Updates and Developments to EPA's
Water Contaminant Information Tool (WCIT)
John Bain, ORISE Participant, and George Gardenier, Chemist, Water Laboratory Alliance
U.S. Environmental Protection Agency, Office of Water, Water Security Division
vvEPA
Background
Recent & Upcoming Updates
What is WCIT?
•	The Water Contaminant Information Tool
(WCIT) is EPA's secure, web-based
informational database on priority
contaminants of concern for drinking water
and wastewater systems.
Information in WCIT
•	There are currently over 800 CBR
contaminants of concern to water systems
housed in the database.
A
I
|—| Field and Laboratory
Methods
~
Drinking Water
Treatment
~	Water Quality Indicators
r—| Environmental
Indicators
~	Wastewater Treatment
Infrastructure
~
Decontamination
0 General Information
0 Contaminant Summary
~	Other Names/Forms
~	Physical Properties
~	Availability
~	Fate and Transport
~	Medical Information
~	Toxicity Information
Who can use WCIT?
•	WCIT access is limited to certain users,
including federal employees, drinking water
and wastewater utilities, public health
officials and laboratories.
How can WCIT be used?
•	WCIT assists users prior to, during, and
after a contamination incident by providing
important information on contaminants
•	For more information on WCIT and
upcoming training webinars visit our
websites:
•	https://www.epa.gov/waterlabnetwork
•	https://www.epa.gov/waterlabnetwork/acc
ess-water-contaminant-information-tool
The Water Contaminant Information Tool (WCIT) is a living tool - constantly being updated
In Progress:
Legionella
Perfluorooctanoic acid (PFOA)
Perfluorooctane sulfonate
(PFOS)
Technology Development
Current WCIT layout & search function
1
WATER CONTAMINANT INFORMATION TOOL

it that has been identified or is suspected, but is not designed to be a
finitive means of identifying an unknown substance.
E WCITS INTENDED USERS?
ended for use by water utilities, EPA Program Offices and Regions,
ral organizations. State drinking water programs, public health officials,
ital laboratories, emergency first responders, and technical assistance
•	Since the Water Contaminant
Information Tool (WCIT)
was first released technology
has advanced significantly.
•	Working on developing
a mobile website.
Example of possible new layout & search function
Condensed
and
improved
search
function
Updated
look for the
homepage

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Exposure and Pathways Analysis of Infectious Livestock Carcass Management Options during Emergency Situations
United States
Environmental Protection
Agency
Margaret McVey:
Sandip Chattopadhyay1, Joshua Cleland2,
1U.S. Environmental Protection Agency, Office of Research and Development; 2ICF; 3U.S
Animal and Plant Health Inspection Service
2 Kaedra Jones2, Paul Lemieux1, Sarah Taft1, Lori Miller3
Department of Agriculture-

Background
Management of livestock carcasses following large-scale mortalities is needed to protect humans, livestock, and wildlife from
hazards; to maintain water, air, and soil resources; to protect ecological resources; and to enhance food and agricultural
security. Previous health and environmental assessments (eg., CAST 2009; Gwyther et al. 2011; NABCC 2004; Pollard et
al. 2008; UKDH 2001) of mass livestock mortality events relied on qualitative evaluation of exposure or observations based
on incident-specific circumstances, which limitstheir usefulness for general decision-making.
To address the need for a quantitative analysis, the U.S. Department of Homeland Security, U.S. Department of Agriculture,
and U.S. Environmental Protection Agency are jointly conducting an exposure assessment for potential releases of chemical
toxicants and microbes of seven livestock carcass management options (Table 1) and associated carcass handling and
transportation activities.
Exposure estimates have ranked for the livestock carcass management options for a hypothetical site. Four mortality
scenarios are being evaluated: natural disaster, foot-and-mouth disease (FMD) outbreak, a chemical contamination incident
and a radiological contamination incident. This presentation focuses on the natural disaster emergency and human exposure
to microbes.
II
Problem Formuiation
The scope of the assessment includes the disaster scenario, scale of mortality, chemical and microbial hazards, and
management options assessed. The management options considered for the exposure assessment are shown in Table 1.
To focus the assessment on differences among the carcass management options, exposures are assessed for each option
using a hypothetical site setting and mortality scenarios. 50 U.S. tons (45,359 kg) of carcasses are considered for ail
management options. Exposure receptors include adult and child residents of the hypothetical farm and workers engaged in
carcass management. Potential exposure pathways include inhalation, ingestion of drinking wafer, and ingestion of
homegrown foods. Homegrown foods include fruits and vegetables; livestock products including beef, dairy, pork, poultry,
and eggs; and fish caught in an on-site lake. Drinking water for the farm family is obtained from an on-site well.
Table 1. Livestock Carcass Management Options for the Exposure Assessment-
Livestock Carcass Management Options
Combustion-based Management
On-site Open Burning — Burning of carcasses in combustible heaps called pyres. It requires combustible material
additives (e.g., straw, hay, coal) to reach necessary temperatures to completely consume animal carcasses. Pyres are
layered with combustible material underneath the carcasses with space for sufficient air circulation.
On-site Air-Curtain Burning - Carcasses are burned in a partially enclosed (partially open on top) refractory-lined
firebox. A forced airflow, driven by a diesel-powered blower, creates a recirculation zone over the burn area that retains
much of the smoke and soot within the fire box. This process allows higher temperatures to be reached and more
complete combustion than open pyre burning.
Off-site Fixed-facility Incineration — Commercial scale incinerators are usually fueled with propane, diesel, or natural
gas, and burn solid waste through a series of stages. To be used for carcass disposal, an incinerator must be able to
handle high moisture content (-70%) and long burn times to allow complete combustion. Most municipal waste-to-
enerov incinerators do not accept carcasses for disposal.	
Land-based Management
On-site Unlined Burial - Carcasses are placed in an unlined trench or a lined pit. After placement, carcasses are
covered with at least 6 feet of soil, including 3 feet of soil mounded above ground level.
On-site Composting - Carcass disposal via composting is highly controlled, managed, and regulated. Additional
materials are required such as carbon sources, bulking agents, and biofilters (layer of carbon source and bulking
material enhances microbial activity by regulating moisture content, pH, temperature, etc.).
Off-site Lined Landfill — Landfills are engineered structures where waste is isolated form the surrounding
environment. Some municipal solid waste landfills (RCRA Subtitle D) are specifically designated for livestock carcass
disposal. Landfills must include composite liners and clay to protect groundwater and soil from the leach ate, and they
must implement leach ate collection and removal systems.
Materials Processing
Off-site Rendering - Rendering occurs in regulated facilities and transforms animal carcasses (including whole animals,
carcass trimmings, inedible offal and bones) into three distinct end-products: carcass meal, melted fat/tallow, and water.
1
Hazard Identification
Hazardous chemicals and microbes of concern that are released directly from decomposing carcasses or from carcass
management (including combustion products and added materials) and post-management processes (e g., compost
application, ash burial) were identified. Microbes of concern were identified as those that could be present in healthy
cattle in the United States. Table 2 identifies the scope of microbial hazards from six gram-positive bacteria, seven gram-
negative bacteria, three protozoa, six viruses, a fungus, and a prion. Even though some of these agents are unlikely to
be present in U.S. cattle herds, they are included as worst-case situation.
Potential for microbial exposures was evaluated for each on-site option based on the likely occurrence, persistence, and
mobility. These microbes are included as potential hazards in the livestock carcass storage, transportation, and handling
stage as well as for the on-site unlined burial option, because there are no initial assumptions on thermal conditions that
eliminate the consideration of any microbes for these stages of carcass management. The temperatures reached during
com bust ion-based management options and composting vary with the duration that these temperatures are reached.
Therefore, heat-resistant prions that cause bovine spongiform encephalopathy (BSE) may remain viable even after
exposure to the temperatures reached during on-site open burning and on-site composting. Spore-forming bacteria may
also survive the composting process.
No microbial hazards are quantitatively evaluated for off-site management options, because all environmental releases
from these options are subject to existing regulations that are assumed to protect human health and the environment.
Although exposures are not quantified for these options, they are included in the relative ranking of options.
Natural Disaster
Mortalities
nnn
On-site Transportation
Diffusion through cover soil
Table 2. Microbial Hazards Possibly Associated with Livestock Carcass Management-
Management
Potential Microbial Hazards
at Each Stage of Livestock Carcass Management
Type and Options
Livestock Management Options
Storage, Transportation, and Handling Activities
Combustion-
based
Management
On-site open burning:
•Prions (PrP3:)3
Identified pathogens:
•	Bacillus anthracis3 • Yersinia enterocolitica
Campylobacter spp. • Cryptosporidium spp.
•	Clostridium perfringens * Giardia spp.
•	Coxlella burnetii • Toxoplasma gondii
•	Dermatophllus congoiensis • Trichophyton verrucosum
•	EschericNa coll 0157:H7 • Rotavirus
and other shiga-toxin • Hepatitis E virus®
producing strains • Influenza D virus®
•	Leptospira spp. • Enteroviruses
•	Listeria monocytogenes • Adenoviruses
•	Mycobacterium avium * Caliciviruses
Paratuberculosis (e.g., norovirus)
•	M, bovis • Prions (PrPSc)a
•	Salmonella spp.
•	SNgelia spp.
On-site air-curtain burning:
•None
Off-site fixed-facility Incineration:
•None
Land-based
Management
On-site unlined burial:
•All identified microbes (listed at right)
On-site composting:
•B, anthracisa;
•C, perfringens;
•Coxieiia burnetii;
•Prions (PrP35)®
Off-site lined landfill:
•None
Rendering
• None
a Microbes with a low likelihood of presence in the U.S.
Conceptual Models - Exposure Pathways
Figure 1. Conceptual Model for Open Burning and Air-curtain Burning.
II
Work-in-progress
•	Quantitative analysis of exposure to FMD. Exposures are being assessed using existing, peer-
reviewed modeling tools, including EPA developed AERMOD and Viruio.
•	Ranking of management options relative to each other based on the following factors: (1) the
number conceptual model pathways; (2) the number of pathways with potential microbial exposure;
(3) the number of quantified pathways; and (4) quantified exposures. Numbers of conceptual model
pathways and potential exposures for each option are included to account for pathways with
unquantified exposure levels.
•	Exposure assessments for the chemical and radiological attacks/release incidents.
Leaching from to
subsurface soil and
Groundwater
Sedimentation,
Resuspension, &
Diffusive exchange
Groundwater	Sediment
Well
Water
Conceptual models were developed for each of the seven livestock carcass management options and associated
handling and transportation activities. Examples are presented in Figures 1 and 2 for the on-site combustion and
carcass burial options, respectively.
These conceptual models were used to identify exposure pathways for microbes and chemical toxicants. Table 3
shows the exposure pathways for microbes. For most exposure pathways, potential exposures were determined to
be negligible based on source conditions or properties related to survival, fate, and transport for a specific microbe.
Exposure pathways determined to have potentially non-negligible exposures are in shaded cells in Table 3.
Natural Disaster
Mortalities
Figure 2. Conceptual Model for Carcass Burial.
Table 3. Exposure Pathways for Livestock Carcass Management Options - Microbes.
Exposure
Exposure Pathways -
Transportation and Handling
Activities

Exposure Pathways - Management Options


Medium
Handling
Temporary
Carcass
Storage
Transport-
Burning
Air-curtain
Burning
Burial
Composting
Off-site
Off-site
Landfilling
Rendering
Inhalation
1) Air —
Inhalation0
1)	Air —•
Inhalation0
2)	Leachate —
Soil —GW —»
1)Aerosolb
1)	Aii*
2)	Ash —GW
— Aerosol11
1)	Air®
2)	Ash — GW
1)	Air6
2)	Leachate
— GW —
Aerosol6
1)	All4
2)	Compost —
GW —
1) Air1
1)Aii«
1) Air0
Ingestion
2) Hand-to-
mouth oral
contact1
-
-
-
-
-
-

-
-
Incidental
Soil
Ingestion
-
-
-
3) Air—Soil"
3) Air — Soil6
-
-
2) Air — Soil1
-
-
Fish
Ingestion
-
3) Leachate -*
Soil-GW-
SW — Fish
ingestion'
-
4)	Air — SW —
Fish"
5)	Air — soil —
SW —Fish6
6)	Ash — GW
— SW — Fish"
4)	Air— SW —
Fish®
5)	Air — Soil —
SW - Fish'
6)	Ash — GW
— SW — Fish0
3) Leachate
-GW —
SW —Fish6
3)	Compost —
Soil — SW —
Fish'
4)	Compost —
GW —SW —
Fish'
3)	Air— SW
—	Fish0
4)	Air—Soil
—	SW —
Fish1
-
-
Ground-

4) Leachate —
Soil — GW —

7) Ash — GW'
7) Ash — GW
4) Leachate
5) Compost



Ingestion

Drinking water
ingestion*



-GW
GWS



Ingestion
of Food
Produced
on the
Farm
-
"
-
8)	Air —Plants/
Livestock1'
9)	Air —Soil —
Plants/
Livestock*'
10)	Ash — GW
— Livestock11
8)	Air—Plants/
livestock'
9)	Air —Soil —
Plants/
Livestock6
10)	Ash — GW
— Livestock6
5)	Air —
Plants/
6)	Leachate
— GW —
Livestock'
6)	Air —
Plants/
Livestock'
7)	Compost —
Soil —GW —
Livestock1'
5)	Air —~
Plants/
6)	Air —Soil
— Plants/
2) Air-
Plants/
Livestock0
2) Air-
Plants/
Livestock0
Dermal
Contact
3jDemial
contact1
-
-
-
-
-
-
-
-
-
Abbreviations:"—¦" = no exposure pathways; SW = surface water; GW = groundwater
Note: Exposure pathways shown in bold were included in the quantitative exposure
a Quantitative assessment conducted; results are presented in Section 6.2.b Potential exposures ar
microbial properties.
c Environmental releases or exposures are assumed to be adequately controlled by existing pollutio
equipment.
¦ assumed to be negligible based on source conditions or
control regulations or use of personal protective
References
CAST (Council for Agricultural Science and Technology) (2009). Issue Paper 41. Ames, Iowa.
Gwyther CL, et al. (2011). Waste Management 31(4): 767-778.
NABCC (National Agricultural Biosecurity Center Consortium) (2004). Report prepared by the NABCC, Carcass
Disposal Working Group, For the USDA Animal & Plant Health Inspection Service, Per Cooperative Agreement 02-
1001-0355-CA.
Pollard S, et al. (2008). Environ. Sci. Technol. 42: 3145-3154.
United Kingdom Department of Health (UKDH) (2001) . A Rapid Qualitative Assessment of possible risks to Public
Health from current Foot & Mouth Disposal Options.

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DAHLGREN
Operational Field Demonstration
Outdoor Biological Simulant Releases in an Operationally Relevant Environment
Amanda Clark, M.S.,
Naval Surface Warfare Center, Dahlgren Division; Department of Homeland Security
Introduction
Collector Siting
In 2003, the Department of Homeland Security (DHS) deployed a nationwide bio surveillance system to
provide early warning of a biological attack by conducting surveillance for aerosolized biological agents in
specific locations across the United States. This effort operates in direct support of DHS Strategic Goals and
Homeland Security Presidential Directive 10. DHS requested Naval Surface Warfare Center Dahlgren
Division (NSWCDD) demonstrate the established system's ability to collect, detect and identify the presence
of a bio-aerosol.
Objectives
Objective #1
Demonstrate that the currently fielded bio surveillance system can detect an intentional
aerosol release of a biological simulant in an operationally relevant environment
Objective #2
Demonstrate current modeling capabilities in an operationally relevant environment
including up-front collector siting and back-end event reconstruction.
Site selection performed as it is operationally for a selected
jurisdiction. NSF-Dahlgren installation was treated as a
representative jurisdiction and ten collection sites were
identified around the installation.
As a follow on to the model's output for collector siting, an
operational process called micro-siting was performed by the
program office where collector location was adjusted to
ensure they were in non-hazardous locations reasonably
Trial 20140904
300 Gram Moving Point (107.6 meters)
Operationally Relevant
Draws a distinction between the Naval Support Facility Dahlgren (NSF-Dahlgren) venue and
other traditional biological detection test venues or laboratory test facilities
NSF-Dahlgren offers more relevant operational factors:
¦	Environment - Littoral
¦	Topography
¦	Infrastructure
NSF-Dahlgren was considered to be more representative cf an urban setting and moderate
climatic conditions as compared to the typical alternative test venues.
Environmental Impact Statement
The Final Environmental Impact Statement (EIS) Outdoor Research, Development, and Test and
Evaluation Activities, Volume 1, prepared by the US Department of the Navy, NSWCDD
evaluated the effects of expanding research, development, test and evaluation (RDT&E)
activities within the Potomac River Test Range and Explosives Experimental Area complexes,
the Mission Area, and special-use airspace at NSF-Dahlgren. One of those activities specifically
covered is the release of biosafety level 1 biological simulants. This demonstration is covered by the
National Environmental Policy Act by the 2013 RDT&E EIS, Record of Decision signed 10/15/1013
Simulant
Bacillus atrophaeus var. globigii (aka Bg)
Dugway Bg ATCC #9372 amended with 5% Aerosil R 812S Fluidizer
Provided by Dugway Proving Ground (DPG)
Biosafety Level 1 (BSL-1) organism
Ubiquitous in the environment, commonly
found in hay, soil and water.
Cultured Bg creates pigmented colonies that
allow for discrimination from other
environmental bacteria.
Light Microscopy
¦ L arge number of single phase bright B g
spores with many large >20|im
conglomerations
Reference System Characterization
Referee System and Dissemination System Development and Characterization
Completed inside negative pressure isolation tent with adjustable
unidirectional airflow.
Objectives of initial indoor characterization:
#1. Demonstrate the ability to create and control biological aerosol
>	Effectively aerosolize a powder to achieve predetermined
concentrations
#2. Collect/detect the aerosolized Bg using the DHS system
>	Assess system performance with selected simulant - qualitative
#3. Collect/quantify the aerosolized Bg using the referee system
>	The ruler by which the system under test's performance is
measured - quantitative
Scanning Electron Microscopy
¦	Estimated spore size 0.6-0.8jrm x 1.1-1.4 jam
Laser Diffraction Analysis
¦	Number % vs. Particle Size
¦	Data aggregated from 0.6-1.4 jrm region
¦ 33% of material fell within this region
¦	Within that region the mean particle size was
0.76 jmi with a median of 0.71 jxm.
Serial Dilution Plating
¦	3.94E+07 CFU/mg

-------
oEPA
www.epa.gov
Life Sciences Dual Use Research of Concern (DURC)
Brendan Doyle1, Cayce Parrish1, Eli Walton2
1U.S. Environmental Protection Agency;2 Contractor, U.S. Environmental Protection Agency
Introduction
DURC and Institutional DURC Policies
EPA Responsibilities
Life sciences research is essential to scientific advances that underpin
improvements in public health and safety, agriculture (including crops and
other plants and animals), the environment, materiel, and national
security.
Despite its values and benefits, certain types of research conducted for
legitimate purposes can be utilized for both benevolent and harmful
purposes. Such research is called dual use research.
Dual Use Research of Concern (DURC):
A subset of dual use research defined as life sciences research that,
based on current understanding, can be reasonably anticipated to provide
knowledge, information, products, or technologies that could be directly
misapplied to pose a significant threat with broad potential consequences
to public health and safety, agricultural crops and other plants, animals,
the environment, materiel, or national security

¦



Background
In 2011, two studies of avian flu virus, funded by the National Institutes of
Health, raised national security concerns when researchers tried to
publish their results, which included methods for making the virus more
transmissible. This research had the potential to meet the definition of
dual use research of concern.
In response, the U.S. Government issued two government-wide policies
for the oversight of life sciences research involving avian flu virus and 14
other high-consequence agents and toxins:
•	Dual Use Research of Concern (DURC) Policy; and, the
*	Institutional Dual Use Research of Concern (iDURC) Policy.
U.S. Environmental Protection Agency
Office of Research and Development
Two policies have been released that seek to enhance oversight, ensure
responsible research, and mitigate the risks associated with dual use
research of concern
United States Government Policy for Oversight of Life Sciences Dual
Use Research of Concern (DURC Policy) March, 2012
~	Complements existing regulations and policies governing the
possession and handling of pathogens and toxins;
~	Sets standards and procedures for departments and agencies to
review federally funded or conducted life sciences research
involving:
~	15 agents or toxins;
~	One or more listed experiments; and that
~	Meets the definition of dual use research of concern.
~	Requires the implementation of risk mitigation measures, if
necessary.
United States Government Policy for Institutional Oversight of Life
Sciences Dual Use Research of Concern (iDURC Policy)
79 Federal Register 57589. effective September 25, 2015.
Sets standards and procedures for institutions that receive funding
from the U.S. Government for life sciences research to review all life
sciences research conducted at their institution and determine
whether it:
~	Involves any of the 15 agents or toxins;
~	Involves one or more listed experiments;
~	Meets the definition of dual use research of concern.
Requires the implementation of risk mitigation measures, if
necessary.
EPA Order 1000.19, Policy and Procedures for Managing Dual Use
Research of Concern (approved September 14, 2016) establishes a
systematic approach for Environmental Protection Agency (EPA) and
extramural researchers to identify and mitigate when and how the risks
that knowledge, information, products, or technologies produced by
certain life sciences research may be misapplied in ways that pose
significant threats with broad, potential consequences to public health and
safety, agricultural crops and other plants, animals, the environment,
materiel, or national security. EPA's Policy is that all life sciences
research involving one or more of the listed select agents or toxins shall
be subject to institutional review and oversight and conducted and
communicated responsibly.
"Life Sciences Research," according to EPA, and based on the
definition of research in 40 CFR § 26.102(d), is a systematic investigation
designed to develop or contribute to generalizable knowledge involving
living organisms (e.g., microbes, human beings, animals, and plants) and
their products. EPA does not consider the following activities to be
research: routine product testing, quality control, mapping, collection of
general-purpose statistics, routine monitoring and evaluation of an
operational program, observational studies, and the training of scientific
and technical personnel. [Note: This is consistent with Office of
Management and Budget Circular A-11.]
DURC Policy
Under the DURC Policy, EPA must conduct a semi-annual inventory of all
funded or conducted research that involves any of the 15 agents or toxins.
•	The Agency has regularly conducted this survey since June 2012 and
reports the results to the National Security Council
iDURC Policy
Under the iDURC Policy, EPA must require institutions that receive life
sciences research funding from EPA to comply with the Policy's provisions.
•	The Agency has developed a solicitation provision and contract clause
for contracts, terms and conditions for grants, and language for
interagency agreements and Federal Technology Transfer Act (FTTA)
agreements that address institutional compliance with the iDURC Policy
•	All EPA funding agreements that involve life sciences research
must contain language addressing iDURC Policy compliance.
DURC Agents, Toxins & Experiments
The following 15 agents and toxins are identified in the DURC and iDURC
Policies:
I.
Avian IrtfluMwta virus (highly
8. Franctselki tuforensis

pathogenic)
9. Marburg virus
z
Bacillus anthrocis
10. Reconstructed 1918 Influenza virus
3.
Botulinum neurotoxin (in any
11. Rinderpest virus

quantity)
12. Toxin'producing strains of Clostridium
4.
BtirkhalderSa mollei
botalinam
5.
HurklwIderSa pseudontalM
13. Variola inafor virus
6.
Ebola virus
14. Variola minor virus
7.
Foot-and-mouth disease virus
IS. Ycninta pestis
Categories of experiments:
a)	Enhances the harmful consequences of the agent or toxin;
b)	Disrupts immunity or the effectiveness of an immunization against the agent or
toxin without clinical and/or agricultural justification;
c)	Confers to the agent or toxin resistance to clinically and/or agriculturally useful
prophylactic or therapeutic interventions against that agent or toxin or facilitates
their ability to evade detection methodologies;
d)	Increases the stability, transmissibility, or the ability to disseminate the agent or
toxin;
e)	Alters the host range or tropism of the agent or toxin;
f)	Enhancesthesu scepti b i I ity of a h ost p opu I ati on to th e ag ent or toxi n;
g)	Generates or reconstitutes an eradicated or extinct agent or toxin listed above.
If a research project involves any of these agents or toxins, Principal
Investigators and their institutions should notify EPA's Institutional Contact
for Dual Use Research at: DURC@epa.gov.

-------
Advanced Decontamination Concepts and National Security Product Development
Brian France (bfrance@tda,com) and William Bell,
TDA Research, Inc.
12345 W 52nd Ave, Wheat Ridge, CO. USA 80033
HA
RESEARCH
Handheld Electrochemical Decontamination Technology - eCI02
In this project, TDA Research, Inc. (TDA) is developing a new liquid decon solution based on electrochemical technology
originally developed at Procter & Gamble. The system consists of a dispenser with an electrochemical cell in which
electrical power applied to an aqueous solution of sodium chlorite produces chlorine dioxide (Cl02), an effective
decontaminant of CW agents that is also highly effective against biological threats, including bacterial spores. A sodium
bromide salt in the same solution produces hypobromite ion (BrO-1) during electrochemical conversion, which decomposes
the G-agents. Both CI02 and BrO-1 are highly reactive and not persistent: they react quickly with any CW or BW agents
present, and decompose in a short period of time, leaving no hazardous residue. The decontaminant has also been
shown to be compatible with military materials.
For electrochemical conversion only solid salts are transported, not reactive species; starting salt solutions can be made
with available on-site water. The solid salts are highly shelf stable and readily transportable. Consumables consist of long
shelf life lithium batteries and solid salt packages. The electrochemical system is safe for the operator because the
active component is generated only as needed, so operators do not mix or carry any highly reactive toxic material. The
chlorine dioxide and hypobromite react and decompose within a few hours, leaving no hazardous residue. This system
benefits.the warfighter by providing an effective decon solution that is readily transported, easy to use, environmentally
friendly and safe for the operator.
Environmental Protection Agency Registration Efficacy Testing
i Efficacy against anthrax spores, by modified AOAC 2008.05 test protocol
i Testing was performed at Naval Surface Warfare Center - Dahlgren Division (NSWCDD)
• Buhr, T., Young, A., Minter, Z., Wells, C. and Shegogue, D. (2011), Decontamination of a hard surface contaminated
with Bacillus anthracisASterne and B. anthracis Ames spores using electrochemically generated liquid-phase
chlorine dioxide (eCI02). Journal of Applied Microbiology. Ill, 1057-1064
Test Organism
Bacillus
anthracis
ASterne
Identification
Number
Date Preformed
BAC1056 01/26/2010
Results
(Log Reduction)
1 min. 5 min. 15 min,
NSWCDD performed tests s
7-log kill within 1 minute.
r to the EPA protocols using Bacillus anthracis Ames and
EPA Registration for Efficacy against Anthrax
i July 23, 2015
' US EPA Reg. No. 85797-1
Advanced Surfactant Blend Designed Specifically
for Decontamination with Multiple uses - SSDX-12
Aircraft are extremely expensive and sensitive assets which are critical for defense and transportation. Chemical products
used on an aircraft must meet strict materials compatibility requirements to ensure they don't degrade, shorten the life
expectancy or cause the failure of any aircraft component.
Aircraft that have been contaminated with toxic chemicals cannot be decontaminated using traditional decon solutions,
which include oxidants such as bleach or hydrogen peroxide. Using these reactive materials would corrode parts;
damaged components would then have to be identified and replaced to prevent catastrophic failure. Decontaminants that
remove the toxic chemicals without harming the aircraft are essential.
The goal of this effort was to develop a detergent formulation that was specifically tailored to remove chemical and
biological agents from contaminated surfaces, and that had excellent compatibility with all the components of an aircraft.
To ensure the product would be successful and widely accepted it was designed as a dual use item, both for
decontamination and as an aerospace equipment cleaning compound qualified under military and commercial standards.
•	A surfactant blend specifically designed to emulsify and lift agents from surfaces
•	Non-reactive, non-corrosive, pH neutral, no-VOC, biodegradable
•	High concentration/ HE formulation (reduced shipping and storage)
•	Dual use
•	Commercially available
•	Non-hazardous, no DOT restrictions
Decontamination Performance - HaMMER ATD
Large panel decontamination testing using SSDX-12 as a decontaminant prewash showed
•	Low or high pressure SSDX-12 prewash provides improved decontamination compared to no prewash
• Low pressure prewash was used in the contact hazard testing but could have additional safety benefits to the
decontamination operator by preventing backsplash.
•	Scrubbing during decontamination is not required if SSDX-12 prewash is performed
•	SSDX-12 prewash removes 99.95% of a VX contact hazard without reactive decontaminants
•	SSDX-12 used prior to a reactive decon during VX decon allows for decontamination to below 0.75 mg/m2
requirements (target objective <0.75 mg/m2)
•	SSDX-12 prewash removed 6 times more VX than a water prewash
•	A 30 minute SSDX-12 prewash can bring the HD contact hazard down from 10g/m2 to below the 15 mg/m2
requirement without the need for expensive reactive decontaminants. (target objective <100 mg/m2)
•	SSDX-12 prewash prevents migration of HD to non-contaminated substrates better than water alone.
Automated Vehicle Decontamination
Compatible with mobile and stationary vehicle decontamination platforms
Aircraft distributor
Unique Challenges of Decontaminating Sensitive
Equipment - Routine Aircraft Cleaner
Qualified to the following Aircraft cleaning specifications
•	U.S. Air Force MIL-PRF-87937D as a Type IV heavy duty water dilutable aerospace clean
• Currently used to clean C-130's, C-17 and B-52's
•	AMS 1626C Cleaner for Aircraft Exterior Surfaces
•	AIRBUS AIM09-00-002 External and General Cleaners
•	Boeing D6-17487 Exterior General Cleaners
•	Douglas Aircraft CSD No.l Type 1 General Cleaning of Painted and Unpainted Surfaces
Heavy Maintenance Facilities/Industrial Cleaner
Hill Air Force Bases uses the product as a facilities cleaner
Industrial wastewater treatment compatible
Prevents migration of heavy metals in aircraft rr
• OSHA Expanded Standards, Cd and Cr
aintenance facilities
Photo-Generated Chlorine Dioxide for Consumer and
National Security Applications
DHS requires the ability to respond to an attack with biological agents. Specifically, it requires a decontaminant to kill
anthrax spores on building interiors, exteriors, and even large outdoor surfaces. TDA is developing an innovative bio-
agent decon technology that is effective for decon of building interiors, exteriors, and is particularly suited for decon of
wide areas, such as airports. This technology uses a stable salt solution containing sodium chlorite with a photocatalyst
that absorbs light; the light-activated process produces chlorine dioxide (CI02), a known biocide and effective anthrax
decontaminant. Chlorine dioxide is reduced back to chlorite after oxidizing an anthrax spore and the photocatalyst can
repeatedly absorb light, allowing continued production of CI02. This makes the technology ideally suited to sustain a low
level of chlorine dioxide in solution over a long time, which has been shown to be effective against bacterial spores,
biofilms and stains. During this project, TDA is working develop an EPA registered formulation for a product with claims
of efficacy against anthrax.
Fundamentals
•	Light activated chemistry
•	Oxjdant/Chlorine Dioxide Is not generated until it Is applied
to the surface
•	Lower concentration of chemical components
•	Does hot require a special applicator
•	Not as effective against all chemical agents
•	Reduced concentration of chlorine dioxide, begins generating
oxidant when applied
•	Requires light
Decontamination Performance
•	Multiple formulations have shown good efficacy under solar simulation light.
•	8 log reductions within 15 minutes of solutions of commercially available B. subtilis spores
•	Decontaminant is quenched to stop reactions at correct time period
•	Surface decontamination is slower
•	Oxidant presumable has to 'dig' through piles of spores
•	Decontaminant is light dependent, lower light levels require longer exposure times
EPA Registration and Efficacy
•	EPA has published product performance test guidelines - 810.2100 Sterilants - Efficacy Data Recommendations
•	AOAC Method 966.04 - Sporicidal Activity of Disinfectants Test
•	15 minute exposure and 7-log exposure challenge
•	Formulation optimization requirements to speed up decontamination
•	Goal is a formulation that is an increment size of a consumer product (4 a
•	Preliminary Results
•	7.34 log reductions, dried on glass slides, 15 minutes exposure
•	7.34 log reductions, dried on Arizona Test Dust, 15 minute exposure
Potential Consumer Applications
•	Non-food contact, hard surface sanitizer
•	Passes ASTM E 1153-03 testing
•	3 log reduction with 5 minute contact period
•	Klebsiella pneumoniae - 99.9992% reduction
•	Staphylococcus aureus - 99.9802% reduction
« Could be EPA approved
•	Outdoor Mold and Mildew Remediation
•	EPA registration has been submitted, expected January 2017
Acknowledgements
eCI02 funding, Army Research Office, STTR Phase II contract: W911NF-13-C-0096
Decontamination Surfactant, US Air Force, SBIR Phase II contract: FA8222-14-C-0001
Photo-catalytic chlorine dioxide, Department of Homeland Security, SBIR Phase II contract: HSHQDC-14-R-00035
Plant and crop protection, US Department of Agriculture, SBIR Phase I contract: 2016-33610-25483
Plant and Crop Protection
•	Plant are susceptible to microbial pathogens too. Very few anti-bacterial products a
•	Photo-CI02 is effective against bacterial leaf spot
•	EPA submission has begun, expected November 2017
Biofilm Control Products
Summary
EPA registration of a biological decon national security product is
•	Required
•	Not simple
•	Can be successfully accomplished
Dual use products are more likely to be successful and sustainable
eClQ2 has been successfully registered
Development and registration of other, dual-use products are in progress

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Characterization of Anthrax Surrogates
by Chromogenic Media
Douglas W. Hamilton1 and Paul Lemieux2
1ORISE Research Participant • 2United States Environmental Protection Agency * 2National Homeland Security Research Center
OAK RIDQE INSTITUTE FOR
aOIENCJ5 AND EDUCATION
Man&sJstyORttJ Mrose
Abstract
The safety threat posed by spores of virulent Bacillus anthracis
precludes its use in many research activities and planning
exercises. Avirulent surrogates belonging to the B. cereus group
have been reported with properties similar to B. anthracis, with
some organisms being useful surrogates for evaluation of
decontamination processes, and other organisms being useful
surrogates for reaerosolization studies. This study compared the
growth characteristics of three members of the B. cereus group [8.
atrophaeus subsp. globigii (BG), B. anthracis Sterne (BAS) and B.
thuringiensis var. kurstaki (BTK)] when cultured on media
containing chromogenic substrates. Culture-based analytical
methods are the "gold standard" for determining the effectiveness
of decontamination efforts and to inform response and recovery
initiatives. Plate counting on chromogenic agar provides
quantitative information about a sample, can determine if a
pathogen is viable, and has the potential to improve laboratory
surge response by evaluating samples based on biochemical
characteristics. Chromogenic agars contain substrates that are
enzymatically cleaved during cellular processes, resulting in the
accumulation of the cleaved products within the vegetative cell and
the ability to visually identify bacterial colonies (by morphology and
color) based on substrate utilization. The chromogenic media
investigated in this study produce visually striking blue colonies
when organisms possessing specific biochemical processes are
cultured.
Three media were selected for this study, including tryptic soy agar
(TSA), R&F anthracis chromogenic agar (ACA) and Brilliance
Bacillus cereus agar (BBCA). In replicate experiments, it was
determined that no statistical difference exists between sample
enumeration on TSA, ACA and BBCA for the selected surrogate
spores. Incubation temperature and time were found to be critical
to the positive identification of surrogate spores on chromogenic
agars. This study identified methods useful in the characterization
and quantification of surrogates routinely used for research and
planning activities. The chromogenic agars are useful tools in the
differentiation of certain types of spores based on biochemical
characteristics, particularly in the presence of background flora
that do not grow blue colonies. These media are readily adaptable
to standard laboratory analytical methods.
Introduction
Background:
Objectives:
Materials and Methods
Media
8. atrophaeus subsp. globigii (BG)
B. anthracis Sterne 34F2 (BAS)
B. thuringiensis var. kurstaki (BTK)
Tryptic Soy Agar (TSA)
Brilliance Bacillus cereus Agar (BBCA)
R&F anthracis Chromogenic Agar (ACA)
Serially dilute spore stocks
Spread plate 100 ^il on media
Incubate @ 36°C for 48 hours
Compare colony formation on
chromogenic media to TSA
(t-testwith a = 0.05)
Results
Three media were selected for this study, including tryptic soy agar
(TSA), R&F anthracis chromogenic agar (ACA) and Brilliance
Bacillus cereus agar (BBCA). TSA is a general nutrient agar and
served as the control media for statistical analyses. ACA is a
selective and differential media that contains the chromogenic
substrate 5-bromo-4-chloro-3-indoxyl-choline phosphate (X-CP)
which identifies the production of phosphatidylcholine-specific
phospholipase C (PC-PLC) produced by BAS and BTK resulting in
blue colony formation. A mutation in the pIcR gene of BAS
inactivates the regulator, PIcR, reducing PC-PLC activity and
allowing BAS to be differentiated from BTK following an additional
24 hours of incubation. BBCA is a differential media that contains
the chromogenic substrate 5-bromo-4-chloro-3-indolyl-p-
glucopyranoside which identifies the production of (3-g|ucosidase
resulting in blue colony formation.
B. anthracis Sterne (BAS)
18 HOURS	34 HOURS	42
B. thuringiensis (BTK)
18 HOURS	24 HOOK	41 HOURS	« HOURS
B. atrophaeus (BG)
IS HOURS	24 HOURS	42 HOURS	48 HOURS

1


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lS
mm
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B. anthracis Steme (BAS)
¦ Exp 1 SExp 2
B. thuringiensis (BTK)
¦ Exp 1 SExp 2
B. atrophaeus (BG)
¦ Exp 1 -frExp 2
Replicate enumeration experiments showing no difference between
colony formation on control (TSA) and chromogenic (BBCA & ACA)
agars [t-tests (a = 0.05)]. (n=5)
Media
Replicate enumeration experiments showing no difference between
colony formation on control (TSA) and chromogenic (BBCA & ACA)
agars [t-tests (a = 0.05)]. (n=5)
Replicate enumeration experiments showing no difference between
colony formation on control (TSA) and chromogenic (BBCA) agars
[t-tests (a = 0.05)]. (n=5)
Discussion
Evaluate the enzymatic expression (blue colony phenotype) of three
common anthrax surrogates using three types of agar growth media.
Determine if there is a difference in the number of colonies formed
based ori the type of media used to culture the spores.
Recommend useful strategies for the culture-based characterization of
each spore type.
Recommended spore enumeration strategies
~	BAS: BBCA or ACA at 48 hours & 36°C
~	BTK: BBCA or ACA at 24 hours & 36 °C
~	BG: BBCA or TSA at 24 hours & 36°C
Enzymatic expression (blue colony phenotype) is temperature dependent.
Lower incubation temperatures yield negative results for BAS & BTK on
both chromogenic media.
Colony development of BAS is slower on ACA (contains antibiotic).
Acknowledgments
is supported through an appointment to the Research Participation Program at the U.S. Environmental
Protection Agency, National Homeland Security Research Center, administered by the Oak Ridge Institute for Science
and Education through an interagency agreement between the U.S. Department of Energy and the Environmental
Protection Agency.
agency's peer and administrative review and has been approved for publication,
srcial products in this report doe
for use.

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Broad-Spectrum Enzyme-Based Decontamination of Chemical Warfare Agents:
A Flexible Decontamination Platform
Anna M. Leech, Jessica L. Mi Ike, Jonita G. Gidel, Scott Donahue, and Jeremy P. Walker, Ph.D.
FUR Detection, Inc. Pittsburgh, PA
Limitations of Current State of the Art
• Many commercial off the shelf (COTS) decontaminants
have the following deficiencies which limit broad use:
Poor materials compatibility (corrosive, caustic)
Large footprint for transport / storage
Non-speafie decon can produce toxic byproducts
Inadequate surfactant packages to extract agents frc
coatings, plastics, eh

• Past enzymatic decontaminant limitations:
•	Poor enzyme kinetic chemistry for V-series Agents or HD
•	Poor shelf-life work investment
•	Lack good solvent package that is compatible with enzymes
'ies from challenging surfaces
Ad vantages of an Enzyme-Based Decontaminant
• Recent improvements in catalytic efficiency
• No need for a gent-specific formulations
•	Removal of persistent agent from challenging surfaces
¦	Enzyme compatible surfactant formulation
¦	Reduces tcocic off-gassing after decontamination procedure
•	Green Technology
¦	Improved economies of scalefor protein production
¦	Bengn ccmponents
¦	Low envircnmental impact & low occupational health hazard
•	Low logistical burden
¦	Catalytic formulations require less mass than stoichiomdric ones
Enzyme Specificity for Broad-Spectrum Decontaminant
OPH variant enzyme 3D stnjcture (left) and hydrolysis reactions for the
detoxification of VX (top) and GB (bottom), The enzyme functions
optimally at pH ~ S, Addic byproducts generated from hydrolysis are
neutralized by buffers.
HaloalkaneDHG 3D protein structure (l^t) and hydrolytic dealkylation of
sulfur mustard (right),. Non-toxic thiodiglycol and hydrochloric acid
byproducts are generated. These byproducts are buffered by salts
present in FLIR's decontaminant.
Soman (GD)
cydosarin (GF)
Formulation for Solubilization of Persistent Agents
• The biggest challenge for
Decon is removing the
agent from porous surfaces:
• G\RC paint
Aqueous Decontamination: Stirred Reactor Decon
Flexible Decon Formulations
• FUR emulsion outperforms
commercial off-the-shelf
(COTS) decontaminant in
solubilization & extraction
of persistent simulants
• Additional Features:
•	Enzyme compatible
•	Minimal agitation required





¦
¦

w
,


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n

Ls^nlL

ASTM Materials Compatibility
• FUR decontaminant was
tested side-by-side with a
commercial off the shelf
(COTS) oxidative
decontaminant
> FUR decontaminant
underwent materials
compatibility testing
sorption and hardness for
various plastics and elastomers
(bottom right)
• Compatibility testing
demonstrate that FLIR's
decontaminant is not
damaging to these materials
• While COTSdecontaminate
severely corrodes some metals
ASTM Materials Compatibility: Corrosion Testing
r-\
decontaminant (coupons 5
6) has excellent material
compatibility
significant corrosion to copper
(coupons 9 & 10).
- A color charge occurs, and a
hole develcps in the rnaal
after immersion in COTS
mr4
0 *• an
FLIR decontaminant (coupons
11 & 12) has excellent material
compatibility with copper
Addition of enzymatic action to
the FLIR emulsion formulation
results in rapid simulant
decontamination in <15
99.9% 94% 69%
99.9% 99.1% 99.9%
>99.9% DFP, C
detoxified by FLIR decontaminant!
Surface Decontamination: Extraction & Decon
1 Liquid Challenge: 10g/m2 cc
1 Chemical agent resistant coating (CARC)
panels were challenged with lQg/m2DEVX
and allowed to dwell for 60 minutes
1 FLIR's enzymatic decontaminant is capable
of solubilizing, extracting, and detoxifying
liquid agents residing on or soaked into
surfaces.
by FLIR decontaminant!
Enzymatic Performance Specifications
~ Liquid challenge: 10g/m2 contact
Joint General Purpose Decontaminant Requirements
16.7mg/m2 3D min 10mg/m2 5 min O.lmg/m2
0.4mg/m2 3D min 0.3mg/m2 5 min O.lmg/m2
FLIR decontaminant exceeds objective performance
specifications with G-and V-series nerve simulants!
Personal Decon
• The components have
been utilized in
beauty items
Replace charcoal-
based sorptive
decontaminants &
Aircraft Decon
• Sufficient material
compatibility to
detoxify aircraft and
sensitive equipment
rapid hydrolysis of
Human Remains Decon
can be applied and remai
Clearance Decon
• Sprayable gel formulation which
decontaminates surfaces and off-
gassed vapors over 72 hrs. through the
addition of polymers to enhance
physical properties
Skin Decontaminant Features
•	Benign components
•	Nonenhancement of
percutaneous agent
absorption
•	Nontoxic and
components
•	Components easily
integrated to gel or cream
•	Ease of app
to affected
1 Neutralization of CWAs
apid hydr
•	Hy droly si s produ ct s ai
non-toxic
•	Enhanced Stability
•	Shelf-life >3 years
Path Forward
K )
•	Complete productization of
decontaminant
¦	3rd-Party live agent
validation of performance
•	Create TDP and product
marketing literature
¦	Evaluate surfactant
extraction performance
with skin surrogates
•	Test for skin aliergenicity
Acknowledgements
vFLIR
•	David Wilson
•	Jason Robosky
•	DTRA
•	Contract #W911NF06C0089
•	Enzyme Providers/
Collaborators
•	Novozymes
•	Dr. Alex Berlin
•	PlantVfcx
•	Dr. Yvonne Rosenberg
•	Prof. Frank Raushel
•	Texas A&M University
$FLIR
Contact:
Anna M. Leech
P:+1412 423 2100 x 101
Email: anna.leech@flir.com | www.flir.com
FLIR Detection
2240 William Pitt Way, Pittsburgh, PA 15238, USA

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On-Site Size Reduction
•	Manual of mechanical
cutting, grinding, crushing
carcass
•	Reduce size and/or volume
to ease handling and further
processing
Encapsulation
•	Encase carcass in protective
matrix
•	Foams
•	Concrete
•	Flyash
•	Lime
•	Limit potential spread of
pathogens during transport
Digestion
•	Liquefy carcasses under acidic
conditions
•	Uses lactic acid or phosphoric
acid
•	Releases methane, C02, water
EPA Technical Fact Sheet - EPA/600/F-16/111
Identification and Screening of Infectious Carcass Pretreatment Alternatives - EPA/600/R-15/053
Feasibility of Selected Infectious Carcass Pretreatment Technologies- EPA/60 0/R-15/301
Physical Inactivation
•	Destroying infectious agents
(other than spores, prions) using
high temperature steam
•	Effective on limited number of
agents (viruses)
Alkaline Hydrolysis
•	Hydrolysis in NaOH or KOH
under moderately high (150 °C
temperatures
•	Produces aqueous hydrolysate
that requires neutralization
•	May or may not be able to be
discharged into wastewater
systems
Freezing
Done in fixed facilities or mobile
units
May not impact pathogens, but
can be effective in extending
storage time and easing
transportation
Bioreduction
• Biodegradation in partially sealed
vessel under mild heat and
aeration
Chemical Inactivation
Destroying infectious agents
using chemical agents (e.g.,
hypochlorite, peroxide)
Can be coupled with other pre-
treatment approaches (e.g., size
reduction)
Could be limited to surface
inactivation
Table 4. Favorable Applications of the
Pretreatment Options
Packaging
•	Encase carcass in
flexible or rigid container
•	Contain liquids
•	Limit potential spread of
pathogens during
transport
v>EPA
United States
Environmental Protection
Agency
Pre-Treatment Technologies to Facilitate Management of Animal
Carcasses from Animal Health Emergencies
Sandip Chattopadhyay, Paul Lemieux; US EPA, Office of Research and Development
Lori Miller; USDA. Animal and Plant Health Inspection Service
Large-Scale Mobile Carcass Grinder
Table 1. Carcass Pretreatment Options Matrix
Carcass in Digester
Alkaline Hydrolysis Unit
Table 3. Advantages and Disadvantages
of Carcass Pretreatment Technologies
Sterilization
•	Destroying infectious agents
using high temperature steam
•	Kills all microorganisms
Table 2. Comparison of Pretreatment
Technologies and Overall Ranking against
Various Carcass Management Options
Ad d itives/sorbents
•	Promote oxygen
transport
•	Dissuade vermin
•	Ab sorb excess I i q u i d
Handling and Processing of Carcasses

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31
/J

Underground Transport Restoration Program
Lab to field studies by EPA researchers
The Department of Homeland Security (DHS) Underground Transport Restoration (UTR) Program is working with EPA and other agencies to identify and evaluate methods for addressing a wide area
contamination incident involving an underground transit system, investigating both physical structures (tunnels and stations) as well as rolling stock (railcars). This project addresses a high-priority
need to improve recovery capabilities in the event of a biological release. Scientists and engineers at EPA are contributing to the project by conducting experiments to evaluate the efficacy of various
decontaminants and procedures for safe, efficient, and cost-friendly remediation of a subway system which has been contaminated by a biological agent.
Ill
Volumetric Decontamination Studies
Methyl Bromide (MB) Fumigation
EPA examined the efficacy of IVIB for the
decontamination of indoor and outdoor materials
contaminated with B. anthracis spores. Results of this study revealed the influence of
relative humidity during fumigation, where greater efficacy was achieved at higher
relative humidity.
Chlorine Dioxide (ClOrj) Fumigation
Researchers evaluated CI02's ability to
decontaminate in diverse environmental conditions
on sample materials with varying levels of surface I
grime obtained from a subway system. Both higher
temperatures and relative humidity created the best I	
conditions for effective inactivation of spores.
Fogging Technology & Sporicidal Decontamination
Tests were conducted in pilot-scale chambers to
assess the efficacy of different foggers and
sporicidal liquids to inactivate B. anthracis and
surrogate spores on subway system- and railcar-
related materials.
W
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Employing microbiological surrogates to compare chlorine dioxide fumigation
SnBRE and heat treatment of commercial poultry barns under field conditions
Julian N. Rosenberg1*, Douwe F. Mason1, M. Worth Calfee2, Eric R. Rhodes2,
Shannon D. Serre3, Madeline C. Bette1, Shawn P. Ryan2, John Y. Mason1
©
bioWALL
Protecting Water. Air, Land & Life
SnSnE ^EPA
United States
Environmental Protection
Agency
1 BioWALL LLC, a Sabre Company, 1891 New Scotland Road, Slingerlands, NY 12159
* To whom correspondence should be addressed: jrosenberg@sabrecompanies.com
2	U.S. EPA, National Homeland Security Research Center, Office of Research and Development
3	U.S. EPA, CBRN Consequence Management & Advisory Division, Office of Land and Emergency Management
Overview
Commercial-scale livestock production
facilities contaminated with highly pathogenic avian influenza
(HPAI) or other biological contaminants pose potential risks to human
and animal health following an outbreak. Current procedures for
decontaminating viruses and bacteria in complex agricultural facilities
are limited. Further, there is a need for rapid facility-clearance methods
for all pathogens in order to return livestock operations to pre-incident
risk levels. Under a cooperative research and development agreement
with the EPA, the virucidal and sporicidal efficacies of chlorine dioxide
(CI02) fumigation were compared to heat treatment of egg layer barns
in a large-scale field test. A test matrix of biological surrogates and
material coupons was devised to compare pathogen inactivation on
diverse surfaces under challenging fall/winter conditions.
"Sentinel" Surrogate Approach Comparative Virucidal & Sporicidal Efficacies
Clearing a barn for repopulation
using currently accepted methods involves physically swabbing
representative surfaces of the barn to collect environmental samples, which
are then subjected to PCR analysis and/or viral isolation. Surface sampling
by swabbing is known to be unreliable, especially on porous surfaces
Moreover, viral isolation takes weeks to process and can overload
laboratories during outbreak events. The rationale for the surrogate
approach stems from the need to rapidly determine disinfection
efficacy on actual building materials in order to efficiently clear
contaminated buildings with a high degree of confidence. As such, the
biological surrogates employed must be representative of the target
pathogens, possess similar resistance to disinfectants as those pathogens,
and have bioassays that can deliver results with relatively short timeframes.
For these reasons, MS2 bacteriophage (ATCC 15597-B1, non-enveloped
viral surrogate) and Bacillus subtilis endospores (ATCC 6633, gram-positive
bacteria) served as surrogates for the field test. These "sentinel" surrogates
(depicted to the right) were inoculated on various material coupons, each
prepared in triplicate, with and without organic soil loading, then sealed
individually in Tyvek/Tyvek pouches to prevent cross-contamination.
In total, over 1,000 coupons were deployed for this field test, including
relevant controls. Following the respective treatments, each set of
biological indicators was retrieved and sent to third party contract labs for
whole coupon extraction and enumeration of plaque or colony forming units
Diverse Material Matrices
Influenza virus
woildoMruses.unl.edu/
categoiy/image-
wall/inside-virusesi
Viable surrogate populations recovered
from clean and soiled coupons revealed anticipated trends in log-reductions,
chiefly organic load negatively impacting the efficacy of each treatment while also
improving surrogate recovery. However, the field test also shed light on some
intriguing data regarding viral persistence. As illustrated in the plots to the right,
white bars represent average CFU/PFU recovered from control coupons; gray bars
show the average CFU/PFU collected from treated coupons (five locations, each in
triplicate). Taken together, the results demonstrate pervasive sporicidal sterilization
on the clean B. subtilis spore coupons following CI02 fumigation (Fig. 1a) and near
6-log reductions on the soiled set (Fig. 1b). Corresponding coupons subjected to
heat treatment did not exhibit any significant reductions in viable spores (Fig. 1c-d).
As for the viral surrogate, MS2 populations recovered from coupons fumigated with
CI02 demonstrated widespread virucidal efficacy (»3-log reductions) on all chlorine
dioxide coupons (Fig. 2a-b). Alternatively, none of the MS2 coupons treated in the
heat barn exhibited >3-log reductions in PFU (Fig. 2c-d). In fact, viable MS2 was
detected in greater abundance on some heat treated coupons for which poor phage
stability was observed on the respective controls (iron, steel, HDPE, and concrete),
suggesting that heat treatment at low humidity may augment the persistence of
non-enveloped MS2 phage on these surfaces relative to winter conditions.
Future Work & Broader Impacts
Figure 1. Bacillus Spore Recovery





a




nb
I
n

ii


1
1

1
1
1
Figure 2. MS2 Phage Recovery
Porous and non-porous materials
representing the varied surfaces and structural components
present in poultry barns were produced as 0.25 in2 reference
coupons and inoculated with either MS2 phage or B. subtilis
spores. The suite of reference materials included unpainted pine
plywood, cotton belt, concrete, black iron, galvanized steel, and
high-density polyethylene. In order to simulate the actual soiling
loads found in working barns, both clean and soiled coupons
were used in the present study (right). Representative organic
dust comprised of fresh poultry manure was sourced from a
commercial chicken farm. The manure was desiccated,
mechanically pulverized, and applied to each coupon as a base
grime layer in a 10% solution of eicosane (C20H42) as a carrier.

Valuable information for stakeholders
in the poultry industry was generated and disseminated
under this CRADA. Although the current study focused on barns
affected by HPAI, these methodologies examined should be
relevant to the protein production industry as a whole. The use of
surrogate microorganisms that represent far more robust
pathogens than an enveloped influenza virus (i.e., spores)
generated working data for more broad decontamination
preparedness. Large-scale sterilization technology will play a
major role in the future of agricultural outbreak response—
providing reliable and rapid decontamination, mitigating further
spread of disease, and enabling producers to return to production
as soon as possible, thus dampening extended economic impacts.
Spatially distributed real-time monitoring of both test barns
during the field test enabled off-site participants to view respective treatment conditions via data link.
In addition to EPA and USDA collaborators, project observers included senior officers of the National Guard,
FEMA, U.S. Air Force, and Homeland Security. The live data feed relayed temperature, relative humidity,
and CI02 concentration (as applicable) from 10 locations in each of the test barns (left).
"Sentinel" surrogates were distributed at 5 of the 10 locations in each
barn. Commercial spore strips were staged at all 20 locations.	Microchem Laboratory - Jason Williams, Don DeClue; Mesa Labs, Inc.
The first head-to-head comparison
of CI02 and heat treatment under field conditions offers
a robust data set of quantified virucidal and sporicidal efficacy
on half a dozen high-challenge surfaces, similar to those
encountered in egg layer barns. The US HPAI outbreak of
summer 2015 resulted in the death of 50 million chickens and
turkeys, billions of dollars in taxpayer expenditures, and
hundreds of millions of dollars in producer losses and
downtime. However, there are limited data confirming that
disinfection processes used in the 2015 response were
efficacious. From an analytical standpoint, the turn-around time
for the surrogate-based approach piloted during this project
requires days, instead of weeks, and is far more reliable than
environmental swabbing. We hope that the results of this work
provide sound scientific evidence for future decisions related to
choice of remediation options, ultimately allowing producers to
confidently return to production in much shorter timeframes.
Follow-up benchtop studies will be pursued under laboratory
conditions to further compare surrogate-pathogen equivalency
and potentially broaden the panel of surrogates.
Acknowledgements:
Paul Nirgenau; Yakibou, Inc. - Joe Dalmasso
Field Assistance: EPA: Marshall Gray, Megan Schuette, Eric Nold, Katrina McConkey, Eric Koglin, Joseph Wood, Rich Rupert
USDA: Craig Ramsey, Deborah Nelson; QA/QC: Eletha Brady-Roberts, Ramona Sherman; Daybreak Foods, Inc. - Rick Roedl
SABRE / BioWALL: Michael Cecchini, Ryan DeGonzague; Thanks to Daniel Griffin for providing the design template for this poster.
Research goals:
© Directly compare the efficacy of two decontamination methods
used for commercial poultry barns: (1) chlorine dioxide fumigation
targeting 25,000 ppmv-hrs at 75'F and 85% RH and (2) heat treatment
following USDA guidelines of 100-120°F for seven days with at least
three consecutive days >100'F.
(D Develop & evaluate mixed panel of non-pathogenic surrogates
for viral and bacterial spore contaminants common to the poultry
industry and more broadly relevant to public health.
(3) Examine material effects of each decontamination process
on visible surfaces and assess structural integrity of each barn
following the specified treatment conditions.
@ Analyze efficacy results with respect to stakeholder needs
including biosecurity implications, cost considerations, and
operational feasibility.
Presented at the 2016 EPA International Decontamination Research & Development Conference ¦ November 1-3, 2016 ¦ Research Triangle Park, NC ¦ U.S. EPA CRADA 893-16
-------
Fate and Transport of VX and Sulfur Mustard across a Permeable Layer into Porous Subsurfaces
Lukas Oudejans1, David See2. Daniel Chappie2, Anthony Eiiingson2, and Katherine Mitchell2.
1 EPA NHSRC, Research Triangle Park, NC; 2 Battelle, Columbus, OH.
Abstract
A release of a chemical warfare agent (CWA) into the urban environment has the
potential to contaminate painted and/or sealed building materials that would require
remediation. This study investigated the fate and transport of two CWAs, VX and sulfur
mustard (HD), deposited on painted/sealed stainless steel and on freestanding
paint/sealant films. In the case of freestanding films, a solid phase extraction (SPE) disk
was placed under the test coupon to mimic a porous material and to collect agent which
permeated through the paint/sealant films. Three types of paints and two types of
sealants were evaluated. Samples were challenged with 2 microliters of VX or HD and
then held at ambient laboratory conditions for durations ranging from 3 hours to 48 hours
(HD) or 72 hours (VX). Following contact of the agent with the surface, residual CWA was
measured via wipe-sampling of the coupon surface, solvent extraction of the steel or
freestanding film, and solvent extraction of the underlying SPE. Extracts were then
analyzed for VX or HD using gas chromatography/mass spectrometry. CWA recoveries
from the film wipe, film extraction, and SPE extraction were measured and reported, as
well as the total CWA recovery based on the summation of the individual recoveries.
Generally, total VX recoveries and film wipe recoveries decreased with increasing
weathering (<62% after 72 hours and <19% after 72 hours, respectively). VX was
generally detected from the film extractions (4.4% to 47% after 72 hours) and SPE
extractions (<2.5% to 19% after 72 hours), but consistently increasing or decreasing
trends were not apparent across ail the paints/sealants. HD recoveries from surface
wiping rapidly decreased (<2.0% after 48 hours), but HD remained recoverable from film
and SPE extractions. On painted surfaces, total HD recoveries remained high (>72%)
after 48 hours, except in the case of oil gloss painted stainless steel (25%). Total HD
recoveries obtained during sealant testing tended to be lower (generally <48% after 48
hours), with the exception of polyurethane sealant film placed over SPE disk, which
yielded a total recovery of 86% after 48 hours (73% of which came from the underlying
SPE disk).
This research clearly demonstrates that VX and HD can penetrate through
paints/sealants and are quite capable of migrating into underlying porous materials.
Surface sampling may capture only a fraction of the VX and HD retained in paint/sealant
layers and/or underlying porous materials, thus sampling and remediation strategies
must address the potential for CWA to be retained within porous materials beneath
painted/sealed surfaces.
Methods
Testing was conducted with the following paints and sealants:
•	Latex flat, latex semi-gloss, and oil gloss paint
•	Epoxy and polyurethane sealant
The paints/sealants were applied to stainless steel or made into freestanding (FS) films
that were placed on top of a porous material, i.e., SPE disk.
Two microliters of VX or HD were spiked onto the paint/sealant surfaces. Figure 1 shows
HD applied to sealed stainless steel and Figure 2 shows a FS paint film being removed
from an SPE disk (after the FS paint film was wipe-sampled).
Figure 1. HD applied to sealed
steel (white is epoxy, grey is
polyurethane).
Figure 2. Removal of FS
paint film from SPE disk.
Lukas Oudejans I Oudeians.Lukas@epa.aov I 919-541-2973
Battelle
flie Business of Innovation
Methods (continued)
Results (continued)
A Low Volatility Agent Permeation (LVAP) setup was used for testing with the SPE disk. The
setup used a latex gasket and weighted washer to ensure close contact between the FS film
(5.0 cm diameter) and SPE disk, while also inhibiting fugitive CWA vapor from reaching the SPE
disk. This ensured that CWA recovered from the SPE disk was attributed to permeation rather
than vapor adsorption. The SPE disk represents the porous material. A photograph of the LVAP
setup is shown in Figure 3 and a diagram is provided in Figure 4.
F$PaM Cation
UtwGaciort
PTt-t Dink
Figure 3. LVAP photograph. sk
Figure 4. LVAP diagram.
The CWA challenged materials were allowed to weather under ambient laboratory conditions
(18-23 °C; 28-49% RH across all tests) for pre-determined times, ranging from 3 to 72 hours.
Residual CWA was then measured via:
•	wipe-sampling of the painted/sealed surface (wipe),
•	solvent extraction of the stainless steel or FS film post-wipe sampling (coupon), and
•	solvent extraction of the underlying SPE disk (SPE).
2"x2" 4-ply rayon/polyester (gauze) wipes were used as the wipe-sampling media. Hexane
was used as the extraction solvent and was used to wet the gauze during wipe sampling.
Samples were extracted by sonication for 10 minutes.
Extracts were analyzed for CWA using gas chromatography/mass spectrometry. The VX and
HD residual mass detection limit was 2.6 pg for wipe samples and 2.5 pg for the other
samples.
CWA recoveries are presented in Figures 5a/5b for VX and 6a/6b for HD. Figure 7 shows the
curve fitting of HD recovery associated with the FS latex semi-gloss paint film test. The curves
were fitted using first order equations that simultaneously accounted for the rate of evaporation,
absorption into the paint, and breakthrough from the paint into the underlying SPE disk.
In general:
•	CWA was recovered from most wipes, coupons, and SPE disks.
•	CWA recoveries from wipes generally decreased over time.
•	CWA recoveries from SPE disks generally increased over time.
For VX:
•	VX appeared to have a greater affinity (higher %VX recoveries from coupons) for latex semi-
gloss paint than the other paints tested.
•	VX recoveries from wipe samples tended to be higher than those for HD.
•	Total VX recoveries decreased faster than total HD recoveries, except when associated with
epoxy sealed steel (steel epoxy) and FS epoxy film (SPE epoxy).
For HD:
•	HD tended to have a greater affinity (higher %HD recoveries) for the paints/sealants
(coupons) and SPE disk than VX, except for epoxy sealed materials.
•	HD recoveries from wipe sampling rapidly decreased to <2% after 48 hours.
•	Total HD recoveries from the painted surfaces and the polyurethane sealed surfaces were
greater than the VX recoveries.
g| '0
11 «
ft -
.1 -
il »
II
¦u •* "0
il »
i! _
ij
ill,
1 ( vn 1 S 2472 i t 2472 S • 2473 S • 34 73 Si 3472
U»l	Um'	(Ml	M	Wt	tff
Time (hourt) and Mat*
Pe^rufthaat lo»Y Psf»wWh««*
Figure 5a. VX recovery from paint tests.
Figure 5b. VX recovery from sealant tests.
ji«
ii
a i »
I § ,
& -
II
Figure 6a. HD recovery from paint tests.
WeMhpring Tim* ((town)
Wejlht-ifcng Time (>khvi) and Mate* Ui
Figure 6b. HD recovery from sealant tests.
Figure 7 (left). Scatter plot with fitted curve of HD
recovery from FS latex semi-gloss paint.
•	Wipe represents non-evaporated HD.
•	Coupon represents HD absorbed into paint.
•	SPE represents HD breakthrough.
The curve fit (R2=0.983) was much better than for
other combinations of VX/HD and paint/sealant.
Error bars represent ± one standard deviation.
Conclusions
VX and HD can penetrate into paints/sealants and migrate into underlying porous materials.
Permeation is influenced by the CWA and paint/sealant and time dependent.
Porous materials might serve as reservoirs for CWA increasing persistence.
Wipe sampling may capture only a fraction of the CWA retained.
Sampling and remediation strategies must account for CWA permeation to materials.
Disclaimer
The U.S. EPA, through its Office of Research and Development, funded and managed this investigation through Contract No. EP-C-
11-038 Task Order 29 with Battelle. This document has been subjected to the Agency's review and has been approved for
presentation. Note that approval does not signify that the contents necessarily reflect the views of the Agency. Mention of trade
names or commercial products, or services does not constitute EPA approval, endorsement or recommendation for use.
-------
dstl
Assessment of the use of Sodium Hydroxide for the destruction of ricin
N. Walker
CBR Division, Dstl, Porton Down, Salisbury, Wiltshire, SP4 OJQ.
Abstract
Ricin is a protein toxin found in the seeds of the castor oil plant Ricinus communis. It can be extracted from the seeds
by a number of different methods producing an extract with high protein content, with ricin typically being 10 % of the
total protein concentration in an aqueous extract. We are interested in methodologies for simple and rapid destruction
of ricin.
Chlorine based decontaminants e.g. sodium or calcium hypochlorite, can be used to decontaminate protein toxins
by denaturing and hydrolysing the toxin. A disadvantage of the addition of these chlorine based decontaminants to
stocks of highly proteinaceous solutions is that they can cause frothing and the release of free chlorine resulting in a
respiratory hazard to the operator.
We have therefore investigated the use of sodium hydroxide as a potential low technology, environmentally friendly
and relatively safe method for the destruction of large quantities of aqueous ricin extract. Being a protein, NaOH will
hydrolyse the amide bond between the amino acids of the ricin resulting in the toxin being broken down into small
peptides and amino acids, thereby destroying the toxin. The efficiency of 10 %, 5 %, 2.5 % or 1 % w/v NaOH to
hydrolyse ricin both in 500mM NaCI and in 500mM NaCI pH4 was assessed by an antibody based Hand Held Assay
and SDS-PAGE.
Introduction
Ricin is a type II ribosome inactivating protein
extracted from the seeds of the castor oil plant,
Ricinus communis. It can be extracted using a
number of different approaches. These include using
solvents to remove the castor oil from the seeds prior
to extraction of the toxin into an aqueous buffer [1]
centrifugation to remove the castor oil from a seed
homogenate in aqueous buffer [2] or using acetone
to remove the castor oil producing a powdered
product[3L
Whichever method is used a highly proteinaceous
solution is produced with ricin being typically 10% of
the total protein content.
A number of chemicals can be used to decontaminate
protein toxins by denaturing and hydrolysing the toxin.
Chlorine based decontaminants for example,sodium or
calcium hypochlorite release free chlorine into solution
which can off gas when added to solutions of high
protein content. If being used to treat large volumes
of proteinaceous material the chlorine released could
pose a hazard to the operator and also leave large
volumes of chlorinated waste to be disposed of.
NaOH although corrosive, does not off gas and
once treatment has been proved effective can be
neutralised by addition of an acid resulting generation
of the acid's salt and water allowing the hydrolysate to
be disposed of to drains.
We have investigated the use of 10 %, 5 %, 2.5 %
or 1 % w/v NaOH to hydrolyse ricin in an aqueous
extract with the efficiency of the hydrolysis being
assessed by the antibody based Hand Held Assay
(HHA) and SDS-PAGE.
Ricinus communis.
Ricinus communis seeds.
Materials and methods
Preparation of Ricin extract
100 g Ricinus communis seeds were blended in
a Waring blender in 200 ml_, 500mM NaCI. The
blended seeds were then centrifuged at 18600 g for
30 minutes, the supernatant removed and centrifuged
again at 27000 g for 20 minutes. The resultant extract
was stored at -80 °C until required.
Determination of total protein content
Total protein content was determined using the BCA
protein assay (Pierce).
Determination of ricin content
The ricin content of the extract was determined
by SDS-PAGE. Samples were run alongside a BSA
standard curve on a 10-15% PhastGel™ (GE
Healthcare), and stained using PhastGel Blue R™
(GE Healthcare). Bands were analysed using a GS-
800 densitometer (Bio-Rad) and the Bio-Rad Quantity
One software. Band densities of the BSA standards
were plotted against concentration and using
Linear Regression within Graph Pad Prism the ricin
concentration calculated from the density of the ricin
band.
Small scale NaOH experiments
Aliquots of 800 pi extract were incubated with a final
concentration of 10 %, 5 %, 2.5 % or 1 % NaOH w/v
added from a 50 % NaOH w/v stock solution. Samples
were taken at 30 minutes, 2, 4 and 24 hours and
analysed by SDS-PAGE and Hand Held Assay.
Medium scale NaOH experiments
Aliquots of 100 mL extract were treated with a final
concentration of 10 % NaOH w/v, added as NaOH
pellets and stirred using a magnetic stirrer until the
pellets had dissolved. Samples were taken at t=0, 30
minutes, 1, 2, 4 and 24 hours and analysed by SDS-
PAGE and HHA. Four Litres of Bovine serum albumin
(BSA) at 40 mg/rnL in 500mM NaCI pH4 was treated
with 10 % NaOH and stirred overhead. Samples were
analysed by SDS-PAGE. after 1 hour and 24 hours.
Hand Held Assay
Samples were diluted 1:100 in the assay buffer (150
rnMNaCI, lOmM HEPES, 0.01 % Tween and 0.095
% sodium azide) provided with the Hand Held Assay
(HHA) and 100 pi added to the test lane. The assay
was read after 20 minutes.
SDS-PAGE
Samples were diluted 1:5 in water before mixing in
a ratio of 2:1 of sample to Laemlli buffer containing
no (B-mercaptoethanol. Samples were run on a 10-
15% PhastGel™ (GE Healthcare), and stained using
PhastGel Blue R™ (GE Healthcare).
References
1.	Olsnes S, Pihl A (1973) Different biological properties of the two constituent peptide chains of ricin, a toxic protein inhibiting protein synthesis. Biochemistry 12: 3121-3126.
2.	Thullier P. Griffiths G (2009) Broad recognition of ricin toxins prepared from a range of Ricinus cultivars using immunochromatographic tests. Clin Toxicol (Phila) 47: 643-650.
3.	Ovenden SP Pigott EJ, Rochfort S, Bourne DJ (2014) Liquid Chromatography-Mass Spectrometry and Chemometric Analysis of Ricinus communis Extracts for Cultivar Identification.
Phytochem Anal.
Results and Discussion
Prior to use both the HHA and SDS-PAGE running conditions were optimised for the presence of 10% NaOH. The
crude ricin extract usedin this work had a total protein concentration of 40rng/rnL and an estimated ricin concentration
of 7mg/mL.
SmaSS Scale Assessment
Initially lrriL small scale reactions were set up to assess the effect of NaOH on the ricin. From a 50 % stock solution,
NaOH was added to the extract with or without the pH adjusted to pH 4 with acetic acid.
A concentration effect of NaOH was seen on the rate of hydrolysis of both the ricin and other proteins within the
extract. In the HHA (Table 1.) which being antibody based detects both the presence, and structural integrity of
the ricin, the Visual Score Card (VSC) score of the test line decreased at each time point with the ricin becoming
undetectable after 4 hours at all NaOH concentrations.
The SDS-PAGE analysis (Figure 1.) complemented the HHA assay data. Within 30 minutes there was significant
hydrolysis of the ricin and other proteins in the extract and by 24 hours in both the 5 % and 10 % NaOH reactions all
protein was completely hydrolysed leaving a single band at the dye front of the gel.

500mM NaCI adjusted to
pl-l 4
% NaOH
500mM NaCI
% NaOH
untreated
control

10%
5% I5
w %.
1 %
10%:
5% 2'8
P %
X %
t=30
min
5
6
7
8
5
6
8
9
10
t=2
hour
2
3
4
5
2
3
4
6
10
t=4
hour
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
10
t=24
hour
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
10
Table 1. Hand Held Assay VSC scores following the addition of
varying concentrations of NaOH to ricin extract. The score is given
in relation to the intensity of the test line in comparison with a VSC
score card and ranges from 10, the highest, to 2 at which point the
test line is only just visible.
KDa
250>	A	B
150>
100>
75 >
50>
37>
25>
20>
15>
10>	
1
Mt|!
III-

1 2 B 4 5 6
Figure 1, 10-15 % SDS-PAGE PhastGel of ricin extract following
addition of varying concentrations of NaOH. (A) 30 minutes post
addition (B) 24 hours post addition. Lane 1 - molecular weight
marker, Lane 2 - 10 % NaOH, Lane 3 - 5 % NaOH, Lane 4 - 2.5 %
NaOH, Lane 5-1% NaOH. Lane 6- Untreated ricin extract. The
ricin band at 66KDa is marked with an arrow in lane 6 on gel B.
Conclusions
•	10% w/v NaOH is the recommended
concentration for the destruction of ricin
•	A minimum incubation time of 4 hours is required
for no structurally intact ricin to be detected.
Medium Scale Assessment
10% NaOH was taken forward asthe optimal
concentration and assessed against a larger volume of
ricin extract and with the NaOH added as a solid.
NaOH pellets were added to three replicates of 100 mL
of crude ricin extract to give a final concentration of 10
% w/v NaOH. Following stirring samples were taken at
time 0, which was considered when no visible pellets
were present and then after 30 minutes, Ihour, 2 hours,
4 hours and 24 hours incubation. Samples were again
analysed by HHA and SDS-PAGE. The temperature of
the extract was also recorded at these time points.
The VSC scores (Table 2.) and SDS-PAGE (data not
shown) results followed the same trend as the small
scale assessment. The VSC results indicated that the
rate of hydrolysis to be faster than in the small scale
studies.
Rep 1 | Rep 2 | Rep 3

HHA VSC
Score
Temp°C
HHAVSC
Score
Temp °C
HHAVSC
Score
Temp °C
Before
treatment
10
16
10
16
10
16
t=0
8
32
8
32
8
33
t=30 min
3
25
3
25
3
25
t= Ihour
2
22
2
22
2
22
t=2 hour
-ve
21
-ve
21
-ve
21
t=4 hour
-ve
20
-ve
20
-ve
20
t=24
hour
n/a
20
n/a
20
n/a
20
Table 2. Hand Held Assay VSC scores following the addition of
NaOH pellets to ricin extract to a final concentration of 10 % w/v.n/a
- assay not run
To assess this method on a larger scale, 4 litres of the
simulant BSA at 40 mg/mL in 500mM NaCI pH4, was
treated with NaOH pellets to a final concentration of
10 % w/v and the solution was stirred.As for the ricin
extract no sign of intact protein was seen by SDS-PAGE
after 1 hour; the reaction yielded a single band at the
dye front after 24 hours.
-------
*>EPA
www.epa.gov/research
Introduction
Analytical detection methods for conventional chemical warfare agents (CWAs) were
mostly derived for military purposes and did not focus on the enviromnental aspect nor
intended for use when dealing with contaminated civilian areas. If civilian areas are
affected (e.g., Tokyo, Japan in 1995 and Syria in 2013), then it is necessary to ensure that
analytical detection methods are appropriate for a particular analyte in the desired matrix.
CWA degradation by-products can be extremely hazardous to humans, with similar
toxicity values as the parent CWA, and environmentally persistent compared to parent
agent. Therefore, it is important to have available analytical methods to detect the parent
CWA and its degradation products in environmental matrices.
Impact
The presence of degradation products can assist decision-makers with identifying the
extent of contamination, potentially high-concentrated areas of the parent and/or
degradation products, and decontamination efficacy and/or remediation completeness
necessary for civilian reoccupation.
Lewisite and VX Degradation By-Product Method Development for Environmental
Remediation by Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS)
Stuart A. Willison1, Terry M. O'Neill2, Sandip Chattopadhyay1, Carolyn Koester3, Deon Anex3, Romy Campisano1, Matthew Magnuson1
1U.S. Environmental Protection Agency, 2MRIGIobal, 3Lawrence Livermore National Laboratory
y Y"
C,Hs0-P-
%
*
¦WL
& ch3
Mi
4
(CVAA)	(CVAOA)
Figure 1. Lewisite 1 hydrolysis and oxidative
pathways under environmental conditions.
Figure 2. VX hydrolysis pathway under
environmental conditions.
Identified Gaps and Method Application
Lewisite 1 is a reactive vesicant agent. Since arsenic is present in a variety of agricultural
chemicals and in natural sources, assaying for elemental arsenic lacks specificity required
for source material identification. Neither Lewisite or degradates (Figure 1) exist in the
environment.
•	Gas chromatographic (GC) analysis of Lewisite or CVAA presents difficulties,
including GC inlet reactivity/corrosion, column deterioration, and clogged
syringes.
•	CVAA and CVAOA are environmentally persistent and unique, unambiguous
indicators for Lewisite.
•	The new method addresses sampling and analysis for CVAA and CVAOA in soil,
wipe extracts, and water by LC-MS/MS.
•	Method directly addresses need for Lewisite and degradate analysis procedures.
VX is considered to be one of the most toxic CWAs ever produced. Under certain pH
conditions, VX will degrade to EA-2192 (Figure 2), which is environmentally persistent
and possesses toxic properties similar to VX. Analysis methods following strict data
quality objectives (DQOs) are needed for detecting EA-2192 in drinking water at
environmentally low and health-based levels.
U.S. EPA Method 538 analytical conditions and were adapted and performance
metrics were applied for the determination of EA2192 in drinking water.
Laboratories are familiar with EPA 500 series methods, thus, the capability to
analyze EA2192 with the rigorous conditions of Method 538 will increase the
analysis capacity following a VX release and subsequent remediation activities.
Method and Results
Lewisite 1 hydrolyzes quickly in the environment to CVAA, then oxidizes more
slowly to CVAOA. The developed method extracts Lewisite 1 and/or degradation
products from the matrices of interest and converts them to CVAOA prior to analysis.
Phenyl arsonous acid (PAA), which can be oxidized to phenyl arsonic acid (PAOA),
was used as a surrogate to show that both extraction and oxidation processes were
successfully implemented. The use of LC-MS/MS provides analytical specificity, by
which degradation products of Lewisite 1 can be more easily measured (Figure 3) in
the presence of interfering compounds.

vv
i r

Figure 3. Chromatograms depicting CVAA analysis and oxidation to CVAOA by LC-MS/MS.
Stock and spike solutions consisted of Lewisite in water. The extraction, sample
preparation, and analysis results are provided in Tables 1 and 2. Transformation of
Lewisite 1 to CVAA and subsequent oxidation to CVAOA, by hydrogen peroxide
(H202) was investigated and concluded that complete conversion was successful.

Water
Wipes
Soil
Extraction
None
~
1:1 Dilution with
30% H202
Shaker table for 30
min with 10 mM
HC1
~
1:1 Dilution with
30% H,02
Shaker table for 30 min with
10.0 mL 50/50 (v/v) 10 mM HC1
/methanol
~
1:1 Dilution with 30% H202
Oxidation
Table 1. Sample extraction (of CVAA) and oxidation (to CVAOA) procedures in tested matrices.
Method detection limits (MDLs) and recoveries for all tested matrices are reported in
Table 2 and compared to risk-based health levels. A 14 day stability study was
performed under refrigerated conditions (4 0 C ± 2 0 C) for Lewisite by-products.
Statistical analysis suggests that Lewisite degradates were stable in water and on
wipes, but significant differences were observed (< 2 days) for soil types.
Matrix
Spiked CVAA
Concentration
Measured CVAOA
Concentration
(avg ± std)
Recovery
(%)
MDL for
CVAOA
ATL*
Water
Og/L)
0.20 e-3
0. 22 e -3 ± 0.01 e-3
110
0.04 e-3
0.03 e-3
Wipe
(Hg/wipe)
3.0
3.0 ± 0.1
101
0.4
-
Sand
(Hg/g)
0.20
0.17 ± 0.02
85
0.07
0.3
NB Soil
Wg)
0.20
0.22 ± 0.01
112
0.03
0.3
VA Soil
Ws)
0.40
0.17 ± 0.01
43
0.03
0.3
GA Soil
(t»g/g)
0.40
0.32 ± 0.02
80
0.05
0.3
Table 2. Analytical recoveries (n=7'), MDLs, and Analytical Target Levels (ATLs) for Lewisite 1
method. "ATL values based on U.S. Army Public Health Command Chemical Agent Health-
Based Standards and Guidelines Summary Table 2: Criteria for Water, Soil, Waste, 7/2011.
Stuart Willison I Willison.stuart@epa.qov
Method and Results
Water samples fortified with VX degradation product (EA-2192) were analyzed by
LC-MS/MS. The compound was evaluated using EPA Method 538 conditions,
including accuracy, precision, reproducibility, linearity, detection limit, and
quantitation limit and results are presented in Table 3. The retention time for EA-2192
was 5.4 minutes. Target chemicals, Methamidophos and Methamidophos-d6 (used as
an internal standard), were used to assess method performance.
EPA Method 538 Performance Metrics Applied to EA-2192
Results j
Calibration Curve Accuracy at Calibration 1 (0.050 fig/L)
96.4-105%
Calibration Curve Accuracy at Calibration 2-7 (< 5.00 jJ.g/L)
92.4-107%
Laboratory Reagent Blank
ND
Method Precision at Calibration 4 (0.480 |xg/L)
9.61%
Method Accuracy at Calibration 4 (0.480 jxg/L)
21.8%
Method Detection Limit (|Xg/L)
0.013
Method Reporting Limit (jig/L)
0.125
Risk-Based Health Level* (f^g/L)
0.021
Table 3. U.S. EPA Method performance criteria applied to EA-2192. *Risk-Based Criteria to
Support Validation of Detection Methods for Drinking Water and Air, EPA/600/R-08/021, 2008.
A stability for EA-2192 was performed using water from four unique water utility
companies, representing four different water types (Table 4). Storage stability of EA-
2192 was evaluated for 28 days under refrigerated conditions (5° C ± 3 ° C). The
average EA-2192 concentration in all four water types after the tested period was
99.2 % of the Time 0 results. Chlorinated water was also tested (CI at ~1 mg/L,
representing a typical Free Chlorine level in drinking water).
Water Type
Day 28 as a % of Day 0
In-house Deionized (DI) Water
Low TOC, chlorinated surface water
86.7 %
91.3 %
High TOC, chlorinated surface water
93.4 %
Low TOC, chlorinated surface water
High hardness, chlorinated surface water
DI Water + 1 mg/L free CI, no preservatives
99.7 %
112%
ND at Day 0
U.S. Environmental Protection Agency
Office of Research and Development
Table 4. Stability studies for EA-2192 in different water sources. TOC = Total Organic Carbon.
Conclusion
A novel LC-MS method was developed for CVAA and CVAOA in environmental
matrices. The presence of CVAOA and/or CVAA is indicative of Lewisite 1
contamination, as there are no known natural sources for these compounds. The
method was successfully tested with real-world samples suspected of containing
Lewisite. In addition to the target analytes, the LC-MS method analyzed for thirteen
additional As by-products, not possible by GC-MS or ICP-MS analysis techniques,
and correctly identified the arsenic contamination.
U.S. EPA Method 538 performance metrics were successfully
applied to EA-2192. Stability studies suggest that the toxic
by-product is stable in drinking water sources. Other
nonvolatile CWAs potentially can be tested using Method 538.
Both methods successfully fulfill an Agency gap by addressing
lab capacity and capability for analyzing environmental samples containing toxic
CWAs and their degradation by-products.
DISCLAIMER: The U.S. Environmental Protection Agency through its Office of Research and Development (funded and managed) or (partially
funded and collaborated in) the research described here under (contract number) or (assistance agreement number) to (contracting company
name). It has been subjected to the Agency's review and has been approved for publication. Note that approval does not signify that the contents
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Chemical Hazard Mitigation Efficacy of Dahlgren Decontaminant
Bruce Campbell, Erin Lamb, Amy Andrews, Melany Corlew, Ellen Engelhart, and George Wrenn, Battelle IHMRC
Kathryn Burns and Bryan Tienes, NSWCDD; Amit Kapoor and Randy Sakowitz, First Line Technology, LLC
ABSTRACT
TECHNICAL APPROACH
RESULTS
Chemical decontamination efficacy tests against persistent nerve agent (VX) and distilled mustard agent (HD)
were performed at the Battelle Hazardous Materials Research Center (HMRC) to assess the hazard mitigation
performance of Dahlgren Decontaminant prepared by First Line Technology, LLC (FLT) and by the Naval
Surface Warfare Center Dahlgren Division (NSWCDD). Contact transfer and remaining contamination tests
were performed on coupon samples of five materials: chemical agent resistant coating, water dispersible
(CARCW), polycarbonate (Lexan), ship deck coating (Nonskid), silicone rubber, and stainless steel (17-4PH)
that are typically representative of military vehicles and equipment.
Test procedures, calculations, and analyses were performed following U.S. Army Test Operations Procedure
(TOP) 8-2-061A "Chemical Decontaminant Testing" [1], using detailed practices described in ECBC-TR-980
"The Chemical Contaminant and Decontaminant Test Methodology Source Document, Second Edition" [2]
Contact transfer results were compared to chemical exposure hazard levels published in USACHPPM 47-EM-
5863-04 "Acute Toxicity Estimation and Operational Risk Management of Chemical Warfare Agent Exposures"
[3] using hand surface area estimates from Yu, et.al., Burns. 2008 Dec;34(8):1183-9 [4] and Sheridan, et.al., J
Burn Care Rehabil. 1S95 Nov-Dec;16(6):605-6 [5],
Dahlgren Decontaminant formulations from FLT and NSWCDD exhibited similar chemical hazard mitigation
performance against each chemical warfare agent on each test material. In general, contamination levels were
reduced by at least two orders of magnitude on coatings and hard materials. Differences in decontamination
performance were observed on polycarbonate (Lexan) with HD and on Navy Nonskid with VX. Differences
were caused by variations in the materials, themselves, not the decontaminant. Tests with the FLT formulation
used SABIC Margard MR-10 polycarbonate and Navy Nonskid Compound-G (Type-I), while tests with the
NSWCDD formulation used SABIC GE Lexan XL-10 and a sprayable Nonskid coating (Type-IV).
The chemical agent contact exposure hazard was reduced to negligible risk levels (i.e., no noticeable effect) on
most materials for both chemical agents, even with repeated direct contact (i.e., ten hand touches). Reduction
of contamination levels on silicone rubber was less than one order of magnitude for both HD and VX. Repeated
direct contact with HD on silicone would be sufficient to produce noticeable but not disabling health effects.
Incidental contact (i.e., one hand touch) with VX on silicone could produce disabling or incapacitating injuries,
and repeated direct contact with VX on silicone or Compound G Nonskid could be sufficient to produce death,
severe disabling, or incapacitating injuries without prompt medical stabilization and access to field or fixed site
hospital facilities.
Logarithmic {hase10| reduction of HD challenge for five materials aged one
hour after contamination in indoor ambient or hot-humid conditions, then
decontaminated with Dahlgren Decontaminant from First Line Techno logy, LLC
or NSWC-Dahlgren Division
Interaction of HD and Dahlgren Decontaminant on Material Surfaces
(samples shown from 2016 test with FLT Dahlgren Decontaminant)


Dahlgren Ce
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Enzyme-Based Disclosure Sprays for Nerve and B lister Chemical Warfare Agents
Scott Donahue, Jason Robosky, Beverly Rogers, Jonita Gidel, Jessica Milke, and Jeremy Walker, PhD
FUR Systems, Inc., Pittsburgh, PA
Agentase™ Disclosure Spray (ADS)
Enzyme-based, aqueous formulation to detect chemical
warfare agents on surfaces
-	Class-specific formulations for G- & V-series nerve agents or HD blister ager
-	Nerve agent formulation operates by inhibition of acetylcholinesterase
-	Colorimetric dyes indicate detection (yellow = clean, red = contaminated)
Decontamination Applications
-	Contamination mapping
-	Enhanced decon efficiency
-	Post-decon assurance
Reconnaissance Applications
-	Consequence management
-	Sensitive site exploitation
Technology Applications
-	Standoff detection of surface contamination
-	Chemical agent sensing science
Performance has been extensively validated via live agent
testing
-	Edgewood Chemical Biological Command (ECBC) and Battelle
-	Various international test labs
Fielded ADS
ADS has been fielded in several kits with protection,
identification, reconnaissance, and decontamination products
Advanced Threat Detection (AT Boxes)
-	US Army 20th Support Command
-	4 fielded
Domestic Response Capability (DRC) Kits
-	National Guard WMD-CSTs
-	57 fielded
Dismounted Reconnaissance Sets Kits & Outfits (DR SKO)
-	Army, Air Force, Navy, Marines, and National Guard WMD-CSTs
-	49 fielded
CIDAS Program of Record
Contamination Indicator Decontamination Assurance
System (CIDAS)
-	Participating services include Army, Air Force, Marines, and
SOCOM
Managed by Joint Project Manager Protection (JPM P)
-	Fields all equipment in support of field decontamination (PPE,
CPE, decontaminants, etc.)
FLIR is prime contractor to provide indicator formulation
(ADS)
-	Small Scale (0.5 L) Systems and Large Scale (2 gal) consumables
-	Nerve and Training indicator formulations and Confidence Check
Cards (Blister formulation expected to transition in 2017)
10 year program
-	Engineering and Manufacturing Development Phase (2015-2017)
-	Low Rate Initial Production Phase (2017-2019)
-	Full Rate Production Phase (2019-2025)
Nerve Agent Disclosure Spray
P5T
Nerve Spray is yellow
upon application
In contaminated areas,
the formulation
enzyme is inhibited by
nerve agents and the
indicator dye turns red
In clean areas, the dye
remains yellow
Blister Agent Disclosure Spray
Blister Spray is red upon application
In contaminated areas, the formulation enzyme is inhibited by
blister agents and spray remains red
In clean areas, the red dye becomes colorless, allowing
visualization of the yellow background dye
After signal development, the color scheme is similar to the
Nerve Spray, with red detections on a yellow background

t «
• •
Decon Triage with ADS
s ,
H ~ $FLIR
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ADS Stability
ADS R&D has focused heavily on formulation stability
Shelf-life of packaged formulations is optimized through
extensive studies involving accelerated aging at elevated
temperatures
Performance of reconstituted sprays remains stable for at least
eight hours at temperatures as high as 40°C
Standard quality control procedures include evaluation of ADS
performance after incubation at 70°C for one week
Nerve ADS est. >5 years
Blister ADS est. >5 years
;t. >3 years >12 months >3 months >6 months
t. >3 years 8 months 3 months 6 months
User Training/Confidence
Training ADS uses a benign simulant to mimic the detection
response of Nerve ADS for end user instruction
is
Confidence Check Cards for Nerve, Blister, and Training Sprays
give the operator increased assurance that sprays are
functional prior to application
ADS is available in several form
factors to fit application needs
-	Screening sensitive equipment
for contamination
-	Personnel screening
-	Sensitive site assessment
-	Decon assurance/verification
-	Equipment/vehicle screening
-	Consequence management
-	Mapping wide area
contamination for
decontamination
UV Low Light Formulation
Enhanced detection on dark
surfaces
Available in two options
-	An additive to standard Nerve
ADS formulation
•	UV dye powder added to
traditional ADS
•	Adds low light capability to
existing product kits
•	No change in detection sensitivity
-	An optically transparent Nerve
ADS formulation
•	Useful for FBI and SOCOM users
•	Looks like water on surface until
illuminated
¦ Leaves no visible residue upon
drying
•	Commercial forensic kit
compatible
•	Ideal for interior decontamination
Human Health Risks/Environmental Impact
Exposure to Liquid Residue During
Typical Use /Accidental Release
(Indirect via Soil)
Blister 6E-01 / 7E-05 /
2E-01 2E-03
Exposure to Liquid Residue in
Surface Water During Typical Use
or Accidental Release (Direct
Powders)
Training 2E-04 1E-07
Nerve 1E-03 3E-07
Blister 4E-03 8E-06
Thorough User Health Risk and Environmental
Impact Study performed by consulting firm
-	Hazard Index (HI) = HQ,ngestion + HQderma, + HQinhalation
-	All HI<1, indicates no potential for adverse health
effects to occur
-	ADS is compliant with all applicable regulations
(scale considerations)
-	ADS does not pose a hazard to users even without
IPE during typical use or accidental release
-	ADS does not pose an ecological hazard to aquatic
or terrestrial model organisms
Acknowledgements
Defense Threat Reduction Agency
Army Research Office
Hazard Mitigation, Materiel, and Equipment Restoration
Advanced Technology Demonstration (HaMMER ATD)
Joint Project Manager for Protection
Warfighters
$FLIR
Contact:
Scott Donahue
Project Manager, FLIR Detection
2240 William Pitt Way, Pittsburgh, PA 15238, USA
Email: scott.donahue@flir.com | www.flir.com

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