EPA/600/R-04/126D September 29, 2008
United States
Environmental Protection
Agency
Standardized Analytical Methods
for Environmental Restoration
Following Homeland Security Events
REVISION 4.0
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EPA/600/R-04/126D| September 2008 www.epa.gov/sam
Standardized Analytical Methods
for Environmental Restoration
Following Homeland Security
Events - Revision 4.0
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
Cincinnati, OH 45268
COMPUTER SCIENCES CORPORATION
Alexandria, VA 22304-3540
Office of Research and Development
National Homeland Security Research Center, Response Capability Enhancement
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Disclaimer
Disclaimer
The U.S. Environmental Protection Agency (EPA) through its Office of Research and Development
funded and managed the research described here under Contract EP-W-06-046 to Computer Sciences
Corporation (CSC). This document has been subjected to the Agency's review and has been approved for
publication. Note that approval does not signify that the contents necessarily reflect the views of the
Agency.
Mention of trade names or commercial products in this document or in the methods referenced in this
document does not constitute endorsement or recommendation for use.
Questions concerning this document or its application should be addressed to:
Oba Vincent
National Homeland Security Research Center
Office of Research and Development (163)
U.S. Environmental Protection Agency
26 West Martin Luther King Drive
Cincinnati, OH 45268
(513)569-7456
vincent.oba@epa.gov
SAM Revision 4.0 ii September 29, 2008
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Use of This Document
Use of This Document
The information contained in this document represents the latest step in an ongoing effort of the
Environmental Protection Agency's (EPA's) National Homeland Security Research Center
(NHSRC) to provide standardized analytical methods for use by those laboratories tasked with
performing confirmatory analyses of environmental samples in support of EPA restoration efforts
following a homeland security incident. Although at this time, some of the methods listed have not
been fully validated for a particular analyte (e.g., analytes not explicitly identified in the method) or
sample type, the methods are considered to contain the most appropriate currently available
techniques. Unless a published method listed in this document states specific applicability to the
analyte/sample type for which it has been selected, it should be assumed that method testing is
needed, and adjustments may be required to accurately account for variations in analyte/sample
type characteristics, environmental samples, and target risk levels. Many of the target analytes
listed in this document have only recently become an environmental concern. EPA is actively
pursuing development and validation of Standardized Analytical Protocols (SAPs) based on the
methods listed, including optimization of procedures for measuring target analytes or agents. In
those cases where method procedures are determined to be insufficient for a particular situation,
EPA will provide guidance regarding appropriate actions. This will be an ongoing process as EPA
will strive to establish a consistent level of validation for all listed analytes.
SAM Revision 4.0 iii September 29, 2008
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Foreword
Foreword
The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting the Nation's
land, air, and water resources. Under a mandate of national environmental laws, EPA strives to formulate
and implement actions leading to a compatible balance between human activities and the ability of natural
systems to support and nurture life. To meet this mandate, EPA's research program is providing data and
technical support for solving environmental problems today and building a scientific base necessary to
manage our ecological resources wisely, understand how pollutants affect our health, and prevent or
reduce environmental risks in the future.
The National Homeland Security Research Center (NHSRC) is EPA's organization for conducting
research to facilitate protection and decontamination of indoor and outdoor areas and water infrastructure
subject to chemical, biological, or radiological (CBR) terror attacks. NHSRC's research is designed to
provide appropriate, effective, and validated technologies, methods, and guidance to understand the risks
posed by CBR agents and to enhance our ability to detect, contain, and clean up in the event of an
incident involving such agents. This document is intended to provide guidance for selecting methods that
have a high likelihood of ensuring analytical consistency when laboratories are faced with a large scale
environmental restoration crisis. At the same time, the document can be used as a tool to identify analytes
that require further methods development and verification to ensure desired performance. NHSRC will
also provide direct technical assistance to response personnel in the event of a CBR attack, as well as
provide related interagency liaisons. This is most effectively accomplished by contacting EPA's
Emergency Operations Center.
This publication has been produced as part of the NHSRC's long-term research plan. It is published and
made available by EPA's Office of Research and Development to assist the user community and to link
researchers with their clients.
x Jonathan G. Herrmann, Director
National Homeland Security Research Center
SAM Revision 4.0 iv September 29, 2008
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Abbreviations and Acronyms
Abbreviations and Acronyms
AA Atomic Absorption
AdV40 Adenovirus 40
AdV41 Adenovirus 41
AEM Applied and Environmental Microbiology
Amp-ELISA Amplified-Enzyme-Linked Immunosorbent Assay
APHA American Public Health Association
APHL Association of Public Health Laboratories
AOAC AOAC International (formerly the Association of Official Analytical Chemists)
ASE Automated Soxhlet Extraction
ASM American Society for Microbiology
ASTM ASTM International (formerly the American Society for Testing and Materials)
ATP 2-aminothiophenol
AWWA American Water Works Association
BCM Biosynth Chromogen Medium
BCYE Buffered Charcoal Yeast Extract
BGMK Buffalo Green Monkey Kidney
BHT Butylated hydroxytoluene
BS Bismuth Sulfite
BLEB Buffered Listeria Enrichment Broth
BMBL Biosafety in Microbiological and Biomedical Laboratories
BSA Bovine Serum Albumin
BSL Biosafety Level
BZ Quinuclidinyl benzilate
°C Degrees Celsius
CA Chocolate Agar
CAS RN Chemical Abstracts Service Registry Number
CBR Chemical, Biological, or Radiological
CCID Coordinating Center for Infectious Diseases
CDC Centers for Disease Control and Prevention
cDNA Complementary Deoxyribonucleic Acid
CFR Code of Federal Regulations
CFSAN Center for Food Safety and Applied Nutrition
CPU Colony Forming Unit
CIEIA Competitive Inhibition Enzyme Immunoassay
CLLE Continuous Liquid-Liquid Extraction
CLP Contract Laboratory Program
CPE Cytopathic Effect
cps counts per second
CS Tear gas; Chlorobenzylidene malonitrile
CVAA Cold Vapor Atomic Absorption or 2-Chlorovinylarsonous acid
CVAFS Cold Vapor Atomic Fluorescence Spectrometry
2,4-D 2,4-Dichlorophenoxyacetic Acid
DAPI 4',6-diamidino-2-phenylindole
DAS Diacetoxyscipenol
DAS-HG-HSA Diacetoxyscipenol Hemiglutarate Human Serum Albumin
DAS-HS-HRP Diacetoxyscipenol Hemisuccinate Horseradish Peroxidase Conjugate
DB-1 100% Dimethylpolysiloxane
DBPR Division of Bioterrorism Preparedness and Response
DHS Department of Homeland Security
DIG Differential Interference Contrast
DIG-ELISA Digoxigenin Labeled Enzyme-Linked Immunosorbent Assay
SAM Revision 4.0
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Abbreviations and Acronyms
DIMP Diisopropyl methylphosphonate
DMT 3,4-Dimercaptotoluene
DNA Deoxyribonucleic Acid
2,4-DNPH 2,4-Dinitrophenylhydrazine
DoD Department of Defense
DOE Department of Energy
DOT Department of Transportation
DPD N.N-diethyl-p-phenylenediamine
DTPA Diethylenetriamine-pentaacetate
EA2192 Diisopropylaminoethyl methylthiophosphonate
BCD Electron Capture Detector
e-CFR Electronic Code of Federal Regulations
ECL Electrochemiluminescence
ED Electron Diffraction or Ethyldichloroarsine
EDEA N-Ethyldiethanolamine
EDL Estimated Detection Limit
EDTA Ethylenediaminetetraacetic acid
EDXA Energy Dispersive X-ray Analysis
EEB Enterohemorrhagic E. coll Enrichment Broth
EIA Enzyme Immunoassay
ELCD Electrolytic Conductivity Detector
ELISA Enzyme-Linked Immunosorbent Assay
EMC Emission Measurement Center
EMJH Ellinghausen-McCullough Johnson Harris Formulation
EML Environmental Measurements Laboratory
EMMI Environmental Monitoring Methods Index
EMPA Ethyl methylphosphonic acid
EMSL Environmental Monitoring and Support Laboratory
EPA Environmental Protection Agency
EQL Estimated Quantitation Limit
ESI Electrospray lonization
ETV Environmental Technology Verification
FA Fluorescence Assay
FBI Federal Bureau of Investigation
FDA Food and Drug Administration
FEMS Federation of European Microbiological Societies
FGI Fluorescein derivative of Conus geographus a-conotoxin
FID Flame lonization Detector
FL Fluorescence Detector
FPD Flame Photometric Detector
FRET Fluorescence Resonance Energy Transfer
FRMAC Federal Radiological Monitoring and Assessment Center
FSIS Food Safety and Inspection Service
GA Tabun
GB Sarin
GC Gas Chromatograph or Gas Chromatography
GC-ECD Gas Chromatography - Electron Capture Detector
GC-FID Gas Chromatography - Flame lonization Detector
GC-FPD Gas Chromatography - Flame Photometric Detector
GC-MS Gas Chromatography - Mass Spectrometry
GC-MD Gas Chromatography - Multi-Detector
GC-NPD Gas Chromatography - Nitrogen-phosphorus Detector
GD Soman
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Abbreviations and Acronyms
GE
Ge(Li)
GESTIS
GF
GFAA
GTC
HAdV
HASL
HAV
HCoV
HEV
HEPA
HD
HHS
HMTD
HMX
HN-1
HN-2
HN-3
HPGe
HPLC
HPLC-FL
HPLC-MS
HPLC-PDA
HPLC-UV
HPLC-vis
HRP
HV
1C
1C 20
1C 50
ICP
ICP-AES
ICP-MS
ICR
ID50
IDL
ILM
IMPA
IMS
INCHEM
IO
i.p.
IRIS
ISE
1-Methylethyl ester ethylphosphonofluoridic acid or Genome Equivalent
Germanium (Lithium)
A German database (Gefahrstoffdaten banken) containing data and information on
hazardous substances and products
Cyclohexyl sarin
Graphite Furnace Atomic Absorption Spectrophotometer or Graphite Furnace Atomic
Absorption Spectrophotometry
Guanidinium Thiocyanate
Human Adenoviruses
Health and Safety Laboratory, currently known as Environmental Measurements
Laboratory (EML)
Hepatitis A Virus
Human Coronavirus
Hepatitis E Virus
High Efficiency Particulate Air (Filter)
Sulfur mustard / mustard gas; bis(2-chloroethyl) sulfide
Health and Human Services
Hexamethylenetriperoxidediamine
Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine
Nitrogen mustard 1; bis(2-chloroethyl)ethylamine
Nitrogen mustard 2; 2,2'-dichloro-N-methyldiethylamine N,N-bis(2-
chloroethyl)methylamine
Nitrogen mustard 3; tris(2-chloroethyl)amine
High Purity Germanium
High Performance Liquid Chromatography
High Performance Liquid Chromatograph - Fluorescence
High Performance Liquid Chromatograph - Mass Spectrometer
High Performance Liquid Chromatography - Photodiode Array Detector
High Performance Liquid Chromatography - Ultraviolet
High Performance Liquid Chromatography - visible
Horseradish Peroxidase
High Volume
Ion Chromatograph or Ion Chromatography
Inhibitory Concentration - Concentration to inhibit 20%
Inhibitory Concentration - Concentration to inhibit 50%
Inductively Coupled Plasma
Inductively Coupled Plasma - Atomic Emission Spectrometry
Inductively Coupled Plasma - Mass Spectrometry
Information Collection Requirements Rule or Information Collection Request
A dose which would be infectious to 50% of the population
Instrument Detection Limit
Inorganic Laboratory Method
Isopropyl methylphosphonic acid
Immunomagnetic Separation
INCHEM is a means of rapid access to internationally peer reviewed information on
chemicals commonly used throughout the world, which may also occur as contaminants
in the environment and food. It consolidates information from a number of
intergovernmental organizations whose goal it is to assist in the sound management of
chemicals, http:7/www.inchcm.org/
Inorganic
Intraperitoneally
Integrated Risk Information System (U.S. EPA)
Ion Specific Electrode
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Abbreviations and Acronyms
ISG Impregnated Silica Gel
ISO International Organization for Standardization
L-l Lewisite 1; 2-Chlorovinyldichloroarsine
L-2 Lewisite 2; bis(2-Chlorovinyl)chloroarsine
L-3 Lewisite 3; tris(2-Chlorovinyl)arsine
LB-M Lim Benyesh-Melnick
LC Liquid Chromatograph
LC/APCI-MS Liquid Chromatography / Atmospheric Pressure Chemical lonization-Mass Spectrometry
LC/ESI-MS Liquid Chromatography / Electrospray lonization-Mass Spectrometry
LC-MS Liquid Chromatography - Mass Spectrometry
LC-MS-MS Liquid Chromatography Tandem Mass Spectrometry
LC-TSP Liquid Chromatography - Thermospray
LFD Lateral Flow Device
LIA Lysine Iron Agar
LLD Lower Limit of Detection
LOD Limit of Detection
LRN Laboratory Response Network
LSE Liquid-solid Extraction
Ltd. A private company limited by shares
mAb Monoclonal Antibody
MARLAP Multi-Agency Radiological Laboratory Analytical Protocols
MCAWW Methods for Chemical Analysis of Water and Waste (EPA/600/4-79/020)
MDL Method Detection Limit
MF Membrane Filtration
MIC Methyl Isocyanate
MLD Minimum Lethal Dose
MOPS Morpholinepropanesulfonic Acid
MPA Methylphosphonic acid
MPN Most Probable Number
mRNA Messenger Ribonucleic Acid
MS Mass Spectrometer or Mass Spectrometry or Matrix Spike
MS-MS Tandem Mass Spectometry
MSD Matrix Spike Duplicate
MSE Microscale Solvent Extraction
MSRV Modified Semisolid Rappaport-Vassiliadis
MTBE Methyl fert-butyl ether
MW Molecular Weight
NA Not Applicable
nAchR Nicotinic Acetylcholine Receptor
Nal(Tl) Thallium-Activated Sodium Iodide
NBD chloride 7-Chloro-4-nitrobenzo-2-oxa-l,3-diazole
NBD-F 7-Fluoro-4-nitro-2,1,3-benzoxadiazole
NCPDCID National Center for the Prevention, Detection, and Control of Infectious Diseases
NCRP National Council on Radiation Protection and Measurements
NCTC National Collection of Type Cultures
NEMI National Environmental Methods Index
NERL National Exposure Research Laboratory
NHSRC National Homeland Security Research Center
NIOSH National Institute for Occupational Safety and Health
NIST National Institute of Standards and Technology
nM Nanomolar
NMAM NIOSH Manual of Analytical Methods
NNSA National Nuclear Security Administration
SAM Revision 4.0
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Abbreviations and Acronyms
NoV Norovirus
NOS Not Otherwise Specified
NPD Nitrogen-phosphorus Detector
NRC Nuclear Regulatory Commission
NRMRL National Risk Management Research Laboratory
nS nano Siemens
NTIS National Technical Information Service
NTU Nephelometric Turbidity Units
OAQPS U.S. EPA Office of Air Quality Planning and Standards
OAR U.S. EPA Office of Air and Radiation
ORAU Oak Ridge Associated Universities
ORISE Oak Ridge Institute for Science and Education
ORD U.S. EPA Office of Research and Development
ORF Open Reading Frame
OSWER U.S. EPA Office of Solid Waste and Emergency Response
OSHA Occupational Safety and Health Administration
OVS OSHA Versatile Sampler
OW U.S. EPA Office of Water
OXA Oxford Medium
PBS Phosphate Buffered Saline
PCDDs Polychlorinated Dibenzo-p-dioxins
PCDFs Polychlorinated Dibenzofurans
PCR Polymerase Chain Reaction
PDA Photodiode Array Detector
PEL Permissible Exposure Limit
PETN Pentaerythritol tetranitrate
PFE Pressurized Fluid Extraction
PFIB Perfluoroisobutylene
PFIB-ATP PFIB with 2-aminothiophenol
PFIB-DMT PFIB with 3,4-Dimercaptotoluene
PID50 50% Pig Infectious Dose
PMPA Pinacolyl methyl phosphonic acid
1,2-PP l-(2-pyridyl)piperazine
PubMED PubMED is a service of the U.S. National Library of Medicine (www.pubmed.gov),
containing citations from scientific journals
PUF Polyurethane Foam
PVC Polyvinyl Chloride
QA Quality Assurance
QAP Quality Assessment Program
QC Quality Control
qPCR Quantitative Polymerase Chain Reaction
® Registered Trademark
R 33 Methylphosphonothioic acid, S-[2-(diethylamino)ethyl] O-2-methylpropyl ester (VR)
RCRA Resource Conservation and Recovery Act
RDX Hexahydro-1,3,5 -trinitro-1,3,5 -triazine
RLAB Regional Laboratory
RNA Ribonucleic Acid
rpm Revolutions per Minute
rRNA Ribosomal Ribonucleic Acid
RTECS Registry of Toxic Effects of Chemical Substances
RT-PCR Reverse Transcription Polymerase Chain Reaction
SAED Select Area Electron Diffraction
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Abbreviations and Acronyms
SAM Standardized Analytical Methods for Environmental Restoration Following Homeland
Security Events
SAP Standardized Analytical Protocol
SARS Severe Acute Respiratory Syndrome
SaV Sapovirus
SBA Sheep Blood Agar
SIM Selective Ion Monitoring
SM Standard Methods for the Examination of Water and Wastewater
SPE Solid-Phase Extraction
spp. Species (plural)
STEC Shiga-toxigenic E. coli
STEL Short Term Exposure Limit
STX Saxitoxin
Stx Shiga Toxin
Stx-1 Shiga Toxin Type 1
Stx-2 Shiga Toxin Type 2
SW Solid Waste
TATP Triacetone triperoxide
TBD To Be Determined
TCBS Thiosulfate-Citrate-Bile Salts-Sucrose
TC-SMAC Tellurite Cefixime Sorbitol MacConkey Agar
TCLP Toxicity Characteristic Leaching Procedure
TDG Thiodiglycol
TEA Triethanolamine
TEM Transmission Electron Microscope or Microscopy
TETR Touchdown Enzyme Time Release
TFA Trifluoroacetic acid
™ Trademark
TM Test Methods or Thayer-Martin
1,3,5-TNB 1,3,5-Trinitrobenzene
2,4,6-TNT 2,4,6-Trinitrotoluene
TO Toxic Organic
TOFMS Time-of-Flight Mass Spectrometry
TOXNET Toxicology Data Network
TRU Transuranic
TSB Tryptic Soy Broth
TSAye Trypticase™ Soy Agar with yeast extract
TSI Triple Sugar Iron
TSP Thermospray
TSP-MS Thermopray - Mass Spectrometry
TTN Technical Transfer Network
TTX Tetrodotoxin
UF Ultrafiltration
U.S. United States
USDA United States Department of Agriculture
USGS United States Geological Survey
UV Ultraviolet
UVM University of Vermont
VCSB Voluntary Consensus Standard Body
VE Phosphonothioic acid, ethyl-, S-(2-(diethylamino)ethyl) O-ethyl ester
VG Phosphonothioic acid, S-(2-(diethylamino)ethyl) O,O-diethyl ester
vis Visible Detector
VM Phosphonothioic acid, methyl-,S-(2-(diethylamino)ethyl) O-ethyl ester
SAM Revision 4.0
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Abbreviations and Acronyms
VOCs Volatile Organic Compounds
VR Methylphosphonothioic acid, S-[2-(diethylamino)ethyl] O-2-methylpropyl ester (R 33)
VX O-ethyl-S-(2-diisopropylaminoethyl)methylphosphonothiolate
WEF Water Environment Federation
WHO World Health Organization
XLD Xylose Lysine Deoxycholate
SAM Revision 4.0 xi September 29, 2008
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Abbreviations and Acronyms
SAM Revision 4.0 xii September 29, 2008
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Acknowledgments
Acknowledgments
Contributions of the following individuals and organizations to the development of the SAM information
are gratefully acknowledged.
United States Environmental Protection Agency (EPA)
• Office of Research and Development, National Homeland Security Research Center
Joan Bursey
Kathy Hall
Romy Lee
Alan Lindquist
Matthew Magnuson
Chandrika Moudgal
Tonya Nichols
Ben Packard
Eugene Rice
Rob Rothman
Frank Schaefer
Sanjiv Shah
Ramona Sherman
Erin Silvestri
Sarah Taft
Oba Vincent
Stuart Willison
Charlena Yoder
• Office of Research and Development, National Exposure Research Laboratory (NERL), Las
Vegas
Don Betowski (Environmental Sciences Division)
Christian Daughton (Environmental Sciences Division)
Jane Denne (Environmental Sciences Division)
Michael Hiatt (Environmental Sciences Division)
Tammy Jones-Lepp (Environmental Sciences Division)
Stephen Pia (Environmental Sciences Division)
Lee Riddick (Environmental Sciences Division)
Charlita Rosal (Environmental Sciences Division)
Brian Schumacher (Environmental Sciences Division)
Wayne Sovocool (Environmental Sciences Division)
Katrina Varner (Environmental Sciences Division)
John Zimmerman (Environmental Sciences Division)
Kim Rogers (Human Exposure and Atmospheric Sciences Division)
Jeannette VanEmon (Human Exposure and Atmospheric Sciences Division)
• Office of Research and Development, National Exposure Research Laboratory (NERL),
Cincinnati
James Owens (Microbiological and Chemical Exposure Assessment Research Division)
Gerard Stelma (Microbiological and Chemical Exposure Assessment Research Division)
Ann Grimm (Microbiological and Chemical Exposure Assessment Research Division)
• Office of Solid Waste and Emergency Response
Michele Burgess (Office of Emergency Management)
Barry Lesnik (Office of Solid Waste)
Scott Hudson (Office of Emergency Management, National Decontamination Team)
SAM Revision 4.0 xiii September 29, 2008
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Acknowledgments
Michael Johnson (Office of Superfund Remediation and Technology Innovation)
Lawrence Kaelin (Office of Emergency Management, National Decontamination Team)
Michael Ottlinger (Office of Emergency Management, National Decontamination Team)
Terry Smith (Office of Emergency Management)
Shen Yi-yang (Office of Emergency Management)
• Office of Air and Radiation, Office of Radiation and Indoor Air
Lindsey Bender (Radiation Protection Division)
George Dilbeck (National Air and Radiation Environmental Laboratory)
Dennis Farmer (Radiation and Indoor Environments National Laboratory)
John Griggs (National Air and Radiation Environmental Laboratory)
Daniel Mackney (National Air and Radiation Environmental Laboratory)
David Musick (Radiation and Indoor Environments National Laboratory)
• Office of Water, Office of Ground Water and Drinking Water
Jessica Pulz (Water Security Division)
James Sinclair (Technical Support Center)
• Office of Prevention, Pesticides, and Toxic Substances, Office of Pesticide Programs
Yaorong Qian (Biological and Economic Analysis Division)
Chuck Stafford (Environmental Science Center)
Dallas Wright (Environmental Science Center)
• Office of Enforcement and Compliance Assurance, Office of Criminal Enforcement,
Forensics and Training
Dan Hurlbut (National Enforcement Investigations Center)
Don Smith (National Enforcement Investigations Center)
Larry Strattan (National Enforcement Investigations Center)
• EPA Regions
John Curry (Region 3)
Joe Dorsey (Region 3)
Diane Gregg (Region 6)
Adrienne Greenlee (Region 7)
Ted Haigh (Region 5)
Stephanie Harris (Region 10)
Peggy Knight (Region 10)
Johnson Mathew (Region 6)
Ed O'Neill (Region 6)
Peter Philbrook (Region 1)
Steve Reimer (Region 10)
Dennis Revell (Region 4)
Mel Ritter (Region 6)
Norman Rodriguez (Region 7)
Sue Warner (Region 3)
Laura Webb (Region 7)
Wayne Whipple (Region 5)
Larry Zintek (Region 5)
United States Department of Health and Human Services
• Centers for Disease Control and Prevention (CDC)
Matt Arduino (National Center for Preparedness, Detection and Control of Infectious Diseases)
Kevin Ashley (National Institute for Occupational Safety and Health)
SAM Revision 4.0 xiv September 29, 2008
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Acknowledgments
Raymond Biagini (National Institute for Occupational Safety and Health)
Erin Black (Coordinating Office for Terrorism Preparedness and Emergency Response)
Cheryl Bopp (National Center for Zoonotic, Vector-Borne, and Enteric Diseases)
Jay Gee (National Center for Zoonotic, Vector-Borne, and Enteric Diseases)
Vincent Hill (National Center for Zoonotic, Vector-Borne, and Enteric Diseases)
Rudolph Johnson (National Center for Environmental Health)
Richard Kellogg (National Center for Preparedness, Detection and Control of Infectious Diseases)
Gene Kennedy (National Institute for Occupational Safety and Health)
Stephen Morse (National Center for Preparedness, Detection and Control of Infectious Diseases)
John Snawder (National Institute for Occupational Safety and Health)
Richard Wang (National Center for Environmental Health)
Betsy Weirich (National Center for Preparedness, Detection and Control of Infectious Diseases)
• United States Food and Drug Administration
Jennifer Brzezinski
David Craft
Eric Garber
Michael McLaughlin
United States Department of Agriculture
Jim Trapp
Jim Trout
Paul Zimba
United States Department of Commerce
Peter Moeller (National Oceanic & Atmospheric Administration)
United States Department of Defense
Johnathan Kiel (U.S. Air Force)
Elaine Strauss (U.S. Navy, Naval Surface Warfare Center Dahlgren Division)
Sina Bavari (U.S. Army, Medical Research Institute of Infectious Diseases)
Tambra Dunams (Shaw Group, Inc. under contract with U.S. Army, Chemical Materials Agency)
Robert Durgin (U.S. Army, Chemical Materials Agency)
Alan Hewitt (U.S. Army, U.S. Army Corps of Engineers)
Mark Poli (U.S. Arm}-, Medical Research Institute of Infectious Diseases)
Ronald Swatski (U.S. Arm}', Center for Health Promotion and Preventive Medicine)
United States Department of Energy
Gary Brown (Sandia National Laboratory)
Steve Goldberg (New Brunswick Laboratory)
Jon Neuhoff (New Brunswick Laboratory)
Chino Srinivasan (New Brunswick Laboratory)
State Agencies
Rick Bokanyi (Ohio Department of Health)
David Degenhardt (Wisconsin State Laboratory of Hygiene)
Timothy Fitzpatrick (Florida Department of Environmental Protection)
Robert Glowacky (Virginia State Department of General Services)
Rebecca Hoffman (Wisconsin State Laboratory of Hygiene)
Christopher Retarides (Virginia State Department of General Services)
Tom York (Virginia State Department of General Services)
SAM Revision 4.0 xv September 29, 2008
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Acknowledgments
Hamilton Sundstrand
Richard Trubey
Environmental Management Support, Inc.
Anna Berne
Computer Sciences Corporation
Eric Boring
Danielle Carter
Joan Cuddeback
Melody Jensen
Larry Umbaugh
SAM Revision 4.0 xvi September 29, 2008
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Table of Contents
Standardized Analytical Methods for Environmental Restoration
Following Homeland Security Events
Revision 4.0
September 29, 2008
Contents
Disclaimer ii
Use of This Document iii
Foreword iv
Abbreviations and Acronyms v
Acknowledgments xiii
Section 1.0: Introduction 1
Section 2.0: Background 3
Section 3.0: Scope and Application 7
Section 4.0: Points of Contact 11
Section 5.0: Selected Chemical Methods 13
5.1 General Guidance 14
5.1.1 Standard Operating Procedures for Identifying Chemical Methods 14
5.1.2 General QC Guidance for Chemical Methods 32
5.1.3 Safety and Waste Management 33
5.2 Method Summaries 34
5.2.1 EPA Method 8: Determination of Sulfuric Acid and Sulfur Dioxide Emissions from
Stationary Sources 34
5.2.2 EPA Method 200.7: Determination of Metals and Trace Elements in Waters and Wastes by
Inductively Coupled Plasma-Atomic Emission Spectrometry 35
5.2.3 EPA Method 200.8: Determination of Trace Elements in Waters and Wastes by Inductively
Coupled Plasma-Mass Spectrometry 36
5.2.4 EPA Method 245.2: Mercury (Automated Cold Vapor Technique) 37
5.2.5 EPA Method 252.2: Osmium (Atomic Absorption, Furnace Technique) 37
5.2.6 EPA Method 300.1, Revision 1.0: Determination of Inorganic Anions in Drinking Water by
Ion Chromatography 38
5.2.7 EPA Method 335.4: Determination of Total Cyanide by Semi-Automated Colorimetry 39
5.2.8 EPA Method 350.1: Nitrogen, Ammonia (Colorimetric, Automated Phenate) 40
5.2.9 EPA Method 507: Determination of Nitrogen- and Phosphorus-Containing Pesticides in
Water by Gas Chromatography with a Nitrogen-Phosphorus Detector 40
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Table of Contents
5.2.10 EPA Method 524.2: Measurement of Purgeable Organic Compounds in Water by Capillary
Column Gas Chromatography / Mass Spectrometry 41
5.2.11 EPA Method 525.2: Determination of Organic Compounds in Drinking Water by Liquid-
Solid Extraction and Capillary Column Gas Chromatography / Mass Spectrometry 41
5.2.12 EPA Method 531.2: Measurement of N-Methylcarbamoyloximes and N-Methylcarbamates
in Water by Direct Aqueous Inj ection HPLC with Postcolumn Derivatization 42
5.2.13 EPA Method 549.2: Determination of Diquat and Paraquat in Drinking Water by Liquid-
Solid Extraction and High Performance Liquid Chromatography with Ultraviolet Detection
43
5.2.14 EPA Method 551.1: Determination of Chlorination Disinfection Byproducts, Chlorinated
Solvents, and Halogenated Pesticides/Herbicides in Drinking Water by Liquid-Liquid
Extraction and Gas Chromatography with Electron-Capture Detection 43
5.2.15 EPA Method 614: The Determination of Organophosphorus Pesticides in Municipal and
Industrial Wastewater 44
5.2.16 EPA Method 3031 (SW-846): Acid Digestion of Oils for Metals Analysis by Atomic
Absorption or ICP Spectrometry 45
5.2.17 EPA Method 3050B (SW-846): Acid Digestion of Sediments, Sludges, and Soils 46
5.2.18 EPA Method 3520C (SW-846): Continuous Liquid-Liquid Extraction 47
5.2.19 EPA Method 3535A (SW-846): Solid-Phase Extraction 48
5.2.20 EPA Method 3541 (SW-846): Automated Soxhlet Extraction 51
5.2.21 EPA Method 3545A (SW-846): Pressurized Fluid Extraction (PFE) 53
5.2.22 EPA Method 3570 (SW-846): Microscale Solvent Extraction (MSB) 56
5.2.23 EPA Method 3571 (SW-846): Extraction of Solid and Aqueous Samples for Chemical
Agents 59
5.2.24 EPA Method 3580A (SW-846): Waste Dilution 60
5.2.25 EPA Method 3585 (SW-846): Waste Dilution for Volatile Organics 62
5.2.26 EPA Method 503OC (SW-846): Purge-and-Trap for Aqueous Samples 63
5.2.27 EPA Method 5035A (SW-846): Closed-System Purge-and-Trap and Extraction for Volatile
Organics in Soil and Waste Samples 64
5.2.28 EPA Method 6010C (SW-846): Inductively Coupled Plasma - Atomic Emission
Spectrometry 65
5.2.29 EPA Method 6020A (SW-846): Inductively Coupled Plasma - Mass Spectrometry 66
5.2.30 EPA Method 7010 (SW-846): Graphite Furnace Atomic Absorption Spectrophotometry... 67
5.2.31 EPA Method 7470A (SW-846): Mercury in Liquid Wastes (Manual Cold-Vapor Technique)
68
5.2.32 EPA Method 747IB (SW-846): Mercury in Solid or Semisolid Wastes (Manual Cold-Vapor
Technique) 68
5.2.33 EPA Method 7473 (SW-846): Mercury in Solids and Solutions by Thermal Decomposition,
Amalgamation, and Atomic Absorption Spectrophotometry 69
5.2.34 EPA Method 7580 (SW-846): White Phosphorus (P4) by Solvent Extraction and Gas
Chromatography 70
5.2.35 EPA Method 8015C (SW-846): Nonhalogenated Organics Using GC/FID 70
5.2.36 EPA Method 8260C (SW-846): Volatile Organic Compounds by Gas Chromatography-
Mass Spectrometry (GC/MS) 71
5.2.37 EPA Method 8270D (SW-846): Semivolatile Organic Compounds by Gas
Chromatography/Mass Spectrometry (GC-MS) 72
5.2.38 EPA Method 8290A, Appendix A (SW-846): Procedure for the Collection, Handling,
Analysis, and Reporting of Wipe Tests Performed within the Laboratory 75
5.2.39 EPA Method 8315A (SW-846): Determination of Carbonyl Compounds by High
Performance Liquid Chromatography (HPLC) 79
5.2.40 EPA Method 8316 (SW-846): Acrylamide, Acrylonitrile and Acrolein by High Performance
Liquid Chromatography (HPLC) 79
SAM Revision 4.0 xviii September 29, 2008
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5.2.41 EPA Method 8318A (SW-846): N-Methylcarbamates by High Performance Liquid
Chromatography (HPLC) 80
5.2.42 EPA Method 832IB (SW-846): Solvent-Extractable Nonvolatile Compounds by High
Performance Liquid Chromatography-Thermospray-Mass Spectrometry (HPLC-TS-MS) or
Ultraviolet (UV) Detection 81
5.2.43 EPA Method 8330B (SW-846): Nitroaromatics and Nitramines by High Performance
Liquid Chromatography (HPLC) 82
5.2.44 EPA ILM05.3 Cyanide: Analytical Methods for Total Cyanide Analysis 83
5.2.45 EPA Region 7 RLAB Method 3135.21: Cyanide, Total and Amenable in Aqueous and Solid
Samples Automated Colorimetric with Manual Digestion 83
5.2.46 IO [Inorganic] Compendium Method IO-3.1: Selection, Preparation, and Extraction of Filter
Material 84
5.2.47 IO [Inorganic] Compendium Method IO-3.4: Determination of Metals in Ambient
Particulate Matter Using Inductively Coupled Plasma (ICP) Spectroscopy 85
5.2.48 IO [Inorganic] Compendium Method IO-3.5: Determination of Metals in Ambient
Particulate Matter Using Inductively Coupled Plasma/Mass Spectrometry (ICP-MS) 86
5.2.49 IO [Inorganic] Compendium Method IO-5: Sampling and Analysis for Vapor and Particle
Phase Mercury in Ambient Air Utilizing Cold Vapor Atomic Fluorescence Spectrometry
(CVAFS) 87
5.2.50 EPA Air Method, Toxic Organics - 10A (TO-10A): Determination of Pesticides and
Polychlorinated Biphenyls in Ambient Air Using Low Volume Polyurethane Foam (PUF)
Sampling Followed by Gas Chromatographic/Multi-Detector Detection (GC/MD) 88
5.2.51 EPA Air Method, Toxic Organics - 15 (TO-15): Determination of Volatile Organic
Compounds (VOCs) in Air Collected in Specially-Prepared Canisters and Analyzed by Gas
Chromatography/Mass Spectrometry (GC/MS) 90
5.2.52 NIOSH Method 1612: Propylene Oxide 92
5.2.53 NIOSH Method 2016: Formaldehyde 92
5.2.54 NIOSH Method 2513: Ethylene Chlorohydrin 93
5.2.55 NIOSH Method 3510: Monomethylhydrazine 93
5.2.56 NIOSH Method 5600: Organophosphorus Pesticides 94
5.2.57 NIOSH Method 5601: Organonitrogen Pesticides 94
5.2.58 NIOSH Method 6001: Arsine 95
5.2.59 NIOSH Method 6002: Phosphine 96
5.2.60 NIOSH Method 6004: Sulfur Dioxide 96
5.2.61 NIOSH Method 6010: Hydrogen Cyanide 97
5.2.62 NIOSH Method 6013: Hydrogen Sulfide 97
5.2.63 NIOSH Method 6015: Ammonia 98
5.2.64 NIOSH Method 6402: Phosphorus Trichloride 98
5.2.65 NIOSH Method 7903: Acids, Inorganic 99
5.2.66 NIOSH Method 7905: Phosphorus 99
5.2.67 NIOSH Method 7906: Fluorides, Aerosol and Gas, by 1C 100
5.2.68 NIOSH Method 9102: Elements on Wipes 100
5.2.69 NIOSH Method S301-1: Fluoroacetate Anion 101
5.2.70 OSHA Method 40: Methylamine 102
5.2.71 OSHA Method 54: Methyl Isocyanate (MIC) 102
5.2.72 OSHA Method 61: Phosgene 103
5.2.73 OSHA Method ID-211: Sodium Azide and Hydrazoic Acid in Workplace Atmospheres . 103
5.2.74 OSHA Method ID216SG: Boron Trifluoride (BF3) 104
5.2.75 OSHA Method PV2004: Acrylamide 104
5.2.76 OSHA Method PV2103: Chloropicrin 105
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5.2.77 ASTM Method D5755-03: Standard Test Method for Microvacuum Sampling and Indirect
Analysis of Dust by Transmission Electron Microscopy for Asbestos Structure Number
Surface Loading 106
5.2.78 ASTM Method D6480-99: Standard Test Method for Wipe Sampling of Surfaces, Indirect
Preparation, and Analysis for Asbestos Structure Number Concentration by Transmission
Electron Microscopy 106
5.2.79 ISO Method 10312:1995: Ambient Air - Determination of Asbestos Fibres - Direct-transfer
Transmission Electron Microscopy Method 107
5.2.80 Standard Method 4500-NH3 B: Nitrogen (Ammonia) Preliminary Distillation Step 107
5.2.81 Standard Method 4500-NH3 G: Nitrogen (Ammonia) Automated Phenate Method 108
5.2.82 Standard Method 4500-C1 G: DPD Colorimetric Method 108
5.2.83 Literature Reference for Chlorine (Analyst, 1999. 124: 1853-1857) 109
5.2.84 Literature Reference for Fluoroacetate salts (Analytical Letters, 1994. 27 (14): 2703-2718)
109
5.2.85 Literature Reference for Perfluoroisobutylene (Journal of Chromatography A, 2005. 1098:
156-165) 110
5.2.86 Literature Reference for Sodium Azide (Journal of Forensic Sciences, 1998. 43(1): 200-
202) Ill
Section 6.0: Selected Radiochemical Methods 113
6.1 General Guidance 114
6.1.1 Standard Operating Procedure s for Identifying Radiochemical Methods 114
6.1.2 General QC Guidance for Radiochemical Methods 116
6.1.3 Safety and Waste Management 117
6.2 Method Summaries 118
6.2.1 EPA Method 111: Determination of Polonium-210 Emissions from Stationary Sources ..118
6.2.2 EPA Method 900.0: Gross Alpha and Gross Beta Radioactivity in Drinking Water 119
6.2.3 EPA Method 901.1: Gamma Emitting Radionuclides in Drinking Water 120
6.2.4 EPA Method 903.0: Alpha-Emitting Radium Isotopes in Drinking Water 121
6.2.5 EPA Method 903.1: Radium-226 in Drinking Water - Radon Emanation Technique 121
6.2.6 EPA Method 908.0: Uranium in Drinking Water- Radiochemical Method 122
6.2.7 EPA Method EMSL-19: Determination of Radium-226 and Radium-228 in Water, Soil, Air
and Biological Tissue 122
6.2.8 EPA Method EMSL-33: Isotopic Determination of Plutonium, Uranium, and Thorium in
Water, Soil, Air, and Biological Tissue 123
6.2.9 EML HASL-300 Method Am-01-RC: Americium in Soil 124
6.2.10 EML HASL-300 Method Am-02-RC: Americium-241 in Soil-Gamma Spectrometry 124
6.2.11 EML HASL-300 Method Am-04-RC: Americium in QAP Water and Air Filters - Eichrom's
TRU Resin ". 125
6.2.12 EML HASL-300 Method Ga-01-R: Gamma Radioassay 126
6.2.13 EML HASL-300 Method Po-02-RC: Polonium in Water, Vegetation, Soil, and Air Filters
126
6.2.14 EML HASL-300 Method Pu-12-RC: Plutonium and/or Americium in Soil or Sediments. 127
6.2.15 EML HASL-300 Method Sr-03-RC: Strontium-90 in Environmental Samples 128
6.2.16 FRMAC Method Volume 2, Page 33: Gross Alpha and Beta in Air 128
6.2.17 ORISE Method AP-1: Gross Alpha and Beta for Various Matrices 129
6.2.18 ORISE Method AP-11: Sequential Determination of the Actinides in Environmental
Samples Using Total Sample Dissolution and Extraction Chromatography 130
6.2.19 ASTM Method D3084: Standard Practice for Alpha Spectrometry in Water 131
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6.2.20 ASTM Method D3972: Standard Test Method for Isotopic Uranium in Water by
Radiochemistry 131
6.2.21 Standard Method 7110 B: Gross Alpha and Gross Beta Radioactivity (Total, Suspended,
and Dissolved) 132
6.2.22 Standard Method 7120: Gamma-Emitting Radionuclides 133
6.2.23 Standard Method 7500-Ra B: Radium: Precipitation Method 134
6.2.24 Standard Method 7500-Ra C: Radium: Emanation Method 134
6.2.25 Standard Method 7500-Sr B: Total Radioactive Strontium and Strontium-90: Precipitation
Method 135
6.2.26 Standard Method 7500-U B: Uranium: Radiochemical Method 135
6.2.27 Standard Method 7500-U C: Uranium: Isotopic Method 136
Section 7.0: Selected Pathogen Methods 137
7.1 General Guidance 138
7.1.1 Standard Operating Procedures for Identifying Pathogen Methods 139
7.1.2 General QC Guidance for Pathogen Methods 142
7.1.3 Safety and Waste Management 143
7.2 Method Summaries 144
7.2.1 Laboratory Response Network (LRN) 144
7.2.2 USEPA Manual of Methods for Virology, EPA/600/4-84/013, April 2001 145
7.2.3 USEPA Environmental Regulations and Technology, Control of Pathogens and Vector
Attraction in Sewage Sludge EPA/625/R-92/013, July 2003: Baylisascarisprocyonis 146
7.2.4 EPA Method 1622: Cryptosporidium in Water by Filtration/IMS/FA 147
7.2.5 EPA Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA 148
7.2.6 EPA Method 1682: Salmonella spp 148
7.2.7 CDC, ASM, APHL: Basic Diagnostic Testing Protocols for Level A Laboratories for the
Presumptive Identification of Bacillus anthracis 149
7.2.8 CDC, ASM, APHL: Basic Protocols for Level A Laboratories for the Presumptive
Identification of Francisella tularensis 150
7.2.9 CDC Laboratory Assay: "Triplex PCR for Detection of & Typhi Using SmartCycler®".. 151
7.2.10 CDC Laboratory Assay: "Detection of Diarrheagenic Eshcerichia coli and Shigella Using
LightCycler®" 151
7.2.11 CDC Laboratory Assay: "TaqMan Assays for Detection of V. cholerae ctxA, Ol rfb, and
O139rfb." 152
7.2.12 USDA Laboratory Guidebook: "FSIS Procedure for the Use ofaListeria monocytogenes
Polymerase Chain Reaction (PCR) Screening Test." MLG 8A.03. 2007 153
7.2.13 U.S. FDA Bacteriological Analytical Manual, Chapter 10, 2003: Listeria monocytogenes\54
7.2.14 Standard Method 9213 B: Staphylococcus aureus 154
7.2.15 Standard Method 9260 B: General Qualitative Isolation and Identification Procedures for
Salmonella 155
7.2.16 Standard Method 9260 E: Shigella 156
7.2.17 Standard Method 9260 F: Pathogenic Escherichia coli 157
7.2.18 Standard Method 9260 G: Campylobacter jejuni 157
7.2.19 Standard Method 9260 H: Vibrio cholerae 158
7.2.20 Standard Method 9260 I: Leptospira 159
7.2.21 ASM Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism: Brucella
species 160
7.2.22 ASM Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism: Burkholderia
mallei and Burkholderia pseudomallei 160
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7.2.23 ASM Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism: Yersiniapestis
161
7.2.24 Literature Reference for Campylobacter jejuni (Molecular and Cellular Probes. 2006. 20:
269-279) 162
7.2.25 Literature Reference for Chlamydophilapsittaci (Journal of Clinical Microbiology. 2000.
38(3): 1085-1093) 163
7.2.26 Literature Reference for Escherichia coll O157:H7 (Applied and Environmental
Microbiology. 2003. 69(10): 6327-6333) 164
7.2.27 Literature Reference for Non-Typhoidal Salmonella (Journal of Applied Microbiology.
2007. 102(2): 516-530) 165
7.2.28 Literature Reference for Adenoviruses (Applied and Environmental Microbiology. 2005.
71(6): 3131-3136) 165
7.2.29 Literature Reference for Astroviruses (Canadian Journal of Microbiology. 2004. 50: 269-
278) 166
7.2.30 Literature Reference for Noroviruses (Journal of Clinical Microbiology. 2004. 42(10):
4679-4685) 167
7.2.31 Literature Reference for Sapoviruses (Journal of Medical Virology. 2006. 78(10): 1347-
1353) 168
7.2.32 Literature Reference for Coronaviruses (SARS) (Journal of Virological Methods. 2004.
122:29-36) 168
7.2.33 Literature Reference for Hepatitis E Virus (Journal of Virological Methods. 2006. 131(1):
65-71) 169
7.2.34 Literature Reference for Influenza H5N1 (Emerging Infectious Diseases. 2005. 11(8):
1303-1305) 170
7.2.35 Literature Reference for Enteric Viruses (Applied and Environmental Microbiology. 2003.
69(6): 3158-3164) 171
7.2.36 Literature Reference for Cryptosporidium spp. (Applied and Environmental Microbiology.
1999. 65(9): 3936-3941) ' 171
7.2.37 Literature Reference for Cryptosporidium spp. (Applied and Environmental Microbiology.
2007. 73(13): 4218-4225) ' 172
7.2.38 Literature Reference for Entamoeba histolytica (Journal of Parasitology. 1972. 58(2): 306-
310) 173
7.2.39 Literature Reference for Entamoeba histolytica (Journal of Clinical Microbiology. 2005.
43(11): 5491-5497) 174
7.2.40 Literature Reference for Giardia spp. (Transactions of the Royal Society of Tropical
Medicine and Hygiene. 1983. 77(4): 487-488) 174
7.2.41 Literature Reference for Toxoplasma gondii (Emerging Infectious Diseases. 2006. 12(2):
326-329) 175
7.2.42 Literature Reference for Toxoplasma gondii (Applied and Environmental Microbiology.
2004. 70(7): 4035-4039) 176
Section 8.0: Selected Biotoxin Methods 177
8.1 General Guidance 178
8.1.1 Standard Operating Procedures for Identifying Biotoxin Methods 179
8.1.2 General QC Guidance for Biotoxin Methods 181
8.1.3 Safety and Waste Management 182
8.2 Method Summaries 183
8.2.1 Laboratory Response Network (LRN) 183
8.2.2 U.S. FDA, Bacteriological Analytical Manual Online, Chapter 17, 2001: Botulinum
Neurotoxins 185
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8.2.3 U.S. FDA, Bacteriological Analytical Manual Online, Appendix 1, 2001: Rapid Methods
for Detecting Foodborne Pathogens 186
8.2.4 AOAC Official Method 991.31: Aflatoxins in Corn, Raw Peanuts, and Peanut Butter 186
8.2.5 AOAC Official Method 993.06: Staphylococcal Enterotoxins in Selected Foods 187
8.2.6 AOAC Official Method 994.08: Aflatoxin in Corn, Almonds, Brazil Nuts, Peanuts, and
Pistachio Nuts 187
8.2.7 Literature Reference for Abrin (119th AOAC Annual Meeting & Exposition, 2005, p. 613)
188
8.2.8 Literature Reference for Abrin and Shiga and Shiga-like Toxins (Pharmacology Toxicology.
2001. 88(5): 255-260) 189
8.2.9 Literature Reference for Abrin and Ricin (Analytical Biochemistry. 2008. 378(1): 87-89)
190
8.2.10 Literature Reference for a-Conotoxin (Biochemical Journal. 1997. 328: 245-250) 191
8.2.11 Literature Reference for a-Conotoxin (Journal of Medicinal Chemistry. 2004. 47(5): 1234-
1241) 191
8.2.12 Literature Reference for a-Amanitin, T-2 Mycotoxin (Journal of Food Protection. 2005.
68(6): 1294-1301) 192
8.2.13 Literature Reference for a-Amanitin (Journal of Chromatography B. 1991. 563(2): 299-
311) 193
8.2.14 Literature Reference for Anatoxin-a (Biomedical Chromatography. 1996. 10: 46-47) 193
8.2.15 Literature Reference for Brevetoxins (Environmental Health Perspectives. 2002. 110(2):
179-185) 194
8.2.16 Literature Reference for Brevetoxins (Toxicon. 2004. 43(4): 455-465) 194
8.2.17 Literature Reference for Cylindrospermopsin (FEMS Microbiology Letters. 2002. 216(2):
159-164) 195
8.2.18 Literature Reference for Diacetoxyscirpenol (DAS) (International Journal of Food
Microbiology. 1988. 6(1): 9-17) 195
8.2.19 Literature Reference for Diacetoxyscirpenol (DAS) and T-2 Mycotoxin (Rapid
Communications in Mass Spectrometry. 2006. 20(9): 1422-1428) 196
8.2.20 Literature Reference for Microcystins (Journal of AOAC International. 2001. 84(4): 1035-
1044) 197
8.2.21 Literature Reference for Microcystins (Analyst. 1994. 119(7): 1525-1530) 197
8.2.22 Literature Reference for Picrotoxin (Journal of Pharmaceutical & Biomedical Analysis.
1989. 7(3): 369-375) 198
8.2.23 Literature Reference for Ricin (Journal of AOAC International. 2008. 91(2): 376-382)... 199
8.2.24 Literature Reference for Ricin by Ricinine detection (Journal of Analytical Toxicology.
2005.29(3): 149-155) 199
8.2.25 Literature Reference for Saxitoxin (Journal of AOAC International. 1995. 78(2): 528-532)
200
8.2.26 Literature Reference for Shiga and Shiga-like Toxin (Journal of Clinical Microbiology.
2007. 45(10): 3377-3380) 201
8.2.27 Literature Reference for Tetrodotoxin (Journal of Clinical Laboratory Analysis. 1992. 6(2):
65-72) 201
8.2.28 Literature Reference for Tetrodotoxin (Analytical Biochemistry. 2001. 290(1): 10-17)... 202
8.2.29 ELISA Kits for Cylindrospermopsin 202
8.2.30 ELISA Kits for Saxitoxins 203
8.2.31 Lateral Flow Immunoassay Kits 204
Section 9.0: Conclusions 205
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Table of Contents
Appendices
Appendix A: Selected Chemical Methods A-l
Appendix B: Selected Radiochemical Methods B-l
Appendix C: Selected Pathogen Methods C-l
Appendix D: Selected Biotoxin Methods D-l
Figures
Figure 1-1. Environmental Evaluation Analytical Process Roadmap for Homeland Security Events 2
Figure 2-1. SAM Method Selection Process 5
Tables
Table 5-1. Chemical Methods and Corresponding Text Section Numbers 14
Table 5-2. Sources of Chemical Methods 31
Table 6-1. Radiochemical Methods and Corresponding Text Section Numbers 114
Table 6-2. Sources of Radiochemical Methods 116
Table 7-1. Pathogen Methods and Corresponding Text Section Numbers 139
Table 7-2. Sources of Pathogen Methods 141
Table 8-1. Biotoxin Methods and Corresponding Text Section Numbers 179
Table 8-2. Sources of Biotoxin Methods 180
SAM Revision 4.0 xxiv September 29, 2008
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Section 1 - Introduction
Section 1.0: Introduction
After the terrorist attacks of September 11, 2001 and the anthrax attacks in the fall of 2001, federal and
state personnel provided response, recovery, and remediation under trying circumstances, including
unprecedented demand on their capabilities to analyze environmental samples. In reviewing these events,
the Environmental Protection Agency (EPA) identified several areas where the country could better
prepare itself in the event of future terrorist incidents. The need to improve the nation's laboratory
capacity and capability to analyze environmental samples following a homeland security event was one of
the most important areas identified.
In response, EPA formed the Homeland Security Laboratory Capacity Workgroup to identify and
implement opportunities for near-term improvements and to develop recommendations for addressing
longer-term laboratory issues. The EPA Homeland Security Laboratory Capacity Workgroup consists of
representatives from the Office of Research and Development (ORD), Office of Air and Radiation
(OAR), Office of Water (OW), Office of Solid Waste and Emergency Response (OSWER), Office of
Environmental Information, Office of Pollution Prevention and Toxics, and several EPA regional offices.
A critical area identified by the workgroup was the need for a list of analytical methods to be used by all
laboratories when analyzing homeland security event samples and, in particular, when analysis of many
samples is required over a short period of time. Having standardized methods would reduce confusion,
permit sharing of sample load between laboratories, improve data comparability, and simplify the task of
outsourcing analytical support to the commercial laboratory sector. Standardized methods would also
improve the follow-up activities of validating results, evaluating data, and making decisions. To this end,
workgroup members formed an Analytical Methods Subteam to address homeland security methods
issues.
The Analytical Methods Subteam recognized that widely different analytical methods are required for
various phases of environmental sample analyses in support of homeland security preparation and
response: (1) ongoing surveillance and monitoring; (2) response and rapid screening for determining
whether an event has occurred; (3) preliminary site characterizations to determine the extent and type of
contamination; and (4) confirmatory laboratory analyses to plan, implement, and evaluate the
effectiveness of site remediation. Figure 1-1 represents these analytical phases. EPA's Standardized
Analytical Methods for Environmental Restoration Following Homeland Security Events (SAM) provides
information for analytical methods to be applied during the "Site Remediation" phase.
SAM Revision 4.0 1 September 29, 2008
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Section 1 - Introduction
Figure 1-1. Environmental Evaluation Analytical Process Roadmap for Homeland
Security Events
SAM
Surveillance and Monitoring
Immediate Response/
Credibility Determination
Preliminary Site
Characterization
Site Remediation
(Standardized Analytical Methods for Environmental
Restoration Following Homeland Security Events)
SAM Revision 4.0
September 29, 2008
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Section 2 - Background
Section 2.0: Background
In support of this document, EPA periodically assembles methods experts from within EPA and other
federal agencies to review methods and, if necessary, revise the methods listed. SAM identifies a single
method or method group per analyte/sample type to ensure a consistent analytical approach across
multiple laboratories when analyzing environmental samples following an event. Method selection is
based on consideration of specific criteria that emphasize method performance and include existing
laboratory capabilities, laboratory capacity, method applicability to multiple environmental sample types,
and method applicability to multiple SAM analytes. For some analytes, the preferred method is a clear
choice; for others, competing criteria make the choice more difficult. Final method selections are based
on technical recommendations from the SAM work groups. For analytes where limited laboratory
testing/experience exists, such as chemical warfare agents, methods were selected based on their
applicability to similar chemicals (e.g., nerve agents and some pesticides). In these cases, laboratory
studies to test the ability of the selected method to measure the target analyte(s) are either underway or
planned. Figure 2-1 summarizes steps and provides the criteria used during the SAM method selection
process. It is important to note that the method selection criteria included in this figure are listed in non-
hierarchical order and, in some cases, only a subset of the criteria was considered.
Since 2004, EPA's National Homeland Security Research Center (NHSRC) has brought together experts
from across EPA and its sister agencies to develop this compendium of analytical methods to be used
when analyzing environmental samples, and to address site characterization, remediation and clearance
following future homeland security events. Participants have included representatives from EPA program
offices, EPA regions, EPA laboratories, Centers for Disease Control and Prevention (CDC), Food and
Drug Administration (FDA), Department of Homeland Security (DHS), Federal Bureau of Investigation
(FBI), Department of Defense (DoD), Department of Agriculture (USDA), and U.S. Geological Survey
(USGS). Methodologies were considered for chemical and biological agents of concern in the types of
environmental samples that would be anticipated. The primary objective of this effort was to identify
appropriate SAM Analytical Methods Subteam consensus methods that represent a balance between
providing existing, documented, determinative techniques and providing consistent and valid analytical
results.
A survey of available confirmatory analytical methods for approximately 120 biological and chemical
analytes was conducted using existing resources including the following:
• National Environmental Methods Index (NEMI) and NEMI-Chemical, Biological, and Radiological
(NEMI-CBR)
• Environmental Monitoring Method Index (EMMI)
• EPA Test Methods Index
EPA Office of Solid Waste SW-846 Methods
• EPA Microbiology Methods
• National Institute for Occupational Safety and Health (NIOSH) Manual of Analytical Methods
(NMAM)
• Occupational Safety and Health Administration (OSHA) Index of Sampling and Analytical
Methods
• AOAC International
• ASTM International
• International Organization for Standardization (ISO) methods
• Standard Methods for the Examination of Water and Wastewater
• PubMED Literature Database
In September 2004, EPA published Standardized Analytical Methods for Use During Homeland Security
Events, Revision 1.0 (SAM, Revision 1.0, EPA/600/R-04/126), which provided a list of analytical and
SAM Revision 4.0 3 September 29, 2008
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Section 2 - Background
sample preparation methods that were selected for measurement of 82 chemical analytes in
aqueous/liquid, solid, oily solid, and air samples, and 27 biological analytes in water, dust, and aerosol
samples. During 2005, SAM was expanded to include radioisotopes, several persistent chemical warfare
agent degradation products, a drinking water sample type, methods for determination of the viability of
biological organisms, and a separate section for biotoxin analytes. Where necessary, the methods
included in SAM Revision 1.0 were updated to reflect more recent or appropriate methodologies. Similar
efforts to those used for method selection during development of SAM Revision 1.0 were undertaken to
select and include methods for measurement of radioisotopes and chemical warfare agent degradation
products in all sample types, for measurement of CBR analytes in drinking water, and to determine the
viability of biological organisms. These additional analytes and the corresponding methods selected were
included in SAM Revision 2.0.
During 2006, SAM was revised further to incorporate analytes included on updated federal agency lists,
provide additional or more current method listings for target analytes, incorporate explosives into the
chemical analytes listing, combine identification and viability methods information for pathogens, and
address comments from EPA Science Advisory Board's Homeland Security Advisory Committee1 to
clarify the intended use of the document. These changes were included in SAM Revision 3.0
(Standardized Analytical Methods for Environmental Restoration Following Homeland Security Events,
February 2007 / EPA/600/R-07/015). SAM Revision 3.0 included a new title to emphasize the intended
use of SAM methods for analysis during environmental restoration activities. Following publication of
SAM Revision 3.0, SAM workgroups updated the document to include the addition of several chemical
analytes, one radionuclide, and one biotoxin, along with corresponding selected methods, and provided
the updated documents as SAM Revision 3.1 (November 2007 / EPA/600/R-07/136). In 2007, NHSRC
also developed a Web-based version of the SAM document to allow users and other stakeholders to
search for specific needs and to submit questions and comments regarding the information.
NHSRC plans to continue convening SAM Technical Workgroups at least once per year, to evaluate and,
if necessary, update the analytes and methods that are listed. This version, SAM Revision 4.0, reflects the
addition of several chemical and radiochemical analytes as well as the addition of a wipe sample type for
chemical analytes and several polymerase chain reaction (PCR) methods for pathogens.
1 EPA Science Advisory Board's Homeland Security Advisory Committee:
hUp://voscmitc.cpa.gov/sab/sabpcoplc.nsf/WcbCommiUccs/BOARD
SAM Revision 4.0 4 September 29, 2008
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Section 2 - Background
Figure 2-1. SAM Method Selection Process
Step 1
Is there an EPA
published method for
measurement of the
analyte in the sample
type of interest?
Is there a method that has been
developed and published by another
federal agency or Voluntary Consensus
Standard Body (VCSB) for measurement
of the analyte in the sample type of
interest?
Is there an EPA,
federal, or VCSB method
that has been developed for
measurement of the analyte
in another environmental
sample type?
Are there procedures
described and supported by
data in a peer-reviewed
journal article for
measurement of the analyte
in the sample type of
interest?
Evaluate method against
selection criteria
Repeat Steps 1 - 4 to identify methods that measure
analytes similarto the analyte of concern
Use the following criteria as guidelines to assess which method is most
appropriate for inclusion in SAM:
• Has the method been tested/approved by issuing program office?
• Has the method been evaluated based on reliability, performance criteria
(e.g., sensitivity, specificity, false positives/false negatives, precision,
recovery)?
• Is the method appropriate for measurement of this analyte in the sample
type of interest to assess extent of contamination and decontamination
effectiveness?
• Has the method been tested for the specific intended use?
• Is the existing lab capacity (i.e., equipment, number of labs, cost) suitable
for implementation of the method?
• Is the required equipment readily available?
• Is the method capable of determining viability of an organism?
• What is the time required for analysis?
• Are reagents, standards, controls, etc., available and accessible?
• Is specific and/or unique training required?
• Are large sample volumes required?
• Are analytical costs high?
• Has the method already been selected for other SAM analytes?
• Are modifications needed to accommodate the analytes or sample types?
( Select method for inclusion in SAM J
Cf no methods are available, prioritize \
for further research J
SAM Revision 4.0
September 29, 2008
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Section 2 - Background
SAM Revision 4.0 6 September 29, 2008
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Section 3 - Scope and Application
Section 3.0: Scope and Application
The premise and purpose of this document is to standardize the analytical methods that will be used in
cases when multiple laboratories are called on to analyze environmental samples following a homeland
security event. The document also is intended as a tool that will be available to assist state and local
laboratories in planning for and analyzing environmental samples following a homeland security event.
The methods presented in this document should be used to:
• Determine the extent of site contamination (assumes early responders have identified contaminants
prior to EPA's remediation effort), and
Confirm effectiveness of decontamination in support of site clearance decisions.
The methods provided are limited to ones that would be used to determine, to the extent possible within
analytical limitations, the presence of chemical, radiochemical, pathogen, and biotoxin analytes of
concern and their concentrations in environmental media. The methods include detailed laboratory
procedures for confirming the identification of analytes and determining their concentrations in
environmental samples. The methods, therefore, are not designed to be used for rapid or immediate
response or for conducting an initial evaluation (triage or screening) of suspected material to determine if
it poses an immediate danger or should be analyzed in specially designed, highly secure facilities. This
document also is not intended to provide information regarding sample collection activities or equipment.
Methods for addressing these needs are and will be the subject of other efforts.
Methods are provided in this document as corresponding to specific analyte/sample type combinations
that are listed in Appendices A (chemical), B (radiochemical), C (pathogen), and D (biotoxin).
Summaries of each method are provided in numerical order by the developing agency, throughout
Sections 5.2 (chemical methods), 6.2 (radiochemical methods), 7.2 (pathogen methods), and 8.2 (biotoxin
methods).
It is important to note that, in some cases, the methods included in this document have not been fully
validated for the analyte/sample type combination(s) for which they have been selected. The
information contained in this document represents the latest step in an ongoing effort by EPA's
NHSRC to provide standardized analytical methods for use by those laboratories tasked with
performing confirmatory analyses on environmental samples in support of EPA restoration efforts
following a homeland security incident. Although at this time, some of the methods listed have not
been fully validated for a particular analyte (e.g., analytes not explicitly identified in the method) or
sample type, the methods are considered to contain the most appropriate currently available
techniques. Unless a published method listed in this document states specific applicability to the
analyte/sample type combination for which it has been selected, it should be assumed that method
testing is needed, and adjustments may be required to accurately account for variations in analyte
characteristics, environmental samples, and target risk levels. Many of the target analytes listed in
this document have only recently become an environmental concern. EPA is actively pursuing
development and validation of Standardized Analytical Protocols (SAPs) based on the methods
listed, including optimization of procedures for measuring target analytes or agents. In those cases
where method procedures are determined to be insufficient for a particular situation, EPA will
provide guidance regarding appropriate actions. This will be an ongoing process as EPA will strive
to establish a consistent level of validation for all listed analytes.
SAM Revision 4.0 7 September 29, 2008
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Section 3 - Scope and Application
EPA recognizes that specification of a single method may limit laboratory capacity and techniques that
may be needed to evaluate difficult samples. In those cases where method procedures are determined to
be insufficient for a particular situation, EPA will provide guidance regarding appropriate actions (see list
of contacts in Section 4). Where further development and testing are necessary, EPA is developing and
validating SAPs based on the methods that are listed in this document. Once validation is complete, data
regarding the resulting method performance and data quality objectives will be available. The SAM
document and corresponding SAPs will be reviewed frequently. EPA plans to continue to update the
SAM document to address the needs of homeland security, to reflect improvements in analytical
methodology and new technologies, and to incorporate changes in analytes based on needs. EPA also
anticipates that addenda may be generated to provide guidance regarding issues that currently are not
addressed by this document. Any deviations from the methods referenced in this document should be
coordinated with the appropriate point(s) of contact identified in Section 4.
Participants in the chemical, radiochemical, pathogen, and biotoxin work groups, including
representatives from the U.S. EPA, CDC, FDA, DHS, FBI, DoD, USD A, and USGS evaluated the
suitability of existing methodologies and selected this set of methods for use by those laboratories that
support EPA environmental restoration efforts in an emergency. EPA recognizes that this advanced
selection of such methods may pose potential risks, including the following:
• Selecting technologies that may not be the most cost-effective technologies currently available for
addressing the particular situation at hand;
• Selecting methodologies that may not be appropriate for use in responding to a particular
emergency because EPA did not anticipate having to analyze for a particular analyte or
analyte/sample type combination; and
Preventing development and adoption of new and better measurement technologies.
To address these potential risks as soon as possible, EPA plans to take several steps. These include the
following:
Developing and specifying measurement quality objectives for all analyte/sample type
combinations listed in this document. This includes required minimum standards of accuracy (bias
and precision) and sensitivity for the analysis of samples that support the data quality needs of the
particular stage of the emergency response/recovery process);
Specifying guidance for ensuring the analytical methods listed provide results that are consistent
with and support their intended use as indicated in SAM;
• Working with other government agencies and the private sector to establish a laboratory network to
ensure that laboratories, selected to assist EPA and its federal, state, and local partners in
responding to homeland security events, have the requisite expertise and systems to perform this
type of testing; and
Continuing to work with multiple agencies and stakeholders to update SAM and supporting
documents periodically.
EPA recognizes that having data of known and documented quality is critical. Public officials can
accurately assess the activities that may be needed in remediating a site during and following emergency
situations. Data must be of sufficient quality to support decision making. Quality control (QC), however,
takes time. Time is often critical in emergency-related activities where there will be tremendous pressure
to conduct sampling and analytical operations quickly and efficiently. While reduced levels of QC might
be tolerated during the rapid screening stage of emergency response, implementation of analytical
methods for risk assessment and site release will require a higher and more appropriate level of QC.
Many of the methods listed in this document include QC requirements for collecting and analyzing
samples. These QC requirements may or may not be appropriate for addressing emergency response
SAM Revision 4.0 8 September 29, 2008
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Section 3 - Scope and Application
situations, and may be adjusted as necessary to maximize data and decision quality. Specific QC
recommendations for analysis of samples for chemical, radiochemical, pathogen, and biotoxin analytes
are provided in each corresponding section of this document (i.e., Sections 5.1.2, 6.1.2, 7.1.2, and 8.1.2,
respectively).
SAM Revision 4.0 9 September 29, 2008
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Section 3 - Scope and Application
SAM Revision 4.0 10 September 29, 2008
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Section 4 - Points of Contact
Section 4.0: Points of Contact
Questions concerning this document, or the methods identified in this document, should be addressed to
the appropriate point(s) of contact identified below. These contacts should be consulted regarding any
method deviations or modifications, sample problems or interferences, QC requirements, or the use of
potential alternative methods. As previously indicated, any deviations from the recommended method(s)
should be reported immediately to ensure data comparability is maintained when responding to homeland
security events.
General
Oba Vincent - Primary
National Homeland Security Research Center
U.S. EPA ORD (NG16)
26 West Martin Luther King Jr. Drive
Cincinnati, OH 45268
(513)569-7456
Rob Rothman - Alternate
National Homeland Security Research Center
U.S. EPA ORD (NG16)
26 West Martin Luther King Jr. Drive
Cincinnati, OH 45268
(513)569-7187
Chemical Methods
Steve Reimer - Primary
U.S. EPA Region 10 - Manchester Laboratory
7411 Beach Drive East
Port Orchard, WA 98366
(360)871-8718
reimer.steve'.«:cpa.gov
Matthew Magnuson - Alternate
National Homeland Security Research Center
U.S. EPA ORD (NG16)
26 West Martin Luther King Jr. Drive
Cincinnati, OH 45268
(513)569-7321
magnusorunatthew@eDa.gov
Radiochemical Methods
John Griggs - Primary
U.S. EPA Office of Radiation and Indoor Air
Environmental Laboratory
540 South Morris Avenue
Montgomery, AL 36115-2601
(334) 270-3450
griggsjghnjt^cpjLSQY
Kathy Hall - Alternate
National Homeland Security Research Center
U.S. EPA ORD (NG16)
26 West Martin Luther King Jr. Drive
Cincinnati, OH 45268
(513)379-5260
Pathogen Methods
Tonya Nichols - Primary
National Homeland Security Research Center
U.S. EPA ORD (NG16)
26 West Martin Luther King Jr. Drive
Cincinnati, OH 45268
(513)569-7805
Sanjiv Shah -Alternate
National Homeland Security Research Center
U.S.EPAORD-8801RR
1200 Pennsylvania Avenue, NW
Washington, DC 20460
(202) 564-9522
Biotoxins Methods
Matthew Magnuson - Primary
National Homeland Security Research Center
U.S. EPA ORD (NG16)
26 West Martin Luther King Jr. Drive
Cincinnati, OH 45268
(513)569-7321
magnuson.matthew@epa.gov
Sanjiv Shah -Alternate
National Homeland Security Research Center
U.S.EPAORD-8801RR
1200 Pennsylvania Avenue, NW
Washington, DC 20460
(202) 564-9522
shah.saniiv@epa.gov
SAM Revision 4.0
11
September 29, 2008
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Section 4 - Points of Contact
SAM Revision 4.0 12 September 29, 2008
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Section 5 - Selected Chemical Methods
Section 5.0: Selected Chemical Methods
Appendix A provides a list of methods to be used in analyzing environmental samples for chemical
contaminants during remediation activities that result from a homeland security event. Methods are listed
for each analyte and for each sample type that potentially may need to be measured and analyzed when
responding to an environmental emergency. Procedures from peer-reviewed journal articles are listed for
those analyte-sample type combinations where methods are not available. Once standard procedures are
available, the literature references will be replaced.
Please note: This section provides guidance for selecting chemical methods that have a high likelihood
of assuring analytical consistency when laboratories are faced with a large scale environmental
restoration crisis. Not all methods have been verified for the analyte/sample type combination listed in
Appendix A. Please refer to the specified method to identify analyte/sample type combinations that
have been verified. Any questions regarding information discussed in this section should be addressed
to the appropriate contact(s) listed in Section 4.
Appendix A is sorted alphabetically by analyte and includes the following information:
Analyte(s). The component, contaminant, or constituent of interest.
• Chemical Abstracts Service Registration Number (CAS RN). A unique identifier for chemical
substances that provides an unambiguous way to identify a chemical or molecular structure when
there are many possible systematic, generic, or trivial names.
• Determinative technique. An analytical instrument or technique used to determine the quantity and
identification of compounds or components in a sample.
• Method type. Two method types (sample preparation and determinative) are used to complete
sample analysis. In some cases, a single method contains information for both sample preparation
and determinative procedures. In most instances, however, two separate methods may need to be
used in conjunction.
• Solid samples. The recommended method/procedure to identify and measure the analyte of interest
in solid phase samples.
Non-aqueous liquid/organic solid samples. The recommended method/procedure to identify and
measure the analyte of interest in non-aqueous liquid/organic phase samples. An organic solid
sample is a solid that completely dissolves in an organic solvent and leaves no solid residue.
• Aqueous liquid samples. The recommended method/procedure to identify and measure the analyte
of interest in aqueous liquid phase samples.
• Drinking water samples. The recommended method/procedure to identify and measure the analyte
of interest in drinking water samples.
• Air samples. The recommended method/procedure to identify and measure the analyte of interest in
air samples.
Wipe samples. The recommended method/procedure to identify and measure the analyte of interest
in wipes used to collect a sample from a surface.
SAM Revision 4.0 13 September 29, 2008
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Section 5 - Selected Chemical Methods
5.1 General Guidance
This section provides a general overview of how to identify the appropriate chemical method(s) for a
given analyte-sample type combination, as well as recommendations for QC procedures.
For additional information on the properties of the chemicals listed in Appendix A, TOXNET
Qittgy/toxncyilnijiih.goWindcjyibtil), a cluster of databases on toxicology, hazardous chemicals, and
related areas maintained by the National Library of Medicine, is an excellent resource. Additional
resources include:
Syracuse Research Corporation's Physprop and Chemfate, part of the Environmental Fate Database
supported by EPA. http://www.syrrcs.com/csc/databascs.htm
• INCHEM at http://www.inchem.org/ contains both chemical and toxicity information.
• The Registry of Toxic Effects of Chemical Substances (RTECS) database can be accessed via the
NIOSH Web site at http://www.cdc.gov/niosh/rtecs/vz72d288.htmltfJWlDAW for toxicity
information.
• EPA's Integrated Risk Information System (IRIS): http://www.cpa.gov/iris/ contains toxicity
information.
• Forensic Science and Communications published by the Laboratory Division of the FBI.
http://www.fbi.gov/hq/lab/fsc/current/backissu.htm
European Chemicals Bureau Toxicology and Chemical Substances: httg^/ecbjrcjt and
http: 7/ccb. j re .it/to sting-methods/ containing information regarding European Directive 67/548/EEC
and Annex V
Additional research on chemical contaminants is ongoing within EPA. Databases to manage this
information are currently under development.
5.1.1 Standard Operating Procedures for Identifying Chemical Methods
To determine the appropriate method to be used on an environmental sample, locate the analyte of
concern under the "Analyte(s)" column in Appendix A: Chemical Methods under. After locating the
analyte of concern, continue across the table to identify the appropriate determinative technique (e.g.,
high performance liquid chromatography [HPLC], gas chromatography - mass spectrometry [GC-MS]),
then identify the appropriate sample preparation and determinative method(s) for the sample type of
interest (solid, non-aqueous liquid/organic solid, aqueous liquid, drinking water, air, or wipe). In some
cases, two methods (sample preparation and determinative) are needed to complete sample analysis.
Sections 5.2.1 through 5.2.86 below provide summaries of the sample preparation and determinative
methods listed in Appendix A. Once a method has been identified in Appendix A, Table 5-1 can be used
to locate the method summary.
Table 5-1. Chemical Methods and Corresponding Text Section Numbers
Analyte
Acrylamide
Acrylonitrile
CASRN
79-06-1
107-13-1
Method
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
831 6 (EPA SW-846)
PV2004 (OSHA)
Section
5.2.22
5.2.38
5.2.40
5.2.75
SAM Revision 4.0
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September 29, 2008
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Section 5 - Selected Chemical Methods
Analyte
Aldicarb (Temik)
Aldicarb sulfone
Aldicarb sulfoxide
Allyl alcohol
4-Aminopyridine
Ammonia
Ammonium metavanadate
Arsenic, Total
Arsenic trioxide
Arsine
Asbestos
Boron trifluoride
Brodifacoum
Bromadiolone
CASRN
116-06-3
1646-88-4
1646-87-3
107-18-6
504-24-5
7664-41-7
7803-55-6
7440-38-2
1327-53-3
7784-42-1
1332-21-4
7637-07-2
56073-10-0
28772-56-7
Method
531. 2 (EPA OW)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
831 8A (EPA SW-846)
5601 (NIOSH)
3585 (EPA SW-846)
5030C (EPA SW-846)
5035A (EPA SW-846)
8260C (EPA SW-846)
TO-15(EPAORD)
3535A (EPA SW-846)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
8330B (EPA SW-846)
350.1 (EPAOW)
6015 (NIOSH)
4500-NHs B (SM)
4500-NHs G (SM)
200.7 (EPA OW)
200.8 (EPA OW)
3031 (EPA SW-846)
3050B (EPA SW-846)
601 OC (EPA SW-846)
6020A (EPA SW-846)
IO-3.1 (EPAORD)
IO-3.4 (EPA ORD)
IO-3.5 (EPAORD)
9102 (NIOSH)
200.7 (EPA OW)
200.8 (EPA OW)
3050B (EPA SW-846)
7010 (EPA SW-846)
6001 (NIOSH)
9102 (NIOSH)
D5755-03 (ASTM)
D6480-99 (ASTM)
10312:1995 (ISO)
ID216SG(OSHA)
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8290A Appendix A (EPA SW-846)
8321 B (EPA SW-846)
Section
5.2.12
5.2.22
5.2.38
5.2.41
5.2.57
5.2.25
5.2.26
5.2.27
5.2.36
5.2.51
5.2.19
5.2.22
5.2.38
5.2.43
5.2.8
5.2.63
5.2.80
5.2.81
5.2.2
5.2.3
5.2.16
5.2.17
5.2.28
5.2.29
5.2.46
5.2.47
5.2.48
5.2.68
5.2.2
5.2.3
5.2.17
5.2.30
5.2.58
5.2.68
5.2.77
5.2.78
5.2.79
5.2.74
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.38
5.2.42
SAM Revision 4.0
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September 29, 2008
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Section 5 - Selected Chemical Methods
Analyte
BZ (Quinuclidinyl benzilate)
Calcium arsenate
Carbofuran (Furadan)
Carbon disulfide
Chlorfenvinphos
Chlorine
2-Chloroethanol
CASRN
6581-06-2
7778-44-1
1563-66-2
75-15-0
470-90-6
7782-50-5
107-07-3
Method
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8290A Appendix A (EPA SW-846)
8321 B (EPA SW-846)
TO-IOA(EPAORD)
200.7 (EPA OW)
200.8 (EPA OW)
3031 (EPA SW-846)
3050B (EPA SW-846)
601 OC (EPA SW-846)
6020A (EPA SW-846)
IO-3.1 (EPAORD)
IO-3.4 (EPA ORD)
IO-3.5 (EPAORD)
9102(NIOSH)
531. 2 (EPA OW)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
831 8A (EPA SW-846)
5601 (NIOSH)
524.2 (EPA OW)
3585 (EPA SW-846)
5030C (EPA SW-846)
5035A (EPA SW-846)
8260C (EPA SW-846)
TO-15(EPAORD)
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
4500-CI G (SM)
Analyst. 1999. 124: 1853-1857
3585 (EPA SW-846)
5030C (EPA SW-846)
5035A (EPA SW-846)
8260C (EPA SW-846)
2513 (NIOSH)
Section
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.38
5.2.42
5.2.50
5.2.2
5.2.3
5.2.16
5.2.17
5.2.28
5.2.29
5.2.46
5.2.47
5.2.48
5.2.68
5.2.12
5.2.22
5.2.38
5.2.41
5.2.57
5.2.10
5.2.25
5.2.26
5.2.27
5.2.36
5.2.51
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
5.2.82
5.2.83
5.2.25
5.2.26
5.2.27
5.2.36
5.2.54
SAM Revision 4.0
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Section 5 - Selected Chemical Methods
Analyte
3-Chloro-1 ,2-propanediol
Chloropicrin
Chlorosarin
Chlorosoman
2-Chlorovinylarsonous acid (CVAA)
Chlorpyrifos
CASRN
96-24-2
76-06-2
1445-76-7
7040-57-5
85090-33-1
2921-88-2
Method
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
551.1 (EPAOW)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
PV2103(OSHA)
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
200.7 (EPA OW)
200.8 (EPA OW)
3031 (EPA SW-846)
3050B (EPA SW-846)
601 OC (EPA SW-846)
6020A (EPA SW-846)
IO-3.1 (EPAORD)
IO-3.4 (EPA ORD)
IO-3.5 (EPAORD)
9102(NIOSH)
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
Section
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
5.2.14
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.76
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
5.2.2
5.2.3
5.2.16
5.2.17
5.2.28
5.2.29
5.2.46
5.2.47
5.2.48
5.2.68
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
SAM Revision 4.0
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Section 5 - Selected Chemical Methods
Analyte
Crimidine
Cyanide, Amenable to chlorination
Cyanide, Total
Cyanogen chloride
Cyclohexyl sarin (GF)
1,2-Dichloroethane
Dichlorvos
Dicrotophos
CASRN
535-89-7
NA
57-12-5
506-77-4
329-99-7
107-06-2
62-73-7
141-66-2
Method
3535A (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
RLAB Method 3135.21
335.4 (EPA OW)
ILM05.3 CN (EPA CLP)
6010(NIOSH)
3585 (EPA SW-846)
5030C (EPA SW-846)
5035A (EPA SW-846)
8260C (EPA SW-846)
TO-15(EPAORD)
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
524.2 (EPA OW)
3585 (EPA SW-846)
5030C (EPA SW-846)
5035A (EPA SW-846)
8260C (EPA SW-846)
TO-15(EPAORD)
525.2 (EPA OW)
3535A (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
3535A (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
Section
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.45
5.2.7
5.2.44
5.2.61
5.2.25
5.2.26
5.2.27
5.2.36
5.2.51
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
5.2.10
5.2.25
5.2.26
5.2.27
5.2.36
5.2.51
5.2.11
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
SAM Revision 4.0
18
September 29, 2008
-------
Section 5 - Selected Chemical Methods
Analyte
Diesel Range Organics
Diisopropyl methylphosphonate (DIMP)
Dimethylphosphite
Dimethylphosphoramidic acid
Diphacinone
Disulfoton
Disulfoton sulfoxide
CASRN
NA
1445-75-6
868-85-9
33876-51-6
82-66-6
298-04-4
2497-07-6
Method
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
801 5C (EPA SW-846)
8290A Appendix A (EPA SW-846)
3535A (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8290A Appendix A (EPA SW-846)
8321 B (EPA SW-846)
TO-IOA(EPAORD)
3535A (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
3535A (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8290A Appendix A (EPA SW-846)
8321 B (EPA SW-846)
TO-IOA(EPAORD)
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8290A Appendix A (EPA SW-846)
8321 B (EPA SW-846)
525.2 (EPA OW)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
5600 (NIOSH)
Section
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.35
5.2.38
5.2.19
5.2.21
5.2.22
5.2.24
5.2.38
5.2.42
5.2.50
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
5.2.19
5.2.21
5.2.22
5.2.24
5.2.38
5.2.42
5.2.50
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.38
5.2.42
5.2.11
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.56
SAM Revision 4.0
19
September 29, 2008
-------
Section 5 - Selected Chemical Methods
Analyte
1,4-Dithiane
EA2192 [Diisopropylaminoethyl methyl-
thiophosphonate]
Ethyl methylphosphonic acid (EMPA)
Ethyldichloroarsine (ED)
N-Ethyldiethanolamine (EDEA)
Ethylene oxide
Fenamiphos
CASRN
505-29-3
73207-98-4
1832-53-7
598-14-1
139-87-7
75-21-8
22224-92-6
Method
3535A (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
3535A (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8290A Appendix A (EPA SW-846)
8321 B (EPA SW-846)
TO-IOA(EPAORD)
3535A (EPA SW-846)
3545A (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
TO-15(EPAORD)
9102(NIOSH)
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8290A Appendix A (EPA SW-846)
8321 B (EPA SW-846)
TO-IOA(EPAORD)
3585 (EPA SW-846)
5030C (EPA SW-846)
5035A (EPA SW-846)
8260C (EPA SW-846)
TO-15(EPAORD)
525.2 (EPA OW)
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
Section
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.19
5.2.21
5.2.22
5.2.24
5.2.38
5.2.42
5.2.50
5.2.19
5.2.21
5.2.24
5.2.37
5.2.51
5.2.68
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.38
5.2.42
5.2.50
5.2.25
5.2.26
5.2.27
5.2.36
5.2.51
5.2.11
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
SAM Revision 4.0
20
September 29, 2008
-------
Section 5 - Selected Chemical Methods
Analyte
Fentanyl
Fluoride
Fluoroacetamide
Fluoroacetic acid and fluoroacetate salts
Formaldehyde
Gasoline Range Organics
Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)
Hexamethylenetriperoxidediamine (HMTD)
Hydrogen bromide
Hydrogen chloride
Hydrogen cyanide
Hydrogen fluoride
Hydrogen sulfide
CASRN
437-38-7
16984-48-8
640-19-7
NA
50-00-0
NA
121-82-4
283-66-9
10035-10-6
7647-01-0
74-90-8
7664-39-3
7783-06-4
Method
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8290A Appendix A (EPA SW-846)
8321 B (EPA SW-846)
TO-IOA(EPAORD)
300.1, Rev 1.0 (EPA OW)
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
300.1, Rev 1.0 (EPA OW)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
S301-1 (NIOSH)
Analytical Letters, 1994, 27 (14),
2703-2718
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
831 5A (EPA SW-846)
2016 (NIOSH)
3570 (EPA SW-846)
3585 (EPA SW-846)
5030C (EPA SW-846)
5035A (EPA SW-846)
801 5C (EPA SW-846)
8290A Appendix A (EPA SW-846)
3535A (EPA SW-846)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
8330B (EPA SW-846)
7903 (NIOSH)
6010 (NIOSH)
7903 (NIOSH)
6013 (NIOSH)
Section
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.38
5.2.42
5.2.50
5.2.6
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
5.2.6
5.2.22
5.2.38
5.2.69
5.2.84
5.2.22
5.2.38
5.2.39
5.2.53
5.2.22
5.2.25
5.2.26
5.2.27
5.2.35
5.2.38
5.2.19
5.2.22
5.2.38
5.2.43
5.2.65
5.2.61
5.2.65
5.2.62
SAM Revision 4.0
21
September 29, 2008
-------
Section 5 - Selected Chemical Methods
Analyte
Isopropyl methylphosphonic acid (IMPA)
Kerosene
Lead arsenate
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine]
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine]
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine]
Lewisite oxide
Mercury, Total
Methamidophos
Methomyl
Methoxyethylmercuric acetate
CASRN
1832-54-8
64742-81-0
7645-25-2
541-25-3
40334-69-8
40334-70-1
1306-02-1
7439-97-6
10265-92-6
16752-77-5
151-38-2
Method
3535A (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8290A Appendix A (EPA SW-846)
8321 B (EPA SW-846)
TO-IOA(EPAORD)
3570 (EPA SW-846)
3585 (EPA SW-846)
5030C (EPA SW-846)
5035A (EPA SW-846)
801 5C (EPA SW-846)
8290A Appendix A (EPA SW-846)
200.7 (EPA OW)
200.8 (EPA OW)
3031 (EPA SW-846)
3050B (EPA SW-846)
601 OC (EPA SW-846)
6020A (EPA SW-846)
IO-3.1 (EPAORD)
IO-3.4 (EPA ORD)
IO-3.5 (EPAORD)
9102(NIOSH)
245.2 (EPA OW)
7473 (EPA SW-846)
IO-5 (EPA ORD)
9102(NIOSH)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
531. 2 (EPA OW)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
831 8A (EPA SW-846)
5601 (NIOSH)
245.2 (EPA OW)
7473 (EPA SW-846)
IO-5 (EPA ORD)
9102 (NIOSH)
Section
5.2.19
5.2.21
5.2.22
5.2.24
5.2.38
5.2.42
5.2.50
5.2.22
5.2.25
5.2.26
5.2.27
5.2.35
5.2.38
5.2.2
5.2.3
5.2.16
5.2.17
5.2.28
5.2.29
5.2.46
5.2.47
5.2.48
5.2.68
5.2.4
5.2.33
5.2.49
5.2.68
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
5.2.12
5.2.22
5.2.38
5.2.41
5.2.57
5.2.4
5.2.33
5.2.49
5.2.68
SAM Revision 4.0
22
September 29, 2008
-------
Section 5 - Selected Chemical Methods
Analyte
Methyl acrylonitrile
Methyl fluoroacetate
Methyl hydrazine
Methyl isocyanate
Methyl parathion
Methylamine
N-Methyldiethanolamine (MDEA)
1-Methylethyl ester ethylphosphonofluoridic
acid (GE)
CASRN
126-98-7
453-18-9
60-34-4
624-83-9
298-00-0
74-89-5
105-59-9
1189-87-3
Method
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
831 6 (EPA SW-846)
PV2004 (OSHA)
300.1, Rev 1.0 (EPA OW)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
S301-1 (NIOSH)
Analytical Letters, 1994, 27 (14):
2703-2718
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
3510 (NIOSH)
OSHA 54
3535A (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
OSHA 40
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8290A Appendix A (EPA SW-846)
8321 B (EPA SW-846)
TO-IOA(EPAORD)
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
Section
5.2.22
5.2.38
5.2.40
5.2.75
5.2.6
5.2.22
5.2.38
5.2.69
5.2.84
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.55
5.2.71
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
5.2.70
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.38
5.2.42
5.2.50
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
SAM Revision 4.0
23
September 29, 2008
-------
Section 5 - Selected Chemical Methods
Analyte
Methylphosphonic acid (MPA)
Mevinphos
Mustard, nitrogen (HN-1) [bis(2-chloroethyl)-
ethylamine]
Mustard, nitrogen (HN-2) [2,2'-dichloro-N-
methyldiethylamine N,N-bis(2-chloroethyl)-
methylamine]
Mustard, nitrogen (HN-3) [tris(2-chloroethyl)-
amine]
Mustard, sulfur/ Mustard gas (HD)
Nicotine compounds
Octahydro-1, 3,5, 7-tetranitro-1, 3,5,7-
tetrazocine (HMX)
CASRN
993-13-5
7786-34-7
538-07-8
51-75-2
555-77-1
505-60-2
54-11-5
2691-41-0
Method
3535A (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8290A Appendix A (EPA SW-846)
8321 B (EPA SW-846)
TO-IOA(EPAORD)
525.2 (EPA OW)
3535A (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
3570 (EPA SW-846)
3571 (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
3535A (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
3535A (EPA SW-846)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
8330B (EPA SW-846)
Section
5.2.19
5.2.21
5.2.22
5.2.24
5.2.38
5.2.42
5.2.50
5.2.11
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
5.2.22
5.2.23
5.2.37
5.2.38
5.2.50
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.19
5.2.22
5.2.38
5.2.43
SAM Revision 4.0
24
September 29, 2008
-------
Section 5 - Selected Chemical Methods
Analyte
Organophosphate pesticides, NOS
Osmium tetroxide
Oxamyl
Paraquat
Parathion
Pentaerythritol tetranitrate (PETN)
Perfluoroisobutylene (PFIB)
Phencyclidine
CASRN
NA
20816-12-0
23135-22-0
4685-14-7
56-38-2
78-11-5
382-21-8
77-10-1
Method
507 (EPA OW)
61 4 (EPA OW)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
5600 (NIOSH)
252.2 (EPA OW)
3050B (EPA SW-846)
601 OC (EPA SW-846)
IO-3.1 (EPAORD)
IO-3.4 (EPA ORD)
9102 (NIOSH)
531. 2 (EPA OW)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
831 8A (EPA SW-846)
5601 (NIOSH)
549.2 (EPA OW)
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
3535A (EPA SW-846)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
8330B (EPA SW-846)
OSHA61
3535A (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
Section
5.2.9
5.2.15
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.56
5.2.5
5.2.17
5.2.28
5.2.46
5.2.47
5.2.68
5.2.12
5.2.22
5.2.38
5.2.41
5.2.57
5.2.13
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
5.2.19
5.2.22
5.2.38
5.2.43
5.2.72
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
SAM Revision 4.0
25
September 29, 2008
-------
Section 5 - Selected Chemical Methods
Analyte
Phenol
Phorate
Phosgene
Phosphamidon
Phosphine
Phosphorus trichloride
Pinacolyl methyl phosphonic acid (PMPA)
Propylene oxide
CASRN
108-95-2
298-02-2
75-44-5
13171-21-6
7803-51-2
7719-12-2
616-52-4
75-56-9
Method
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
3535A (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
OSHA61
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
6002 (NIOSH)
6402 (NIOSH)
3535A (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8290A Appendix A (EPA SW-846)
8321 B (EPA SW-846)
TO-IOA(EPAORD)
3585 (EPA SW-846)
5030C (EPA SW-846)
5035A (EPA SW-846)
8260C (EPA SW-846)
1612 (NIOSH)
Section
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
5.2.72
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
5.2.59
5.2.64
5.2.19
5.2.21
5.2.22
5.2.24
5.2.38
5.2.42
5.2.50
5.2.25
5.2.26
5.2.27
5.2.36
5.2.52
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Section 5 - Selected Chemical Methods
Analyte
R 33 (VR) [methylphosphonothioic acid, S-[2-
(diethylamino)ethyl] O-2-methylpropyl ester]
Sarin
Semivolatile Organic Compounds, NOS
Sodium arsenite
Sodium azide
Soman (GD)
CASRN
159939-87-4
107-44-8
NA
7784-46-5
26628-22-8
96-64-0
Method
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
3570 (EPA SW-846)
3571 (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
525.2 (EPA OW)
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
200.7 (EPA OW)
200.8 (EPA OW)
3031 (EPA SW-846)
3050B (EPA SW-846)
601 OC (EPA SW-846)
6020A (EPA SW-846)
IO-3.1 (EPAORD)
IO-3.4 (EPA ORD)
IO-3.5 (EPAORD)
9102(NIOSH)
300.1, Rev 1.0 (EPA OW)
3580A (EPA SW-846)
ID-211 (OSHA)
Journal of Forensic Sciences,
1998. 43(1): 200-202
3535A (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
Section
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
5.2.22
5.2.23
5.2.37
5.2.38
5.2.50
5.2.11
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
5.2.2
5.2.3
5.2.16
5.2.17
5.2.28
5.2.29
5.2.46
5.2.47
5.2.48
5.2.68
5.2.6
5.2.24
5.2.73
5.2.86
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
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Section 5 - Selected Chemical Methods
Analyte
Strychnine
Sulfur dioxide
Sulfur trioxide
Tabun (GA)
Tear gas (CS) [chlorobenzylidene malonitrile]
Tetraethyl pyrophosphate
Tetramethylenedisulfotetramine
CASRN
57-24-9
7446-09-5
7446-11-9
77-81-6
2698-41-1
107-49-3
80-12-6
Method
3535A (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
6004 (NIOSH)
Method 8 (EPA)
3535A (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
3535A (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
Section
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.60
5.2.1
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
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Section 5 - Selected Chemical Methods
Analyte
Thallium sulfate
Thiodiglycol (TDG)
Thiofanox
1,4-Thioxane
Titanium tetrachloride
Triethanolamine (TEA)
CASRN
10031-59-1
111-48-8
39196-18-4
15980-15-1
7550-45-0
102-71-6
Method
200.7 (EPA OW)
200.8 (EPA OW)
3031 (EPA SW-846)
3050B (EPA SW-846)
601 OC (EPA SW-846)
6020A (EPA SW-846)
IO-3.1 (EPAORD)
IO-3.4 (EPA ORD)
IO-3.5 (EPAORD)
9102(NIOSH)
3535A (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
531. 2 (EPA OW)
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8290A Appendix A (EPA SW-846)
8321 B (EPA SW-846)
5601 (NIOSH)
3535A (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
3050B (EPA SW-846)
601 OC (EPA SW-846)
6020A (EPA SW-846)
9102 (NIOSH)
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8290A Appendix A (EPA SW-846)
8321 B (EPA SW-846)
TO-IOA(EPAORD)
Section
5.2.2
5.2.3
5.2.16
5.2.17
5.2.28
5.2.29
5.2.46
5.2.47
5.2.48
5.2.68
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
5.2.12
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.38
5.2.42
5.2.57
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.17
5.2.28
5.2.29
5.2.68
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.38
5.2.42
5.2.50
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Section 5 - Selected Chemical Methods
Analyte
Trimethyl phosphite
1,3,5-Trinitrobenzene (1,3,5-TNB)
2,4,6-Trinitrotoluene(2,4,6-TNT)
Vanadium pentoxide
VE [phosphonothioic acid, ethyl-, S-(2-
(diethylamino)ethyl) O-ethyl ester]
VG [phosphonothioic acid, S-(2-
(diethylamino)ethyl) O,O-diethyl ester]
VM [phosphonothioic acid, methyl-,S-(2-
(diethylamino)ethyl) O-ethyl ester]
VX [O-ethyl-S-(2-
diisopropylaminoethyl)methyl-
phosphonothiolate]
White phosphorus
CASRN
121-45-9
99-35-4
118-96-7
1314-62-1
21738-25-0
78-53-5
21770-86-5
50782-69-9
12185-10-3
Method
3535A (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
3535A (EPA SW-846)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
8330B (EPA SW-846)
200.7 (EPA OW)
200.8 (EPA OW)
3031 (EPA SW-846)
3050B (EPA SW-846)
601 OC (EPA SW-846)
6020A (EPA SW-846)
IO-3.1 (EPAORD)
IO-3.4 (EPA ORD)
IO-3.5 (EPAORD)
9102(NIOSH)
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
3580A (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
3570 (EPA SW-846)
3571 (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
3570 (EPA SW-846)
7580 (EPA SW-846)
8290A Appendix A (EPA SW-846)
7905(NIOSH)
Section
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
5.2.19
5.2.22
5.2.38
5.2.43
5.2.2
5.2.3
5.2.16
5.2.17
5.2.28
5.2.29
5.2.46
5.2.47
5.2.48
5.2.68
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.50
5.2.22
5.2.23
5.2.37
5.2.38
5.2.50
5.2.22
5.2.34
5.2.38
5.2.66
The following analytes should be prepared and/or analyzed by the following methods only if problems (e.g.,
insufficient recovery, Interferences) occur when using the sample preparation/determinative techniques Identified for
these analytes in Appendix A.
Allyl alcohol
BZ (Quinuclidinyl benzilate)
3-Chloro-1 ,2-propanediol
Chlorosarin
Chlorosoman
Crimidine
107-18-6
6581-06-2
96-24-2
1445-76-7
7040-57-5
535-89-7
TO-IOA(EPAORD)
8270D (EPA SW-846)
TO-15(EPAORD)
TO-15(EPAORD)
8321 B (EPA SW-846)
5.2.50
5.2.37
5.2.51
5.2.51
5.2.42
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Section 5 - Selected Chemical Methods
Analyte
Diisopropyl methylphosphonate (DIMP)
Dimethylphosphoramidic acid
1,4-Dithiane
EA2192 [Diisopropylaminoethyl methyl-
thiophosphonate]
Ethyl methylphosphonic acid (EMPA)
Hydrogen fluoride
Isopropyl methylphosphonic acid (IMPA)
Mercury, Total
Methamidophos
Methoxymercuric acetate
1-Methylethyl ester ethylphosphonofluoridic
acid (GE)
Methylphosphonic acid (MPA)
Perfluoroisobutylene (PFIB)
Pinacolyl methyl phosphonic acid (PMPA)
Sarin
Soman (GD)
1,4-Thioxane
CASRN
1445-75-6
33876-51-6
505-29-3
73207-98-4
1832-53-7
7664-39-3
1832-54-8
7439-97-6
10265-92-6
151-38-2
1189-87-3
993-13-5
382-21-8
616-52-4
107-44-8
96-64-0
15980-15-1
Method
8270D (EPA SW-846)
TO-15(EPAORD)
8270D (EPA SW-846)
3585 (EPA SW-846)
5030C (EPA SW-846)
5035A (EPA SW-846)
8260C (EPA SW-846)
8270D (EPA SW-846)
7906 (NIOSH)
8270D (EPA SW-846)
7470A (EPA SW-846)
7471 B (EPA SW-846)
5600 (NIOSH)
7470A (EPA SW-846)
7471 B (EPA SW-846)
TO-15(EPAORD)
8270D (EPA SW-846)
Journal of Chromatography A.
2005. 1098: 156-165
8270D (EPA SW-846)
TO-15(EPAORD)
3585 (EPA SW-846)
5030C (EPA SW-846)
5035A (EPA SW-846)
8260C (EPA SW-846)
Section
5.2.37
5.2.51
5.2.37
5.2.25
5.2.26
5.2.27
5.2.36
5.2.37
5.2.67
5.2.37
5.2.31
5.2.32
5.2.56
5.2.31
5.2.32
5.2.51
5.2.37
5.2.85
5.2.37
5.2.51
5.2.25
5.2.26
5.2.27
5.2.36
Method summaries are listed in order of method selection hierarchy (see Figure 2-1), starting with EPA
methods, followed by methods from other federal agencies, voluntary consensus standard bodies
(VCSBs), and literature references. Methods are listed in numerical order under each publisher. Where
available, a direct link to the full text of the method is provided in the method summary. For additional
information on preparation procedures and methods available through consensus standards organizations,
please use the contact information provided in Table 5-2.
Table 5-2. Sources of Chemical Methods
Name
NEMI
U.S. EPA OW Methods
U.S. EPA SW-846 Methods
U.S. EPA ORD Methods
U.S. EPA Air Toxics Methods
Publisher
EPA, USGS
EPAOW
EPA OSWER
EPA ORD
EPA OAR
Reference
http://vwvw.nemi.cjov
http://vwvw.epa.qov/safewater/methods/
sourcalt.html
http://www.epa.qov/epaoswer/hazwaste
/test/rrjairUTtm
http://www.epa.qov/ttnamti1/
http://www.epa.qov/ttn/amtic/airtox.html
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Section 5 - Selected Chemical Methods
Name
OSHA Methods
NIOSH Methods
Standard Methods for the Examination
of Water and Wastewater(SM), 21st
Edition, 2005*
Annual Book of ASTM Standards*
European GESTIS database
ISO Methods*
Official Methods of Analysis of AOAC
International*
Analyst
Analytical Letters*
Journal of Chromatography A*
Journal of Forensic Sciences*
Publisher
OSHA
NIOSH
American Public Health
Association (APHA),
American Water Works
Association (AWWA), and
Water Environment
Federation (WEF)
ASTM International
HVBG
ISO
AOAC International
Royal Society of Chemistry
Taylor & Francis
Elsevier Science Publishers
ASTM International
Reference
IrttjD^/www^oshcLqovY^^
dex.html
http://www.cdc.qov/niosh/nmam/
http://www.standardmethods.orq
http://www.astm.org
http://www.hvbq. de/e/bia/qestis/analytic
al methods/index. html
http://www.iso.orq
http://www.aoac.orq
http://www.rsc.orq/Publishinq/Journals/
AM
MtjD^/WWWJJTf^^
http://www.elsevier.com/
http://www.astm.orq
' Subscription and/or purchase required.
5.1.2 General Quality Control (QC) Guidance for Chemical Methods
Having analytical data of appropriate quality requires that laboratories: (1) conduct the necessary QC
activities to ensure that measurement systems are in control and operating correctly; (2) properly
document results of the analyses; and (3) properly document measurement system evaluation of the
analysis-specific QC, including corrective actions. In addition to the laboratories being capable of
generating accurate and precise data during an emergency situation, they must be able to deliver results in
a timely and efficienct manner. Therefore, laboratories must be prepared with calibrated instruments, the
proper standards, standard analytical procedures, standard operating procedures, and qualified and trained
technicians. Moreover, laboratories also must be capable of providing rapid turnaround of sample
analyses and data reporting.
The level or amount of QC needed during sample analysis and reporting depends on the intended purpose
of the data that are generated (e.g., the decision(s) to be made). The specific needs for data generation
should be identified. QC requirements and data quality objectives should be derived based on those
needs, and should be applied consistently across laboratories when multiple laboratories are used. For
almost all of the chemical warfare agents, most laboratories will not have access to analytical standards
for calibration and QC. Use of these agents is strictly controlled by the DoD and access is limited. For
information regarding purchase and distribution of ultradilute agents for laboratory use, please contact
Terry Smith, EPA's Office of Emergency Management, at (202) 564-2908.
A minimum set of analytical QC procedures should be planned, documented, and conducted for all
chemical testing. Some method-specific QC requirements are described in many of the individual
methods that are cited in this document and will be referenced in any SAPs developed to address specific
analytes and sample types of concern. Individual methods, sampling and analysis protocols, or
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Section 5 - Selected Chemical Methods
contractual statements of work should also be consulted to determine if any additional QC might be
needed. Analytical QC requirements generally consist of analysis of laboratory control samples to
document whether the analytical system is in control; matrix spikes (MS) to identify and quantify
measurement system accuracy for the media of concern and, at the levels of concern, various blanks as a
measure of freedom from contamination; as well as matrix spike duplicates (MSB) or sample replicates to
assess data precision.
In general, for measurement of chemical analytes, appropriate QC includes an initial demonstration of
measurement system capability, as well as ongoing analysis of standards and other samples to ensure the
continued reliability of the analytical results. Examples of appropriate QC include:
• Demonstration that the measurement system is operating properly:
>• Initial calibration; and
>• Method blanks.
• Demonstration of analytical method suitability for intended use:
>• Detection and quantitation limits;
>• Precision and recovery (verify measurement system has adequate accuracy); and
>• Analyte/matrix/level of concern-specific QC samples (verify that measurement system has
adequate sensitivity at levels of concern).
• Demonstration of continued analytical method reliability:
>• MS/MSDs (recovery and precision);
>• QC samples (system accuracy and sensitivity at levels of concern);
>• Surrogate spikes (where appropriate);
>• Continuing calibration verification; and
>• Method blanks.
QC tests should be consistent with Good Laboratory Practices and be run as frequently as necessary to
ensure the reliability of analytical results. As with the identification of needed QC samples, the frequency
of QC sampling should be established based on an evaluation of data quality objectives. The type and
frequency of QC tests can be refined over time.
Ensuring data quality also requires that laboratory results are properly assessed and documented. The
results of the data quality assessment are transmitted to decision makers. This evaluation is as important
as the data for ensuring informed and effective decisions. While some degree of data evaluation is
necessary in order to be able to confirm data quality, 100% verification and/or validation is neither
necessary nor conducive to efficient decision making in emergency situations. The level of such reviews
should be determined based on the specific situation being assessed and on the corresponding data quality
objectives. In every case, the levels of QC and data review necessary to support decision making should
be determined as much in advance of data collection as possible.
Please note: The appropriate point of contact identified in Section 4 should be consulted regarding
appropriate quality assurance (QA) and QC procedures prior to sample analysis. These contacts will
consult with the EPA OSWER coordinator responsible for laboratory activities during the specific event
to ensure QA/QC procedures are performed consistently across laboratories. OSWER is planning to
develop QA/QC guidance for laboratory support. EPA program offices will be responsible for ensuring
that the QA/QC practices are implemented.
5.1.3 Safety and Waste Management
It is imperative that safety precautions are used during collection, processing, and analysis of
environmental samples. Laboratories should have a documented health and safety plan for handling
samples that may contain the target CBR contaminants. Laboratory staff should be trained in, and need to
SAM Revision 4.0 33 September 29, 2008
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Section 5 - Selected Chemical Methods
implement, the safety procedures included in the plan. In addition, many of the methods summarized or
cited in Section 5.2 contain some specific requirements, guidance, or information regarding safety
precautions that should be followed when handling or processing environmental samples and reagents.
These methods also provide information regarding waste management. Other resources that can be
consulted for additional information include the following:
• CDC - Title 42 of the Code of Federal Regulations part 72 (42 CFR 72). Interstate Shipment of
Etiological Agents.
• CDC - 42 CFR part 73. Select Agents and Toxins.
• U.S. Department of Transportation (DOT) - 49 CFR part 172. Hazardous Materials Table, Special
Provisions, Hazardous Materials Communications, Emergency Response Information, and Training
Requirements.
• EPA - 40 CFR part 260. Hazardous Waste Management System: General.
• EPA - 40 CFR part 270. EPA Administered Permit Programs: The Hazardous Waste Permit
Program.
• OSHA - 29 CFR part 1910.1450. Occupational Exposure to Hazardous Chemicals in Laboratories.
• OSHA - 29 CFR part 1910.120. Hazardous Waste Operations and Emergency Response.
Please note that the Electronic Code of Federal Regulations (e-CFR) is available at
http://ecfr.gpoaccess.gov/.
5.2 Method Summaries
Summaries for the analytical methods listed in Appendix A are provided in Sections 5.2.1 through 5.2.86.
These sections contain summary information only, extracted from the selected methods. Each method
summary contains a table identifying the contaminants in Appendix A to which the method applies, a
brief description of the analytical method, and a link to, or source for, obtaining a full version of the
method. The full version of the method should be consulted prior to sample analysis.
5.2.1 EPA Method 8: Determination of Sulfuric Acid and Sulfur Dioxide Emissions from
Stationary Sources
Analyte(s)
Sulfur Trioxide
CASRN
7446-11-9
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Isokinetic extraction
Determinative Technique: Titrimetry
Method Developed for: Sulfuric acid, sulfur trioxide, and sulfur dioxide in air
Method Selected for: SAM lists this method for preparation and analysis of air samples.
Detection and Quantitation: Collaborative tests have shown that the minimum detectable limit of the
method is 0.06 mg/m3 for sulfuric acid.
Description of Method: A gas sample is extracted isokinetically. Sulfuric acid and sulfur dioxide are
separated, and both fractions are measured separately by the barium-thorin titration method. Sulfur
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Section 5 - Selected Chemical Methods
trioxide is measured by the analysis of sulfuric acid. Possible interfering agents include fluorides, free
ammonia, and dimethyl aniline.
Special Considerations: Possible interfering agents include fluorides, free ammonia, and dimethyl
aniline.
Source: EPA Emission Measurement Center (EMC) of the Office of Air Quality Planning and Standards
(OAQPS). "Method 8: Determination of Sulfuric Acid and Sulfur Dioxide Emissions from Stationary
Sources." httii^/wiwj^
5.2.2 EPA Method 200.7: Determination of Metals and Trace Elements in Waters and
Wastes by Inductively Coupled Plasma-Atomic Emission Spectrometry
Analyte(s)
Ammonium metavanadate
Arsenic, Total
Arsenic trioxide
Arsine
Calcium arsenate
2-Chlorovinylarsonous acid (CVAA)
Lead arsenate
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine]
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine]
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine]
Lewisite Oxide
Sodium arsenite
Thallium sulfate
Vanadium pentoxide
CASRN
7803-55-6
740-38-2
1327-53-3
7784-42-1
7778-44-1
85090-33-1
7645-25-2
541-25-3
40334-69-8
40334-70-1
1306-02-1
7784-46-5
10031-59-1
1314-62-1
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Acid digestion
Determinative Technique: Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES)
Method Developed for: Determination of metals in solution. This method is a consolidation of existing
methods for water, wastewater, and solid wastes.
Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid and drinking
water samples.
Detection and Quantitation: Method detection limits (MDLs) in aqueous samples have been found to
be 0.008 mg/L for arsenic, 0.003 mg/L for vanadium, and 0.001 mg/L for thallium.
Description of Method: This method will determine metals in aqueous samples. An aliquot of a well-
mixed, homogeneous sample is accurately weighed or measured for sample processing. For total
recoverable analysis of a sample containing undissolved material, analytes are first solubilized by gentle
refluxing with nitric and hydrochloric acids. After cooling, the sample is made up to volume, mixed, and
centrifuged or allowed to settle overnight prior to analysis. For determination of dissolved analytes in a
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Section 5 - Selected Chemical Methods
filtered aqueous sample aliquot, or for the "direct analysis" total recoverable determination of analytes in
drinking water where sample turbidity is < 1 nephelometric turbidity units (NTU), the sample is made
ready for analysis by the addition of nitric acid, and then diluted to a predetermined volume and mixed
before analysis. The prepared sample is analyzed using ICP-AES. Specific analytes targeted by Method
200.7 are listed in Section 1.1 of the method.
Special Considerations: Laboratory testing is currently underway for speciation of lewisite 1 using GC-
MS techniques.
Source: EPA. 1994. "Method 200.7: Determination of Metals and Trace Elements in Water and Wastes
by Inductively Coupled Plasma-Atomic Emission Spectrometry," Revision 4.4.
^
5.2.3 EPA Method 200.8: Determination of Trace Elements in Waters and Wastes by
Inductively Coupled Plasma-Mass Spectrometry
Analyte(s)
Ammonium metavanadate
Arsenic, Total
Arsenic trioxide
Arsine
Calcium arsenate
2-Chlorovinylarsonous acid (CVAA)
Lead arsenate
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine]
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine]
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine]
Lewisite Oxide
Sodium arsenite
Thallium sulfate
Vanadium pentoxide
CASRN
7803-55-6
740-38-2
1327-53-3
7784-42-1
7778-44-1
85090-33-1
7645-25-2
541-25-3
40334-69-8
40334-70-1
1306-02-1
7784-46-5
10031-59-1
1314-62-1
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Acid digestion
Determinative Technique: Inductively Coupled Plasma-Mass Spectrometry (ICP-MS)
Method Developed for: Dissolved and total elements in ground water, surface water, drinking water,
wastewater, sludges, and soils.
Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid and drinking
water samples.
Detection and Quantitation: MDLs for arsenic in aqueous samples have been found to be 1.4 ug/L in
scanning mode, and 0.4 ug/L in selected ion monitoring mode. The recommended calibration range is 10
to 200 i_ig/L.
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Section 5 - Selected Chemical Methods
Description of Method: This method will determine metal-containing compounds only as the total metal
(e.g., total arsenic). An aliquot of a well-mixed, homogeneous sample is accurately weighed or measured
for sample processing. For total recoverable analysis of a sample containing undissolved material,
analytes are first solubilized by gentle refluxing with nitric and hydrochloric acids. After cooling, the
sample is made up to volume, mixed, and centrifuged or allowed to settle overnight prior to analysis. For
determination of dissolved analytes in a filtered aqueous sample aliquot, or for the "direct analysis" total
recoverable determination of analytes in drinking water where sample turbidity is < 1 NTU, the sample is
made ready for analysis by the addition of nitric acid, and then diluted to a predetermined volume and
mixed before analysis. The prepared sample is analyzed using ICP-MS. Specific analytes targeted by
Method 200.8 are listed in Section 1.1 of the method.
Special Considerations: Laboratory testing is currently underway for speciation of lewisite 1 using GC-
MS techniques.
Source: EPA. 1994. "Method 200.8: Determination of Trace Elements in Waters and Wastes by
Inductively Coupled Plasma-Mass Spectrometry," Revision 5.4.
200.8.pdf
5.2.4 EPA Method 245.2: Mercury (Automated Cold Vapor Technique)
Analyte(s)
Mercury, Total
Methoxyethylmercuric acetate
CASRN
7439-97-6
151-38-2
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Acid digestion
Determinative Technique: Cold vapor atomic absorption (CVAA)
Method Developed for: Mercury in surface waters. It may be applicable to saline waters, wastewaters,
effluents, and domestic sewages providing potential interferences are not present.
Method Selected for: SAM lists this method for preparation and analysis of drinking water samples.
Detection and Quantitation: Applicable concentration range is 0.2 to 20.0 iig/L.
Description of Method: This method will determine methoxyethylmercuric acetate as total mercury. If
dissolved mercury is targeted, the sample is filtered prior to acidification. To detect total mercury
(inorganic and organic mercury), the sample is treated with potassium permanganate and potassium
persulfate to oxidize organic mercury compounds prior to analysis. Inorganic mercury is reduced to the
elemental state (using tin sulfate or tin chloride) and aerated from solution. The mercury vapor passes
through a cell positioned in the light path of a CVAA spectrophotometer. The concentration of mercury
is measured using the CVAA spectrophotometer.
Source: EPA. 1974. "Method 245.2: Mercury (Automated Cold Vapor Technique)."
http://www.epa.gOv/sani/pdfs/EPA-245.2.pdf
5.2.5 EPA Method 252.2: Osmium (Atomic Absorption, Furnace Technique)
Analyte(s)
Osmium tetroxide
CASRN
20816-12-0
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Section 5 - Selected Chemical Methods
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Direct aspiration
Determinative Technique: Graphite furnace atomic absorption spectrophotometry (GFAA)
Method Developed for: Osmium in drinking, surface, and saline waters, and domestic and industrial
wastes
Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid and drinking
water samples.
Detection and Quantitation: Detection limit for osmium is 20 jig/L. The optimal applicable
concentration range is 50 to 500 \igfL.
Description of Method: This method will determine osmium tetroxide as osmium. Method 252.2 is not
a stand alone method in that sections of the method reference "Methods of Chemical Analysis of Water
and Waste", EPA/600/4-79/020, March 1983 (MCAWW). Samples are prepared according to the "direct
aspiration method" (See Section 9.1 of the Atomic Absorption Methods section of MCAWW) except that
the addition of sulfuric acid is omitted in the final adjustment. If only dissolved osmium is determined,
the sample is filtered before acidification with nitric acid. For total osmium, the sample is digested with
nitric and hydrochloric acids and made up to volume. Samples are analyzed according to the "furnace
procedure" (see Section 9.3 of the Atomic Absorption Methods section of MCAWW), using GFAA. A
representative aliquot of sample is placed in the graphite tube in the furnace, evaporated to dryness,
chaffed, and atomized. Radiation from an excited element is passed through the vapor containing ground
state atoms of the element. The intensity of the transmitted radiation decreases in proportion to the
amount of the ground state element in the vapor. A monochromator isolates the characteristic radiation
from the hollow cathode lamp and a photosensitive device measures the attenuated transmitted radiation.
Source: EPA. 1978. "Method 252.2: Osmium (Atomic Absorption, Furnace Technique)."
http://www.epa.gOV/sam/pdfs/EPA-252.2.pdf
5.2.6 EPA Method 300.1, Revision 1.0: Determination of Inorganic Anions in Drinking
Water by Ion Chromatography
Analyte(s)
Fluoride
Fluoroacetic acid and fluoroacetate salts
Methyl fluoroacetate
Sodium azide (analyze for hydrazoic acid)
CASRN
16984-48-8
NA
453-18-9
26628-22-8
Analysis Purpose: Sample preparation and/or analysis
Sample Preparation Technique: For fluoride, use direct injection. For fluoroacetic acid, fluoroacetate
salts, and methyl fluoroacetate, use Analytical Letters, 1994, 27(14): 2703-2718 (solid and non-aqueous
liquid/organic solid samples), NIOSH Method S301-1 (air samples), and EPA SW-846 Method
3570/8290A Appendix A (wipe samples). For sodium azide, use water extraction, filtration, and
acidification steps from the Journal of Forensic Science, 1998. 43(1):200-202 (solid samples), filtration
and acidification steps from this journal (aqueous liquid and drinking water samples), and the
acidification step from the journal with EPA SW-846 Method 3580A (non-aqueous liquid/organic solid
samples).
Determinative Technique: Ion chromatography (1C)
Method Developed for: Inorganic anions in reagent water, surface water, ground water, and finished
drinking water
SAM Revision 4.0 38 September 29, 2008
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Section 5 - Selected Chemical Methods
Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid and drinking
water samples for fluoride, fluoroacetic acid, fluoroacetate salts, and methyl fluoroacetate. It also should
be used for analysis of solid and non-aqueous liquid/organic solid, air, and/or wipe samples for
fluoroacetic acid, fluoroacetate salts, methyl fluoroacetate, and sodium azide when appropriate sample
preparation techniques have been applied.
Detection and Quantitation: The detection limit for fluoride in reagent water is 0.009 mg/L. The MDL
varies depending upon the nature of the sample and the specific instrumentation employed. The estimated
calibration range should not extend over more than 2 orders of magnitude in concentration over the
expected concentration range of the samples.
Description of Method: This method was developed for analysis of aqueous samples, and can be
adapted for analysis of prepared non-aqueous liquid/organic solid, solid, and air samples when
appropriate sample preparation techniques have been applied (see Appendix A). A small volume of an
aqueous liquid sample (10 \iL or 50 \iL) is introduced into an ion chromatograph. The volume selected
depends on the concentration of fluoroacetate ion in the sample. The anions of interest are separated and
measured, using a system comprising a guard column, analytical column, suppressor device, and
conductivity detector. The separator columns and guard columns, as well as eluent conditions, are
identical. To achieve comparable detection limits, an ion chromatographic system must use suppressed
conductivity detection, be properly maintained, and be capable of yielding a baseline with no more than 5
nS noise/drift per minute of monitored response over the background conductivity.
Source: EPA. 1997. "Method 300.1: Determination of Inorganic Anions in Drinking Water by Ion
Chromatography," Revision 1.0. http://wmny.epa.gov/sam/pdfs/EPA-300.1 .pdf
5.2.7 EPA Method 335.4: Determination of Total Cyanide by Semi-Auto mated
Colorimetry
Analyte(s)
Cyanide, Total
CASRN
57-12-5
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Reflux-distillation
Determinative Technique: Spectrophotometry
Method Developed for: Cyanide in drinking, ground, surface, and saline waters, and domestic and
industrial wastes
Method Selected for: SAM lists this method for preparation and analysis of drinking water samples.
Detection and Quantitation: The applicable range is 5 to 500 jig/L.
Description of Method: Cyanide is released from cyanide complexes as hydrocyanic acid by manual
reflux-distillation, and absorbed in a scrubber containing sodium hydroxide solution. The cyanide ion in
the absorbing solution is converted to cyanogen chloride by reaction with chloramine-T, which
subsequently reacts with pyridine and barbituric acid to give a red-colored complex.
Special Considerations: Some interferences, such as aldehydes, nitrate-nitrite, oxidizing agents,
thiocyanate, thiosulfate, and sulfide, are eliminated or reduced by distillation.
Source: EPA. 1993. "Method 335.4: Determination of Total Cyanide by Semi-Automated Colorimetry,"
Revision 1.0. liltp://www.epa.gov/sam/pdfs/EPA-335.4.pdf
SAM Revision 4.0 39 September 29, 2008
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Section 5 - Selected Chemical Methods
5.2.8 EPA Method 350.1: Nitrogen, Ammonia (Colorimetric, Automated Phenate)
Analyte(s)
Ammonia
CASRN
7664-41-7
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Distillation
Determinative Technique: Spectrophotometry
Method Developed for: Ammonia in drinking, ground, surface, and saline waters, and domestic and
industrial wastes
Method Selected for: SAM lists this method for preparation and analysis of drinking water samples.
Detection and Quantitation: The working range for ammonia is 0.01 to 2.0 mg/L.
Description of Method: This method identifies and determines the concentration of ammonia in
drinking water samples by spectrophotometry. Samples are buffered at a pH of 9.5 with borate buffer to
decrease hydrolysis of cyanates and organic nitrogen compounds, and are distilled into a solution of boric
acid. Alkaline phenol and hypochlorite react with ammonia to form indophenol blue that is proportional
to the ammonia concentration. The blue color formed is intensified with sodium nitroprusside and
measured spectrophotometrically.
Source: EPA. 1993. "Method 350.1: Nitrogen, Ammonia (Colorimetric, Automated Phenate)," Revision
2.0. http://www.epa.gov/sam/pdfs/EPA-350.1 .pdf
5.2.9 EPA Method 507: Determination of Nitrogen- and Phosphorus-Containing
Pesticides in Water by Gas Chromatography with a Nitrogen-Phosphorus Detector
Analyte(s)
Organophosphate pesticides, NOS
CASRN
NA
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Solvent extraction
Determinative Technique: Gas chromatography - Nitrogen-phosphorus detector (GC-NPD)
Method Developed for: Nitrogen- and phosphorus-containing pesticides in ground water and finished
drinking water
Method Selected for: SAM lists this method for preparation and analysis of drinking water samples.
Detection and Quantitation: Estimated detection limits (EDLs) and MDLs differ depending on the
specific pesticide.
Description of Method: A 1-L sample is extracted with methylene chloride by shaking in a separatory
funnel or mechanical tumbling in a bottle. The methylene chloride extract is isolated, dried, and
concentrated to a volume of 5 mL during a solvent exchange to methyl fert-butyl ether (MTBE). The
concentrations of pesticides in the extract are measured using a capillary column gas chromatography
(GC) system equipped with a nitrogen-phosphorus detector (NPD). Specific analytes targeted by Method
507 are listed in Section 1.1 of the method.
Special Considerations: The presence of organophosphate pesticides should be confirmed by either a
secondary GC column or by an MS.
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Section 5 - Selected Chemical Methods
Source: EPA. 1995. "Method 507: Determination of Nitrogen- and Phosphorus-Containing Pesticides in
Water by Gas Chromatography with a Nitrogen-Phosphorus Detector," Revision 2.1.
http://www.cpa.gov/sam/pdfs/EPA-507.pdf
5.2.10 EPA Method 524.2: Measurement of Purgeable Organic Compounds in Water by
Capillary Column Gas Chromatography / Mass Spectrometry
Analyte(s)
Carbon disulfide
1,2-Dichloroethane
CASRN
75-15-0
107-06-2
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Purge and trap
Determinative Technique: GC-MS
Method Developed for: Purgeable volatile organic compounds (VOCs) in surface water, ground water,
and drinking water in any stage of treatment
Method Selected for: SAM lists this method for preparation and analysis of drinking water samples.
Detection and Quantitation: Detection levels for carbon disulfide and 1,2-dichloroethane in reagent
water have been found to be 0.093 (ig/L and 0.02 (ig/L, respectively. The applicable concentration range
of this method is primarily column and matrix dependent, and is approximately 0.02 to 200 (ig/L when a
wide-bore thick-film capillary column is used. Narrow-bore thin-film columns may have a capacity,
which limits the range to approximately 0.02 to 20 (ig/L.
Description of Method: VOCs and surrogates with low water solubility are extracted (purged) from the
sample matrix by bubbling an inert gas through the aqueous sample. Purged sample components are
trapped in a tube containing suitable sorbent materials. When purging is complete, the sorbent tube is
heated and backflushed with helium to desorb the trapped sample components into a capillary GC column
interfaced to a mass spectrometer (MS). The column is temperature programmed to facilitate the
separation of the method analytes, which are then detected with the MS. Specific analytes targeted by
Method 524.2 are listed in Section 1.1 of the method.
Source: EPA. 1992. "Method 524.2: Measurement of Purgeable Organic Compounds in Water by
Capillary Column Gas Chromatography/Mass Spectrometry," Revision 4.0.
5.2.11 EPA Method 525.2: Determination of Organic Compounds in Drinking Water by
Liquid-Solid Extraction and Capillary Column Gas Chromatography / Mass
Spectrometry
Analyte(s)
Dichlorvos
Disulfoton
Disulfoton sulfoxide
Fenamiphos
Mevinphos
Semivolatile Organic Compounds, NOS
CASRN
62-73-7
298-04-4
2497-07-6
22224-92-6
7786-34-7
NA
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Section 5 - Selected Chemical Methods
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Liquid-solid extraction (LSE) or solid-phase extraction (SPE)
Determinative Technique: GC-MS
Method Developed for: Organic compounds in finished drinking water, source water, or drinking water
in any treatment stage
Method Selected for: SAM lists this method for preparation and analysis of drinking water samples.
Detection and Quantitation: The applicable concentration range for most analytes is 0.1 to 10 ug/L.
Description of Method: Organic compounds, internal standards, and surrogates are extracted from a
water sample by passing 1 L of sample through a cartridge or disk containing a solid matrix with
chemically bonded Ci8 organic phase (LSE or SPE). The organic compounds are eluted from the LSE
(SPE) cartridge or disk with small quantities of ethyl acetate followed by methylene chloride. The
resulting extract is concentrated further by evaporation of some of the solvent. Sample components are
separated, identified, and measured by injecting an aliquot of the concentrated extract into a high
resolution fused silica capillary column of a GC-MS system. Specific analytes targeted by Method 525.2
are listed in Section 1.1 of the method.
Source: EPA. 1995. "Method 525.2: Determination of Organic Compounds in Drinking Water by
Liquid-Solid Extraction and Capillary Column Gas Chromatography/Mass Spectrometry," Revision 2.0.
http://www.epa.gOv/sam/pdfs/EPA-525.2.pdf
5.2.12 EPA Method 531.2: Measurement of N-Methylcarbamoyloximes and N-
Methylcarbamates in Water by Direct Aqueous Injection HPLC with Postcolumn
Derivatization
Analyte(s)
Aldicarb (Temik)
Aldicarb sulfone
Aldicarb sulfoxide
Carbofuran (Furadan)
Methomyl
Oxamyl
Thiofanox
CASRN
116-06-3
1646-88-4
1646-87-3
1563-66-2
16752-77-5
23135-22-0
39196-18-4
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Direct injection
Determinative Technique: High performance liquid chromatography (HPLC)
Method Developed for: N-methylcarbamoyloximes and N-methylcarbamates in finished drinking
water
Method Selected for: SAM lists this method for preparation and analysis of drinking water samples.
Detection and Quantitation: Detection limits range from 0.026 to 0.115 ug/L. The concentration
range for target analytes in this method was evaluated between 0.2 j^g/L and 10 ^ig/L.
Description of Method: An aliquot of sample is measured in a volumetric flask. Samples are preserved,
spiked with appropriate surrogates and then filtered. Analytes are chromatographically separated by
SAM Revision 4.0 42 September 29, 2008
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Section 5 - Selected Chemical Methods
injecting a sample aliquot (up to 1000 (iL) into a HPLC system equipped with a reverse phase (Ci8)
column. After elution from the column, the analytes are hydrolyzed in a post column reaction to form
methylamine, which is in turn reacted to form a fluorescent isoindole that is detected by a fluorescence
(FL) detector. Analytes also are quantitated using the external standard technique.
Source: EPA. 2001. "Method 531.2: Measurement ofN-Methylcarbamoyloximes andN-
Methylcarbamates in Water by Direct Aqueous Injection HPLC with Postcolumn Derivatization,"
Revision 1.0. http://www.epa.gov/sam/pdfs/EPA-531.2.pdf
5.2.13 EPA Method 549.2: Determination of Diquat and Paraquat in Drinking Water by
Liquid-Solid Extraction and High Performance Liquid Chromatography with
Ultraviolet Detection
Analyte(s)
Paraquat
CASRN
4685-14-7
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: LSE or SPE
Determinative Technique: HPLC
Method Developed for: Diquat and paraquat in drinking water sources and finished drinking water
Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid and drinking
water samples.
Detection and Quantitation: MDL for paraquat is 0.68 (ig/L. The analytical range depends on the
sample matrix and the instrumentation used.
Description of Method: A 250-mL sample is extracted using a C8 LSE cartridge or a C8 disk that has
been specially prepared for the reversed-phase, ion-pair mode. The LSE disk or cartridge is eluted with
acidic aqueous solvent to yield the eluate/extract. An ion-pair reagent is added to the eluate/extract. The
concentrations of paraquat in the eluate/extract are measured using a HPLC system equipped with an
ultraviolet (UV) absorbance detector. A photodiode array detector is used to provide simultaneous
detection and confirmation of the method analytes.
Source: EPA. 1997. "Method 549.2: Determination of Diquat and Paraquat in Drinking Water by
Liquid-Solid Extraction and High Performance Liquid Chromatography with Ultraviolet Detection,"
Revision 1.0. htti)://wjTO\CTa4>w/sa^
5.2.14 EPA Method 551.1: Determination of Chlorination Disinfection Byproducts,
Chlorinated Solvents, and Halogenated Pesticides/Herbicides in Drinking Water by
Liquid-Liquid Extraction and Gas Chromatography with Electron-Capture
Detection
Analyte(s)
Chloropicrin
CASRN
79-06-2
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Solvent extraction
Determinative Technique: Gas Chromatography - Electron capture detector (GC-ECD)
SAM Revision 4.0 43 September 29, 2008
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Section 5 - Selected Chemical Methods
Method Developed for: Chlorination disinfection byproducts, chlorinated solvents, and halogenated
pesticides/herbicides in finished drinking water, drinking water during intermediate stages of treatment,
and raw source water
Method Selected for: SAM lists this method for preparation and analysis of drinking water samples.
Detection and Quantitation: The EDL using MTBE and ammonium chloride-preserved reagent water
on a 100% dimethylpolysiloxane (DB-1) column has been found to be 0.014 (ig/L.
Description of Method: This is a GC-ECD method applicable to the determination of halogenated
analytes in finished drinking water, drinking water during intermediate stages of treatment, and raw
source water. A 50-mL sample aliquot is extracted with 3 mL of MTBE or 5 mL of pentane. Two uL of
the extract is then injected into a GC equipped with a fused silica capillary column and linearized ECD
for separation and analysis. This liquid/liquid extraction technique efficiently extracts a wide boiling
range of non-polar and polar organic components of the sample. Thus, confirmation is quite important,
particularly at lower analyte concentrations. A confirmatory column is suggested for this purpose.
Special Considerations: The presence of chloropicrin should be confirmed by either a secondary GC
column or by an MS.
Source: EPA. 1995. "Method 551.1: Determination of Chlorination Disinfection Byproducts,
Chlorinated Solvents, and Halogenated Pesticides/Herbicides in Drinking Water by Liquid-Liquid
Extraction and Gas Chromatography with Electron-Capture Detection," Revision 1.0.
http://www.epa.gov/sam/pdfs/EPA-551.1 .pdf
5.2.15 EPA Method 614: The Determination of Organophosphorus Pesticides in
Municipal and Industrial Wastewater
Analyte(s)
Organophosphate pesticides, NOS
CASRN
NA
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Solvent extraction
Determinative Technique: Gas chromatography - Flame photometric detector (GC-FPD)
Method Developed for: Organophosphorus pesticides in municipal and industrial wastewater
Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid samples.
Description of Method: This is a GC method applicable to the determination of organophosphate
pesticides in industrial and municipal discharges using a GC with a phosphorus-specific flame
photometric detector (FPD) or thermionic bead detector in the nitrogen mode. A measured volume of
sample, approximately 1 L, is extracted with 15% methylene chloride in hexane using a separatory funnel.
The extract is dried and concentrated to a volume of 10 mL or less. GC conditions are described for the
separation and measurement of the compounds in the extract by flame photometric or thermionic bead
GC. Specific analytes targeted by Method 614 are listed in Section 1.1 of the method.
Special Considerations: The presence of organophosphate pesticides should be confirmed by either a
secondary GC column or by an MS.
Source: EPA. "Method 614: The Determination of Organophosphorus Pesticides in Municipal and
Industrial Wastewater." http://www.epa.gov/sam/pdfs/EPA-614.pdf
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Section 5 - Selected Chemical Methods
5.2.16 EPA Method 3031 (SW-846): Acid Digestion of Oils for Metals Analysis by Atomic
Absorption or ICP Spectrometry
Analyte(s)
Ammonium metavanadate
Arsenic, Total
Arsenic trioxide
Calcium arsenate
2-Chlorovinylarsonous acid (CVAA)
Lead arsenate
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine]
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine]
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine]
Lewisite Oxide
Sodium arsenite
Thallium sulfate
Vanadium pentoxide
CASRN
7803-55-6
740-38-2
1327-53-3
7778-44-1
85090-33-1
7645-25-2
541-25-3
40334-69-8
40334-70-1
1306-02-1
7784-46-5
10031-59-1
1314-62-1
Analysis Purpose: Sample preparation
Sample Preparation Technique: Acid digestion
Determinative Technique: EPA SW-846 Method 6010C or Method 6020A
Method Developed for: Metals in oils, oil sludges, tars, waxes, paints, paint sludges and other viscous
petroleum products
Method Selected for: SAM lists this method for preparation of non-aqueous liquid/organic solid
samples.
Description of Method: This method is used to prepare samples for the determination of arsenic
trioxide, lewisite, lewisite degradation products, and sodium arsenite as total arsenic; thallium sulfate as
total thallium; and ammonium metavanadate and vanadium pentoxide as total vanadium. A 0.5-g sample
of oil, oil sludge, tar, wax, paint, or paint sludge is mixed with potassium permanganate and sulfuric acid.
The mixture is then treated with nitric and hydrochloric acids, filtered, and diluted to volume. Excess
manganese may be removed with ammonium hydroxide. Digestates are analyzed by Method 6020A or
6010C (SW-846).
Source: EPA. 1996. "Method 3031 (SW-846): Acid Digestion of Oils for Metals Analysis by Atomic
Absorption or ICP Spectrometry," Revision 0. http://www.epa.gov/sam/pdfs/EPA-3031 .pdf
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Section 5 - Selected Chemical Methods
5.2.17 EPA Method 3050B (SW-846): Acid Digestion of Sediments, Sludges, and Soils
Analyte(s)
Ammonium metavanadate
Arsenic, Total
Arsenic trioxide
Arsine
Calcium arsenate
2-Chlorovinylarsonous acid (CVAA)
Lead arsenate
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine]
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine]
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine]
Lewisite Oxide
Osmium tetroxide
Sodium arsenite
Thallium sulfate
Titanium tetrachloride
Vanadium pentoxide
CASRN
7803-55-6
740-38-2
1327-53-3
7784-42-1
7778-44-1
85090-33-1
7645-25-2
541-25-3
40334-69-8
40334-70-1
1306-02-1
20816-12-0
7784-46-5
10031-59-1
7550-45-0
1314-62-1
Analysis Purpose: Sample preparation
Sample Preparation Technique: Acid digestion
Determinative Technique: EPA SW-846 Method 6010C, Method 6020A, or Method 7010. Refer to
Appendix A for which of these determinative methods should be used for a particular analyte.
Method Developed for: Metals in sediments, sludges, and soil samples
Method Selected for: SAM lists this method for preparation of solid samples.
Description of Method: This method is used to prepare samples for the determination of arsenic
trioxide, arsine, lewisite, lewisite degradation products, and sodium arsenite as total arsenic; thallium
sulfate as total thallium; titanium tetrachloride as titanium; osmium tetroxide as osmium; and ammonium
metavanadate and vanadium pentoxide as total vanadium. A 1-g to 2-g sample is digested with nitric acid
and hydrogen peroxide. Sample volumes are reduced, then brought up to a final volume of 100 mL.
Samples are analyzed for total arsenic, total thallium, total titanium, or total vanadium by Method 60IOC
or 6020A (SW-846); use Method 6010C (SW-846) for total osmium.
Source: EPA. 1996. "Method 3050B (SW-846): Acid Digestion of Sediments, Sludges, and Soils,"
Revision 2. http://www.cpa.gov/sam/pdfs/EPA-3050b.pdf
SAM Revision 4.0
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Section 5 - Selected Chemical Methods
5.2.18 EPA Method 3520C (SW-846): Continuous Liquid-Liquid Extraction
Analyte(s)
Brodifacoum
Bromadiolone
BZ (Quinuclidinyl benzilate)
Chlorfenvinphos
3-Chloro-1 ,2-propanediol
Chlorosarin
Chlorosoman
Chlorpyrifos
Cyclohexyl sarin (GF)
Diesel Range Organics
Diphacinone
N-Ethyldiethanolamine (EDEA)
Fenamiphos
Fentanyl
Fluoroacetamide
Methyl hydrazine
N-Methyldiethanolamine (MDEA)
1-Methylethyl ester ethylphosphonofluoridic acid (GE)
Mustard, nitrogen (HN-1) [bis(2-chloroethyl)ethylamine]
Mustard, nitrogen (HN-2) [2,2'-dichloro-N-
methyldiethylamine N,N-bis(2-chloroethyl)methylamine]
Mustard, nitrogen (HN-3) [tris(2-chloroethyl)amine]
Parathion
Phenol
Phosphamidon
R 33 (VR) [methylphosphonothioic acid, S-[2-
(diethylamino)ethyl] O-2-methylpropyl ester]
Semivolatile Organic Compounds, NOS
Tear gas (CS) [chlorobenzylidene malonitrile]
Tetramethylenedisulfotetramine
Thiofanox
CASRN
56073-10-0
28772-56-7
6581-06-2
470-90-6
96-24-2
1445-76-7
7040-57-5
2921-88-2
329-99-7
NA
82-66-6
139-87-7
22224-92-6
437-38-7
640-19-7
60-34-4
105-59-9
1189-87-3
538-07-8
51-75-2
555-77-1
56-38-2
108-95-2
13171-21-6
159939-87-4
NA
2698-41-1
80-12-6
39196-18-4
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Section 5 - Selected Chemical Methods
Analyte(s)
Triethanolamine (TEA)
VE [phosphonothioic acid, ethyl-, S-(2-
(diethylamino)ethyl) O-ethyl ester]
VG [phosphonothioic acid, S-(2-(diethylamino)ethyl)
O,O-diethyl ester]
VM [phosphonothioic acid, methyl-, S-(2-
(diethylamino)ethyl) O-ethyl ester]
CASRN
102-71-6
21738-25-0
78-53-5
21770-86-5
Analysis Purpose: Sample preparation
Sample Preparation Technique: Continuous liquid-liquid extraction (CLLE)
Determinative Technique: EPA SW-846 Method 8015C, Method 8270D, or Method 832 IB. Refer to
Appendix A for which of these determinative methods should be used for a particular analyte.
Method Developed for: Organic compounds in aqueous samples
Method Selected for: SAM lists this method for preparation of aqueous liquid and/or drinking water
samples. Please note: Drinking water samples for fenamiphos and semivolatile organic compounds
should be prepared and analyzed by EPA Method 525.2; drinking water samples for thiofanox should be
prepared and analyzed by EPA Method 531.2; all other drinking water samples and all aqueous liquid
samples should be prepared using this method (SW-846 Method 3520C).
Description of Method: This method is applicable to the isolation and concentration of water-insoluble
and slightly soluble organics in preparation for a variety of chromatographic procedures. A measured
volume of sample, usually 1 L, is placed into a continuous liquid-liquid extractor, adjusted, if necessary,
to a specific pH and extracted with organic solvent for 18 to 24 hours. The extract is filtered through
sodium sulfate to remove residual moisture, concentrated, and exchanged as necessary into a solvent
compatible with the cleanup or determinative procedure used for analysis.
Source: EPA. 1996. "Method 3520C (SW-846): Continuous Liquid-Liquid Extraction," Revision 3.
|2^^
5.2.19 EPA Method 3535A (SW-846): Solid-Phase Extraction
Analyte(s)
4-Aminopyridine
Brodifacoum
Bromadiolone
BZ (Quinuclidinyl benzilate)
Chlorfenvinphos
3-Chloro-1 ,2-propanediol
Chlorosarin
Chlorosoman
Chlorpyrifos
CASRN
504-24-5
56073-10-0
28772-56-7
6581-06-2
470-90-6
96-24-2
1445-76-7
7040-57-5
2921-88-2
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Section 5 - Selected Chemical Methods
Analyte(s)
Crimidine
Cyclohexyl sarin (GF)
Dichlorvos
Dicrotophos
Diesel Range Organics
Diisopropyl methylphosphonate (DIMP)
Dimethylphosphite
Dimethylphosphoramidic acid
Diphacinone
1,4-Dithiane
EA2192 [Diisopropylaminoethyl
methylthiolophosphonate]
Ethyl methylphosphonic acid (EMPA)
Ethyldichloroarsine (ED)
N-Ethyldiethanolamine (EDEA)
Fenamiphos
Fentanyl
Fluoroacetamide
Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)
Hexamethylenetriperoxidediamine (HMTD)
Isopropyl methylphosphonic acid (IMPA)
Methamidophos
Methyl hydrazine
Methyl parathion
N-Methyldiethanolamine (MDEA)
1-Methylethyl ester ethylphosphonofluoridic acid (GE)
Methylphosphonic acid (MPA)
Mevinphos
Mustard, nitrogen (HN-1) [bis(2-chloroethyl)ethylamine]
Mustard, nitrogen (HN-2) [2,2'-dichloro-N-
methyldiethylamine N,N-bis(2-chloroethyl)methylamine]
Mustard, nitrogen (HN-3) [tris(2-chloroethyl)amine]
CASRN
535-89-7
329-99-7
62-73-7
141-66-2
NA
1445-75-6
868-85-9
33876-51-6
82-66-6
505-29-3
73207-98-4
1832-53-7
598-14-1
139-87-7
22224-92-6
437-38-7
640-19-7
121-82-4
283-66-9
1832-54-8
10265-92-6
60-34-4
298-00-0
105-59-9
1189-87-3
993-13-5
7786-34-7
538-07-8
51-75-2
555-77-1
SAM Revision 4.0
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Section 5 - Selected Chemical Methods
Analyte(s)
Nicotine compounds
Octahydro-1 ,3,5,7-tetranitro-1 ,3,5,7-tetrazocine (HMX)
Parathion
Pentaerythritol tetranitrate (PETN)
Phencyclidine
Phenol
Phorate
Phosphamidon
Pinacolyl methyl phosphonic acid (PMPA)
R 33 (VR) [methylphosphonothioic acid, S-[2-
(diethylamino)ethyl] O-2-methylpropyl ester]
Semivolatile Organic Compounds, NOS
Soman (GD)
Strychnine
Tabun (GA)
Tear gas (CS) [chlorobenzylidene malonitrile]
Tetraethyl pyrophosphate
Tetramethylenedisulfotetramine
Thiodiglycol (TDG)
Thiofanox
1,4-Thioxane
Triethanolamine (TEA)
Trimethyl phosphite
1,3,5-Trinitrobenzene (1,3,5-TNB)
2,4,6-Trinitrotoluene(2,4,6-TNT)
VE [phosphonothioic acid, ethyl-, S-(2-
(diethylamino)ethyl) O-ethyl ester]
VG [phosphonothioic acid, S-(2-(diethylamino)ethyl)
O,O-diethyl ester]
VM [phosphonothioic acid, methyl-, S-(2-
(diethylamino)ethyl) O-ethyl ester]
CASRN
54-11-5
2691-41-0
56-38-2
78-11-5
77-10-1
108-95-2
298-02-2
13171-21-6
616-52-4
159939-87-4
NA
96-64-0
57-24-9
77-81-6
2698-41-1
107-49-3
80-12-6
111-48-8
39196-18-4
15980-15-1
102-71-6
121-45-9
99-35-4
118-96-7
21738-25-0
78-53-5
21770-86-5
Analysis Purpose: Sample preparation
Sample Preparation Technique: SPE
SAM Revision 4.0
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Section 5 - Selected Chemical Methods
Determinative Technique: EPA SW-846 Method 8015C, Method 8270D, Method 832IB, or Method
8330B. Refer to Appendix A for which of these determinative methods should be used for a particular
analyte.
Method Developed for: Organic compounds in ground water, wastewater, and Toxicity Characteristic
Leaching Procedure (TCLP, Method 1311) leachates
Method Selected for: SAM lists this method for preparation of aqueous liquid and/or drinking water
samples. Please note: Drinking water samples for dichlorvos, fenamiphos, mevinphos, and semivolatile
organic compounds NOS should be prepared and analyzed by EPA Method 525.2; drinking water
samples for polychlorinated biphenyls should be prepared and analyzed by EPA Method 508; drinking
water samples for thiofanox should be prepared and analyzed by EPA Method 531.2; all other drinking
water samples and all aqueous liquid samples should be prepared using this method (SW-846 Method
3535 A).
Description of Method: This method describes a procedure for isolating target organic analytes from
aqueous and liquid samples using SPE media. Sample preparation procedures vary by analyte group.
Following any necessary pH adjustment, a measured volume of sample is extracted by passing it through
the SPE medium (disks or cartridges), which is held in an extraction device designed for vacuum filtration
of the sample. Target analytes are eluted from the solid-phase media using an appropriate solvent which
is collected in a receiving vessel. The resulting solvent extract is dried using sodium sulfate and
concentrated, as needed.
Special Considerations: Tetramethylenedisulfotetramine may require SPE extraction using acetone or
methyl ethylketone.
Source: EPA. 1998. "Method 3535A (SW-846): Solid-Phase Extraction (SPE)," Revision 1.
http://www.epa.gov/sam/pdfs/EPA-3535a.pdf
5.2.20 EPA Method 3541 (SW-846): Automated Soxhlet Extraction
Analyte(s)
Brodifacoum
Bromadiolone
BZ (Quinuclidinyl benzilate)
Chlorfenvinphos
3-Chloro-1 ,2-propanediol
Chlorosarin
Chlorosoman
Chlorpyrifos
Cyclohexyl sarin (GF)
Diesel Range Organics
Diphacinone
Disulfoton
Disulfoton sulfoxide
CASRN
56073-10-0
28772-56-7
6581-06-2
470-90-6
96-24-2
1445-76-7
7040-57-5
2921-88-2
329-99-7
NA
82-66-6
298-04-4
2497-07-6
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Section 5 - Selected Chemical Methods
Analyte(s)
N-Ethyldiethanolamine (EDEA)
Fenamiphos
Fentanyl
Fluoroacetamide
Methamidophos
Methyl hydrazine
N-Methyldiethanolamine (MDEA)
1-Methylethyl ester ethylphosphonofluoridic acid (GE)
Mustard, nitrogen (HN-1) [bis(2-chloroethyl)ethylamine]
Mustard, nitrogen (HN-2) [2,2'-dichloro-N-
methyldiethylamine N,N-bis(2-chloroethyl)methylamine]
Mustard, nitrogen (HN-3) [tris(2-chloroethyl)amine]
Organophosphate pesticides, NOS
Parathion
Phenol
Phosphamidon
R 33 (VR) [methylphosphonothioic acid, S-[2-
(diethylamino)ethyl] O-2-methylpropyl ester]
Semivolatile Organic Compounds, NOS
Tear gas (CS) [chlorobenzylidene malonitrile]
Tetramethylenedisulfotetramine
Thiofanox
Triethanolamine (TEA)
VE [phosphonothioic acid, ethyl-, S-(2-
(diethylamino)ethyl) O-ethyl ester]
VG [phosphonothioic acid, S-(2-(diethylamino)ethyl)
O,O-diethyl ester]
VM [phosphonothioic acid, methyl-, S-(2-
(diethylamino)ethyl) O-ethyl ester]
CASRN
139-87-7
22224-92-6
437-38-7
640-19-7
10265-92-6
60-34-4
105-59-9
1189-87-3
538-07-8
51-75-2
555-77-1
NA
56-38-2
108-95-2
13171-21-6
159939-87-4
NA
2698-41-1
80-12-6
39196-18-4
102-71-6
21738-25-0
78-53-5
21770-86-5
Analysis Purpose: Sample preparation
Sample Preparation Technique: Automated Soxhlet extraction (ASE)
Determinative Technique: EPA SW-846 Method 8015C, Method 8270D, or Method 832 IB. Refer to
Appendix A for which of these determinative methods should be used for a particular analyte.
SAM Revision 4.0
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Section 5 - Selected Chemical Methods
Method Developed for: Organic compounds in soil, sediment, sludges, and waste solids
Method Selected for: SAM lists this method for preparation of solid samples.
Description of Method: Approximately 10 g of solid sample is mixed with an equal amount of
anhydrous sodium sulfate and placed in an extraction thimble or between two plugs of glass wool. After
adding the appropriate surrogate amount, the sample is extracted using an appropriate solvent in an
automated Soxhlet extractor. The extract is dried with sodium sulfate to remove residual moisture,
concentrated and exchanged, as necessary, into a solvent compatible with the cleanup or determinative
procedure for analysis.
Source: EPA. 1994. "Method 3541 (SW-846): Automated Soxhlet Extraction," Revision 0.
5.2.21 EPA Method 3545A (SW-846): Pressurized Fluid Extraction (PFE)
Analyte(s)
Brodifacoum
Bromadiolone
BZ (Quinuclidinyl benzilate)
Chlorfenvinphos
3-Chloro-1 ,2-propanediol
Chloropicrin
Chlorosarin
Chlorosoman
Chlorpyrifos
Crimidine
Cyclohexyl sarin (GF)
Dichlorvos
Dicrotophos
Diesel Range Organics
Diisopropyl methylphosphonate (DIMP)
Dimethylphosphite
Dimethylphosphoramidic acid
Diphacinone
Disulfoton
Disulfoton sulfoxide
1,4-Dithiane
CASRN
56073-10-0
28772-56-7
6581-06-2
470-90-6
96-24-2
76-06-2
1445-76-7
7040-57-5
2921-88-2
535-89-7
329-99-7
62-73-7
141-66-2
NA
1445-75-6
868-85-9
33876-51-6
82-66-6
298-04-4
2497-07-6
505-29-3
SAM Revision 4.0
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Section 5 - Selected Chemical Methods
Analyte(s)
EA2192 [Diisopropylaminoethyl
methylthiolophosphonate]
Ethyl methylphosphonic acid (EMPA)
Ethyldichloroarsine (ED)
N-Ethyldiethanolamine (EDEA)
Fenamiphos
Fentanyl
Fluoroacetamide
Isopropyl methylphosphonic acid (IMPA)
Methamidophos
Methyl hydrazine
Methyl parathion
N-Methyldiethanolamine (MDEA)
1-Methylethyl ester ethylphosphonofluoridic acid (GE)
Methylphosphonic acid (MPA)
Mevinphos
Mustard, nitrogen (HN-1) [bis(2-chloroethyl)ethylamine]
Mustard, nitrogen (HN-2) [2,2'-dichloro-N-
methyldiethylamine N,N-bis(2-chloroethyl)methylamine]
Mustard, nitrogen (HN-3) [tris(2-chloroethyl)amine]
Nicotine compounds
Organophosphate pesticides, NOS
Parathion
Phencyclidine
Phenol
Phorate
Phosphamidon
Pinacolyl methyl phosphonic acid (PMPA)
R 33 (VR) [methylphosphonothioic acid, S-[2-
(diethylamino)ethyl] O-2-methylpropyl ester]
Semivolatile Organic Compounds, NOS
Soman (GD)
CASRN
73207-98-4
1832-53-7
598-14-1
139-87-7
22224-92-6
437-38-7
640-19-7
1832-54-8
10265-92-6
60-34-4
298-00-0
105-59-9
1189-87-3
993-13-5
7786-34-7
538-07-8
51-75-2
555-77-1
54-11-5
NA
56-38-2
77-10-1
108-95-2
298-02-2
13171-21-6
616-52-4
159939-87-4
NA
96-64-0
SAM Revision 4.0
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Section 5 - Selected Chemical Methods
Analyte(s)
Strychnine
Tabun (GA)
Tear gas (CS) [chlorobenzylidene malonitrile]
Tetraethyl pyrophosphate
Tetramethylenedisulfotetramine
Thiodiglycol (TDG)
Thiofanox
1,4-Thioxane
Triethanolamine (TEA)
Trimethyl phosphite
VE [phosphonothioic acid, ethyl-, S-(2-
(diethylamino)ethyl) O-ethyl ester]
VG [phosphonothioic acid, S-(2-(diethylamino)ethyl)
O,O-diethyl ester]
VM [phosphonothioic acid, methyl-, S-(2-
(diethylamino)ethyl) O-ethyl ester]
CASRN
57-24-9
77-81-6
2698-41-1
107-49-3
80-12-6
111-48-8
39196-18-4
15980-15-1
102-71-6
121-45-9
21738-25-0
78-53-5
21770-86-5
Analysis Purpose: Sample preparation
Sample Preparation Technique: PFE
Determinative Technique: EPA SW-846 Method 8015C, Method 8270D, or Method 832 IB. Refer to
Appendix A for which of these determinative methods should be used for a particular analyte.
Method Developed for: Organic compounds in soils, clays, sediments, sludges, and waste solids
Method Selected for: SAM lists this method for preparation of solid samples.
Detection and Quantitation: This method has been validated for solid matrices containing 250 to
12,500 ug/kg of semivolatile organic compounds, 250 to 2500 ug/kg of organophosphorus pesticides, 5 to
250 ug/kg of organochlorine pesticides, 50 to 5000 ug/kg of chlorinated herbicides, and 1 to 2500 ng/kg
of polychlorinated dibenzo-p-dioxins (PCDDs) / polychlorinated dibenzofurans (PCDFs).
Description of Method: Approximately 10 to 30 g of soil sample is prepared for extraction either by air
drying the sample, or by mixing the sample with anhydrous sodium sulfate or pelletized diatomaceous
earth. The sample is then ground and loaded into the extraction cell. The extraction cell containing the
sample is heated to the extraction temperature, pressurized with the appropriate solvent system, and
extracted for 5 minutes (or as recommended by the instrument manufacturer). The extract may be
concentrated, if necessary, and exchanged into a solvent compatible with the cleanup or determinative
step being employed.
Special Considerations: Sodium sulfate can cause clogging, and air drying or palletized diatomaceous
earth may be preferred.
Source: EPA. 1998. "Method 3545A (SW-846): Pressurized Fluid Extraction (PFE)," Revision 1.
j?pjy|2^^
SAM Revision 4.0
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Section 5 - Selected Chemical Methods
5.2.22 EPA Method 3570 (SW-846): Microscale Solvent Extraction (MSE)
Analyte(s)
Acrylamide
Acrylonitrile
Aldicarb (Temik)
Aldicarb sulfone
Aldicarb sulfoxide
4-Aminopyridine
BZ (Quinuclidinyl benzilate)
Brodifacoum
Bromadiolone
Carbofuran (Furadan)
Chlorfenvinphos
3-Chloro-1 ,2-propanediol
Chloropicrin
Chlorosarin
Chlorosoman
Chlorpyrifos
Crimidine
Cyclohexyl sarin (GF)
Dichlorvos
Dicrotophos
Diesel Range Organics
Diisopropyl methylphosphonate (DIMP)
Dimethylphosphite
Dimethylphosphoramidic acid
Diphacinone
Disulfoton
Disulfoton sulfoxide
1,4-Dithiane
EA2192 [Diisopropylaminoethyl
methylthiolophosphonate]
Ethylmethyl phosphonate (EMPA)
CASRN
79-06-1
107-13-1
116-06-3
1646-88-4
1646-87-3
504-24-5
6581-06-2
56073-10-0
28772-56-7
1563-66-2
470-90-6
96-24-2
76-06-2
1445-76-7
7040-57-5
2921-88-2
535-89-7
329-99-7
62-73-7
141-66-2
NA
1445-75-6
868-85-9
33876-51-6
82-66-6
298-04-4
2497-07-6
505-29-3
73207-98-4
1832-53-7
SAM Revision 4.0
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Section 5 - Selected Chemical Methods
Analyte(s)
N-Ethyldiethanolamine (EDEA)
Fenamiphos
Fentanyl
Fluoroacetamide
Fluoroacetic acid and fluoroacetate salts
Formaldehyde
Gasoline Range Organics
Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)
Hexamethylenetriperoxidediamine (HMTD)
Isopropyl methylphosphonic acid (IMPA)
Kerosene
Methamidophos
Methomyl
Methyl acrylonitrile
Methyl fluoroacetate
Methyl hydrazine
Methyl parathion
N-Methyldiethanolamine (MDEA)
1-Methylethyl ester ethylphosphonofluoridic acid (GE)
Methylphosphonic acid (MPA)
Mevinphos
Mustard, nitrogen (HN-1) [bis(2-chloroethyl)ethylamine]
Mustard, nitrogen (HN-2) [2,2'-dichloro-N-
methyldiethylamine N,N-bis(2-chloroethyl)methylamine]
Mustard, nitrogen (HN-3) [tris(2-chloroethyl)amine]
Mustard, sulfur/ Mustard gas (HD)
Nicotine compounds
Octahydro-1 ,3,5,7-tetranitro-1 ,3,5,7-tetrazocine (HMX)
Organophosphate pesticides, NOS
Oxamyl
Parathion
Pentaerythritol tetranitrate (PETN)
CASRN
139-87-7
22224-92-6
437-38-7
640-19-7
NA
50-00-0
NA
121-82-4
283-66-9
1832-54-8
64742-81-0
10265-92-6
16752-77-5
126-98-7
453-18-9
60-34-4
298-00-0
105-59-9
1189-87-3
993-13-5
7786-34-7
538-07-8
51-75-2
555-77-1
505-60-2
54-11-5
2691-41-0
NA
23135-22-0
56-38-2
78-11-5
SAM Revision 4.0
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Section 5 - Selected Chemical Methods
Analyte(s)
Phencyclidine
Phenol
Phorate
Phosphamidon
Pinacolyl methyl phosphonic acid (PMPA)
R 33 (VR) [methylphosphonothioic acid, S-[2-
(diethylamino)ethyl] O-2-methylpropyl ester]
Sarin (GB)
Semivolatile Organic Compounds, NOS
Soman (GD)
Strychnine
Tabun (GA)
Tear gas (CS) [chlorobenzylidene malonitrile]
Tetraethyl pyrophosphate
Tetramethylenedisulfotetramine
Thiodiglycol (TDG)
Thiofanox
1,4-Thioxane
Triethanolamine (TEA)
Trimethyl phosphite
1 ,3,5-Trinitrobenzene (1 ,3,5-TNB)
2,4,6-Trinitrotoluene(2,4,6-TNT)
VE [phosphonothioic acid, ethyl-, S-(2-
(diethylamino)ethyl) O-ethyl ester]
VG [phosphonothioic acid, S-(2-(diethylamino)ethyl)
O,O-diethyl ester]
VM [phosphonothioic acid, methyl-, S-(2-
(diethylamino)ethyl) O-ethyl ester]
VX[O-ethyl-S-(2-diisopropylaminoethyl)methyl-
phosphonothiolate]
White phosphorus
CASRN
77-10-1
108-95-2
298-02-2
13171-21-6
616-52-4
159939-87-4
107-44-8
NA
96-64-0
57-24-9
77-81-6
2698-41-1
107-49-3
80-12-6
111-48-8
39196-18-4
15980-15-1
102-71-6
121-45-9
99-35-4
118-96-7
21738-25-0
78-53-5
21770-86-5
50782-69-9
12185-10-3
Analysis Purpose: Sample preparation
Sample Preparation Technique: MSB
SAM Revision 4.0
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Section 5 - Selected Chemical Methods
Determinative Technique: EPA OW Method 300.1 Revision 1.0; EPA SW-846 Methods 7580, 8015C,
8270D, 8315A, 8316, 8318A, 8321B, and 8330B. Refer to Appendix A for which of these determinative
methods should be used for a particular analyte.
Method Developed for: Extracting volatile, semivolatile, and nonvolatile organic compounds from
solids such as soils, sludges, and wastes
Method Selected for: SAM lists this method for preparation of wipe samples.
Description of Method: Samples are prepared by shake extraction with an organic solvent in sealed
extraction tubes. Careful manipulation of the sample, solvent, drying agent, and spiking solutions during
the procedure minimizes loss of volatile compounds while maximizing extraction of volatile,
semivolatile, and nonvolatile compounds. Sample extracts are collected, dried, and concentrated using a
modification of the Kuderna-Danish concentration method or other appropriate concentration technique.
By increasing the number of theoretical plates and reducing the distillation temperature, extracts are
concentrated without loss of volatile constituents. Samples should be prepared one at a time to the point
of solvent addition (i.e., do not pre-weigh a number of samples then add the solvent). Samples should be
extracted as soon after collection as possible, and exposure to air before sample extraction is minimized
as much as possible.
Source: EPA. 2002. "Method 3570 (SW-846): Microscale Solvent Extraction (MSB)," Revision 0.
5.2.23 EPA Method 3571 (SW-846): Extraction of Solid and Aqueous Samples for
Chemical Agents
Analyte(s)
Mustard, sulfur/ Mustard gas (HD)
Sarin (GB)
VX [O-ethyl-S-(2-diisopropylaminoethyl)methyl-
phosphonothiolate]
CASRN
505-60-2
107-44-8
50782-69-9
Analysis Purpose: Sample preparation
Sample Preparation Technique: Solvent microextraction
Determinative Technique: EPA SW-846 Method 8270D
Method Developed for: HD, GB, and VX in concrete, charcoal, wood, water, brine, ash, coral, sand, and
soil
Method Selected for: SAM lists this method for preparation of solid, non-aqueous liquid/organic solid,
aqueous liquid, and drinking water samples.
Description of Method: This method provides procedures for sample collection and extraction of the
referenced compounds from solids and aqueous samples. A separate extract is required for each agent to
be measured. Glacial acetic acid is added as a preservative to samples being assayed for GB and glacial
acetic acid/sodium chloride is a preservative for samples assayed for HD. No preservative is added for
VX. Samples are extracted with 10% isopropanol in dichloromethane by vortex mixing and filtered, if
necessary. An optional water wash is included for VX that back-extracts the compound from heavy
organics that could interfere with the assay. An optional column cleanup procedure is described to
separate GB from heavy organics, if needed. Solvents are used to elute the extract first through the
CarboprepQO column, then the silica column.
SAM Revision 4.0 59 September 29, 2008
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Section 5 - Selected Chemical Methods
Source: EPA. 2007. "Method 3571 (SW-846): Extraction of Solid and Aqueous Samples for Chemical
Agents," Revision 0. httjr//v\mw1ej3^
5.2.24 EPA Method 3580A (SW-846): Waste Dilution
Analyte(s)
Brodifacoum
Bromadiolone
BZ (Quinuclidinyl benzilate)
Chlorfenvinphos
3-Chloro-1 ,2-propanediol
Chloropicrin
Chlorosarin
Chlorosoman
Chlorpyrifos
Crimidine
Cyclohexyl sarin (GF)
Dichlorvos
Dicrotophos
Diesel Range Organics
Diisopropyl methylphosphonate (DIMP)
Dimethylphosphite
Dimethylphosphoramidic acid
Diphacinone
Disulfoton
Disulfoton sulfoxide
1,4-Dithiane
EA2192 [Diisopropylaminoethyl
methylthiolophosphonate]
Ethyl methylphosphonic acid (EMPA)
Ethyldichloroarsine (ED)
N-Ethyldiethanolamine (EDEA)
Fenamiphos
Fentanyl
CASRN
56073-10-0
28772-56-7
6581-06-2
470-90-6
96-24-2
76-06-2
1445-76-7
7040-57-5
2921-88-2
535-89-7
329-99-7
62-73-7
141-66-2
NA
1445-75-6
868-85-9
33876-51-6
82-66-6
298-04-4
2497-07-6
505-29-3
73207-98-4
1832-53-7
598-14-1
139-87-7
22224-92-6
437-38-7
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Section 5 - Selected Chemical Methods
Analyte(s)
Fluoroacetamide
Isopropyl methylphosphonic acid (IMPA)
Methamidophos
Methyl hydrazine
Methyl parathion
N-Methyldiethanolamine (MDEA)
1-Methylethyl ester ethylphosphonofluoridic acid (GE)
Methylphosphonic acid (MPA)
Mevinphos
Mustard, nitrogen (HN-1) [bis(2-chloroethyl)ethylamine]
Mustard, nitrogen (HN-2) [2,2'-dichloro-N-
methyldiethylamine N,N-bis(2-chloroethyl)methylamine]
Mustard, nitrogen (HN-3) [tris(2-chloroethyl)amine]
Nicotine compounds
Organophosphate pesticides, NOS
Parathion
Phencyclidine
Phenol
Phorate
Phosphamidon
Pinacolyl methyl phosphonic acid (PMPA)
R 33 (VR) [methylphosphonothioic acid, S-[2-
(diethylamino)ethyl] O-2-methylpropyl ester]
Semivolatile Organic Compounds, NOS
Sodium azide (analyze for hydrazoic acid)
Soman (GD)
Strychnine
Tabun (GA)
Tear gas (CS) [chlorobenzylidene malonitrile]
Tetraethyl pyrophosphate
Tetramethylenedisulfotetramine
Thiodiglycol (TDG)
CASRN
640-19-7
1832-54-8
10265-92-6
60-34-4
298-00-0
105-59-9
1189-87-3
993-13-5
7786-34-7
538-07-8
51-75-2
555-77-1
54-11-5
NA
56-38-2
77-10-1
108-95-2
298-02-2
13171-21-6
616-52-4
159939-87-4
NA
26628-22-8
96-64-0
57-24-9
77-81-6
2698-41-1
107-49-3
80-12-6
111-48-8
SAM Revision 4.0
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Section 5 - Selected Chemical Methods
Analyte(s)
Thiofanox
1,4-Thioxane
Triethanolamine (TEA)
Trimethyl phosphite
VE [phosphonothioic acid, ethyl-, S-(2-
(diethylamino)ethyl) O-ethyl ester]
VG [phosphonothioic acid, S-(2-(diethylamino)ethyl)
O,O-diethyl ester]
VM [phosphonothioic acid, methyl-, S-(2-
(diethylamino)ethyl) O-ethyl ester]
CASRN
39196-18-4
15980-15-1
102-71-6
121-45-9
21738-25-0
78-53-5
21770-86-5
Analysis Purpose: Sample preparation
Sample Preparation Technique: Waste dilution
Determinative Technique: EPA SW-846 Method 8015C, Method 8270D, or Method 832 IB. Refer to
Appendix A for which of these determinative methods should be used for a particular analyte.
Method Developed for: Organic compounds in non-aqueous waste samples
Method Selected for: SAM lists this method for preparation of non-aqueous liquid/organic solid
samples.
Description of Method: This method describes solvent dilution of a non-aqueous waste sample prior to
cleanup and/or analysis. One gram of sample is weighed into a capped tube and the sample is diluted to
10.0 mL with an appropriate solvent.
Special Considerations: The method is designed for wastes that may contain organic chemicals at a
concentration greater than 20,000 mg/kg and that are soluble in the dilution solvent.
Source: EPA. 1992. "Method 3580A (SW-846): Waste Dilution," Revision 1.
http://www.epa.gov/sam/pdfs/EPA-3580a.pdf
5.2.25 EPA Method 3585 (SW-846): Waste Dilution for Volatile Organics
Analyte(s)
Allyl alcohol
Carbon disulfide
2-Chloroethanol
Cyanogen chloride
1,2-Dichloroethane
Ethylene oxide
Gasoline Range Organics
Kerosene
CASRN
107-18-6
75-15-0
107-07-3
506-77-4
107-06-2
75-21-8
NA
64742-81-0
SAM Revision 4.0
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Section 5 - Selected Chemical Methods
Analyte(s)
Propylene oxide
CASRN
75-56-9
The following analytes should be prepared by this method (and determined by the corresponding SW-846 Method
826QC) only if problems (e.g., insufficient recovery, interferences) occur when using the sample
preparation/determinative techniques identified for these analytes in Appendix A.
1,4-Dithiane
1,4-Thioxane
505-29-3
15980-15-1
Analysis Purpose: Sample preparation
Sample Preparation Technique: Waste dilution
Determinative Technique: EPA SW-846 Method 8015C or Method 8260C. Refer to Appendix A for
which of these determinative methods should be used for a particular analyte.
Method Developed for: VOCs in non-aqueous waste
Method Selected for: SAM lists this method for preparation of non-aqueous liquid/organic solid
samples.
Description of Method: This method describes solvent dilution of a non-aqueous waste sample prior to
direct injection analysis. It is designed for use in conjunction with GC or GC-MS analysis of wastes that
may contain organic chemicals at a concentration greater than 1 mg/kg and that are soluble in the dilution
solvent. Highly contaminated or highly complex samples may be diluted prior to analysis for volatiles
using direct injection. One gram of sample is weighed into a capped tube or volumetric flask. The
sample is diluted to 2.0 to 10.0 mL with «-hexadecane or other appropriate solvent. Diluted samples are
injected into the GC or GC-MS for analysis.
Special Considerations: For use in analysis of wastes that may contain organic chemicals at a
concentration greater than 1 mg/kg and that are soluble in the dilution solvent.
Source: EPA. 1996. "Method 3585 (SW-846): Waste Dilution for Volatile Organics," Revision 0.
http://www.epa.gov/sam/pclfs/EP A-3585.pdf
5.2.26 EPA Method 5030C (SW-846): Purge-and-Trap for Aqueous Samples
Analyte(s)
Allyl alcohol
Carbon disulfide
2-Chloroethanol
Cyanogen chloride
1,2-Dichloroethane
Ethylene oxide
Gasoline Range Organics
Kerosene
Propylene oxide
CASRN
107-18-6
75-15-0
107-07-3
506-77-4
107-06-2
75-21-8
NA
64742-81-0
75-56-9
SAM Revision 4.0
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Section 5 - Selected Chemical Methods
Analyte(s)
CASRN
The following analytes should be prepared by this method (and determined by the corresponding SW-846 Method
8260C) only if problems (e.g., insufficient recovery, interferences) occur when using the sample
preparation/determinative techniques identified for these anaiytes in Appendix A.
1,4-Dithiane
1,4-Thioxane
505-29-3
15980-15-1
Analysis Purpose: Sample preparation
Sample Preparation Technique: Purge and trap
Determinative Technique: EPA SW-846 Method 8015C or Method 8260C. Refer to Appendix A for
which of these determinative methods should be used for a particular analyte.
Method Developed for: VOCs in aqueous and water miscible liquid samples
Method Selected for: SAM lists this method for preparation of aqueous liquid and/or drinking water
samples. For carbon disulfide, and 1,2-dichloroethane, EPA Method 524.2 (rather than Method 5030C)
should be used for preparation of drinking water samples.
Description of Method: This method describes a purge-and-trap procedure for the analysis of VOCs in
aqueous liquid samples and water miscible liquid samples. An inert gas is bubbled through a portion of
the aqueous liquid sample at ambient temperature, and the volatile components are transferred from the
aqueous liquid phase to the vapor phase. The vapor is swept through a sorbent column where the volatile
components are adsorbed. After purging is completed, the sorbent column is heated and backflushed with
inert gas to desorb the components onto a GC column.
Source: EPA. 2003. "Method 5030C (SW-846): Purge-and-Trap for Aqueous Samples, Revision 3.
http://www.epa.gov/sam/pdfs/EPA-5030c.pdf
5.2.27 EPA Method 5035A (SW-846): Closed-System Purge-and-Trap and Extraction for
Volatile Organics in Soil and Waste Samples
Analyte(s)
Allyl alcohol
Carbon disulfide
2-Chloroethanol
Cyanogen chloride
1,2-Dichloroethane
Ethylene oxide
Gasoline Range Organics
Kerosene
Propylene oxide
CASRN
107-18-6
75-15-0
107-07-3
506-77-4
107-06-2
75-21-8
NA
64742-81-0
75-56-9
SAM Revision 4.0
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Section 5 - Selected Chemical Methods
Analyte(s)
CASRN
The following analytes should be prepared by this method (and determined by the corresponding SW-846 Method
8260C) only if problems (e.g., insufficient recovery, interferences) occur when using the sample
preparation/determinative techniques identified for these anaiytes in Appendix A.
1,4-Dithiane
1,4-Thioxane
505-29-3
15980-15-1
Analysis Purpose: Sample preparation
Sample Preparation Technique: Purge and trap
Determinative Technique: EPA SW-846 Method 8015C or Method 8260C. Refer to Appendix A for
which of these determinative methods should be used for a particular analyte.
Method Developed for: VOCs in solid materials (e.g., soils, sediments, and solid waste) and oily wastes
Method Selected for: SAM lists this method for preparation of solid samples.
Description of Method: This method describes a closed-system purge-and-trap process for analysis of
VOCs in solid samples containing low levels (0.5 to 200 ug/kg) of VOCs. The method also provides
specific procedures for preparation of samples containing high levels (>200 ug/kg) of VOCs. For low-
level VOCs, a 5-g sample is collected into a vial that is placed into an autosampler device. Reagent
water, surrogates, and internal standards are added automatically, and the vial is heated to 40°C. The
volatiles are purged into an appropriate trap using an inert gas combined with sample agitation. When
purging is complete, the trap is heated and backflushed with helium to desorb the trapped sample
components into a GC for analysis. For high-level VOCs, samples are either collected into a vial that
contains a water-miscible organic solvent or a portion of sample is removed from the vial and dispersed in
a water-miscible solvent. An aliquot of the solvent is added to reagent water, along with surrogates and
internal standards, then purged and analyzed using an appropriate determinative method (e.g., Method
8015C or 8260C (SW-846)).
Source: EPA. 2002. "Method 5035A (SW-846): Closed-System Purge-and-Trap and Extraction for
Volatile Organics in Soil and Waste Samples," Draft Revision 1. http://www.epa.gov/sam/pdfs/EPA-
5.2.28 EPA Method 6010C (SW-846): Inductively Coupled Plasma - Atomic Emission
Spectrometry
Analyte(s)
Ammonium metavanadate
Arsenic, Total
Arsenic trioxide
Calcium arsenate
2-Chlorovinylarsonous acid (CVAA)
Lead arsenate
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine]
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine]
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine]
CASRN
7803-55-6
740-38-2
1327-53-3
7778-44-1
85090-33-1
7645-25-2
541-25-3
40334-69-8
40334-70-1
SAM Revision 4.0
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Section 5 - Selected Chemical Methods
Analyte(s)
Lewisite Oxide
Osmium tetroxide
Sodium arsenite
Thallium sulfate
Titanium tetrachloride
Vanadium pentoxide
CASRN
1306-02-1
20816-12-0
7784-46-5
10031-59-1
7550-45-0
1314-62-1
Analysis Purpose: Analysis
Sample Preparation Technique: EPA SW-846 Method 3050B (solid samples), Method 3031 (non-
aqueous liquid/organic solid samples), and NIOSH Method 9102 (wipe samples)
Determinative Technique: ICP-AES
Method Developed for: Trace elements in solution
Method Selected for: SAM lists this method for analysis of solid, non-aqueous liquid/organic solid, and
wipe samples. Osmium tetroxide and titanium tetrachloride are not of concern in non-aqueous
liquid/organic solid samples.
Detection and Quantitation: Detection limits vary with each analyte. Estimated instrument detection
limits (IDLs) for arsenic and titanium are 30 \ig/L and 5.0 jig/L, respectively. The upper end of the
analytical range may be extended by sample dilution.
Description of Method: This method determines arsenic trioxide, lewisite, lewisite degradation
products, and sodium arsenite as total arsenic; osmium tetroxide as osmium; thallium sulfate as thallium;
titanium tetrachloride as titanium; and ammonium metavanadate and vanadium pentoxide as total
vanadium. Soil samples (prepared using SW-846 Methods 3050B), and non-aqueous liquid/organic solid
samples (prepared using SW-846 Methods 3031) are analyzed by ICP-AES.
Special Considerations: Laboratory testing is currently underway for speciation of lewisite 1 using GC-
MS techniques.
Source: EPA. 2000. "Method 6010C (SW-846): Inductively Coupled Plasma-Atomic Emission
Spectrometry," Revision 3. http://www.epa.gov/sam/pdfs/EPA-601 Oc.pdf
5.2.29 EPA Method 6020A (SW-846): Inductively Coupled Plasma - Mass Spectrometry
Analyte(s)
Ammonium metavanadate
Arsenic, Total
Arsenic trioxide
Calcium arsenate
2-Chlorovinylarsonous acid (CVAA)
Lead arsenate
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine]
CASRN
7803-55-6
740-38-2
1327-53-3
7778-44-1
85090-33-1
7645-25-2
541-25-3
SAM Revision 4.0
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Section 5 - Selected Chemical Methods
Analyte(s)
Lewisite 2
(L-2) [bis(2-chlorovinyl)chloroarsine]
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine]
Lewisite oxide
Sodium arsenite
Thallium sulfate
Titanium tetrachloride
Vanadium pentoxide
CASRN
40334-69-8
40334-70-1
1306-02-1
7784-46-5
10031-59-1
7550-45-0
1314-62-1
Analysis Purpose: Analysis
Sample Preparation Technique: EPA SW-846 Method 3050B (solid samples), Method 3031 (non-
aqueous liquid/organic solid samples), and NIOSH Method 9102 (wipe samples)
Determinative Technique: ICP-MS
Method Developed for: Elements in water samples and in waste extracts or digests
Method Selected for: SAM lists this method for analysis of solid, non-aqueous liquid/organic solid, and
wipe samples. Titanium tetrachloride is not of concern in non-aqueous liquid/organic solid samples.
Detection and Quantitation: In relatively simple sample types, detection limits will generally be below
0.1 (ig/L. Less sensitive elements, such as arsenic, may have detection limits of 1.0 (ig/L or higher. The
upper end of the analytical range may be extended by sample dilution.
Description of Method: This method will determine arsenic trioxide, lewisite, lewisite degradation
products, and sodium arsenite as total arsenic. The method also will determine thallium sulfate as total
thallium, titanium tetrachloride as titanium, and ammonium metavanadate and vanadium pentoxide as
total vanadium. Aqueous samples (prepared using SW-846 Method 5050), soil samples (prepared using
SW-846 Methods 3050B or 5050), non-aqueous liquid/organic solid samples (prepared using SW-846
Methods 3050B or 3031), and air filter/particle samples (prepared using IO Method 3.5) are analyzed by
ICP-MS. IDLs, sensitivities, and linear ranges vary with sample type, instrumentation, and operation
conditions.
Special Considerations: Laboratory testing is currently underway for speciation of lewisite 1 using GC-
MS techniques.
Source: EPA. 1998. "Method 6020A (SW-846): Inductively Coupled Plasma-Mass Spectrometry,"
Revision 1. http://www.cpa.gov/sam/pdfh/inPA-6020a.pdf
5.2.30 EPA Method 7010 (SW-846): Graphite Furnace Atomic Absorption
Spectrophotometry
Analyte(s)
Arsine
CASRN
7784-42-1
Analysis Purpose: Analysis
Sample Preparation Technique: EPA SW-846 Method 3050B (solid samples) and NIOSH Method
9102 (wipe samples)
Determinative Technique: GFAA
SAM Revision 4.0
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Section 5 - Selected Chemical Methods
Method Developed for: Metals in environmental samples including, but not limited to, ground water,
domestic and industrial wastes, extracts, soils, sludges, sediments, and similar wastes
Method Selected for: SAM lists this method for analysis of solid and wipe samples.
Detection and Quantitation: Detection limits vary with each sample type and instrument used. The
analytical range may be extended by sample dilution.
Description of Method: This method determines arsine as arsenic in environmental samples. Soil
samples (prepared using SW-846 Method 3050B) are analyzed by GFAA. A representative aliquot of the
sample is placed in the graphite tube in the furnace, evaporated to dryness, charred, and atomized.
Source: EPA. 1998. "Method 7010 (SW-846): Graphite Furnace Atomic Absorption
Spectrophotometry," Revision 0. http://www.epa.gov/sam/pdfs/EPA-7010.pdf
5.2.31 EPA Method 7470A (SW-846): Mercury in Liquid Wastes (Manual Cold-Vapor
Technique)
Analyte(s)
Mercury, Total
Methoxyethylmercuric acetate
CASRN
7439-97-6
151-38-2
Analysis Purpose: Sample preparation and/or analysis
Sample Preparation Technique: Acid digestion (solid and aqueous liquid samples) and NIOSH Method
9102 (wipe samples)
Determinative Technique: CVAA
Method Developed for: Mercury in mobility-procedure extracts, aqueous wastes, and ground waters
Method Selected for: SAM lists this method for use if problems occur when using EPA SW-846
Method 7473 for these analytes during preparation and analysis of aqueous liquid samples. (See Footnote
13 of Appendix A.)
Detection and Quantitation: The detection limit for the method is 0.2 p:g/L.
Description of Method: A 100-mL aqueous sample is digested with acids, permanganate solution,
persulfate solution, and heat. The sample is cooled and reduced with hydroxylamine-sodium chloride
solution. Just prior to analysis, the sample is treated with Sn(II), reducing the mercury to Hg(0). The
reduced sample is sparged and the mercury vapor is analyzed by CVAA.
Special Considerations: Chloride and copper are potential interferences.
Source: EPA. 1994. "Method 7470A (SW-846): Mercury in Liquid Waste (Manual Cold-Vapor
Technique)," Revision 1. http://www.epa.gov/sam/pdfs/EPA-7470a.pdf
5.2.32 EPA Method 7471B (SW-846): Mercury in Solid or Semisolid Wastes (Manual Cold-
Vapor Technique)
Analyte(s)
Mercury, Total
Methoxyethylmercuric acetate
CASRN
7439-97-6
151-38-2
SAM Revision 4.0 68 September 29, 2008
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Section 5 - Selected Chemical Methods
Analysis Purpose: Sample preparation and/or analysis
Sample Preparation Technique: Acid digestion (solid and aqueous liquid samples) and NIOSH Method
9102 (wipe samples)
Determinative Technique: CVAA
Method Developed for: Total mercury in soils, sediments, bottom deposits, and sludge-type materials
Method Selected for: SAM lists this method for use if problems occur when using EPA SW-846
Method 7473 for these analytes during preparation and analysis of solid and wipe samples. (See Footnote
13 of Appendix A.)
Description of Method: A 0.5-g to 0.6-g sample is digested with aqua regia, permanganate solution, and
heat. The sample is cooled and reduced with hydroxylamine-sodium chloride solution. Just prior to
analysis, the sample is treated with Sn(II), reducing the mercury to Hg(0). The reduced sample is sparged
and the mercury vapor is analyzed by CVAA.
Special Considerations: Chloride and copper are potential interferences.
Source: EPA. 1998. "Method 7471B (SW-846): Mercury in Solid or Semisolid Waste (Manual Cold-
Vapor Technique)," Revision 2. hjt]i;//wwvj^
5.2.33 EPA Method 7473 (SW-846): Mercury in Solids and Solutions by Thermal
Decomposition, Amalgamation, and Atomic Absorption Spectrophotometry
Analyte(s)
Mercury, Total
Methoxyethylmercuric acetate
CASRN
7439-97-6
151-38-2
Analysis Purpose: Sample preparation and/or analysis
Sample Preparation Technique: Thermal decomposition (solid and aqueous liquid samples) and
NIOSH Method 9102 (wipe samples)
Determinative Technique: Spectrophotometry
Method Developed for: Total mercury in solids, aqueous samples, and digested solutions
Method Selected for: SAM lists this method for preparation and analysis of solid, aqueous liquid, and
wipe samples.
Detection and Quantitation: The IDL is 0.01 ng total mercury. The typical working range for this
method is 0.05 to 600 ng.
Description of Method: Controlled heating in an oxygenated decomposition furnace is used to liberate
mercury from solid and aqueous samples. The sample is dried and then thermally and chemically
decomposed within the furnace. The decomposition products are carried by flowing oxygen to the
catalytic section of the furnace, where oxidation is completed and halogens and nitrogen/sulfur oxides are
trapped. The remaining decomposition products are then carried to an amalgamator that selectively traps
mercury. After the system is flushed with oxygen to remove any remaining gases or decomposition
products, the amalgamator is rapidly heated, releasing mercury vapor. Flowing oxygen carries the
mercury vapor through absorbance cells positioned in the light path of a single wavelength atomic
absorption spectrophotometer. Absorbance (peak height or peak area) is measured at 253.7 nm as a
function of mercury concentration.
Special Considerations: If equipment is not available, use CVAA Methods 747IB (EPA SW-846) for
solid samples and 7470A (EPA SW-846) for aqueous liquid samples.
SAM Revision 4.0 69 September 29, 2008
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Section 5 - Selected Chemical Methods
Source: EPA. 1998. "Method 7473 (SW-846): Mercury in Solids and Solutions by Thermal
Decomposition, Amalgamation, and Atomic Absorption Spectrophotometry," Revision 0.
^
5.2.34 EPA Method 7580 (SW-846): White Phosphorus (P4) by Solvent Extraction and Gas
Chromatography
Analyte(s)
White Phosphorus
CASRN
12185-10-3
Analysis Purpose: Sample preparation and/or analysis
Sample Preparation Technique: Solvent extraction (solid, non-aqueous liquid/organic solid, aqueous
liquid, and drinking water samples) and EPA SW-846 Method 3570/8290A Appendix A (wipe samples)
Determinative Technique: GC-NPD
Method Developed for: White phosphorus in soil, sediment, and water
Method Selected for: SAM lists this method for preparation and analysis of solid, non-aqueous
liquid/organic solid, aqueous liquid, drinking water, and wipe samples.
Detection and Quantitation: MDLs for reagent water, well water, and pond water were calculated to be
0.008, 0.009, 0.008 jig/L, respectively. MDLs for sand, a sandy loam soil (Lebanon soil), and soil from
the Rocky Mountain Arsenal (USAEC soil) were calculated to be 0.02, 0.43, 0.07 jig/kg, respectively.
This procedure provides sensitivity on the order of 0.01 \igfL.
Description of Method: Method 7580 may be used to determine the concentration of white phosphorus
in soil, sediment, and water samples using solvent extraction and GC. Water samples are extracted by
one of two procedures, depending on the sensitivity required. For the more sensitive procedure, a 500-
mL water sample is extracted with 50 mL of diethyl ether. The extract is concentrated by back extraction
with reagent water, yielding a final extract volume of approximately 1.0 mL. A 1.0 \iL aliquot of this
extract is injected into a GC equipped with an NPD. Wet soil or sediment samples are analyzed by
extracting a 40 g wet-weight aliquot of the sample with a mixture of 10.0 mL degassed reagent water and
10.0 mL isooctane. The extraction is performed in a glass jar on a platform shaker for 18 hours. A 1.0
\iL aliquot of the extract is analyzed by GC-NPD, providing sensitivity on the order of 1 jig/kg.
Special Considerations: The presence of white phosphorus should be confirmed by either a secondary
GC column or by an MS.
Source: EPA. 1996. "Method 7580 (SW-846): White Phosphorus (P4) by Solvent Extraction and Gas
Chromatography," Revision 0. http://wmw.epa.gov/sam/pdfs/EPA-7580.pdf
5.2.35 EPA Method 8015C (SW-846): Nonhalogenated Organics Using GC/FID
Analyte(s)
Diesel Range Organics
Gasoline Range Organics
Kerosene
CASRN
NA
NA
64742-81-0
SAM Revision 4.0 70 September 29, 2008
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Section 5 - Selected Chemical Methods
Analysis Purpose: Analysis
Sample Preparation Technique: EPA SW-846 Method 3545A/3541 or Method 5035A (solid samples),
Method 3580A or Method 3585 (non-aqueous liquid/organic solid samples), Method 3535A or 5030C
(aqueous liquid and drinking water samples), and Method 3570/8290A Appendix A (wipe samples).
Refer to Appendix A for which of these preparation methods should be used for a particular
analyte/sample type combination.
Determinative Technique: Gas chromatograph - Flame ionization detector (GC-FID)
Method Developed for: Various nonhalogenated VOCs and semivolatile organic compounds in water
samples
Method Selected for: SAM lists this method for analysis of solid, non-aqueous liquid/organic solid,
aqueous liquid, drinking water, and wipe samples.
Detection and Quantitation: The estimated MDLs vary with each analyte and range between 2 and 48
Vig/L for aqueous liquid samples. The MDLs in other matrices have not been evaluated. The analytical
range depends on the target analyte (s) and the instrument used.
Description of Method: This method provides GC conditions for the detection of certain
nonhalogenated volatile and semivolatile organic compounds. Depending on the analytes of interest,
samples may be introduced into the GC by a variety of techniques including purge-and-trap, direct
injection of aqueous liquid samples, and solvent extraction. An appropriate column and temperature
program are used in the GC to separate the organic compounds. Detection is achieved by a flame
ionization detector (FID). The method allows the use of packed or capillary columns for the analysis and
confirmation of the non-halogenated individual analytes.
Special Considerations: The presence of the analytes listed in the table above should be confirmed by
either a secondary GC column or by an MS.
Source: EPA. 2000. "Method 8015C (SW-846): Nonhalogenated Organics Using GC/FID," Revision 3.
http://www.epa.gov/sam/pdfs/EPA-8015c.pdf
5.2.36 EPA Method 8260C (SW-846): Volatile Organic Compounds by Gas
Chromatography-Mass Spectrometry (GC/MS)
Analyte(s)
Allyl alcohol
Carbon disulfide
2-Chloroethanol
Cyanogen chloride
1,2-Dichloroethane
Ethylene oxide
Propylene oxide
CASRN
107-18-6
75-15-0
107-07-3
506-77-4
107-06-2
75-21-8
75-56-9
The following analytes should be determined by this method (and corresponding sample preparation methods)
only if problems (e.g., insufficient recovery, interferences) occur when using the sample preparation/determinative
techniques identified for these analytes in Appendix A.
1,4-Dithiane
1,4-Thioxane
505-29-3
15980-15-1
SAM Revision 4.0 71 September 29, 2008
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Section 5 - Selected Chemical Methods
Analysis Purpose: Analysis
Sample Preparation Technique: EPA SW-846 Method 5035A (solid samples), Method 3585 (non-
aqueous liquid/organic solid samples), and Method 5030C (aqueous liquid and drinking water samples)
Determinative Technique: GC-MS
Method Developed for: Applicable to nearly all types of samples, regardless of water content, including
various air sampling trapping media, ground and surface water, aqueous sludges, caustic liquors, acid
liquors, waste solvents, oily wastes, mousses, tars, fibrous wastes, polymeric emulsions, filter cakes, spent
carbons, spent catalysts, soils, and sediments.
Method Selected for: SAM lists this method for analysis of solid, non-aqueous liquid/organic solid,
aqueous liquid, and drinking water samples. For carbon disulfide and 1,2-dichloroethane only, EPA
Method 524.2 (rather than 8260C) should be used for analysis of drinking water samples.
Detection and Quantitation: Using standard quadrupole instrumentation and the purge-and-trap
technique, estimated quantitation limits are 5 jig/kg (wet weight) for soil/sediment samples and 5 \ig/L for
ground water. Somewhat lower limits may be achieved using an ion trap MS or other instrumentation of
improved design. No matter which instrument is used, estimated quantitation limits (EQLs) will be
proportionately higher for sample extracts and samples that require dilution or when a reduced sample
size is used to avoid saturation of the detector. The EQL for an individual analyte is dependent on the
instrument as well as the choice of sample preparation/introduction method.
Description of Method: Volatile compounds are introduced into a GC by purge-and-trap or other
procedures (see Section 1.2 in Method 8260C). The analytes can be introduced directly to a wide-bore
capillary column or cryofocused on a capillary pre-column before being flash evaporated to a narrow-bore
capillary for analysis. Alternatively, the effluent from the trap is sent to an injection port operating in the
split mode for injection to a narrow-bore capillary column. The column is temperature-programmed to
separate the analytes, which are then detected with a MS interfaced to the GC. Analytes eluted from the
capillary column are introduced into the MS via a jet separator or a direct connection.
Source: EPA. 2006. "Method 8260C (SW-846): Volatile Organic Compounds by Gas
Chromatography/Mass Spectrometry (GC/MS)," Revision 3. httjK//wjroyp^
8260c.pdf
5.2.37 EPA Method 8270D (SW-846): Semivolatile Organic Compounds by Gas
Chromatography/Mass Spectrometry (GC-MS)
Analyte(s)
Chlorfenvinphos
3-Chloro-1 ,2-propanediol1
Chloropicrin2
Chlorosarin
Chlorosoman
Chlorpyrifos
Crimidine3
Cyclohexyl sarin (GF)
Dichlorvos
Dicrotophos
CASRN
470-90-6
96-24-2
76-06-2
1445-76-7
7040-57-5
2921-88-2
535-89-7
329-99-7
62-73-7
141-66-2
SAM Revision 4.0 72 September 29, 2008
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Section 5 - Selected Chemical Methods
Analyte(s)
Dimethylphosphite
Disulfoton
Disulfoton sulfoxide
1,4-Dithiane4
Ethyldichloroarsine (ED)
Fenamiphos
Fluoroacetamide
Methamidophos
Methyl hydrazine
Methyl parathion
1-Methylethyl ester ethylphosphonofluoridic acid (GE)
Mevinphos
Mustard, nitrogen (HN-1) [bis(2-chloroethyl)ethylamine]
Mustard, nitrogen (HN-2) [2,2'-dichloro-N-
methyldiethylamine N,N-bis(2-chloroethyl)methylamine]
Mustard, nitrogen (HN-3) [tris(2-chloroethyl)amine]
Mustard, sulfur/ Mustard gas (HD)5
Nicotine compounds
Organophosphate pesticides, NOS
Parathion
Phencyclidine
Phenol
Phorate
Phosphamidon
R 33 (VR) [methylphosphonothioic acid, S-[2-
(diethylamino)ethyl] O-2-methylpropyl ester]
Sarin (GB)5
Semivolatile Organic Compounds, NOS
Soman (GD)
Strychnine
Tabun (GA)
Tear gas (CS) [chlorobenzylidene malonitrile]
CASRN
868-85-9
298-04-4
2497-07-6
505-29-3
598-14-1
22224-92-6
640-19-7
10265-92-6
60-34-4
298-00-0
1189-87-3
7786-34-7
538-07-8
51-75-2
555-77-1
505-60-2
54-11-5
NA
56-38-2
77-10-1
108-95-2
298-02-2
13171-21-6
159939-87-4
107-44-8
NA
96-64-0
57-24-9
77-81-6
2698-41-1
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Section 5 - Selected Chemical Methods
Analyte(s)
Tetraethyl pyrophosphate
Tetramethylenedisulfotetramine2'6
Thiodiglycol (TDG)
1,4-Thioxane4
Trimethyl phosphite2
VE [phosphonothioic acid, ethyl-, S-(2-
(diethylamino)ethyl) O-ethyl ester]
VG [phosphonothioic acid, S-(2-(diethylamino)ethyl)
O,O-diethyl ester]
VM [phosphonothioic acid, methyl-, S-(2-
(diethylamino)ethyl) O-ethyl ester]
VX [O-ethyl-S-(2-diisopropylaminoethyl)methyl-
phosphonothiolate]5
CASRN
107-49-3
80-12-6
111-48-8
15980-15-1
121-45-9
21738-25-0
78-53-5
21770-86-5
50782-69-9
The following analyte should be determined by this method only if liquid chromatography-mass spectrometry (LC-
MS) [electros pray] procedures are not available to the laboratory. Sample preparation methods should remain the
same.
BZ (Quinuclidinyl benzilate)1
Diisopropyl methylphosphonate (DIMP)
Dimethylphosphoramidic acid1
EA2192 [Diisopropylaminoethyl
methylthiolophosphonate]1
Ethylmethyl phosphonate (EMPA)1
Isopropyl methylphosphonic acid (IMPA)1
Methylphosphonic acid (MPA)1
Pinacolyl methyl phosphonic acid (PMPA)1
6581-06-2
1445-75-6
33876-51-6
73207-98-4
1832-53-7
1832-54-8
993-13-5
616-52-4
1 For this analyte, SW-846 Method 8270D must be modified to include a derivatization step.
2 If proplems occur with analyses, lower the injection temperature.
3 If problems occur when using this method, it is recommended that SW-846 Method 8321B be used. Sample
preparation methods should remain the same.
4 If problems occur when using this method, it is recommended that SW-846 Method 8260C and appropriate
corresponding sample preparation procedures (i.e., Method 5035A for solid samples, Method 3585 for non-aqueous
liquid/organic solid samples, and Method 5030C for aqueous liquid and drinking water samples) be used.
5 For this analyte, refer to EPA SW-846 Method 8271 forGC-MS conditions.
6 This analyte may require selective ion monitoring (SIM) analyses.
Analysis Purpose: Analysis
Sample Preparation Technique: EPA SW-846 Method 3545A/3541 (solid samples), Method 3580A
(non-aqueous liquid/organic solid samples), Method 3520C/3535A (aqueous liquid and drinking water
samples), and Method 3570/8290A Appendix A or NIOSH 9102 (wipe samples). Refer to Appendix A
for which of these preparation methods should be used for a particular analyte/sample type combination.
Determinative Technique: GC-MS
SAM Revision 4.0
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Section 5 - Selected Chemical Methods
Method Developed for: Semivolatile organic compounds in extracts prepared from many types of solid
waste matrices, soils, air sampling media and water samples
Method Selected for: SAM lists this method for analysis of solid, non-aqueous liquid/organic solid,
aqueous liquid, drinking water, and/or wipe samples. Please note: drinking water samples for dichlorvos,
disulfoton, disulfoton sulfoxide, fenamiphos, mevinphos, and semivolatile organic compounds NOS
should be prepared and analyzed by EPA Method 525.2; aqueous liquid and drinking water samples for
organophosphate pesticides NOS should be prepared and analyzed by EPA Methods 614 and 507,
respectively; aqueous liquid and drinking water samples for chloropicrin should be prepared and analyzed
by EPA Method 551.1; all other analyte/sample type combinations should be analyzed by this method
(SW-8468270D).
Detection and Quantitation: The EDLs vary with each analyte and range between 10 and 1000 ug/L for
aqueous liquid samples and 660 and 3300 ug/kg for soil samples. The analytical range depends on the
target analyte (s) and the instrument used.
Description of Method: Samples are prepared for analysis by GC-MS using the appropriate sample
preparation and, if necessary, sample cleanup procedures. Semivolatile compounds are introduced into
the GC-MS by injecting the sample extract into a GC with a narrow-bore fused-silica capillary column.
The GC column is temperature-programmed to separate the analytes, which are then detected with a MS
connected to the GC. Analytes eluted from the capillary column are introduced into the MS. For the
determination of 3-chloro-l,2-propanediol, dimethylphosphoramidic acid, EA2192, EMPA, IMPA, MPA,
and pinacolyl methyl phosphonic acid, a derivatization step is required prior to injection into the GC-MS.
The phosphonic acids require derivatization with a trimethylsilyl agent and 3-chloro-l,2-propanediol
requires derivatization with a heptafluorobutyryl agent.
Special Considerations: Refer to footnotes provided in analyte table above for special considerations
that should be applied when measuring specific analytes.
Source: EPA. 1998. "Method 8270D (SW-846): Semivolatile Organic Compounds by Gas
Chromatography/ Mass Spectrometry (GC/MS)," Revision 4. http://www.epa.gov/sam/pdfs/EPA-
ggTOcLpdf
5.2.38 EPA Method 8290A, Appendix A (SW-846): Procedure for the Collection, Handling,
Analysis, and Reporting of Wipe Tests Performed within the Laboratory
Analyte(s)
Acrylamide
Acrylonitrile
Aldicarb (Temik)
Aldicarb sulfone
Aldicarb sulfoxide
4-Aminopyridine
BZ (Quinuclidinyl benzilate)
Brodifacoum
Bromadiolone
Carbofuran (Furadan)
CASRN
79-06-1
107-13-1
116-06-3
1646-88-4
1646-87-3
504-24-5
6581-06-2
56073-10-0
28772-56-7
1563-66-2
SAM Revision 4.0 75 September 29, 2008
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Section 5 - Selected Chemical Methods
Analyte(s)
Chlorfenvinphos
3-Chloro-1 ,2-propanediol
Chloropicrin
Chlorosarin
Chlorosoman
Chlorpyrifos
Crimidine
Cyclohexyl sarin (GF)
Dichlorvos
Dicrotophos
Diesel Range Organics
Diisopropyl methylphosphonate (DIMP)
Dimethylphosphite
Dimethylphosphoramidic acid
Diphacinone
Disulfoton
Disulfoton sulfoxide
1,4-Dithiane
EA2192 [Diisopropylaminoethyl
methylthiolophosphonate]
Ethylmethyl phosphonate (EMPA)
N-Ethyldiethanolamine (EDEA)
Fenamiphos
Fentanyl
Fluoroacetamide
Fluoroacetic acid and fluoroacetate salts
Formaldehyde
Gasoline Range Organics
Hexahydro-1 ,3,5-trinitro-1 ,3,5-triazine (RDX)
Hexamethylenetriperoxidediamine (HMTD)
Isopropyl methylphosphonic acid (IMPA)
Kerosene
CASRN
470-90-6
96-24-2
76-06-2
1445-76-7
7040-57-5
2921-88-2
535-89-7
329-99-7
62-73-7
141-66-2
NA
1445-75-6
868-85-9
33876-51-6
82-66-6
298-04-4
2497-07-6
505-29-3
73207-98-4
1832-53-7
139-87-7
22224-92-6
437-38-7
640-19-7
NA
50-00-0
NA
121-82-4
283-66-9
1832-54-8
64742-81-0
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Section 5 - Selected Chemical Methods
Analyte(s)
Methamidophos
Methomyl
Methyl acrylonitrile
Methyl fluoroacetate
Methyl hydrazine
Methyl parathion
N-Methyldiethanolamine (MDEA)
1-Methylethyl ester ethylphosphonofluoridic acid (GE)
Methylphosphonic acid (MPA)
Mevinphos
Mustard, nitrogen (HN-1) [bis(2-chloroethyl)ethylamine]
Mustard, nitrogen (HN-2) [2,2'-dichloro-N-
methyldiethylamine N,N-bis(2-chloroethyl)methylamine]
Mustard, nitrogen (HN-3) [tris(2-chloroethyl)amine]
Mustard, sulfur/ Mustard gas (HD)
Nicotine compounds
Octahydro-1 ,3,5,7-tetranitro-1 ,3,5,7-tetrazocine (HMX)
Organophosphate pesticides, NOS
Oxamyl
Parathion
Pentaerythritol tetranitrate (PETN)
Phencyclidine
Phenol
Phorate
Phosphamidon
Pinacolyl methyl phosphonic acid (PMPA)
R 33 (VR) [methylphosphonothioic acid, S-[2-
(diethylamino)ethyl] O-2-methylpropyl ester]
Sarin (GB)
Semivolatile Organic Compounds, NOS
Soman (GD)
Strychnine
CASRN
10265-92-6
16752-77-5
126-98-7
453-18-9
60-34-4
298-00-0
105-59-9
1189-87-3
993-13-5
7786-34-7
538-07-8
51-75-2
555-77-1
505-60-2
54-11-5
2691-41-0
NA
23135-22-0
56-38-2
78-11-5
77-10-1
108-95-2
298-02-2
13171-21-6
616-52-4
159939-87-4
107-44-8
NA
96-64-0
57-24-9
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Section 5 - Selected Chemical Methods
Analyte(s)
Tabun (GA)
Tear gas (CS) [chlorobenzylidene malonitrile]
Tetraethyl pyrophosphate
Tetramethylenedisulfotetramine
Thiodiglycol (TDG)
Thiofanox
1,4-Thioxane
Triethanolamine (TEA)
Trimethyl phosphite
1 ,3,5-Trinitrobenzene (1 ,3,5-TNB)
2,4,6-Trinitrotoluene(2,4,6-TNT)
VE [phosphonothioic acid, ethyl-, S-(2-
(diethylamino)ethyl) O-ethyl ester]
VG [phosphonothioic acid, S-(2-(diethylamino)ethyl)
O,O-diethyl ester]
VM [phosphonothioic acid, methyl-, S-(2-
(diethylamino)ethyl) O-ethyl ester]
VX [O-ethyl-S-(2-diisopropylaminoethyl)methyl-
phosphonothiolate]
White phosphorus
CASRN
77-81-6
2698-41-1
107-49-3
80-12-6
111-48-8
39196-18-4
15980-15-1
102-71-6
121-45-9
99-35-4
118-96-7
21738-25-0
78-53-5
21770-86-5
50782-69-9
12185-10-3
Analysis Purpose: Sample preparation
Sample Preparation Technique: Solvent extraction
Determinative Technique: EPA OW Method 300.1 Revision 1.0; EPA SW-846 Methods 7580, 8015C,
8270D, 8315A, 8316, 8318A, 8321B, and 8330B. Refer to Appendix A for which of these determinative
methods should be used for a particular analyte.
Method Developed for: Evaluation of surface contamination by 2,3,7,8-substituted PCDD and PCDF
congeners
Method Selected for: SAM lists this method for preparation of wipe samples.
Description of Method: A surface area of 2 inches by 1 foot is wiped with glass fiber paper saturated
with distilled-in-glass acetone. One wipe is used per designated area. Wipes are combined into a single
composite sample in an extraction jar and solvent extracted using a wrist action shaker.
Special Considerations: The solvent systems described in this method extraction have been evaluated
for PCDD and PCDF congeners only. Other analytes may require different solvent systems for optimal
sample extraction.
Source: EPA. 2007. "Method 8290A, Appendix A (SW-846): Procedure for the Collection, Handling,
Analysis, and Reporting of Wipe Tests Performed within the Laboratory," Revision 1.
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Section 5 - Selected Chemical Methods
5.2.39 EPA Method 8315A (SW-846): Determination of Carbonyl Compounds by High
Performance Liquid Chromatography (HPLC)
Analyte(s)
Formaldehyde
CASRN
50-00-0
Analysis Purpose: Sample preparation and/or analysis
Sample Preparation Technique: Solvent extraction (solid, aqueous liquid, and drinking water samples)
and EPA SW-846 Method 3570/8290A Appendix A (wipe samples)
Determinative Technique: HPLC
Method Developed for: Free carbonyl compounds in aqueous, soil, waste, and stack samples
Method Selected for: SAM lists this method for preparation and analysis of solid, aqueous liquid,
drinking water, and wipe samples.
Detection and Quantitation: The MDL for formaldehyde varies depending on sample conditions and
instrumentation, but is approximately 6.2 \ig/L for aqueous liquid samples.
Description of Method: A measured volume of aqueous liquid sample (approximately 100 mL), or an
appropriate amount of solids extract (approximately 25 g), is buffered to pH 3 and derivatized with 2,4-
dinitrophenylhydrazine (2,4-DNPH). Using the appropriate extraction technique, the derivatives are
extracted using methylene chloride and the extracts are exchanged with acetonitrile prior to HPLC
analysis. HPLC conditions are described permitting the separation and measurement of various carbonyl
compounds in the extract by absorbance detection at 360 nm. If formaldehyde is the only analyte of
interest, the aqueous liquid sample and/or solid sample extract should be buffered to pH 5.0 to minimize
the formation of artifact formaldehyde.
Source: EPA. 1996. "Method 8315A (SW-846): Determination of Carbonyl Compounds by High
Performance Liquid Chromatography (HPLC)," Revision 1. Mtpi//m%wjpa±gQY/gam/ptfg/EPA:,
8315a.pdf
5.2.40 EPA Method 8316 (SW-846): Acrylamide, Acrylonitrile and Acrolein by High
Performance Liquid Chromatography (HPLC)
Analyte(s)
Acrylamide
Acrylonitrile
Methyl acrylonitrile
CASRN
79-06-1
107-13-1
126-98-7
Analysis Purpose: Sample preparation and/or analysis
Sample Preparation Technique: Direct injection (aqueous liquid and drinking water samples), water
extraction (solid and non-aqueous liquid/organic solid samples), and EPA SW-846 Method 3570/8290A
Appendix A (wipe samples)
Determinative Technique: HPLC
Method Developed for: Acrylamide, acrylonitrile, and acrolein in water samples
Method Selected for: SAM lists this method for preparation and/or analysis of solid, non-aqueous
liquid/organic solid, aqueous liquid, drinking water, and wipe samples.
Detection and Quantitation: Acrylamide has an MDL of 10 iig/L; acrylonitrile has an MDL of 20 jig/L.
SAM Revision 4.0 79 September 29, 2008
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Section 5 - Selected Chemical Methods
Description of Method: Samples are analyzed by HPLC. A 200-(jL aliquot is injected onto a Ci8
reverse-phase column, and compounds in the effluent are detected with a UV detector. Solid and non-
aqueous liquid/organic solid samples should be water extracted prior to injection. Aqueous liquid and
drinking water samples can be directly injected.
Source: EPA. 1994. "Method 8316 (SW-846): Acrylamide, Acrylonitrile and Acrolein by High
Performance Liquid Chromatography (HPLC)," Revision 0. _httgj//w^m¥_._ep_^
5.2.41 EPA Method 8318A (SW-846): A/-Methylcarbamates by High Performance Liquid
Chromatography (HPLC)
Analyte(s)
Aldicarb (Temik)
Aldicarb sulfone
Aldicarb sulfoxide
Carbofuran (Furadan)
Methomyl
Oxamyl
CASRN
116-06-3
1646-88-4
1646-87-3
1563-66-2
16752-77-5
23135-22-0
Analysis Purpose: Sample preparation and/or analysis
Sample Preparation Technique: Solvent extraction (solid, non-aqueous liquid/organic solid, and
aqueous liquid samples), and EPA SW-846 Method 3570/8290A Appendix A (wipe samples)
Determinative Technique: HPLC
Method Developed for: 7V-methylcarbamates in soil, water, and waste matrices
Method Selected for: SAM lists this method for preparation and/or analysis of solid, non-aqueous
liquid/organic solid, aqueous liquid, and wipe samples.
Detection and Quantitation: The estimated MDLs vary with each analyte and range from 1.7 to 9.4
Vig/L for aqueous liquid samples and 10 to 50 jig/kg for soil samples.
Description of Method: 7V-methylcarbamates are extracted from aqueous liquid samples with methylene
chloride, and from soils, oily solid waste, and oils with acetonitrile. The extract solvent is exchanged to
methanol/ethylene glycol, and the extract is cleaned using a Ci8 cartridge, filtered, and eluted on a Ci8
analytical column. After separation, the target analytes are hydrolyzed and derivatized post-column, then
quantified fluorometrically. The sensitivity of the method usually depends on the level of interferences
present, rather than on instrument conditions. Waste samples with a high level of extractable fluorescent
compounds are expected to yield significantly higher detection limits.
Source: EPA. 2000. "Method 8318A (SW-846): N-Methylcarbamates by High Performance Liquid
Chromatography (HPLC)," Revision 1.
SAM Revision 4.0 80 September 29, 2008
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Section 5 - Selected Chemical Methods
5.2.42 EPA Method 8321B (SW-846): Solvent-Extractable Nonvolatile Compounds by
High Performance Liquid Chromatography-Thermospray-Mass Spectrometry
(HPLC-TS-MS) or Ultraviolet (UV) Detection
Analyte(s)
Brodifacoum
Bromadiolone
BZ (Quinuclidinyl benzilate)1
Diisopropyl methylphosphonate (DIMP)2
Dimethylphosphoramidic acid1
Diphacinone
EA2192 [Diisopropylaminoethyl
methylthiolophosphonate]1
Ethylmethyl phosphonate (EMPA)1
N-Ethyldiethanolamine (EDEA)
Fentanyl
Isopropyl methylphosphonic acid (IMPA)1
N-Methyldiethanolamine (MDEA)
Methylphosphonic acid (MPA)1
Pinacolyl methyl phosphonic acid (PMPA)1
Thiofanox
Triethanolamine (TEA)
CASRN
56073-10-0
28772-56-7
6581-06-2
1445-75-6
33876-51-6
82-66-6
73207-98-4
1832-53-7
139-87-7
437-38-7
1832-54-8
105-59-9
993-13-5
616-52-4
39196-18-4
102-71-6
The following analyte should be determined by this method only if problems (e.g., insufficient recovery,
interferences) occur when using SW-846 Method 8270D. Sample preparation methods should remain the same as
those listed in Appendix A.
Crimidine3
535-89-7
LC-MS (electrospray) procedures are preferred for these analytes; however, if this technique is not available to the
laboratory, GC-MS procedures using derivatization based on SW-846 Method 8270D may be used. Sample
preparation methods should remain the same. Both electrospray LC-MS and GC-MS derivatization procedures are
currently under development.
2 If problems occur with the analysis of DIMP using EPA SW-846 Method 8321 B, use SW-846 Method 8270D.
3 This analyte needs to be determined using a wavelength of 230 nm.
Analysis Purpose: Analysis
Sample Preparation Technique: EPA SW-846 Method 3545A/3541 (solid samples), 3580A (non-
aqueous liquid/organic solid samples), 3520C/3535A (aqueous liquid and drinking water samples), and
Method 3570/8290A Appendix A (wipe samples). For thiofanox. EPA Method 531.2 (rather than
Method 3520C/3535A) should be used for preparation of drinking water samples. Refer to Appendix A
for which of these preparation methods should be used for a particular analyte/sample type combination.
Determinative Technique: HPLC
SAM Revision 4.0
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Section 5 - Selected Chemical Methods
Method Developed for: Solvent-extractable nonvolatile compounds, including dyes, organophosphorus
compounds, phenoxyacid herbicides, and carbamates in solid, water, aqueous, and non-aqueous samples
Method Selected for: SAM lists this method for analysis of solid, non-aqueous liquid/organic solid,
aqueous liquid, drinking water, and wipe samples.
Description of Method: This method provides reversed-phase HPLC, thermospray (TSP) MS, and UV
conditions for detection of the target analytes. Sample extracts can be analyzed by direct injection into
the TSP or onto a LC-TSP interface. A gradient elution program is used to separate the compounds.
Primary analysis may be performed by UV detection; however, positive results should be confirmed by
TSP-MS. Quantitative analysis may be performed by either TSP-MS or UV detection, using either an
external or internal standard approach. TSP-MS detection may be performed in either a negative
ionization (discharge electrode) mode or a positive ionization mode, with a single quadrupole MS. The
use of MS-MS techniques is an option. The analytical range and detection limits vary depending on the
target analyte and instrument used.
Special Considerations: Refer to footnotes provided in analyte table above for special considerations
that should be applied when measuring specific analytes.
Source: EPA. 1998. "Method 832 IB (SW-846): Solvent-Extractable Nonvolatile Compounds by High
Performance Liquid Chromatography-Thermospray-Mass Spectrometry (HPLC-TSP-MS) or Ultraviolet
(UV) Detection," Revision 2.
5.2.43 EPA Method 8330B (SW-846): Nitroaromatics and Nitramines by High Performance
Liquid Chromatography (HPLC)
Analyte(s)
4-Aminopyridine
Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)
Hexamethylenetriperoxidediamine (HMTD)
Octahydro-1 ,3,5,7-tetranitro-1 ,3,5,7-tetrazocine (HMX)
Pentaerythritol tetranitrate (PETN)
1 ,3,5-Trinitrobenzene (1 ,3,5-TNB)
2,4,6-Trinitrotoluene(2,4,6-TNT)
CASRN
504-24-5
121-82-4
283-66-9
2691-41-0
78-11-5
99-35-4
118-96-7
Analysis Purpose: Sample preparation and/or analysis
Sample Preparation Technique: Solvent extraction or direct injection (solid and non-aqueous
liquid/organic solid samples), EPA SW-846 Method 3535A (aqueous liquid and drinking water samples),
and EPA SW-846 Method 3570/8290A Appendix A (wipe samples)
Determinative Technique: HPLC
Method Developed for: Trace analysis of explosives and propellant residues in water, soil, or sediment
Method Selected for: SAM lists this method for preparation and/or analysis of solid, non-aqueous
liquid/organic solid, aqueous liquid, drinking water, and wipe samples. Aqueous liquid and drinking
water samples are prepared using Methods 3535A or 8330B prior to analysis.
Detection and Quantitation: The detection limits, ranges, and interferences depend on the target
compound
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Section 5 - Selected Chemical Methods
Description of Method: This method is intended for the trace analysis of explosives and propellant
residues by HPLC using a dual wavelength UV detector in a water, soil, or sediment matrix. All of the
compounds listed in this method are either used in the manufacture of explosives or propellants, or they
are the degradation products of compounds used for that purpose. Samples are prepared for analysis by
high performance liquid chromatography - ultraviolet (HPLC-UV) using the appropriate sample
preparation technique (solid phase extraction by 3535A or solvent extraction by 8330B) and, if necessary,
sample cleanup procedures. Method 8330 provides a salting-out extraction procedure for low
concentration (ppt or ng/L) of explosives residues in surface or ground water. Direct injection of diluted
and filtered water samples can be used for water samples of higher concentration. Soil and sediment
samples are extracted using acetonitrile in an ultrasonic bath, filtered and chromatographed.
Source: EPA. 2006. "Method 8330B (SW-846): Nitroaromatics, Nitramines, and Nitrate Esters by High
Performance Liquid Chromatography (HPLC)," Revision 2. http://www.epa.gov/sam/pdfs/EPA-
5.2.44 EPA ILM05.3 Cyanide: Analytical Methods for Total Cyanide Analysis
Analyte(s)
Cyanide, Total
CASRN
57-12-5
Analysis Purpose: Sample preparation and/or analysis
Sample Preparation Technique: Acid digestion followed by distillation
Determinative Technique: Spectrophotometry
Method Developed for: Metals in water, sediment, sludge, and soil
Method Selected for: SAM lists this method for preparation and/or analysis of solid, aqueous liquid, and
wipe samples.
Detection and Quantitation: The method quantitation limits are 10 pig/L or 2.5 mg/kg
Description of Method: The method allows for either large volume (500-mL aqueous liquid samples or
1-g to 5-g solid samples mixed with 500 mL of reagent water) or medium volume (50-mL aqueous liquid
samples or 1-g solid samples mixed with 50 mL of reagent water) sample preparation. Aqueous liquid
samples are tested for sulfides and oxidizing agents prior to preparation. Sulfides are removed with
cadmium carbonate or lead carbonate. Samples are treated with sulfuric acid and magnesium chloride
and distilled into a sodium hydroxide solution. The solution is treated with color agents and the cyanide
determined as an ion complex by visible Spectrophotometry.
Special Considerations: Surfactants may interfere with the distillation procedure.
Source: EPA. "ILM05.3: Exhibit D - Part D: Analytical Methods for Total Cyanide Analysis."
http://www.epa.gOV/sam/pdfs/EPA-ILM05.3.pdf
5.2.45 EPA Region 7 RLAB Method 3135.21: Cyanide, Total and Amenable in Aqueous
and Solid Samples Automated Colorimetric with Manual Digestion
Analyte(s)
Cyanide, Amenable to chlorination
CASRN
NA
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Acid digestion followed by distillation
SAM Revision 4.0 83 September 29, 2008
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Section 5 - Selected Chemical Methods
Determinative Technique: Spectrophotometry
Method Developed for: Cyanide in drinking, ground, and surface waters, domestic and industrial waste
waters, sediments and solid waste
Method Selected for: SAM lists this method for preparation and analysis of solid, aqueous liquid,
drinking water, and wipe samples.
Detection and Quantitation: The applicable range is 0.003 to 0.500 mg/L cyanide in the distillate. This
range can be expanded by sample dilution, either by using less sample for distillation or diluting the
distillate.
Description of Method: This method detects inorganic cyanides that are present as either simple soluble
salts or complex radicals. It may be used to determine values for both total cyanide and cyanide
amenable to chlorination (also known as available cyanide). Cyanide in the sample released as
hydrocyanic acid by refluxing the sample with strong acid. The hydrocyanic acid is distilled and
collected in an absorber-scrubber containing sodium hydroxide solution. The cyanide ion in the
absorbing solution is then determined by automated colorimetry. For determination of cyanide amenable
to chlorination, a portion of the sample is chlorinated using sodium hypochlorite at a pH > 1 1 to
decompose the cyanide. Cyanide levels are then determined in both the chlorinated sample portion of the
sample and a portion of the sample that has not been chlorinated using the total cyanide method.
Cyanides amenable to chlorination are then calculated by difference between unchlorinated and the
chlorinated aliquots of the sample.
Special Considerations: Alternate cyanide analyzer equipment may be used, provided it is used
according to the procedures described and the laboratory can demonstrate equivalent performance.
Source: EPA Region 7. 2008. "RLAB Method 3135.21: Cyanide, Total and Amenable in Aqueous and
Soil Samples Automated Colorimetric with Manual Digestion." htjjK//wgw^e|)^^
3135.2I.pdf
5.2.46 IO [Inorganic] Compendium Method IO-3.1: Selection, Preparation, and Extraction
of Filter Material
Analyte(s)
Ammonium metavanadate
Arsenic, Total
Arsenic trioxide
Calcium arsenate
2-Chlorovinylarsonous acid (CVAA)
Lead arsenate
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine]
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine]
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine]
Lewisite Oxide
Osmium tetroxide
Sodium arsenite
CASRN
7803-55-6
740-38-2
1327-53-3
7778-44-1
85090-33-1
7645-25-2
541-25-3
40334-69-8
40334-70-1
1306-02-1
20816-12-0
7784-46-5
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Section 5 - Selected Chemical Methods
Analyte(s)
Thallium sulfate
Vanadium pentoxide
CASRN
10031-59-1
1314-62-1
Analysis Purpose: Sample preparation
Sample Preparation Technique: Acid extraction
Determinative Technique: EPA Method IO-3.4 or Method IO-3.5. Osmium tetroxide should be
analyzed by Method IO-3.4.
Method Developed for: Particulate metals in air.
Method Selected for: SAM lists this method for preparation of air samples.
Description of Method: This method supports determination of arsenic trioxide, lewisite compounds,
lewisite oxide, CVAA, and sodium arsenite as total arsenic. Thallium sulfate is determined as total
thallium and ammonium metavanadate and vanadium pentoxide are determined as total vanadium. A
subsample (one-ninth of the overall filter) is obtained by cutting a strip from the filter used to collect the
sample. The filter strip is extracted using a hydrochloric/nitric acid mix and microwave or hotplate
heating. The extract is filtered, worked up to 20 mL, and analyzed using either Method IO-3.4 or Method
IO-3.5.
Source: EPA. 1999. "IO Compendium Method IO-3.1: Compendium of Methods for the Determination
of Inorganic Compounds in Ambient Air: Selection, Preparation and Extraction of Filter Material."
http://www.epa.gov/sam/pdfs/EPA-IO-3.1 .pelf
5.2.47 IO [Inorganic] Compendium Method IO-3.4: Determination of Metals in Ambient
Particulate Matter Using Inductively Coupled Plasma (ICP) Spectroscopy
Analyte(s)
Ammonium metavanadate
Arsenic, Total
Arsenic trioxide
Calcium arsenate
2-Chlorovinylarsonous acid (CVAA)
Lead arsenate
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine]
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine]
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine]
Lewisite Oxide
Osmium tetroxide
Sodium arsenite
Thallium sulfate
Vanadium pentoxide
CASRN
7803-55-6
740-38-2
1327-53-3
7778-44-1
85090-33-1
7645-25-2
541-25-3
40334-69-8
40334-70-1
1306-02-1
20816-12-0
7784-46-5
10031-59-1
1314-62-1
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Section 5 - Selected Chemical Methods
Analysis Purpose: Analysis
Sample Preparation Technique: EPA Method IO-3.1
Determinative Technique: ICP-AES
Method Developed for: Metals in ambient particulate matter
Method Selected for: SAM lists this method for analysis of air samples.
Description of Method: This method determines arsenic trioxide, lewisite, lewisite degradation
products, and sodium arsenite as total arsenic. Osmium tetroxide is determined as total osmium, thallium
sulfate is determined as total thallium, and ammonium metavanadate and vanadium pentoxide are
determined as total vanadium. Ambient air is sampled by high-volume filters using Method IO-2.1 (a
sampling method) and the filters are extracted by Method IO-3.1. The extracts are analyzed by ICP-AES
or ICP-MS (see Method IO-3.5 in Section 5.2.48). Detection limits, ranges, and interference corrections
are dependent on the analyte and the instrument used.
Special Considerations: Laboratory testing is currently underway for speciation of lewisite 1 using GC-
MS techniques.
Source: EPA. 1999. "IO Compendium Method IO-3.4: Compendium of Methods for the Determination
of Inorganic Compounds in Ambient Air: Determination of Metals in Ambient Particulate Matter Using
Inductively Coupled Plasma (ICP) Spectroscopy." http://www.epa.gov/sam/pdfs/EPA-IO-3.4.pdf
EPA. 1999. "IO Compendium Method IO-2.1: Compendium of Methods for the Determination of
Inorganic Compounds in Ambient Air: Sampling of Ambient Air for Total Suspended Particulate Matter
(SPM) and PMio Using High Volume (HV) Sampler." http://www.epa.gOv/sam/pdfs/EPA-IO-2.l.pdf
5.2.48 IO [Inorganic] Compendium Method IO-3.5: Determination of Metals in Ambient
Particulate Matter Using Inductively Coupled Plasma/Mass Spectrometry (ICP-MS)
Analyte(s)
Ammonium metavanadate
Arsenic, Total
Arsenic trioxide
Calcium arsenate
2-Chlorovinylarsonous acid (CVAA)
Lead arsenate
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine]
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine]
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine]
Lewisite Oxide
Sodium arsenite
Thallium sulfate
Vanadium pentoxide
CASRN
7803-55-6
740-38-2
1327-53-3
7778-44-1
85090-33-1
7645-25-2
541-25-3
40334-69-8
40334-70-1
1306-02-1
7784-46-5
10031-59-1
1314-62-1
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Section 5 - Selected Chemical Methods
Analysis Purpose: Analysis
Sample Preparation Technique: EPA Method IO-3.1
Determinative Technique: ICP-MS
Method Developed for: Metals in ambient participate matter
Method Selected for: SAM lists this method for analysis of air samples.
Description of Method: This method determines arsenic trioxide, lewisite, lewisite degradation
products, and sodium arsenite as total arsenic. Thallium sulfate is determined as total thallium and
ammonium metavanadate and vanadium pentoxide are determined as total vanadium. Ambient air is
sampled by high-volume filters using Method IO-2.1 (a sampling method). The filters are extracted by
Method IO-3.1 and the extracts analyzed by ICP-MS or ICP-AES (see Method IO-3.4 in Section 5.2.47).
Detection limits, ranges, and interference corrections are dependent on the analyte and the instrument
used.
Special Considerations: Laboratory testing is currently underway for speciation of lewisite 1 using
GC-MS techniques.
Source: EPA. 1999. "IO Compendium Method IO-3.5: Compendium of Methods for the Determination
of Inorganic Compounds in Ambient Air: Determination of Metals in Ambient Particulate Matter Using
Inductively Coupled Plasma/Mass Spectrometry (ICP/MS)." http://www.epa.gov/sam/pdfs/EPA-IO-
3.5.pdf
EPA. 1999. "IO Compendium Method IO-2.1: Compendium of Methods for the Determination of
Inorganic Compounds in Ambient Air: Sampling of Ambient Air for Total Suspended Particulate Matter
(SPM) and PM10 Using High Volume (HV) Sampler." http://www.epa.gov/sam/pdfs/EPA-IO-2.1 .pdf
5.2.49 IO [Inorganic] Compendium Method IO-5: Sampling and Analysis for Vapor and
Particle Phase Mercury in Ambient Air Utilizing Cold Vapor Atomic Fluorescence
Spectrometry (CVAFS)
Analyte(s)
Mercury, Total
Methoxyethylmercuric acetate
CASRN
7439-97-6
151-38-2
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Acid digestion for particulate mercury
Determinative Technique: CVAFS
Method Developed for: Vapor and particle phase mercury in ambient air
Method Selected for: SAM lists this method for preparation and analysis of air samples.
Detection and Quantitation: The detection limits are 30 pg/m3 for particulate mercury and 45 pg/m3 for
vapor phase mercury. Detection limits, analytical range, and interferences are dependent on the
instrument used.
Description of Method: Vapor phase mercury is collected using gold-coated glass bead traps at a flow
rate of 0.3 L/min. The traps are directly desorbed onto a second (analytical) trap. The mercury desorbed
from the analytical trap is determined by Atomic Fluorescence Spectrometry. Particulate mercury is
sampled on glass-fiber filters at a flow rate of 30 L/min. The filters are extracted with nitric acid and
microwave heating. The extract is oxidized with bromine chloride, then reduced with stannous chloride
and purged from solution onto a gold-coated glass bead trap. This trap is desorbed onto a second trap, the
second trap is desorbed, and the mercury is determined by CVAFS.
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Section 5 - Selected Chemical Methods
Special Considerations: There are no known positive interferences at 253.7 nm wavelength. Water
vapor will cause a negative interference.
Source: EPA. 1999. "IO Compendium Method IO-5: Compendium of Methods for the Determination of
Inorganic Compounds in Ambient Air: Sampling and Analysis for Vapor and Particle Phase Mercury in
Ambient Air Utilizing Cold Vapor Atomic Fluorescence Spectrometry (CVAFS)."
5.2.50 EPA Air Method, Toxic Organics - 10A (TO-10A): Determination of Pesticides and
Polychlorinated Biphenyls in Ambient Air Using Low Volume Polyurethane Foam
(PDF) Sampling Followed by Gas Chromatographic/Multi-Detector Detection
(GC/MD)
Analyte(s)
BZ (Quinuclidinyl benzilate)1
Chlorfenvinphos
3-Chloro-1 ,2-propanediol1'2
Chlorosarin2
Chlorosoman2
Chlorpyrifos
Cyclohexyl sarin (GF)
Dichlorvos
Dicrotophos
Diisopropyl methylphosphonate (DIMP)2
Dimethylphosphite
Dimethylphosphoramidic acid1
EA2192 [Diisopropylaminoethyl
methylthiolophosphonate]1
Ethyl methylphosphonic acid (EMPA)1
N-Ethyldiethanolamine (EDEA)
Fenamiphos
Fentanyl
Fluoroacetamide
Isopropyl methylphosphonic acid (IMPA)1
Methamidophos3
Methyl parathion
N-Methyldiethanolamine (MDEA)
CASRN
6581-06-2
470-90-6
96-24-2
1445-76-7
7040-57-5
2921-88-2
329-99-7
62-73-7
141-66-2
1445-75-6
868-85-9
33876-51-6
73207-98-4
1832-53-7
139-87-7
22224-92-6
437-38-7
640-19-7
1832-54-8
10265-92-6
298-00-0
105-59-9
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Section 5 - Selected Chemical Methods
Analyte(s)
1-Methylethyl ester ethylphosphonofluoridic acid (GE)2
Methylphosphonic acid (MPA)1
Mevinphos
Mustard, nitrogen (HN-1) [bis(2-chloroethyl)ethylamine]
Mustard, nitrogen (HN-2) [2,2'-dichloro-N-
methyldiethylamine N,N-bis(2-chloroethyl)methylamine]
Mustard, nitrogen (HN-3) [tris(2-chloroethyl)amine]
Mustard, sulfur/ Mustard gas (HD)
Parathion
Phencyclidine
Phenol
Phorate
Phosphamidon
Pinacolyl methyl phosphonic acid (PMPA)1
R 33 (VR) [methylphosphonothioic acid, S-[2-
(diethylamino)ethyl] O-2-methylpropyl ester]
Sarin (GB)2
Semivolatile Organic Compounds, NOS
Soman (GD)2
Tabun (GA)
Tear gas (CS) [chlorobenzylidene malonitrile]
Tetraethyl pyrophosphate
Tetramethylenedisulfotetramine
Thiodiglycol (TDG)
Triethanolamine (TEA)
Trimethyl phosphite
VE [phosphonothioic acid, ethyl-, S-(2-
(diethylamino)ethyl) O-ethyl ester]
VG [phosphonothioic acid, S-(2-(diethylamino)ethyl)
O,O-diethyl ester]
VM [phosphonothioic acid, methyl-, S-(2-
(diethylamino)ethyl) O-ethyl ester]
VX [O-ethyl-S-(2-diisopropylaminoethyl)methyl-
phosphonothiolate]
CASRN
1189-87-3
993-13-5
7786-34-7
538-07-8
51-75-2
555-77-1
505-60-2
56-38-2
77-10-1
108-95-2
298-02-2
13171-21-6
616-52-4
159939-87-4
107-44-8
NA
96-64-0
77-81-6
2698-41-1
107-49-3
80-12-6
111-48-8
102-71-6
121-45-9
21738-25-0
78-53-5
21770-86-5
50782-69-9
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Section 5 - Selected Chemical Methods
Analyte(s)
CASRN
The following analyte should be determined by this method only if problems (e.g., insufficient recovery,
interferences) occur when using Method TO-15.
Allyl alcohol
107-18-6
1 For this analyte, HPLC is the preferred technique; however, if problems occur, Method TO-10A must be modified to
include a derivatization step prior to analysis by GC-MS.
2 If problems occur when using this method, it is recommended that the canister Method TO-1 5 be used.
3 If problems occur when using this method, it is recommended that NIOSH Method 5600 be used.
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Solvent extraction
Determinative Technique: GC-MS or HPLC
Method Developed for: Pesticides and polychlorinated biphenyls in ambient air
Method Selected for: SAM lists this method for preparation and analysis of air samples.
Detection and Quantitation: The limit of detection (LOD) will depend on the specific compounds
measured, the concentration level, and the degree of specificity required. This method is applicable to
multicomponent atmospheres, 0.001 to 50 vig/m3 concentrations, and 4 to 24-hour sampling periods.
Description of Method: A low-volume (1 to 5 L/minute) sample collection rate is used to collect vapors
on a sorbent cartridge containing PUF in combination with another solid sorbent. Airborne particles also
are collected, but the sampling efficiency is not known. Pesticides and other chemicals are extracted from
the sorbent cartridge with 5% diethyl ether in hexane and determined by GC-MS. For common
pesticides, HPLC coupled with a UV detector or electrochemical detector is preferable. If analyzed by
GC-MS, BZ, dimethylphosphoramidic acid, EA2192, EMPA, IMPA, MPA, and PMPA require
derivatization with a trimethylsilyl agent prior to injection into the GC.
Special Considerations: Refer to footnotes provided in analyte table above for special considerations
that should be applied when measuring specific analytes.
Source: EPA. 1999. "Air Method, Toxic Organics-lOA (TO-10A): Compendium of Methods for the
Determination of Inorganic Compounds in Ambient Air: Determination of Pesticides and Polychlorinated
Biphenyls in Ambient Air Using Low Volume Polyurethane Foam (PUF) Sampling Followed by Gas
Chromatographic/Multi-Detector Detection (GC/MD)." httg:llw\v\^
5.2.51 EPA Air Method, Toxic Organics - 15 (TO-15): Determination of Volatile Organic
Compounds (VOCs) in Air Collected in Specially-Prepared Canisters and Analyzed
by Gas Chromatography/Mass Spectrometry (GC/MS)
Analyte(s)
Allyl alcohol
Carbon disulfide
Cyanogen chloride
1,2-Dichloroethane
Ethyldichloroarsine (ED)
Ethylene oxide
CASRN
107-18-6
75-15-0
506-77-4
107-06-2
598-14-1
75-21-8
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Section 5 - Selected Chemical Methods
Analyte(s)
CASRN
The following analytes should be determined by this method only if problems (e.g., insufficient recovery,
interferences) occur when using Method TO-10A.
3-Chloro-1 ,2-propanediol
Chlorosarin
Chlorosoman
Diisopropyl methylphosphonate (DIMP)
1-Methylethyl ester ethylphosphonofluoridic acid (GE)
Sarin (GB)
Soman (GD)
96-24-2
1445-76-7
7040-57-5
1445-75-6
1189-87-3
107-44-8
96-64-0
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Samples are collected using canisters.
Determinative Technique: GC-MS
Method Developed for: VOCs in air
Method Selected for: SAM lists this method for preparation and analysis of air samples.
Detection and Quantitation: This method applies to ambient concentrations of VOCs above 0.5 ppbv
and typically requires VOC enrichment by concentrating up to 1 L of a sample volume; however, when
using current technologies, quantifications of approximately 100 pptv have been achieved with 0.5-L
sample volumes.
Description of Method: The atmosphere is sampled by introduction of air into a specially prepared
stainless steel canister (electropolished or silica-coated). A sample of air is drawn through a sampling
train comprising components that regulate the rate and duration of sampling into the pre-evacuated and
passivated canister. Grab samples also may be collected. After the air sample is collected, the canister
valve is closed, an identification tag is attached to the canister, and the canister is transported to the
laboratory for analysis. To analyze the sample, a known volume of sample is directed from the canister
through a solid multisorbent concentrator. Recovery of less volatile compounds may require heating the
canister.
After the concentration and drying steps are completed, VOCs are thermally desorbed, entrained in a
carrier gas stream, and then focused in a small volume by trapping on a cryo-focusing (ultra-low
temperature) trap or small volume multisorbent trap. The sample is then released by thermal desorption
and analyzed by GC-MS.
Special Considerations: If problems occur when using this method for determination of allyl alcohol, it
is recommended that Method TO-10A be used. For 3-chloro-l,2-propanediol, Method TO-15 must be
modified to include a derivatization step.
Source: EPA. 1999. "Air Method, Toxic Organics-15 (TO-15): Compendium of Methods for the
Determination of Toxic Organic Compounds in Ambient Air, Second Edition: Determination of Volatile
Organic Compounds (VOCs) in Air Collected in Specially-Prepared Canisters and Analyzed by Gas
Chromatography/Mass Spectrometry (GC/MS)." http://www.epa.gov/sam/pdfs/EPA-TO-15.pdf
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Section 5 - Selected Chemical Methods
5.2.52 NIOSH Method 1612: Propylene Oxide
Analyte(s)
Propylene oxide
CASRN
75-56-9
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Coconut shell charcoal solid sorbent tube
Determinative Technique: GC-FID
Method Developed for: Propylene oxide in air
Method Selected for: SAM lists this method for preparation and analysis of air samples.
Detection and Quantitation: The working range is between 8 and 295 ppm for air samples of 5 L.
Description of Method: A sample tube containing coconut shell charcoal is used for sample collection
with a flow rate of 0.01 to 0.2 L/min. One milliliter of carbon disulfide is added to the vial and allowed to
sit for 30 minutes prior to analysis with occasional agitation. No interferences have been found.
Special Considerations: The presence of propylene oxide should be confirmed by either a secondary
GC column or by an MS.
Source: NIOSH. 1994. "Method 1612: Propylene Oxide," Issue 2.
http://wmw.epa.gov/sain/pdfs/NIOSH-1612.pdf
5.2.53 NIOSH Method 2016: Formaldehyde
Analyte(s)
Formaldehyde
CASRN
50-00-0
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Solvent extraction
Determinative Technique: HPLC
Method Developed for: Formaldehyde in air
Method Selected for: SAM lists this method for preparation and analysis of air samples.
Detection and Quantitation: The detection limit for formaldehyde is 0.07 jig/sample. The working
range is 0.015 to 2.5 mg/m3 (0.012 to 2.0 ppm) for a 15-L sample.
Description of Method: This method can be used for the determination of formaldehyde using HPLC
with a UV detector. Air is sampled onto a cartridge containing silica gel coated with 2,4-DNPH, at a rate
of 0.03 to 1.5 L/min. The cartridge is extracted with 10 mL of acetonitrile and analyzed by HPLC-UV at
a wavelength of 360 nm. Ozone has been observed to consume the 2,4-DNPH reagent and to degrade the
formaldehyde derivative. Ketones and other aldehydes can react with 2,4-DNPH; the derivatives
produced, however, are separated chromatographically from the formaldehyde derivative.
Source: NIOSH. 2003. "Method 2016: Formaldehyde," Issue 2.
http://mwv.epa.gov/sam/pdfs/NIOSH-2016.pdf
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Section 5 - Selected Chemical Methods
5.2.54 NIOSH Method 2513: Ethylene Chlorohydrin
Analyte(s)
2-Chloroethanol (ethylene chlorohydrin)
CASRN
107-07-3
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Solvent desorption
Determinative Technique: GC-FID
Method Developed for: Ethylene chlorohydrin in air
Method Selected for: SAM lists this method for preparation and analysis of air samples.
Detection and Quantitation: The working range of the method is 0.5 to 15 ppm for a 20-L air sample.
Description of Method: Samples are drawn into a tube containing petroleum charcoal at a rate of 0.01 to
0.2 L/min and transferred into vials containing eluent (carbon disulfide, 2-propanol, and «-pentadiene as
an internal standard). Vials must sit for 30 minutes prior to analysis by GC-FID. No interferences have
been identified. Humidity may decrease the breakthrough volume during sample collection.
Special Considerations: The presence of 2-chloroethanol should be confirmed by either a secondary
GC column or by an MS.
Source: NIOSH. 1994. "Method 2513: Ethylene Chlorohydrin," Issue 2.
5.2.55 NIOSH Method 3510: Monomethylhydrazine
Analyte(s)
Methyl hydrazine (monomethylhydrazine)
CASRN
60-34-4
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Samples are collected into a bubbler containing hydrochloric acid.
Determinative Technique: Spectrophotometry
Method Developed for: Monomethylhydrazine in air
Method Selected for: SAM lists this method for preparation and analysis of air samples.
Detection and Quantitation: The working range of the method is 0.027 to 2.7 ppm for a 20-L air
sample.
Description of Method: Samples are collected into a bubbler containing hydrochloric acid using a flow
rate of 0.5 to 1.5 L/min. Samples are then mixed with phosphomolybdic acid solution and transferred to a
large test tube for spectrophotometric analysis. Positive interferences that have been noted include
stannous ion, ferrous ion, zinc, sulfur dioxide, and hydrogen sulfide. Negative interferences may occur by
oxidation of the monomethylhydrazine by halogens, oxygen (especially in the presence of copper (I) ions)
and hydrogen dioxide.
Source: NIOSH. 1994. "Method 3510: Monomethylhydrazine," Issue 1.
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Section 5 - Selected Chemical Methods
5.2.56 NIOSH Method 5600: Organophosphorus Pesticides
Analyte(s)
Disulfoton
Disulfoton sulfoxide
Organophosphate pesticides, NOS
CASRN
298-04-4
2497-07-6
NA
The following analyte should be determined by this method only if problems (e.g., insufficient recovery,
interferences) occur when using Method TO-10A.
Methamidophos
10265-92-6
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Solvent desorption
Determinative Technique: GC-FPD
Method Developed for: Organophosphorus pesticides in air
Method Selected for: SAM lists this method for preparation and analysis of air samples.
Detection and Quantitation: The detection limit depends on the compound being measured. The
working range for each analyte is provided in Table 5 of the method. These ranges cover from 0.1 to 2
times the OSHA Permissible Exposure Limits (PELs).
Description of Method: This method is used for the detection of Organophosphorus pesticides using GC
with a FPD. The method also may be applicable to the determination of other Organophosphorus
compounds after evaluation for desorption efficiency, sample capacity, sample stability, and precision and
accuracy. The working range for each analyte is provided in Table 5 of the method. These ranges cover
from 0.1 to 2 times the OSHA PELs (see Table 5 of the method). The method also is applicable to Short
Term Exposure Limit (STEL) measurements using 12-L samples.
Special Considerations: Several organophosphates may co-elute with either target analytes or internal
standards causing integration errors. These include other pesticides, and the following: tributyl
phosphate, tris-(2-butoxy ethyl) phosphate, tricresyl phosphate, and triphenyl phosphate.
Source: NIOSH. 1994. "Method 5600: Organophosphorus Pesticides," Issue 1.
5.2.57 NIOSH Method 5601: Organonitrogen Pesticides
Analyte(s)
Aldicarb
Aldicarb sulfone
Aldicarb sulfoxide
Carbofuran
Methomyl
Oxamyl
CASRN
116-06-3
1646-88-4
1646-87-3
1563-66-2
16752-77-5
23135-22-0
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Section 5 - Selected Chemical Methods
Analyte(s)
Thiofanox
CASRN
39196-18-4
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Solvent desorption
Determinative Technique: HPLC
Method Developed for: Organonitrogen pesticides in air
Method Selected for: SAM lists this method for preparation and analysis of air samples.
Detection and Quantitation: The detection limit for aldicarb is 1.2 \ig per sample and 0.6 \ig per sample
for carbofuran, methyomyl, and oxamyl. The working ranges for aldicarb, carbofuran, and oxamyl are
listed in Table 2 of the method, and range from 0.5 to 10 times the OSHA PEL.
Description of Method: This method can be used for the determination of organonitrogen pesticides
using HPLC with a UV detector. The method also may be applicable to the determination of other
organonitrogen compounds and to a broad range of pesticides having UV chromophores, e.g.,
acetanilides, acid herbicides, organophosphates, phenols, pyrethroids, sulfonyl ureas, sulfonamides,
triazines, and uracil pesticides. Because of the broad response of the UV detector at shorter wavelengths,
there are many potential interferences. Those tested include solvents (chloroform and toluene),
antioxidants (betylated hydroxytoluene [BHT]), plasticizers (dialkyl phthalates), nitrogen compounds
(nicotine and caffeine), impurities in HPLC reagents (e.g., in triethylamine), other pesticides (2,4-
Dichlorophenoxyacetic acid [2,4-D], atrazine, parathion, etc.), and pesticide hydrolysis products (1-
naphthol). Confirmation techniques are recommended when analyte identity is uncertain.
Special Considerations: The presence of the analytes listed in the table above should be confirmed by
either a secondary GC column or by an MS.
Source: NIOSH. 1998. "Method 5601: Organonitrogen Pesticides," Issue 1.
5.2.58 NIOSH Method 6001: Arsine
Analyte(s)
Arsine
CASRN
7784-42-1
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Coconut shell charcoal solid sorbent tube
Determinative Technique: GFAA
Method Developed for: Arsine in air
Method Selected for: SAM lists this method for preparation and analysis of air samples.
Detection and Quantitation: The working range of the method is 0.001 to 0.2 mg/m3 for a 10-L sample.
Description of Method: Arsine is determined as arsenic. A 0.1- to 10-L volume of air is drawn through
a sorbent tube containing activated charcoal. The sorbent is extracted with a nitric acid solution, and
arsenic is determined by GFAA.
Special Considerations: The method is subject to interferences from other arsenic compounds.
Source: NIOSH. 1994. "Method 6001: Arsine," Issue 2. http://www.epa.gov/sam/pdfs/NIOSH-6001 .pdf
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Section 5 - Selected Chemical Methods
5.2.59 NIOSH Method 6002: Phosphine
Analyte(s)
Phosphine
CASRN
7803-51-2
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Solvent desorption with hot acidic permanganate solution
Determinative Technique: Spectrophotometry
Method Developed for: Phosphine in air
Method Selected for: SAM lists this method for preparation and analysis of air samples.
Detection and Quantitation: The working range of the method is 0.02 to 0.9 mg/m3 for a 16-L sample.
Description of Method: In this method, phosphine is determined as phosphate. A volume of 1 to 16 L
of air is drawn through a sorbent tube containing silica gel coated with mercuric cyanide. The sorbent is
extracted with a potassium permanganate/sulfuric acid solution and washed with reagent water.
Following treatment with the color agent and extraction into organic solvent, phosphate is determined by
visible spectrometry.
Special Considerations: The method is subject to interferences from phosphorus trichloride,
phosphorus pentachloride, and organic phosphorus compounds.
Source: NIOSH. 1994. "Method 6002: Phosphine," Issue 2.
http://wmw.epa.gov/sain/pdfs/NIOSH-6002.pdf
5.2.60 NIOSH Method 6004: Sulfur Dioxide
Analyte(s)
Sulfur dioxide
CASRN
7446-09-5
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Treated filter extracted with carbonate/bicarbonate solution
Determinative Technique: 1C
Method Developed for: Sulfur dioxide in air
Method Selected for: SAM lists this method for preparation and analysis of air samples.
Detection and Quantitation: The working range of the method is 0.5 to 20 mg/m3 for a 100-L sample.
Description of Method: In this method, sulfur dioxide is determined as sulfite plus sulfate. A volume of
40 to 200 L of air is drawn through a sodium carbonate-treated filter that is preceded by a 0.8 pirn filter to
remove particulates and sulfuric acid. The treated filter is extracted with a carbonate/bicarbonate solution
and the extract analyzed by 1C for sulfite and sulfate. The sulfur dioxide is present as sulfite on the filter;
however, because sulfite oxidizes to sulfate, both ions must be determined and the results summed.
Special Considerations: The method is subject to interference from sulfur trioxide in dry conditions.
Source: NIOSH. 1994. "Method 6004: Sulfur Dioxide," Issue 2.
http://www.epa.gov/sam/pdfs/NIOSH-6004.pdf
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Section 5 - Selected Chemical Methods
5.2.61 NIOSH Method 6010: Hydrogen Cyanide
Analyte(s)
Cyanide, Total
Hydrogen cyanide
CASRN
57-12-5
74-90-8
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Solvent desorption
Determinative Technique: Spectrophotometry
Method Developed for: Hydrogen cyanide in air
Method Selected for: SAM lists this method for preparation and analysis of air samples.
Detection and Quantitation: The working range of the method is 3 to 260 mg/m3 for a 3-L sample.
Description of Method: Hydrogen cyanide is determined as a cyanide ion complex by this method. A
volume of 2 to 90 L of air is drawn through a soda lime sorbent tube. A glass-fiber filter is used to
remove particulate cyanides prior to the sorbent tube. Cyanide is extracted from the sorbent with reagent
water treated with sodium hydroxide. The extract is pH adjusted with hydrochloric acid, oxidized with N-
chlorosuccinimide/succinimide, and treated with the coupling-color agent (barbituric acid/pyridine). The
cyanide ion is determined by visible Spectrophotometry using a wavelength of 580 nm.
Special Considerations: The method is subject to interference from high concentrations of hydrogen
sulfide.
Source: NIOSH. 1994. "Method 6010: Hydrogen Cyanide," Issue 2.
5.2.62 NIOSH Method 6013: Hydrogen Sulfide
Analyte(s)
Hydrogen sulfide
CASRN
7783-06-4
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Solvent extraction
Determinative Technique: 1C
Method Developed for: Hydrogen sulfide in air
Method Selected for: SAM lists this method for preparation and analysis of air samples.
Detection and Quantitation: The working range of the method is 0.9 to 20 mg/m3 for a 20-L sample.
Description of Method: Hydrogen sulfide is determined as sulfate by this method. A volume of 1.2 to
40 L of air is drawn through charcoal sorbent. A prefilter is used to remove particulates. The sorbent
portions are extracted with an ammonium hydroxide/hydrogen peroxide solution and the extract is
analyzed for sulfate by 1C.
Special Considerations: The method is subject to interference from sulfur dioxide.
Source: NIOSH. 1994. "Method 6013: Hydrogen Sulfide," Issue 1.
http://www.epa.gov/sani/pdfs/NIOSH-6013.pdf
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Section 5 - Selected Chemical Methods
5.2.63 NIOSH Method 6015: Ammonia
Analyte(s)
Ammonia
CASRN
7664-41-7
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Water extraction
Determinative Technique: Spectrophotometry
Method Developed for: Ammonia in air
Method Selected for: SAM lists this method for preparation and analysis of air samples.
Detection and Quantitation: The working range of the method is 0.15 to 300 mg/m for a 10-L sample.
Twice the recommended sample volume should be collected in order to achieve an action level of 70
IJg/m3.
Description of Method: Ammonia is determined as indophenol blue by this method. A volume of 0.1 to
96 L of air is drawn through a sulfuric acid-treated silca gel sorbent. A prefilter is used to remove
particulates. The sorbent is extracted with reagent water, the pH adjusted, and reagents are added to
generate the indophenol blue compound in the presence of ammonium. The extract is analyzed by visible
Spectrophotometry. No interferences have been identified.
Source: NIOSH. 1994. "Method 6015: Ammonia," Issue 2.
http://www.epa.gov/sam/pdfs/NIOSH-6015.pdf
5.2.64 NIOSH Method 6402: Phosphorus Trichloride
Analyte(s)
Phosphorus trichloride
CASRN
7719-12-2
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Add reagent to samples in bubbler solution and heat
Determinative Technique: Spectrophotometry
Method Developed for: Phosphorus trichloride in air
Method Selected for: SAM lists this method for preparation and analysis of air samples.
Detection and Quantitation: The working range of the method is 1.2 to 80 mg/m3 for a 25-L sample.
Description of Method: In this method, phosphorus trichloride is determined as phosphate. A volume
of 11 to 100 L of air is drawn through a bubbler containing reagent water. The resulting phosphorus acid
solution is oxidized to phosphoric acid and color agents are added. The solution is analyzed by visible
Spectrophotometry. Phosphorus (V) compounds do not interfere. The sample solutions are stable to
oxidation by air during sampling.
Source: NIOSH. 1994. "Method 6402: Phosphorus Trichloride," Issue 2.
http://miw.epa.gov/sam/pdfs/NIOSH-6402.pdf
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Section 5 - Selected Chemical Methods
5.2.65 NIOSH Method 7903: Acids, Inorganic
Analyte(s)
Hydrogen bromide
Hydrogen chloride
Hydrogen fluoride
CASRN
10035-10-6
7647-01-0
7664-39-3
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Solvent desorption
Determinative Technique: 1C
Method Developed for: Inorganic acids in air
Method Selected for: SAM lists this method for preparation and analysis of air samples.
Detection and Quantitation: The working range of this method is 0.01 to 5 mg/m3 for a 50-L sample.
Description of Method: Acids are analyzed as bromide, chloride, and fluoride. A volume of 3 to 100 L
of air is drawn through a silica gel sorbent. The sorbent portions are extracted with a buffered
carbonate/bicarbonate solution and the extract is analyzed by 1C.
Special Considerations: Particulate salts of the acids are an interference (trapped on the glass wool
filter plug in the sorbent tube). Chlorine and bromine are also interferences. Acetate, formate, and
propionate interferences may be reduced by use of a weaker eluent. If problems occur when using this
method for analysis of hydrogen fluoride, it is recommended that NIOSH Method 7906 be used.
Source: NIOSH. 1994. "Method 7903: Acids, Inorganic," Issue 2.
http://mwv.epa.gov/sam/pdfs/NIOSH-7903.pdf
5.2.66 NIOSH Method 7905: Phosphorus
Analyte(s)
White phosphorus
CASRN
12185-10-3
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: GC solid sorbent tube and solvent extracted (desorbed)
Determinative Technique: GC-FPD or GC-NPD
Method Developed for: Phosphorus in air
Method Selected for: SAM lists this method for preparation and analysis of air samples.
Detection and Quantitation: The LOD for samples analyzed by GC-FPD is 0.005 ug per sample. The
working range for samples analyzed by GC-FPD is 0.056 to 0.24 mg/m3 for a 12-L sample.
Description of Method: This method identifies and determines the concentration of white phosphorus in
air by using a GC-FPD. Samples prepared by this method can be analyzed alternatively by GC-NPD.
Five to 100 L of air is drawn through a GC solid sorbent tube, and the sorbent is extracted (desorbed) with
xylene. Phosphorus is determined by GC-FPD or GC-NPD. The method is applicable to vapor-phase
phosphorus only; if particulate phosphorus is expected, a filter could be used in the sampling train.
Special Considerations: The presence of white phosphorus should be confirmed by either a secondary
GC column or by an MS.
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Section 5 - Selected Chemical Methods
Source: NIOSH. 1994. "Method 7905: Phosphorus," Issue 2.
htto://www:epa1goy/samM^
5.2.67 NIOSH Method 7906: Fluorides, Aerosol and Gas, by 1C
Analyte(s)
Hydrogen fluoride
CASRN
7664-39-3
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Water extraction
Determinative Technique: 1C
Method Developed for: Fluorides in aerosol and gas
Method Selected for: SAM lists this method for use if problems occur when using NIOSH Method 7903
for the analysis of hydrogen fluoride during preparation and analysis of air samples. (See Footnote 11 of
Appendix A.)
Detection and Quantitation: The working range of the method is 0.04 to 8 mg/m3 for 250-L samples.
Description of Method: Hydrogen fluoride is determined as fluoride ion by this method. A volume of 1
to 800 L of air is drawn through a 0.8-iJm cellulose ester membrane (to trap particulate fluorides) and a
cellulose pad treated with sodium carbonate (to trap gaseous fluoride). The pad is extracted with reagent
water and the extract is analyzed for fluoride by 1C.
Special Considerations: If other aerosols are present, gaseous fluoride may be slightly underestimated
due to adsorption onto or reaction with particles, with concurrent overestimation of particulate/gaseous
fluoride ratio.
Source: NIOSH. 1994. "Method 7906: Fluorides, Aerosol and Gas by 1C," Issue 1.
http://www.epa.8Qv/sam/pdfs/NIOSH-7906.pdf
5.2.68 NIOSH Method 9102: Elements on Wipes
Analyte(s)
Ammonium metavanadate
Arsenic, Total
Arsenic trioxide
Arsine
Calcium arsenate
2-Chlorovinylarsonous acid (CVAA)
Ethyldichloroarsine (ED)
Lead arsenate
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine]
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine]
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine]
CASRN
7803-55-6
740-38-2
1327-53-3
7784-42-1
7778-44-1
85090-33-1
598-14-1
7645-25-2
541-25-3
40334-69-8
40334-70-1
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Section 5 - Selected Chemical Methods
Analyte(s)
Lewisite Oxide
Mercury, Total
Methoxyethylmercuric acetate
Osmium tetroxide
Sodium arsenite
Thallium sulfate
Titanium tetrachloride
Vanadium pentoxide
CASRN
1306-02-1
7439-97-6
151-38-2
20816-12-0
7784-46-5
10031-59-1
7550-45-0
1314-62-1
Analysis Purpose: Sample preparation
Sample Preparation Technique: Acid digestion
Determinative Technique: EPA SW-846 Methods 6010C, 6020A, 7010, 7473, and 8270D. Refer to
Appendix A for which of these determinative methods should be used for a particular analyte.
Method Developed for: Measurement of metals on wipe surfaces using ICP-AES
Method Selected for: SAM lists this method for preparation of wipe samples.
Detection and Quantitation: The range for arsenic is 0.261-105 jig/wipe; for thallium 0.136-50.0
jig/wipe; for vanadium 0.0333-25.0 jig/wipe.
Description of Method: Surface wipe samples are transferred to a clean beaker, followed by the addition
of concentrated nitric and perchloric acids. The beaker contents are held at room temperature for 30
minutes, then heated at 150°C for 8 hours. Additional nitric acid is added until the wipe media is
completely destroyed. The sample is then taken to near dryness and the residue dissolved and diluted
before being analyzed.
Special Considerations: ICP-MS may also be used for the analysis of wipe samples; however, at this
time, this technique has not been evaluated for wipes.
Source: NIOSH. 2003. "Method 9102, Issue 1: Elements on Wipes."
http://wvw.epa.gov/sam/pdfs/NIOSH-9102.pdf
5.2.69 NIOSH Method S301-1: Fluoroacetate Anion
Analyte(s)
Fluoroacetic acid and fluoroacetate salts
Methyl fluoroacetate
CASRN
NA
453-18-9
Analysis Purpose: Sample preparation
Sample Preparation Technique: Water extraction
Determinative Technique: EPA Method 300.1 Rev 1.0
Method Developed for: Fluoroacetate anion in air
Method Selected for: SAM lists this method for preparation of air samples.
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Section 5 - Selected Chemical Methods
Detection and Quantitation: The detection limit is estimated to be 20 ng of sodium fluoroacetate per
injection, corresponding to a 100-uL aliquot of a 0.2-ug/mL standard. The analytical range of this
method is estimated to be 0.01 to 0.16 mg/m3.
Description of Method: This method was developed specifically for sodium fluoroacetate, but also may
be applicable to other fluoroacetate salts. The method determines fluoroacetate salts as fluoroacetate
anion. A known volume of air (e.g., 480 L was used in validation of this method) is drawn through a
cellulose ester membrane filter to collect sodium fluoroacetate. Sodium fluoroacetate is extracted from
the filter with 5 mL of deionized water, and the resulting sample is analyzed by 1C using electrolytic
conductivity detection.
Special Considerations: When analyzing samples for methyl fluoroacetate (as fluoroacetate ion),
addition of base is required to assist dissociation into fluoroacetate anion.
Source: NIOSH. 1977. "Method S301-1: Sodium Fluoroacetate."
http://www.epa.gov/sam/pdfs/NIOSH-S301-l.pdf
5.2.70 OSHA Method 40: Methylamine
Analyte(s)
Methylamine
CASRN
74-89-5
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Solvent desorption
Determinative Technique: HPLC
Method Developed for: Methylamine in air
Method Selected for: SAM lists this method for preparation and analysis of air samples.
Detection and Quantitation: The detection limit of the overall procedure is 0.35 \ig per sample (28 ppb
or 35 iig/m3). Quantitation limits of 28 ppb (35 jig/m3) have been achieved. This is the smallest amount
of methylamine that can be quantified within the requirements of a recovery of at least 75% and a
precision (standard deviation of 1.96) of ± 25% or better.
Description of Method: This method is used for detection of methylamine using HPLC with a FL or
visible (vis) detector. Samples are collected by drawing 10-L volumes of air at a rate of 0.2 L/min
through standard size sampling tubes containing XAD-7 resin coated with 10% 7-chloro-4-nitrobenzo-2-
oxa-l,3-diazole (NBD chloride) by weight. Samples are desorbed with 5% (w/v) NBD chloride in
tetrahydrofuran (with a small amount of sodium bicarbonate present), heated in a hot water bath, and
analyzed by high performance liquid chromatography - fluorescence (HPLC-FL) or high performance
liquid chromatography - visible (HPLC-vis).
Source: OSHA. 1982. "Method 40: Methylamine." Method originally obtained from www.osha.gov.
but is provided here for reference. httj3;//wwv.j^
5.2.71 OSHA Method 54: Methyl Isocyanate (MIC)
Analyte(s)
Methyl isocyanate
CASRN
624-83-9
Analysis Purpose: Sample preparation and analysis
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Section 5 - Selected Chemical Methods
Sample Preparation Technique: Solvent desorption
Determinative Technique: HPLC
Method Developed for: Methyl isocyanate in air
Method Selected for: SAM lists this method for preparation and analysis of air samples.
Description of Method: This method determines the concentration of methyl isocyanate in air by using
HPLC with a FL or UV detector. Samples are collected by drawing a known volume of air through
XAD-7 tubes coated with 0.3 mg of l-(2-pyridyl)piperazine (1-2PP). Samples are desorbed with
acetonitrile and analyzed by HPLC using a FL or UV detector.
Source: OSHA. 1985. "Method 54: Methyl Isocyanate (MIC)." Method originally obtained from
_goi, but is provided here for reference. htljLi//wj¥^^
5.2.72 OSHA Method 61: Phosgene
Analyte(s)
Perfluoroisobutylene (PFIB)
Phosgene
CASRN
382-21-8
75-44-5
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Solvent desorption
Determinative Technique: GC-NPD
Method Developed for: Phosgene in air samples
Method Selected for: SAM lists this method for preparation and analysis of air samples.
Description of Method: This method determines the concentration of phosgene in air by using GC with
an NPD. Air samples are collected by drawing known volumes of air through sampling tubes containing
XAD-2 adsorbent that has been coated with 2-(hydroxymethyl)piperidine. The samples are desorbed with
toluene and then analyzed by GC using an NPD.
Special Considerations: The presence of PFIB and phosgene should be confirmed by either a
secondary GC column or by an MS. If problems occur when using this method for determination of
PFIB, it is recommended that a method based on the following journal article be used: Journal of
Chromatography A. 1098: (2005) 156-165.
Source: OSHA. 1986. "Method 61: Phosgene." Method originally obtained from wmnv.osha.gov, but is
provided here for reference. htt]3i/Mlwj|^^
5.2.73 OSHA Method ID-211: Sodium Azide and Hydrazoic Acid in Workplace
Atmospheres
Analyte(s)
Sodium azide
CASRN
26628-22-8
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Buffer desorption
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Section 5 - Selected Chemical Methods
Determinative Technique: 1C
Method Developed for: Sodium azide and hydrazoic acid in workplace atmospheres
Method Selected for: SAM lists this method for preparation and analysis of air and wipe samples.
Detection and Quantitation: The detection limit was found to be 0.001 ppm as hydrazoic acid (HN3) or
0.003 mg/m3 as sodium azide (NaN3) for a 5-L air sample. The quantitation limit was found to be 0.004
ppm as HN3 or 0.01 1 mg/m3 as NaN3 for a 5-L air sample.
Description of Method: This method describes sample collection and analysis of airborne azides [as
NaN3 and hydrazoic acid HN3] . Particulate NaN3 is collected on a PVC filter or in the glass wool plug of
the sampling tube. Gaseous HN3 is collected and converted to NaN3 by the impregnated silica gel (ISO)
sorbent within the sampling tube. The collected azide on either media is desorbed in a weak buffer
solution, and the resultant anion (N3~) is analyzed by 1C using a variable wavelength UV detector at 210
nm. A gravimetric conversion is used to calculate the amount of NaN3 or HN3 collected.
Source: OSHA. 1992. "Method ID-21 1: Sodium Azide and Hydrazoic Acid in Workplace
Atmospheres."
5.2.74 OSHA Method ID216SG: Boron Trifluoride (BF3)
Analyte(s)
Boron trifluoride
CASRN
7637-07-2
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Sample collected in bubbler (no sample preparation required)
Determinative Technique: Ion specific electrode (ISE)
Method Developed for: Boron trifluoride in air samples
Method Selected for: SAM lists this method for preparation and analysis of air samples.
Detection and Quantitation: The detection limit is 10 \\.g in a 30-L sample.
Description of Method: Boron trifluoride is determined as fluoroborate. A volume of 30 to 480 L of air
is drawn through a bubbler containing 0.1 M ammonium fluoride. The solution is diluted and analyzed
with a fluoroborate ISE.
Source: OSHA. 1989. "Method ID216SG: Boron Trifluoride (BF3)." Method originally obtained from
www.gshg,go\. but is provided here for reference.
5.2.75 OSHA Method PV2004: Acrylamide
Analyte(s)
Acrylamide
Acrylonitrile
Methylacrylonitrile
CASRN
79-06-1
107-13-1
126-98-7
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Solvent desorption
Determinative Technique: HPLC
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Section 5 - Selected Chemical Methods
Method Developed for: Acrylamide in air
Method Selected for: SAM lists this method for preparation and analysis of air samples.
Detection and Quantitation: The detection limit was found to be 0.7 pig/mL (0.006 mg/m3 for a 1-mL
desorption volume or 0.029 mg/m3 for a 5-mL desorption volume based on a 120-L air volume).
Applicable working ranges for a 1-mL and 5-mL desorption volume are 0.017 - 1.5 mg/m3 and 0.083 - 7.5
mg/m3, respectively.
Description of Method: This method determines the concentration of acrylamide in air by using HPLC
with a UV detector. Samples are collected by drawing known volumes of air through OSHA versatile
sampler (OVS-7) tubes, each containing a glass fiber filter and two sections of XAD-7 adsorbent.
Samples are desorbed with a solution of 5% methanol/95% water and analyzed by HPLC using a UV
detector.
Source: OSHA. 1991. "Method PV2004: Acrylamide." http://www.epa.gov/sam/pdfs/OSHA-
PV2004.pdf
5.2.76 OSHA Method PV2103: Chloropicrin
Analyte(s)
Chloropicrin
CASRN
79-06-2
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Solvent desorption
Determinative Technique: GC-ECD
Method Developed for: Chloropicrin in air
Method Selected for: SAM lists this method for preparation and analysis of air samples.
Detection and Quantitation: The detection limit is 0.01 ng, with a l-p:L injection volume. This is the
smallest amount that could be detected under normal operating conditions. The working range is 33.2-
1330
Description of Method: This method determines the concentration of Chloropicrin in air by GC-ECD.
Samples are collected by drawing a known volume of air through two XAD-4 tubes in series. Samples
are desorbed with ethyl acetate and analyzed by GC-ECD.
Special Considerations: The presence of Chloropicrin should be confirmed by either a secondary GC
column or by an MS. Chloropicrin is light sensitive, and samples should be protected from light.
Source: OSHA. 1991. "Method PV2103: Chloropicrin." http://www.epa.gov/sam/pdfs/OSHA-
PV2103.pdf
SAM Revision 4.0 105 September 29, 2008
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Section 5 - Selected Chemical Methods
5.2.77 ASTM Method D5755-03: Standard Test Method for Microvacuum Sampling and
Indirect Analysis of Dust by Transmission Electron Microscopy for Asbestos
Structure Number Surface Loading
Analyte(s)
Asbestos
CASRN
1332-21-4
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Direct transfer
Determinative Technique: Transmission electron microscopy (TEM)
Method Developed for: Asbestos in dust
Method Selected for: SAM lists this method for preparation and analysis of solid (e.g., soft surfaces-
microvac) samples.
Description of Method: This method describes procedures to identify asbestos in dust and provide an
estimate of the surface loading of asbestos reported as the number of asbestos structures per unit area of
sampled surface. The sample is collected by vacuuming a known surface area with a standard 25- or 37-
mm air sampling cassette using a plastic tube that is attached to the inlet orifice, which acts as a nozzle.
The sample is transferred from inside the cassette to an aqueous suspension of known volume. Aliquots
of the suspension are then filtered through a membrane, and a section of the membrane is prepared and
transferred to a TEM grid using a direct transfer method. The asbestiform structures are identified, sized,
and counted by TEM, using select area electron diffraction (SAED) and energy dispersive X-ray analysis
(EDXA) at a magnification of 15,000 to 20,OOOX.
Source: ASTM. 2003. "Method D5755-03: Standard Test Method for Microvacuum Sampling and
Indirect Analysis of Dust by Transmission Electron Microscopy for Asbestos Structure Number Surface
Loading." http://www.astm.org/Standards/D5755.htm
5.2.78 ASTM Method D6480-99: Standard Test Method for Wipe Sampling of Surfaces,
Indirect Preparation, and Analysis for Asbestos Structure Number Concentration
by Transmission Electron Microscopy
Analyte(s)
Asbestos
CASRN
1332-21-4
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Direct transfer
Determinative Technique: TEM
Method Developed for: Asbestos in samples wiped from surfaces
Method Selected for: SAM lists this method for preparation and analysis of wipe (e.g., hard surfaces-
wipes) samples.
Description of Method: This method describes a procedure to identify asbestos in samples wiped from
surfaces and to provide an estimate of the concentration of asbestos reported as the number of asbestos
structures per unit area of sampled surface. A sample is collected by wiping a surface of known area with
a wipe material. The sample is transferred from the wipe material to an aqueous suspension of known
volume. Aliquots of the suspension are then filtered through a membrane filter, and a section of the
membrane filter is prepared and transferred to a TEM grid, using the direct transfer method. The
asbestiform structures are identified, sized, and counted by TEM, using electron diffraction (ED) and
EDXA at a magnification from 15,000 to 20,OOOX.
SAM Revision 4.0 106 September 29, 2008
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Section 5 - Selected Chemical Methods
Source: ASTM. 1999. "Method D6480-99: Standard Test Method for Wipe Sampling of Surfaces,
Indirect Preparation, and Analysis for Asbestos Structure Number Concentration by Transmission
Electron Microscopy." http://www.astm.org/DATABASE.CART/HISTORICAL/D6480-99.htm
5.2.79 ISO Method 10312:1995: Ambient Air - Determination of Asbestos Fibres - Direct-
transfer Transmission Electron Microscopy Method
Analyte(s)
Asbestos
CASRN
1332-21-4
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Direct transfer
Determinative Technique: TEM
Method Developed for: Asbestos in ambient air
Method Selected for: SAM lists this method for preparation and analysis of air samples.
Detection and Quantitation: In a 4000-L air sample with approximately 10 pg/m3 (typical of clean or
rural atmospheres), an analytical sensitivity of 0.5 structure/L can be obtained. This is equivalent to a
detection limit of 1.8 structure/L when an area of 0.195 mm of the TEM specimen is examined. The
range of concentrations that can be determined is 50 to 7,000 structures/mm2 on the filter.
Description of Method: This method determines the type(s) of asbestos fibers present, but cannot
discriminate between individual fibers of the asbestos and non-asbestos analogues of the same amphibole
mineral. The method is defined for polycarbonate capillan/pore filters or cellulose ester (either mixed
esters of cellulose or cellulose nitrate) filters through which a known volume of air has been drawn. The
method is suitable for determination of asbestos in both exterior and building atmospheres.
Source: ISO. 2005. "Method 10312: 1995: Ambient Air - Determination of Asbestos Fibres - Direct
Transfer Transmission Electron Microscopy Method."
5.2.80 Standard Method 4500-NH3 B: Nitrogen (Ammonia) Preliminary Distillation Step
Analyte(s)
Ammonia
CASRN
7664-41-7
Analysis Purpose: Sample preparation
Sample Preparation Technique: Distillation
Determinative Technique: Standard Method 4500-NH3 G
Method Developed for: Nitrogen (ammonia) in drinking waters, clean surface or groundwater, and
good-quality nitrified wastewater effluent
Method Selected for: SAM lists this method for preparation of aqueous liquid samples.
Description of Method: A 0.5- to 1-L sample is dechlorinated, buffered, adjusted to pH 9.5, and distilled
into a sulfuric acid solution. The distillate is brought up to volume, neutralized with sodium hydroxide,
and analyzed by Method 4500-NH3 G.
SAM Revision 4.0 107 September 29, 2008
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Section 5 - Selected Chemical Methods
Source: APHA, AWWA, and WEF. 2005. "Method 4500-NH3 B: Nitrogen (Ammonia) Preliminary
Distillation Step." Standard Methods for the Examination of Water and Wastewater. 21st Edition.
http://www.standardmethods.org/
5.2.81 Standard Method 4500-NH3 G: Nitrogen (Ammonia) Automated Phenate Method
Analyte(s)
Ammonia
CASRN
7664-41-7
Analysis Purpose: Analysis
Sample Preparation Technique: Standard Method 4500-NH3 B
Determinative Technique: Spectrophotometry
Method Developed for: Nitrogen (ammonia) in drinking waters, clean surface or groundwater, and
good-quality nitrified wastewater effluent
Method Selected for: SAM lists this method for analysis of aqueous liquid samples.
Detection and Quantitation: The range of the method is 0.02 to 2.0 mg/L.
Description of Method: Ammonia is determined as indophenol blue by this method. A portion of the
neutralized sample distillate (from procedure 4500-NH3 B) is run through a manifold. The ammonium in
the distillate reacts with solutions of disodium ethylenediaminetetraacetic acid (EDTA), sodium phenate,
sodium hypochlorite, and sodium nitroprusside. The resulting indophenol blue is detected by colorimetry
in a flow cell. Photometric measurement is made between the wavelengths of 630 and 660 nm.
Source: APHA, AWWA, and WEF. 2005. "Method 4500-NH3 G: Nitrogen (Ammonia) Automated
Phenate Method." Standard Methods for the Examination of Water and Wastewater. 21st Edition.
http://www staii' laidiuotlioi ls.org/
5.2.82 Standard Method 4500-CI G: DPD Colorimetric Method
Analyte(s)
Chlorine
CASRN
7782-50-5
Analysis Purpose: Sample preparation and/or analysis
Sample Preparation Technique: Water samples are buffered and colorimetric agent is added.
Procedures in Analyst, 1999. 124: 1853-1857 are used for preparation of air samples.
Determinative Technique: Spectrophotometry
Method Developed for: Chlorine in water and wastewater
Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid and drinking
water samples. It also should be used for analysis of air samples when appropriate sample preparation
techniques have been applied.
Detection and Quantitation: The method can detect 10 \ig/L chlorine.
Description of Method: A portion of aqueous liquid sample is buffered and reacted with N,N-diethyl-/>
phenylenediamine (DPD) color agent. The resulting free chlorine is determined by colorimetry. If total
chlorine (including chloroamines and nitrogen trichloride) is to be determined, potassium iodide crystals
are added. Results for chromate and manganese are blank corrected using thioacetamide solution.
Special Considerations: Organic contaminants and strong oxidizers may cause interference.
SAM Revision 4.0 108 September 29, 2008
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Section 5 - Selected Chemical Methods
Source: APHA, AWWA, and WEF. 2005. "Method 4500-C1 G: DPD Colorimetric Method." Standard
Methods for the Examination of Water and Wastewater. 21st Edition, http://www.standardmethods.org/
5.2.83 Literature Reference for Chlorine (Analyst, 1999. 124: 1853-1857)
Analyte(s)
Chlorine
CASRN
7782-50-5
Analysis Purpose: Sample preparation
Sample Preparation Technique: Buffered water extraction
Determinative Technique: Standard Method 4500-C1 G
Method Developed for: Active chlorine in air
Method Selected for: SAM lists this procedure for preparation of air samples.
Detection and Quantitation: Detection limit of 0.1 mg of chlorine; the collection efficiency was >90%;
recovery of chlorine spikes from 0.05-g aliquots of the sorbent was not quantitative (-60%) but was
reproducible.
Description of Method: A procedure is described for determination of total combined gas-phase active
chlorine (i.e., C12) hypochlorous acid [HOC1], and chloramines) and is based on a sulfonamide-
functionalized silica gel sorbent. For determination of the collected chlorine, a modified version of the
DPD colorimetric procedure is used, which yielded a detection limit of 0.1 mg of chlorine. At flow rates
ranging from 31 to 294 mL/min, the collection efficiency was >90% based on breakthrough analysis.
Recovery of chlorine spikes from 0.05-g aliquots of the sorbent was not quantitative (-60%) but was
reproducible; the recovery is accounted for in samples by adding weighed amounts of sorbent to the
standards.
Source: Johnson, B.J., Emerson, D.W., Song, L., Floyd, J., and Tadepalli, B. 1999. "Determination of
active chlorine in air by bonded phase sorbent collection and spectophotometric analysis." Analyst.
124(12): 1853-1857. wmw.epa.gov/sam/pdfs/Analystl24 pgl853-1857.pdf
5.2.84 Literature Reference for Fluoroacetate salts (Analytical Letters, 1994. 27 (14):
2703-2718)
Analyte(s)
Fluoroacetic acid and fluoroacetate salts
Methyl fluoroacetate
CASRN
NA
453-18-9
Analysis Purpose: Sample preparation
Sample Preparation Technique: Ultrasonic extraction
Determinative Technique: EPA Method 300.1, Revision 1.0
Method Developed for: Sodium fluoroacetate in soil
Method Selected for: SAM lists this procedure for preparation of solid and non-aqueous liquid/organic
solid samples.
Description of Method: Sodium fluoroacetate is determined at sub-microgram per gram concentrations
in small (-1 g) soil samples. Samples are ultrasonically extracted with water, filtered, and analyzed by
Method 300.1.
SAM Revision 4.0 109 September 29, 2008
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Section 5 - Selected Chemical Methods
Source: Tomkins, B.A. 1994. "Screening-Procedure for Sodium Fluoroacetate (Compound 1080) at
Sub-Microgram/Gram Concentrations in Soils." Analytical Letters. 27(14): 2703-2718.
jjifa^
5.2.85 Literature Reference for Perfluoroisobutylene (Journal of Chromatography A,
2005. 1098: 156-165)
Analyte(s)
Perfluoroisobutylene (PFIB)
CASRN
382-21-8
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Thermal desorption
Determinative Technique: GC-FPD / GC-NPD / GC-MS
Method Developed for: Phosgene and PFIB in air
Method Selected for: SAM lists this method for use if problems occur when using OSHA Method 61
for the analysis of perfluoroisobutylene during preparation and analysis of air samples. (See Footnote 16
of Appendix A.)
Detection and Quantitation: Limits of detection for PFIB with 2-aminothiophenol (PFIB-ATP) and
PFIB with 3,4-dimercaptotoluene (PFIB-DMT), using 10-L air samples (typical sampling volume) by
GC-MS analyses were determined to be 2 and 19 ng/m3, respectively.
Description of Method: This procedure is for the identification and measurement of phosgene and
perfluoroisobutylene in air using GC-MS, GC-NPD, or GC-FPD. This procedure is to be used in the
event that OSHA Method 61 is problematic for the determination of PFIB. Air samples are collected by
drawing known volumes of air through sampling tubes containing Tenax® TA sorbent coated with 2-
aminothiophenol (ATP) or 3,4-dimercaptotoluene (DMT). The derivatized samples are thermally
desorbed and analyzed with one of the GC techniques. The 2-aminothiophenol derivative can be
analyzed by either of the three techniques, whereas the 3,4-dimercaptotoluene is not suitable for analysis
by GC-NPD or GC-FPD.
Special Considerations: If a determinative technique other than GC-MS is used, the presence of PFIB
should be confirmed by either a secondary GC column or by an MS.
Source: Muir, B., Cooper, D.B., Carrie, W.A., Timperley, C.M., Slater, B.J., and Quick, S. 2005.
"Analysis of Chemical Warfare Agents III. Use of bis-Nucleophiles in the Trace Level Determination of
Phosgene and Perfluoroisobutylene." Journal of Chromatography A. 1098: 156-165.
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SAM Revision 4.0 110 September 29, 2008
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Section 5 - Selected Chemical Methods
5.2.86 Literature Reference for Sodium Azide (Journal of Forensic Sciences, 1998. 43(1):
200-202)
Analyte(s)
Sodium azide
CASRN
26628-22-8
Analysis Purpose: Sample preparation
Sample Preparation Technique: Water extraction, filtration, and/or acidification
Determinative Technique: EPA Method 300.1, Revision 1.0
Method Developed for: Sodium azide in blood
Method Selected for: SAM lists this procedure for preparation of solid, non-aqueous liquid/organic
solid, aqueous liquid, and drinking water samples.
Detection and Quantitation: This method can routinely quantify to at least 100 jig/L, and the detection
limit is estimated to be 30
Description of Method: Samples are analyzed by 1C using suppressed conductivity detection. Water
extraction and filtration steps should be used for the preparation of solid samples. Filtration steps should
be used for preparation of aqueous liquid and drinking water samples. An acidification step may be
required prior to using EPA SW-846 Method 3580A for preparation of non-aqueous liquid/organic solid
samples.
Special Considerations: The procedure described above has been developed for the analysis of sodium
azide in blood samples.
Source: Kruszyna, R., Smith, R.P., and Kruszyna, H. 1998. "Determining Sodium Azide Concentration
in the Blood by Ion Chromatography." Journal of Forensic Sciences. 43(1) : 200-202.
http://iournalsip.astin.org/TOURNALS/FORENSIC/PAGES/2933.htm
SAM Revision 4.0 111 September 29, 2008
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Section 5 - Selected Chemical Methods
SAM Revision 4.0 112 September 29, 2008
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Section 6 - Selected Radiochemical Methods
Section 6.0: Selected Radiochemical Methods
A list of analytical methods to be used in analyzing environmental samples for radiochemical
contaminants during homeland security events is provided in Appendix B. Methods are listed for each
isotope and for each sample type that potentially may need to be measured and analyzed when responding
to an environmental emergency.
Please note: This section provides guidance for selecting radiochemical methods that have a high
likelihood of assuring analytical consistency when laboratories are faced with a large scale
environmental restoration crisis. Not all methods have been verified for the analyte/sample type
combination listed in Appendix B. Please refer to the specified method to identify analyte/sample type
combinations that have been verified. Any questions regarding information discussed in this section
should be addressed to the appropriate contact(s) listed in Section 4.
Appendix B is sorted alphabetically by analyte and includes the following information:
• Analyte(s). The radionuclide(s) or contaminant (s) of interest.
• CAS RN. A unique identifier for chemical substances that provides an unambiguous way to identify
a chemical or molecular structure when there are many possible systematic, generic, or trivial names.
In this section (Section 6.0) and Appendix B, the CAS RNs correspond to the specific radionuclide
identified.
• Determinative technique. An analytical instrument or technique used for qualitative and
confirmatory determination of compounds or components in a sample.
• Drinking water sample methods. The recommended methods/procedures for sample preparation
and analysis to measure the analyte of interest in drinking water samples. Methods have been
identified for qualitative and confirmatory determination.
• Aqueous and liquid phase sample methods. The recommended methods/procedures for sample
preparation and analysis to measure the analyte of interest in aqueous and/or non-aqueous liquid
phase samples. Methods have been identified for qualitative and confirmatory determination.
• Soil and sediment phase sample methods. The recommended methods/procedures for sample
preparation and analysis to measure the analyte of interest in soil and sediment samples. Methods
have been identified for qualitative and confirmatory determination.
• Surface wipe sample methods. The recommended methods/procedures for sample preparation and
analysis to measure the analyte of interest in surface wipe samples. Methods have been identified for
qualitative and confirmatory determination.
• Air filter sample methods. The recommended methods/procedures for sample preparation and
analysis to measure the analyte of interest in air filter samples. Methods have been identified for
qualitative and confirmatory determination.
• Qualitative determination method identifier. A unique identifier or number assigned to an
analytical method by the method publisher. The identified method is intended to determine the
presence of a radiological element or isotope. These methods are less precise than confirmatory
methods, and are used when greater sample throughput and more rapid reporting of results is
required.
• Confirmatory method identifier. A unique identifier or number assigned to an analytical method by
the method publisher. The identified method is for measurement of the activity from a particular
radioisotope per unit of mass, volume, or area sampled.
SAM Revision 4.0 113 September 29, 2008
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Section 6 - Selected Radiochemical Methods
6.1 General Guidance
The guidance summarized in this section provides a general overview of how to identify the appropriate
radiochemical method (s) for a given analyte-sample type combination, as well as recommendations for
QC procedures.
For additional information on the properties of the radionuclides listed in Appendix B, TOXNET
(http://toxnet.nlm.nih.gov/index.html), a cluster of databases on toxicology, hazardous chemicals, and
related areas maintained by the National Library of Medicine, is an excellent resource. EPA's Radiation
Protection (http://www.epa.gov/radiation/radionuclides/index.html) and the Multi-Agency Radiological
Laboratory Analytical Protocols Manual (MARLAP) (http://www.epa.gov/radiation/marlap/manual.html)
Web sites provide some additional information pertaining to radionuclides of interest and selection of
radiochemical methods.
6.1.1 Standard Operating Procedures for Identifying Radiochemical Methods
To determine the appropriate method to be used on an environmental sample, locate the analyte of
concern in Appendix B: Radiochemical Methods under the "Analyte Class" or "Analyte(s)" column.
After locating the analyte of concern, continue across the table to identify the appropriate determinative
technique (e.g., alpha spectrometry), then identify the appropriate qualitative and/or confirmatory method
for the sample type of interest (drinking water, aqueous and liquid phase, soil and sediment, surface
wipes, and air filters) for the particular analyte.
Sections 6.2.1 through 6.2.27, below, provide summaries of the qualitative and confirmatory methods
listed in Appendix B. Once a method has been identified in Appendix B, Table 6-1 can be used to locate
the method summary.
Table 6-1. Radiochemical Methods and Corresponding Text Section Numbers
Analyte / Analyte Class
Gross Alpha
Gross Beta
Gamma
Americium-241
Californium-252
CASRN
NA
NA
NA
14596-10-2
13981-17-4
Method
900.0 (EPA)
FRMAC, Vol 2, pg. 33
AP1 (ORISE)
7110B(SM)
901.1 (EPA)
Ga-01-R(HASL-300)
Am-01-RC (HASL-300)
Am-02-RC (HASL-300)
Am-04-RC (HASL-300)
Pu-12-RC (HASL-300)
AP11 (ORISE)
D3084 (ASTM)
Am-01-RC (HASL-300)
Am-04-RC (HASL-300)
Pu-12-RC (HASL-300)
AP11 (ORISE)
D3084 (ASTM)
Section
6.2.2
6.2.16
6.2.17
6.2.21
6.2.3
6.2.12
6.2.9
6.2.10
6.2.11
6.2.14
6.2.18
6.2.19
6.2.9
6.2.11
6.2.14
6.2.18
6.2.19
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Section 6 - Selected Radiochemical Methods
Analyte / Analyte Class
Cesium-137
Cobalt-60
Curium-244
Europium-154
lridium-192
Plutonium-238
Plutonium-239
Ruthenium-103
Ruthenium-106
Selenium-75
Uraniurn-234
Uranium-235
Uranium-238
CASRN
10045-97-3
10198-40-0
13981-15-2
15585-10-1
1 r\r\A T ££ n
14694-69-0
13981-16-3
15117-48-3
1 TQR1 c;9 7
1 QQQO CQ Q
13968-53-1
13967-48-1
14265-71-5
1 nnQR Q7 o
I oybb-^y-o
15117-96-1
7440-61-1
Method
901.1 (EPA)
Ga-01-R(HASL-300)
7120(SM)
Am-01-RC (HASL-300)
Am-04-RC (HASL-300)
Pu-12-RC (HASL-300)
AP11 (ORISE)
D3084 (ASTM)
901.1 (EPA)
Ga-01-R (HASL-300)
7120(SM)
901.1 (EPA)
Ga-01-R (HASL-300)
901.1 (EPA)
Ga-01-R (HASL-300)
7120(SM)
EMSL-33 (EPA)
AP11 (ORISE)
D3084 (ASTM)
Method 111 (EPA)
Po-02-RC (HASL-300)
903.0 (EPA)
903.1 (EPA)
EMSL-19(EPA)
D3084 (ASTM)
7500-Ra B (SM)
7500-Ra C (SM)
901.1 (EPA)
Ga-01-R (HASL-300)
7120(SM)
Sr-03-RC (HASL-300)
7500-Sr B (SM)
908.0 (EPA)
EMSL-33 (EPA)
AP11 (ORISE)
D3084 (ASTM)
D3972 (ASTM)
7500-U B (SM)
7500-U C (SM)
Section
6.2.3
6.2.12
6.2.22
6.2.9
6.2.11
6.2.14
6.2.18
6.2.19
6.2.3
6.2.12
6.2.22
6.2.3
6.2.12
6.2.3
6.2.12
6.2.22
6.2.8
6.2.18
6.2.19
6.2.1
6.2.13
6.2.4
6.2.5
6.2.7
6.2.19
6.2.23
6.2.24
6.2.3
6.2.12
6.2.22
6.2.15
6.2.25
6.2.6
6.2.8
6.2.18
6.2.19
6.2.20
6.2.26
6.2.27
The method summaries are listed in order of method selection hierarchy (see Figure 2-1), starting with
EPA methods, followed by methods from other federal agencies and VCSBs. Methods are listed in
SAM Revision 4.0
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Section 6 - Selected Radiochemical Methods
numerical order under each publisher. Where available, a direct link to the full text of the selected
analytical method is provided in the method summary. For additional information regarding sample
preparation and analysis procedures and on methods available through consensus standards organizations,
please use the contact information provided in Table 6-2.
Table 6-2. Sources of Radiochemical Methods
Name
NEMI
CFR Promulgated Test
Methods (TM)
Prescribed Procedures for
Measurement of Radioactivity
in Drinking Water (EPA-600 4-
80-032, August 1980)
Radiochemical Analytical
Procedures for Analysis of
Environmental Samples, March
1979. EMSL-LV-0539-17
EML Procedures Manual,
Health and Safety Laboratory
(HASL-300), 28th Edition,
February, 1997
Federal Radiological Monitoring
and Assessment Center
(FRMAC) Laboratory Manual
Oak Ridge Institute for Science
and Education (ORISE)
Laboratory Procedures Manual
Annual Book of AST M
Standards, Vol. 11.02*
Standard Methods for the
Examination of Water and
Wastewater, 21st Edition, 2005*
Publisher
U.S. EPA, USGS
U.S. EPA, Technical Transfer
Network (TTN) EMC
U.S. EPA, ORD, Environmental
Monitoring and Support Laboratory
(EMSL)
U.S. EPA, EMSL
U.S. Department of Energy (DOE),
Environmental Measurements
Laboratory (EML) / Now U.S. DHS
United States DOE
National Nuclear Security
Administration (NNSA)
ORISE
Independent Environmental
Assessment and Verification
ASTM International
APHA, AWWA, and WEF
Reference
http://www.nemi.gov
Jltt|)^/WWJ/y||)|L§OV^
http://www.sld.state.nm.us/Documents/for
ewd.pdf
Also available from National Technical
Information Service (NTIS)*, U.S.
Department of Commerce, 5285 Port
Royal Road, Springfield, VA 22161, (703)
605-6000.
Available NTIS*, U.S. Department of
Commerce, 5285 Port Royal Road,
Springfield, VA 22161, (703) 605-6000.
http://www.eml.st.dhs.qov/publications/pro
cman.cfm
Also available from NTIS*, U.S.
Department of Commerce, 5285 Port
Royal Road, Springfield, VA 22161, (703)
605-6000.
http://www.nv.doe.aov/nationalsecuritv/ho
mejandjecujlty/fjTnac/m^iTu^is^aspx
http://orise.orau.qov/ieav/survev-
£!I!JeMs/!iiy]T|M3MMMlD
http://www.astm.orq
http://www.standardrnethods.orq
Subscription and/or purchase required.
6.1.2 General QC Guidance for Radiochemical Methods
Having data of known and documented quality is critical so that public officials can accurately assess the
activities that may be needed in responding to emergency situations. Having such data requires that
laboratories: (1) conduct the necessary QC to ensure that measurement systems are in control and
operating correctly; (2) properly document results of the analyses; and (3) properly document
measurement system evaluation of the analysis-specific QC. Ensuring data quality also requires that
laboratory results are properly evaluated and the results of the data quality evaluation are transmitted to
decision makers.
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Section 6 - Selected Radiochemical Methods
The level or amount of QC needed often depends on the intended purpose of the data that are generated.
Various levels of QC may be required if the data are generated during contaminant presence/absence
qualitative determinations versus confirmatory analyses. The specific needs for data generation should be
identified. QC requirements and data quality objectives should be derived based on those needs, and
should be applied consistently across laboratories when multiple laboratories are used. For example,
during rapid sample screening analyses, minimal QC samples (e.g., blanks, duplicates) and
documentation might be required to ensure data quality. Implementation of the analytical methods for
evaluation of environmental samples during site assessment through site clearance, such as those
identified in this document, might require increased QC.
Some method-specific QC requirements are described in many of the individual methods that are cited in
this manual. QC requirements will be referenced in SAPs developed to address specific analytes and
sample types of concern. Individual methods, sampling and analysis protocols, or contractual statements
of work also should be consulted to determine any additional QC that may be needed.
QC samples are required to assess the precision, bias, and reliability of sample results. All QC results are
tracked on control charts and reviewed for acceptability and trends in analysis or instrument operation.
QC parameters are measured as required per method at the prescribed frequency. QC of laboratory
analyses using radiochemical methods includes ongoing analysis of QC samples and tracking QC
parameters including, but not limited to the following:
• Method blanks;
• Calibration checks;
• Sample and sample duplicates;
• Laboratory control sample recoveries
• MS/MSD recoveries; and
• Tracer and/or carrier yield.
Please note: The appropriate point of contact identified in Section 4 should be consulted regarding
appropriate QA/QC procedures prior to sample analysis. These contacts will consult with the EPA
coordinator responsible for laboratory activities during the specific event to ensure QA/QC procedures are
performed consistently across laboratories. EPA program offices will be responsible for ensuring that the
QA/QC practices are implemented.
6.1.3 Safety and Waste Management
It is imperative that safety precautions be used during collection, processing, and analysis of
environmental samples. Laboratories should have a documented health and safety plan for handling
samples that may contain target CBR contaminants, and laboratory staff should be trained in and
implement the safety procedures included in the plan. In addition, many of the methods summarized or
cited in Section 6.2 contain specific requirements, guidance, or information regarding safety precautions
that should be followed when handling or processing environmental samples and reagents. These
methods also provide information regarding waste management. Other resources that can be consulted
for additional information include the following:
. OSHA - 29 CFR part 1910.1450. Occupational Exposure to Hazardous Chemicals in Laboratories.
http ://www.access. gpo.gov/nara/cfr/waisidx 06/29cfr 1 9 1 Oa 06.html
. EPA - 40 CFR part 260. Hazardous Waste Management System: General.
http://www.access.gpo.gov/nara/cfr/waisidx 07/40cfr260 07.html
. EPA - 40 CFR part 270. EPA Administered Permit Programs: The Hazardous Waste Permit Program.
...... 07/40cfr270 ..... OJJitml
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Section 6 - Selected Radiochemical Methods
NRC - 10 CFR part 20. Standards for Protection Against Radiation
http://www.access.gpo.gov/nara/cfr/waisidx 00/10cfr20 00. html
U.S. DOE. Order O 435.1: Radioactive Waste Management. July 1, 1999. Available at:
U.S. DOE. M 435.1-1. Radioactive Waste Management Manual. Office of Environmental
Management. July 9, 1999. Available at:
wj^¥jte
U.S. DOE. Compendium of EPA-Approved Analytical Methods for Measuring Radionuclides in
Drinking Water. Prepared by the Office of Environmental Policy and Assistance Air, Water and
Radiation Division (EH-412). June 1998
U.S. EPA. 1996. Profile and Management Options for EPA Laboratory Generated Mixed Waste.
Office of Radiation and Indoor Air, Washington, DC
EPA 402-R-96-015. August. Profile and Management Options for EPA Laboratory Generated
Mixed Waste. Available at: hjt]3://ep_a.j_w/^
. U.S. EPA. 2001. Changes to 40 CFR 266 (Storage, Treatment, Transportation, and Disposal of
Mixed Waste), Federal Register 66:27217-27266, May 16
. U.S. EPA. 2002. RCRA Orientation Manual OSWER, Washington, DC. EPA530-R-02-016. 259
pp. Available at: hJtpi/Mww^pa.gQWepaQswef^general/Qrientat/
. Waste Management in a Radioanalytical Laboratory, Chapter 17 MARLAP Manual, July 2004
. National Research Council. 1995. Prudent Practices in the Laboratory: Handling and Disposal of
Chemicals, National Academy Press, Washington, DC
. National Council on Radiation Protection and Measurements (NCRP). 2002. Risk-Based
Classification of Radioactive and Hazardous Chemical Wastes, 7910 Woodmont Avenue, Suite 400,
Bethesda, MD 20814-3095
. U.S. Nuclear Regulatory Commission (NRC) / U.S. EPA. 1995. Low-Level Mixed Waste Storage
Guidance, Federal Register 60:40204-402 1 1
6.2 Method Summaries
Summaries for the analytical methods listed in Appendix B are provided in Sections 6.2.1 through 6.2.27.
These summaries contain information that has been extracted from the selected methods. Each method
summary contains a table identifying the contaminants in Appendix B to which the method applies, a
brief description of the analytical method, and a link to the full version of the method or a source for
obtaining a full version of the method. The full version of the method should be consulted prior to
sample analysis.
6.2.1 EPA Method 111: Determination of Polonium-210 Emissions from Stationary
Sources
Analyte(s)
Polonium-210
CASRN
13981-52-7
Analysis Purpose: Qualitative and confirmatory determination
Determinative Technique: Alpha spectrometry
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Method Developed for: Polonium-210 in particulate matter samples collected from stationary source
exhaust stacks
Method Selected for: SAM lists this method for qualitative and confirmatory analysis of surface wipes
and air filters.
Description of Method: This method covers the determination of polonium-210 in particulate matter
samples collected from stationary sources such as exhaust stacks. Polonium-210 in the sample is put in
solution, deposited on a metal disc, and the radioactive disintegration rate measured. Polonium in acid
solution spontaneously deposits on surface metals that are more electropositive than polonium.
Source: EPA EMC, prepared by the OAQPS. 2000. "Method 111: Determination of Polonium-210
Emissions from Stationary Sources."
6.2.2 EPA Method 900.0: Gross Alpha and Gross Beta Radioactivity in Drinking Water
Analysis Purpose: Gross alpha and gross beta determination
Determinative Technique: Alpha/Beta counting
Method Developed for: Gross alpha and gross beta particle activities in drinking water
Method Selected for: SAM lists this method for gross alpha and gross beta determination in drinking
water samples.
Description of Method: The method provides an indication of the presence of alpha and beta emitters,
including the following SAM analytes:
Americium-241 (CAS RN 14596-10-2) Alpha emitter
Californium-252 (CAS RN 13981-17-4) Alpha emitter
Cesium-137 (CAS RN 10045-97-3) Beta emitter
Cobalt-60 (CAS RN 10198-40-0) Beta emitter
Curium-244 (CAS RN 13981-15-2) Alpha emitter
Europium-154 (CAS RN 15585-10-1) Beta emitter
Iridium-192 (CAS RN 14694-69-0) Beta emitter
Plutonium-238 (CAS RN 13981-16-3) Alpha emitter
Plutonium-239 (CAS RN 15117-48-3) Alpha emitter
Polonium-210 (CAS RN 13981-52-7) Alpha emitter
Radium-226 (CAS RN 13982-63-3) Alpha emitter
Ruthenium-103 (CAS RN 13968-53-1) Beta emitter
Ruthenium-106 (CAS RN 13967-48-1) Beta emitter
Strontium-90 (CAS RN 10098-97-2) Beta emitter
Uranium-234 (CAS RN 13966-29-5) Alpha emitter
Uranium-235 (CAS RN 15117-96-1) Alpha emitter
Uranium-238 (CAS RN 7440-16-1) Alpha emitter
An aliquot of a preserved drinking water sample is evaporated to a small volume (3 to 5 mL) and
transferred quantitatively to a tarred 2-inch planchet. The aliquot volume is determined based on a
maximum total solids content of 100 mg. The sample aliquot is evaporated to dryness in the planchet to a
constant weight, cooled, and counted using a gas proportional or scintillation counting system. The
counting system is calibrated with thorium-230 for gross alpha, and with strontium-90 for gross beta
analysis . A traceable standards-based efficiency curve must be developed for each calibration nuclide
(Th-230 and Sr-90) based on a range of total solids content in the 2-inch planchet from 0 to 100 mg (see
method for specific recommendations and requirements for the use of cesium-137).
2 EPA lists standards for use when analyzing drinking water in the table at 40 CFR 141.25 (footnote 11).
SAM Revision 4.0 119 September 29, 2008
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Section 6 - Selected Radiochemical Methods
Special Considerations: Long counting time and increased sample size may be required to meet
detection limits. Sensitivity is limited by the concentration of solids in the sample.
Source: EPA, EMSL. 1980. "Method 900.0: Gross Alpha and Gross Beta Radioactivity in Drinking
Water." Prescribed Procedures for Measurement of Radioactivity in Drinking Water, EPA/600/4/80/032.
6.2.3 EPA Method 901.1: Gamma Emitting Radionuclides in Drinking Water
Analyte(s)
Cesium-137
Cobalt-60
Europium-154
lodine-131
lridium-192
Ruthenium-103
Ruthenium-106
Selenium-75
CASRN
10045-97-3
10198-40-0
15585-10-1
10043-66-0
14694-69-0
13968-53-1
13967-48-1
14265-71-5
Analysis Purpose: Qualitative and confirmatory analysis
Determinative Technique: Gamma spectrometry
Method Developed for: Gamma emitting radionuclides in drinking water
Method Selected for: SAM lists this method for qualitative and confirmatory analysis of drinking water
samples.
Description of Method: This method is applicable for analysis of water samples that contain
radionuclides that emit gamma photons with energies ranging from approximately 60 to 2000 keV. The
method uses gamma spectroscopy for measurement of gamma photons emitted from radionuclides
without separating them from the sample matrix. A homogeneous aliquot of water is placed into a
standard geometry (normally a Marinelli beaker) for gamma counting, typically using a high purity
germanium (HPGe) detector. Detectors such as Germanium (Lithium) (Ge(Li)) or thallium-activated
sodium iodide (Nal(Tl)) also can be used. Sample aliquots are counted long enough to meet the required
sensitivity of measurement. To reduce adsorbance of radionuclides on the walls of the counting
container, the sample is acidified at collection time. Due to its lower resolution, significant interference
can occur using the Nal(Tl) detector when counting a sample containing radionuclides that emit gamma
photons of similar energies. When using this method, shielding is needed to reduce background
interference. Detection limits are dependent on sample volume, geometry (physical shape), and counting
time.
Source: EPA, EMSL. 1980. "Method 901.1: Gamma Emitting Radionuclides in Drinking Water."
Prescribed Procedures for Measurement of Radioactivity in Drinking Water, EPA/600/4/80/032.
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6.2.4 EPA Method 903.0: Alpha-Emitting Radium Isotopes in Drinking Water
Analyte(s)
Radium-226
CASRN
13982-63-3
Analysis Purpose: Qualitative determination
Determinative Technique: Alpha counting
Method Developed for: Total soluble alpha emitting radioisotopes of radium, namely radium-223,
radium-224 and radium-226 in drinking water
Method Selected for: SAM lists this method for qualitative determination in drinking water samples.
Description of Method: This method covers measurement of the total soluble alpha emitting
radioisotopes of radium, namely radium-223, radium-224 and radium-226 in drinking water. The method
does not give an accurate measurement of radium-226 content in the sample when other alpha emitters are
present. If radium-223 and radium-224 are present, the results can be used to provide a gross
determination of radium-226. When the total radium alpha activity of a drinking water sample is greater
than 5 pCi/L, use of Method 903.1 (Radium-226 in Drinking Water) is preferred. Radium in the water
sample is collected by coprecipitation with barium and lead sulfate, and purified by re-precipitation from
EDTA solution. Citric acid is added to ensure that complete interchange occurs before the first
precipitation step. The final barium sulfate precipitate is alpha counted to determine the total
disintegration rate of the radium isotopes. By making a correction for the ingrowth of radon and its alpha
emitting progeny for the elapsed time after separation, one can determine radium activity in the sample.
Presence of significant natural barium in the sample can result in a falsely high yield. Based on a 1000-
mL sample and 100-minute counting time, the minimum detectable level for this method is 0.5 pCi/L.
Source: EPA, EMSL. 1980. "Method 903.0: Alpha-Emitting Radium Isotopes in Drinking Water."
Prescribed Procedures for Measurement of Radioactivity in Drinking Water, EPA/600/4/80/032.
http://www.epj.gov/sam/pdfs/EP A-903.0.pdf
6.2.5 EPA Method 903.1: Radium-226 in Drinking Water- Radon Emanation Technique
Analyte(s)
Radium-226
CASRN
13982-63-3
Analysis Purpose: Confirmatory analysis
Determinative Technique: Alpha counting
Method Developed for: Radium-226 in drinking water
Method Selected for: SAM lists this method for confirmatory analysis of drinking water samples.
Description of Method: This method is specific for radium-226, and is based on the emanation and
scintillation counting of radon-222, a daughter product of radium-226. Radium-226 is concentrated and
separated from the water sample by coprecipitation on barium sulfate. The precipitate is dissolved in
EDTA reagent, placed in a sealed bubbler and stored for ingrowth of radon-222. After ingrowth, the
radon-222 gas is purged into a scintillation cell. When the short-lived radon-222 daughters are in
equilibrium with the parent (after ~4h), the scintillation cell is counted for activity. The absolute
measurement of radium-226 is effected by calibrating the scintillation cell system with a standard solution
of the nuclide. There are no radioactive interferences in this method. Based on a 1000-mL sample and
100-minute counting time, the minimum detectable level for this method is 0.5 pCi/L.
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Source: EPA, EMSL. 1980. "Method 903.1: Radium-226 in Drinking Water - Radon Emanation
Technique." Prescribed Procedures for Measurement of Radioactivity in Drinking Water,
EPA/600/4/80/032. http://www.epa.gov/sam/pdfs/EPA-903.1 .pdf
6.2.6 EPA Method 908.0: Uranium in Drinking Water- Radiochemical Method
Analyte(s)
Uranium-234
Uranium-235
Uranium-238
CASRN
13966-29-5
15117-96-1
7440-61-1
Analysis Purpose: Qualitative determination
Determinative Technique: Alpha counting
Method Developed for: Total uranium alpha particle activity in drinking water
Method Selected for: SAM lists this method for qualitative determination in drinking water samples.
Description of Method: This method measures total uranium alpha activity of a sample, without doing
an isotopic uranium analysis. The sample is acidified with hydrochloric acid and boiled to eliminate
carbonate and bicarbonate ions. Uranium is coprecipitated with ferric hydroxide and separated from the
sample. The uranium is then separated from other radionuclides that were carried down with the ferric
hydroxide by dissolving the hydroxide precipitate in hydrochloric acid, putting the solution through an
anion exchange column, washing the column with hydrochloric acid, and finally eluting the uranium with
hydrochloric acid. The uranium eluate is evaporated and the uranium chemical form is converted to
nitrate. The residue is transferred to a stainless steel planchet, dried, flamed, and counted for alpha
particle activity. Since uranium is a naturally occurring radionuclide, reagents must be checked for
uranium contamination by analyzing a complete reagent blank by the same procedure as used for the
samples. Based on a 1000- mL sample and 100-minute counting time in a single laboratory study, the
minimum detectable level for this method is 1.0 pCi/L.
Special Considerations: If it is suspected that the sample exists in refractory form (i.e., non-digestible
or dissolvable material after normal digestion methods) or if there is a matrix interference problem, use
ORISE Method API 1.
Source: EPA, EMSL. 1980. "Method 908.0: Uranium in Drinking Water - Radiochemical Method."
Prescribed Procedures for Measurement of Radioactivity in Drinking Water, EPA/600/4/80/032.
http://www.epa.gOv/sam/pdfs/EPA-908.0.pdf
6.2.7 EPA Method EMSL-19: Determination of Radium-226 and Radium-228 in Water,
Soil, Air and Biological Tissue
Analyte(s)
Radium-226
CASRN
13982-63-3
Analysis Purpose: Confirmatory analysis
Determinative Technique: Alpha counting
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Method Developed for: Radium-226 and radium-228 in water, soil, air, biological tissues, and
biological fluids
Method Selected for: SAM lists this method for confirmatory analysis of soil/sediment, surface wipe,
and air filter samples.
Description of Method: Following acid digestion and filtration of soil, sediment, surface wipe, or air
filter samples, radium is precipitated with barium sulfate. Barium-radium-sulfate is dissolved in a
pentasodium diethylenetriamine-pentaacetate (DTPA) solution and transferred to an emanation tube. The
radon is allowed to come to equilibrium for approximately 30 days. Radium-226 decays by alpha
emission to radon-222. Radon-222 is separated and collected from the liquid by a de-emanation
technique. The radon is counted by alpha scintillation 4.5 hours after de-emanation, at which time the
short-lived progeny have reached 97% of equilibrium. An applicable measurement range has not been
determined; however, samples that contain 0.1 pCi of Radium-226 have been analyzed.
Source: EPA, EMSL. 1979. "EMSL-19: Determination of Radium-226 and Radium-228 in Water, Soil,
Air and Biological Tissue." Radiochemical Analytical Procedures for Analysis of Environmental
Samples. http://www.epa.gov/sam/pdfs/EPA-EMSL-19.pdf
6.2.8 EPA Method EMSL-33: Isotopic Determination of Plutonium, Uranium, and
Thorium in Water, Soil, Air, and Biological Tissue
Analyte(s)
Plutonium-238
Plutonium-239
Uranium-234
Uranium-235
Uranium-238
CASRN
13981-16-3
15117-48-3
13966-29-5
15117-96-1
7440-61-1
Analysis Purpose: Confirmatory analysis
Determinative Technique: Alpha spectrometry
Method Developed for: Isotopic plutonium, uranium, and thorium, together or individually, in soil,
water, air filters, urine, or ashed residues of vegetation, animal tissues, and bone
Method Selected for: SAM lists this method for confirmatory analysis of drinking water,
aqueous/liquid, soil/sediment, surface wipe, and/or air filter samples.
Description of Method: This method is appropriate for the analysis of isotopic plutonium, uranium, and
thorium, together or individually, by alpha spectrometry. Plutonium-236, uranium-232, and thorium-234
tracer standards are added for the determination of chemical yields. Samples are decomposed by nitric-
hydrofluoric acid digestion or ignition to assure that all of the plutonium is dissolved and chemically
separated from the sample by coprecipitation with sodium and ammonium hydroxide, anion exchange,
and electrodeposition. The residues are dissolved in dilute nitric acid and successive sodium and
ammonium hydroxide precipitations are performed in the presence of boric acid to remove fluoride and
soluble salts. The hydroxide precipitate is dissolved, the solution is pH-adjusted with hydrochloric acid,
and plutonium and uranium are adsorbed on an anion exchange column, separating them from thorium.
Plutonium is eluted with hydrobromic acid. The actinides are electrodeposited on stainless steel discs
from an ammonium sulfate solution and subsequently counted by alpha spectrometry. This method is
designed to detect environmental levels of activity as low as 0.02 pCi per sample. To avoid possible
cross-contamination, sample aliquot activities should be limited to 25 pCi or less.
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Section 6 - Selected Radiochemical Methods
Special Considerations: If it is suspected that the sample exists in refractory form (i.e., non-digestible
or dissolvable material after normal digestion methods) or if there is a matrix interference problem, use
ORISE Method API 1.
Source: EPA, EMSL. 1979. "EMSL-33: Isotopic Determination of Plutonium, Uranium, and Thorium in
Water, Soil, Air, and Biological Tissue." Radiochemical Analytical Procedures for Analysis of
Environmental Sample. http://www.epa.gov/sam/pdfs/EPA-EMSL-33.pdf
6.2.9 EML HASL-300 Method Am-01-RC: Americium in Soil
Analyte(s)
Americium-241
Californium-252
Curium-244
CASRN
14596-10-2
13981-17-4
13981-15-2
Analysis Purpose: Confirmatory analysis
Determinative Technique: Alpha spectrometry
Method Developed for: Americium in soil
Method Selected for: SAM lists this method for confirmatory analysis of soil/sediment samples.
Description of Method: This method uses alpha spectrometry for determination of americium-241 in
soil, and also can be applied for determination of californium. Americium is leached from soil with nitric
acid and hydrochloric acid. Americium-243 is added as a tracer to determine chemical yield. The soil is
processed through the plutonium separation steps using ion exchange resin according to Method Pu-11-
RC. Americium is collected with a calcium oxalate precipitation and finally isolated and purified by ion
exchange. Californium-252 and curium-244 are eluted with americium as americium is stripped off the
column. After source preparation by microprecipitation, americium-241, californium-252, and curium-
244 are determined by alpha spectrometry analysis. The counting period chosen depends on the
sensitivity required of the measurement and the degree of uncertainty in the result that is acceptable. The
lower limit of detection (LLD) for americium-241 is 0.5 mBq when counted for 1000 minutes. In cases
where less than 100 g of sample is available, use of Pu-12-RC is recommended.
Special Considerations: If it is suspected that the sample exists in refractory form (i.e., non-digestible
or dissolvable material after normal digestion methods) or if there is a matrix interference problem, use
ORISE Method API 1.
Source: EML, U.S. DOE (EML is currently part of the U.S. DHS). 1997. "HASL-300 Method Am-01-
RC: Americium in Soil." EML Procedures Manual, HASL-300, 28th Edition.
http://www.epa.gov/sam/pdfs/EML-Am-01-RC.pdf
6.2.10 EML HASL-300 Method Am-02-RC: Americium-241 in Soil-Gamma Spectrometry
Analyte(s)
Americium-241
CASRN
14596-10-2
Analysis Purpose: Qualitative determination
Determinative Technique: Gamma spectrometry
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Method Developed for: Americium-241 in large volume soil samples
Method Selected for: SAM lists this method for qualitative determination in soil/sediment samples.
Description of Method: This method uses gamma spectrometry for determination of americium-241 in
soil. Americium-241 decays with the emission of a gamma ray at 59.5 keV with a decay frequency
(abundance or yield) of 35.9%. The sample is placed into an appropriately sized standard geometry
(normally a Marinelli beaker) after drying and grinding the sample for homogenization. Gamma-ray
attenuation corrections are required if the calibration source and the sample are in a different matrix or are
of different densities. The LLD for 600 to 800 g of soil in a Marinelli beaker is 0.74 mBq for a 1000-
minute count.
Source: EML, U.S. DOE (EML is currently part of the U.S. DHS). 1997. "HASL-300 Method Am-02-
RC: Americium-241 in Soil-Gamma Spectrometry." EML Procedures Manual, HASL-300, 28th Edition.
http://www.epa.gov/sam/pdfs/EML-Am-02-RC.pdf
6.2.11 EML HASL-300 Method Am-04-RC: Americium in QAP Water and Air Filters
Eichrom's TRU Resin
Analyte(s)
Americium-241
Californium-252
Curium-244
CASRN
14596-10-2
13981-17-4
13981-15-2
Analysis Purpose: Confirmatory analysis
Determinative Technique: Alpha spectrometry
Method Developed for: Americium (but not lanthanides) in water and air filters
Method Selected for: SAM lists this method for confirmatory analysis of drinking water, aqueous/liquid
samples, surface wipes, and air filters.
Description of Method: This method is specific to measurement of americium isotopes in samples that
do not contain lanthanides, but also can be used for measurement of californium and curium. The method
uses microprecipitation and determination by alpha spectrometry. Americium-243 is added to the sample
to determine chemical yield. The sample is processed through separation steps using ion exchange resins.
The eluate from the ion exchange column containing americium (and all other ions, except plutonium) is
evaporated, redissolved, and loaded onto a Transuranic (TRU) Resin extraction column. Americium (and
curium and californium, if present) is separated and purified on the column and finally stripped with
dilute nitric acid stripping solution. Microprecipitation is used to prepare for alpha spectrometry. The
method involves sample preparation steps from EML HASL-300 Method Pu-10-RC for water samples.
The LLD for total americium is 0.3 mBq when counted for 1000 minutes.
Special Considerations: If it is suspected that the sample exists in refractory form (i.e., non-digestible
or dissolvable material after normal digestion methods) or if there is a matrix interference problem, use
ORISE Method API 1.
Source: EML, U.S. DOE (EML is currently part of the U.S. DHS). 1997. "HASL-300 Method Am-04-
RC: Americium in QAP Water and Air Filters - Eichrom's TRU Resin." EML Procedures Manual,
HASL-300, 28th Edition. http://www.epa.gov/sam/pdfs/EML-Am-04-RC.pdf
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6.2.12 EML HASL-300 Method Ga-01-R: Gamma Radioassay
Analyte(s)
Cesium-137
Cobalt-60
Europium-154
lodine-131
lridium-192
Ruthenium-103
Ruthenium-106
Selenium-75
CASRN
10045-97-3
10198-40-0
15585-10-1
10043-66-0
14694-69-0
13968-53-1
13967-48-1
14265-71-5
Analysis Purpose: Qualitative and confirmatory analysis or gross gamma determination
Determinative Technique: Gamma spectrometry
Method Developed for: Gamma-ray emitting radionuclides in a variety of environmental matrices
Method Selected for: SAM lists this method for qualitative and/or confirmatory analysis of
aqueous/liquid, soil/sediment, surface wipes, and/or air filter samples.
Description of Method: This method uses gamma spectroscopy for the measurement of gamma photons
emitted from radionuclides without separating them from the sample matrix. Samples are placed into a
standard geometry for gamma counting, typically using an HPGe detector. Detectors such as Ge(Li) or
Nal(Tl) also can be used. The sample is placed into a standard geometry for gamma counting. Soil
samples and sludge are placed into an appropriately sized Marinelli beaker after drying and grinding the
sample for homogenization. Air filters and surface wipes can be counted directly or pressed into a
planchet and counted. Samples are counted long enough to meet the required sensitivity of measurement.
For typical counting systems and sample types, activity levels of approximately 40 Bq are measured, and
sensitivities as low as 0.002 Bq can be achieved for many nuclides. Because of electronic limitations,
count rates higher than 2000 counts per second (cps) should be avoided. High activity samples may be
diluted, reduced in size, or moved away from the detector (a limited distance) to reduce the count rate and
allow for analysis. The method is applicable for analysis of samples that contain radionuclides emitting
gamma photons with energies above approximately 20 keV for germanium (Ge) (both HPGe and GeLi)
detectors and above 50 keV for Nal(Tl) detectors.
Source: EML, U.S. DOE (EML is currently part of the U.S. DHS). 1997. "HASL-300 Method Ga-01-R:
Gamma Radioassay." EML Procedures Manual, HASL-300, 28th Edition.
http://www.epa.gov/sam/pdfs/KML-Ga-01-R.pdf
6.2.13 EML HASL-300 Method Po-02-RC: Polonium in Water, Vegetation, Soil, and Air
Filters
Analyte(s)
Polonium-210
CASRN
1-3981-52-7
Analysis Purpose: Qualitative and confirmatory analysis
Determinative Technique: Alpha spectrometry
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Method Developed for: Polonium in water, vegetation, soil, and air filters
Method Selected for: SAM lists this method for qualitative and confirmatory analysis of drinking water,
aqueous/liquid, and soil/sediment samples.
Description of Method: This method uses alpha spectrometry for determination of polonium in water,
vegetation, soil, and air filter samples. Polonium equilibrated with Po-208 or Po-209 tracer is isolated
from most other elements by coprecipitation with lead sulfide. The sulfide precipitate is dissolved in
weak hydrochloric acid solution. Polonium is quantitatively deposited on a nickel disc, and the plated
disc is counted on an alpha spectrometer to measure chemical yield and activity of the sample. The
solution from the deposition may be retained and analyzed for Pb-210. When counted for 1000 minutes,
the LLD for polonium is 1.0 mBq for water and 1.3 mBq for vegetation, soil and filters.
Source: EML, U.S. DOE (EML is currently part of the U.S. DHS). 1997. "HASL-300 Method Po-02-
RC: Polonium in Water, Vegetation, Soil, and Air Filters." EML Procedures Manual, HASL-300, 28th
Edition.
6.2.14 EML HASL-300 Method Pu-12-RC: Plutonium and/or Americium in Soil or
Sediments
Analyte(s)
Americium-241
Californium-252
Curium-244
CASRN
14596-10-2
13981-17-4
13981-15-2
Analysis Purpose: Confirmatory analysis
Determinative Technique: Alpha spectrometry
Method Developed for: Plutonium and americium in soil
Method Selected for: This method is listed in SAM for use when small soil and sediment sample sizes
(<100 g) will be analyzed.
Description of Method: A sample of soil of up to 100 g in size is equilibrated with Am-243 tracer.
Contaminant isotopes are leached with nitric and hydrochloric acid. Plutonium is removed by ion
exchange. The eluent from the plutonium separation is saved for determination of americium, curium,
and californium. Americium, curium, and californium are collected with a calcium oxalate
coprecipitation, isolated and purified by extraction chromatography. Microprecipitation is used to prepare
the sample for analysis by alpha spectrometry of americium, curium, and californium. The LLD for
americium is 0.5 mBq when counted for 1000 minutes.
Special Considerations: In cases where only small sample sizes (<100 g) will be analyzed, this method
is recommended for confirmatory analysis. If it is suspected that the sample exists in refractory form (i.e.,
non-digestible or dissolvable material after normal digestion methods) or if there is a matrix interference
problem, use ORISE Method API 1.
Source: EML, U.S. DOE (EML is currently part of the U.S. DHS). 1997. "HASL-300 Method Pu-12-
RC: Plutonium and/or Americium in Soil or Sediments." EML Procedures Manual, HASL-300, 28th
Edition. http://wmny.epa.gov/sain/pdfs/EML-Pu-12-RC.pdf
SAM Revision 4.0 127 September 29, 2008
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Section 6 - Selected Radiochemical Methods
6.2.15 EML HASL-300 Method Sr-03-RC: Strontium-90 in Environmental Samples
Analyte(s)
Strontium-90
CASRN
10098-97-2
Analysis Purpose: Qualitative and confirmatory analysis
Determinative Technique: Beta counting by low-background gas flow proportional detector
Method Developed for: Strontium-90 in vegetation, water, air filters and soil
Method Selected for: SAM lists this method for qualitative and confirmatory analysis of soil/sediment,
surface wipe, and air filter samples.
Description of Method: Strontium is separated from calcium, other fission products, and natural
radioactive elements. Fuming nitric acid separations remove the calcium and most other interfering ions.
Radium, lead and barium are removed with barium chromate. Traces of other fission products are
scavenged with iron hydroxide. After strontium-90 and yttrium-90 equilibrium has been attained, yttrium-
90 is precipitated as the hydroxide and converted to oxalate for counting on a low-background gas
proportional beta counter. Chemical yield is determined with strontium-85 tracer by counting in a gamma
well detector.
Source: EML, U.S. DOE (EML is currently part of the U.S. DHS). 1997. "HASL-300 Method Sr-03-
RC: Strontium-90 in Environmental Samples." EML Procedures Manual, HASL-300, 28th Edition.
http://wvw.epa.gov/sani/pdfs/EML-Sr-03-RC.pdf
6.2.16 FRMAC Method Volume 2, Page 33: Gross Alpha and Beta in Air
Analysis Purpose: Gross alpha and gross beta determination
Determinative Technique: Alpha/Beta counting
Method Developed for: Gross alpha and beta in air
Method Selected for: SAM lists this method for gross alpha and gross beta determination in air filters,
and for direct counting of surface wipes.
Description of Method: A thin-window gas-flow proportional counter is used for counting gross alpha
and beta radioactivity. The method supplies an approximation of the alpha and beta activity present in the
air or the removable surface activity dependent on the sample type. The method provides an indication of
the presence of alpha and beta emitters, including the following SAM analytes:
Americium-241
Californium-252
Cesium- 137
Cobalt-60
Curium-244
Europium- 154
Iridium-192
Plutonium-238
Plutonium-239
Polonium-210
Radium-226
Ruthenium- 103
Ruthenium- 106
Strontium-90
Uranium-234
SAM Revision 4. 0
(CASRN 14596-10-2)
(CASRN 13981-17-4)
(CAS RN 10045-97-3)
(CASRN 10198-40-0)
(CASRN 13981-15-2)
(CASRN 15585-10-1)
(CAS RN 14694-69-0)
(CASRN 13981-16-3)
(CASRN 15117-48-3)
(CASRN 13981-52-7)
(CAS RN 13982-63-3)
(CASRN 13968-53-1)
(CASRN 13967-48-1)
(CAS RN 10098-97-2)
(CAS RN 13966-29-5)
128
Alpha emitter
Alpha emitter
Beta emitter
Beta emitter
Alpha emitter
Beta emitter
Beta emitter
Alpha emitter
Alpha emitter
Alpha emitter
Alpha emitter
Beta emitter
Beta emitter
Beta emitter
Alpha emitter
September 29, 2008
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Section 6 - Selected Radiochemical Methods
• Uranium-235 (CAS RN 15117-96-1) Alpha emitter
• Uranium-238 (CAS RN 7440-16-1) Alpha emitter
For this application, the procedure requires the use of thorium-230 for alpha counting efficiency and
cesium-137 for beta counting efficiency in the calibration of the detector. An air filter or swipe sample is
placed onto a planchet then counted for alpha and beta radioactivity. Activity is reported in activity units
per volume of air sampled, as units of activity per surface area sampled, or as total units of activity in
cases where sample collection information is not available.
Source: FRMAC. 1998. "Gross Alpha and Beta in Air." FRMAC Monitoring and Analysis Manual -
Sample Preparation and Analysis - Volume 2, DOE/NV/11718-181 Vol. 2, UC-707, p. 33.
http://www.epa.gov/sam/pdfs/FRMAC-Vol2-pR33.pdf
6.2.17 ORISE Method AP-1: Gross Alpha and Beta for Various Matrices
Analysis Purpose: Gross alpha and gross beta determination
Determinative Technique: Alpha/Beta counting
Method Developed for: Gross alpha and beta in water, soil, vegetation, and other solids
Method Selected for: SAM lists this method for gross alpha and gross beta determination in
soil/sediment samples.
Description of Method: This method provides an indication of the presence of alpha and beta emitters,
including the following SAM analytes:
Americium-241 (CAS RN 14596-10-2) Alpha emitter
Californium-252 (CAS RN 13981-17-4) Alpha emitter
Cesium-137 (CAS RN 10045-97-3) Beta emitter
Cobalt-60 (CAS RN 10198-40-0) Beta emitter
Curium-244 (CAS RN 13981-15-2) Alpha emitter
Europium-154 (CAS RN 15585-10-1) Beta emitter
Iridium-192 (CAS RN 14694-69-0) Beta emitter
Plutonium-238 (CAS RN 13981-16-3) Alpha emitter
Plutonium-239 (CAS RN 15117-48-3) Alpha emitter
Polonium-210 (CAS RN 13981-52-7) Alpha emitter
Radium-226 (CAS RN 13982-63-3) Alpha emitter
Ruthenium-103 (CAS RN 13968-53-1) Beta emitter
Ruthenium-106 (CAS RN 13967-48-1) Beta emitter
Strontium-90 (CAS RN 10098-97-2) Beta emitter
Uranium-234 (CAS RN 13966-29-5) Alpha emitter
Uranium-235 (CAS RN 15117-96-1) Alpha emitter
Uranium-238 (CAS RN 7440-16-1) Alpha emitter
This procedure provides screening measurements to indicate whether specific chemical analyses are
required for water, soil, vegetation, and other solids. Liquid samples are acidified, concentrated, dried in
a planchet, and counted in a low-background proportional counter. Solid samples are dried and processed
to provide homogeneity, and a known quantity is transferred to a planchet and counted in a low-
background proportional counter.
Special Considerations: Volatile radionuclides will not be accurately determined using this procedure.
SAM Revision 4.0 129 September 29, 2008
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Section 6 - Selected Radiochemical Methods
Source: ORISE, Oak Ridge Associated Universities (ORAU). 2001. "Method AP-1: Gross Alpha and
Beta for Various Matrices." Laboratory Procedures Manual for the Environmental Survey and Site
Assessment Program. http://www.epa.gov/sam/pdfs/ORISE-AP-1 .pdf
6.2.18 ORISE Method AP-11: Sequential Determination of the Actinides in Environmental
Samples Using Total Sample Dissolution and Extraction Chromatography
Analyte(s)
Americium-241
Californium-252
Curium-244
Plutonium-238
Plutonium-239
Uranium-234
Uranium-235
Uranium-238
CASRN
14596-10-2
13981-17-4
13981-15-2
13981-16-3
15117-48-3
13966-29-5
15117-96-1
7440-61-1
Analysis Purpose: Qualitative and confirmatory analysis
Determinative Technique: Alpha spectrometry
Method Developed for: Americium, curium, plutonium, neptunium, thorium, and/or uranium in water
and solid samples
Method Selected for: SAM recommends this method for confirmatory analysis when a sample exists in
a refractory form (i.e., non-digestible or dissolvable material after normal digestion methods) or if there is
a matrix interference problem. In the event of refractory radioactive material, SAM recommends this
method for both qualitative determination and confirmatory analysis of drinking water, aqueous/liquid,
soil/sediment, surface wipes, and air filter samples.
Description of Method: Solid and unfiltered aqueous samples are dissolved completely by a
combination of potassium hydrogen fluoride and pyrosulfate fusions. Filtered aqueous samples are
evaporated to dryness followed by a pyrosulfate fusion. The fusion cake is dissolved and, for analyses
requiring uranium only, two barium sulfate precipitations are performed, and the uranium is separated
using EDTA. For all other analyses, one barium sulfate precipitation is performed and all alpha emitters
are coprecipitated on barium sulfate. The barium sulfate is dissolved and the actinides are separated by
extraction chromatography. An optional section is presented for the separation of americium from the
lanthanides. All actinides are coprecipitated on cerium fluoride and counted with an alpha spectrometer
system.
Source: ORISE, ORAU. 2001. "Method AP-11: Sequential Determination of the Actinides in
Environmental Samples Using Total Sample Dissolution and Extraction Chromatography." Laboratory
Procedures Manual for the Environmental Survey and Site Assessment Program.
http://www.epa.gov/sam/pdfs/ORISE-AP-ll.pdf
SAM Revision 4.0 130 September 29, 2008
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Section 6 - Selected Radiochemical Methods
6.2.19 ASTM Method D3084: Standard Practice for Alpha Spectrometry in Water
Analyte(s)
Americium-241
Californium-252
Curium-244
Plutonium-238
Plutonium-239
Radium-226
Uranium-234
Uranium-235
Uranium-238
CASRN
14596-10-2
13981-17-4
13981-15-2
13981-16-3
15117-48-3
13982-63-3
13966-29-5
15117-96-1
7440-61-1
Analysis Purpose: Qualitative determination
Determinative Technique: Alpha Spectrometry
Method Developed for: Alpha particle spectra in water
Method Selected for: SAM lists this method for qualitative determination in drinking water,
aqueous/liquid, soil and sediment, surface wipes, and/or air filter samples.
Description of Method: This standard practice covers the process that is required to obtain well-
resolved alpha spectra from water samples and discusses the associated problems. This practice is
typically preceded with specific chemical separations and mounting techniques that are included in
referenced methods. A chemical procedure is required to isolate and purify the radionuclides (see ASTM
Methods D3865, Standard Test Method for Plutonium in Water and D3972, Standard Test Method for
Isotopic Uranium in Water by Radiochemistry), and a radioactive tracer is added to determine yield. A
source is prepared by employing electrodeposition, microprecipitation, or evaporation (depositing the
solution onto a stainless steel or platinum disc). Electrodeposition and microprecipitation are preferred.
The source's radioactivity is then measured in an alpha spectrometer according to manufacturer's
operating instructions. The counting period chosen depends on the sensitivity required of the
measurement and the degree of uncertainty in the result that is acceptable.
Special Considerations: If it is suspected that the sample exists in refractory form (i.e., non-digestible
or dissolvable material after normal digestion methods) or if there is a matrix interference problem, use
ORISE Method API 1 for sample preparation instead of the methods referenced in ASTM Method D3084.
Source: ASTM. 1996. "Method D3084: Standard Practice for Alpha Spectrometry in Water." Annual
Book of ASTM Standards, Vol. 11.02. http://mw.astin.org/DATABASE.CART/HISTORICAL/D3084-
96.htm
6.2.20 ASTM Method D3972: Standard Test Method for Isotopic Uranium in Water by
Radiochemistry
Analyte(s)
Uranium-234
Uranium-235
CASRN
13966-29-5
15117-96-1
SAM Revision 4.0
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September 29, 2008
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Section 6 - Selected Radiochemical Methods
Analyte(s)
Uranium-238
CASRN
7440-61-1
Analysis Purpose: Confirmatory analysis
Determinative Technique: Alpha spectrometry
Method Developed for: Alpha-particle-emitting isotopes of uranium in water
Method Selected for: SAM lists this method for confirmatory analysis of drinking water samples.
Description of Method: Uranium is chemically separated from a water sample by coprecipitation with
ferrous hydroxide followed by anion exchange, and electrodeposition. When suspended matter is present,
an acid dissolution step is added to ensure that all of the uranium dissolves. The sample is acidified, and
uranium-232 is added as an isotopic tracer to determine chemical yield. Uranium is coprecipitated from
the sample with ferrous hydroxide. This precipitate is dissolved in concentrated hydrochloric acid, or is
subjected to acid dissolution with concentrated nitric and hydrofluoric acids, if the hydrochloric acid fails
to dissolve the precipitate. Uranium is separated from other radionuclides by adsorption on anion
exchange resin, followed by elution with hydrochloric acid. The uranium is finally electrodeposited onto
a stainless steel disc and counted using alpha spectrometry.
Special Considerations: If it is suspected that the sample exists in refractory form (i.e., non-digestible
or dissolvable material after normal digestion methods) or if there is a matrix interference problem, use
ORISE Method API 1.
Source: ASTM. 2002. "Method D3972: Standard Test Method for Isotopic Uranium in Water by
Radiochemistry." Annual Book of ASTMStandards, Vol. 11.02.
http://www.astm.org/Standards/D3972.htm
6.2.21 Standard Method 7110 B: Gross Alpha and Gross Beta Radioactivity (Total,
Suspended, and Dissolved)
Analysis Purpose: Gross alpha and gross beta determination
Determinative Technique: Alpha/Beta counting
Method Developed for: Gross alpha and gross beta activity in water
Method Selected for: SAM lists this method for gross alpha and gross beta determination in
aqueous/liquid samples.
Description of Method: This method allows for measurement of gross alpha and gross beta radiation in
water samples. The method provides an indication of the presence of alpha and beta emitters, including
the following SAM analytes:
Americium-241 (CAS RN 14596-10-2) Alpha emitter
Californium-252 (CAS RN 13981-17-4) Alpha emitter
Cesium-137 (CAS RN 10045-97-3) Beta emitter
Cobalt-60 (CASRN 10198-40-0) Beta emitter
Curium-244 (CAS RN 13981-15-2) Alpha emitter
Europium-154 (CAS RN 15585-10-1) Beta emitter
Iridium-192 (CAS RN 14694-69-0) Beta emitter
Plutonium-238 (CAS RN 13981-16-3) Alpha emitter
Plutonium-239 (CAS RN 15117-48-3) Alpha emitter
Polonium-210 (CAS RN 13981-52-7) Alpha emitter
Radium-226 (CAS RN 13982-63-3) Alpha emitter
SAM Revision 4.0 132 September 29, 2008
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Section 6 - Selected Radiochemical Methods
• Ruthenium- 103
• Ruthenium- 106
• Strontium-90
• Uranium-234
• Uranium-235
• Uranium-238
(CASRN 13968-53-1)
(CASRN 13967-48-1)
(CAS RN 10098-97-2)
(CAS RN 13966-29-5)
(CASRN 151 17-96-1)
(CASRN 7440-16-1)
Beta emitter
Beta emitter
Beta emitter
Alpha emitter
Alpha emitter
Alpha emitter
This method recommends using a thin-window gas-flow proportional counter for counting gross alpha
and beta radioactivity. An internal proportional or Geiger counter may also be used. An aliquot of
sample is evaporated to a small volume and transferred to a tared counting pan. The sample residue is
dried to constant weight, cooled, and reweighed to determine dry residue weight, then counted for alpha
and beta radioactivity.
Special Considerations: Ground water samples containing elevated levels of dissolved solids will
require use of smaller sample volumes.
Source: APHA, AWWA, and WEF. 2005. "Method 7110 B: Gross Alpha and Gross Beta Radioactivity
(Total, Suspended, and Dissolved)." Standard Methods for the Examination of Water and Wastewater.
21st Edition. http://wwv.stand^^
6.2.22 Standard Method 7120: Gamma-Emitting Radionuclides
Analyte(s)
Cesium-137
Cobalt-60
Europium-154
lridium-192
Ruthenium-103
Ruthenium-106
Selenium-75
CASRN
10045-97-3
10198-40-0
15585-10-1
14694-69-0
13968-53-1
13967-48-1
14265-71-5
Analysis Purpose: Qualitative and confirmatory determination
Determinative Technique: Gamma spectrometry
Method Developed for: Gamma emitting radionuclides in water
Method Selected for: SAM lists this method for qualitative and confirmatory analysis of aqueous/liquid
samples.
Description of Method: The method uses gamma spectroscopy using either Ge detectors or NaI(TD
crystals for the measurement of gamma photons emitted from radionuclides present in water. The
method can be used for qualitative and confirmatory determinations with Ge detectors or semi-qualitative
and semi-quantitative determinations (using Nal(Tl) detectors). Exact confirmation using Nal is possible
for single nuclides or when the gamma emissions are limited to a few well-separated energies. A
homogeneous water sample is placed into a standard geometry (normally a Marinelli beaker) for gamma
counting. Sample portions are counted long enough to meet the required sensitivity of measurement. A
standard containing a mixture of gamma energies from approximately 100 to 2000 keV is used for energy
calibration.
SAM Revision 4.0
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September 29, 2008
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Section 6 - Selected Radiochemical Methods
Source: APHA, AWWA, and WEF. 2005. "Method 7120: Gamma-Emitting Radionuclides." Standard
Methods for the Examination of Water and Wastewater. 21st Edition, http://www.standardmethods.org/
6.2.23 Standard Method 7500-Ra B: Radium: Precipitation Method
Analyte(s)
Radium-226
CASRN
13982-63-3
Analysis Purpose: Qualitative determination
Determinative Technique: Alpha counting
Method Developed for: Alpha-emitting isotopes of radium in water
Method Selected for: SAM lists this method for qualitative determination in aqueous/liquid samples.
Description of Method: This method is for determination of all alpha-emitting radium isotopes by alpha
decay analysis. Lead and barium carriers are added to the sample containing alkaline citrate, then sulfuric
acid is added to precipitate radium, barium, and lead as sulfates. The precipitate is purified by washing
with nitric acid, dissolving in alkaline EDTA, and re-precipitating as radium-barium sulfate after pH
adjustment to 4.5. This slightly acidic EDTA keeps other naturally occurring alpha-emitters and the lead
carrier in solution. Radium-223, -224, and -226 are identified by the rate of ingrowth of their daughter
products in barium sulfate precipitate. The results are corrected by the rate of ingrowth of daughter
products to determine radium activity. This method involves alpha counting by a gas-flow internal
proportional counter, scintillation counter, or thin end-window gas-flow proportional counter.
Source: APHA, AWWA, and WEF. 2005. "Method 7500-Ra B: Radium: Precipitation Method."
Standard Methods for the Examination of Water and Wastewater. 21st Edition.
http^/wvvw.standarjmethods.org/
6.2.24 Standard Method 7500-Ra C: Radium: Emanation Method
Analyte(s)
Radium-226
CASRN
13982-63-3
Analysis Purpose: Confirmatory determination
Determinative Technique: Alpha counting
Method Developed for: Soluble, suspended, and total radium-226 in water
Method Selected for: SAM lists this method for confirmatory analysis of aqueous/liquid samples.
Description of Method: Radium in water is concentrated and separated from sample solids by
coprecipitation with a relatively large amount of barium as the sulfate. The precipitate is treated to
remove silicates, if present, and to decompose insoluble radium compounds, fumed with phosphoric acid
to remove sulfite, and dissolved in hydrochloric acid. The completely dissolved radium is placed in a
bubbler, which is then closed and stored for a period of several days to 4 weeks for ingrowth of radon.
The bubbler is connected to an evacuation system and the radon gas is removed from the liquid by
aeration and helium, dried with a desiccant, and collected in a counting cell. Four hours after radon
collection, the cell is counted. The activity of the radon is equal to the radium concentration. The
minimum detectable concentration depends on counter characteristics, background-counting rate of
scintillation cell, cell efficiency, length of counting period, and contamination of apparatus and
environment by radium-226. Without reagent purification, the overall reagent blank (excluding
SAM Revision 4.0 134 September 29, 2008
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Section 6 - Selected Radiochemical Methods
background) should be between 0.03 and 0.05 pCi radium-226, which may be considered the minimum
detectable amount under routine conditions.
Source: APHA, AWWA, and WEF. 2005. "Method 7500-Ra C: Radium: Emanation Method." Standard
Methods for the Examination of Water and Wastewater. 21st Edition. httjii//wij^
6.2.25 Standard Method 7500-Sr B: Total Radioactive Strontium and Strontium-90:
Precipitation Method
Analyte(s)
Strontium-90
CASRN
10098-97-2
Analysis Purpose: Qualitative and confirmatory analysis
Determinative Technique: Beta counting by low-background gas flow proportional detector
Method Developed for: Strontium-90 or total radioactive strontium in drinking water or filtered raw
water
Method Selected for: SAM lists this method for qualitative and confirmatory analysis of drinking water
and aqueous/liquid samples.
Description of Method: A known amount of inactive strontium ions, in the form of strontium nitrate, is
added as a "carrier." The carrier, alkaline earths, and rare earths are precipitated as the carbonate to
concentrate the radiostrontium. The carrier, along with the radionuclides of strontium, is separated from
other radioactive elements and inactive sample solids by precipitation as strontium nitrate using fuming
nitric acid solution. The carrier and radionuclides of strontium are precipitated as strontium carbonate,
which is dried, weighed to determine recovery of carrier, and measured for radioactivity. The activity of
the final precipitate is due to radioactive strontium only, because all other radioactive elements have been
removed. Because it is impossible to separate the isotopes of strontium-89 and strontium-90 by any
chemical procedure, the amount of strontium-90 is determined by separating and measuring the activity of
yttrium-90, its daughter product. This method involves beta counting by a gas-flow internal proportional
counter or thin end-window low-background proportional counter. A correction is applied to compensate
for loss of carriers and activity during the various purification steps.
Source: APHA, AWWA, and WEF. 2005. "Method 7500-Sr B: Total Radioactive Strontium and
Strontium-90: Precipitation Method." Standard Methods for the Examination of Water and Wastewater.
21st Edition. http://www.standardmethods.org/
6.2.26 Standard Method 7500-U B: Uranium: Radiochemical Method
Analyte(s)
Uranium-234
Uranium-235
Uranium-238
CASRN
13966-29-5
15117-96-1
7440-61-1
Analysis Purpose: Qualitative determination
Determinative Technique: Alpha counting
Method Developed for: Total uranium alpha activity in water
Method Selected for: SAM lists this method for qualitative determination in aqueous/liquid samples.
SAM Revision 4.0 135 September 29, 2008
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Section 6 - Selected Radiochemical Methods
Description of Method: The sample is acidified with hydrochloric or nitric acid and boiled to eliminate
carbonate and bicarbonate ions. Uranium is coprecipitated with ferric hydroxide and subsequently
separated. The ferric hydroxide is dissolved, passed through an anion-exchange column, and washed with
acid, and the uranium is eluted with dilute hydrochloric acid. The acid eluate is evaporated to near
dryness, the residual salt is converted to nitrate, and the alpha activity is counted by a gas-flow
proportional counter or alpha scintillation counter.
Special Considerations: If it is suspected that the sample exists in refractory form (i.e., non-digestible
or dissolvable material after normal digestion methods) or if there is a matrix interference problem, use
ORISE Method API 1.
Source: APHA, AWWA, and WEF. 2005. "Method 7500-U B: Uranium: Radiochemical Method."
Standard Methods for the Examination of Water and Wastewater. 21st Edition.
http://www.standardmethods.org/
6.2.27 Standard Method 7500-U C: Uranium: Isotopic Method
Analyte(s)
Uranium-234
Uranium-235
Uranium-238
CASRN
13966-29-5
15117-96-1
7440-61-1
Analysis Purpose: Confirmatory determination
Determinative Technique: Alpha spectrometry
Method Developed for: Isotopic content of the uranium alpha activity; determining the differences
among naturally occurring, depleted, and enriched uranium in water
Method Selected for: SAM lists this method for confirmatory analysis of aqueous/liquid samples.
Description of Method: This method is a radiochemical procedure for determination of the isotopic
content of uranium alpha activity. The sample is acidified with hydrochloric or nitric acid, and uranium-
232 is added as an isotopic tracer. Uranium is coprecipitated with ferric hydroxide and subsequently
separated from the sample. The ferric hydroxide precipitate is dissolved and the solution passed through
an anion-exchange column. The uranium is eluted with dilute hydrochloric acid. The acid eluate is
evaporated to near dryness, and the residual salt is converted to nitrate and electrodeposited onto a
stainless steel disc and counted by alpha spectrometry.
Special Considerations: If it is suspected that the sample exists in refractory form (i.e., non-digestible
or dissolvable material after normal digestion methods) or if there is a matrix interference problem, use
ORISE Method API 1.
Source: APHA, AWWA, and WEF. 2005. "Method 7500-U C: Uranium: Isotopic Method." Standard
Methods for the Examination of Water and Wastewater. 21st Edition, http://wmny.standardmethods.org/
SAM Revision 4.0 136 September 29, 2008
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Section 7 - Selected Pathosen Methods
Section 7.0: Selected Pathogen Methods
A list of the most appropriate methods currently available for use in analyzing environmental samples for
pathogens is provided in Appendix C. This list represents an initial effort towards the goal of providing
standardized analytical procedures. These methods should be used to support remediation activities (site
assessment through clearance) following a homeland security event. The purpose of this section is to
provide summary information regarding the procedures listed. Methods are listed for each pathogen that
may need to be measured and analyzed following an event. Appendix C is sorted alphabetically within
pathogen categories (i.e., bacteria, viruses, protozoa, and helminths).
Protocols from peer-reviewed journal articles are listed where standardized methods for pathogens are not
currently available. Future steps include the development and validation of standardized methods. The
literature references will be replaced as standardized, validated protocols become available.
Pathogens that are categorized as Biosafety Level 4 (BSL-4), such as hemorrhagic fever viruses and
smallpox, will be handled only by reference laboratories with BSL-4 capability and are not included in
this document. All other pathogens are to be handled using BSL-2 or BSL-3 containment and practices
(as appropriate) using SAM procedures. If known, the BSL classification for each pathogen is provided
in the method summaries in Sections 7.2.2 through 7.2.42). Pathogens that are considered to be solely of
agricultural concern (i.e., animal and plant pathogens) are not currently included. Such pathogens may be
considered for possible inclusion in future document revisions. Some of the pathogens addressed by this
document are commonly found in the environment, and the methods listed in Appendix C assume that
analyses will be used to evaluate contamination levels that are above background levels. If possible, an
investigation of initial background levels, as well as appropriate controls for background levels, should be
performed. It is anticipated that additional site- or event-specific information also will be required to
support the analytical results.
Selection of methods from Appendix C should be based on specific data and information needs, including
whether there is a need to determine either the presence of a pathogen, the viability of a pathogen, or both.
Although culture-based methods have been selected for many of the pathogens, molecular techniques
such as PCR will likely be used for viruses because of the difficulty and time required to propagate these
agents in host cell cultures. Prior to the start of site decontamination, viability may not be an issue and
rapid mass screening techniques, such as PCR, may be more appropriate. After decontamination,
viability may be required to evaluate the efficacy of decontamination procedures; thus, a technique that
determines viability should be chosen. In such cases, methods that combine rapid sample processing and
viability determination, such as culture confirmed by PCR, should be considered for processing large
numbers of samples in a timely manner. Viability procedures are listed for each pathogen where
available. Commercially available spore strips also may be used as general indicators that a
decontamination process (e.g., fumigation) has successfully been applied. Spore strips, however, cannot
replace negative-culture results as an indicator of decontamination efficacy.
Users of this document should be aware that analysis of environmental samples poses specific problems,
and it is likely that a single analytical procedure will not be applicable to all sample types. The methods
listed in this document attempt to address a wide range of environmental samples (e.g., drinking water,
soil), each with specific physical and biological properties (e.g., pH, inhibitory substances, background
microorganisms). Within each sample type, a high level of variability may also exist. For example, soils
sampled in one geographical area may have qualities affecting analysis that are not present in soils from
other areas. Sample preparatory techniques, such as immunomagnetic separation (IMS), may be useful
for reducing the impact of these variables. Analyzing representative quantities of environmental samples
also presents a problem and can result in reduced analytical sensitivity. In these cases, sample
concentration techniques, such as membrane filtration (MF) and ultrafiltration (UF), may be useful. In
addition to removing background microorganisms and inhibitory substances, IMS may also be useful for
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concentrating sample aliquots to ensure that the full sample volume can be assayed. This may allow
methods such as PCR to be more sensitive.
Appendix C includes the following information:
• Pathogen(s). A specific causative agent (e.g., viruses, bacteria) of disease.
• Viability. Ability to grow and/or reproduce.
• Analytical technique. An analytical instrument or procedure used to determine the identity,
quantity, and/or viability of a pathogen.
• Analytical method. The unique identifier or number assigned to an analytical method by the method
publisher.
• Solid (soil, powder). The recommended method/procedure for the pathogen of interest in solid
samples such as soil and powders.
• Particulate (swabs, wipes, filters). The recommended method/procedure to measure the pathogen of
interest in particulate sample collection devices such as swabs, wipes and high efficiency particulate
air (HEPA) filters.
• Liquid/water (filter, grab). The recommended method/procedure for the pathogen of interest in
liquid or aqueous samples that have been concentrated or grab samples.
• Drinking water (filter, grab). The recommended method/procedure for the pathogen of interest in
potable water samples that have been concentrated or grab samples.
• Aerosol (growth media, filter, liquid). The recommended method/procedure for the pathogen of
interest in air sample collectors such as growth media, filters, or liquid.
Some of the methods in Appendix C include multiple analytical techniques by inference. The analytical
technique listed in Appendix C is intended to be a description of the predominant technique that is
required to provide the data quality parameter (viability or detection and identification). This description
does not preclude the use of other techniques that are within or referenced by the method. For example, a
viability test listed as "culture" may include immunochemical or PCR based assays for the identification
of isolates.
Several of the methods listed in Appendix C also include options such as the potential for use of multiple
cell culture media for primary isolation, allowance for selection of a defined subset of a larger number of
biochemical tests for biochemical testing, or use of alternative devices for sample concentration. The
method may provide guidance as to which options should be used under particular circumstances, or this
may be left to the discretion of the laboratory.
7.1 General Guidance
This section provides a general overview of how to identify the appropriate pathogen method (s) for a
given pathogen as well as recommendations for QC procedures.
For additional information on the properties of the pathogens listed in Appendix C, TOXNET
(http://toxnet.nlm.nih.gov/index.html), a cluster of databases on toxicology, hazardous chemicals, and
related areas maintained by the National Library of Medicine, is an excellent resource. Also informative
are CDC's Emergency Preparedness and Response Web site (httg^/wvm.bt.cdc.gov/) and the FDA
Center for Food Safety and Applied Nutrition (CFSAN) "Bad Bug Book"
(lit^)i//wwwxfea^^ . Further research on pathogens is ongoing within EPA.
Databases to manage this information are currently under development.
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7.1.1 Standard Operating Procedures for Identifying Pathogen Methods
To determine the appropriate method that is to be used on an environmental sample, locate the pathogen
in Appendix C: Pathogen Methods under the " Pathogen (s)" column. After locating the pathogen,
continue across the table and select an analytical technique. After an analytical technique has been
chosen (e.g., culture, PCR, immunoassay), select the analytical method applicable to the sample type of
interest (solid, particulate, liquid/drinking water or aerosol).
Sections 7.2.1 through 7.2.42 below provide summaries of the analytical methods listed in Appendix C.
Once a method has been identified in Appendix C, Table 7-1 can be used to locate the method summary.
Table 7-1. Pathogen Methods and Corresponding Text Section Numbers
Pathogen
Bacteria [Disease]
Bacillus anthracis [Anthrax]
Brucella spp. [Brucellosis]
Burkholderia mallei [Glanders]
Burkholderia pseudomallei
[Melioidosis]
Campylobacterjejuni
[Campylobacteriosis]
Chlamydophila psittaci [Psittacosis]
Coxiella burnetii [Q-fever]
Escherichia coli 0 1 5 7 : H 7
Fr and sell a tularensis [Tu I are mi a]
Leptospira spp. [Leptospirosis]
Usteria monocytogenes
[Listeriosis]
Non-typhoidal Salmonella
[Salmonellosis]
(Method not applicable for
Salmonella Typhi)
Salmonella Typhi [Typhoid fever]
Method
LRN
CDC Basic Diagnostic Testing Protocols for Level A
Laboratories for the Presumptive Identification of Bacillus
anthracis
LRN
ASM Sentinel Laboratory Guidelines for Suspected Agents
of Bioterrorism: Brucella species
LRN
ASM Sentinel Laboratory Guidelines for Suspected Agents
of Bioterrorism: Burkholderia mallei and Burkholderia
pseudomallei
SM 9260 G
Molecular and Cellular Probes. 2006. 20: 269-279
Journal of Clinical Microbiology. 2000. 38: 1085-1093
LRN
SM 9260 F
Applied and Environmental Microbiology. 2003. 69(10):
6327-6333
LRN
CDC, ASM, APHL Basic Protocols for Level A Laboratories
for the Presumptive Identification of Francisella tularensis
SM 9260 I
USDA Laboratory Guidebook MLG 8A.03
FDA/Bacteriological Analytical Manual Chapter 10, 2003
Method 1682
Journal of Applied Microbiology. 2007. 102(2): 516-530
CDC Laboratory Assay: S. Typhi
SM 9260 B
Section
7.2.1
7.2.7
7.2.1
7.2.21
7.2.1
7.2.22
7.2.18
7.2.24
7.2.25
7.2.1
7.2.17
7.2.26
7.2.1
7.2.8
7.2.20
7.2.12
7.2.13
7.2.6
7.2.27
7.2.9
7.2.15
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Pathogen
Shigella spp. [Shigellosis]
Staphylococcus aureus
Vibrio cholerae O1 and O1 39
[Cholera]
Yersinia pestis [Plague]
Method
CDC Laboratory Assay: Shigella
SM 9260 E
SM9213 B
CDC Laboratory Assay: V. cholerae
SM 9260 H
LRN
ASM Sentinel Laboratory Guidelines for Suspected Agents
of Bioterrorism: Yersinia pestis
Section
7.2.10
7.2.16
7.2.14
7.2.11
7.2.19
7.2.1
7.2.23
Viruses
Adenoviruses: A-F
Astroviruses
Caliciviruses: Noroviruses
Caliciviruses: Sapoviruses
Coronaviruses: SARS
Hepatitis E Virus
Influenza H5N1 Virus
Picornaviruses: Enteroviruses
Picornaviruses: Hepatitis A Virus
Reoviruses: Rotaviruses
Applied and Environmental Microbiology. 2005. 71(6):
3131-3136
Canadian Journal of Microbiology. 2004. 50: 269-278
Journal of Clinical Microbiology. 2004.42(10): 4679-4685
Journal of Medical Virology, 2006. 78(10): 1347-1353
Journal of Virological Methods. 2004. 122: 29-36
Journal of Virological Methods. 2006. 131(1): 65-71
Emerging Infectious Diseases. 2005. 11(8): 1303-1305
USEPA Manual of Methods for Virology EPA/600/4-84/01 3,
2001
Applied and Environmental Microbiology. 2003. 69(6):
3158-3164
Applied and Environmental Microbiology. 2003. 69(6):
3158-3164
7.2.28
7.2.29
7.2.30
7.2.31
7.2.32
7.2.33
7.2.34
7.2.2
7.2.35
7.2.35
Protozoa
Cryptosporidium spp.
[Cryptosporidiosis]
Entamoeba histolytica
Giardia spp. [Giardiasis]
Toxoplasma gondii
[Toxoplasmosis]
Method 1622
Method 1623
Applied and Environmental Microbiology. 1999. 65(9):
3936-3941
Applied and Environmental Microbiology. 2007. 73(13):
4218-4225
Journal of Parasitology. 1972.
58(2): 306-310
Journal of Clinical Microbiology. 2005. 43(11): 5491-5497
Method 1623
Transactions of the Royal Society of Tropical Medicine and
Hygiene. 1983. 77(4): 487-488
Emerging Infectious Diseases. 2006. 12(2): 326-329
Applied and Environmental Microbiology. 2004. 70(7):
4035-4039
7.2.4
7.2.5
7.2.36
7.2.37
7.2.38
7.2.39
7.2.5
7.2.40
7.2.41
7.2.42
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Pathogen
Method
Section
Helminths
Baylisascaris procyonis
[Raccoon roundworm fever]
EPA/625/R92/013
7.2.3
Method summaries are listed in order of method selection hierarchy (see Figure 2-1), starting with EPA
methods, followed by methods from other federal agencies, VCSBs, and journal articles. Methods are
listed in numerical order under each publisher. For additional information regarding sample preparation
and analysis procedures available through consensus standards organizations, please use the contact
information provided in Table 7-2.
Table 7-2. Sources of Pathogen Methods
Name
NEMI
U.S. EPA Microbiology Methods
Information Collection Requirements
Rule (ICR) Microbial Laboratory Manual
USEPA Manual of Methods for Virology
Environmental Regulations and
Technology: Control of Pathogens and
Vector Attraction in Sewage and Sludge
CDC Laboratory Assays
USDA / Food Safety and Inspection
Service (FSIS) Microbiology Laboratory
Guidebook
Bacteriological Analytical Manual
OSHA Methods
NIOSH Methods
Standard Methods for the Examination
of Water and Wastewater, 21st Edition,
2005*
Annual Book ofASTM Standards"
Applied and Environmental Microbiology
(AEM)*
Journal of Clinical Microbiology*
Clinical Microbiology Procedures
Handbook, 2nd Edition, 2004*
Molecular and Cellular Probes*
Canadian Journal of Microbiology*
Journal of Medical Virology*
Publisher
U.S. EPA, USGS
U.S. EPA
U.S. EPAORD
U.S. EPA
U.S. EPA, National Risk
Management Research
Laboratory (NRMRL)
HHS, CDC
USDA FSIS
U.S. FDA, CFSAN
OSHA
NIOSH
APHA, AWWA, and WEF
ASTM International
American Society for Microbiology
(ASM)
ASM
ASM
Elsevier
NRC Research Press
Wiley InterScience
Reference
http://www.nemi.qov
llttfil/ZMMMMLIMJlQ^^
http://www.epa.qov/nerlcwww/icrmi
cro.pdf
llSfil/ZlMVMLBMI^^
ut.htm
http://www.epa.qov/nrmrl/pubs/625
r92013/625r92013.htm
eiia.trees@cdc.hhs.qov
http://www.fsis.usda.qov/Science/
MiCTtMologioaM^^
dex.asp
http://www,cfsan.fda.qov/~ebam/b
am-toc.html
http://www.osha.qov
http://www.cdc.qov/niosh/nmam/
llMl/ZMMW-Manto
http ://www. astm. orq
httE^/aem.asrrLorg/
http://jcm.asm.orq/
http://estore.asm.ora/viewltemDeta
ils^sc?JteoiiJ>i323
http://www.elsevier.com
http://pubs.nrc-cnrc.qc.ca/
http://www3.interscience.wilev.com
/cfliiMD/honie
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Section 7 - Selected Pathosen Methods
Name
Journal of Virological Methods*
Emerging Infectious Diseases
Journal of Parasitology*
Transactions of the Royal Society of
Tropical Medicine and Hygiene*
Diagnostic Procedures in Veterinary
Bacteriology and Mycology
Sentinel Level Clinical Microbiology
Laboratory Guidelines
Journal of Applied Microbiology*
Publisher
Elsevier
CDC
American Society of
Parasitologists
The Royal Society of Tropical
Medicine and Hygiene
Academic Press
ASM
Blackwell Publishing
Reference
Jrtti3]//wwvj||sj3vieLCi3m
JTttBI/ZMMVLaJcjiOT/n^
http://www.bioone.orq
http://www,rstmh.Qrg/
http://www.pubmedcentral.nih.gov/
articlerender.fcqi?artid=1 481 267
http://www.asm.org/Policv/index.as
p?bid=6342
http://www.blackwellpublishinq.co
m/iournal.asp?ref=1 364-
5072&site=l
Subscription and/or purchase required.
publication date.
ASM does not require a subscription or purchase 6 months after the
7.1.2 General QC Guidance for Pathogen Methods
Generation of analytical data of known and documented quality is a critical factor in the accurate
assessment of and appropriate response to emergency situations. The generation of data of sufficient
quality requires that analytical laboratories: (1) have appropriately trained personnel; (2) acquire and
maintain required supplies, equipment, and reagents; (3) conduct the appropriate QC procedures to ensure
that all measurement systems are in control and operating properly; (4) properly document all analytical
results; and (5) properly document analytical QC procedures and corrective actions.
The level or amount of QC needed depends on the intended purpose of the data generated. Various levels
of QC may be required if the data are generated for presence/absence determinations versus quantitative
results. Specific data needs should be identified, and QC requirements should be based on those needs,
and should be applied consistently across laboratories when multiple laboratories are used. The
individual methods listed, sampling and analytical protocols, or contractual statements of work should be
consulted to determine if additional QC procedures are required.
Method-specific QC requirements are described in many of the methods cited in this manual and will be
included in protocols developed to address specific pathogen/sample type combinations of concern. In
general, analytical QC requirements for pathogen methods include an initial demonstration of
measurement system capability, as well as the capability of the laboratory and the analyst to perform the
method with the required precision and accuracy.
Ongoing analysis of control samples to ensure the continued reliability of the analytical results should
also be performed. At a minimum, the following QC analyses should be conducted on an ongoing basis:
• Media and reagent sterility checks;
• Positive and negative controls;
• Method blanks;
• Reference matrix spikes to evaluate initial and ongoing method/analyst performance, if available;
• Matrix spikes to evaluate method performance in the sample type of interest;
• MSB and/or sample replicates to assess method precision; and
• Instrument calibration checks and temperature controls.
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QC procedures should be performed as frequently as necessary to ensure the reliability of analytical
results.
Please note: The appropriate point of contact identified in Section 4 should be consulted regarding
appropriate QA/QC procedures prior to sample analysis. These contacts will consult with the EPA
OSWER coordinator responsible for laboratory activities during the specific event to ensure QA/QC
procedures are performed consistently across laboratories. OSWER is planning to develop QA/QC
guidance for laboratory support. EPA program offices will be responsible for ensuring that the QA/QC
practices are implemented.
7.1.3 Safety and Waste Management
It is imperative that safety precautions be used during collection, processing, and analysis of
environmental samples. Laboratories should have a documented health and safety plan for handling
samples that may contain target CBR contaminants, and laboratory staff should be trained in and
implement the safety procedures included in the plan. Pathogens in samples taken from areas
contaminated as the result of a homeland security event may be more hazardous than naturally occurring
pathogens of the same genus and species. The pathogens may have been manufactured, engineered, or
treated in such a manner as to enhance dispersion or virulence characteristics. These conditions may
warrant special handling for samples arising from intentional contamination incidents. A laboratory must
be made aware of these potential circumstances, and should carefully consider implementing additional
safety measures before agreeing to accept these samples.
In addition, many of the methods listed in Appendix C and summarized or cited in Section 7.2 contain
specific requirements, guidance, or information regarding safety precautions that should be followed
when handling or processing environmental samples and reagents. BSL-2 is suitable for work involving
agents that pose moderate hazards to personnel and the environment. BSL-3 is applicable when
performing manipulations of indigenous or exotic agents that may cause serious or potentially lethal
disease and also have the potential for aerosol transmission. BSLs are provided in the method summaries
in Section 7.2 whenever available. It is important to note, however, that some pathogens that are
normally handled at BSL-2 may require BSL-3 procedures and facilities if large volumes, high
concentrations, or potential aerosols are expected as a part of the analytical process. For more
information on BSL practices and procedures, the following references should be consulted:
• Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th Edition, found at
littp:/M^w.cdc.gov/OD/ohs/biosfty/binbl5/bmbl5toc.htin
• "Laboratory Security and Emergency Response Guidance for Laboratories Working with Select
Agents, " Morbidity and Mortality Weekly Report, Vol. 51, No. RR-19, 1-6, December 6, 2002, found
at http://www.cdc.gov/mmwr/pdf/rr/rr51 19.pdf.
• Microbiology Biosafety for Level A Laboratories, found at
• Select Agent Rules and Regulations (42 CFR part 73 and 9 CFR part 121) found at
llttp;//wjwj:fc^
http://ecfr.gpoaccess.gov/cgi/t/text/text-idx?c=ecfr&tpl=/ecfrbrowse/TitleQ9/9cfrl21 main OZ.tpl
The following methods provide information regarding waste management:
• EPA - Hazardous Waste Management (40 CFR parts 260) and EPA Administered Permit Programs
(40 CFR part 270), found at
idx?sid=cac9da30cd241fa70d461e4a917eb75e&c=ecfr&tpl=/ecfrbrowse/Title40/40tab OZ.tpl
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Other resources that can be consulted for additional information include the following:
• OSHA - Hazardous Waste Operations and Emergency Response (29 CFR part 1910.120) found at
http://www.osha.gov/pls/oshaweb/owadisp.show document?p table=STANDARDS&p id=9765
• OSHA - Occupational Exposure to Hazardous Chemicals in Laboratories (29 CFR part 1910.1450)
found at
http://www.osha.gov/pls/oshaweb/owadisp.show document?p table=STANDARDS&p id=10106
• OSHA - Respiratory Protection (29 CFR part 1910.134) found at
..... document?j) ...... id^!2716&p ...... taMe=SXANDARDS
DOT Hazardous Materials Shipment and Packaging (49 CFR parts 171-180)
hj^V/ecJLgpQaccess.gQY/cgi/t/text/tejcti
idxVsid=585c275eel9254ba07625d8c92fe925f&c=ecfr&tpl=/ecfrbrowse/Title49/49cfrv2 O2.tpl
7.2 Method Summaries
Summaries of the analytical methods listed in Appendix C are provided in Sections 7.2.1 through 7.2.42.
Each summary contains a table identifying the pathogen(s) and sample type to which the method applies,
a brief description of the method, performance data (if available), and a link to, or source for, obtaining a
full version of the method.
7.2.1 Laboratory Response Network (LRN)
The LRN was created in accordance with Presidential Directive 39, which established terrorism
preparedness responsibilities for federal agencies. The LRN is primarily a national network of local,
state, federal, military, food, agricultural, veterinary, and environmental laboratories; however, additional
LRN laboratories are located in strategic international locations. The CDC provides technical and
scientific support to member laboratories as well as secure access to standardized procedures and reagents
for rapid (within 4-6 hours) presumptive detection of biothreat agents and emerging infectious disease
agents. These rapid presumptive assays are part of sample type/analyte specific algorithms of assays
which lead to a confirmed result. The algorithm for a confirmed result is often a combination of one or
more presumptive positive results from a rapid assay in combination with a positive result from one of the
"gold standard" methods, such as culture. The standardized procedures, reagents, and agent-specific
algorithms are considered to be sensitive and are available only to LRN member laboratories. Thus, these
procedures are not available to the general public and are not discussed in this document.
It is important to note that, in some cases, the procedures may not be fully developed or validated for each
environmental sample type/pathogen combination listed in Appendix C, nor are all LRN member
laboratories necessarily capable of analyzing all of the sample type/pathogen combinations. Additional
LRN comparable assays (e.g., PCR) also are being developed or acquired that may be used in place of
LRN assays. Except for Coxiella burnetii, culture methods are available for all of these pathogens as
Sentinel Laboratory Guidelines
The agents identified below and listed in Appendix C are included in the HHS/USDA select agent list and
should be analyzed in accordance with appropriate regulatory compliance (42 CFR parts 72 and 73, and 9
CFR part 121) and safety and BSL requirements (see CDC's BMBL, 5th Edition,
cj^
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Pathogen(s) [Disease]
Bacillus anthracis [Anthrax]
Brucella spp. [Brucellosis]
Burkholderia mallei [Glanders]
Burkholderia pseudomallei [Melioidosis]
Coxiella burnetii [Q-fever]
Francisella tularensis [Tularemia]
Yersinia pestis [Plague]
Agent Category
Bacteria
Bacteria
Bacteria
Bacteria
Bacteria
Bacteria
Bacteria
For additional information on the LRN, including selection of a laboratory capable of receiving and
processing the specified sample type/pathogen, please use the contact information provided below or visit
Centers for Disease Control and Prevention
Laboratory Response Branch
Division of Bioterrorism Preparedness and Response (DBPR)
National Center for the Prevention, Detection, and Control of Infectious Diseases (NCPDCID)
Coordinating Center for Infectious Diseases (CCID)
Centers for Disease Control and Prevention (CDC)
1600 Clifton Road NE, Mailstop C-18
Atlanta, GA 30333
Telephone: (404) 639-2790
E-mail: lrn@cdc.gov
Local public health laboratories, private laboratories, and commercial laboratories with questions about
the LRN should contact their state public health laboratory director or the Association of Public Health
Laboratories (APHL) (contact information provided below) .
Association of Public Health Laboratories
8515 Georgia Avenue, Suite 700
Silver Spring, MD 20910 Telephone: (240) 485-2745
Fax: (240) 485-2700
Web site: www.aphl.org
E-mail: info@aphl.org
7.2.2 USEPA Manual of Methods for Virology, EPA/600/4-84/013, April 2001
Pathogen(s)
Picornaviruses: Enteroviruses
Agent Category
Viruses
BSL
Not specified
Analysis Purpose: Detection and viability
Sample Preparation: Samples should be prepared according to the procedures described in the USEPA
Manual of Methods for Virology (EPA/600/4-84/013, April 2001).
Analytical Technique: Tissue culture with serum neutralization
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Method Developed for: Enteroviruses in water
Method Selected for: SAM lists this manual for detection and viability assessment in solid, particulate,
aerosol, liquid, and water samples. Further research is needed to develop and standardize the procedures
for environmental sample types other than water. See Appendix C or Table 7-1 for additional methods
that should be used for this pathogen.
Description of Method: This manual describes procedures for determining the infectivity of
enteroviruses, including a neutralization test used to identify these viruses. The test uses reference-typing
sera directed against isolated waterborne viruses, and consists of simultaneously inoculating virus and
antiserum into a microtiter plate, incubating the virus-antibody mixture for 2 hours, adding a suspension
of host cells to the mixture, incubating the host cells-virus-antibody mixture for 3 days, and then
examining the cells daily for 5 more days for the presence/absence of a cytopathic effect (CPE). The test
uses the Lim Benyesh-Melnick (LB-M) antiserum pools, which consist of 61 equine antisera, including
LB-M antiserum pools A-H for the identification of 41 enteroviruses. Chapters 7 and 14 in this manual
describe procedures for the collection and preparation of virus samples. Sample preparation procedures
described include concentration and processing of waterborne viruses by positively charged 1MDS
cartridge filters and flocculation. These general procedures can be used for many viruses and may be
adapted for analysis of particulate, liquid, water, and aerosol samples.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank.
Special Considerations: This manual also describes procedures for preparation of samples for
adenovirus, astrovirus, norovirus, sapovirus, coronavirus - severe acute respiratory syndrome (SARS),
hepatitis E virus, influenza H5N1 virus, picornaviruses (enterovirus and hepatitis A virus), and reovirus
(rotavirus).
Source: EPA. 2001. "Chapter 12 - Identification of Enteroviruses." USEPA Manual of Methods for
Virology, EPA/600/4-84/013. http://wmw.epa.gov/sam/pdfs/EPA-600-4-84-013.pdf
7.2.3 USEPA Environmental Regulations and Technology, Control of Pathogens and
Vector Attraction in Sewage Sludge EPA/625/R-92/013, July 2003: Baylisascaris
procyonis
Pathogen(s) [Disease]
Baylisascaris procyonis
[Raccoon roundworm fever]
Agent Category
Helminths
BSL
2
Analysis Purpose: Detection and viability
Sample Preparation: Samples should be prepared according to the procedures in EPA/625/R-92/013.
Analytical Technique: Microscopy and embryonation of eggs
Method Developed for: Baylisascaris procyonis in wastewater, sludge, and compost samples
Method Selected for: SAM lists these protocols for detection and viability assessment of Baylisascaris
procyonis in solid, particulate, liquid, and water samples. Further research is needed to develop and
standardize the procedures for environmental sample types other than wastewater, sludge, and compost.
See Appendix C or Table 7-1 for additional methods that should be used for this pathogen.
Description of Method: The protocol describes procedures for analysis of solid and wastewater samples
and may be adapted to analysis of particulate, liquid, water, and aerosol samples. Samples are processed
by blending with buffered water containing a surfactant. The blend is screened to remove large particles,
the solids in the screened portion are allowed to settle out, and the supernatant is decanted. The sediment
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Section 7 - Selected Pathosen Methods
is subjected to density gradient centrifugation using magnesium sulfate. This flotation procedure yields a
layer likely to contain Ascaris and other parasite ova, if present in the sample. Small particulates are
removed by a second screening on a small mesh size screen. The resulting concentrate is incubated until
control Ascaris eggs are fully embryonated. The concentrate is then microscopically examined for the
categories of Ascaris ova on a counting chamber.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank.
Source: EPA. 2003. "Appendix I: Test Method for Detecting, Enumerating, and Determining the
Viability of Ascaris Ova in Sludge." USEPA Environmental Regulations and Technology: Control of
Pathogens and Vector Attractions in Sewage Sludge, EPA/625/R-92/013.
7.2.4 EPA Method 1622: Cryptosporidium in Water by Filtration/IMS/FA
Pathogen(s) [Disease]
Cryptosporidium spp. [Cryptosporidiosis]
Agent Category
Protozoa
BSL
2
Analysis Purpose: Detection, not suitable for viability
Sample Preparation: Samples should be prepared according to procedures in EPA Method 1622.
Analytical Technique: IMS and fluorescence assay (FA) microscopy
Method Developed for: Cryptosporidium in surface water
Method Selected for: SAM lists this method for detection in solid, particulate, liquid, and water
samples. Further research is needed to develop and standardize the procedures for environmental sample
types other than surface water. See Appendix C or Table 7-1 for additional methods that should be used
for this pathogen.
Description of Method: This method describes procedures for analysis of drinking water samples and
may be adapted for analysis of solid and particulate samples. A water sample is filtered and the oocysts
and extraneous materials are retained on the filter. Materials on the filter are eluted, the eluate is
centrifuged to pellet the oocysts, and the supernatant fluid is aspirated. A solution containing anti-
Cryptosporidium antibodies conjugated to magnetic beads is added to the pellet and mixed. The oocyst
magnetic bead complex is separated from the extraneous materials using a magnet, and the extraneous
materials are discarded. The magnetic bead complex is then detached from the oocysts. The oocysts are
stained on well slides with fluorescently labeled monoclonal antibodies (mAbs) and 4',6-diamidino-2-
phenylindole (DAPI). The stained sample is examined using fluorescence and differential interference
contrast (DIG) microscopy. Qualitative analysis is performed by scanning each slide well for objects that
meet the size, shape, and fluorescence characteristics of Cryptosporidium oocysts. Quantitative analysis
is performed by counting the total number of objects on the slide confirmed as oocysts. This method is
not intended to determine viability of the oocysts.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, MS/MSD, and blank.
Source: EPA. 2005. "Method 1622: Cryptosporidium in Water by Filtration/IMS/FA."
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7.2.5 EPA Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA
Pathogen(s) [Disease]
Cryptosporidium spp. [Cryptosporidiosis]
Giardia spp. [Giardiasis]
Agent Category
Protozoa
Protozoa
BSL
2
2
Analysis Purpose: Detection, not suitable for viability
Sample Preparation: Samples should be prepared according to procedures in EPA Method 1623.
Analytical Technique: IMS and FA microscopy
Method Developed for: Cryptosporidium and Giardia in surface water
Method Selected for: SAM lists this method for the detection of Cryptosporidium spp. in solid,
particulate, liquid, and water samples and Giardia spp. in solid, particulate, liquid, and water samples.
Further research is needed to develop and standardize the procedures for environmental sample types
other than surface water. See Appendix C or Table 7-1 for additional methods that should be used for this
pathogen.
Description of Method: This method describes procedures for analysis of water samples and may be
adapted for assessment of solid, particulate, and liquid samples. A water sample is filtered and the
oocysts and cysts and extraneous materials are retained on the filter. Materials on the filter are eluted, the
eluate is centrifuged to pellet the oocysts and cysts, and the supernatant fluid is aspirated. A solution
containing anti-Cryptosporidium or anti-Giardia antibodies conjugated to magnetic beads is added to the
pellet and mixed. The oocyst and cyst magnetic bead complex is separated from the extraneous materials
using a magnet, and the extraneous materials are discarded. The magnetic bead complex is then detached
from the oocysts and cysts. The oocysts and cysts are stained on well slides with fluorescently labeled
mAbs and DAPI. The stained sample is examined using fluorescence and DIG microscopy. Qualitative
analysis is performed by scanning each slide well for objects that meet the size, shape, and fluorescence
characteristics of Cryptosporidium oocysts and Giardia cysts. Quantitative analysis is performed by
counting the total number of objects on the slide confirmed as oocysts or cysts. This method is not
intended to determine viability of the parasites.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, MS/MSD, and blank.
Source: EPA. 2001. "Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA."
http^/wvvw.epa.goy/sam/pdjs/EP_Ajj^23.pdf
7.2.6 EPA Method 1682: Salmonella spp.
Pathogen(s) [Disease]
Non-typhoidal Salmonella [Salmonellosis]
(Method not applicable to Salmonella Typhi)
Agent Category
Bacteria
BSL
2
Analysis Purpose: Detection and viability
Sample Preparation: Solid samples should be prepared according to EPA Method 1680; particulate
samples should be prepared according to "Swab Materials and Bacillus anthracis Spore Recovery from
Nonporous Surfaces," Rose, L., Jensen, B., Peterson, A., Banerjee, S.N., and Arduino, MJ. 2004.
Emerging Infectious Diseases. 10(6):1023-1029 (www.epa.gov/sam/pdfs/EID-10(6)-pgsl023-1029.pdf)
and "Evaluation of a Macro foam Swab Protocol for the Recovery of Bacillus anthracis Spores from a
Steel Surface," Hodges, L.R., Rose, L.J., Peterson, A., Nobel-Wang, J., and Arduino, MJ. 2006. Applied
and Environmental Microbiology. 72(6): 4429-4430 (www.epa.gov/sam/pdfs/AEM-72(6)-pgs4429-
4430.pdf).
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Analytical Technique: Culture and immunoassay
Method Developed for: Non-typhoidal Salmonella in biosolids
Method Selected for: SAM lists this method for detection and viability assessment in solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types other than biosolids. See Appendix C or Table 7-1 for additional methods
that should be used for this pathogen.
Description of Method: This method describes procedures for analysis of solid samples (biosolids) and
may be adapted for assessment of water, liquid, particulate, and aerosol samples. Prepared samples are
inoculated into tubes of tryptic soy broth (TSB) and incubated for 24 hours. Positive (turbid) tubes are
spotted onto plates of modified semisolid Rappaport-Vassiliadis (MSRV) medium and incubated at 42°C
for 16 to 18 hours. The MSRV medium uses novobiocin and malachite green to inhibit non-Salmonella
species, while allowing most Salmonella species to grow. Presumptive colonies are isolated on xylose
lysine deoxycholate (XLD) agar and confirmed using lysine iron agar (LIA), triple sugar iron (TSI) agar,
and urea broth, followed by serological typing using polyvalent O antisera.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, matrix spike, and blank.
Special Considerations: This method will not detect Salmonella Typhi. MSRV and the elevated
incubation temperature (42°C) are inhibitory for S. Typhi.
Source: EPA. 2006. "Method 1682: Salmonella in Sewage Sludge (Biosolids) by MSRV Medium."
http://www.epa.gov/sam/pdfs/EPA-1682.pdf
7.2.7 CDC, ASM, APHL: Basic Diagnostic Testing Protocols for Level A Laboratories for
the Presumptive Identification of Bacillus anthracis
Pathogen(s) [Disease]
Bacillus anthracis [Anthrax]
Agent Category
Bacteria
BSL
3
Analysis Purpose: Detection and viability
Sample Preparation: Solid samples should be prepared according to EPA Method 1680; particulate
samples should be prepared according to "Swab Materials and Bacillus anthracis Spore Recovery from
Nonporous Surfaces," Rose, L., Jensen, B., Peterson, A., Banerjee, S.N., and Arduino, MJ. 2004.
Emerging Infectious Diseases. 10(6): 1023-1029 (www.epa.gov/sam/pdfs/EID-10(6)-pgsl023-1029.pdf)
and "Evaluation of a Macro foam Swab Protocol for the Recovery of Bacillus anthracis Spores from a
Steel Surface," Hodges, L.R., Rose, L.J., Peterson, A., Nobel-Wang, J., and Arduino, MJ. 2006. Applied
and Environmental Microbiology. 72(6): 4429-4430 (vm^Lm^S^lhMilm^MM^M^msMl^L
4430.pdf).
Analytical Technique: Culture
Method Developed for: B. anthracis in clinical samples
Method Selected for: SAM lists this protocol for detection and viability assessment in solid, particulate,
aerosol, liquid, and water samples. Further research is needed to develop and standardize the procedures
for environmental sample types. See Appendix C or Table 7-1 for additional methods that should be used
for this pathogen.
Description of Method: This method describes procedures for analysis of clinical samples and may be
adapted for assessment of solid, particulate, and liquid samples. Samples are plated directly on sheep
blood agar (SBA) or first enriched in a nutrient broth (e.g., TSB). Incubation is for at least 3 days at
35°C-37°C. B. anthracis forms 2-5 mm, flat or slightly convex colonies with irregular edges on SBA,
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with no 6-hemolysis. Presumptive identification is made by culture examination, microscopy, motility
testing, and biochemical testing. B. anthracis is a non-motile, Gram-positive rod with central to sub-
terminal spores, and is catalase-negative. Cultures (isolates) that cannot be ruled out as B. anthracis
based on the characteristics noted above may be referred to an appropriate reference laboratory for
confirmation.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank.
Special Considerations: B. anthracis is a select agent requiring regulatory compliance (42 CFR parts
72 and 73, and 9 CFR part 121); appropriate safety and BSL requirements should be followed (see CDC's
BMBL, 5th Edition, littp://www.cdc.gov/OD/ohs/biosfty/binbl5/bmbl5toc.htm).
Source: CDC, ASM, and APHL. 2002. "Basic Diagnostic Testing Protocols for Level A Laboratories
for the Presumptive Identification of Bacillus anthracis." http://www.epa.gov/sam/pdfs/CDC-
Anthrax.pdf
7.2.8 CDC, ASM, APHL: Basic Protocols for Level A Laboratories for the Presumptive
Identification of Francisella tularensis
Pathogen(s) [Disease]
Francisella tularensis [Tularemia]
Agent Category
Bacteria
BSL
3
Analysis Purpose: Detection and viability
Sample Preparation: Solid samples should be prepared according to EPA Method 1680; particulate
samples should be prepared according to "Swab Materials and Bacillus anthracis Spore Recovery from
Nonporous Surfaces," Rose, L., Jensen, B., Peterson, A., Banerjee, S.N., and Arduino, MJ. 2004.
Emerging Infectious Diseases. 10(6): 1023-1029 (www.epa.gov/sam/pdfs/EID-10(6)-pgs 1023-1029.pdf)
and "Evaluation of a Macro foam Swab Protocol for the Recovery of Bacillus anthracis Spores from a
Steel Surface," Hodges, L.R., Rose, L.J., Peterson, A., Nobel-Wang, J., and Arduino, MJ. 2006. Applied
and Environmental Microbiology. 72(6): 4429-4430 (www.epa.gov/sam/pdfs/AEM-72(6)-pgs4429-
4430.pdf).
Analytical Technique: Culture
Method Developed for: F. tularensis in clinical samples
Method Selected for: SAM lists this protocol for detection and viability assessment in solid, particulate,
aerosol, liquid, and water samples. Further research is needed to develop and standardize the procedures
for environmental sample types. See Appendix C or Table 7-1 for additional methods that should be used
for this pathogen.
Description of Method: This method describes procedures for analysis of clinical samples and may be
adapted for assessment of solid, particulate, and liquid samples. Samples are plated directly on a
cysteine-supplemented media such as chocolate agar (CA), Thayer-Martin (TM) agar, or buffered
charcoal yeast extract (BCYE) agar. After incubation at 35°C-37°C for at least 48 hours, F. tularensis
forms small, gray-white to opaque colonies. Presumptive identification is made by culture examination,
microscopy, motility testing, and biochemical testing. F. tularensis is a minute, pleomorphic, faint-
straining, Gram-negative coccobacillus and is weakly catalase-positive, oxidase-negative, 6-lactamase-
positive, and urease-negative. Cultures (isolates) that cannot be ruled out as F. tularensis based on the
characteristics noted above may be referred to an appropriate reference laboratory for confirmation.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank.
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Special Considerations: F. tularensis is a select agent requiring regulatory compliance (42 CFR parts
72 and 73, and 9 CFR part 121); appropriate safety and BSL requirements should also be followed (see
CDC's BMBL, 5th Edition, http://www.cdc.gov/QD/ohs/biostty/bmbl5/bmbl5toc.htm).
Source: CDC, ASM, and APHL. 2001. "Basic Protocols for Level A Laboratories for the Presumptive
Identification of Francisella tularensis." http://www.epa.gov/sam/pdfs/CDC-Ftularemia.pdf
7.2.9 CDC Laboratory Assay: "Triplex PCR for Detection of S. Typhi Using
SmartCycler®"
Pathogen(s) [Disease]
Salmonella Typhi [Typhoid fever]
Agent Category
Bacteria
BSL
2/3 (aerosol release)
Analysis Purpose: Detection, not suitable for viability
Sample Preparation: Solid samples should be prepared according to "Quantification of Bias Related to
the Extraction of DNA Directly from Soils," Frostegard, A., Courtois, S., Ramisse, V., Clerc, S.,
Bernillon, D., Le Gall, F., Jeannin, P., Nesme, X., and Simonet, P. 1999. Applied and Environmental
Microbiology. 65(12): 5409-5420 (www.epa.gov/sam/pdfs/AEM-65(12)-pgs5409-5420.pdf).
Analytical Technique: Real-time PCR
Method Developed for: S. Typhi from cultures or isolates
Method Selected for: SAM lists these procedures for detection in solid, particulate, aerosol, liquid,
and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types. See Appendix C or Table 7-1 for additional methods that should be used for
this pathogen.
Description of Method: Procedures are described for analysis of isolates and may be adapted for
assessment of solid, particulate, aerosol, liquid, and water samples. The assay uses real-time PCR for
identification of S. Typhi. Cell lysate templates are prepared by suspending a portion of a colony in 300
|iL of distilled water and boiling for 10 minutes. After centrifugation at 4,500 rpm for 2 minutes, 1 (_iL of
the supernatant is used in the PCR reaction. Alternatively, deoxyribonucleic acid (DNA) may be purified
using a commercially available kit or automated DNA extraction system. PCR is performed on a
SmartCycler® using primers and probes designed for the Vi capsular gene (viaB), the H antigen gene
(fliC-d), and the tyvelose epimerase gene (tyv). This assay is also available for the LightCycler® platform
as three single target PCRs.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank. PCR QC checks should be performed according to EPA
Draft Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on
Environmental Samples document wjyw.epjLgQy^m/p_dfs^PA=Q_AiQ_C PCR Qct2Q04,,pdf or consult
the point of contact identified in Section 4.
Source: HHS, CDC, Laboratory Assay. "Triplex PCR for Detection of S. Typhi Using SmartCycler®."
Contact: Dr. Eija Trees, CDC, email: eija.trees@cdc.hhs.goY.
7.2.10 CDC Laboratory Assay: "Detection of Diarrheagenic Eshcerichia coli and Shigella
Using LightCycler®"
Pathogen(s) [Disease]
Shigella spp. [Shigellosis]
Agent Category
Bacteria
BSL
2
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Analysis Purpose: Detection, not suitable for viability
Sample Preparation: Solid samples should be prepared according to "Quantification of Bias Related to
the Extraction of DNA Directly from Soils," Frostegard, A., Courtois, S., Ramisse, V., Clerc, S.,
Bernillon, D., Le Gall, F., Jeannin, P., Nesme, X., and Simonet, P. 1999. Applied and Environmental
Microbiology. 65(12): 5409-5420 (www.epa.gov/sam/pdfs/AEM-65(12)-pgs5409-5420.pdf).
Analytical Technique: Real-time PCR
Method Developed for: Shigella from cultures or isolates
Method Selected for: SAM lists these procedures for detection in solid, particulate, aerosol, liquid, and
water samples. Further research is needed to develop and standardize the procedures for environmental
sample types. See Appendix C or Table 7-1 for additional methods that should be used for this pathogen.
Description of Method: Procedures are described for analysis of isolates and may be adapted for
assessment of solid, particulate, aerosol, liquid, and water samples. The assay uses real-time PCR for
identification of Shigella. Cell lysate templates are prepared by suspending a portion of a colony in 300
\iL of distilled water and boiling for 10 minutes. After centrifugation at 4,500 rpm for 2 minutes, 1 \iL of
the supernatant is used in the PCR reaction. Alternatively, DNA may be purified using a commercially
available kit or automated DNA extraction system. PCR is performed on a LightCycler® using primers
and probes designed for the /paH-plasmid, which encodes the invasive plasmid antigen H. This gene can
be found on both the chromosome and a plasmid for Shigella spp. An alternative multiplex PCR is
described targeting both ipaH and Stx\ (Shiga toxin) genes using TaqMan® chemistry and SmartCycler®.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank. PCR QC checks should be performed according to EPA
Draft Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on
Environmental Samples document w^wj^.gov/san^ or consult
the point of contact identified in Section 4.
Source: HHS, CDC, Laboratory Assay. "Detection of Diarrheagenic Eshcerichia coli and Shigella
Using LightCycler®." Contact: Dr. Eija Trees, CDC, email:
7.2.11 CDC Laboratory Assay: "TaqMan Assays for Detection of V. cholerae ctxA, O1 rfb,
andO139rflb."
Pathogen(s) [Disease]
Vibrio cholerae [Cholera]
Agent Category
Bacteria
BSL
2
Analysis Purpose: Detection, not suitable for viability
Sample Preparation: Solid samples should be prepared according to "Quantification of Bias Related to
the Extraction of DNA Directly from Soils," Frostegard, A., Courtois, S., Ramisse, V., Clerc, S.,
Bernillon, D., Le Gall, F., Jeannin, P., Nesme, X., and Simonet, P. 1999. Applied and Environmental
Microbiology. 65(12): 5409-5420 (wwvjp^gw/sam^^
Analytical Technique: Real-time PCR
Method Developed for: V. cholerae Ol and O139 from cultures or isolates
Method Selected for: SAM lists these procedures for detection in solid, particulate, aerosol, liquid, and
water samples. Further research is needed to develop and standardize the procedures for environmental
sample types. See Appendix C or Table 7-1 for additional methods that should be used for this pathogen.
Description of Method: Procedures are described for analysis of isolates and may be adapted for
assessment of solid, particulate, aerosol, liquid, and water samples. The assay uses real-time PCR for
identification of V. cholerae. Cell lysate templates are prepared by suspending a portion of a colony in
300 |iL of 0.01M Tris-EDTA (TE) buffer and boiling for 10 minutes. After centrifugation at 4,500 rpm
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for 2 minutes, 1 \iL of the supernatant is used in the PCR reaction. Alternatively, DNA may be purified
using a commercially available kit or automated DNA extraction system. PCR is performed on a
LightCycler® as single target assays or on a SmartCycler® as multiplex PCR using primers and probes
designed for the cholera toxin ctx gene, the Ol antigen Olrfb gene, and the O139 antigen O139 rfb gene.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank. PCR QC checks should be performed according to EPA
Draft Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on
Environmental Samples document WAWjga.gov/sani/pdfe^PAj^Aj^C_^CR_Qcj2004.gdl or consult
the point of contact identified in Section 4.
Source: HHS, CDC, Laboratory Assay. "TaqMan Assays for Detection of V. cholerae ctxA, Ol rfb, and
O139 rfb." Contact: Dr. Eija Trees, CDC, email:
7.2.12 USDA Laboratory Guidebook: "FSIS Procedure for the Use of a Listeria
monocytogenes Polymerase Chain Reaction (PCR) Screening Test." MLG 8A.03.
2007.
Pathogen(s) [Disease]
Listeria monocytogenes [Listeriosis]
Agent Category
Bacteria
BSL
2
Analysis Purpose: Detection, not suitable for viability
Sample Preparation: Solid samples should be prepared according to "Quantification of Bias Related to
the Extraction of DNA Directly from Soils," Frostegard, A., Courtois, S., Ramisse, V., Clerc, S.,
Bernillon, D., Le Gall, F., Jeannin, P., Nesme, X., and Simonet, P. 1999. Applied and Environmental
Microbiology. 65(12): 5409-5420 (www.epa.gov/sam/pdfs/AEM-65(12)-pgs5409-5420.pdf).
Analytical Technique: Real-time PCR
Method Developed for: L. monocytogenes in food samples
Method Selected for: SAM lists these procedures for detection in solid, particulate, aerosol, liquid, and
water samples. Further research is needed to develop and standardize the procedures for environmental
sample types. See Appendix C or Table 7-1 for additional methods that should be used for this pathogen.
Description of Method: Procedures are described for analysis of food samples and may be adapted for
assessment of solid, particulate, aerosol, liquid, and water samples. The method uses real-time PCR for
identification of L. moncytogenes. Samples are homogenized in modified University of Vermont (UVM)
broth and incubated at 30.0°C ± 2.0°C for 8-24 hours. PCR is performed with the BAX® System.
Additional enrichment may be required at 35.0°C ± 2.0°C for 18-24 hours, using
morpholinepropanesulfonic acid (MOPS) buffered Listeria enrichment broth (BLEB). LODs were
determined to be better than 1 colony forming unit (CFU)/g in a 25 g meat or poultry sample,
approximately 4.5 CFU/g in a 25 g pasteurized liquid whole egg blend sample, and 1.0 x 10-2 CFU/mL in
a 500 mL brine sample.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank. PCR QC checks should be performed according to EPA
Draft Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on
Environmental Samples document www.epa.gov/sain/pdfs/EPA-QA-OC PCR Oct2004.pdf or consult
the point of contact identified in Section 4.
Source: USDA, FSIS. 2007. "FSIS Procedure for the Use of a Listeria monocytogenes Polymerase
Chain Reaction (PCR) Screening Test." Laboratory Guidebook MLG 8A.03.
http://www.epa.gov/sam/pdfs/USD A-MLG-8A.03.pdf
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7.2.13 U.S. FDA Bacteriological Analytical Manual, Chapter 10, 2003: Listeria
monocytogenes
Pathogen(s) [Disease]
Listeria monocytogenes [Listeriosis]
Agent Category
Bacteria
BSL
2
Analysis Purpose: Detection and viability
Sample Preparation: Solid samples should be prepared according to EPA Method 1680; particulate
samples should be prepared according to "Swab Materials and Bacillus anthracis Spore Recovery from
Nonporous Surfaces," Rose, L., Jensen, B., Peterson, A., Banerjee, S.N., and Arduino, MJ. 2004.
Emerging Infectious Diseases. 10(6): 1023-1029 (www.epa.gov/sam/pdfs/EID- 10(6)-pgs 1023-1029.pdf)
and "Evaluation of a Macro foam Swab Protocol for the Recovery of Bacillus anthracis Spores from a
Steel Surface," Hodges, L.R., Rose, L.J., Peterson, A., Nobel-Wang, J., and Arduino, MJ. 2006. Applied
and Environmental Microbiology. 72(6): 4429-4430 (ww^.ega.gw/sam/gi^^
4430.pdf).
Analytical Technique: Culture and immunoassay
Method Developed for: Listeria monocytogenes in food
Method Selected for: SAM lists this manual for detection and viability assessment in solid, particulate,
aerosol, liquid, and water samples. Further research is needed to develop and standardize the procedures
for environmental sample types. See Appendix C or Table 7-1 for additional methods that should be used
for this pathogen.
Description of Method: Procedures are described for analysis of food samples and may be adapted for
assessment of solid, particulate, aerosol, liquid, and water samples. Prepared samples are incubated for 4
hours in BLEB without selective agents. Cycloheximide is added and incubation continued. At 24 and
48 hours, BLEB cultures are streaked onto esculin-containing selective isolation agar (i.e., Oxford
Medium [OXA]) and incubated for an additional 24 to 48 hours. AnZ. monocytogenes-L. ivanovii
differential selective agar, such as Biosynth Chromogen Medium (BCM), is streaked at 48 hours.
Presumptive Listeria colonies are black with a black halo on esculin-containing media and blue on BCM
plates. Isolated colonies are streaked onto Trypticase™ soy agar with yeast extract (TSAye), incubated
for 24 to 48 hours, and examined for morphological and biochemical characteristics. L. monocytogenes is
a rod-shaped Gram-positive, motile bacterium. It is catalase-positive, ramnose-positive, and mannitol-
and xylose-negative. Purified isolates may be rapidly identified using commercially available
biochemical typing kits. Confirmation is performed with commercially available sera.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank.
Source: U.S. FDA, CFSAN. 2003. "Chapter 10 - Detection and Enumeration of Listeria monocytogenes
in Foods." Bacteriological Analytical Manual Online.
CJiaMOjldf
7.2.14 Standard Method 9213 B: Staphylococcus aureus
Pathogen(s)
Staphylococcus aureus
Agent Category
Bacteria
BSL
2
Analysis Purpose: Detection and viability
Sample Preparation: Solid samples should be prepared according to EPA Method 1680; particulate
samples should be prepared according to "Swab Materials and Bacillus anthracis Spore Recovery from
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Nonporous Surfaces," Rose, L., Jensen, B., Peterson, A., Banerjee, S.N., and Arduino, MJ. 2004.
Emerging Infectious Diseases. 10(6): 1023-1029 (ww^eEa^
and "Evaluation of a Macro foam Swab Protocol for the Recovery of Bacillus anthracis Spores from a
Steel Surface," Hodges, L.R., Rose, L.J., Peterson, A., Nobel-Wang, J., and Arduino, MJ. 2006. Applied
and Environmental Microbiology. 72(6): 4429-4430 (www.epa.gov/sam/pdfs/AEM-72(6)-pgs4429-
4430.pdf).
Analytical Technique: Culture
Method Developed for: Staphylococcus aureus in water
Method Selected for: SAM lists this method for detection and viability assessment in solid, particulate,
aerosol, liquid, and water samples. Further research is needed to develop and standardize the procedures
for environmental sample types other than water. See Appendix C or Table 7-1 for additional methods
that should be used for this pathogen.
Description of Method: Procedures are described for analysis of water samples and may be adapted for
assessment of solid, liquid, particulate, and aerosol samples. Prepared samples are inoculated into tubes
of M-staphylococcus broth and incubated for 24 hours. Positive (turbid) tubes are streaked onto plates of
Baird-Parker agar and incubated for 48 hours. Presumptive S. aureus colonies are tested for mannitol
fermentation by the addition of a drop of bromthymol blue, a pH indicator. Isolated colonies are
examined for morphological and biochemical characteristics. S. aureus is a Gram-positive coccus.
Biochemical characterizations include catalase-positive, coagulase-positive, fermentation of mannitol,
and anaerobic fermentation of glucose.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank.
Source: APHA, AWWA, and WEF. 2005. "Method 9213 B: Staphylococcus aureus." Standard Methods
for the Examination of Water and Wastewater. 21st Edition, http://wviny.standardmetliods.org/
7.2.15 Standard Method 9260 B: General Qualitative Isolation and Identification
Procedures for Salmonella
Pathogen(s) [Disease]
Salmonella Typhi [Typhoid fever]
Agent Category
Bacteria
BSL
2/3 (aerosol release)
Analysis Purpose: Detection and viability
Sample Preparation: Solid samples should be prepared according to EPA Method 1680; particulate
samples should be prepared according to "Swab Materials and Bacillus anthracis Spore Recovery from
Nonporous Surfaces," Rose, L., Jensen, B., Peterson, A., Banerjee, S.N., and Arduino, MJ. 2004.
Emerging Infectious Diseases. 10(6): 1023-1029 (myw.epa.gov/sam/pdfs/EID-10(6)-pgs 1023-1029.pdf)
and "Evaluation of a Macro foam Swab Protocol for the Recovery of Bacillus anthracis Spores from a
Steel Surface," Hodges, L.R., Rose, L.J., Peterson, A., Nobel-Wang, J., and Arduino, MJ. 2006. Applied
and Environmental Microbiology. 72(6): 4429-4430 (www.epa.gov/sam/pdfs/AEM-72(6)-pgs4429-
4430.pdf).
Analytical Technique: Culture and immunoassay
Method Developed for: Salmonella Typhi in water
Method Selected for: SAM lists this method for detection and viability assessment in solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types other than water. See Appendix C or Table 7-1 for additional methods that
should be used for this pathogen.
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Description of Method: This method describes procedures for analysis of water samples and may be
adapted for assessment of solid, particulate, and liquid samples. Concentrated samples are enriched in
either selenite cystine, selenite-F, or tetrathionate broths and incubated at 35 °C to 37°C for up to 5 days.
An aliquot from each turbid tube is streaked onto bismuth sulfite (BS) plates and incubated at 35 °C to
37°C for 24-48 hours. Presumptive positive colonies are then subjected to biochemical characterization.
Confirmation is through serological testing using polyvalent O and Vi antiserum.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank.
Special Considerations: This method is not preferred for non-typhoidal Salmonella.
Source: APHA, AWWA, and WEF. 2005. "Method 9260 B: General Qualitative Isolation and
Identification Procedures for Salmonella." Standard Methods for the Examination of Water and
Wastewater. 21st Edition, http://www.standardmethods.org/
7.2.16 Standard Method 9260 E: Shigella
Pathogen(s) [Disease]
Shigella spp. [Shigellosis]
Agent Category
Bacteria
BSL
2
Analysis Purpose: Detection and viability
Sample Preparation: Solid samples should be prepared according to EPA Method 1680; particulate
samples should be prepared according to "Swab Materials and Bacillus anthracis Spore Recovery from
Nonporous Surfaces," Rose, L., Jensen, B., Peterson, A., Banerjee, S.N., and Arduino, MJ. 2004.
Emerging Infectious Diseases. 10(6): 1023-1029 (www.epa.gov/sam/pdfs/EID-10(6)-pgs 1023-1029.pdf)
and "Evaluation of a Macro foam Swab Protocol for the Recovery of Bacillus anthracis Spores from a
Steel Surface," Hodges, L.R., Rose, L.J., Peterson, A., Nobel-Wang, J., and Arduino, MJ. 2006. Applied
and Environmental Microbiology. 72(6): 4429-4430 (www^epa^ov/sam/p^MAEMjTZMzBgsMZgz
4430.pdf).
Analytical Technique: Culture and immunoassay
Method Developed for: Shigella spp. in water and solids
Method Selected for: SAM lists this method for detection and viability assessment in solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types other than water. See Appendix C or Table 7-1 for additional methods that
should be used for this pathogen.
Description of Method: This method describes procedures for analysis of water samples and may be
adapted for assessment of solid, particulate, and liquid samples. This method contains two options for
sample concentration: MF (liquid samples) and centrifugation (liquid and solid samples) for analyses.
Both options include inoculation of an enrichment medium (Selenite F broth). Isolation of the target
pathogen is achieved by plating onto XLD and/or MacConkey agar. Biochemical identification consists
of inoculating TSI and LIA slants. Confirmation is performed by slide agglutination tests using
polyvalent antisera.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank.
Source: APHA, AWWA, and WEF. 2005. "Method 9260 E: Shigella." Standard Methods for the
Examination of Water and Wastewater. 21st Edition. htt|X/MwyLMlll3Mfe
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7.2.17 Standard Method 9260 F: Pathogenic Escherichia coli
Pathogen(s)
Escherichia coli O157:H7
Agent Category
Bacteria
BSL
2
Analysis Purpose: Detection and viability
Sample Preparation: Solid samples should be prepared according to EPA Method 1680; particulate
samples should be prepared according to "Swab Materials and Bacillus anthracis Spore Recovery from
Nonporous Surfaces," Rose, L., Jensen, B., Peterson, A., Banerjee, S.N., and Arduino, MJ. 2004.
Emerging Infectious Diseases. 10(6): 1023-1029 (www.epa.gov/sam/pdfs/EID-10(6)-pgs 1023-1029.pdf)
and "Evaluation of a Macro foam Swab Protocol for the Recovery of Bacillus anthracis Spores from a
Steel Surface," Hodges, L.R., Rose, L.J., Peterson, A., Nobel-Wang, J., and Arduino, MJ. 2006. Applied
and Environmental Microbiology. 72(6): 4429-4430 (www^epa±gQWsam/pdMAEll=72MzBgs4429=
4430.pdf).
Analytical Technique: Culture and immunoassay
Method Developed for: Escherichia coli O157:H7 in water and apple juice
Method Selected for: SAM lists this method for detection and viability assessment in solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types other than water. See Appendix C or Table 7-1 for additional methods that
should be used for this pathogen.
Description of Method: This method describes procedures for analysis of water and apple juice samples
and may be adapted for assessment of solid, particulate, and liquid samples. The method allows for two
options, one being a modification of SM 922 IB followed by plating and biochemical identification. The
second option, modification of a food method, allows for the analysis of large sample volumes. A 200-
mL water sample is centrifuged; the pellet is resuspended in E. coli enrichment broth (EEB) and
incubated for 6 hours. Tellurite Cefixime Sorbitol MacConkey (TC-SMAC) plates are inoculated with
the enriched EEB culture, and incubated for up to 24 hours. Colorless colonies on TC-SMAC are tested
for indole production. Presumptive positive colonies are then subjected to biochemical characterization.
Confirmation is through serological testing.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank.
Source: APHA, AWWA, and WEF. 2005. "Method 9260 F: Pathogenic Escherichia coli." Standard
Methods for the Examination of Water and Wastewater. 21st Edition. httg^/WAW.gtandarjniethods.org/
7.2.18 Standard Method 9260 G: Campylobacterjejuni
Pathogen(s) [Disease]
Campylobacterjejuni [Campylobacteriosis]
Agent Category
Bacteria
BSL
2
Analysis Purpose: Detection and viability
Sample Preparation: Solid samples should be prepared according to EPA Method 1680; particulate
samples should be prepared according to "Swab Materials and Bacillus anthracis Spore Recovery from
Nonporous Surfaces," Rose, L., Jensen, B., Peterson, A., Banerjee, S.N., and Arduino, MJ. 2004.
Emerging Infectious Diseases. 10(6): 1023-1029 (www.epa.gov/sam/pdfs/EID-10(6)-pgs 1023-1029.pdf)
and "Evaluation of a Macro foam Swab Protocol for the Recovery of Bacillus anthracis Spores from a
Steel Surface," Hodges, L.R., Rose, L.J., Peterson, A., Nobel-Wang, J., and Arduino, MJ. 2006. Applied
SAM Revision 4.0 157 September 29, 2008
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and Environmental Microbiology. 72(6): 4429-4430 (www.epa.gov/saiii/pdfs/AEM-72(6)-pgs4429-
Analytical Technique: Culture and immunoassay
Method Developed for: Campylobacter jejuni in water
Method Selected for: SAM lists this method for detection and viability assessment in solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types other than water. See Appendix C or Table 7-1 for additional methods that
should be used for this pathogen.
Description of Method: This method describes procedures for analysis of water samples and may be
adapted for assessment of solid, particulate, and liquid samples. Water samples (1 to several liter
volumes) are filtered using a cellulose nitrate membrane filter. Filters are placed on Skirrow's medium
and incubated for 24 hours at 42°C under microaerophilic conditions. Alternatively, samples are enriched
in Campylobacter broth supplemented with antibiotics and lysed horse blood under microaerophilic
conditions at 37°C for 4 hours, then at 42°C for 24-48 hours prior to streaking on Skirrow's medium.
Identification is made by culture examination, microscopy, motility testing, and biochemical testing.
Biochemical tests include oxidase, catalase, nitrite and nitrate reduction, hydrogen sulfide production, and
hippurate hydrolysis. Confirmation is performed using commercially available rapid serological test kits.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank.
Special Considerations: Skirrow's and other selective media containing antibiotics (trimethoprim,
vancomycin, polymixin) may prevent the growth of injured organisms.
Source: APHA, AWWA, and WEF. 2005. "Method 9260 G: Campylobacter jejuni." Standard Methods
for the Examination of Water and Wastewater. 21st Edition, http://www.standardmethods.org/
7.2.19 Standard Method 9260 H: Vibrio cholerae
Pathogen(s) [Disease]
Vibrio cholerae [Cholera]
Agent Category
Bacteria
BSL
2
Analysis Purpose: Detection and viability
Sample Preparation: Solid samples should be prepared according to EPA Method 1680; particulate
samples should be prepared according to "Swab Materials and Bacillus anthracis Spore Recovery from
Nonporous Surfaces," Rose, L., Jensen, B., Peterson, A., Banerjee, S.N., and Arduino, MJ. 2004.
Emerging Infectious Diseases. 10(6): 1023-1029 (www.epa.gov/sam/pdfs/EID-10(6)-pgsl023-1029.pdf)
and "Evaluation of a Macrofoam Swab Protocol for the Recovery of Bacillus anthracis Spores from a
Steel Surface," Hodges, L.R., Rose, L.J., Peterson, A., Nobel-Wang, J., and Arduino, MJ. 2006. Applied
and Environmental Microbiology. 72(6): 4429-4430 (ww^.ega.gw/sam/gi^^
4430.pdf).
Analytical Technique: Culture and immunoassay
Method Developed for: Vibrio cholerae in water
Method Selected for: SAM lists this method for detection and viability assessment in solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types other than water. See Appendix C or Table 7-1 for additional methods that
should be used for this pathogen.
Description of Method: This method describes procedures for analysis of water samples and may be
adapted for assessment of solid, particulate, and liquid samples. Samples are enriched in alkaline peptone
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broth and incubated for up to 8 hours. Thiosulfate-citrate-bile salts-sucrose (TCBS) agar plates are
inoculated with the incubated broth and incubated for 24 hours. Yellow sucrose-fermenting colonies are
presumptive for V. cholerae and are plated on tryptic soy agar with 0.5% sodium chloride. Presumptive
positive colonies are subjected to biochemical characterization. Confirmation is performed using slide
agglutination assays for serological identification.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank.
Source: APHA, AWWA, and WEF. 2005. "Method 9260 H: Vibrio cholerae." Standard Methods for
the Examination of Water and Wastewater. 21st Edition, http://www.standardmethods.org/
7.2.20 Standard Method 9260 I: Leptospira
Pathogen(s) [Disease]
Leptospira interrogans [Leptospirosis]
Agent Category
Bacteria
BSL
2
Analysis Purpose: Detection and viability
Sample Preparation: Solid samples should be prepared according to EPA Method 1680; particulate
samples should be prepared according to "Swab Materials and Bacillus anthracis Spore Recovery from
Nonporous Surfaces," Rose, L., Jensen, B., Peterson, A., Banerjee, S.N., and Arduino, MJ. 2004
Emerging Infectious Diseases. 10(6): 1023-1029 (www.epa.gov/sam/pdfs/EID-10(6)-pgs 1023-1029.pdf)
and "Evaluation of a Macro foam Swab Protocol for the Recovery of Bacillus anthracis Spores from a
Steel Surface," Hodges, L.R., Rose, L.J., Peterson, A., Nobel-Wang, J., and Arduino, MJ. 2006. Applied
and Environmental Microbiology. 72(6): 4429-4430 (www^epa^ov/sam/pg^AEMjTZMzBgsMZgz
4430.pdf).
Analytical Technique: Culture and immunoassay
Method Developed for: Leptospira interrogans in water
Method Selected for: SAM lists this method for detection and viability assessment in solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types other than water. See Appendix C or Table 7-1 for additional methods that
should be used for this pathogen.
Description of Method: This method describes procedures for analysis of water samples and may be
adapted for assessment of solid, particulate, and liquid samples. Filter samples through a 0.22-um filter,
retaining the filtrate as inoculum. If the sample is turbid, a succession of filters of decreasing pore size
may be used prior to the 0.22-um filter. A tube of Leptospira Medium Base (Ellinghausen-McCullough
Johnson Harris formulation [EMJH], supplemented with bovine serum albumin [BSA] and Tween®), is
inoculated with the sample and incubated at 30°C for up to 6 weeks. Alternatively, the sample is added
directly to the media, incubated overnight, passed through a 0.22-um membrane filter, and incubation
continued for up to 6 weeks. Cultures are examined by darkfield microscopy for motile leptospires.
Confirmation is performed by microscopic agglutination test using reference antisera.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank.
Source: APHA, AWWA, and WEF. 2005. "Method 9260 I: Leptospira." Standard Methods for the
Examination of Water and Wastewater. 21st Edition, http://www.standardmethods.org/
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7.2.21 ASM Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism:
Brucella species
Pathogen(s) [Disease]
Brucella spp. [Burcellosis]
Agent Category
Bacteria
BSL
3
Analysis Purpose: Detection and viability
Sample Preparation: Solid samples should be prepared according to EPA Method 1680; particulate
samples should be prepared according to "Swab Materials and Bacillus anthracis Spore Recovery from
Nonporous Surfaces," Rose, L., Jensen, B., Peterson, A., Banerjee, S.N., and Arduino, MJ. 2004.
Emerging Infectious Diseases. 10(6): 1023-1029 (www.epa.gov/sam/pdfs/EID-10(6)-pgs 1023-1029.pdf)
and "Evaluation of a Macro foam Swab Protocol for the Recovery of Bacillus anthracis Spores from a
Steel Surface," Hodges, L.R., Rose, L.J., Peterson, A., Nobel-Wang, J., and Arduino, MJ. 2006. Applied
and Environmental Microbiology. 72(6): 4429-4430 (www.epa.gov/sam/pdfs/AEM-72(6)-pgs4429-
Analytical Technique: Culture
Method Developed for: Brucella spp. in clinical samples
Method Selected for: SAM lists these guidelines for detection and viability assessment in solid,
particulate, aerosol, liquid, and water samples. Further research is needed to develop and standardize the
procedures for environmental sample types. See Appendix C or Table 7-1 for additional methods that
should be used for this pathogen.
Description of Method: This method describes procedures for analysis of clinical samples and may be
adapted for assessment of solid, particulate, and liquid samples. Samples are plated directly on C A or
SBA and incubated at 35°C (5-10% carbon dioxide) for up to 7 days. Colonies are punctuate (minute),
non-pigmented, and non-hemolytic at 48 hours. Presumptive identification is made by culture
examination, microscopy, and biochemical testing. Brucella spp. are small, non-motile, Gram-negative
coccobacilli and are catalase-, oxidase-, and urease-positive. Cultures (isolates) that cannot be ruled out
as Brucella spp. based on the characteristics noted above may be referred to an appropriate reference
laboratory for confirmation. Confirmation is performed using specific antiserum.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank.
Special Considerations: Brucella spp. are select agents requiring regulatory compliance (42 CFR parts
72 and 73, and 9 CFR part 121); appropriate safety and BSL requirements should also be followed (see
CDC's BMBL, 5th Edition, http://www.cdc.gov/OD/ohs/biosfty/bmbl5/bmbl5toc.htm).
Source: ASM. 2004. "Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism: Brucella
species." http://www.epa.gov/sam/pdfs/ASM-Brucella.pdf
7.2.22 ASM Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism:
Burkholderia mallei and Burkholderia pseudomallei
Pathogen(s) [Disease]
Burkholderia mallei [Glanders]
Burkholderia pseudomallei [Melioidosis]
Agent Category
Bacteria
Bacteria
BSL
3
3
Analysis Purpose: Detection and viability
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Sample Preparation: Solid samples should be prepared according to EPA Method 1680; particulate
samples should be prepared according to "Swab Materials and Bacillus anthracis Spore Recovery from
Nonporous Surfaces," Rose, L., Jensen, B., Peterson, A., Banerjee, S.N., and Arduino, MJ. 2004.
Emerging Infectious Diseases. 10(6): 1023-1029 (www.epa.gov/sam/pdfs/EID- 1 0 (6) -pgs 1023- 1 029.pdf)
and "Evaluation of a Macro foam Swab Protocol for the Recovery of Bacillus anthracis Spores from a
Steel Surface," Hodges, L.R., Rose, L.J., Peterson, A., Nobel- Wang, J., and Arduino, MJ. 2006. Applied
and Environmental Microbiology. 72(6): 4429-4430 (www. epa.gov/sam/pdfs/AEM- 7 2 (6) -pgs44 29-
4430.pdf).
Analytical Technique: Culture
Method Developed for: B. mallei and B. pseudomallei in clinical samples
Method Selected for: SAM lists these guidelines for detection and viability assessment in solid,
particulate, aerosol, liquid, and water samples. Further research is needed to develop and standardize the
procedures for environmental sample types. See Appendix C or Table 7-1 for additional methods that
should be used for this pathogen.
Description of Method: This method describes procedures for analysis of clinical samples and may be
adapted for assessment of solid, particulate, and liquid samples. Samples are plated directly on SBA and
incubated at 35°C-37°C. At 48 hours, B. mallei forms gray, translucent colonies and B. pseudomallei
forms small, smooth, creamy colonies with a musty odor that gradually change to dry, wrinkled colonies.
Presumptive identification is made by culture examination, microscopy, motility testing, and biochemical
testing. B. mallei are small, non-motile, Gram-negative coccobacilli or small rods and are oxidase-
variable, catalase-positive, colistin-resistant, indole-negative, nitrate reductase-positive, and arginine
dihydrolase-positive. B. pseudomallei are small, motile, Gram-negative rods and are oxidase-positive,
catalase-positive, colistin-resistant, indole-negative, nitrate reductase-positive, and arginine dihydrolase-
positive. Cultures (isolates) that cannot be ruled out as B. mallei or B. pseudomallei based on the
characteristics noted above may be referred to an appropriate reference laboratory for confirmation.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank.
Special Considerations: B. mallei and B. pseudomallei are select agents requiring regulatory compliance
(42 CFR parts 72 and 73, and 9 CFR part 121); appropriate safety and BSL requirements should also be
followed (see CDC's BMBL, 5th Edition, Mt2i//w^mcticg^
Source: ASM. 2008. "Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism:
Burkholderia mallei and Burkholderia pseudomallei ." http://www.epa.gov/sam/pdfs/ASM-Bmallei-
7.2.23 ASM Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism:
Yersinia pestis
Pathogen(s) [Disease]
Yersinia pestis [Plague]
Agent Category
Bacteria
BSL
3
Analysis Purpose: Detection and viability
Sample Preparation: Solid samples should be prepared according to EPA Method 1680; particulate
samples should be prepared according to "Swab Materials and Bacillus anthracis Spore Recovery from
Nonporous Surfaces," Rose, L., Jensen, B., Peterson, A., Banerjee, S.N., and Arduino, MJ. 2004.
Emerging Infectious Diseases. 10(6): 1023-1029 (www.epa.gov/sam/pdfs/EID-10(6)-pgs 1023-1029.pdf)
and "Evaluation of a Macro foam Swab Protocol for the Recovery of Bacillus anthracis Spores from a
Steel Surface," Hodges, L.R., Rose, L.J., Peterson, A., Nobel-Wang, J., and Arduino, MJ. 2006. Applied
SAM Revision 4.0 161 September 29, 2008
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and Environmental Microbiology. 72(6): 4429-4430 (www.epa.gov/saiii/pdfs/AEM-72(6)-pgs4429-
Analytical Technique: Culture
Method Developed for: Y. pestis in clinical samples
Method Selected for: SAM lists these guidelines for detection and viability assessment in solid,
particulate, aerosol, liquid, and water samples. Further research is needed to develop and standardize the
procedures for environmental sample types. See Appendix C or Table 7-1 for additional methods that
should be used for this pathogen.
Description of Method: This method describes procedures for analysis of clinical samples and may be
adapted for assessment of solid, particulate, and liquid samples. Samples are either plated directly on
SBA or first enriched in a nutrient broth (e.g., TSB) prior to plating. Incubation is for at least 3 days at
28°C-30°C. Y. pestis produces 1.0-2.0 mm, gray-white to opaque colonies on SBA at 24 hours, with a
"fried egg" appearance with longer incubation. There is little to no hemolysis on SBA. In broth, Y. pestis
grows in flocculent clumps. Presumptive identification is made by culture examination, microscopy, and
biochemical testing. Y. pestis is a bi-polar staining, Gram-negative rod and is oxidase-negative, catalase-
positive, urease-negative, and indole-negative. Cultures (isolates) that cannot be ruled out as Y. pestis
based on the characteristics noted above are referred to an appropriate reference laboratory for
confirmation. Cultures (isolates) that cannot be ruled out as Y. pestis based on the characteristics noted
above may be referred to an appropriate reference laboratory for confirmation.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank.
Special Considerations: Y. pestis is a select agent requiring regulatory compliance (42 CFR parts 72
and 73, and 9 CFR part 121); appropriate safety and BSL requirements should also be followed (see
CDC's BMBL, 5th Edition, http://www.cdc.gov/OD/ohs/biosfty/bmbl5/bmbl5toc.htm).
Source: ASM. 2005. "Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism: Yersinia
pestis." http://www.epa.gov/sam/pdfs/ASM-Ypestis.pdf
7.2.24 Literature Reference for Campylobacterjejuni (Molecular and Cellular Probes.
2006. 20: 269-279)
Pathogen(s) [Disease]
Campylobacterjejuni [Campylobacteriosis]
Agent Category
Bacteria
BSL
2
Analysis Purpose: Detection, not suitable for viability
Sample Preparation: Solid samples should be prepared according to "Quantification of Bias Related to
the Extraction of DNA Directly from Soils," Frostegard, A., Courtois, S., Ramisse, V., Clerc, S.,
Bernillon, D., Le Gall, F., Jeannin, P., Nesme, X., and Simonet, P. 1999. Applied and Environmental
Microbiology. 65(12): 5409-5420 (www.epa.gov/sam/pdfs/AEM-65(12)-pgs5409-5420.pdf).
Analytical Technique: Real-time PCR
Method Developed for: Campylobacterjejuni in clinical samples
Method Selected for: SAM lists these procedures for detection in solid, particulate, aerosol, liquid, and
water samples. Further research is needed to develop and standardize the procedures for environmental
sample types. See Appendix C or Table 7-1 for additional methods that should be used for this pathogen.
Description of Method: Procedures are described for analysis of clinical samples and may be adapted
for assessment of solid, particulate, aerosol, liquid, and water samples. The method uses real-time
quantitative PCR (qPCR) for identification of C. jejuni that can be used in conjuction with either the ABI
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Prism® 7700 or 7900 HT Thermal Cyclers. A high through-put method using guanidinium thiocyanate
(GTC) and glass beads is used for extraction of DNA. C. jejuni uses the CJ F primer, CJ R primer, and
CJ probe for real-time quantitative PCR. Results are evaluated against standard curves made with a 10-
fold dilution series of C. jejuni NCTC 1 1 168 DNA in a background of cecum (intestinal) DNA.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank. PCR QC checks should be performed according to EPA
Draft Quality Assurance /Quality Control Guidance for Laboratories Performing PCR Analyses on
Environmental Samples document WAWj^jajrov/sajn/^^ or consult
the point of contact identified in Section 4.
Source: Skanseng, B., Kaldhusdal, M., and Rudi, K. 2006. "Comparison of Chicken Gut Colonization
by the Pathogens Campylobacter jejuni and Clostridium perfringens by the Real-time Quantitative PCR.
Molecular and Cellular Probes. 20: 269-279.
http://www.sciencedirect.com/science? ob=ArticleURL& udi=B6WNC-4JDN6C7-
1& ..... user=10& ...... rdo_c=l& ...... fmt=& ...... _ong=search& ...... sort=c[&yiew=c& ..... version^l& ..... uriVerston=0& ...... userid=
10&md5=dcdea60d7ab66cd57b8ffef7d59ebd39
7.2.25 Literature Reference for Chlamydophila psittaci (Journal of Clinical Microbiology.
2000. 38(3): 1085-1093)
Pathogen(s) [Disease]
Chlamydophila psittaci [Psittacosis]
(formerly known as Chlamydia psittaci)
Agent Category
Bacteria
BSL
2/3
(aerosols and large
volumes)
Analysis Purpose: Detection and viability
Sample Preparation: Solid samples should be prepared according to "Quantification of Bias Related to
the Extraction of DNA Directly from Soils," Frostegard, A., Courtois, S., Ramisse, V., Clerc, S.,
Bernillon, D., Le Gall, F., Jeannin, P., Nesme, X., and Simonet, P. 1999. Applied and Environmental
Microbiology. 65(12): 5409-5420 (www.epa.gov/sam/pdfs/AEM-65(12)-pgs5409-5420.pdf).
Analytical Technique: Tissue culture and PCR
Method Developed for: Chlamydophila psittaci in clinical samples
Method Selected for: SAM lists these procedures for detection and viability assessment in solid,
particulate, aerosol, liquid, and water samples. Further research is needed to develop and standardize the
procedures for environmental sample types. See Appendix C or Table 7-1 for additional methods that
should be used for this pathogen.
Description of Method: Procedures are described for analysis of clinical samples and may be adapted
for assessment of solid, particulate, aerosol, liquid, and water samples. The method uses touchdown
enzyme time release (TETR)-PCR for detection and identification of Chlamydophila psittaci. DNA is
extracted from chlamydia cultures (Buffalo Green Monkey Kidney [BGMK] cells), minced clinical
tissues, and respiratory samples by mixing with a chelating resin, heating first to 56°C for 15-30 minutes,
then 100°C for 8-10 minutes. Primer sets specific for C. psittaci are designed based on the DNA
sequences of the 16S ribosomal ribonucleic acid (rRNA) and 16S-23S rRNA genes (CPS 100/101). PCR
is conducted on a Perkin-Elmer 480 with products separated by electrophoresis in 12% polyacrylamide
gels with Tris-borate-EDTA buffer and visualized with ethidium bromide. Chlamydophila psittaci
samples are to be handled with BSL-3 containment and practices.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank. PCR QC checks should be performed according to EPA
Draft Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on
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Environmental Samples document ww.!Le]iy|o^^ or consult
the point of contact identified in Section 4.
Source: Madico, G., Quinn, T.C., Boman, J., and Gaydos, C.A. 2000. "Touchdown Enzyme Time
Release-PCR for Detection and Identification of Chlamydia trachomatis, C. pneumoniae, and C. psittaci
Using the 16S and 16S-23S Spacer rRNA Genes." Journal of Clinical Microbiology. 38(3): 1085-1093.
http://www.epa.gov/sam/pdfs/.TCM-38(3)-pgsl085-1093.pdf
7.2.26 Literature Reference for Escherichia coli O157:H7 (Applied and Environmental
Microbiology. 2003. 69(10): 6327-6333)
Pathogen(s)
Escherichia coli O1 57:H7
Agent Category
Bacteria
BSL
2
Analysis Purpose: Detection, not suitable for viability
Sample Preparation: Solid samples should be prepared according to "Quantification of Bias Related to
the Extraction of DNA Directly from Soils," Frostegard, A., Courtois, S., Ramisse, V., Clerc, S.,
Bernillon, D., Le Gall, F., Jeannin, P., Nesme, X., and Simonet, P. 1999. Applied and Environmental
Microbiology. 65(12): 5409-5420 (wvtw.epa.gov/sam/pdfs/AEM-65(12)-pgs5409-5420.pdf).
Analytical Technique: Real-time PCR
Method Developed for: E. coli O157:H7 in cultures or isolates
Method Selected for: SAM lists these procedures for detection in solid, particulate, aerosol, liquid,
and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types. See Appendix C or Table 7-1 for additional methods that should be used
for this pathogen.
Description of Method: Procedures are described for analysis of isolates and may be adapted for
assessment of solid, particulate, aerosol, liquid, and water samples. The method uses multiplex real-time
PCR performed on a Smart Cycler® with fluorogenic probes for identification of cultured E. coli
O157:H7 isolates. Sample template (0.5 p:L) is added to a total reaction volume of 25 p:L. Primers and
probes were designed to target the stxl and stx2 (Shiga toxin) genes and the single-nucleotide
polymorphism at position 93 of the uidA (6-glucoronidase) gene, using 6-carboxy-X-rhodamine (ROX),
6-caraboxyfluorescein (FAM), and 6-carboxy-4,7,2',7'-tetrachlorofluorescein (TET) as respective
fluorogens. The method was tested with 138 isolates, including 52 E. coli O157:H7 and two E. coli
O157:H7~ organisms. Specificity was 100% for all three target genes. Sensitivities were 98.6%, 100%,
and 100% for stxl, stx2, and uidA O157:H7~ targets, respectively. This assay did not detect two strains of
the closely related E. coli O55:H7/H~ serotype.
At a minimum, the following quality control checks should be performed and evaluated before using this
protocol: positive control, negative control, and blank. PCR quality control checks should be performed
according to EPA Draft Quality Assurance/Quality Control Guidance for Laboratories Performing PCR
Analyses on Environmental Samples document, ww«yym^w/ne^^ or consult
the point of contact identified in Section 4.
Source: Jinneman, K.C., Yoshitomi, K.J., and Weagant, S.D. 2003. "Multiplex Real-Time PCR Method
to Identify Shiga Toxin Genes stxl and stx2 and Escherichia coli O157:H7/H~Serotype." Applied and
Environmental Microbiology. 69(10): 6327-6333. http://www.epa.gov/sam/pdfs/AEM-69(lU) pgs6327-
6333.pdf.
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7.2.27 Literature Reference for Non-Typhoidal Salmonella (Journal of Applied
Microbiology. 2007. 102(2): 516-530)
Pathogen(s) [Disease]
Non-typhoidal Salmonella [Salmonellosis]
Agent Category
Bacteria
BSL
2
Analysis Purpose: Detection, not suitable for viability
Sample Preparation: Solid samples should be prepared according to "Quantification of Bias Related to
the Extraction of DNA Directly from Soils," Frostegard, A., Courtois, S., Ramisse, V., Clerc, S.,
Bernillon, D., Le Gall, F., Jeannin, P., Nesme, X., and Simonet, P. 1999. Applied and Environmental
Microbiology. 65(12): 5409-5420 (www.epa.gov/sam/pdfs/AEM-65(12)-pgs5409-5420.pdf).
Analytical Technique: Real-time PCR
Method Developed for: Non-typhoidal Salmonella from cultures or isolates
Method Selected for: SAM lists these procedures for detection in solid, particulate, aerosol, liquid, and
water samples. Further research is needed to develop and standardize the procedures for environmental
sample types. See Appendix C or Table 7-1 for additional methods that should be used for this pathogen.
Description of Method: Procedures are described for analysis of isolates and may be adapted for
assessment of solid, particulate, aerosol, liquid, and water samples. The method uses real-time PCR for
identification of Salmonella spp. PCR templates are prepared from 1.0 mL of overnight bacterial cultures
propagated overnight in TSB at 37.0°C. Cells are pelleted, washed twice with physiological saline,
resuspended in molecular grade water, and boiled for 10 minutes. PCR is performed on a SmartCycler®
II System using primers and probes designed for the stn gene. The protocol has been evaluated against
353 isolates, including 255 S. enterica representing 158 serotypes, 14 S. bongori representing 12
serotypes, and 84 non-Salmonella representing 56 species from 31 genera. The PCR method had 100%
inclusivity, 96.4% exclusivity, and a level of detection of 3 CFUs per reaction for cultured Salmonella
spp.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank. PCR QC checks should be performed according to EPA
Draft Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on
Environmental Samples document %^Yft;,jja,gQV/sam/p,dfs/EPAr.QA:QC PCR Qct2Q04.-..pdf or consult
the point of contact identified in Section 4.
Source: Moore, M.M., and Feist, M.D. 2007. "Real-time PCR Method for Salmonella spp. Targeting the
stn Gene." Journal of Applied Microbiology. 102(2): 516-530.
http://www3.interscience.wiley.com/iournal/118490299/abstract
7.2.28 Literature Reference for Adenoviruses (Applied and Environmental Microbiology.
2005. 71(6): 3131-3136)
Pathogen(s)
Adenoviruses: Subgroups A-F
Agent Category
Viruses
BSL
2
Analysis Purpose: Detection and viability
Sample Preparation: Samples should be prepared according to the procedures described in the USEPA
Manual of Methods for Virology (EPA/600/4-84/013, April 2001).
Analytical Technique: Tissue culture and real-time PCR
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Method Developed for: Human adenoviruses (HAdV) in cell culture lysate samples
Method Selected for: SAM lists these procedures for detection and viability assessment in solid,
particulate, aerosol, liquid, and water samples. Further research is needed to develop and standardize the
procedures for environmental sample types. See Appendix C or Table 7-1 for additional methods that
should be used for this pathogen.
Description of Method: Procedures are described for analysis of cell culture lysates and may be adapted
for assessment of solid, particulate, aerosol, liquid, and water samples. The detection procedure uses a
broadly reactive fluorogenic 5' nuclease (TaqMan®) quantitative real-time PCR assay for the detection of
all six species (A-F) of HAdV on a R.A.P.I.D.® PCR system. Sensitive detection and discrimination of
adenovirus F species (Adenovirus 40 [AdV40] and Adenovirus 41 [AdV41]) can be achieved by using a
real-time fluorescence resonance energy transfer (FRET)-based PCR assay.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank. PCR QC checks should be performed according to EPA
Draft Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on
Environmental Samples document www.epa.gov/sam/pdfs/EPA-OA-QC PCR Oct2004.pdf or consult
the point of contact identified in Section 4.
Special Considerations: For the viability assessment of adenovirus 40 and 41, given that they can be
difficult to grow in culture, cell lines such as G293 (Journal of Medical Virology, 11 (3): 215-231) or
CaCo-2 (Journal of Medical Virology. 1994. 44(3): 310-315) may be considered when these viruses are
suspected to be present. As detection of adenovirus in environmental samples can be difficult, additional
methods such as described in Effect of Adenovirus Resistance on UV Disinfection Experiments: A Report
on the State of Adenovirus Science (]. AWWA. 2006. 98(6):93-106) also may be useful.
Source: Jothikumar, N., Cromeans, T.L., Hill, V.R., Lu, X., Sobsey, M., and Erdman, D.D. 2005.
"Quantitative Real-Time PCR Assays for Detection of Human Adenoviruses and Identification of
Serotypes 40 and 41." Applied and Environmental Microbiology. 71(6): 3131-3136.
htto://w^w;ei)a1goy/s
7.2.29 Literature Reference for Astroviruses (Canadian Journal of Microbiology. 2004. 50:
269-278)
Pathogen(s)
Astroviruses
Agent Category
Viruses
BSL
Not specified
Analysis Purpose: Detection and viability
Sample Preparation: Samples should be prepared according to the procedures described in the USEPA
Manual of Methods for Virology (EPA/600/4-84/013, April 2001).
Analytical Technique: Integrated cell culture/real-time reverse transcription-PCR
Method Developed for: Astroviruses in clinical samples
Method Selected for: SAM lists these procedures for detection and viability assessment in solid,
particulate, aerosol, liquid, and water samples. Further research is needed to develop and standardize the
procedures for environmental sample types. See Appendix C or Table 7-1 for additional methods that
should be used for this pathogen.
Description of Method: Procedures are described for analysis of clinical samples and may be adapted
for assessment of solid, particulate, aerosol, liquid, and water samples. The method detects eight
astrovirus serotypes. The method is a reverse transcription-PCR procedure optimized for use in a real-
time PCR assay using an ABI Prism® 7000 and can be integrated with sample-cell culture (CaCo-2 cells)
to enhance sensitivity. Water samples are collected by filtration (1MDS filter), and viruses are eluted
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using a beef extract solution (1.5%, pH 9.5). Viruses are concentrated using Celite® adsorption (pH 4.0),
filtration, and Celite®-elution with sodium phosphate (0.15 M, pH 9.0), followed by further concentration
and processing to remove inhibitors (ultracentrifugation, solvent extraction, and molecular weight [MW]-
exclusion filtration). Concentrated samples are analyzed directly or indirectly (following cell culture) by
a two-step reverse transcription-PCR (i.e., reverse transcription followed by PCR) assay using astrovirus-
specific primer sets. Detection of amplicons is by gel electrophoresis with subsequent confirmation by
hybridization (dot-blot) using digoxigenin-labeled internal (nested) probes or by real-time detection using
fluorogenic probes.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank. PCR QC checks should be performed according to EPA
Draft Quality Assurance, Quality Control Guidance for Laboratories Performing PCR Analyses on
Environmental Samples document wjroy|]iy|ov/g^^ or consult
the point of contact identified in Section 4.
Source: Grimm, A.C., Cashdollar, J.L., Williams, P.P., and Fout, G.S. 2004. "Development of an
Astrovirus RT-PCR Detection Assay for Use with Conventional, Real-Time, and Integrated Cell
Culture/RT-PCR." Canadian Journal of Microbiology. 50(4): 269-278. http://pubs.nrc-cnrc.gc.ca/rp-
ps/articleQptions.jsp?icode=cim&ftl=w04-012&lang=eng
7.2.30 Literature Reference for Noroviruses (Journal of Clinical Microbiology. 2004.
42(10): 4679-4685)
Pathogen(s)
Caliciviruses: Noroviruses
Agent Category
Viruses
BSL
Not specified
Analysis Purpose: Detection, not suitable for viability
Sample Preparation: Samples should be prepared according to procedures described in the USEPA
Manual of Methods for Virology (EPA/600/4-84/013, April 2001).
Analytical Technique: Real-time reverse transcription-PCR
Method Developed for: Noroviruses in clinical samples
Method Selected for: SAM lists these procedures for detection in solid, particulate, aerosol, liquid, and
water samples. Further research is needed to develop and standardize the procedures for environmental
sample types. See Appendix C or Table 7-1 for additional methods that should be used for this pathogen.
Description of Method: Procedures are described for analysis of clinical samples and may be adapted
for assessment of solid, particulate, aerosol, liquid, and water samples. This method is an assay for the
detection and quantitation of norovirus using LightCycler® real-time reverse transcription-PCR
technology. Viral ribonucleic acid (RNA) is extracted using either a commercial kit or a silica-based
method. For Norovirus G-l, primers based on the capsid gene sequence are used, and for Norovirus G-II,
primers based on the polymerase gene sequence are used. A SYBR® Green I system is used in the
reaction for visualization. External standard curves for the quantification of norovirus are established
using RNA transcripts from strains S5 and S19, corresponding to G-I/4 and G-II/12, respectively.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank. PCR QC checks should be performed according to EPA
Draft Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on
Environmental Samples document www.epa.gov/sam/pdfs/EPA-QA-QC PCR Oct2004.pdf or consult
the point of contact identified in Section 4.
Source: Pang, X., Lee, B., Chui, L., Preiksaitis, J.K., and Monroe, S.S. 2004. "Evaluation and
Validation of Real-Time Reverse Transcription-PCR Assay Using the LightCycler System for Detection
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and Quantitation of Norovirus." Journal of Clinical Microbiology. 42(10): 4679-4685.
^^^
7.2.31 Literature Reference for Sapoviruses (Journal of Medical Virology. 2006. 78(10):
1347-1353)
Pathogen(s)
Caliciviruses: Sapoviruses
Agent Category
Viruses
BSL
Not specified
Analysis Purpose: Detection, not suitable for viability
Sample Preparation: Samples should be prepared according to procedures described in the USEPA
Manual of Methods for Virology (EPA/600/4-84/013, April 2001).
Analytical Technique: Real-time reverse transcription-PCR
Method Developed for: Sapoviruses in clinical samples
Method Selected for: SAM lists these procedures for detection in solid, particulate, aerosol, liquid, and
water samples. Further research is needed to develop and standardize the procedures for environmental
sample types. See Appendix C or Table 7-1 for additional methods that should be used for this pathogen.
Description of Method: Procedures are described for analysis of clinical samples and may be adapted
for assessment of solid, particulate, aerosol, liquid, and water samples. The method is a TaqMan®-based
real-time reverse transcriptase PCR assay that uses an ABI 7500 system. The assay has the ability to
detect four of the five distinct sapovirus (SaV) genogroups (GI—GV). Sets of primers, based on the
multiple alignment of 27 gene sequences for the polymerase-capsid junction in open reading frame 1
(ORF1), are used to detect human SaV GI, Gil, GIV, and GV sequences in a single tube. Sensitivity
using control plasmids range from 2.5X101to2.5X107 copies per tube. No cross-reactivity is observed
against other enteric viruses, including norovirus (NoV), rotavirus, astrovirus, and adenovirus.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank. PCR QC checks should be performed according to EPA
Draft Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on
Environmental Samples document www1ega.gov/gani/pdfe^P_A=QA=QC PCR OctZOO^jidf or consult
the point of contact identified in Section 4.
Source: Oka, T., Katayama, K., Hansman, G.S., Kageyama, T., Ogawa, S., Wu, F.T., White, P.A., and
Takeda, N. 2006. "Detection of Human Sapovirus by Real-time Reverse Transcription-Polymerase Chain
Reaction." Journal of Medical Virology. 78(10): 1347-1353.
http://cat.inist.ff/?aModele=afficheN&cpsidt=18099754
7.2.32 Literature Reference for Coronaviruses (SARS) (Journal of Virological Methods.
2004. 122:29-36)
Pathogen(s)
Coronaviruses: SARS-associated human
coronavirus
Agent Category
Viruses
BSL
2/3 (propagation)
Analysis Purpose: Detection, not suitable for viability
Sample Preparation: Samples should be prepared according to procedures described in the USEPA
Manual of Methods for Virology (EPA/600/4-84/013, April 2001).
Analytical Technique: Reverse transcription-PCR
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Method Developed for: SARS-associated human coronavirus in clinical samples
Method Selected for: SAM lists these procedures for detection in solid, particulate, aerosol, liquid, and
water samples. Further research is needed to develop and standardize the procedures for environmental
sample types. See Appendix C or Table 7-1 for additional methods that should be used for this pathogen.
Description of Method: Procedures are described for analysis of clinical samples and may be adapted
for assessment of solid, particulate, aerosol, liquid, and water samples. This method uses a conventional
single-tube reverse transcription-PCR procedure conducted on a Stratagene Robocycler® and based on
consensus primer sequences targeting conserved regions of coronavirus genome sequences. End-point
amplicon analysis is by electrophoresis and subsequent visualization. The assay can detect the SARS-
HCoV as well as several other human respiratory coronaviruses (HCoV-OC43 and HCoV-229E). Species
identification is provided by sequencing the amplicon, although rapid screening can be performed by
restriction enzyme analysis.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank. PCR QC checks should be performed according to EPA
Draft Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on
Environmental Samples document www.epa.gov/sam/pdfs/EPA-QA-OC PCR Oct2004.pdf or consult
the point of contact identified in Section 4.
Source: Adachi, D., Johnson. G., Draker, R., Ayers, M., Mazzulli, T., Talbot, P.J., and Tellier, R. 2004.
"Comprehensive Detection and Identification of Human Coronaviruses, Including the SARS-associated
Coronavirus, with a Single RT-PCR Assay." Journal of Virological Methods. 122: 29-36.
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2& user=10& rdoc=l& fmt=& orig=search& sort=d&view=c& version=l& urlVersion=0& userid=
10&md5=b711c55f4966e87bffb977bffa49f8db
7.2.33 Literature Reference for Hepatitis E Virus (Journal of Virological Methods. 2006.
131(1): 65-71)
Pathogen(s)
Hepatitis E virus (HEV)
Agent Category
Viruses
BSL
2
Analysis Purpose: Detection, not suitable for viability
Sample Preparation: Samples should be prepared according to procedures described in the USEPA
Manual of Methods for Virology (EPA/600/4-84/013, April 2001).
Analytical Technique: Real-time reverse transcription-PCR
Method Developed for: Hepatitis E virus (HEV) in spiked water samples
Method Selected for: SAM lists these procedures for detection in solid, particulate, aerosol, liquid, and
water samples. Further research is needed to develop and standardize the procedures for environmental
sample types other than water. See Appendix C or Table 7-1 for additional methods that should be used
for this pathogen.
Description of Method: Procedures are described for analysis of spiked water samples and may be
adapted for assessment of solid, particulate, aerosol, and liquid samples. The method uses a TaqMan®
real-time reverse transcriptase-PCR assay using the R.A.P.I.D.® PCR systems to detect and quantitate all
four major HEV genotypes that may be present in clinical and environmental samples. Primers and
probes are based on the multiple sequence alignments of 27 gene sequences for the ORF3 region.
Thirteen HEV isolates representing genotypes 1-4 were used to assess assay specificity, all thirteen
isolates were positive.
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At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank. PCR QC checks should be performed according to EPA
Draft Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on
Environmental Samples document WAW1ega.gov^am/pdfe^PA:QA:QC PCR Oct2004.gdf or consult
the point of contact identified in Section 4.
Source: Jothikumar, N., Cromeans, T.L., Robertson, B.H., Meng, X.J., and Hill, V.R. 2006. "A Broadly
Reactive One-step Real-time RT-PCR Assay for Rapid and Sensitive Detection of Hepatitis E Virus."
Journal of Virological Methods, Vol. 131(1): 65-71.
http://cat.inist.fr/?aModele=afficlieN&cpsidt= 17367357
7.2.34 Literature Reference for Influenza H5N1 (Emerging Infectious Diseases. 2005.
11(8): 1303-1305)
Pathogen(s)
Influenza H5N1 virus
Agent Category
Viruses
BSL
3
Analysis Purpose: Detection, not suitable for viability
Sample Preparation: Samples should be prepared according to procedures described in the USEPA
Manual of Methods for Virology (EPA/600/4-84/013, April 2001).
Analytical Technique: Real-time reverse transcription-PCR
Method Developed for: Influenza H5N1 virus in clinical samples
Method Selected for: SAM lists these procedures for detection in solid, particulate, aerosol, liquid, and
water samples. Further research is needed to develop and standardize the procedures for environmental
sample types. See Appendix C or Table 7-1 for additional methods that should be used for this pathogen.
Description of Method: Procedures are described for analysis of clinical samples and may be adapted
for assessment of solid, particulate, aerosol, liquid, and water samples. This is a two-step, real-time
reverse transcriptase-PCR multiplex assay. It employs a mixture of two sets of primers and dual-labeled
fluorescent probes that specifically target two different regions of the HA gene of H5N1. Viral
ribonucleic acid (RNA) is extracted using a commercial viral RNA extraction kit and reverse transcribed
with random hexamers. Five microliters of the complementary (cDNA) is used for PCR which is
conducted on a LightCycler®. At the end of each annealing step, the fluorescent signal of each reaction
is measured at a wavelength of 530 nm with the fluorimeter. The assay is specific for the H5 subtype.
Influenza H5N1 virus samples are to be handled with BSL-3 containment and practices.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank. PCR QC checks should be performed according to EPA
Draft Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on
Environmental Samples document www.epa.gov/sam/pdfs/EPA-QA-QC PCR Oct2004.pdf or consult
the point of contact identified in Section 4.
Source: Ng, E.K.O., Cheng, P.K.C., Ng, A.Y.Y., Hoang, T.L., and Lim, W.W.L. 2005.
"Influenza A H5N1 Detection." Emerging Infectious Diseases. 11(8): 1303-1305.
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7.2.35 Literature Reference for Enteric Viruses (Applied and Environmental Microbiology.
2003. 69(6): 3158-3164)
Pathogen(s)
Picornaviruses: Enteroviruses
Picornaviruses: Hepatitis A virus (HAV)
Reoviruses: Rotaviruses (Group A)
Agent Category
Viruses
Viruses
Viruses
BSL
Not specified
2
Not specified
Analysis Purpose: Detection of Enteroviruses and HAV; Detection and viability of Rotoviruses
Sample Preparation: Samples should be prepared according to procedures described in the USEPA
Manual of Methods for Virology (EPA/600/4-84/013, April 2001).
Analytical Technique: Reverse transcription-PCR for detection and tissue culture for viability
Method Developed for: Enteroviruses, HAV, and Group A Rotaviruses in water
Method Selected for: SAM lists these procedures for detection of enterovirus and HAV in solid,
particulate, aerosol, liquid, and water samples, but the procedures are not suitable for determining
viability of these pathogens. These procedures should also be used for detection and viability assessment
of rotavirus (Group A) in solid, particulate, aerosol, liquid, and water samples, although rotavirus is not
likely to be viable in aerosol samples. Further research is needed to develop and standardize the
procedures for environmental sample types other than water. See Appendix C or Table 7-1 for additional
methods that should be used for this pathogen.
Description of Method: Procedures are described for analysis of water samples and may be adapted for
assessment of solid, particulate, aerosol, and liquid samples. The method is used to detect human enteric
viruses (enteroviruses, HAV, rotavirus) in ground water samples. It is a multiplex reverse-transcription
PCR procedure optimized for the simultaneous detection of enteroviruses, HAV, reoviruses, and
rotaviruses. Water samples are collected by filtration and viruses are eluted using a beef extract solution
(1.5%, pH 9.5). Viruses are concentrated using Celite® adsorption (pH 4.0), filtration, and Celite®-
elution with sodium phosphate (0.15 M, pH 9.0), followed by further concentration and processing to
remove inhibitors (ultracentrifugation, solvent extraction, and MW-exclusion filtration). Concentrated
samples are analyzed by a two-step multiplex reverse transcription-PCR using virus-specific primer sets.
Detection of amplicons is by gel electrophoresis with subsequent confirmation by hybridization (dot-blot)
using digoxigenin-labeled internal (nested) probes.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank. PCR QC checks should be performed according to EPA
Draft Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on
Environmental Samples document www.epa.gov/sam/pdfs/EPA-OA-QC PCR Oct2004.pdf or consult
the point of contact identified in Section 4.
Source: Fout, G.S., Martinson, B.C., Moyer, M.W.N., and Dahling, D.R. 2003. "A Multiplex Reverse
Transcription-PCR Method for Detection of Human Enteric Viruses in Groundwater." Applied and
Environmental Microbiology. 69(6): 3158-3164. htt|K//wvw^^^
3164.pdf
7.2.36 Literature Reference for Cryptosporidium spp. (Applied and Environmental
Microbiology. 1999. 65(9): 3936-3941)
Pathogen(s) [Disease]
Cryptosporidium spp. [Cryptosporidiosis]
Agent Category
Protozoa
BSL
2
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Analysis Purpose: Detection and viability
Sample Preparation: Samples should be prepared according to the procedures in Applied and
Environmental Microbiology. 1999. 65(9): 3939-3941.
Analytical Technique: Tissue culture
Method Developed for: Cryptosporidium in animal samples
Method Selected for: SAM lists these procedures for detection and viability assessment in solid,
particulate, liquid, and water samples. Further research is needed to develop and standardize the
procedures for environmental sample types. See Appendix C or Table 7-1 for additional methods that
should be used for this pathogen.
Description of Method: Procedures are described for analysis of animal samples and may be adapted for
assessment of solid, particulate, liquid, and water samples. A cell culture infectivity assay capable of
detecting infectious oocysts is used to quantify viable oocysts through sporozoite invasion and clustering
of foci. Oocysts diluted in a standard 5- or 10-fold multiple dilution format are inoculated onto human
ileocecal adenocarcinoma (HCT-8) cell monolayers. After incubation for 48 hours, anti-sporozoite
polyclonal antibody is used to detect sporozoite invasion, and microscopy is used to confirm replication
(life stages present). Levels of infection and clustering are used to determine the most probable number
(MPN) of infectious oocysts in the stock suspension. For oocysts less than 30 days of age, the correlation
between the initial oocyst inoculum and the MPN calculation is 0.9726. The relationship between the
oocyst inoculum and the MPN diverge as the oocysts age. The 50% infective dose (ID50) in the cell
culture system is approximately 10 oocysts.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank.
Source: Slifko, T.R., Huffman, D.E., and Rose, J.B. 1999. "A Most-Probable-Number Assay for
Enumeration of Infectious Cryptosporidiumparvum Oocysts." Applied and Environmental Microbiology.
65(9): 3936-3941.
7.2.37 Literature Reference for Cryptosporidium spp. (Applied and Environmental
Microbiology. 2007. 73(13): 4218-4225)
Pathogen(s)
Cryptosporidium spp. [Cryptosporidiosis]
Agent Category
Protozoa
BSL
2
Analysis Purpose: Detection, not suitable for viability
Sample Preparation: Solid samples should be prepared according to "Quantification of Bias Related to
the Extraction of DNA Directly from Soils," Frostegard, A., Courtois, S., Ramisse, V., Clerc, S.,
Bernillion, D., Le Gall, F., Jeannin, P., Nesme, X., and Simonet, P. 1999. Applied and Environmental
Microbiology. 65(12): 5409-5420 (www.epa.gov/sam/pdfs/AEM-65(12)-pgs5409-5420.pdf).
Analytical Technique: Real-time PCR
Method Developed for: Cryptosporidium spp. in drinking water samples
Method Selected for: SAM lists this protocol for detection and viability assessment in solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types other than drinking water. See Appendix C or Table 7-1 for additional
methods that should be used for this pathogen.
Description of Method: Procedures are described for analysis of drinking water samples and may be
adapted for assessment of solid, particulate, aerosol, and liquid samples. The method uses real-time PCR
for identification of Cryptosporidium spp. C. parvum oocysts are seeded into 100-L water samples at an
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average concentration of 590,000 ± 84,000 oocysts. One hundred liters of the seeded drinking water is
concentrated by hollow-fiber UF, with the filter eluted with a surfactant solution that is added to the
retentate. A portion of the combined retentate is further concentrated with a 0.2 \m\ filter and one quarter
of the filter used for PCR analysis. DNA for PCR analyses is recovered by bead beating, lysis of cells
with a guanidine thiocyanate based buffer, and recovery and concentration of nucleic acids using spin
columns. Real-time PCR is performed on an iCycler iQ4 detection system using primer sequences and a
TaqMan® probe specific for Cryptosporidium spp. Samples from 8 sites were examined, with an average
recovery efficiency of between 81% and 98%, with a cross-site average and standard deviation of 88%
and 10% respectively.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank.
Special Considerations: This method is only for genus-specific determination of Cryptosporidium and
will not determine if the microorganism is infectious to humans.
Source: Hill, V.R., Kahler, A.M., Jothikumar, N., Johnson, T.B., Hahn, D., and Cromeans, T.L. 2007.
"Multistate Evaluation of an Ultrafiltration-Based Procedure for Simultaneous Recovery of Enteric
Microbes in 100-Liter Tap Water Samples." Applied and Environmental Microbiology. 73(13): 4218-
4225. http://www.epa.gov/sam/pdfs/AEM-73(13)-pgs4218-4225.pdf
7.2.38 Literature Reference for Entamoeba histolytica (Journal of Parasitology. 1972.
58(2): 306-310)
Pathogen(s)
Entamoeba histolytica
Agent Category
Protozoa
BSL
2
Analysis Purpose: Detection and viability
Sample Preparation: Samples should be prepared according to the procedures in Journal of
Parasitology. 1972. 58(2): 306-310.
Analytical Technique: Culture
Method Developed for: Entamoeba histolytica in clinical samples
Method Selected for: SAM lists these procedures for detection and viability assessment in solid,
particulate, liquid, and water samples. Further research is needed to develop and standardize the
procedures for environmental sample types. See Appendix C or Table 7-1 for additional methods that
should be used for this pathogen.
Description of Method: Procedures are described for analysis of clinical samples and may be adapted
for assessment of solid, particulate, liquid, and water samples. Entamoeba histolytica cysts are placed in
a modified TP-S-1 medium and incubated for 10 hours. Live amoebae excyst through a rupture in the
cyst wall, whereas non-viable amoebae remain encysted. Microscopic examination of an aliquot of the
incubated excystation culture allows calculation of the percent of empty (live) cysts and full (dead) cysts
in a population.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank.
Source: Stringert, R.P. 1972. "New Bioassay System for Evaluating Percent Survival of Entamoeba
histolytica Cysts." The Journal of Parasitology. 58(2): 306-310. http://www.epa.gov/sam/pdfs/JP-58(2)-
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7.2.39 Literature Reference for Entamoeba histolytica (Journal of Clinical Microbiology.
2005. 43(11): 5491-5497)
Pathogen(s)
Entamoeba histolytica
Agent Category
Protozoa
BSL
2
Analysis Purpose: Detection, not suitable for viability
Sample Preparation: Samples should be prepared according to the procedures in Journal of Clinical
Microbiology. 2005. 43(11): 5491-5497.
Analytical Technique: Real-time PCR
Method Developed for: Entamoeba histolytica in clinical (fecal and liver abscess) samples
Method Selected for: SAM lists these procedures for detection in solid, particulate, liquid, and water
samples. Further research is needed to develop and standardize the procedures for environmental sample
types. See Appendix C or Table 7-1 for additional methods that should be used for this pathogen.
Description of Method: Procedures are described for analysis of clinical samples and may be adapted
for assessment of solid, particulate, liquid, and water samples. The method is a real-time PCR assay that
targets the 18S rRNA gene sequence of E. histolytica. DNA is extracted using cell disruption and a
commercial DNA extraction kit, with a second commercial kit used to remove potential PCR inhibitors.
TaqMan® real-time PCR is conducted on a GeneAmp® 9700 Thermal Cycler the purified product. The
LOD is 1 cell per mL of sample (SD ± 4). The method differentiates between E. histolytica and E. dispar.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank. PCR QC checks should be performed according to EPA
Draft Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on
Environmental Samples document www.epa.gov/sam/pdfs/EPA-QA-QC PCR Oct2004.pdf or consult
the point of contact identified in Section 4.
Source: Qvarnstrom, Y., James, C., Xayavong, M., Holloway, B.P., Visvesvara, G.S., Sriram, R., and da
Silva, AJ. 2005. "Comparison of Real-time PCR Protocols for Differential Laboratory Diagnosis of
Amebiasis." Journal of Clinical Microbiology. 43(11): 5491-5497. http://www.epa.gov/sam/pdfs/JCM-
7.2.40 Literature Reference for Giardia spp. (Transactions of the Royal Society of
Tropical Medicine and Hygiene. 1983. 77(4): 487-488)
Pathogen(s) [Disease]
Giardia spp. [Giardiasis]
Agent Category
Protozoa
BSL
2
Analysis Purpose: Detection and viability
Analytical Technique: Culture
Method Developed for: Giardia in cell culture samples
Method Selected for: SAM lists these procedures for detection and viability assessment in solid,
particulate, liquid, and water samples. Further research is needed to develop and standardize the
procedures for environmental sample types. See Appendix C or Table 7-1 for additional methods that
should be used for this pathogen.
Description of Method: Procedures are described for analysis of cell culture samples and may be
adapted for assessment of solid, particulate, liquid, and water samples. TYI-S-33 medium supplemented
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with bovine bile and additional cysteine is used to isolate and culture Giardia lamblia. G. lamblia is
incubated for intervals of 72 and 96 hours at 36°C in borosilicate glass tubes. The cells form a dense,
adherent monolayer on the surface of the glass.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank.
Source: Keister, D. 1983. "Axenic Culture of Giardia lamblia in TYI-S-33 Medium Supplemented with
Bile." Transactions of the Royal Society of Tropical Medicine and Hygiene. 77(4): 487-488.
http://wmw.sciencedirect.coin/science? ob=ArticleURL& udi=B75GP-4CODS6C-
user=10& coverDate=12%2F31%2F1983& alid=785177083& rdoc=l& orig=searc
h& cdi=13100& sort=d& docanchor=&view=c& ct=l& acct=C000050221& version=l& urlVersion
=0&
7.2.41 Literature Reference for Toxoplasma gondii (Emerging Infectious Diseases. 2006.
12(2): 326-329)
Pathogen(s) [Disease]
Toxoplasma gondii [Toxoplasmosis]
Agent Category
Protozoa
BSL
2
Analysis Purpose: Detection and viability
Sample Preparation: Samples should be prepared according to the procedures in Emerging Infectious
Diseases 12(2): 326-329.
Analytical Technique: Animal infectivity
Method Developed for: Toxoplasma gondii in water
Method Selected for: SAM lists these procedures for detection and viability assessment in solid,
particulate, liquid, and water samples. Further research is needed to develop and standardize the
procedures for environmental sample types other than water. See Appendix C or Table 7-1 for additional
methods that should be used for this pathogen.
Description of Method: Procedures are described for analysis of water samples and may be adapted for
assessment of solid, particulate, liquid, and water samples. Water samples are filtered through fluoropore
membrane filters and concentrated by centrifugation. The filters can be assayed by any of three methods.
The first method involves performing a bioassay in T. go«t///-seronegative chickens. Serum samples are
tested by enzyme-linked immunosorbent assay (ELISA) and/or modified agglutination test until
seroconversion, with the organs from seropositive animals examined microscopically for T. gondii. Mice
are injected with brain and heart tissue of seropositive chickens, with parasites found in the lungs of mice
being confirmatory for T. gondii. The second method is a similar bioassay with pigs and cats. For the
third assay, DNA is extracted from the fluoropore membranes for PCR identification of isolates.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank.
Source: de Moura, L., Bahia-Oliveira, L.M.G., Wada, M.Y., Jones, J.L., Tuboi, S.H., Carmo, E.H.,
Ramalho, W.M., Camargo, N.J., Trevisan, R., Graca, R.M.T., da Silva, A.J., Moura, L, Dubey, J.P., and
Garrett, D.O. 2006. "Waterborne Toxoplasmosis, Brazil, from Field to Gene." Emerging Infectious
Diseases. 12(2): 326-329. http://www.epa.gov/sam/pdfs/EID-12 (2) -pgs326-329.pdf
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7.2.42 Literature Reference for Toxoplasma gondii (Applied and Environmental
Microbiology. 2004. 70(7): 4035-4039)
Pathogen(s) [Disease]
Toxoplasma gondii [Toxoplasmosis]
Agent Category
Protozoa
BSL
2
Analysis Purpose: Detection, not suitable for viability
Sample Preparation: Samples should be prepared according to the procedures in Applied and
Environmental Microbiology 70(7): 4035-4039.
Analytical Technique: Real-time PCR
Method Developed for: Toxoplasma gondii in water
Method Selected for: SAM lists these procedures for detection of in solid, particulate, liquid, and water
samples. Further research is needed to develop and standardize the procedures for environmental sample
types other than water. See Appendix C or Table 7-1 for additional methods that should be used for this
pathogen.
Description of Method: Procedures are described for analysis of water samples and may be adapted for
assessment of solid, particulate, and liquid samples. The method uses a fluorogenic 5' nuclease
(TaqMan®) real-time PCR assay for the detection of T. gondii oocyst DNA using gene-specific (Bl gene)
primers and probe. The assay uses an iCycler Real-Time PCR Detection System. Water samples (10 to
100 L) are filtered to concentrate oocysts. Filters are eluted and recovered oocysts are further purified and
concentrated by differential flotation and centrifugation. Final sample pellets are split and subjected to
PCR detection and mouse bioassay. In experimental seeding assays, a parasite density of 10 oocysts/L is
detectable in 100% of the cases, and a density of 1 oocyst/L is observable in 60% of the cases.
At a minimum, the following QC checks should be performed and evaluated before using this protocol:
positive control, negative control, and blank. PCR QC checks should be performed according to EPA
Draft Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on
Environmental Samples document www.epa.gov/sam/pdfs/EPA-QA-QC PCR Oct2004.pdf or consult
the point of contact identified in Section 4.
Source: Villena, L, Aubert, D., Gomis, P., Ferte, H., Inglard, J-C., Denise-Bisiaux, H., Dondon, J-M.,
Pisano, E., Ortis, N., and Pinon, J.M. 2004. "Evaluation of a Strategy for Toxoplasma gondii Oocyst
Detection in Water." Applied and Environmental Microbiology. 70(7): 4035-4039.
http://wmw.epa.gov/sain/pdfs/ AEM-70(7)-pgs4035-4039.pdf
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Section 8.0: Selected Biotoxin Methods
A list of methods or procedures to be used in analyzing environmental samples for biotoxin contaminants
is provided in Appendix D. These methods should be used to support remediation activities (site
assessment through clearance) following a homeland security event. Procedures have been compiled for
each biotoxin that may need to be identified and/or quantified following a contamination incident.
Analytical procedures are not currently available for all the analyte-sample type combinations included in
this document. Future research needs include identification of additional methods and development and
validation of the procedures listed. Appendix D is sorted alphabetically by analyte, within each of two
analyte types (i.e., protein and small molecule).
Please note: This section provides guidance for selecting biotoxin methods that have a high likelihood
of assuring analytical consistency when laboratories are faced with a large-scale environmental
restoration crisis. Not all methods have been verified for the analyte/sample type combination listed in
Appendix D. Please refer to the specified method to identify analyte/sample type combinations that
have been verified. Any questions regarding information discussed in this section should be addressed
to the appropriate contact (s) listed in Section 4.
Appendix D provides the following information:
• Analyte(s). The compound or compound (s) of interest.
• CAS RN / Description. A unique identifier for substances that provides an unambiguous way to
identify a toxin or toxin isoform when there are many possible systematic, generic, or trivial names
and/or a brief statement describing the toxin.
• Analysis type. Tests are either for presumptive identification, confirmatory identification, or
biological activity determination.
• Analytical Technique. An analytical instrument or technique used to determine the quantity and
identification of compounds or components in a sample.
• Analytical Method. The recommended method or procedure, and the corresponding publisher.
• Aerosol (filter/cassette or liquid impinger). The recommended method/procedure to measure the
analyte of interest in air sample collection media such as filter cassettes and liquid impingers.
• Solid (soil, powder). The recommended method/procedure to measure the analyte of interest in solid
samples such as soil and powders.
• Particulate (swabs, wipes, filters). The recommended method/procedure to measure the analyte of
interest in particulate sample collection media such as swabs, wipes and HEPA filters.
• Liquid/drinking water. The recommended method/procedure to measure the analyte of interest in
liquid and drinking water samples.
Some of the biotoxins addressed in this document are commonly found in the environment, and the
methods listed in Appendix D assume that analysis will be used to evaluate contamination levels that are
above those of background. If possible, an investigation of initial background levels, as well as controls
for background levels, should be performed.
Procedures listed in Appendix D for protein biotoxins are intended to address presumptive, confirmatory,
and biological activity determinations. Because the confirmatory procedures listed for the small molecule
biotoxins involve a determination of intact compound structure (an indication of biological activity
capability), only presumptive and confirmatory methods are listed for these biotoxins. In terms of this
document, presumptive methods, or methods that support a reasonable basis for accurate results, should
be used in situations that require a large number of samples to be processed. Most of the presumptive
methods listed in Appendix D use immunoassay techniques and are designed for large scale sample
processing. The confirmatory method, or the method that corroborates the presumptive results, should be
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used on the smaller subset of samples for which presumptive analysis indicates the presence of the
biotoxin. Several techniques are listed in Appendix D as confirmatory and generally are more time
consuming and expensive. The use of these terms in this document is not intended to redefine LRN usage
of these terms. The terms presumptive and confirmatory as used by the LRN are described in Section
8.2. 1. If it is necessary to determine the biological activity of a toxin, either an assay (for proteins) or a
technique such as HPLC that determines whether the structure of the biotoxin is intact and likely to be
biologically active (for small molecules) may be used. Biological activity analysis should be applied on
an as-needed basis following analysis with the confirmatory technique.
Numerous analytical techniques using a variety of instrumentation (e.g., HPLC-MS, HPLC-FL,
immunoassay [ELISA], immunoassay [lateral flow device (LFD)], etc.) have been cited in Appendix D.
It is expected that a reduced number of these analytical techniques and instrumentation will be necessary
after method verification and validation. In addition, it is recognized that new reports detailing advances
in biotoxin analysis appear in the literature frequently. Accordingly, the individual techniques and
methods listed in Appendix D are to be regarded as a starting point; after thoughtful consideration of
current technologies at the time of remediation and consultation with the authority in charge of the
remediation activity, these techniques and methods can be modified as necessary for analysis of a
particular sample.
EPA's NHSRC is working on a sample collection document that is intended as a companion to SAM.
This sample collection document will provide information regarding sampling container/media,
preservation, holding time, sample size, and shipping and is intended to complement the laboratory
analytical methods that are the focus of the SAM document.
8.1 General Guidance
This section provides a general overview of how to identify the appropriate method (s) for a given
biotoxin as well as recommendations for QC procedures.
For additional information on the properties of the biotoxins listed in Appendix D, TOXNET
) , a cluster of databases on toxicology, hazardous chemicals, and
related areas maintained by the National Library of Medicine, is an excellent resource.
Additional resources include:
• A U.S. Army Medical Research Institute of Infectious Diseases' document at
httpj//w^wjJsaMdM information regarding
sample collection, toxin analysis and identification, as well as decontamination and water treatment.
• CDC regulations - Select Agents and Toxins, 42 CFR part 73, found at
The CDC has additional information regarding select agent toxins at the following Web site:
http://www.cdc.gov/od/sap/sap/toxinamt.htm
Syracuse Research Corporation's Physprop and Chemfate, part of the Environmental Fate Database
supported by EPA. See http://www.syrres.com/esc/databases.htm.
INCHEM at http : //www. inchem . org/ contains both chemical and toxicity information.
The RTECS database can be accessed via the NIOSH Web site at
http://www.cdc.gov/niosh/rtecs/vz72d288.htmlfJWIDAW for toxicity information.
EPA's IRIS: http : //www. epa . gov/iris/ contains toxicity information.
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• The Forensic Science and Communications Journal published by the Laboratory Division of the FBI.
See http ://www. fbi.gov/hq/lab/fsc/current/backissu.htm.
Additional research on biotoxin contaminants is ongoing within EPA.
8.1.1 Standard Operating Procedures for Identifying Biotoxin Methods
To determine the appropriate method that is to be used on an environmental sample, locate the biotoxin of
concern in Appendix D: Biotoxin Methods under the "Analyte(s)" column. After locating the biotoxin,
continue across the table and identify the appropriate analysis type. After an analysis type has been
chosen, find the analytical technique (e.g., immunoassay) and analytical method applicable to the sample
type of interest (solid, particulate, liquid/drinking water, or aerosol) corresponding to that particular
analyte.
Sections 8.2.1 through 8.2.31 below provide summaries of the analytical methods listed in Appendix D.
Once a method has been identified in Appendix D, Table 8-1 can be used to locate the method summary.
Table 8-1. Biotoxin Methods and Corresponding Text Section Numbers
Biotoxin
Method
Section
Proteins
Abrin
Botulinum neurotoxins
Ricin
Shiga and Shiga-like toxins
(Stx, Stx-1 , Stx-2)
Staphylococcal enterotoxins
(SEB)
Staphylococcal enterotoxins
(SEA, SEC)
1 1 9th AOAC Annual Meeting & Exposition, 2005, p. 61 3
Pharmacology & Toxicology. 2001. 88(5): 255-260
Analytical Biochemistry. 2008. 378: 87-89
LRN
U.S. FDA, Bacteriological Analytical Manual Online, January
2001 , Chapter 1 7, Clostridium botulinum
Lateral Flow Immunoassay Kits
LRN
Analytical Biochemistry. 2008. 378: 87-89
Journal of AOAC International. 2008. 91(2): 376-382
Journal of Analytical Toxicology. 2005. 29: 149-155
Lateral Flow Immunoassay Kits
U.S. FDA, Bacteriological Analytical Manual Online, January
2001, Appendix 1, Rapid Methods for Detecting Foodborne
Pathogens
Pharmacology & Toxicology. 2001 . 88(5): 255-260
Journal of Clinical Microbiology. 2007. 45(10): 3377-3380
LRN
AOAC Official Method 993.06
AOAC Official Method 993.06
8.2.7
8.2.8
8.2.9
8.2.1
8.2.2
8.2.31
8.2.1
8.2.9
8.2.23
8.2.24
8.2.31
8.2.3
8.2.8
8.2.26
8.2.1
8.2.5
8.2.5
Small Molecules
Aflatoxin (Type B1)
a-Amanitin
Anatoxin-a
AOAC Official Method 991.31
AOAC Official Method 994.08
Journal of Food Protection. 2005. 68(6): 1294-1301
Journal of Chromatography B. 1991. 563(2): 299-311
Biomedical Chromatography. 1996. 10: 46-47
8.2.4
8.2.6
8.2.12
8.2.13
8.2.14
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Biotoxin
Brevetoxins (B form)
a-Conotoxin
Cylindrospermopsin
Diacetoxyscirpenol (DAS)
Microcystins (Principal
isoforms: LA, LR, YR, RR,
LW)
Picrotoxin
Saxitoxins (Principal
isoforms SIX, NEOSTX,
GTX, dcGTX, dcSTX)
T-2 Mycotoxin
Tetrodotoxin
Method
Environmental Health Perspectives. 2002. 110(2): 179-185
Toxicon. 2004. 43(4): 455-465
Biochemical Journal. 1997. 328: 245-250
Journal of Medicinal Chemistry. 2004. 47(5): 1 234-1 241
FEMS Microbiology Letters. 2002. 216: 159-164
ELISA Kits for Cylindrospermopsin
International Journal of Food Microbiology. 1988. 6(1): 9-17
Rapid Communications in Mass Spectrometry. 2006. 20(9):
1422-1428
Journal of AOAC International. 2001. 84(4): 1035-1044
Analyst. 1994. 119(7): 1525-1530
Journal of Pharmaceutical and Biomedical Analysis. 1989.
7(3): 369-375
Journal of AOAC International. 1995. 78: 528-532
ELISA Kits for Saxitoxin
Journal of Food Protection. 2005. 68(6): 1294-1301
Rapid Communications in Mass Spectrometry. 2006. 20(9):
1422-1428
Journal of Clinical Laboratory Analysis. 1992. 6: 65-72
Analytical Biochemistry. 2001. 290: 10-17
Section
8.2.15
8.2.16
8.2.10
8.2.11
8.2.17
8.2.29
8.2.18
8.2.19
8.2.20
8.2.21
8.2.22
8.2.25
8.2.30
8.2.12
8.2.19
8.2.27
8.2.28
Method summaries are listed in order of method selection hierarchy (see Figure 2-1), starting with EPA
methods, followed by methods from other federal agencies, VCSBs, and journal articles. Methods are
listed in numerical order under each publisher. Where available, a direct link to the full text of the
method is provided with the method summary. For additional information on sample preparation
procedures and methods available through consensus standards organizations, please use the contact
information provided in Table 8-2.
Table 8-2. Sources of Biotoxin Methods
Name
U.S. FDA, Bacteriological Analytical
Manual Online
Official Methods of Analysis of AOAC
International*
NEMI
Pharmacology & Toxicology*
Analytical Biochemistry*
Biochemical Journal*
Journal of Medicinal Chemistry*
Journal of Food Protection*
Journal of Chromatography B*
Biomedical Chromatography*
Publisher
U.S. FDA
AOAC International
U.S. EPA, USGS
Blackwell Synergy
Science Direct
Portland Press Ltd.
American Chemical Society
International Association for
Food Protection
Elsevier Science Publishers
John Wiley And Sons Ltd
Reference
http://www.cfsan.fda.qov/~ebam/bam-
toc.html
http://www.aoac.orq
http://www.nemi.qov/
http://www.blackwell-SYnerqv.com/loi/Dto
http://www.sciencedirect.com/
http://www.biochemj.orq/
http://www.acs.orq/
http://www.foodprotection.orq/
http://www.elsevier.com/
http://www.wilev.com/
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Name
Environmental Health Perspectives*
Toxicon*
Federation of European Microbiological
Societies (FEMS) Microbiology Letters*
International Journal of Food
Microbiology*
Rapid Communications in Mass
Spectrometry *
Journal of AOAC International*
Analyst*
Journal of Pharmaceutical and
Biomedical Analysis*
Journal of Clinical Microbiology
Journal of Clinical Laboratory Analysis*
Journal of Analytical Toxicology*
Lateral Flow Immunoassay Kits
Publisher
National Institute of
Environmental Health
Sciences
Elsevier Science Publishers
Blackwell Publishing
Elsevier Science Publishers
John Wiley And Sons Ltd.
AOAC International
Royal Society of Chemistry
Elsevier Science Publishers
ASM
John Wiley And Sons Ltd.
S. Tinsley Preston
Environmental Technology
Verification (ETV) Program
Reference
http://www.niehs.nih.qov/
http://www.elsevier.com/
http://www.blackwellpublishinq.com/
http://www.elsevier.com/
http://www.wiley.com/
http://www.aoac.org
http://www.rsc.orq/
http://www.elsevier.com/
http://www.asm.orq/
http://www.wiley.com/
http://www.jatox.com/
http://www.epa.qov/etv/
Subscription and/or purchase required.
8.1.2 General QC Guidance for Biotoxin Methods
Having data of known and documented quality is critical so that public officials can accurately assess the
activities that may be needed in remediating a site during and following emergency situations. Having
such data requires that laboratories: (1) conduct the necessary QC to ensure that measurement systems are
in control and operating properly; (2) properly document results of the analyses; and (3) properly
document measurement system evaluation of the analysis-specific QC. Ensuring data quality also
requires that laboratory results are properly evaluated and the results of the data quality evaluation are
transmitted to decision makers.
The level or amount of QC needed often depends on the intended purpose of the data that are generated.
Various levels of QC may be required if the data are generated during presence/absence determinations
versus confirmatory analyses. The specific needs for data generation should be identified. QC
requirements and data quality objectives should be derived based on those needs and should be applied
consistently across laboratories when multiple laboratories are used. For example, during rapid sample
screening, minimal QC samples (e.g., blanks, duplicates) and documentation might be required to ensure
data quality. Sample analyses for environmental evaluation during site assessment through site clearance,
such as those identified in this document, might require increased QC (e.g., demonstrations of method
sensitivity, precision, and accuracy).
While method-specific QC requirements may be included in many of the procedures that are cited in this
document, and will be referenced in any SAPs developed to address specific analytes and sample types of
concern, the following describes a minimum set of QC samples and procedures that should be conducted
for all analyses. Individual methods, sampling and analysis protocols, or contractual statements of work
also should be consulted to determine any additional QC that may be needed. QC tests should be run as
frequently as necessary to ensure the reliability of analytical results. In general, sufficient QC includes an
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initial demonstration of measurement system capability as well as ongoing assessments to ensure the
continued reliability of the analytical results.
Examples of sufficient QC for the presumptive tests listed in Appendix D include:
• Method blanks;
• Positive test samples / negative test samples;
• Calibration check samples;
• Use of test kits and reagents prior to expiration; and
• Accurate temperature controls.
Examples of sufficient QC for the confirmatory tests listed in Appendix D include:
• Demonstration that the measurement system is operating properly
> Initial calibration
> Method blanks
• Demonstration of measurement system suitability for intended use
> Precision and recovery (verify measurement system has adequate accuracy)
> Analyte/sample type/level of concern-specific QC samples (verify that measurement system has
adequate sensitivity at levels of concern)
• Demonstration of continued measurement system reliability
*• MS/MSDs (recovery and precision)
*• QC samples (system accuracy and sensitivity at levels of concern)
*• Continuing calibration verification
*• Method blanks
Please note: The appropriate point of contact identified in Section 4 should be consulted regarding
appropriate QA/QC procedures prior to sample analysis. These contacts will consult with the EPA
OSWER coordinator responsible for laboratory activities during the specific event to ensure QA/QC
procedures are performed consistently across laboratories. OSWER is planning to develop QA/QC
guidance for laboratory support. EPA program offices will be responsible for ensuring that the QA/QC
practices are implemented.
8.1.3 Safety and Waste Management
It is imperative that safety precautions be used during collection, processing, and analysis of
environmental samples. Laboratories should have a documented health and safety plan for handling
samples that may contain target CBR contaminants, and laboratory staff should be trained in and
implement the safety procedures included in the plan. In addition, many of the methods summarized or
cited in Section 8.2 contain some specific requirements, guidance, or information regarding safety
precautions that should be followed when handling or processing environmental samples and reagents.
These methods also provide information regarding waste management. Other resources that can be
consulted for additional information include the following:
. American Biological Safety Association, Risk Group Classifications for Infectious Agents
(httJl^Mw^Llbsaj^
. Biological Safety: Principles and Practices, 4th Ed. ASM.Press (http://estore.asm.org/);
. CDC - 42 CFR part 72. Interstate Shipment of Etiological Agents.
. CDC - 42 CFR part 73. Select Agents and Toxins.
. DOT - 49 CFR part 172. Hazardous Materials Table, Special Provisions, Hazardous Materials
Communications, Emergency Response Information, and Training Requirements.
. EPA - 40 CFR part 260. Hazardous Waste Management System: General.
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• EPA - 40 CFR part 270. EPA Administered Permit Programs: The Hazardous Waste Permit
Program.
. OSHA - 29 CFR part 1910.1450. Occupational Exposure to Hazardous Chemicals in Laboratories.
. OSHA - 29 CFR part 1910.120. Hazardous Waste Operations and Emergency Response.
. USDA - 9 CFR part 121. Possession, Use, and Transfer of Select Agents and Toxins.
Please note that the e-CFR is available at http://ecfr.gpoaccess.gov/.
8.2 Method Summaries
Summaries for the analytical methods listed in Appendix D are provided in Sections 8.2.1 through 8.2.31.
These sections contain summary information only, extracted from the selected methods. The full version
of the method should be consulted prior to sample analysis.
Each summary contains a table identifying the biotoxin(s) and sample type to which the method applies, a
brief description of the method, performance data (if available), and a link to or source for obtaining a full
version of the method.
8.2.1 Laboratory Response Network (LRN)
The agents and sample types identified below and listed in Appendix D are included in the HHS/USDA
select agent list and should be analyzed in accordance with the appropriate LRN protocols.
The LRN was created in accordance with Presidential Directive 39, which established terrorism
preparedness responsibilities for federal agencies. The LRN is primarily a national network of local,
state, federal, military, food, agricultural, veterinary, and environmental laboratories; however, additional
LRN laboratories are located in strategic international locations. The CDC provides technical and
scientific support to member laboratories as well as secure access to standardized procedures and reagents
for rapid (within 4 to 6 hours) presumptive detection of biothreat agents and emerging infectious disease
agents. These rapid presumptive assays are part of agent-specific algorithms of assays which lead to a
confirmed result. The algorithm for a confirmed result is often a combination of one or more presumptive
positive results from a rapid assay in combination with a positive result from one of the "gold standard"
methods, such as culture. The standardized procedures, reagents, and agent-specific algorithms are
considered to be sensitive and are available only to LRN member laboratories. Thus, these procedures are
not available to the general public and are not discussed in this document.
It is important to note that, in some cases, the procedures may not be fully developed or validated for each
environmental sample type/analyte combination listed in Appendix D, nor are all LRN member
laboratories necessarily capable of analyzing all of the sample type/analyte combinations.
Analyte(s)
Sample Type
CAS RN / Description
Botulinum neurotoxins (Serotypes A, B, E, F)
Solid, Paniculate, Liquid/Water
Protein composed of -100 kDa
heavy chain and -50 kDa light
chain; can be complexed with
hemagglutinin and non-
hemagglutinin components for total
MW of -900 kDa
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Analyte(s)
Ricin
Staphylococcal enterotoxin B (SEB)
Sample Type
Solid, Particulate, Liquid/Water
Solid, Particulate, Liquid/Water
CAS RN / Description
9009-86-3 (ricin) / 60 kDa
glycoprotein composed of two
subunits (-32 kDa A chain and -34
kDa B chain); an agglutinin of MW
1 20 kDa may be present in crude
preparations
39424-53-8 (SEB) / Monomeric
protein of almost 28 kDa
Please note: Not all methods have been verified for the biotoxin/sample type combination listed in
Appendix D. Please refer to the agent-specific method to identify the biotoxin/sample type combinations
that have been validated. Any questions regarding information discussed in this section should be
referred to the appropriate contact (s) listed in Section 4.
For additional information on the LRN, including selection of a laboratory capable of receiving and
processing the specified sample type/analyte, please use the contact information provided below or visit
Centers for Disease Control and Prevention
Laboratory Response Branch
Division of Bioterrorism Preparedness and Response (DBPR)
National Center for Prevention, Detection, and Control of Infectious Diseases (NCPDCID)
Coordinating Center for Infectious Diseases (CCID)
Centers for Disease Control and Prevention (CDC)
1600 Clifton Road NE, Mailstop C-18
Atlanta, GA 30333
Telephone: (404) 639-2790
E-mail: lrn@cdc.gov
Local public health laboratories, private, and commercial laboratories with questions about the LRN
should contact their state public health laboratory director or the APHL (contact information provided
below).
Association of Public Health Laboratories
8515 Georgia Avenue, Suite 700
Silver Spring, MD 209 10
Telephone: (240) 485-2745
Fax: (240) 485-2700
Web site: wmny.aphl.org
E-mail: info@aphl.org
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8.2.2 U.S. FDA, Bacteriological Analytical Manual Online, Chapter 17, 2001: Botulinum
Neurotoxins
Analyte(s)
Botulinum neurotoxins
(Serotypes A, B, E, F)
Agent Category
Protein
Description
Protein composed of -100 kDa heavy
chain and -50 kDa light chain; can be
complexed with hemagglutinin and non-
hemagglutinin components for total MW of
-900 kDa
Analysis Purpose: Confirmatory and biological activity
Analytical Technique: Immunoassay (ELISA) and mouse bioassay
Method Developed for: Botulinum neurotoxins (Serotypes A, B, E, F) in food
Method Selected for: SAM lists this manual for confirmation and biological activity assessment in
aerosol samples. Further research is needed to develop and standardize the procedures for environmental
sample types. See Appendix D or Table 8-1 for additional methods that should be used for this analyte.
Description of Method: An amplified-enzyme-linked immunosorbent assay (amp-ELISA) and a
digoxigenin-labeled enzyme-linked immunosorbent assay (DIG-ELISA) are described for the detection of
Types A, B, E, and F botulinum neurotoxins in food products. The amp-ELISA method uses goat anti-A
or E, rabbit anti-B, or horse anti-F serum to capture the toxins in a 96-well plate, and a corresponding
biotinylated goat antitoxin to detect the toxin. Visualization is with streptavidin-alkaline phosphatase.
The DIG-ELISA method is a modification of the amp-ELISA method, with digoxigenin-labeled antitoxin
IgG's substituted for the streptavidin-alkaline phosphatase. Toxin can be detected at approximately 10
minimum lethal doses (MLD)/mL (0.12 to 0.25 ng/mL). High concentration samples (greater than 10,000
MLD/mL) may give a positive absorbance for more than one toxin type. Further dilution of the sample
will remove cross-reactivity.
The mouse bioassay detects biologically active toxin using a three part approach: toxin screening; toxin
titer; and finally, toxin neutralization using monovalent antitoxin sera. Samples are prepared by
centrifugation for suspended solids under refrigeration, or solids are extracted with an equal volume of pH
6.2 gel-phosphate buffer and then centrifuged. Toxins from nonproteolytic strains of C. botulinum may
need trypsin activation to be detected. Serial dilutions of untreated and trypsin-treated sample fluids are
injected in separate pairs of mice intraperitoneally (i.p.). Mice are also injected with heated, untreated,
undiluted sample. Death of mice, along with symptoms of botulism, confirms presence of botulinum
toxin. After calculation of an MLD, dilute monovalent antitoxin sera types A, B, E, and F are injected
into mice 30 minutes to 1 hour before challenging them with the i.p. injection of each dilution that gave
the highest MLD from the toxic preparation.
Special Considerations: Immunoassays with botulinum toxins may produce variable results with
uncomplexed form of toxin.
Source: U.S. FDA, CFSAN. 2001. "Chapter 17 - Clostridium botulinum." Bacteriological Analytical
Manual Online. http.//www.opa.gov/sam/pdfs/FDA-BAM-Chapl7.pdf
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8.2.3 U.S. FDA, Bacteriological Analytical Manual Online, Appendix 1, 2001: Rapid
Methods for Detecting Foodborne Pathogens
Analyte(s)
Shiga and Shiga-like toxins
(Stx, Stx-1, Stx-2)
Agent Category
Protein
CAS RN / Description
75757-64-1 (Stx) / Protein
composed of one -32 kDa A chain
and five 7.7 kDa B chains
Analysis Purpose: Confirmatory
Analytical Technique: Immunoassay (ELISA)
Method Developed for: Shiga and shiga-like toxins in food
Method Selected for: SAM lists this manual for presumptive analysis in aerosol, solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental samples. See Appendix D or Table 8-1 for additional methods that should be used for this
analyte.
Description of Method: Shiga toxin (Stx) is produced by Shigella dysenteriae and Shiga-like toxins
(Shiga toxin Types 1 [Stx-1] and 2 [Stx-2]) are produced by various Shiga-toxigenic E. coli (STEC). An
ELISA is described for the detection of these toxins. Diluted samples are added to microwells coated
with an anti-Shiga toxin capture antibody. After incubation at room temperature, a wash is performed to
remove unbound material. A second anti-Shiga toxin antibody is added for detection and incubation
continued at room temperature. A wash is performed to remove unbound antibody. Enzyme conjugated
anti-IgG visualization antibody, directed against the species from which the second anti-Shiga toxin
antibody was derived, is added and the plate incubated then rinsed. Substrate is added, and after
incubation to develop the color, stop solution is added. The results are interpreted
spectrophotometrically.
Source: U.S. FDA, CFSAN. 2001. "Rapid Methods for Detecting Foodborne Pathogens."
Bacteriological Analytical Manual Online. http://www.epa.gov/sam/pdfs/FDA-BAM-Appendixl.pdf
8.2.4 AOAC Official Method 991.31: Aflatoxins in Corn, Raw Peanuts, and Peanut Butter
Analyte(s)
Aflatoxin (Type B1)
Agent Category
Small Molecule
CASRN
27261-02-5
Analysis Purpose: Presumptive
Analytical Technique: Immunoassay
Method Developed for: Aflatoxins (Type Bl) in corn, raw peanuts, and peanut butter
Method Selected for: SAM lists this method for presumptive analysis in aerosol, solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types. See Appendix D or Table 8-1 for additional methods that should be used for
this analyte.
Description of Method: This method is for the detection of aflatoxins in agricultural products. The
sample is extracted with methanol-water (7 + 3), filtered, diluted with water, and applied to an affinity
column containing mAb specific for aflatoxins Bl, B2 (CAS RN 22040-96-6), Gl (CAS RN 1385-95-1),
and G2 (CAS RN 7241-98-7). Antibody-bound aflatoxins are removed from the column with methanol.
For detection and quantitation of total aflatoxins, fluorescence measurement after reaction with bromine
solution is performed. For individual aflatoxins, fluorescence detection and postcolumn iodine
derivatization are performed and quantitation is by LC. Method performance was characterized using
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various commodities (e.g., corn) at aflatoxin levels over a range of 10 to 30 ng/g. This method was
originally designed for the analysis of aflatoxins (Bi, 62, GI, and 62) in samples where cleanup was
necessary to remove food components, such as fats and proteins; the cleanup procedure may not be
necessary for analysis of water samples.
Source: AOAC International. 1994. "Method 991.31: Aflatoxins in Corn, Raw Peanuts, and Peanut
Butter." Official Methods of Analysis of AOAC International. 16th Edition, 4th Revision; Vol. II.
http;//\TOW.aoa_c.org/
8.2.5 AOAC Official Method 993.06: Staphylococcal Enterotoxins in Selected Foods
Analyte(s)
Staphylococcal enterotoxins
(SEE, SEA, SEC)
Agent Category
Protein
CAS RN / Description
39424-53-8 (SEE) / Monomeric
protein of ~ 28 kDa; 37337-57-8
(SEA) & 39424-54-9 (SEC) /
Monomeric proteins of ~ 27-27.5
kDa
Analysis Purpose: Presumptive
Analytical Technique: Immunoassay
Method Developed for: Staphylococcal enterotoxins in selected foods
Method Selected for: SAM lists this method for presumptive analysis of Staphylococcal enterotoxins
Type B in aerosol samples, and Types A and C in aerosol, solid, particulate, liquid, and water samples.
Further research is needed to develop and standardize the procedures for environmental sample types.
See Appendix D or Table 8-1 for additional methods that should be used for this analyte.
Description of Method: This method is an enzyme immunoassay (EIA) using a mixture of high-affinity
capture antibodies for identification of toxin(s) in food samples. Samples are prepared by dilution in Tris
buffer, centrifugation, and filtration of the supernatant through a syringe, with adjustment to a final pH of
7.0 to 8.0. Samples are incubated in 96-well plates with the mixture of antibodies conjugated to
horseradish peroxidase (HRP), and visualized with a peroxidase substrate. Assay results are determined
visually or using a microtiter plate reader. Test is considered positive for Staphylococcal enterotoxins if
absorbance is >0.200 and is considered negative if absorbance is <0.200. Specific toxin serotypes are not
differentiated. This method detects from 1.3 to 3.3 ng/mL Staphylococcal enterotoxin in extracts prepared
from food containing 4 to 10 ng/mL Staphylococcal enterotoxin.
Source: AOAC International. 1994. "Method 991.06: Staphylococcal Enterotoxins in Selected Foods."
Official Methods of Analysis of AOAC International. 16th Edition, 4th Revision; Vol. I.
http^/www.aoac.org/
8.2.6 AOAC Official Method 994.08: Aflatoxin in Corn, Almonds, Brazil Nuts, Peanuts,
and Pistachio Nuts
Analyte(s)
Aflatoxin (Type B1)
Agent Category
Small Molecule
CASRN
27261-02-5
Analysis Purpose: Confirmatory
Analytical Technique: HPLC-FL
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Method Developed for: Aflatoxins in corn, almonds, brazil nuts, peanuts, and pistachio nuts
Method Selected for: SAM lists this method for confirmatory analysis in aerosol, solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types. See Appendix D or Table 8-1 for additional methods that should be used for
this analyte.
Description of Method: This method is for the identification of aflatoxins in agricultural products.
Samples are extracted using an acetonitrile-water (9 + 1) solution. Sample extracts are then run through a
multifunctional cleanup column. The purified extract and standards are derivatized with trifluoroacetic
acid, and then analyzed using a HPLC system with a FL detector. Specific aflatoxins can be identified by
their retention time and quantified using standard curves. Method performance was characterized using
various commodities (e.g., corn) at aflatoxin levels over a range of 5-30 ng/g. This method was
originally designed for the analysis of aflatoxins (Bl, B2 [CAS RN 22040-96-6], Gl [CAS RN 1385-95-
1], and G2 [CAS RN 7241-98-7]) in commodities where cleanup was necessary to remove other food
components, such as fats and proteins; the cleanup procedure may not be necessary for water analyses.
Coupling the procedures, or a modification of the procedures, included in this method with an
immunoassay and/or biological activity test (where available) will provide more information regarding
specificity and toxicity.
Source: AOAC International. 1998. "Method 994.08: Aflatoxin in Corn, Almonds, Brazil Nuts, Peanuts,
and Pistachio Nuts." Official Methods of Analysis of AOAC International. 16th Edition, 4th Revision; Vol.
II. http://www.aoac.org/
8.2.7 Literature Reference for Abrin (119th AOAC Annual Meeting & Exposition, 2005, p.
613)
Analyte(s)
Abrin
Agent Category
Protein
CAS RN / Description
1 393-62-0 (abrin) / Glycoprotein
consisting of a deadenylase (25-32
kDa A chain) and lectin (35 kDa B
chain); an agglutinin (A2B2) may be
present in the crude preparations
Analysis Purpose: Presumptive
Analytical Technique: Immunoassay
Method Developed for: Abrin in food
Method Selected for: SAM lists these procedures for presumptive analysis in aerosol, solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types. See Appendix D or Table 8-1 for additional methods that should be used for
this analyte.
Description of Method: Procedures are described for using mouse mAbs and rabbit derived polyclonal
antibodies prepared against a mixture of abrin isozymes for three separate ELIS A and
electrochemiluminescence (ECL)-based assays in food products. The three assays vary by use of
antibody combination: (1) polyclonal (capture)/polyclonal (detection) ELISA, (2) polyclonal/monoclonal
ELISA, and (3) polyclonal/monoclonal ECL assay. The LODs with purified Abrin C and various abrin
extracts in buffer are between 0.1 and 0.5 ng/mL for all three assays. The LOD for abrin dissolved into
food products ranges from 0.1 to 0.5 ng/mL, using the ECL assay. The LOD for abrin dissolved into food
products for the ELISA assays range between 1 and 4 ng/mL depending on the assay configuration. In all
cases, the LODs are considerably less than the concentration at which abrin may pose a health concern.
Special Considerations: Crude preparations of abrin may also contain agglutinins that are unique to
rosary peas and that can cross-react in the immunoassays.
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Source: Garber, E.A., Aldrich, J.L., Wang, J., Brewer, V.A., O'Brien, T.W., and Sigal, G. 2005.
"Detection of Abrin in Foods Using ELISA and Electrochemiluminescence (ECL) Technologies." 119th
AOAC Annual Meeting & Exposition, p. 613.
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kto=author
8.2.8 Literature Reference for Abrin and Shiga and Shiga-like Toxins (Pharmacology
Toxicology. 2001. 88(5): 255-260)
Analyte(s)
Abrin
Shiga and Shiga-like Toxins
(Stx, Stx-1, Stx-2)
Agent Category
Protein
Protein
CAS RN / Description
1 393-62-0 (abrin) / Glycoprotein consisting
of a deadenylase (25-32 kDa A chain) and
lectin (35 kDa B chain); an agglutinin (A2B2)
may be present in the crude preparations
75757-64-1 (Stx) / Protein composed of one
-32 kDa A chain and five 7.7 kDa B chains
Analysis Purpose: Confirmatory for abrin; biological activity for shiga and shiga-like toxins
Analytical Technique: Ribosome inactivation assay
Method Developed for: Abrin in phosphate buffered saline (PBS)
Method Selected for: SAM lists these procedures for confirmatory analysis in aerosol, solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types. See Appendix D or Table 8-1 for additional methods that should be used for
this analyte.
Description of Method: Procedures are described for measuring the biological activity of ribosome-
inactivating proteins using a microtiter plate format for detection of abrin in PBS. Nuclease-treated rabbit
reticulocyte lysate containing luciferase messenger ribonucleic acid (mRNA) is used to measure toxin
activity via inhibition of protein synthesis. The relative biological activity is determined by comparing
luminescence levels in treated samples versus those of untreated controls. The amount of luciferase
translated, as measured by luminescence, is inversely proportional to the toxin concentration. Linear dose
response curves are generated for abrin, with a 50% inhibition of translation at 0.5 nM. Coupling this
procedure, or a modification of this procedure, with an immunoassay will provide more information
regarding the specificity and toxicity of the target biotoxin.
Special Considerations: For abrin, as well as shiga and shiga-like toxins, this assay does not test for
cell binding; cell culture assays are being developed to test for cell binding but are not currently available.
The only readily available assay to test for both the cell binding and enzymatic activity of the intact
(whole) toxin is the mouse bioassay.
Source: Hale, M.L. 2001. "Microtiter-based Assay for Evaluating the Biological Activity of Ribosome-
inactivation Proteins." Pharmacology Toxicology. 88(5): 255-260.
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8.2.9 Literature Reference for Abrin and Ricin (Analytical Biochemistry. 2008. 378(1): 87-
89)
Analyte(s)
Agent Category
CAS RN / Description
Abrin
Protein
1393-62-0 (abrin) / Glycoprotein consisting of
a deadenylase (25-32 kDa A chain) and
lectin (35 kDa B chain); an agglutinin (A2B2)
may be present in the crude preparations
Ricin
Protein
9009-86-3 (ricin) / 60 kDa glycoprotein
composed of two subunits (-32 kDa A chain
and -34 kDa B chain); an agglutinin of MW
120 kDa may be present in crude
preparations
Analysis Purpose: Biological activity
Analytical Technique: Enzyme activity
Method Developed for: Jequirity seed (abrin) and castor bean (ricin) extracts in buffer
Method Selected for: SAM lists these procedures for biological activity analysis in aerosol, solid,
particulate, liquid, and water samples. Further research is needed to develop and standardize the
procedures for environmental sample types. See Appendix D or Table 8-1 for additional methods that
should be used for this analyte.
Description of Method: This in vitro assay is an RNA N-glycosidase enzyme activity assay for the
detection of purified abrin and ricin toxins (Types I and II) and jequirity seed (abrin) and castor bean
(ricin) extracts. Synthetic biotinylated RNA substrates with varied loop sequences are cleaved by either
the ricin or abrin toxin and the RNA products are hybridized to ruthenylated-oligodeoxynucleotides to
generate an ECL signal. Assays are for 2 hours at 48°C. Commercially available ECL-based reagents
and RNase inactivators are used. Control experiments for the jequirity seed experiments and the distinct
GdAA/GdAGA ratio for the castor bean assay demonstrate lack of non-specific cleavage for the assay.
The undiluted castor bean extract contained 22.0 ± 0.7 mg/mL total protein and 4.1 ± 0.3 mg/mL ricin
equivalents as determined by standard protein determination and by ECL immunoassay assays
respectively. The undiluted jequirity seed extract was similarly assayed, with a resultant 21.6 ± 0.6
mg/mL total protein and 3.7 ± 0.3 jig/mL equivalents of toxin. Dilutions were performed to determine
effective signal-to-background ratio and the linear range for calculation of toxin activity. Resultant
calculations for ricin activity equivalents in the undiluted castor bean extract were equivalent to those
obtained with the ECL immunoassays: 4.4 ± 0.2 mg/mL activity equivalents. In contrast, the undiluted
jequirity seed extract contained a calculated level of 740 ± 50 jig/mL activity equivalents, which greatly
exceeded the immunoassay-based value.
Special Considerations: This enzyme activity assay does not test for cell binding; cell culture assays
are being developed to test for cell binding but are not currently available. The only readily available
assay to test for both the cell binding and enzymatic activity of the intact (whole) toxin is the mouse
bioassay.
Source: Keener, W.K., Rivera, V.R., Cho, C.R., Hale, M.L., Garber, E.A.E., and Poll, M.A. 2006.
"Identification of the RNA N-glycosidase Activity of Ricin in Castor bean extracts by an
Electrochemiluminescence-based Assay." Analytical Biochemistry. 378(1): 87-89.
http://www.sciencedirect.com/science? ob=ArticleURL& udi=B6W9V-4S2F5PT-
3& user=10& rdoc=l& fmt=& orig=search& sort=d&view=c& version=l& uiiVersion=0& userid=
10&md5=b70dOd8cd9ebl5819cabl064f80c2acf
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8.2.10 Literature Reference for a-Conotoxin (Biochemical Journal. 1997. 328: 245-250)
Analyte(s)
a-Conotoxin
Agent Category
Small Molecule
CASRN
156467-85-5
Analysis Purpose: Presumptive
Analytical Technique: Immunoassay
Method Developed for: Purified a-Conotoxin GI in phosphate buffer
Method Selected for: SAM lists these procedures for presumptive analysis in aerosol, solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types. See Appendix D or Table 8-1 for additional methods that should be used for
this analyte.
Description of Method: A biologically active fluorescein derivative of Conus geographus a-conotoxin
(FGI) is used in solution-phase-binding assays with two purified Torpedo californica monoclonal
antibodies (mAbs) to detect the toxin in laboratory samples. For competitive ligand-displacement spin-
column assays, FGI was premixed with various dilutions of unlabelled ligands and then incubated with
the two mAbs (5A1 and 8D2) at room temperature. Fluorescence is measured in ratio mode using
cuvettes with excitation and emission monochromators set at gamma = 490 nm and gamma = 525 nm,
respectively. The binding of FGI to the mAbs had apparent dissociation constants of 10 to 100 nM. The
binding specificity and epitopes recognized by the two mAbs against a-conotoxin GI are also
characterized. Competitive displacement assays showed that both mAbs specifically bound a-conotoxin
GI with high avidity. Cross-reactivity with a-conotoxins Ml and SI was not observed for either mAb in a
direct ELISA. With spin-column assay, however, 5A1, but not 8D2, cross-reacted at a low level (100 -
300-fold less avid) with these a-conotoxins. An antibody/a-conotoxin GI molar ratio of 1:1 afforded
complete protection in mouse lethal assays.
Source: Ashcom, J.D., and Stiles, B.C. 1997. "Characterization of a-Conotoxin Interactions with the
Nicotinic Acetylcholine Receptor and Monoclonal Antibodies." Biochemical Journal. 328: 245-250.
http://www.epa.gov/sam/pdfs/BJ-328-pgs245-250.pdf
8.2.11 Literature Reference for a-Conotoxin (Journal of Medicinal Chemistry. 2004. 47(5):
1234-1241)
Analyte(s)
a-Conotoxin
Agent Category
Small Molecule
CASRN
156467-85-5
Analysis Purpose: Confirmatory
Analytical Technique: High performance liquid chromatography - Mass spectrometer (HPLC-MS)
Method Developed for: Conus anemone venom (a-Conotoxins AnIA, AnIB, and AnIC) in buffer
Method Selected for: SAM lists these procedures for confirmatory analysis in aerosol, solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types. See Appendix D or Table 8-1 for additional methods that should be used for
this analyte.
Description of Method: Procedures are discussed for the detection of peptides within the a-conotoxin
molecular mass range using an HPLC-MS. A crude extract of Conus anemone venom sample is made
using 30% acetonitrile/water acidified with 0.1% trifluoroacetic acid (TFA), with the insoluble portion of
the sample removed by centrifugation. A portion of the sample extract is fractionated by size-exclusion
chromatography in order to prepare a sample containing small peptides in the range of 1000 to 2500 Da.
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Chromatography conditions are elution with 30% acetonitrile / 0.048% TFA at a flow rate of 0.5
mL/minute, with detection at 214 nm. Three sulfated a-conotoxins (AnIA, AnIB, and AnIC) can be
identified by LC-MS that are within the molecular mass range of other a-conotoxins (i.e., 1400-2200 Da).
Peptides can be quantified by reversed-phase HPLC using an external reference standard for each peptide.
Source: Loughnan, ML., Nicke, A., Jones, A., Adams, D.J., Alewood, P.P., and Lewis, RJ. 2004.
"Chemical and Functional Identification and Characterization of Novel Sulfated Alpha-conotoxins from
the Cone Snail Conus anemone." Journal of Medicinal Chemistry. 47(5): 1234-1241.
j^^
8.2.12 Literature Reference for a-Amanitin, T-2 Mycotoxin (Journal of Food Protection.
2005.68(6): 1294-1301)
Analyte(s)
a-Amanitin
T-2 Mycotoxin
Agent Category
Small Molecule
Small Molecule
CAS RN / Description
23109-05-9
21259-20-1
Analysis Purpose: Presumptive
Analytical Technique: Immunoassay
Method Developed for: a-Amanitin, ricin, and T-2 mycotoxin in food and beverages
Method Selected for: SAM lists these procedures for presumptive analysis of a-amanitin and T-2 toxin
in aerosol, solid, particulate, liquid, and water samples and for confirmatory analysis of ricin in aerosol,
solid, particulate, liquid, and water samples. Further research is needed to develop and standardize the
procedures for environmental sample types. See Appendix D or Table 8-1 for additional methods that
should be used for this analyte.
Description of Method: Commercially available ELISAs are described and assessed for detection of
ricin, amanitin, and T-2 toxin at levels below those described as a health concern in food samples. Solid
food samples are prepared by washing the sample with sodium phosphate buffer followed by dilution
with phosphate-buffered saline. Liquid beverage samples are prepared by dilution in sodium phosphate
buffer. Amanitin samples are similarly prepared using water instead of buffer, and T-2 toxin samples are
similarly prepared using 35% methanol in water instead of buffer. The prepared samples are used with
commercially obtained ELISA kits according to the manufacturer's directions, except for the
incorporation of an eight-point calibration curve and reading the plates at both 405 and 650 nm after 26
minutes of incubation at 37°C. This assay detects ricin in food products at 0.01 ug/mL with acceptable
background levels. Amanitin can be detected in food products at 1 ug/g with the ELISA. Background
responses occurred, but at less than the equivalent of 0.5 ppm for amanitin. The ELISA kit will
successfully detect T-2 toxin at targeted levels of 0.2 ug/g. The ELISA kit successfully detects T-2 toxin
at targeted levels of 0.2 ug/g; the immunoassay for T-2 toxin, however, shows significant background
responses and varies up to 0.1 ppm.
Source: Garber, E.A., Eppley, R.M., Stack, M.E., McLaughlin, M.A., and Park, D.L. 2005. "Feasibility
of Immunodiagnostic Devices for the Detection of Ricin, Amanitin, and T-2 Toxin in Food." Journal of
Food Protection. 68(6): 1294-1301.
http://wmw.ingeiitacoiinect.coin/content/iafp/ifp/2005/00000068/00000006/art00027
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8.2.13 Literature Reference for a-Amanitin (Journal of Chromatography B. 1991. 563(2):
299-311)
Analyte(s)
a-Amanitin
Agent Category
Small Molecule
CASRN
23109-05-9
Analysis Purpose: Confirmatory
Analytical Technique: HPLC with amperometric detection
Method Developed for: a-Amanitin in plasma
Method Selected for: SAM lists these procedures for confirmatory analysis in aerosol, solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types. See Appendix D or Table 8-1 for additional methods that should be used for
this analyte.
Description of Method: Procedures are described for the selective determination in human plasma of a-
amanitin using HPLC with amperometric detection. After extraction of plasma with disposable Ci8 silica
cartridges, the extracts are separated by isocratic reversed-phase chromatography using a macroporous
polystyrene-divinylbenzene column and a mobile phase of 0.05 M phosphate buffer-acetonitrile (91:9) at
pH 9.5. Amperometric detection is performed by applying an oxidation potential as low as +350 mV (vs.
Ag/AgCl) to a glassy carbon electrode, in a thin-layer flow-cell. The linear range for alpha-amanitin is 3
to 200 ng/mL, and the relative LOD in plasma is 2 ng/mL at a signal-to-noise ratio of 2. The intra-assay
precision has been evaluated at levels of 10 and 200 ng/mL.
Source: Tagliaro, F., Schiavon, G., Bontempelli, G., Carli, G., and Marigo, M. 1991. "Improved High-
performance Liquid Chromatographic Determination with Amperometric Detection of Alpha-amanitin in
Human Plasma Based on its Voltammetric Study." Journal of Chromatography B. 563(2): 299-311.
^
8.2.14 Literature Reference for Anatoxin-a (Biomedical Chromatography. 1996.10: 46-47)
Analyte(s)
Anatoxin-a
Agent Category
Small Molecule
CASRN
64285-06-9
Analysis Purpose: Confirmatory
Analytical Technique: HPLC-FL (precolumn derivatization)
Method Developed for: Anatoxin-a in potable water
Method Selected for: SAM lists these procedures for confirmatory analysis in aerosol, solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types other than water. See Appendix D or Table 8-1 for additional methods that
should be used for this analyte.
Description of Method: Procedures are described for HPLC analysis with fluorimetric detection of
anatoxin-a in water samples after derivatization with 7-fluoro-4-nitro-2,l,3-benzoxadiazole (NBD-F).
Samples are extracted at pH 7 with SPE using a weak cation exchanger. The toxin is eluted with
methanol containing 0.2% TFA. Samples are evaporated, reconstituted with acetonitrile, and re-
evaporated prior to derivatization. This procedure detects anatoxin-a at concentrations of 0.1 ug/L with a
good linear calibration.
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Source: James, K.J., and Sherlock, I.R. 1996. "Determination of the Cyanobacterial Neurotoxin,
Anatoxin-a, by Derivatisation Using 7-Fluoro-4-Nitro-2,l,3-Benzoxadiazole (NBD-F) and HPLC
Analysis with Fluorimetric Detection." Biomedical Chromatography. 10: 46-47.
il^^
8.2.15 Literature Reference for Brevetoxins (Environmental Health Perspectives. 2002.
110(2): 179-185)
Analyte(s)
Brevetoxins (B form)
Agent Category
Small Molecule
CASRN
79580-28-2
Analysis Purpose: Presumptive
Analytical Technique: Immunoassay
Method Developed for: Brevetoxins in shellfish
Method Selected for: SAM lists these procedures for presumptive analysis in aerosol, solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types. See Appendix D or Table 8-1 for additional methods that should be used for
this analyte.
Description of Method: Procedures are described for a competitive ELISA used to detect brevetoxins in
shellfish. The assay uses goat anti-brevetoxin antibodies in combination with a three-step signal
amplification process: (1) secondary biotinylated antibodies; (2) streptavidin-HRP conjugate; and (3)
chromogenic enzyme substrate. Sample preparation for liquids is dilution in PBS. Sample preparation
for solids (oysters) is homogenization in PBS, or extraction in acetone. The working range for the assay
is 0.2 to 2.0 ng/mL for diluted and undiluted liquid samples, and 0.2 to 2.0 ng/mL for solid samples,
corresponding to 0.8 to 8.0 ug brevetoxins/100.0 g shellfish. The method has been compared to the
mouse bioassay and is equivalent in sensitivity.
Source: Naar, J., Bourdelais, A., Tomas, C., Kubanek, J., Whitney, P.L., Flewelling, L., Steidinger, K.,
Lancaster, J., and Badan, D.G. 2002. "A Competitive ELISA to Detect Brevetoxins from Karenia brevis
(Formerly Gymnodinium breve) in Seawater, Shellfish, and Mammalian Body Fluid." Environmental
Health Perspectives. 110(2): 179-185. http://www.epa.gov/sam/pdfs/EHP-l 10(2)-pgsl79-185.pdf
8.2.16 Literature Reference for Brevetoxins (Toxicon. 2004. 43(4): 455-465)
Analyte(s)
Brevetoxins (B form)
Agent Category
Small Molecule
CASRN
79580-28-2
Analysis Purpose: Confirmatory
Analytical Technique: High performance liquid chromatography tandem mass spectrometers (HPLC-
MS-MS)
Method Developed for: Brevetoxins in shellfish
Method Selected for: SAM lists these procedures for confirmatory analysis in aerosol, solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types. See Appendix D or Table 8-1 for additional methods that should be used for
this analyte.
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Description of Method: Shellfish sample homogenates are extracted with acetone, and centrifuged. The
supernatants are combined, evaporated, and re-solubilized in 80% methanol. Following a wash with 95%
n-hexane, the methanolic layer is evaporated, and the residue re-solubilized in 25% methanol and applied
to a Ci8 SPE column. Analytes are eluted with 100% methanol, evaporated, and re-solubilized in
methanol for analysis. Analysis of prepared samples is performed using HPLC-MS-MS with a mobile
phase of water and acetonitrile with acetic acid. Analytes are detected by an MS with electrospray
ionization (ESI) interface. Brevetoxins are extensively metabolized, with many sub-forms. This method
describes multiple liquid chromatography/electrospray ionization mass spectrometry (LC-ESI-MS)
profiles for metabolites of brevetoxins from oysters.
Source: Wang, Z., Plakas, S.M., El Said, K.R., Jester, E.L., Granade, H.R., and Dickey, R.W. 2004.
"LC/MS Analysis of Brevetoxin Metabolites in the Eastern Oyster (Crassostrea virginica}." Toxicon.
43(4): 455-465. ht^caUnMJVM^Qdele^affidieN&cpsidt=lMMlII
8.2.17 Literature Reference for Cylindrospermopsin (FEMS Microbiology Letters. 2002.
216(2): 159-164)
Analyte(s)
Cylindrospermopsin
Agent Category
Small Molecule
CASRN
143545-90-8
Analysis Purpose: Confirmatory
Analytical Technique: High performance liquid chromatography - Photodiode array detector (HPLC-
PDA)
Method Developed for: Cylindrospermopsin in eutrophic waters
Method Selected for: SAM lists these procedures for confirmatory analysis in aerosol, solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types other than water. See Appendix D or Table 8-1 for additional methods that
should be used for this analyte.
Description of Method: Cylindrospermopsin is detected using HPLC with photodiode array detector
(PDA) in environmental waters. The suggested solvent range for Cylindrospermopsin is below 50%
methanol and 30% acetonitrile. Complex samples (culture medium) are purified on a Ci8 column with a
linear gradient of 1 to 12% (v/v) methanol/water over 24 minutes at 40°C, with monitoring at 262 nm.
The use of Ci8 columns for environmental waters is suggested for removal of the large number of organic
compounds that may be present. This method detects and recovers Cylindrospermopsin from spiked
environmental water samples at 1 ug/L.
Source: Metcalf, J.S., Beattie, K.A., Saker, M.L., and Codd, G.A. 2002. "Effects of Organic Solvents on
the High Performance Liquid Chromatographic Analysis of the Cyanobacterial Toxin
Cylindrospermopsin and Its Recovery from Environmental Eutrophic Waters by Solid Phase Extraction."
FEMS Microbiology Letters. 216(2): 159-164. http://cat.inist.fr/?aModele=afficheN&cpsidt=14002569
8.2.18 Literature Reference for Diacetoxyscirpenol (DAS) (International Journal of Food
Microbiology. 1988. 6(1): 9-17)
Analyte(s)
Diacetoxyscirpenol (DAS)
Agent Category
Small Molecule
CASRN
2270-40-8
Analysis Purpose: Presumptive
Analytical Technique: Immunoassay
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Method Developed for: DAS in food
Method Selected for: SAM lists these procedures for presumptive analysis in aerosol, solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types. See Appendix D or Table 8-1 for additional methods that should be used for
this analyte.
Description of Method: An ELISA is used for the detection of DAS in food samples. Antibodies
against DAS are obtained after immunization of rabbits with DAS-hemiglutarate-human serum albumin
(DAS-HG-HSA), and a DAS-hemisuccinate-HRP conjugate (DAS-HS-HRP) is prepared by an ester
method for use as enzyme-labeled toxin in the competitive assay. The detection limit for DAS using this
assay is approximately 10 pg/mL. This assay will cross-react related toxins. The relative cross-
reactivities of the assay are 597.5, 5.2, 100.0, 2.5, and 1.5% for 3 alpha-acetyl-DAS, DAS, T-2 toxin,
neosolaniol, and 15-acetoxyscirpenol, respectively.
Source: Klaffer, U., Martlbauer, E., and Terplan, G. 1988. "Development of a Sensitive Enzyme-linked
Immunosorbent Assay for the Detection of Diacetoxyscirpenol." International Journal of Food
Microbiology. 6(1): 9-17. Mt^Z/w^wjciei^
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59& user=10& coverDate=02%2F29%2F1988& rdoc=3& orig=browse& srch=doc-
cdi=506
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8.2.19 Literature Reference for Diacetoxyscirpenol (DAS) and T-2 Mycotoxin (Rapid
Communications in Mass Spectrometry. 2006. 20(9): 1422-1428)
Analyte(s)
Diacetoxyscirpenol (DAS)
T-2 Mycotoxin
Agent Category
Small Molecule
Small Molecule
CASRN
2270-40-8
21259-20-1
Analysis Purpose: Confirmatory
Analytical Technique: Liquid chromatography/atmospheric pressure chemical ionization mass
Spectrometry (LC/APCI-MS)
Method Developed for: DAS and T-2 mycotoxin in food
Method Selected for: SAM lists these procedures for confirmatory analysis in aerosol, solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types. See Appendix D or Table 8-1 for additional methods that should be used for
this analyte.
Description of Method: A LC/APCI-MS procedure based on time-of-flight mass Spectrometry
(TOFMS), with a real-time reference mass correction, is used for simultaneous determination of
Fusarium mycotoxins (to include DAS and T-2 mycotoxin) in foodstuffs. Mycotoxin samples are
extracted with acetonitrile/water (85:15) and centrifuged, and the supernatant is applied to a column for
cleanup. Prepared samples are separated by liquid chromatography with an aqueous mobile phase of
ammonium acetate and methanol detection is provided in exact mass chromatograms with a mass window
of 0.03 Th. The limits of detection range from 0.1 to 6.1 ng/g in analyzed foodstuffs.
SAM Revision 4.0 196 September 29, 2008
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Source: Tanaka, H., Takino, M., Sugita-Konishi, Y., and Tanaka, T. 2006. "Development of Liquid
Chromatography/Time-of-flight Mass Spectrometric Method for the Simultaneous Determination of
Trichothecenes, Zearalenone, and Aflatoxins in Foodstuffs." Rapid Communications in Mass
Spectrometry. 20(9): 1422-1428. littp://cat.inist.fr/?aModele=afficheN&cpsidt= 17697070
8.2.20 Literature Reference for Microcystins (Journal of AOAC International. 2001. 84(4):
1035-1044)
Analyte(s)
Microcystins
(Principal isoforms: LA, LR, LW, RR, YR)
Agent Category
Small Molecule
CASRN
961 80-79-9 (LA)
101 043-37-2 (LR)
157622-02-1 (LW)
11 1755-37-4 (RR)
101064-48-6 (YR)
Analysis Purpose: Presumptive
Analytical Technique: Immunoassay/Phosphatase assay
Method Developed for: Microcystins (LA, LR, LW, RR, YR) in algae products
Method Selected for: SAM lists these procedures for presumptive analysis in aerosol, solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types. See Appendix D or Table 8-1 for additional methods that should be used for
this analyte.
Description of Method: ELISA and protein phosphatase inhibition assays are used to detect
microcystins in blue-green algae products. Solid samples are prepared by homogenization in methanol
(75% in water), with centrifugation to remove solids. Immunoassays are performed on the prepared
samples using a commercially available ELISA test kit as described by the manufacturer. Samples are
quantitated by comparison with a microcystin LR standard curve. Quantitation with the colorimetric
protein phosphatase inhibition assay is based on a comparison with a microcystin LR standard curve.
ELISA and phosphatase assay results agree over a concentration range of 0.5 to 35 ug/g. Neither assay is
specific for a particular isoform.
Source: Lawrence, J.F., Niedzwiadek, B., Menard, C., Lau, B.P., Lewis, D., Kuper-Goodman, T.,
Carbone, S., and Holmes, C. 2001. "Comparison of Liquid Chromatography/Mass Spectrometry, ELISA,
and Phosphatase Assay for the Determination of Microcystins in Blue-green Algae Products." Journal of
AOAC International. 84(4): 1035-1044. http://cat.inist.fr/?aModele=afficheN&cpsidt=l 135453
8.2.21 Literature Reference for Microcystins (Analyst. 1994. 119(7): 1525-1530)
Analyte(s)
Microcystins
(Principal isoforms: LA, LR, LW, RR, YR)
Agent Category
Small Molecule
CASRN
961 80-79-9 (LA)
101 043-37-2 (LR)
157622-02-1 (LW)
11 1755-37-4 (RR)
101064-48-6 (YR)
Analysis Purpose: Confirmatory
Analytical Technique: HPLC-PDA
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Method Developed for: Microcystins (LA, LR, LW, RR, YR) in raw and treated waters
Method Selected for: SAM lists these procedures for confirmatory analysis in aerosol, solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types other than water. See Appendix D or Table 8-1 for additional methods that
should be used for this analyte.
Description of Method: Procedures are discussed to test the presence of microcystin-LR, -LY, -LW, -
LF (CAS RN 154037-70-4), and -RR in treated and untreated water samples. Cyanobacterial cells are
separated from the water by filtration through 110-mm glass fiber grade C (GF/C) discs. The cellular
components collected on the discs are extracted three times with methanol; the collected extraction fluids
are combined and dried. The residue is resuspended in methanol and analyzed by HPLC-PDA. The
liquid portion of the filtered water sample is subjected to trace enrichment using a Ci8 SPE cartridge,
followed by identification and determination by HPLC-PDA. This procedure can detect microcystin
concentrations as low as 250 ng/L and is the basis of the World Health Organization (WHO) method for
the detection of microcystins.
Source: Lawton, L.A., Edwards, C., and Codd, G.A. 1994. "Extraction and High-performance Liquid
Chromatographic Method for the Determination of Microcystins in Raw and Untreated Waters." Analyst.
119(7): 1525-1530. http://www.rsc.org/Publishing/.Tournals/AN/article.asp?doi=AN9941901525
8.2.22 Literature Reference for Picrotoxin (Journal of Pharmaceutical & Biomedical
Analysis. 1989. 7(3): 369-375)
Analyte(s)
Picrotoxin
Agent Category
Small Molecule
CASRN
124-87-8
Analysis Purpose: Confirmatory
Analytical Technique: HPLC
Method Developed for: Picrotoxin in serum
Method Selected for: SAM lists these procedures for confirmatory analysis in aerosol, solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types. See Appendix D or Table 8-1 for additional methods that should be used for
this analyte.
Description of Method: Procedures are described for quantification of the two components of picrotoxin
(picrotin [CAS RN 21416-53-5] and picrotoxinin [CAS RN 17617-45-7]) in serum samples. Serum
samples are prepared by washing with «-hexane, followed by extraction with chloroform. The
chloroform is evaporated and the sample is reconstituted in acetonitrile- 1 mM ammonium acetate buffer
(pH 6.4) 34:66 (v/v) for assay by reversed-phase HPLC. The effluent is monitored at 200 nm, and
quantification is based on peak-height ratio of analyte to the internal standard. A linear response is
obtained for both analytes (picrotin and picrotoxinin) in the range 0.2 to 20.0 jig/mL.
Source: Soto-Otero, R., Mendez-Alvarez, E., Sierra-Paredes, G., Galan-Valiente, J., Aguilar-Veiga, E.,
and Sierra-Marcuno, G. 1989. "Simultaneous Determination of the Two Components of Picrotoxin in
Serum by Reversed-phase High-performance Liquid Chromatography with Application to a
Pharmacokinetic Study in Rats." Journal of Pharmaceutical & Biomedical Analysis. 7(3): 369-375.
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rlVersion=0& Userid=10&md5=de051acb377a75cf755435f7d23dfdf7
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8.2.23 Literature Reference for Ricin (Journal of AOAC International. 2008. 91(2): 376-
382)
Analyte(s)
Agent Category
CAS RN / Description
Ricin
Protein
9009-86-3 (ricin) / 60 kDa
glycoprotein composed of two
subunits (-32 kDa A chain and -34
kDa B chain); an agglutinin of MW
120 kDa may be present in crude
preparations
Analysis Purpose: Confirmatory
Analytical Technique: Immunoassay
Method Developed for: Ricin for food products
Method Selected for: SAM lists these procedures for confirmatory analysis in aerosol, solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types. See Appendix D or Table 8-1 for additional methods that should be used for
this analyte.
Description of Method: This immunoassay is for the detection of various concentrations of purified
ricin in food products (e.g., juice, dairy products, vegetables, bakery products, condiments). The
immunoassay uses ECL detection in a 96-well plate format with a monoclonal capture antibody against
ricin (19A-2C6) and either a polyclonal or monoclonal detector antibody. The samples and detector
antibodies can be added sequentially or in combination during the capture step. Using the polyclonal
antibody, ricin was detected at concentrations as low as 0.04 ng/mL. Simultaneous addition of sample
and detector antibody allowed for a shortened procedure with only a single 20 minute incubation with no
false negatives caused by "hook" effects at high concentrations of ricin. Quantitation can be performed
either with the sequential procedure or with the simultaneous procedure if it is know that the ricin
concentration is not in the "hook" region. The simultaneous procedure should not be used when a sample
contains constituents that may react with the ruthenium tag. Polyclonal/monoclonal antibodies are
commercially available as an ELISA test kit.
Special Considerations: Crude preparations of ricin may also contain agglutinins that are unique to
castor beans and that can cross-react in the immunoassays.
Source: Garber, E.A.E., and O'Brien, T. W. 2008. "Detection of Ricin in Food Using
Electrochemiluminescence-Based Technology." Journal of AOAC International. 91(2): 376-382.
8.2.24 Literature Reference for Ricin by Ricinine detection (Journal of Analytical
Toxicology. 2005. 29(3): 149-155)
Analyte(s)
Ricinine (Ricin marker)
Agent Category
Small Molecule
CAS RN / Description
5254-40-3 (ricinine) / small
molecule, ricin marker
Analysis Purpose: Complementary presumptive for ricin
Analytical Technique: LC-MS
Method Developed for: Ricinine in human and rat urine samples
Method Selected for: SAM lists these procedures for complementary presumptive analysis of ricin by
ricinine detection in aerosol, solid, particulate, liquid, and water samples. Ricinine, an alkaloid
SAM Revision 4.0
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component of castor beans, is found in crude preparations of ricin, and may be an indicator of ricin
contamination. Further research is needed to develop and standardize the procedures for environmental
sample types. See Appendix D or Table 8-1 for additional methods that should be used for this analyte.
Description of Method: Procedures are described for sample extraction by SPE, isocratic HPLC,
followed by ESI tandem mass spectrometry. For MS analyses, protonated molecular ions are selected in
the multiple reaction monitoring mode and quantified by isotope dilution with 13C6-labeled ricinine as the
internal reference. Urine pools enriched with ricinine at two concentrations were used as quality controls
for validation of the method in urine samples. The calculated limit of detection was 0.04 ng/mL. In
addition to the validation with urine samples, testing was performed on a single human urine sample
(forensic), a crude ricin preparation, and urine samples from an animal ricinine exposure study. For the
human urine sample, the concentration of ricinine was measured to be 4.24 ng/mL. After a series of
simple extraction and filtration steps to provide a crude castor bean preparation, the final ricinine level
was 502 ng/mL. For the animal exposure study, rats were injected with ricinine at 1, 5, and 10 mg/kg,
with mean 24-hour urine concentrations of 1010, 6364, and 17, 152 ng/mL, respectively. Mean 48-hour
urine concentrations were 40, 324, and 610 mg/mL. Stability of ricinine in human urine was also tested,
with ricinine found to be stable in human urine samples when heated at 90°C for 1 hour, and when stored
at 25°C and 5°C for 3 weeks.
Source: Johnson, R.C., Lemire, S.W., Woolfitt, Ospina, M., Preston, K.P, Olson, C.T., and Barr, J.R.
2005. "Quantification of Ricinine in Rat and Human Urine: A Biomarker for Ricin Exposure." Journal of
Analytical Toxicology. 29(3): 149-155. http://www.jatox.com/abstracts/2005/April/149-johnson.html
8.2.25 Literature Reference for Saxitoxin (Journal of AOAC International. 1995. 78(2):
528-532)
Analyte(s)
Agent Category
CASRN
Saxitoxins
Principal isoforms:
Saxitoxin (SIX)
Neosaxitoxin (NEOSTX)
Gonyautoxin (GTX)
Decarbamoylgonyautoxin (dcGTX)
Decarbamoylsaxitoxin (dcSTX)
Small Molecule
35523-89-8 (SIX)
64296-20-4 (NEOSTX)
77462-64-7 (GTX)
None given (dcGTX)
58911-04-9 (dcSTX)
Analysis Purpose: Confirmatory
Analytical Technique: HPLC-FL (post column derivatization)
Method Developed for: Saxitoxins (STX, NEOSTX, GTX, dcGTX, dcSTX) in shellfish
Method Selected for: SAM lists these procedures for confirmatory analysis in aerosol, solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types. See Appendix D or Table 8-1 for additional methods that should be used for
this analyte.
Description of Method: Procedures are described to detect multiple analogues of saxitoxin in shellfish
using ion-interaction chromatography on a silica-based reversed-phase (C8) column with postcolumn
periodate oxidation and FL detection. Toxin groups of different net charges are determined separately by
isocratic elution using either sodium 1-heptanesulfonate in ammonium phosphate (GTX-1, GTX-6,
dcGTX2, dcGTXS) or sodium 1-heplanesulfonate in ammonium phosphate and acetonitrile (STX [CAS
RN 35523-89-8], neoSTX [CAS RN 64296-20-4], dcSTX [CAS RN 58911-04-9]). For biological sample
types, a cleanup procedure using a Ci8 SPE cartridge is effective in preventing false peaks. High
sensitivity with detection limits ranging from 20 to 110 fmol are achieved as a result of reduced band
broadening and optimized reaction conditions. This method, when applied to low-toxicity shellfish, gives
higher values than the standard mouse bioassay.
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Source: Oshima, Y. 1995. "Postcolumn Derivatization Liquid Chromatographic Method for Paralytic
Shellfish Toxins." Journal of AOAC International. 78(2): 528-532.
http://cat.inist.fr/?aModele=afficheN&cpsidt=3469391
8.2.26 Literature Reference for Shiga and Shiga-like Toxin (Journal of Clinical
Microbiology. 2007. 45(10): 3377-3380)
Analyte(s)
Shiga and Shiga-like Toxins
(Stx, Stx-1, Stx-2)
Agent Category
Protein
CAS RN / Description
75757-64-1 (Stx) / Protein
composed of one -32 kDa A chain
and five 7.7 kDa B chains
Analysis Purpose: Presumptive
Analytical Technique: Optical immunoassay
Method Developed for: Shiga toxin in foods
Method Selected for: SAM lists these procedures for presumptive analysis in aerosol, solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types. See Appendix D or Table 8-1 for additional methods that should be used for
this analyte.
Description of Method: Procedures are described for a rapid optical immunoassay for the detection of
Stx-1 and Stx-2 using a commercially available kit. Fecal samples (742 specimens) are assayed for Shiga
toxins with and without enrichment of the specimens in broth. Duplicate assays are applied using either
the rapid optical immunoassay or a commercially available ELISA kit. Samples producing positive
results by immunoassay are confirmed by Vero cell cytotoxicity assay. Sensitivities of 96.8% are
achieved for direct stool sample assays.
Source: Teel, L.D., Daly, J.A., Jerris, R.C., Maul, D., Svanas, G., O'Brien, A.D., and Park, C.H. 2007.
"Rapid Detection of Shiga Toxin-Producing Escherichia coli by Optical Immunoassay." Journal of
Clinical Microbiology. 45(10): 3377-3380. www.epa.gov/sam/pdfs/.TCM-45(10)-pgs3377-3380.pdf
8.2.27 Literature Reference for Tetrodotoxin (Journal of Clinical Laboratory Analysis.
1992. 6(2): 65-72)
Analyte(s)
Tetrodotoxin
Agent Category
Small Molecule
CASRN
9014-39-5
Analysis Purpose: Presumptive
Analytical Technique: Immunoassay
Method Developed for: Tetrodotoxin in buffer
Method Selected for: SAM lists these procedures for presumptive analysis in aerosol, solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types. See Appendix D or Table 8-1 for additional methods that should be used for
this analyte.
Description of Method: Procedures are described for a competitive inhibition enzyme immunoassay
(CIEIA) for tetrodotoxin (TTX) in biological samples. An anti-TTX mAb, designated T20G10, is
directly labeled with alkaline phosphatase for use in the assay. Sensitivities of 6 to 7 ng/mL (1C 50) and 2
to 3 ng/mL (1C 20) are achieved.
SAM Revision 4.0 201 September 29, 2008
-------
Section 8 - Selected Biotoxin Methods
Source: Raybould, T.J., Bignami, G.S., Inouye, L.K., Simpson, S.B., Byrnes, J.B., Grothaus, P.G., and
Vann, B.C. 1992. "A Monoclonal Antibody-based Immunoassay for Detecting Tetrodotoxin in Biological
Samples." Journal of Clinical Laboratory Analysis. 6(2): 65-72.
8.2.28 Literature Reference for Tetrodotoxin (Analytical Biochemistry. 2001. 290(1): 10-
17)
Analyte(s)
Tetrodotoxin
Agent Category
Small Molecule
CASRN
9014-39-5
Analysis Purpose: Confirmatory
Analytical Technique: LC/ESI-MS
Method Developed for: Tetrodotoxin from puffer fish and newt tissues
Method Selected for: SAM lists these procedures for confirmatory analysis in aerosol, solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types. See Appendix D or Table 8-1 for additional methods that should be used for
this analyte.
Description of Method: Procedures are described for LC/ESI-MS analysis of TTXs in tissue samples
from puffer fish and newts by a combination of chromatography on a reversed-phase column with long
carbon chains (C30) and with the mobile phase containing an ion pair reagent (ammonium
heptafluorobutyrate). The relationship between the amount of applied standard TTX and its peak area on
the mass chromatogram (m/z 320) shows good linearity over a range of 50 to 1000 pmol. The detection
limit of TTX in the SIM mode is estimated to be 0.7 pmol, with a signal to noise ratio of 2:1.
Source: Shoji, Y., Yotsu-Yamashita, M., Miyazawa, T., and Yasumoto, T. 2001. "Electrospray
lonization Mass Spectrometry of Tetrodotoxin and its Analogs: Liquid Chromatography/Mass
Spectrometry, Tandem Mass Spectrometry, and Liquid Chromatography/Tandem Mass Spectrometry."
Analytical Biochemistry. 290(1): 10-17.
http://wmw.sciencedirect.coin/science? ob=ArticleURL& udi=B6W9V-45BC729-
B£&jisei=10^^^
= 10&md5=c01fdd21eOd9fl04431816fd41869fle
8.2.29 ELISA Kits for Cylindrospermopsin
Analyte(s)
Cylindrospermopsin
Agent Category
Small Molecule
CASRN
143545-90-8
Analysis Purpose: Presumptive
Analytical Technique: Immunoassay
Method Developed for: Cylindrospermopsin in ground water, surface water, and well water
Method Selected for: SAM lists these procedures for presumptive analysis in aerosol, solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types other than water. See Appendix D or Table 8-1 for additional methods that
should be used for this analyte.
Description of Method: Cylindrospermopsin is detected using a colorimetric immunoassay (competitive
ELISA) procedure. A sample (0.05 mL), enzyme conjugate (cylindrospermopsin-HRP), and an antibody
SAM Revision 4.0 202 September 29, 2008
-------
Section 8 - Selected Biotoxin Methods
solution containing rabbit anti-cylindrospermopsin antibodies are added to plate wells containing
immobilized sheep anti-rabbit antibodies. Both the cylindrospermopsin (if present) in the sample and
cylindrospermopsin-HRP conjugate compete in solution to bind to the rabbit anti-cylindrospermopsin
antibodies in proportion to their respective concentrations. The anti-cylindrospermopsin antibody-target
complexes are then bound to the immobilized sheep anti-rabbit antibodies on the plate. After incubation,
the unbound molecules are washed and decanted. A specific substrate is then added which is converted
from a colorless to a blue solution by the HRP enzyme conjugate solution. The reaction is terminated with
the addition of a dilute acid. The concentration of cylindrospermopsin in the sample is determined
photometrically by comparing sample absorbance to the absorbance of the calibrators (standards) at a
specific wavelength (450 nm). The applicable concentration range is 0.4-2.0 \igfL, with a minimum
detection level of 0.4
Source: NEMI 2006.
http://infotiti .»i u*cs.gov/pls/apex/f?p=119:38:7526698938332159::::P38 METHOD
8.2.30 ELISA Kits for Saxitoxins
Analyte(s)
Agent Category
CASRN
Saxitoxins
Principal isoforms:
Saxitoxin (SIX)
Neosaxitoxin (NEOSTX)
Gonyautoxin (GTX)
Decarbamoylgonyautoxin (dcGTX)
Decarbamoylsaxitoxin (dcSTX)
Small Molecule
35523-89-8 (SIX)
64296-20-4 (NEOSTX)
77462-64-7 (GTX)
None given (dcGTX)
58911-04-9 (dcSTX)
Analysis Purpose: Presumptive
Analytical Technique: Immunoassay
Method Developed for: STX in water and solid samples (e.g., shellfish)
Method Selected for: SAM lists these procedures for confirmatory analysis in aerosol, solid, particulate,
liquid, and water samples. Further research is needed to develop and standardize the procedures for
environmental sample types other than water. See Appendix D or Table 8-1 for additional methods that
should be used for this analyte.
Description of Method: Saxitoxin is detected using a colorimetric immunoassay (competitive ELISA)
procedure. A sample (0.05 mL), enzyme conjugate (saxitoxin-HRP), and an antibody solution containing
rabbit anti-saxitoxin antibodies are added to plate wells containing immobilized sheep anti-rabbit
antibodies. Both the saxitoxin (if present) in the sample and saxitoxin-HRP conjugate compete in solution
to bind to the rabbit anti-saxitoxin antibodies in proportion to their respective concentrations. The anti-
saxitoxin antibody-target complexes are then bound to the immobilized sheep anti-rabbit antibodies on
the plate. After incubation, the unbound molecules are washed and decanted. A specific substrate is then
added which is converted from a colorless to a blue solution by the HRP enzyme conjugate solution. The
reaction is terminated with the addition of a dilute acid. The concentration of saxitoxin in the sample is
determined photometrically by comparing sample absorbance to the absorbance of the calibrators
(standards) at a specific wavelength (450 nm). The applicable concentration range is 0.015-0.4 ng/mL,
with a minimum detection level of 0.015 ng/mL.
Special Considerations: This kit is not intended for other types of saxitoxin. Cross-reactivity is
observed with the following saxitoxin types: dcSTX (29%), GTX 2, 3, and 5B (23%), sulfo GTX 1 and 2
(2.0%, dcGTX 2 and 3 (1.4%), NEOSTX (1.3%), dcNEOSTX (0.6%), GTX 1 and 4 (<0.2%). High
concentrations (e.g., above 0.1 ng/mL for toxins with >20% cross-reactivity) of these other types of
saxitoxin may produce false positive responses.
SAM Revision 4.0 203 September 29, 2008
-------
Section 8 - Selected Biotoxin Methods
Source: NEMI. 2006.
http://infotrek.er.usss.gov/pls/apex/f?p=l 19:38:8989971104293493::::P38 METHOD
8.2.31 Lateral Flow Immunoassay Kits
Analyte(s)
Agent Category
CAS RN / Description
Botulinum neurotoxins
(Types A, B)
Protein
Protein composed of -100 kDa heavy
chain and -50 kDa light chain; can be
complexed with hemagglutinin and
non-hemagglutinin components for
total MW of -900 kDa
Ricin
Protein
9009-86-3 (ricin)/60 kDa
glycoprotein composed of two
subunits (-32 kDa A chain and -34
kDa B chain); an agglutinin of MW
120 kDa may be present in crude
preparations
Analysis Purpose: Presumptive
Analytical Technique: Immunoassay
Method Developed for: Botulinum neurotoxins (Types A, B) and ricin in buffer or water samples
Method Selected for: SAM lists these procedures for presumptive analysis in aerosol samples. Further
research is needed to develop and standardize the procedures for environmental sample types other than
water. See Appendix D or Table 8-1 for additional methods that should be used for this analyte.
Description of Method: Test strips are self-contained, qualitative assays for screening environmental
samples for the presence of botulinum toxin and ricin. After the sample is collected, it is transferred onto
the test strip where dye-labeled antibodies detect trace amounts of the contaminant, as indicated by the
presence of two bands in the test result window. After 15 minutes, the results are read visually.
Botulinum neurotoxin Type A can be detected at 5 mg/L and Type B at 4 mg/L, 33% of the time. Ricin
toxin can be detected at 20 mg/L, with no cross-reactivity to certain substances (i.e., lectin from
soybeans).
An alternative lateral flow immunochromatographic device also can be used. This device uses two
antibodies in combination to specifically detect target antigen in solution. When a sufficient amount of
target antigen is present, the colloidal gold label accumulates in the sample window on a test strip,
forming a visible reddish-brown colored line. The presence of two bands indicates a positive reading.
Botulinum neurotoxin Type A can be detected at 0.01 mg/L and Type B at 0.5 mg/L, with no false
negatives detected when interferents are present. Ricin toxin can be detected at 0.035 mg/L, with 88%
accuracy.
These two lateral flow immunoassay kits have been evaluated by the U.S. EPA ETV Program
and
BioThreat092104.pdf) for the detection of botulinum neurotoxins Types A and B and ricin. Information
regarding the evaluation of test strips can be accessed at these sites.
Special Considerations: Crude preparations of ricin may also contain agglutinins that are unique to castor
beans and that can cross-react in the immunoassays. Immunoassays with botulinum toxins may produce
variable results with uncomplexed form of toxin.
Source: ETV. 2006. http ://www. epa.gov/etv/
SAM Revision 4.0
204
September 29, 2008
-------
Section 9 -Conclusions
Section 9.0: Conclusions
Methods listed in Appendix A (chemical methods), Appendix B (radiochemical methods), Appendix C
(pathogen methods), and Appendix D (biotoxin methods) are recommended for use in assessment of the
extent of contamination and the effectiveness of decontamination following a homeland security event.
The primary objective of this document is not necessarily to identify the "best" analytical methods, but
rather to identify appropriate methods that represent a balance between providing existing, documented,
determinative techniques and providing consistent and valid analytical results. The method selected for
each analyte/sample type combination was deemed the most general, appropriate, and broadly applicable
of available methods. This is a living guidance document for use by EPA and EPA-contracted
laboratories tasked with analysis of environmental samples following a homeland security event.
Recommended methods are subject to change based on procedure testing and advances in technology.
Any questions concerning the information in this document should be directed to the appropriate point (s)
of contact listed in Section 4.
SAM Revision 4.0 205 September 29, 2008
-------
Section 9 -Conclusions
SAM Revision 4.0 206 September 29, 2008
-------
Appendix A - Selected Chemical Methods
Appendix A: Selected Chemical Methods
SAM Revision 4.0 September 29, 2008
-------
Appendix A - Selected Chemical Methods
SAM Revision 4.0 September 29, 2008
-------
Appendix A: Selected Chemical Methods
Analyte(s)
Ally! alcohol
Ammonium metavanadate
(analyze for total vanadium)
CASRN
79 06 1
107 13 1
116 06 3
1646 88 4
1646 87 3
107 18 6
504 24 5
7RO? ^ Ft
IddCt ?R 7
Determinative
Technique
HPLC
HPLC
HPLC
HPLC
HPLC
GC MS
HPLC
IPP M^ / IPP AF^
IPP M^ / IPP AF^
Method Type
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Solid Samples
Water extraction
8316
(EPASW-846)
Water extraction
8316
(EPASW-846)
831 8A
(EPASW-846)
831 8A
(EPASW-846)
831 8A
(EPASW-846)
5035A
(EPA SW-846)
8260C
(EPASW-846)
8330B
(EPASW-846)
3050B
(EPASW-846)
6010C/6020A
(EPASW-846)
3050B
(EPASW-846)
6010C/6020A
(EPASW-846)
Non-aqueous
Liquid/Organic
Solid Samples1
Water extraction
8316
(EPA SW-846)
Water extraction
8316
(EPA SW-846)
8318A
(EPA SW-846)
831 8A
(EPA SW-846)
831 8A
(EPA SW-846)
3585
(EPA SW-846)
8260C
(EPA SW-846)
8330B
(EPA SW-846)
3031
(EPA SW-846)
6010C/6020A
(EPA SW-846)
3031
(EPA SW-846)
6010C/6020A
(EPA SW-846)
Aqueous Liquid
Samples
8316
(EPA SW-846)
8316
(EPA SW-846)
8318A
(EPA SW-846)
831 8A
(EPA SW-846)
831 8A
(EPA SW-846)
5030C
(EPA SW-846)
8260C
(EPA SW-846)
3535A/8330B
(EPA SW-846)
8330B
(EPA SW-846)
4500- NH3 B
(SM)
4500- NH3 G
(SM)
200.7/200.8
(EPAOW)
200.7/200.8
(EPAOW)
Drinking Water
Samples
8316
(EPASW-846)
8316
(EPASW-846)
531.2
(EPA OW)
531.2
(EPA OW)
531.2
(EPA OW)
5030C
(EPASW-846)
8260C
(EPASW-846)
3535A/8330B
(EPASW-846)
8330B
(EPASW-846)
350.1
(EPA OW)
200.7/200.8
(EPA OW)
200.7/200.8
(EPA OW)
Air Samples
PV2004
(OS HA)
PV2004
(OS HA)
5601
(NIOSH)
5601
(NIOSH)
5601
(NIOSH)
TO-152
(EPA ORD)
6015
(NIOSH)
IO-3.1
(EPA ORD)
IO-3.4/IO-3.5
(EPA ORD)
IO-3.1
(EPA ORD)
IO-3.4/IO-3.5
(EPA ORD)
Wipes
3570/8290A Appendix A
(EPA SW-846)
8316
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8316
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
831 8A
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
831 8A
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
831 8A
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8330B
(EPASW-846)
9102
(NIOSH)
6010C/6020A
(EPASW-846)
9102
(NIOSH)
6010C/6020A
(EPASW-846)
SAM Revision 4.0, Appendix A
A-1
September 29, 2008
-------
Analyte(s)
Arsenic trioxide
(analyze for total arsenic)
Arsine
Asbestos
Boron trifluoride
Brodifacoum
Bromadiolone
BZ (Quinuclidinyl benzilate)
Calcium arsenate
(analyze as total arsenic)
Carbofuran (Furadan)
Carbon disulfide
Chlorfenvinphos
CASRN
1 ?97 ^? ?
7704 40 1
1 ??9 91 4
7CQ7 H7 9
ccn7Q inn
00770 cc 7
cco-1 r\fi 9
7770 44 i
1 ^fi? fifi 9
7c i c n
47O QO fi
Determinative
Technique
IPP M*^ / IPP AF^
GFAA
TEM
ISE
HPLC
HPLC
HPLC
IPP M^ / IPP AF^
HPLC
C.C M*^
C.C M*^
Method Type
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Solid Samples
3050B
(EPASW-846)
6010C/6020A
(EPASW-846)
3050B
(EPA SW-846)
7010
(EPA SW-846)
D5755-03 (soft
surfaces-
microvac)
(ASTM)
Not of concern
3541/3545A
(EPA SW-846)
8321B
(EPA SW-846)
3541/3545A
(EPA SW-846)
8321B
(EPA SW-846)
3541/3545A
(EPA SW-846)
8321 B3
(EPASW-846)
3050B
(EPASW-846)
6010C/6020A
(EPASW-846)
831 8A
(EPASW-846)
5035A
(EPA SW-846)
8260C
(EPASW-846)
3541/3545A
(EPASW-846)
8270D
(EPASW-846)
Non-aqueous
Liquid/Organic
Solid Samples1
3031
(EPA SW-846)
6010C/6020A
(EPA SW-846)
Not of concern
Not of concern
Not of concern
3580A
(EPA SW-846)
8321 B
(EPASW-846)
3580A
(EPASW-846)
8321 B
(EPASW-846)
3580A
(EPASW-846)
8321 B3
(EPA SW-846)
3031
(EPA SW-846)
6010C/6020A
(EPA SW-846)
831 8A
(EPA SW-846)
3585
(EPA SW-846)
8260C
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
Aqueous Liquid
Samples
200.7/200.8
(EPAOW)
200.7/200.8
(EPA OW)
Not of concern
Not of concern
3520C/3535A
(EPA SW-846)
8321 B
(EPASW-846)
3520C/3535A
(EPASW-846)
8321 B
(EPASW-846)
3520C/3535A
(EPASW-846)
8321 B3
(EPA SW-846)
200.7/200.8
(EPAOW)
831 8A
(EPA SW-846)
5030C
(EPA SW-846)
8260C
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
Drinking Water
Samples
200.7/200.8
(EPA OW)
200.7/200.8
(EPA OW)
Not of concern
Not of concern
3520C/3535A
(EPA SW-846)
8321B
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8321 B
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8321 B3
(EPASW-846)
200.7/200.8
(EPA OW)
531.2
(EPA OW)
524.2
(EPA OW)
3520C/3535A
(EPASW-846)
8270D
(EPASW-846)
Air Samples
IO-3.1
(EPA ORD)
IO-3.4/IO-3.5
(EPA ORD)
6001
(NIOSH)
10312:1995
(ISO)
ID216SG
(OS HA)
Not of concern
Not of concern
TO-10A4
(EPA ORD)
IO-3.1
(EPA ORD)
IO-3.4/IO-3.5
(EPA ORD)
5601
(NIOSH)
TO-15
(EPA ORD)
TO-10A
(EPA ORD)
Wipes
9102
(NIOSH)
6010C/6020A
(EPA SW-846)
9102
(NIOSH)
7010
(EPA SW-846)
D6480-99
(ASTM)
Not of concern
3570/8290A Appendix A
(EPA SW-846)
8321B
(EPA SW-846)
3570/8290A Appendix A
(EPASW-846)
8321 B
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8321 B3
(EPASW-846)
9102
(NIOSH)
6010C/6020A
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
831 8A
(EPASW-846)
Not of concern
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPASW-846)
SAM Revision 4.0, Appendix A
A-2
September 29, 2008
-------
Analyte(s)
Chlorine
Chloropicrin
Chlorosarin
Chlorosoman
(degradation product of Lewisite)
Chlorpyrifos
Crimidine
Cyanide, Amenable to chlorination
Cyanide, Total
CASRN
7782-50-5
1O7 O7 ?
qc 04 o
7C r\p. o
1 44R 7C 7
7O4O ^7 £.
ocnqn ?? i
OQO1 QQ O
CQC oq 7
NA
C7 i o c
Determinative
Technique
Spectrophotometry
r;p iwic; / r;p pin
r;p iwic
r;p iwic; / r;p ppn
r;p iwic;
r;p iwic;
IPP M*^ / IPP AF^
C.C M*^
r;p iwic;
Spectrophotometry
Spectrophotometry
Method Type
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Solid Samples
Not of concern
5035A
(EPASW-846)
8260C
(EPASW-846)
3541/3545A
(EPASW-846)
8270D5
(EPASW-846)
3545A
(EPASW-846)
8270D7
(EPASW-846)
3541/3545A
(EPASW-846)
8270D
(EPASW-846)
3541/3545A
(EPASW-846)
8270D
(EPASW-846)
3050B
(EPASW-846)
6010C/6020A
(EPASW-846)
3541/3545A
(EPASW-846)
8270D
(EPASW-846)
3545A
(EPASW-846)
8270D8
(EPASW-846)
3135.21
(EPA RLAB)
ILM05.3 CN
(EPA CLP)
Non-aqueous
Liquid/Organic
Solid Samples1
Not of concern
3585
(EPA SW-846)
8260C
(EPA SW-846)
3580A
(EPA SW-846)
8270D5
(EPA SW-846)
3580A
(EPA SW-846)
8270D7
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3031
(EPA SW-846)
6010C/6020A
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
8270D8
(EPA SW-846)
Not of concern
Not of concern
Aqueous Liquid
Samples
4500-CI G
(SM)
5030C
(EPA SW-846)
8260C
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D5
(EPA SW-846)
551.1
(EPAOW)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
200.7/200.8
(EPAOW)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
8270D8
(EPA SW-846)
3135.21
(EPA RLAB)
ILM05.3CN
(EPA CLP)
Drinking Water
Samples
4500-CI G
(SM)
5030C
(EPASW-846)
8260C
(EPASW-846)
3520C/3535A
(EPASW-846)
8270D5
(EPASW-846)
551.1
(EPA OW)
3520C/3535A
(EPASW-846)
8270D
(EPASW-846)
3520C/3535A
(EPASW-846)
8270D
(EPASW-846)
200.7/200.8
(EPA OW)
3520C/3535A
(EPASW-846)
8270D
(EPASW-846)
3535A
(EPA SW-846)
8270D8
(EPASW-846)
3135.21
(EPA RLAB)
335.4
(EPA OW)
Air Samples
Analyst, 124(12):
1853-1857
4500-CI G
(SM)
2513
(NIOSH)
TO-10A6
(EPA ORD)
PV2103 (OSHA)
TO-10A6
(EPA ORD)
TO-10A6
(EPA ORD)
IO-3.1
(EPA ORD)
IO-3.4/IO-3.5
(EPA ORD)
TO-10A
(EPA ORD)
Not of concern
Not of concern
6010
(NIOSH)
Wipes
Not of concern
Not of concern
3570/8290A Appendix A
(EPA SW-846)
8270D5
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D7
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPASW-846)
9102
(NIOSH)
6010C/6020A
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D8
(EPASW-846)
3135.21
(EPA RLAB)
ILM05.3CN
(EPA CLP)
SAM Revision 4.0, Appendix A
A-3
September 29, 2008
-------
Analyte(s)
1,2-Dichloroethane
(degradation product of HD)
Diisopropyl methylphosphonate
fniiwiP^
(degradation product of GB)
Dimethylphosphoramidic acid
(degradation product of GA)
Disulfoton
CASRN
cnc 77 A
Qoq qq 7
1O7 OP. 9
co 7-3 7
1 41 P.P. 9
MA
1 44^ 7c c
ope oc q
QQQ7C C1 C
82-66-6
298-04-4
Determinative
Technique
C.C M*^
r;p iwic;
r;p iwic;
r;p iwic;
C.C M*^
r;p Fin
upi p
r;p iwic;
upi p
upi p
GC-MS / GC-FPD
Method Type
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Solid Samples
5035A
(EPASW-846)
8260C
(EPASW-846)
3541/3545A
(EPASW-846)
8270D
(EPASW-846)
5035A
(EPA SW-846)
8260C
(EPASW-846)
3545A
(EPASW-846)
8270D
(EPA SW-846)
3545A
(EPA SW-846)
8270D
(EPA SW-846)
3541/3545A
(EPA SW-846)
801 5C
(EPA SW-846)
3545A
(EPA SW-846)
8321 B9
(EPASW-846)
3545A
(EPASW-846)
8270D
(EPA SW-846)
3545A
(EPASW-846)
8321 B3
(EPASW-846)
3541/3545A
(EPASW-846)
8321 B
(EPASW-846)
3541/3545A
(EPA SW-846)
8270D
(EPA SW-846)
Non-aqueous
Liquid/Organic
Solid Samples1
3585
(EPA SW-846)
8260C
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3585
(EPA SW-846)
8260C
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
801 5C
(EPA SW-846)
3580A
(EPA SW-846)
8321 B9
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
8321 B3
(EPA SW-846)
3580A
(EPA SW-846)
8321 B
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
Aqueous Liquid
Samples
5030C
(EPA SW-846)
8260C
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
5030C
(EPA SW-846)
8260C
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
801 5C
(EPA SW-846)
3535A
(EPA SW-846)
8321 B9
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
8321 B3
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8321 B
(EPA SW-846)
525.2
(EPA OW)
Drinking Water
Samples
5030C
(EPASW-846)
8260C
(EPASW-846)
3520C/3535A
(EPASW-846)
8270D
(EPASW-846)
524.2
(EPA OW)
525.2
(EPA OW)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
801 5C
(EPA SW-846)
3535A
(EPA SW-846)
8321 B9
(EPASW-846)
3535A
(EPA SW-846)
8270D
(EPASW-846)
3535A
(EPA SW-846)
8321 B3
(EPASW-846)
3520C/3535A
(EPASW-846)
8321 B
(EPA SW-846)
525.2
(EPA OW)
Air Samples
TO-15
(EPA ORD)
TO-10A
(EPA ORD)
TO-15
(EPA ORD)
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
TO-10A6
(EPA ORD)
TO-10A
(EPA ORD)
TO-10A4
(EPA ORD)
5600
(NIOSH)
Wipes
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
801 5C
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8321 B9
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8321 B3
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8321 B
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
SAM Revision 4.0, Appendix A
A-4
September 29, 2008
-------
Analyte(s)
Disulfoton sulfoxide
1,4-Dithiane
(degradation product of HD)
EA2192 [Diisopropylaminoethyl
methylthiolophosphonate]
(hydrolysis product of VX)
Ethyl methylphosphonic acid
(EM PA)
(degradation product of VX)
Ethyldichloroarsine (ED)
N-Ethyldiethanolamine (EDEA)
(degradation product of HN-1)
Ethylene oxide
Fenamiphos
Fentanyl
Fluoride
Fluoroacetamide
CASRN
94Q7 O7 Ft
cnc; on o
7?9O7 QR 4
1832-53-7
598-14-1
139-87-7
75-21-8
22224-92-6
437-38-7
16984-48-8
640-19-7
Determinative
Technique
np M^ / np Fpn
HP M^
HPLC
HPLC
GC-MS
HPLC
GC-MS
GC-MS
HPLC
1C
GC-MS
Method Type
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Solid Samples
3541/3545A
fFPA ^W RdFi\
8270D
(EPASW-846)
3545A
(EPASW-846)
8270D10
(EPASW-846)
3545A
(EPASW-846)
8321 B3
(EPASW-846)
3545A
(EPASW-846)
8321 B3
(EPASW-846)
3545A
(EPASW-846)
8270D
(EPA SW-846)
3541/3545A
(EPA SW-846)
8321 B
(EPA SW-846)
5035A
(EPA SW-846)
8260C
(EPA SW-846)
3541/3545A
(EPA SW-846)
8270D
(EPA SW-846)
3541/3545A
(EPASW-846)
8321 B
(EPASW-846)
Not of concern
3541/3545A
(EPA SW-846)
8270D
(EPASW-846)
Non-aqueous
Liquid/Organic
Solid Samples1
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
8270D10
(EPA SW-846)
3580A
(EPA SW-846)
8321 B3
(EPA SW-846)
3580A
(EPA SW-846)
8321 B3
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
8321 B
(EPA SW-846)
3585
(EPA SW-846)
8260C
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
8321 B
(EPA SW-846)
Not of concern
3580A
(EPA SW-846)
8270D
(EPA SW-846)
Aqueous Liquid
Samples
525.2
(EPAOW)
3535A
(EPA SW-846)
8270D10
(EPA SW-846)
3535A
(EPA SW-846)
8321 B3
(EPA SW-846)
3535A
(EPA SW-846)
8321 B3
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8321 B
(EPA SW-846)
5030C
(EPA SW-846)
8260C
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8321 B
(EPA SW-846)
300.1, Rev 1.0
(EPA OW)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
Drinking Water
Samples
525.2
(EPA OW)
3535A
(EPA SW-846)
8270D10
(EPASW-846)
3535A
(EPA SW-846)
8321 B3
(EPASW-846)
3535A
(EPASW-846)
8321 B3
(EPASW-846)
3535A
(EPASW-846)
8270D
(EPASW-846)
3520C/3535A
(EPA SW-846)
8321 B
(EPA SW-846)
5030C
(EPA SW-846)
8260C
(EPA SW-846)
525.2
(EPA OW)
3520C/3535A
(EPASW-846)
8321 B
(EPA SW-846)
300.1, Rev 1.0
(EPA OW)
3520C/3535A
(EPA SW-846)
8270D
(EPASW-846)
Air Samples
5600
(NIOSH)
Not of concern
TO-10A4
(EPA ORD)
TO-10A4
(EPA ORD)
TO-15
(EPA ORD)
TO-10A
(EPA ORD)
TO-15
(EPA ORD)
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
Not of concern
TO-10A
(EPA ORD)
Wipes
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D10
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8321 B3
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8321 B3
(EPASW-846)
9102
(NIOSH)
8270D
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8321 B
(EPA SW-846)
Not of concern
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8321 B
(EPA SW-846)
Not of concern
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPASW-846)
SAM Revision 4.0, Appendix A
A-5
September 29, 2008
-------
Analyte(s)
Fluoroacetic acid and fluoroacetate
salts (analyze for fluoroacetate ion)
Formaldehyde
Gasoline Range Organics
triazine (RDX)
Hexamethylenetriperoxidediamine
(HMTD)
Hydrogen bromide
Hydrogen chloride
Hydrogen cyanide
Hydrogen fluoride
Hydrogen sulfide
Isopropyl methylphosphonic acid
(IMPA) (degradation product of
GB)
CASRN
NA
en on o
NA
191 R9 4
OQQ CC Q
100?^ 1 o R
7R47 O1 O
74 QO R
7CC4 QQ Q
77QQ r\p. A
1 R?9 ^4 R
Determinative
Technique
1C
HPLC
np Fin
HPLC
HPLC
1C
1C
Spectrophotometry
1C
1C
HPLC
Method Type
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Solid Samples
Analytical
Letters, 27(14):
2703-2718
300.1, Rev 1.0
(EPA OW)
831 5A
(EPASW-846)
5035A
(EPASW-846)
801 5C
(EPASW-846)
8330B
(EPA SW-846)
8330B
(EPA SW-846)
Not of concern
Not of concern
Not of concern
Not of concern
Not of concern
3545A
(EPASW-846)
8321 B3
(EPASW-846)
Non-aqueous
Liquid/Organic
Solid Samples1
Analytical Letters,
27(14):
2703-2718
300.1, Rev 1.0
(EPAOW)
Not of concern
3585
(EPA SW-846)
801 5C
(EPA SW-846)
8330B
(EPA SW-846)
8330B
(EPA SW-846)
Not of concern
Not of concern
Not of concern
Not of concern
Not of concern
3580A
(EPA SW-846)
8321 B3
(EPA SW-846)
Aqueous Liquid
Samples
300.1, Rev 1.0
(EPAOW)
831 5A
(EPA SW-846)
5030C
(EPA SW-846)
801 5C
(EPA SW-846)
3535A/8330B
(EPA SW-846)
8330B
(EPA SW-846)
3535A/8330B
(EPA SW-846)
8330B
(EPA SW-846)
Not of concern
Not of concern
Not of concern
Not of concern
Not of concern
3535A
(EPA SW-846)
8321 B3
(EPA SW-846)
Drinking Water
Samples
300.1, Rev 1.0
(EPA OW)
831 5A
(EPASW-846)
5030C
(EPASW-846)
801 5C
(EPASW-846)
3535A/8330B
(EPASW-846)
8330B
(EPA SW-846)
3535A/8330B
(EPA SW-846)
8330B
(EPA SW-846)
Not of concern
Not of concern
Not of concern
Not of concern
Not of concern
3535A
(EPASW-846)
8321 B3
(EPASW-846)
Air Samples
S301-1
(NIOSH)
300.1, Rev 1.0
(EPAOW)
2016
(NIOSH)
Not of concern
Not of concern
Not of concern
7903
(NIOSH)
7903
(NIOSH)
6010
(NIOSH)
790311
(NIOSH)
6013
(NIOSH)
TO-10A4
(EPA ORD)
Wipes
3570/8290A Appendix A
(EPA SW-846)
300.1, Rev 1.0
(EPA OW)
3570/8290A Appendix A
(EPA SW-846)
831 5A
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
801 5C
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8330B
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8330B
(EPA SW-846)
Not of concern
Not of concern
Not of concern
Not of concern
Not of concern
3570/8290A Appendix A
(EPA SW-846)
8321 B3
(EPASW-846)
SAM Revision 4.0, Appendix A
A-6
September 29, 2008
-------
Analyte(s)
Kerosene
Lead arsenate
(analyze as total arsenic)
Lewisite 1 (L-1)12
[2-chlorovinyldichloroarsine]
(analyze for total arsenic)
Lewisite 2 (L-2)
[bis(2-chlorovinyl)chloroarsine]
(analyze for total arsenic)
Lewisite 3 (L-3)
(analyze for total arsenic)
Lewisite oxide
(degradation product of Lewisite)
Mercury, Total
Methamidophos
Methomyl
Methoxyethylmercuric acetate
(analyze for total mercury)
Methyl acrylonitrile
CASRN
£4740 Q-| n
7C4C oc o
541-25-3
40334-69-8
4O??4 7O 1
1 ?Ofi O9 1
74QQ 07 c
in9fic; no c
1 £7^9 77 ^
1 C1 QQ 0
1 9fi QR 7
Determinative
Technique
HP Fin
IPP M*^ / IPP AF^
ICP-MS/ICP-AES
ICP-MS/ICP-AES
IPP M*^ / IPP AF^
IPP M*^ / IPP AF^
Spectrophotometry
/CVAA/CVAFS
C.C M*^
HPLC
Spectrophotometry
/ CVAA / CVAFS
HPLC
Method Type
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Solid Samples
5035A
(EPASW-846)
801 5C
(EPASW-846)
3050B
(EPASW-846)
6010C/6020A
(EPASW-846)
3050B
(EPASW-846)
6010C/6020A
(EPASW-846)
3050B
(EPASW-846)
6010C/6020A
(EPASW-846)
3050B
(EPA SW-846)
6010C/6020A
(EPA SW-846)
3050B
(EPA SW-846)
6010C/6020A
(EPA SW-846)
747313
(EPASW-846)
3541/3545A
(EPASW-846)
8270D
(EPASW-846)
831 8A
(EPASW-846)
747313
(EPASW-846)
Water extraction
8316
(EPASW-846)
Non-aqueous
Liquid/Organic
Solid Samples1
3585
(EPA SW-846)
801 5C
(EPA SW-846)
3031
(EPA SW-846)
6010C/6020A
(EPA SW-846)
3031
(EPA SW-846)
6010C/6020A
(EPA SW-846)
3031
(EPA SW-846)
6010C/6020A
(EPA SW-846)
3031
(EPA SW-846)
6010C/6020A
(EPA SW-846)
3031
(EPA SW-846)
6010C/6020A
(EPA SW-846)
Not of concern
3580A
(EPA SW-846)
8270D
(EPA SW-846)
831 8A
(EPA SW-846)
Not of concern
Water extraction
8316
(EPA SW-846)
Aqueous Liquid
Samples
5030C
(EPA SW-846)
801 5C
(EPA SW-846)
200.7/200.8
(EPAOW)
200.7/200.8
(EPAOW)
200.7/200.8
(EPAOW)
200.7/200.8
(EPA OW)
200.7/200.8
(EPA OW)
747313
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
831 8A
(EPA SW-846)
747313
(EPA SW-846)
8316
(EPA SW-846)
Drinking Water
Samples
5030C
(EPASW-846)
801 5C
(EPASW-846)
200.7/200.8
(EPA OW)
200.7/200.8
(EPA OW)
200.7/200.8
(EPA OW)
200.7/200.8
(EPA OW)
200.7/200.8
(EPA OW)
245.2
(EPA OW)
3535A
(EPASW-846)
8270D
(EPASW-846)
531.2
(EPA OW)
245.2
(EPA OW)
8316
(EPASW-846)
Air Samples
Not of concern
IO-3.1
(EPA ORD)
IO-3.4/IO-3.5
(EPA ORD)
IO-3.1
(EPA ORD)
IO-3.4/IO-3.5
(EPA ORD)
IO-3.1
(EPA ORD)
IO-3.4/IO-3.5
(EPA ORD)
IO-3.1
(EPA ORD)
IO-3.4/IO-3.5
(EPA ORD)
IO-3.1
(EPA ORD)
IO-3.4/IO-3.5
(EPA ORD)
IO-5
(EPA ORD)
TO-10A14
(EPA ORD)
5601
(NIOSH)
IO-5
(EPA ORD)
PV2004
(OS HA)
Wipes
3570/8290A Appendix A
(EPA SW-846)
801 5C
(EPASW-846)
9102
(NIOSH)
6010C/6020A
(EPASW-846)
9102
(NIOSH)
6010C/6020A
(EPASW-846)
9102
(NIOSH)
6010C/6020A
(EPA SW-846)
9102
(NIOSH)
6010C/6020A
(EPA SW-846)
9102
(NIOSH)
6010C/6020A
(EPA SW-846)
9102
(NIOSH)
747313
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
831 8A
(EPASW-846)
9102
(NIOSH)
747313
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8316
(EPASW-846)
SAM Revision 4.0, Appendix A
A-7
September 29, 2008
-------
Analyte(s)
Methyl fluoroacetate
(analyze for fluoroacetate ion)
Methyl hydrazine
Methyl isocyanate
Methyl parathion
Methylamine
N-Methyldiethanolamine (MDEA)
(degradation product of HN-2)
1 -Methylethyl ester
ethylphosphonofluoridic acid (GE)
Methylphosphonic acid (MPA)
(degradation product of VX, GB, &
GD)
Mevinphos
[bis(2-chloroethyl)ethylamine]
Mustard, nitrogen (HN-2)
N,N-bis(2-chloroethyl)methylamine]
CASRN
4CQ i Q Q
en ?4 A
624-83-9
OQQ no o
74 RQ £.
irm ^Q Q
1 1 RQ R7 ?
QQQ 1 Q C
77QC -24 7
CQQ f\7 Q
c-i 7C o
Determinative
Technique
1C
HP M^ /
Spectrophotometry
HPLC
C.C M*^
HPLC
HPLC
GC-MS
HPLC
nr M*^
C.C M*^
r;p iwic;
Method Type
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Solid Samples
Analytical
Letters, 27(14):
2703-2718
300.1, Rev 1.0
(EPA OW)
3541/3545A
(EPASW-846)
8270D
(EPASW-846)
Not of concern
3545A
(EPASW-846)
8270D
(EPA SW-846)
Not of concern
3541/3545A
(EPA SW-846)
8321 B
(EPA SW-846)
3541/3545A
(EPA SW-846)
8270D
(EPASW-846)
3545A
(EPASW-846)
8321 B3
(EPASW-846)
3545A
(EPASW-846)
8270D
(EPASW-846)
3541/3545A
(EPASW-846)
8270D
(EPASW-846)
3541/3545A
(EPASW-846)
8270D
(EPASW-846)
Non-aqueous
Liquid/Organic
Solid Samples1
Analytical Letters,
27(14):
2703-2718
300.1, Rev 1.0
(EPAOW)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
3580A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
3580A
(EPA SW-846)
8321 B
(EPA SW-846)
3580A
(EPASW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
8321 B3
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
Aqueous Liquid
Samples
300.1, Rev 1.0
(EPAOW)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
3535A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
3520C/3535A
(EPA SW-846)
8321 B
(EPA SW-846)
3520C/3535A
(EPASW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
8321 B3
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
Drinking Water
Samples
300.1, Rev 1.0
(EPA OW)
3520C/3535A
(EPASW-846)
8270D
(EPASW-846)
Not of concern
3535A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
3520C/3535A
(EPA SW-846)
8321B
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPASW-846)
3535A
(EPASW-846)
8321 B3
(EPASW-846)
525.2
(EPA OW)
3520C/3535A
(EPASW-846)
8270D
(EPASW-846)
3520C/3535A
(EPASW-846)
8270D
(EPASW-846)
Air Samples
S301-1
(NIOSH)
300.1, Rev 1.0
(EPAOW)
3510
(NIOSH)
OSHA 54
TO-10A
(EPA ORD)
OSHA 40
TO-10A
(EPA ORD)
TO-10A6
(EPA ORD)
TO-10A4
(EPA ORD)
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
Wipes
3570/8290A Appendix A
(EPA SW-846)
300.1, Rev 1.0
(EPA OW)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPASW-846)
Not of concern
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
3570/8290A Appendix A
(EPA SW-846)
8321B
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8321 B3
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPASW-846)
SAM Revision 4.0, Appendix A
A-8
September 29, 2008
-------
Analyte(s)
[tris(2-chloroethyl)amine]
Mustard, sulfur/ Mustard gas (HD)
Nicotine compounds
(analyze as nicotine)
tetrazocine (HMX)
Organophosphate pesticides, NOS
Osmium tetroxide
(analyze for total osmium)
Oxamyl
Paraquat
Parathion
Pentaerythritol tetranitrate (PETN)
Perfluoroisobutylene (PFIB)
CASRN
ccc 77 -i
cnc; en 9
C.A 1 1 C.
ocqi 41 n
NA
9OR1 fi 1 9 O
OQ1 QC OO n
4coc 147
CC QQ 0
70 1 1 c
QQO O1 Q
Determinative
Technique
r;p iwic;
r;p iwic;
r;p iwic;
HPLC
HP M*^ / c.c MPn
/ c.c FPn
IPP AF^ / HFAA
HPLC
HPLC
C.C M*^
HPLC
HP M*^ / HP MPn
Method Type
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Solid Samples
3541/3545A
(EPASW-846)
8270D
(EPASW-846)
3571
(EPASW-846)
8270D15
(EPASW-846)
3545A
(EPASW-846)
8270D
(EPA SW-846)
8330B
(EPASW-846)
3541/3545A
(EPASW-846)
8270D
(EPASW-846)
3050B
(EPASW-846)
601 OC
(EPA SW-846)
831 8A
(EPA SW-846)
Not of concern
3541/3545A
(EPA SW-846)
8270D
(EPA SW-846)
8330B
(EPA SW-846)
Not of concern
Non-aqueous
Liquid/Organic
Solid Samples1
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3571
(EPA SW-846)
8270D15
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
8330B
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
831 8A
(EPA SW-846)
Not of concern
3580A
(EPA SW-846)
8270D
(EPA SW-846)
8330B
(EPA SW-846)
Not of concern
Aqueous Liquid
Samples
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3571
(EPA SW-846)
8270D15
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A/8330B
(EPA SW-846)
8330B
(EPA SW-846)
614
(EPAOW)
252.2
(EPAOW)
831 8A
(EPA SW-846)
549.2
(EPA OW)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A/8330B
(EPA SW-846)
8330B
(EPA SW-846)
Not of concern
Drinking Water
Samples
3520C/3535A
(EPASW-846)
8270D
(EPASW-846)
3571
(EPASW-846)
8270D15
(EPASW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A/8330B
(EPASW-846)
8330B
(EPASW-846)
507
(EPA OW)
252.2
(EPA OW)
531.2
(EPA OW)
549.2
(EPA OW)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A/8330B
(EPA SW-846)
8330B
(EPA SW-846)
Not of concern
Air Samples
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
Not of concern
Not of concern
5600
(NIOSH)
IO-3.1
(EPA ORD)
IO-3.4
(EPA ORD)
5601
(NIOSH)
Not of concern
TO-10A
(EPA ORD)
Not of concern
OSHA6116
Wipes
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D15
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8330B
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPASW-846)
9102
(NIOSH)
601 OC
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
831 8A
(EPA SW-846)
Not of concern
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8330B
(EPA SW-846)
Not of concern
SAM Revision 4.0, Appendix A
A-9
September 29, 2008
-------
Analyte(s)
Phencyclidine
Phenol
Phorate
Phosgene
Phosphamidon
Phosphine
Phosphorus trichloride
Pinacolyl methyl phosphonic acid
(PMPA)
(degradation product of GD)
Propylene oxide
R 33 (VR)
methylpropyl ester]
Sarin (GB)
CASRN
77 1O 1
TOR cm 9
OQQ H9 9
7C AA C
1 ?1 71 91 fi
7RO? m 9
771 Q 1 9 9
ci c co 4
jc cc n
1 RQQ?Q R7 4
1O7 44 R
Determinative
Technique
C.C M*^
r;p iwic;
r;p iwic;
r;p MPn
r;p iwic;
Spectrophotometry
Spectrophotometry
HPLC
c.c M*^ / nr Fin
r;p iwic;
C.C M*^
Method Type
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Solid Samples
3545A
(EPASW-846)
8270D
(EPASW-846)
3541/3545A
(EPASW-846)
8270D
(EPASW-846)
3545A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
3541/3545A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
Not of concern
3545A
(EPA SW-846)
8321 B3
(EPASW-846)
5035A
(EPASW-846)
8260C
(EPASW-846)
3541/3545A
(EPASW-846)
8270D
(EPASW-846)
3571
(EPASW-846)
8270D15
(EPASW-846)
Non-aqueous
Liquid/Organic
Solid Samples1
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
3580A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
Not of concern
3580A
(EPA SW-846)
8321 B3
(EPA SW-846)
3585
(EPA SW-846)
8260C
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3571
(EPA SW-846)
8270D15
(EPA SW-846)
Aqueous Liquid
Samples
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
Not of concern
3535A
(EPASW-846)
8321 B3
(EPA SW-846)
5030C
(EPA SW-846)
8260C
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3571
(EPA SW-846)
8270D15
(EPA SW-846)
Drinking Water
Samples
3535A
(EPASW-846)
8270D
(EPASW-846)
3520C/3535A
(EPASW-846)
8270D
(EPASW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
Not of concern
3535A
(EPA SW-846)
8321 B3
(EPASW-846)
5030C
(EPASW-846)
8260C
(EPASW-846)
3520C/3535A
(EPASW-846)
8270D
(EPASW-846)
3571
(EPASW-846)
8270D15
(EPASW-846)
Air Samples
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
OSHA 61
TO-10A
(EPA ORD)
6002
(NIOSH)
6402
(NIOSH)
TO-10A4
(EPA ORD)
1612
(NIOSH)
TO-10A
(EPA ORD)
TO-10A6
(EPA ORD)
Wipes
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
Not of concern
3570/8290A Appendix A
(EPA SW-846)
8321 B3
(EPASW-846)
Not of concern
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D15
(EPASW-846)
SAM Revision 4.0, Appendix A
A-10
September 29, 2008
-------
Analyte(s)
Semivolatile Organic Compounds,
NOS
Sodium arsenite
(analyze for total arsenic)
Sodium azide
Tohl in (C.&\
Tear gas (CS) [chlorobenzylidene
malonitrile]
CASRN
MA
77R4 AC. c
26628-22-8
qc C.A n
C7 04 q
744fi nq c;
744fi 1 1 q
77 P.1 fi
ocqo 41 i
1O7 4Q ?
on 1 9 £
Determinative
Technique
r;p iwic;
IPP M*^ / IPP AF^
1C
C.C M*^
r;p iwic;
IP
HP M*^
HP M*^
C.C M*^
HP M*^
Method Type
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Solid Samples
3541/3545A
(EPASW-846)
8270D
(EPASW-846)
3050B
(EPASW-846)
6010C/6020A
(EPASW-846)
J. of Forensic
Sciences, 43(1):
200-202"
300.1, Revl.O18
(EPA OW)
3545A
(EPASW-846)
8270D
(EPASW-846)
3545A
(EPASW-846)
8270D
(EPA SW-846)
3545A
(EPA SW-846)
8270D
(EPA SW-846)
3541/3545A
(EPASW-846)
8270D
(EPASW-846)
3545A
(EPASW-846)
8270D
(EPA SW-846)
3541/3545A
(EPASW-846)
8270D7
(EPASW-846)
Non-aqueous
Liquid/Organic
Solid Samples1
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3031
(EPA SW-846)
6010C/6020A
(EPA SW-846)
3580A17
(EPA SW-846)
300.1, Revl.O18
(EPAOW)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
8270D7
(EPA SW-846)
Aqueous Liquid
Samples
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
200.7/200.8
(EPAOW)
J. of Forensic
Sciences, 43(1):
200-202"
300.1, Revl.O18
(EPAOW)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D7
(EPA SW-846)
Drinking Water
Samples
525.2
(EPA OW)
200.7/200.8
(EPA OW)
J. of Forensic
Sciences, 43(1):
200-202"
300.1, Revl.O18
(EPA OW)
3535A
(EPA SW-846)
8270D
(EPASW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPASW-846)
8270D
(EPASW-846)
3535A
(EPA SW-846)
8270D
(EPASW-846)
3520C/3535A
(EPASW-846)
8270D7
(EPASW-846)
Air Samples
TO-10A
(EPA ORD)
IO-3.1
(EPA ORD)
IO-3.4/IO-3.5
(EPA ORD)
ID-211 (OSHA)
TO-10A6
(EPA ORD)
6004
(NIOSH)
Method 8
(EPA OAQPS)
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
Wipes
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPASW-846)
9102
(NIOSH)
6010C/6020A
(EPASW-846)
ID-211 (OSHA)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D7
(EPASW-846)
SAM Revision 4.0, Appendix A
A-11
September 29, 2008
-------
Analyte(s)
Thallium sulfate
(analyze for total thallium)
Thiodiglycol (TDG)
(degradation product of HD)
Thiofanox
1,4-Thioxane
(degradation product of HD)
Titanium tetrachloride
(analyze for total titanium)
Triethanolamine (TEA)
Trimethyl phosphite
Vanadium pentoxide
(analyze for total vanadium)
VE [phosphonothioic acid, ethyl-, S
ester]
CASRN
1On?1 ^Q 1
111 4 P. P.
QQI qc 104
1 ^QRO 1^1
jccn AC. n
1O9 71 P.
191 4R Q
QQ ?£; A
iiQ qc 7
1 Q1 A CO 1
91 7?P. 9^ O
Determinative
Technique
IPP M*^ / IPP AF^
C.C M*^
HPLC
r;p iwic;
IPP M*^ / IPP AF^
HPLC
C.C M*^
HPLC
HPLC
IPP M*^ / IPP AF^
C.C M*^
Method Type
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Solid Samples
3050B
(EPASW-846)
6010C/6020A
(EPASW-846)
3545A
(EPA SW-846)
8270D
(EPA SW-846)
3541/3545A
(EPA SW-846)
8321 B
(EPA SW-846)
3545A
(EPA SW-846)
8270D10
(EPASW-846)
3050B
(EPASW-846)
6010C/6020A
(EPASW-846)
3541/3545A
(EPASW-846)
8321 B
(EPASW-846)
3545A
(EPA SW-846)
8270D7
(EPASW-846)
8330B
(EPASW-846)
8330B
(EPASW-846)
3050B
(EPA SW-846)
6010C/6020A
(EPA SW-846)
3541/3545A
(EPA SW-846)
8270D
(EPA SW-846)
Non-aqueous
Liquid/Organic
Solid Samples1
3031
(EPA SW-846)
6010C/6020A
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
8321 B
(EPA SW-846)
3580A
(EPA SW-846)
8270D10
(EPA SW-846)
Not of concern
3580A
(EPA SW-846)
8321 B
(EPA SW-846)
3580A
(EPA SW-846)
8270D7
(EPA SW-846)
8330B
(EPA SW-846)
8330B
(EPA SW-846)
3031
(EPA SW-846)
6010C/6020A
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
Aqueous Liquid
Samples
200.7/200.8
(EPAOW)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8321 B
(EPA SW-846)
3535A
(EPA SW-846)
8270D10
(EPA SW-846)
Not of concern
3520C/3535A
(EPA SW-846)
8321 B
(EPA SW-846)
3535A
(EPA SW-846)
8270D7
(EPA SW-846)
3535A/8330B
(EPA SW-846)
8330B
(EPA SW-846)
3535A/8330B
(EPA SW-846)
8330B
(EPA SW-846)
200.7/200.8
(EPA OW)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
Drinking Water
Samples
200.7/200.8
(EPA OW)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
531.2
(EPA OW)
3535A
(EPA SW-846)
8270D10
(EPASW-846)
Not of concern
3520C/3535A
(EPASW-846)
8321 B
(EPA SW-846)
3535A
(EPA SW-846)
8270D7
(EPASW-846)
3535A/8330B
(EPASW-846)
8330B
(EPASW-846)
3535A/8330B
(EPASW-846)
8330B
(EPASW-846)
200.7/200.8
(EPA OW)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
Air Samples
IO-3.1
(EPA ORD)
IO-3.4/IO-3.5
(EPA ORD)
TO-10A
(EPA ORD)
5601
(NIOSH)
Not of concern
Not of concern
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
Not of concern
Not of concern
IO-3.1
(EPA ORD)
IO-3.4/IO-3.5
(EPA ORD)
TO-10A
(EPA ORD)
Wipes
9102
(NIOSH)
6020A/6010C
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8321 B
(EPA SW-846)
3570/8290A Appendix A
(EPASW-846)
8270D10
(EPASW-846)
9102
(NIOSH)
6010C/6020A
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8321 B
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D7
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8330B
(EPASW-846)
3570/8290A Appendix A
(EPA SW-846)
8330B
(EPA SW-846)
9102
(NIOSH)
6010C/6020A
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
SAM Revision 4.0, Appendix A
A-12
September 29, 2008
-------
Analyte(s)
VG [phosphonothioic acid, S-(2-
(diethylamino)ethyl) O,O-diethyl
ester]
VM [phosphonothioic acid, methyl-,
S-(2-(diethylamino)ethyl) O-ethyl
ester]
VX [0-ethyl-S-(2-
diisopropylaminoethyl)methyl-
phosphonothiolate]
White phosphorus
CASRN
78-53-5
21770-86-5
50782-69-9
12185-10-3
Determinative
Technique
GC-MS
GC-MS
GC-MS
GC-NPD/
GC-FPD
Method Type
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Solid Samples
3541/3545A
(EPA SW-846)
8270D
(EPA SW-846)
3541/3545A
(EPA SW-846)
8270D
(EPA SW-846)
3571
(EPA SW-846)
8270D15
(EPA SW-846)
7580
(EPA SW-846)
Non-aqueous
Liquid/Organic
Solid Samples1
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3571
(EPA SW-846)
8270D15
(EPA SW-846)
7580
(EPA SW-846)
Aqueous Liquid
Samples
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3571
(EPA SW-846)
8270D15
(EPA SW-846)
7580
(EPA SW-846)
Drinking Water
Samples
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3571
(EPA SW-846)
8270D15
(EPA SW-846)
7580
(EPA SW-846)
Air Samples
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
7905
(NIOSH)
Wipes
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D15
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
7580
(EPA SW-846)
Footnotes
An organic solid sample is a solid that completely dissolves in an organic solvent and leaves no solid residue.
If problems occur when using this method, it is recommended that TO-1OA be used.
3 LC-MS (electrospray) procedures are preferred for these analytes; however, if this technique is not available to the laboratory, GC-MS procedures using derivatization based on SW-846 Method 8270D
may be used. Sample preparation methods should remain the same. Both electrospray LC-MS and GC-MS derivatization procedures are currently under development.
4 For this analyte, HPLC is the preferred technique; however, if problems occur, Method TO-1 OA must be modified to include a derivatization step prior to analysis by GC-MS.
For this analyte, SW-846 Method 8270D must be modified to include a derivatization step.
If problems occur when using this method, it is recommended that the canister Method TO-15 be used.
If problems occur with analyses, lower the injection temperature.
8 If problems occur when using this method, it is recommended that SW-846 Method 8321B be used as the determinative method. Sample preparation methods should remain the same.
9 If problems occur with the analysis of DIMP using EPA SW-846 Method 8321 B, use SW-846 Method 8270D.
10 If problems occur when using this method, it is recommended that SW-846 Method 8260C and appropriate corresponding sample preparation procedures (i.e., 5035A for solid samples, 3585 for non-
aqueous liquid/organic solid samples, and 5030C for aqueous liquid and drinking water samples) be used.
If problems occur when using this method, it is recommended that NIOSH Method 7906 be used.
12
Laboratory testing is currently underway for speciation of Lewisite 1 using GC-MS techniques.
13 If equipment is not available, use CVAA Methods 7471B (EPA SW-846) for solid samples and 7470A (EPA SW-846) for aqueous liquid samples.
If problems occur when using this method, it is recommended that NIOSH Method 5600 be used.
For this analyte, refer to EPA SW-846 Method 8271 for GC-MS conditions.
16 If problems occur when using this method, it is recommended that a method based on the following journal article be used: J. Chrom. A, 1098: (2005) 156-165.
17 Water extraction, filtration, and acidification steps from the Journal of Forensic Science, 1998. 43(1 ):200-202 should be used for the preparation of solid samples. Filtration and acidification steps from
this journal should be used for preparation of aqueous liquid and drinking water samples. The acidification step from the journal should be used with EPA SW-846 Method 3580A for preparation of non-
aqueous liquid/organic solid samples.
18 If analyses are problematic, refer to column manufacturer for alternate conditions
SAM Revision 4.0, Appendix A
A-13
September 29, 2008
-------
SAM Revision 4.0, Appendix A A -14 September 29, 2008
-------
Appendix B - Selected Radiochemical Methods
Appendix B: Selected Radiochemical Methods
SAM Revision 4.0 September 29, 2008
-------
Appendix B - Selected Radiochemical Methods
SAM Revision 4.0 September 29, 2008
-------
Appendix B: Selected Radiochemical Methods
Analyte Class
Gross Alpha
Gross Beta
Gamma
Analyte(s)
Americium-2412
Californium-2522
Cesium-137
Cobalt-60
Curium-2442
Europium-154
lodine-131
lridium-192
Plutonium-2382
Plutonium-2392
CASRN
14596-10-2
13981-17-4
10045-97-3
10198-40-0
13981-15-2
15585-10-1
10043-66-0
14694-69-0
13981-16-3
15117-48-3
Determinative
Technique
Alpha/Beta
counting
Alpha/Beta
counting
Gamma
spectrometry
Determinative
Technique
Alpha/Gamma
spectrometry
Alpha
spectrometry
Gamma
spectrometry
Gamma
spectrometry
Alpha
spectrometry
Gamma
spectrometry
Gamma
spectrometry
Gamma
spectrometry
Alpha
spectrometry
Alpha
spectrometry
Drinking Water Samples
900.0 (EPA)
900.0 (EPA)
901.1 (EPA)
Drinking Water Samples
Qualitative
Determination1
D3084
(ASTM)
D3084
(ASTM)
901.1
(EPA)
901.1
(EPA)
D3084
(ASTM)
901.1
(EPA)
901.1
(EPA)
901.1
(EPA)
D3084
(ASTM)
D3084
(ASTM)
Confirmatory
Am-04-RC
(HASL-300)
Am-04-RC
(HASL-300)
901.1
(EPA)
901.1
(EPA)
Am-04-RC
(HASL-300)
901.1
(EPA)
901.1
(EPA)
901.1
(EPA)
EMSL-33
(EPA)
EMSL-33
(EPA)
Aqueous and Liquid Phase
Samples
7110B(SM)
7110B(SM)
Ga-01-R
(HASL-300)
Aqueous and Liquid Phase
Samples
Qualitative
Determination1
D3084
(ASTM)
D3084
(ASTM)
7120
(SM)
7120
(SM)
D3084
(ASTM)
7120
(SM)
Ga-01-R
(HASL-300)
7120
(SM)
D3084
(ASTM)
D3084
(ASTM)
Confirmatory
Am-04-RC
(HASL-300)
Am-04-RC
(HASL-300)
7120
(SM)
7120
(SM)
Am-04-RC
(HASL-300)
7120
(SM)
Ga-01-R
(HASL-300)
7120
(SM)
EMSL-33
(EPA)
EMSL-33
(EPA)
Soil and Sediment Samples
AP1
(ORISE)
AP1
(ORISE)
Ga-01-R
(HASL-300)
Soil and Sediment Samples
Qualitative
Determination1
Am-02-RC
(HASL-300)
D3084
(ASTM)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
D3084
(ASTM)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
D3084
(ASTM)
D3084
(ASTM)
Confirmatory
Am-01-RC3
(HASL-300)
Am-01-RC3
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Am-01-RC3
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
EMSL-33
(EPA)
EMSL-33
(EPA)
Surface Wipes
FRMAC, Vol 2, pg. 33
FRMAC, Vol 2, pg. 33
Ga-01-R
(HASL-300)
Surface Wipes
Qualitative
Determination1
D3084
(ASTM)
D3084
(ASTM)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
D3084
(ASTM)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
D3084
(ASTM)
D3084
(ASTM)
Confirmatory
Am-04-RC
(HASL-300)
Am-04-RC
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Am-04-RC
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
EMSL-33
(EPA)
EMSL-33
(EPA)
Air Filters
FRMAC, Vol 2, pg. 33
FRMAC, Vol 2, pg. 33
Ga-01-R
(HASL-300)
Air Filters
Qualitative
Determination1
D3084
(ASTM)
D3084
(ASTM)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
D3084
(ASTM)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
D3084
(ASTM)
D3084
(ASTM)
Confirmatory
Am-04-RC
(HASL-300)
Am-04-RC
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Am-04-RC
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
EMSL-33
(EPA)
EMSL-33
(EPA)
SAM Revision 4.0, Appendix B
B-1
September 29, 2008
-------
Analyte(s)
Polonium-210
Radium-226
Ruthenium-103
Ruthenium-106
Selenium-75
Strontium-90
Uranium-2342
Uranium-2352
Uranium-2382
CASRN
13981-52-7
13982-63-3
13968-53-1
13967-48-1
14265-71-5
10098-97-2
13966-29-5
15117-96-1
7440-61-1
Determinative
Technique
Alpha
spectrometry
Alpha counting /
spectrometry
Gamma
spectrometry
Gamma
spectrometry
Gamma
spectrometry
Beta counting by
low-background
gas flow
proportional
detector
Alpha counting /
spectrometry
Alpha counting /
spectrometry
Alpha counting /
spectrometry
Drinking Water Samples
Qualitative
Determination1
Po-02-RC
(HASL-300)
903.0
(EPA)
901.1
(EPA)
901.1
(EPA)
901.1
(EPA)
7500-Sr B
(SM)
908.04
(EPA)
908.04
(EPA)
908.04
(EPA)
Confirmatory
Po-02-RC
(HASL-300)
903.1
(EPA)
901.1
(EPA)
901.1
(EPA)
901.1
(EPA)
7500-Sr B
(SM)
D3972
(ASTM)
D3972
(ASTM)
D3972
(ASTM)
Aqueous and Liquid Phase
Samples
Qualitative
Determination1
Po-02-RC
(HASL-300)
7500-Ra B
(SM)
7120
(SM)
7120
(SM)
7120
(SM)
7500-Sr B
(SM)
7500-U B4
(SM)
7500-U B4
(SM)
7500-U B4
(SM)
Confirmatory
Po-02-RC
(HASL-300)
7500-Ra C
(SM)
7120
(SM)
7120
(SM)
7120
(SM)
7500-Sr B
(SM)
7500-U C
(SM)
7500-U C
(SM)
7500-U C
(SM)
Soil and Sediment Samples
Qualitative
Determination1
Po-02-RC
(HASL-300)
D3084
(ASTM)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Sr-03-RC
(HASL-300)
D3084
(ASTM)
D3084
(ASTM)
D3084
(ASTM)
Confirmatory
Po-02-RC
(HASL-300)
EMSL-19
(EPA)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Sr-03-RC
(HASL-300)
EMSL-33
(EPA)
EMSL-33
(EPA)
EMSL-33
(EPA)
Surface Wipes
Qualitative
Determination1
Method 1 1 1
(EPA)
D3084
(ASTM)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Sr-03-RC
(HASL-300)
D3084
(ASTM)
D3084
(ASTM)
D3084
(ASTM)
Confirmatory
Method 1 1 1
(EPA)
EMSL-19
(EPA)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Sr-03-RC
(HASL-300)
EMSL-33
(EPA)
EMSL-33
(EPA)
EMSL-33
(EPA)
Air Filters
Qualitative
Determination1
Method 1 1 1
(EPA)
D3084
(ASTM)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Sr-03-RC
(HASL-300)
D3084
(ASTM)
D3084
(ASTM)
D3084
(ASTM)
Confirmatory
Method 1 1 1
(EPA)
EMSL-19
(EPA)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Sr-03-RC
(HASL-300)
EMSL-33
(EPA)
EMSL-33
(EPA)
EMSL-33
(EPA)
1 In those cases where the same method is listed for qualitative determination and confirmatory analysis, qualitative determination can be performed by application of the method over a shorter count time than
that used for confirmatory analysis.
2 If it is suspected that the sample exists in refractory form (i.e., non-digestible or dissolvable material after normal digestion methods) or if there is a matrix interference problem, use ORISE Method AP11 for
qualitative determination or confirmatory analysis of alpha radioactivity.
3 In cases where only small sample volumes (£100 g) are available, use HASL-300 Method Pu-12-RC.
This method was developed for measurement of total uranium and does not distinguish between uranium isotopes.
SAM Revision 4.0, Appendix B
B-2
September 29, 2008
-------
Appendix C - Selected Pathosen Methods
Appendix C: Selected Pathogen Methods
SAM Revision 4.0 September 29, 2008
-------
Appendix C - Selected Pathosen Methods
SAM Revision 4.0 September 29, 2008
-------
Appendix C: Selected Pathogen Methods
Not all methods have been evaluated for each pathogen/sample type/environmental matrix combination in Appendix C. Each laboratory using these methods must
operate a formal quality assurance program and, at a minimum, analyze appropriate quality control samples (Section 7.1.2). Also, if required, a modification or a
replacement of a sample preparation method may be warranted for a specific pathogen/sample type/environmental matrix or a combination thereof. Additionally, the SAM
Pathogen primary and alternate points of contact should be consulted for additional guidance (Section 4.0, Points of Contact).
Note: If viability determinations are needed (e.g., evaluation of the efficacy of disinfection), a viability-based procedure (such as culture) should be used. Rapid analyis
techniques (such as PCR, some immunoassays) are preferred for estimating the extent of contamination and should be used in conjunction with culture for confirmation
of identification. In cases where a method is listed as "requires modification of analytical method," the analytical method should be used as a starting point and modified
as necessary for analysis of a particular sample type.
Pathogen(s)
[Disease]
Analytical
Technique
Analytical
Method
Solid1
(soil, powder)
Particulate2
(swabs, wipes,
HEPA)
Liquid/Water
(filter, grab)
Drinking Water
(filter, grab)
Aerosol
(growth media,
filter, liquid)
Bacteria
Bacillus anthracis
[Anthrax]
Brucella spp.
(6. abortus, B. melitensis, B. suis)
[Brucellosis]
Burkholderia mallei
[Glanders]
Burkholderia pseudomallei
[Melioidosis]
Campylobacterjejuni
[Campylobacteriosis]
Culture
Real-time PCR/
Immunoassay
Culture
Real-time PCR/
Immunoassay
Culture
Real-time PCR/
Immunoassay
Culture
Real-time PCR/
Immunoassay
Culture
Immunoassay
Real-time PCR
Basic Diagnostic Testing Protocols for Level A Laboratories for the Presumptive Identification of Bacillus anthracis
CDC/ASM/APHL
LRN comparable assays
Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism: Brucella species
ASM
LRN comparable assays
Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism: Burkholderia mallei and B. pseudomallei
ASM
LRN comparable assays
Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism: Burkholderia mallei and B. pseudomallei
ASM
LRN comparable assays
SM 9260 G
SM 9260 G
Molecular and Cellular
Probes 20: 269-279
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
SM 9260 G
SM 9260 G
Requires
modification of
analytical method
SM 9260 G
SM 9260 G
Requires
modification of
analytical method
Unlikely to be viable
Unlikely to be viable
Requires
modification of
analytical method
SAM Revision 4.0, Appendix C
C-1
September 29, 2008
-------
Pathogen(s)
[Disease]
Chlamydophila psittaci
(formerly known as Chlamydia psittaci)
[Psittacosis]
Coxiella burnetii
[Q-fever]
Escherichia coli O157:H7
Francisella tularensis
[Tularemia]
Leptospira
(L. interrogans Serovars
Icteroheamorrhagiae, Autralis, Balum,
Bataviae, Bejro, Pomona)
[Leptospirosis]
Listeria monocytogenes
[Listeriosis]
Analytical
Technique
Tissue culture
PCR
Culture
Real-time PCR/
Immunoassay
Culture
Immunoassay
Real-time PCR
Culture
Real-time PCR/
Immunoassay
Culture
Immunoassay
Culture
Immunoassay
Real-time PCR
Analytical
Method
Journal of Clinical
Microbiology
38(3): 1085-1093
Journal of Clinical
Microbiology
38(3): 1085-1093
Solid1
(soil, powder)
Requires
modification of
analytical method
Requires
modification of
analytical method
Particulate2
(swabs, wipes,
HEPA)
Requires
modification of
analytical method
Requires
modification of
analytical method
Liquid/Water
(filter, grab)
Requires
modification of
analytical method
Requires
modification of
analytical method
Drinking Water
(filter, grab)
Requires
modification of
analytical method
Requires
modification of
analytical method
Aerosol
(growth media,
filter, liquid)
Requires
modification of
analytical method
Requires
modification of
analytical method
If analysis of this pathogen is required, contact the LRN (404-639-2790) for information on the LRN laboratory capable of receiving and
processing the sample. In some cases, methods that apply to environmental samples may not be fully developed or validated.
LRN comparable assays
SM 9260 F
SM 9260 F
Applied &
Environmental
Microbiology 69(10):
6327-6333
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
SM 9260 F
SM 9260 F
Requires
modification of
analytical method
SM 9260 F
SM 9260 F
Requires
modification of
analytical method
Unlikely to be viable
Unlikely to be viable
Requires
modification of
analytical method
Basic Protocols for Level A Laboratories for the Presumptive Identification of Francisella tularensis
CDC/ASM/APHL
LRN comparable assays
SM 9260 I
SM 9260 I
FDA/Bacteriological
Analytical Manual
Chapter 10, 2003
FDA/Bacteriological
Analytical Manual
Chapter 10, 2003
USDA Laboratory
Guidebook
MLG 8A.03
SM 9260 I
SM 9260 I
FDA/Bacteriological
Analytical Manual
Chapter 10, 2003
FDA/Bacteriological
Analytical Manual
Chapter 10, 2003
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
SM 9260 I
SM 9260 I
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
SM 9260 I
SM 9260 I
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Unlikely to be viable
Unlikely to be viable
Unlikely to be viable
Unlikely to be viable
Requires
modification of
analytical method
SAM Revision 4.0, Appendix C
C-2
September 29, 2008
-------
Pathogen(s)
[Disease]
Non-typhoidal Salmonella
(Not applicable to S. Typhi)
[Salmonellosis]
Salmonella Typhi
[Typhoid fever]
Shigella spp.
[Shigellosis]
Staphylococcus aureus
Vibrio cholerae O1 and O1 39
[Cholera]
Analytical
Technique
Culture
Immunoassay
Real-time PCR
Culture
Immunoassay
Real-time PCR
Culture
Immunoassay
Real-time PCR
Culture
Culture
Immunoassay
Real-time PCR
Analytical
Method
EPA Method 1682
EPA Method 1682
Journal of Applied
Microbiology
102(2): 516-530
SM 9260 B
SM 9260 B
CDC Laboratory Assay:
S. Typhi
SM 9260 E
SM 9260 E
CDC Laboratory Assay:
Shigella
SM9213 B
SM 9260 H
SM 9260 H
CDC Laboratory Assay:
V. cholerae
Solid1
(soil, powder)
EPA Method 1682
EPA Method 1682
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Particulate2
(swabs, wipes,
HEPA)
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Liquid/Water
(filter, grab)
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
SM 9260 B
SM 9260 B
Requires
modification of
analytical method
SM 9260 E
SM 9260 E
Requires
modification of
analytical method
SM9213 B
SM 9260 H
SM 9260 H
Requires
modification of
analytical method
Drinking Water
(filter, grab)
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
SM 9260 B
SM 9260 B
Requires
modification of
analytical method
SM 9260 E
SM 9260 E
Requires
modification of
analytical method
SM9213 B
SM 9260 H
SM 9260 H
Requires
modification of
analytical method
Aerosol
(growth media,
filter, liquid)
Unlikely to be viable
Unlikely to be viable
Requires
modification of
analytical method
Unlikely to be viable
Unlikely to be viable
Requires
modification of
analytical method
Unlikely to be viable
Unlikely to be viable
Requires
modification of
analytical method
Requires
modification of
analytical method
Unlikely to be viable
Unlikely to be viable
Requires
modification of
analytical method
SAM Revision 4.0, Appendix C
C-3
September 29, 2008
-------
Pathogen(s)
[Disease]
Yersinia pestis
[Plague]
Analytical
Technique
Culture
Real-time PCR/
Immunoassay
Analytical
Method
Solid1
(soil, powder)
Particulate2
(swabs, wipes,
HEPA)
Liquid/Water
(filter, grab)
Drinking Water
(filter, grab)
Aerosol
(growth media,
filter, liquid)
Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism: Yersinia pestis
ASM
LRN comparable assays
Viruses
Adenoviruses:
Enteric and non-enteric (A-F)
Astroviruses
Caliciviruses: Noroviruses
Caliciviruses: Sapovirus
Coronavi ruses:
SARS-associated human coronavirus
Hepatitis E virus (HEV)
Influenza H5N1 virus
Tissue culture3
Real-time PCR
Integrated Cell
Culture/Reverse
transcription-PCR
Real-time reverse
transcription-PCR
Real-time reverse
transcription-PCR
Real-time reverse
transcription-PCR
Reverse
transcription-PCR
Real-time reverse
transcription-PCR
Real-time reverse
transcription-PCR
Applied &
Environmental
Microbiology 71(6):
3131-3136
Applied &
Environmental
Microbiology 71(6):
3131-3136
Canadian Journal of
Microbiology 50:
269-278
Canadian Journal of
Microbiology 50:
269-278
Journal of Clinical
Microbiology 42(10):
4679-4685
Journal of Medical
Virology 78(10):
1347-1353
Journal of Virological
Methods 122: 29-36
Journal of Virological
Methods
131(1): 65-71
Emerging Infectious
Diseases 11(8):
1303-1305
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Journal of Clinical
Microbiology Vol.
42(10): 4679-46S55
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
SAM Revision 4.0, Appendix C
C-4
September 29, 2008
-------
Pathogen(s)
[Disease]
Picornaviruses: Enteroviruses
Picornavi ruses:
Hepatitis A virus (HAV)
Reo viruses:
Rotavirus (Group A)
Analytical
Technique
Tissue culture
Reverse
transcription-PCR
Reverse transcription
PCR
Tissue culture
Reverse
transcription-PCR
Analytical
Method
USEPA Manual of
Methods for Virology
EPA/600/4-84/013,
April 2001
Applied &
Environmental
Microbiology 69(6):
3158-3164
Applied &
Environmental
Microbiology 69(6):
3158-3164
Applied &
Environmental
Microbiology 69(6):
3158-3164
Applied &
Environmental
Microbiology 69(6):
3158-3164
Solid1
(soil, powder)
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Particulate2
(swabs, wipes,
HEPA)
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Liquid/Water
(filter, grab)
USEPA Manual of
Methods for Virology
EPA/600/4-84/013,
April 2001
Applied &
Environmental
Microbiology 69(6):
31 58-31 644
Applied &
Environmental
Microbiology 69(6):
31 58-31 644
Applied &
Environmental
Microbiology 69(6):
31 58-31 644
Applied &
Environmental
Microbiology 69(6):
31 58-31 644
Drinking Water
(filter, grab)
USEPA Manual of
Methods for Virology
EPA/600/4-84/013,
April 2001
Applied &
Environmental
Microbiology 69(6):
31 58-31 644
Applied &
Environmental
Microbiology 69(6):
31 58-31 644
Applied &
Environmental
Microbiology 69(6):
31 58-31 644
Applied &
Environmental
Microbiology 69(6):
31 58-31 644
Aerosol
(growth media,
filter, liquid)
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Requires
modification of
analytical method4
Protozoa
Cryptosporidium spp.
[Cryptosporidiosis]
Entamoeba histolytica
Tissue culture
IMS/FA
Real-time PCR
Culture
Real-time PCR
Applied &
Environmental
Microbiology 65(9):
3936-3941
EPA Method 1622
and/or Method 1 623
Applied &
Environmental
Microbiology 73(13):
4218-4225
Journal of Parasitology
58(2): 306-310
Journal of Clinical
Microbiology
43(11): 5491-5497
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
EPA Method 1622
and/or Method 1 623
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
EPA Method 1622
and/or Method 1 623
Applied &
Environmental
Microbiology 73(13):
4218-4225
Requires
modification of
analytical method
Requires
modification of
analytical method
Unlikely to be found
Unlikely to be found
Unlikely to be found
Unlikely to be found
Unlikely to be found
SAM Revision 4.0, Appendix C
C-5
September 29, 2008
-------
Pathogen(s)
[Disease]
Giardiaspp.
[Giardiasis]
Toxoplasma gondii
[Toxoplasmosis]
Analytical
Technique
Culture
IMS/FA
Animal infectivity
Real-time PCR
Analytical
Method
Trans. R. Soc. Trap.
Med. Hyg. 77(4):
487-488
EPA Method 1623
Emerging Infectious
Diseases 12(2):
326-329
Applied &
Environmental
Microbiology 70(7):
4035-4039
Solid1
(soil, powder)
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Particulate2
(swabs, wipes,
HEPA)
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Liquid/Water
(filter, grab)
Requires
modification of
analytical method
EPA Method 1623
Emerging Infectious
Diseases 1 2(2):
326-329
Applied &
Environmental
Microbiology 70(7):
4035-4039
Drinking Water
(filter, grab)
Requires
modification of
analytical method
EPA Method 1623
Emerging Infectious
Diseases 1 2(2):
326-329
Applied &
Environmental
Microbiology 70(7):
4035-4039
Aerosol
(growth media,
filter, liquid)
Unlikely to be found
Unlikely to be found
Unlikely to be found
Unlikely to be found
Helminths
Baylisascaris procyonis
[Raccoon roundworm infection]
Embryonation of eggs
and microscopy
EPA/625/R-92/013
EPA/625/R-92/013
Requires
modification of
analytical method
EPA/625/R-92/013
Requires
modification of
analytical method
Unlikely to be found
General Remediation Efficacy
Biological indicator (spore) strips
Culture
Manufacturers' Instructions
1 Solid samples (except those containing viruses) should be (1) prepared for culture according to EPA Method 1680, Fecal Coliforms in Sewage Sludge (Biosolids) by Multiple-Tube Fermentation using
Lauryl Tryptose Broth (LTB) and EC Medium, and (2) prepared for PCR according to "Quantification of Bias Related to the Extraction of DMA Directly from Soils," Frostegard, A., Courtois, S., Ramisse, V.,
Clerc, S., Bernillon, D., Le Gall, F., Jeannin, P., Nesme, X., and Simonet, P. 1999. Applied and Environmental Microbiology. 65(12): 5409-5420.
2 Particulate samples (except those containing viruses) should be prepared for culture according to "Swab Materials and Bacillus anthracis Spore Recovery from Nonporous Surfaces," Rose, L, Jensen,
B., Peterson, A., Banerjee, S.N., and Arduino, M.J. 2004. Emerging Infectious Diseases. 10(6): 1023-1029 and "Evaluation of a Macrofoam Swab Protocol for the Recovery of Bacillus anthracis Spores
from a Steel Surface," Hodges, L.R., Rose, L.J., Peterson, A., Nobel-Wang, J., and Arduino, M.J. 2006. Applied and Environmental Microbiology. 72(6): 4429-4430.
3 Given that adenovirus 40 and 41 can be difficult to grow in culture, additional cell lines such as G293 (Journal of Medical Virology. 1983. 11 (3): 215-231) or Caco-2 (Journal of Medical Virology. 1994.
44(3): 310-315) may be considered when these viruses are suspected to be present.
4 Samples should be prepared according to procedures found in USEPA Manual of Methods for Virology EPA/600/4-84/013, April 2001.
SAM Revision 4.0, Appendix C
C-6
September 29, 2008
-------
Appendix D - SelectedBiotoxin Methods
Appendix D: Selected Biotoxin Methods
SAM Revision 4.0 September 29, 2008
-------
Appendix D - SelectedBiotoxin Methods
SAM Revision 4.0 September 29, 2008
-------
Appendix D: Selected Biotoxin Methods
Analyte(s)
CAS RN / Description
Analysis Type
Analytical
Technique
Aerosol
(filter/cassette, liquid
impinger)
Solid
(soil, powder)
Particulate
(swabs, wipes, filters)
Liquid/Drinking Water
Protein
Abrin
Botulinum neurotoxins
(Serotoypes A, B, E, F)
Ricin
Shiga and Shiga-like Toxins
(Stx, Stx-1 , Stx-2)
1393-62-0 (abrin)/
Glycoprotein consisting of a
deadenylase (25-32 kDa A
chain) and lectin (35 kDa B
chain); an agglutinin (A2B2)
may be present in the crude
preparations
Protein composed of -100
kDa heavy chain and -50
kDa light chain; can be
complexed with
hemagglutinin and non-
hemagglutinin components
for total MW of -900 kDa
9009-86-3 (ricin) /
60 kDa glycoprotein
composed of two subunits
(-32 kDa A chain and -34
kDa B chain); an agglutinin
of MW 120 kDa may be
present in crude
preparations
5254-40-3 (ricinine) /
small molecule, ricin marker
75757-64-1 (Stx) /
Protein composed of one
-32 kDa A chain and five
7.7 kDa B chains
Presumptive
Confirmatory
Biological
Activity
Presumptive
Confirmatory
Biological
Activity
Presumptive
Complementary
Presumptive
(ricinine)
Confirmatory
Biological
Activity
Presumptive
Confirmatory
Biological
Activity
Immunoassay1
Ribosome
inactivation assay
Enzyme activity2
Immunoassay3
Immunoassay3
(ELISA)
Mouse Bioassay
Immunoassay1
LC-MS
Immunoassay
Enzyme activity2
Optical
immunoassay
Immunoassay
(ELISA)
Ribosome
inactivation
assav2
Adapted from 1 1 9th AOAC
Annual Meeting &
Exposition, 2005, p. 613
Adapted from
Pharmacology &
Toxicology 88(5): 255-260
Adapted from Analytical
Biochemistry 378(1): 87-89
Adapted from lateral flow
immunassay kits
Adapted from FDA
Bacteriological Analytical
Manual, Chapter 17
Adapted from FDA
Bacteriological Analytical
Manual, Chapter 17
Adapted from lateral flow
immunassay kits
Adapted from Journal of
Analytical Toxicology
29(3): 149-155
Adapted from Journal of
AOAC International 91 (2):
376-382
Adapted from Analytical
Biochemistry 378(1): 87-89
Adapted from Journal of
Clinical Microbiology
45(10): 3377-3380
Adapted from FDA
Bacteriological Analytical
Manual, Appendix 1
Adapted from
Pharmacology &
Toxicology 88(5): 255-260
Adapted from 1 1 9th AOAC
Annual Meeting &
Exposition, 2005, p. 613
Adapted from
Pharmacology &
Toxicology 88(5): 255-260
Adapted from Analytical
Biochemistry 378(1): 87-89
Adapted from 1 1 9th AOAC
Annual Meeting &
Exposition, 2005, p. 613
Adapted from
Pharmacology &
Toxicology 88(5): 255-260
Adapted from Analytical
Biochemistry 378(1): 87-89
Adapted from 1 1 9th AOAC
Annual Meeting &
Exposition, 2005, p. 613
Adapted from
Pharmacology &
Toxicology 88(5): 255-260
Adapted from Analytical
Biochemistry 378(1): 87-89
LRN
If analysis for this agent is required in solid, particulate, or liquid samples, contact
the LRN at (404) 639-2790 for information of the closest LRN laboratory capable
of receiving and processing the sample. The terms presumptive and confirmatory
as used for LRN methods are described in Section 8.2.1 .
LRN
If analysis for this agent is required in solid, particulate, or liquid samples, contact
the LRN at (404) 639-2790 for information of the closest LRN laboratory capable
of receiving and processing the sample. The terms presumptive and confirmatory
as used for LRN methods are described in Section 8.2.1 .
Adapted from Journal of
Analytical Toxicology
29(3): 149-155
Adapted from Journal of
AOAC International 91 (2):
376-382
Adapted from Analytical
Biochemistry 378(1): 87-89
Adapted from Journal of
Clinical Microbiology
45(10): 3377-3380
Adapted from FDA
Bacteriological Analytical
Manual, Appendix 1
Adapted from
Pharmacology &
Toxicology 88(5): 255-260
Adapted from Journal of
Analytical Toxicology
29(3): 149-155
Adapted from Journal of
AOAC International 91 (2):
376-382
Adapted from Analytical
Biochemistry 378(1): 87-89
Adapted from Journal of
Clinical Microbiology
45(10): 3377-3380
Adapted from FDA
Bacteriological Analytical
Manual, Appendix 1
Adapted from
Pharmacology &
Toxicology 88(5): 255-260
Adapted from Journal of
Analytical Toxicology
29(3): 149-155
Adapted from Journal of
AOAC International 91 (2):
376-382
Adapted from Analytical
Biochemistry 378(1): 87-89
Adapted from Journal of
Clinical Microbiology
45(10): 3377-3380
Adapted from FDA
Bacteriological Analytical
Manual, Appendix 1
Adapted from
Pharmacology &
Toxicology 88(5): 255-260
SAM Revision 4.0, Appendix D
D-1
September 29, 2008
-------
Analyte(s)
Staphylococcal enterotoxins
(SEB)
Staphylococcal enterotoxins
(SEA, SEC)
CAS RN / Description
39424-53-8 (SEB)/
Monomeric protein of
- 28 kDa
37337-57-8 (SEA)
39424-54-9 (SEC) /
Monomeric proteins of
27 27 5 kDa
Analysis Type
Presumptive
Confirmatory
Biological
Activity
Presumptive
Confirmatory
Biological
Activity
Analytical
Technique
Immunoassay
TBD
TBD
Immunoassay
TBD
TBD
Aerosol
(filter/cassette, liquid
impinger)
Adapted from 993.06
(AOAC)
TBD
TBD
Adapted from 993.06
(AOAC)
TBD
TBD
Solid
(soil, powder)
Particulate
(swabs, wipes, filters)
Liquid/Drinking Water
LRN
If analysis for this agent is required in solid, particulate, or liquid samples, contact
the LRN at (404) 639-2790 for information of the closest LRN laboratory capable
of receiving and processing the sample. The terms presumptive and confirmatory
as used for LRN methods are described in Section 8.2.1 .
TBD
TBD
Adapted from 993.06
(AOAC)
TBD
TBD
TBD
TBD
Adapted from 993.06
(AOAC)
TBD
TBD
TBD
TBD
Adapted from 993.06
(AOAC)
TBD
TBD
Small Molecule
Aflatoxin
(Type B1)
a Amanitin
Anatoxin-a
Brevetoxins
(B form)
27261-02-5
64285-06-9
79580-28-2
Presumptive
Confirmatory
Presumptive
Confirmatory
Presumptive
Confirmatory
Presumptive
Confirmatory
Immunoassay
HPLC-FL
Immunoassay
HPLC
ampero metric
detection
TBD
HPLC-FL
(precolumn
derivatization)
Immunoassay
HPLC-MS-MS
Adapted from 991. 31
(AOAC)
Adapted from 994. 08
(AOAC)
Adapted from Journal of
Food Protection 68(6):
1294-1301
Adapted from Journal of
Chromatography
563(2): 299-311
TBD
Adapted from Biomedical
Chromatography B
10:46-47
Adapted from
Environmental Health
Perspectives
110(2): 179-185
Adapted from Toxicon
43(4): 455-465
Adapted from 991. 31
(AOAC)
Adapted from 994. 08
(AOAC)
Adapted from Journal of
Food Protection 68(6):
1294-1301
Adapted from Journal of
Chromatography
563(2): 299-311
TBD
Adapted from Biomedical
Chromatography B
10:46-47
Adapted from
Environmental Health
Perspectives
110(2): 179-185
Adapted from Toxicon
43(4): 455-465
Adapted from 991. 31
(AOAC)
Adapted from 994. 08
(AOAC)
Adapted from Journal of
Food Protection 68(6):
1294-1301
Adapted from Journal of
Chromatography
563(2): 299-311
TBD
Adapted from Biomedical
Chromatography B
10:46-47
Adapted from
Environmental Health
Perspectives
110(2): 179-185
Adapted from Toxicon
43(4): 455-465
Adapted from 991. 31
(AOAC)
Adapted from 994. 08
(AOAC)
Adapted from Journal of
Food Protection 68(6):
1294-1301
Adapted from Journal of
Chromatography
563(2): 299-311
TBD
Adapted from Biomedical
Chromatography B
10:46-47
Adapted from
Environmental Health
Perspectives
110(2): 179-185
Adapted from Toxicon
43(4): 455-465
SAM Revision 4.0, Appendix D
D-2
September 29, 2008
-------
Analyte(s)
a Conotoxin
Cylindrospermopsin
Diacetoxyscirpenol (DAS)
Microcystins
Principal isoforms: LA, LR, LW,
RR, YR
Picrotoxin
Saxitoxins
Principal isoforms:
Saxitoxin (SIX)
Neosaxitoxin (NEOSTX)
Gonyautoxin (GTX)
Decarbamoylgonyautoxin (dcGTX)
Decarbamoylsaxitoxin (dcSTX)
T-2 Mycotoxin
CAS RN / Description
156467-85-5
2270-40-8
96180-79-9 (LA)
101043-37-2 (LR)
157622-02-1 (LW)
111755-37-4 (RR)
101 064-48-6 (YR)
124-87-8
35523-89-8 (SIX)
64296-20-4 (NEOSTX)
77462-64-7 (GTX)
None given (dcGTX)
58911-04-9 (dcSTX)
21259-20-1
Analysis Type
Presumptive
Confirmatory
Presumptive
Confirmatory
Presumptive
Confirmatory
Presumptive
Confirmatory
Presumptive
Confirmatory
Presumptive
Confirmatory
Presumptive
Confirmatory
Analytical
Technique
Immunoassay
HPLC-MS
Immunoassay
HPLC-PDA
Immunoassay
LC/APCI-MS
Immunoassay/
Phosphatase
assay
HPLC-PDA
Immunoassay
HPLC
Immunoassay
HPLC-FL
(post column
derivatization)
Immunoassay
LC/APCI-MS
Aerosol
(filter/cassette, liquid
impinger)
Adapted from Biochemical
Journal 328: 245-250
Adapted from Journal of
Medicinal Chemistry
47(5): 1234-1241
Adapted from ELISAkits
for Cylindrospermopsin
Adapted from FEMS
Microbiology Letters
216(2): 159-164
Adapted from International
Journal of Food
Microbiology 6(1): 9-17
Adapted from Rapid
Communications in Mass
Spectra metry
20(9): 1422-1428
Adapted from Journal of
AOAC International 84(4):
1035-1044
Adapted from Analyst
119(7): 1525-1530
TBD
Adapted from Journal of
Pharmaceutical and
Biomedical Analysis
7(3): 369-375
Adapted from ELISA kits
for Saxitoxins
Adapted from Journal of
AOAC International 78(2):
528-532
Adapted from Journal of
Food Protection 68(6):
1294-1301
Adapted from Rapid
Communications in Mass
Spectra metry
20(9): 1422-1428
Solid
(soil, powder)
Adapted from Biochemical
Journal 328: 245-250
Adapted from Journal of
Medicinal Chemistry
47(5): 1234-1241
Adapted from ELISA kits
for Cylindrospermopsin
Adapted from FEMS
Microbiology Letters
216(2): 159-164
Adapted from International
Journal of Food
Microbiology 6(1): 9-17
Adapted from Rapid
Communications in Mass
Spectra metry
20(9): 1422-1428
Adapted from Journal of
AOAC International 84(4):
1035-1044
Adapted from Analyst
119(7): 1525-1530
TBD
Adapted from Journal of
Pharmaceutical and
Biomedical Analysis
7(3): 369-375
Adapted from ELISAkits
for Saxitoxins
Adapted from Journal of
AOAC International 78(2):
528-532
Adapted from Journal of
Food Protection 68(6):
1294-1301
Adapted from Rapid
Communications in Mass
Spectra metry
20(9): 1422-1428
Particulate
(swabs, wipes, filters)
Adapted from Biochemical
Journal 328: 245-250
Adapted from Journal of
Medicinal Chemistry
47(5): 1234-1241
Adapted from ELISAkits
for Cylindrospermopsin
Adapted from FEMS
Microbiology Letters
216(2): 159-164
Adapted from International
Journal of Food
Microbiology 6(1): 9-17
Adapted from Rapid
Communications in Mass
Spectra metry
20(9): 1422-1428
Adapted from Journal of
AOAC International 84(4):
1035-1044
Adapted from Analyst
119(7): 1525-1530
TBD
Adapted from Journal of
Pharmaceutical and
Biomedical Analysis
7(3): 369-375
Adapted from ELISAkits
for Saxitoxins
Adapted from Journal of
AOAC International 78(2):
528-532
Adapted from Journal of
Food Protection 68(6):
1294-1301
Adapted from Rapid
Communications in Mass
Spectra metry
20(9): 1422-1428
Liquid/Drinking Water
Adapted from Biochemical
Journal 328: 245-250
Adapted from Journal of
Medicinal Chemistry
47(5): 1234-1241
Adapted from ELISA kits
for Cylindrospermopsin
Adapted from FEMS
Microbiology Letters
216(2): 159-164
Adapted from International
Journal of Food
Microbiology 6(1): 9-17
Adapted from Rapid
Communications in Mass
Spectra metry
20(9): 1422-1428
Adapted from Journal of
AOAC International 84(4):
1035-1044
Adapted from Analyst
119(7): 1525-1530
TBD
Adapted from Journal of
Pharmaceutical and
Biomedical Analysis
7(3): 369-375
Adapted from ELISAkits
for Saxitoxins
Adapted from Journal of
AOAC International 78(2):
528-532
Adapted from Journal of
Food Protection 68(6):
1294-1301
Adapted from Rapid
Communications in Mass
Spectra metry
20(9): 1422-1428
SAM Revision 4.0, Appendix D
D-3
September 29, 2008
-------
Analyte(s)
Tetrodotoxin
CAS RN / Description
9014-39-5
Analysis Type
Presumptive
Confirmatory
Analytical
Technique
Immunoassay
LC/ESI-MS
Aerosol
(filter/cassette, liquid
impinger)
Adapted from Journal of
Clinical Laboratory
Analysis 6(2): 65-72
Adapted from Analytical
Biochemistry 290(1): 10-17
Solid
(soil, powder)
Adapted from Journal of
Clinical Laboratory
Analysis 6(2): 65-72
Adapted from Analytical
Biochemistry 290(1): 10-17
Particulate
(swabs, wipes, filters)
Adapted from Journal of
Clinical Laboratory
Analysis 6(2): 65-72
Adapted from Analytical
Biochemistry 290(1): 10-17
Liquid/Drinking Water
Adapted from Journal of
Clinical Laboratory
Analysis 6(2): 65-72
Adapted from Analytical
Biochemistry 290(1): 10-17
1 Crude preparations of ricin and abrin may also contain agglutinins that are unique to castor beans and rosary peas, respectively, and that can cross-react in the immunoassays.
2 This assay does not test for cell binding: cell culture assays are being developed to test for cell binding but are not currently available. The only readily available assay to test for both the cell binding and
enzymatic activity of the intact (whole) toxin is a mouse bioassay.
Immunoassays may produce variable results with uncomplexed form of toxin.
SAM Revision 4.0, Appendix D
D-4
September 29, 2008
------- |