EPA/600/R-04/126E September 29, 2009
United States
Environmental Protection
Agency
Standardized Analytical Methods
for Environmental Restoration
Following Homeland Security Events
REVISION 5.0
~f 4
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Office of Research and Development
National Homeland Security Research Center
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EPA/600/R-04/126E| September 2009 www.epa.gov/sam
Standardized Analytical Methods
for Environmental Restoration
Following Homeland Security
Events - Revision 5.0
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
Cincinnati, OH 45268
Office of Research and Development
National Homeland Security Research Center, Response Capability Enhancement
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Disclaimer
Disclaimer
The 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:
Rob Rothman
National Homeland Security Research Center
Office of Research and Development (NG16)
U.S. Environmental Protection Agency
26 West Martin Luther King Drive
Cincinnati, OH 45268
(513)569-7187
rothman.rob@epa.gov
Questions concerning this document and its information also can be submitted using the SAM Web site:
www.epa.gov/sam.
SAM Revision 5.0 ii September 29, 2009
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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. The information also can be found on the SAM Web site
(www.epa.gov/sam), which provides searchable links to supporting information based on SAM
analytes and the analytical methods listed.
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, analytical interferences, and target risk levels.
Many of the SAM analytes have only recently become an environmental concern. EPA is actively
pursuing development and validation of Standard 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 guidlines 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 5.0 iii September 29, 2009
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Foreword
Foreword
The U.S. Environmental Protection Agency (EPA) is charged by Congress to protect human health and
the environment and specifically, to protect the Nation's land, air, and water resources. Since 1970, EPA
has been working toward a cleaner and healthier environment for the American people. To help meet this
mandate, EPA's research program provides data and technical support for solving environmental
problems today and for building the scientific base necessary to manage our ecological resources wisely,
understand how pollutants affect our health and prevent or reduce environmental risks in the future.
Following the terrorist attacks of September 11, 2001, and the subsequent mailing of anthrax-tainted
letters, EPA's role with respect to homeland security was expanded. Presidential Directives identified
EPA both as the primary federal agency responsible for the country's water supplies and for
decontamination following a chemical, biological, and/or radiological (CBR) attack. To provide
scientific and technical support to help EPA meet this expanded role, EPA's National Homeland Security
Research Center (NHSRC) was established. The NHSRC research program is focused on developing and
delivering reliable, responsive expertise and products based on scientific research and evaluations of
technology. NHSRC's research is conducted to help protect the country's water infrastructure and to
facilitate decontamination of indoor and outdoor areas in the event of CBR attacks.
One specific goal of NHSRC's research is to support the Environmental Response Laboratory Network
(ERLN), a nationwide network of federal, state and local environmental laboratories. Toward this end,
NHSRC has undertaken research designed to provide appropriate, effective, and verified technologies and
methods to understand the risks posed by CBR agents and to enhance EPA's ability to detect, contain, and
clean up in the event of an incident involving such agents. This document provides a compendium of
methods that can be used when laboratories are faced with analytical demands associated with an
environmental restoration crisis involving CBR contaminants. Additionally, this document can be used as
a tool to identify analytes that require further method development and verification to ensure desired
performance.
This publication represents the fifth version and update to the analytical methods available for use in an
environmental crisis event requiring decontamination and restoration. The information contained within
this document will continue to be revised as our research progress and new information becomes
available.
Cynthia Sonich-Mullin, Acting Director
National Homeland Security Research Center
SAM Revision 5.0 iv September 29, 2009
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Abbreviations and Acronyms
Abbreviations and Acronyms
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)
ASM American Society for Microbiology
ASTM ASTM International (formerly the American Society for Testing and Materials)
AWWA American Water Works Association
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, and/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
CRL Chicago Regional Laboratory
CVAA Cold vapor atomic absorption
2-CVAA 2-Chlorovinylarsonous acid
CVAFS Cold vapor atomic fluorescence spectrometry
2,4-D 2,4-Dichlorophenoxyacetic acid
DAPI 4',6-Diamidino-2-phenylindole
DAS Diacetoxyscirpenol
DAS-HG-HSA Diacetoxyscirpenol hemiglutarate human serum albumin
DAS-HS-HRP Diacetoxyscirpenol hemisuccinate horseradish peroxidase conjugate
DB-1 100%Dimethylpolysiloxane
DBPR Division of Bioterrorism Preparedness and Response
DHS U.S. Department of Homeland Security
DIC Differential interference contrast
DIG-ELISA Digoxigenin labeled enzyme-linked immunosorbent assay
DIMP Diisopropyl methylphosphonate
DNA Deoxyribonucleic acid
2,4-DNPH 2,4-Dinitrophenylhydrazine
SAM Revision 5.0
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Abbreviations and Acronyms
DoD U.S. Department of Defense
DOE U.S. Department of Energy
DOT U.S. Department of Transportation
DPD N,N-Diethyl-p-phenylenediamine
DQO Data quality objective
DTPA Diethylenetriamine-pentaacetate
EA2192 Diisopropylaminoethyl methylthiolophosphonate
ECD Electron capture detector
e-CFR Electronic Code of Federal Regulations
ECL Electrochemiluminescence
ED 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
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 U.S. Environmental Protection Agency
EQL Estimated quantitation limit
ESI Electrospray ionization
ETV Environmental Technology Verification
FA Fluorescence assay
FBI U.S. Federal Bureau of Investigation
FDA U.S. Food and Drug Administration
FEMS Federation of European Microbiological Societies
FGI Fluorescein derivative of Conus geographus a-conotoxin
FID Flame ionization 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 ionization 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
GE 1-Methylethyl ester ethylphosphonofluoridic acid
Ge Germanium
Ge(Li) Germanium (Lithium)
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Abbreviations and Acronyms
GESTIS
GF
GFAA
GTC
HAdV
HASL
HAV
HCoV
HEV
HD
HHS
HMTD
HMX
HN-1
HN-2
HN-3
HPGe
HPLC
HPLC-FL
HPLC-MS
HPLC-MS-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
ISO
ISO
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
Sulfur mustard / mustard gas; bis(2-chloroethyl) sulfide
U.S. 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 chromatography - fluorescence
High performance liquid chromatography - mass spectrometery
High performance liquid chromatography tandem mass spectrometry
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
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://www.inchem.org/
Inorganic
Intraperitoneally
Integrated Risk Information System (EPA)
Ion specific electrode
Impregnated silica gel
International Organization for Standardization
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Abbreviations and Acronyms
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 or Liquid chromatography
LC/APCI-MS Liquid chromatography / atmospheric pressure chemical ionization - mass spectrometry
LC/ESI-MS Liquid chromatography / electrospray ionization - 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
mAbs Monoclonal antibodies
MARLAP Multi-Agency Radiological Laboratory Analytical Protocols (EPA/402/3-04/001 A, B, C)
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
MRM Multiple reaction monitoring
mRNA Messenger ribonucleic acid
MS Mass spectrometer or Mass spectrometry
MS-MS Tandem mass spectrometry
MS/MSD Matrix spike/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 EPA 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
NoV Norovirus
SAM Revision 5.0
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Abbreviations and Acronyms
NOS Not otherwise specified
NPD Nitrogen-phosphorus detector
NRC U.S. Nuclear Regulatory Commission
NRMRL EPA National Risk Management Research Laboratory
nS nano Siemens
NTIS National Technical Information Service
NTU Nephelometric turbidity units
OAQPS EPA Office of Air Quality Planning and Standards
OAR EPA Office of Air and Radiation
ORAU Oak Ridge Associated Universities
ORD EPA Office of Research and Development
ORF Open reading frame
ORIA Office of Radiation and Indoor Air
ORISE Oak Ridge Institute for Science and Education
OSWER EPA Office of Solid Waste and Emergency Response
OSHA Occupational Safety and Health Administration
OVS OSHA versatile sampler
OW 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
PID50 50% Pig infectious dose
PLET Polymyxin, lysozyme, EDTA, thallous acetate
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 Qittp: //www .pubmed. govX
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
SAM Standardized Analytical Methods for Environmental Restoration Following Homeland
Security Events
SAP Standard Analytical Protocol
SAM Revision 5.0
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Abbreviations and Acronyms
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)
SRM Single reaction monitoring
STEC Shiga-toxigenic E. coli
STEL Short term exposure limit
STX Saxitoxin
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 Transmission electron microscopy
TETR Touchdown enzyme time release
TFA Trifluoroacetic acid
™ Unregistered trademark
TM 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
TP-S-1 Trypticase-panmede liver digest-serum
TRU Transuranic
TSB Tryptic soy broth
TSAye Trypticase™ soy agar with yeast extract
TSI Triple sugar iron
TSP Thermospray
TSP-MS Thermospray -mass spectrometry
TTN Technical Transfer Network
TTX Tetrodotoxin
TYI-S-33 Trypticase-yeast-iron-serum
UF Ultrafiltration
U.S. United States
USDA U.S. Department of Agriculture
USGS U.S. 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
VOCs Volatile organic compounds
SAM Revision 5.0
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Abbreviations and Acronyms
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 5.0 xi September 29, 2009
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Abbreviations and Acronyms
SAM Revision 5.0 xii September 29, 2009
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Acknowledgments
Acknowledgments
Contributions of the following individuals and organizations to the development of SAM Revision 5.0 are
gratefully acknowledged. Please refer to older versions of SAM for historical acknowledgments.
United States Environmental Protection Agency (EPA)
• Office of Research and Development, National Homeland Security Research Center
Kathy Hall
Romy Lee
Alan Lindquist
Matthew Magnuson
Tonya Nichols
Eugene Rice
Rob Rothman
Frank Schaefer
Sanjiv Shah
Sarah Taft
Oba Vincent
Stuart Willison
• Office of Research and Development, National Exposure Research Laboratory (NERL), Las
Vegas
Stephen Pia (Environmental Sciences Division)
• Office of Research and Development, National Exposure Research Laboratory (NERL),
Cincinnati
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)
Jeanelle Martinez (Office of Emergency Management)
Marissa Mullins (Office of Emergency Management)
Terry Smith (Office of Emergency Management)
• Office of Air and Radiation, Office of Radiation and Indoor Air
George Dilbeck (National Air and Radiation Environmental Laboratory)
John Griggs (National Air and Radiation Environmental Laboratory)
Daniel Mackney (National Air and Radiation Environmental Laboratory)
• Office of Water, Office of Ground Water and Drinking Water
Malik Raynor (Water Security Division)
• Office of Prevention, Pesticides, and Toxic Substances, Office of Pesticide Programs
Elizabeth Flynt (Stennis Space Center)
• Office of Enforcement and Compliance Assurance, Office of Criminal Enforcement,
Forensics and Training
Don Smith (National Enforcement Investigations Center)
SAM Revision 5.0 xiii September 29, 2009
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Acknowledgments
• EPA Regions
Katie Adams (Region 10)
Jack Berges (Region 9)
Diane Gregg (Region 6)
Ted Haigh (Region 5)
Stephanie Harris (Region 10)
Jesse Kiernan (Region 8)
Ed O'Neill (Region 6)
Steve Reimer (Region 10)
Michael Wasko (Region 4)
Larry Zintek (Region 5)
United States Department of Health and Human Services
• Centers for Disease Control and Prevention (CDC)
Kevin Ashley (National Institute for Occupational Safety and Health)
Clayton B'Hymer (National Institute for Occupational Safety and Health)
Raymond Biagini (National Institute for Occupational Safety and Health)
Barun De (National Center for Zoonotic, Vector-Borne, and Enteric Diseases)
Jay Gee (National Center for Zoonotic, Vector-Borne, and Enteric Diseases)
Mindy Glass (National Center for Zoonotic, Vector-Borne, and Enteric Diseases)
Vincent Hill (National Center for Zoonotic, Vector-Borne, and Enteric Diseases)
Alex Hoffmaster (National Center for Zoonotic, Vector-Borne, and Enteric Diseases)
Rudolph Johnson (National Center for Environmental Health)
Jennifer Links (National Center for Environmental Health)
Chung Marston (National Center for Zoonotic, Vector-Borne, and Enteric Diseases)
Stephen Morse (National Center for Preparedness, Detection and Control of Infectious Diseases)
Michele Parsons (National Center for Zoonotic, Vector-Borne, and Enteric Diseases)
Laura Rose (National Center for Preparedness, Detection and Control of Infectious Diseases)
Richard Wang (National Center for Environmental Health)
Betsy Weirich (National Center for Preparedness, Detection and Control of Infectious Diseases)
Lihua Xiao (National Center for Zoonotic, Vector-Borne, and Enteric Diseases)
• United States Food and Drug Administration
Eric Garber
Michael McLaughlin
Socrates Trujillo
United States Department of Agriculture
Mark Jenkins
Robert Phillips
United States Department of Commerce
Peter Moeller (National Oceanic & Atmospheric Administration)
United States Department of Defense
Jon Davis (U.S. Army, Medical Research Institute of Infectious Diseases)
Paul Grasso (U.S. Army)
Johnathan Kiel (U.S. Air Force)
Jose-Luis Sagripanti (U.S. Army Research, Development, and Engineering Command - Edgewood
Chemical Biological Center)
Elaine Strauss (U.S. Navy, Naval Surface Warfare Center Dahlgren Division)
Ronald Swatski (U.S. Army, Center for Health Promotion and Preventive Medicine)
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Acknowledgments
United States Department of Energy
Steve Goldberg (New Brunswick Laboratory)
United States Geological Survey
Donna Francy
State Agencies
Jack Bennett (State of Connecticut Department of Public Health)
Timothy Fitzpatrick (Florida Department of Environmental Protection)
Liang Lin (Florida Department of Environmental Protection)
Christopher Retarides (Virginia State Department of General Services)
Colin Wright (Florida Department of Environmental Protection)
National Laboratories
Staci Kane (Lawrence Livermore National Laboratory)
Carolyn Koester (Lawrence Livermore National Laboratory)
Timothy Straub (Pacific Northwest National Laboratory)
Environmental Management Support, Inc.
Anna Berne
G. Fred Lee & Associates
Fred Lee
Hamilton Sundstrand
Richard Trubey
Texas A&M University - Corpus Christi
Paul Zimba
Computer Sciences Corporation
Eric Boring
Joan Cuddeback
Danielle Garozzo
Melody Jensen
Larry Umbaugh
Joshua Vinson
SAM Revision 5.0 xv September 29, 2009
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Acknowledgments
SAM Revision 5.0 xvi September 29, 2009
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Table of Contents
Standardized Analytical Methods for Environmental Restoration
Following Homeland Security Events
Revision 5.0
September 29, 2009
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 Guidelines 14
5.1.1 Standard Operating Procedures for Identifying Chemical Methods 14
5.1.2 General Quality Control (QC) Guidelines for Chemical Methods 33
5.1.3 Safety and Waste Management 34
5.2 Method Summaries 35
5.2.1 EPA Method 8: Determination of Sulfuric Acid and Sulfur Dioxide Emissions from
Stationary Sources 35
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 245.1: Determination of Mercury in Water by Cold Vapor Atomic Absorption
Spectrometry (CVAA) 38
5.2.5 EPA Method 252.2: Osmium (Atomic Absorption, Furnace Technique) 38
5.2.6 EPA Method 300.1, Revision 1.0: Determination of Inorganic Anions in Drinking Water by
Ion Chromatography 39
5.2.7 EPA Method 335.4: Determination of Total Cyanide by Semi-Automated Colorimetry 40
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 41
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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 42
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 42
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 43
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
44
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 44
5.2.15 EPA Method 614: The Determination of Organophosphorus Pesticides in Municipal and
Industrial Wastewater 45
5.2.16 EPA Method 3031 (SW-846): Acid Digestion of Oils for Metals Analysis by Atomic
Absorption or ICP Spectrometry 46
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 48
5.2.19 EPA Method 3535A (SW-846): Solid-Phase Extraction 49
5.2.20 EPA Method 3541 (SW-846): Automated Soxhlet Extraction 52
5.2.21 EPA Method 3545A (SW-846): Pressurized Fluid Extraction (PFE) 54
5.2.22 EPA Method 3570 (SW-846): Microscale Solvent Extraction (MSB) 57
5.2.23 EPA Method 3571 (SW-846): Extraction of Solid and Aqueous Samples for Chemical
Agents 61
5.2.24 EPA Method 3580A (SW-846): Waste Dilution 61
5.2.25 EPA Method 3585 (SW-846): Waste Dilution for Volatile Organics 64
5.2.26 EPA Method 503OC (SW-846): Purge-and-Trap for Aqueous Samples 65
5.2.27 EPA Method 5035A (SW-846): Closed-System Purge-and-Trap and Extraction for Volatile
Organics in Soil and Waste Samples 66
5.2.28 EPA Method 6010C (SW-846): Inductively Coupled Plasma - Atomic Emission
Spectrometry 67
5.2.29 EPA Method 6020A (SW-846): Inductively Coupled Plasma - Mass Spectrometry 68
5.2.30 EPA Method 7010 (SW-846): Graphite Furnace Atomic Absorption Spectrophotometry... 69
5.2.31 EPA Method 7470A (SW-846): Mercury in Liquid Wastes (Manual Cold-Vapor Technique)
69
5.2.32 EPA Method 747IB (SW-846): Mercury in Solid or Semisolid Wastes (Manual Cold-Vapor
Technique) 70
5.2.33 EPA Method 7473 (SW-846): Mercury in Solids and Solutions by Thermal Decomposition,
Amalgamation, and Atomic Absorption Spectrophotometry 71
5.2.34 EPA Method 7580 (SW-846): White Phosphorus (P4) by Solvent Extraction and Gas
Chromatography 71
5.2.35 EPA Method 8015C (SW-846): Nonhalogenated Organics Using GC/FID 72
5.2.36 EPA Method 8260C (SW-846): Volatile Organic Compounds by Gas Chromatography-
Mass Spectrometry (GC/MS) 73
5.2.37 EPA Method 8270D (SW-846): Semivolatile Organic Compounds by Gas
Chromatography/Mass Spectrometry (GC-MS) 74
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 77
5.2.39 EPA Method 8315A (SW-846): Determination of Carbonyl Compounds by High
Performance Liquid Chromatography (HPLC) 81
5.2.40 EPA Method 8316 (SW-846): Acrylamide, Acrylonitrile and Acrolein by High Performance
Liquid Chromatography (HPLC) 81
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5.2.41 EPA Method 8318A (SW-846): N-Methylcarbamates by High Performance Liquid
Chromatography (HPLC) 82
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 83
5.2.43 EPA Method 8330B (SW-846): Nitroaromatics and Nitramines by High Performance
Liquid Chromatography (HPLC) 84
5.2.44 EPA CRL MS014: Analysis of Aldicarb, Bromadiolone, Carbofuran, Oxamyl and
Methomyl in Water by Multiple Reaction Monitoring Liquid Chromatography / Tandem
Mass Spectrometry (LC/MS/MS) 85
5.2.45 EPA CRL MS015: Analysis of Thiodiglycol in Water by Single Reaction Monitoring
Liquid Chromatography / Tandem Mass Spectrometry (LC/MS/MS) 86
5.2.46 EPA CRL MS016: Analysis of Diethanolamine, Triethanolamine, n-Methyldiethanolamine
and n-Ethyldiethanolamine in Water by Single Reaction Monitoring Liquid
Chromatography / Tandem Mass Spectrometry (LC/MS/MS) 86
5.2.47 EPA CRL MS017: Analysis of Diisopropyl Methylphosphonate, Ethyl Hydrogen
Dimethylamidophosphate, Isopropyl Methylphosphonic Acid, Methylphosphonic Acid and
Pinacolyl Methylphosphonic Acid in Water by Multiple Reaction Monitoring Liquid
Chromatography /Tandem Mass Spectrometry (LC/MS/MS) 87
5.2.48 EPAILM05.3 Cyanide: Analytical Methods for Total Cyanide Analysis 88
5.2.49 EPA Region 7 RLAB Method 3135.21: Cyanide, Total and Amenable in Aqueous and Solid
Samples Automated Colorimetric with Manual Digestion 88
5.2.50 IO [Inorganic] Compendium Method IO-3.1: Selection, Preparation, and Extraction of Filter
Material 89
5.2.51 IO [Inorganic] Compendium Method IO-3.4: Determination of Metals in Ambient
Particulate Matter Using Inductively Coupled Plasma (ICP) Spectroscopy 90
5.2.52 IO [Inorganic] Compendium Method IO-3.5: Determination of Metals in Ambient
Particulate Matter Using Inductively Coupled Plasma/Mass Spectrometry (ICP-MS) 91
5.2.53 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) 92
5.2.54 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) 93
5.2.55 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) 95
5.2.56 NIOSH Method 1612: Propylene Oxide 96
5.2.57 NIOSH Method 2016: Formaldehyde 97
5.2.58 NIOSH Method 2513: Ethylene Chlorohydrin 97
5.2.59 NIOSH Method 3510: Monomethylhydrazine 98
5.2.60 NIOSH Method 5600: Organophosphorus Pesticides 98
5.2.61 NIOSH Method 5601: Organonitrogen Pesticides 99
5.2.62 NIOSH Method 6001: Arsine 100
5.2.63 NIOSH Method 6002: Phosphine 100
5.2.64 NIOSH Method 6004: Sulfur Dioxide 101
5.2.65 NIOSH Method 6010: Hydrogen Cyanide 101
5.2.66 NIOSH Method 6013: Hydrogen Sulfide 102
5.2.67 NIOSH Method 6015: Ammonia 102
5.2.68 NIOSH Method 6402: Phosphorus Trichloride 103
5.2.69 NIOSH Method 7903: Acids, Inorganic 103
5.2.70 NIOSH Method 7905: Phosphorus 104
5.2.71 NIOSH Method 7906: Fluorides, Aerosol and Gas, by 1C 104
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5.2.72 NIOSH Method 9102: Elements on Wipes 105
5.2.73 NIOSH Method S301-1: Fluoroacetate Anion 106
5.2.74 OSHA Method 40: Methylamine 106
5.2.75 OSHA Method 54: Methyl Isocyanate (MIC) 107
5.2.76 OSHA Method 61: Phosgene 107
5.2.77 OSHA Method ID-211: Sodium Azide and Hydrazoic Acid in Workplace Atmospheres . 108
5.2.78 OSHA Method ID216SG: Boron Trifluoride (BF3) 108
5.2.79 OSHA Method PV2004: Acrylamide 109
5.2.80 OSHA Method PV2103: Chloropicrin 109
5.2.81 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 110
5.2.82 ASTM Method D6480-05: Standard Test Method for Wipe Sampling of Surfaces, Indirect
Preparation, and Analysis for Asbestos Structure Number Concentration by Transmission
Electron Microscopy 110
5.2.83 ISO Method 10312:1995: Ambient Air - Determination of Asbestos Fibres - Direct-transfer
Transmission Electron Microscopy Method Ill
5.2.84 Standard Method 45 00-NH3 B: Nitrogen (Ammonia) Preliminary Distillation Step Ill
5.2.85 Standard Method 4500-NH3 G: Nitrogen (Ammonia) Automated Phenate Method 112
5.2.86 Standard Method 4500-C1 G: DPD Colorimetric Method 112
5.2.87 Literature Reference for Chlorine (Analyst, 1999. 124: 1853-1857) 113
5.2.88 Literature Reference for Fluoroacetate salts (Analytical Letters, 1994. 27 (14): 2703-2718)
113
5.2.89 Literature Reference for Methamidophos (Chromatographia. 2006. 63(5/6): 233-237).... 114
5.2.90 Literature Reference for Methamidophos (Journal of Chromatography A, 2007. 1154: 3-25)
114
5.2.91 Literature Reference for Fluoroacetamide (Journal of Chromatography B, 2008. 876(1):
103-108) 115
5.2.92 Literature Reference for Sodium Azide (Journal of Forensic Sciences, 1998. 43(1): 200-
202) 116
Section 6.0: Selected Radiochemical Methods 117
6.1 General Guidelines 118
6.1.1 Standard Operating Procedure s for Identifying Radiochemical Methods 118
6.1.2 General QC Guidelines for Radiochemical Methods 121
6.1.3 Safety and Waste Management 122
6.2 Method Summaries 123
6.2.1 EPA Method 111: Determination of Polonium-210 Emissions from Stationary Sources .. 123
6.2.2 EPA Method 900.0: Gross Alpha and Gross Beta Radioactivity in Drinking Water 123
6.2.3 EPA Method 901.1: Gamma Emitting Radionuclides in Drinking Water 124
6.2.4 EPA Method 903.0: Alpha-Emitting Radium Isotopes in Drinking Water 125
6.2.5 EPA Method 903.1: Radium-226 in Drinking Water - Radon Emanation Technique 126
6.2.6 EPA Method 906.0: Tritium in Drinking Water 126
6.2.7 EPA Method 908.0: Uranium in Drinking Water- Radiochemical Method 127
6.2.8 EPA Method EMSL-19: Determination of Radium-226 and Radium-228 in Water, Soil, Air
and Biological Tissue 127
6.2.9 EPA Method EMSL-33: Isotopic Determination of Plutonium, Uranium, and Thorium in
Water, Soil, Air, and Biological Tissue 128
6.2.10 EML HASL-300 Method Am-01-RC: Americium in Soil 129
6.2.11 EML HASL-300 Method Am-02-RC: Americium-241 in Soil-Gamma Spectrometry 129
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6.2.12 EML HASL-300 Method Am-04-RC: Americium in QAP Water and Air Filters - Eichrom's
TRU Resin 130
6.2.13 EML HASL-300 Method Ga-01-R: Gamma Radioassay 130
6.2.14 EML HASL-300 Method Po-02-RC: Polonium in Water, Vegetation, Soil, and Air Filters
131
6.2.15 EML HASL-300 Method Pu-12-RC: Plutonium and/or Americium in Soil or Sediments. 132
6.2.16 EML HASL-300 Method Sr-03-RC: Strontium-90 in Environmental Samples 132
6.2.17 EML HASL-300 Method Tc-02-RC: Technetium-99 in Water - TEVA® Resin 133
6.2.18 FRMAC Method Volume 2, Page 33: Gross Alpha and Beta in Air 133
6.2.19 ORISE Method API: Gross Alpha and Beta for Various Matrices 134
6.2.20 ORISE Method AP2: Determination of Tritium 135
6.2.21 ORISE Method AP5: Determination of Technetium-99 136
6.2.22 ORISE Method API 1: Sequential Determination of the Actinides in Environmental Samples
Using Total Sample Dissolution and Extraction Chromatography 136
6.2.23 ORISE Method Procedure #9: Determination of 1-125 in Environmental Samples 137
6.2.24 ASTM Method D3084-05: Standard Practice for Alpha Spectrometry in Water 138
6.2.25 ASTM Method D3972-02: Standard Test Method for Isotopic Uranium in Water by
Radiochemistry 138
6.2.26 Standard Method 7110 B: Gross Alpha and Gross Beta Radioactivity (Total, Suspended,
and Dissolved) 139
6.2.27 Standard Method 7120: Gamma-Emitting Radionuclides 140
6.2.28 Standard Method 7500-Ra B: Radium: Precipitation Method 141
6.2.29 Standard Method 7500-Ra C: Radium: Emanation Method 141
6.2.30 Standard Method 7500-Sr B: Total Radioactive Strontium and Strontium-90: Precipitation
Method 142
6.2.31 Standard Method 7500-U B: Uranium: Radiochemical Method 142
6.2.32 Standard Method 7500-U C: Uranium: Isotopic Method 143
Section 7.0: Selected Pathogen Methods 145
7.1 General Guidelines 146
7.1.1 Standard Operating Procedures for Identifying Pathogen Methods 147
7.1.2 General QC Guidelines for Pathogen Methods 148
7.1.3 Safety and Waste Management 149
7.1.4 Laboratory Response Network (LRN) 150
7.2 Method Summaries for Bacteria 151
7.2.1 Bacillus anthracis [Anthrax] - BSL-3 151
7.2.1.1 Literature Reference for B. anthracis (Public Health Reports. 1977. 92(2): 176-186)
152
7.2.2 Brucella spp. [Brucellosis] - BSL-3 152
7.2.2.1 ASM Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism:
Brucella species 153
7.2.3 Burkholderia mallei [Glanders] - BSL-3 and Burkholderiapseudomallei [Melioidosis] -
BSL-3 153
7.2.3.1 ASM Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism:
Burkholderia mallei and Burkholderia pseudomallei 153
7.2.4 Campylobacter jejuni [Campylobacteriosis] - BSL-2 154
7.2.4.1 Standard Method 9260 G: Campylobacter jejuni 154
7.2.4.2 Literature Reference for Campylobacter jejuni (Molecular and Cellular Probes.
2006. 20(5): 269-279) 155
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7.2.5 Chlamydophilapsittaci [Psittacosis] (formerly known as Chlamydia psittaci) - BSL-2;
BSL-3 for aerosols and large volumes 156
7.2.5.1 Literature Reference for Chlamydophila psittaci (Journal of Clinical Microbiology.
2000.38(3): 1085-1093) 156
7.2.6 Coxiella burnetii [Q-fever] - BSL- 3 157
7.2.7 Escherichia coli O157:H7 - BSL-2 157
7.2.7.1 Standard Method 9260 F: Pathogenic Escherichia coli 157
7.2.7.2 Literature Reference for Escherichia coli O157:H7 (Applied and Environmental
Microbiology. 2003. 69(10): 6327-6333) 158
7.2.8 Francisella tularensis [Tularemia] - BSL-3 158
7.2.8.1 CDC, ASM, APHL: Basic Protocols for Level A Laboratories for the Presumptive
Identification of Francisella tularensis 159
7.2.9 Leptospira interrogans [Leptospirosis] - BSL-2 159
7.2.9.1 Standard Method 9260 I: Leptospira 159
7.2.9.2 Literature Reference for Leptospira spp. (Diagnostic Microbiology and Infectious
Diseases. 2009. 64(3): 247-255) 160
7.2.10 Listeria monocytogenes [Listeriosis] - BSL-2 161
7.2.10.1 USDA Laboratory Guidebook: "FSIS Procedure for the Use of a Listeria
monocytogenes Polymerase Chain Reaction (PCR) Screening Test." MLG 8A.03.
2007 161
7.2.10.2 U.S. FDA Bacteriological Analytical Manual, Chapter 10, 2003: Listeria
monocytogenes 162
7.2.11 Non-typhoidal Salmonella (Not applicable to S. Typhi) [Salmonellosis] - BSL-2 162
7.2.11.1 EPA Method 1682: Salmonella spp 163
7.2.11.2 Literature Reference for Non-Typhoidal Salmonella (Journal of Applied
Microbiology. 2007. 102(2): 516-530) 163
7.2.12 Salmonella Typhi [Typhoid fever] - BSL-2; BSL-3 for aerosol release 164
7.2.12.1 CDC Laboratory Assay: "Triplex PCR for Detection of S. Typhi Using
SmartCycler®" 164
7.2.12.2 Standard Method 9260 B: General Qualitative Isolation and Identification
Procedures for Salmonella 165
7.2.13 Shigella spp. [Shigellosis] - BSL-2 165
7.2.13.1 CDC Laboratory Assay: "Detection of Diarrheagenic Escherichia coli and Shigella
Using LightCycler®" 165
7.2.13.2 Standard Method 9260 E: Shigella 166
7.2.14 Staphylococcus aureus - BSL-2 167
7.2.14.1 Standard Method 9213 B: Staphylococcus aureus 167
7.2.15 Vibrio cholerae [Cholera] - BSL-2 167
7.2.15.1 CDC Laboratory Assay: "TaqMan Assays for Detection of V. cholerae ctxA, 01
rfb, andO139rfb." 168
7.2.15.2 Standard Method 9260 H: Vibrio cholerae 168
7.2.16 Yersiniapestis [Plague] - BSL-3 169
7.2.16.1 ASM Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism:
Yersinia pestis 169
7.3 Method Summaries for Viruses 170
7.3.1 Adenoviruses: Enteric and non-enteric (A-F) - BSL-2 170
7.3.1.1 Literature Reference for Adenoviruses (Applied and Environmental Microbiology.
2005. 71(6): 3131-3136) 170
7.3.2 Astroviruses - BSL not specified 171
7.3.2.1 Literature Reference for Astroviruses (Canadian Journal of Microbiology. 2004. 50:
269-278) 171
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7.3.3 Caliciviruses: Noroviruses - BSL-2 171
7.3.3.1 Literature Reference for Noroviruses (Journal of Clinical Microbiology. 2004.
42(10): 4679-4685) 172
7.3.4 Caliciviruses: Sapovirus - BSL-2 172
7.3.4.1 Literature Reference for Sapoviruses (Journal of Medical Virology. 2006. 78(10):
1347-1353) 172
7.3.5 Coronaviruses: SARS-associated human coronavirus - BSL-2; BSL-3 for propagation ... 173
7.3.5.1 Literature Reference for Coronaviruses (SARS) (Journal of Virological Methods.
2004. 122: 29-36) 173
7.3.6 Hepatitis E virus (HEV) - BSL-2 174
7.3.6.1 Literature Reference for Hepatitis E Virus (Journal of Virological Methods. 2006.
131(1): 65-71) 174
7.3.7 InfluenzaH5N1 virus-BSL-3 174
7.3.7.1 Literature Reference for Influenza H5N1 (Emerging Infectious Diseases. 2005.
11(8): 1303-1305) 175
7.3.8 Picornaviruses: Enteroviruses - BSL-2 175
7.3.8.1 USEPA Manual of Methods for Virology, EPA/600/4-84/013, April 2001 175
7.3.8.2 Literature Reference for Enteric Viruses (Applied and Environmental Microbiology.
2003. 69(6): 3158-3164) 176
7.3.9 Picornaviruses: Hepatitis A virus (HAV) - BSL-2 177
7.3.10 Reoviruses: Rotavirus (Group A) - BSL not specified 177
7.3.10.1 Literature Reference for Reoviruses: Rotavirus (Group A) (Journal of Virological
Methods. 2009. 155: 126-131) 177
7.4 Method Summaries for Protozoa 178
7.4.1 Cryptosporidium spp. [Cryptosporidiosis] - BSL-2 178
7.4.1.1 EPA Method 1622: Cryptosporidium in Water by Filtration/IMS/FA 178
7.4.1.2 EPA Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA
179
7.4.1.3 Literature Reference for Cryptosporidium spp. (Applied and Environmental
Microbiology. 1999. 65(9): 3936-3941) 180
7.4.1.4 Literature Reference for Cryptosporidium spp. (Applied and Environmental
Microbiology. 2007. 73(13): 4218-4225) 180
7.4.2 Entamoeba histolytica - BSL-2 181
7.4.2.1 Literature Reference for Entamoeba histolytica (Journal of Parasitology. 1972.
58(2): 306-310) 181
7.4.2.2 Literature Reference for Entamoeba histolytica (Journal of Clinical Microbiology.
2005. 43(11): 5491-5497) 182
7.4.3 Giardia spp. [Giardiasis] - BSL-2 182
7.4.3.1 Literature Reference for Giardia spp. (Transactions of the Royal Society of
Tropical Medicine and Hygiene. 1983. 77(4): 487-488) 182
7.4.4 Toxoplasma gondii [Toxoplasmosis] - BSL-2 183
7.4.4.1 Literature Reference for Toxoplasma gondii (Emerging Infectious Diseases. 2006.
12(2): 326-329) 183
7.4.4.2 Literature Reference for Toxoplasma gondii (Applied and Environmental
Microbiology. 2004. 70(7): 4035-4039) 184
7.5 Method Summaries for Helminths 184
7.5.1 Baylisascarisprocyonis [Raccoon roundworm fever] - BSL-2 184
7.5.1.1 USEPA Environmental Regulations and Technology, Control of Pathogens and
Vector Attraction in Sewage Sludge EPA/625/R-92/013, July 2003: Baylisascaris
procyonis 185
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Section 8.0: Selected Biotoxin Methods 187
8.1 General Guidelines 188
8.1.1 Standard Operating Procedures for Identifying Biotoxin Methods 189
8.1.2 General QC Guidelines for Biotoxin Methods 190
8.1.3 Safety and Waste Management 191
8.1.4 Laboratory Response Network (LRN) 192
8.2 Method Summaries for Protein Biotoxins 193
8.2.1 Abrin 193
8.2.1.1 Literature Reference for Abrin (Journal of Food Protection. 2008. 71(9): 1868-
1874) 193
8.2.1.2 Literature Reference for Abrin and Ricin (Analytical Biochemistry. 2008. 378(1):
87-89) 194
8.2.1.3 Literature Reference for Abrin and Shiga and Shiga-like Toxins (Pharmacology
Toxicology. 2001. 88(5): 255-260) 195
8.2.2 Botulinum neurotoxins (Serotypes A, B, E, F) 195
8.2.2.1 U.S. FDA, Bacteriological Analytical Manual Online, Chapter 17, 2001: Botulinum
Neurotoxins 196
8.2.2.2 Lateral Flow Immunoassay Kits 196
8.2.3 Ricin (Ricinine) 197
8.2.3.1 Literature Reference for Ricin (Journal of AOAC International. 2008. 91(2): 376-
382) 197
8.2.3.2 Literature Reference for Ricin by Ricinine detection (Journal of Analytical
Toxicology. 2005. 29(3): 149-155) 198
8.2.4 Shiga and Shiga-like toxins (Stx, Stx-1, Stx-2) 199
8.2.4.1 U.S. FDA, Bacteriological Analytical Manual Online, Appendix 1, 2001: Rapid
Methods for Detecting Foodborne Pathogens 199
8.2.4.2 Literature Reference for Shiga and Shiga-like Toxin (Journal of Clinical
Microbiology. 2007. 45(10): 3377-3380) 199
8.2.5 Staphylococcal enterotoxins (SEA, SEE, SEC) 200
8.2.5.1 AOAC Official Method 993.06: Staphylococcal Enterotoxins in Selected Foods 200
8.3 Method Summaries for Small Molecule Biotoxins 200
8.3.1 Aflatoxin(TypeBl) 201
8.3.1.1 AOAC Official Method 991.31: Aflatoxins in Corn, Raw Peanuts, and Peanut
Butter 201
8.3.2 a-Amanitin 201
8.3.2.1 Literature Reference for a-Amanitin (Journal of Chromatography B. 1991. 563(2):
299-311) 201
8.3.2.2 Literature Reference for a-Amanitin, T-2 Mycotoxin (Journal of Food Protection.
2005.68(6): 1294-1301) 202
8.3.3 Anatoxin-a 203
8.3.3.1 Literature Reference for Anatoxin-a (Biomedical Chromatography. 1996. 10(1):
46-47) 203
8.3.4 Brevetoxins (B form) 203
8.3.4.1 Literature Reference for Brevetoxins (Environmental Health Perspectives. 2002.
110(2): 179-185) 203
8.3.4.2 Literature Reference for Brevetoxins (Toxicon. 2004. 43(4): 455-465) 204
8.3.5 a-Conotoxin 204
8.3.5.1 Literature Reference for a-Conotoxin (Biochemical Journal. 1997. 328(1): 245-
250) 204
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8.3.5.2 Literature Reference for a-Conotoxin (Journal of Medicinal Chemistry. 2004.
47(5): 1234-1241) 205
8.3.6 Cylindrospermopsin 206
8.3.6.1 Literature Reference for Cylindrospermopsin (FEMS Microbiology Letters. 2002.
216(2): 159-164) 206
8.3.6.2 ELISA Kits for Cylindrospermopsin 206
8.3.7 Diacetoxyscirpenol (DAS) 207
8.3.7.1 Literature Reference for Diacetoxyscirpenol (DAS) (International Journal of Food
Microbiology. 1988. 6(1): 9-17) 207
8.3.7.2 Literature Reference for Diacetoxyscirpenol (DAS) and T-2 Mycotoxin (Rapid
Communications in Mass Spectrometry. 2006. 20(9): 1422-1428) 207
8.3.8 Microcystins (Principal isoforms: LA, LR, LW, RR, YR) 208
8.3.8.1 Literature Reference for Microcystins (Journal of AOAC International. 2001.
84(4): 1035-1044) 208
8.3.8.2 Literature Reference for Microcystins (Analyst. 1994. 119(7): 1525-1530) 208
8.3.9 Picrotoxin 209
8.3.9.1 Literature Reference for Picrotoxin (Journal of Pharmaceutical & Biomedical
Analysis. 1989. 7(3): 369-375) 209
8.3.10 Saxitoxins (Principal isoforms: STX, NEOSTX, GTX, dcGTX, dcSTX) 210
8.3.10.1 Literature Reference for Saxitoxin (Journal of AOAC International. 1995. 78(2):
528-532) 210
8.3.10.2 ELISA Kits for Saxitoxins 210
8.3.11 T-2 Mycotoxin 211
8.3.12 Tetrodotoxin 211
8.3.12.1 Literature Reference for Tetrodotoxin (Analytical Biochemistry. 2001. 290(1): 10-
17) 211
8.3.12.2 Literature Reference for Tetrodotoxin (Journal of Clinical Laboratory Analysis.
1992. 6(2): 65-72) 212
Section 9.0: Conclusions 213
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
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Tables
Table 5-1. Chemical Methods and Corresponding Text Section Numbers 14
Table 5-2. Sources of Chemical Methods 32
Table 6-1. Radiochemical Methods and Corresponding Text Section Numbers 118
Table 6-2. Sources of Radiochemical Methods 120
Table 7-1. Sources of Pathogen Methods 147
Table 8-1. Sources of Biotoxin Methods 189
SAM Revision 5.0 xxvi September 29, 2009
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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 (i.e.,
chemical, biological, and/or radiological [CBR] crime/attack) 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 5.0 1 September 29, 2009
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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
SAM
(Standardized Analytical Methods for Environmental
Restoration Following Homeland Security Events)
SAM Revision 5.0
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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 for Chemical, Biological, and
Radiological Methods (NEMI-CBR)
• Environmental Monitoring Method Index (EMMI)
• EPA Test Methods Index
EPA Office of Solid Waste SW-846 Methods
• EPA Microbiological 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
sample preparation methods that were selected for measurement of 82 chemical analytes in
SAM Revision 5.0 3 September 29, 2009
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Section 2 - Background
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 is continuing to convene SAM Technical Workgroups at least once per year, to evaluate and, if
necessary, update the analytes and methods that are listed. SAM Revision 4.0 included the addition of a
wipe sample type for chemical analytes and several polymerase chain reaction (PCR) methods for
pathogens. SAM Revision 5.0 reflects the addition of a drinking water sample type for biotoxins. Both
Revisions 4.0 and 5.0 also reflect the addition of several chemical and radiochemical analytes.
JEPA Science Advisory Board's Homeland Security Advisory Committee:
http://vosemite.epa.gov/sab/sabpeople.nsfAVebCommittees/BOARD
SAM Revision 5.0 4 September 29, 2009
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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 similario 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 )
/ If no methods are available, prioritize
V for further research
SAM Revision 5.0
September 29, 2009
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Section 2 - Background
SAM Revision 5.0 6 September 29, 2009
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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 (i.e., CBR crime/attack). 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 those 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. The information also can be found on the SAM Web site
(www.epa.gov/sam), which provides searchable links to supporting information based on SAM
analytes and the analytical methods listed.
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, analytical interferences, and target risk levels.
Many of the SAM analytes have only recently become an environmental concern. EPA is actively
pursuing development and validation of Standard 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 guidelines 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 5.0 7 September 29, 2009
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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 guidelines 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 guidelines 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 EPA, CDC, FDA, DHS, FBI, DoD, USDA, 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 guidelines 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.
Public officials must accurately assess all of the activities that are needed concerning site contamination
following an emergency situation. These activities include initial assessment of potential site
contamination for determination of immediate public and environmental risk, determination of the extent
of contamination, and full remediation of the site. EPA recognizes that having data of known and
documented quality is critical in making proper decisions during each of these activities. Data quality
SAM Revision 5.0 8 September 29, 2009
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Section 3 - Scope and Application
objectives (DQOs) must be established for each response activity2. These DQOs are based upon needs
for both quality and response time. During initial assessments, time is of utmost importance and DQOs
must be established that weigh the need for rapid analytical response (e.g., using screening methods)
against the need for very high quality data (confirmational methods such as those listed in SAM). Many
of the methods listed in this document include quality control (QC) requirements for collecting and
analyzing samples. EPA will assess these QC requirements to ensure analytical data quality supports
decisions concerning site remediation and release. These QC requirements may be adjusted as necessary
to maximize data and decision quality. Specific QC considerations and 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).
Information regarding EPA's DQO process, considerations, and planning is available at:
http://www.epa.gov/QUALITY/dqos.html.
SAM Revision 5.0 9 September 29, 2009
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Section 3 - Scope and Application
SAM Revision 5.0 10 September 29, 2009
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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. In addition, general questions and comments can be submitted via the SAM Web site
(www. epa. gov/sam).
General
Rob Rothman - Primary
National Homeland Security Research Center
U.S. EPA ORD (NG16)
26 West Martin Luther King Jr. Drive
Cincinnati, OH 45268
(513)569-7187
rothman.rob@epa.gov
Romy Lee - Alternate
National Homeland Security Research Center
U.S. EPA ORD (NG16)
26 West Martin Luther King Jr. Drive
Cincinnati, OH 45268
(513)569-7016
lee.romy@epa.gov
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@epa.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
magnuson .matthew@epa. 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
griggs.iohn@epa.gov
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
hall.kathy@epa.gov
Pathogen Methods
Michele Burgess - Primary
U.S. EPA OSWER
Ariel Rios Building - 5104A
1200 Pennsylvania Avenue, NW
Washington, DC 20460
(202) 564-8006
burgess.michele@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. sani iv@epa. gov
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.sanjiv@epa.gov
SAM Revision 5.0
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September 29, 2009
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Section 4 - Points of Contact
SAM Revision 5.0 12 September 29, 2009
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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.
Following a homeland security event, it is assumed that only those areas with contamination greater than
pre-existing/naturally prevalent levels commonly found in the environment would be subject to
remediation. Dependent on site- and event-specific goals, investigation of background levels using
methods listed in Appendix A is recommended.
SAM Revision 5.0 13 September 29, 2009
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Section 5 - Selected Chemical Methods
5.1 General Guidelines
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
(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. Additional
resources include:
• SRC's PHYSPROP (http://srcinc.com/what-we-do/product.aspx?id=133) and CHEMFATE, part of
the Environmental Fate Database supported by EPA (http://srcinc.com/what-we-
do/product.aspx?id=132&terms=Environmental+Fate+and+Exposure).
• 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/default.html for toxicity information.
EPA's Integrated Risk Information System (IRIS): http://www.epa.gov/iris/ contains toxicity
information (searchable on TOXNET).
• Forensic Science and Communications published by the Laboratory Division of the FBI.
http: //www .fbi. gov/hq/lab/fsc/current/backissu .htm.
• Joint Research Centre/Institute for Health & Consumer Protection: http://ecb.jrc.it and
http://ecb.jrc.it/testing-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 5.2.92 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
Ace p hate
CASRN
30560-19-1
Method
Chromatographia. 2006. 63(5/6):
233-237
Journal of Chromatography A.
2007. 1154(1): 3-25
Section
5.2.89
5.2.90
SAM Revision 5.0 14 September 29, 2009
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Section 5 - Selected Chemical Methods
Analyte
Acrylamide
Acrylonitrile
Aldicarb (Temik)
Aldicarb sulfone
Aldicarb sulfoxide
Allyl alcohol
4-Aminopyridine
Ammonia
Ammonium metavanadate
Arsenic, Total
Arsenic trioxide
Arsine
Asbestos
Boron trifluoride
CASRN
79-06-1
107-13-1
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
Method
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
831 6 (EPA SW-846)
PV2004 (OSHA)
531. 2 (EPA OW)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
831 8A (EPA SW-846)
MS014(EPACRL)
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-NH3G(SM)
200.7 (EPAOW)
200.8 (EPAOW)
3031 (EPA SW-846)
3050B (EPA SW-846)
601 OC (EPA SW-846)
6020A (EPA SW-846)
IO-3.1 (EPAORD)
IO-3.4(EPAORD)
IO-3.5 (EPAORD)
9102 (NIOSH)
200.7 (EPAOW)
200.8 (EPAOW)
3050B (EPA SW-846)
7010 (EPA SW-846)
6001 (NIOSH)
9102 (NIOSH)
D5755-03 (ASTM)
D6480-05 (ASTM)
10312:1995 (ISO)
ID216SG(OSHA)
Section
5.2.22
5.2.38
5.2.40
5.2.79
5.2.12
5.2.22
5.2.38
5.2.41
5.2.44
5.2.61
5.2.25
5.2.26
5.2.27
5.2.36
5.2.55
5.2.19
5.2.22
5.2.38
5.2.43
5.2.8
5.2.67
5.2.84
5.2.85
5.2.2
5.2.3
5.2.16
5.2.17
5.2.28
5.2.29
5.2.50
5.2.51
5.2.52
5.2.72
5.2.2
5.2.3
5.2.17
5.2.30
5.2.62
5.2.72
5.2.81
5.2.82
5.2.83
5.2.78
SAM Revision 5.0
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September 29, 2009
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Section 5 - Selected Chemical Methods
Analyte
Brodifacoum
Bromadiolone
BZ [Quinuclidinyl benzilate]
Calcium arsenate
Carbofuran (Furadan)
CASRN
56073-10-0
28772-56-7
6581-06-2
7778-44-1
1563-66-2
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)
8290A Appendix A (EPA SW-846)
8321 B (EPA SW-846)
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)
MS014(EPACRL)
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(EPAORD)
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)
MS014(EPACRL)
5601 (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
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.38
5.2.42
5.2.44
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.38
5.2.42
5.2.54
5.2.2
5.2.3
5.2.16
5.2.17
5.2.28
5.2.29
5.2.50
5.2.51
5.2.52
5.2.72
5.2.12
5.2.22
5.2.38
5.2.41
5.2.44
5.2.61
SAM Revision 5.0
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Section 5 - Selected Chemical Methods
Analyte
Carfentanil
Carbon disulfide
Chlorfenvinphos
Chlorine
2-Chloroethanol
3-Chloro-1 ,2-propanediol
Chloropicrin
CASRN
59708-52-0
75-15-0
470-90-6
7782-50-5
107-07-3
96-24-2
76-06-2
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)
8290A Appendix A (EPA SW-846)
8321 B (EPA SW-846)
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)
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)
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
5.2.10
5.2.25
5.2.26
5.2.27
5.2.36
5.2.55
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.54
5.2.86
5.2.87
5.2.25
5.2.26
5.2.27
5.2.36
5.2.58
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.54
5.2.14
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.80
SAM Revision 5.0
17
September 29, 2009
-------
Section 5 - Selected Chemical Methods
Analyte
Chlorosarin
Chlorosoman
2-Chlorovinylarsonous acid (2-CVAA)
Chlorpyrifos
Chlorpyrifos oxon
Crimidine
Cyanide, Amenable to chlorination
Cyanide, Total
Cyanogen chloride
CASRN
1445-76-7
7040-57-5
85090-33-1
2921-88-2
5598-15-2
535-89-7
NA
57-12-5
506-77-4
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)
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(EPAORD)
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-10A (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)
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-1 5 (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
5.2.54
5.2.2
5.2.3
5.2.16
5.2.17
5.2.28
5.2.29
5.2.50
5.2.51
5.2.52
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
5.2.54
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.49
5.2.7
5.2.48
5.2.65
5.2.25
5.2.26
5.2.27
5.2.36
5.2.55
SAM Revision 5.0
18
September 29, 2009
-------
Section 5 - Selected Chemical Methods
Analyte
Cyclohexyl sarin (GF)
1,2-Dichloroethane
Dichlorvos
Dicrotophos
Diesel range organics
Diisopropyl methylphosphonate (DIMP)
CASRN
329-99-7
107-06-2
62-73-7
141-66-2
NA
1445-75-6
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)
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)
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)
MS017(EPACRL)
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
5.2.54
5.2.10
5.2.25
5.2.26
5.2.27
5.2.36
5.2.55
5.2.11
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.54
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.54
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
5.2.47
5.2.54
SAM Revision 5.0
19
September 29, 2009
-------
Section 5 - Selected Chemical Methods
Analyte
Dimethylphosphite
Dimethylphosphoramidic acid
Diphacinone
Disulfoton
Disulfoton sulfoxide
1,4-Dithiane
EA2192 [Diisopropylaminoethyl methyl-
thiolophosphonate]
CASRN
868-85-9
33876-51-6
82-66-6
298-04-4
2497-07-6
505-29-3
73207-98-4
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)
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)
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)
Section
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.54
5.2.19
5.2.21
5.2.22
5.2.24
5.2.38
5.2.42
5.2.54
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
5.2.60
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
5.2.54
SAM Revision 5.0
20
September 29, 2009
-------
Section 5 - Selected Chemical Methods
Analyte
Ethyl methylphosphonic acid (EMPA)
Ethyldichloroarsine (ED)
N-Ethyldiethanolamine (EDEA)
Ethylene oxide
Fenamiphos
Fentanyl
CASRN
1832-53-7
598-14-1
139-87-7
75-21-8
22224-92-6
437-38-7
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)
MS017(EPACRL)
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)
MS016(EPACRL)
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)
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
5.2.19
5.2.21
5.2.22
5.2.24
5.2.38
5.2.42
5.2.47
5.2.54
5.2.19
5.2.21
5.2.24
5.2.37
5.2.55
5.2.72
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.38
5.2.42
5.2.46
5.2.54
5.2.25
5.2.26
5.2.27
5.2.36
5.2.55
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
5.2.54
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 5.0
21
September 29, 2009
-------
Section 5 - Selected Chemical Methods
Analyte
Fluoride
Fluoroacetamide
Fluoroacetic acid and fluoroacetate salts
2-Fluoroethanol
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
Isopropyl methylphosphonic acid (IMPA)
Kerosene
CASRN
16984-48-8
640-19-7
NA
371-62-0
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
1832-54-8
64742-81-0
Method
300.1, Rev 1.0 (EPA OW)
Journal of Chromatography B.
2008. 876, 103-108
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
3585 (EPA SW-846)
5030C (EPA SW-846)
5035A (EPA SW-846)
8260C (EPA SW-846)
2513 (NIOSH)
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)
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)
MS017(EPACRL)
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)
Section
5.2.6
5.2.91
5.2.6
5.2.22
5.2.38
5.2.73
5.2.88
5.2.25
5.2.26
5.2.27
5.2.36
5.2.58
5.2.22
5.2.38
5.2.39
5.2.57
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
5.2.69
5.2.65
5.2.69
5.2.66
5.2.19
5.2.21
5.2.22
5.2.24
5.2.38
5.2.42
5.2.47
5.2.54
5.2.22
5.2.25
5.2.26
5.2.27
5.2.35
5.2.38
SAM Revision 5.0
22
September 29, 2009
-------
Section 5 - Selected Chemical Methods
Analyte
Lead arsenate
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine]
Lewisite 2 (L-2) [bis(2-
chlorovinyljchloroarsine]
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine]
Lewisite oxide
Mercuric chloride
Mercury, Total
Methamidophos
Methomyl
Methoxyethylmercuric acetate
Methyl acrylonitrile
Methyl fluoroacetate
CASRN
7645-25-2
541-25-3
40334-69-8
40334-70-1
1306-02-1
7487-94-7
7439-97-6
10265-92-6
16752-77-5
151-38-2
126-98-7
453-18-9
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(EPAORD)
IO-3.5 (EPAORD)
9102(NIOSH)
245.1 (EPAOW)
7473 (EPA SW-846)
9102(NIOSH)
245.1 (EPAOW)
7473 (EPA SW-846)
IO-5 (EPAORD)
9102(NIOSH)
Chromatographia. 2006. 63(5/6):
233-237
Journal of Chromatography A.
2007. 1154(1): 3-25
531. 2 (EPAOW)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
831 8A (EPA SW-846)
MS014(EPACRL)
5601 (NIOSH)
245.1 (EPAOW)
7473 (EPA SW-846)
IO-5 (EPAORD)
9102 (NIOSH)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
831 6 (EPA SW-846
PV2004 (OSHA)
300.1, Rev 1.0 (EPAOW)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
S301-1 (NIOSH)
Analytical Letters. 1994. 27(14):
2703-2718
Section
5.2.2
5.2.3
5.2.16
5.2.17
5.2.28
5.2.29
5.2.50
5.2.51
5.2.52
5.2.72
5.2.4
5.2.33
5.2.72
5.2.4
5.2.33
5.2.53
5.2.72
5.2.89
5.2.90
5.2.12
5.2.22
5.2.38
5.2.41
5.2.44
5.2.61
5.2.4
5.2.33
5.2.53
5.2.72
5.2.22
5.2.38
5.2.40
5.2.79
5.2.6
5.2.22
5.2.38
5.2.73
5.2.88
SAM Revision 5.0
23
September 29, 2009
-------
Section 5 - Selected Chemical Methods
Analyte
Methyl hydrazine
Methyl isocyanate
Methyl paraoxon
Methyl parathion
Methylamine
N-Methyldiethanolamine (MDEA)
1-Methylethyl ester ethylphosphonofluoridic
acid (GE)
Methylphosphonic acid (MPA)
CASRN
60-34-4
624-83-9
950-35-6
298-00-0
74-89-5
105-59-9
1189-87-3
993-13-5
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)
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)
MS016(EPACRL)
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)
8290A Appendix A (EPA SW-846)
8321 B (EPA SW-846)
MS017(EPACRL)
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
5.2.59
5.2.75
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.54
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
5.2.46
5.2.54
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.54
5.2.19
5.2.21
5.2.22
5.2.24
5.2.38
5.2.42
5.2.47
5.2.54
SAM Revision 5.0
24
September 29, 2009
-------
Section 5 - Selected Chemical Methods
Analyte
Mevinphos
Monocrotophos
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
CASRN
7786-34-7
6923-22-4
538-07-8
51-75-2
555-77-1
505-60-2
54-11-5
2691-41-0
NA
Method
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)
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)
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)
Section
5.2.11
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.54
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.54
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.54
5.2.22
5.2.23
5.2.37
5.2.38
5.2.54
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
5.2.9
5.2.15
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.60
SAM Revision 5.0
25
September 29, 2009
-------
Section 5 - Selected Chemical Methods
Analyte
Osmium tetroxide
Oxamyl
Paraquat
Paraoxon
Parathion
Pentaerythritol tetranitrate (PETN)
Phencyclidine
Phenol
CASRN
20816-12-0
23135-22-0
4685-14-7
311-45-5
56-38-2
78-11-5
77-10-1
108-95-2
Method
252.2 (EPA OW)
3050B (EPA SW-846)
6010C(EPASW-846)
IO-3.1 (EPAORD)
IO-3.4(EPAORD)
9102(NIOSH)
531. 2 (EPA OW)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
831 8A (EPA SW-846)
MS014(EPACRL)
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-10A (EPAORD)
3535A (EPA SW-846)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
8330B (EPA SW-846)
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-10A (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-10A (EPAORD)
Section
5.2.5
5.2.17
5.2.28
5.2.50
5.2.51
5.2.72
5.2.12
5.2.22
5.2.38
5.2.41
5.2.44
5.2.61
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
5.2.54
5.2.19
5.2.22
5.2.38
5.2.43
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.54
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.54
SAM Revision 5.0
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September 29, 2009
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Section 5 - Selected Chemical Methods
Analyte
Phorate
Phorate sulfone
Phorate sulfoxide
Phosgene
Phosphamidon
Phosphine
Phosphorus trichloride
Pinacolyl methyl phosphonic acid (PMPA)
Propylene oxide
R 33 (VR) [methylphosphonothioic acid, S-
[2-(diethylamino)ethyl] O-2-methylpropyl
ester]
Sarin (GB)
CASRN
298-02-2
2588-04-7
2588-03-6
75-44-5
13171-21-6
7803-51-2
7719-12-2
616-52-4
75-56-9
159939-87-4
107-44-8
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)
OSHA 61
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)
MS017(EPACRL)
TO-IOA(EPAORD)
3585 (EPA SW-846)
5030C (EPA SW-846)
5035A (EPA SW-846)
8260C (EPA SW-846)
1612 (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)
Section
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.54
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
5.2.54
5.2.63
5.2.68
5.2.19
5.2.21
5.2.22
5.2.24
5.2.38
5.2.42
5.2.47
5.2.54
5.2.25
5.2.26
5.2.27
5.2.36
5.2.56
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.54
5.2.22
5.2.23
5.2.37
5.2.38
5.2.54
SAM Revision 5.0
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Section 5 - Selected Chemical Methods
Analyte
Semivolatile organic compounds, NOS
Sodium arsenite
Sodium azide
Soman (GD)
Strychnine
Sulfur dioxide
Sulfur trioxide
Tabun (GA)
CASRN
NA
7784-46-5
26628-22-8
96-64-0
57-24-9
7446-09-5
7446-11-g
77-81-6
Method
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(EPAORD)
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-10A (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)
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-10A (EPAORD)
Section
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
5.2.54
5.2.2
5.2.3
5.2.16
5.2.17
5.2.28
5.2.29
5.2.50
5.2.51
5.2.52
5.2.72
5.2.6
5.2.24
5.2.77
5.2.92
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.54
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.64
5.2.1
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.54
SAM Revision 5.0
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Section 5 - Selected Chemical Methods
Analyte
Tetraethyl pyrophosphate
Tetramethylenedisulfotetramine
Thallium sulfate
Thiodiglycol (TDG)
Thiofanox
CASRN
107-49-3
80-12-6
10031-59-1
111-48-8
39196-18-4
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)
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(EPAORD)
IO-3.5 (EPAORD)
9102(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)
MS015(EPACRL)
TO-10A (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)
Section
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.54
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.54
5.2.2
5.2.3
5.2.16
5.2.17
5.2.28
5.2.29
5.2.50
5.2.51
5.2.52
5.2.72
5.2.19
5.2.21
5.2.22
5.2.24
5.2.38
5.2.42
5.2.45
5.2.54
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
5.2.61
SAM Revision 5.0
29
September 29, 2009
-------
Section 5 - Selected Chemical Methods
Analyte
1,4-Thioxane
Titanium tetrachloride
Triethanolamine (TEA)
Trimethyl phosphite
1 ,3,5-Trinitrobenzene (1 ,3,5-TNB)
2,4,6-Trinitrotoluene(2,4,6-TNT)
Vanadium pentoxide
CASRN
15980-15-1
7550-45-0
102-71-6
121-45-9
99-35-4
118-96-7
1314-62-1
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)
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)
MS016(EPACRL)
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)
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(EPAORD)
IO-3.5 (EPAORD)
9102(NIOSH)
Section
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
5.2.72
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.38
5.2.42
5.2.46
5.2.54
5.2.19
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.54
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
5.2.50
5.2.51
5.2.52
5.2.72
SAM Revision 5.0
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Section 5 - Selected Chemical Methods
Analyte
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
21738-25-0
78-53-5
21770-86-5
50782-69-9
12185-10-3
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)
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
5.2.18
5.2.19
5.2.20
5.2.21
5.2.22
5.2.24
5.2.37
5.2.38
5.2.54
5.2.22
5.2.23
5.2.37
5.2.38
5.2.54
5.2.22
5.2.34
5.2.38
5.2.70
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
Diisopropyl methylphosphonate (DIMP)
Dimethylphosphoramidic acid
EA2192 [Diisopropylaminoethyl methyl-
thiolophosphonate]
Ethyl methylphosphonic acid (EMPA)
Hydrogen fluoride
Isopropyl methylphosphonic acid (IMPA)
Mercuric chloride
Mercury, Total
Methamidophos
Methoxyethylmercuric acetate
1-Methylethyl ester ethylphosphonofluoridic
acid (GE)
Methylphosphonic acid (MPA)
Pinacolyl methyl phosphonic acid (PMPA)
Sarin (GB)
Soman (GD)
107-18-6
6581-06-2
96-24-2
1445-76-7
7040-57-5
535-89-7
1445-75-6
33876-51-6
73207-98-4
1832-53-7
7664-39-3
1832-54-8
7487-94-7
7439-97-6
10265-92-6
151-38-2
1189-87-3
993-13-5
616-52-4
107-44-8
96-64-0
TO-IOA(EPAORD)
8270D (EPA SW-846)
TO-15(EPAORD)
TO-15(EPAORD)
8321 B (EPA SW-846)
8270D (EPA SW-846)
TO-15(EPAORD)
8270D (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)
8270D (EPA SW-846)
TO-15(EPAORD)
5.2.54
5.2.37
5.2.55
5.2.55
5.2.42
5.2.37
5.2.55
5.2.37
5.2.37
5.2.71
5.2.37
5.2.31
5.2.32
5.2.60
5.2.31
5.2.32
5.2.55
5.2.37
5.2.37
5.2.55
SAM Revision 5.0
31
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-------
Section 5 - Selected Chemical Methods
Analyte
1,4-Thioxane
CASRN
15980-15-1
Method
3585 (EPA SW-846)
5030C (EPA SW-846)
5035A (EPA SW-846)
8260C (EPA SW-846)
Section
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
EPA OW Methods
EPA SW-846 Methods
EPA ORD Methods
EPA Air Toxics Methods
OSHA Methods
NIOSH Methods
Standard Methods for the Examination
of Water and Wastewater(SM), 21st
Edition, 2005*
Annual Book ofASTM Standards*
GESTIS Substance Database
ISO Methods*
Official Methods of Analysis of AOAC
International*
Analyst
Analytical Letters*
Journal of Chromatography A*
Journal of Forensic Sciences*
Chromatographia
Publisher
EPA, USGS
EPAOW
EPA OSWER
EPA ORD
EPA OAR
OSHA
NIOSH
American Public Health
Association (APHA),
American Water Works
Association (AWWA), and
Water Environment
Federation (WEF)
ASTM International
BGIA
ISO
AOAC International
Royal Society of Chemistry
Taylor & Francis
Elsevier Science Publishers
ASTM International
Vieweg+Teubner
Reference
http://www.nemi.qov
http://www.epa.qov/safewater/methods/
sourcalt.html
http://www.epa.qov/epaoswer/hazwaste
/test/main. htm
http://www.epa.gov/ttnamti1/
http://www.epa.qov/ttn/amtic/airtox.html
http://www.osha.qov/dts/sltc/methods/in
dex.html
http://www.cdc.gov/niosh/nmam/
http://www.standardmethods.org
http://www.astm.org
http://www.dguv.de/bgia/en/gestis/stoffd
b/index.isp
http://www.iso.org
http://www.aoac.org
http://www.rsc.org/Publishing/Journals/
AN/
http://www.informaworld.com/smpp/title
~content=t71 3597227
http://www.elsevier.com/
http://www.astm.org
http://www.chromatographia.de/
' Subscription and/or purchase required.
SAM Revision 5.0
32
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Section 5 - Selected Chemical Methods
5.1.2 General QC Guidelines 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 efficient manner. Therefore, laboratories must be prepared with calibrated instruments, the
proper standards, standard analytical procedures, standard operating procedures, and qualified and trained
staff. 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
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 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 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:
>• Matrix spike/matrix spike duplicates (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.
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QC tests should be consistent with EPA's Good Laboratory Practice Standards
(http://www.epa.gov/oecaerth/monitoring/programs/fifra/glp.html) 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
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, guidelines, 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
Etiologic Agents.
• CDC-42 CFR part 73. Select Agents and Toxins.
• 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/.
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5.2 Method Summaries
Summaries for the analytical methods listed in Appendix A are provided in Sections 5.2.1 through 5.2.92.
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
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." http://www.epa.gov/sam/pdfs/EPA-Method8.pdf
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 (2-CVAA)
Lead arsenate
CASRN
7803-55-6
7440-38-2
1327-53-3
7784-42-1
7778-44-1
85090-33-1
7645-25-2
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Analyte(s)
Lewisite
Lewisite 2
1 (L-1) [2-chlorovinyldichloroarsine]
(L-2) [bis(2-chlorovinyl)chloroarsine]
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine]
Lewisite oxide
Sodium arsenite
Thallium sulfate
Vanadium pentoxide
CASRN
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 metal-containing compounds only as the total metal
(e.g., total arsenic) 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 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.
http://www.epa.gOv/sam/pdfs/EPA-200.7.pdf
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
CASRN
7803-55-6
7440-38-2
1327-53-3
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Analyte(s)
Arsine
Calcium arsenate
2-Chlorovinylarsonous acid (2-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
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 ug/L.
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. http://www.epa.gov/sam/pdfs/EPA-
200.8.pdf
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Section 5 - Selected Chemical Methods
5.2.4 EPA Method 245.1: Determination of Mercury in Water by Cold Vapor Atomic
Absorption Spectrometry (CVAA)
Analyte(s)
Mercuric chloride
Mercury, Total
Methoxyethylmercuric acetate
CASRN
7487-94-7
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 10.0 (ig Hg/L. The detection limit
for this method is 0.2 (ig Hg/L.
Description of Method: This method will determine mercuric chloride and 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 stannous 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. 1994. "Method 245.1: Determination of Mercury in Water by Cold Vapor Atomic
Absorption Spectrometry CVAA)." http://www.epa.gov/sam/pdfs/EPA-245.1 .pdf
5.2.5 EPA Method 252.2: Osmium (Atomic Absorption, Furnace Technique)
Analyte(s)
Osmium tetroxide
CASRN
20816-12-0
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 (ig/L. The optimal applicable
concentration range is 50 to 500 (ig/L.
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,
http://www.epa.gov/sam/pdfs/MCAWW-9.1 .pdf) except that the addition of sulfuric acid is omitted in the
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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, http://www.epa.gOv/sam/pdfs/MCAWW-9.3.pdf). 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.
Special Considerations: Concerns have been raised regarding the use of nitric acid when analyzing
samples for osmium tetroxide; hydrochloric acid should be considered and evaluated as a possible
alternative.
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
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.
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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.
Special Considerations: For sodium azide, if analyses are problematic, refer to column manufacturer
for alternate conditions.
Source: EPA. 1997. "Method 300.1: Determination of Inorganic Anions in Drinking Water by Ion
Chromatography," Revision 1.0. http://www.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 (ig/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 include aldehydes, nitrate-nitrite, and oxidizing agents,
such as chlorine, thiocyanate, thiosulfate, and sulfide. These interferences can be eliminated or reduced
by distillation.
Source: EPA. 1993. "Method 335.4: Determination of Total Cyanide by Semi-Automated Colorimetry,"
Revision 1.0. http://www.epa.gov/sam/pdfs/EPA-335.4.pdf
5.2.8 EPA Method 350.1: Nitrogen, Ammonia (Colorimetric, Automated Phenate)
Analyte(s)
Ammonia
CASRN
7664-41-7
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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.
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.epa.gov/sam/pdfs/EPA-507.pdf
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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 lower
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.
http://www.epa.gOv/sam/pdfs/EPA-524.2.pdf
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
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Liquid-solid extraction (LSE) or solid-phase extraction (SPE)
Determinative Technique: GC-MS
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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 aqueous liquid and/or
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.
Special Considerations: SPE using Ci8 resin may not work for certain compounds having high water
solubility. In these cases, other sample preparation techniques or different SPE resins may be required.
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: 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 |^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
injecting a sample aliquot (up to 1000 uL) 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
SAM Revision 5.0 43 September 29, 2009
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Section 5 - Selected Chemical Methods
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. http://www.epa.gOv/sam/pdfs/EPA-549.2.pdf
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
76-06-2
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Solvent extraction
Determinative Technique: Gas Chromatography -electron capture detector (GC-ECD)
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
SAM Revision 5.0 44 September 29, 2009
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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.
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
SAM Revision 5.0 45 September 29, 2009
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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 (2-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
7440-38-2
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, calcium and lead arsenate, 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 60IOC (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
5.2.17 EPA Method 3050B (SW-846): Acid Digestion of Sediments, Sludges, and Soils
Analyte(s)
Ammonium metavanadate
Arsenic, Total
CASRN
7803-55-6
7440-38-2
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Section 5 - Selected Chemical Methods
Analyte(s)
Arsenic trioxide
Arsine
Calcium arsenate
2-Chlorovinylarsonous acid (2-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
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, calcium and lead arsenate, 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 6010C or 6020A (SW-846); use Method 6010C (SW-846) for total osmium; use
Method 7010 (SW-846) for arsine.
Special Considerations: Concerns have been raised regarding the use of nitric acid when analyzing
samples for osmium tetroxide; hydrochloric acid should be considered and evaluated as a possible
alternative.
Source: EPA. 1996. "Method 3050B (SW-846): Acid Digestion of Sediments, Sludges, and Soils,"
Revision 2. http://www.epa. gov/sam/pdfs/EPA-3 05 Ob .pdf
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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]
Carfentanil
Chlorfenvinphos
3-Chloro-1 ,2-propanediol
Chlorosarin
Chlorosoman
Chlorpyrifos
Chlorpyrifos oxon
Cyclohexyl sarin (GF)
Diesel range organics
Diphacinone
N-Ethyldiethanolamine (EDEA)
Fenamiphos
Fentanyl
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]
Paraoxon
Parathion
Phenol
Phosphamidon
R 33 (VR) [methylphosphonothioic acid, S-[2-
(diethylamino)ethyl] O-2-methylpropyl ester]
Semivolatile organic compounds, NOS
Tetramethylenedisulfotetramine
CASRN
56073-10-0
28772-56-7
6581-06-2
59708-52-0
470-90-6
96-24-2
1445-76-7
7040-57-5
2921-88-2
5598-15-2
329-99-7
NA
82-66-6
139-87-7
22224-92-6
437-38-7
60-34-4
105-59-9
1189-87-3
538-07-8
51-75-2
555-77-1
311-45-5
56-38-2
108-95-2
13171-21-6
159939-87-4
NA
80-12-6
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Section 5 - Selected Chemical Methods
Analyte(s)
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
39196-18-4
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 8321B. 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; aqueous/liquid samples for bromadiolone should be
analyzed using EPA CRL MS014; aqueous liquid samples for EDEA, MDEA, and TEA should be
analyzed using EPA CRL MS016. All other drinking water and 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.
Special Considerations: Some of the target compounds will hydrolyze in water, with hydrolysis rates
dependant on various factors such as sample pH and temperature. For more information on the
preparation and analysis of thiofanox, see application note:
http://www.pickeringlabs.com/catalog/pdfs/MA112%20expanded%20Carbamates.pdf
Source: EPA. 1996. "Method 3520C (SW-846): Continuous Liquid-Liquid Extraction," Revision 3.
http://www.epa.gov/sam/pdfs/EPA-3520c.pdf
5.2.19 EPA Method 3535A (SW-846): Solid-Phase Extraction
Analyte(s)
4-Aminopyridine
Brodifacoum
Bromadiolone
BZ [Quinuclidinyl benzilate]
CASRN
504-24-5
56073-10-0
28772-56-7
6581-06-2
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Section 5 - Selected Chemical Methods
Analyte(s)
Carfentanil
Chlorfenvinphos
3-Chloro-1 ,2-propanediol
Chlorosarin
Chlorosoman
Chlorpyrifos
Chlorpyrifos oxon
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
Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)
Hexamethylenetriperoxidediamine (HMTD)
Isopropyl methylphosphonic acid (IMPA)
Methyl hydrazine
Methyl paraoxon
Methyl parathion
N-Methyldiethanolamine (MDEA)
1-Methylethyl ester ethylphosphonofluoridic acid (GE)
CASRN
59708-52-0
470-90-6
96-24-2
1445-76-7
7040-57-5
2921-88-2
5598-15-2
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
505-29-3
73207-98-4
1832-53-7
598-14-1
139-87-7
22224-92-6
437-38-7
121-82-4
283-66-9
1832-54-8
60-34-4
950-35-6
298-00-0
105-59-9
1189-87-3
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Section 5 - Selected Chemical Methods
Analyte(s)
Methylphosphonic acid (MPA)
Mevinphos
Monocrotophos
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
Octahydro-1 ,3,5,7-tetranitro-1 ,3,5,7-tetrazocine (HMX)
Paraoxon
Parathion
Pentaerythritol tetranitrate (PETN)
Phencyclidine
Phenol
Phorate
Phorate sulfone
Phorate sulfoxide
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)
Tetraethyl pyrophosphate
Tetramethylenedisulfotetramine
Thiodiglycol (TDG)
Thiofanox
1,4-Thioxane
Triethanolamine (TEA)
Trimethyl phosphite
1 ,3,5-Trinitrobenzene (1 ,3,5-TNB)
CASRN
993-13-5
7786-34-7
6923-22-4
538-07-8
51-75-2
555-77-1
54-11-5
2691-41-0
311-45-5
56-38-2
78-11-5
77-10-1
108-95-2
298-02-2
2588-04-7
2588-03-6
13171-21-6
616-52-4
159939-87-4
NA
96-64-0
57-24-9
77-81-6
107-49-3
80-12-6
111-48-8
39196-18-4
15980-15-1
102-71-6
121-45-9
99-35-4
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Section 5 - Selected Chemical Methods
Analyte(s)
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
118-96-7
21738-25-0
78-53-5
21770-86-5
Analysis Purpose: Sample preparation
Sample Preparation Technique: SPE
Determinative Technique: EPA SW-846 Method 8015C, Method 8270D, Method 8321B, 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 thiofanox should be prepared and analyzed by EPA Method 531.2; aqueous liquid samples
for DIMP, EMPA, IMPA, MPA, and PMPA should be prepared and analyzed using EPA CRL MS017;
aqueous liquid samples for EDEA, MDEA and TEA should be prepared and analyzed using EPA CRL
MS016; aqueous liquid samples for bromadiolone should be prepared and analyzed using EPA CRL
MS014; aqueous liquid samples for thiodiglycol should be prepared and analyzed using EPA CRL
MS015. All other drinking water samples and all aqueous liquid samples should be prepared using this
method (SW-846 Method 3535A).
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. Water samples that contain a high level of particulates or a large amount of humic
products may not be extractable by SPE. Some of the target compounds will hydrolyze in water, with
hydrolysis rates dependant on various factors such as sample pH and temperature.
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
CASRN
56073-10-0
28772-56-7
SAM Revision 5.0 52 September 29, 2009
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Section 5 - Selected Chemical Methods
Analyte(s)
BZ [Quinuclidinyl benzilate]
Carfentanil
Chlorfenvinphos
3-Chloro-1 ,2-propanediol
Chlorosarin
Chlorosoman
Chlorpyrifos
Chlorpyrifos oxon
Cyclohexyl sarin (GF)
Diesel range organics
Diphacinone
Disulfoton
Disulfoton sulfoxide
N-Ethyldiethanolamine (EDEA)
Fenamiphos
Fentanyl
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
Paraoxon
Parathion
Phenol
Phosphamidon
R 33 (VR) [methylphosphonothioic acid, S-[2-
(diethylamino)ethyl] O-2-methylpropyl ester]
Semivolatile organic compounds, NOS
Tetramethylenedisulfotetramine
CASRN
6581-06-2
59708-52-0
470-90-6
96-24-2
1445-76-7
7040-57-5
2921-88-2
5598-15-2
329-99-7
NA
82-66-6
298-04-4
2497-07-6
139-87-7
22224-92-6
437-38-7
60-34-4
105-59-9
1189-87-3
538-07-8
51-75-2
555-77-1
NA
311-45-5
56-38-2
108-95-2
13171-21-6
159939-87-4
NA
80-12-6
SAM Revision 5.0
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Section 5 - Selected Chemical Methods
Analyte(s)
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
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
Determinative Technique: EPA SW-846 Method 8015C, Method 8270D, or Method 8321B. Refer to
Appendix A for which of these determinative methods should be used for a particular analyte.
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.
Special Considerations: Some of the target compounds will hydrolyze in water, with hydrolysis rates
dependant on various factors such as sample pH and temperature.
Source: EPA. 1994. "Method 3541 (SW-846): Automated Soxhlet Extraction," Revision 0.
http://www.epa.gov/sam/pdfs/EPA-3541 .pdf
5.2.21 EPA Method 3545A (SW-846): Pressurized Fluid Extraction (PFE)
Analyte(s)
Brodifacoum
Bromadiolone
BZ [Quinuclidinyl benzilate]
Carfentanil
Chlorfenvinphos
3-Chloro-1 ,2-propanediol
Chloropicrin
Chlorosarin
Chlorosoman
CASRN
56073-10-0
28772-56-7
6581-06-2
59708-52-0
470-90-6
96-24-2
76-06-2
1445-76-7
7040-57-5
SAM Revision 5.0
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Section 5 - Selected Chemical Methods
Analyte(s)
Chlorpyrifos
Chlorpyrifos oxon
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
Isopropyl methylphosphonic acid (IMPA)
Methyl hydrazine
Methyl paraoxon
Methyl parathion
N-Methyldiethanolamine (MDEA)
1-Methylethyl ester ethylphosphonofluoridic acid (GE)
Methylphosphonic acid (MPA)
Mevinphos
Monocrotophos
Mustard, nitrogen (HN-1) [bis(2-chloroethyl)ethylamine]
CASRN
2921-88-2
5598-15-2
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
1832-54-8
60-34-4
950-35-6
298-00-0
105-59-9
1189-87-3
993-13-5
7786-34-7
6923-22-4
538-07-8
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Section 5 - Selected Chemical Methods
Analyte(s)
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
Paraoxon
Parathion
Phencyclidine
Phenol
Phorate
Phorate sulfone
Phorate sulfoxide
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)
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
51-75-2
555-77-1
54-11-5
NA
311-45-5
56-38-2
77-10-1
108-95-2
298-02-2
2588-04-7
2588-03-6
13171-21-6
616-52-4
159939-87-4
NA
96-64-0
57-24-9
77-81-6
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
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Section 5 - Selected Chemical Methods
Analysis Purpose: Sample preparation
Sample Preparation Technique: Pressurized Fluid Extraction (PFE)
Determinative Technique: EPA SW-846 Method 8015C, Method 8270D, or Method 8321B. 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 poly chlorinated dibenzo-p-dioxins (PCDDs) / poly chlorinated 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. Phencyclidine and VX require extraction with 5% triethylamine in ethyl acetate.
Some of the target compounds will hydrolyze in water, with hydrolysis rates dependant on various factors
such as sample pH and temperature.
Source: EPA. 1998. "Method 3545A (SW-846): Pressurized Fluid Extraction (PFE)," Revision 1.
http://www.epa.gov/sam/pdfs/EPA-3545a.pdf
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
Carfentanil
Carbofuran (Furadan)
Chlorfenvinphos
3-Chloro-1 ,2-propanediol
Chloropicrin
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
59708-52-0
1563-66-2
470-90-6
96-24-2
76-06-2
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Section 5 - Selected Chemical Methods
Analyte(s)
Chlorosarin
Chlorosoman
Chlorpyrifos
Chlorpyrifos oxon
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)
N-Ethyldiethanolamine (EDEA)
Fenamiphos
Fentanyl
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
Methomyl
Methyl acrylonitrile
Methyl fluoroacetate
CASRN
1445-76-7
7040-57-5
2921-88-2
5598-15-2
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
NA
50-00-0
NA
121-82-4
283-66-9
1832-54-8
64742-81-0
16752-77-5
126-98-7
453-18-9
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Section 5 - Selected Chemical Methods
Analyte(s)
Methyl hydrazine
Methyl paraoxon
Methyl parathion
N-Methyldiethanolamine (MDEA)
1-Methylethyl ester ethylphosphonofluoridic acid (GE)
Methylphosphonic acid (MPA)
Mevinphos
Monocrotophos
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
Paraoxon
Parathion
Pentaerythritol tetranitrate (PETN)
Phencyclidine
Phenol
Phorate
Phorate sulfone
Phorate sulfoxide
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)
CASRN
60-34-4
950-35-6
298-00-0
105-59-9
1189-87-3
993-13-5
7786-34-7
6923-22-4
538-07-8
51-75-2
555-77-1
505-60-2
54-11-5
2691-41-0
NA
23135-22-0
311-45-5
56-38-2
78-11-5
77-10-1
108-95-2
298-02-2
2588-04-7
2588-03-6
13171-21-6
616-52-4
159939-87-4
107-44-8
NA
96-64-0
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Section 5 - Selected Chemical Methods
Analyte(s)
Strychnine
Tabun (GA)
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
57-24-9
77-81-6
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
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.
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Source: EPA. 2002. "Method 3570 (SW-846): Microscale Solvent Extraction (MSB)," Revision 0.
http://www.epa.gov/sam/pdfs/EPA-3570.pdf
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: MSE
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
Carboprep90 column, then the silica column.
Source: EPA. 2007. "Method 3571 (SW-846): Extraction of Solid and Aqueous Samples for Chemical
Agents," Revision 0. http://www.epa.gov/sam/pdfs/EPA-3571 .pdf
5.2.24 EPA Method 3580A (SW-846): Waste Dilution
Analyte(s)
Brodifacoum
Bromadiolone
BZ [Quinuclidinyl benzilate]
Carfentanil
Chlorfenvinphos
3-Chloro-1 ,2-propanediol
Chloropicrin
CASRN
56073-10-0
28772-56-7
6581-06-2
59708-52-0
470-90-6
96-24-2
76-06-2
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Section 5 - Selected Chemical Methods
Analyte(s)
Chlorosarin
Chlorosoman
Chlorpyrifos
Chlorpyrifos oxon
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
Isopropyl methylphosphonic acid (IMPA)
Methyl hydrazine
Methyl paraoxon
Methyl parathion
N-Methyldiethanolamine (MDEA)
1-Methylethyl ester ethylphosphonofluoridic acid (GE)
Methylphosphonic acid (MPA)
Mevinphos
Monocrotophos
CASRN
1445-76-7
7040-57-5
2921-88-2
5598-15-2
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
1832-54-8
60-34-4
950-35-6
298-00-0
105-59-9
1189-87-3
993-13-5
7786-34-7
6923-22-4
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Section 5 - Selected Chemical Methods
Analyte(s)
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
Paraoxon
Parathion
Phencyclidine
Phenol
Phorate
Phorate sulfone
Phorate sulfoxide
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)
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]
CASRN
538-07-8
51-75-2
555-77-1
54-11-5
NA
311-45-5
56-38-2
77-10-1
108-95-2
298-02-2
2588-04-7
2588-03-6
13171-21-6
616-52-4
159939-87-4
NA
26628-22-8
96-64-0
57-24-9
77-81-6
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
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Section 5 - Selected Chemical Methods
Analyte(s)
VM [phosphonothioic acid, methyl-, S-(2-
(diethylamino)ethyl) O-ethyl ester]
CASRN
21770-86-5
Analysis Purpose: Sample preparation
Sample Preparation Technique: Waste dilution
Determinative Technique: EPA SW-846 Method 8015C, Method 8270D, or Method 8321B. 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
2-Fluoroethanol
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
371-62-0
NA
64742-81-0
75-56-9
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 analytes in Appendix A.
1,4-Thioxane
15980-15-1
Analysis Purpose: Sample preparation
Sample Preparation Technique: Waste dilution
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Section 5 - Selected Chemical Methods
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/pdfs/EPA-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
2-Fluoroethanol
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
371-62-0
NA
64742-81-0
75-56-9
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 analytes in Appendix A.
1,4-Thioxane
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.
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Section 5 - Selected Chemical Methods
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.
Special Considerations: Heated purge may be required for poor-purging analytes.
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
2-Fluoroethanol
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
371-62-0
NA
64742-81-0
75-56-9
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 analytes in Appendix A.
1,4-Thioxane
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
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Section 5 - Selected Chemical Methods
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
8015Cor8260C(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-
5035a.pdf
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 (2-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
7440-38-2
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
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.
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Section 5 - Selected Chemical Methods
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 (ig/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, calcium and lead arsenate, 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 Method 3050B), non-
aqueous liquid/organic solid samples (prepared using SW-846 Method 3031), and wipe samples (prepared
using NIOSH Method 9102) are analyzed by ICP-AES.
Special Considerations: Laboratory testing is currently underway for speciation of lewisite 1 using GC-
MS techniques.
Source: EPA. 2007. "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 (2-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
Titanium tetrachloride
Vanadium pentoxide
CASRN
7803-55-6
7440-38-2
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
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.
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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, calcium and lead arsenate, 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. Soil samples (prepared using SW-846 Method 3050B), non-
aqueous liquid/organic solid samples (prepared using SW-846 Method 3031), and wipe samples (prepared
using NIOSH Method 9102) 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.epa.gov/sam/pdfs/EPA-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
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 total arsenic in environmental samples. Soil
samples (prepared using SW-846 Method 3050B) and wipe samples (prepared using NIOSH Method
1902) 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)
Mercuric chloride
Mercury, Total
Methoxyethylmercuric acetate
CASRN
7487-94-7
7439-97-6
151-38-2
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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 (ig/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)
Mercuric chloride
Mercury, Total
Methoxyethylmercuric acetate
CASRN
7487-94-7
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: 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. http://www.epa.gov/sam/pdfs/EPA-7471b.pdf
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5.2.33 EPA Method 7473 (SW-846): Mercury in Solids and Solutions by Thermal
Decomposition, Amalgamation, and Atomic Absorption Spectrophotometry
Analyte(s)
Mercuric chloride
Mercury, Total
Methoxyethylmercuric acetate
CASRN
7487-94-7
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 747 IB (EPA SW-846) for
solid samples and 7470A (EPA SW-846) for aqueous liquid samples.
Source: EPA. 1998. "Method 7473 (SW-846): Mercury in Solids and Solutions by Thermal
Decomposition, Amalgamation, and Atomic Absorption Spectrophotometry," Revision 0.
http://www.epa.gov/sam/pdfs/EPA-7473.pdf
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
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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 (ig/L, respectively. MDLs for sand, a sandy loam soil (Lebanon soil), and soil from
the Rocky Mountain Arsenal (U.S. Army Environmental Center soil) were calculated to be 0.02, 0.43,
0.07 (ig/kg, respectively. This procedure provides sensitivity on the order of 0.01 (ig/L for water samples
and 1 (ig/kg for soil samples.
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.
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://www.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
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
(ig/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,
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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
2-Fluoroethanol
Propylene oxide
CASRN
107-18-6
75-15-0
107-07-3
506-77-4
107-06-2
75-21-8
371-62-0
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-Thioxane
15980-15-1
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 (emulsified oil), 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 i
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
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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. http://www.epa.gov/sam/pdfs/EPA-
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
Chlorpyrifos oxon
Crimidine3
Cyclohexyl sarin (GF)
Dichlorvos
Dicrotophos
Dimethylphosphite
Disulfoton
Disulfoton sulfoxide
1,4-Dithiane
Ethyldichloroarsine (ED)
Fenamiphos
Methyl hydrazine
Methyl paraoxon
Methyl parathion
1-Methylethyl ester ethylphosphonofluoridic acid (GE)
Mevinphos
Monocrotophos
Mustard, nitrogen (HN-1) [bis(2-chloroethyl)ethylamine]
CASRN
470-90-6
96-24-2
76-06-2
1445-76-7
7040-57-5
2921-88-2
5598-15-2
535-89-7
329-99-7
62-73-7
141-66-2
868-85-9
298-04-4
2497-07-6
505-29-3
598-14-1
22224-92-6
60-34-4
950-35-6
298-00-0
1189-87-3
7786-34-7
6923-22-4
538-07-8
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Analyte(s)
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)4
Nicotine compounds
Organophosphate pesticides, NOS
Paraoxon
Parathion
Phencyclidine
Phenol
Phorate
Phorate sulfone
Phorate sulfoxide
Phosphamidon
R 33 (VR) [methylphosphonothioic acid, S-[2-
(diethylamino)ethyl] O-2-methylpropyl ester]
Sarin (GB)4
Semivolatile organic compounds, NOS
Soman (GD)
Strychnine
Tabun (GA)
Tetraethyl pyrophosphate
Tetramethylenedisulfotetramine2
1,4-Thioxane5
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]4
CASRN
51-75-2
555-77-1
505-60-2
54-11-5
NA
311-45-5
56-38-2
77-10-1
108-95-2
298-02-2
2588-04-7
2588-03-6
13171-21-6
159939-87-4
107-44-8
NA
96-64-0
57-24-9
77-81-6
107-49-3
80-12-6
15980-15-1
121-45-9
21738-25-0
78-53-5
21770-86-5
50782-69-9
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Analyte(s)
CASRN
The following analyte should be determined by this method only if liquid chromatography-mass spectrometry (LC-
MS) [electrospray] 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
Ethyl methylphosphonic acid (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 problems 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 For this analyte, refer to EPA SW-846 Method 8271 forGC-MS conditions.
5 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.
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
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-846 8270D).
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
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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. Procedures for derivatization are described in
the following references:
Black et al. 1994. "Application of gas chromatography-mass spectrometry and gas chromatography-
tandem mass spectrometry to the analysis of chemical warfare samples, found to contain residues of the
nerve agent sarin, sulphur mustard and their degradation products." Journal of Chromatography A.
662(2): 301-321. http://www.sciencedirect.com/science/journal/00219673
Brereton, P., Kelly, J., Crews, C., Honour, S., and Wood, R. 2001. "Determination of 3-Chloro-l,2-
Propanediol in Foods and Food Ingredients by Gas Chromatography with Mass Spectrometric Detection:
Collaborative Study." Journal of AOAC International. 84(2): 455-465. http://www.atypon-
link.com/AOAC/doi/abs/10.5555/iaoi.2001.84.2.455
Divinova, V., Svejkovska, B., Dolezal, M., and Velisek, J. 2004. "Determination of Free and Bound 3-
Chloropropane-l,2-diol by Gas Chromatography with Mass Spectrometric Detection using Deuterated 3-
Chloropropane-l,2-diol as Internal Standard." Czech Journal of Food Sciences. 22(5): 182-189.
http://www.epa.gov/sam/pdfs/Czech J Food Sci-22(5)_pg 182-189.pdf
Retho, C., and Blanchard, F. 2005. "Determination of 3-chloropropane-l,2-diol as its 1,3-dioxolane
derivative at the (ig kg-1 level: Application to a wide range of foods." Food Additives & Contaminants:
Part A Chemistry, Analysis, Control, Exposure & Risk Assessment. 22(12): 1189-1197.
http: //www. informaworld. com/smpp/content~db=all~content=a727751832
White et al. 1992. "Determination of 3-Quinuclidinyl Benzilate (QNB) and Its Major Methoabolites in
Urine by Isotope Dilution Gas Chromatography/Mass Spectrometry." Journal of Analytical Toxicology.
16: 182-187. http://www.jatox.com/shop/shopexd.asp?id=4062
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-
8270d.pdf
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
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
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Analyte(s)
Bromadiolone
Carfentanil
Carbofuran (Furadan)
Chlorfenvinphos
3-Chloro-1 ,2-propanediol
Chloropicrin
Chlorosarin
Chlorosoman
Chlorpyrifos
Chlorpyrifos oxon
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)
N-Ethyldiethanolamine (EDEA)
Fenamiphos
Fentanyl
Fluoroacetic acid and fluoroacetate salts
Formaldehyde
Gasoline range organics
Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)
CASRN
28772-56-7
59708-52-0
1563-66-2
470-90-6
96-24-2
76-06-2
1445-76-7
7040-57-5
2921-88-2
5598-15-2
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
NA
50-00-0
NA
121-82-4
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Section 5 - Selected Chemical Methods
Analyte(s)
Hexamethylenetriperoxidediamine (HMTD)
Isopropyl methylphosphonic acid (IMPA)
Kerosene
Methomyl
Methyl acrylonitrile
Methyl fluoroacetate
Methyl hydrazine
Methyl paraoxon
Methyl parathion
N-Methyldiethanolamine (MDEA)
1-Methylethyl ester ethylphosphonofluoridic acid (GE)
Methylphosphonic acid (MPA)
Mevinphos
Monocrotophos
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
Paraoxon
Parathion
Pentaerythritol tetranitrate (PETN)
Phencyclidine
Phenol
Phorate
Phorate sulfone
Phorate sulfoxide
Phosphamidon
CASRN
283-66-9
1832-54-8
64742-81-0
16752-77-5
126-98-7
453-18-9
60-34-4
950-35-6
298-00-0
105-59-9
1189-87-3
993-13-5
7786-34-7
6923-22-4
538-07-8
51-75-2
555-77-1
505-60-2
54-11-5
2691-41-0
NA
23135-22-0
311-45-5
56-38-2
78-11-5
77-10-1
108-95-2
298-02-2
2588-04-7
2588-03-6
13171-21-6
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Section 5 - Selected Chemical Methods
Analyte(s)
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)
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
616-52-4
159939-87-4
107-44-8
NA
96-64-0
57-24-9
77-81-6
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.
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Section 5 - Selected Chemical Methods
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.
http://www.epa.gov/sam/pdfs/EPA-8290a.pdf
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. http://www.epa. gov/sam/pdfs/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
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Section 5 - Selected Chemical Methods
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 (ig/L; acrylonitrile has an MDL of 20 (ig/L.
Description of Method: Samples are analyzed by HPLC. A 200-uL 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. http://www.epa.gov/sam/pdfs/EPA-8316.pdf
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 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, and wipe samples.
Detection and Quantitation: The estimated MDLs vary with each analyte and range from 1.7 to 9.4
(ig/L for aqueous samples and 10 to 50 (ig/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 ds
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.
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Section 5 - Selected Chemical Methods
Source: EPA. 2000. "Method 83ISA (SW-846): N-Methylcarbamates by High Performance Liquid
Chromatography (HPLC)," Revision 1. http://www.epa.gov/sam/pdfs/EPA-8318a.pdf
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
Carfentanil
Diisopropyl methylphosphonate (DIMP)2
Dimethylphosphoramidic acid1
Diphacinone
EA2192 [Diisopropylaminoethyl
methylthiolophosphonate]1
Ethyl methylphosphonic acid (EMPA)1
N-Ethyldiethanolamine (EDEA)
Fentanyl
Isopropyl methylphosphonic acid (IMPA)1
N-Methyldiethanolamine (MDEA)
Methylphosphonic acid (MPA)1
Pinacolyl methyl phosphonic acid (PMPA)1
Thiodiglycol (TDG)
Thiofanox
Triethanolamine (TEA)
CASRN
56073-10-0
28772-56-7
6581-06-2
59708-52-0
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
111-48-8
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
1 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
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Section 5 - Selected Chemical Methods
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
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. Aqueous liquid samples for DIMP, EMPA, IMPA,
MPA, and PMPA should be analyzed using EPA Chicago Regional Laboratory (CRL) MS017; aqueous
liquid samples for EDEA, MDEA, and TEA should be prepared and analyzed using EPA CRL MS016;
aqueous liquid samples for bromadiolone should be prepared and analyzed using EPA CRL MS014;
aqueous liquid samples for thiodiglycol should be prepared and analyzed using EPA CRL MS015.
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 8321B (SW-846): Solvent-Extractable Nonvolatile Compounds by High
Performance Liquid Chromatography-Thermospray-Mass Spectrometry (HPLC-TSP-MS) or Ultraviolet
(UV) Detection," Revision 2. http://www.epa.gov/sam/pdfs/EPA-8321b.pdf
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
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Section 5 - Selected Chemical Methods
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
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. 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-
8330b.pdf
5.2.44 EPA CRL MS014: Analysis of Aldicarb, Bromadiolone, Carbofuran, Oxamyl and
Methomyl in Water by Multiple Reaction Monitoring Liquid Chromatography /
Tandem Mass Spectrometry (LC/MS/MS)
Analyte(s)
Aldicarb (Temik)
Aldicarb sulfone
Aldicarb sulfoxide
Bromadiolone
Carbofuran (Furadan)
Methomyl
Oxamyl
CASRN
116-06-3
1646-88-4
1646-87-3
28772-56-7
1563-66-2
16752-77-5
23135-22-0
Analysis Purpose: Sample preparation and/or analysis
Sample Preparation Technique: Solvent extraction
Determinative Technique: Liquid Chromatography Tandem Mass Spectrometry (LC-MS-MS)
Method Developed for: Determination of aldicarb, bromadiolone, carbofuran, oxamyl and methomyl in
water
Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid samples.
Detection and Quantitation: The limit of detection for aldicarb, bromadiolone, carbofuran, methomyl
and oxamyl is 100 ng/L. The reporting limit for aldicarb, carbofuran, methomyl and oxamyl is 1 (ig/L.
Description of Method: Target compounds are analyzed by direct injection without derivatization by
LC-MS-MS. Samples are shipped to the laboratory at 4°C (± 2°C), spiked with surrogates, filtered using
a syringe driven filter unit, and analyzed directly by LC-MS-MS within 7 days. If stored prior to analysis,
samples must be stored at < 4°C (± 2°C). The target compounds are identified by comparing the sample
SAM Revision 5.0 85 September 29, 2009
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Section 5 - Selected Chemical Methods
primary and confirmatory multiple reaction monitoring (MRM) transitions to the known standard primary
and confirmatory MRM transitions. The retention time for the analytes of interest must also fall within
the retention time of the standard by ± 5%. The target compounds are quantitated using the primary
single reaction monitoring (SRM) transition and external standard calibration.
Source: EPA, Chicago Regional Laboratory (CRL). 2008. "MS014: Analysis of Aldicarb,
Bromadiolone, Carbofuran, Oxamyl and Methomyl in Water by Multiple Reaction Monitoring Liquid
Chromatography/ Tandem Mass Spectrometry (LC/MS/MS)." http:\\www.epa.gov/sam/pdfs/EPA-
MS014.pdf
5.2.45 EPA CRL MS015: Analysis of Thiodiglycol in Water by Single Reaction Monitoring
Liquid Chromatography / Tandem Mass Spectrometry (LC/MS/MS)
Analyte(s)
Thiodiglycol
CASRN
111-48-8
Analysis Purpose: Sample preparation and/or analysis
Sample Preparation Technique: Solvent extraction
Determinative Technique: LC-MS-MS
Method Developed for: Determination of thiodiglycol in water samples
Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid samples.
Detection and Quantitation: The limit of detection for thiodiglycol is 50 (ig/L; the reporting limit is 250
ug/L.
Description of Method: Thiodiglycol is analyzed by direct injection without derivatization by LC-MS-
MS. Samples are shipped to the laboratory at 4°C (± 2°C), spiked with surrogates, filtered using a syringe
driven filter unit and analyzed directly by LC-MS-MS within 7 days. The target compound is identified
by comparing the sample primary SRM transition to the known standard SRM transition. The retention
time must fall within the retention time of the standard by ± 5%. Thiodiglycol is quantitated using the
primary SRM transition utilizing an external standard calibration.
Source: EPA, CRL. 2008. "MS015: Analysis of Thiodiglycol in Water by Single Reaction Monitoring
Liquid Chromatography / Tandem Mass Spectrometry (LC/MS/MS)."
http:\\www.epa.gov/sam/pdfs/EPA-MSO 15.pdf
5.2.46 EPA CRL MS016: Analysis of Diethanolamine, Triethanolamine, n-
Methyldiethanolamine and n-Ethyldiethanolamine in Water by Single Reaction
Monitoring Liquid Chromatography / Tandem Mass Spectrometry (LC/MS/MS)
Analyte(s)
N-Ethyldiethanolamine (EDEA)
N-Methyldiethanolamine (MDEA)
Triethanolamine (TEA)
CASRN
139-87-7
105-59-9
102-71-6
Analysis Purpose: Sample preparation and/or analysis
Sample Preparation Technique: Solvent extraction
Determinative Technique: LC-MS-MS
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Method Developed for: Determination of diethanolamine, triethanolamine, «-methyldiethanolamine and
«-ethyldiethanolamine in water samples
Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid samples.
Detection and Quantitation: The limit of detection for «-ethyldiethanolamine and triethanolamine is 5
(ig/L; the reporting limit is 25 (ig/L. The limit of detection for n-methyldiethanolamine is 20 (ig/L, with a
reporting limit of 25 (ig/L.
Description of Method: Target compounds are analyzed by direct injection without derivatization by
LC-MS-MS. Samples are shipped to the laboratory at 4°C (± 2°C), spiked with surrogates, filtered using
a syringe driven filter unit and analyzed directly by LC-MS-MS within 7 days. Target compounds are
identified by comparing sample SRM transitions to the known standard SRM transitions. The retention
time for the analytes of interest must also fall within the retention time of the standard by ± 5%. The
target compounds are quantitated using the SRM transition and external standard calibration.
Source: EPA, CRL. 2008. "MS016: Analysis of Diethanolamine, Triethanolamine, n-
Methyldiethanolamine and «-Ethyldiethanolamine in Water by Single Reaction Monitoring Liquid
Chromatography / Tandem Mass Spectrometry (LC/MS/MS)." http:\\www.epa.gov/sam/pdfs/EPA-
MS016.pdf
5.2.47 EPA CRL MS017: Analysis of Diisopropyl Methylphosphonate, Ethyl Hydrogen
Dimethylamidophosphate, Isopropyl Methylphosphonic Acid, Methylphosphonic
Acid and Pinacolyl Methylphosphonic Acid in Water by Multiple Reaction
Monitoring Liquid Chromatography / Tandem Mass Spectrometry (LC/MS/MS)
Analyte(s)
Diisopropyl methylphosphonate (DIMP)
Ethyl methylphosphonic acid (EMPA)
Isopropyl methylphosphonic acid (IMPA)
Methylphosphonic acid (MPA)
Pinacolyl methyl phosphonic acid (PMPA)
CASRN
1445-75-6
1832-53-7
1832-54-8
993-13-5
616-52-4
Analysis Purpose: Sample preparation and/or analysis
Sample Preparation Technique: Solvent extraction
Determinative Technique: LC-MS-MS
Method Developed for: Determination of diisopropyl methylphosphonate, ethyl hydrogen
dimethylamidophosphate, isopropyl methylphosphonic acid, methylphosphonic acid and pinacolyl
methylphosphonic acid in water
Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid samples.
Detection and Quantitation: The limits of detection and reporting limits for this method vary for each
analyte and range between 0.10 to 20 (ig/L and 5 to 100 (ig/L, respectively.
Description of Method: Target compounds are analyzed by direct injection without derivatization by
LC-MS-MS. Samples are shipped to the laboratory at 4°C (± 2°C), spiked with surrogates, filtered using
a syringe driven filter unit and analyzed directly by LC-MS-MS within 1 day. The target compounds are
identified by comparing the sample SRM transitions to the known standard SRM transitions. The
retention time for the analytes of interest must also fall within the retention time of the standard by ± 5%.
Target compounds are quantitated using the SRM transition of the target compounds and external
standard calibration.
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Source: EPA, CRL. 2008. "MS017: Analysis of Diisopropyl Methylphosphonate, Ethyl Hydrogen
Dimethylamidophosphate, Isoproyl Methylphosphonic Acid, Methylphosphonic Acid and Pinacolyl
Methylphosphonic Acid in Water by Multiple Reaction Monitoring Liquid Chromatography / Tandem
Mass Spectrometry (LC/MS/MS)." http:\\www.epa.gov/sam/pdfs/EPA-MS017.pdf
5.2.48 EPA CLP 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 (ig/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 Contract Laboratory Program (CLP). "ILM05.3: Exhibit D - Part D: Analytical Methods
for Total Cyanide Analysis." http://www.epa.gov/sam/pdfs/EPA-ILM05.3.pdf
5.2.49 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
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.
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Section 5 - Selected Chemical Methods
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 > 11 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." http://www.epa.gov/sam/pdfs/EPA-
3135.2I.pdf
5.2.50 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 (2-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
7440-38-2
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
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.
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Description of Method: This method supports determination of arsenic trioxide, lewisite, lewisite
degradation products, calcium and lead arsenate, 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 .pdf
5.2.51 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 (2-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
7440-38-2
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
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, calcium and lead arsenate, 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. Detection limits, ranges, and interference corrections are dependent on the analyte and the
instrument used.
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Special Considerations: Laboratory testing is currently underway for speciation of lewisite 1 using GC-
MS techniques. Concerns have been raised regarding the use of nitric acid when analyzing samples for
osmium tetroxide; hydrochloric acid should be considered and evaluated as a possible alternative.
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 PM10 Using High Volume (HV) Sampler." http://www.epa.gov/sam/pdfs/EPA-IO-2.1 .pdf
5.2.52 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 (2-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
7440-38-2
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: Analysis
Sample Preparation Technique: EPA Method IO-3.1
Determinative Technique: ICP-MS
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, calcium and lead arsenate, 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. Detection limits, ranges, and interference corrections are dependent on the
analyte and the instrument used.
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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
5.2.53 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: Cold vapor atomic fluorescence Spectrometry (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.
Special Considerations: There are no known positive interferences at 253.7 run 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)."
http://www.epa.gov/sam/pdfs/EPA-IO-5.pdf
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5.2.54 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-propanediol2
Chlorosarin2
Chlorosoman2
Chlorpyrifos
Chlorpyrifos oxon
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
Isopropyl methylphosphonic acid (IMPA)1
Methyl paraoxon
Methyl parathion
N-Methyldiethanolamine (MDEA)
1-Methylethyl ester ethylphosphonofluoridic acid (GE)2
Methylphosphonic acid (MPA)1
Mevinphos
Monocrotophos
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
6581-06-2
470-90-6
96-24-2
1445-76-7
7040-57-5
2921-88-2
5598-15-2
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
1832-54-8
950-35-6
298-00-0
105-59-9
1189-87-3
993-13-5
7786-34-7
6923-22-4
538-07-8
51-75-2
555-77-1
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Analyte(s)
Mustard, sulfur/ Mustard gas (HD)
Paraoxon
Parathion
Phencyclidine
Phenol
Phorate
Phorate sulfone
Phorate sulfoxide
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)
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
505-60-2
311-45-5
56-38-2
77-10-1
108-95-2
298-02-2
2588-04-7
2588-03-6
13171-21-6
616-52-4
159939-87-4
107-44-8
NA
96-64-0
77-81-6
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
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-15 be used.
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Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Solvent extraction
Determinative Technique: GC-MS orHPLC
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 (ig/m3 concentrations, and 4 to 24-hour sampling periods.
Description of Method: A low-volume (1 to 5 L/min) 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. See Special Considerations in Section 5.2.37
for information regarding derivatization of compounds.
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)." http://www.epa.gov/sam/pdfs/EPA-TO-10a.pdf
5.2.55 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
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)
96-24-2
1445-76-7
7040-57-5
1445-75-6
1189-87-3
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Analyte(s)
Sarin (GB)
Soman (GD)
CASRN
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.
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
5.2.56 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 sorbenttube
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.
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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. Analysis is performed on a GC-FID. 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://www.epa.gov/sam/pdfs/NIOSH-l 612.pdf
5.2.57 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 (ig/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.
htto://www.epa.gov/sam/pdfs/NIOSH-2016.pdf
5.2.58 NIOSH Method 2513: Ethylene Chlorohydrin
Analyte(s)
2-Chloroethanol
2-Fluoroethanol
CASRN
107-07-3
371-62-0
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Solvent desorption
Determinative Technique: GC-FID
Method Developed for: Ethylene chlorohydrin (2-chloroethanol) 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.
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Description of Method: Samples are drawn into atube 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.
htto://www.epa.gov/sam/pdfs/NIOSH-2513.pdf
5.2.59 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 transferred to a 25-mL flask, mixed with phosphomolybdic acid
solution, diluted to the mark with 0.1 M hydrochloric acid, and then transferred to a large test tube for
spectrophotometric analysis. Positive interferences include other hydrazines, as well as 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.
http://www.epa.gov/sam/pdfs/NIOSH-3510.pdf
5.2.60 NIOSH Method 5600: Organophosphorus Pesticides
Analyte(s)
Disulfoton
Disulfoton sulfoxide
Organophosphate pesticides, NOS
CASRN
298-04-4
2497-07-6
NA
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.
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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. Samples are prepared by desorbing the XAD-2 resin with 2 mL of toluene/acetone (90/10
v/v) solution. 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.
http://www.epa.gov/sam/pdfs/NIOSH-5600.pdf
5.2.61 NIOSH Method 5601: Organonitrogen Pesticides
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: 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, methomyl, 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. Samples are prepared by desorbing the XAD-2 resin with 2 mL of
triethylamine-phosphate solution, rotating end-over-end for 45 minutes, and filtering. 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 (butylated hydroxytoluene [BHT]), plasticizers (dialkyl
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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 HPLC column or by an MS.
Source: NIOSH. 1998. "Method 5601: Organonitrogen Pesticides," Issue 1.
http://www.epa.gov/sam/pdfs/NIOSH-5601.pdf
5.2.62 NIOSH Method 6001: Arsine
Analyte(s)
Arsine
CASRN
7784-42-1
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Coconut shell charcoal solid sorbenttube
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
5.2.63 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.
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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://www.epa.gov/sam/pdfs/NIOSH-6002.pdf
5.2.64 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 (im 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
5.2.65 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 0.6 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.
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Special Considerations: The method is subject to interference from high concentrations of hydrogen
sulfide. Two liters is the minimum volume required to measure concentration of 5 ppm.
Source: NIOSH. 1994. "Method 6010: Hydrogen Cyanide," Issue 2.
http://www.epa.gov/sam/pdfs/NIOSH-6010.pdf
5.2.66 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 15 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/sam/pdfs/NIOSH-6013.pdf
5.2.67 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/m3 for a 10-L sample.
Twice the recommended sample volume should be collected in order to achieve an action level of 70
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 silica 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.
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Source: NIOSH. 1994. "Method 6015: Ammonia," Issue 2.
htto://www.epa.gov/sam/pdfs/NIOSH-6015.pdf
5.2.68 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 with bromine to phosphoric acid and color agent (sodium molybdate) and reducing
agent (hydrazine sulfate) are added. The solution is analyzed for the resulting molybdenum blue complex
by visible Spectrophotometry. Phosphorus (V) compounds do not interfere. Sample solutions are stable
to oxidation by air during sampling.
Source: NIOSH. 1994. "Method 6402: Phosphorus Trichloride," Issue 2.
http://www.epa.gov/sam/pdfs/NIOSH-6402.pdf
5.2.69 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.
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Source: NIOSH. 1994. "Method 7903: Acids, Inorganic," Issue 2.
htto://www.epa.gov/sam/pdfs/NIOSH-7903.pdf
5.2.70 NIOSH Method 7905: Phosphorus
Analyte(s)
White phosphorus
CASRN
12185-10-3
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: GC solid sorbenttube and solvent extracted (desorbed)
Determinative Technique: GC-FPD
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. Five to 100 L of air is drawn through a GC solid sorbent tube, and the sorbent is
extracted (desorbed) with xylene. 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.
Source: NIOSH. 1994. "Method 7905: Phosphorus," Issue 2.
http://www.epa.gov/sam/pdfs/NIOSH-7905.pdf
5.2.71 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-(im 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.
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Source: NIOSH. 1994. "Method 7906: Fluorides, Aerosol and Gas by 1C," Issue 1.
htto://www.epa.gov/sam/pdfs/NIOSH-7906.pdf
5.2.72 NIOSH Method 9102: Elements on Wipes
Analyte(s)
Ammonium metavanadate
Arsenic, Total
Arsenic trioxide
Arsine
Calcium arsenate
2-Chlorovinylarsonous acid (2-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]
Lewisite oxide
Mercuric chloride
Mercury, Total
Methoxyethylmercuric acetate
Osmium tetroxide
Sodium arsenite
Thallium sulfate
Titanium tetrachloride
Vanadium pentoxide
CASRN
7803-55-6
7440-38-2
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
1306-02-1
7487-94-7
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, and 7473. 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 tolOS (ig/wipe; forthallium 0.136 to 50.0
(ig/wipe; for vanadium 0.0333 to 25.0 (ig/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
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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."
htto://www.epa.gov/sam/pdfs/NIOSH-9102.pdf
5.2.73 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.
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
5.2.74 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.
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Detection and Quantitation: The detection limit of the overall procedure is 0.35 (ig per sample (28 ppb
or 35 (ig/m3). Quantitation limits of 28 ppb (35 (ig/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: OSF£A. 1982. "Method 40: Methylamine." Method originally obtained from www.osha.gov.
but is provided here for reference. http://www.epa.gov/sam/pdfs/OSFIA-Method40.pdf
5.2.75 OSHA Method 54: Methyl Isocyanate (MIC)
Analyte(s)
Methyl isocyanate
CASRN
624-83-9
Analysis Purpose: Sample preparation and analysis
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
www.osha.gov. but is provided here for reference. http://www.epa.gov/sam/pdfs/OSHA-Method54.pdf
5.2.76 OSHA Method 61: Phosgene
Analyte(s)
Phosgene
CASRN
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
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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 phosgene should be confirmed by either a secondary GC
column or by MS
Source: OSHA. 1986. "Method 61: Phosgene." Method originally obtained from www.osha.gov. but is
provided here for reference. http://www.epa.gov/sam/pdfs/OSHA-Method61 .pdf
5.2.77 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
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 0.011 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 (ISG)
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-211: Sodium Azide and Hydrazoic Acid in Workplace
Atmospheres." http://www.epa.gov/sam/pdfs/OSHA-ID-211 .pdf
5.2.78 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 (ig in a 30-L sample.
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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.osha.gov. but is provided here for reference. http://www.epa.gov/sam/pdfs/OSHA-ID216SG.pdf
5.2.79 OSHA Method PV2004: Acrylamide
Analyte(s)
Acrylamide
Acrylonitrile
Methyl acrylonitrile
CASRN
79-06-1
107-13-1
126-98-7
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Solvent desorption
Determinative Technique: HPLC
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 (ig/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
5.2.80 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-(iL injection volume. This is the
smallest amount that could be detected under normal operating conditions. The working range is 33.2 to
1330 (ig/m3.
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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
5.2.81 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
5.2.82 ASTM Method D6480-05: 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
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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 and EDXA at
a magnification from 15,000 to 20,OOOX.
Source: ASTM. 2005. "Method D6480-05: 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/Standards/D6480.htm
5.2.83 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."
http://www.iso.org/iso/iso catalogue/catalogue tc/catalogue detail.htm?csnumber=18358
5.2.84 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
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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.
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/
5.2.85 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. standardmethods.org/
5.2.86 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
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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.
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/
5.2.87 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 (ig 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 (ig 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 spectropnotometric analysis." Analyst
124(12): 1853-1857. www.epa.gov/sam/pdfs/Analystl24 pgl853-1857.pdf
5.2.88 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
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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.
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.
http://www.informaworld.com/smpp/content~content=a747219004~db=all~order=page
5.2.89 Literature Reference for Methamidophos (Chromatographia. 2006. 63(5/6): 233-
237)
Analyte(s)
Acephate
Methamidophos
CASRN
30560-19-1
10265-92-6
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: SPE
Determinative Technique: LC-MS-MS
Method Developed for: Pesticides (methamidophos) in water samples
Method Selected for: SAM lists this procedure for preparation and analysis of aqueous liquid and
drinking water samples.
Detection and Quantitation: The limit of detection for this limit is 30 (ig/L.
Description of Method: A multi-residue analytical method is described for monitoring polar pesticides,
such as acephate and methamidophos, in water with SPE (solid-phase extraction) and LC-MS-MS.
Samples are analyzed using a Ci8 analytical column (150 mm x 3.2 mm I.D., 5(im particle size) coupled
with a Cis guard cartridge system (4 mm x 3.0 mm I.D.).
Special Considerations: The procedure described above has been developed for the analysis of various
pesticides (methamidophos) in reagent water by LC-MS-MS. Modifications may be needed for
application to drinking water samples.
Source: Liu, F., Bischoff, G., Pestemer, W., Xu, W., and Kofoet, A. 2006. "Multi-residue Analysis of
Some Polar Pesticides in Water Samples with SPE and LC/MS/MS." Chromatographia. 63(5/6): 233-
237. http://www.epa.gov/sam/pdfs/Chromatographia-63 pg233-237.pdf
5.2.90 Literature Reference for Methamidophos (Journal of Chromatography A, 2007.
1154:3-25)
Analyte(s)
Acephate
CASRN
30560-19-1
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Analyte(s)
Methamidophos
CASRN
10265-92-6
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Solvent extraction
Determinative Technique: LC-MS-MS
Method Developed for: Pesticides (methamidophos) in crops
Method Selected for: SAM lists this procedure for preparation and analysis of solid, non-aqueous
liquid/organic solid, air, and wipe samples.
Detection and Quantitation: The limit of detection for this method is 0.01 mg/kg.
Description of Method: A liquid chromatography-tandem quadrupole mass spectrometry (LC-MS-MS)
multi-residue method for the simultaneous target analysis of a wide range of pesticides and metabolites in
fruit, vegetables and cereals is described. Gradient elution has been used in conjunction with positive
mode electrospray ionization tandem mass spectrometry to detect up to 171 pesticides and/or metabolites
in different crop matrices using a single chromatographic run. Pesticide residues are extracted/partitioned
from the samples with acetone/dichloromethane/light petroleum. Samples are analyzed by LC-MS-MS
using a Qg analytical column (150 mm x 3.2 mm I.D., 5(im particle size) coupled with a Cig guard
cartridge system (4 mm x 3.0 mm I.D.).
Special Considerations: The procedure has been developed for the analysis of various pesticides
(methamidophos) in crops using LC-MS-MS; modifications will be needed for application to
environmental samples such as soils, wipes, and air samples collected on sorbent/filters.
Source: Hiemstra, M., de Kok, A. 2007. "Comprehensive Multi-residue Method for the Target Analysis
of Pesticides in Crops Using Liquid Chromatography-tandem Mass Spectrometry." Journal of
Chromatography A. 1154(1): 3-25. http://www.sciencedirect.com/science/iournal/00219673
5.2.91 Literature Reference for Fluoroacetamide (Journal of Chromatography B, 2008.
876(1): 103-108)
Analyte(s)
Fluoroacetamide
CASRN
640-19-7
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Water extraction
Determinative Technique: GC/MS
Method Developed for: Fluoroacetamide and tetramine in blood, urine and stomach contents
Method Selected for: SAM lists this procedure for preparation and analysis of solid, non-aqueous
liquid/organic solid, aqueous liquid, drinking water, air, and wipe samples.
Detection and Quantitation: The detection limit of this method for fluoroacetamide is 0.01 ug/g.
Description of Method: Samples are extracted by microscale liquid-liquid extraction using acetonitrile,
ENVI-CARB, and sodium chloride. Samples are analyzed by GC/MS using a 30-m DB-5MS capillary
column (or equivalent) coupled with a 1.5 m Innowax capillary column (or equivalent) by a quartz
capillary column connector. If analyzing for fluoroacetamide alone, only the Innowax capillary column is
needed.
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Special Considerations: The procedure has been developed for the analysis of fluoroacetamide and
tetramine in blood, urine and stomach fluid samples; modifications will be needed for application to
environmental samples.
Source: Xu, X., Song, G., Zhu, Y., Zhang, J., Zhao, Y., Shen, H., Cai, Z., Han, J., and Ren, Y. 2008.
"Simultaneous Determination of two Accute Poisoning Rodenticides Tetramine and Fluoroacetamide
with a Coupled Column in Poisoning Cases." Journal of Chromatography B. 876(1): 103-108.
http://www.sciencedirect.com/science/journal/15700232
5.2.92 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, aqueous liquid, and drinking
water samples.
Detection and Quantitation: This method can routinely quantify to at least 100 (ig/L, and the detection
limit is estimated to be 30 (ig/L.
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://www.astm.org/JOURNALS/FORENSIC/PAGES/2933.htm
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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.
Following a homeland security event, it is assumed that only those areas with contamination greater than
pre-existing/naturally prevalent levels commonly found in the environment would be subject to
SAM Revision 5.0 111 September 29, 2009
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Section 6 - Selected Radiochemical Methods
remediation. Dependent on site- and event-specific goals, investigation of background levels using
methods listed in Appendix B is recommended.
6.1 General Guidelines
The guidelines summarized in this section provide 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. Emergency response documents recently developed by EPA's Office of Radiation
and Indoor Air (ORIA) may be found at http://www.epa.gov/narel/incident_guides.html.
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 6.2.32, 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
Select Mixed Fission Products
Americium-241
CASRN
NA
NA
NA
14596-10-2
Method
900.0 (EPA)
FRMAC, Vol 2, pg. 33
AP1 (ORISE)
7110 B(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)
D3084-05 (ASTM)
Section
6.2.2
6.2.18
6.2.19
6.2.26
6.2.3
6.2.13
6.2.10
6.2.11
6.2.12
6.2.15
6.2.22
6.2.24
Please note that this category does not cover all fission products.
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Analyte / Analyte Class
Californium-252
Cesium-137
Cobalt-60
Curium-244
Europium-154
lodine-125
lodine-131
lridium-192
Molybdenum-99
Plutonium-238
Plutonium-239
Polonium-210
Radium-226
Ruthenium-103
Ruthenium-106
Selenium-75
Strontium-90
CASRN
13981-17-4
10045-97-3
10198-40-0
13981-15-2
15585-10-1
14158-31-7
10043-66-0
14694-69-0
14119-15-4
13981-16-3
15117-48-3
13981-52-7
13982-63-3
13968-53-1
13967-48-1
14265-71-5
10098-97-2
Method
Am-01-RC(HASL-300)
Am-04-RC (HASL-300)
Pu-12-RC (HASL-300)
AP11 (ORISE)
D3084-05 (ASTM)
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)
D3084-05 (ASTM)
901.1 (EPA)
Ga-01-R (HASL-300)
7120 (SM)
Procedure #9 (ORISE)
901.1 (EPA)
Ga-01-R (HASL-300)
901.1 (EPA)
Ga-01-R (HASL-300)
7120 (SM)
901.1 (EPA)
Ga-01-R (HASL-300)
EMSL-33 (EPA)
AP11 (ORISE)
D3084-05 (ASTM)
Method 111 (EPA)
Po-02-RC (HASL-300)
903.0 (EPA)
903.1 (EPA)
EMSL-19(EPA)
D3084-05 (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-SrB(SM)
Section
6.2.10
6.2.12
6.2.15
6.2.22
6.2.24
6.2.3
6.2.13
6.2.27
6.2.10
6.2.12
6.2.15
6.2.22
6.2.24
6.2.3
6.2.13
6.2.27
6.2.23
6.2.3
6.2.13
6.2.3
6.2.13
6.2.27
6.2.3
6.2.12
6.2.9
6.2.22
6.2.24
6.2.1
6.2.14
6.2.4
6.2.5
6.2.8
6.2.24
6.2.28
6.2.29
6.2.3
6.2.13
6.2.27
6.2.16
6.2.30
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Analyte / Analyte Class
Technetium-99
Tritium (Hydrogen-3)
Uranium-234
Uranium-235
Uranium-238
CASRN
14133-76-7
10028-17-8
13966-29-5
15117-96-1
7440-61-1
Method
Tc-02-RC (HASL-300)
APS (ORISE)
906.0 (EPA)
AP2 (ORISE)
908.0 (EPA)
EMSL-33 (EPA)
AP11 (ORISE)
D3084-05 (ASTM)
D3972-02 (ASTM)
7500-U B (SM)
7500-U C (SM)
Section
6.2.17
6.2.21
6.2.6
6.2.20
6.2.7
6.2.9
6.2.22
6.2.24
6.2.25
6.2.31
6.2.32
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
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 6-1. Sources of Radiochemical Methods
Name
NEMI
CFR Promulgated Test
Methods
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
Publisher
EPA, USGS
EPA, Technical Transfer Network
(TTN) EMC
EPA, ORD, Environmental
Monitoring and Support Laboratory
(EMSL)
EPA, EMSL
Department of Energy (DOE),
Environmental Measurements
Laboratory (EML) / Now DHS
DOE, National Nuclear Security
Administration (NNSA)
ORISE, Independent
Environmental Assessment and
Verification
Reference
http://www.nemi.qov
http://www.epa.aov/ttn/emc/promaate.html
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.aov/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
melandsecuritv/frmac/manuals.aspx
http://orise.orau.aov/ieav/survev-
proiects/lab-manual.htm
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Name
Annual Book of AST M
Standards, Vol. 11.02*
Standard Methods for the
Examination of Water and
Wastewater, 21st Edition, 2005*
Publisher
ASTM International
APHA, AWWA, and WEF
Reference
http://www.astm.org
http://www.standardmethods.org
' Subscription and/or purchase required.
6.1.2 General QC Guidelines 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.
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.
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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, guidelines, or information regarding safety precautions
that should be followed when handling or processing environmental samples and reagents. These
methods may also provide information regarding waste management. Laboratories should consult with
the responsible government agencies prior to disposal of waste materials. Other resources that can be
consulted for additional information include the following:
• OSHA - 29 CFRpart 1910.1450. Occupational Exposure to Hazardous Chemicals in Laboratories.
http://www.access.gpo.gov/nara/cfr/waisidx 06/29cfrl910a 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.
http://www.access.gpo.gov/nara/cfr/waisidx 07/40cfr270 07.html
. NRC - 10 CFR part 20. Standards for Protection Against Radiation
http://www.access.gpo.gov/nara/cfr/waisidx 00/10cfr20 OO.html
. DOE. Order O 435.1: Radioactive Waste Management. July 1, 1999. Available at:
www.directives.doe.gov/pdfs/doe/doetext/neword/435/o4351 .html
. DOE. M 435.1-1. Radioactive Waste Management Manual. Office of Environmental Management.
July 9, 1999. Available at: http://www.directives.doe.gov/pdfs/doe/doetext/neword/435/m4351-
Lhtml
. 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. Available at:
httD://www.orau.org/ptp/PTP%20Librarv/librarv/DOE/Misc/radmeth3.pdf
. 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. Available at:
http://www.epa.gov/rpdwebOO/docs/mixed-waste/402-r-96-015.pdf
. EPA. 2001. Changes to 40 CFR 266 (Storage, Treatment, Transportation, and Disposal of Mixed
Waste), Federal Register 66:27217-27266, May 16. Available at:
http://frwebgate.access.gpo.gov/cgi-bin/getdoc.cgi?dbname=2001register&docid=01-11408-
filed.pdf
. EPA. 2008. Resource Conservation and Recovery Act (RCRA) Orientation Manual. OSWER,
Washington, DC. EPA530-R-02-016. 259 pp. Available at:
http://www.epa.gov/osw/inforesources/pubs/orientat/
. MARLAP Manual. 2004. Chapter 17. Waste Management in a Radioanalytical Laboratory.
Available at: http://www.epa.gov/rpdwebOO/docs/marlap/402-b-04-001b-17-final.pdf
• National Research Council. 1995. Prudent Practices in the Laboratory; Handling and Disposal of
Chemicals, National Academy Press, Washington, DC. Available at:
http://books.nap.edu/openbook.php?isbn=0309052297
• National Council on Radiation Protection and Measurements (NCRP). 2002. Risk-Based
Classification of Radioactive and Hazardous Chemical Wastes, Report Number 139. 7910
Woodmont Avenue, Suite 400, Bethesda, MD 20814-3095
• Nuclear Regulatory Commission (NRC) / EPA. 1995. Joint Nuclear Regulatory
Commission/Environmental Protection Agency Guidance on the Storage of Mixed Radioactive and
Hazardous Waste., Federal Register 60:40204-40211
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6.2 Method Summaries
Summaries for the analytical methods listed in Appendix B are provided in Sections 6.2.1 through 6.2.32.
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
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." http://www.epa.gov/sam/pdfs/EPA-l 11 .pdf
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 (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
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• 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
analysis3. 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 forthe use of cesium-137).
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.
http://www.epa.gOv/sam/pdfs/EPA-900.0.pdf
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
Molybdenum-99
Ruthenium-103
Ruthenium-106
Selenium-75
CASRN
10045-97-3
10198-40-0
15585-10-1
10043-66-0
14694-69-0
14119-15-4
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 select gamma
emitters in drinking water samples.
3 EPA lists standards for use when analyzing drinking water in the table at 40 CFR 141.25 (footnote 11).
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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.
http://www.epa.gov/sam/pdfs/EPA-901.1 .pdf
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.epa.gOv/sam/pdfs/EPA-903.0.pdf
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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.
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 906.0: Tritium in Drinking Water
Analyte(s)
Tritium (Hydrogen-3)
CASRN
10028-17-8
Analysis Purpose: Qualitative and confirmatory analysis
Determinative Technique: Liquid scintillation
Method Developed for: Tritium (as T2O or HTO) in drinking water
Method Selected for: SAM lists this method for qualitative and confirmatory analysis of drinking water
and aqueous/liquid phase samples.
Description of Method: An unpreserved 100-mL aliquot of a drinking water sample is distilled after
adjusting pH with a small amount of sodium hydroxide and adding potassium permanganate. The
alkaline treatment prevents other radionuclides, such as radioiodine and radiocarbon, from distilling with
the tritium. The permanganate treatment oxidizes trace organics that may be present in the sample and
prevents their appearance in the distillate. To determine the concentration of tritium, the middle fraction
of the distillate is used, because the early and late fractions are more apt to contain materials interfering
with the liquid scintillation counting process. A portion of this collected fraction is added to a liquid
scintillator cocktail, and the solution is mixed, dark adapted and counted for beta particle activity. The
efficiency of the system can be determined by the use of prepared tritiated water standards having the
same density and color as the sample.
Source: EPA, EMSL. 1980. "Method 906.0: Tritium in Drinking Water:'Prescribed Procedures for
Measurement of Radioactivity in Drinking Water, EPA/600/4/80/032, http: //www .epa. gov/sam/pdfs/EPA-
906.0.pdf
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6.2.7 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
6.2.8 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
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
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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
6.2.9 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.
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
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6.2.10 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, DOE (EML is currently part of the 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
6.2.11 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
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
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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, DOE (EML is currently part of the 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
6.2.12 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, DOE (EML is currently part of the 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
6.2.13 EML HASL-300 Method Ga-01-R: Gamma Radioassay
Analyte(s)
Cesium-137
Cobalt-60
Europium-154
lodine-131
CASRN
10045-97-3
10198-40-0
15585-10-1
10043-66-0
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Analyte(s)
lridium-192
Molybdenum-99
Ruthenium-103
Ruthenium-106
Selenium-75
CASRN
14694-69-0
14119-15-4
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 select
gamma emitters in 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 forNal(Tl) detectors.
Source: EML, DOE (EML is currently part of the DHS). 1997. "HASL-300 Method Ga-01-R: Gamma
Radioassay." EML Procedures Manual, HASL-300, 28th Edition. http://www.epa.gov/sam/pdfs/EML-Ga-
01-R.pdf
6.2.14 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
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
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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, DOE (EML is currently part of the DHS). 1997. "HASL-300 Method Po-02-RC:
Polonium in Water, Vegetation, Soil, and Air Filters." EML Procedures Manual, HASL-300, 28th Edition.
http://www.epa.gov/sam/pdfs/EML-Po-02-RC.pdf
6.2.15 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, DOE (EML is currently part of the DHS). 1997. "HASL-300 Method Pu-12-RC:
Plutonium and/or Americium in Soil or Sediments.'
http://www.epa.gov/sam/pdfs/EML-Pu-12-RC.pdf
Plutonium and/or Americium in Soil or Sediments." EML Procedures Manual, HASL-300, 28th Edition.
6.2.16 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
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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, DOE (EML is currently part of the DHS). 1997. "HASL-300 Method Sr-03-RC:
Strontium-90 in Environmental Samples." EML Procedures Manual, HASL-300, 28th Edition.
http://www.epa.gov/sam/pdfs/EML-Sr-03-RC.pdf
6.2.17 EML HASL-300 Method Tc-02-RC: Technetium-99 in Water- TEVA® Resin
Analyte(s)
Technetium-99
CASRN
14133-76-7
Analysis Purpose: Qualitative and confirmatory analysis
Determinative Technique: Liquid scintillation
Method Developed for: Technetium-99 (Tc-99) in water
Method Selected for: SAM lists this method for qualitative and confirmatory analysis of drinking water
and aqueous/liquid phase samples.
Description of Method: The sample containing Tc-99 is mixed with Technetium-95m (Tc-95m) added
as a gamma-emitting tracer. The two isotopes of technetium are brought to an isotopic equilibrium and
separated from other elements by ferrous and ferric hydroxide coprecipitation. The precipitate is dissolved
with dilute nitric acid and passed through a commercially available resin column (TEVA® Resin) which is
highly specific for technetium in the pertechnatate form. The resin is washed with dilute nitric acid to
remove possible interferences and then it is extruded directly into a suitable liquid scintillation cocktail.
The sample is typically counted for 1 hour to simultaneously determine Tc-99 activity and the Tc-95m
radiochemical yield. Quench/efficiency calibration curves need to be established for the liquid
scintillation spectrometer for both Tc-95m and Tc-99.
Source: EML, DOE (EML is currently part of the DHS). 1997. "HASL-300 Method Tc-02-RC:
Technetium-99 in Water - TEVA® Resin." EML Procedures Manual, HASL-300, 28th Edition.
http://www.epa.gov/sam/pdfs/EML-Tc-02-RC.pdf
6.2.18 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.
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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 (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
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-pg33.pdf
6.2.19 ORISE Method AP1: 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
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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.
Source: ORISE, Oak Ridge Associated Universities (ORAU). 2001. "Method API: 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-APl.pdf
6.2.20 ORISE Method AP2: Determination of Tritium
Analyte(s)
Tritium (Hydrogen-3)
CASRN
10028-17-8
Analysis Purpose: Qualitative and confirmatory analysis
Determinative Technique: Liquid scintillation
Method Developed for: Tritium in soil, sediment, animal tissue, vegetation, smears, and water samples
Method Selected for: SAM lists this method for qualitative and confirmatory analysis of soil/sediment
and surface wipe samples.
Description of Method: The tritium in aqueous and solid samples is distilled using an Allihn condenser.
For solid samples, an appropriate volume of water is added to facilitate distillation. Certain solid samples
may be refluxed to ensure distribution of any tritium that may be in the sample. The sample may be
spiked with a standard tritium solution to evaluate quenching and counting efficiency. After the sample
has been distilled, an aliquot of the distillate is added to a scintillation cocktail and the sample is counted
using a liquid scintillation analyzer.
Special Considerations: Other volatile radionuclides such as iodine and carbon isotopes may interfere
and may require that the sample be made alkaline using solid sodium hydroxide before distillation.
Organic impurities may interfere and may require the addition of an oxidizing agent to the sample as well
as spiking the samples with a standard tritium solution. The addition of a standard tritium solution to
each sample allows for counting efficiencies to be calculated for each individual sample.
Source: ORISE, ORAU. 2001. "Method AP2: Determination of Tritium." Laboratory Procedures
Manual for the Environmental Survey and Site Assessment Program.
http://www.epa.gov/sam/pdfs/ORISE-AP2.pdf
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6.2.21 ORISE Method APS: Determination of Technetium-99
Analyte(s)
Technetium-99
CASRN
14133-76-7
Analysis Purpose: Qualitative and confirmatory analysis
Determinative Technique: Liquid scintillation
Method Developed for: Technetium-99 in sediment, soil, smears, and water at environmental levels
Method Selected for: SAM lists this method for qualitative and confirmatory analysis of soil/sediment,
surface wipe, and air filter samples.
Description of Method: Solid samples are leached with dilute nitric acid. The leachates are passed
through a commercially available resin column (TEVA® resin) which is highly specific for technetium in
the pertechnatate form. The technetium is absorbed onto the extraction resin. The resin is added to a
scintillation vial containing an appropriate cocktail and counted using a liquid scintillation analyzer.
Most interfering beta emitting radionuclides (including C-14, P-32, S-35, Sr-90, Y-90, and Th-234) are
effectively removed using TEVA® resin under the conditions in this procedure.
Special Considerations: Tritium may follow technetium due to the absorption of some tritium-labeled
compounds by the resin. Possible tritium interferences are eliminated by setting the technetium counting
window above the maximum energy of tritium beta particles.
Source: ORISE, ORAU. 2001. "Method APS: Determination of Technetium-99." Laboratory
Procedures Manual for the Environmental Survey and Site Assessment Program.
http://www.epa.gov/sam/pdfs/ORISE-AP5.pdf
6.2.22 ORISE Method AP11: 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
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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 API 1: 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-APl 1 .pdf
6.2.23 ORISE Method Procedure #9: Determination of 1-125 in Environmental Samples
Analyte(s)
lodine-125
CASRN
14158-31-7
Analysis Purpose: Qualitative and confirmatory analysis
Determinative Technique: Gamma spectrometry
Method Developed for: Iodine-125 in environmental samples, such as soil, sediment, vegetation, water,
milk, filters (air or water), etc.
Method Selected for: SAM lists this method for qualitative and confirmatory analysis of drinking water,
aqueous/liquid, soil/sediment, surface wipe, and air filter samples.
Description of Method: In this method a direct comparison is made between the sample and a source
prepared from a National Institute of Standards and Technology (NIST) traceable standard. If it is
known, either by the sample preparation procedure or by a qualitative analysis on some device (high
resolution intrinsic planar detector) that 1-125 is the only radionuclide contributing to the observed peak,
then this method can be used as a rapid quantitative method.
The sample is prepared by matrix specific techniques and the final sample is placed in a 16 millimeter
culture tube and counted in a 3" x 3" thin window sodium iodide (Nal) well detector attached to a pulse
height analyzer. 1-125 gamma counting rate is determined in the 25 to 35 keV energy range by pulse
height analysis. NIST traceable liquid standards are also counted in the same geometric configuration as
the samples to determine 1-125 counting efficiency.
Special Considerations: Due to the low photon energy of 1-125, the Compton scattering and x-ray
photons from other radionuclides may cause significant interferences in this procedure.
Source: ORISE, ORAU. 1995. "Procedure #9: Determination of 1-125 in Environmental Samples."
Laboratory Procedures Manual for the Environmental Survey and Site Assessment Program.
http://www.epa.gov/sam/pdfs/ORISE-Procedure9-1995.pdf
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6.2.24 ASTM Method D3084-05: 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. 2005. "Method D3084-05: Standard Practice for Alpha Spectrometry in Water." Annual
Book of ASTM Standards, Vol. 11.02. http://www.astm.org/Standards/D3084.htm
6.2.25 ASTM Method D3972-02: Standard Test Method for Isotopic Uranium in Water by
Radiochemistry
Analyte(s)
Uranium-234
Uranium-235
Uranium-238
CASRN
13966-29-5
15117-96-1
7440-61-1
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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 D3972-02: Standard Test Method for Isotopic Uranium in Water by
Radiochemistry." Annual Book of ASTM Standards, Vol. 11.02.
httD://www.astm.org/DATABASE.CART/HISTORICAL/D3972-02.htm
6.2.26 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
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
(CAS RN 14596-10-2)
(CAS RN 1398 1-17-4)
(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)
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
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• Uranium-234
• Uranium-235
• Uranium-238
(CAS RN 13966-29-5)
(CAS RN 15 117-96-1)
(CAS RN 7440-16-1)
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://www.standardmethods.org/
6.2.27 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 select gamma
emitters in aqueous/liquid samples.
Description of Method: The method uses gamma spectroscopy using either Ge detectors or Nal(Tl)
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.
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/
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6.2.28 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.
htto: //www. standardmethods.org/
6.2.29 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
background) should be between 0.03 and 0.05 pCi radium-226, which may be considered the minimum
detectable amount under routine conditions.
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Source: APHA, AWWA, and WEF. 2005. "Method 7500-Ra C: Radium: Emanation Method." Standard
Methods for the Examination of Water and Wastewater. 21st Edition, http: //www. standardmethods. org/
6.2.30 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
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/
6.2.31 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.
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
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Section 6 - Selected Radiochemical Methods
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.
htto: //www. standardmethods.org/
6.2.32 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: //www. standardmethods. org/
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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 through 7.5. 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. Following a homeland security event, it is
assumed that only those areas with contamination greater than pre-existing levels commonly found in the
environment would be subject to remediation. Dependent on site- and event-specific goals, investigation
of background levels, using methods listed in Appendix C, should be performed.
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.
In addition to analytical methods, Appendix C lists commercially available spore strips. Commercially
available spore strips may be used as general indicators that a decontamination process (e.g., fumigation)
has been successful. Spore strips, however, cannot replace negative-culture results as an indicator of
decontamination efficacy. Culture-based methods have been selected for many of the pathogens;
however, due to technical difficulty and time constraints, molecular techniques such as PCR will likely be
used for viruses. 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. 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.
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
concentrating sample aliquots to ensure that the full sample volume can be assayed. This may increase
the sensitivity of methods such as PCR.
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Appendix C includes the following information:
• Pathogen(s). A specific causative agent (e.g., viruses, bacteria) of disease.
Viability. Ability to 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 dust-collecting socks used
with vacuum collection.
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 guidelines as to which options should be used under particular circumstances, or this
may be left to the discretion of the laboratory.
7.1 General Guidelines
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 (http://www.bt.cdc.gov/) and the FDA
Center for Food Safety and Applied Nutrition (CFSAN) "Bad Bug Book"
(http://www.cfsan.fda.gov/~mow/intro.html). 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).
Once a method has been identified in Appendix C, the corresponding method summary can be found in
Sections 7.2.1 through 7.5. Method summaries are listed first by alphabetical order within each pathogen
subcategory (i.e., bacteria, virus, protozoa, helminths) and then 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. Where available, a direct link to the full text of the method is provided with the
method summary. For additional information regarding sample preparation and analysis procedures
available through consensus standards organizations, other federal agencies, and journals please use the
contact information provided in Table 7-1.
Table 7-1. Sources of Pathogen Methods
Name
NEMI
EPA Microbiology Home Page
Information Collection Requirements
Rule (ICR) Microbial Laboratory Manual
EPA 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 of AST M Standards*
Applied and Environmental Microbiology
(AEM)*
Journal of Clinical Microbiology*
Clinical Microbiology Procedures
Handbook, 2nd Edition, 2004*
Molecular and Cellular Probes*
Publisher
EPA, USGS
EPA
EPA ORD
EPA
EPA, National Risk Management
Research Laboratory (NRMRL)
HHS, CDC
USDA FSIS
FDA, CFSAN
OSHA
NIOSH
APHA, AWWA, and WEF
ASTM International
American Society for Microbiology
(ASM)
ASM
ASM
Elsevier
Reference
http://www.nemi.qov
http://www.epa.qov/microbes/
http://www.epa.qov/nerlcwww/icrmi
cro.pdf
http://www.epa.qov/nerlcwww/abo
ut.htm
http://www.epa.qov/nrmrl/pubs/625
r92013/625r9201 3.htm
eiia.trees(5).cdc.hhs.qov
http://www.fsis.usda.qov/Science/
Microbioloqical Lab Guidebook/in
dex.asp
http://www.cfsan.fda.qov/~ebam/b
am-toc.html
http://www.osha.qov
http://www.cdc.qov/niosh/nmam/
http://www.standardmethods.orq
http://www.astm.orq
http://aem.asm.orq/
http://icm.asm.orq/
http://estore.asm.orq/viewltemDeta
ils.asp?ltemlD=323
http://www.elsevier.com
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Name
Canadian Journal of Microbiology*
Journal of Medical Virology*
Journal of Virological Methods*
Diagnostics Microbiology and Infectious
Disease
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
NRC Research Press
Wiley InterScience
Elsevier
Elsevier
CDC
American Society of
Parasitologists
The Royal Society of Tropical
Medicine and Hygiene
Academic Press
ASM
Blackwell Publishing
Reference
http://pubs.nrc-cnrc.ac.ca/
http://vvww3.interscience.wilev.com
/cai-b in/ho me
http://www.elsevier.com
http://www.elsevier.com
http://www.cdc.aov/ncidod/EID/
http://www.bioone.ora
http://www.rstmh.ora/
http://www.pubmedcentral.nih.aov/
articlerender.fcai?artid=1481267
http://www.asm.ora/index.php7opti
on=com content&view=article&id=
6342
http://www.blackwellpublishina.co
m/iournal.asp?ref=1 364-
5072&site=1
* Subscription and/or purchase required. ASM does not require a subscription or purchase 6 months after the
publication date.
7.1.2 General QC Guidelines 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, based on those needs, 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.
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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.
QC procedures and proper maintenance of ancillary laboratory equipment (e.g., thermometers,
autoclaves) 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, guidelines, 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
http://www.cdc.gov/OD/ohs/biosfty/bmbl5/bmbl5toc.htm
"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/rr5119.pdf
• Microbiology Biosafety for Level A Laboratories, found at
http://www.bt.cdc.gov/documents/PPTResponse/table3bbiosafetv.pdf
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Select Agent Rules and Regulations found at the National Select Agent Registry
(http://www.selectagents.gov/)and http://ecfr.gpoaccess.gov/cgi/t/text/text-
idx?c=ecfr&tpl=/ecfrbrowse/Title09/9cfrl21 main _02.tpl
The following sources provide information regarding waste management:
• EPA - Hazardous Waste Management (40 CFR parts 260) and EPA Administered Permit Programs
(40 CFR part 270), found at http://ecfr.gpoaccess.gov/cgi/tAext/text-
idx?sid=cac9da30cd241fa70d461e4a917eb75e&c=ecfr&tpl=/ecfrbrowse/Title40/40tab O2.tpl
Other resources that can be consulted for additional information include the following:
OSF£A - 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?ptable=STANDARDS&pid=10106
• OSHA - Respiratory Protection (29 CFR part 1910.134) found at
http://www.osha.gov/pls/oshaweb/owadisp.show document?pid=12716&ptable=STANDARDS
• DOT Hazardous Materials Shipment and Packaging (49 CFR parts 171-180)
http://ecfr.gpoaccess.gov/cgi/t/text/text-
idx?sid=585c275eel9254ba07625d8c92fe925f&c=ecfr&tpl=/ecfrbrowse/Title49/49cfrv2 O2.tpl
7.1.4 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
(http://www.asm.org/index.php?option=com content&view=article&id=6342).
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,
http://www.cdc.gov/OD/ohs/biosftv/bmbl5/bmbl5toc.htm).
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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
http://www.bt.cdc.gov/lrn/.
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 Method Summaries for Bacteria
Summaries of the analytical methods for bacteria listed in Appendix C are provided in Sections 7.2.1
through 7.2.16. Each summary contains a brief description of the method, intended method application,
performance data (if available), and a link to, or source for, obtaining a full version of the method.
7.2.1 Bacillus anthracis [Anthrax] - BSL-3
Method
LRN
Public Health Reports. 1977. 92(2): 176-186.
Analytical Technique
Real-time PCR/lmmunoassay
Culture
Section
7.1.4
7.2.1.1
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7.2.1.1 Literature Reference for B. anthracis (Public Health Reports. 1977. 92(2):
176-186)
Analysis Purpose: Detection and viability
Sample Preparation: Solid samples should be prepared according to EPA Method 1680;
participate samples should be prepared according to "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 (www.epa.gov/sam/pdfs/AEM-72(6)-pgs4429-4430.pdf).
Analytical Technique: Culture
Method Developed for: B. anthracis in soil and water 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.
Description of Method: This method describes procedures for analysis of water and solid
samples and may be adapted for assessment of particulate and aerosol samples. After dilution
and suspension in phosphate buffer with Tween® 80, soil samples are centrifuged at low speed
(400 x g) for 5 minutes to remove sediment, followed by a high-speed (6,000 x g) centrifugation
of the supernatant for 30 minutes to pellet spores. The heat-shocked pellet is plated directly on
Polymyxin, Lysozyme, EDTA, Thallous acetate (PLET) and sheep blood agar (SBA). Water
samples are subjected only to the low speed centrifugation, followed by passing the supernatant
through a 0.45 \am filter. The filter is placed in buffer, heat-shocked, and the supernatant plated
on PLET and SB A. PLET and SB A plates are examined after 24 and 48 hours incubation at 37°C
for colonies with typical morphology. Cultures (isolates) that cannot be ruled out as B. anthracis
based on the characteristics noted above should be referred to an appropriate reference laboratory
for confirmation.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed.
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,
http://www.cdc.gov/OD/ohs/biosfty/bmbl5/bmbl5toc.htm).
Source: Rees, H.B., Smith, M.A., Spendlove, J.C., Fraser, R.S., Fukushima, T., Barbour, A.G.,
Schoenfeld, F.J. 1977. "Epidemiologic and Laboratory Investigations of Bovine Anthrax in Two
Utah Counties in 1975." Public Health Reports. 92(2): 176-186.
http://www.epa.gov/sam/pdfs/PHR-92(2)-pgsl76-186.pdf
7.2.2 Brucella spp. [Brucellosis] - BSL-3
Method
LRN
ASM Sentinel Laboratory Guidelines for Suspected
Agents of Bioterrorism: Brucella species
Analytical Technique
Real-time PCR/lmmunoassay
Culture
Section
7.1.4
7.2.2.1
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7.2.2.1 ASM Sentinel Laboratory Guidelines for Suspected Agents of
Bioterrorism: Brucella species
Analysis Purpose: Detection and viability
Sample Preparation: Solid samples should be prepared according to EPA Method 1680;
participate samples should be prepared according to "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 (www.epa.gov/sam/pdfs/AEM-72(6)-pgs4429-4430.pdf).
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.
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 chocolate agar (CA) 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 a battery of biochemical tests specific for
Brucella spp.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed.
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
7.2.3 Burkholderia mallei [Glanders] - BSL-3 and Burkholderia pseudomallei
[Melioidosis] - BSL-3
Method
LRN
ASM Sentinel Laboratory Guidelines for Suspected
Agents of Bioterrorism: Burkholderia mallei and
Burkholderia pseudomallei
Analytical Technique
Real-time PCR/lmmunoassay
Culture
Section
7.1.4
7.2.3.1
7.2.3.1 ASM Sentinel Laboratory Guidelines for Suspected Agents of
Bioterrorism: Burkholderia mallei and Burkholderia pseudomallei
Analysis Purpose: Detection and viability
Sample Preparation: Solid samples should be prepared according to EPA Method 1680;
particulate samples should be prepared according to "Evaluation of a Macrofoam Swab Protocol
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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 (www.epa.gov/sam/pdfs/AEM-72(6)-pgs4429-4430.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.
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 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 when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed.
Special Considerations: B. mallei and B. pseudomallei are select agents requiring regulatory
compliance (42 CFR parts 72 and 73, and 9 CFRpart 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. 2008. "Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism:
Burkholderia mallei and Burkholderiapseudomallei." http://www.epa.gov/sam/pdfs/ASM-
Bmallei-Bpseudomallei.pdf
7.2.4 Campylobacterjejuni [Campylobacteriosis] - BSL-2
Method
SM 9260 G
Molecular and Cellular Probes. 2006. 20: 269-279
Analytical Technique
Culture and Immunoassay
Real-time PCR
Section
7.2.4.1
7.2.4.2
7.2.4.1 Standard Method 9260 G: Campylobacterjejuni
Analysis Purpose: Detection and viability
Sample Preparation: Solid samples should be prepared according to EPA Method 1680;
particulate samples should be prepared according to "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 (www.epa.gov/sam/pdfs/AEM-72(6)-pgs4429-4430.pdf).
Analytical Technique: Culture and immunoassay
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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.
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 when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed.
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/
7.2.4.2 Literature Reference for Campylobacter jejuni (Molecular and Cellular
Probes. 2006. 20(5): 269-279)
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: Campylobacter jejuni 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.
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 conjunction
with either the ABI Prism® 7700 or 7900 HT Thermal Cyclers. A high through-put method
using guanidinium thiocyanate (GTC) and glass beads is used for extraction of deoxyribonucleic
acid (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 national collection of type cultures (NCTC) 11168 DNA in a background of cecum
(intestinal) DNA.
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At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed. 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: 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(5): 269-279.
htto: //www. sciencedirect. com/science/i ournal/0 8908508
7.2.5 Chlamydophila psittaci [Psittacosis] (formerly known as Chlamydia psittaci) -
BSL-2; BSL-3 for aerosols and large volumes
Method
Journal of Clinical Microbiology. 2000.
1093
38(3): 1085-
Analytical Technique
Tissue culture and PCR
Section
7.2.5.1
7.2.5.1 Literature Reference for Chlamydophila psittaci (Journal of Clinical
Microbiology. 2000. 38(3): 1085-1093)
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-65d2)-
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.
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 when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed. 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.
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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/JCM-38(3)-pgsl085-1093.pdf
7.2.6 Coxiella burnetii [Q-fever] - BSL- 3
Method
LRN (Sentinel Laboratory Guideline not available)
Analytical Technique
Culture and Real-time
PCR/lmmunoassay
Section
7.1.4
7.2.7 Escherichia coli O157:H7 - BSL-2
Method
SM 9260 F
Applied and Environmental Microbiology. 2003.
69(10): 6327-6333
Analytical Technique
Culture and Immunoassay
Real-time PCR
Section
7.2.7.1
7.2.7.2
7.2.7.1 Standard Method 9260 F: Pathogenic Escherichia coli
Analysis Purpose: Detection and viability
Sample Preparation: Solid samples should be prepared according to EPA Method 1680;
particulate samples should be prepared according to "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 (www.epa.gov/sam/pdfs/AEM-72(6)-pgs4429-4430.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.
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 922IB 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 when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed.
Source: APHA, AWWA, and WEF. 2005. "Method 9260 F: Pathogenic Escherichia coli"
Standard Methods for the Examination of Water and Wastewater. 21st Edition.
http: //www. standardmethods.org/
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7.2.7.2 Literature Reference for Escherichia coli O157:H7 (Applied and
Environmental Microbiology. 2003. 69(10): 6327-6333)
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-65d2)-
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.
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 (iL) is added to a total
reaction volume of 25 (iL. 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 (B-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/H" 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/FT serotype.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed. 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,
www.epa.gov/sam/pdfs/EPA-QA-QC_PCR_Oct2004.pdf. 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-69d 0)-pgs6327-6333.pdf.
7.2.8 Francisella tularensis [Tularemia] - BSL-3
Method
LRN
CDC, ASM, APHL Basic Protocols for Level A
Laboratories for the Presumptive Identification of
Francisella tularensis
Analytical Technique
Real-time PCR /Immunoassay
Culture
Section
7.1.4
7.2.8.1
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7.2.8.1 CDC, ASM, APHL: Basic Protocols for Level A Laboratories for the
Presumptive Identification of Francisella tularensis
Analysis Purpose: Detection and viability
Sample Preparation: Solid samples should be prepared according to EPA Method 1680;
participate samples should be prepared according to "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 (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.
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 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, B-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 when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed.
Special Considerations: F. tularensis is a select agent requiring regulatory compliance (42
CFR parts 72 and 73, and 9 CFRpart 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: 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 Leptospira interrogans [Leptospirosis] - BSL-2
Method
SM 9260 1
Diagnostic Microbiology and Infectious Disease.
2009. 64(3): 247-255
Analytical Technique
Culture and Immunoassay
Real-time PCR
Section
7.2.9.1
7.2.9.2
7.2.9.1 Standard Method 9260 I: Leptospira
Analysis Purpose: Detection and viability
Sample Preparation: Solid samples should be prepared according to EPA Method 1680;
particulate samples should be prepared according to "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 (www.epa.gov/sam/pdfs/AEM-72(6)-pgs4429-4430.pdf).
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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.
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 either with
leptospire antibody free serum or with bovine serum albumin (BSA) and polysorbates, 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 when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed.
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/
7.2.9.2 Literature Reference for Leptospira spp. (Diagnostic Microbiology and
Infectious Diseases. 2009. 64(3): 247-255)
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: Leptospira spp. 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.
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. A TaqMan®
probe for the lipL32 gene, which is present only in pathogenic strains of Leptospira, is used to
identify pathogenic leptospires and to exclude intermediate and non-pathogenic Leptospira
species. The assay correctly identified 32 pathogenic strains and excluded 6 intermediate and 13
non-pathogenic strains. Clinical samples (blood and urine) were spiked withZ. interrogans
serovar Icterohaemorrhagiae cultures to provide a final concentration of 1 * 106 leptospires/mL
sample. Samples were serially diluted and DNA extracted from 200 (iL of the sample using a
QIAmp DNA blood minikit (QIAGEN). DNA eluted into water was analyzed on both the
Lightcycler® 1.2 and the ABI 7500, and the protocol was found to be 100% sensitive and specific
for pathogenic Leptospira spp. The assay was able to detect as low as 1 * 101 leptospires/mL
whole blood with a Ct value of 40.55 and down to 1 * 102 leptospires/mL in plasma with a Ct
value of 38.88. Using DNA from L. interrogans serovar Icterohaemorrhagiae, the lower limit of
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detection was found to be 20 (Lightcycler® 1.2) and 50 (ABI 7500) genomic equivalents per
reaction using the concentration at which 95% or more of the replicated reactions yielded a
positive result.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed. 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: Stoddard, R.A., Gee, J.E., Wilkins, P.P., McCaustland, K., and A.R. Hoffmaster. 2009.
"Detection of Pathogenic Leptospira spp. Through TaqMan Polymerase Chain Reaction
Targeting the LipL32 Gene." Diagnostic Microbiology and Infectious Disease. 64(3): 247-255.
http://www.sciencedirect.com/science/iournal/07328893
7.2.10 Listeria monocytogenes [Listeriosis] - BSL-2
Method
USDA Laboratory Guidebook MLG 8A.03
FDA/Bacteriological Analytical Manual Chapter 10,
2003
Analytical Technique
Real-time PCR
Culture and Immunoassay
Section
7.2.10.1
7.2.10.2
7.2.10.1 USDA Laboratory Guidebook: "FSIS Procedure for the Use of a Listeria
monocytogenes Polymerase Chain Reaction (PCR) Screening Test." MLG
8A.03. 2007.
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.
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. monocytogenes. 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 * 10"2 CFU/mL in a 500 mL brine sample.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed. PCR QC checks should be
performed according to EPA Draft Quality Assurance/Quality Control Guidance for Laboratories
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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: USDA, FSIS. 2007. "FSIS Procedure for the Use ofaListeria monocytogenes
Polymerase Chain Reaction (PCR) Screening Test." Laboratory GuidebookMLG 8A.03.
htto://www.epa.gov/sam/pdfs/USDA-MLG-8A.03.pdf
7.2.10.2 FDA Bacteriological Analytical Manual, Chapter 10, 2003: Listeria
monocytogenes
Analysis Purpose: Detection and viability
Sample Preparation: Solid samples should be prepared according to EPA Method 1680;
particulate samples should be prepared according to "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 (www.epa.gov/sam/pdfs/AEM-72(6)-pgs4429-4430.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.
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® Listeria monocytes Detection System), is streaked at 48 hours. Presumptive
Listeria colonies are black with a black halo on esculin-containing media and blue on Biosynth
Chromogen Medium 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 when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed.
Source: FDA, CFSAN. 2003. "Chapter 10 - Detection and Enumeration of Listeria
monocytogenes in Foods." Bacteriological Analytical Manual Online.
http://www.epa.gov/sam/pdfs/FDA-BAM-ChaplO.pdf
7.2.11 Non-typhoidal Salmonella (Not applicable to S. Typhi) [Salmonellosis] - BSL-2
Method
EPA Method 1682
Journal of Applied Microbiology. 2007. 102(2):
516-530
Analytical Technique
Culture and Immunoassay
Real-time PCR
Section
7.2.11.1
7.2.11.2
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7.2.11.1 EPA Method 1682: Salmonella spp.
Analysis Purpose: Detection and viability
Sample Preparation: Solid samples should be prepared according to EPA Method 1680;
participate samples should be prepared according to "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 (www.epa.gov/sam/pdfs/AEM-72(6)-pgs4429-4430.pdf).
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.
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 when using this
protocol: positive control, negative control, matrix spike and blank. Ongoing analysis of QC
samples to ensure reliability of the analytical results should also be performed.
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
7.2.11.2 Literature Reference for Non-Typhoidal Salmonella (Journal of Applied
Microbiology. 2007. 102(2): 516-530)
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-65d2)-
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.
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
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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 CPUs per reaction for cultured Salmonella spp.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed. 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: 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.
htto://www3 .interscience.wilev.com/iournal/118490299/abstract
7.2.12 Salmonella Typhi [Typhoid fever] - BSL-2; BSL-3 for aerosol release
Method
CDC Laboratory Assay: S. Typhi
SM 9260 B
Analytical Technique
Real-time PCR
Culture and Immunoassay
Section
7.2.12.1
7.2.12.2
7.2.12.1 CDC Laboratory Assay: "Triplex PCR for Detection of S. Typhi Using
SmartCycler®"
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.
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 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 SmartCycler® using primers and probes designed for the Vi capsular gene (yiaB),
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 when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed. PCR QC checks should be
performed according to EPA Draft Quality Assurance/Quality Control Guidance for Laboratories
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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: HHS, CDC, Laboratory Assay. "Triplex PCR for Detection of S. Typhi Using
SmartCycler®." Contact: Dr. Eija Trees, CDC, email: eija.trees(gicdc .hhs.gov.
7.2.12.2 Standard Method 9260 B: General Qualitative Isolation and Identification
Procedures for Salmonella
Analysis Purpose: Detection and viability
Sample Preparation: Solid samples should be prepared according to EPA Method 1680;
particulate samples should be prepared according to "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 (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.
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 when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed.
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/
7.2.13 Shigella spp. [Shigellosis] - BSL-2
Method
CDC Laboratory Assay: Shigella
SM 9260 E
Analytical Technique
Real-time PCR
Culture and Immunoassay
Section
7.2.13.1
7.2.13.2
7.2.13.1 CDC Laboratory Assay: "Detection of Diarrheagenic Escherichia coli and
Shigella Using LightCycler®"
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
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Environmental Microbiology. 65(12): 5409-5420 (www.epa.gov/sam/pdfs/AEM-65d2)-
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.
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 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 when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed. 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: HHS, CDC, Laboratory Assay. "Detection of Diarrheagenic Escherichia coll and
Shigella Using LightCycler®." Contact: Dr. Eija Trees, CDC, email: eija.trees@cdc.hhs.gov.
7.2.13.2 Standard Method 9260 E: Shigella
Analysis Purpose: Detection and viability
Sample Preparation: Solid samples should be prepared according to EPA Method 1680;
particulate samples should be prepared according to "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 (www.epa.gov/sam/pdfs/AEM-72(6)-pgs4429-4430.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.
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.
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At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed.
Source: APHA, AWWA, and WEF. 2005. "Method 9260 E: Shigella" Standard Methods for
the Examination of Water and Wastewater. 21st Edition, http://www.standardmethods.org/
7.2.14 Staphylococcus aureus - BSL-2
Method
SM9213B
Analytical Technique
Culture
Section
7.2.14.1
7.2.14.1 Standard Method 9213 B: Staphylococcus aureus
Analysis Purpose: Detection and viability
Sample Preparation: Solid samples should be prepared according to EPA Method 1680;
particulate samples should be prepared according to "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 (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.
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 when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed.
Source: APHA, AWWA, and WEF. 2005. "Method 9213 B: Staphylococcus aureus" Standard
Methods for the Examination of Water and Wastewater. 21st Edition.
http: //www. standardmethods.org/
7.2.15 Vibrio cholerae [Cholera] - BSL-2
Method
CDC Laboratory Assay: V. cholerae
SM 9260 H
Analytical Technique
Real-time PCR
Culture and Immunoassay
Section
7.2.15.1
7.2.15.2
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7.2.15.1 CDC Laboratory Assay: "TaqMan Assays for Detection of V. cholerae
cfxA, O1 rfb, and O139 rfb."
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-65d2)-
pgs5409-5420.pdf).
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.
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 for 2 minutes, 1 \\L 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 Olr/fr
gene, and the O139 antigen O139 rfb gene.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed. 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/EP A-
QA-QC_PCR_Oct2004.pdf or consult the point of contact identified in Section 4.
Source: FiHS, CDC, Laboratory Assay. "TaqMan Assays for Detection of V. cholerae ctxA, Ol
rfb, and O139 rfb" Contact: Dr. Eija Trees, CDC, email: eija.trees@cdc.hhs.gov.
7.2.15.2 Standard Method 9260 H: Vibrio cholerae
Analysis Purpose: Detection and viability
Sample Preparation: Solid samples should be prepared according to EPA Method 1680;
particulate samples should be prepared according to "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 (www.epa.gov/sam/pdfs/AEM-72(6)-pgs4429-4430.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.
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 broth by incubation 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
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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 when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed.
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/
7.2.16 Yersinia pestis [Plague] - BSL-3
Method
LRN
ASM Sentinel Laboratory Guidelines for Suspected
Agents of Bioterrorism: Yersinia pestis
Analytical Technique
Real-time PCR/lmmunoassay
Culture
Section
7.1.4
7.2.16.1
7.2.16.1 ASM Sentinel Laboratory Guidelines for Suspected Agents of
Bioterrorism: Yersinia pestis
Analysis Purpose: Detection and viability
Sample Preparation: Solid samples should be prepared according to EPA Method 1680;
particulate samples should be prepared according "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 (www.epa.gov/sam/pdfs/AEM-72(6')-pgs4429-4430.pdf).
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.
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.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed.
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/biosftv/bmbl5/bmbl5toc.htm).
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Source: ASM. 2005. "Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism:
Yersiniapestis" http://www.epa.gov/sam/pdfs/ASM-Ypestis.pdf
7.3 Method Summaries for Viruses
Summaries of the analytical methods for viruses listed in Appendix C are provided in Sections 7.3.1
through 7.3.10. Each summary contains sample preparation information, intended method application, a
brief description of the method, performance data (if available), and a link to, or source for, obtaining a
full version of the method.
7.3.1 Adenoviruses: Enteric and non-enteric (A-F) - BSL-2
Method
Applied and Environmental Microbiology. 2005.
71(6): 3131-3136
Analytical Technique
Tissue culture and Real-time PCR
Section
7.3.1.1
7.3.1.1 Literature Reference for Adenoviruses (Applied and Environmental
Microbiology. 2005. 71(6): 3131-3136)
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
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.
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 when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed. 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.
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 (J. 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
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Identification of Serotypes 40 and 41." Applied and Environmental Microbiology. 71(6): 3131-
3136. http://www.epa.gov/sam/pdfs/AEM-7K6Vpgs3131-3136.pdf
7.3.2 Astroviruses - BSL not specified
Method
Canadian Journal of Microbiology. 2004. 50: 269-
278
Analytical Technique
Integrated cell culture/Real-time
reverse transcription-PCR
Section
7.3.2.1
7.3.2.1 Literature Reference for Astroviruses (Canadian Journal of Microbiology.
2004. 50: 269-278)
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.
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 real-time 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 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 real-time reverse transcription-PCR (i.e., reverse transcription followed by real-time
PCR) assay using astrovirus-specific primer sets.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed. 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: 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/inDetail.isp?icode=cim&lang=eng&vol=50&is=4
7.3.3 Caliciviruses: Noroviruses - BSL-2
Method
Journal of Clinical Microbiology.
4685
2004. 42(10): 4679-
Analytical Technique
Real-time reverse transcription-
PCR
Section
7.3.3.1
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7.3.3.1 Literature Reference for Noroviruses (Journal of Clinical Microbiology.
2004. 42(10): 4679-4685)
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.
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 when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed. 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 and Quantitation of Norovirus." Journal of Clinical Microbiology. 42(10): 4679-4685.
http://www.epa.gov/sam/pdfs/JCM-42(10')-pgs4679-4685.pdf
7.3.4 Caliciviruses: Sapovirus - BSL-2
Method
Journal of Medical Virology, 2006. 78 (10): 1347-
1353
Analytical Technique
Real-time reverse transcription-
PCR
Section
7.3.4.1
7.3.4.1 Literature Reference for Sapoviruses (Journal of Medical Virology. 2006.
78(10): 1347-1353)
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.
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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 when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed. 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: 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.
httD://cat.inist.fr/?aModele=afficheN&cpsidt=18099754
7.3.5 Coronaviruses: SARS-associated human coronavirus - BSL-2; BSL-3 for
propagation
Method
Journal of Virological Methods. 2004. 122: 29-36
Analytical Technique
Reverse transcription-PCR
Section
7.3.5.1
7.3.5.1 Literature Reference for Coronaviruses (SARS) (Journal of Virological
Methods. 2004. 122: 29-36)
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
Method Developed for: Severe acute respiratory syndrome (SARS)-associated human
coronavirus (HCoV) 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.
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 when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
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ensure reliability of the analytical results should also be performed. 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: 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. http://www.sciencedirect.com/science/iournal/01660934
7.3.6 Hepatitis E virus (HEV) - BSL-2
Method
Journal of Virological Methods. 2006.
131(1): 65-71
Analytical Technique
Real-time reverse transcription-
PCR
Section
7.3.6.1
7.3.6.1 Literature Reference for Hepatitis E Virus (Journal of Virological
Methods. 2006. 131(1): 65-71)
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.
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.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed. 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: 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, 131(1): 65-71.
http://cat.inist.fr/?aModele=afficheN&cpsidt=17367357
7.3.7 Influenza H5N1 virus - BSL-3
Method
Emerging Infectious Diseases. 2005.
1303-1305
11(8):
Analytical Technique
Real-time reverse transcription-
PCR
Section
7.3.7.1
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7.3.7.1 Literature Reference for Influenza H5N1 (Emerging Infectious Diseases.
2005. 11(8): 1303-1305)
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.
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 when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed. 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.
http://www.epa.gov/sam/pdfs/EID-l U8Vpgsl303-1305.pdf
7.3.8 Picornaviruses: Enteroviruses - BSL-2
Method
USEPA Manual of Methods for Virology
EPA/600/4-84/013,2001
Applied and Environmental Microbiology. 2003.
69(6): 3158-3164
Analytical Technique
Tissue culture
Reverse transcription-PCR
Section
7.3.8.1
7.3.8.2
7.3.8.1 USEPA Manual of Methods for Virology, EPA/600/4-84/013, April 2001
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
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.
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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 1 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 when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed.
Special Considerations: This manual also describes procedures for preparation of samples for
adenovirus, astrovirus, norovirus, sapovirus, coronavirus - 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://www.epa.gov/sam/pdfs/EPA-600-4-84-013.pdf
7.3.8.2 Literature Reference for Enteric Viruses (Applied and Environmental
Microbiology. 2003. 69(6): 3158-3164)
Analysis Purpose: Detection of Enteroviruses and Hepatitis A virus (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 liquid and water samples. Further research is needed to
develop and standardize the procedures for environmental sample types other than water.
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.
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At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed. 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: 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.
htto://www.epa.gov/sam/pdfs/AEM-69(6')-pgs3158-3164.pdf
7.3.9 Picornaviruses: Hepatitis A virus (HAV) - BSL-2
Method
Applied and Environmental Microbiology. 2003.
69(6): 3158-3164
Analytical Technique
Reverse transcription-PCR
Section
7.3.8.2
See Section 7.3.8.2 for information on reverse transcription-PCR procedures for Picornaviruses:
Hepatitis A virus.
7.3.10 Reoviruses: Rotavirus (Group A) - BSL not specified
Method
Applied and Environmental Microbiology. 2003.
69(6): 3158-3164
Journal of Virological Methods. 2009. 155: 126-
131
Analytical Technique
Tissue culture
Real-time reverse transcription-
PCR
Section
7.3.8.2
7.3.10.1
7.3.10.1 Literature Reference for Reoviruses: Rotavirus (Group A) (Journal of
Virological Methods. 2009. 155(2): 126-131)
Analysis Purpose: Detection
Sample Preparation: Samples should be prepared according to procedures described in Journal
of Virological Methods. 2009. 155: 126-131.
Analytical Technique: Real-time reverse transcription-PCR
Method Developed for: Group A Rotaviruses in water
Method Selected for: SAM lists these procedures for detection of rotavirus (Group A) in solid,
particulate, aerosol, and drinking water samples, but the procedures are not suitable for
determining viability of these pathogens. Further research is needed to develop and standardize
the procedures for environmental sample types other than drinking water.
Description of Method: Procedures are described for analysis of drinking water samples and
may be adapted for assessment of solid, particulate, and aerosol samples. The method is used to
detect rotavirus in drinking water samples using one-step real-time reverse-transcription PCR.
Water samples (100 L) are concentrated up to 400 mL by UF and 100-mL volumes are further
concentrated using a 30-kDa Centricon® Plus-70 unit. Concentrated samples (approximately 1
mL each) are subjected to nucleic acid extraction using a guanidine thiocyantate-based buffer.
Ten percent of the RNA extract (corresponding to 2.5 L of the original water sample) is analyzed
using RT-PCR performed on an iCylcer iQ™ Real-Time Detection System with primers and
probe specific for the rotavirus NSP3 gene. The sensitivity of the assay was found to be six virus
particles per reaction (nuclease-free water) as determined using quantified rotavirus stocks and a
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plasmid DNA stock. Specificity testing did not identify any cross-reactivity of the assay with a
panel of 36 non-rotavirus enteric viruses.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed. 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.
Special Considerations: Complex matrices such as solid, particulate, source water, and aerosol
samples may require more extensive inhibitor removal procedures than those described in this
method. Sample preparation procedures as described by Fout et al. (Section 7.3.8.2) may be
used.
Source: Jothikumar, N., Kang, G., and V.R. Hill. 2009. "Broadly Reactive TaqMan® Assay for
Real-time RT-PCR Detection of Rotavirus in Clinical and Environmental Samples." Journal of
Virological Methods. 155(2): 126-131. http://www.sciencedirect.com/science/journal/01660934
7.4 Method Summaries for Protozoa
Summaries of the analytical methods for protozoa listed in Appendix C are provided in Sections 7.4.1
through 7.4.4. Each summary contains sample type for which the method was developed, sample type to
which the method applies, sample preparation information, a brief description of the method, performance
data (if available), and a link to, or source for, obtaining a full version of the method.
7.4.1 Cryptosporidium spp. [Cryptosporidiosis] - BSL-2
Method
EPA Method 1622
EPA Method 1623
Applied and Environmental Microbiology. 1999.
65(9): 3936-3941
Applied and Environmental Microbiology. 2007.
73(1 3): 421 8-4225
Analytical Technique
IMS/FA
IMS/FA
Tissue culture
Real-time PCR
Section
7.4.1.1
7.4.1.2
7.4.1.3
7.4.1.4
7.4.1.1 EPA Method 1622: Cryptosporidium in Water by Filtration/IMS/FA
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.
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
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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, species, or infectivity of the oocysts.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, MS/MSD and blank. Ongoing analysis of QC
samples to ensure reliability of the analytical results should also be performed.
Source: EPA. 2005. "Method 1622: Cryptosporidium in Water by Filtration/IMS/FA."
http://www.epa.gov/sam/pdfs/EPA-1622.pdf
7.4.1.2 EPA Method 1623: Cryptosporidium and Giardia in Water by
Filtration/IMS/FA
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. 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.
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 and 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 DIC 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, species, or infectivity of the parasites.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, MS/MSD and blank. Ongoing analysis of QC
samples to ensure reliability of the analytical results should also be performed.
Source: EPA. 2001. "Method 1623: Cryptosporidium and Giardia in Water by
Filtration/IMS/FA." http://www.epa.gov/sam/pdfs/EPA-1623.pdf
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7.4.1.3 Literature Reference for Cryptosporidium spp. (Applied and
Environmental Microbiology. 1999. 65(9): 3936-3941)
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.
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 when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed.
Source: Slifko, T.R., Huffman, D.E., and Rose, J.B. 1999. "A Most-Probable-Number Assay for
Enumeration of Infectious Cryptosporidium parvum Oocysts." Applied and Environmental
Microbiology. 65(9): 3936-3941. http://www.epa.gov/sam/pdfs/AEM-65(9)-pgs3936-3941.pdf
7.4.1.4 Literature Reference for Cryptosporidium spp. (Applied and
Environmental Microbiology. 2007. 73(13): 4218-4225)
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-65d2)-
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 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.
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 average concentration of 590,000 ± 84,000 oocysts. One hundred
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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 (im 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 when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed.
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
7.4.2 Entamoeba histolytica - BSL-2
Method
Journal of Parasitology. 1972. 58(2): 306-310
Journal of Clinical Microbiology. 2005. 43(11):
5491-5497
Analytical Technique
Culture
Real-time PCR
Section
7.4.2.1
7.4.2.2
7.4.2.1 Literature Reference for Entamoeba histolytica (Journal of Parasitology.
1972.58(2): 306-310)
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.
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 trypticase-panmede liver digest-serum (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 when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed.
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Source: Stringert, R.P. 1972. "New Bioassay System for Evaluating Percent Survival of
Entamoeba histolytica Cysts." The Journal of Parasitology. 58(2): 306-310.
htto://www.epa.gov/sam/pdfs/JP-58(2')-pgs306-310.pdf
7.4.2.2 Literature Reference for Entamoeba histolytica (Journal of Clinical
Microbiology. 2005. 43(11): 5491-5497)
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.
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 Gene Amp® 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 when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed. 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, A.J. 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-43an-pgs5491-5497.pdf
7.4.3 Giardia spp. [Giardiasis] - BSL-2
Method
EPA Method 1623
Transactions of the Royal Society of Tropical
Medicine and Hygiene. 1983. 77(4): 487^88
Analytical Technique
IMS/FA
Culture
Section
7.4.1.2
7.4.3.1
7.4.3.1 Literature Reference for Giardia spp. (Transactions of the Royal Society
of Tropical Medicine and Hygiene. 1983. 77(4): 487-488)
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.
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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. Trypticase-yeast-iron-
serum (TYI-S-33) medium supplemented 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 when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed.
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://www.sciencedirect.com/science/iournal/00359203
7.4.4 Toxoplasma gondii [Toxoplasmosis] - BSL-2
Method
Emerging Infectious Diseases. 2006. 12(2): 326-329
Applied and Environmental Microbiology. 2004.
70(7): 4035-4039
Analytical Technique
Animal infectivity
Real-time PCR
Section
7.4.4.1
7.4.4.2
7.4.4.1 Literature Reference for Toxoplasma gondii (Emerging Infectious
Diseases. 2006. 12(2): 326-329)
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.
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. gondii-
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 when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed.
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, I.,
Dubey, J.P., and Garrett, D.O. 2006. "Waterborne Toxoplasmosis, Brazil, from Field to Gene."
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Emerging Infectious Diseases. 12(2): 326-329. http://www.epa.gov/sam/pdfs/EID-12(2)-pgs326-
329.pdf
7.4.4.2 Literature Reference for Toxoplasma gondii (Applied and Environmental
Microbiology. 2004. 70(7): 4035-4039)
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.
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 when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed. 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, I., 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://www.epa.gov/sam/pdfs/AEM-70(7)-pgs4035-4039.pdf
7.5 Method Summaries for Helminths
A summary of the analytical method for helminths listed in Appendix C is provided in Section 7.5.1. The
summary contains sample preparation information, intended method application, a brief description of the
method, performance data (if available), and a link to, or source for, obtaining a full version of the
method.
7.5.1 Baylisascaris procyonis [Raccoon roundworm fever] - BSL-2
Method
EPA/625/R92/013
Analytical Technique
Embryonation of eggs and
microscopy
Section
7.5.1.1
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7.5.1.1 USEPA Environmental Regulations and Technology, Control of
Pathogens and Vector Attraction in Sewage Sludge EPA/625/R-92/013,
July 2003: Baylisascaris procyonis
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.
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 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 when using this
protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to
ensure reliability of the analytical results should also be performed.
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.
http://www.epa.gov/sam/pdfs/EPA-625-R-92-013.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 dust-collecting socks used
with vacuum collection.
Liquid/water. The recommended method/procedure to measure the analyte of interest in liquid and
water samples.
• Drinking water. The recommended method/procedure to measure the analyte of interest in drinking
water samples.
Following a homeland security event, it is assumed that only those areas with contamination greater than
pre-existing, naturally prevalent levels commonly found in the environment would be subject to
remediation. Dependent on site- and event-specific goals, investigation of background levels using
methods listed in Appendix D is recommended.
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
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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, may be
used when 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., high performance liquid
chromatography - mass spectrometer [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.
The presence of disinfectants (e.g., chlorine) and/or preservatives added during water sample collection to
slow degradation (e.g., pH adjusters, de-chlorinating agents) could possibly affect analytical results.
When present, the impact of these agents on method performance should be evaluated if not previously
determined. 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 Guidelines
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
(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.
Additional resources include:
• Defense Against Toxin Weapons, published by the U.S. Army Medical Research Institute of
Infectious Diseases (http://www.usamriid.armv.mil/education/defensetox/toxdefbook.pdf) contains
information regarding sample collection, toxin analysis and identification, as well as decontamination
and water treatment.
Select Agent Rules and Regulations found at the National Select Agent Registry
(http://www.selectagents.gov/)
• The CDC has additional information regarding select agent toxins at the following Web site:
http://www.cdc.gov/od/sap/sap/toxinamt.htm
SRC's PHYSPROP and Chemfate, part of the Environmental Fate Database supported by EPA. See
http://srcinc.com/what-we-do/product.aspx?id=133.
• INCHEM at http://www.inchem.org/ contains both chemical and toxicity information.
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The RTECS database can be accessed via the NIOSH Web site at
http://www.cdc.gov/niosh/rtecs/default.html for toxicity information.
• 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.
Once a method has been identified in Appendix D, the corresponding method summary can be found in
Sections 8.2.1 through 8.3.12. Method summaries are listed first by alphabetical order within each
biotoxin subcategory (i.e., protein and small molecule) and then 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. 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 8-1.
Table 8-1. Sources of Biotoxin Methods
Name
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*
Environmental Health Perspectives*
Toxicon*
Federation of European Microbiological
Societies (FEMS) Microbiology Letters*
Publisher
FDA
AOAC International
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
National Institute of
Environmental Health
Sciences
Elsevier Science Publishers
Wiley-Blackwell
Reference
http://www.cfsan.fda.gov/~ebam/bam-
toc.html
http://www.aoac.org
http://www.nemi.qov/
http://www.blackwell-svnerav.com/loi/pto
http://www.sciencedirect.com/
http://www.biochemi.org/
http://www.acs.org/
http://www.foodprotection.org/
http://www.elsevier.com/
http://www.wiley.com/
http://www.niehs.nih.gov/
http://www.elsevier.com/
http://www.wiley.com/
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Name
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
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.elsevier.com/
http://www.wilev.com/
http://www.aoac.org
http://www.rsc.orq/
http://www.elsevier.com/
http://www.asm.org/
http://www.wilev.com/
http://www.iatox.com/
http://www.epa.gov/etv/
' Subscription and/or purchase required.
8.1.2 General QC Guidelines 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
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;
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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, guidelines, 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, available
at http://www.absa.org/riskgroups/index.html.
. BMBL, 5th Edition, found at http://www.cdc.gov/OD/ohs/biosfty/bmbl5/bmbl5toc.htm.
• Biological Safety: Principles and Practices, 4th Ed. ASMPress (http://estore.asm.org/).
. CDC - 42 CFR part 72. Interstate Shipment of Etiologic 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.
. 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.
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• 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/.
8.1.4 Laboratory Response Network (LRN)
The agents and sample types identified below and listed in Appendix D are included in the FiHS/USDA
select agent list and should be analyzed in accordance with the appropriate LRN protocols. Additional
information on select agents and regulations may be obtained at the National Select Agent Registry at:
http://www.selectagents.gov/.
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)
Botulinum neurotoxins (Serotypes A, B, E, F)
Ricin
Staphylococcal enterotoxin B (SEB)
Sample Type
Solid, Participate,
Liquid/Water, Drinking Water
Solid, Particulate,
Liquid/Water, Drinking Water
Solid, Particulate,
Liquid/Water, Drinking Water
CAS RN / 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
9009-86-3 (ricin) 760 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
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.
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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
http://www.bt.cdc.gov/lrn/.
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 20910
Telephone: (240) 485-2745
Fax: (240) 485-2700
Web site: www.aphl.org
E-mail: info@aphl.org
8.2 Method Summaries for Protein Biotoxins
Summaries of the analytical methods for protein biotoxins listed in Appendix D are provided in Sections
8.2.1 through 8.2.5. 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 brief description of the method, intended method application, performance data
(if available), and a link to or source for obtaining a full version of the method.
8.2.1 Abrin
CAS RN: 1393-62-0.
Description: Glycoprotein consisting of a deadenylase (25-32 kDa A chain) and lectin (35 kDa
B chain); an agglutinin (A2B2) may be present in crude preparations.
Method
Journal of Food Protection. 2008. 71(9): 1868-1874
Pharmacology & Toxicology. 2001. 88(5): 255-260
Analytical Biochemistry. 2008. 378: 87-89
Analytical Technique
Immunoassay
Ribosome inactivation assay
Enzyme activity
Section
8.2.1.1
8.2.1.2
8.2.1.3
8.2.1.1 Literature Reference for Abrin (Journal of Food Protection. 2008. 71(9):
1868-1874)
Analysis Purpose: Presumptive
Analytical Technique: Immunoassay
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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.
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 ELISA and
electrochemiluminescence (ECL)-based assays in food products. The three assays vary by use of
antibody combination (e.g, assay configuration): (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 spiked into food products ranged from 0.1 to 0.5 ng/mL,
using the ECL assay. The LOD for abrin spiked into food products for the ELISA assays ranged
between 0.5 and 10 ng/mL depending on the antibody combination. In all cases, the LODs were
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. Addition of non-fat milk
powder to the sample buffer may eliminate false-positive results (Dayan-Kenigsberg, J.,
Bertocchi, A., and Garber, E.A. 2008. "Rapid Detection of Ricin in Cosmetics and Elimination of
Artifacts Associated with Wheat Lectin." Journal of Immunological Methods. 336(2): 251-254).
http: //www. sciencedirect. com/science/j ournal/00221759
Source: Garber, E.A., Walker, J.L., and O'Brien, T.W. 2008. "Detection of Abrin in Foods
Using Enzyme-Linked Immunosorbent Assay and Electrochemiluminescence Technologies."
Journal of Food Protection. 71(9): 1868-1874.
httD://www.ingentaconnect.com/content/iafb/ifp/2008/00000071/00000009/art00015
8.2.1.2 Literature Reference for Abrin and Ricin (Analytical Biochemistry. 2008.
378(1): 87-89)
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.
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) or in 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 require incubation 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 (ig/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
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equivalents. In contrast, the undiluted jequirity seed extract contained a calculated level of 740 ±
50 (ig/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 Poli, M.A.
2008. "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/iournal/00032697
8.2.1.3 Literature Reference for Abrin and Shiga and Shiga-like Toxins
(Pharmacology Toxicology. 2001. 88(5): 255-260)
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.
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.
http ://www3 .interscience. wiley .com/j ournal/120703798/abstract
8.2.2 Botulinum neurotoxins (Serotypes A, B, E, F)
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.
Method
LRN
FDA, Bacteriological Analytical Manual Online, January
2001, Chapter 17, Clostridium botulinum
Lateral Flow Immunoassay Kits
Analytical Technique
Immunoassay, Immunoassay
(ELISA) and Mouse bioassay
Immunoassay (ELISA) and
Mouse bioassay
Immunoassay
Section
8.1.4
8.2.2.1
8.2.2.2
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8.2.2.1 FDA, Bacteriological Analytical Manual Online, Chapter 17, 2001:
Botulinum Neurotoxins
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 procedure for confirmation and biological activity
assessment in aerosol samples. Further research is needed to develop and standardize the
procedures for environmental sample types.
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: FDA, CFSAN. 2001. "Chapter 17 - Clostridium botulinum" Bacteriological Analytical
Manual Online. http://www.epa.gov/sam/pdfs/FDA-BAM-Chapl7.pdf
8.2.2.2 Lateral Flow Immunoassay Kits
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.
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
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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 EPA ETV Program
(http://www.epa.gov/sam/pdfs/ETV-BADD091904.pdf and http://www.epa.gov/sam/pdfs/ETV-
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: Immunoassays with botulinum toxins may produce variable results with
uncomplexed form of toxin. Addition of non-fat milk powder to the sample buffer may eliminate
false-positive results (Dayan-Kenigsberg, Bertocchi, J.A., and Garber, E.A.E. 2008. "Rapid
Detection of Ricin in Cosmetics and Elimination of Artifacts Associated with Wheat Lectin."
Journal of Immunological Methods. 336(2): 251-254).
http: //www. sciencedirect. com/science/j ournal/00221759
Source: ETV. 2006. http://www.epa.gov/etv/
8.2.3 Ricin (Ricinine)
Ricin - CAS RN: 9009-86-3.
Description: 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.
Ricinine - CAS RN: 5254-40-3.
Description: Small molecule, ricin marker.
Method
LRN
Analytical Biochemistry. 2008. 378: 87-89
Lateral Flow Immunoassay Kits
Journal of AOAC International. 2008. 91(2): 376-382
Journal of Analytical Toxicology. 2005. 29: 149-155
Analytical Technique
Immunoassay
Enzyme activity
Immunoassay
Immunoassay
LC-MS
Section
8.1.4
8.2.1.3
8.2.2.2
8.2.3.1
8.2.3.2
8.2.3.1 Literature Reference for Ricin (Journal of AOAC International. 2008.
91 (2): 376-382)
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.
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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.
http://www.atvpon-link.eom/AOAC/doi/abs/10.5555/iaoi.91.2.376
8.2.3.2 Literature Reference for Ricin by Ricinine detection (Journal of Analytical
Toxicology. 2005. 29(3): 149-155)
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 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.
Description of Method: Procedures are described for sample extraction by SPE, isocratic
HPLC, followed by electrospray ionization (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.iatox.com/abstracts/2005/April/149-johnson.html
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8.2.4 Shiga and Shiga-like toxins (Stx, Stx-1, Stx-2)
CAS RN: 75757-64-1 (Stx).
Description: Protein composed of one
-32 kDa A chain and five 7.7 kDa B chains.
Method
Pharmacology & Toxicology. 2001. 88(5): 255-260
FDA, Bacteriological Analytical Manual Online,
January 2001 , Appendix 1 , Rapid Methods for
Detecting Foodborne Pathogens
Journal of Clinical Microbiology. 2007. 45(10): 3377-
3380
Analytical Technique
Ribosome inactivation assay
Immunoassay (ELISA)
Optical immunoassay
Section
8.2.1.3
8.2.4.1
8.2.4.2
8.2.4.1 FDA, Bacteriological Analytical Manual Online, Appendix 1, 2001: Rapid
Methods for Detecting Foodborne Pathogens
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.
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: FDA, CFSAN. 2001. "Rapid Methods for Detecting Foodborne Pathogens."
Bacteriological Analytical Manual Online. http://www.epa.gov/sam/pdfs/FDA-BAM-
Appendixl.pdf
8.2.4.2 Literature Reference for Shiga and Shiga-like Toxin (Journal of Clinical
Microbiology. 2007. 45(10): 3377-3380)
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.
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
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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/JCM-45qOV
pgs3377-3380.pdf
8.2.5 Staphylococcal enterotoxins (SEA, SEB, SEC)
CAS RNs: 39424-53-8 (SEB), 37337-57-8 (SEA), 39424-54-9 (SEC)
Description: Monomeric protein of- 28 kDa (SEB), monomeric proteins of
(SEA and SEC)
27-27.5 kDa
Method
LRN
AOAC Official Method 993.06
Analytical Technique
Immunoassay
Immunoassay
Section
8.1.4
8.2.5.1
8.2.5.1 AOAC Official Method 993.06: Staphylococcal Enterotoxins in Selected
Foods
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.
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, centrirugation, 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/
8.3 Method Summaries for Small Molecule Biotoxins
Summaries of the analytical methods for small molecule biotoxins listed in Appendix D are provided in
Sections 8.3.1 through 8.3.12. 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 brief description of the method, intended method application, performance data
(if available), and a link to or source for obtaining a full version of the method.
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8.3.1 Aflatoxin (TypeBI)
CAS RN: 27261-02-5
Method
AOAC Official Method 991.31
Analytical Technique
Immunoassay and HPLC-FL
Section
8.3.1.1
8.3.1.1 AOAC Official Method 991.31: Aflatoxins in Corn, Raw Peanuts, and
Peanut Butter
Analysis Purpose: Presumptive and confirmatory
Analytical Technique: Immunoassay and HPLC-FL
Method Developed for: Aflatoxins (Type Bl) in corn, raw peanuts, and peanut butter
Method Selected for: SAM lists this method for presumptive and confirmatory analyses in
aerosol, solid, particulate, liquid, and water samples. Further research is needed to develop and
standardize the procedures for environmental sample types.
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 mAbs 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 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, B2, GI, and G2) 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.
Special Considerations: AOAC Official Method 994.08: Aflatoxin in Corn, Almonds, Brazil
Nuts, Peanuts, and Pistachio Nuts, (AOAC International. 1998. Official Methods of Analysis of
AOAC International, 16th Edition, 4th Revision, Vol. II. http://www.aoac.org/) may be used as a
complementary HPLC-FL method in order to provide more flexibility for analyses.
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://www.aoac.org/
8.3.2 a-Amanitin
CAS RN: 23109-05-9
Method
Journal of Chromatography B. 1991. 563(2): 299-311
Journal of Food Protection. 2005. 68(6): 1294-1301
Analytical Technique
HPLC amperometric detection
Immunoassay
Section
8.3.2.1
8.3.2.2
8.3.2.1 Literature Reference for a-Amanitin (Journal of Chromatography B. 1991.
563(2): 299-311)
Analysis Purpose: Confirmatory
Analytical Technique: HPLC with amperometric detection
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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.
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. http://www.ncbi.nlm.nih.gov/pubmed/2055993
8.3.2.2 Literature Reference for a-Amanitin, T-2 Mycotoxin (Journal of Food
Protection. 2005. 68(6): 1294-1301)
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.
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://www.ingentaconnect.com/content/iafp/ifp/2005/00000068/00000006/art00027
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8.3.3 Anatoxin-a
CAS RN: 64285-06-9
Method
Biomedical Chromatography. 1996. 10:46-47
Analytical Technique
HPLC-FL (precolumn
derivatization)
Section
8.3.3.1
8.3.3.1 Literature Reference for Anatoxin-a (Biomedical Chromatography. 1996.
10(1): 46-47)
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.
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.
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(1): 46-47.
http://www3 .interscience.wiley.com/journal/18562/abstract
8.3.4 Brevetoxins (B form)
CAS RN: 79580-28-2
Method
Environmental Health Perspectives. 2002. 110(2):
179-185
lexicon. 2004. 43(4): 455-465
Analytical Technique
Immunoassay
HPLC-MS-MS
Section
8.3.4.1
8.3.4.2
8.3.4.1 Literature Reference for Brevetoxins (Environmental Health
Perspectives. 2002. 110(2): 179-185)
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.
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
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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)-pgs 179-185.pdf
8.3.4.2 Literature Reference for Brevetoxins (Toxicon. 2004. 43(4): 455-465)
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.
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 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. http://cat.inist.fr/?aModele=afficheN&cpsidt=15668117
8.3.5 a-Conotoxin
CAS RN: 156467-85-5
Method
Biochemical Journal. 1997. 328: 245-250
Journal of Medicinal Chemistry. 2004. 47(5): 1234-
1241
Analytical Technique
Immunoassay
HPLC-MS
Section
8.3.5.1
8.3.5.2
8.3.5.1 Literature Reference for a-Conotoxin (Biochemical Journal. 1997. 328(1):
245-250)
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.
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Description of Method: A biologically active fluorescein derivative ofConus 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, E.G. 1997. "Characterization of a-Conotoxin Interactions
with the Nicotinic Acetylcholine Receptor and Monoclonal Antibodies." Biochemical Journal.
328(1): 245-250. http://www.epa.gov/sam/pdfs/BJ-328-pgs245-250.pdf
8.3.5.2 Literature Reference for a-Conotoxin (Journal of Medicinal Chemistry.
2004.47(5): 1234-1241)
Analysis Purpose: Confirmatory
Analytical Technique: 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.
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 centrirugation. 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. 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, M.L., Nicke, A., Jones, A., Adams, D.J., Alewood, P.P., and Lewis, R.J.
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. http://pubs.acs.org/cgi-bin/abstract.cgi/imcmar/2004/47/i05/abs/im031010o.html
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8.3.6 Cylindrospermopsin
CAS RN: 143545-90-8
Method
FEMS Microbiology Letters. 2002. 216: 159-164
ELISA Kits for Cylindrospermopsin
Analytical Technique
HPLC-PDA
Immunoassay
Section
8.3.6.1
8.3.6.2
8.3.6.1 Literature Reference for Cylindrospermopsin (FEMS Microbiology
Letters. 2002. 216(2): 159-164)
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.
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
Cig 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 Qg 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.
htto://cat.inist.fr/?aModele=afficheN&cpsidt=14002569
8.3.6.2 ELISA Kits for Cylindrospermopsin
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.
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 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
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sample absorbance to the absorbance of the calibrators (standards) at a specific wavelength (450
nm). The applicable concentration range is 0.4-2.0 (ig/L, with a minimum detection level of 0.4
ug/L.
Source: NEMI. 2006.
http://infotrek.er.usgs.gov/pls/apex/f?p=119:38:7526698938332159::::P38 METHOD ID:9507
8.3.7 Diacetoxyscirpenol (DAS)
CAS RIM: 2270-40-8
Method
International Journal of Food Microbiology. 1988.
6(1): 9-17
Rapid Communications in Mass Spectrometry.
2006. 20(9): 1422-1428
Analytical Technique
Immunoassay
LC/APCI-MS
Section
8.3.7.1
8.3.7.2
8.3.7.1 Literature Reference for Diacetoxyscirpenol (DAS) (International Journal
of Food Microbiology. 1988. 6(1): 9-17)
Analysis Purpose: Presumptive
Analytical Technique: Immunoassay
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.
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.
http: //www. sciencedirect. com/science/i ournal/01681605
8.3.7.2 Literature Reference for Diacetoxyscirpenol (DAS) and T-2 Mycotoxin
(Rapid Communications in Mass Spectrometry. 2006. 20(9): 1422-1428)
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.
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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.
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.
htto://cat.inist.fr/?aModele=afficheN&cpsidt=17697070
8.3.8 Microcystins (Principal isoforms: LA, LR, LW, RR, YR)
CAS RNs: 96180-79-9 (LA), 101043-37-2 (LR), 157622-02-1 (LW), 111755-37-4 (RR),
101064-48-6 (YR)
Method
Journal of AOAC International. 2001. 84(4): 1035-
1044
Analyst. 1994. 119(7): 1525-1530
Analytical Technique
Immunoassay/Phosphatase
assay
HPLC-PDA
Section
8.3.8.1
8.3.8.2
8.3.8.1 Literature Reference for Microcystins (Journal of AOAC International.
2001.84(4): 1035-1044)
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.
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 microcystins-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
8.3.8.2 Literature Reference for Microcystins (Analyst. 1994. 119(7): 1525-1530)
Analysis Purpose: Confirmatory
Analytical Technique: HPLC-PDA
SAM Revision 5.0
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September 29, 2009
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Section 8- Selected Biotoxin Methods
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.
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 ds 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/Journals/AN/article.asp?doi=AN9941901525
8.3.9 Picrotoxin
CAS RN: 124-87-8
Method
Journal of Pharmaceutical and Biomedical Analysis.
1989. 7(3): 369-375
Analytical Technique
HPLC
Section
8.3.9.1
8.3.9.1 Literature Reference for Picrotoxin (Journal of Pharmaceutical &
Biomedical Analysis. 1989. 7(3): 369-375)
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.
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 ug/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. http://www.sciencedirect.com/science/journal/07317085
SAM Revision 5.0
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September 29, 2009
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Section 8- Selected Biotoxin Methods
8.3.10 Saxitoxins (Principal isoforms: SIX, NEOSTX, GTX, dcGTX, dcSTX)
CAS RNs: 35523-89-8 (STX), 64296-20-4 (NEOSTX), 77462-64-7 (GTX), 58911-04-9
(dcSTX)
Method
Journal of AOAC International. 1995. 78: 528-
532
ELISA Kits for Saxitoxin
Analytical Technique
HPLC-FL (post column
derivatization)
Immunoassay
Section
8.3.10.1
8.3.10.2
8.3.10.1 Literature Reference for Saxitoxin (Journal of AOAC International. 1995.
78(2): 528-532)
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.
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, dcGTX3) 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 finol 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.
Source: Oshima, Y. 1995. "Postcolumn Derivatization Liquid Chromatographic Method for
Paralytic Shellfish Toxins." Journal of AOAC International. 78(2): 528-532.
htto://cat.inist.fr/?aModele=afficheN&cpsidt=3469391
8.3.10.2 ELISA Kits for Saxitoxins
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.
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
SAM Revision 5.0 210 September 29, 2009
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Section 8- Selected Biotoxin Methods
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.
Source: NEMI. 2006.
http://infotrek.er.usgs.gov/pls/apex/f?p=l 19:38:8989971104293493:
:P38 METHOD ID:9512
8.3.11 T-2 Mycotoxin
CAS RN: 21259-20-1
Method
Journal of Food Protection. 2005. 68(6): 1294-1301
Rapid Communications in Mass Spectrometry. 2006.
20(9): 1422-1428
Analytical Technique
Immunoassay
LC/APCI-MS
Section
8.3.2.2
8.3.7.2
See Sections 8.3.2.2 and 8.3.7.2 for information on immunoassay and LC/APCI-MS procedures
for T-2 Mycotoxin.
8.3.12 Tetrodotoxin
CAS RN: 9014-39-5
Method
Analytical Biochemistry. 2001. 290: 10-17
Journal of Clinical Laboratory Analysis. 1992. 6: 65-
72
Analytical Technique
LC/ESI-MS
Immunoassay
Section
8.3.12.1
8.3.12.2
8.3.12.1 Literature Reference for Tetrodotoxin (Analytical Biochemistry. 2001.
290(1): 10-17)
Analysis Purpose: Confirmatory
Analytical Technique: LC/ESI-MS
Method Developed for: Tetrodotoxin (TTX) 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.
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.
SAM Revision 5.0
211
September 29, 2009
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Section 8- Selected Biotoxin Methods
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://www.sciencedirect.com/science/iournal/00032697
8.3.12.2 Literature Reference for Tetrodotoxin (Journal of Clinical Laboratory
Analysis. 1992. 6(2): 65-72)
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.
Description of Method: Procedures are described for a competitive inhibition enzyme
immunoassay (CIEIA) for tetrodotoxin 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.
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.
http://www3.interscience.wilev.eom/iournal/l 12131435/abstract
SAM Revision 5.0 212 September 29, 2009
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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 document, which can be used as a guide 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 5.0 213 September 29, 2009
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Section 9 - Conclusions
SAM Revision 5.0 214 September 29, 2009
-------
Appendix A - Selected Chemical Methods
Appendix A: Selected Chemical Methods
SAM Revision 5.0 September 29, 2009
-------
Appendix A - Selected Chemical Methods
SAM Revision 5.0 September 29, 2009
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Appendix A: Selected Chemical Methods
Analyte(s)
Acephate
Acrylamide
Acrylonitrile
Aldicarb (Temik)
Aldicarb sulfone
Aldicarb sulfoxide
Allyl alcohol
4-Aminopyridine
Ammonia
Ammonium metavanadate
(analyze for total vanadium)
Arsenic, Total
CASRN
30560-19-1
79-06-1
107-13-1
116-06-3
1646-88-4
1646-87-3
107-18-6
504-24-5
7664-41 -7
7803-55-6
7440-38-2
Determinative
Technique
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
GC-MS
HPLC
Spectrophotometry
ICP-AES / ICP-MS
ICP-AES / ICP-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
Adapted from
Journal of
Chroma tography A,
1154(1): 3-25
Water extraction
8316
(EPA SW-846)
Water extraction
8316
(EPA SW-846)
831 8A
(EPA SW-846)
831 8A
(EPA SW-846)
831 8A
(EPA SW-846)
5035A
(EPA SW-846)
8260C
(EPA SW-846)
8330B
(EPA SW-846)
Not of concern
3050B
(EPA SW-846)
6010C/6020A
(EPA SW-846)
3050B
(EPA SW-846)
6010C/6020A
(EPA SW-846)
Non-aqueous
Liquid/Organic
Solid Samples1
Adapted from
Journal of
Chromatography A,
1154(1): 3-25
Water extraction
8316
(EPA SW-846)
Water extraction
8316
(EPA SW-846)
831 8A
(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)
Not of concern
3031
(EPA SW-846)
6010C/6020A
(EPA SW-846)
3031
(EPA SW-846)
6010C/6020A
(EPA SW-846)
Aqueous Liquid
Samples
Adapted from
Chromatographia,
63(5/6): 233-237
8316
(EPA SW-846)
8316
(EPA SW-846)
MS014
(EPA CRL)
MS014
(EPA CRL)
MS014
(EPA CRL)
5030C
(EPA SW-846)
8260C
(EPA SW-846)
3535A/8330B
(EPA SW-846)
8330B
(EPA SW-846)
4500- NH3 B
(SM)
4500- NH3G
(SM)
200.7/200.8
(EPAOW)
200.7/200.8
(EPA OW)
Drinking Water
Samples
Adapted from
Chromatographia,
63(5/6): 233-237
8316
(EPA SW-846)
8316
(EPA SW-846)
531.2
(EPAOW)
531.2
(EPAOW)
531.2
(EPAOW)
5030C
(EPA SW-846)
8260C
(EPA SW-846)
3535A/8330B
(EPA SW-846)
8330B
(EPA SW-846)
350.1
(EPAOW)
200.7/200.8
(EPAOW)
200.7/200.8
(EPA OW)
Air Samples
Adapted from
Journal of
Chromatography A,
1154(1): 3-25
PV2004
(OS HA)
PV2004
(OS HA)
5601
(NIOSH)
5601
(NIOSH)
5601
(NIOSH)
TO-152
(EPAORD)
Not of concern
6015
(NIOSH)
IO-3.1
(EPAORD)
IO-3.4/IO-3.5
(EPAORD)
10-3.1
(EPAORD)
IO-3.4/IO-3.5
(EPAORD)
Wipes
Adapted from
Journal of
Chromatography A,
11 54(1): 3-25
3570/8290A Appendix A
(EPA SW-846)
8316
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8316
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
831 8A
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
831 8A
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
831 8A
(EPA SW-846)
Not of concern
3570/8290A Appendix A
(EPA SW-846)
8330B
(EPA SW-846)
Not of concern
9102
(NIOSH)
6010C/6020A
(EPA SW-846)
9102
(NIOSH)
6010C/6020A
(EPA SW-846)
SAM Revision 5.0, Appendix A
A-1
September 29, 2009
-------
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)
Carfentanil
Carbon disulfide
Chlorfenvinphos
CASRN
1327-53-3
7784-42-1
1332-21-4
7637-07-2
56073-10-0
28772-56-7
6581-06-2
7778-44-1
1563-66-2
59708-52-0
75-15-0
470-90-6
Determinative
Technique
ICP-AES / ICP-MS
GFAA/ICP-AES/
ICP-MS
TEM
ISE
HPLC
HPLC/LC-MS-MS
HPLC
ICP-AES / ICP-MS
HPLC/LC-MS-MS
HPLC
GC-MS
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
Sample Prep
Determinative
Solid Samples
3050B
(EPA SW-846)
601 OC/6020A
(EPA SW-846)
3050B
(EPA SW-846)
7010
(EPA SW-846)
D5755-03 (soft
surfaces-microvac)
(ASTM)
Not of concern
3541/3545A
(EPA SW-846)
832 1B
(EPA SW-846)
3541/3545A
(EPA SW-846)
832 1B
(EPA SW-846)
3541/3545A
(EPA SW-846)
832 1B3
(EPA SW-846)
3050B
(EPA SW-846)
6010C/6020A
(EPA SW-846)
831 8A
(EPA SW-846)
354 1/3545 A
(EPA SW-846)
832 1B
(EPA SW-846)
5035A
(EPA SW-846)
8260C
(EPA SW-846)
3541/3545A
(EPA SW-846)
8270D
(EPA SW-846)
Non-aqueous
Liquid/Organic
Solid Samples1
3031
(EPA SW-846)
601 OC/6020A
(EPA SW-846)
Not of concern
Not of concern
Not of concern
3580A
(EPA SW-846)
832 1B
(EPA SW-846)
3580A
(EPA SW-846)
832 1B
(EPA SW-846)
3580A
(EPA SW-846)
832 1B3
(EPA SW-846)
3031
(EPA SW-846)
6010C/6020A
(EPA SW-846)
831 8A
(EPA SW-846)
3580A
(EPA SW-846)
832 1B
(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
(EPA OW)
200.7/200.8
(EPA OW)
Not of concern
Not of concern
3520C/3535A
(EPA SW-846)
832 1B
(EPA SW-846)
MS014
(EPACRL)
3520C/3535A
(EPA SW-846)
832 1B3
(EPA SW-846)
200.7/200.8
(EPAOW)
MS014
(EPACRL)
3520C/3535A
(EPA SW-846)
832 1B
(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)
832 1B
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8321 B
(EPA SW-846)
3520C/3535A
(EPA SW-846)
832 1B3
(EPA SW-846)
200.7/200.8
(EPAOW)
531.2
(EPAOW)
3520C/3535A
(EPA SW-846)
832 1B
(EPA SW-846)
524.2
(EPAOW)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
Air Samples
10-3.1
(EPAORD)
IO-3.4/IO-3.5
(EPAORD)
6001
(NIOSH)
10312:1995
(ISO)
ID216SG
(OSHA)
Not of concern
Not of concern
TO-10A4
(EPAORD)
IO-3.1
(EPAORD)
IO-3.4/IO-3.5
(EPAORD)
5601
(NIOSH)
Not of concern
TO-15
(EPAORD)
TO-10A
(EPAORD)
Wipes
9102
(NIOSH)
6010C/6020A
(EPA SW-846)
9102
(NIOSH)
7010
(EPA SW-846)
D6480-05
(hard surfaces-wipes)
(ASTM)
Not of concern
3570/8290A Appendix A
(EPA SW-846)
832 1B
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8321 B
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
832 1B3
(EPA SW-846)
9102
(NIOSH)
6010C/6020A
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
831 8A
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
832 1B
(EPA SW-846)
Not of concern
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
SAM Revision 5.0, Appendix A
A-2
September 29, 2009
-------
Analyte(s)
Chlorine
2-Chloroethanol
3-Chloro-1 ,2-propanediol
Chloropicrin
Chlorosarin
Chlorosoman
2-Chlorovinylarsonous acid
(2-CVAA) (degradation product of
Lewisite)
Chlorpyrifos
Chlorpyrifos oxon
Crimidine
Cyanide, Amenable to chlorination
CASRN
7782-50-5
107-07-3
96-24-2
76-06-2
1445-76-7
7040-57-5
85090-33-1
2921-88-2
5598-15-2
535-89-7
NA
Determinative
Technique
Spectrophotometry
GC-MS/GC-FID
GC-MS
GC-MS / GC-ECD
GC-MS
GC-MS
ICP-AES / ICP-MS
GC-MS
GC-MS
GC-MS
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
(EPA SW-846)
8260C
(EPA SW-846)
354 1/3545 A
(EPA SW-846)
8270D5
(EPA SW-846)
3545A
(EPA SW-846)
8270D7
(EPA SW-846)
354 1/3545 A
(EPA SW-846)
8270D
(EPA SW-846)
3541/3545A
(EPA SW-846)
8270D
(EPA SW-846)
3050B
(EPA SW-846)
6010C/6020A
(EPA SW-846)
3541/3545A
(EPA SW-846)
8270D
(EPA SW-846)
3541/3545A
(EPA SW-846)
8270D
(EPA SW-846)
3545A
(EPA SW-846)
8270D8
(EPA SW-846)
3135.21
(EPA RLAB)
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)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
8270D8
(EPA SW-846)
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
(EPA OW)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
8270D8
(EPA SW-846)
3135.21
(EPA RLAB)
Drinking Water
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
(EPA OW)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270 D
(EPA SW-846)
3535A
(EPA SW-846)
8270D8
(EPA SW-846)
3135.21
(EPA RLAB)
Air Samples
Adapted from
Analyst, 124(12):
1853-1857
4500-CI G
(SM)
2513
(NIOSH)
TO-10A6
(EPAORD)
PV2103(OSHA)
TO-10A6
(EPAORD)
TO-1 OA6
(EPAORD)
10-3.1
(EPAORD)
IO-3.4/IO-3.5
(EPAORD)
TO-10A
(EPAORD)
TO-10A
(EPAORD)
Not of concern
Not of concern
Wipes
Not of concern
Not of concern
3570/8290A Appendix A
(EPA SW-846)
8270D5
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D7
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
9102
(NIOSH)
6010C/6020A
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8270 D
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D8
(EPA SW-846)
3135.21
(EPA RLAB)
SAM Revision 5.0, Appendix A
A-3
September 29, 2009
-------
Analyte(s)
Cyanide, Total
Cyanogen chloride
Cyclohexyl sarin (GF)
1,2-Dichloroethane
(degradation product of HD)
Dichlorvos
Dicrotophos
Diesel range organics
Diisopropyl methylphosphonate
(DIMP)
(degradation product of GB)
Dimethylphosphite
Dimethylphosphoramidic acid
(degradation product of GA)
Diphacinone
Disulfoton
CASRN
57-12-5
506-77-4
329-99-7
107-06-2
62-73-7
141-66-2
NA
1445-75-6
868-85-9
33876-51-6
82-66-6
298-04-4
Determinative
Technique
Spectrophotometry
GC-MS
GC-MS
GC-MS
GC-MS
GC-MS
GC-FID
HPLC/LC-MS-MS
GC-MS
HPLC
HPLC
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
Sample Prep
Determinative
Solid Samples
ILM05.3CN
(EPA CLP)
5035A
(EPA SW-846)
8260C
(EPA SW-846)
3541/3545A
(EPA SW-846)
8270 D
(EPA SW-846)
5035A
(EPA SW-846)
8260C
(EPA SW-846)
3545A
(EPA SW-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)
832 1B9
(EPA SW-846)
3545A
(EPA SW-846)
8270D
(EPA SW-846)
3545A
(EPA SW-846)
832 1B3
(EPA SW-846)
354 1/3545 A
(EPA SW-846)
832 1B
(EPA SW-846)
3541/3545A
(EPA SW-846)
8270D
(EPA SW-846)
Non-aqueous
Liquid/Organic
Solid Samples1
Not of concern
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)
832 1B9
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
832 1B3
(EPA SW-846)
3580A
(EPA SW-846)
832 1B
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
Aqueous Liquid
Samples
ILM05.3CN
(EPA CLP)
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)
MS017
(EPACRL)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
832 1B3
(EPA SW-846)
3520C/3535A
(EPA SW-846)
832 1B
(EPA SW-846)
525.2
(EPAOW)
Drinking Water
Samples
335.4
(EPAOW)
5030C
(EPA SW-846)
8260C
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
524.2
(EPAOW)
525.2
(EPAOW)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
801 5C
(EPA SW-846)
3535A
(EPA SW-846)
832 1B9
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
832 1B3
(EPA SW-846)
3520C/3535A
(EPA SW-846)
832 1B
(EPA SW-846)
525.2
(EPAOW)
Air Samples
6010
(NIOSH)
TO-15
(EPA ORD)
TO-10A
(EPA ORD)
TO-15
(EPA ORD)
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
Not of concern
TO-1 OA6
(EPA ORD)
TO-10A
(EPA ORD)
TO-1 OA4
(EPA ORD)
Not of concern
5600
(NIOSH)
Wipes
ILM05.3CN
(EPA CLP)
Not of concern
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)
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)
832 1B9
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
832 1B3
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
832 1B
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
SAM Revision 5.0, Appendix A
A-4
September 29, 2009
-------
Analyte(s)
Disulfoton sulfoxide
1 ,4-Dithiane
(degradation product of HD)
EA2192 [Diisopropylaminoethyl
methy Ith io lophospho nate]
(hydrolysis product of VX)
Ethyl methylphosphonic acid
(EMPA)
(degradation product of VX)
Ethyldichloroarsine (ED)
N-Ethyldiethanolamine (EDEA)
(degradation product of HN-1)
Ethylene oxide
Fenamiphos
Fentanyl
Fluoride
Fluoroacetamide
CASRN
2497-07-6
505-29-3
73207-98-4
1832-53-7
598-14-1
1 39-87-7
75-21-8
22224-92-6
437-38-7
1 6984-48-8
640-19-7
Determinative
Technique
GC-MS / GC-FPD
GC-MS
HPLC
HPLC/LC-MS-MS
GC-MS
HPLC/LC-MS-MS
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
(EPA SW-846)
8270D
(EPA SW-846)
3545A
(EPA SW-846)
8270D
(EPA SW-846)
3545A
(EPA SW-846)
832 1B3
(EPA SW-846)
3545A
(EPA SW-846)
832 1B3
(EPA SW-846)
3545A
(EPA SW-846)
8270D
(EPA SW-846)
3541/3545A
(EPA SW-846)
832 1B
(EPA SW-846)
5035A
(EPA SW-846)
8260C
(EPA SW-846)
3541/3545A
(EPA SW-846)
8270D
(EPA SW-846)
3541/3545A
(EPA SW-846)
832 1B
(EPA SW-846)
Not of concern
Adapted from
Journal of
Chromatography B,
876(1): 103-108
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)
832 1B3
(EPA SW-846)
3580A
(EPA SW-846)
832 1B3
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
832 1B
(EPA SW-846)
3585
(EPA SW-846)
8260C
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
832 1B
(EPA SW-846)
Not of concern
Adapted from
Journal of
Chromatography B,
876(1): 103-108
Aqueous Liquid
Samples
525.2
(EPAOW)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
832 1B3
(EPA SW-846)
MS017
(EPACRL)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
MS016
(EPACRL)
5030C
(EPA SW-846)
8260C
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
832 1B
(EPA SW-846)
300.1, Rev 1.0
(EPA OW)
Adapted from
Journal of
Chromatography B,
876(1): 103-108
Drinking Water
Samples
525.2
(EPAOW)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
832 1B3
(EPA SW-846)
3535A
(EPA SW-846)
832 1B3
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
832 1B
(EPA SW-846)
5030C
(EPA SW-846)
8260C
(EPA SW-846)
525.2
(EPAOW)
3520C/3535A
(EPA SW-846)
832 1B
(EPA SW-846)
300.1, Rev 1.0
(EPA OW)
Adapted from
Journal of
Chromatography B,
876(1): 103-108
Air Samples
5600
(NIOSH)
Not of concern
TO-1 OA4
(EPAORD)
TO-1 OA4
(EPAORD)
TO-1 5
(EPA ORD)
TO-10A
(EPAORD)
TO-1 5
(EPAORD)
TO-10A
(EPAORD)
Not of concern
Not of concern
Adapted from
Journal of
Chromatography B,
876(1): 103-108
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)
832 1B3
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
832 1B3
(EPA SW-846)
9102
(NIOSH)
8270D
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
832 1B
(EPA SW-846)
Not of concern
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
832 1B
(EPA SW-846)
Not of concern
Adapted from
Journal of
Chromatography B,
876(1): 103-108
SAM Revision 5.0, Appendix A
A-5
September 29, 2009
-------
Analyte(s)
Fluoroacetic acid and fluoroacetate
salts (analyze for fluoroacetate ion)
2-Fluoroethanol
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
NA
371-62-0
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
Determinative
Technique
1C
GC-MS/GC-FID
HPLC
GC-FID
HPLC
HPLC
1C
1C
Spectrophotometry
1C
1C
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
Adapted from
Analytical Letters,
27(1 4): 2703-271 8
300.1, Rev 1.0
(EPAOW)
5035A
(EPA SW-846)
8260C
(EPA SW-846)
831 5A
(EPA SW-846)
5035A
(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
Non-aqueous
Liquid/Organic
Solid Samples1
Adapted from
Analytical Letters,
27(1 4): 2703-271 8
300.1, Rev 1.0
(EPAOW)
3585
(EPA SW-846)
8260C
(EPA SW-846)
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
Aqueous Liquid
Samples
300.1, Rev 1.0
(EPA OW)
5030C
(EPA SW-846)
8260C
(EPA SW-846)
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)
8330 B
(EPA SW-846)
Not of concern
Not of concern
Not of concern
Not of concern
Not of concern
Drinking Water
Samples
300.1, Rev 1.0
(EPA OW)
5030C
(EPA SW-846)
8260C
(EPA SW-846)
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)
8330 B
(EPA SW-846)
Not of concern
Not of concern
Not of concern
Not of concern
Not of concern
Air Samples
S301-1
(NIOSH)
300.1, Rev 1.0
(EPAOW)
2513
(NIOSH)
2016
(NIOSH)
Not of concern
Not of concern
Not of concern
7903
(NIOSH)
7903
(NIOSH)
6010
(NIOSH)
790310
(NIOSH)
6013
(NIOSH)
Wipes
3570/8290A Appendix A
(EPA SW-846)
300.1 , Rev 1 .0
(EPAOW)
Not of concern
3570/8290A Appendix A
(EPA SW-846)
831 5A
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
801 5C
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8330B
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8330 B
(EPA SW-846)
Not of concern
Not of concern
Not of concern
Not of concern
Not of concern
SAM Revision 5.0, Appendix A
A-6
September 29, 2009
-------
Analyte(s)
Isopropyl methylphosphonic acid
(IMPA) (degradation product of
GB)
Kerosene
Lead arsenate
(analyze as total arsenic)
Lewisite 1 (L-1)11
[2-chlorovinyldichloroarsine]
(analyze for total arsenic)
Lewisite 2 (L-2)
[bis(2-chlorovinyl)chloroarsine]
(analyze for total arsenic)
Lewisite 3 (L-3)
[tris(2-chlorovinyl)arsine]
(analyze for total arsenic)
Lewisite oxide
(degradation product of Lewisite)
Mercuric chloride (analyze for total
mercury)
Mercury, Total
Methamidophos
Methomyl
Methoxyethylmercuric acetate
(analyze for total mercury)
CASRN
1832-54-8
64742-81-0
7645-25-2
541-25-3
40334-69-8
40334-70-1
1306-02-1
7487-94-7
7439-97-6
1 0265-92-6
16752-77-5
151-38-2
Determinative
Technique
HPLC/LC-MS-MS
GC-FID
ICP-AES / ICP-MS
ICP-AES / ICP-MS
ICP-AES / ICP-MS
ICP-AES / ICP-MS
ICP-AES / ICP-MS
Spectrophotometry
/CVAA/CVAFS
Spectrophotometry
/ CVAA / CVAFS
LC-MS-MS
HPLC/LC-MS-MS
Spectrophotometry
/ CVAA / CVAFS
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
Sample Prep
Determinative
Solid Samples
3545A
(EPA SW-846)
832 1B3
(EPA SW-846)
5035A
(EPA SW-846)
801 5C
(EPA SW-846)
3050B
(EPA SW-846)
6010C/6020A
(EPA SW-846)
3050B
(EPA SW-846)
601 OC/6020A
(EPA SW-846)
3050B
(EPA SW-846)
601 OC/6020A
(EPA SW-846)
3050B
(EPA SW-846)
6010C/6020A
(EPA SW-846)
3050B
(EPA SW-846)
6010C/6020A
(EPA SW-846)
747312
(EPA SW-846)
747312
(EPA SW-846)
Adapted from
Journal of
Chroma tography A,
1154(1): 3-25
831 8A
(EPA SW-846)
747312
(EPA SW-846)
Non-aqueous
Liquid/Organic
Solid Samples1
3580A
(EPA SW-846)
832 1B3
(EPA SW-846)
3585
(EPA SW-846)
801 5C
(EPA SW-846)
3031
(EPA SW-846)
6010C/6020A
(EPA SW-846)
3031
(EPA SW-846)
601 OC/6020A
(EPA SW-846)
3031
(EPA SW-846)
601 OC/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
Not of concern
Adapted from
Journal of
Chromatography A,
1154(1): 3-25
831 8A
(EPA SW-846)
Not of concern
Aqueous Liquid
Samples
MS017
(EPACRL)
5030C
(EPA SW-846)
801 5C
(EPA SW-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)
747312
(EPA SW-846)
747312
(EPA SW-846)
Adapted from
Chromatographia,
63(5/6): 233-237
MS014
(EPA CRL)
747312
(EPA SW-846)
Drinking Water
Samples
3535A
(EPA SW-846)
832 1B3
(EPA SW-846)
5030C
(EPA SW-846)
801 5C
(EPA SW-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)
245.1
(EPAOW)
245.1
(EPAOW)
Adapted from
Chromatographia,
63(5/6): 233-237
531.2
(EPAOW)
245.1
(EPAOW)
Air Samples
TO-10A4
(EPAORD)
Not of concern
IO-3.1
(EPAORD)
IO-3.4/IO-3.5
(EPAORD)
10-3.1
(EPAORD)
IO-3.4/IO-3.5
(EPAORD)
IO-3.1
(EPAORD)
IO-3.4/IO-3.5
(EPAORD)
IO-3.1
(EPAORD)
IO-3.4/IO-3.5
(EPAORD)
10-3.1
(EPA ORD)
IO-3.4/IO-3.5
(EPAORD)
Not of concern
IO-5
(EPAORD)
Adapted from
Journal of
Chromatography A,
1154(1): 3-25
5601
(NIOSH)
IO-5
(EPAORD)
Wipes
3570/8290A Appendix A
(EPA SW-846)
832 1B3
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
801 5C
(EPA SW-846)
9102
(NIOSH)
6010C/6020A
(EPA SW-846)
9102
(NIOSH)
6010C/6020A
(EPA SW-846)
9102
(NIOSH)
6010C/6020A
(EPA SW-846)
9102
(NIOSH)
6010C/6020A
(EPA SW-846)
9102
(NIOSH)
6010C/6020A
(EPA SW-846)
9102
(NIOSH)
747312
(EPA SW-846)
9102
(NIOSH)
747312
(EPA SW-846)
Adapted from
Journal of
Chromatography A,
11 54(1): 3-25
3570/8290A Appendix A
(EPA SW-846)
831 8A
(EPA SW-846)
9102
(NIOSH)
747312
(EPA SW-846)
SAM Revision 5.0, Appendix A
A-7
September 29, 2009
-------
Analyte(s)
Methyl acrylonitrile
Methyl fluoroacetate
(analyze for fluoroacetate ion)
Methyl hydrazine
Methyl isocyanate
Methyl paraoxon
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
CASRN
126-98-7
453-18-9
60-34-4
624-83-9
950-35-6
298-00-0
74-89-5
105-59-9
1 1 89-87-3
993-13-5
7786-34-7
Determinative
Technique
HPLC
1C
GC-MS /
Spectrophotometry
HPLC
GC-MS
GC-MS
HPLC
HPLC/LC-MS-MS
GC-MS
HPLC
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
Water extraction
8316
(EPASW-846)
Adapted from
Analytical Letters,
27(1 4): 2703-271 8
300.1, Rev 1.0
(EPAOW)
3541/3545A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
3545A
(EPA SW-846)
8270D
(EPA SW-846)
3545A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
3541/3545A
(EPA SW-846)
832 1B
(EPA SW-846)
3541/3545A
(EPA SW-846)
8270D
(EPA SW-846)
3545A
(EPA SW-846)
832 1B3
(EPASW-846)
3545A
(EPASW-846)
8270D
(EPA SW-846)
Non-aqueous
Liquid/Organic
Solid Samples1
Water extraction
8316
(EPA SW-846)
Adapted from
Analytical Letters,
27(1 4): 2703-271 8
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)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
3580A
(EPA SW-846)
832 1B
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
832 1B3
(EPASW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
Aqueous Liquid
Samples
8316
(EPASW-846)
300.1, Rev 1.0
(EPA OW)
3520C/3535A
(EPASW-846)
8270D
(EPASW-846)
Not of concern
3535A
(EPASW-846)
8270D
(EPASW-846)
3535A
(EPASW-846)
8270 D
(EPA SW-846)
Not of concern
MS016
(EPACRL)
3520C/3535A
(EPA SW-846)
8270D
(EPASW-846)
MS017
(EPACRL)
3535A
(EPASW-846)
8270D
(EPASW-846)
Drinking Water
Samples
8316
(EPASW-846)
300.1, Rev 1.0
(EPA OW)
3520C/3535A
(EPASW-846)
8270D
(EPASW-846)
Not of concern
3535A
(EPASW-846)
8270D
(EPA SW-846)
3535A
(EPASW-846)
8270 D
(EPA SW-846)
Not of concern
3520C/3535A
(EPA SW-846)
8321 B
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPASW-846)
3535A
(EPASW-846)
832 1B3
(EPASW-846)
525.2
(EPAOW)
Air Samples
PV2004
(OS HA)
S301-1
(NIOSH)
300.1, Rev 1.0
(EPAOW)
3510
(NIOSH)
OSHA54
TO-10A
(EPA OR D)
TO-10A
(EPA OR D)
OS HA 40
TO-10A
(EPA OR D)
TO-10A6
(EPAORD)
TO-1 OA4
(EPAORD)
TO-10A
(EPAORD)
Wipes
3570/8290A Appendix A
(EPASW-846)
8316
(EPASW-846)
3570/8290A Appendix A
(EPASW-846)
300.1 , Rev 1 .0
(EPAOW)
3570/8290A Appendix A
(EPASW-846)
8270D
(EPASW-846)
Not of concern
3570/8290A Appendix A
(EPASW-846)
8270D
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8270 D
(EPA SW-846)
Not of concern
3570/8290A Appendix A
(EPA SW-846)
8321 B
(EPA SW-846)
3570/8290A Appendix A
(EPASW-846)
8270D
(EPASW-846)
3570/8290A Appendix A
(EPASW-846)
832 1B3
(EPASW-846)
3570/8290A Appendix A
(EPASW-846)
8270D
(EPASW-846)
SAM Revision 5.0, Appendix A
A-8
September 29, 2009
-------
Analyte(s)
Monocrotophos
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
(analyze as nicotine)
Octahydro-1 ,3,5,7-tetranitro-1 ,3,5,7
tetrazocine (HMX)
Organophosphate pesticides, NOS
Osmium tetroxide
(analyze for total osmium)
Oxamyl
Paraquat
Paraoxon
CASRN
6923-22-4
538-07-8
51-75-2
555-77-1
505-60-2
54-11-5
2691-41-0
NA
20816-12-0
23135-22-0
4685-14-7
311-45-5
Determinative
Technique
GC-MS
GC-MS
GC-MS
GC-MS
GC-MS
GC-MS
HPLC
GC-MS /GC-NPD
/ GC-FPD
ICP-AES / GFAA
HPLC/LC-MS-MS
HPLC
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
Sample Prep
Determinative
Solid Samples
3545A
(EPASW-846)
8270D
(EPA SW-846)
3541/3545A
(EPA SW-846)
8270D
(EPA SW-846)
3541/3545A
(EPA SW-846)
8270D
(EPASW-846)
354 1/3545 A
(EPASW-846)
8270D
(EPASW-846)
3571
(EPASW-846)
8270D13
(EPASW-846)
3545A
(EPA SW-846)
8270D
(EPASW-846)
8330B
(EPA SW-846)
3541/3545A
(EPA SW-846)
8270D
(EPA SW-846)
3050B
(EPA SW-846)
601 OC
(EPA SW-846)
831 8A
(EPA SW-846)
Not of concern
3541/3545A
(EPA SW-846)
8270D
(EPA SW-846)
Non-aqueous
Liquid/Organic
Solid Samples1
3580A
(EPASW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3580A
(EPASW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3571
(EPASW-846)
8270D13
(EPASW-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)
Aqueous Liquid
Samples
3535A
(EPASW-846)
8270D
(EPASW-846)
3520C/3535A
(EPASW-846)
8270D
(EPASW-846)
3520C/3535A
(EPASW-846)
8270D
(EPASW-846)
3520C/3535A
(EPASW-846)
8270D
(EPASW-846)
3571
(EPASW-846)
8270D13
(EPASW-846)
3535A
(EPASW-846)
8270D
(EPASW-846)
3535A/8330B
(EPASW-846)
8330B
(EPASW-846)
614
(EPAOW)
252.2
(EPAOW)
MS014
(EPACRL)
549.2
(EPAOW)
3520C/3535A
(EPASW-846)
8270D
(EPA SW-846)
Drinking Water
Samples
3535A
(EPASW-846)
8270D
(EPASW-846)
3520C/3535A
(EPASW-846)
8270D
(EPASW-846)
3520C/3535A
(EPASW-846)
8270D
(EPASW-846)
3520C/3535A
(EPASW-846)
8270D
(EPASW-846)
3571
(EPASW-846)
8270D13
(EPASW-846)
3535A
(EPASW-846)
8270D
(EPASW-846)
3535A/8330B
(EPASW-846)
8330B
(EPASW-846)
507
(EPAOW)
252.2
(EPAOW)
531.2
(EPAOW)
549.2
(EPAOW)
3520C/3535A
(EPASW-846)
8270 D
(EPA SW-846)
Air Samples
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
Not of concern
Not of concern
5600
(NIOSH)
10-3.1
(EPA ORD)
IO-3.4
(EPA ORD)
5601
(NIOSH)
Not of concern
TO-10A
(EPA ORD)
Wipes
3570/8290A Appendix A
(EPASW-846)
8270D
(EPASW-846)
3570/8290A Appendix A
(EPASW-846)
8270D
(EPASW-846)
3570/8290A Appendix A
(EPASW-846)
8270D
(EPASW-846)
3570/8290A Appendix A
(EPASW-846)
8270D
(EPASW-846)
3570/8290A Appendix A
(EPASW-846)
8270D13
(EPASW-846)
3570/8290A Appendix A
(EPASW-846)
8270D
(EPASW-846)
3570/8290A Appendix A
(EPASW-846)
8330B
(EPASW-846)
3570/8290A Appendix A
(EPASW-846)
8270D
(EPASW-846)
9102
(NIOSH)
601 OC
(EPASW-846)
3570/8290A Appendix A
(EPASW-846)
831 8A
(EPASW-846)
Not of concern
3570/8290A Appendix A
(EPASW-846)
8270 D
(EPA SW-846)
SAM Revision 5.0, Appendix A
A-9
September 29, 2009
-------
Analyte(s)
Parathion
Pentaerythritol tetranitrate (PETN)
Phencyclidine
Phenol
Phorate
Phorate sulfone
Phorate sulfoxide
Phosgene
Phosphamidon
Phosphine
Phosphorus trichloride
Pinacolyl methyl phosphonic acid
(PMPA)
(degradation product of GD)
CASRN
56-38-2
78-11-5
77-10-1
1 08-95-2
298-02-2
2588-04-7
2588-03-6
75-44-5
13171-21-6
7803-51 -2
7719-12-2
616-52-4
Determinative
Technique
GC-MS
HPLC
GC-MS
GC-MS
GC-MS
GC-MS
GC-MS
GC-NPD
GC-MS
Spectrophotometry
Spectrophotometry
HPLC/LC-MS-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
Sample Prep
Determinative
Solid Samples
3541/3545A
(EPA SW-846)
8270D
(EPA SW-846)
8330B
(EPA SW-846)
3545A
(EPA SW-846)
8270D
(EPA SW-846)
3541/3545A
(EPA SW-846)
8270D
(EPA SW-846)
3545A
(EPA SW-846)
8270D
(EPA SW-846)
3545A
(EPA SW-846)
8270D
(EPA SW-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)
832 1B3
(EPA SW-846)
Non-aqueous
Liquid/Organic
Solid Samples1
3580A
(EPA SW-846)
8270D
(EPA SW-846)
8330B
(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)
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)
832 1B3
(EPA SW-846)
Aqueous Liquid
Samples
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A/8330B
(EPA SW-846)
8330B
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270 D
(EPA SW-846)
3535A
(EPA SW-846)
8270 D
(EPA SW-846)
3535A
(EPA SW-846)
8270 D
(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
MS017
(EPACRL)
Drinking Water
Samples
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A/8330B
(EPA SW-846)
8330B
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270 D
(EPA SW-846)
3535A
(EPA SW-846)
8270 D
(EPA SW-846)
3535A
(EPA SW-846)
8270 D
(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
(EPA SW-846)
832 1B3
(EPA SW-846)
Air Samples
TO-10A
(EPAORD)
Not of concern
TO-10A
(EPAORD)
TO-10A
(EPAORD)
TO-10A
(EPAORD)
TO-10A
(EPAORD)
TO-1 OA
(EPAORD)
OSHA61
TO-1 OA
(EPAORD)
6002
(NIOSH)
6402
(NIOSH)
TO-1 OA4
(EPAORD)
Wipes
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8330B
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8270 D
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8270 D
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8270 D
(EPA SW-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)
832 1B3
(EPA SW-846)
SAM Revision 5.0, Appendix A
A-10
September 29, 2009
-------
Analyte(s)
Propylene oxide
R 33 (VR)
[methylphosphonothioic acid, S-[2-
(diethylamino)ethyl] O-2-
methylpropyl ester]
Sarin (GB)
Semivolatile organic compounds,
NOS
Sodium arsenite
(analyze for total arsenic)
Sodium azide
(analyze as azide ion)
So man (GD)
Strychnine
Sulfur dioxide
Sulfur trioxide
Tabun (GA)
CASRN
75-56-9
159939-87-4
107-44-8
NA
7784-46-5
26628-22-8
96-64-0
57-24-9
7446-09-5
7446-11-9
77-81-6
Determinative
Technique
GC-MS/GC-FID
GC-MS
GC-MS
GC-MS
ICP-AES / ICP-MS
1C
GC-MS
GC-MS
1C
Titrimetry
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
5035A
(EPA SW-846)
8260C
(EPA SW-846)
3541/3545A
(EPA SW-846)
8270D
(EPA SW-846)
3571
(EPA SW-846)
8270D13
(EPA SW-846)
354 1/3545 A
(EPA SW-846)
8270D
(EPA SW-846)
3050B
(EPA SW-846)
6010C/6020A
(EPA SW-846)
Adapted from J. of
Forensic Sciences,
43(1): 200-20214
300.1, Revl.O15
(EPAOW)
3545A
(EPA SW-846)
8270D
(EPA SW-846)
3545A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
Not of concern
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)
3571
(EPA SW-846)
8270D13
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3031
(EPA SW-846)
6010C/6020A
(EPA SW-846)
3580A14
(EPA SW-846)
300.1, Revl.O15
(EPAOW)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
Not of concern
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)
3571
(EPA SW-846)
8270D13
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
200.7/200.8
(EPA OW)
Adapted from J. of
Forensic Sciences,
43(1): 200-20214
300.1, Rev 1.015
(EPAOW)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
Not of concern
3535A
(EPA SW-846)
8270D
(EPA SW-846)
Drinking Water
Samples
5030C
(EPA SW-846)
8260C
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3571
(EPA SW-846)
8270D13
(EPA SW-846)
525.2
(EPAOW)
200.7/200.8
(EPA OW)
Adapted from J. of
Forensic Sciences,
43(1): 200-20214
300.1, Rev 1.015
(EPAOW)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
Not of concern
3535A
(EPA SW-846)
8270D
(EPA SW-846)
Air Samples
1612
(NIOSH)
TO-10A
(EPAORD)
TO-10A6
(EPAORD)
TO-10A
(EPAORD)
10-3.1
(EPAORD)
IO-3.4/IO-3.5
(EPAORD)
ID-211 (OS HA)
TO-1 OA6
(EPAORD)
Not of concern
6004
(NIOSH)
Method 8
(EPA OAQPS)
TO-10A
(EPAORD)
Wipes
Not of concern
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D13
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
9102
(NIOSH)
6010C/6020A
(EPA SW-846)
ID-211 (OS HA)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
Not of concern
3570/8290A Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
SAM Revision 5.0, Appendix A
A-11
September 29, 2009
-------
Analyte(s)
Tetraethyl pyrophosphate
Tetramethylenedisulfotetramine
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)
(degradation product of HN-3)
Trimethyl phosphite
1,3,5-Trinitrobenzene (1,3,5-TNB)
2,4,6-Trinitrotoluene (2,4,6-TNT)
Vanadium pentoxide
(analyze for total vanadium)
CASRN
107-49-3
80-12-6
10031-59-1
111-48-8
39196-18-4
15980-15-1
7550-45-0
102-71-6
121-45-9
99-35-4
118-96-7
1314-62-1
Determinative
Technique
GC-MS
GC-MS
ICP-AES / ICP-MS
HPLC/LC-MS-MS
HPLC
GC-MS
ICP-AES / ICP-MS
HPLC/LC-MS-MS
GC-MS
HPLC
HPLC
ICP-AES / ICP-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
Sample Prep
Determinative
Solid Samples
3545A
(EPASW-846)
8270D
(EPA SW-846)
3541/3545A
(EPA SW-846)
8270D7
(EPASW-846)
3050B
(EPASW-846)
601 OC/6020A
(EPASW-846)
3545A
(EPA SW-846)
832 1B
(EPASW-846)
3541/3545A
(EPA SW-846)
832 1B
(EPA SW-846)
3545A
(EPA SW-846)
8270D16
(EPASW-846)
3050B
(EPA SW-846)
6010C/6020A
(EPA SW-846)
3541/3545A
(EPA SW-846)
832 1B
(EPA SW-846)
3545A
(EPA SW-846)
8270D7
(EPASW-846)
8330B
(EPASW-846)
8330B
(EPA SW-846)
3050 B
(EPA SW-846)
6010C/6020A
(EPA SW-846)
Non-aqueous
Liquid/Organic
Solid Samples1
3580A
(EPASW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
8270D7
(EPASW-846)
3031
(EPA SW-846)
601 OC/6020A
(EPA SW-846)
3580A
(EPA SW-846)
832 1B
(EPA SW-846)
3580A
(EPA SW-846)
832 1B
(EPA SW-846)
3580A
(EPA SW-846)
8270D16
(EPASW-846)
Not of concern
3580A
(EPA SW-846)
832 1B
(EPA SW-846)
3580A
(EPASW-846)
8270D7
(EPASW-846)
8330B
(EPA SW-846)
8330B
(EPA SW-846)
3031
(EPA SW-846)
6010C/6020A
(EPA SW-846)
Aqueous Liquid
Samples
3535A
(EPASW-846)
8270D
(EPASW-846)
3520C/3535A
(EPASW-846)
8270D7
(EPASW-846)
200.7/200.8
(EPAOW)
MS015
(EPACRL)
3520C/3535A
(EPASW-846)
832 1B
(EPASW-846)
3535A
(EPASW-846)
8270D16
(EPASW-846)
Not of concern
MS016
(EPACRL)
3535A
(EPASW-846)
8270D7
(EPASW-846)
3535A/8330B
(EPASW-846)
8330B
(EPASW-846)
3535A/8330B
(EPASW-846)
8330B
(EPASW-846)
200.7/200.8
(EPA OW)
Drinking Water
Samples
3535A
(EPASW-846)
8270D
(EPASW-846)
3520C/3535A
(EPASW-846)
8270D7
(EPASW-846)
200.7/200.8
(EPA OW)
3535A
(EPASW-846)
832 1B
(EPASW-846)
531.2
(EPAOW)
3535A
(EPASW-846)
8270D16
(EPASW-846)
Not of concern
3520C/3535A
(EPASW-846)
832 1B
(EPASW-846)
3535A
(EPASW-846)
8270D7
(EPASW-846)
3535A/8330B
(EPASW-846)
8330B
(EPASW-846)
3535A/8330B
(EPASW-846)
8330B
(EPASW-846)
200.7/200.8
(EPA OW)
Air Samples
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
10-3.1
(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)
Wipes
3570/8290A Appendix A
(EPASW-846)
8270D
(EPASW-846)
3570/8290A Appendix A
(EPASW-846)
8270D7
(EPASW-846)
9102
(NIOSH)
6020A/6010C
(EPASW-846)
3570/8290A Appendix A
(EPASW-846)
832 1B
(EPASW-846)
3570/8290A Appendix A
(EPASW-846)
832 1B
(EPASW-846)
3570/8290A Appendix A
(EPASW-846)
8270D16
(EPASW-846)
9102
(NIOSH)
6010C/6020A
(EPASW-846)
3570/8290A Appendix A
(EPASW-846)
832 1B
(EPASW-846)
3570/8290A Appendix A
(EPASW-846)
8270D7
(EPASW-846)
3570/8290A Appendix A
(EPASW-846)
8330B
(EPASW-846)
3570/8290A Appendix A
(EPASW-846)
8330B
(EPASW-846)
9102
(NIOSH)
6010C/6020A
(EPASW-846)
SAM Revision 5.0, Appendix A
A-12
September 29, 2009
-------
Analyte(s)
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
21738-25-0
78-53-5
21770-86-5
50782-69-9
12185-10-3
Determinative
Technique
GC-MS
GC-MS
GC-MS
GC-MS
GC-NPD/GC-FPD
Method Type
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Solid Samples
354 1/3545 A
(EPASW-846)
8270D
(EPA SW-846)
3541/3545A
(EPA SW-846)
8270D
(EPA SW-846)
3541/3545A
(EPA SW-846)
8270D
(EPA SW-846)
3571
(EPA SW-846)
8270D13
(EPASW-846)
7580
(EPASW-846)
Non-aqueous
Liquid/Organic
Solid Samples1
3580A
(EPASW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3580A
(EPA SW-846)
8270D
(EPA SW-846)
3571
(EPA SW-846)
8270D13
(EPASW-846)
7580
(EPA SW-846)
Aqueous Liquid
Samples
3520C/3535A
(EPASW-846)
8270D
(EPASW-846)
3520C/3535A
(EPASW-846)
8270D
(EPASW-846)
3520C/3535A
(EPASW-846)
8270D
(EPASW-846)
3571
(EPASW-846)
8270D13
(EPASW-846)
7580
(EPASW-846)
Drinking Water
Samples
3520C/3535A
(EPASW-846)
8270D
(EPASW-846)
3520C/3535A
(EPASW-846)
8270D
(EPASW-846)
3520C/3535A
(EPASW-846)
8270D
(EPA SW-846)
3571
(EPASW-846)
8270D13
(EPASW-846)
7580
(EPASW-846)
Air Samples
TO-10A
(EPAORD)
TO-10A
(EPAORD)
TO-10A
(EPAORD)
TO-10A
(EPAORD)
7905
(NIOSH)
Wipes
3570/8290A Appendix A
(EPASW-846)
8270D
(EPASW-846)
3570/8290A Appendix A
(EPASW-846)
8270D
(EPASW-846)
3570/8290A Appendix A
(EPASW-846)
8270D
(EPA SW-846)
3570/8290A Appendix A
(EPASW-846)
8270D13
(EPASW-846)
3570/8290A Appendix A
(EPASW-846)
7580
(EPASW-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-10A 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-10A must be modified to include a derivatization step prior to analysis by GC-MS.
5
For this analyte, SW-846 Method 8270D must be modified to include a derivatization step.
6
If problems occur when using this method, it is recommended that the canister Method TO-15 be used.
7
If problems occur with analyses, lower the injection temperature.
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 NIOSH Method 7906 be used.
11
Laboratory testing is currently under way for speciation of Lewisite 1 using GC-MS techniques.
12 If equipment is not available, use CVAA Methods 7471B (EPA SW-846) for solid samples and 7470A (EPA SW-846) for aqueous liquid samples.
13
For this analyte, refer to EPA SW-846 Method 8271 for GC-MS conditions.
14 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.
If analyses are problematic, refer to column manufacturer for alternate conditions
16 If problems occur when using this method, it is recommended that SW-846 Method 8260C and appropriate corresponding sample preparation procedures (i.e., 5035Afor solid samples, 3585 for non-aqueous
liquid/organic solid samples, and 5030C for aqueous liquid and drinking water samples) be used.
SAM Revision 5.0, Appendix A
A-13
September 29, 2009
-------
SAM Revision 5.0, Appendix A A - 14 September 29, 2009
-------
Appendix B - Selected Radiochemical Methods
Appendix B: Selected Radiochemical Methods
SAM Revision 5.0 September 29, 2009
-------
Appendix B - Selected Radiochemical Methods
SAM Revision 5.0 September 29, 2009
-------
Appendix B: Selected Radiochemical Methods
Analyte Class
Gross Alpha
Gross Beta
Gamma
Select Mixed Fission
Products1
Analyte(s)
Americium-2413
Californium-2523
Cesium-137
Cobalt-60
Curium-2443
Europium-154
lodine-125
lodine-131
lridium-192
CASRN
14596-10-2
13981-17-4
10045-97-3
10198-40-0
13981-15-2
15585-10-1
14158-31-7
10043-66-0
14694-69-0
Determinative
Technique
Alpha/Beta
counting
Alpha/Beta
counting
Gamma
spectrometry
Gamma
spectrometry
Determinative
Technique
Alpha/Gamma
spectrometry
Alpha
spectrometry
Gamma
spectrometry
Gamma
spectrometry
Alpha
spectrometry
Gamma
spectrometry
Gamma
spectrometry
Gamma
spectrometry
Gamma
spectrometry
Drinking Water Samples
900.0 (EPA)
900.0 (EPA)
901.1 (EPA)
901.1 (EPA)
Drinking Water Samples
Qualitative
Determination2
D3084-05
(ASTM)
D3084-05
(ASTM)
901.1
(EPA)
901.1
(EPA)
D3084-05
(ASTM)
901.1
(EPA)
Procedure #9
(ORISE)
901.1
(EPA)
901.1
(EPA)
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)
Procedure #9
(ORISE)
901.1
(EPA)
901.1
(EPA)
Aqueous and Liquid Phase
Samples
7110 B(SM)
7110B(SM)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Aqueous and Liquid Phase
Samples
Qualitative
Determination2
D3084-05
(ASTM)
D3084-05
(ASTM)
7120
(SM)
7120
(SM)
D3084-05
(ASTM)
7120
(SM)
Procedure #9
(ORISE)
Ga-01-R
(HASL-300)
7120
(SM)
Confirmatory
Am-04-RC
(HASL-300)
Am-04-RC
(HASL-300)
7120
(SM)
7120
(SM)
Am-04-RC
(HASL-300)
7120
(SM)
Procedure #9
(ORISE)
Ga-01-R
(HASL-300)
7120
(SM)
Soil and Sediment Samples
AP1
(ORISE)
AP1
(ORISE)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Soil and Sediment Samples
Qualitative
Determination2
Am-02-RC
(HASL-300)
D3084-05
(ASTM)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
D3084-05
(ASTM)
Ga-01-R
(HASL-300)
Procedure #9
(ORISE)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Confirmatory
Am-01-RC4
(HASL-300)
Am-01-RC4
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Am-01-RC4
(HASL-300)
Ga-01-R
(HASL-300)
Procedure #9
(ORISE)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Surface Wipes
FRMAC, Vol2, pg.33
FRMAC, Vol2, pg.33
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Surface Wipes
Qualitative
Determination2
D3084-05
(ASTM)
D3084-05
(ASTM)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
D3084-05
(ASTM)
Ga-01-R
(HASL-300)
Procedure #9
(ORISE)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
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)
Procedure #9
(ORISE)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Air Filters
FRMAC, Vol2, pg.33
FRMAC, Vol2, pg.33
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Air Filters
Qualitative
Determination2
D3084-05
(ASTM)
D3084-05
(ASTM)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
D3084-05
(ASTM)
Ga-01-R
(HASL-300)
Procedure #95
(ORISE)
Ga-01-R5
(HASL-300)
Ga-01-R
(HASL-300)
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)
Procedure #95
(ORISE)
Ga-01-R5
(HASL-300)
Ga-01-R
(HASL-300)
SAM Revision 5.0, Appendix B
B- 1
September 29, 2009
-------
Analyte(s)
Molybdenum-99
Plutonium-2383
Plutonium-2393
Polonium-210
Radium-226
Ruthenium-103
Ruthenium-106
Selenium-75
Strontium-90
Technetium-99
Tritium
(Hydrogen-3)
Uranium-2343
Uranium-2353
CAS RN
14119-15-4
13981-16-3
15117-48-3
13981-52-7
13982-63-3
13968-53-1
13967-48-1
14265-71-5
10098-97-2
14133-76-7
10028-17-8
13966-29-5
15117-96-1
Determinative
Technique
Gamma
spectrometry
Alpha
spectrometry
Alpha
spectrometry
Alpha
spectrometry
Alpha counting /
spectrometry
Gamma
spectrometry
Gamma
spectrometry
Gamma
spectrometry
Beta counting
Liquid
scintillation
Liquid
scintillation
Alpha counting /
spectrometry
Alpha counting /
spectrometry
Drinking Water Samples
Qualitative
Determination2
901.1
(EPA)
D3084-05
(ASTM)
D3084-05
(ASTM)
Po-02-RC
(HASL-300)
903.0
(EPA)
901.1
(EPA)
901.1
(EPA)
901.1
(EPA)
7500-Sr B
(SM)
Tc-02-RC
(HASL-300)
906.0
(EPA)
908. 07
(EPA)
908. 07
(EPA)
Confirmatory
901.1
(EPA)
EMSL-33
(EPA)
EMSL-33
(EPA)
Po-02-RC
(HASL-300)
903.1
(EPA)
901.1
(EPA)
901.1
(EPA)
901.1
(EPA)
7500-Sr B
(SM)
Tc-02-RC
(HASL-300)
906.0
(EPA)
D3972-02
(ASTM)
D3972-02
(ASTM)
Aqueous and Liquid Phase
Samples
Qualitative
Determination2
Ga-01-R
(HASL-300)
D3084-05
(ASTM)
D3084-05
(ASTM)
Po-02-RC
(HASL-300)
7500- Ra B
(SM)
7120
(SM)
7120
(SM)
7120
(SM)
7500-Sr B
(SM)
Tc-02-RC
(HASL-300)
906.0
(EPA)
7500-U B7
(SM)
7500-U B7
(SM)
Confirmatory
Ga-01-R
(HASL-300)
EMSL-33
(EPA)
EMSL-33
(EPA)
Po-02-RC
(HASL-300)
7500- Ra C
(SM)
7120
(SM)
7120
(SM)
7120
(SM)
7500-Sr B
(SM)
Tc-02-RC
(HASL-300)
906.0
(EPA)
7500-U C
(SM)
7500-U C
(SM)
Soil and Sediment Samples
Qualitative
Determination2
Ga-01-R
(HASL-300)
D3084-05
(ASTM)
D3084-05
(ASTM)
Po-02-RC
(HASL-300)
D3084-05
(ASTM)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Sr-03-RC
(HASL-300)
APS
(ORISE)
AP2
(ORISE)
D3084-05
(ASTM)
D3084-05
(ASTM)
Confirmatory
Ga-01-R
(HASL-300)
EMSL-33
(EPA)
EMSL-33
(EPA)
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)
APS
(ORISE)
AP2
(ORISE)
EMSL-33
(EPA)
EMSL-33
(EPA)
Surface Wipes
Qualitative
Determination2
Ga-01-R
(HASL-300)
D3084-05
(ASTM)
D3084-05
(ASTM)
Method 1 1 1
(EPA)
D3084-05
(ASTM)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Sr-03-RC
(HASL-300)
APS
(ORISE)
AP2
(ORISE)
D3084-05
(ASTM)
D3084-05
(ASTM)
Confirmatory
Ga-01-R
(HASL-300)
EMSL-33
(EPA)
EMSL-33
(EPA)
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)
APS
(ORISE)
AP2
(ORISE)
EMSL-33
(EPA)
EMSL-33
(EPA)
Air Filters
Qualitative
Determination2
Ga-01-R
(HASL-300)
D3084-05
(ASTM)
D3084-05
(ASTM)
Method 1 1 1
(EPA)
D3084-05
(ASTM)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Sr-03-RC
(HASL-300)
APS
(ORISE)
Not
applicable6
D3084-05
(ASTM)
D3084-05
(ASTM)
Confirmatory
Ga-01-R
(HASL-300)
EMSL-33
(EPA)
EMSL-33
(EPA)
Method 111
(EPA)
EMSL-19
(EPA)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Sr-03-RC
(HASL-300)
APS
(ORISE)
Not
applicable6
EMSL-33
(EPA)
EMSL-33
(EPA)
SAM Revision 5.0, Appendix B
B-2
September 29, 2009
-------
Analyte(s)
Uranium-2383
CAS RN
7440-61-1
Determinative
Technique
Alpha counting /
spectrometry
Drinking Water Samples
Qualitative
Determination2
908. 07
(EPA)
Confirmatory
D3972-02
(ASTM)
Aqueous and Liquid Phase
Samples
Qualitative
Determination2
7500-U B7
(SM)
Confirmatory
7500-U C
(SM)
Soil and Sediment Samples
Qualitative
Determination2
D3084-05
(ASTM)
Confirmatory
EMSL-33
(EPA)
Surface Wipes
Qualitative
Determination2
D3084-05
(ASTM)
Confirmatory
EMSL-33
(EPA)
Air Filters
Qualitative
Determination2
D3084-05
(ASTM)
Confirmatory
EMSL-33
(EPA)
Footnotes
1
Please note that this category does not cover all fission products.
2 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.
3 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.
4 In cases where only small sample volumes (£1°° 9) are available, use HASL-300 Method Pu-12-RC.
5
This procedure should be used only for filters specifically designed for iodine.
6
Because tritium is not sampled using traditional air filters, this matrix is not applicable.
7
This method was developed for measurement of total uranium and does not distinguish between uranium isotopes.
SAM Revision 5.0, Appendix B
B-3
September 29, 2009
-------
SAM Revision 5.0, Appendix B B - 4 September 29, 2009
-------
Appendix C - Selected Pathosen Methods
Appendix C: Selected Pathogen Methods
SAM Revision 5.0 September 29, 2009
-------
Appendix C - Selected Pathosen Methods
SAM Revision 5.0 September 29, 2009
-------
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, dust
socks)
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
[Campy lobacteriosis]
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
Public Health Reports
92(2): 176-1 86
Public Health
Reports 92(2):
176-186
Requires
modification of
analytical method
Public Health
Reports 92(2):
176-186
Public Health
Reports 92(2):
176-186
Requires
modification of
analytical method
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(5): 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 5.0, Appendix C
C-1
September 29, 2009
-------
Pathogen(s)
[Disease]
Chlamydophila psittaci
( formerly known as Chlamydia psittaci)
[Psittacosis]
Coxiella bumetii
[Q-fever]
Escherichia coli O157:H7
Francisella tularensis
[Tularemia]
Leptospira
(L. interrogans Serovars
Icteroheamorrhagiae, Autralis, Balum,
Bataviae, Bejro, Pomona)
[Leptospirosis]
Analytical
Technique
Tissue culture
PCR
Culture
Real-time PCR/
Immunoassay
Culture
Immunoassay
Real-time PCR
Culture
Real-time PCR/
Immunoassay
Culture
Immunoassay
Real-time PCR
Analytical
Method
Journal of Clinical
Microbiology
38(3): 1085-1 093
Journal of Clinical
Microbiology
38(3): 1085-1093
Solid1
(soil, powder)
Requires
modification of
analytical method
Requires
modification of
analytical method
Particulate2
(swabs, wipes, dust
socks)
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
Diagnostic Microbiology
and Infectious Disease
64(3): 247-255
SM 9260 I
SM 9260 I
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
SM 9260 I
SM 9260 I
Requires
modification of
analytical method
Unlikely to be viable
Unlikely to be viable
Requires
modification of
analytical method
SAM Revision 5.0, Appendix C
C-2
September 29, 2009
-------
Pathogen(s)
[Disease]
Listeria monocytogenes
[Listeriosis]
Non-typhoidal Salmonella
(Not applicable to S. Typhi)
[Salmonellosis]
Salmonella Typhi
[Typhoid fever]
Shigella spp.
[Shigellosis]
Staphylococcus aureus
Analytical
Technique
Culture
Immunoassay
Real-time PCR
Culture
Immunoassay
Real-time PCR
Culture
Immunoassay
Real-time PCR
Culture
Immunoassay
Real-time PCR
Culture
Analytical
Method
FDA/Bacteriological
Analytical Manual
Chapter 10, 2003
FDA/Bacteriological
Analytical Manual
Chapter 10, 2003
US DA Laboratory
Guidebook
MLG 8A.03
EPA Method 1682
EPA Method 1682
Journal of Applied
Microbiology
102(2): 51 6-530
SM 9260 B
SM 9260 B
CDC Laboratory Assay:
S. Typhi
SM 9260 E
SM 9260 E
CDC Laboratory Assay:
Shigella
SM9213B
Solid1
(soil, powder)
FDA/Bacteriological
Analytical Manual
Chapter 10, 2003
FDA/Bacteriological
Analytical Manual
Chapter 10, 2003
Requires
modification of
analytical method
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
Particulate2
(swabs, wipes, dust
socks)
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
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
SM9213B
Drinking Water
(filter, grab)
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 B
SM 9260 B
Requires
modification of
analytical method
SM 9260 E
SM 9260 E
Requires
modification of
analytical method
SM9213B
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
Unlikely to be viable
Unlikely to be viable
Requires
modification of
analytical method
Requires
modification of
analytical method
SAM Revision 5.0, Appendix C
C-3
September 29, 2009
-------
Pathogen(s)
[Disease]
Vibrio cholerae O1 and O1 39
[Cholera]
Yersinia pestis
[Plague]
Analytical
Technique
Culture
Immunoassay
Real-time PCR
Culture
Real-time PCR/
Immunoassay
Analytical
Method
SM 9260 H
SM 9260 H
CDC Laboratory Assay:
V. cholerae
Solid1
(soil, powder)
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Particulate2
(swabs, wipes, dust
socks)
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
Liquid/Water
(filter, grab)
SM 9260 H
SM 9260 H
Requires
modification of
analytical method
Drinking Water
(filter, grab)
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
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
Coronaviruses:
SARS-associated human coronavirus
Hepatitis E virus (HEV)
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
Applied &
Environmental
Microbiology 71 (6):
3131-3136
Applied &
Environmental
Microbiology 71(6):
3131-3136
Canadian Journal of
Microbiology 50(4):
269-278
Canadian Journal of
Microbiology 50(4):
269-278
Journal of Clinical
Microbiology 42(10):
4679-4685
Journal of Medical
Virology 78(10):
1347-1353
Journal of Virological
Methods 122(1): 29-36
Journal of Virological
Methods 131(1): 65-71
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(1 0): 4679-4685"
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 5.0, Appendix C
C-4
September 29, 2009
-------
Pathogen(s)
[Disease]
Influenza H5N1 virus
Picornaviruses: Enteroviruses
Picornaviruses:
Hepatitis A virus (HAV)
Reovi ruses:
Rotavirus (Group A)
Analytical
Technique
Real-time reverse
transcription-PCR
Tissue culture
Reverse
transcription-PCR
Reverse transcription-
PCR
Tissue culture
Real-time reverse
transcription-PCR
Analytical
Method
Emerging Infectious
Diseases 11 (8):
1303-1305
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
Journal of Virological
Methods 155(2):
126-131
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
Requires
modification of
analytical method4
Particulate2
(swabs, wipes, dust
socks)
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
Liquid/Water
(filter, grab)
Requires
modification of
analytical method4
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)
Requires
modification of
analytical method4
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
Journal of Virological
Methods
155(2): 126-131
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
Requires
modification of
analytical method4
Protozoa
Cryptosporidium spp.
[Cryptosporidiosis]
Tissue culture
IMS/FA
Real-time PCR
Applied &
Environmental
Microbiology 65(9):
3936-3941
EPA Method 1622
and/or Method 1623
Applied &
Environmental
Microbiology 73(13):
4218-4225
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 1623
Requires
modification of
analytical method
Requires
modification of
analytical method
EPA Method 1622
and/or Method 1623
Applied &
Environmental
Microbiology 73(13):
4218-4225
Unlikely to be found
Unlikely to be found
Unlikely to be found
SAM Revision 5.0, Appendix C
C-5
September 29, 2009
-------
Pathogen(s)
[Disease]
Entamoeba histolytica
Giardiaspp.
[Giardiasis]
Toxoplasma gondii
[Toxoplasmosis]
Analytical
Technique
Culture
Real-time PCR
Culture
IMS/FA
Animal infectivity
Real-time PCR
Analytical
Method
Journal of Parasitology
58(2): 306-310
Journal of Clinical
Microbiology
43(11): 5491 -5497
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
Requires
modification of
analytical method
Requires
modification of
analytical method
Particulate2
(swabs, wipes, dust
socks)
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
EPA Method 1623
Emerging Infectious
Diseases 12(2):
326-329
Applied &
Environmental
Microbiology 70(7):
4035-4039
Drinking Water
(filter, grab)
Requires
modification of
analytical method
Requires
modification of
analytical method
Requires
modification of
analytical method
EPA Method 1623
Emerging Infectious
Diseases 12(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
Unlikely to be found
Unlikely to be found
Helminths
Baylisascaris procyonis
[Raccoon roundworm infection]
Embryonation of eggs
and microscopy
EPA/625/R-92/01 3
EPA/625/R-92/013
Requires
modification of
analytical method
EPA/625/R-92/013
EPA/625/R-92/013
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 5.0, Appendix C
C-6
September 29, 2009
-------
Appendix D - SelectedBiotoxin Methods
Appendix D: Selected Biotoxin Methods
SAM Revision 5.0 September 29, 2009
-------
Appendix D - SelectedBiotoxin Methods
SAM Revision 5.0 September 29, 2009
-------
Appendix D: Selected Biotoxin Methods
Note: The presence of disinfectants (e.g., chlorine) and/or preservatives added during water sample collection to slow degradation (e.g., pH adjusters, de-chlorinating agents)
could possibly affect analytical results. When present, the impact of these agents on method performance should be evaluated if not previously determined.
Analyte(s)
Protein
Abrin
Botulinum neurotoxins
(Serotoypes A, B, E, F)
Ricin
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 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
chain and -34 kDa B
chain); an agglutinin of
MW 120 kDa maybe
present in crude
preparations
5254-40-3 (ricinine) /
small molecule, ricin
marker
Analysis Type
Presumptive
Confirmatory
Biological
Activity
Presumptive
Confirmatory
Biological
Activity
Presumptive
Complementary
Presumptive
(ricinine)
Confirmatory
Biological
Activity
Analytical
Technique
Immunoassay1
Ribosome
inactivation
assay
Enzyme activity2
Immunoassay3
Immunoassay3
(ELISA)
Mouse Bioassay
Immunoassay1
LC-MS
Immunoassay
Enzyme activity2
Aerosol
(filter/cassette, liquid
impinger)
Adapted from Journal
of Food Protection
71(9): 1868-1874
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
Solid
(soil, powder)
Adapted from Journal
of Food Protection
71(9): 1868-1874
Adapted from
Pharmacology &
Toxicology
88(5): 255-260
Adapted from
Analytical
Biochemistry
378(1): 87-89
Particulate
(swabs, wipes, dust
socks)
Adapted from Journal
of Food Protection
71(9): 1868-1874
Adapted from
Pharmacology &
Toxicology
88(5): 255-260
Adapted from
Analytical
Biochemistry
378(1): 87-89
Liquid Water
Adapted from Journal
of Food Protection
71(9): 1868-1874
Adapted from
Pharmacology &
Toxicology
88(5): 255-260
Adapted from
Analytical
Biochemistry
378(1): 87-89
Drinking Water
Adapted from Journal
of Food Protection
71(9): 1868-1874
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 8.1 .4.
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 8.1 .4.
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 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 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 Analytical
Toxicology
29(3): 149-155
Adapted from Journal
of AOAC International
91(2): 376-382
Adapted from
Analytical
Biochemistry
378(1): 87-89
SAM Revision 5.0, Appendix D
D- 1
September 29, 2009
-------
Analyte(s)
Shiga and Shiga-like Toxins
(Stx, Stx-1, Stx-2)
Staphylococcal enterotoxins
(SEE)
Staphylococcal enterotoxins
(SEA, SEC)
CAS RN /
Description
75757-64-1 (Stx) /
Protein composed of
one -32 kDa A chain
and five 7.7 kDa B
chains
39424-53-8 (SEE) /
Monomeric protein of
~ 28 kDa
37337-57-8 (SEA)
39424-54-9 (SEC) /
Monomeric proteins of
Analysis Type
Presumptive
Confirmatory
Biological
Activity
Presumptive
Confirmatory
Biological
Activity
Presumptive
Confirmatory
Biological
Activity
Analytical
Technique
Optical
immunoassay
Immunoassay
(ELISA)
Ribosome
inactivation
assay2
Immunoassay
TBD
TBD
Immunoassay
TBD
TBD
Aerosol
(filter/cassette, liquid
impinger)
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 993.06
(AOAC)
TBD
TBD
Adapted from 993.06
(AOAC)
TBD
TBD
Solid
(soil, powder)
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
Particulate
(swabs, wipes, dust
socks)
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
Liquid Water
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
Drinking Water
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
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 8.1 .4.
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
TBD
TBD
Adapted from 993.06
(AOAC)
TBD
TBD
Small Molecule
Aflatoxin
(Type B1)
a-Amanitin
27261-02-5
23109-05-9
Presumptive
Confirmatory
Presumptive
Confirmatory
Immunoassay
HPLC-FL
Immunoassay
HPLC
amperometric
detection
Adapted from 991. 31
(AOAC)
Adapted from 991. 31
(AOAC)
Adapted from Journal
of Food Protection
68(6): 1294-1301
Adapted from Journal
of Chromatography
563(2): 299-3 11
Adapted from 991. 31
(AOAC)
Adapted from 991. 31
(AOAC)
Adapted from Journal
of Food Protection
68(6): 1294-1301
Adapted from Journal
of Chromatography
563(2): 299-31 1
Adapted from 991. 31
(AOAC)
Adapted from 991. 31
(AOAC)
Adapted from Journal
of Food Protection
68(6): 1294-1301
Adapted from Journal
of Chromatography
563(2): 299-31 1
Adapted from 991. 31
(AOAC)
Adapted from 991. 31
(AOAC)
Adapted from Journal
of Food Protection
68(6): 1294-1301
Adapted from Journal
of Chromatography
563(2): 299-31 1
Adapted from 991. 31
(AOAC)
Adapted from 991. 31
(AOAC)
Adapted from Journal
of Food Protection
68(6): 1294-1301
Adapted from Journal
of Chromatography
563(2): 299-31 1
SAM Revision 5.0, Appendix D
D-2
September 29, 2009
-------
Analyte(s)
Anatoxin-a
Brevetoxins
(B form)
a-Conotoxin
Cylindrospermopsin
Diacetoxyscirpenol (DAS)
Microcystins
Principal isoforms: LA, LR, LW,
RR, YR
Picrotoxin
CAS RN /
Description
64285-06-9
79580-28-2
156467-85-5
143545-90-8
2270-40-8
961 80-79-9 (LA)
101 043-37-2 (LR)
157622-02-1 (LW)
11 1755-37-4 (RR)
101 064-48-6 (YR)
124-87-8
Analysis Type
Presumptive
Confirmatory
Presumptive
Confirmatory
Presumptive
Confirmatory
Presumptive
Confirmatory
Presumptive
Confirmatory
Presumptive
Confirmatory
Presumptive
Confirmatory
Analytical
Technique
TBD
HPLC-FL
(precolumn
derivatization)
Immunoassay
HPLC-MS-MS
Immunoassay
HPLC-MS
Immunoassay
HPLC-PDA
Immunoassay
LC/APCI-MS
Immunoassay/
Phosphatase
assay
HPLC-PDA
Immunoassay
HPLC
Aerosol
(filter/cassette, liquid
impinger)
TBD
Adapted from
Biomedical
Chromatography B
10(1): 46-47
Adapted from
Environmental Health
Perspectives
110(2): 179-185
Adapted from Toxicon
43(4): 455-465
Adapted from
Biochemical Journal
328(1): 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 Spectrometry
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
Solid
(soil, powder)
TBD
Adapted from
Biomedical
Chromatography B
10(1): 46-47
Adapted from
Environmental Health
Perspectives
110(2): 179-185
Adapted from Toxicon
43(4): 455-465
Adapted from
Biochemical Journal
328(1): 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 Spectrometry
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
Particulate
(swabs, wipes, dust
socks)
TBD
Adapted from
Biomedical
Chromatography B
10(1): 46-47
Adapted from
Environmental Health
Perspectives
110(2): 179-185
Adapted from Toxicon
43(4): 455-465
Adapted from
Biochemical Journal
328(1): 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 Spectrometry
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
Liquid Water
TBD
Adapted from
Biomedical
Chromatography B
10(1): 46-47
Adapted from
Environmental Health
Perspectives
110(2): 179-185
Adapted from Toxicon
43(4): 455-465
Adapted from
Biochemical Journal
328(1): 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 Spectrometry
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
Drinking Water
TBD
Adapted from
Biomedical
Chromatography B
10(1): 46-47
Adapted from
Environmental Health
Perspectives
110(2): 179-185
Adapted from Toxicon
43(4): 455-465
Adapted from
Biochemical Journal
328(1): 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 Spectrometry
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
SAM Revision 5.0, Appendix D
D-3
September 29, 2009
-------
Analyte(s)
Saxitoxins
Principal isoforms:
Saxitoxin (SIX)
Neosaxitoxin (NEOSTX)
Gonyautoxin (GTX)
Decarbamoylgonyautoxin (dcGTX)
Decarbamoylsaxitoxin (dcSTX)
T-2 Mycotoxin
Tetrodotoxin
CAS RN /
Description
35523-89-8 (SIX)
64296-20-4 (NEOSTX)
77462-64-7 (GTX)
None given (dcGTX)
58911 -04-9 (dcSTX)
21259-20-1
9014-39-5
Analysis Type
Presumptive
Confirmatory
Presumptive
Confirmatory
Presumptive
Confirmatory
Analytical
Technique
Immunoassay
HPLC-FL
(post column
derivatization)
Immunoassay
LC/APCI-MS
Immunoassay
LC/ESI-MS
Aerosol
(filter/cassette, liquid
impinger)
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 Spectrometry
20(9): 1422-1428
Adapted from Journal
of Clinical Laboratory
Analysis 6(2): 65-72
Adapted from
Analytical
Biochemistry
290(1): 10-17
Solid
(soil, powder)
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 Spectrometry
20(9): 1422-1428
Adapted from Journal
of Clinical Laboratory
Analysis 6(2): 65-72
Adapted from
Analytical
Biochemistry
290(1): 10-17
Particulate
(swabs, wipes, dust
socks)
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 Spectrometry
20(9): 1422-1428
Adapted from Journal
of Clinical Laboratory
Analysis 6(2): 65-72
Adapted from
Analytical
Biochemistry
290(1): 10-17
Liquid Water
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 Spectrometry
20(9): 1422-1428
Adapted from Journal
of Clinical Laboratory
Analysis 6(2): 65-72
Adapted from
Analytical
Biochemistry
290(1): 10-17
Drinking Water
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 Spectrometry
20(9): 1422-1428
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 5.0, Appendix D
D-4
September 29, 2009
-------
-------
vvEPA
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development
National Homeland Security Research Center
Cincinnati, OH 45268
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