Transcript Document

Selected Analytical Methods: Chemical
Warfare Agent and Degradation
Analytes
Stuart A. Willison, Ph.D.
Threat & Consequence Assessment Division
National Homeland Security Research Center
US Environmental Protection Agency
7/7/2015
U.S. Environmental Protection Agency
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Partner/Stakeholder Priorities
Primary research areas for Chemical Warfare
Agents (CWAs) and their degradation
products in environmental matrices include:
• Analytical protocols (all matrices)
• Surface sampling (porous surfaces)
• Stability studies for CWA and degradate standards
Increased Environmental Response
Laboratory Network (ERLN) capability and
capacity for CWA and degradate sampling and
analysis
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Sampling and Analytical Method Evaluation,
Selection and Development
Method
Development
Sample
Collection
•
•
Transportation
to Laboratory
Sample Collection
Protocols (SCPs)
Matrix Specific-e.g.,
Wipe Efficiency
Studies
Sample
Processing
• Selected Analytical
Methods (SAM)
• Sample Analytical
Protocols (SAPs)Matrix Specific
Field
Application
of Results
Exposure
Assessment
Goal: Include ALL sample collection
and processing steps for method
development
Optimize Instrumentation to meet DQOs
(chromatography, mass calibration,
tune, evaluate instrument stability)
Determine Best Calibration
(linear/quadratic, internal
standards)
Determine Preservatives
(e.g. select antimicrobial & dechlorinating
agent)
Determine Interferences (Are DQOs
met in various difficult matrices?)
YES
Determine
Holding Time
Does Method Meet
DQOs?
Write Method
NO
Revise Technical Approach
Use by ERLN
Adapted from J. Shoemaker and B. Boutin, USEPA/ORD at WQTC 2008
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Sample Collection During an
Incident
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Selected Analytical Methods (SAM)
Website: www.epa.gov/sam
SAM 2012
Published: July 2012
Chemical Methods
142 analytes
5 matrices
CWAs
CWA, precursor, and degradates
TICs
Semi-volatile organics,
inorganics
Pesticides
Organophosphate, carbamate
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SAM Methods: Tiered
Approach
Tier I
Analyte/sample type is a target of the method.
Multi-laboratory evaluated will allow implementation for the
analyte/sample type with no modifications.
Example: EPA Method 500 series for pesticides in water
Tier II
Tier III
Method has been used by laboratories to address the
analyte/sample type, but not multi-lab validated.
(1) The analyte/sample type is a target of a single-lab
verified method, or
(2) the analyte/sample type is not a target of a Tier I
method.
Example: Wipe method for vesicant/nerve agent degradates
Analyte/sample type is not a target of the method, and/or
no reliable data supporting the method's fitness for its
intended use are available.
Example: EPA Method 500 series for EA2192 in water
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Japan Subway Attack by Use of a
Nerve Agent
Kasumigaseki station was one of the many subway stations
affected during the attack
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Meeting Lab Throughput
Requirements
Potential approaches
• Methods which can be applied to multiple labs for
workload distribution
• Environmental Response Laboratory Network
(ERLN)
• High throughput sampling techniques and laboratory
analysis techniques
• Automated techniques or rapid method
capabilities
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Examples of Chemical Warfare
Agents Available in SAM
Document
• Analytes
– Tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), sulfur
mustard (HD), nitrogen mustards (HN-1 & HN-3), lewisite (L), VX
and Russian VX (R-VX)
– Nitrogen mustard degradates
– Nerve agent degradates
• Sample Types
– Soil
– Water
– Wipes
• Analytical Techniques
– Gas Chromatography/Time-of-Flight Mass Spectrometry
(GC/TOF-MS)
– Liquid Chromatography/Tandem Mass Spectrometry
(LC/MS/MS)
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Method Detection Levels (MDL)
for G-series, HD and VX
Residential Soil
(µg/kg)
Water
(µg/L)
Wipe
(ng/kg)
Agent
MDL
GC/TOF-MS*
MDL
GC/TOF-MS*
MDL
GC/TOF-MS
GB
0.22
0.14
0.0030
GD
0.67
0.11
0.0017
GF
0.12
0.14
0.0047
HD
0.12
0.10
0.0015
VX
0.41
0.23
0.008
* MDL for reagent sand or reagent water for soil
and water, respectively.
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Method Detection Levels (MDL)
for GA, HN-1, HN-3 and R-VX
Residential Soil
(mg/kg)
Water (µg/L)
Wipe (ng)
Agent
MDL
GC/TOF-MS*
MDL
GC/TOF-MS*
TOF-MDL
GC/TOF-MS*
GA
0.00033
0.13
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HN1
0.00057
0.084
2.3
HN3
0.00 16
0.72
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RVX
0.015
22
441
* MDL for reagent sand or reagent water for soil
and water, respectively.
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Stability Study for Ultra-Dilute
CWA Standards
• One year stability study for 10 ppm standards of
GB, GD, GF, HD and VX
• Flame-sealed ampoules - One year shelf-life
• Opened ampoules ~ 2-6 months depending on analyte
• Additional preservation study with only VX
IMPACT:
• Dilute concentrations allow for shipment to ERLN labs
without special requirements.
• Provides stability of analyte standards over time in
order to help with preparation for an incident
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Immunomagnetic Scavenging and
LC/MS Detection of VX in Water
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Immunomagnetic Scavenging and
LC/MS Detection of VX in Water
1) Conjugation of
antibody to beads
4) LC/MS/MS Analysis
2) Binding of
antibody to
BuChE
Expose
to water
Magnetic Bead
Antibody
3) Protein
digestion
BuChE
Peptides
IMSc LC-MS/MS Method
for Detection of VX in Water
IMPACT:
• Method sensitivity down to the ppt level
– Calculated method detection limit in HPLC-grade water = 5.6
ng/L
– Minimum reportable level = 25 ng/L
– Small sample size (100 uL)
•
Can be used to analyze up to 500 samples per day
• Low concentrations of VX can be detected in preserved tap
water 91 days after spiking
– Suggests applicability of this method for determining
water contamination with VX and utility during
environmental remediation
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Detection of other Chemicals/Toxins
in Water
Versatility:
• Robust and versatile process that can be applied to
other nerve agents
• Adapted for Ricin, and other toxins use automated
methods for rapid throughput and determination
• Allows for 100’s of samples to be processed in a
single day because of short chromatography run
times and automated processing of samples
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Persistence of VX and Degradation
Product on Asphalt
Degradation profile of VX on ground asphalt.
Columbus et. al., Environ. Sci. Technol., 2012, 46, 3921−3927
31P
NMR results: (a) 0.1 days, (b) 6 days, (c) 10 days,
(d) 14 days, (e) 17 days, (f) 20 days, and (g) 24 days
after the contamination.
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Notable Degradates Available in
SAM Document
• Nerve agents: VX, VR, VE, VG, GA, GB, GD, GF
– Notable degradation products: EA2192
• Vesicant agents – HD, Lewisite (L-1, L-2, L-3), HN-1,
HN-2, HN-3
– Notable degradation products: CVAA
IMPACT:
• Identifies potentially toxic degradates, which may
persist
• Degradate analysis allows samplers to identify
potentially concentrated areas of concern for parent
compound
• Another step towards ensuring remediation efforts
are completed or effective
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Sampling and Analysis Procedure
for Degradation Products
• Most CWA degradation products are not amenable to
gas-chromatography/mass spectrometry (GC-MS)
analysis without a derivatization step
• Liquid chromatography/mass spectrometry (LC-MS)
offers advantages over GC-MS because polar analytes
can be directly analyzed
• Selected CWA degradation products were analyzed by
LC-MS/MS in different matrix samples
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Hydrolysis Pathway of Nitrogen
Mustards in the Environment
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Sampling and Analysis Procedure
for Degradation Products
• Optimal eluents, chromatography separation, mass
spectrometric parameters were characterized with
low (ppb) detection limits*
• Optimal wipe investigated: cotton gauze, non-woven
polyester fiber cloth, pre-cleaned textile wipe, glass
fiber filter, and filter paper
• Surface evaluation included the following surfaces:
galvanized steel, glass, laminate, vinyl tile, treated
wood, painted drywall
* EPA Reports: EPA 600/R-11/143 and EPA/600/R-12/581
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LC-MS/MS Chromatogram of Nitrogen Mustard
Degradation Products*
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* S.A. Willison, J. Chromatogr. A, 1270 (2012) 72– 79
LC-MS/MS Chromatogram of Nitrogen Mustard
Degradation Products
Wipe 1
Wipe 2
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MDL for Nitrogen Mustard
Degradation Products
LAMINATE SURFACE WIPE*
MDL Calculation:
LC-MS/MS
MDL = n x SD
Analyte
MDL †
(ng/cm2)
MDL
(ng /mL)
TEA
0.2
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Where:
EDEA
0.03
3.1
MDEA
0.1
12.4
SD = the standard
deviation for the analytical results
DEA
0.1
14.0
†ng/cm2
units calculated by dividing
concentration on surface by surface area
(100 cm2)
and
n = 3.14 = the Student’s tvalue for seven replicate samples
* S.A. Willison, J. Chromatogr. A, 1270 (2012) 72– 79
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Analyte Recoveries from Surface
Wiping
Analyte
TEA1
EDEA
MDEA
DEA1
Surface
% Recovery
(n=7)
% Recovery
(n=7)
% Recovery
(n=7)
% Recovery
(n=7)
Laminate
81-99
47-71
66-80
71-81
Metal
20-46
49-56
54-74
62-79
Glass
91-149
31-36
52-61
59-81
Vinyl Tile
41-79
7-22
51-61
25-39
Painted
Drywall2
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8
17
13
Wood2
2
1
2
1
1
Recoveries were subtracted from surface blanks for a
representative recovery of target analytes
2 Recoveries were only possible at highest spike concentration
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Enhancing Throughput for LC-MS/MS
Analytical Methods
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Enhancing Throughput for LC-MS/MS
Analytical Methods*
* manuscript in preparation
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Fate of CWAs in Wastewater
• Investigate nerve agent degradation products in a
laboratory fortified bio-sludge reactor (simulating a
wastewater treatment plant) followed by LC-MS/MS
analysis
• If treatment from the biodegradation process is
incomplete, persistent toxic agents and/or their toxic byproducts could be released back into the environment
and eventually into a water source
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Wastewater Treatment Process
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Sorption Kinetics of EMPA to
Activated Sludge
Determine time for EMPA sorption to occur:
• EMPA spiked onto sludge solids (3 mg/L)
• Vials rotated and analyzed (5, 10, 20, 40, 60 minutes)
• Extracted from solid and liquid phases and compared
to standard (EMPA on filter paper (3 mg/L))
Results:
• Recovery of EMPA was 83-93%
• Statistical analysis (a = 0.05) indicated an
insignificant difference between EMPA sorption on
activated sludge and the standard (EMPA on filter
paper)
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Sorption Kinetics of EMPA to
Activated Sludge
Determine EMPA sorption rates by varying total
suspended solids (TSS) concentrations:
• TSS concentrations (1235, 820, 795, 655, 585, 175
mg/L)
Results:
• Recovery of EMPA from all TSS concentrations was
95-106%
• TSS sludge concentrations did not have an impact on
EMPA’s ability to sorb to the activated sludge [statistical
analysis did not suggest a difference between EMPA
sorption to activated sludge and standard (EMPA on
filter paper)]
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Biodegradation in Activated Sludge with
Active Nitrifiers at 25°C
Liquid Phase EMPA Concentration (µg L-1)
1000
80
EMPA
NH3-N
COD
800
70
60
50
600
40
400
30
20
200
10
0
0
0
2
4
6
Time (Hrs)
8
10
12
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1400
14
1200
12
1000
10
800
8
600
6
400
4
EMPA
200
NH3-N Concentration (mg L-1)
Liquid Phase EMPA Concentration (µg L-1)
Biodegradation in Activated Sludge
with Allylthiourea (ATU) at 25°C
2
NH3-N
0
0
0
2
4
6
Time (Hrs)
8
10
12
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Impact
• If threshold and pH effects do apply (with respect to microbial
population), then large quantities of the proper activated
sludge content must be present
• Unless a suitable microbial population is present, possible
ionized acid CWA degradation products, like EMPA, and
similarly sorbed and biodegraded compounds may pass
through an activated sludge wastewater treatment plant
largely unchanged
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Future Direction
•
•
•
•
•
•
Analysis approaches for other matrices adapted from Tier I methods
Refinement of existing methods
Degradates and by-products
Decontamination-focused studies (and associated detection
challenges)
Improving recovery on porous surfaces
Enhanced federal collaborations – agreement between EPA, DHS, and
DOD.
Method verification
• Many analytes in SAM have not be verified in light of potential DQOs
for large scale environmental remediation.
• Methods may be evaluated in 2nd or more labs.
• May be compatible with current public health lab operations
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Acknowledgements
SAM 2012
Co-authors:
Published: July 2012
Romy Campisano1
Matthew Magnuson1
Erin Silvestri1
Terry Smith2
Carolyn Koester3
Hiba Ernst1
1U.S.
EPA, National Homeland Security Research Center
EPA, Office of Emergency Management
3Lawrence Livermore National Laboratory
2U.S.
DISCLAIMER: The U.S. EPA through its Office of
Research and Development partially funded the
research described in this presentation. It has been
reviewed by the Agency but does not necessarily
reflect the Agency’s views. No official endorsement
should be inferred. EPA does not endorse the
purchase or sale of any commercial products or
services.
Website: www.epa.gov/sam
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