides
had comparable numbers of shed leaves and growth compared to the control plants (sprayed with
water); see Tables 5-18 and 5-19, respectively. Although somewhat unexpected, the DI-, PAA-,
and dichlor-sprayed plants showed a decrease in height over the four week period; this may be
due to loss of leaves, height measurement variability, or some other factors.

Table 5-17. Average Number of Damaged Leaves for Indian Hawthorne Plant (± SD)

Test
Solution

Baseline

Week 1

Week 2

Week 3

Week 4

Percent Change from Baseline

DI Water

17 ±7.3

44 ± 16

45 ± 18.3

47 ± 15.7

71 ±30.6

316%

Dichlor

22 ± 10.9

28 ±5.9

40 ±23.2

45 ± 22.5

68 ±39.4

204%

PAA

24 ± 14.1

42 ±29

40 ± 14

43 ± 26.7

70 ± 29.5

193%

Table 5-18. Average Number of Shed Leaves per Indian Hawthorne Plant

Sporicide

Week 1

Week 2

Week 3

Week 4

Percent change from Week 1

DI Water

12.75 ± 3

30.25 ± 10.5

21.75 ± 11.73

7.25 ±6.13

-43.14%

Dichlor

12.25 ±4.99

25.75 ±4.99

12.25 ±5.85

6.25 ±3.3

-48.98%

PAA

7.25 ± 2.22

23.75 ± 12.42

13 ±6.06

2.75 ± 1.5

-62.07%

50

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Table 5-19. Average Height for Indian Hawthorne Plant (Inches)

Sporicide

Baseline

Week 1

Week 2

Week 3

Week 4

Percent
Change
from

Baseline

DI Water

12.88 ± 1.76

13.31 ± 1.89

12.75

12.31 ± 1.52

12.56 ± 1.14

-2.0%

Dichlor

12.69 ±2.29

13.38 ± 1.36

12.63 ±0.92

13.25 ± 1.94

12.94 ± 1.95

2.0%

PAA

12.94 ±0.66

12.63 ±0.78

12.25 ±0.61

11.88 ±0.72

11.94 ±0.66

-8.0%

The Creeping Jenny phytotoxicity results are summarized in Tables 5-20 to 5-22. The
plants that received the dichlor treatment showed a substantial increase in the average number of
damaged leaves (500% increase over the 4-week period) compared to plants sprayed with only
deionized water (230% increase). Plants treated with dichlor also showed a 266% increase in the
number of shed leaves compared to the 64% increase for the water controls (Table 5-21). For the
PAA, there appeared to be less effect of PAA on the number of damaged leaves but the results
showed a substantial increase in the number of shed leaves (340%) compared to the plants
sprayed with water. As indicated in Table 5-22, both sporicides did not appear to affect the
height of the plants compared to the water.

Table 5-20. Average Number of Damaged Leaves for Each Rinsed Creeping Jenny

Sporicide

Baseline

Week 1

Week 2

Week 3

Week 4

Percent Change from
Baseline

DI Water

8 ±2.4

80 ±51.7

27 ±26.8

28 ±28

25 ± 24.8

230%

Dichlor

11 ±7.2

70 ± 42.7

30 ±29.5

29 ±29

65 ± 64.5

500%

PAA

8 ±6.9

25 ± 7.3

19 ± 19.3

27 ±26.8

26 ±25.8

212%

Table 5-21. Total Shed Leaves for Each Rinsed Creeping Jenny Plant

Sporicide

Week 1

Week 2

Week 3

Week 4

Percent Change from Week 1

DI Water

3.5 ±5

0.25 ±0.5

4.5 ± 1.73

5.7 ±2.22

64.0%

Dichlor

1.5 ± 1.73

1.25 ± 1.5

5.75 ±2.22

5.5 ±4.2

266%

PAA

1.25 ±0.5

1 ± 0.82

7 ± 4.08

5.5 ± 1.7

340%

51

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Table 5-22. Average Height of Rinsed Creeping Jenny Plants (Inches±SD)

Sporicide

Baseline

Week 1

Week 2

Week 3

Week 4

Percent
Change from

Baseline

DI Water

4.25 ±0.29

4.44 ± 0.66

4.00

4.25 ±0.65

3.88 ±0.63

-9.0%

Dichlor

4.38 ±0.52

3.81 ±0.24

3.88 ±0.25

4 ± 0

4.38 ±0.48

0.0%

PAA

4.31 ±0.55

4.5 ± 1.02

4.81 ±0.63

5 ±0.35

4.13 ±0.63

-4.0%

The phytotoxicity results for the rinsed blueberry plants are summarized in Tables 5-23 to
5-25. With respect to the average number of damaged leaves, at week 4, all three
decontaminants exhibited a similar number of damaged leaves (113-166). But in terms of the
percent change in number of damaged leaves over the course of the 4-week observation period,
the PAA had the greatest impact on the plants (a 132% increase). A similar trend was observed
for the number of shed leaves: the PAA increased the number of shed leaves by 325% by week 4
while the plants exposed to dichlor and deionized water had decreases of 51% and 22%
respectively. These results are caveated however by the fact that the total number of shed leaves
each week for each decontaminant was relatively low, and at week 4, the plants averaged only 4-
5 leaves shed per week (Table 5-24). With respect to plant growth, plants exposed to DI water
had the greatest percent change in height (24%), compared to 7% and 13% growth for the dichlor
and PAA plants, respectively.

Table 5-23. Average Number of Damaged Leaves for Each Rinsed Blueberry Plant (±SD)

Sporicide

Baseline

Week 1

Week 2

Week 3

Week 4

Percent Change from
Baseline

DI Water

93 ±45.4

95 ± 54.2

86 ± 48.8

109 ±46.2

113 ±46.5

22%

Dichlor

109 ±21.7

109 ± 13.3

128 ±47.1

156 ±32.5

166 ±41.9

51%

PAA

58 ±21.3

137 ±45.4

145 ±33.6

126 ± 10.5

134 ±33.7

132%

Table 5-24. Total Number of Shed Leaves for Each Rinsed Blueberry Plant (±SD)

Sporicide

Week 1

Week 2

Week 3

Week 4

Percent Change from
Week 1

DI Water

11.75 ± 16.96

20.25 ± 38.53

5.25 ±9.18

4.25 ± 5.44

-63.83%

Dichlor

6.5 ±5.2

5 ± 4.69

3 ± 2.45

5 ±3.74

-23.08%

PAA

1 ± 1.15

8.25 ± 9.22

5.75 ±6.65

4.25 ± 5.44

325.00%

52

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Table 5-25. Height for Rinsed Blueberry Plant (Inches)

Sporicide

Baseline

Week 1

Week 2

Week 3

Week 4

Percent
Change from
Baseline

DI Water

23.81 ± 1.8

26.75 ±2.37

26.31 ±0.94

28.06 ± 1.05

29.63 ± 1.48

24.0%

Dichlor

25.5 ±2.04

26.69 ±3.99

29.88 ±5.6

27.5 ± 1.08

27.35 ± 1.08

7.0%

PAA

24.38 ± 1.09

24.13 ± 1.31

23.25 ±2.4

24.56 ± 2.92

27.5 ±3.85

13.0%

Phytotoxicity results for the unrinsed blueberry shrubs are presented in Tables 5-26
through 5-28. Plant observations for damaged and shed leaves were inadvertently not recorded
for the unrinsed blueberry plants during the initial weeks of the experiment, so an additional two
weeks of evaluations were performed. Between Weeks 5 and 6, plants exposed to water or
dichlor did not show a substantial change in the average number of damaged leaves, while the
average number of damaged leaves for the plants exposed to the PAA decreased by over 50%.
The PAA plants also had the least average number of shed leaves in Weeks 5 and 6 (an average
of 8 leaves, compared to 25 and 42 leaves shed for the control and dichlor plants, respectively, in
Week 6). Lastly, as shown in Table 5-28, the average plant height did not appear to be affected
by the decontaminant; the dichlor and PAA plants grew more (percent growth) than the control
(water) plants over the 5-week period.

Table 5-26. Average Number of Damaged Leaves for Each Unrinsed Blueberry Plant (±SD)

Sporicide

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

DI Water

N/A

N/A

N/A

N/A

24.5 ±21.95

25.25 ±33.18

Dichlor

N/A

8.67 ± 8.62

N/A

N/A

41.25 ±56.01

42.25 ± 55.7

PAA

N/A

N/A

N/A

N/A

20 ± 11.22

8.25 ± 10.59

Table 5-27. Total Number of Shed Leaves for Each Unrinsed Blueberry Plant(±SD)

Sporicide

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

DI Water

N/A

N/A

N/A

N/A

24 ±21.95

25 ±33.18

Dichlor

N/A

8.67 ± 8.62

N/A

N/A

41 ±56.01

42 ±55.7

PAA

N/A

N/A

N/A

N/A

20 ± 11.22

8.2 ± 10.59

53

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Table 5-28. Height for Unrinsed Blueberry Plant (Inches ± SD)

Sporicide

Baseline

Week 1

Week 2

Week 3

Week 4

Week 5

Percent Change
from Baseline

DI Water

25.38 ±3.1

25 ±3

24.88 ±2.93

25.13 ±3.42

25.63 ±3.09

24.44 ± 2.9

-4.0%

Dichlor

24.44 ±2.14

25 ± 1.58

25.25 ±2.06

25.5 ±2.38

25.5 ±2.06

25.3 ± 1.8

4.0%

PAA

25.25 ± 1.81

26.38 ± 1.97

26.63 ± 1.8

26.5 ± 2.04

25.83 ±2.47

25.58 ± 1.91

1.0%

Although there are some data missing from the unrinsed blueberry shrub observations,
some limited comparisons can be made for the effects on the rinsed and unrinsed blueberry
plants. In terms of plant growth in height, the rinsed blueberry shrubs appeared to have grown
more over the observation period than the unrinsed plants. Over a 4-week period, the rinsed
dichlor and PAA plants grew an average of 7 and 13%, respectively, compared to only a 4 and
1% growth over a 5-week period for the unrinsed plants. These differences, however, are most
likely not significant.

Tables 5-29 and 5-30 summarize the phytotoxicity results for the fescue sod coupons. The
sod treated with dichlor had a 374% increase in unhealthy areas, compared to a 163% increase for
PAA and a 110% increase for the water controls. In addition, the sod coupons sprayed with dichlor
had the least amount of growth over the 4-week period, whereas the PAA and control sod showed
similar growth.

Table 5-29. Percent of Fescue Sod Coupon Measured as Damaged by Photoanalysis (±SD)

Sporicide

Baseline

Week 1

Week 2

Week 3

Week 4

Percent Change from Baseline

DI Water

37 ± 16.5

37 ±41.5

58 ±37.8

75 ±38.4

78 ±32.8

110%

Dichlor

19 ±6.7

74 ± 15.4

89 ± 14

94 ±3.7

90 ±8

374%

PAA

28 ±8.9

87 ±3.5

77 ± 13.6

79 ± 13.4

73 ±5

163%

Table 5-30. Height of Fescue Grass in the Center of the Coupon (inches ± SD)

Sporicide

Baseline

Week 1

Week 2

Week 3

Week 4

Percent Change
from Baseline

DI Water

N/A

2.81 ± 1.07

3.5 ± 1.78

2.75 ± 1.55

5.94 ±3.47

111.0%

Dichlor

NA

1.81 ±0.43

1.69 ±0.55

1.31 ±0.55

2.7 ±3.11

49.0%

PAA

N/A

1.25 ±0.29

2.69 ± 1.57

2 ±0.91

2.5 ±2.29

100.0%

Table 5-31 provides a summary of the average percentage of unhealthy grass, and Table
5-32 provides the average height at the center of each sod coupon, for the zoysia grass. The
zoysia sod coupons that were treated with dichlor or PAA showed less diseased area compared to

54

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baseline conditions, whereas the sod sprayed with water showed a slight increase in diseased
area over that same time period. Data for each of the 3 treatments suggests the health of the sod
deteriorated substantially over Weeks 1 and 2 but then improved during Weeks 3 and 4. Over
the 4-week period, grass height grew at similar percentages for the DI and PAA sod, but the sod
coupons sprayed with dichlor exhibited the least amount of growth (only 33%).

Table 5-31. Average Percent of Zoysia Sod Coupon Measured as Damaged per Photo Test (±SD)

Sporicide

Baseline

Week 1

Week 2

Week 3

Week 4

Percent
Change from
Baseline

DI Water

37 ±2.4

94 ±5.2

89 ±3.2

55 ±2.6

38 ±4.3

2.7%

Dichlor

47 ± 6.6

71 ± 12.2

70 ± 7.3

36 ±3.6

45 ±8.1

-5.1%

PAA

44 ± 7.4

77 ± 13.2

72 ± 10.4

42 ± 10.4

18 ± 14.9

-58.0%

Table 5-32. Height of Zoysia Grass in the Center of the Sod Coupon (inches ±SD)

Sporicide

Baseline

Week 1

Week 2

Week 3

Week 4

Percent
Change from
Baseline

DI Water

3.25 ±0.61

3.13 ±0.48

2.63 ±0.83

4.5 ±0.94

6.56 ± 1.14

102%

Dichlor

3.88 ±0.63

3.75 ±0.29

4.5 ±0.65

6.38 ± 1.65

5.15 ± 1.34

33%

PAA

3.33 ±0.59

3.81 ± 1.25

4.88 ± 1.13

6.88 ±0.95

6.08 ±0.52

83%

55

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6 Summary and Conclusions

Over the course of this three-year study, dozens of experiments were conducted at the
bench- and pilot-scale to assess options to decontaminate vegetation in the event it becomes
contaminated with B. anthracis spores. The results presented here as part of the AnCOR
project help to advance the state of the science and engineering to build capabilities and
capacity for the remediation and recovery at a USCG base in the event of a bioterrorism
incident. While this study was performed for the USCG, the results discussed in this report
can be applied to any event involving the wide-area release of B. anthracis spores. The
following is a summary of the findings and conclusions from the study:

• At the time the search was conducted, there was essentially no publicly available
scientific literature related to the decontamination of vegetation contaminated with B.
anthracis spores or other biological agents.

• Some commercial off-the-shelf (COTS) pesticides registered with EPA for use on
plant foliage, crops, or turf have PAA as their active ingredient; PAA is one of the
most effective liquid sporicidal chemicals available for inactivation of B. anthracis
spores. For this reason, PAA-based decontaminants were included in this study.
However, the concentrations of PAA used in this study were generally higher than
the concentrations prescribed on the label.

• Inoculation and recovery of B.g. spores from plant material did not present any
difficulties, and in some cases, recovery of spores from leaf material was better than
the recovery from the reference material (galvanized steel).

• In the bench-scale decontamination efficacy tests using the PAA-based
decontaminants, efficacy for the plant material generally improved (but not always)
with increasing PAA concentration. PAA concentrations of approximately 0.5%
were required to obtain a nearly 6 LR or higher of B.g. In the same experiments and
where data are available, decontamination efficacy of the RMC coupons was usually
substantially higher than the decontamination efficacy of the leaf coupons.

• Of the six bench-scale decontamination efficacy tests using either dichlor or pAB,
none resulted in a LR of 6.0 or greater for the leaf material, although one test
achieved a 5.98 LR with pAB. The highest LR achieved with dichlor was 3.8.

• In the pilot-scale small plant decontamination efficacy tests, typical positive control
recoveries from samples ranged from approximately 2-4 log CFU. Because of the
low spore loading, results were reported in terms of the number of samples positive
for B.g. (of 15 for an experiment), rather than LR. Samples were scored as positive
if only one CFU were detected in the filter plate sample, which occurred not
infrequently. Most of the samples scored as positive typically showed fewer than 10
CFU.

• Of the 11 experiments conducted with small plants, four experiments resulted in
having no samples positive for B.g., and this occurred using either dichlor or PAA.

-------
Generally, PAA was somewhat more effective than dichlor if test conditions (e.g.,
same type of plant, same volume of decontaminant) were similar.

•	All the four decontamination experiments conducted with pine bark resulted in
having at least one sample positive for E.g., signifying the difficulty in
decontaminating this type of material. The test with PAA concentration at 0.55%
resulted in only 2 samples of 15 testing positive for E.g. Dichlor performed poorly
against the bark matrix, returning nearly all 15 samples positive for E.g. spores in
both experiments.

•	With respect to the sod decontamination results, there was no apparent association
between increasing the spray volumes of the decontaminant within each experiment
and improved decontamination efficacy. Overall, the zoysia grass was the most
easily decontaminated, in that it had the least number of samples that were positive
(9) following treatment. The Bermuda grass had the most positive samples (35). In
addition, the average number of positive samples per test (of 15) for the PAA was
5.25, while for dichlor, the average number of positive samples per test was 9.8.

•	Simple phytotoxicity tests were conducted to assess any detrimental impacts (such as
leaf discoloration, leaf loss, and lack of growth) on the plants due to their being
sprayed with the decontaminants. While none of the small plants died during the
month-long observations following exposure to the decontaminant, there were some
mixed results with respect to other phytotoxic effects that varied by plant type,
decontaminant, and the type of phytotoxic effect. No obvious trends in effects were
noted.

•	The phytotoxic effects of the decontaminants on the Indian Hawthorn plants were
generally indistinguishable from water. For the Creeping Jenny plants, there did
appear to be more damage to leaf material by both decontaminants, compared to the
water controls, with the dichlor impact likely being more pronounced than the PAA.
For the rinsed and unrinsed blueberry shrubs, the phytotoxic effect results were
inconclusive or at best counterintuitive, e.g., the PAA seemed to have more of an
effect on the rinsed blueberry plants compared to the unrinsed plants (in terms of leaf
damage and leaf shedding), although plant growth was greater for the rinsed
blueberry plants.

•	Based on the efficacy and phytotoxicity results of this study, we recommend that a
PAA-based decontaminant be used for the vegetative materials in the full-scale
AnCOR Wide Area Demonstration project. In addition, PAA-based decontaminants
are already registered for use as biocides on plants, albeit at lower concentrations.
The Wide Area Demonstration will provide the opportunity to evaluate the
decontamination of trees and grass at full-scale, in terms of efficacy as well as the
sprayer technology used to apply the decontaminants at a realistic scale.

57

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7 References

Brown, G. S., Betty, R.G., Brockmann, J.E., Lucero, D.A., Souza, C.A., Walsh, K.A.,
Boucher, R.M., Tezak, M., Wilson, M.C. and Rudolph, T. (2007A) Evaluation of a
wipe surface sample method for collection of Bacillus spores from nonporous surfaces.
Appl Environ Microbiol 73(3): 706-710.

Calfee, M.W.,Lee, S.D. and Ryan, S.P. (2013) A rapid and repeatable method to deposit
bioaerosols on material surfaces. J Microbiol Methods, 92(3): 375-380.

Canter, D.A., Gunning, D., Rodgers, P., O'Connor, L., Traunero, C., and Kempter, C.J.
(2005) Remediation of Bacillus anthracis contamination in the U.S. Department of
Justice mail facility, Biosecur Bioterror 3: 119-127.

Kabashima, J.,Giles, D.K., and . Parrella, M.P. (1995) Electrostatic sprayers improve

pesticide efficacy in greenhouses. California Agriculture. July - August 1995. 49(4):
31-35.

Lee, S.D., Ryan, S.P., and Snyder, E.G. (2011). Development of an aerosol surface

inoculation method for Bacillus spores. ApplEnviron Microbiol. 77(5): 1638-1645.

Mullins, J. C.; Garofolo, G., Van Ert, M., Fasanella, A., Lukhnova, L., Hugh-Jones, M. E.,
and Blackburn, J. K. (2013) Ecological niche modeling of Bacillus anthracis on three
continents: evidence for genetic-ecological divergence? PLoS One8(8): e72451.

Pethybridge, S. J., and Nelson, S. C. (2015). Leaf doctor: A new portable application for
quantifying plant disease severity. Plant Disease, 99(10): 1310-1316.
https://doi.org/10.1094/pdis-03-15-0319-re

Rastogi,V.K., Ryan, S.P., Wallace, L., Smith, L.S., Shah, S.S. and Martin, G.B. (2010)
Systematic evaluation of the efficacy of chlorine dioxide in decontamination of
building interior surfaces contaminated with anthrax spores, Appl Environ Microbiol
76: 3343-3351.

Ryan, S.P., Lee, S.D., Calfee, M.W., Wood, J.P., McDonald, S., Clayton, M., and Griffin-
Gatchalian, N. (2014) Effect of inoculation method on the determination of
decontamination efficacy against Bacillus spores. World J Microbiol Biotechnol
30(10): 2609-2623. DOI 10.1007/sl 1274-014-1684-2.

Schmitt, K., and Zacchia, N.A.(2012) Total decontamination cost of the anthrax letter
attacks. Biosecur Bioterror 10: 98-107.

U.S. Department of Defense; U.S. Department of Health and Human Services; U.S.
Department of Homeland Security; U.S. Department of Agriculture National
Biodefense Strategy (2018).

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U.S. EPA (2010) Determining the efficacy of liquids and fumigants in systematic

decontamination studies for Bacillus cmthracis using multiple test methods. U.S.
Environmental Protection Agency, Washington, DC, EPA/600/R-10/088.

U.S. EPA (2013). Bio-response operational testing and evaluation (BOTE) project - Phase 1:
Decontamination assessment. U.S. Environmental Protection Agency, Washington, DC,
EPA/600/R-13/168.

U.S. EPA (2012) Assessment of liquid and physical decontamination methods for

environmental surfaces contaminated with bacterial spores: Evaluation of spray method
parameters and impact of surface grime. U.S. Environmental Protection Agency,
Washington, DC, EPA/600/R/12/591.

U.S. EPA (2006) Evaluation of spray-applied sporicidal decontamination technologies. U.S.
Environmental Protection Agency, Washington, DC, EPA/600/R-06/146.

U.S. EPA (2015) Surface decontamination methodologies for a wide-areaB. cmthracis

incident. U.S. Environmental Protection Agency, Washington, DC. EPA/600/S-15/172.

U.S. EPA. U.S. Coast Guard Vessel Decontamination Demonstration. 2021A.

U.S. EPA (2021) Inactivation of a Bacillus anthracis spore surrogate on outdoor materials
via the spray application of sodium dichloro-s-triazinetrione and other chlorine-based
decontaminant solutions. U.S. Environmental Protection Agency, Washington, DC.
EPA/600/R-21/004

Wood, J., Calfee, W., M. Clayton, M., Griffin-Gatchalian, N. and Touati, D. (2011)
Optimizing acidified bleach solutions to improve sporicidal efficacy on building
materials. Lett Appl Microbiol 53(6):666-672.

Wood, J.P., Calfee, M.W., Clayton, M., Griffin-Gatchalian, N., Touati, A., Ryan, S.,

Mickelsen, L., Smith, L. and Rastogi, V. (2016), A simple decontamination approach
using hydrogen peroxide vapor for Bacillus anthracis spore inactivation. J Appl
Microbiol doi: 10.1111/jam. 13284. (Accepted manuscript)

Wood, J.P., Calfee, M.W., Clayton, M., Griffin-Gatchalian. N., Touati, A. and Egler, K.
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59

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Appendix A

BENCH-SCALE TESTING

60

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A.l Preliminary Test Results

A. 1.1 Recovery Efficiency from Plant Leaves—Test Results

The objective of the preliminary test was to determine the recovery efficiency of the
target organism using the established 18-mm coupon extraction method for a variety of plant
leaves. Materials tested included magnolia leaves, pear tree leaves, and ivy leaves. Galvanized-
steel coupons were also included as reference material coupons (RMCs). Relative to RMCs, the
extraction method recovered 145%, 134%, and 141% of spores from the magnolia, pear tree, and
ivy leaf coupons, respectively. Sample recoveries were sufficient to move forward with testing
using the current extraction procedure as it was. Table A-l details the average CFU recovered
per sample and the percent recovery for each plant material relative to RMCs.

Table A-l. Recovery Efficiency from Leaves

Coupon Material

Average CFU/Sample

Percent Recovery1
(%)

Magnolia Tree Leaf

4.95 X105 ± 7.79 xlO4

145.4

Pear Tree Leaf

4.58 XlO5 ± 7.43 xlO4

134.4

Ivy Leaf

4.83 XlO5 ± 7.86 xlO4

141.9

Galvanized-steel RMC

3.40 XlO5 ±8.16 xlO4

N/A

Relative to galvanized-steel RMCs

A. 1.2 Characterization of Spore Deposition onto Plant Material Using the LV-ADA

The LV-ADA was designed and fabricated for the aerosol deposition of spores onto one
or more full-sized plants. Prior to deploying the LV-ADA for use during testing, preliminary
testing was conducted to characterize the spore distribution throughout foliage of multiple plants
during inoculation.

Three (3) Manhattan Euonymus plants were transferred to the LV-ADA, then it was
sealed, and the plants inoculated with 5x10s B.g. After completing an 18-hour settling period, the
leaves of each plant were sampled using a 12.7-mm circular punch. The centers of 3 leaves were
selected from predetermined sample areas and combined in a single sample tube. Sample areas
were established by dividing each plant into 3 sections along the vertical y-axis and the
horizontal x-axis as shown in Figure A-l.

-------
X3 . . X2 . . XI

Figure A-l. Sample areas for spore distribution tests

Average CPU recoveries ranged from 9.77E+02 (± 6.55E-01 SD) and 8.52E+03 (±
1.15E+00 SD). Inoculation was completed without apparent bias for any particular sample area.
Sample recoveries were sufficient to demonstrate the effective use of the LV-ADA for aerosol
inoculation of full-sized plants. Table A-2 shows the average spore recoveries for each sample
area.

Table A-2. Average CFU Recoveries from Horizontal and Vertical Sample Sections

Average CFU/sample

Vertical Axis

Horizontal Axis

XI

X2

X3

Average CFU

Y1

1.20E+03 ± 5.66E-01

6.25E+02 ± 8.49E-01

8.52E+03 ± 1.15E+00

3.45E+03 ± 1.80E+00

Y2

1.67E+03 ± 1.05E+00

2.64E+03 ± 1.52E+00

5.56E+03 ± 8.91E-01

3.29E+03 ± 1.14E+00

Y3

7.51E+02 ± 7.34E-01

9.77E+02 ± 6.55E-01

1.63E+03 ± 1.12E+00

1.12E+03 ± 9.64E-01

Average CFU

1.21E+03 ± 8.76E-01

1.41E+03 ± 1.59E+00

5.23E+03 ± 1.20E+00

N/A

N/A = not applicable

62

-------
A. 1.3 Neutralization testing

Initial screening tests to assess whether or how much neutralizer was needed to quench
the active ingredient in the residual sporicidal solution remaining on the 18-mm leaf coupon
surfaces was conducted. Refer to Table A-3, which shows the percent CFU recovery (%) after
decontaminant-exposed negative leaf coupons were neutralized with DE broth, then spiked with
inoculum. For the tests with pAB, diluting with 10 mL of PBST preloaded in the sample tubes,
with no DE broth, was found to provide 26% recovery of spores, compared to 83% recovery
when using 2-ml DE broth. Subsequent testing with high concentration dichlor solution showed
CFU recoveries of 93% and greater when the sample containers were preloaded with DE broth.
Preloading the sample containers with neutralizer was an effective way to stop residual kill from
residual chlorine-based solutions.

Table A-3. Residual pAB and Dichlor decontaminant evaluation

Test II)

Decontaminant

mg/L FAC

DE Broth (ml)

CFU Recovery (%) with Neutralizer3

pAB

6240

None

26.1

4.2

pAB

6080

83.2

Dichlor

20231

100.1

Dichlor

10516

93.1

pAB

6173

N/A

Dichlor

20320

N/A

"Relative to positive controls

Table A-4 presents the CFU recovery for each PAA-based neutralization test. PAA-based
solutions with concentrations < 0.25% did not require the additional neutralization step to stop
residual inactivation. The 10 ml of PBST preloaded in the sample container provided sufficient
dilution to neutralize the active ingredient. However, decontaminant formulations with
concentrations that were > 0.25% PAA required an effective neutralizer during sampling. Sample
neutralization was attempted for Test 8, resulting in small improvements in CFU recovery. A
4.1% recovery was observed for the 0.57% PAA formulation of Jet Ag and 4.9% recovery for
the 0.46% PAA formulation of Oxidate 2.0.

-------
Table A-4. Residual Decontamination Evaluation for PAA

Test II)

Decontaminant

%PAA

DE Broth (ml)

CFU Recovery (%) with Neutralizer

4

Jet Ag 15

0.13

None

96.6

4

Oxidate 2.0

0.18

None

87.3

5

Jet Ag 15

0.25

None

50

5

Jet Ag 15

0.5

None

0.05

5

Oxidate 2.0

0.25

None

50

5

Oxidate 2.0

0.5

None

0.05

8

Jet Ag 15

0.57

0.8

4.1

8

Oxidate 2.0

0.46

7

4.9

64

-------
Appendix B

DETAILED TEST CONDITIONS FOR BENCH AND PILOT SCALE TESTS

65

-------
Table B-l. Bench-scale decontamination efficacy test matrix with Dichlor

Test ID

Plant Type

FAC
(ppm)

pH

Temperature

(°C)

Contact time
minutes

Neutralizer

6

Gold Dust Aucuba

10516

6.36

24.4

15

DE broth (3 ml)

6

Gold Dust Aucuba

20231

6.35

24.2

15

DE broth (6 ml)

7

White Indian
Hawthorn

20320

N/A

N/A

15

DE broth (6 ml)

Table B-2. Bench-scale decontamination efficacy test matrix with Jet-Ag

Test ID

Plant Type

PAA

(%)



HP

(%)



pH

Temperature

(°C)



Contact time
minutes



Neutralizer

4

Gold Dust Aucuba

0.039

0.046

N/A

N/A

15

None

4

Gold Dust Aucuba

0.072

0.093

N/A

N/A

15

None

4

Gold Dust Aucuba

0.13

0.13

N/A

N/A

15

None

5

Gold Dust Aucuba

0.28

0.27

2.59

22.4

15

None

5

Gold Dust Aucuba

0.58

0.58

2.50

22.4

15

None

8

White Indian
Hawthorn

0.57

1.47

N/A

N/A

15

DE broth (0.8 ml)

Table B-3. Bench-scale decontamination efficacy test matrix with Oxidate 2.0

Test ID

Plant Type

PAA

(%)



HP

(%)



pH

Temperature

(°C)



Contact time
minutes



Neutralizer

4

Gold Dust Aucuba

0.020

0.078

N/A

N/A

15

None

4

Gold Dust Aucuba

0.079

0.30

N/A

N/A

15

None

4

Gold Dust Aucuba

0.11

0.71

N/A

N/A

15

None

4

Gold Dust Aucuba

0.18

1.16

N/A

N/A

15

None

5

Gold Dust Aucuba

0.30

1.80

2.79

22.4

15

None

5

Gold Dust Aucuba

0.52

3.32

2.57

22.4

15

None

8

White Indian
Hawthorn

0.44

2.49

2.59

26.8

15

DE broth (7 ml)

Table B-4. Bench-scale decontamination efficacy test matrix with pAB

Test II)

Plant Type

Decon
Solution

FAC
(ppm)

pH

Temperature

(°C)

Contact time
minutes

Neutralizer

4

Gold Dust Aucuba

pAB

6240

6.92

23.8

15

None

4.2

Gold Dust Aucuba

pAB

6080

6.95

22.1

15

DE broth (2 mL)

66

-------
7

White Indian

pAB

6173

N/A

N/A

15

DE broth (2 ml)



Hawthorn













A control test with deionized water was performed to assess the physical removal of
spores from the procedure. The results of this test provided a baseline to determine the reduction
of spores due to the active ingredient for tests using sporicidal solutions. Table B-5 summarizes
the test conditions for the deionized waster tests.

Table B-5. Pilot-Scale Control Test with Deionized Water

Test
ID

Plant Type

Sprayer
Used

Total

B.g. spores

Volume
Disseminated
(mL)

Contact

time
minutes

DI Water Rinse
Duration (min)

P-03

White Indian
Hawthorn

Electrostatic
Sprayer

5xl07

198

59.35

2

67

-------
Table B-6. Pilot-scale decontamination efficacy test matrix with Jet-Ag

Test
ID

Plant Type

Sprayer
Used

MDIID

Measured
PAA

(%)

Measured
HP

(%)

Measured
pH

Measured
Temperature

(°C)

Volume
Disseminated
(mL)

Contact

time
minutes

DI Water Rinse
Duration (min)

P-01

Wliite Indian Hawthorn

ES

5 x 105

0.088

0.095

3.05

26.4

90

30

2

P-02

Wliite Indian Hawthorn

BP

5 x 107

0.58

0.725

2.28

26.1

2232

60

2

P-04

White Indian Hawthorn

ES

5 x 107

0.53

0.66

2.32

23.4

214

60.5

2

P-05

Wliite Indian Hawthorn

ES

5 x 107

0.306

0.381

2.39

22.7

236

60

2

P-06

Blueberry Shrub

ES

5 x 107

0.306

0.382

2.49

19.9

242

60

2

P-07

Japanese Spurge

ES

5 x 107

0.31

0.38

7.09

20.3

121

60

2

P-08

Pine Tree Bark

ES

5 x 107

0.3

0.38

7.06

21.9

260

60

2

P-09

Pine Tree Bark

ES

5 x 107

0.55

0.7

6.96

19.5

260

60

2

P-15.1

Zenith Zoysia Sod

ES

5 x 105

0.58

0.76

NA

NA

26.1

>18 h

none

P-15.2

Zenith Zoysia Sod

ES

5 x 105

0.58

0.76

NA

NA

52.3

>18 h

none

P-15.3

Zenith Zoysia Sod

ES

5 x 105

0.58

0.76

NA

NA

78.4

>18 h

none

P-15.4

Zenith Zoysia Sod

ES

5 x 105

0.58

0.76

NA

NA

104.5

>18 h

none

P-15.5

Zenith Zoysia Sod

ES

5 x 105

0.58

0.76

NA

NA

130.7

>18 h

none

P-19.1

Zenith Zoysia Sod

BP

5 x 107

0.57

0.72

2.29

23.7

16.5

>18 h

none

P-19.2

Zenith Zoysia Sod

BP

5 x 107

0.57

0.72

2.29

23.7

33

>18 h

none

P-19.3

Zenith Zoysia Sod

BP

5 x 107

0.57

0.72

2.29

23.7

49.5

>18 h

none

P-19.4

Zenith Zoysia Sod

BP

5 x 107

0.57

0.72

2.29

23.7

66

>18 h

none

P-19.5

Zenith Zoysia Sod

BP

5 x 107

0.57

0.72

2.29

23.7

82.5

>18 h

none

P-21.1

Tall Fescue Sod

BP

5 x 107

0.57

0.78

2.13

24.3

18

>18 h

none

P-21.2

Tall Fescue Sod

BP

5 x 107

0.57

0.78

2.13

24.3

36

>18 h

none

P-21.3

Tall Fescue Sod

BP

5 x 107

0.57

0.78

2.13

24.3

54

>18 h

none

68

-------
P-21.4

Tall Fescue Sod

BP

5 x 107

0.57

0.78

2.13

24.3

72

>18 h

none

P-21.5

Tall Fescue Sod

BP

5 x 107

0.57

0.78

2.13

24.3

90

>18 h

none

P-24.1

Bermuda Sod

BP

5 x 107

0.46

0.67

2.23

18.9

18.7

>18 h

none

P-24.2

Bermuda Sod

BP

5 x 107

0.46

0.67

2.23

18.9

37.4

>18 h

none

P-24.3

Bermuda Sod

BP

5 x 107

0.46

0.67

2.23

18.9

56.1

>18 h

none

P-24.4

Bermuda Sod

BP

5 x 107

0.46

0.67

2.23

18.9

74.8

>18 h

none

P-24.5

Bermuda Sod

BP

5 x 107

0.46

0.67

2.23

18.9

93.5

>18 h

none

69

-------
Table B-7. Pilot-scale decontamination efficacy test matrix with Dichlor

Test ID

Plant Type

Sprayer1

B.g.
Spores

FAC
(ppm)

pH

Temperature

(°C)

Volume
Disseminated
(mL)

Contact time
mminutes2

DI Water

Rinse
Duration
minutes

P-26

Blueberry shrubs

BP

5xl07

20609

6.61

20.2

240.5

60

2

P-ll

Eleanor Taber Indian Hawthorn

ES

5xl07

20,030

7.08

22.5

260

60

2

P-13

Goldilocks Creeping Jenny

ES

5xl07

20832

6.58

20.8

260

60

2

P-14

Nena Ivy

ES

5xl07

19,403

6.65

16.3

131

60

2

P-10

Pine Tree Bark

ES

5xl07

20,431

6.51

21.2

264

60

2

P-12

Pine Tree Bark

BP

5xl07

20431

6.57

22.6

2148

60

2

P-23.1

Bermuda Sod

BP

5xl07

20030

6.55

23.8

19

>18 h

none

P-23.2

Bermuda Sod

BP

5xl07

20030

6.55

23.8

38

>18 h

none

P-23.3

Bermuda Sod

BP

5xl07

20030

6.55

23.8

57

>18 h

none

P-23.4

Bermuda Sod

BP

5xl07

20030

6.55

23.8

76

>18 h

none

P-23.5

Bermuda Sod

BP

5xl07

20030

6.55

23.8

95

>18 h

none

P-25.1

Bermuda Sod

BP

5xl07

19630

6.22

24.1

18.1

>18 h

none

P-25.1

Bermuda Sod

BP

5xl07

19630

6.22

24.1

36.2

>18 h

none

P-25.1

Bermuda Sod

BP

5xl07

19630

6.22

24.1

54.3

>18 h

none

P-25.1

Bermuda Sod

BP

5xl07

19630

6.22

24.1

72.4

>18 h

none

P-25.1

Bermuda Sod

BP

5xl07

19630

6.22

24.1

90.5

>18 h

none

P-20.1

Tall Fescue Sod

BP

5xl07

21400

6.37

23.9

18

>18 h

none

P-20.2

Tall Fescue Sod

BP

5xl07

21400

6.37

23.9

36

>18 h

none

P-20.3

Tall Fescue Sod

BP

5xl07

21400

6.37

23.9

54

>18 h

none

P-20.4

Tall Fescue Sod

BP

5xl07

21400

6.37

23.9

72

>18 h

none

70

-------
P-20.5

Tall Fescue Sod

BP

5xl07

21400

6.37

23.9

90

>18 h

none

P-22.1

Tall Fescue Sod

BP

5xl07

20151

6.52

23.5

57

>18 h

none

P-22.2

Tall Fescue Sod

BP

5xl07

20151

6.52

23.5

76

>18 h

none

P-22.3

Tall Fescue Sod

BP

5xl07

20151

6.52

23.5

95

>18 h

none

P-22.4

Tall Fescue Sod

BP

5xl07

20151

6.52

23.5

114

>18 h

none

P-22.5

Tall Fescue Sod

BP

5xl07

20151

6.52

23.5

133

>18 h

none

P-17.1

Zenith Zoysia Sod

ES

5xl05

19830

6.61

24.7

25.7

>18 h

none

P-17.2

Zenith Zoysia Sod

ES

5xl05

19830

6.61

24.7

51.3

>18 h

none

P-17.3

Zenith Zoysia Sod

ES

5xl05

19830

6.61

24.7

77

>18 h

none

P-17.4

Zenith Zoysia Sod

ES

5xl05

19830

6.61

24.7

102.7

>18 h

none

P-17.5

Zenith Zoysia Sod

ES

5xl05

19830

6.61

24.7

128.3

>18 h

none

P-18.1

Zenith Zoysia Sod

BP

5xl07

20030

6.54

22.9

18

>18 h

none

P-18.2

Zenith Zoysia Sod

BP

5xl07

20030

6.54

22.9

54

>18 h

none

P-18.3

Zenith Zoysia Sod

BP

5xl07

20030

6.54

22.9

72

>18 h

none

P-18.4

Zenith Zoysia Sod

BP

5xl07

20030

6.54

22.9

108

>18 h

none

P-18.5

Zenith Zoysia Sod

BP

5xl07

20030

6.54

22.9

126

>18 h

none

1 Backpack sprayer (BP) or electrostatic sprayer (ES),2 Tests with >18-h contact times remained unrinsed and were allowed to air dry overnight

71

-------
Table B-8. Phytotoxicity test matrix with deionized water

Test II)

Plant type

Measured
spray rate
(mL/second)

Spray Duration
(seconds)

Contact time
(minutes)

DI Water Rinse
Duration
(minutes)

1

White Indian Hawthorne

2.2

120

60

2

4

Goldilocks Creeping
Jenny

18.6

7

60

2

7

Blueberry Shrub

19

7

60

2

10

Tall Fescue Sod

18.5

5

until dry

no rinse

13

Zenith Zoysia Sod

18.5

5

until dry

no rinse

16

Blueberry Shrub

18

5

until dry

no rinse

Table B-9. Phytotoxicity test matrix with Dichlor

Test
ID

Plant type

FAC
(ppm)

pH

Temperature

(°C)

spray rate
(mL/second)

Spray
Duration
(seconds)

Contact

time
(minutes)

DI Water Rinse
Duration
(minutes)

2

White
Indian
Hawthorne

19830

6.61

24.7

2.2

120

60

2

5

Goldilocks
Creeping
Jenny

20331

6.59

21.2

16.8

8

60

2

8

Blueberry
Shrub

20200

6.53

23.1

18.5

7

60

2

11

Tall Fescue
Sod

19500

6.5

23.8

18.5

5

until dry

no rinse

14

Zenith
Zoysia Sod

19830

6.47

22.4

19

5

until dry

no rinse

17

Blueberry
Shrub

19830

6.56

23.1

18

5

until dry

no rinse

Table B-10. Phytotoxicity Test Matrix with Jet Ag

Test
ID

Plant type

PAA

(%)

HP

(%)

pH

Temperature

(°C)

Spray Rate
(mL/second)

Spray
Duration
(seconds)

Contact

time
(minutes)

DI Water

Rinse
Duration
(minutes)

3

White Indian
Hawthorne

0.47

0.66

2.35

23

2.1

120

60

2

6

Goldilocks
Creeping Jenny

0.48

0.46

2.25

22.8

15.5

8

60

2

9

Blueberry Shrub

0.52

0.697

2.35

23.6

18.5

7

60

2

72

-------
12

Tall Fescue Sod

0.57

0.71

2.18

24.9

18.2

5

until dry

no rinse

15

Zenith Zoysia Sod

0.51

0.68

2.27

22.9

18.5

5

until dry

no rinse

18

Blueberry Shrub

0.49

0.72

2.16

25.4

18

5

until dry

no rinse

73

-------
Appendix C

PHYTOTOXICITY DETAILED RESULTS

74

-------
Table C-l. Average Damaged Leaves Per Indian Hawthorne Plant (Unrinsed)

Test II)

Baseline

Week 1

Week 2

Week 3

Week 4

Percent
Change

DI water

17

44

45

47

71

317

Dichlor

22

28

40

45

68

209

Jet Ag®

24

42

40

43

70

192

Table C-2. Total Shed Leaves per Indian Hawthorne Plant (Unrinsed)

Test ID

Plant ID

Week 1

Week 2

Week 3

Week 4

Total Shed
Leaves

Average Shed
Leaves



1

14

39

38

16

107



DI Water

2

9

34

19

3

65

288

3

14

33

20

7

74



4

14

15

10

3

42





5

13

33

19

4

69



Dichlor

6

9

25

11

8

53

226

7

8

22

14

10

54



8

19

23

5

3

50





9

8

21

10

2

41



JetAg®

10

10

39

22

2

73

187

11

5

9

9

5

28



12

6

26

11

2

45



Table C-3 Height for Unrinsed Indian Hawthorne Plant (Inches)

Test ID

Plant ID

Baseline

Week 1

Week 2

Week 3

Week 4

Average Plant
Height



1

11.00

12

NA

11.75

11.75



DI Water

2

15.25

16

NA

14.5

14.25

12.6

3

12.50

12

NA

11

12.25



4

12.75

13.25

12.75

12

12





5

14.25

13.75

13.75

15

14.5



Dichlor

6

15.00

15.00

12.75

14

14.25

12.9

7

11.25

11.75

11.5

10.5

10.25



8

10.25

13.00

12.5

13.5

12.75



JetAg®

9

12.00

12.25

12.25

12

12.5

11.9

10

13.25

13.50

13

12.75

12.5

75

-------


11

13.5

11.75

11.5

11

11.25



12

13.00

13.00

12.25

11.75

11.5

Table C-4. Average Damaged Leaves for Each Rinsed Creeping Jenny

Test II)

Baseline

Week 1

Week 2

Week 3

Week 4

Percent
Change

DI water

8

80

27

28

25

212

Dichlor

11

70

30

29

65

491

Jet Ag

8

25

19

27

26

225

Table C-5. Total Shed Leaves for Each Rinsed Creeping Jenny Plant

Test ID

Plant ID

Week 1

Week 2

Week 3

Week 4

Total Shed
Leaves



Average Shed
Leaves



1

10

0

3

3

16



DI

2

1

0

3

5

9

56

Water

3

0

1

6

7

14



4

3

0

6

8

17





5

0

0

5

6

11



Dichlor

6

1

3

9

11

24

56

7

1

0

4

1

6



8

4

2

5

4

15





9

2

1

11

8

22



Jet Ag

10

1

1

10

4

16

59

11

1

0

3

5

9



12

1

2

4

5

12



Table C-6. Height of Rinsed Creeping Jenny Plants (Inches)

Test II)

Plant ID

Baseline

Week 1

Week 2

Week 3

Week 4



1

4.5

3.5

3.5

3.5

4

DI Water

2

4

5

3

4

3

3

4.5

4.75

5.5

5

4.5



4

4

4.5

4.5

4.5

4



5

5

4

4

4

4

Dichlor

6

4.25

3.5

4

4

4.5



7

3.75

4

3.5

4

5

76

-------


8

4.5

3.75

4

4

4



9

4.5

3.75

5

4.75

5

Jet Ag

10

3.5

3.5

4

4.75

4

11

4.5

5.25

4.75

5.5

3.5



12

4.75

5.5

5.5

5

4

Table C-7. Average Number of Damaged Leaves for Each Rinsed Blueberry Plant

Test ID

Baseline

Week 1

Week 2

Week 3

Week 4

DI water

93

95

86

86

109

Dichlor

109

109

128

128

156

Jet Ag

58

137

145

145

126

Table C-8. Total Number of Shed Leaves for Each Rinsed Blueberry Plant

Test II)

Plant ID

Week 1

Week 2

Week 3

Week 4

Total Shed
Leaves

Average Shed
Leaves



1

1

3

0

0

4



DI Water

2

6

78

19

4

107

166

3

3

0

1

1

5



4

37

0

1

12

50





5

3

6

0

5

14



Dichlor

6

14

0

2

0

16

78

7

3

11

5

9

28



8

6

3

5

6

20





9

2

9

15

12

38



Jet Ag

10

0

21

6

1

28

77

11

2

1

2

4

9



12

0

2

0

0

2



77

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Table C-9. Height for Rinsed Blueberry Plant (Inches)

Test ID

Plant ID

Baseline

Week 1

Week 2

Week 3

Week 4

Average Plant
Height



1

24.5

24

26

26.5

27.5



DI Water

2

22

26.25

27.5

28.5

29.75

28.1

3

26

27

26.5

28.75

30.5



4

22.75

29.75

25.25

28.5

30.75





5

25.5

28

37

28

26.5



Dichlor

6

28

23.5

27.25

27.5

27.5

27.5

7

23

23.5

24

26

27.5



8

25.5

31.75

31.25

28.5

29





9

23.5

23

23

24

26.25



Jet Ag

10

25.75

25.5

24.5

27.5

30.75

24.6

11

24.75

25

25.5

26

28.5



12

23.5

23

20

20.75

23.25



Table C-10. Percent of Unrinsed Fescue Sod Coupon Measured as Damaged by Photoanalysis

Test ID

Plant ID

Baseline

Week 1

Week 2

Week 3

Week 4

DI

1

57.84

16.9

91.2

92.98

94.23

Water

2

42.96

15.02

65.19

95.55

96.34



3

25.09

15.95

16.79

17.71

28.67



4

22.62

99.02

Not available

95.01

92.25

Dichlor

5

16.48

96.15

95.6

90.65

79.13



6

17.84

64.97

67.89

92

88.68



7

12.98

71.72

93.72

97.38

92.87



8

28.45

62.52

97.58

97.89

98.15

Jet Ag

9

30.64

82.33

66.09

81.73

71.91



10

33.74

90.37

66.94

63.87

67.04



11

14.74

88.48

78.9

73.37

75.89



12

32.47

88.28

95.09

95.42

78.42

78

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Table C-ll. Height of Grass at the Center of the Unrinsed Fescue Sod Coupon (Inches)

Test ID Plant Replicate Week 1 Week 2 Week 3 Week 4



1

3.5

3.5

2.5

5

DI

2

3.5

2.5

2

10.75

Water

3

3

6

5

5.5



4

1.25

2

1.5

2.5



5

1.25

2

2

3

Dichlor

6

2.25

2.25

1.5

1

7

1.75

1

0.75

1



8

2

1.5

1

0.5



9

1.5

5

3

8

Jet Ag

10

1.5

1.5

1

5

11

1

2.25

2.5

2



12

1

2

1.5

0.5

Table C-12. Average Percent of Zoysia Sod Coupon Measured as Damaged per Photo Test

Test ID

Baseline

Week 1

Week 2

Week 3

Week 4

DI water

37

94

89

55

38

Dichlor

47

71

70

36

45

Jet Ag

44

77

72

42

18

Table C-13. Height of Grass at the Center of the Fescue Sod Coupon (Inches)

Sporicide

Test II)

baseline

Wkl

Wk 2

Wk 3

Wk4



1

3.25

3

1.75

3.5

6.75

DI Water

2

3.25

3.5

2.5

4.5

8

3

4

3.5

3.75

5.75

6.25



4

2.5

2.5

2.5

4.25

5.25



5

4

4

5.25

8.5

4

Dichlor

6

3

3.5

3.75

4.5

5.5

7

4

3.5

4.75

6

4



8

4.5

4

4.25

6.5

5



9

2.58

3

3.5

7

7.25

Jet Ag

10

3.25

5.5

5

5.5

6.25

11

3.5

4

6.25

7.5

6.5



12

4

2.75

4.75

7.5

5.5

79

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Table C-14. Decontaminant Effect Substantially Worse than Baseline Plants



Indian
Hawthorne
(rinsed)

Creeping Jenny
(rinsed)

Blueberry Shrub
(rinsed) *

Blueberry

Shrub
(unrinsed)

Fescue Sod
(unrinsed)

Zoysia Sod
(unrinsed)

Compared to DI Water
plants:

DC

PAA

DC

PAA

DC

PAA

DC

PAA

DC

PAA

DC

PAA

>100 Percent change in
average # of damaged leaves,
from baseline to week 4

N

N

Y

N

N

Y

Y

N

NA

NA

NA

NA

Percent change in average #
shed leaves over 4-week
period

-27%

-54%

0%

5%

-113%

-116%

NA

NA

NA

NA

NA

NA

>100 Percent change of
average percentage of sod
coupon damaged from
baseline to week 4

NA

NA

NA

NA

NA

NA

NA

NA

Y

N

N

N

Percent plant height change
from baseline to week 4

4%

-6%

9%

5%

-17%

-12%

7%

5%

-62%

-11%

-69%

-19%

DC=dichlor

80

-------
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