&EPA
United States
Environmental Protection
Agency
EPA/600/R-12/555 July 16, 2012
Selected Analytical Methods
for Environmental
Remediation and Recovery
[SAM)-2012
Office of Research and Development
National Homeland Security Research Center
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EPA/600/R-12/555 | July 2012 | www.epa.gov/sam
Selected Analytical Methods for
Environmental Remediation and
Recovery (SAM) 2012
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
Cincinnati, OH 45268
I Office of Research and Development
| National Homeland Security Research Center
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Disclaimer
Disclaimer
The U.S. Environmental Protection Agency (EPA) through its Office of Research and Development
funded and managed the research described here under Contract EP-C-10-060 to Computer Sciences
Corporation (CSC). This document has been subjected to the Agency's review and has been approved for
publication. The contents of this document reflect the views of the contributors and technical work
groups and do not 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:
Kathy Hall
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)379-5260
hall .kathyigjepa. gov
SAM 2012 ii July 16, 2012
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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 selected analytical methods for use by those laboratories tasked with
performing confirmatory analyses of environmental samples in support of EPA remediation and
recovery efforts following a homeland security incident. SAM is intended for use by EPA and EPA-
contracted and -subcontracted laboratories; it also can be used by other agencies and laboratory
networks, such as the Integrated Consortium of Laboratory Networks (ICLN). The information also
can be found on the SAM website (www.epa.gov/sam'), which provides searchable links to
supporting information based on SAM analytes and the analytical methods listed.
At this time, only some of the methods selected have been validated for the listed analyte and
sample type. However, the methods are considered to contain the most appropriate currently
available techniques based on expert judgment. 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 evaluation is needed, and adjustments may be required to
accurately account for variations in analyte/sample type characteristics, environmental samples,
analytical interferences, and data quality objectives (DQOs).
EPA will strive to continue development and evaluation of analytical protocols, including
optimization of procedures for measuring target analytes or agents in specific sample types, as
appropriate. In those cases where method procedures are determined to be insufficient for a
particular situation, NHSRC will continue to provide technical support regarding appropriate
actions. NHSRC has also compiled information and published documents regarding field
screening equipment, sample collection materials, rapid screening/ preliminary identification
equipment, and disposal of samples corresponding to SAM analytes and sample types. These
documents are available at www.epa.gov/sam/samcomp.htm.
SAM 2012 iii July 16, 2012
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Abbreviations and Acronyms
ACS
amp-ELISA
APCI
APHA
APHL
AOAC
API
ASM
ASR
ASTM
AWWA
BAM
BGMK
BHT
BMBL
BSL
BZ
ฐC
CASRN
CBR
CCID
CDC
CFR
CFSAN
CIEIA
CLLE
CLP
CPE
cps
CT
CVAA
2-CVAA
CVAFS
CWA
2,4-D
DAPI
DAS
DAS-HG-HSA
DAS-HS-HRP
DB-1
DBPR
DHS
DIG
DIG-ELISA
DIMP
DL
DNA
2,4-DNPH
DoD
DOE
Abbreviations and Acronyms
American Chemical Society
Amplified-enzyme-linked immunosorbent assay
Atmospheric Pressure Chemical lonization
American Public Health Association
Association of Public Health Laboratories
AOAC International (formerly the Association of Official Analytical Chemists)
Atmospheric pressure ionization
American Society for Microbiology
Analytical Service Requests
ASTM International (formerly the American Society for Testing and Materials)
American Water Works Association
Bacteriological Analytical Manual
Buffalo green monkey kidney
Butylated hydroxytoluene
Biosafety in Microbiological and Biomedical Laboratories
Biosafety level
Quinuclidinyl benzilate
Degree Celsius
Chemical Abstracts Service Registry Number
Chemical, biological and/or radiological
Coordinating Center for Infectious Diseases
Centers for Disease Control and Prevention
Code of Federal Regulations
Center for Food Safety and Applied Nutrition
Competitive inhibition enzyme immunoassay
Continuous liquid-liquid extraction
Contract Laboratory Program
Cytopathic effect
Counts per second
Cycle threshold
Cold vapor atomic absorption
2-Chlorovinylarsonous acid
Cold vapor atomic fluorescence spectrometry
Chemical Warfare Agent
2,4-Dichlorophenoxyacetic acid
4',6-Diamidino-2-phenylindole
Diacetoxyscirpenol
Diacetoxyscirpenol hemiglutarate human serum albumin
Diacetoxyscirpenol hemisuccinate horseradish peroxidase conjugate
100% Dimethylpolysiloxane
Division of Bioterrorism Preparedness and Response
U.S. Department of Homeland Security
Differential interference contrast
Digoxigenin labeled enzyme-linked immunosorbent assay
Diisopropyl methylphosphonate
Detection limit
Deoxyribonucleic acid
2,4-Dinitrophenylhydrazine
U.S. Department of Defense
U.S. Department of Energy
SAM 2012
IV
July 16, 2012
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Abbreviations and Acronyms
DOT U.S. Department of Transportation
DPD N,N-Diethyl-/>-phenylenediamine
DQO Data quality objective
DTPA Diethylenetriamine-pentaacetate
DVL Detection verification level
EA2192 S-2-(Diisopropylamino)ethyl methylphosphonothioic acid
BCD Electron capture detector
e-CFR Electronic Code of Federal Regulations
ECL Electrochemiluminescence
ED Ethyldichloroarsine
EDC l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
EDEA N-Ethyldiethanolamine
EDL Estimated detection limit
EDTA Ethylenediaminetetraacetic acid
EDXA Energy dispersive X-ray analysis
EIA Enzyme immunoassay
ELISA Enzyme-Linked Immunosorbent Assay
EMC Emission Measurement Center
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
ERLN Environmental Response Laboratory Network
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
FGC-ECD Fast gas chromatography with electron capture detection
FGI Fluorescein derivative of Conus geographus a-conotoxin
FID Flame ionization detector
FL Fluorescence detector
FPD Flame photometric detector
FRET Fluorescence resonance energy transfer
FRhK-4 Fetal rhesus monkey kidney
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-NPD Gas chromatography-nitrogen-phosphorus detector
GD Soman
GE 1-Methylethyl ester ethylphosphonofluoridic acid
Ge Germanium
Ge(Li) Germanium (Lithium)
SAM 2012
July 16, 2012
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Abbreviations and Acronyms
GESTIS
GF
GFAA
HASL
HAV
HCoV
HEV
HD
HFBA
HFBI
HHS
HLB
HMTD
HMX
HN-1
HN-2
HN-3
HP(Ge)
HPLC
HPLC-FL
HPLC-MS
HPLC-MS-MS
HPLC-PDA
HPLC-TSP-MS
HPLC-UV
HPLC-vis
HRP
HTO
HV
IBRD
1C
1C 20
1C 50
ICLN
ICP
ICP-AES
ICP-MS
ICR
IDL
IFA
ILM
IMPA
IMS
INCHEM
A German database (Gefahrstoffdatenbanken) containing data and information on
hazardous substances and products
Cyclohexyl sarin
Graphite furnace atomic absorption spectrophotometer or Graphite furnace atomic
absorption spectrophotometry
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
Heptafluorobutyric anhydride
Heptafluorobutyrylimidazole
U.S. Health and Human Services
Hydrophilic-lipophilic-balanced
Hexamethylenetriperoxidediamine
Octahydro-l,3,5,7-tetranitro-l,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 spectrometry
High performance liquid chromatography tandem mass spectrometry
High performance liquid chromatography-photodiode array detector
High performance liquid chromatography-thermospray-mass spectrometry
High performance liquid chromatography-ultraviolet
High performance liquid chromatography-visible
Horseradish peroxidase
Tritiated water
High volume
Interagency Biological Restoration Demonstration
Ion chromatograph or Ion chromatography
Inhibitory concentration - Concentration to inhibit 20%
Inhibitory concentration - Concentration to inhibit 50%
Integrated Consortium of Laboratory Networks
Intestinal contents preparation (pathogens); Inductively coupled plasma (chemistry)
Inductively coupled plasma - atomic emission spectrometry
Inductively coupled plasma - mass spectrometry
Information Collection Requirements Rule
Instrument detection limit
Immunofluorescence
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://wwwinchem.org/
SAM 2012
VI
July 16, 2012
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Abbreviations and Acronyms
IO Inorganic
i.p. Intraperitoneally
IRIS Integrated Risk Information System (EPA)
ISE Ion specific electrode
ISO Impregnated silica gel
ISO International Organization for Standardization
KHP Potassium hydrogen phthalate
L-l Lewisite 1; 2-Chlorovinyldichloroarsine
L-2 Lewisite 2; bis(2-Chlorovinyl)chloroarsine
L-3 Lewisite 3; tris(2-Chlorovinyl)arsine
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
LCMRL Lowest common minimum reporting level
LC-MS Liquid chromatography-mass spectrometry
LC-MS-MS Liquid chromatography tandem mass spectrometry
LC-TSP Liquid chromatography-thermospray
LFD Lateral flow device
LLD Lower limit of detection
LOD Limit of detection
LOQ Limit of quantitation
LRN Laboratory Response Network
LSC Liquid scintillation counter
LSE Liquid-solid extraction
Ltd. A private company limited by shares
M Molar
mAbs Monoclonal antibodies
MAE Microwave-assisted extraction
MALDI Matrix-assisted laser-desorption ionization
MARLAP Multi-Agency Radiological Laboratory Analytical Protocols (EPA/402/B-04/001 A,
B, C)
MDEA N-Methyldiethanolamine
MDL Method detection limit
MFA Monofluoroacetate
MIC Methyl isocyanate
MLD Minimum lethal dose
MPA Methylphosphonic acid
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
MTBE Methyl fert-butyl ether
MW Molecular weight
NA Not applicable
Nal(Tl) Thallium-activated sodium iodide
NAREL National Air and Radiation Environmental Laboratory
NBD chloride 7-Chloro-4-nitrobenzo-2-oxa-l,3-diazole
NBD-F 7-Fluoro-4-nitro-2,l,3-benzoxadiazole
NCPDCID National Center for the Prevention, Detection, and Control of Infectious Diseases
SAM 2012
VII
July 16, 2012
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Abbreviations and Acronyms
NCRP
NEMI
NERL
NHSRC
NIOSH
NIST
nM
NMAM
NNSA
NPD
NRC
NRMRL
nS
NTIS
NTU
OAQPS
OAR
ORAU
ORD
ORIA
ORISE
OSWER
OSHA
OVS
OW
PBS
PCDDs
PCDFs
PCR
PDA
PEL
PETN
PFBHA
PFE
PMPA
1,2-PP
ppbv
pptv
PTFE
PubMED
PUF
PVC
PVDF
QA
QAP
QC
ฎ
R33
RCRA
RDX
RESL
RLAB
National Council on Radiation Protection and Measurements
National Environmental Methods Index
National Exposure Research Laboratory (EPA)
National Homeland Security Research Center (EPA)
National Institute for Occupational Safety and Health
National Institute of Standards and Technology
Nanomolar
NIOSH Manual of Analytical Methods
National Nuclear Security Administration
Nitrogen-phosphorus detector
U.S. Nuclear Regulatory Commission
National Risk Management Research Laboratory (EPA)
Nano Siemens
National Technical Information Service
Nephelometric turbidity units
Office of Air Quality Planning and Standards (EPA)
Office of Air and Radiation (EPA)
Oak Ridge Associated Universities
Office of Research and Development (EPA)
Office of Radiation and Indoor Air (EPA)
Oak Ridge Institute for Science and Education
Office of Solid Waste and Emergency Response (EPA)
Occupational Safety and Health Administration
OSHA versatile sampler
Office of Water (EPA)
Phosphate buffered saline
Polychlorinated dibenzo-/?-dioxins
Polychlorinated dibenzofurans
Polymerase chain reaction
Photodiode array detector
Permissible exposure limit
Pentaerythritol tetranitrate
O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine
Pressurized fluid extraction
Pinacolyl methyl phosphonic acid
1 -(2-Pyridyl)piperazine
Parts per billion by volume
Parts per trillion by volume
Polytetrafluoroethylene
U.S. National Library of Medicine (http: //www .pubmed. gov)
Polyurethane foam
Polyvinyl chloride
Polyvinylidene fluoride
Quality assurance
Quality assessment program
Quality control
Registered trademark
Methylphosphonothioic acid, S-[2-(diethylamino)ethyl] O-2-methylpropyl ester (VR)
Resource Conservation and Recovery Act
Hexahydro-1,3,5-trinitro-1,3,5-triazine
Radiological and Environmental Sciences Laboratory
Regional laboratory method
SAM 2012
Vlll
July 16, 2012
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Abbreviations and Acronyms
RNA Ribonucleic acid
RTECS Registry of Toxic Effects of Chemical Substances
RV-PCR Rapid Viability-Polymerase Chain Reaction
SAED Select area electron diffraction
SAM Selected Analytical Methods for Environmental Remediation and Recovery
SARS Severe Acute Respiratory Syndrome
SEA Staphylococcal enterotoxin type A
SEE Staphylococcal enterotoxin type B
SEC Staphylococcal enterotoxin type C
SIM Selective ion monitoring
SM Standard Methods for the Examination of Water and Wastewater
sMac sorbitol-MacConkey's
SPE Solid-phase extraction
SRC Syracuse Research Corporation
SRM Single reaction monitoring
SRS Savannah River National Laboratory, Savannah River Site
STEC Shiga-toxigenic E. coll
STEL Short term exposure limit
STX Saxitoxin
Stx-1 Shiga toxin Type 1
Stx-2 Shiga toxin Type 2
SW Solid Waste
TBD To be determined
TCLP Toxicity Characteristic Leaching Procedure
TDG Thiodiglycol
TEA Triethanolamine
TEM Transmission electron microscope or Transmission electron microscopy
TEPP Tetraethylpyrophosphate
TETS Tetramethylenedisulfotetramine or tetramine
TFA Trifluoroacetic acid
THF Tetrahydrofuran
Unregistered trademark
1,3,5-TNB 1,3,5-Trinitrobenzene
2,4,6-TNT 2,4,6-Trinitrotoluene
T2O Tritium oxide
TO Toxic Organic
TOP Time-of-flight
TOXNET Toxicology Data Network
TRU Transuranic
TSP Thermospray
TTN Technical Transfer Network
TTX Tetrodotoxin
U.S. United States
USDA U.S. Department of Agriculture
USGS U.S. Geological Survey
UV Ultraviolet
VBNC Viable but non-culturable
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
SAM2012
IX
July 16, 2012
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Abbreviations and Acronyms
VOC Volatile organic compound
VR Methylphosphonothioic acid, S-[2-(diethylamino)ethyl] O-2-methylpropyl ester (R
33)
VX O-Ethyl-S-(2-diisopropylaminoethyl)methylphosphonothiolate
WCIT Water Contaminant Information Tool
WEF Water Environment Federation
WLA Water Laboratory Alliance
WHO World Health Organization
WSD Water Security Division (EPA, Office of Water)
SAM 2012 x July 16, 2012
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Acknowledgments
Acknowledgments
Contributions of the following individuals and organizations to the development of SAM 2012 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
(NHSRC)
Joan Bursey (Senior Environmental Employment Program Grantee)
Hiba Ernst
Kathy Hall
Romy Lee
Alan Lindquist
Matthew Magnuson
Tonya Nichols
Eugene Rice
Frank Schaefer
Sanjiv Shah
Erin Silvestri
Emily Snyder
Stuart Willison
Office of Air and Radiation, Office of Radiation and Indoor Air (ORIA)
John Griggs
Daniel Mackney
Office of Solid Waste and Emergency Response, Office of Emergency Management (OEM)
Schatzi Fitz-James
Scott Hudson
Lawrence Kaelin
Terry Smith
Office of Water, Office of Ground Water and Drinking Water (OGWDW)
Pamela Barnes (Water Security Division)
Elizabeth Hedrick (Water Security Division)
Ouida Holmes (Water Security Division)
James Sinclair (Technical Support Center)
Office of Research and Development, National Exposure Research Laboratory (NERL)
Jennifer Cashdollar
Ann Grimm
Gerard Stelma
Office of Research and Development, National Health and Environmental Effects Research
Laboratory (NHEERL)
Denise Macmillan
Office of Pesticide Programs (OPP)
Chuck Stafford
SAM 2012 xi July 16, 2011
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Acknowledgments
EPA Regions
Jack Berges (Region 9)
Diane Gregg (Region 6)
Stephanie Harris (Region 10)
Steve Reimer (Region 10)
Sue Warner (Region 3)
Dennis Wesolowski (Region 5)
Larry Zintek (Region 5)
United States Department of Commerce (DOC)
Peter Moeller (National Oceanic & Atmospheric Administration)
United States Department of Defense (DoD)
Bob Durgin (U.S. Army, Chemical Materials Agency)
Marty Johnson (U.S. Army, Radiation Standards Laboratory)
Elaine Strauss (U.S. Navy, Naval Surface Warfare Center Dahlgren Division)
Dick Ward (U.S. Army, Chemical Materials Agency)
United States Department of Energy (DOE)
Sherrod Maxwell (Savannah River Site)
United States Department of Health and Human Services (DHHS)
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)
Tambra Dunams (National Center for Environmental Health)
Jay Gee (National Center for Zoonotic and Emerging Infectious Diseases)
Vincent Hill (National Center for Zoonotic, Vector-Borne, and Enteric Diseases)
Jennifer Links (National Center for Environmental Health)
Stephen Morse (National Center for Preparedness, Detection and Control of Infectious Diseases)
Laura Rose (National Center for Preparedness, Detection and Control of Infectious Diseases)
Richard Wang (National Center for Environmental Health)
Lihua Xiao (National Center for Zoonotic, Vector-Borne, and Enteric Diseases)
United States Food and Drug Administration (FDA)
Eric Garber
Sherwood Hall
Shashi Sharma
United States Department of Homeland Security (DHS)
Linda Beck (Department of Homeland Security, Office of Health Affairs)
Mark Whitmire (Chemical Security Analysis Center)
United States Geological Survey (USGS)
Rebecca Bushon
State Agencies
Jack Bennett (Connecticut Department of Public Health, Division of Laboratory Services)
Sanwat Chaudhuri (Unified Utah State Laboratories)
Patrick Dhooge (State of New Mexico, Department of Health)
Christopher Retarides (Virginia Division of Consolidated Laboratories)
Michael Wichman (State Hygienic Laboratory at the University of Iowa)
SAM 2012 xii July 16, 2011
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Acknowledgments
Municipalities
Akin Babatola (City of Santa Cruz Wastewater Treatment Facility Laboratory)
Ian Hurley (New York City Department of Environmental Protection)
David Nehrkorn (San Francisco Public Utility Commission)
Earl Peterkin (Philadelphia Water Department)
Anthony Rattonetti (San Francisco Public Utility Commission)
Associations
Michael Heintz (Association of Public Health Laboratories)
Jack Krueger (Consultant to the Association of Public Health Laboratories)
National Laboratories
Staci Kane (Lawrence Livermore National Laboratory)
Carolyn Koester (Lawrence Livermore National Laboratory)
Rich Ozanich (Pacific Northwest National Laboratory)
Sonoya Shanks (Sandia National Laboratories)
Tim Straub (Pacific Northwest National Laboratory)
Carolyn Wong (Lawrence Livermore National Laboratory)
National Institute of Standards and Technology (NIST)
Jayne Morrow
Bioanalysis Consulting LLC
Johnathan Kiel (formerly, U.S. Air Force)
Environmental Management Support, Inc.
Anna Berne
Computer Sciences Corporation (CSC)
Eric Boring
Yildiz Chambers
Joan Cuddeback
Emily King
Dan Montgomery
Larry Umbaugh
Joshua Vinson
SAM 2012 xiii July 16, 2011
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SAM 2012
xiv
July 16, 2011
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Table of Contents
Selected Analytical Methods for
Environmental Remediation and Recovery (SAM)
SAM 2012
Contents
Disclaimer ii
Use of This Document iii
Abbreviations and Acronyms iv
Acknowledgments xi
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 QC Guidelines for Chemical Methods 31
5.1.3 Safety and Waste Management 32
5.2 Method Summaries 33
5.2.1 EPA Method 200.7: Determination of Metals and Trace Elements in Waters and
Wastes by Inductively Coupled Plasma-Atomic Emission Spectrometry 33
5.2.2 EPA Method 200.8: Determination of Trace Elements in Waters and Wastes by
Inductively Coupled Plasma-Mass Spectrometry 34
5.2.3 EPA Method 245.1: Determination of Mercury in Water by Cold Vapor Atomic
Absorption Spectrometry 35
5.2.4 EPA Method 300.1, Revision 1.0: Determination of Inorganic Anions in Drinking
Water by Ion Chromatography 36
5.2.5 EPA Method 335.4: Determination of Total Cyanide by Semi-Automated
Colorimetry 37
5.2.6 EPA Method 350.1: Nitrogen, Ammonia (Colorimetric, Automated Phenate) 37
5.2.7 EPA Method 524.2: Measurement of Purgeable Organic Compounds in Water by
Capillary Column Gas Chromatography / Mass Spectrometry 38
5.2.8 EPA Method 525.2: Determination of Organic Compounds in Drinking Water by
Liquid-Solid Extraction and Capillary Column Gas Chromatography / Mass
Spectrometry 39
5.2.9 EPA Method 531.2: Measurement of N-Methylcarbamoyloximes and N-
Methylcarbamates in Water by Direct Aqueous Injection HPLC With Postcolumn
Derivatization 40
5.2.10 EPA Method 538: Determination of Selected Organic Contaminants in Drinking
Water by Direct Aqueous Injection-Liquid Chromatography/Tandem Mass
Spectrometry (DAI-LC/MS/MS) 40
5.2.11 EPA Method 549.2: Determination of Diquat and Paraquat in Drinking Water by
Liquid-Solid Extraction and High Performance Liquid Chromatography With
Ultraviolet Detection 41
SAM 2012 xv July 16, 2011
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Table of Contents
5.2.12 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 42
5.2.13 EPA Method 556.1: Determination of Carbonyl Compounds in Drinking Water by
Fast Gas Chromatography 42
5.2.14 EPA Method 3050B (SW-846): Acid Digestion of Sediments, Sludges, and Soils 43
5.2.15 EPAMethod3511 (SW-846): Organic Compounds in Water by Microextraction 44
5.2.16 EPA Method 3520C (SW-846): Continuous Liquid-Liquid Extraction 45
5.2.17 EPA Method 3535A (SW-846): Solid-Phase Extraction 46
5.2.18 EPA Method 3541 (SW-846): Automated Soxhlet Extraction 47
5.2.19 EPA Method 3545A (SW-846): Pressurized Fluid Extraction (PFE) 49
5.2.20 EPA Method 3570 (SW-846): Microscale Solvent Extraction (MSB) 50
5.2.21 EPA Method 5030C (SW-846): Purge-and-Trap for Aqueous Samples 53
5.2.22 EPA Method 5035A (SW-846): Closed-System Purge-and-Trap and Extraction for
Volatile Organics in Soil and Waste Samples 54
5.2.23 EPA Method 6010C (SW-846): Inductively Coupled Plasma - Atomic Emission
Spectrometry 54
5.2.24 EPA Method 6020A (SW-846): Inductively Coupled Plasma - Mass Spectrometry 55
5.2.25 EPA Method 7470A (SW-846): Mercury in Liquid Wastes (Manual Cold-Vapor
Technique) 56
5.2.26 EPA Method 7471B (SW-846): Mercury in Solid or Semisolid Wastes (Manual Cold-
Vapor Technique) 57
5.2.27 EPA Method 7473 (SW-846): Mercury in Solids and Solutions by Thermal
Decomposition, Amalgamation, and Atomic Absorption Spectrophotometry 57
5.2.28 EPA Method 7580 (SW-846): White Phosphorus (P4) by Solvent Extraction and Gas
Chromatography 58
5.2.29 EPA Method 8015C (SW-846): Nonhalogenated Organics Using GC/FID 59
5.2.30 EPA Method 8260C (SW-846): Volatile Organic Compounds by Gas
Chromatography-Mass Spectrometry (GC/MS) 60
5.2.31 EPA Method 8270D (SW-846): Semivolatile Organic Compounds by Gas
Chromatography/Mass Spectrometry (GC-MS) 61
5.2.32 EPA Method 8290A, Appendix A (SW-846): Procedure for the Collection, Handling,
Analysis, and Reporting of Wipe Tests Performed Within the Laboratory 62
5.2.33 EPA Method 8315A (SW-846): Determination of Carbonyl Compounds by High
Performance Liquid Chromatography (HPLC) 65
5.2.34 EPA Method 8316 (SW-846): Acrylamide, Acrylonitrile and Acrolein by High
Performance Liquid Chromatography (HPLC) 65
5.2.35 EPA Method 8318A (SW-846): 7V-Methylcarbamates by High Performance Liquid
Chromatography (HPLC) 66
5.2.36 EPA Method 832IB (SW-846): Solvent-Extractable Nonvolatile Compounds by High
Performance Liquid Chromatography-Thermospray-Mass Spectrometry (HPLC-TS-
MS) or Ultraviolet (UV) Detection 67
5.2.37 EPA Method 8330B (SW-846): Nitroaromatics, Nitramines, and Nitrate Esters by
High Performance Liquid Chromatography (HPLC) 68
5.2.38 EPA CLP ISM01.3 Cyanide: Analytical Methods for Total Cyanide Analysis 69
5.2.39 EPA Method 3135.21: Cyanide, Total and Amenable in Aqueous and Solid Samples
Automated Colorimetric With Manual Digestion 69
5.2.40 EPA IO [Inorganic] Compendium Method IO-3.1: Selection, Preparation, and
Extraction of Filter Material 70
5.2.41 EPA IO [Inorganic] Compendium Method IO-3.4: Determination of Metals in
Ambient Particulate Matter Using Inductively Coupled Plasma (ICP) Spectroscopy 71
SAM 2012 xvi July 16, 2011
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Table of Contents
5.2.42 EPA IO [Inorganic] Compendium Method IO-3.5: Determination of Metals in
Ambient Particulate Matter Using Inductively Coupled Plasma/Mass Spectrometry
(ICP-MS) 72
5.2.43 EPA 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) 73
5.2.44 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) 73
5.2.45 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) 75
5.2.46 EPA 600/R-l 1/091: High Throughput Determination of Tetramine in Drinking Water
by Solid Phase Extraction and Isotope Dilution Gas Chromatography/Mass
Spectrometry (GC/MS) 76
5.2.47 EPA/600/R-11/143: Surface Analysis Using Wipes for the Determination of
Nitrogen Mustard Degradation Products by Liquid Chromatography/Tandem Mass
Spectrometry (LC/MS/MS) 77
5.2.48 Analytical Protocol for Chemical Warfare Agents in Water, Soil, and Wipes 78
5.2.49 NIOSH Method 1612: Propylene Oxide 78
5.2.50 NIOSH Method 2016: Formaldehyde 79
5.2.51 NIOSH Method 2513: Ethylene Chlorohydrin 79
5.2.52 NIOSH Method 3510: Monomethylhydrazine 80
5.2.53 NIOSH Method 5600: Organophosphorus Pesticides 80
5.2.54 NIOSH Method 5601: Organonitrogen Pesticides 81
5.2.55 NIOSH Method 6001: Arsine 82
5.2.56 NIOSH Method 6002: Phosphine 82
5.2.57 NIOSH Method 6010: Hydrogen Cyanide 83
5.2.58 NIOSH Method 6013: Hydrogen Sulfide 83
5.2.59 NIOSH Method 6015: Ammonia 84
5.2.60 NIOSH Method 6402: Phosphorus Trichloride 84
5.2.61 NIOSH Method 7903: Acids, Inorganic 85
5.2.62 NIOSH Method 7905: Phosphorus 85
5.2.63 NIOSH Method 7906: Fluorides, Aerosol and Gas, by 1C 86
5.2.64 NIOSH Method 9102: Elements on Wipes 86
5.2.65 NIOSH Method S301-1: Fluoroacetate Anion 87
5.2.66 OSHA Method 40: Methylamine 88
5.2.67 OSHA Method 54: Methyl Isocyanate (MIC) 88
5.2.68 OSHA Method 61: Phosgene 89
5.2.69 OSHA Method ID-211: Sodium Azide and Hydrazoic Acid in Workplace
Atmospheres 89
5.2.70 OSHA Method ID216SG: Boron Trifluoride (BF3) 90
5.2.71 OSHA Method PV2004: Acrylamide 90
5.2.72 OSHA Method PV2103: Chloropicrin 91
5.2.73 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 91
5.2.74 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 92
SAM 2012 xvii July 16, 2011
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Table of Contents
5.2.75 ASTM Method D7597-09: Standard Test Method for Determination of Diisopropyl
Methylphosphonate, Ethyl Hydrogen Dimethylamidophosphate, Ethyl
Methylphosphonic Acid, Isopropyl Methylphosphonic Acid, Methylphosphonic Acid
and Pinacolyl Methylphosphonic Acid in Water by Liquid Chromatography/Tandem
Mass Spectrometry 93
5.2.76 ASTM Method D7598-09: Standard Test Method for Determination of Thiodiglycol
in Water by Single Reaction Monitoring Liquid Chromatography/Tandem Mass
Spectrometry 93
5.2.77 ASTM Method D7599-09: Standard Test Method for Determination of
Diethanolamine, Triethanolamine, 7V-Methyldiethanolamine and 7V-
Ethyldiethanolamine in Water by Single Reaction Monitoring Liquid
Chromatography/Tandem Mass Spectrometry (LC/MS/MS) 94
5.2.78 ASTM Method D7644-10: Standard Test Method for Determination of
Bromadiolone, Brodifacoum, Diphacinone and Warfarin in Water by Liquid
Chromatography/Tandem Mass Spectrometry (LC/MS/MS) 95
5.2.79 ASTM Method D7645-10: Standard Test Method for Determination of Aldicarb,
Aldicarb Sulfone, Aldicarb Sulfoxide, Carbofuran, Methomyl, Oxamyl and
Thiofanox in Water by Liquid Chromatography/Tandem Mass Spectrometry
(LC/MS/MS) 95
5.2.80 ASTM Method E2787-11: Standard Test Method for Determination of Thiodiglycol
in Soil Using Pressurized Fluid Extraction Followed by Single Reaction Monitoring
Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS) 96
5.2.81 ASTM Method E2838-11: Standard Test Method for Determination of Thiodiglycol
on Wipes by Solvent Extraction Followed by Liquid Chromatography/Tandem Mass
Spectrometry (LC/MS/MS) 97
5.2.82 ASTM Method E2866-12: Standard Test Method for Determination of Diisopropyl
Methylphosphonate, Ethyl Methylphosphonic Acid, Isopropyl Methylphosphonic
Acid, Methylphosphonic Acid and Pinacolyl Methylphosphonic Acid in Soil by
Pressurized Fluid Extraction and Analyzed by Liquid Chromatography/Tandem Mass
Spectrometry 98
5.2.83 ISO Method 10312:1995: Ambient Air - Determination of Asbestos Fibres - Direct-
Transfer Transmission Electron Microscopy Method 98
5.2.84 Standard Method 4500-NH3 B: Nitrogen (Ammonia) Preliminary Distillation Step 99
5.2.85 Standard Method 4500-NH3 G: Nitrogen (Ammonia) Automated Phenate Method 99
5.2.86 Standard Method 4500-C1 G: Chlorine (Residual) DPD Colorimetric Method 100
5.2.87 Literature Reference for Hexamethylenetriperoxidediamine (HMTD) (Analyst, 2001.
126:1689-1693) 100
5.2.88 Literature Reference for Chlorine in Air (Analyst, 1999. 124(12): 1853-1857) 101
5.2.89 Literature Reference for Methamidophos (Chromatographia. 2006. 63(5/6): 233-237).... 102
5.2.90 Literature Reference for Cyanogen Chloride (Encyclopedia of Anal. Chem. 2006 DOI:
10.1002/9780470027318.a0809) 102
5.2.91 Literature Reference for 3-Chloro-l,2-propanediol (Eur. J. Lipid Sci. Technol. 2011,
113: 345-355) 103
5.2.92 Literature Reference for Methyl Hydrazine (Journal of Chromatography 1993 (617),
157-162) 104
5.2.93 Literature Reference for Paraquat (Journal of Chromatography A, 2008, 1196-1197,
110-116) 104
5.2.94 Literature Reference for Methamidophos (Journal of Chromatography A, 2007. 1154:
3-25) 105
5.2.95 Literature Reference for Fluoroacetic Acid/Fluoroacetate Salts/Methyl Fluoroacetate
(Journal of Chromatography A, 1139(2007)271-278) 106
SAM 2012 xviii July 16, 2011
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5.2.96 Literature Reference for 3-Chloro-l,2-propanediol (Journal of Chromatography A,
2000.866:65-77) 106
5.2.97 Literature Reference for Fluoroacetic Acid/Fluoroacetate Salts/Methyl Fluoroacetate
(Journal of Chromatography B, 2010, 878: 1045-1050) 107
5.2.98 Literature Reference for Fluoroacetamide (Journal of Chromatography B, 2008.
876(1): 103-108) 108
5.2.99 Literature Reference for Sodium Azide (Journal of Forensic Sciences, 1998. 43(1):
200-202) 108
Section 6.0: Selected Radiochemical Methods 110
6.1 General Guidelines Ill
6.1.1 Standard Operating Procedures for Identifying Radiochemical Methods Ill
6.1.2 General QC Guidelines for Radiochemical Methods 115
6.1.3 Safety and Waste Management 116
6.2 Method Summaries 118
6.2.1 EPA Method 111: Determination of Polonium-210 Emissions from Stationary
Sources 118
6.2.2 EPA Method 900.0: Gross Alpha and Gross Beta Radioactivity in Drinking Water 118
6.2.3 EPA Method 901.1: Gamma Emitting Radionuclides in Drinking Water 119
6.2.4 EPA Method 903.1: Radium-226 in Drinking Water - Radon Emanation Technique 120
6.2.5 EPA Method 905.0: Radioactive Strontium in Drinking Water 120
6.2.6 EPA Method 906.0: Tritium in Drinking Water 121
6.2.7 EPA Method EMSL-19: Determination of Radium-226 and Radium-228 in Water,
Soil, Air and Biological Tissue 122
6.2.8 EPA Method EMSL-33: Isotopic Determination of Plutonium, Uranium, and
Thorium in Water, Soil, Air, and Biological Tissue 122
6.2.9 EPA Method Rapid Radiochemical Method for Phosphorus-32 in Water for
Environmental Restoration Following Homeland Security Events 123
6.2.10 EPA Method R4-73-014: Radioactive Phosphorus 123
6.2.11 EPA Method: Determination of Radiostrontium in Food and Bioenvironmental
Samples 124
6.2.12 EPA Method: Rapid Radiochemical Method for Americium-241 in Water for
Environmental Restoration Following Homeland Security Events 125
6.2.13 EPA Method: Rapid Radiochemical Method for Plutonium-238 and Plutonium-
239/240 in Water for Environmental Restoration Following Homeland Security
Events 125
6.2.14 EPA Method: Rapid Radiochemical Method for Radium-226 in Water for
Environmental Restoration Following Homeland Security Events 126
6.2.15 EPA Method: Rapid Radiochemical Method for Radiostrontium in Water for
Environmental Restoration Following Homeland Security Events 126
6.2.16 EPA Method: Rapid Radiochemical Method for Isotopic Uranium in Water for
Environmental Restoration Following Homeland Security Events 127
6.2.17 EPA Method: Rapid Method for Acid Digestion of Glass-Fiber and
Organic/Polymeric Composition Filters and Swipes Prior to Isotopic Uranium,
Plutonium, Americium, Strontium, and Radium Analyses for Environmental
Remediation Following Homeland Security Events 128
6.2.18 EPA Method: Rapid Method for Sodium Carbonate Fusion of Glass-Fiber and
Organic/Polymeric Composition Filters and Swipes Prior to Isotopic Uranium,
Plutonium, Americium, Strontium, and Radium Analyses for Environmental
Remediation Following Homeland Security Events 129
6.2.19 EML HASL-300 Method Am-01-RC: Americium in Soil 130
SAM 2012 xix July 16, 2011
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6.2.20 EML HASL-300 Method Am-04-RC: Americium in QAP Water and Air Filters -
Eichrom's TRU Resin 131
6.2.21 EML HASL-300 Method Am-06-RC: Americium and/or Plutonium in Vegetation 131
6.2.22 EML HASL-300 Method Ga-01-R: Gamma Radioassay 132
6.2.23 EML HASL-300 Method Po-02-RC: Polonium in Water, Vegetation, Soil, and Air
Filters 133
6.2.24 EML HASL-300 Method Pu-12-RC: Plutonium and/or Americium in Soil or
Sediments 133
6.2.25 EML HASL-300 Method Ra-03-RC: Radium-226 in Soil, Vegetable Ash, and Ion
Exchange Resin 134
6.2.26 EML HASL-300 Method Sr-03-RC: Strontium-90 in Environmental Samples 134
6.2.27 EML HASL-300 Method Tc-01-RC: Technetium-99 in Water and Vegetation 135
6.2.28 EML HASL-300 Method Tc-02-RC: Technetium-99 in Water - TEVAฎ Resin 135
6.2.29 EML HASL-300 Method U-02-RC: Isotopic Uranium in Biological and
Environmental Materials 136
6.2.30 DOE FRMAC Method Volume 2, Page 33: Gross Alpha and Beta in Air 136
6.2.31 DOE RESL Method P-2: 32P Fish, Vegetation, Dry Ash, Ion Exchange 137
6.2.32 DOE SRS Actinides and Sr-89/90 in Soil Samples 138
6.2.33 DOE SRS Actinides and Sr-89/90 in Vegetation: Fusion Method 138
6.2.34 ORISE Method API: Gross Alpha and Beta for Various Matrices 139
6.2.35 ORISE Method AP2: Determination of Tritium 140
6.2.36 ORISE Method AP5: Determination of Technetium-99 140
6.2.37 ORISE Method API 1: Sequential Determination of the Actinides in Environmental
Samples Using Total Sample Dissolution and Extraction Chromatography 141
6.2.38 ORISE Method Procedure #9: Determination of 1-125 in Environmental Samples 142
6.2.39 ASTM Method D3084-05: Standard Practice for Alpha Spectrometry in Water 142
6.2.40 ASTM Method D3972-02: Standard Test Method for Isotopic Uranium in Water by
Radiochemistry 143
6.2.41 ASTM Method D5811-08: Standard Test Method for Strontium-90 in Water 144
6.2.42 ASTM Method D7168-05: Standard Test Method for Technetium-99 in Water by
Solid Phase Extraction Disk 144
6.2.43 Standard Method 7110 B: Gross Alpha and Gross Beta Radioactivity (Total,
Suspended, and Dissolved) 145
6.2.44 Standard Method 7120: Gamma-Emitting Radionuclides 146
6.2.45 Standard Method 7500-Ra B: Radium: Precipitation Method 146
6.2.46 Standard Method 7500-Ra C: Radium: Emanation Method 147
6.2.47 Standard Method 7500-U B: Uranium: Radiochemical Method 147
6.2.48 Standard Method 7500-U C: Uranium: Isotopic Method 148
6.2.49 Y-12 (DOE) Preparation of Samples for Total Activity Screening 148
Section 7.0: Selected Pathogen Methods 151
7.1 General Guidelines 155
7.1.1 Standard Operating Procedures for Identifying Pathogen Methods 155
7.1.2 General QC Guidelines for Pathogen Methods 157
7.1.3 Safety and Waste Management 158
7.1.4 Laboratory Response Network (LRN) 159
7.2 Method Summaries for Bacteria 161
7.2.1 Bacillus anthracis [Anthrax] - BSL-3 161
7.2.2 Brucella spp. [Brucellosis] - BSL-3 165
7.2.3 Burkholderia mallei [Glanders] - BSL-3 and Burkholderiapseudomallei
[Melioidosis] - BSL-3 168
SAM 2012 xx July 16, 2011
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7.2.4 Campylobacter jejuni [Campylobacteriosis] - BSL-2 172
7.2.5 Chlamydophilapsittaci [Psittacosis] (formerly known as Chlamydiapsittaci) -
BSL-2; BSL-3 for Aerosols and Large Volumes 175
7.2.6 Coxiella burnetii [Q-fever] - BSL- 3 177
7.2.7 Escherichia coli O157:H7 - BSL-2 180
7.2.8 Francisella tularensis [Tularemia] - BSL-3 183
7.2.9 Leptospira interrogans [Leptospirosis] - BSL-2 186
7.2.10 Listeria monocytogenes [Listeriosis] - BSL-2 189
7.2.11 Non-typhoidal Salmonella (Not applicable to S. Typhi) [Salmonellosis] - BSL-2 192
7.2.12 Salmonella Typhi [Typhoid fever] - BSL-2; BSL-3 for Aerosol Release 195
7.2.13 Shigella spp. [Shigellosis] - BSL-2 198
7.2.14 Staphylococcus aureus - BSL-2 200
7.2.15 Vibrio cholerae [Cholera] - BSL-2 203
7.2.16 Yersiniapestis [Plague] - BSL-3 206
7.3 Method Summaries for Viruses 209
7.3.1 Adenoviruses: Enteric and Non-enteric (A-F) - BSL-2 209
7.3.2 Astroviruses - BSL not specified 213
7.3.3 Caliciviruses: Noroviruses - BSL-2 216
7.3.4 Caliciviruses: Sapovirus- BSL-2 217
7.3.5 Coronaviruses: Severe Acute Respiratory Syndrome (SARS) -associated Human
Coronavirus (HCoV) - BSL-2; BSL-3 for Propagation 220
7.3.6 Hepatitis E Virus (HEV) - BSL-2 224
7.3.7 InfluenzaH5N1 virus-BSL-3 227
7.3.8 Picornaviruses: Enteroviruses - BSL-2 228
7.3.9 Picornaviruses: Hepatitis A Virus (HAV) - BSL-2 231
7.3.10 Reoviruses: Rotavirus (Group A) - BSL-2 234
7.4 Method Summaries for Protozoa 237
7.4.1 Cryptosporidium spp. [Cryptosporidiosis] - BSL-2 237
7.4.2 Entamoeba histolytica - BSL-2 243
7.4.3 Giardiaspp. [Giardiasis] - BSL-2 245
7.4.4 Toxoplasma gondii [Toxoplasmosis] - BSL-2 249
7.5 Method Summaries for Helminths 252
7.5.1 Baylisascarisprocyonis [Raccoon roundworm fever] - BSL-2 252
Section 8.0: Selected Biotoxin Methods 255
8.1 General Guidelines 257
8.1.1 Standard Operating Procedures for Identifying Biotoxin Methods 257
8.1.2 General QC Guidelines for Biotoxin Methods 259
8.1.3 Safety and Waste Management 260
8.1.4 Laboratory Response Network (LRN) 261
8.2 Method Summaries for Protein Biotoxins 262
8.2.1 Abrin 262
8.2.2 Botulinum Neurotoxins (Serotypes A, B, E, F) 265
8.2.3 Ricin (Ricinine) 268
8.2.4 Shiga and Shiga-like Toxins (Stx, Stx-1, Stx-2) 269
8.2.5 Staphylococcal Enterotoxins (SEA, SEE, SEC) 270
8.3 Method Summaries for Small Molecule Biotoxins 272
SAM 2012 xxi July 16, 2011
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8.3.1 Aflatoxin(TypeBl) 272
8.3.2 a-Amanitin 273
8.3.3 Anatoxin-a 274
8.3.4 Brevetoxins (B form) 275
8.3.5 a-Conotoxin 276
8.3.6 Cylindrospermopsin 277
8.3.7 Diacetoxyscirpenol (DAS) 278
8.3.8 Microcystins (Principal isoforms: LA, LR, LW, RR, YR) 279
8.3.9 Picrotoxin 280
8.3.10 Saxitoxins 281
8.3.11 T-2 Mycotoxin 282
8.3.12 Tetrodotoxin 283
Section 9.0: Conclusions 284
Figures
Figure 1-1. Environmental Evaluation Analytical Process Roadmap for Homeland Security Events 2
Figure 2-1. SAM Method Selection Process 5
Figure 7-1. Sample Analysis During Site Characterization and Post Decontamination Phases
Following a Biological Contamination Event 152
Tables
Table 5-1. Chemical Methods and Corresponding Section Numbers 15
Table 5-2. Sources of Chemical Methods 29
Table 6-1. Radiochemical Methods and Corresponding Section Numbers Ill
Table 6-2 Sources of Radiochemical Methods 116
Table 7-1. Sources of Pathogen Methods 156
Table 8-1. Sources of Biotoxin Methods 260
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
Attachments
DRAFT Attachment 1: SAM Supporting Documents 1-1
SAM 2012 xxii July 16, 2011
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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 laboratory capabilities to analyze environmental samples. In reviewing these
events, the Environmental Protection Agency (EPA) identified several areas to enhance the resiliency of
the nation following homeland security events related to intentional and unintentional contamination. The
need to improve the nation's laboratory capacity and capability to analyze environmental samples
following such events (i.e., chemical, biological and/or radiological [CBR] contamination) was one of the
most important areas identified.
To address this need, EPA formed the Homeland Security Laboratory Capacity Work Group 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 Work Group consisted
of representatives from the EPA's 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 Chemical Safety and Pollution Prevention, and several
EPA regional offices.
A critical area identified by the work group was the need for a list of selected analytical methods to be
used (ideally) by all laboratories when analyzing contamination event samples and, in particular, when
analysis of many samples is required over a short period of time. Using the same selected 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. Use of such
methods would also improve the follow-up activities of validating results, evaluating data and making
decisions. To this end, work group 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 Selected
Analytical Methods for Environmental Remediation and Recovery (SAM)1 provides information for
analytical methods to be applied during the "Site Remediation" phase. Methods have been selected to
support activities related to site assessment (including preliminary, qualitative analyses to characterize the
extent of contamination), site cleanup (to evaluate the efficacy of remediation efforts), and site clearance
(releasing a site, including water and wastewater systems, for its intended use) decisions.
1 Formerly EPA's Standardized Analytical Methods for Environmental Restoration Following Homeland Security
Events (SAM). SAM and its methods are available at: www.epa.gov/sam
SAM 2012 1 July 16, 2011
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Section 1 - Introduction
Figure 1-1. Environmental Evaluation Analytical Process Roadmap for Homeland
Security Events
Surveillance and Monitoring
(if appropriate)
Immediate Response/
Credibility Determination
Preliminary Site
Characterization
SAM
Site Remediation
SAM
(Selected Analytical Methods for Environmental
Remediation and Recovery)
^^ Assessment *)
Cleanup
( Clearance
SAM 2012
July 16, 2011
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Section 2 - Background
Section 2.0: Background
In support of this document, EPA periodically assembles methods experts from within EPA, as well as
other federal, state and local agencies; public utilities; national laboratories; and academia, to review
methods and, if necessary, revise the methods listed. SAM analytes are included based on selection
criteria that address the needs and priorities of EPA as well as other federal agencies (e.g., environmental
persistence, half lives, availability, toxicity). The sample types listed in SAM are specific to each
technical section and have been determined by the SAM technical work groups to be a concern during site
remediation. 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
under the direction of EPA's National Homeland Security Research Center (NHSRC). For analytes
where limited laboratory capacity exists, such as chemical warfare agents (CWAs), methods were
selected based on their applicability to similar chemicals (e.g., nerve agents and some pesticides). In
these cases, laboratory studies to evaluate the ability of the selected method to measure the target
analyte(s) are either underway or needed. 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, 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 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), U.S. Geological Survey (USGS), numerous state agencies and universities. Methodologies are
considered for chemical, radiochemical, biological and biotoxin agents of concern in the types of
environmental samples that would be anticipated. The primary objective of this effort is to support EPA's
Environmental Response Laboratory Network (ERLN) and Water Laboratory Alliance (WLA) by
identifying appropriate SAM methods that represent a balance between providing existing, documented
techniques and providing consistent and valid analytical results.
Surveys of available analytical methods are 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 Water Methods
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
SAM 2012 3 July 16, 2011
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Section 2 - Background
Standard Methods for the Examination of Water and Wastewater
Scientific Literature
Since publication of SAM Revision 1.0 in September 2004, NHSRC has continued to convene technical
work groups to evaluate and, if necessary, update the analytes and methods that are listed. Details
regarding changes that have been incorporated into each revision of SAM are provided in Attachment 1.
SAM 2012 also reflects a title change agreed to by stakeholders (i.e., EPA's NHSRC; Office of
Groundwater and Drinking Water, Water Security Division (WSD) and WLA; Office of Emergency
Management and ERLN; Office of Radiation and Indoor Air (ORIA); and Regional Offices) during a
2010 SAM Summit, to better reflect SAM's focus on providing selected analytical methods for use across
multiple laboratories during environmental remediation and recovery. This current revision (SAM 2012)
includes the addition of vegetation as a sample type under the radiochemistry sections, the addition of
method applicability tiers to Appendix A (Selected Chemical Methods), several new methods added or
replaced for currently listed chemical analytes, clarification of immunoassay methods listed for biotoxin
analytes, and the addition of restructured pathogen sections to more clearly define scope and application.
In addition to updating SAM analytes and methods, SAM work groups have identified four areas for
development of SAM companion documents to provide information regarding field screening equipment,
sample collection, rapid screening and preliminary analysis equipment, and sample disposal to
supplement the analytical methods included in SAM. The information listed in these documents
generally corresponds to the analytes and methods in SAM and will be updated as resources allow to
reflect revisions to SAM. Currently available SAM companion documents are listed in Attachment 1.
SAM 2012 4 July 16, 2011
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Section 2 - Background
Figure 2-1. SAM Method Selection Process
Step 1
/"Is there an EPA\^
published method for ^x
measurement of the
analyte in the sample /
\type of interest?'/
Y
NO
Step 2 ,/ \,
./ \.
,/ 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
\x interest? //
NO
-YES-
StepS
/ Is there an EPA,
federal, or VCSB method
that has been developed for
measurement of the analyte
in another environmental
sample type? /
-YES
Step 4
/
/ 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? /
-YES
Evaluate method against
selection criteria
Repeat Steps 1 - 4 to identify methods that measure
analytes similarto the analyte of concern
If no methods are available, prioritize
for further research
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 and
trained personnel, 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
SAM 2012
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SAM 2012
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Section 3 - Scope and Application
Section 3.0: Scope and Application
The premise and purpose of this document is to select the analytical methods that will be used in cases
when multiple laboratories are called on to analyze environmental samples following an intentional or
unintentional incident such as a homeland security event (e.g., CBR contamination). The document is
intended to support the ERLN and WLA, and also can be used as a tool to assist state and local
laboratories in planning for and analyzing environmental samples following such events. 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);
Evaluate the efficacy of remediation efforts during site cleanup; 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 and viability, when applicable, 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. This document also is not intended to provide information
regarding sample collection activities or equipment. In conjunction with SAM, NHSRC has developed
SAM companion documents that are intended to provide information regarding field screening
equipment, sample collection, laboratory rapid screening/preliminary identification equipment, and
sample disposal in support of the confirmatory methods and analytes listed in SAM. Currently available
SAM companion documents are listed in Attachment 1.
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 throughout Sections 5.2 (chemical methods), 6.2 (radiochemical methods),
7.2 (pathogen methods) and 8.2 (biotoxin methods).
SAM 2012 7 July 16, 2011
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Section 3 - Scope and Application
The information contained in this document represents the latest step in an ongoing effort by EPA's
NHSRC to provide selected analytical methods for use by those laboratories tasked with performing
confirmatory analyses of environmental samples in support of EPA remediation and recovery efforts
following a homeland security incident. SAM is intended for use by EPA and EPA-contracted and
-subcontracted laboratories; it also can be used by other agencies and laboratory networks, such as
the Integrated Consortium of Laboratory Networks (ICLN). The information also can be found on
the SAM website (www.epa.gov/sam). which provides searchable links to supporting information
based on SAM analytes and the analytical methods listed.
At this time, only some of the methods selected have been validated for the listed analyte and sample
type. However, the methods are considered to contain the most appropriate currently available
techniques based on expert judgment. 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 evaluation is needed, and adjustments may be required to accurately account for
variations in analyte/sample type characteristics, environmental samples, analytical interferences, and
data quality objectives (DQOs).
EPA will strive to continue development and evaluation of analytical protocols, including
optimization of procedures for measuring target analytes or agents in specific sample types, as
appropriate. In those cases where method procedures are determined to be insufficient for a
particular situation, NHSRC will continue to provide technical support regarding appropriate actions.
NHSRC has also compiled information and published documents regarding field screening
equipment, sample collection materials, rapid screening/preliminary identification equipment, and
disposal of samples corresponding to SAM analytes and sample types. These documents are
available at www.epa.gov/sam/samcomp.htm.
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 technical advice regarding appropriate actions
(see list of contacts in Section 4). Additional information is also provided in the Agency Policy Directive
Number FEM-2010-01.2 Where further development and testing are necessary, EPA is continuing to
develop and evaluate analytical protocols based on the methods that are listed in this document and on
current EPA policies for validating analytical methods. Once validation is complete, data regarding the
resulting method performance and data quality objectives (DQOs) will be available. EPA plans to
continue to update the SAM document as appropriate 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 updates regarding
information and issues that currently are not addressed by this document. Information regarding the use
of deviations from the methods referenced in this document is provided in Section 4.
Participants in the chemical, radiochemical, pathogen and biotoxin work groups evaluated the suitability
of existing methodologies and selected this set of methods for use by those laboratories that support EPA
environmental remediation efforts following an intentional or unintentional incident such as a homeland
security event. 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;
2 U.S. EPA, Forum of Environmental Measurements, July 21, 2010, Ensuring the Validity of Agency Methods
Validation and Peer Review Guidelines: Methods of Analysis Developed for Emergency Response Situations,
Agency Policy Directive Number FEM-2010-01.
SAM 2012 8 July 16, 2011
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Section 3 - Scope and Application
Selecting methodologies that may not be appropriate for use in responding to a particular event
because EPA did not anticipate having to analyze for a particular analyte or analyte/sample type
combination; and
Discouraging development and adoption of new and better measurement technologies.
To address these potential risks, the following measures are taken:
Using an established SAM method selection process (Figure 2-1) to help ensure that the analytical
methods listed provide results that are consistent with and support their intended use;
Collaborating with the ERLN, which includes the WLA and is part of the Integrated Consortium of
Laboratory Networks (ICLN), to ensure that the methods selected meet the network's needs for
consistent analytical capabilities, to address capacity, and to provide quality data to inform
remediation decisions; and
Continuing to work with multiple agencies and stakeholders to update methods in SAM as needed.
Public officials need to accurately assess and characterize site contamination following an emergency
situation. This assessment includes initial characterization of potential site contamination for
determination of immediate public and environmental risk, determination of the extent of contamination,
and effective approaches for site remediation. EPA recognizes that having data of known and
documented quality is critical in making proper decisions during each of these activities, and strives to
establish DQOs for each response activity.3 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. 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). EPA's ERLN, which is tasked with providing laboratory support following homeland
security-related contamination events, also has established data reporting procedures. Requirements for
receiving, tracking, storing, preparing, analyzing and reporting data are specified in the U.S. EPA (2011)
Environmental Response Laboratory Network Laboratory Requirements Document at:
http://epa.gov/erln/techsupport.html; project-specific requirements also are included in individual
Analytical Service Requests (ASRs).
3 Information regarding EPA's DQO process, considerations, and planning is available at:
http://www.epa.gov/QUALITY/dqos.html.
SAM 2012 9 July 16, 2011
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SAM 2012
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July 16, 2011
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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. EPA recommends that these contacts be consulted
regarding any method deviations or modifications, sample problems or interferences, QC requirements,
the use of potential alternative methods, or the need to address analytes or sample types other than those
listed in SAM. 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 cases where laboratories are specifically tasked by EPA to use these methods following an
event, method deviations or modifications must be approved by the Analytical Service Requestor (as
defined by ERLN) prior to use. In addition, general questions and comments can be submitted via the
SAM website (www.epa.gov/sam).
General
Kathy Hall - Primary
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
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.john@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
Sanjiv Shah -Primary
National Homeland Security Research Center
U.S. EPAORD-8801RR
1300 Pennsylvania Avenue, NW
Washington, DC 20460
(202) 564-9522 shah.sanjiv@epa.gov
Erin Silvestri -Alternate
National Homeland Security Research Center
U.S. EPA ORD (NG16)
26 West Martin Luther King Jr. Drive
Cincinnati, OH 45268
(513)569-7619 silvestri.erin@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
1300 Pennsylvania Avenue, NW
Washington, DC 20460
(202) 564-9522 shah.sanjiv@epa.gov
SAM 2012
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SAM 2012
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SAM 2012 Section 5.0- 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 contamination incident. 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.
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 2012 13 July 16, 2011
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SAM 2012 Section 5.0- 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 quality control (QC) procedures.
For additional information on the properties of the chemicals listed in Appendix A, Toxicology Data
Network (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:
Syracuse Research Corporation's (SRC) PHYSPROP (http://srcinc.com/what-we-
do/product.aspx?id=133) and CHEMFATE (http://srcinc.com/what-we-
do/product.aspx?id= 132&terms=Environmental+Fate+and+Exposure) contain information pertaining
to chemical structures, names, physical properties and persistence. PFiYSPROP and CHEMFATE are
sponsored by EPA.
INCHEM (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
National Institute for Occupational Safety and Health (NIOSH) website
(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).
EPA's Water Contaminant Information Tool (WCIT) (http://www.epa.gov/wcit) can be accessed by
registered users.
Forensic Science and Communications (http://www.fbi.gov/about-us/lab/forensic-science-
communications) is published by the Laboratory Division of the Federal Bureau of Investigation
(FBI).
Joint Research Centre / Institute for Health & Consumer Protection (http ://ihcp j re .ec .europa.eu/)
contains information regarding European Directive 67/548/EEC and Annex V.
Agency of Toxic Substances & Disease Registry (ATSDR) Toxic Substances Portal
(http://www.atsdr.cdc.gov/toxprofiles/index.asp) provides Toxicological Profiles.
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: Selected Chemical Methods. 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, aqueous liquid, drinking water, air or wipe). In some cases, two methods (sample
preparation and determinative) are needed to complete sample analysis.
The fitness of a method for an intended use is related to site-specific data quality objectives (DQOs) for a
particular environmental remediation activity. These selected chemical methods have been assigned the
tiers (below) to indicate a level of method usability for the specific analyte and sample type. The
assigned tiers reflect the conservative view for DQOs involving timely implementation of methods for
SAM 2012 14 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
analysis of a high number of samples (such that multiple laboratories are necessary), low limits of
identification and quantification, and appropriate QC:
Tier I: Analyte/sample type is a target of the method(s). Data are available for all aspects of method
performance and QC measures supporting its use for analysis of environmental samples
following a contamination event. Evaluation and/or use of the method(s) in multiple
laboratories indicate that the method can be implemented with no additional modifications for
the analyte/sample type.
Tier II: (1) The analyte/sample type is a target of the method(s) and the method(s) has been evaluated for
the analyte/sample type by one or more laboratories, or (2) the analyte/sample type is not a
target of the method(s), but the method has been used by laboratories to address the
analyte/sample type. In either case, available data and/or information indicate that
modifications will likely be needed for use of the method(s) to address the analyte/sample
type.
Tier III: The analyte/sample type is not a target of the method(s), and/or no reliable data supporting
the method's fitness for its intended use are available. Data from other analytes or sample
types, however, suggest that the method(s), with significant modification, may be applicable.
Once a method has been identified in Appendix A, Table 5-1 can be used to locate the method summary.
Sections 5.2.1 through 5.2.99 below provide summaries of the sample preparation and determinative
methods listed in Appendix A.
Table 5-1. Chemical Methods and Corresponding Section Numbers
Analyte
Acephate
Acrylamide
Acrylonitrile
Aldicarb (Temik)
Aldicarb sulfone
Aldicarb sulfoxide
CASRN
30560-19-1
79-06-1
107-13-1
116-06-3
1646-88-4
1646-87-3
Method
538 (EPA OW)
Chromatographia (2006) 63(5/6):
233-237
J. Chromatogr. A (2007) 1 1 54(1 ):
3-25
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
831 6 (EPA SW-846)
PV2004 (OSHA)
524.2 (EPA OW)
3570 (EPA SW-846)
5035A (EPA SW-846)
8260C (EPA-SW846)
8290A Appendix A (EPA SW-846)
PV2004 (OSHA)
531. 2 (EPA OW)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
831 8A (EPA SW-846)
5601 (NIOSH)
D7645-10(ASTM)
Section
5.2.10
5.2.89
5.2.94
5.2.20
5.2.32
5.2.34
5.2.71
5.2.7
5.2.20
5.2.22
5.2.30
5.2.32
5.2.71
5.2.9
5.2.20
5.2.32
5.2.35
5.2.54
5.2.79
SAM 2012
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SAM 2012 Section 5.0- Selected Chemical Methods
Analyte
Allyl alcohol
4-Aminopyridine
Ammonia
Ammonium metavanadate (analyze as total
vanadium)
Arsenic, Total
Arsenic trioxide (analyze as total arsenic)
Arsine (analyze as total arsenic in non-air
samples)
Asbestos
Boron trifluoride
Brodifacoum
Bromadiolone
BZ [Quinuclidinyl benzilate]
CASRN
107-18-6
504-24-5
7664-41-7
7803-55-6
7440-38-2
1327-53-3
7784-42-1
1332-21-4
2095581
56073-10-0
28772-56-7
1709855
Method
5030C (EPA SW-846)
5035A (EPA SW-846)
8260C (EPA-SW846)
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-NH3 B (SM)
4500-NH3 G (SM)
200.7 (EPA OW)
200.8 (EPA OW)
3050B (EPA SW-846)
601 OC (EPA SW-846)
6020A (EPA SW-846)
IO-3.1 (EPAORD)
IO-3.4 (EPA ORD)
IO-3.5 (EPAORD)
9102(NIOSH)
200.7 (EPAOW)
200.8 (EPA OW)
3050B (EPA SW-846)
601 OC (EPA SW-846)
6020A (EPA SW-846)
6001 (NIOSH)
9102(NIOSH)
D5755-03 (ASTM)
D6480-05 (ASTM)
10312:1995 (ISO)
ID216SG(OSHA)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
8321 B (EPA SW-846)
D7644-10(ASTM)
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
8321 B (EPA SW-846)
TO-IOA(EPAORD)
Section
5.2.21
5.2.22
5.2.30
5.2.45
5.2.17
5.2.20
5.2.32
5.2.37
5.2.6
5.2.59
5.2.84
5.2.85
5.2.1
5.2.2
5.2.14
5.2.23
5.2.24
5.2.40
5.2.41
5.2.42
5.2.64
5.2.1
5.2.2
5.2.14
5.2.23
5.2.24
5.2.55
5.2.64
5.2.73
5.2.74
5.2.83
5.2.70
5.2.18
5.2.19
5.2.20
5.2.32
5.2.36
5.2.78
5.2.16
5.2.17
5.2.18
5.2.19
5.2.20
5.2.32
5.2.36
5.2.44
SAM 2012
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SAM 2012 Section 5.0- Selected Chemical Methods
Analyte
Calcium arsenate (analyze as total arsenic)
Carbofuran (Furadan)
Carbon disulfide
Carfentanil
Chlorfenvinphos
Chlorine
2-Chloroethanol
3-Chloro-1 ,2-propanediol
CASRN
7778-44-1
1563-66-2
75-15-0
59708-52-0
470-90-6
7782-50-5
107-07-3
96-24-2
Method
200.7 (EPA OW)
200.8 (EPA OW)
3050B (EPA SW-846)
6010C(EPASW-846)
6020A (EPA SW-846)
IO-3.1 (EPAORD)
IO-3.4 (EPA ORD)
IO-3.5 (EPAORD)
9102(NIOSH)
531. 2 (EPA OW)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
831 8A (EPA SW-846)
5601 (NIOSH)
D7645-10(ASTM)
524.2 (EPA OW)
5030C (EPA SW-846)
5035A (EPA SW-846)
8260C (EPA-SW846)
TO-15(EPAORD)
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (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)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
4500-CI G (SM)
Analyst (1999) 124(12): 1853-1857
5030C (EPA SW-846)
5035A (EPA SW-846)
8260C (EPA-SW846)
2513 (NIOSH)
TO-IOA(EPAORD)
Eur. J. Lipid Sci. Technol. (2011)
113: 345-355
J. Chromatogr. A (2000) 866:
65-77
Section
5.2.1
5.2.2
5.2.14
5.2.23
5.2.24
5.2.40
5.2.41
5.2.42
5.2.64
5.2.9
5.2.20
5.2.32
5.2.35
5.2.54
5.2.79
5.2.7
5.2.21
5.2.22
5.2.30
5.2.45
5.2.16
5.2.17
5.2.18
5.2.19
5.2.20
5.2.32
5.2.36
5.2.16
5.2.17
5.2.18
5.2.19
5.2.20
5.2.31
5.2.32
5.2.44
5.2.86
5.2.88
5.2.21
5.2.22
5.2.30
5.2.51
5.2.44
5.2.91
5.2.96
SAM 2012
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SAM 2012 Section 5.0- Selected Chemical Methods
Analyte
Chloropicrin
Chlorosarin
Chlorosoman
2-Chlorovinylarsonous acid (2-CVAA)
(analyze as total arsenic)
Chlorpyrifos
Chlorpyrifos oxon
Crimidine
Cyanide, Amenable to chlorination
Cyanide, Total
Cyanogen chloride
CASRN
76-06-2
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
551.1 (EPAOW)
3570 (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
PV2103(OSHA)
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
200.7 (EPAOW)
200.8 (EPA OW)
3050B (EPA SW-846)
601 OC (EPA SW-846)
6020A (EPA SW-846)
IO-3.1 (EPAORD)
IO-3.4 (EPA ORD)
IO-3.5 (EPAORD)
9102(NIOSH)
525.2 (EPA OW)
3511 (EPA SW-846)
3570 (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
525.2 (EPA OW)
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
3511 (EPA SW-846)
3570 (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
RLAB Method 3135.21
335.4 (EPA OW)
ISM01.3CN(EPACLP)
6010(NIOSH)
TO-15(EPAORD)
Encyclopedia of Anal. Chem. (2006)
DOI: 1 0. 1 002/978047002731 8.a0809
Section
5.2.12
5.2.20
5.2.31
5.2.32
5.2.72
5.2.16
5.2.17
5.2.18
5.2.19
5.2.20
5.2.31
5.2.32
5.2.44
5.2.1
5.2.2
5.2.14
5.2.23
5.2.24
5.2.40
5.2.41
5.2.42
5.2.64
5.2.8
5.2.15
5.2.20
5.2.31
5.2.32
5.2.44
5.2.8
5.2.16
5.2.17
5.2.18
5.2.19
5.2.20
5.2.31
5.2.32
5.2.44
5.2.15
5.2.20
5.2.31
5.2.32
5.2.39
5.2.5
5.2.38
5.2.57
5.2.45
5.2.90
SAM 2012
18
July 16, 2011
-------
SAM 2012 Section 5.0- Selected Chemical Methods
Analyte
Cyclohexyl sarin (GF)
1,2-Dichloroethane
Dichlorvos
Dicrotophos
Diesel range organics
Diisopropyl methylphosphonate (DIMP)
Dimethylphosphite
Dimethylphosphoramidic acid
CASRN
329-99-7
107-06-2
62-73-7
141-66-2
NA
1445-75-6
868-85-9
33876-51-6
Method
CWA Protocol (EPA NHSRC)
524.2 (EPA OW)
5030C (EPA SW-846)
5035A (EPA SW-846)
8260C (EPA-SW846)
TO-15(EPAORD)
525.2 (EPA OW)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (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)
801 5C (EPA SW-846)
8290A Appendix A (EPA SW-846)
538 (EPA OW)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
8321 B (EPA SW-846)
TO-IOA(EPAORD)
D7597-09 (ASTM)
E-2866-12(ASTM)
3570 (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
5035A (EPA SW-846)
8290A Appendix A (EPA SW-846)
8321 B (EPA SW-846)
TO-IOA(EPAORD)
Section
5.2.48
5.2.7
5.2.21
5.2.22
5.2.30
5.2.45
5.2.8
5.2.17
5.2.18
5.2.19
5.2.20
5.2.31
5.2.32
5.2.44
5.2.17
5.2.18
5.2.19
5.2.20
5.2.31
5.2.32
5.2.44
5.2.16
5.2.17
5.2.18
5.2.19
5.2.20
5.2.29
5.2.32
5.2.10
5.2.20
5.2.32
5.2.36
5.2.44
5.2.75
5.2.82
5.2.20
5.2.31
5.2.32
5.2.44
5.2.17
5.2.18
5.2.19
5.2.20
5.2.22
5.2.32
5.2.36
5.2.44
SAM 2012
19
July 16, 2011
-------
SAM 2012 Section 5.0- Selected Chemical Methods
Analyte
Diphacinone
Disulfoton
Disulfoton sulfone oxon
Disulfoton sulfoxide
Disulfoton sulfoxide oxon
1,4-Dithiane
EA21 92 [S-2-(diisopropylamino)ethyl
methylphosphonothioic acid]
Ethyl methylphosphonic acid (EMPA)
Ethyldichloroarsine (ED)
N-Ethyldiethanolamine (EDEA)
CASRN
82-66-6
298-04-4
2496-91-5
2497-07-6
2496-92-6
505-29-3
73207-98-4
1832-53-7
598-14-1
139-87-7
Method
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
8321 B (EPA SW-846)
D7644-10(ASTM)
525.2 (EPA OW)
3570 (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
5600(NIOSH)
525.2 (EPA OW)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
5600(NIOSH)
3511 (EPA SW-846)
3570 (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
8321 B (EPA SW-846)
TO-IOA(EPAORD)
8290A Appendix A (EPA SW-846)
8321 B (EPA SW-846)
TO-IOA(EPAORD)
D7597-09 (ASTM)
E-2866-12(ASTM)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
8270D (EPA SW-846)
TO-15(EPAORD)
9102(NIOSH)
3541 (EPA SW-846)
3545A (EPA SW-846)
8321 B (EPA SW-846)
TO-IOA(EPAORD)
EPA 600/R-1 1/143 (EPA / NIOSH)
D7599-09 (ASTM)
Section
5.2.18
5.2.19
5.2.20
5.2.32
5.2.36
5.2.79
5.2.8
5.2.20
5.2.31
5.2.32
5.2.53
5.2.8
5.2.18
5.2.19
5.2.20
5.2.31
5.2.32
5.2.53
5.2.15
5.2.20
5.2.31
5.2.32
5.2.17
5.2.18
5.2.19
5.2.20
5.2.32
5.2.36
5.2.44
5.2.32
5.2.36
5.2.44
5.2.75
5.2.82
5.2.17
5.2.18
5.2.19
5.2.31
5.2.45
5.2.64
5.2.18
5.2.19
5.2.36
5.2.44
5.2.47
5.2.77
SAM 2012
20
July 16, 2011
-------
SAM 2012 Section 5.0- Selected Chemical Methods
Analyte
Ethylene oxide
Fenamiphos
Fentanyl
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)
CASRN
75-21-8
22224-92-6
437-38-7
16984-48-8
640-19-7
NA
371-62-0
50-00-0
NA
121-82-4
283-66-9
Method
5030C (EPA SW-846)
5035A (EPA SW-846)
8260C (EPA SW-846)
TO-15(EPAORD)
525.2 (EPA OW)
3511 (EPA SW-846)
3570 (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)
8290A Appendix A (EPA SW-846)
8321 B (EPA SW-846)
300.1, Rev 1.0 (EPA OW)
J. Chromatogr. B (2008) 876(1):
103-108
S301-1 (NIOSH)
J. Chromatogr. A (2007) 1139:
271 -278
J. Chromatogr. B. (2010) 878:
1045-1050
5030C (EPA SW-846)
5035A (EPA SW-846)
8260C (EPA-SW846)
2513 (NIOSH)
556.1 (EPAOW)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
831 5A (EPA SW-846)
2016 (NIOSH)
3570 (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)
3535A (EPA SW-846)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
8330B (EPA SW-846)
Analyst (2001) 126: 1689-1693
Section
5.2.21
5.2.22
5.2.30
5.2.45
5.2.8
5.2.15
5.2.20
5.2.31
5.2.32
5.2.44
5.2.16
5.2.17
5.2.18
5.2.19
5.2.20
5.2.32
5.2.36
5.2.4
5.2.98
5.2.65
5.2.95
5.2.97
5.2.21
5.2.22
5.2.30
5.2.51
5.2.13
5.2.20
5.2.32
5.2.33
5.2.50
5.2.20
5.2.21
5.2.22
5.2.29
5.2.32
5.2.17
5.2.20
5.2.32
5.2.37
5.2.17
5.2.20
5.2.32
5.2.37
5.2.87
SAM 2012
21
July 16, 2011
-------
SAM 2012 Section 5.0- Selected Chemical Methods
Analyte
Hydrogen bromide
Hydrogen chloride
Hydrogen cyanide
Hydrogen fluoride
Hydrogen sulfide
Isopropyl methylphosphonic acid (IMPA)
Kerosene
Lead arsenate (analyze as total arsenic)
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine]
(analyze as total arsenic)
Lewisite 2 (L-2) [bis(2-
chlorovinyljchloroarsine] (analyze as total
arsenic)
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine]
(analyze as total arsenic)
Lewisite oxide (analyze as total arsenic)
Mercuric chloride (analyze as total mercury)
Mercury, Total
Methamidophos
Methomyl
CASRN
10035-10-6
7647-01-0
74-90-8
7664-39-3
2148878
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
10265-92-6
16752-77-5
Method
7903 (NIOSH)
6010(NIOSH)
7903 (NIOSH)
6013 (NIOSH)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
8321 B (EPA SW-846)
TO-IOA(EPAORD)
D7597-09 (ASTM)
E-2866-12(ASTM)
3570 (EPA SW-846)
5030C (EPA SW-846)
5035A (EPA SW-846)
801 5C (EPA SW-846)
8290A Appendix A (EPA SW-846)
200.7 (EPA OW)
200.8 (EPA OW)
3050B (EPA SW-846)
601 OC (EPA SW-846)
6020A (EPA SW-846)
IO-3.1 (EPAORD)
IO-3.4 (EPA ORD)
IO-3.5 (EPAORD)
9102 (NIOSH)
245.1 (EPAOW)
7473 (EPA SW-846)
9102 (NIOSH)
245.1 (EPAOW)
7473 (EPA SW-846)
IO-5 (EPA ORD)
9102 (NIOSH)
538 (EPA OW)
Chromatographia (2006) 63(5/6):
233-237
J. Chromatogr. A (2007) 1 1 54(1 ):
3-25
531. 2 (EPAOW)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
831 8A (EPA SW-846)
5601 (NIOSH)
D7645-10(ASTM)
Section
5.2.61
5.2.57
5.2.61
5.2.58
5.2.20
5.2.32
5.2.36
5.2.44
5.2.75
5.2.82
5.2.20
5.2.21
5.2.22
5.2.29
5.2.32
5.2.1
5.2.2
5.2.14
5.2.23
5.2.24
5.2.40
5.2.41
5.2.42
5.2.64
5.2.3
5.2.27
5.2.64
5.2.3
5.2.27
5.2.43
5.2.64
5.2.10
5.2.89
5.2.94
5.2.9
5.2.20
5.2.32
5.2.35
5.2.54
5.2.79
SAM 2012
22
July 16, 2011
-------
SAM 2012 Section 5.0- Selected Chemical Methods
Analyte
Methoxyethylmercuric acetate (analyze as
total mercury)
Methyl acrylonitrile
Methyl fluoroacetate (analyze as
fluoroacetate ion)
Methyl hydrazine
Methyl isocyanate
Methyl paraoxon
Methyl parathion
Methylamine
N-Methyldiethanolamine (MDEA)
1-Methylethyl ester ethylphosphonofluoridic
acid (GE)
CASRN
151-38-2
126-98-7
453-18-9
60-34-4
624-83-9
950-35-6
298-00-0
74-89-5
105-59-9
1189-87-3
Method
245.1 (EPAOW)
7473 (EPA SW-846)
IO-5 (EPA ORD)
9102(NIOSH)
524.2 (EPA OW)
3570 (EPA SW-846)
5035A (EPA SW-846)
8260C (EPA-SW846)
8290A Appendix A (EPA SW-846)
PV2004 (OSHA)
S301-1 (NIOSH)
J. Chromatogr. A (2007) 1139:
271 -278
J. Chromatogr. B (2010) 878:
1045-1050
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
3510 (NIOSH)
J. Chromatogr. (1993)617:
157-162
OSHA 54
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
OSHA 40
3541 (EPA SW-846)
3545A (EPA SW-846)
8321 B (EPA SW-846)
TO-IOA(EPAORD)
EPA 600/R-1 1/143 (EPA / NIOSH)
D7599-09 (ASTM)
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
Section
5.2.3
5.2.27
5.2.43
5.2.64
5.2.7
5.2.20
5.2.22
5.2.30
5.2.32
5.2.71
5.2.65
5.2.95
5.2.97
5.2.18
5.2.19
5.2.20
5.2.32
5.2.52
5.2.92
5.2.67
5.2.17
5.2.18
5.2.19
5.2.20
5.2.31
5.2.32
5.2.44
5.2.66
5.2.18
5.2.19
5.2.36
5.2.44
5.2.47
5.2.77
5.2.16
5.2.17
5.2.18
5.2.19
5.2.20
5.2.31
5.2.32
5.2.44
SAM 2012
23
July 16, 2011
-------
SAM 2012 Section 5.0- Selected Chemical Methods
Analyte
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)
CASRN
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
Method
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
8321 B (EPA SW-846)
TO-IOA(EPAORD)
D7597-09 (ASTM)
E-2866-12(ASTM)
525.2 (EPA OW)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (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)
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)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
CWA Protocol (EPA NHSRC)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
3535A (EPA SW-846)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
8330B (EPA SW-846)
Section
5.2.20
5.2.32
5.2.36
5.2.44
5.2.75
5.2.82
5.2.8
5.2.17
5.2.18
5.2.19
5.2.20
5.2.31
5.2.32
5.2.44
5.2.17
5.2.18
5.2.19
5.2.20
5.2.31
5.2.32
5.2.44
5.2.16
5.2.17
5.2.18
5.2.19
5.2.20
5.2.31
5.2.32
5.2.44
5.2.16
5.2.17
5.2.18
5.2.19
5.2.20
5.2.31
5.2.32
5.2.44
5.2.48
5.2.17
5.2.18
5.2.19
5.2.20
5.2.31
5.2.32
5.2.17
5.2.20
5.2.32
5.2.37
SAM 2012
24
July 16, 2011
-------
SAM 2012 Section 5.0- Selected Chemical Methods
Analyte
Osmium tetroxide (analyze as total osmium)
Oxamyl
Paraoxon
Parathion
Paraquat
Pentaerythritol tetranitrate (PETN)
Phencyclidine
Phorate
Phorate sulfone
Phorate sulfone oxon
Phorate sulfoxide
Phorate sulfoxide oxon
Phosgene
CASRN
20816-12-0
23135-22-0
311-45-5
56-38-2
4685-14-7
78-11-5
77-10-1
298-02-2
2588-04-7
2588-06-9
2588-03-6
2588-05-8
75-44-5
Method
200.7 (EPA OW)
200.8 (EPA OW)
3050B (EPA SW-846)
6010C(EPASW-846)
IO-3.1 (EPAORD)
IO-3.4 (EPA ORD)
9102(NIOSH)
531. 2 (EPA OW)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
831 8A (EPA SW-846)
5601 (NIOSH)
D7645-10(ASTM)
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
549.2 (EPA OW)
J. Chromatogr. A (2008) 1196-97:
110-116
3535A (EPA SW-846)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
8330B (EPA SW-846)
3511 (EPA SW-846)
3570 (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
OSHA61
Section
5.2.1
5.2.2
5.2.14
5.2.23
5.2.40
5.2.41
5.2.64
5.2.9
5.2.20
5.2.32
5.2.35
5.2.54
5.2.79
5.2.16
5.2.17
5.2.18
5.2.19
5.2.20
5.2.31
5.2.32
5.2.44
5.2.11
5.2.93
5.2.17
5.2.20
5.2.32
5.2.37
5.2.15
5.2.20
5.2.31
5.2.32
5.2.44
5.2.17
5.2.18
5.2.19
5.2.20
5.2.31
5.2.32
5.2.44
5.2.68
SAM 2012
25
July 16, 2011
-------
SAM 2012 Section 5.0- Selected Chemical Methods
Analyte
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)
Soman (GD)
Sodium arsenite (analyze as total arsenic)
Sodium azide (analyze as azide ion)
CASRN
13171-21-6
7803-51-2
2125683
616-52-4
75-56-9
159939-87-4
107-44-8
96-64-0
7784-46-5
26628-22-8
Method
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
6002 (NIOSH)
6402 (NIOSH)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
8321 B (EPA SW-846)
TO-IOA(EPAORD)
D7597-09 (ASTM)
E-2866-12(ASTM)
5030C (EPA SW-846)
5035A (EPA SW-846)
8260C (EPA-SW846)
1612 (NIOSH)
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
CWA Protocol (EPA NHSRC)
200.7 (EPA OW)
200.8 (EPA OW)
3050B (EPA SW-846)
601 OC (EPA SW-846)
6020A (EPA SW-846)
IO-3.1 (EPAORD)
IO-3.4 (EPA ORD)
IO-3.5 (EPAORD)
9102 (NIOSH)
300.1, Rev 1.0 (EPA OW)
ID-211 (OSHA)
J. Forensic Sci. (1998)43(1):
200-202
Section
5.2.16
5.2.17
5.2.18
5.2.19
5.2.20
5.2.31
5.2.32
5.2.44
5.2.56
5.2.60
5.2.20
5.2.32
5.2.36
5.2.44
5.2.75
5.2.82
5.2.21
5.2.22
5.2.30
5.2.49
5.2.16
5.2.17
5.2.18
5.2.19
5.2.20
5.2.31
5.2.32
5.2.44
5.2.48
5.2.1
5.2.2
5.2.14
5.2.23
5.2.24
5.2.40
5.2.41
5.2.42
5.2.64
5.2.4
5.2.69
5.2.99
SAM 2012
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SAM 2012 Section 5.0- Selected Chemical Methods
Analyte
Strychnine
Tabun (GA)
Tetraethyl pyrophosphate (TEPP)
Tetramethylenedisulfotetramine (TETS)
Thallium sulfate (analyze as total thallium)
Thiodiglycol (TDG)
Thiofanox
CASRN
57-24-9
77-81-6
107-49-3
80-12-6
10031-59-1
111-48-8
39196-18-4
Method
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
3511 (EPA SW-846)
3570 (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
3511 (EPA SW-846)
3570 (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
EPA 600/R-1 1/091 (EPA /CDC)
200.7 (EPA OW)
200.8 (EPA OW)
3050B (EPA SW-846)
601 OC (EPA SW-846)
6020A (EPA SW-846)
IO-3.1 (EPAORD)
IO-3.4 (EPA ORD)
IO-3.5 (EPAORD)
9102(NIOSH)
TO-IOA(EPAORD)
D7598-09 (ASTM)
E2787-11 (ASTM)
E2838-11 (ASTM)
538 (EPA OW)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
8290A Appendix A (EPA SW-846)
8321 B (EPA SW-846)
5601 (NIOSH)
D7645-10(ASTM)
Section
5.2.17
5.2.18
5.2.19
5.2.20
5.2.31
5.2.32
5.2.17
5.2.18
5.2.19
5.2.20
5.2.31
5.2.32
5.2.44
5.2.15
5.2.20
5.2.31
5.2.32
5.2.44
5.2.15
5.2.20
5.2.31
5.2.32
5.2.44
5.2.46
5.2.1
5.2.2
5.2.14
5.2.23
5.2.24
5.2.40
5.2.41
5.2.42
5.2.64
5.2.44
5.2.76
5.2.80
5.2.81
5.2.10
5.2.18
5.2.19
5.2.20
5.2.32
5.2.36
5.2.54
5.2.79
SAM 2012
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SAM 2012 Section 5.0- Selected Chemical Methods
Analyte
1,4-Thioxane
Titanium tetrachloride (analyze as total
titanium)
Triethanolamine (TEA)
Trimethyl phosphite
1 ,3,5-Trinitrobenzene (1 ,3,5-TNB)
2,4,6-Trinitrotoluene(2,4,6-TNT)
Vanadium pentoxide (analyze as total
vanadium)
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
15980-15-1
7550-45-0
102-71-6
121-45-9
99-35-4
118-96-7
1314-62-1
21738-25-0
78-53-5
21770-86-5
50782-69-9
12185-10-3
Method
3511 (EPASW-846)
3570 (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)
3541 (EPA SW-846)
3545A (EPA SW-846)
8321 B (EPA SW-846)
TO-IOA(EPAORD)
EPA 600/R-1 1/143 (EPA / NIOSH)
D7599-09 (ASTM)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (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)
3050B (EPA SW-846)
601 OC (EPA SW-846)
6020A (EPA SW-846)
IO-3.1 (EPAORD)
IO-3.4 (EPA ORD)
IO-3.5 (EPAORD)
9102 (NIOSH)
3520C (EPA SW-846)
3535A (EPA SW-846)
3541 (EPA SW-846)
3545A (EPA SW-846)
3570 (EPA SW-846)
8270D (EPA SW-846)
8290A Appendix A (EPA SW-846)
TO-IOA(EPAORD)
CWA Protocol (EPA NHSRC)
3570 (EPA SW-846)
7580 (EPA SW-846)
8290A Appendix A (EPA SW-846)
7905 (NIOSH)
Section
5.2.15
5.2.20
5.2.31
5.2.32
5.2.14
5.2.24
5.2.25
5.2.64
5.2.18
5.2.19
5.2.36
5.2.44
5.2.47
5.2.77
5.2.18
5.2.19
5.2.20
5.2.31
5.2.32
5.2.44
5.2.17
5.2.20
5.2.32
5.2.37
5.2.1
5.2.2
5.2.14
5.2.23
5.2.24
5.2.40
5.2.41
5.2.42
5.2.64
5.2.16
5.2.17
5.2.18
5.2.19
5.2.20
5.2.31
5.2.32
5.2.44
5.2.48
5.2.20
5.2.28
5.2.32
5.2.62
SAM 2012
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SAM 2012 Section 5.0- Selected Chemical Methods
Analyte | CAS RN | Method | Section
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
3-Chloro-1 ,2-propanediol
Chlorosarin
Chlorosoman
Crimidine
Diisopropyl methylphosphonate (DIMP)
Dimethylphosphoramidic acid
EA21 92 [S-2-(diisopropylamino)ethyl
methylphosphonothioic acid]
Hydrogen fluoride
Mercuric chloride (analyze as total mercury)
Mercury, Total
Methamidophos
Methoxyethylmercuric acetate (analyze as
total mercury)
1-Methylethyl ester ethylphosphonofluoridic
acid (GE)
Sarin (GB)
Soman (GD)
1,4-Thioxane
107-18-6
96-24-2
1445-76-7
7040-57-5
535-89-7
1445-75-6
33876-51-6
73207-98-4
7664-39-3
7487-94-7
7439-97-6
10265-92-6
151-38-2
1189-87-3
107-44-8
96-64-0
15980-15-1
TO-IOA(EPAORD)
TO-15(EPAORD)
TO-15(EPAORD)
8321 B (EPA SW-846)
TO-15(EPAORD)
8270D (EPA SW-846)
8270D (EPA SW-846)
7906 (NIOSH)
7470A (EPA SW-846)
7471 B (EPA SW-846)
5600 (NIOSH)
7470A (EPA SW-846)
7471 B (EPA SW-846)
TO-15(EPAORD)
TO-15(EPAORD)
5030C (EPA SW-846)
5035A (EPA SW-846)
8260C (EPA SW-846)
5.2.44
5.2.45
5.2.45
5.2.36
5.2.45
5.2.31
5.2.31
5.2.63
5.2.25
5.2.26
5.2.53
5.2.25
5.2.26
5.2.45
5.2.45
5.2.21
5.2.22
5.2.30
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
National Environmental Methods Index
(NEMI)
EPA Contract Laboratory Program
(CLP) Methods
EPA Office of Water (OW) Methods
EPA Solid Waste (SW)-846 Methods
EPA Office of Research and
Development (ORD) Methods
EPA Air Toxics Methods
Publisher
EPA, U.S. Geological Survey
(USGS)
EPA, CLP
EPAOW
EPA Office of Solid Waste and
Emergency Response
(OSWER)
EPA ORD
EPA Office of Air and Radiation
(OAR)
Reference
http://www.nemi.gov
http://www.epa.gov/superfund/proqr
ams/clp/
http://www.epa.aov/safewater/metho
ds/sourcalt.html
http://www.epa.gov/epaoswer/hazw
aste/test/main.htm
http://www.epa.gov/ttnamti1/
http://www.epa.gOV/ttn/amtic/airtox.h
trnl
SAM 2012
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SAM 2012 Section 5.0- Selected Chemical Methods
Name
EPA / Centers for Disease Control and
Prevention (CDC) / NIOSH Reports
EPA Analytical Protocols
Occupational Safety and Health
Administration (OSHA) Methods
NIOSH Methods
Standard Methods for the Examination
of Water and Wastewater (SM), 21st
Edition, 2005*
Annual Book ofASTM Standards*
GESTIS Substance Database
International Organization for
Standardization (ISO) Methods*
Official Methods of Analysis of AOAC
International*
Analyst*
Analytical Letters*
Journal of Chromatography A and B*
Journal of Forensic Sciences*
Chromatographia*
Encyclopedia of Analytical Chemistry*
European Journal of Lipid Science and
Technology*
EPA Water Contamination Information
Tool (WCIT)
Publisher
EPAORD, CDC
EPANHSRC
OSHA
NIOSH
American Public Health
Association (APHA), American
Waterworks Association
(AWWA) and Water
Environment Federation (WEF)
ASTM International
Institut fur Arbeitsschutz der
Deutschen Gesetzlichen
Unfallversicherung (IFA)
ISO
AOAC International
Royal Society of Chemistry
Taylor & Francis
Elsevier Science Publishers
ASTM International
Vieweg+Teubner
Wiley
Wiley
EPA OW Water Security
Division (WSD)
Reference
http://vwvw.epa.gov/nhsrc/news/new
s042407.html
and
http://cfpub.epa.qov/si/si public rec
ord_Report.cfm?dirEntrvld=2381 03
http://www.epa.gov/sam/contact us.
htm
http://www.osha.gov/dts/sltc/method
s/index.html
http://www.cdc.gov/niosh/nmam/
http://www.standardmethods.org
http://www.astm.org
http://www.dguv.de/ifa/en/gestis/stof
fdb/index.isp
http://www.iso.org
http://www.aoac.org
http://www.rsc.org/Publishing/Journ
als/AN/
http://www.informaworld.eom/smpp/t
itle~content=t71 3597227
http://www.iournals.elsevier.com/iou
rnal-of-chromatographv-a/
http://www.astm.org/DIGITAL LIBR
ARY/JOURNALS/FORENSIC/PAGE
S/JFS16113J.htm
http://www.springer.eom/chemistry/a
nalvtical+chemistrv/iournal/10337
http://www.wilev.com/WilevCDA/Wil
evTitle/productCd-0471 976709. html
http://www.wHev-
vch.de/publish/en/journals/alphabeti
clndex/2114/
http://www.epa.gov/wcit
SAM 2012
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SAM 2012 Section 5.0- Selected Chemical Methods
Name
Analytical Chemistry*
Journal of Agricultural and Food
Chemistry*
Publisher
American Chemical
Society(ACS)
ACS
Reference
http://pubs.acs.org/iournal/ancham
http://pubs.acs.org/iournal/iafcau
' Subscription and/or purchase required.
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.4 In addition to the laboratories being capable of
generating accurate and precise data during site remediation, 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 DQOs 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 (CWAs), most laboratories will not have access to analytical standards for
calibration and QC. Use of these agents is strictly controlled by the Department of Defense (DoD) and
access is limited. For information regarding laboratory analysis of samples containing CWAs or
laboratory requirements to possess and use ultra-dilute agent standards, please use the contact information
provided on the Environmental Response Laboratory Network (ERLN) website at:
http: //www. epa.gov/oemerln 1 /contact .html.
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 analytical protocols 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
> Method blanks
Information regarding EPA's DQO process, considerations, and planning is available at:
http://www.epa.gov/QUALITY/dqos.html.
SAM 2012
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July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Demonstration of analytical method suitability for intended use
> Detection and quantitation limits
> Precision and recovery (verify measurement system has adequate accuracy)
> 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
> Method blanks
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. Additional guidance can be found at:
www.epa.gov/qualitv/qatools.html; in Chapter 1 of EPA SW-846 "Test Methods for Evaluating Solid
Waste, Physical/Chemical Methods,"
(http://www.epa.gov/epawaste/hazard/testmethods/sw846/pdfs/chapl.pdf): and in EPA's 2005 "Manual
for the Certification of Laboratories Analyzing Drinking Water," (EPA 815-R-05-004)
(http://www.epa.gov/ogwdwOOO/methods/pdfs/manual labcertification.pdf). As with the identification of
needed QC samples, the frequency of QC sampling should be established based on an evaluation of
DQOs. 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 included within the data report when 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 DQOs. 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 ERLN or Water Laboratory Alliance (WLA) 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.
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 chemical, biological and/or radiological (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.
SAM2012 32 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
These methods also provide information regarding waste management. Other resources that can be
consulted for additional information include the following:
Centers for Disease Control and Prevention (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. Available at:
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. Available at: http://www.access.gpo.gov/nara/cfr/waisidx_07/40cfr270_07.html
OSHA - 29 CFR part 1910.1450. Occupational Exposure to Hazardous Chemicals in Laboratories.
Available at: http://www.access.gpo.gov/nara/cfr/waisidx_06/29cfrl910a_06.html
OSHA - 29 CFR part 1910.120. Hazardous Waste Operations and Emergency Response.
Please note that the Electronic Code of Federal Regulations (e-CFR) is available at
http://ecfr.gpoaccess.gov/.
5.2 Method Summaries
Summaries for the analytical methods listed in Appendix A are provided in Sections 5.2.1 through 5.2.99.
These sections contain summary information 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. Summaries are provided for informational use. Tiers that have been assigned to each
method/analyte pair (see Section 5.1.1) also are provided in Appendix A. The full version of the method
should be consulted prior to sample analysis.
5.2.1 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 (analyze as total vanadium)
Arsenic, Total
Arsenic trioxide (analyze as total arsenic)
Arsine (analyze as total arsenic in non-air samples)
Calcium arsenate (analyze as total arsenic)
2-Chlorovinylarsonous acid (2-CVAA) (analyze as total arsenic)
Lead arsenate (analyze as total arsenic)
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] (analyze as total arsenic)
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] (analyze as total arsenic)
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] (analyze as total arsenic)
Lewisite oxide (analyze as total arsenic)
Osmium tetroxide (analyze as total osmium)
Sodium arsenite (analyze as total arsenic)
Thallium sulfate (analyze as total thallium)
Vanadium pentoxide (analyze as total vanadium)
CASRN
7803-55-6
7440-38-2
1327-53-3
7784-42-1
7778-44-1
85090-33-1
7645-25-2
541-25-3
40334-69-8
40334-70-1
1306-02-1
20816-12-0
7784-46-5
10031-59-1
1314-62-1
SAM2012 33 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Analysis Purpose: Sample preparation, and analyte determination and measurement
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. See Appendix A for corresponding method usability tiers.
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. Users should consult with the appropriate point of contact listed in Section 4.0 regarding
use of graphite furnace atomic absorption spectrophotometry (GFAA) as a back-up or for additional
confirmatory analyses.
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
5.2.2 EPA Method 200.8: Determination of Trace Elements in Waters and Wastes by
Inductively Coupled Plasma-Mass Spectrometry
Analyte(s)
Ammonium metavanadate (analyze as total vanadium)
Arsenic, Total
Arsenic trioxide (analyze as total arsenic)
Arsine (analyze as total arsenic in non-air samples)
Calcium arsenate (analyze as total arsenic)
2-Chlorovinylarsonous acid (2-CVAA) (analyze as total arsenic)
Lead arsenate (analyze as total arsenic)
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] (analyze as total arsenic)
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] (analyze as total arsenic)
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] (analyze as total arsenic)
Lewisite oxide (analyze as total arsenic)
Osmium tetroxide (analyze as total osmium)
Sodium arsenite (analyze as total arsenic)
Thallium sulfate (analyze as total thallium)
Vanadium pentoxide (analyze as total vanadium)
CASRN
7803-55-6
7440-38-2
1327-53-3
7784-42-1
7778-44-1
85090-33-1
7645-25-2
541-25-3
40334-69-8
40334-70-1
1306-02-1
20816-12-0
7784-46-5
10031-59-1
1314-62-1
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SAM 2012 Section 5.0- Selected Chemical Methods
Analysis Purpose: Sample preparation, and analyte determination and measurement
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. See Appendix A for corresponding method usability tiers.
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. Users should consult with the appropriate point of contact listed in Section 4.0 regarding
use of GFAA as a back-up or for additional confirmatory analyses.
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
5.2.3 EPA Method 245.1: Determination of Mercury in Water by Cold Vapor Atomic
Absorption Spectrometry
Analyte(s)
Mercuric chloride (analyze as total mercury)
Mercury, Total
Methoxyethylmercuric acetate (analyze as total mercury)
CASRN
7487-94-7
7439-97-6
151-38-2
Analysis Purpose: Sample preparation, and analyte determination and measurement
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 aqueous liquid and drinking
water samples. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: Applicable concentration range is 0.2 to 10.0 ug Hg/L. The detection limit
for this method is 0.2 ug 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
SAM2012 35 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
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.
Special Considerations: If problems occur during analysis of aqueous liquid samples, refer to CVAA
Method 7470A (EPA SW-846).
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
5.2.4 EPA Method 300.1, Revision 1.0: Determination of Inorganic Anions in Drinking
Water by Ion Chromatography
Analyte(s)
Fluoride
Sodium azide (analyze as azide ion)
CASRN
16984-48-8
26628-22-8
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: For fluoride, use direct injection. For sodium azide, use water
extraction, filtration and acidification steps from the Journal of Forensic Science, 1998. 43(1):200-202
(solid samples), and filtration and acidification steps from this journal (aqueous liquid and drinking water
samples).
Determinative Technique: Ion chromatography (1C) with conductivity detection
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. It also should be used for analysis of solid, air and/or wipe samples for
sodium azide when appropriate sample preparation techniques have been applied. See Appendix A for
corresponding method usability tiers.
Detection and Quantitation: The detection limit for fluoride in reagent water is 0.009 mg/L. The MDL
varies depending upon the nature of the sample and the specific instrumentation employed. The estimated
calibration range should not extend over more than 2 orders of magnitude in concentration over the
expected concentration range of the samples.
Description of Method: This method was developed for analysis of aqueous samples, and can be
adapted for analysis of prepared 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 nano Siemens (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.
SAM2012 36 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
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
5.2.5 EPA Method 335.4: Determination of Total Cyanide by Semi-Automated
Colorimetry
Analyte(s)
Cyanide, Total
CASRN
57-12-5
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Reflux-distillation
Determinative Technique: Visible 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.
See Appendix A for corresponding method usability tiers.
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
5.2.6 EPA Method 350.1: Nitrogen, Ammonia (Colorimetric, Automated Phenate)
Analyte(s)
Ammonia
CASRN
7664-41-7
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Distillation
Determinative Technique: Visible 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.
See Appendix A for corresponding method usability tiers.
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
SAM2012 37 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
to the ammonia concentration. The blue color formed is intensified with sodium nitroprusside and
measured spectrophotometrically.
Special Considerations: Reduced volume distillation techniques, such as midi-distillation or micro-
distillation, can be used in place of traditional macro-distillation techniques.
Source: EPA. 1993. "Method 350.1: Nitrogen, Ammonia (Colorimetric, Automated Phenate)," Revision
2.0. http://www.epa.gov/sam/pdfs/EPA-350.1 .pdf
5.2.7 EPA Method 524.2: Measurement of Purgeable Organic Compounds in Water by
Capillary Column Gas Chromatography / Mass Spectrometry
Analyte(s)
Acrylonitrile
Carbon disulfide
1,2-Dichloroethane
Methyl acrylonitrile
CASRN
107-13-1
75-15-0
107-06-2
126-98-7
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Purge-and-trap
Determinative Technique: Gas chromatography-mass spectrometry (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 for
carbon disulfide and 1,2-dichloroethane, and preparation and analysis of drinking and aqueous/liquid
samples for acrylonitrile and methyl acrylonitrile. See Appendix A for corresponding method usability
tiers.
Detection and Quantitation: Detection levels for acrylonitrile, carbon disulfide, 1,2-dichloroethane and
methyl acrylonitrile in reagent water have been found to be 0.22, 0.093, 0.02 and 0.11 (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 gas
chromatography (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.
Special Considerations: The most recent version of this method (Method 524.3) requires
instrumentation, such as cryogenic auto samplers, which are not currently in common use. If laboratory
use of this equipment increases, Method 524.3 may be considered for SAM applications.
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
SAM2012 38 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
5.2.8 EPA Method 525.2: Determination of Organic Compounds in Drinking Water by
Liquid-Solid Extraction and Capillary Column Gas Chromatography / Mass
Spectrometry
Analyte(s)
Chlorpyrifos
Chlorpyrifos oxon1
Dichlorvos
Disulfoton
Disulfoton sulfone oxon1
Disulfoton sulfoxide
Disulfoton sulfoxide oxon1
Fenamiphos
Mevinphos
CASRN
2921-88-2
5598-15-2
62-73-7
298-04-4
2496-91-5
2497-07-6
2496-92-6
22224-92-6
7786-34-7
1 If problems occur when using this method for measurement of oxon compounds, analysts should consider use of
procedures included in "Oxidation of Selected Organophosphate Pesticides During Chlorination of Simulated Drinking
Water." Water Research. 2009. 43(2): 522-534. http://www.sciencedirect.com/science/iournal/00431354
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Liquid-solid extraction (LSE) or solid-phase extraction (SPE)
Determinative Technique: GC-MS
Method Developed for: Organic compounds in finished drinking water, source water or drinking water
in any treatment stage
Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid and/or
drinking water samples. For Chlorpyrifos in aqueous liquids, use Method 3511 for sample preparation
and Method 8270D for analysis. For Chlorpyrifos oxon in aqueous liquids, use Methods 3520C/3535A
for sample preparation and Method 8270D for analysis. See Appendix A for corresponding method
usability tiers.
Detection and Quantitation: The applicable concentration range for most analytes is 0.1 to 10 (ig/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: Refer to footnote provided in analyte table above for special considerations
that should be applied when measuring specific analytes. SPE using d8 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
SAM2012 39 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
5.2.9 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
CASRN
116-06-3
1646-88-4
1646-87-3
1563-66-2
16752-77-5
23135-22-0
Analysis Purpose: Sample preparation, and analyte determination and measurement
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.
See Appendix A for corresponding method usability tiers.
Detection and Quantitation: Detection limits range from 0.026 to 0.1 15 ug/L. The concentration
range for target analytes in this method was evaluated between 0.2 |^g/L and 10
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 (Qg)
column. After elution from the column, the analytes are hydrolyzed in a post column reaction to form
methylamine, which is in turn reacted to form a fluorescent isoindole that is detected by a fluorescence
(FL) detector. Analytes also are quantitated using the external standard technique.
Source: EPA. 2001. "Method 531.2: Measurement of N-Methylcarbamoyloximes and N-
Methylcarbamates in Water by Direct Aqueous Injection HPLC With Postcolumn Derivatization,"
Revision 1.0. http://www.epa.gov/sam/pdfs/EPA-53 1 .2.pdf
5.2.10 EPA Method 538: Determination of Selected Organic Contaminants in Drinking
Water by Direct Aqueous Injection-Liquid Chromatography/Tandem Mass
Spectrometry (DAI-LC/MS/MS)
Analyte(s)
Ace p hate
Diisopropyl methylphosphonate (DIMP)
Methamidophos
Thiofanox
CASRN
30560-19-1
1445-75-6
10265-92-6
39196-18-4
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Direct injection
Determinative Technique: Liquid Chromatography Tandem Mass Spectrometry (LC-MS-MS)
Method Developed for: Acephate, DIMP, methamidophos and thiofanox in drinking water samples
Method Selected for: SAM lists this method for preparation and analysis of drinking water samples.
See Appendix A for corresponding method usability tiers.
SAM 2012 40 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Detection and Quantitation: The MDLs for acephate, DIMP, methamidophos and thiofanox in reagent
water were calculated to be 0.019, 0.014, 0.017 and 0.090 (ig/L, respectively. The Lowest Common
Minimum Reporting Levels (LCMRLs) in reagent water were calculated to be 0.044, 0.022, 0.032 and
0.18 (ig/L, respectively
Description of Method: A 40-mL water sample is collected in a bottle containing sodium omadine and
ammonium acetate. An aliquot of the sample is placed in an autosampler vial and internal standards are
added. A 50-uL or larger injection is made into a liquid chromatograph (LC) equipped with a Qg column
that is interfaced to an MS-MS operated in the electrospray ionization (ESI) mode. The analytes are
separated and identified by comparing the acquired mass spectra and retention times to reference spectra
and retention times for calibration standards acquired under identical LC-MS-MS conditions. The
concentration of each analyte is determined by internal standard calibration using procedural standards.
Source: EPA. 2009. "Method 538: Determination of Selected Organic Contaminants in Drinking Water
by Direct Aqueous Injection-Liquid Chromatography/Tandem Mass Spectrometry (DAI-LC/MS/MS),"
Revision 1.0. http://www. epa.gov/sam/pdfs/EPA-538.pdf
5.2.11 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 analyte determination and measurement
Sample Preparation Technique: LSE or SPE
Determinative Technique: HPLC-ultraviolet (UV)
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. See Appendix A for corresponding method usability tiers.
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 a 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
SAM 2012 41 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
5.2.12 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 analyte determination and measurement
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
Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid and drinking
water samples. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The estimated detection limit (EDL) using methyl fert-butyl ether (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
5.2.13 EPA Method 556.1: Determination of Carbonyl Compounds in Drinking Water by
Fast Gas Chromatography
Analyte(s)
Formaldehyde
CASRN
50-00-0
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Liquid-liquid extraction with hexane
Determinative Technique: Fast gas Chromatography with electron capture detection (FGC-ECD)
Method Developed for: Formaldehyde in drinking water samples
Method Selected for: SAM lists this method for preparation and analysis of drinking water samples.
See Appendix A for corresponding method usability tiers.
SAM 2012 42 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Detection and Quantitation: MDLs for formaldehyde in reagent water were calculated as 0.09 and 0.08
(ig/L for primary and secondary columns, respectively. The applicable concentration range is
approximately 5 to 40 (ig/L.
Description of Method: A 20-mL volume of water sample is adjusted to pH 4 with potassium hydrogen
phthalate (KHP) and the analytes are derivatized at 35 ฐC for 2 hours with 15 mg of O-
(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine (PFBHA) reagent. The oxime derivatives are extracted
from the water with 4 mL of hexane. The extract is processed through an acidic wash step, and analyzed
by FGC-ECD. The target analytes are identified and quantified by comparison to a procedural standard.
Two chromatographic peaks will be observed for many of the target analytes. Both (E) and (Z) isomers
are formed for carbonyl compounds that are asymmetrical, and that are not sterically hindered. The (E)
and (Z) isomers may not be chromatographically resolved in a few cases. Compounds with two carbonyl
groups, such as glyoxal and methyl glyoxal, can produce even more isomers. Chromatographic peaks
used for analyte identification are provided in Section 17, Table 1 and Figure 1 of the method.
Special Considerations: All results should be confirmed on a second, dissimilar capillary GC column.
Source: EPA. 1999. "Method 556.1: Determination of Carbonyl Compounds in Drinking Water by Fast
Gas Chromatography," Revision 1.0. http://www.epa.gov/sam/pdfs/EPA-556.1 .pdf
5.2.14 EPA Method 3050B (SW-846): Acid Digestion of Sediments, Sludges, and Soils
Analyte(s)
Ammonium metavanadate (analyze as total vanadium)
Arsenic, Total
Arsenic trioxide (analyze as total arsenic)
Arsine (analyze as total arsenic in non-air samples)
Calcium arsenate (analyze as total arsenic)
2-Chlorovinylarsonous acid (2-CVAA) (analyze as total arsenic)
Lead arsenate (analyze as total arsenic)
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] (analyze as total arsenic)
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] (analyze as total arsenic)
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] (analyze as total arsenic)
Lewisite oxide (analyze as total arsenic)
Osmium tetroxide (analyze as total osmium)
Sodium arsenite (analyze as total arsenic)
Thallium sulfate (analyze as total thallium)
Titanium tetrachloride (analyze as total titanium)
Vanadium pentoxide (analyze as total vanadium)
CASRN
7803-55-6
7440-38-2
1327-53-3
7784-42-1
7778-44-1
85090-33-1
7645-25-2
541-25-3
40334-69-8
40334-70-1
1306-02-1
20816-12-0
7784-46-5
10031-59-1
7550-45-0
1314-62-1
Analysis Purpose: Sample preparation
Sample Preparation Technique: Acid digestion
Determinative Technique: ICP-AES / ICP-MS
Determinative Method: EPA SW-846 Method 6010C or Method 6020A. 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. See Appendix A for
corresponding method usability tiers.
SAM 2012
43
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SAM 2012 Section 5.0- Selected Chemical Methods
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
5.2.15 EPA Method 3511 (SW-846): Organic Compounds in Water by Microextraction
Analyte(s)
Chlorpyrifos
Crimidine
1,4-Dithiane
Fenamiphos
Phencyclidine
Tetraethyl pyrophosphate (TEPP)
Tetramethylenedisulfotetramine (TETS)
1,4-Thioxane
CASRN
2921-88-2
535-89-7
505-29-3
22224-92-6
77-10-1
107-49-3
80-12-6
15980-15-1
Analysis Purpose: Sample preparation
Sample Preparation Technique: Microextraction
Determinative Technique: GC-MS
Determinative Method: EPA SW-846 Method 8270D
Method Developed for: Volatile, semivolatile and nonvolatile organic compounds in water
Method Selected for: SAM lists this method for preparation of aqueous liquid and drinking water
samples. Drinking water samples for TETS should be prepared and analyzed using EPA 600/R-l 1/091.
Drinking water samples for chlorpyrifos and fenamiphos should be prepared and analyzed using EPA
Method 525.2. See Appendix A for corresponding method usability tiers.
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, but no longer than 14 days from the date of collection for acid
preserved samples. If samples are not acidified, the extraction should be performed within 7 days from
the date of collection. Exposure to air before sample extraction should be minimized as much as possible.
SAM 2012 44 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Source: EPA. 2002. "Method 3511 (SW-846): Organic Compounds in Water by Microextraction,"
Revision 0. http://www.epa.gov/sam/EPA-3511 .pdf
5.2.16 EPA Method 3520C (SW-846): Continuous Liquid-Liquid Extraction
Analyte(s)
BZ [Quinuclidinyl benzilate]
Carfentanil
Chlorfenvinphos
Chlorosarin
Chlorosoman
Chlorpyrifos oxon
Diesel range organics
Fentanyl
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
Phosphamidon
R 33 (VR) [methylphosphonothioic acid, S-[2-(diethylamino)ethyl] O-2-methylpropyl ester]
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
6581-06-2
59708-52-0
470-90-6
1445-76-7
7040-57-5
5598-15-2
NA
437-38-7
1189-87-3
538-07-8
51-75-2
555-77-1
311-45-5
56-38-2
13171-21-6
159939-87-4
21738-25-0
78-53-5
21770-86-5
Analysis Purpose: Sample preparation
Sample Preparation Technique: Continuous liquid-liquid extraction (CLLE)
Determinative Technique: Gas chromatography-flame ionization detector (GC-FID) / GC-MS / HPLC
Determinative Method: 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 chlorpyrifos oxon should be prepared and analyzed
using EPA Method 525.2. See Appendix A for corresponding method usability tiers.
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.
Source: EPA. 1996. "Method 3520C (SW-846): Continuous Liquid-Liquid Extraction," Revision 3.
http://www.epa.gov/sam/pdfs/EPA-3520c.pdf
SAM 2012
45
July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
5.2.17 EPA Method 3535A (SW-846): Solid-Phase Extraction
Analyte(s)
4-Aminopyridine
BZ [Quinuclidinyl benzilate]
Carfentanil
Chlorfenvinphos
Chlorosarin
Chlorosoman
Chlorpyrifos oxon
Dichlorvos
Dicrotophos
Diesel range organics
Dimethylphosphoramidic acid
EA2192 [S-2-(diisopropylamino)ethyl methylphosphonothioic acid]
Ethyldichloroarsine (ED)
Fentanyl
Hexamethylenetriperoxidediamine (HMTD)
Hexahydro-1 ,3,5-trinitro-1 ,3,5-triazine (RDX)
Methyl paraoxon
Methyl parathion
1-Methylethyl ester ethylphosphonofluoridic acid (GE)
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)
Phorate
Phorate sulfone
Phorate sulfone oxon1
Phorate sulfoxide
Phorate sulfoxide oxon1
Phosphamidon
R 33 (VR) [methylphosphonothioic acid, S-[2-(diethylamino)ethyl] O-2-methylpropyl ester]
Strychnine
Tabun (GA)
1 ,3,5-Trinitrobenzene (1 ,3,5-TNB)
2,4,6-Trinitrotoluene(2,4,6-TNT)
VE [phosphonothioic acid, ethyl-, S-(2-(diethylamino)ethyl) O-ethyl ester]
VG [phosphonothioic acid, S-(2-(diethylamino)ethyl) O,O-diethyl ester]
VM [phosphonothioic acid, methyl-, S-(2-(diethylamino)ethyl) O-ethyl ester]
CASRN
504-24-5
6581-06-2
59708-52-0
470-90-6
1445-76-7
7040-57-5
5598-15-2
62-73-7
141-66-2
NA
33876-51-6
73207-98-4
598-14-1
437-38-7
283-66-9
121-82-4
950-35-6
298-00-0
1189-87-3
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
298-02-2
2588-04-7
2588-06-9
2588-03-6
2588-05-8
13171-21-6
159939-87-4
57-24-9
77-81-6
99-35-4
118-96-7
21738-25-0
78-53-5
21770-86-5
1 If problems occur when using this method for measurement of oxon compounds, analysts should consider use of
procedures included in "Oxidation of Selected Organophosphate Pesticides During Chlorination of Simulated Drinking
Water." Water Research. 2009. 43(2): 522-534. http://www.sciencedirect.com/science/iournal/00431354
Analysis Purpose: Sample preparation
Sample Preparation Technique: SPE
SAM 2012
46
July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Determinative Technique: GC-FID / GC-MS / HPLC
Determinative Method: 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 chlorpyrifos oxon, dichlorvos and mevinphos should
be prepared and analyzed by EPA Method 525.2. All other drinking water samples and all aqueous liquid
samples should be prepared using this method (SW-846 Method 3535A). See Appendix A for
corresponding method usability tiers.
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: Refer to footnote provided in analyte table above for special considerations
that should be applied when measuring specific analytes. 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-3 5 3 5 a.pdf
5.2.18 EPA Method 3541 (SW-846): Automated Soxhlet Extraction
Analyte(s)
Brodifacoum
Bromadiolone
BZ [Quinuclidinyl benzilate]
Carfentanil
Chlorfenvinphos
Chlorosarin
Chlorosoman
Chlorpyrifos oxon
Dichlorvos
Dicrotophos
Diesel range organics
Dimethylphosphoramidic acid
Diphacinone
Disulfoton sulfone oxon1
Disulfoton sulfoxide
Disulfoton sulfoxide oxon1
EA2192 [S-2-(diisopropylamino)ethyl methylphosphonothioic acid]
Ethyldichloroarsine (ED)
N-Ethyldiethanolamine (EDEA)
Fentanyl
Methyl hydrazine
CASRN
56073-10-0
28772-56-7
6581-06-2
59708-52-0
470-90-6
1445-76-7
7040-57-5
5598-15-2
62-73-7
141-66-2
NA
33876-51-6
82-66-6
2496-91-5
2497-07-6
2496-92-6
73207-98-4
598-14-1
139-87-7
437-38-7
60-34-4
SAM 2012
47
July 16, 2011
-------
SAM 2012 Section 5.0- Selected Chemical Methods
Analyte(s)
Methyl paraoxon
Methyl parathion
N-Methyldiethanolamine (MDEA)
1-Methylethyl ester ethylphosphonofluoridic acid (GE)
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
Paraoxon
Parathion
Phorate
Phorate sulfone
Phorate sulfone oxon1
Phorate sulfoxide
Phorate sulfoxide oxon1
Phosphamidon
R 33 (VR) [methylphosphonothioic acid, S-[2-(diethylamino)ethyl] O-2-methylpropyl ester]
Strychnine
Tabun (GA)
Thiofanox
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
950-35-6
298-00-0
105-59-9
1189-87-3
7786-34-7
6923-22-4
538-07-8
51-75-2
555-77-1
54-11-5
311-45-5
56-38-2
298-02-2
2588-04-7
2588-06-9
2588-03-6
2588-05-8
13171-21-6
159939-87-4
57-24-9
77-81-6
39196-18-4
102-71-6
121-45-9
21738-25-0
78-53-5
21770-86-5
1 If problems occur when using this method for measurement of oxon compounds, analysts should consider use of
procedures included in "Oxidation of Selected Organophosphate Pesticides During Chlorination of Simulated Drinking
Water." Water Research. 2009. 43(2): 522-534. http://www.sciencedirect.com/science/iournal/00431354
Analysis Purpose: Sample preparation
Sample Preparation Technique: Automated Soxhlet extraction
Determinative Technique: GC-FID / GC-MS / HPLC
Determinative Method: 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. See Appendix A for
corresponding method usability tiers.
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: Refer to footnote provided in analyte table above for special considerations
that should be applied when measuring specific analytes. Some of the target compounds will hydrolyze
in water, with hydrolysis rates dependant on various factors such as sample pH and temperature.
SAM 2012
48
July 16, 2011
-------
SAM 2012 Section 5.0- Selected Chemical Methods
Source: EPA. 1994. "Method 3541 (SW-846): Automated Soxhlet Extraction," Revision 0.
http://www.epa.gov/sam/pdfs/EPA-3541 .pdf
5.2.19 EPA Method 3545A (SW-846): Pressurized Fluid Extraction (PFE)
Analyte(s)
Brodifacoum
Bromadiolone
BZ [Quinuclidinyl benzilate]
Carfentanil
Chlorfenvinphos
Chlorosarin
Chlorosoman
Chlorpyrifos oxon
Dichlorvos
Dicrotophos
Diesel range organics
Dimethylphosphoramidic acid
Diphacinone
Disulfoton sulfone oxon1
Disulfoton sulfoxide
Disulfoton sulfoxide oxon1
EA2192 [S-2-(diisopropylamino)ethyl methylphosphonothioic acid]
Ethyldichloroarsine (ED)
N-Ethyldiethanolamine (EDEA)
Fentanyl
Methyl hydrazine
Methyl paraoxon
Methyl parathion
N-Methyldiethanolamine (MDEA)
1-Methylethyl ester ethylphosphonofluoridic acid (GE)
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
Paraoxon
Parathion
Phorate
Phorate sulfone
Phorate sulfone oxon1
Phorate sulfoxide
Phorate sulfoxide oxon1
Phosphamidon
R 33 (VR) [methylphosphonothioic acid, S-[2-(diethylamino)ethyl] O-2-methylpropyl ester]
Strychnine
Tabun (GA)
Thiofanox
CASRN
56073-10-0
28772-56-7
6581-06-2
59708-52-0
470-90-6
1445-76-7
7040-57-5
5598-15-2
62-73-7
141-66-2
NA
33876-51-6
82-66-6
2496-91-5
2497-07-6
2496-92-6
73207-98-4
598-14-1
139-87-7
437-38-7
60-34-4
950-35-6
298-00-0
105-59-9
1189-87-3
7786-34-7
6923-22-4
538-07-8
51-75-2
555-77-1
54-11-5
311-45-5
56-38-2
298-02-2
2588-04-7
2588-06-9
2588-03-6
2588-05-8
13171-21-6
159939-87-4
57-24-9
77-81-6
39196-18-4
SAM 2012
49
July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Analyte(s)
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
102-71-6
121-45-9
21738-25-0
78-53-5
21770-86-5
1 If problems occur when using this method for measurement of oxon compounds, analysts should consider use of
procedures included in "Oxidation of Selected Organophosphate Pesticides During Chlorination of Simulated Drinking
Water." Water Research. 2009. 43(2): 522-534. http://www.sciencedirect.com/science/iournal/00431354
Analysis Purpose: Sample preparation
Sample Preparation Technique: PFE
Determinative Technique: GC-FID / GC-MS / HPLC
Determinative Method: 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. See Appendix A for
corresponding method usability tiers.
Detection and Quantitation: This method has been validated for solid matrices containing 250 to
12,500 ug/kg of semivolatile organic compounds, 250 to 2500 ug/kg of organophosphorus pesticides, 5 to
250 ug/kg of organochlorine pesticides, 50 to 5000 ug/kg of chlorinated herbicides, and 1 to 2500 ng/kg
of polychlorinated dibenzo-/?-dioxins (PCDDs) /polychlorinated dibenzofurans (PCDFs).
Description of Method: Approximately 10 to 30 g of soil sample is prepared for extraction either by air
drying the sample, or by mixing the sample with anhydrous sodium sulfate or pelletized diatomaceous
earth. The sample is then ground and loaded into the extraction cell. The extraction cell containing the
sample is heated to the extraction temperature, pressurized with the appropriate solvent system, and
extracted for 5 minutes (or as recommended by the instrument manufacturer). The extract may be
concentrated, if necessary, and exchanged into a solvent compatible with the cleanup or determinative
step being employed.
Special Considerations: Refer to footnote provided in analyte table above for special considerations
that should be applied when measuring specific analytes. Sodium sulfate can cause clogging, and air-
drying or pelletized diatomaceous earth may be preferred. 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
5.2.20 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
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
SAM 2012
50
July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Analyte(s)
Carbofuran (Furadan)
Chlorfenvinphos
Chloropicrin
Chlorosarin
Chlorosoman
Chlorpyrifos
Chlorpyrifos oxon
Crimidine
Dichlorvos
Dicrotophos
Diesel range organics
Diisopropyl methylphosphonate (DIMP)
Dimethylphosphite
Dimethylphosphoramidic acid
Diphacinone
Disulfoton
Disulfoton sulfone oxon1
Disulfoton sulfoxide
Disulfoton sulfoxide oxon1
1,4-Dithiane
EA2192 [S-2-(diisopropylamino)ethyl methylphosphonothioic acid]
Ethyl methylphosphonic acid (EMPA)
Fenamiphos
Fentanyl
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 hydrazine
Methyl paraoxon
Methyl parathion
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]
Nicotine compounds
Octahydro-1 ,3,5,7-tetranitro-1 ,3,5,7-tetrazocine (HMX)
Oxamyl
Paraoxon
Parathion
Pentaerythritol tetranitrate (PETN)
Phencyclidine
Phorate
CASRN
1563-66-2
470-90-6
76-06-2
1445-76-7
7040-57-5
2921-88-2
5598-15-2
535-89-7
62-73-7
141-66-2
NA
1445-75-6
868-85-9
33876-51-6
82-66-6
298-04-4
2496-91-5
2497-07-6
2496-92-6
505-29-3
73207-98-4
1832-53-7
22224-92-6
437-38-7
50-00-0
NA
121-82-4
283-66-9
1832-54-8
64742-81-0
16752-77-5
126-98-7
60-34-4
950-35-6
298-00-0
1189-87-3
993-13-5
7786-34-7
6923-22-4
538-07-8
51-75-2
555-77-1
54-11-5
2691-41-0
23135-22-0
311-45-5
56-38-2
78-11-5
77-10-1
298-02-2
SAM 2012
51
July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Analyte(s)
Phorate sulfone
Phorate sulfone oxon1
Phorate sulfoxide
Phorate sulfoxide oxon1
Phosphamidon
Pinacolyl methyl phosphonic acid (PMPA)
R 33 (VR) [methylphosphonothioic acid, S-[2-(diethylamino)ethyl] O-2-methylpropyl ester]
Strychnine
Tabun (GA)
Tetraethyl pyrophosphate
Tetramethylenedisulfotetramine
Thiofanox
1,4-Thioxane
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]
White phosphorus
CASRN
2588-04-7
2588-06-9
2588-03-6
2588-05-8
13171-21-6
616-52-4
159939-87-4
57-24-9
77-81-6
107-49-3
80-12-6
39196-18-4
15980-15-1
121-45-9
99-35-4
118-96-7
21738-25-0
78-53-5
21770-86-5
12185-10-3
1 If problems occur when using this method for measurement of oxon compounds, analysts should consider use of
procedures included in "Oxidation of Selected Organophosphate Pesticides During Chlorination of Simulated Drinking
Water." Water Research. 2009. 43(2): 522-534. http://www.sciencedirect.com/science/iournal/00431354
Analysis Purpose: Sample preparation
Sample Preparation Technique: MSB
Determinative Technique: Gas chromatography - nitrogen-phosphorus detector (GC-NPD) / GC-FID /
GC-MS / HPLC
Determinative Method: 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 solid and wipe samples. Some of the
analytes listed in the table above have been assigned a different method for preparation of soil samples.
See Appendix A for appropriate soil sample preparation methods and for corresponding method usability
tiers.
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.
SAM 2012
52
July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Special Considerations: Refer to footnote provided in analyte table above for special considerations
that should be applied when measuring specific analytes.
Source: EPA. 2002. "Method 3570 (SW-846): Microscale Solvent Extraction (MSB)," Revision 0.
http: //www. epa.gov/sam/pdfs/EPA-3 5 70 .pdf
5.2.21 EPA Method 5030C (SW-846): Purge-and-Trap for Aqueous Samples
Analyte(s)
Allyl alcohol
Carbon disulfide
2-Chloroethanol
1,2-Dichloroethane
Ethylene oxide
2-Fluoroethanol
Gasoline range organics
Kerosene
Propylene oxide
CASRN
107-18-6
75-15-0
107-07-3
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: GC-FID / GC-MS
Determinative Method: EPA SW-846 Method 8015C or Method 8260C. Refer to Appendix A for
which of these determinative methods should be used for a particular analyte.
Method Developed for: VOCs in aqueous and water miscible liquid samples
Method Selected for: SAM lists this method for preparation of aqueous liquid and/or drinking water
samples. For carbon disulfide and 1,2-dichloroethane, EPA Method 524.2 (rather than Method 5030C)
should be used for preparation of drinking water samples. See Appendix A for corresponding method
usability tiers.
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
SAM2012 53 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
5.2.22 EPA Method 5035A (SW-846): Closed-System Purge-and-Trap and Extraction for
Volatile Organics in Soil and Waste Samples
Analyte(s)
Acrylonitrile
Allyl alcohol
Carbon disulfide
2-Chloroethanol
1,2-Dichloroethane
Ethylene oxide
2-Fluoroethanol
Gasoline range organics
Kerosene
Methyl acrylonitrile
Propylene oxide
CASRN
107-13-1
107-18-6
75-15-0
107-07-3
107-06-2
75-21-8
371-62-0
NA
64742-81-0
126-98-7
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: GC-FID / GC-MS
Determinative Method: 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. See Appendix A for
corresponding method usability tiers.
Description of Method: This method describes a closed-system purge-and-trap process for analysis of
VOCs in solid samples containing low levels (0.5 to 200 ug/kg) of VOCs. The method also provides
specific procedures for preparation of samples containing high levels (>200 ug/kg) of VOCs. For low-
level VOCs, a 5-g sample is collected into a vial that is placed into an autosampler device. Reagent
water, surrogates and internal standards are added automatically, and the vial is heated to 40 degrees
Celsius (ฐC). The volatiles are purged into an appropriate trap using an inert gas combined with sample
agitation. When purging is complete, the trap is heated and backflushed with helium to desorb the
trapped sample components into a GC for analysis. For high-level VOCs, samples are either collected
into a vial that contains a water-miscible organic solvent or a portion of sample is removed from the vial
and dispersed in a water-miscible solvent. An aliquot of the solvent is added to reagent water, along with
surrogates and internal standards, then purged and analyzed using an appropriate determinative method
[e.g., Method 8015C or 8260C (SW-846)].
Source: EPA. 2002. "Method 5035A (SW-846): Closed-System Purge-and-Trap and Extraction for
Volatile Organics in Soil and Waste Samples," Draft Revision 1. http://www.epa.gov/sam/pdfs/EPA-
5035a.pdf
5.2.23 EPA Method 601OC (SW-846): Inductively Coupled Plasma - Atomic Emission
Spectrometry
Analyte(s)
Ammonium metavanadate (analyze as total vanadium)
Arsenic, Total
CASRN
7803-55-6
7440-38-2
SAM 2012 54 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Analyte(s)
Arsenic trioxide (analyze as total arsenic)
Arsine (analyze as total arsenic in non-air samples)
Calcium arsenate (analyze as total arsenic)
2-Chlorovinylarsonous acid (2-CVAA) (analyze as total arsenic)
Lead arsenate (analyze as total arsenic)
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] (analyze as total arsenic)
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] (analyze as total arsenic)
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] (analyze as total arsenic)
Lewisite oxide (analyze as total arsenic)
Osmium tetroxide (analyze as total osmium)
Sodium arsenite (analyze as total arsenic)
Thallium sulfate (analyze as total thallium)
Titanium tetrachloride (analyze as total titanium)
Vanadium pentoxide (analyze as total vanadium)
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: Analyte determination and measurement
Determinative Technique: ICP-AES
Sample Preparation Method: EPA SW-846 Method 3050B (solid samples) and NIOSH Method 9102
(wipe samples)
Sample Preparation Technique: Acid digestion
Method Developed for: Trace elements in solution
Method Selected for: SAM lists this method for analysis of solid and wipe samples. See Appendix A
for corresponding method usability tiers.
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) 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. Users should consult with the appropriate point of contact listed in Section 4.0 regarding
use of GFAA as a back-up or for additional confirmatory analyses.
Source: EPA. 2007. "Method 6010C (SW-846): Inductively Coupled Plasma-Atomic Emission
Spectrometry," Revision 3. http://www.epa.gov/sam/pdfs/EPA-6010c.pdf
5.2.24 EPA Method 6020A (SW-846): Inductively Coupled Plasma - Mass Spectrometry
Analyte(s)
Ammonium metavanadate (analyze as total vanadium)
Arsenic, Total
Arsenic trioxide (analyze as total arsenic)
Arsine (analyze as total arsenic in non-air samples)
Calcium arsenate (analyze as total arsenic)
2-Chlorovinylarsonous acid (2-CVAA) (analyze as total arsenic)
CASRN
7803-55-6
7440-38-2
1327-53-3
7784-42-1
7778-44-1
85090-33-1
SAM 2012
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July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Analyte(s)
Lead arsenate (analyze as total arsenic)
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] (analyze as total arsenic)
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] (analyze as total arsenic)
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] (analyze as total arsenic)
Lewisite oxide (analyze as total arsenic)
Sodium arsenite (analyze as total arsenic)
Thallium sulfate (analyze as total thallium)
Titanium tetrachloride (analyze as total titanium)
Vanadium pentoxide (analyze as total vanadium)
CASRN
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: Analyte determination and measurement
Determinative Technique: ICP-MS
Sample Preparation Method: EPA SW-846 Method 3050B (solid samples) and NIOSH Method 9102
(wipe samples)
Sample Preparation Technique: Acid digestion
Method Developed for: Elements in water samples and in waste extracts or digests
Method Selected for: SAM lists this method for analysis of solid and wipe samples. See Appendix A
for corresponding method usability tiers.
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) 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. Users should consult with the appropriate point of contact listed in Section 4.0 regarding
use of GFAA as a back-up or for additional confirmatory analyses.
Source: EPA. 1998. "Method 6020A (SW-846): Inductively Coupled Plasma-Mass Spectrometry,"
Revision 1. http://www.epa.gov/sam/pdfs/EPA-6020a.pdf
5.2.25 EPA Method 7470A (SW-846): Mercury in Liquid Wastes (Manual Cold-Vapor
Technique)
Analyte(s)
Mercuric chloride (analyze as total mercury)
Mercury, Total
Methoxyethylmercuric acetate (analyze as total mercury)
CASRN
7487-94-7
7439-97-6
151-38-2
Analysis Purpose: Sample preparation, and/or analyte determination and measurement
Sample Preparation Technique: Acid digestion (solid and aqueous liquid samples) and acid digestion
by NIOSH Method 9102 (wipe samples)
Determinative Technique: CVAA
SAM 2012 56 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Method Developed for: Mercury in mobility-procedure extracts, aqueous wastes and ground waters
Method Selected for: SAM lists this method for reference if problems occur when using EPA Method
245.1 for these analytes during preparation and analysis of aqueous liquid samples. (See Footnote 9 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
5.2.26 EPA Method 7471B (SW-846): Mercury in Solid or Semisolid Wastes (Manual Cold-
Vapor Technique)
Analyte(s)
Mercuric chloride (analyze as total mercury)
Mercury, Total
Methoxyethylmercuric acetate (analyze as total mercury)
CASRN
7487-94-7
7439-97-6
151-38-2
Analysis Purpose: Sample preparation, and/or analyte determination and measurement
Sample Preparation Technique: Acid digestion (solid and aqueous liquid samples) and acid digestion
by 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
8 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
5.2.27 EPA Method 7473 (SW-846): Mercury in Solids and Solutions by Thermal
Decomposition, Amalgamation, and Atomic Absorption Spectrophotometry
Analyte(s)
Mercuric chloride (analyze as total mercury)
Mercury, Total
Methoxyethylmercuric acetate (analyze as total mercury)
CASRN
7487-94-7
7439-97-6
151-38-2
SAM 2012 57 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Analysis Purpose: Sample preparation, and/or analyte determination and measurement
Sample Preparation Technique: Thermal decomposition (solid and aqueous liquid samples) and acid
digestion by NIOSH Method 9102 (wipe samples)
Determinative Technique: Visible 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 and wipe samples.
See Appendix A for corresponding method usability tiers.
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 7471B (EPA SW-846) for
solid 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
5.2.28 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 analyte determination and measurement
Sample Preparation Technique: Solvent extraction (solid, aqueous liquid and drinking water samples)
and MSB / solvent extraction by EPA SW-846 Method 3570/8290A Appendix A (wipe samples)
Determinative Technique: GC-NPD
Method Developed for: White phosphorus in soil, sediment and water
Method Selected for: SAM lists this method for preparation and analysis of solid, aqueous liquid,
drinking water and wipe samples. See Appendix A for corresponding method usability tiers.
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
SAM2012 58 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
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 a nitrogen-phosphorus detector (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 \\L 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
5.2.29 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: Analyte determination and measurement
Determinative Technique: GC-FID
Sample Preparation Method: EPA SW-846 Method 3541/3545A or Method 5035A (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.
Sample Preparation Technique: Automated Soxhlet extraction / PFE / purge-and-trap (solid samples),
SPE / purge-and-trap (aqueous liquid and drinking water samples), and MSE / solvent extraction (wipe
samples).
Method Developed for: Various nonhalogenated VOCs and semivolatile organic compounds in water
samples
Method Selected for: SAM lists this method for analysis of solid, aqueous liquid, drinking water and
wipe samples. See Appendix A for corresponding method usability tiers.
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,
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
SAM2012 59 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
5.2.30 EPA Method 8260C (SW-846): Volatile Organic Compounds by Gas
Chromatography-Mass Spectrometry (GC/MS)
Analyte(s)
Acrylonitrile
Allyl alcohol
Carbon disulfide
2-Chloroethanol
1,2-Dichloroethane
Ethylene oxide
2-Fluoroethanol
Methyl acrylonitrile
Propylene oxide
CASRN
107-13-1
107-18-6
75-15-0
107-07-3
107-06-2
75-21-8
371-62-0
126-98-7
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: Analyte determination and measurement
Determinative Technique: GC-MS
Sample Preparation Method: EPA SW-846 Method 5035A (solid samples), Method 5030C (aqueous
liquid and drinking water samples), and Method 3570/8290A Appendix A (wipe samples).
Sample Preparation Technique: Purge-and-trap (solid samples, aqueous liquid and drinking water
samples) and MSB / solvent extraction (wipe samples).
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, aqueous liquid, drinking water and/or
wipe samples. For acrylonitrile, carbon disulfide, 1,2-dichloroethane and methyl acrylonitrile only, EPA
Method 524.2 (rather than 8260C) should be used for analysis of drinking water samples. See Appendix
A for corresponding method usability tiers.
Detection and Quantitation: Using standard quadrupole instrumentation and the purge-and-trap,
estimated quantitation limits (EQLs) are 5 (ig/kg (wet weight) for soil/sediment samples and 5 (ig/L for
ground water. Somewhat lower limits may be achieved using an ion trap MS or other instrumentation of
improved design. No matter which instrument is used, EQLs will be proportionately higher for sample
extracts and samples that require dilution or when a reduced sample size is used to avoid saturation of the
detector. The EQL for an individual analyte is dependent on the instrument as well as the choice of
sample preparation/introduction method.
Description of Method: Volatile compounds are introduced into a GC by purge-and-trap or other
procedures (see Section 1.2 in Method 8260C). The analytes can be introduced directly to a wide-bore
capillary column or cryofocused on a capillary pre-column before being flash evaporated to a narrow-bore
capillary for analysis. Alternatively, the effluent from the trap is sent to an injection port operating in the
split mode for injection to a narrow-bore capillary column. The column is temperature-programmed to
separate the analytes, which are then detected with a MS interfaced to the GC. Analytes eluted from the
capillary column are introduced into the MS via a jet separator or a direct connection.
Source: EPA. 2006. "Method 8260C (SW-846): Volatile Organic Compounds by Gas
Chromatography/Mass Spectrometry (GC/MS)," Revision 3. http ://www.epa. gov/sam/pdfs/EPA-
8260c.pdf
SAM 2012 60 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
5.2.31 EPA Method 8270D (SW-846): Semivolatile Organic Compounds by Gas
Chromatography/Mass Spectrometry (GC-MS)
Analyte(s)
Chlorfenvinphos
Chloropicrin1
Chlorosarin
Chlorosoman
Chlorpyrifos
Chlorpyrifos oxon
Crimidine2
Dichlorvos
Dicrotophos
Dimethylphosphite
Disulfoton
Disulfoton sulfone oxon3
Disulfoton sulfoxide
Disulfoton sulfoxide oxon3
1,4-Dithiane
Ethyldichloroarsine (ED)
Fenamiphos
Methyl paraoxon
Methyl parathion
1-Methylethyl ester ethylphosphonofluoridic acid (GE)
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
Paraoxon
Parathion
Phencyclidine
Phorate
Phorate sulfone
Phorate sulfone oxon3
Phorate sulfoxide
Phorate sulfoxide oxon3
Phosphamidon
R 33 (VR) [methylphosphonothioic acid, S-[2-(diethylamino)ethyl] O-2-methylpropyl ester]
Strychnine
Tabun (GA)
Tetraethyl pyrophosphate
Tetramethylenedisulfotetramine1
1,4-Thioxane4
Trimethyl phosphite1
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
470-90-6
76-06-2
1445-76-7
7040-57-5
2921-88-2
5598-15-2
535-89-7
62-73-7
141-66-2
868-85-9
298-04-4
2496-91-5
2497-07-6
2496-92-6
505-29-3
598-14-1
22224-92-6
950-35-6
298-00-0
1189-87-3
7786-34-7
6923-22-4
538-07-8
51-75-2
555-77-1
54-11-5
311-45-5
56-38-2
77-10-1
298-02-2
2588-04-7
2588-06-9
2588-03-6
2588-05-8
13171-21-6
159939-87-4
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
1 If problems occur with analyses, lower the injection temperature.
SAM 2012
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July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
2 If problems occur when using this method, it is recommended that SW-846 Method 8321B be used. Sample
preparation methods should remain the same.
3 If problems occur when using this method for measurement of oxon compounds, analysts should consider use of
procedures included in "Oxidation of Selected Organophosphate Pesticides During Chlorination of Simulated
Drinking Water." Water Research. 2009. 43(2): 522-534. http://www.sciencedirect.com/science/iournal/00431354
4 If problems occur when using this method, it is recommended that SW-846 Method 8260C and appropriate
corresponding sample preparation procedures (i.e., Method 5035A for solid samples and Method 5030C for
aqueous liquid and drinking water samples) be used.
Analysis Purpose: Analyte determination and measurement
Determinative Technique: GC-MS
Sample Preparation Method: EPA SW-846 Method 3541/3545A/3570(solid samples), Method
3511/3520C/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.
Sample Preparation Technique: Automated Soxhlet extraction / PFE/MSE (solid samples), CLLE /
SPE/MSE (aqueous liquid and drinking water samples), and MSE / solvent extraction / acid digestion
(wipe samples).
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, aqueous liquid, drinking water and/or
wipe samples. Please note: Drinking water samples for chlorpyrifos, chlorpyrifos oxon, dichlorvos,
disulfoton, disulfoton sulfoxide, fenamiphos and mevinphos should be prepared and analyzed by EPA
Method 525.2; aqueous liquid and drinking water samples for chloropicrin should be prepared and
analyzed by EPA Method 551.1; drinking water samples for TETS should be analyzed using EPA 600/R-
11/091; all other analyte/sample type combinations should be analyzed by this method (SW-846 8270D).
See Appendix A for corresponding method usability tiers.
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.
Special Considerations: Refer to footnotes provided in analyte table above for special considerations
that should be applied when measuring specific analytes.
Source: EPA. 1998. "Method 8270D (SW-846): Semivolatile Organic Compounds by Gas
Chromatography/Mass Spectrometry (GC/MS)," Revision 4. http://www.epa.gov/sam/pdfs/EPA-
8270d.pdf
5.2.32 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
CASRN
79-06-1
107-13-1
116-06-3
1646-88-4
1646-87-3
SAM 2012 62 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Analyte(s)
4-Aminopyridine
BZ [Quinuclidinyl benzilate]
Brodifacoum
Bromadiolone
Carfentanil
Carbofuran (Furadan)
Chlorfenvinphos
Chloropicrin
Chlorosarin
Chlorosoman
Chlorpyrifos
Chlorpyrifos oxon
Crimidine
Dichlorvos
Dicrotophos
Diesel range organics
Diisopropyl methylphosphonate (DIMP)
Dimethylphosphite
Dimethylphosphoramidic acid
Diphacinone
Disulfoton
Disulfoton sulfone oxon1
Disulfoton sulfoxide
Disulfoton sulfoxide oxon1
1,4-Dithiane
EA2192 [S-2-(diisopropylamino)ethyl methylphosphonothioic acid]
Ethyl methylphosphonic acid (EMPA)
Fenamiphos
Fentanyl
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 hydrazine
Methyl paraoxon
Methyl parathion
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]
Nicotine compounds
Octahydro-1 ,3,5,7-tetranitro-1 ,3,5,7-tetrazocine (HMX)
Oxamyl
CASRN
504-24-5
6581-06-2
56073-10-0
28772-56-7
59708-52-0
1563-66-2
470-90-6
76-06-2
1445-76-7
7040-57-5
2921-88-2
5598-15-2
535-89-7
62-73-7
141-66-2
NA
1445-75-6
868-85-9
33876-51-6
82-66-6
298-04-4
2496-91-5
2497-07-6
2496-92-6
505-29-3
73207-98-4
1832-53-7
22224-92-6
437-38-7
50-00-0
NA
121-82-4
283-66-9
1832-54-8
64742-81-0
16752-77-5
126-98-7
60-34-4
950-35-6
298-00-0
1189-87-3
993-13-5
7786-34-7
6923-22-4
538-07-8
51-75-2
555-77-1
54-11-5
2691-41-0
23135-22-0
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SAM 2012 Section 5.0- Selected Chemical Methods
Analyte(s)
Paraoxon
Parathion
Pentaerythritol tetranitrate (PETN)
Phencyclidine
Phorate
Phorate sulfone
Phorate sulfone oxon1
Phorate sulfoxide
Phorate sulfoxide oxon1
Phosphamidon
Pinacolyl methyl phosphonic acid (PMPA)
R 33 (VR) [methylphosphonothioic acid, S-[2-(diethylamino)ethyl] O-2-methylpropyl ester]
Strychnine
Tabun (GA)
Tetraethyl pyrophosphate
Tetramethylenedisulfotetramine
Thiofanox
1,4-Thioxane
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]
White phosphorus
CASRN
311-45-5
56-38-2
78-11-5
77-10-1
298-02-2
2588-04-7
2588-06-9
2588-03-6
2588-05-8
13171-21-6
616-52-4
159939-87-4
57-24-9
77-81-6
107-49-3
80-12-6
39196-18-4
15980-15-1
121-45-9
99-35-4
118-96-7
21738-25-0
78-53-5
21770-86-5
12185-10-3
1 If problems occur when using this method for measurement of oxon compounds, analysts should consider use of
procedures included in "Oxidation of Selected Organophosphate Pesticides During Chlorination of Simulated Drinking
Water." Water Research. 2009. 43(2): 522-534. http://www.sciencedirect.com/science/iournal/00431354
Analysis Purpose: Sample preparation
Sample Preparation Technique: Solvent extraction
Determinative Technique: GC-NPD / GC-FID / GC-MS / HPLC
Determinative Method: 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. See Appendix A for
corresponding method usability tiers.
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: Refer to footnote provided in analyte table above for special considerations
that should be applied when measuring specific analytes. 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.
SAM 2012
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SAM 2012 Section 5.0- Selected Chemical Methods
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
5.2.33 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 analyte determination and measurement
Sample Preparation Technique: Solvent extraction (solid and aqueous liquid samples) and MSE /
solvent extraction by 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 and
wipe samples. See Appendix A for corresponding method usability tiers.
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
5.2.34 EPA Method 8316 (SW-846): Acrylamide, Acrylonitrile and Acrolein by High
Performance Liquid Chromatography (HPLC)
Analyte(s)
Acrylamide
CASRN
79-06-1
Analysis Purpose: Sample preparation, and/or analyte determination and measurement
Sample Preparation Technique: Direct injection (aqueous liquid and drinking water samples), water
extraction (solid), and MSE / solvent extraction by 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, aqueous liquid,
drinking water and wipe samples. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: Acrylamide has an MDL of 10 (ig/L; acrylonitrile has an MDL of 20 (ig/L.
SAM 2012 65 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Description of Method: Samples are analyzed by HPLC. A 200-(j,L aliquot is injected onto a Ci8
reverse-phase column, and compounds in the effluent are detected with a UV detector. Solid samples
should be water extracted prior to injection. Aqueous liquid and drinking water samples can be directly
injected.
Special Considerations: For details on method modifications allowing for the use of LC-MS-MS
detection, please refer to the points of contact in Section 4.0.
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
5.2.35 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 analyte determination and measurement
Sample Preparation Technique: Solvent extraction (solid samples), and MSE / solvent extraction by
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 and wipe samples.
See Appendix A for corresponding method usability tiers.
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 Ci8
analytical column. After separation, the target analytes are hydrolyzed and derivatized post-column, then
quantified fluorometrically. The sensitivity of the method usually depends on the level of interferences
present, rather than on instrument conditions. Waste samples with a high level of extractable fluorescent
compounds are expected to yield significantly higher detection limits.
Special Considerations: Techniques for analysis of these compounds in soil have been moving towards
the use of LC/MS. Laboratories that are routinely using LC/MS for analysis of these compounds should
consult with an appropriate contact in Section 4.0 regarding its use.
Source: EPA. 2000. "Method 8318A (SW-846): N-Methylcarbamates by High Performance Liquid
Chromatography (HPLC)," Revision 1. http://www.epa.gov/sam/pdfs/EPA-8318a.pdf
SAM 2012 66 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
5.2.36 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]
Carfentanil
Diisopropyl methylphosphonate (DIMP)
Dimethylphosphoramidic acid1
Diphacinone
EA2192 [S-2-(diisopropylamino)ethyl methylphosphonothioic acid]
Ethyl methylphosphonic acid (EMPA)
N-Ethyldiethanolamine (EDEA)
Fentanyl
Isopropyl methylphosphonic acid (IMPA)
N-Methyldiethanolamine (MDEA)
Methylphosphonic acid (MPA)
Pinacolyl methyl phosphonic acid (PMPA)
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
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.
Crimidine1
535-89-7
1 This analyte is determined using a wavelength of 230 nm.
Analysis Purpose: Analyte determination and measurement
Determinative Technique: High performance liquid chromatography-mass spectrometry (HPLC-MS);
HPLC (using electrospray or other atmospheric pressure ionization interface)
Sample Preparation Method: EPA SW-846 Method 3541/3545A (solid samples), 3520C/3535A
(aqueous liquid and drinking water samples), and Method 3570/8290A Appendix A (wipe samples). For
thiofanox, EPA Method 538 (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.
Sample Preparation Technique: Varies with analyte/sample type (see Appendix A).
Method Developed for: Solvent-extractable nonvolatile compounds, including dyes, organophosphorus
compounds, phenoxyacid herbicides and carbamates in solid and water samples
Method Selected for: SAM lists this method for analysis of solid, aqueous liquid, drinking water and
wipe samples. It is intended to serve as a general-purpose HPLC method; SAM users should refer to
other LC methods listed in Appendix A for these analytes for specific instrument conditions. See
Appendix A for corresponding method usability tiers.
Description of Method: As published, Method 832IB provides reversed-phase HPLC, thermospray
(TSP) MS and UV conditions for detection of the target analytes. TSP instrumentation is obsolete,
however, and currently available electrospray or atmospheric pressure ionization (API) techniques are
recommended. SAM users should refer to other LC methods listed in Appendix A for these analytes for
specific instrument conditions. Quantitative analysis may be performed by MS detection, using either an
external or internal standard approach. Primary analysis of some analytes (e.g., carbamates) may be
performed by UV detection; however, results should be confirmed using MS. The instrument conditions,
SAM 2012
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SAM 2012 Section 5.0- Selected Chemical Methods
analytical range and detection limits vary depending on the target analyte, sample type and instrument
used.
Special Considerations: Refer to footnote 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-8321 b.pdf
5.2.37 EPA Method 8330B (SW-846): Nitroaromatics, Nitramines, and Nitrate Esters 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 analyte determination and measurement
Sample Preparation Technique: Solvent extraction or direct injection (solid samples), SPE by EPA
SW-846 Method 3535A (aqueous liquid and drinking water samples), and MSE / solvent extraction by
EPA SW-846 Method 3570/8290A Appendix A (wipe samples)
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, aqueous liquid,
drinking water and wipe samples. Aqueous liquid and drinking water samples are prepared using
Methods 3535A or 8330B prior to analysis. For HMTD, procedures adapted from Analyst (2001)
126:1689 - 1693 are used for sample analysis. See Appendix A for corresponding method usability tiers.
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) detection using the appropriate sample
preparation technique (SPE by Method 3535A or solvent extraction by Method 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
SAM 2012 68 July 16, 2011
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5.2.38 EPA CLP ISM01.3 Cyanide: Analytical Methods for Total Cyanide Analysis
Analyte(s)
Cyanide, Total
CASRN
57-12-5
Analysis Purpose: Sample preparation, and/or analyte determination and measurement
Sample Preparation Technique: Midi-or micro-distillation
Determinative Technique: Visible spectrophotometry
Method Developed for: Metals in water, sediment, sludge and soil
Method Selected for: SAM lists this method for preparation and analysis of solid, aqueous liquid and
wipe samples. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The method quantitation limits are 10 (ig/L for aqueous samples and 0.5
mg/kg for solid samples.
Description of Method: Cyanide is released as hydrocyanic acid from cyanide complexes by means of
reflux-distillation, using either a midi- or micro-distillation process, and absorbed in a scrubber containing
sodium hydroxide solution. The cyanide ion in the absorbing solution is then determined
spectrophotometrically. In the semi-automated spectrophotometric measurement, cyanide is converted to
cyanogen chloride without hydrolyzing to cyanate, by reaction with chloramine-T, at a pH less than 8.
After the reaction is complete, color is formed on the addition of pyridine-barbituric acid reagent, and
absorbance is read between 570 and 580 nanometers (nm). To obtain colors of comparable intensity, it is
essential to have the same salt content in both the sample and the standards.
Source: EPA Contract Laboratory Program (CLP). "ISM01.2: Exhibit D - Part D: Analytical Methods
for Total Cyanide Analysis." http://www.epa.gov/sam/EPA-ISMO 1.3.pdf
5.2.39 EPA 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 analyte determination and measurement
Sample Preparation Technique: Acid digestion followed by distillation
Determinative Technique: Visible spectrophotometry
Method Developed for: Cyanide in drinking, ground and surface waters, domestic and industrial
wastewaters, sediments and solid waste
Method Selected for: SAM lists this method for preparation and analysis of solid, aqueous liquid,
drinking water and wipe samples. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The applicable range is 0.003 to 0.500 mg/L cyanide in the distillate. This
range can be expanded by sample dilution, either by using less sample for distillation or diluting the
distillate.
Description of Method: This method detects inorganic cyanides that are present as either simple soluble
salts or complex radicals. It may be used to determine values for both total cyanide and cyanide
amenable to chlorination (also known as available cyanide). Cyanide in the sample released as
hydrocyanic acid by refluxing the sample with strong acid. The hydrocyanic acid is distilled and
collected in an absorber-scrubber containing sodium hydroxide solution. The cyanide ion in the
absorbing solution is then determined by automated colorimetry. For determination of cyanide amenable
to chlorination, a portion of the sample is chlorinated using sodium hypochlorite at a pH > 11 to
SAM 2012 69 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
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. 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
5.2.40 EPA IO [Inorganic] Compendium Method IO-3.1: Selection, Preparation, and
Extraction of Filter Material
Analyte(s)
Ammonium metavanadate (analyze as total vanadium)
Arsenic, Total
Arsenic trioxide (analyze as total arsenic)
Calcium arsenate (analyze as total arsenic)
2-Chlorovinylarsonous acid (2-CVAA) (analyze as total arsenic)
Lead arsenate (analyze as total arsenic)
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] (analyze as total arsenic)
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] (analyze as total arsenic)
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] (analyze as total arsenic)
Lewisite oxide (analyze as total arsenic)
Osmium tetroxide (analyze as total osmium)
Sodium arsenite (analyze as total arsenic)
Thallium sulfate (analyze as total thallium)
Vanadium pentoxide (analyze as total vanadium)
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: ICP-AES / ICP-MS
Determinative Method: EPA Method IO-3.4 or Method IO-3.5. Osmium tetroxide should be analyzed
byMethodIO-3.4.
Method Developed for: Particulate metals in air.
Method Selected for: SAM lists this method for preparation of air samples. See Appendix A for
corresponding method usability tiers.
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
SAM 2012 70 July 16, 2011
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5.2.41 EPA IO [Inorganic] Compendium Method IO-3.4: Determination of Metals in
Ambient Particulate Matter Using Inductively Coupled Plasma (ICP) Spectroscopy
Analyte(s)
Ammonium metavanadate (analyze as total vanadium)
Arsenic, Total
Arsenic trioxide (analyze as total arsenic)
Calcium arsenate (analyze as total arsenic)
2-Chlorovinylarsonous acid (2-CVAA) (analyze as total arsenic)
Lead arsenate (analyze as total arsenic)
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] (analyze as total arsenic)
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] (analyze as total arsenic)
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] (analyze as total arsenic)
Lewisite oxide (analyze as total arsenic)
Osmium tetroxide (analyze as total osmium)
Sodium arsenite (analyze as total arsenic)
Thallium sulfate (analyze as total thallium)
Vanadium pentoxide (analyze as total vanadium)
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: Analyte determination and measurement
Determinative Technique: ICP-AES
Sample Preparation Method: EPA Method IO-3.1
Sample Preparation Technique: Acid extraction
Method Developed for: Metals in ambient particulate matter
Method Selected for: SAM lists this method for analysis of air samples. See Appendix A for
corresponding method usability tiers.
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.
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." EPA/625/R-96/010a.
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." EPA/625/R-96/010a.
http://www.epa.gov/sam/pdfs/EPA-IO-2.1 .pdf
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SAM 2012 Section 5.0- Selected Chemical Methods
5.2.42 EPA 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 (analyze as total vanadium)
Arsenic, Total
Arsenic trioxide (analyze as total arsenic)
Calcium arsenate (analyze as total arsenic)
2-Chlorovinylarsonous acid (2-CVAA) (analyze as total arsenic)
Lead arsenate (analyze as total arsenic)
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] (analyze as total arsenic)
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] (analyze as total arsenic)
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] (analyze as total arsenic)
Lewisite oxide (analyze as total arsenic)
Sodium arsenite=(analyze as total arsenic)
Thallium sulfate (analyze as total thallium)
Vanadium pentoxide (analyze as total vanadium)
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: Analyte determination and measurement
Determinative Technique: ICP-MS
Sample Preparation Method: EPA Method IO-3.1
Sample Preparation Technique: Acid extraction
Method Developed for: Metals in ambient particulate matter
Method Selected for: SAM lists this method for analysis of air samples. See Appendix A for
corresponding method usability tiers.
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.
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)." EPA/625/R-96/010a.
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." EPA/625/R-96/010a.
http://www.epa.gov/sam/pdfs/EPA-IO-2.1 .pdf
SAM 2012 72 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
5.2.43 EPA 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 (analyze as total mercury)
CASRN
7439-97-6
151-38-2
Analysis Purpose: Sample preparation, and analyte determination and measurement
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. See Appendix
A for corresponding method usability tiers.
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/minute. The traps are directly desorbed onto a second (analytical) trap. The mercury
desorbed from the analytical trap is determined by CVAFS. Particulate mercury is sampled on glass-fiber
filters at a flow rate of 30 L/minute. 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 nm wavelength. Water
vapor will cause a negative interference.
Source: EPA. 1999. "IO Compendium Method IO-5: Compendium of Methods for the Determination of
Inorganic Compounds in Ambient Air: Sampling and Analysis for Vapor and Particle Phase Mercury in
Ambient Air Utilizing Cold Vapor Atomic Fluorescence Spectrometry (CVAFS)." EPA/625/R-96/010a.
http://www.epa.gov/sam/pdfs/EPA-IO-5.pdf
5.2.44 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)
Analyte(s)
BZ [Quinuclidinyl benzilate]1
Chlorfenvinphos
3-Chloro-1 ,2-propanediol2
Chlorosarin2
Chlorosoman2
Chlorpyrifos
Chlorpyrifos oxon
Dichlorvos
Dicrotophos
Diisopropyl methylphosphonate (DIMP)2
Dimethylphosphite
Dimethylphosphoramidic acid1
CASRN
6581-06-2
470-90-6
96-24-2
1445-76-7
7040-57-5
2921-88-2
5598-15-2
62-73-7
141-66-2
1445-75-6
868-85-9
33876-51-6
SAM 2012 73 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Analyte(s)
EA2192 [S-2-(diisopropylamino)ethyl methylphosphonothioic acid]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]
Paraoxon
Parathion
Phencyclidine
Phorate
Phorate sulfone
Phorate sulfone oxon3
Phorate sulfoxide
Phorate sulfoxide oxon3
Phosphamidon
Pinacolyl methyl phosphonic acid (PMPA)1
R 33 (VR) [methylphosphonothioic acid, S-[2-(diethylamino)ethyl] O-2-methylpropyl ester]
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]
CASRN
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
311-45-5
56-38-2
77-10-1
298-02-2
2588-04-7
2588-06-9
2588-03-6
2588-05-8
13171-21-6
616-52-4
159939-87-4
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
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 (see references listed under Special Considerations in
Section 5.2.31).
2 If problems occur when using this method, it is recommended that the canister Method TO-15 be used.
3 If problems occur when using this method for measurement of oxon compounds, analysts should consider use of
procedures included in "Oxidation of Selected Organophosphate Pesticides During Chlorination of Simulated Drinking
Water." Water Research. 2009. 43(2): 522-534. http://www.sciencedirect.com/science/iournal/00431354
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Solvent extraction
Determinative Technique: GC-MS or HPLC
Method Developed for: Pesticides and polychlorinated biphenyls in ambient air
SAM 2012
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SAM 2012 Section 5.0- Selected Chemical Methods
Method Selected for: SAM lists this method for preparation and analysis of air samples. See Appendix
A for corresponding method usability tiers.
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/minute) sample collection rate is used to collect vapors
on a sorbent cartridge containing PUF in combination with another solid sorbent. Airborne particles also
are collected, but the sampling efficiency is not known. Pesticides and other chemicals are extracted from
the sorbent cartridge with 5% diethyl ether in hexane and determined by GC-MS. For common
pesticides, HPLC coupled with a UV detector or electrochemical detector is preferable. If analyzed by
GC-MS, BZ, dimethylphosphoramidic acid, EA2192, EMPA, IMP A, 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 for EPA SW-846
8270D in Section 5.2.31 for information regarding derivatization of compounds.
Source: EPA. 1999. "Air Method, Toxic Organics-lOA (TO-10A): Compendium of Methods for the
Determination of Toxic Organic 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)." EPA 625/R-96/010b.
http://www.epa.gov/sam/pdfs/EPA-TO-10a.pdf
5.2.45 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
Analysis Purpose: Sample preparation, and analyte determination and measurement
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. See Appendix
A for corresponding method usability tiers.
Detection and Quantitation: This method applies to ambient concentrations of VOCs above 0.5 parts
per billion by volume (ppbv) and typically requires VOC enrichment by concentrating up to 1 L of a
SAM 2012 15 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
sample volume; however, when using current technologies, quantifications of approximately 100 parts per
trillion by volume (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. In cases where lower detection levels are needed, use
procedures included in EPA Compendium Method TO-15: Reduction of Method Detection Limits to
Meet Vapor Intrusion Monitoring Needs (http://www.epa.gov/ttnamtil/files/ambient/airtox/TO-15-
Supplement.pdf).
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)." EPA 625/R-96/010b.
http: //www. epa. gov/sam/pdfs/EPA-TO-15 .pdf
5.2.46 EPA 600/R-11/091: High Throughput Determination of Tetramine in Drinking Water
by Solid Phase Extraction and Isotope Dilution Gas Chromatography/Mass
Spectrometry (GC/MS)
Analyte(s)
Tetramethylenedisulfotetramine (TETS)
CASRN
80-12-6
Analysis Purpose: Sample preparation, and/or analyte determination and measurement
Sample Preparation Technique: SPE using 96 well-plates
Determinative Technique: GC-MS
Method Developed for: TETS in drinking water
Method Selected for: SAM lists this method for preparation and analysis of drinking water samples.
See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The MDL for TETS is 0.15 ug/L. The reporting range is 0.5 - 250 ug/L.
Description of Method: A 50-mL water sample is collected, and a preservative and/or dechlorinating
agent is added as required by site-specific conditions. An aliquot is pipetted into a well of a
preconditioned 96-well solid phase extraction plate, and isotopically labeled TETS is added. Following a
wash step with 5% methanol/95%water, tetramine is eluted in acetonitrile. The extract is concentrated to
dryness under nitrogen and heat, and then adjusted to a 100-uL volume in acetonitrile. TETS is separated
from the sample matrix and identified by GC-MS analysis, operated in selective ion monitoring (SIM)
mode or equivalent. Analyte identification is accomplished by comparing the acquired mass spectra,
SAM 2012 76 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
including ion ratios, and retention times to reference spectra and retention times for calibration standards
acquired under identical GC-MS conditions. Quantitation is performed using the internal standard
technique. Utilization of an isotopically-labeled internal standard provides a high degree of accuracy and
precision for sample quantitation by accounting for analyte recovery and analytical efficiency.
Source: EPA and CDC. 2011. "High Throughput Determination of Tetramine in Drinking Water by
Solid Phase Extraction and Isotope Dilution Gas Chromatography/Mass Spectrometry (GC/MS)." EPA
600/R-11/091. http://www.epa.gov/sam/EPA-600-R-ll-091.pdf
5.2.47 EPA/600/R-11/143: Surface Analysis Using Wipes for the Determination of
Nitrogen Mustard Degradation Products by Liquid Chromatography/Tandem Mass
Spectrometry (LC/MS/MS)
Analyte(s)
A/-Ethyldiethanolamine (EDEA)
A/-Methyldiethanolamine (MDEA)
Triethanolamine (TEA)
CASRN
139-87-7
105-59-9
102-71-6
Analysis Purpose: Sample preparation, and/or analyte determination and measurement
Sample Preparation Technique: Extracted using sonication and filtered using a syringe-poly vinylidene
fluoride (PVDF) filter unit
Determinative Technique: LC-MS-MS
Method Developed for: TEA, EDEA and MDEA in wipe surfaces
Method Selected for: SAM lists this method for preparation and analysis of wipe samples. See
Appendix A for corresponding method usability tiers.
Detection and Quantitation: Detection limits (DL) for EDEA, MDEA and TEA are 0.06, 0.07 and 0.12
ng/cm2, respectively. The limit of quantitation (LOQs) for EDEA, MDEA and TEA are 6.25, 6.85 and
12.3 ng/cm2,respectively. The reporting range for all three target compounds is 0.1 - 5.0 ng/cm2.
Description of Method: Samples are collected from surfaces with wipes and stored at 4 ฐC (ฑ 2 ฐC) if
not analyzed within 24-hours. Samples are brought to ambient temperature, then spiked with a surrogate
compound and solvent. Samples are then sonicated, extracted with a syringe filter unit, concentrated, and
analyzed directly by LC-MS-MS operated in the positive electrospray ionization (ESI+) mode. Each
target compound is separated and identified by retention time and by comparing the sample primary
multiple reaction monitoring (MRM) transition to the known standard MRM transition from reference
spectra under identical LC-MS-MS conditions. The retention time for the analytes in the sample must fall
within ฑ 5% of the retention time of the analytes in standard solution. The concentration of each analyte
is determined by the instrumentation software using external calibration.
Source: EPA and CDC. 2011. "Surface Analysis Using Wipes for the Determination of Nitrogen
Mustard Degradation Products by Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS)."
EPA/600/R-11/143. http://www.epa.gov/sam/EPA-600-R-ll-143.pdf
SAM 2012 77 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
5.2.48 Analytical Protocol for Chemical Warfare Agents in Water, Soil, and
Wipes
Analyte(s)
Cyclohexyl sarin (GF)
Mustard, sulfur / Mustard gas (HD)
Sarin (GB)
Soman (GD)
VX
CASRN
329-99-7
505-60-2
107-44-8
96-64-0
50782-69-9
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Microscale extraction
Determinative Technique: GC-MS
Method Developed for: Determination of GF, GB, GD, HD and VX in water, soil and wipes
Method Selected for: SAM lists this procedure for preparation and analysis of aqueous liquid, drinking
water, solid, wipe and air samples. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The calibration ranges in full scan mode are 11.4 - 114 (ig/L (GB, GF and
VX) and 5.7 - 57 ug/L (GD and HD) for water samples; 20 - 200 (ig/kg (GB, GF and VX) and 10 - 100
Hg/kg (GD and HD) for soil samples; 0.02 - 0.2 (ig/cm2 (GB, GF and VX) and 0.01 - 0.1 (ig/cm2 (GD
and HD) for wipes.
Description of Method: The method involves solvent extraction of the sample followed by GC/MS
analysis to determine semivolatile CWAs. Prior to analysis, samples must be prepared using sample
preparation techniques appropriate for each matrix type. Aqueous, solid and wipe samples are spiked
with surrogates and extracted by microscale extraction. All target compounds in aqueous and wipe
samples and all target compounds except for VX in solid samples are extracted with methylene chloride.
VX in solid samples is extracted first using a tris buffer solution, followed by extraction with methylene
chloride. Extracts are dried, concentrated (solids and wipe extracts only) by nitrogen evaporation, then
analyzed by GC-MS, using either a mass selective detector in full scan mode or time-of-flight (TOP).
Special Considerations: The method has been extensively tested in reagent water, sand, wipes, drinking
water and groundwater, and is currently undergoing multi-laboratory testing and validation in additional
environmental matrices. The procedures are specifically for use by laboratories with EPA approval for
handling and analysis of samples containing CWAs, and may require modifications for application to
environmental sample types.
Source: EPA. March 2011. "DRAFT Analytical Protocol for Chemical Warfare Agents in Water, Soil
and Wipes." Copies of this analytical protocol may be requested from NHSRC at
http://www.epa.gov/sam/contact us.htm
5.2.49 NIOSH Method 1612: Propylene Oxide
Analyte(s)
Propylene oxide
CASRN
75-56-9
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Coconut shell charcoal solid sorbenttube
Determinative Technique: GC-FID
SAM 2012 78 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Method Developed for: Propylene oxide in air
Method Selected for: SAM lists this method for preparation and analysis of air samples. See Appendix
A for corresponding method usability tiers.
Detection and Quantitation: The working range is between 8 and 295 ppm for air samples of 5 L.
Description of Method: A sample tube containing coconut shell charcoal is used for sample collection
with a flow rate of 0.01 to 0.2 L/minute. A 1-mL volume 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-1612.pdf
5.2.50 NIOSH Method 2016: Formaldehyde
Analyte(s)
Formaldehyde
CASRN
50-00-0
Analysis Purpose: Sample preparation, and analyte determination and measurement
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. See Appendix
A for corresponding method usability tiers.
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/minute. The cartridge is extracted with 10 mL of acetonitrile and analyzed by HPLC-UV
at a wavelength of 360 nm. Ozone has been observed to consume the 2,4-DNPH reagent and to degrade
the formaldehyde derivative. Ketones and other aldehydes can react with 2,4-DNPH; the derivatives
produced, however, are separated chromatographically from the formaldehyde derivative.
Source: NIOSH. 2003. "Method 2016: Formaldehyde," Issue 2.
http://www.epa.gov/sam/pdfs/NIOSH-2016.pdf
5.2.51 NIOSH Method 2513: Ethylene Chlorohydrin
Analyte(s)
2-Chloroethanol
2-Fluoroethanol
CASRN
107-07-3
371-62-0
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Solvent desorption
Determinative Technique: GC-FID
SAM 2012 79 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Method Developed for: Ethylene chlorohydrin (2-chloroethanol) in air
Method Selected for: SAM lists this method for preparation and analysis of air samples. See Appendix
A for corresponding method usability tiers.
Detection and Quantitation: The working range of the method is 0.5 to 15 ppm for a 20-L air sample.
Description of Method: Samples are drawn into a tube containing petroleum charcoal at a rate of 0.01 to
0.2 L/minute and transferred into vials containing eluent (carbon disulfide, 2-propanol and ซ-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.
http://www.epa.gov/sam/pdfs/NIOSH-2513.pdf
5.2.52 NIOSH Method 3510: Monomethylhydrazine
Analyte(s)
Methyl hydrazine (monomethylhydrazine)
CASRN
60-34-4
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Samples are collected into a bubbler containing hydrochloric acid.
Determinative Technique: Visible spectrophotometry
Method Developed for: Monomethylhydrazine in air
Method Selected for: SAM lists this method for preparation and analysis of air samples. See Appendix
A for corresponding method usability tiers.
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/minute. 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
5.2.53 NIOSH Method 5600: Organophosphorus Pesticides
Analyte(s)
Disulfoton
Disulfoton sulfone oxon1
Disulfoton sulfoxide
Disulfoton sulfoxide oxon1
CASRN
298-04-4
2496-91-5
2497-07-6
2496-92-6
1 If problems occur when using this method for measurement of oxon compounds, analysts should consider use of
procedures included in "Oxidation of Selected Organophosphate Pesticides During Chlorination of Simulated Drinking
Water." Water Research. 2009. 43(2): 522-534. http://www.sciencedirect.com/science/iournal/00431354
SAM 2012 80 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Solvent desorption
Determinative Technique: Gas chromatography-flame photometric detector (GC-FPD)
Method Developed for: Organophosphorus pesticides in air
Method Selected for: SAM lists this method for preparation and analysis of air samples. See Appendix
A for corresponding method usability tiers.
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 flame photometric detector (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: Refer to footnote provided in analyte table above for special considerations
that should be applied when measuring specific analytes. 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
5.2.54 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 analyte determination and measurement
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. See Appendix
A for corresponding method usability tiers.
Detection and Quantitation: The detection limit for aldicarb is 1.2 \\.g 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.
SAM 2012 81 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
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
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
5.2.55 NIOSH Method 6001: Arsine
Analyte(s)
Arsine (analyze as total arsenic in non-air samples)
CASRN
7784-42-1
Analysis Purpose: Sample preparation, and analyte determination and measurement
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. See Appendix
A for corresponding method usability tiers.
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
5.2.56 NIOSH Method 6002: Phosphine
Analyte(s)
Phosphine
CASRN
7803-51-2
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Solvent desorption with hot acidic permanganate solution
Determinative Technique: Visible spectrophotometry
Method Developed for: Phosphine in air
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SAM 2012 Section 5.0- Selected Chemical Methods
Method Selected for: SAM lists this method for preparation and analysis of air samples. See Appendix
A for corresponding method usability tiers.
Detection and Quantitation: The working range of the method is 0.02 to 0.9 mg/m3 for a 16-L sample.
Description of Method: In this method, phosphine is determined as phosphate. A volume of 1 to 16 L
of air is drawn through a sorbent tube containing silica gel coated with mercuric cyanide. The sorbent is
extracted with a potassium permanganate/sulfuric acid solution and washed with reagent water.
Following treatment with the color agent and extraction into organic solvent, phosphate is determined by
visible spectrometry.
Special Considerations: The method is subject to interferences from phosphorus trichloride,
phosphorus pentachloride and organic phosphorus compounds.
Source: NIOSH. 1994. "Method 6002: Phosphine," Issue 2.
http://www.epa.gov/sam/pdfs/NIOSH-6002.pdf
5.2.57 NIOSH Method 6010: Hydrogen Cyanide
Analyte(s)
Cyanide, Total
Hydrogen cyanide
CASRN
57-12-5
74-90-8
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Solvent desorption
Determinative Technique: Visible spectrophotometry
Method Developed for: Hydrogen cyanide in air
Method Selected for: SAM lists this method for preparation and analysis of air samples. See Appendix
A for corresponding method usability tiers.
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.
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
5.2.58 NIOSH Method 6013: Hydrogen Sulfide
Analyte(s)
Hydrogen sulfide
CASRN
7783-06-4
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Solvent extraction
Determinative Technique: 1C with conductivity detection
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SAM 2012 Section 5.0- Selected Chemical Methods
Method Developed for: Hydrogen sulfide in air
Method Selected for: SAM lists this method for preparation and analysis of air samples. See Appendix
A for corresponding method usability tiers.
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
5.2.59 NIOSH Method 6015: Ammonia
Analyte(s)
Ammonia
CASRN
7664-41-7
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Water extraction
Determinative Technique: Visible spectrophotometry
Method Developed for: Ammonia in air
Method Selected for: SAM lists this method for preparation and analysis of air samples. See Appendix
A for corresponding method usability tiers.
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.
Source: NIOSH. 1994. "Method 6015: Ammonia," Issue 2.
http://www.epa.gov/sam/pdfs/NIOSH-6015.pdf
5.2.60 NIOSH Method 6402: Phosphorus Trichloride
Analyte(s)
Phosphorus trichloride
CASRN
7719-12-2
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Add reagent to samples in bubbler solution and heat
Determinative Technique: Visible spectrophotometry
Method Developed for: Phosphorus trichloride in air
Method Selected for: SAM lists this method for preparation and analysis of air samples. See Appendix
A for corresponding method usability tiers.
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SAM 2012 Section 5.0- Selected Chemical Methods
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
5.2.61 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 analyte determination and measurement
Sample Preparation Technique: Solvent desorption
Determinative Technique: 1C with conductivity detection
Method Developed for: Inorganic acids in air
Method Selected for: SAM lists this method for preparation and analysis of air samples. See Appendix
A for corresponding method usability tiers.
Detection and Quantitation: The working range of this method is 0.01 to 5 mg/m3 for a 50-L sample.
Description of Method: Acids are analyzed as bromide, chloride and fluoride. A volume of 3 to 100 L
of air is drawn through a silica gel sorbent. The sorbent portions are extracted with a buffered
carbonate/bicarbonate solution and the extract is analyzed by 1C.
Special Considerations: Particulate salts of the acids are an interference (trapped on the glass wool
filter plug in the sorbent tube). Chlorine and bromine are also interferences. Acetate, formate and
propionate interferences may be reduced by use of a weaker eluent. If problems occur when using this
method for analysis of hydrogen fluoride, it is recommended that NIOSH Method 7906 be used.
Source: NIOSH. 1994. "Method 7903: Acids, Inorganic," Issue 2.
http://www.epa.gov/sam/pdfs/NIOSH-7903.pdf
5.2.62 NIOSH Method 7905: Phosphorus
Analyte(s)
White phosphorus
CASRN
12185-10-3
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: GC solid sorbent tube 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. See Appendix
A for corresponding method usability tiers.
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SAM 2012 Section 5.0- Selected Chemical Methods
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
5.2.63 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 with conductivity detection
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 6 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.
Source: NIOSH. 1994. "Method 7906: Fluorides, Aerosol and Gas by 1C," Issue 1.
http://www.epa.gov/sam/pdfs/NIOSH-7906.pdf
5.2.64 NIOSH Method 9102: Elements on Wipes
Analyte(s)
Ammonium metavanadate (analyze as total vanadium)
Arsenic, Total
Arsenic trioxide (analyze as total arsenic)
Arsine (analyze as total arsenic in non-air samples)
Calcium arsenate (analyze as total arsenic)
2-Chlorovinylarsonous acid (2-CVAA) (analyze as total arsenic)
Ethyldichloroarsine (ED)
Lead arsenate (analyze as total arsenic)
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
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Analyte(s)
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] (analyze as total arsenic)
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] (analyze as total arsenic)
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] (analyze as total arsenic)
Lewisite oxide (analyze as total arsenic)
Mercuric chloride (analyze as total mercury)
Mercury, Total
Methoxyethylmercuric acetate (analyze as total mercury)
Osmium tetroxide (analyze as total osmium)
Sodium arsenite (analyze as total arsenic)
Thallium sulfate (analyze as total thallium)
Titanium tetrachloride (analyze as total titanium)
Vanadium pentoxide (analyze as total vanadium)
CASRN
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: ICP-AES / ICP-MS / Spectrophotometry
Determinative Method: EPA SW-846 Methods 6010C, 6020A 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. See Appendix A for
corresponding method usability tiers.
Detection and Quantitation: The range for arsenic is 0.261 tolOS (ig/wipe; for thallium 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 ฐC for 8 hours. Additional nitric acid is added until the wipe media is
completely destroyed. The sample is then taken to near dryness and the residue dissolved and diluted
before being analyzed.
Special Considerations: ICP-MS may also be used for the analysis of wipe samples; however, at this
time, this technique has not been evaluated for wipes. Nitric and perchloric acids are strong oxidizers and
extremely corrosive. Perform all perchloric acid digestions in a perchloric acid hood. When working
with acids, use gloves and avoid inhalation or contact with skin or clothing.
Source: NIOSH. 2003. "Method 9102, Issue 1: Elements on Wipes."
http://www.epa.gov/sam/pdfs/NIOSH-9102.pdf
5.2.65 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: LC-MS
Determinative Method: Adapted from J. Chromatogr. A, 1139 (2002) 271 - 278.
Method Developed for: Fluoroacetate anion in air
SAM 2012 87 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Method Selected for: SAM lists this method for preparation of air samples. See Appendix A for
corresponding method usability tiers.
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 LC-MS.
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
5.2.66 OSHA Method 40: Methylamine
Analyte(s)
Methylamine
CASRN
74-89-5
Analysis Purpose: Sample preparation, and analyte determination and measurement
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. See Appendix
A for corresponding method usability tiers.
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 ฑ 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/minute
through standard size sampling tubes containing XAD-7 resin coated with 10% 7-chloro-4-nitrobenzo-2-
oxa-l,3-diazole (NBD chloride) by weight. Samples are desorbed with 5% (w/v) NBD chloride in
tetrahydrofuran (with a small amount of sodium bicarbonate present), heated in a hot water bath, and
analyzed by high performance liquid chromatography-fluorescence (HPLC-FL) or high performance
liquid chromatography-visible (HPLC-vis).
Source: OSHA. 1982. "Method 40: Methylamine." Method originally obtained from www.osha.gov.
but is provided here for reference. http://www.epa.gov/sam/pdfs/OSHA-Method40.pdf
5.2.67 OSHA Method 54: Methyl Isocyanate (MIC)
Analyte(s)
Methyl isocyanate
CASRN
624-83-9
Analysis Purpose: Sample preparation, and analyte determination and measurement
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SAM 2012 Section 5.0- Selected Chemical Methods
Sample Preparation Technique: Solvent desorption
Determinative Technique: HPLC
Method Developed for: Methyl isocyanate in air
Method Selected for: SAM lists this method for preparation and analysis of air samples. See Appendix
A for corresponding method usability tiers.
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
5.2.68 OSHA Method 61: Phosgene
Analyte(s)
Phosgene
CASRN
75-44-5
Analysis Purpose: Sample preparation, and analyte determination and measurement
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. See Appendix
A for corresponding method usability tiers.
Description of Method: This method determines the concentration of phosgene in air by using GC with
an NPD. Air samples are collected by drawing known volumes of air through sampling tubes containing
XAD-2 adsorbent that has been coated with 2-(hydroxymethyl)piperidine. The samples are desorbed with
toluene and then analyzed by GC using an NPD.
Special Considerations: The presence of 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
5.2.69 OSHA Method ID-211: Sodium Azide and Hydrazoic Acid in Workplace
Atmospheres
Analyte(s)
Sodium azide (analyze as azide ion)
CASRN
26628-22-8
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Buffer desorption
Determinative Technique: IC-UV
Method Developed for: Sodium azide and hydrazoic acid in workplace atmospheres
SAM 2012 89 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Method Selected for: SAM lists this method for preparation and analysis of air and wipe samples. See
Appendix A for corresponding method usability tiers.
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 polyvinyl chloride (PVC) filter or in
the glass wool plug of the sampling tube. Gaseous HN3 is collected and converted to NaN3 by the
impregnated silica gel (ISO) sorbent within the sampling tube. The collected azide on either media is
desorbed in a weak buffer solution, and the resultant anion (N3~) is analyzed by 1C using a variable
wavelength UV detector at 210 nm. A gravimetric conversion is used to calculate the amount of NaN3 or
HN3 collected.
Source: OSHA. 1992. "Method ID-211: Sodium Azide and Hydrazoic Acid in Workplace
Atmospheres." http://www.epa.gov/sam/pdfs/OSHA-ID-211 .pdf
5.2.70 OSHA Method ID216SG: Boron Trifluoride (BF3)
Analyte(s)
Boron trifluoride
CASRN
7637-07-2
Analysis Purpose: Sample preparation, and analyte determination and measurement
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. See Appendix
A for corresponding method usability tiers.
Detection and Quantitation: The detection limit is 10 (ig in a 30-L sample.
Description of Method: Boron trifluoride is determined as fluoroborate. A volume of 30 to 480 L of air
is drawn through a bubbler containing 0.1 M ammonium fluoride. The solution is diluted and analyzed
with a fluoroborate ISE.
Source: OSHA. 1989. "Method ID216SG: Boron Trifluoride (BF3)." Method originally obtained from
www.osha.gov. but is provided here for reference. http://www.epa.gov/sam/pdfs/OSHA-ID216SG.pdf
5.2.71 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 analyte determination and measurement
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. See Appendix
A for corresponding method usability tiers.
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SAM 2012 Section 5.0- Selected Chemical Methods
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
5.2.72 OSHA Method PV2103: Chloropicrin
Analyte(s)
Chloropicrin
CASRN
79-06-2
Analysis Purpose: Sample preparation, and analyte determination and measurement
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. See Appendix
A for corresponding method usability tiers.
Detection and Quantitation: The detection limit is 0.01 ng, with a \-\\L 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.
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
5.2.73 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 analyte determination and measurement
Sample Preparation Technique: Direct transfer
Determinative Technique: Transmission electron microscopy (TEM)
SAM 2012 91 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
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. See Appendix A for corresponding method usability tiers.
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
5.2.74 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 analyte determination and measurement
Sample Preparation Technique: Direct transfer
Determinative Technique: TEM
Method Developed for: Asbestos in samples wiped from surfaces
Method Selected for: SAM lists this method for preparation and analysis of wipe (e.g., hard surfaces-
wipes) samples. See Appendix A for corresponding method usability tiers.
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
SAM 2012 92 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
5.2.75 ASTM Method D7597-09: Standard Test Method for Determination of Diisopropyl
Methylphosphonate, Ethyl Hydrogen Dimethylamidophosphate, Ethyl
Methylphosphonic Acid, Isopropyl Methylphosphonic Acid, Methylphosphonic
Acid and Pinacolyl Methylphosphonic Acid in Water by Liquid
Chromatography/Tandem Mass Spectrometry
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 analyte determination and measurement
Sample Preparation Technique: Filtered using a syringe-driven Millexฎ-HV PVDF filter unit
Determinative Technique: LC-MS-MS
Method Developed for: Diisopropyl methylphosphonate, ethyl hydrogen dimethylamidophosphate,
isopropyl methylphosphonic acid, methylphosphonic acid and pinacolyl methylphosphonic acid in surface
water
Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid and drinking
water samples. For DIMP in drinking water samples, use EPA Method 538 for sample preparation and
analysis. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The detection verification levels (DVLs) and reporting range for this
method vary for each analyte and range from 0.25 to 20 (ig/L and 5 to 1500 (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 0 to 6 ฐC, 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 single reaction monitoring (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 ฑ 5%. Target compounds are quantitated using the SRM transition of the target
compounds and external standard calibration.
Source: ASTM. 2009. "Method D7597-09: Standard Test Method for Determination of Diisopropyl
Methylphosphonate, Ethyl Hydrogen Dimethylamidophosphate, Ethyl Methylphosphonic Acid, Isopropyl
Methylphosphonic Acid, Methylphosphonic Acid and Pinacolyl Methylphosphonic Acid in Water by
Liquid Chromatography/Tandem Mass Spectrometry." http://www.astm.org/Standards/D7597.htm
5.2.76 ASTM Method D7598-09: Standard Test Method for Determination of Thiodiglycol
in Water by Single Reaction Monitoring Liquid Chromatography/Tandem Mass
Spectrometry
Analyte(s)
Thiodiglycol
CASRN
111-48-8
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Filtered using a syringe-driven Millexฎ HV PVDF filter unit
Determinative Technique: LC-MS-MS
Method Developed for: Thiodiglycol in surface water samples
SAM 2012 93 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid and drinking
water samples. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The DVL for thiodiglycol is 20 (ig/L; the reporting range is 100 to 10000
ug/L.
Description of Method: Thiodiglycol is analyzed by direct injection without derivatization by LC-MS-
MS. Samples are shipped to the laboratory at 0 to 6 ฐC, 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 ฑ 5%. Thiodiglycol is quantitated using the
primary SRM transition and external standard calibration.
Source: ASTM. 2009. "Method D7598-09: Standard Test Method for Determination of Thiodiglycol in
Water by Single Reaction Monitoring Liquid Chromatography/Tandem Mass Spectrometry."
http://www.astm.org/Standards/D7598.htm
5.2.77 ASTM Method D7599-09: Standard Test Method for Determination of
Diethanolamine, Triethanolamine, A/-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 analyte determination and measurement
Sample Preparation Technique: Filtered using a syringe-driven Millexฎ HV PVDF filter unit
Determinative Technique: LC-MS-MS
Method Developed for: Diethanolamine, triethanolamine, ซ-methyldiethanolamine and n-
ethyldiethanolamine in surface water samples
Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid and drinking
water samples. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The DVL and reporting range for EDEA and TEA are 5 (ig/L and 25 to
500 (ig/L, respectively. The DVL and reporting range for MDEA are 10 (ig/L and 50 to 500 (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 0 to 6 ฐC, 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 ฑ 5%. Target
compounds are quantitated using the SRM transition and external standard calibration.
Source: ASTM. 2009. "Method D7599-09: Standard Test Method for Determination of Diethanolamine,
Triethanolamine, 7V-Methyldiethanolamine and jV-Ethyldiethanolamine in Water by Single Reaction
Monitoring Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS)."
http://www.astm.org/Standards/D7599.htm
SAM 2012 94 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
5.2.78 ASTM Method D7644-10: Standard Test Method for Determination of
Bromadiolone, Brodifacoum, Diphacinone and Warfarin in Water by Liquid
Chromatography/Tandem Mass Spectrometry (LC/MS/MS)
Analyte(s)
Brodifacoum
Bromadiolone
Diphacinone
CASRN
56073-10-0
28772-56-7
82-66-6
Analysis Purpose: Sample preparation, and/or analyte determination and measurement
Sample Preparation Technique: Filtered using a syringe-driven PVDF filter unit
Determinative Technique: LC-MS-MS
Method Developed for: Bromadiolone, brodifacoum and diphacinone in water
Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid and drinking
water samples. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The DVLs and reporting range for each analyte are 0.020 (ig/L and 0.125 -
2.5 (ig/L, respectively.
Description of Method: Target compounds are analyzed by direct injection without derivatization using
LC-MS-MS. Samples are shipped to the laboratory at 0 to 6 ฐC, spiked with surrogates, filtered using a
syringe-driven filter unit, and analyzed directly by LC-MS-MS within 14 days. The target analytes are
identified by retention time and two SRM transitions. The retention time for the analytes in the sample
must fall within ฑ 5% of the retention time of the analytes in standard solution. Target analytes are
measured using the primary SRM transition of the analytes and external standard calibration. Analytes
are confirmed using the confirmatory SRM transitions.
Source: ASTM. 2009. "Method D7644-10: Standard Test Method for Determination of Bromadiolone,
Brodifacoum, Diphacinone and Warfarin in Water by Liquid Chromatography/Tandem Mass
Spectrometry." http: //www. astm. org/Standards/D7644 .htm. Note: If this method is no longer available
through ASTM, please refer to the appropriate point of contact listed in Section 4 for information on
obtaining the method.
5.2.79 ASTM Method D7645-10: Standard Test Method for Determination of Aldicarb,
Aldicarb Sulfone, Aldicarb Sulfoxide, Carbofuran, Methomyl, Oxamyl and
Thiofanox in Water by Liquid Chromatography/Tandem Mass Spectrometry
(LC/MS/MS)
Analyte(s)
Aldicarb
Aldicarb sulfone
Aldicarb sulfoxide
Carbofuran
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/or analyte determination and measurement
Sample Preparation Technique: Filtered using a syringe-driven PVDF filter unit
Determinative Technique: LC-MS-MS
SAM 2012 95 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Method Developed for: Aldicarb, aldicarb sulfone, aldicarb sulfoxide, carbofuran, oxamyl, methomyl
and thiofanox in water
Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid samples for
aldicarb, aldicarb sulfone, aldicarb sulfoxide, carbofuran, methomyl, oxamyl and thiofanox. See
Appendix A for corresponding method usability tiers.
Detection and Quantitation: The DVL for aldicarb sulfone, aldicarb sulfoxide and thiofanox is 250
ng/L. The reporting range is 1 - 100 (ig/L.
Description of Method: Target compounds are analyzed by direct injection without derivatization using
LC-MS-MS. Samples are shipped to the laboratory at 0 to 6 ฐC, spiked with surrogates, filtered using a
syringe-driven filter unit, and analyzed directly by LC-MS-MS within 14 days. The target analytes are
identified by comparing primary and confirmatory MRM transitions to known standard primary and
confirmatory MRM transitions. The retention time for the analytes in the sample must fall within ฑ 5% of
the retention time of the analytes in standard solution. Target analytes are measured using the primary
SRM transition and external standard calibration.
Source: ASTM. 2010. "Method D7645: Standard Test Method for Determination of Aldicarb, Aldicarb
Sulfone, Aldicarb Sulfoxide, Carbofuran, Methomyl, Oxamyl and Thiofanox in Water by Liquid
Chromatography/Tandem Mass Spectrometry." http://www.astm.org/Standards/D7645.htm. Note: If
this method is no longer available through ASTM, please refer to the appropriate point of contact listed in
Section 4 for information on obtaining the method.
5.2.80 ASTM Method E2787-11: Standard Test Method for Determination of Thiodiglycol
in Soil Using Pressurized Fluid Extraction Followed 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 analyte determination and measurement
Sample Preparation Technique: Extracted using PFE, and filtered using a syringe-driven PVDF filter
unit
Determinative Technique: LC-MS-MS
Method Developed for: Thiodiglycol in solid samples
Method Selected for: SAM lists this method for preparation and analysis of solid samples. See
Appendix A for corresponding method usability tiers.
Detection and Quantitation: The MDL for thiodiglycol is 54 (ig/kg. The reporting range is 200 -
16,000 (ig/kg.
Description of Method: Samples are shipped to the laboratory at 0 to 6 ฐC and must be extracted,
concentrated, and analyzed by LC-MS-MS within 7 days. Approximately 5 - 30 g of soil is mixed with
an appropriate amount (depending on the wetness of the soil) of drying agent (diatomaceous earth),
spiked with a surrogate, and extracted in a PFE system using methanol. Extracts are filtered using a 0.2-
micron filter and concentrated to a final volume of 0.4 mL using a nitrogen evaporation device. The
volume of the extract is brought up to 2 mL with HPLC-grade water and analyzed by LC-MS-MS. The
target analytes are identified by comparing the sample SRM transitions to the known standard SRM
transitions. The retention time for the analytes in the sample must fall within ฑ5% of the retention time
of the analytes in standard solution. Target analytes are measured using the SRM transition and external
standard calibration.
SAM 2012 96 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Source: ASTM. 2011. "Method E2787: Standard Test Method for Determination of Thiodiglycol in Soil
Using Pressurized Fluid Extraction Followed by Single Reaction Monitoring Liquid Chromatography/
Tandem Mass Spectrometry." http://www.astm.org/Standards/E2787.htm. Note: If this method is no
longer available through ASTM, please refer to the appropriate point of contact listed in Section 4 for
information on obtaining the method.
5.2.81 ASTM Method E2838-11: Standard Test Method for Determination of Thiodiglycol
on Wipes by Solvent Extraction Followed by Liquid Chromatography/Tandem
Mass Spectrometry (LC/MS/MS)
Analyte(s)
Thiodiglycol
CASRN
111-48-8
Analysis Purpose: Sample preparation, and/or analyte determination and measurement
Sample Preparation Technique: Extracted using sonication or PFE and filtered using a syringe-driven
PVDF filter unit
Determinative Technique: LC-MS-MS
Method Developed for: Thiodiglycol in wipes
Method Selected for: SAM lists this method for preparation and analysis of wipe samples. See
Appendix A for corresponding method usability tiers.
Detection and Quantitation: The MDL for thiodiglycol is 0.085 (ig/wipe. The reporting range is 1 - 80
(ig/wipe.
Description of Method: Wipe samples are shipped to the laboratory at 0 to 6 ฐC, and must be extracted,
concentrated, and analyzed by LC-MS-MS within 7 days. Extraction may be performed using sonication
or PFE. Extracts are filtered using a 0.2 micron filter and concentrated to a final volume of 2 mL when
using sonication or 4 mL when using PFE. If sample throughput is less of a concern, the PFE extracts can
be concentrated down to 2 mL. Extracts are analyzed by LC-MS-MS. The target analytes are identified
by comparing the SRM transitions to the known standard SRM transitions. The retention time for the
analytes in the sample must fall within ฑ 5% of the retention time of the analytes in standard solution.
Target analytes are measured using the SRM transition and external standard calibration.
Source: ASTM. 2011. "Method E2383: Standard Test Method for Determination of Thiodiglycol on
Wipes Using Pressurized Fluid Extraction Followed by Single Reaction Monitoring Liquid
Chromatography/Tandem Mass Spectrometry." http://www.astm.org/Standards/E2838.htm. Note: If
this method is no longer available through ASTM, please refer to the appropriate point of contact listed in
Section 4 for information on obtaining the method.
SAM 2012 97 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
5.2.82 ASTM Method E2866-12: Standard Test Method for Determination of Diisopropyl
Methylphosphonate, Ethyl Methylphosphonic Acid, Isopropyl Methylphosphonic
Acid, Methylphosphonic Acid and Pinacolyl Methylphosphonic Acid in Soil by
Pressurized Fluid Extraction and Analyzed by Liquid Chromatography/Tandem
Mass Spectrometry
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 analyte determination and measurement
Sample Preparation Technique: Extracted using PFE and filtered using a syringe-driven Millexฎ-HV
PVDF filter unit
Determinative Technique: LC-MS-MS
Method Developed for: Diisopropyl methylphosphonate, ethyl hydrogen dimethylamidophosphate,
isopropyl methylphosphonic acid, methylphosphonic acid and pinacolyl methylphosphonic acid in soil
Method Selected for: SAM lists this method for preparation and analysis of solid samples. See
Appendix A for corresponding method usability tiers.
Detection and Quantitation: The MDLs range from 1.3 to 8.7 (ig/kg. The reporting range for all
analytes is 40 to 2000 ug/kg.
Description of Method: Target compounds are analyzed by direct injection without derivatization by
LC-MS-MS. Samples are shipped to the laboratory at 0 to 6 ฐC and must be extracted, concentrated, and
analyzed by LC-MS-MS within 7 days. Approximately 5 - 30 g of soil are mixed with an appropriate
amount (depending on the wetness of the soil) of drying agent (diatomaceous earth), spiked with a
surrogate, and extracted in a PFE system using water. Extracts are filtered using a 0.2-micron filter and
analyzed by LC-MS-MS. 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 ฑ 5%. Target compounds are quantitated using the SRM
transition of the target compounds and external standard calibration.
Source: ASTM. 2012. "Method E2866-12: Standard Test Method for Determination of Diisopropyl
Methylphosphonate, Ethyl Hydrogen Dimethylamidophosphate, Ethyl Methylphosphonic Acid, Isopropyl
Methylphosphonic Acid, Methylphosphonic Acid and Pinacolyl Methylphosphonic Acid in Water by
Liquid Chromatography/Tandem Mass Spectrometry." http://www.astm.org/Standards/E2866.htm.
Note: If this method is no longer available through ASTM, please refer to the appropriate point of
contact listed in Section 4 for information on obtaining the method.
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 analyte determination and measurement
Sample Preparation Technique: Direct transfer
Determinative Technique: TEM
SAM 2012 98 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Method Developed for: Asbestos in ambient air
Method Selected for: SAM lists this method for preparation and analysis of air samples. See Appendix
A for corresponding method usability tiers.
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
Determinative Technique: Visible spectrophotometry
Determinative Method: 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. See Appendix
A for corresponding method usability tiers.
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: Analyte determination and measurement
Determinative Technique: Visible spectrophotometry
Sample Preparation Method: Standard Method 4500-NH3 B
Sample Preparation Technique: Distillation
SAM 2012 99 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
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. See Appendix A
for corresponding method usability tiers.
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: Chlorine (Residual) DPD Colorimetric Method
Analyte(s)
Chlorine
CASRN
7782-50-5
Analysis Purpose: Sample preparation, and/or analyte determination and measurement
Sample Preparation Technique: Water samples are buffered and colorimetric agent is added. Buffered
water extraction by Analyst, 1999. 124: 1853-1857 are used for preparation of air samples.
Determinative Technique: Visible spectrophotometry
Method Developed for: Chlorine in water and wastewater
Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid and drinking
water samples. It also should be used for analysis of air samples when appropriate sample preparation
techniques have been applied. See Appendix A for corresponding method usability tiers.
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-CI 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 Hexamethylenetriperoxidediamine (HMTD) (Analyst, 2001.
126:1689-1693)
Analyte(s)
Hexamethylenetriperoxidediamine (HMTD)
CASRN
283-66-9
Analysis Purpose: Analyte determination and measurement
Sample Preparation Technique: SW-846 Methods 8330B/3535A (solid samples, aqueous liquid and
drinking water samples), and 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.
SAM 2012 100 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Determinative Technique: LC-MS-MS
Method Developed for: Trace quantities of HMTD in explosives or explosive mixtures
Method Selected for: SAM lists this procedure for analysis of solid, aqueous liquid, drinking water and
wipe samples. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The LOD is 20 (ig/L.
Description of Method: Prepared samples are analyzed by positive mode atmospheric pressure chemical
ionization (APCI) LC-MS-MS using a Ci8 analytical column (150 mm x 2.0 mm I.D., 5(im particle size)
coupled with a Ci8 guard cartridge system (10 mm x 2.0 mm I.D.). Elution using a 95/5 water/methanol
solution detects HMTD at m/z = 209 and a retention time of- 15.5 minutes.
Special Considerations: The procedure has been developed for the determination of HMTD in
explosives or explosive mixtures; modifications will be needed for application to environmental samples
such as soils, wipes and water samples. Until modifications can be developed and tested, it is
recommended that the sample preparation procedures described in SW-846 Methods 8330B and 3535A
(solid samples, aqueous liquid and drinking water samples) and SW-846 Methods 3570 and 8290A
Appendix A (wipe samples) be used.
Source: Crowson, A. and Berardah, M.S. 2001. "Development of an LC/MS Method for the Trace
Analysis of Hexamethylenetriperoxidediamine (HMTD)." Analyst 126(10): 1689-1693.
http://pubs.rsc.org/en/Content/ArticleLanding/2001/AN/bl07354k
5.2.88 Literature Reference for Chlorine in Air (Analyst, 1999. 124(12): 1853-1857)
Analyte(s)
Chlorine
CASRN
7782-50-5
Analysis Purpose: Sample preparation
Sample Preparation Technique: Buffered water extraction
Determinative Technique: Visible spectrophotometry
Determinative Method: Standard Method 4500-C1 G
Method Developed for: Active chlorine in air
Method Selected for: SAM lists this procedure for preparation of air samples. See Appendix A for
corresponding method usability tiers.
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/minute, 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 Spectrophotometric Analysis." Analyst.
124(12): 1853-1857. http://pubs.rsc.org/en/content/articlelanding/1999/an/a906305f
SAM 2012 101 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
5.2.89 Literature Reference for Methamidophos (Chromatographia. 2006. 63(5/6): 233-
237)
Analyte(s)
Ace p hate
Methamidophos
CASRN
30560-19-1
10265-92-6
Analysis Purpose: Sample preparation, and analyte determination and measurement
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 samples.
See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The LOD 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 and LC-MS-MS. Samples are analyzed using a
Cig analytical column (150 mm x 3.2 mm I.D., 5(im particle size) coupled with a Qg guard cartridge
system (4 mm x 3.0 mm I.D.).
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.springerlink.com/content/gg871501126390x6/
5.2.90 Literature Reference for Cyanogen Chloride (Encyclopedia of Anal. Chem. 2006
DOI: 10.1002/9780470027318.a0809)
Analyte(s)
Cyanogen chloride
CASRN
506-77-4
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Purge-and-trap, headspace, liquid-liquid microextraction
Determinative Technique: GC-MS, GC-ECD
Method Developed for: Determination of cyanogen chloride in drinking water
Method Selected for: SAM lists this procedure for preparation and analysis of aqueous liquid, drinking
water and solid samples. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: In drinking water, the MDL is 0.13 (ig/L when using purge-and-trap
GC-MS or liquid-liquid microextraction GC-ECD, and 0.04 (ig/L when using headspace GC-ECD.
Description of Method: The method describes three different sample preparation techniques (purge-
and-trap, headspace and micro liquid-liquid extraction) and two different determinative techniques (GC-
MS and GC-ECD). Using the purge-and-trap technique, cyanogen chloride and an internal standard are
extracted (purged) from the sample matrix by bubbling an inert gas through the sample. Purged sample
components are trapped in a tube containing suitable sorbent materials. When purging is complete, the
sorbent tube is heated. Simultaneously, a short piece of deactivated fused silica precolumn is cooled with
liquid nitrogen to refocus the analytes. The cryotrap is heated to inject the sample onto a GC-MS.
For headspace GC-ECD analyses, a 40-mL vial is filled with sample without headspace. With the vial
upside down, a volume of nitrogen is forced into the sample using a syringe, and an equivalent sample
SAM 2012 102 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
volume is dispelled through a second syringe. The sample is shaken by hand and, after settling, a volume
of the headspace is sampled by syringe and injected into a split-mode GC-ECD. For liquid-liquid
microextraction GC-ECD analyses, 30 mL of water sample is extracted in a 40-mL vial, with 10 g of
Na2SO4, 4 mL of MTBE and an internal standard. The sample is shaken by mechanical shaker or by
hand. After allowing the phases to separate, the MTBE layer is transferred to another vial and injected
into a GC-ECD.
Special Considerations: This procedure has been developed for water samples; modifications may be
needed for application to environmental samples such as solid samples.
Source: Xie, Y. 2006. "Cyanogen Chloride and Cyanogen Bromide Analysis in Drinking Water."
Encyclopedia of Analytical Chemistry, 1-11.
http://onlinelibrarv.wilev.com/doi/10.1002/9780470027318.a0809/abstract
5.2.91 Literature Reference for 3-Chloro-1,2-propanediol (Eur. J. Lipid Sci. Technol. 2011,
113:345-355)
Analyte(s)
3-Chloro-1 ,2-propanediol
CASRN
96-24-2
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Solvent extraction, followed by solid phase extraction cleanup and
derivatization
Determinative Technique: GC-MS
Method Developed for: Trace quantities of 3-chloro-l,2-propanediol in foodstuffs
Method Selected for: SAM lists this procedure for preparation and analysis of solid and wipe samples.
See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The low calibration standard is 5 (ig/L. The MDL in food ranges from 4 to
16 (ig/kg. The working range is 4 - 4,000 (ig/kg.
Description of Method: Foodstuffs (olive oil, cereal and potato products) are solvent extracted with
hexane/diethyl ether and centrifuged. The resulting organic layer is washed several times (by adding
water, vortexing and then centrifuging), then dried with sodium sulfate. The extract is concentrated to
dryness, and redissolved in tetrahydrofuran (THF) to which acidified methanol is added. The reaction
mixture is neutralized with sodium bicarbonate and washed with 3 aliquots of hexane, and the residue is
quantitatively transferred to a sodium chloride solution. This solution is mixed with the contents of a
highly pure diatomaceous earth based solid phase refill sachet, transferred to a chromatography column,
and then eluted with diethyl ether. The collected eluent is concentrated by rotary evaporation and
derivatized with heptaflurobutyrylimidazole (HFBI) at 70 ฐC for 15-20 minutes. After washing with
water, the extracts are analyzed using a GC-MS.
Special Considerations: The procedure has been developed for the determination of 3-chloro-l,2-
propanediol in foodstuffs only; modifications may be needed for application to environmental samples.
Source: Hamlet, C. G. and Asuncion, L. 2011. "Single-Laboratory Validation of a Method to Quantify
Bound 2-Chloropropane-l,3-diol and 3-Chloropropane-l,2-diol in Foodstuffs Using Acid Catalysed
Transesterification, HFBI Derivatisation and GC/MS Detection." Eur. J. Lipid Sci. Technol., 113(3):
345-355. http://onlinelibrarv.wilev.eom/doi/10.1002/eilt.vll3.3/issuetoc
SAM 2012 103 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
5.2.92 Literature Reference for Methyl Hydrazine (Journal of Chromatography 1993 (617),
157-162)
Analyte(s)
Methyl hydrazine
CASRN
60-34-4
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: SW-846 Method 3541/3545 (for solids), SW-846 Methods 3570/8290
Appendix A (for wipes), filtration for water samples, followed by derivatization for all sample types
Determinative Technique: HPLC-UV
Method Developed for: Determination of hydrazine in human plasma
Method Selected for: SAM lists this procedure for preparation and analysis of aqueous liquid and
drinking water samples, and for the analysis of solid and wipe samples. See Appendix A for
corresponding method usability tiers.
Detection and Quantitation: Detection limit in pooled plasma is 1 (ig/L. The reporting range is 5 -
1000 ug/L.
Description of Method: Samples are prepared in a single-step reaction by protein denaturation with
trichloroacetic acid, and derivatization to a stable azine with 4-hydroxybenzaldehyde. Chromatographic
separation is carried out on a reversed-phase (octadecylsilane) column with methanol:water (60:40) as the
mobile phase and UV detection at 340 nm. Retention time of the azine derivative of methyl hydrazine is
3.5 minutes.
Special Considerations: This procedure has been developed for human plasma; modifications may be
needed for application to environmental samples such as aqueous liquid, drinking water, solid and wipes
samples.
Source: Kircherr, H. 1993. "Determination of Hydrazine in Human Plasma by High Performance Liquid
Chromatography." Journal of Chromatography B, 617(1): 157-162.
http://www.sciencedirect.com/science/article/pii/0378434793804368
5.2.93 Literature Reference for Paraquat (Journal of Chromatography A, 2008, 1196-
1197, 110-116)
Analyte(s)
Paraquat
CASRN
4685-14-7
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Extraction by digestion, shaking or microwave-assisted extraction
(MAE) followed by SPE cleanup
Determinative Technique: LC-UV or LC-MS-MS
Method Developed for: Determination of quaternary ammonium herbicides in soil
Method Selected for: SAM lists this procedure for preparation and analysis of solid and wipe samples.
See Appendix A for corresponding method usability tiers.
Detection and Quantitation: Limits of detection are 10 (ig/kg by digestion and 50 (ig/kg by MAE when
using LC-UV and 1.0 (ig/kg by digestion and 3.0 (ig/kg by MAE when using LC-MS-MS. Estimated
quantification limits are 20 (ig/kg and 100 (ig/kg when using LC-UV and 2.0 (ig/kg by digestion and
7.5(ig/kg by MAE when using LC-MS-MS.
SAM 2012 104 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Description of Method: Soil matrices can be extracted using one of the following three procedures: (1)
digestion with an acidic methanol/ EDTA solution, (2) shaking in an EDTA/ammonium formate solution,
or (3) using a microwave assisted extraction system in a benzalkonium chloride/acid solution. Cleanup of
extracts is performed by SPE using silica cartridges for all three extraction procedures. Detection of these
herbicides is carried out by either LC-UV or LC-MS-MS.
Special Considerations: This procedure has been developed for soil samples; modifications may be
needed for application to environmental samples such as wipes samples.
Source: Pateiro-Moure, M., Martinez-Carballo, E., Arias-Estevez, M. and Simal-Gandara, J. 2008.
"Determination of Quaternary Ammonium Herbicides in Soils. Comparison of Digestion, Shaking and
Microwave-Assisted Extractions." Journal of Chromatography A, 1196-1197, 110-116.
http://www.elsevier.com/locate/chroma
5.2.94 Literature Reference for Methamidophos (Journal of Chromatography A, 2007.
1154:3-25)
Analyte(s)
Ace p hate
Methamidophos
CASRN
30560-19-1
10265-92-6
Analysis Purpose: Sample preparation, and analyte determination and measurement
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, air and wipe
samples. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The LOD 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 ESI+
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 Ci8 analytical
column (150 mm x 3.2 mm I.D., 5(im particle size) coupled with a Ci8 guard cartridge system (4 mm x
S.Omml.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. and 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/journal/00219673
SAM 2012 105 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
5.2.95 Literature Reference for Fluoroacetic Acid/Fluoroacetate Salts/Methyl
Fluoroacetate (Journal of Chromatography A, 1139 (2007) 271-278)
Analyte(s)
Fluoroacetic acid and fluoroacetate salts (analyze as fluoroacetate ion)
Methyl fluoroacetate (analyze as fluoroacetate ion)
CASRN
NA
453-18-9
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Water extraction followed by SPE cleanup and derivatization for solid
and wipe samples. Use NIOSH Method S301-1 for air samples.
Determinative Technique: LC-MS
Method Developed for: Determination of fluoroacetate in food
Method Selected for: SAM lists this procedure for preparation and analysis of solids and wipes and for
the analysis of air samples. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The LOD is 0.8 (ig/L. The calibration range is 20 - 10,000 (ig/L.
Description of Method: The method utilizes a water extraction, SPE cleanup, and LC-MS for
determination of fluoroacetate as monofluoroacetate (MFA). SPE is performed using Ci8 cartridges. The
LC-MS system utilizes a Ci8 column and the MS is operated in atmospheric pressurized chemical
ionization (APCI) negative mode. If significant interferences are observed, the method describes a
qualitative procedure that can be used to confirm the presence of fluoroacetate. The sample is first
prepared as described in the quantitative method. Then an aliquot is derivatized by adding 2-
nitrophenylhydrazine, l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and pyridine
buffer, and heating at 65 ฐC for 15 minutes. The extract is then cleaned by putting it through a Ci8
cartridge. The extracts are then blown to dryness, reconstituted in 2 mL of water/methanol (20/80), and
filtered through a 0.2 (im filter. Analysis of the cleaned extract is performed on an LC-MS using a C8
column and gradient elution, beginning with 25% methanol for the first 3 minutes, followed by 80%
methanol over the next 10 minutes. A post run equilibration (7 minutes) is used prior to the next
injection.
Special Considerations: This procedure has been developed for food; modifications may be needed for
application to environmental samples such as solid and wipe samples. In addition, the air filter extraction
procedure (described in NIOSH Method S301-1) was not developed for the LC-MS-MS detector, and it
may be necessary to alter the extraction method if interferences arising from the extraction are observed.
Source: Noonan, G.O., Begley, T.H. and Diachenko, G.W. 2007. "Rapid Quantitative and Qualitative
Confirmatory Method for the Determination of Monofluoroacetic Acid in Foods by Liquid
Chromatography-Mass Spectrometry." Journal of Chromatography A, 1139: 271-278.
http://www.elsevier.com/locate/chroma
5.2.96 Literature Reference for 3-Chloro-1,2-propanediol (Journal of Chromatography A,
2000. 866: 65-77)
Analyte(s)
3-Chloro-1 ,2-propanediol
CASRN
96-24-2
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Solvent extraction followed by derivatization
Determinative Technique: GC-ECD
SAM 2012 106 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Method Developed for: Determination of 3-chloro-l,2-propanediol in water
Method Selected for: SAM lists this procedure for preparation and analysis of aqueous liquid and
drinking water samples. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The MDL is 0.73 (ig/L. The reporting range is 11 - 169 (ig/L.
Description of Method: Sodium sulfate, sodium bisulfate and a surrogate are added to a 5-mL sample
and extracted twice with 5 mL of ethyl acetate. The two ethyl acetate extracts are combined and
concentrated to 50 (iL under nitrogen evaporation. Then 100 (iL of acetonitrile is added, and the solution
is mixed and transferred to a drying column containing sodium sulfate. An additional 100 \\L of
acetonitrile is used to rinse the sample vial and the rinse is transferred to the drying column. After letting
the sample sit on the column for 10 minutes, it is eluted with 2 mL of acetonitrile. The dried extract is
derivatized by adding 50 \\L of heptafluorobutyric anhydride (HFBA) and heating at 75 ฐC for 30
minutes. The derivatized sample is extracted with water, then hexane, followed by a saturated sodium
bicarbonate solution. The aqueous layer is discarded, and the hexane layer is washed twice with sodium
bicarbonate solution and shaking for 30 seconds. The hexane extract is then transferred to a GC vial and
analyzed by GC-ECD with a DB5-MS column.
Special Considerations: The procedure has been tested for reagent grade water and seawater;
modifications may be needed for application to environmental samples.
Source: Matthew, B.M. and Anastasio, C. 2000. "Determination of Halogenated Mono-alcohols and
Diols in Water by Gas Chromatography With Electron-Capture Detection." Journal of Chromatography
A, 866(1): 65-77. http://www.elsevier.com/locate/chroma
5.2.97 Literature Reference for Fluoroacetic Acid/Fluoroacetate Salts/Methyl
Fluoroacetate (Journal of Chromatography B, 2010, 878: 1045-1050)
Analyte(s)
Fluoroacetic acid and fluoroacetate salts (analyze as fluoroacetate ion)
Methyl fluoroacetate (analyze as fluoroacetate ion)
CASRN
NA
453-18-9
Analysis Purpose: Sample preparation (aqueous liquid and drinking water only), and analyte
determination and measurement
Sample Preparation Technique: SPE using 96-well plates (see Special Considerations)
Determinative Technique: LC-MS-MS
Method Developed for: Determination of fluoroacetate in urine
Method Selected for: SAM lists this procedure for preparation and analysis of drinking water and
aqueous liquid samples. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The detection limit for fluoroacetate in human urine is 0.9 (ig/L. The
reporting range is 50 to 5000 (ig/L.
Description of Method: Fluoroacetate, in the form of monofluoroacetate, is extracted from urine by
placing aliquots onto a 96-well plate and performing SPE using hydrophilic-lipophilic-balanced (HLB)
reverse-phase sorbent plates (or equivalent). The extracts are then separated with isocratic high-
performance liquid Chromatography with a reverse-phase analytical column with embedded basic ion-
pairing groups. Target compounds are identified using ESI tandem mass spectrometry. The retention
time, when using the conditions described in this journal article, are expected to be ~1.4 minutes.
Special Considerations: This procedure has been developed for urine samples; modifications may be
needed for application to environmental samples. For drinking water samples and relatively clean
SAM 2012 107 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
aqueous liquid samples, direct injection may be suitable. If the laboratory has the capability of
performing IC-MS, they may consider using the following method, which has been developed specifically
for water samples: http://www.dionex.com/en-us/webdocs/110767-AN276-IC-Fluoroacetate-Water-
28Dec2011-LPN2807.pdf
Source: Hamelin, E., Mawhinney, D.B., Parry R. and Kobelski, R.J. 2010. "Quantification of
Monofluoroacetate and Monochloroacetate in Human Urine by Isotope Dilution Liquid Chromatography
Tandem Mass Spectrometry." Journal of Chromatography B, 878(15-16): 1045-1050.
http://www.elsevier.com/locate/chromb
5.2.98 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 analyte determination and measurement
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, aqueous liquid,
drinking water, air and wipe samples. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The detection limit of this method for fluoroacetamide is 0.01 (ig/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.
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 Acute 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/article/pii/S1570023208007757
5.2.99 Literature Reference for Sodium Azide (Journal of Forensic Sciences, 1998.
43(1): 200-202)
Analyte(s)
Sodium azide (analyze as azide ion)
CASRN
26628-22-8
Analysis Purpose: Sample preparation
Sample Preparation Technique: Water extraction, filtration and/or acidification
Determinative Technique: 1C with conductivity detection
Determinative Method: EPA Method 300.1, Revision 1.0
SAM 2012 108 July 16, 2011
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SAM 2012 Section 5.0- Selected Chemical Methods
Method Developed for: Sodium azide in blood
Method Selected for: SAM lists this procedure for preparation of solid, aqueous liquid and drinking
water samples. See Appendix A for corresponding method usability tiers.
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.
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/DIGITAL LIBRARY/JOURNALS/FORENSIC/PAGES/JFS16113J.htm
SAM 2012 109 July 16, 2011
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SAM 2012 Section 6.0- Selected Radiochemical Methods
Section 6.0: Selected Radiochemical Methods
A list of analytical methods to be used in analyzing environmental samples for radiochemical
contaminants following a contamination incident 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.
Chemical Abstracts Service Registry 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. 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.
Vegetation sample methods. The recommended methods/procedures for sample preparation and
analysis to measure the analyte of interest in vegetation (i.e., grasses, leaves, trees, etc.) not intended
for human consumption. 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 radionuclide. 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
radionuclide per unit of mass, volume or area sampled.
SAM 2012 110 July 16, 2011
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SAM 2012 Section 6.0- Selected Radiochemical Methods
Following a contamination incident, it is assumed that only those areas with contamination greater than
pre-existing/naturally prevalent levels (i.e., background) 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 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
quality control (QC) procedures.
For additional information on the properties of the radionuclides listed in Appendix B, Toxicology Data
Network (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 Program (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) websites provide some additional information
pertaining to radionuclides of interest and selection of radiochemical methods. Documents for emergency
response operations for laboratories, recently developed by EPA's Office of Radiation and Indoor Air
(ORIA), describe the likely analytical decision paths that would be required by personnel at a
radioanalytical laboratory following a radiological or nuclear contamination incident. These documents
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: Selected 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.
Once a method has been identified in Appendix B, Table 6-1 can be used to locate the method summary.
Sections 6.2.1 through 6.2.36, below, provide summaries of the qualitative and confirmatory methods
listed in Appendix B.
Table 6-1. Radiochemical Methods and Corresponding Section Numbers
Analyte / Analyte Class
Gross Alpha
Gross Beta
Gamma
Select Mixed Fission Products
Total Activity Screening
CASRN
NA
NA
NA
NA
Method
900.0 (EPA)
FRMAC, Vol 2, pg. 33 (DOE)
AP1 (ORISE)
7110 B(SM)
901.1 (EPA)
Ga-01-R(HASL-300)
Y-12 Preparation of Samples
for Total Activity Screening
(DoD)
Section
6.2.2
6.2.30
6.2.34
6.2.43
6.2.3
6.2.22
6.2.49
Please note that this category does not cover all fission products.
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Analyte / Analyte Class
Americium-241
Californium-252
Cesium-137
Cobalt-60
Curium-244
Europium-154
lodine-125
lodine-131
lridium-192
Molybdenum-99
Phosphorus-32
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
14119-15-4
14596-37-3
Method
Rapid Radiochemical Method
forAm-241 (EPA)
Am-01-RC(HASL-300)
Am-04-RC (HASL-300)
Am-06-RC (HASL-300)
Pu-12-RC (HASL-300)
Actinides and Sr-89/90 in Soil
Samples (SRS)
Actinides and Sr-89/90 in
Vegetation (SRS)
AP11 (ORISE)
D3084-05 (ASTM)
7120 (SM)
Rapid methods for acid or
fusion digestion (EPA)
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)
Rapid Radiochemical Method
for P-32 (EPA)
R4-73-014(EPA)
RESL P-2 (DOE)
Section
6.2.12
6.2.19
6.2.20
6.2.21
6.2.24
6.2. 32
6.2. 33
6.2.37
6.2.39
6.2.44
6.2.17 and 6.2.18
6.2.19
6.2.20
6.2.24
6.2.37
6.2.39
6.2.3
6.2.22
6.2.44
6.2.19
6.2.20
6.2.24
6.2.37
6.2.39
6.2.3
6.2.22
6.2.44
6.2.38
6.2.3
6.2.22
6.2.3
6.2.22
6.2.44
6.2.3
6.2.22
6.2.9
6.2.10
6.2.31
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Analyte / Analyte Class
Plutonium-238
Plutonium-239
Polonium-210
Radium-226
Ruthenium-103
Ruthenium-106
Selenium-75
Strontium-89
Strontium-90
Technetium-99
Tritium (Hydrogen-3)
CASRN
13981-16-3
15117-48-3
13981-52-7
13982-63-3
13968-53-1
13967-48-1
14265-71-5
14158-27-1
10098-97-2
14133-76-7
10028-17-8
Method
EMSL-33 (EPA)
Rapid Radiochemical Method
for Pu-238 and -239/240 (EPA)
Actinides and Sr-89/90 in Soil
Samples (SRS)
Actinides and Sr-89/90 in
Vegetation (SRS)
AP11 (ORISE)
D3084-05 (ASTM)
Rapid methods for acid or
fusion digestion (EPA)
Method 111 (EPA)
Po-02-RC (HASL-300)
903.1 (EPA)
EMSL-19(EPA)
Rapid Radiochemical Method
for Ra-226 (EPA)
Ra-03-RC (HASL-300)
D3084-05 (ASTM)
7500-Ra B (SM)
7500-Ra C (SM)
Rapid methods for acid or
fusion digestion (EPA)
901.1 (EPA)
Ga-01-R (HASL-300)
7120 (SM)
905.0 (EPA)
Strontium in Food and
Bioenvironmental Samples
Actinides and Sr-89/90 in Soil
Samples (SRS)
Actinides and Sr-89/90 in
Vegetation (SRS)
905.0 (EPA)
Rapid Radiochemical Method
for Radiostrontium (EPA)
Sr-03-RC (HASL-300)
Actinides and Sr-89/90 in Soil
Samples (SRS)
Actinides and Sr-89/90 in
Vegetation (SRS)
D581 1-08 (ASTM)
Rapid methods for acid or
fusion digestion (EPA)
Tc-01-RC (HASL-300)
Tc-02-RC (HASL-300)
APS (ORISE)
D71 68-05 (ASTM
906.0 (EPA)
AP2 (ORISE)
Section
6.2.8
6.2. 13
6.2.29
6.2. 33
6.2.37
6.2.39
6.2.17 and 6.2.18
6.2.1
6.2.23
6.2.4
6.2.7
6.2. 14
6.2.25
6.2. 39
6.2.45
6.2.46
6.2.17 and 6.2.18
6.2.3
6.2.22
6.2.44
6.2.5
6.2.11
6.2.32
6.2.33
6.2.5
6.2.15
6.2.26
6.2. 32
6.2. 33
6.2.41
6.2.17 and 6.2.18
6.2.27
6.2.28
6.2.36
6.2.42
6.2.6
6.2.35
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SAM 2012 Section 6.0- Selected Radiochemical Methods
Analyte / Analyte Class
Uranium-234
Uranium-235
Uranium-238
CASRN
13966-29-5
15117-96-1
7440-61-1
Method
EMSL-33 (EPA)
Rapid Radiochemical Method
for Isotopic Uranium (EPA)
U-02-RC (HASL-300)
Actinides and Sr-89/90 in Soil
Samples (SRS)
Actinides and Sr-89/90 in
Vegetation(SRS)
AP11 (ORISE)
D3972-02 (ASTM)
7500-U B (SM)
7500-U C (SM)
Rapid methods for acid or
fusion digestion (EPA)
Section
6.2.8
6.2. 16
6.2. 29
6.2. 32
6.2. 33
6.2.37
6.2.40
6.2.47
6.2.48
6.2.17 and 6.2.18
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 voluntary consensus standard bodies
(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 6-2 Sources of Radiochemical Methods
Name
National Environmental
Methods Index (NEMI)
Code of Federal Regulations
(CFR) Promulgated Test
Methods
Prescribed Procedures for
Measurement of Radioactivity
in Drinking Water (EPA-600 4-
80-032, August 1980)
Rapid Radiochemical Methods
for Selected Radionuclides in
Water for Environmental
Restoration Following
Homeland Security Events
(EPA 402-R-1 0-001)
Rapid Radiochemical Method
for Phosphorus-32 in Water
Rapid Radiochemical Methods
for acid digestion and sodium
carbonate fusion of filters and
swipes
Publisher
EPA, U.S. Geological Survey
(USGS)
EPA, Technical Transfer Network
(TTN) Emission Measurement
Center (EMC)
EPA, ORD, Environmental
Monitoring and Support Laboratory
(EMSL)
EPA, ORIA, National Air and
Radiation Environmental
Laboratory (NAREL)
EPA, ORIA, NAREL
EPA, ORIA, NAREL
Reference
http://vwvw.nemi.gov
http://wvvw.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.
http://www.epa.gov/narel/
When published, method will be available
at:
http://www.epa.gov/narel/new docs.html
When published, methods will be available
at:
http://www.epa.aov/narel/new docs.html
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SAM 2012 Section 6.0- Selected Radiochemical Methods
Name
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
Y-12 National Security Complex
(Y-12)
Radiological and Environmental
Sciences Laboratory (RESL)
Analytical Chemistry Branch
Procedures Manual
Savannah River Site (SRS)
Methods
Oak Ridge Institute for Science
and Education (ORISE)
Laboratory Procedures Manual
Annual Book of AST M
Standards, Vol. 11.02*
Standard Methods for the
Examination of Water and
Wastewater, 21st Edition, 2005*
Publisher
EPA, EMSL
Department of Energy (DOE),
Environmental Measurements
Laboratory (EML) / Now DHS
DOE, National Nuclear Security
Administration (NNSA)
DOE, NNSA
DOE, RESL
DOE, SRS
ORISE, Independent
Environmental Assessment and
Verification
ASTM International
American Public Health
Association (APHA), American
Waterworks Association
(AWWA), and Water Environment
Federation (WEF)
Reference
Available NTIS*, U.S. Department of
Commerce, 5285 Port Royal Road,
Springfield, VA 22161, (703)605-6000.
http://vwvw.nbl.doe.qov/htm/EML Leqacv
Website/ProcMan/Start. htm
Also available from NTIS*, U.S.
Department of Commerce, 5285 Port
Royal Road, Springfield, VA 22161, (703)
605-6000.
http://www.nv.doe.qov/nationalsecuritv/ho
melandsecuritv/frmac/manuals.aspx
http://www.v12.doe.gov/
Available from NTIS, U.S. Department of
Commerce, 5285 Port Royal Road,
Springfield, VA 22161, (703)605-6000.
Savannah River National Laboratory
Savannah River Site
Aiken, SC 29808,
(803)725-6211
http://orise.orau.qov/ieav/survev-
proiects/lab-manual.htm
http://www.astm.orq
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 remediating a site and determine effectiveness following remediation.
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 included within the data report when transmitted to decision makers.
5 Information regarding EPA's DQO process, considerations, and planning is available at:
http://www.epa.gov/QUALITY/dqos.html.
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SAM 2012 Section 6.0- Selected Radiochemical Methods
The level or amount of QC needed often depends on the intended purpose of the data that are generated.
Various levels of QC may be required if the data are generated during contaminant presence/absence
qualitative determinations versus confirmatory analyses. The specific needs for data generation should be
identified. QC requirements and data quality objectives (DQOs) 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 needed 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 frequency.
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 analytical protocols developed to address specific
analytes and sample types of concern. Additional information regarding QC requirements specific to
radiochemical methods is included in the MARLAP manual at:
http://www.epa.gov/radiation/marlap/manual.html. Individual methods, sampling and analysis protocols
or contractual statements of work should also 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
Matrix spike/matrix spike duplicate (MS/MSD) recoveries and precision
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
Environmental Response Laboratory Network (ERLN) or Water Laboratory Alliance (WLA) coordinator
responsible for laboratory activities during the specific event to ensure QA/QC procedures are performed
consistently across laboratories. EPA program offices will be responsible for ensuring that the QA/QC
practices are implemented.
6.1.3 Safety and Waste Management
It is imperative that safety precautions be used during collection, processing and analysis of
environmental samples. Laboratories should have a documented radiation safety plan or manual in
addition to a health and safety plan for handling samples that may contain target chemical, biological
and/or radiological (CBR) contaminants, and laboratory staff should be trained in and implement the
safety procedures in the plan or manual. 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:
SAM 2012 116 July 16, 2011
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SAM 2012 Section 6.0- Selected Radiochemical Methods
Occupational Safety and Health Administration (OSHA) - 29 CFRpart 1910.1450. Occupational
Exposure to Hazardous Chemicals in Laboratories. Available at:
http://www. access. gpo.gov/nara/cfr/waisidx_06/29cfrl910a_06.html
EPA - 40 CFRpart 260. Hazardous Waste Management System: General. Available at:
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.
Available at: http://www.access.gpo.gov/nara/cfr/waisidx_07/40cfr270_07.html
U.S. Nuclear Regulatory Commission (NRC) - 10 CFR part 20. Standards for Protection Against
Radiation. Available at: http://www.access.gpo.gov/nara/cfr/waisidx_00/10cfr20_00.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-
l.html
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:
http://www.orau.org/ptp/PTP%20Librarv/librarv/DOE/Misc/radmeth3.pdf
EPA. 1996. Profile and Management Options for EPA Laboratory Generated Mixed Waste. ORIA,
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=2001_register&docid=01-11408-
filed.pdf
EPA. 2008. Resource Conservation and Recovery Act (RCRA) Orientation Manual. Office of Solid
Waste and Emergency Response (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. EPA
402-B-04-001B. 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
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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SAM 2012 Section 6.0- Selected Radiochemical Methods
6.2 Method Summaries
Summaries for the analytical methods listed in Appendix B are provided in Sections 6.2.1 through 6.2.49.
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. Summaries are provided for informational use. 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
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.
Polonium-209 tracers should be added to determine the chemical yield.
Source: EPA EMC, prepared by the Office of Air Quality Planning and Standards (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
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
SAM 2012 118 July 16, 2011
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SAM 2012 Section 6.0- Selected Radiochemical Methods
Plutonium-238 (CAS RN 13981-16-3) Alpha emitter
Plutonium-239 (CAS RN 15117-48-3) Alpha emitter
Polonium-210 (CAS RN 13981-52-7) Alpha emitter
Radium-226 (CAS RN 13982-63-3) Alpha emitter
Ruthenium-103 (CAS RN 13968-53-1) Beta emitter
Ruthenium-106 (CAS RN 13967-48-1) Beta emitter
Strontium-90 (CAS RN 10098-97-2) Beta emitter
Uranium-234 (CAS RN 13966-29-5) Alpha emitter
Uranium-235 (CAS RN 15117-96-1) Alpha emitter
Uranium-238 (CAS RN 7440-16-1) Alpha emitter
An aliquot of a preserved drinking water sample is evaporated to a small volume (3 to 5 mL) and
transferred quantitatively to a tarred 2-inch planchet. The aliquot volume is determined based on a
maximum total solids content of 100 mg. The sample aliquot is evaporated to dryness in the planchet to a
constant weight, cooled, and counted using a gas proportional or scintillation counting system. The
counting system is calibrated with thorium-230 for gross alpha, and with strontium-90 for gross beta
analysis. A traceable standards-based efficiency curve must be developed for each calibration nuclide
(thorium-230 and strontium-90) based on a range of total solids content in the 2-inch planchet from 0 to
100 mg (see method for specific recommendations and requirements for the use of cesium-13 7).
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. The method provides
an overall measure of alpha and beta activity, including activity for the radionuclides listed above, but
does not permit the specific identification of any alpha or beta emitting radionuclides.
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)
Americium-241
Cesium-137
Cobalt-60
Europium-154
lodine-131
lridium-192
Molybdenum-99
Ruthenium-103
Ruthenium-106
Selenium-75
Select Mixed Fission Products
CASRN
14596-10-2
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
NA
Analysis Purpose: Qualitative and confirmatory analysis
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.
6 EPA lists standards for use when analyzing drinking water in the table at 40 CFR 141.25 (Footnote 11).
SAM 2012 119 July 16, 2011
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SAM 2012 Section 6.0- Selected Radiochemical Methods
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 detector. Detectors such as Germanium (Lithium) or thallium-activated sodium iodide 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 thallium-
activated sodium iodide 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, in general, dependent on analyte radionuclide gamma-ray abundance,
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 903.1: Radium-226 in Drinking Water- Radon Emanation Technique
Analyte(s)
Radium-226
CASRN
13982-63-3
Analysis Purpose: Confirmatory analysis
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, the immediate decay product of radium-226. Radium-226 is
concentrated and separated from the water sample by coprecipitation on barium sulfate. The precipitate is
dissolved in ethylenediamine tetraacetic acid (EOTA) 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
6.2.5 EPA Method 905.0: Radioactive Strontium in Drinking Water
Analyte(s)
Strontium-89
Strontium-90
CASRN
14158-27-1
10098-97-2
SAM 2012 120 July 16, 2011
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SAM 2012 Section 6.0- Selected Radiochemical Methods
Analysis Purpose: Qualitative and confirmatory analysis
Technique: Beta counting
Method Developed for: Strontium-89, strontium-90 and total strontium in drinking water
Method Selected for: SAM lists this method for qualitative and confirmatory analysis of aqueous/liquid
and drinking water samples for strontium-89 and confirmatory analysis of drinking water samples for
strontium-90.
Description of Method: Stable strontium carrier is added to the water sample. Both strontium-89 and
strontium-90 are precipitated from the solution as insoluble carbonates. Interferences from calcium and
from some radionuclides are removed by one or more precipitations of the strontium carrier as strontium
nitrate. Barium and radium are removed by precipitation as chromates. The yttrium-90 decay product of
strontium-90 is removed by a hydroxide precipitation step. The separated strontium-89 and strontium-90
are precipitated as carbonates, weighed for determination of the chemical recovery, and counted for beta
particle activity. The counting result, ascertained immediately after separation, represents the total
strontium activity (strontium-89 and strontium-90) plus an insignificant fraction of the yttrium-90 that has
grown into the separated strontium-90. The yttrium-90 decay product is allowed to in-grow for
approximately two weeks and then is separated with stable yttrium carrier as hydroxide and finally
precipitated as the oxalate, weighed for chemical recovery, and mounted for beta counting. The
strontium-90 concentration is determined from the yttrium-90 activity; strontium-89 concentration is
determined from the difference.
Source: EPA, EMSL. 1980. "Method 905.0: Radioactive Strontium in Drinking Water,
Prescribed Procedures for Measurement of Radioactivity in Drinking Water, EPA/600/4/80/032.
http://www.epa.gOv/sam/pdfs/EPA-905.0.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
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 (HTO) 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
SAM 2012 121 July 16, 2011
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6.2.7 EPA Method EMSL-19: Determination of Radium-226 and Radium-228 in Water,
Soil, Air and Biological Tissue
Analyte(s)
Radium-226
CASRN
13982-63-3
Analysis Purpose: Confirmatory analysis
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
pentasodium diethylenetriamine-pentaacetate (DTPA) solution and transferred to an emanation tube. The
radon is allowed to come to equilibrium for approximately 30 days. Radium-226 decays by alpha
emission to radon-222. Radon-222 is separated and collected from the liquid by a de-emanation
technique. The radon is counted by alpha scintillation 4.5 hours after de-emanation, at which time the
short-lived progeny have reached 97% of equilibrium. An applicable measurement range has not been
determined; however, samples that contain 0.1 pCi of Radium-226 have been analyzed.
Source: EPA, EMSL. 1979. "EMSL-19: Determination of Radium-226 and Radium-228 in Water, Soil,
Air and Biological Tissue." Radiochemical Analytical Procedures for Analysis of Environmental
Samples. http://www.epa.gov/sam/pdfs/EPA-EMSL-19.pdf
6.2.8 EPA Method EMSL-33: Isotopic Determination of Plutonium, Uranium, and
Thorium in Water, Soil, Air, and Biological Tissue
Analyte(s)
Plutonium-238
Plutonium-239
Uranium-234
Uranium-235
Uranium-238
CASRN
13981-16-3
15117-48-3
13966-29-5
15117-96-1
7440-61-1
Analysis Purpose: Confirmatory analysis
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.
SAM 2012 122 July 16, 2011
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SAM 2012 Section 6.0- Selected Radiochemical Methods
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 Samples. http://www.epa.gov/sam/pdfs/EPA-EMSL-33.pdf
6.2.9 EPA Method Rapid Radiochemical Method for Phosphorus-32 in Water for
Environmental Restoration Following Homeland Security Events
Analyte(s)
Phosphorus-32
CASRN
14596-37-3
Analysis Purpose: Qualitative analysis
Technique: Liquid scintillation
Method Developed for: Phosporus-32 in water
Method Selected for: SAM lists this method for qualitative analysis of drinking water samples.
Description of Method: A 100-mL water sample is filtered and phosphate carrier is added to the filtered
sample. The solution is then passed through a cation exchange resin, followed by a Diphonixฎ resin, to
remove interferences from cation radionuclides. The eluent is treated with a mixture of 10 mL of 30 %
hydrogen peroxide and 10 mL of concentrated nitric acid, reduced to approximately 2-5 mL by heating,
and quantitatively transferred to a liquid scintillation vial for counting. The Cerenkov photons from the
P-32 beta (1710 keV, Emax) decay are detected using a calibrated liquid scintillation counter (LSC).
Following counting of the sample, an aliquot of the final solution is used for yield determination by the
inductively coupled plasma-atomic emission spectrometry (ICP-AES) method.
Special Considerations: SAM lists this method for rapid qualitative screening of drinking water
samples. The method is not intended for use in compliance monitoring of drinking water.
Source: EPA, Office of Radiation and Indoor Air, National Air and Radiation Environmental Laboratory
(NAREL). "Rapid Radiochemical Method for Phosphorus-32 in Water for Environmental Restoration
Following Homeland Security Events." Rapid Radiochemical Methods for Selected Radionuclides in
Water for Environmental Restoration Following Homeland Security Events. When published, this
method will be posted at http://www.epa.gov/narel/new docs.html.
6.2.10 EPA Method R4-73-014: Radioactive Phosphorus
Analyte(s)
Phosphorus-32
CASRN
14596-37-3
SAM 2012 123 July 16, 2011
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SAM 2012 Section 6.0- Selected Radiochemical Methods
Analysis Purpose: Qualitative and confirmatory analysis
Technique: Low background alpha/beta counter
Method Developed for: Phosporus-32 in nuclear reactor solutions
Method Selected for: SAM lists this method for qualitative and confirmatory analysis of water samples.
Description of Method: 200 mL or less of a water sample is acidified with nitric acid and carriers of
phosphorus (standardized), cobalt, zirconium, silver and manganese are added. Hydroxides are
precipitated by the addition of hydrogen peroxide and potassium hydroxide, and the hot solution is
filtered through filter paper. Carriers of cobalt and zirconium are added to the filtrate, and the hydroxides
are precipitated by the addition of hydrogen peroxide and potassium hydroxide. The solution is filtered
and the hydroxides are discarded. The filtrate is acidified with hydrochloric acid, and phosphorous is
precipitated as magnesium ammonium phosphate by the addition of a magnesium mixture and ammonium
hydroxide. The magnesium ammonium phosphate is collected on a tared filter, dried, and weighed to
determine the chemical yield. The precipitate is mounted and beta counted with a gas-flow proportional
counter.
Source: EPA, EMSL. 1980. "Method R4-73-14: Radioactive Phosphorus." Prescribed Procedures for
Radiochemical Analysis of Nuclear Reactor Solutions. http://www.epa.gov/sam/EPA-R4-73-014.pdf
6.2.11 EPA Method: Determination of Radiostrontium in Food and Bioenvironmental
Samples
Analyte(s)
Strontium-89
CASRN
14158-27-1
Analysis Purpose: Qualitative and confirmatory analysis
Technique: Low background alpha/beta counter
Method Developed for: Strontium-89 and strontium-90 in food, vegetation and tissue samples
Method Selected for: SAM lists this method for qualitative analysis of wipes and air filters and
confirmatory analysis of wipes, air filters, soil and sediment and vegetation.
Description of Method: This method is use for the determination of strontium-89 and strontium-90 in
various bio-environmental samples. A sample of 10 g or less is placed in a nickel crucible. Barium and
strontium (standardized) carriers are added to the sample. Sodium hydroxide pellets and anhydrous
sodium carbonate are added and mixed, and the sample is fused as a carbonate. The strontium-calcium
carbonates are dissolved in hydrochloric acid, complexed with di-sodium EDTA and passed through a
cation column where the strontium is absorbed, and the complexed calcium passes through. The
strontium is eluted from the column and precipitated as a carbonate. The strontium carbonate is weighed
and mounted on a planchet for beta counting with a low background gas-flow alpha beta counter. The
chemical yield is determined gravimetrically, using calculations provided in the method.
Special Considerations: This method was developed for analysis of food, vegetation and tissue.
Additional laboratory development and testing is necessary for application to soil, sediment, air filters and
wipes.
Source: EPA, National Environmental Research Center. 1975. "Determination of Radiostrontium in
Food and Bioenvironmental Samples" Handbook of Radiochemical Methods, EPA-680/4-75-001.
http://www.epa.gov/sam/RadioStrontium in food.pdf
SAM 2012 124 July 16, 2011
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SAM 2012 Section 6.0- Selected Radiochemical Methods
6.2.12 EPA Method: Rapid Radiochemical Method for Americium-241 in Water for
Environmental Restoration Following Homeland Security Events
Analyte(s)
Americium-241
CASRN
14158-27-1
Analysis Purpose: Qualitative analysis
Technique: Alpha spectrometry
Method Developed for: Americium-241 in water
Method Selected for: SAM lists this method for qualitative analysis of drinking water samples.
Description of Method: The method is based on a sequence of two chromatographic extraction resins.
Americium is concentrated, isolated, and purified by removing interfering radionuclides as well as other
components of the sample in order to prepare the americium fraction for counting by alpha spectrometry.
The method utilizes vacuum-assisted flow to improve the speed of the separations. Prior to use of the
extraction resins, the water sample is filtered as necessary to remove any insoluble fractions, equilibrated
with americium-243 tracer, and concentrated by evaporation or calcium phosphate precipitation. The
sample test source is prepared by microprecipitation with neodymium fluoride. Standard laboratory
protocol for the use of an alpha spectrometer is used when the sample is ready for counting.
Special Considerations: SAM lists this method for rapid qualitative screening of drinking water
samples. The method is not intended for use in compliance monitoring of drinking water.
Source: EPA, Office of Radiation and Indoor Air National Air and Radiation Environmental Laboratory
(NAREL). 2010. "Rapid Radiochemical Method for Americium-241 in Water for Environmental
Restoration Following Homeland Security Events." Rapid Radiochemical Methods for Selected
Radionuclides in Water for Environmental Restoration Following Homeland Security Events, EPA 402-
R-10-001. http://www.epa.gov/sam/EPA-402-R-10-001 .pdf
6.2.13 EPA Method: Rapid Radiochemical Method for Plutonium-238 and Plutonium-
239/240 in Water for Environmental Restoration Following Homeland Security Events
Analyte(s)
Plutonium-238
Plutonium-239
CASRN
13981-16-3
15117-48-3
Analysis Purpose: Qualitative analysis
Technique: Alpha spectrometry
Method Developed for: Plutonium-238 and -239 in water
Method Selected for: SAM lists this method for qualitative analysis of drinking water samples.
Description of Method: This method is based on the sequential use of two chromatographic extraction
resins to isolate and purify plutonium by removing interfering radionuclides as well as other components
of the matrix in order to prepare the plutonium fraction for counting by alpha spectrometry. The method
utilizes vacuum-assisted flow to improve the speed of the separations. Prior to using the extraction resins,
a water sample is filtered as necessary to remove any insoluble fractions, equilibrated with plutonium-242
tracer, and concentrated by either evaporation or coprecipitation with calcium phosphate. The sample test
source is prepared by microprecipitation with neodymium fluoride. Standard laboratory protocol for the
use of an alpha spectrometer is used when the sample is ready for counting.
SAM 2012 125 July 16, 2011
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SAM 2012 Section 6.0- Selected Radiochemical Methods
Special Considerations: SAM lists this method for rapid qualitative screening of drinking water
samples. The method is not intended for use in compliance monitoring of drinking water.
Source: EPA, Office of Radiation and Indoor Air National Air and Radiation Environmental Laboratory
(NAREL). 2010. "Rapid Radiochemical Method for Plutonium-238 and -239 in Water for Environmental
Restoration Following Homeland Security Events." Rapid Radiochemical Methods for Selected
Radionuclides in Water for Environmental Restoration Following Homeland Security Events, EPA 402-
R-10-001. http://www.epa.gov/sam/EPA-402-R-10-001 .pdf
6.2.14 EPA Method: Rapid Radiochemical Method for Radium-226 in Water for
Environmental Restoration Following Homeland Security Events
Analyte(s)
Radium-226
CASRN
13982-63-3
Analysis Purpose: Qualitative analysis
Technique: Alpha spectrometry
Method Developed for: Radium-226 in water
Method Selected for: SAM lists this method for qualitative analysis of drinking water samples.
Description of Method: A known quantity of radium-225 is used as the yield determinant in this
analysis. The sample is initially digested using concentrated nitric acid, followed by volume reduction
and conversion to the chloride salt using concentrated hydrochloric acid. The solution is adjusted to a
neutral pH and batch equilibrated with manganese resin to separate radium from any radioactive and/or
non-radioactive matrix constituents. Further selectivity is achieved using a column containing Diphonixฎ
resin. The radium (including radium-226) eluted from the column is prepared for counting by
microprecipitation with barium sulfate. Low-level measurements are performed by alpha spectrometry.
The activity measured in the radium-226 region of interest is corrected for chemical yield based on the
observed activity of the alpha peak at 7.07 MeV.
Special Considerations: SAM lists this method for rapid qualitative screening of drinking water
samples. The method is not intended for use in compliance monitoring of drinking water.
Source: EPA, Office of Radiation and Indoor Air National Air and Radiation Environmental Laboratory
(NAREL). 2010. "Rapid Radiochemical Method for Radium-226 in Water for Environmental Restoration
Following Homeland Security Events." Rapid Radiochemical Methods for Selected Radionuclides in
Water for Environmental Restoration Following Homeland Security Events, EPA 402-R-10-001.
http://www.epa.gov/sam/EPA-402-R-10-001.pdf
6.2.15 EPA Method: Rapid Radiochemical Method for Radiostrontium in Water for
Environmental Restoration Following Homeland Security Events
Analyte(s)
Strontium-90
CASRN
10098-97-2
Analysis Purpose: Qualitative analysis
Technique: Beta counting
Method Developed for: Strontium-90 in water
Method Selected for: SAM lists this method for qualitative analysis of drinking water samples.
SAM 2012 126 July 16, 2011
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SAM 2012 Section 6.0- Selected Radiochemical Methods
Description of Method: Strontium is isolated from the sample matrix and purified from potentially
interfering radionuclides and matrix constituents using a strontium-specific, rapid chemical separation
procedure. The sample is equilibrated with strontium carrier, and concentrated by coprecipitation with
strontium/barium carbonate. If insoluble residues are noted during acid dissolution steps, the residue and
precipitate mixture is digested in 8 M nitric acid to solubilize strontium. The solution is passed through a
Sr-Resin extraction chromatography column that selectively retains strontium while allowing most
interfering radionuclides and matrix constituents to pass through to waste. If present in the sample,
residual plutonium and several interfering tetravalent radionuclides are stripped from the column using an
oxalic acid/ nitric acid rinse. Strontium is eluted from the column with 0.05 M nitric acid and taken to
dryness in a tared, stainless steel planchet. The planchet containing the strontium nitrate precipitate is
weighed to determine the strontium yield. The sample test source is promptly counted on a gas flow
proportional counter to determine the beta emission rate, which is used to calculate the total
radiostrontium activity.
Special Considerations: SAM lists this method for rapid qualitative screening of drinking water
samples. The method is not intended for use in compliance monitoring of drinking water.
Source: EPA, Office of Radiation and Indoor Air National Air and Radiation Environmental Laboratory
(NAREL). 2010. "Rapid Radiochemical Method for Radiostrontium in Water for Environmental
Restoration Following Homeland Security Events." Rapid Radiochemical Methods for Selected
Radionuclides in Water for Environmental Restoration Following Homeland Security Events, EPA 402-
R-10-001. http://www.epa.gov/sam/EPA-402-R-10-001 .pdf
6.2.16 EPA Method: Rapid Radiochemical Method for Isotopic Uranium in Water for
Environmental Restoration Following Homeland Security Events
Analyte(s)
Uranium-234
Uranium-235
Uranium-238
CASRN
13966-29-5
15117-96-1
7440-61-1
Analysis Purpose: Qualitative analysis
Technique: Alpha spectrometry
Method Developed for: Uranium-234, -235 and -238 in water
Method Selected for: SAM lists this method for qualitative analysis of drinking water samples.
Description of Method: This method is based on the sequential elution of interfering radionuclides as
well as other components of the sample matrix by extraction chromatography to isolate and purify
uranium for counting by alpha spectrometry. The method utilizes vacuum assisted flow to improve the
speed of the separations. Prior to the use of the extraction resins, a water sample is filtered as necessary
to remove any insoluble fractions, equilibrated with uranium-232 tracer, and concentrated by either
evaporation or coprecipitation with calcium phosphate. The sample test source is prepared by
microprecipitation with neodymium fluoride. Standard laboratory protocol for the use of an alpha
spectrometer is used when the sample is ready for counting.
Special Considerations: SAM lists this method for rapid qualitative screening of drinking water
samples. The method is not intended for use in compliance monitoring of drinking water.
Source: EPA, Office of Radiation and Indoor Air National Air and Radiation Environmental Laboratory
(NAREL). "Rapid Radiochemical Method for Isotopic Uranium in Water for Environmental Restoration
Following Homeland Security Events." Rapid Radiochemical Methods for Selected Radionuclides in
SAM 2012 127 July 16, 2011
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SAM 2012 Section 6.0- Selected Radiochemical Methods
Water for Environmental Restoration Following Homeland Security Events, EPA 402-R-10-001.
http://www.epa.gov/sam/EPA-402-R-10-001.pdf
6.2.17 EPA Method: Rapid Method for Acid Digestion of Glass-Fiber and
Organic/Polymeric Composition Filters and Swipes Prior to Isotopic Uranium,
Plutonium, Americium, Strontium, and Radium Analyses for Environmental
Remediation Following Homeland Security Events
Analyte(s)
Americium-241
Plutonium-238
Plutonium-239
Radium-226
Strontium-90
Uranium-234
Uranium-235
Uranium-238
CASRN
14158-27-1
13981-16-3
15117-48-3
13982-63-3
10098-97-2
13966-29-5
15117-96-1
7440-61-1
Analysis Purpose: Qualitative analysis
Technique: Alpha spectrometry
Method Developed for: Americium-241, plutonium-238 and -239, radium-226, strontium-90, uranium-
234, -235 and -238 in surface wipes and air filters
Method Selected for: SAM lists this method for qualitative analysis of surface wipe and air filter
samples.
Description of Method: The method is based on the complete dissolution of both the filter material and
deposited particulates. Glass-fiber filters (the siliceous filter as well as deposited silicates) are dissolved
with direct application of hydrofluoric acid. The addition of nitric and hydrochloric acids facilitates
dissolution of remaining solids. The sample digestate is taken to dryness and re-dissolved in nitric acid.
Filters composed of organic materials, such as cellulose or polypropylene, are dry ashed in a 450 ฐC
muffle furnace to destroy the organic filter material, then processed through the acid dissolution steps
referenced above for non-organic filter material. Once sample dissolution is complete, it is re-dissolved
in nitric acid solution. The sample is then processed for specific analyte determination using one of the
following rapid methods contained in Rapid Radiochemical Methods for Selected Radionuclides in Water
for Environmental Restoration Following Homeland Security Events (http://www.epa.gov/sam/EPA-402-
R-10-001.pdf):
Rapid Radiochemical Method for Americium-241 in Water for Environmental Remediation
Following Homeland Security Events
Rapid Radiochemical Method for Plutonium-238 and Plutonium-239/240 in Water for
Environmental Remediation Following Homeland Security Events
Rapid Radiochemical Method for Isotopic Uranium in Water for Environmental Remediation
Following Homeland Security Events
Rapid Radiochemical Method for Radium-226 in Water for Environmental Remediation
Following Homeland Security Events
Rapid Radiochemical Method for Total Radiostrontium (Sr-90) in Water for Environmental
Remediation Following Homeland Security Events
Special Considerations: This method is a gross pre-treatment technique, to be used prior to use of the
appropriate rapid separation methods cited above. Filters that contain large amounts of particulate
material may result in persistent undissolved particulates in the digestion process. These samples may
SAM 2012 128 July 16, 2011
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SAM 2012 Section 6.0- Selected Radiochemical Methods
require repeated application of the digestion procedure to cause a complete dissolution of the particulates.
If refractory constituents are suspected in the sampled particulates or the acidic digestion procedure is
otherwise deemed to be ineffective because of refractory residuals or constituents, the alternate "Rapid
Method for Sodium Carbonate Fusion of Glass-Fiber and Organic/Polymeric Composition Filters and
Swipes Prior to Isotopic Uranium, Plutonium, Americium, Strontium, and Radium Analyses for
Environmental Remediation Following Homeland Security Events" (Section 6.2.18) should be considered
for sample preparation.
Source: EPA, Office of Radiation and Indoor Air National Air and Radiation Environmental Laboratory
(NAREL). 2010. "Rapid Method for Acid Digestion of Glass-Fiber and Organic/Polymeric Composition
Filters and Swipes Prior to Isotopic Uranium, Plutonium, Americium, Strontium, and Radium Analyses
for Environmental Remediation Following Homeland Security Events." When published, this method
will be posted at http://www.epa.gov/narel/new_docs.html.
6.2.18 EPA Method: Rapid Method for Sodium Carbonate Fusion of Glass-Fiber and
Organic/Polymeric Composition Filters and Swipes Prior to Isotopic Uranium,
Plutonium, Americium, Strontium, and Radium Analyses for Environmental
Remediation Following Homeland Security Events
Analyte(s)
Americium-241
Plutonium-238
Plutonium-239
Radium-226
Strontium-90
Uranium-234
Uranium-235
Uranium-238
CASRN
14158-27-1
13981-16-3
15117-48-3
13982-63-3
10098-97-2
13966-29-5
15117-96-1
7440-61-1
Analysis Purpose: Qualitative analysis
Technique: Alpha spectrometry
Method Developed for: Americium-241, plutonium-238 and -239, radium-226, strontium-90, uranium-
234, -235 and -238 in surface wipes and air filters
Method Selected for: SAM lists this method for qualitative analysis of surface wipe and air filter
samples.
Description of Method: The method is based on the complete dissolution of both the filter or swipe
material and the deposited particulates. Glass-fiber media and deposited particulates are destroyed by
fusion with molten sodium carbonate in a nickel or platinum crucible. The resulting fusion cake is
dissolved in hydrochloric acid. Filters composed of organic materials, such as cellulose or
polypropylene, are charred in a crucible to destroy the organic filter material. The resulting charred
media and deposited particulates are destroyed by fusion with molten sodium carbonate in a nickel or
platinum crucible. The resulting fusion cake is dissolved in hydrochloric acid. Once sample fusion is
complete and the fusion cake is dissolved in hydrochloric acid, the sample is processed for specific
analyte determination using one of the following rapid methods:
Rapid Radiochemical Method for Americium-241 in Water for Environmental Remediation
Following Homeland Security Events
Rapid Radiochemical Method for Plutonium-238 and Plutonium-239/240 in Water for
Environmental Remediation Following Homeland Security Events
SAM 2012 129 July 16, 2011
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SAM 2012 Section 6.0- Selected Radiochemical Methods
Rapid Radiochemical Method for Isotopic Uranium in Water for Environmental Remediation
Following Homeland Security Events
Rapid Radiochemical Method for Radium-226 in Water for Environmental Remediation
Following Homeland Security Events
Rapid Radiochemical Method for Total Radiostrontium (Sr-90) in Water for Environmental
Remediation Following Homeland Security Events
Special Considerations: This method is a gross pre-treatment technique, to be used prior to use of the
appropriate rapid separation methods cited. Filters that contain large amounts of particulate material may
result in persistent undissolved particulates in the digestion process. These samples may require repeated
application of the digestion procedure to cause a complete dissolution of the particulates.
Source: EPA, Office of Radiation and Indoor Air National Air and Radiation Environmental Laboratory
(NAREL). 2010. "Rapid Method for Sodium Carbonate Fusion of Glass-Fiber and Organic/Polymeric
Composition Filters and Swipes Prior to Isotopic Uranium, Plutonium, Americium, Strontium, and
Radium Analyses for Environmental Remediation Following Homeland Security Events." When
published, this method will be posted at http://www.epa.gov/narel/new_docs.html.
6.2.19 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
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-252 and curium-244. 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 EML HASL-300 Method 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
SAM2012 130 July 16, 2011
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SAM 2012 Section 6.0- Selected Radiochemical Methods
6.2.20 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
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, air filters and vegetation.
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
6.2.21 EML HASL-300 Method Am-06-RC: Americium and/or Plutonium in Vegetation
Analyte(s)
Americium-241
Californium-252
Curium-244
Plutonium-238
Plutonium-239
CASRN
14596-10-2
13981-17-4
13971-52-2
13981-16-3
15117-48-3
Analysis Purpose: Confirmatory analysis
Technique: Alpha spectrometry
Method Developed for: Americium and/or plutonium in vegetation
Method Selected for: SAM lists this method for confirmatory analysis of vegetation.
Description of Method: Vegetation is either dry ashed in a ceramic crucible using a muffle furnace or
wet ashed with nitric acid. Plutonium-236 and americium-243 tracers are added after dry ashing or before
wet ashing. Wet ashing requires considerably more time and must be carefully monitored due to the
SAM 2012 131 July 16, 2011
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SAM 2012 Section 6.0- Selected Radiochemical Methods
highly reactive nature of vegetation. The sample is further digested with hydrofluoric acid to dissolve
silicate compounds. Plutonium is separated by ion exchange and determined by alpha spectrometry using
the plutonium-236 tracer to determine the recovery. Americium (and californium-252 and curium-244, if
present) is collected with a calcium oxalate precipitation and finally isolated and purified by ion
exchange. After source preparation by microprecipitation, americium -241 (and californium-252 and
curium-244, if present) is determined by alpha spectrometry using americium-243 tracer to provide
recovery data.
Special Consideration: Polytetrafluoroethylene (PTFE) beakers must be used when digesting samples
with hydrofluoric acid.
Source: EML, DOE (EML is currently part of the DHS). 1997. "HASL-300 Method Am-06-RC:
Americium and/or Plutonium in Vegetation." EML Procedures Manual, HASL-300, 28th Edition.
http://www.epa.gov/sam/EML-Am-06-RC.pdf
6.2.22 EML HASL-300 Method Ga-01-R: Gamma Radioassay
Analyte(s)
Americium-241
Cesium-137
Cobalt-60
Europium-154
lodine-131
lridium-192
Molybdenum-99
Ruthenium-103
Ruthenium-106
Selenium-75
Select Mixed Fission Products
CASRN
14596-10-2
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
NA
Analysis Purpose: Qualitative and confirmatory analysis or gross gamma determination
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 wipe, air filters and/or vegetation.
Description of Method: This method uses gamma spectrometry 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 high purity Germanium [HP(Ge)] 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 HP(Ge) and GeLi) detectors and above 50 keV forNal(Tl) detectors.
SAM 2012
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SAM 2012 Section 6.0- Selected Radiochemical Methods
Source: EML, DOE (EML is currently part of the DHS). 1997. "HASL-300 Method Ga-01-R: Gamma
Radioassa
01-R.pdf
Radioassay." EML Procedures Manual, HASL-300, 28th Edition, http://www.epa.gov/sam/pdfs/EML-Ga-
6.2.23 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
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, soil/sediment and/or vegetation samples.
Description of Method: This method uses alpha spectrometry for determination of polonium in water,
vegetation, soil and air filter samples. Polonium equilibrated with polonium-208 or polonium-209 tracer
is isolated from most other elements by coprecipitation with lead sulfide. The sulfide precipitate is
dissolved in weak hydrochloric acid solution. Polonium is quantitatively deposited on a nickel disc, and
the plated disc is counted on an alpha spectrometer to measure chemical yield and activity of the sample.
The solution from the deposition may be retained and analyzed for polonium-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
6.2.24 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
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 americium-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.
SAM2012 133 July 16, 2011
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SAM 2012 Section 6.0- Selected Radiochemical Methods
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." EML Procedures Manual, HASL-300, 28th Edition.
http://www.epa.gov/sam/pdfs/EML-Pu-12-RC.pdf
6.2.25 EML HASL-300 Method Ra-03-RC: Radium-226 in Soil, Vegetable Ash, and Ion
Exchange Resin
Analyte(s)
Radium-226
CASRN
13982-63-3
Analysis Purpose: Qualitative and confirmatory analysis
Technique: Radon emanation
Method Developed for: Radium-226 in soil, vegetation ash and ion exchange resin
Method Selected for: SAM lists this method for qualitative and confirmatory analysis of vegetation.
Description of Method: Soil, vegetation ash or ion exchange resin are prepared for radon-222 emanation
measurement. The sample is pretreated with nitric acid-hydrogen fluoride, fused with potassium fluoride
and transposed to pyrosulfate. The cake is dissolved in dilute hydrochloric acid. Radium-barium sulfate
is precipitated, filtered, and dissolved in alkaline EDTA. The chemical yield is determined with the y-
emitting tracer barium-133. The solution is transferred to a radon bubbler. Radon is de-emanated into an
ionization chamber or scintillation cell, and counted using a counter with a photomultiplier.
Special Consideration: Use of platinum crucibles is required in this method.
Source: EML, DOE (EML is currently part of the DHS). 1997. "HASL-300 Method Ra-03-RC: Radium
226 in Soil, Vegetable Ash, and Ion Exchange resin." EML Procedures Manual, HASL-300, 28th Edition.
http: //www. epa.gov/sam/EML-Ra-03 -RC .pdf
6.2.26 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
Technique: Beta counting
Method Developed for: Strontium-90 in vegetation, water, air filters and soil
Method Selected for: SAM lists this method for confirmatory analysis of soil and sediment samples,
vegetation, surface wipes and air filters.
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
SAM2012 134 July 16, 2011
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SAM 2012 Section 6.0- Selected Radiochemical Methods
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." EMLPn
http://www.epa.gov/sam/pdfs/EML-Sr-03-RC.pdf
Strontium-90 in Environmental Samples." EML Procedures Manual, HASL-300, 28th Edition.
6.2.27 EML HASL-300 Method Tc-01-RC: Technetium-99 in Water and Vegetation
Analyte(s)
Technetium-99
CASRN
14133-76-7
Analysis Purpose: Qualitative and confirmatory analysis
Technique: Beta counting / Gamma spectrometry
Method Developed for: Technetium-99 in water and vegetation
Method Selected for: SAM lists this method for confirmatory analysis of vegetation.
Description of Method: Samples are wet ashed with nitric acid. After wet ashing is complete, samples
are evaporated to the smallest volume possible with no salting out. The resulting solution is cooled,
transferred to a 1-L beaker, and diluted to 800 mL with reagent water. The sample solution is then stirred
and filtered with suction through a 15-cm glass fiber filter, and the filter is washed with water. The filter
containing the silica and insoluble material is discarded. Technetium-99 is equilibrated with technetium-
95m tracer in the wet ashing step. Technetium is separated from other elements by anion exchange and
electro-deposition, and technetium-99 is beta counted. Gamma spectrometry measurement of technetium-
95m tracer provides the chemical yield.
Special Consideration: Technetium-95m tracer is no longer readily available from the source cited in
the method. If technetium-95m can not be obtained, technetium-99m tracer may be substituted.
Source: EML, DOE (EML is currently part of the DHS). 1997. "HASL-300 Method Tc-01-RC:
Technetium-99 in Water and Vegetation." EML Procedures Manual, HASL-300, 28th Edition.
http://www.epa.gov/sam/EML-Tc-01 -RC.pdf
6.2.28 EML HASL-300 Method Tc-02-RC: Technetium-99 in Water- TEVAฎ Resin
Analyte(s)
Technetium-99
CASRN
14133-76-7
Analysis Purpose: Qualitative and confirmatory analysis
Technique: Liquid scintillation
Method Developed for: Technetium-99 in water
Method Selected for: SAM lists this method for qualitative and confirmatory analysis of drinking water
samples.
Description of Method: The sample containing technetium-99 is mixed with technetium-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 (TEVAฎ
Resin) which is highly specific for technetium in the pertechnetate form. The resin is washed with dilute
SAM2012 135 July 16, 2011
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SAM 2012 Section 6.0- Selected Radiochemical Methods
nitric acid to remove possible interferences and then it is eluted directly into a suitable liquid scintillation
cocktail. The sample is typically counted for 1 hour to simultaneously determine technetium-99 activity
and the technetium-95m radiochemical yield. Quench/efficiency calibration curves need to be established
for the liquid scintillation spectrometer for both technetium-95m and technetium-99.
Source: EML, DOE (EML is currently part of the DHS). 1997. "HASL-300 Method Tc-02-RC:
Technetium-99 in Water - TEVAฎ Resin." EML Procedures Manual, HASL-300, 28th Edition.
http://www.epa.gov/sam/pdfs/EML-Tc-02-RC.pdf
6.2.29 EML HASL-300 Method U-02-RC: Isotopic Uranium in Biological and
Environmental Materials
Analyte(s)
Uranium-234
Uranium-235
Uranium-238
CASRN
13966-29-5
15117-96-1
7440-61-1
Analysis Purpose: Qualitative and confirmatory analysis
Technique: Alpha spectrometry
Method Developed for: Isotopic uranium in biological and environmental materials
Method Selected for: SAM lists this method for confirmatory analysis of vegetation.
Description of Method: Uranium from acid leached, dry-ashed and wet-ashed materials is equilibrated
with uranium-232 tracer, and isolated by anion exchange chromatography. The separated uranium
isotopes are microprecipitated for alpha spectrometry.
Special Considerations: For microprecipitation procedures, refer to HASL-300 Method G-03.
Source: EML, DOE (EML is currently part of the DHS). 1997. "HASL-300 Method U-02-RC: Isotopic
Uranium in Biological and Environmental ME
http://www.epa.gov/sam/EML-U-02-RC.pdf
Uranium in Biological and Environmental Materials." EML Procedures Manual, HASL-300, 28th Edition.
6.2.30 DOE FRMAC Method Volume 2, Page 33: Gross Alpha and Beta in Air
Analysis Purpose: Gross alpha and gross beta determination
Technique: Alpha/Beta counting
Method Developed for: Gross alpha and beta in air
Method Selected for: SAM lists this method for gross alpha and gross beta determination in air filters,
and for direct counting of surface wipes.
Description of Method: A thin-window gas-flow proportional counter is used for counting gross alpha
and beta radioactivity. The method supplies an approximation of the alpha and beta activity present in the
air or the removable surface activity dependent on the sample type. The method provides an indication of
the presence of alpha and beta emitters, including the following SAM analytes:
Americium-241 (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
SAM2012 136 July 16, 2011
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SAM 2012 Section 6.0- Selected Radiochemical Methods
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
6.2.31 DOE RESL Method P-2:32P Fish, Vegetation, Dry Ash, Ion Exchange
Analyte(s)
Phosphorus-32
CASRN
14596-37-3
Analysis Purpose: Qualitative and confirmatory analysis
Technique: Cerenkov counting with Liquid Scintillation
Method Developed for: Phosphorus-32 in fish and vegetation
Method Selected for: SAM lists this method for qualitative and confirmatory analysis of soil, sediment,
wipes, air filters and vegetation.
Description of Method: Samples up to 500 g are dry ashed at 550 ฐC and dissolved in two portions of
nitric acid. The sample is evaporated to half volume and transferred to a perchloric acid hood.
Concentrated nitric acid and concentrated perchloric acid are added, and the sample is evaporated to
dryness. The residue is dissolved in hydrochloric acid and filtered through a glass fiber filter. Iron-55 is
removed by precipitation with cupferron. The solution containing phosphate is purified by passing it
through anion and cation columns to remove possible contaminants. The purified phosphate is
precipitated as magnesium ammonium phosphate, filtered onto a glass fiber filter, and dried. The
magnesium ammonium phosphate is dissolved in nitric acid and transferred to a counting vial.
Phopsphorus-32 is assayed by counting the Cerenkov radiation with a liquid scintillation counter.
Special Considerations: Laboratories using this method must have a designated perchloric acid fume
hood. This method was developed for analysis offish and vegetation. Additional development and
testing is necessary for application to soil, sediment, wipes and air filters. Phosphorus and iron carrier
must be added to matrices that do not contain mg quantities of both elements.
SAM2012 137 July 16, 2011
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SAM 2012 Section 6.0- Selected Radiochemical Methods
Source: RESL, DOE. 1977. "Method P-2: 32P Fish, Vegetation, Dry Ash, Ion Exchange." RESL
Analytical Chemistry Branch Procedures Manual, IDO-12096. http://www.epa.gov/sam/RESL-P-2.pdf
6.2.32 DOE SRS Actinides and Sr-89/90 in Soil Samples
Analyte(s)
Americium-241
Plutonium-238
Plutonium-239
Strontium-89
Strontium-90
Uranium-234
Uranium-235
Uranium-238
CASRN
14596-10-2
13981-16-3
15517-48-3
14158-27-1
10098-97-2
13966-29-5
15117-96-1
7440-61-1
Analysis Purpose: Qualitative analysis
Technique: Alpha spectrometry and beta counting
Method Developed for: Actinides and strontium-89 and -90 in soil samples
Method Selected for: SAM lists this method for qualitative analysis of soil and sediment samples.
Description of Method: Radioactive tracers are added to samples prior to wet ashing. Samples are fused
at 600 ฐC using sodium hydroxide in zirconium crucibles. An iron hydroxide precipitation is performed.
After dissolution by acidification of the precipitate, a lanthanum fluoride precipitation is used to further
eliminate the sample matrix. The lanthanum fluoride precipitate is redissolved in nitric acid, boric acid,
and aluminum nitrate. A column separation using TEVAฎ, TRU and DGA resins is applied to separate
the actinides into three fractions: plutonium-neptunium, uranium and americium/curium. Plutonium-242
(or plutonium-236 if neptunium-237 is measured), thorium-229, americium-243 and uranium-232 are
used as tracers to determine yield. The various fractions of actinides are eluted from the resin columns
and precipitated with cesium fluoride, dried, and counted by alpha spectrometry. Strontium resin is used
to separate strontium-89/90 for measurement by beta counting.
Special Considerations: Thorium-228, if present as a daughter of the uranium-232 tracer, will interfere
with thorium-228 analysis. Self-cleaning uranium-232 tracer, with thorium-228 removed, is required if
thorium isotopes are separated and measured with uranium.
Source: SRS, DOE. 2011. "Actinides and Sr-89/90 in Soil." SRSManualL3.23, Procedure L3.23-10054.
http://www.epa.gov/sam/L3.23-10054.pdf
6.2.33 DOE SRS Actinides and Sr-89/90 in Vegetation: Fusion Method
Analyte(s)
Americium-241
Plutonium-238
Plutonium-239
Strontium-89
Strontium-90
Uranium-234
Uranium-235
Uranium-238
CASRN
14596-10-2
13981-16-3
15517-48-3
14158-27-1
10098-97-2
13966-29-5
15117-96-1
7440-61-1
SAM 2012
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SAM 2012 Section 6.0- Selected Radiochemical Methods
Analysis Purpose: Qualitative analysis
Technique: Alpha spectrometry
Method Developed for: Actinides and strontium-89 and -90 in vegetation
Method Selected for: SAM lists this method for qualitative analysis of vegetation.
Description of Method: Radioactive tracers are added to samples prior to wet ashing. Samples are fused
at 600 ฐC using sodium hydroxide in zirconium crucibles. An iron hydroxide precipitation is performed.
After dissolution by acidification of the precipitate, a lanthanum fluoride precipitation is used to further
eliminate the sample matrix. The lanthanum fluoride precipitate is redissolved in nitric acid, boric acid
and aluminum nitrate. A column separation using commercially available resins (TEVAฎ, TRU and
DGA) is applied to separate the actinides into three fractions: plutonium/neptunium, uranium and
americium/curium. Plutonium-242 (or Plutonium-236 if neptunium-237 is measured), thorium -229,
americium-243 and uranium-232 are used as tracers to determine yield. The various fractions of actinides
are eluted from the resin columns and precipitated with cesium fluoride, dried, and counted by alpha
spectrometry. Strontium resin is used to separate strontium-89/90 for measurement by beta counting.
Special Considerations: Thorium-228, if present as a daughter of uranium-232 tracer, will interfere
with thorium-228 analysis. Self-cleaning uranium-232 tracer, with thorium-228 removed, is required if
thorium isotopes are separated and measured with uranium.
Source: SRS, DOE. 2011. "Actinides and Sr-89/90 in Vegetation: Fusion Method." SRSManual L3.23,
Procedure L3.23-10055. http://www.epa.gov/sam/L3.23-10055.pdf
6.2.34 ORISE Method AP1: Gross Alpha and Beta for Various Matrices
Analysis Purpose: Gross alpha and gross beta determination
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 and vegetation.
Description of Method: This method provides an indication of the presence of alpha and beta emitters,
including the following SAM analytes:
Americium-241
Californium-252
Cesium-137
Cobalt-60
Curium-244
Europium-154
Iridium-192
Plutonium-238
Plutonium-239
Polonium-210
Radium-226
Ruthenium-103
Ruthenium-106
Strontium-90
Uranium-234
Uranium-235
Uranium-238
(CAS RN 14596-10-2)
(CASRN 13981-17-4)
(CAS RN 10045-97-3)
(CASRN 10198-40-0)
(CASRN 13981-15-2)
(CASRN 15585-10-1)
(CAS RN 14694-69-0)
(CASRN 13981-16-3)
(CASRN 15117-48-3)
(CASRN 13981-52-7)
(CAS RN 13982-63-3)
(CAS RN 13968-53-1)
(CAS RN 13967-48-1)
(CAS RN 10098-97-2)
(CAS RN 13966-29-5)
(CASRN 15117-96-1)
(CAS RN 7440-16-1)
Alpha emitter
Alpha emitter
Beta emitter
Beta emitter
Alpha emitter
Beta emitter
Beta emitter
Alpha emitter
Alpha emitter
Alpha emitter
Alpha emitter
Beta emitter
Beta emitter
Beta emitter
Alpha emitter
Alpha emitter
Alpha emitter
SAM 2012
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SAM 2012 Section 6.0- Selected Radiochemical Methods
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-AP 1 .pdf
6.2.35 ORISE Method AP2: Determination of Tritium
Analyte(s)
Tritium (Hydrogen-3)
CASRN
10028-17-8
Analysis Purpose: Qualitative and confirmatory analysis
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,
surface wipes and vegetation.
Description of Method: The tritium in aqueous and solid samples is distilled using an Allihn condenser.
For solid samples, an appropriate volume of laboratory reagent 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
6.2.36 ORISE Method APS: Determination of Technetium-99
Analyte(s)
Technetium-99
CASRN
14133-76-7
Analysis Purpose: Qualitative and confirmatory analysis
Technique: Liquid scintillation
Method Developed for: Technetium-99 in sediment, soil, smears and water at environmental levels
SAM 2012 140 July 16, 2011
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SAM 2012 Section 6.0- Selected Radiochemical Methods
Method Selected for: SAM lists this method for qualitative and confirmatory analysis of soil/sediment,
surface wipe and air filter samples; and qualitative analysis of vegetation.
Description of Method: Solid samples are leached with dilute nitric acid. The leachates are passed
through a commercially available resin column (TEVAฎ resin) which is highly specific for technetium in
the pertechnetate 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 carbon-14, phosphorus-32, sulfur-35, strontium-
90, yttrium-90 and thorium-234) are effectively removed using TEVAฎ 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
6.2.37 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
Technique: Alpha spectrometry
Method Developed for: Americium, curium, plutonium, neptunium, thorium and/or uranium in water
and solid samples
Method Selected for: SAM recommends this method for confirmatory analysis when a sample exists in
a refractory form (i.e., non-digestible or dissolvable material after normal digestion methods) or if there is
a matrix interference problem. In the event of refractory radioactive material, SAM recommends this
method for both qualitative determination and confirmatory analysis of drinking water, aqueous/liquid,
soil/sediment, surface wipes and air filter samples.
Description of Method: Solid and unfiltered aqueous samples (after evaporation to dryness) 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
SAM 2012 141 July 16, 2011
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SAM 2012 Section 6.0- Selected Radiochemical Methods
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
6.2.38 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
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, air filter and vegetation 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 iodine-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-mL culture
tube and counted in a 3" x 3" thin window sodium iodide (Nal) well detector attached to a pulse height
analyzer. Iodine-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 iodine-125 counting efficiency.
Special Considerations: Due to the low photon energy of iodine-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
6.2.39 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
CASRN
14596-10-2
13981-17-4
13981-15-2
13981-16-3
15117-48-3
13982-63-3
Analysis Purpose: Qualitative determination
SAM 2012 142 July 16, 2011
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SAM 2012 Section 6.0- Selected Radiochemical Methods
Technique: Alpha spectrometry
Method Developed for: Alpha particle spectra in water
Method Selected for: SAM lists this method for qualitative determination of californium-252 and
curium-244 in drinking water, surface wipes, air filters and vegetation; americium-241, californium-252,
curium-244, and plutonium-238 and -239 in aqueous and liquid phase samples; and californium-252,
curium-244 and radium-226 in soil and sediment.
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 Methodfor 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
6.2.40 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
Analysis Purpose: Confirmatory analysis
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.
SAM 2012 143 July 16, 2011
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SAM 2012 Section 6.0- Selected Radiochemical Methods
Special Considerations: If it is suspected that the sample exists in refractory form (i.e., non-digestible
or dissolvable material after normal digestion methods) or if there is a matrix interference problem, use
ORISE Method API 1.
Source: ASTM. 2002. "Method D3972-02: Standard Test Method for Isotopic Uranium in Water by
Radiochemistry." Annual Book of ASTM Standards, Vol. 11.02.
http://www.astm.org/DATABASE.CART/HISTORICAL/D3972-02.htm
6.2.41 ASTM Method D5811-08: Standard Test Method for Strontium-90 in Water
Analyte(s)
Strontium-90
CASRN
10098-97-2
Analysis Purpose: Qualitative and confirmatory analysis
Technique: Beta counting
Method Developed for: Strontium-90 in water samples
Method Selected for: SAM lists this method for qualitative and confirmatory analysis of aqueous and
liquid phase samples.
Description of Method: An aliquot of the sample is measured into a beaker, and strontium carrier is
added. The sample is digested with nitric acid, sorbed on an ion exchange column, eluted, and evaporated
to dryness. The residue is redissolved in nitric acid and then is selectively sorbed on a solid phase
extraction column. Strontium is eluted with dilute nitric acid dried on a planchet, weighed, and counted
for beta radiation.
Special Considerations: Significant amounts of stable strontium, if present in the sample, will interfere
with the yield determination.
Source: ASTM. 2008. "Method D5811-08: Standard Test Method for Strontium-90 in Water:'Annual
Book of ASTM Standards, Vol. 11.02. http://www.astm.org/Standards/D5811 .htm
6.2.42 ASTM Method D7168-05: Standard Test Method for Technetium-99 in Water by
Solid Phase Extraction Disk
Analyte(s)
Technetium-99
CASRN
14133-76-7
Analysis Purpose: Qualitative and confirmatory analysis
Technique: Liquid scintillation
Method Developed for: Technetium-99 in water samples
Method Selected for: SAM lists this method for qualitative and confirmatory analysis of aqueous and
liquid phase samples.
Description of Method: A measured aliquot of sample is transferred to a beaker and hydrogen peroxide
is added to facilitate the formation of the extractable pertechnetate ion. The sample may be heated to
oxidize organics, if suspected to be present. The entire sample is passed through a technetium-selective
solid-phase extraction (SPE) disk onto which the pertechnetate is adsorbed. The disk is transferred to a
liquid scintillation vial, scintillation cocktail is added, and the contents are well mixed. The beta-emission
SAM 2012 144 July 16, 2011
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SAM 2012 Section 6.0- Selected Radiochemical Methods
rate of the sample is determined by liquid scintillation spectrometry. Chemical yield corrections are
determined by the method of standard additions.
Special Considerations: Suspended materials must be removed by filtration or centrifuging prior to
processing the sample.
Source: ASTM. 2005. "Method D7168-05: Standard Test Method for Technetium-99 in Water by Solid
Phase Extraction Disk." Annual Book of ASTM Standards, Vol. 11.02.
http://www.astm.org/Standards/D7168.htm
6.2.43 Standard Method 7110 B: Gross Alpha and Gross Beta Radioactivity (Total,
Suspended, and Dissolved)
Analysis Purpose: Gross alpha and gross beta determination
Technique: Alpha/Beta counting
Method Developed for: Gross alpha and gross beta activity in water
Method Selected for: SAM lists this method for gross alpha and gross beta determination in
aqueous/liquid samples.
Description of Method: This method allows for measurement of gross alpha and gross beta radiation in
water samples. The method provides an indication of the presence of alpha and beta emitters, including
the following SAM analytes:
Americium-241 (CAS
Californium-252 (CAS
Cesium-137 (CAS
Cobalt-60 (CAS
Curium-244 (CAS
Europium-154 (CAS
Iridium-192 (CAS
Plutonium-238 (CAS
Plutonium-239 (CAS
Polonium-210 (CAS
Radium-226 (CAS
Ruthenium-103 (CAS
Ruthenium-106 (CAS
Strontium-90 (CAS
Uranium-234 (CAS
Uranium-235 (CAS
Uranium-238 (CAS
RN 14596-10-2)
RN 13981-17-4)
RN 10045-97-3)
RN 10198-40-0)
RN 13981-15-2)
RN 15585-10-1)
RN 14694-69-0)
RN 13981-16-3)
RN 15117-48-3)
RN 13981-52-7)
RN 13982-63-3)
RN 13968-53-1)
RN 13967-48-1)
RN 10098-97-2)
RN 13966-29-5)
RN 15117-96-1)
RN 7440-16-1)
Alpha emitter
Alpha emitter
Beta emitter
Beta emitter
Alpha emitter
Beta emitter
Beta emitter
Alpha emitter
Alpha emitter
Alpha emitter
Alpha emitter
Beta emitter
Beta emitter
Beta emitter
Alpha emitter
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.
SAM 2012
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SAM 2012 Section 6.0- Selected Radiochemical Methods
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/
6.2.44 Standard Method 7120: Gamma-Emitting Radionuclides
Analyte(s)
Americium-241
Cesium-137
Cobalt-60
Europium-154
lridium-192
Ruthenium-103
Ruthenium-106
Selenium-75
CASRN
14596-10-2
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
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
radioactive standard, in the same geometry as the samples, containing a mixture of gamma energies from
approximately 50 to 2000 keV is used for energy and efficiency 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/
6.2.45 Standard Method 7500-Ra B: Radium: Precipitation Method
Analyte(s)
Radium-226
CASRN
13982-63-3
Analysis Purpose: Qualitative determination
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
SAM 2012 146 July 16, 2011
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SAM 2012 Section 6.0- Selected Radiochemical Methods
adjustment to 4.5. This slightly acidic EDTA keeps other naturally occurring alpha-emitters and the lead
carrier in solution. Radium-223, -224 and -226 are identified by the rate of ingrowth of their daughter
products in barium sulfate precipitate. The results are corrected by the rate of ingrowth of daughter
products to determine radium activity. This method involves alpha counting by a gas-flow internal
proportional counter, scintillation counter or thin end-window gas-flow proportional counter.
Source: APHA, AWWA, and WEF. 2005. "Method 7500-Ra B: Radium: Precipitation Method."
Standard Methods for the Examination of Water and Wastewater. 21st Edition.
http: //www. standardmethods. org/
6.2.46 Standard Method 7500-Ra C: Radium: Emanation Method
Analyte(s)
Radium-226
CASRN
13982-63-3
Analysis Purpose: Confirmatory determination
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.
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/
6.2.47 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
Technique: Alpha counting
Method Developed for: Total uranium alpha activity in water
Method Selected for: SAM lists this method for qualitative determination in aqueous/liquid samples.
SAM 2012 147 July 16, 2011
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SAM 2012 Section 6.0- Selected Radiochemical Methods
Description of Method: The sample is acidified with hydrochloric or nitric acid and boiled to eliminate
carbonate and bicarbonate ions. Uranium is coprecipitated with ferric hydroxide and subsequently
separated. The ferric hydroxide is dissolved, passed through an anion-exchange column, washed with
acid, and the uranium is eluted with dilute hydrochloric acid. The acid eluate is evaporated to near
dryness, the residual salt is converted to nitrate, and the alpha activity is counted by a gas-flow
proportional counter or alpha scintillation counter.
Special Considerations: If it is suspected that the sample exists in refractory form (i.e., non-digestible
or dissolvable material after normal digestion methods) or if there is a matrix interference problem, use
ORISE Method API 1.
Source: APHA, AWWA, and WEF. 2005. "Method 7500-U B: Uranium: Radiochemical Method."
Standard Methods for the Examination of Water and Wastewater. 21st Edition.
http: //www. standardmethods. org/
6.2.48 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
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/
6.2.49 Y-12 (DOE) Preparation of Samples for Total Activity Screening
Analyte(s)
Total Activity Screening
CASRN
NA
Analysis Purpose: Total activity screening
Technique: Liquid scintillation
SAM 2012 148 July 16, 2011
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SAM 2012 Section 6.0- Selected Radiochemical Methods
Method Developed for: Total activity screening
Method Selected for: SAM lists this method for gross total activity screening of drinking water, liquid
and aqueous phase, soil and sediment, wipe, air filter and vegetation samples.
Description of Method: Aqueous sample aliquots that require no preparation are added directly to the
scintillation cocktail. Solid and semi-solid sample aliquots are digested in nitric acid on a hot plate,
cooled, filtered, and diluted to a specified volume. Oil sample aliquots are weighed directly into a tared
counting vial. A specified volume of liquid scintillation cocktail is added to each vial and mixed with the
sample aliquot. The samples are then counted for total activity.
Special Considerations: The method assumes 100% counting efficiencies for both beta and alpha
emitters. Low energy beta emitters will not be counted at 100% efficiency, which can introduce a
negative bias in the measurement.
Source: Y-12 (DOE). 2005. "Preparation of Samples for Total Activity Screening." Procedure Y50-
AC-65-7230. http://www.epa.gov/sam/Y50-AC-65-7230.pdf
SAM 2012 149 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
Section 7.0: Selected Pathogen Methods
Legislation and Presidential Directives give the U.S. EPA the responsibility to lead environmental and
water clean up efforts associated with terrorist events and other disasters. This responsibility is listed in
the Integrated Consortium of Laboratory Networks (ICLN) Federal Responsible Agency Matrix.
Following a contamination event, it is assumed that the U.S. Centers for Disease Control and Prevention
(CDC) and the Federal Bureau of Investigation (FBI) have completed the identification, confirmation and
strain-level characterization of the bioagent/pathogen contaminant before EPA's remediation begins. The
first phase of EPA's actions includes site characterization, to determine the extent and magnitude of
contamination and guide remediation planning. Based on the results of sample analyses for site
characterization, EPA will determine the approach for site decontamination. During the post
decontamination (clearance) phase of remediation, samples are collected and analyzed to determine the
efficacy of the decontamination treatment.
Based on lessons learned and experience gained through various interagency bioterrorism counter-
measures programs, including the Interagency Biological Restoration Demonstration (IBRD), decision
making during remediation includes scientific, public health, social, economic and political concerns. As
part of this process, the analytical methods selected for use should aid in decision making in an effective
and time-sensitive manner. The purpose of this section is to provide guidance to stakeholders in
determining the appropriate methods for each remedial phase (site characterization and/or post
decontamination) of a response to a biological event. Emphasis is given to environmental sample types
that are most prevalently used to fulfill EPA's homeland security responsibilities (e.g., aerosols, surface
wipes or swabs, drinking water and post decontamination waste water).
Selection of methods from Appendix C should be based on specific data and information needs, including
consideration of the remediation phase and whether there is a need to determine either the presence of a
pathogen, the viability of a pathogen or both. The flow chart in Figure 7-1 presents a summary of the
sample types, overall steps in sample analysis, and analytical techniques that should be used to address
pathogens during EPA site remediation activities following a contamination event. As depicted in Figure
7-1, for SAM Pathogens, site characterization refers to the assessment phase, decontamination refers to
the cleanup phase, and post decontamination refers to the clearance phase.
SAM 2012 151 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
Figure 7-1. Sample Analysis During Site Characterization and Post
Decontamination Phases Following a Biological Contamination Event
/ Site Remediation \
(See Figure 1-1)
SAM
(Selected Analytical Methods for Environmental
Remediation and Recovery)
\
\ (^Assessment) (^ Cleanup J) (^ Clearance^)/
f For SAM Pathogens, site characterization refers to the assessment phase and post decontamination '
refers to the clearance phase. Methods included in SAM Section 7 and Appendix C may be used during
I the cleanup phase, if needed. ,
Sample
(Aerosol, Wipe, Swab, Drinking Water,
Post Decontamination Waste Water)
BSL-2 or BSL-3 Bioacient
1
Drinking Water/
Post-Decon Waste Water
Concentration
(As necessary)
Characterization
Rapid Analytical Method
(Real-time PCR, ELISA, other
immunoassay)
Post Decontamination
Culture Followed by
Rapid Confirmation
(Real-time PCR, ELISA, other
immunoassay)
OR
^^ RV-PCR (if available) ^^-
* Neutralization of decontamination agents may be required for post decontamination phase samples.
SAM 2012
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July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
Methods for Site Characterization Phase: Since decontamination of the affected site has to quickly
follow the site characterization phase, rapid analytical methods should be selected to determine the extent
and magnitude of contamination. It is assumed here that, prior to site characterization, the identity and
viability of the pathogen have been determined by the CDC and FBI. Therefore, in most cases, the
analytical methods selected for this phase may not have to determine whether the pathogen is viable. The
methods should also provide a high throughput analytical capability, so that a large number of samples
can be rapidly analyzed to determine the presence or absence of the pathogen and allow for site
decontamination planning in a time-efficient manner. For most pathogens, such methods routinely
include polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA) or other
immunoassay-based methods. Depending on the pathogen, type of event and response, culture methods
could be appropriate for use during site characterization. In certain cases, the viability determination of
the pathogen within this phase may drive decontamination planning.
Methods for Post Decontamination Phase: It is extremely critical that the analytical methods used for
sample analysis during the post decontamination phase be highly sensitive, specific, rapid, and able to
determine pathogen viability. For post decontamination phase samples, neutralization or removal of the
decontamination agent may be required prior to analysis. Traditional microbiological culture methods
typically include plating on selective medium to determine the viability of the pathogen and to minimize
or eliminate non-target growth. The absence of growth on the medium generally indicates the absence of
live pathogen in the sample [with the exception of some pathogens which may become viable but non-
culturable (VBNC)]. To minimize the analytical time needed to obtain results, typical colonies should be
quickly analyzed to confirm the presence of the pathogen using reliable and rapid methods such as PCR,
ELISA or other immunoassay-based methods, as opposed to time and labor intensive traditional
biochemical and serological procedures. For Bacillus anthracis, the recently published Rapid Viability-
PCR (RV-PCR) method may be used because it provides rapid and high throughput results in addition to
viability determination (Letant et al. 20117, U.S. EPA Report 2010 [EPA/600/R-10/156]8). However, if
pathogen viability and concentration determinations are required for any samples, the culture method
followed by rapid identification techniques (e.g., PCR, ELISA) should be used.
A list of methods that have been selected by the SAM Pathogen Methods Work Group for use in
analyzing environmental samples for pathogens is provided in Appendix C. These methods should be
used during remediation activities following a biological 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 verified and/or validated methods for pathogens are not currently
available. The literature references will be replaced as fully developed and validated protocols become
available.
Please note: This section provides guidance for selecting pathogen methods that have a high likelihood
of assuring analytical consistency when laboratories analyze a large number of samples during
remediation. Not all methods have been verified for the pathogen/sample type combination listed in
Appendix C. Please refer to the specified method to identify analyte/sample type combinations for
which the method has been verified. Any questions regarding information discussed in this section
should be addressed to the appropriate contact(s) listed in Section 4.
Pathogens that are categorized as biosafety level (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
7 Letant, S. E., Murphy, G.A., Alfaro, T. M, Avila, J. R., Kane, S. R., Raber, E., Bunt, T. M. and Shah, S. R. 2011.
Rapid-Viability PCR Method for Detection of Live, Virulent Bacillus anthracis in Environmental Samples."
Applied Environmental Microbiology, 77(18): 6570-6578.
8 U.S. EPA. 2010. "Development and Verification of Rapid Viability Polymerase Chain Reaction (RV-PCR)
Protocols for Bacillus anthracis - For Aapplication to Air Filters, Water and Surface Samples." EPA/600/R-10/156.
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this document. All other pathogens should be handled using BSL-2 or BSL-3 containment and practices,
as appropriate. 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. However, such pathogens may be considered for
possible inclusion in future revisions of SAM.
In addition to analytical methods, Appendix C lists commercially available spore strips, which may be
used as general indicators that a decontamination process (e.g., fumigation) has been successful. Spore
strip results, 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.
Some of the methods in Appendix C include multiple analytical techniques by inference. The analytical
technique listed in Appendix C for each pathogen 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 method or procedure listed as "culture" might 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 use of multiple cell culture media for primary isolation and a selection of a defined
subset of biochemical tests for confirmation. To expedite time-to-results, however, isolates should be
confirmed using rapid techniques (e.g., PCR, ELISA).
Appendix C includes the following information:
Pathogen(s). A specific causative agent (e.g., viruses, bacteria) of disease.
Viability. A microorganism's ability to grow and reproduce and/or cause infection.
Analytical technique. An analytical procedure used to determine the identity, quantity and/or
viability of a pathogen.
Analytical method. A series of techniques which together isolate, concentrate and detect a
microorganism or group of microorganisms. In some cases, a unique identifier or number is assigned
to an analytical method by the method publisher. Analytical methods can be developed for various
sample types, including:
Aerosol (growth media, filters, liquids). The recommended method/procedure for the pathogen
of interest in air sample collectors such as growth media, filters or liquids.
Particulate (swabs, wipes, filters). The recommended method/procedure for the pathogen of
interest in particulate sample collection tools such as swabs, wipes, Sponge-Sticks, and high-
efficiency particulate air (HEPA) collecting socks and filters used for vacuum collection.
Drinking water. The recommended method/procedure for the pathogen of interest in potable
water (concentrated and small volume grab samples).
Post decontamination waste water. The recommended method/procedure for the pathogen of
interest in post decontamination waste water (concentrated and small volume grab samples).
Sample Processing: It is widely recognized in the scientific community that the processing of
biologically contaminated environmental samples is one of the most challenging issues prior to sample
analysis. Although details regarding sample processing are not within the scope of SAM, it is critical that
end users and stakeholders select the most appropriate sample processing procedure for a given sample
type and analytical method. It is highly unlikely that a single procedure will be applicable to all sample
types and analytical methods. Inadequate sample processing may not only decrease recovery efficiency
of biological targets (e.g., pathogen, deoxiribonucleic acid/ribonucleic acid [DNA/RNA], antigen/protein)
from the samples, but also prevent accurate quantitation and high throughput. Note: For post
decontamination samples it may be necessary to neutralize the decontamination agent.
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The methods listed in SAM attempt to address multiple environmental sample types, each with different
physical, chemical and biological properties (e.g., pH, inhibitory substances and background
microorganisms). In this edition, major emphasis is given to the environmental sample types that are
most prevalently used to fulfill EPA's homeland security responsibilities following an event involving
pathogen contaminants (e.g., aerosols, surface wipes or swabs, drinking water and post decontamination
waste water). Other sample types may have to be analyzed, and for those sample types, specific requests
should be sent to the SAM Pathogen Methods Lead and Alternate Lead (see Section 4).
7.1 General Guidelines
This section provides a general overview of how to identify the appropriate method(s) for a given
pathogen as well as recommendations for quality control (QC) procedures.
For additional information on the properties of the pathogens listed in Appendix C, Toxicology Data
Network (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 website
(http://www.bt.cdc.gov/) and the U.S. Food and Drug Administration (FDA) Center for Food Safety and
Applied Nutrition (CFSAN) 2012, "Bad Bug Book"
(http://www.fda.gov/food/foodsafetv/foodborneillness/foodborneillnessfoodbornepathogensnaturaltoxins/
badbugbook/default.htm). Further research on pathogens is ongoing within EPA. Databases to manage
this information are currently under development.
7.1.1 Standard Operating Procedures for Identifying Pathogen Methods
To determine the appropriate analytical method that is to be used for an environmental sample, locate the
pathogen in Appendix C: Selected 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 (aerosols, surface wipes or swabs, drinking water and post decontamination waste water).
Once a method has been identified in Appendix C, the corresponding method summary can be found in
Sections 7.2 through 7.5. Method summaries are listed in alphabetical order within each pathogen
subcategory (i.e., bacteria, virus, protozoa, helminthes) and then by 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. 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 source information provided in Table 7-1.
Table 7-1. Sources of Pathogen Methods
Name
National Environmental Methods Index
(NEMI)
EPA Microbiology Home Page
Information Collection Requirements
Rule (ICR) Microbial Laboratory Manual
EPA Manual of Methods for Virology
Publisher
EPA, U.S. Geological
Survey (USGS)
EPA
EPA ORD
EPA
Reference
http://vwvw.nemi.gov
http://vwvw.epa.qov/nerlcvwvw/epamicrobiol
oqv.html
http://www.epa.aov/nerlcwww/documents/ic
rmicro.pdf
http://www.epa.aov/sam/EPA Manual of
Methods for Viroloav.pdf
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Name
Environmental Regulations and
Technology: Control of Pathogens and
Vector Attraction in Sewage and Sludge
U.S. Department of Agriculture (USDA)
Food Safety and Inspection Service
(FSIS) Microbiology Laboratory
Guidebook
Bacteriological Analytical Manual (BAM)
Occupational Safety and Health
Administration (OSHA) Methods
National Institute for Occupational
Safety and Health (NIOSH) Methods
Standard Methods for the Examination
of Water and Wastewater, 21st Edition,
2005*
Annual Book ofASTM Standards*
Applied and Environmental
Microbiology*
Journal of Clinical Microbiology*
Clinical Microbiology Procedures
Handbook, 3rd Edition, 2010*
Molecular and Cellular Probes*
Canadian Journal of Microbiology*
Food and Environmental Virology*
Journal of Medical Virology*
Journal of Virological Methods*
Diagnostics Microbiology and Infectious
Disease
Emerging Infectious Diseases
Parasitology Research*
Journal of Parasitology*
Transactions of the Royal Society of
Tropical Medicine and Hygiene*
Diagnostic Procedures in Veterinary
Bacteriology and Mycology
Publisher
EPA, National Risk
Management Research
Laboratory (NRMRL)
USDA FSIS
FDA, CFSAN
OSHA
NIOSH
American Public Health
Association (APHA),
American Waterworks
Association (AWWA) and
Water Environment
Federation (WEF)
ASTM International
American Society for
Microbiology (ASM)
ASM
ASM
Elsevier
NRC Research Press
Springer
Wiley InterScience
Elsevier
Elsevier
CDC
Springer
American Society of
Parasitologists
The Royal Society of
Tropical Medicine and
Hygiene
Academic Press
Reference
http://vwvw.eDa. aov/nrmrl/Dubs/625r92013/
625R92013.pdf
http://www.fsis.usda.qov/Science/Microbiol
oqical Lab Guidebook/index.asp
http://www.fda.aov/Food/ScienceResearch/
LaboratorvMethods/BacterioloaicalAnalvtic
alManualBAM/default.htm
http://www.osha.qov
http://www.cdc.qov/niosh/nmam/
http://www.standardmethods.org
http://www.astm.org
http://aem.asm.org/
http://icm.asm.org/
http://estore.asm.org/viewitemdetails.asp7it
emid=908
http://www.iournals.elsevier.com/molecular-
and-cellular-probes/
http://www.nrcresearchpress.com/loi/cim
http://www.springer.com/biomed/virologv/io
urnal/12560
http://onlinelibrarv.wilev.com/iournal/10.100
2/(ISSN)1 096-9071
http://www.scimagoir.com/iournalsearch.ph
p?g=20241&tip=sid
http://www.elsevier.com/wps/find/iournalde
scription.cws home/505759/description#de
http://wwwnc.cdc.gov/eid/
http://www.springer.com/biomed/medical+
microbiology/iournal/436
http://www.iournalofparasitologv.org/
http://www.elsevier.com/wps/find/iournalde
scription.cws home/681 01 9/description#de
scription
http://www.pubmedcentral.nih.gov/articlere
nder.fcgi?artid=1481267
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Name
Sentinel Level Clinical Microbiology
Laboratory Guidelines for Suspected
Agents of Bioterrorism and Emerging
Infectious Diseases
Journal of Applied Microbiology*
Journal of Microbiological Methods*
Clinical Chemistry
Antimicrobial Agents and
Chemotherapy*
Environmental Science and
Technology*
Publisher
ASM
Blackwell Publishing
Elsevier
American Association for
Clinical Chemistry
ASM
American Chemical
Society (ACS)
Reference
http://www.asm.ora/index.php/policv/sentin
el-level-clinical-microbioloqv-laboratorv-
quidelines.html
http://onlinelibrarv.wilev.eom/iournal/10.1 1 1
1/(ISSN)1 365-2672
http://www.sciencedirect.com/science/iourn
al/01677012
http://www.clinchem.orq/content/bv/vear
http://aac.asm.org/
http://pubs.acs.org/iournal/esthaq
Subscription and/or purchase required.
publication date.
ASM does not require a subscription or purchase 6 months after the
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 and proficient 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; (5) properly document analytical QC procedures and corrective actions;
and (6) maintain personnel training and proficiency testing records.9
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. In addition, for molecular techniques (e.g., PCR)
general guidelines are provided in EPA's Quality Assurance/Quality Control Guidance for Laboratories
Performing PCR Analyses on Environmental Samples.
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
Information regarding EPA's DQO process, considerations, and planning is available at:
http://www.epa.gov/QUALITY/dqos.html.
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Reference matrix spikes to evaluate initial and ongoing method/analyst performance, if available
Matrix spikes (where possible) to evaluate method performance in the sample type of interest
Matrix spike duplicates (MSB) and/or sample replicates to assess method precision
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 points of contact identified in Section 4 should be consulted regarding
appropriate quality assurance (QA)/QC procedures prior to sample analysis. These contacts should be
consulted regarding any method deviations or modifications, sample problems or interferences, QC
requirements, the use of alternative methods, or the need to address analytes or sample types other than
those listed in SAM. As previously indicated, any deviations from the recommended method(s) should be
reported immediately to ensure data comparability is maintained. Method deviations or modifications
must be approved by the Environmental Response Laboratory Network (ERLN) or Water Laboratory
Alliance (WLA) prior to use.
7.1.3 Safety and Waste Management
Laboratories should have a documented health and safety plan for handling samples that might contain
target chemical, biological and/or radiological (CBR) contaminants. Laboratory staff should be trained in
the safety procedures included in the plan and implement those procedures. 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 to enhance dispersion or virulence characteristics. Laboratories 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 Sections 7.2 through
7.5 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 can cause serious or potentially lethal
disease and also have the potential for aerosol transmission. Whenever available, BSLs are provided in
the method summaries in Sections 7.2 through 7.5. However, 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:
CDC. 2009. Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th Edition.
Available at: http://www.cdc.gov/biosafety/publications/bmbl5/
CDC.2002. "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. Available at: http://www.cdc.gov/mmwr/pdf/rr/rr5119.pdf
Microbiology Biosafety for Level A Laboratories. Available at:
http://www.bt.cdc.gov/documents/PPTResponse/table3bbiosafety.pdf
Select Agent Rules and Regulations found at the National Select Agent Registry. Available at:
http: //www. selectagents. gov/ and http ://ecfr. gpoacce ss. gov/cgi/t/text/text-
idx?c=ecfr&tpl=/ecfrbrowse/Title09/9cfrl21 main O2.tpl
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The following sources provide information regarding waste management:
EPA - Hazardous Waste Management (40 CFR part 260) and EPA Administered Permit Programs
(40 CFR part 270). Available at: http://ecfr.gpoaccess.gov/cgi/t/text/text-
idx?sid=cac9da30cd241fa70d461e4a917eb75e&c=ecfr&tpl=/ecfrbrowse/Title40/40tab O2.tpl
EPA. 2010. Laboratory Environmental Sample Disposal Information Document Companion to
Standardized Analytical Methods for Environmental Restoration Following Homeland Security
Events (SAM) Revision 5. EPA/600/R-10/092. Available at: http://www.epa.gov/sam/LESDID.pdf
Other resources that can be consulted for additional information include the following:
OSHA - Hazardous Waste Operations and Emergency Response (29 CFR part 1910.120). Available
at: http://www.osha.gov/pls/oshaweb/owadisp.show document?ptable=STANDARDS&pid=9765
OSHA - Occupational Exposure to Hazardous Chemicals in Laboratories (29 CFR part 1910.1450).
Available at:
http://www.osha.gov/pls/oshaweb/owadisp.show document?ptable=STANDARDS&pid=10106
OSHA - Respiratory Protection (29 CFR part 1910.134). Available at:
http://www.osha.gov/pls/oshaweb/owadisp.show_document?p_id=12716&p_table=STANDARDS
DOT Hazardous Materials Shipment and Packaging (49 CFR parts 171-180). Available at:
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 the 1995 Presidential Directive 39, Policy on Counterterrorism,
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 (4-6 hours) presumptive detection of biothreat agents
and emerging infectious disease agents. 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. Except for
Coxiella burnetii, culture methods are available for all of these pathogens as American Society for
Microbiology's (ASM) 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 U.S. Health and Human Services
(HHS)/U.S. Department of Agriculture (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/biosafety/publications/bmbl5/).
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Select Agents Listed in Appendix C
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)
CDC
1600 Clifton Road NE, Mailstop C-18
Atlanta, GA 30333
Telephone: (866) 576-5227
E-mail: lrn(g),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
Website: http://www.aphl.org
E-mail: info@aphl.org
The following references and information sources provide additional information regarding Select Agents
Culture Methods - LRN Sentinel Labs (website references for individual pathogens are included in their
respective summaries):
Avian Influenzae: http://www.asm.org/images/pdf/Clinical/Protocols/avianiinfluenzall-2008.pdf
Brucella: http://www.asm.org/images/pdf/Clinical/Protocols/brucellalO-15-04.pdf
Burkholderia mallei and B. pseudomallei:
http://www.asm.org/images/pdf/Clinical/Protocols/bpseudomallei2008.pdf
Coxiella burnetii: http://www.asm.org/images/pdf/Clinical/Protocols/qfever36-23-03.pdf
Fransicella tularensis: http://www.asm.org/images/pdf/Clinical/Protocols/tularemia.pdf
Yersinia pestis: http://www.asm.org/images/pdf/Clinical/Protocols/vpestis06-ll-10.pdf
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Sources:
ASM. 2010. Sentinel Level Clinical Microbiology Laboratory Guidelines for Suspected Agents of
Bioterrorism and Emerging Infectious Diseases.
http: //www. asm. org/index.php ?option=com_content&view=article&id=63 42
CDC. 2009. Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th Edition.
http://www.cdc. gov/biosafetv/publications/bmbl5/
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.
7.2.1 Bacillus anthracis [Anthrax] - BSL-3
Remediation Phase
Site Characterization
Post Decontamination
Analytical Technique
Real-Time PCR
RV-PCR
Culture and Real-Time PCR
Section
7.2.1.1
7.2.1.2
7.2.1.3
7.2.1.1 Site Characterization Sample Analyses (Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011).
All other environmental sample types should be processed according to procedures within the
"Protocol for Detection of Bacillus anthracis in Environmental Samples During the Remediation
Phase of an Anthrax Event" (U.S. EPA, anticipated publication October 2012), referred to as the
EPA BA Protocol for the remainder of SAM.
Nucleic acid extraction by bead beating and purification for all sample types should be performed
according to procedures within the EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use real-time PCR [EPA BA Protocol (U.S. EPA, anticipated
publication October 2012)].
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), the target nucleic acid should be extracted, purified (EPA BA
Protocol, Sections 9 and 11), and analyzed using the referenced target-specific PCR primers and
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SAM 2012 Section 7.0- Selected Pathogen Methods
probes and assay parameters. The use of real-time PCR analyses directly on samples (e.g., no
culture component) allows for rapid detection of B. anthracis spores.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control (purified nucleic acid), negative control, external inhibition 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's Quality
Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on
Environmental Samples (EPA 815-B-04-001) document at: http://www.epa.gov/sam/EPA-
QAQC-PCR.pdf. or consult the points of contact identified in Section 4.
Special Considerations: Bacillus anthracis is a select agent requiring regulatory compliance
(42 CFR parts 72 and 73, and 9 CFRpart 121); appropriate safety and BSL requirements should
be followed [see CDC's BMBL, 5th Edition (CDC 2009)].
http://www.cdc.gov/biosafety/publications/bmbl5/).
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
7.2.1.2 Post Decontamination Sample Analyses (RV-PCR)
Note. Laboratories without RV-PCR capability should analyze samples according to the culture
procedure provided in Section 7.2.1.3.
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011).
SAM 2012 162 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction and purification should be performed according to procedures within the
EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use RV-PCR [EPA BA Protocol (U.S. EPA, anticipated publication
October 2012)].
Description of Method: The RV-PCR procedure is a combination of a broth culture and real-
time PCR. Culturing the sample allows the germination of Bacillus anthracis spores recovered
from a processed sample. The real-time PCR provides rapid detection of Bacillus anthracis. By
combining both culture and PCR the protocol allows for the detection of viable Bacillus anthracis
spores. Prior to analysis, samples (air filter, wipe, Sponge-Stick, vacuum socks or filter, water)
are processed using multiple extraction and wash steps. After brain heart infusion broth is added
to the spores, an aliquot (Time 0 [TO]) is removed and stored at 4 ฐC. The remaining broth is
then incubated for 9 hours at 36 ฐC. After the 9-hour incubation, an aliquot is removed (Time 9
[T9]). Both TO and T9 aliquots then go through DNA extraction and purification followed by
real-time PCR analysis. The cycle threshold (CT) values for the TO and T9 aliquots are then
compared. The difference in CT values between the TO and T9 is used to detect viable Bacillus
anthracis spores. A change (decrease) in the PCR CT > 9 represents increased DNA
amplification in the T9 aliquot relative to the TO aliquot, which in turn, represents an increase in
DNA as a result of the germination and growth of viable spores in the sample during the
incubation period.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Special Considerations: Bacillus anthracis is a select agent requiring regulatory compliance
(42 CFR parts 72 and 73, and 9 CFRpart 121); appropriate safety and BSL requirements should
be followed [see BMBL, 5th Edition (CDC 2009)].
http://www.cdc.gov/biosafety/publications/bmbl5/).
Some laboratories may not have access to a positive control for this agent for culture analyses.
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
SAM 2012 163 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http: //oaspub .epa. gov/eims/eimscomm. getfile ?p_download_id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
7.2.1.3 Post Decontamination Sample Analyses (Culture and Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction by bead beating and purification for all sample types should be performed
according to procedures within the EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use culture and real-time PCR [EPA Protocol (U.S. EPA, anticipated
publication October 2012)].
Description of Method: The culture procedure is based on the CDC Sentinel Procedure for
Bacillus anthracis. Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), the sample is streaked for isolation onto tryptic soy agar with 5%
sheep's blood. Plates are incubated at 35 ฐC - 37 ฐC for 18 - 24 hours. Isolated typical colonies
are resuspended in sterile distilled water. The bacterial suspensions are then heated at 95 ฐC - 98
ฐC to release the DNA from the cells (see Section 11). DNA extracts are then used in real-time
PCR to confirm the presence of Bacillus anthracis. Combining the culture component with
confirmation using real-time PCR analyses allows for detection and viability results within 24 -
30 hours as compared to traditional culture procedures that require a minimum of 48 hours.
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's Quality Assurance/Quality Control Guidance for Laboratories
Performing PCR Analyses on Environmental Samples (EPA 815-B-04-001) document at:
http://www.epa.gov/sam/EPA-OAOC-PCR.pdf. or consult the points of contact identified in
Section 4.
SAM 2012 164 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
Special Considerations: Bacillus anthracis is a select agent requiring regulatory compliance
(42 CFR parts 72 and 73, and 9 CFRpart 121); appropriate safety and BSL requirements should
be followed [see BMBL, 5th Edition (CDC 2009)]
http: //www. cdc. gov/biosafety/publications/bmbl5
Some laboratories may not have access to a positive control for this agent for culture analyses.
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
7.2.2 Brucella spp. [Brucellosis] - BSL-3
Remediation Phase
Site Characterization
Post Decontamination
Analytical Technique
Real-Time PCR
Real-Time PCR/lmmunoassay
Culture and Real-Time PCR
Section
7.2.2.1
7.1.41
7.2.2.2
1 Standardized procedures, reagents and agent-specific algorithms are available to LRN member
laboratories.
7.2.2.1 Site Characterization Sample Analyses (Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (swabs and wipes) should be prepared according to
"National Validation Study of a Swab Protocol for the Recovery of Bacillus anthracis Spores
From Surfaces" (Hodges etal. 2010) or "National Validation Study of a Cellulose Sponge-Wipe
Processing Method for Use After Sampling Bacillus anthracis Spores From Surfaces" (Rose etal.
2011).
SAM 2012 165 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011). All other environmental
sample types should be processed according to procedures within the EPA BA Protocol [U.S.
EPA (anticipated publication October 2012)].
Nucleic acid extraction by bead beating and purification for all sample types should be performed
according to procedures within the EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use real-time PCR (Literature reference for Brucella spp. [Journal of
Microbiological Methods, 2008, 75(2): 375-378]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), the target nucleic acid should be extracted, purified (EPA BA
Protocol, Section 9.2), and analyzed using the referenced target-specific PCR primers and probes
and assay parameters (Journal of Microbiological Methods, 2008, 75(2): 375-378). The use of
real-time PCR analyses directly on samples (e.g., no culture component) allows for rapid
detection of Brucella spp.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control (purified nucleic acid), negative control, external inhibition 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's Quality
Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on
Environmental Samples (EPA 815-B-04-001) document at: http://www.epa.gov/sam/EPA-
QAQC-PCR.pdf. or consult the points of contact identified in Section 4.
Special Considerations: Brucella spp. 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 BMBL, 5th Edition (CDC 2009)].
http://www.cdc.gov/biosafety/publications/bmbl5/
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
Hinic, V., Brodard, I., Thomann,A., Cvetnic, Z., Makaya, P.V., Frey, J. and Abril, C. 2008.
"Novel Identification and Differentiation of Brucella melitensis, B. abortus, B. suis, B. ovis, B.
SAM 2012 166 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
cams, and B. neotomae Suitable for Both Conventional and Real-time PCR Systems." Journal of
Microbiological Methods, 75(2): 375-378.
http://www.sciencedirect.com/science/article/pii/S0167701208002522
7.2.2.2 Post Decontamination Sample Analyses (Culture and Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction and purification should be performed according to procedures within the
EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use culture ("Sentinel Level Clinical Microbiology Laboratory
Guidelines for Suspected Agents of Bioterrorism and Emerging Infectious Diseases: Brucella
species") and real-time PCR (Literature reference for Brucella spp. [Journal of Microbiological
Methods, 2008 75(2): 375-378]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), samples are plated directly on selective and non-selective agars
and incubated at 35 ฐC (5-10% carbon dioxide) for up to 7 days. Confirmation is performed
using real-time PCR. Target nucleic acid should be extracted, purified (EPA BA Protocol,
Section 9.2), and analyzed using the referenced target-specific PCR primers and probes and assay
parameters (Journal of Microbiological Methods, 2008 75(2): 375-378). The use of real-time
PCR analyses directly on isolates (e.g., no biochemical/serological component) allows for rapid
confirmation of Brucella spp.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-OAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Special Considerations: Brucella spp. 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 BMBL, 5th Edition (CDC 2009)].
http://www.cdc.gov/biosafety/publications/bmbl5/
SAM 2012 167 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
Some laboratories may not have access to a positive control for this agent for culture analyses.
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http: //oaspub .epa. gov/eims/eimscomm. getfile ?p_download_id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
ASM. 2004. "Sentinel Level Clinical Microbiology Laboratory Guidelines for Suspected Agents
of Bioterrorism and Emerging Infectious Diseases: Brucella species."
http: //www. asm. org/images/pdf/Clinical/Protocols/brucella 10-15-04.pdf
Hinic, V., Brodard, I., Thomann, A., Cvetnic, Z., Makaya, P.V., Frey, J. and Abril, C. 2008.
"Novel Identification and Differentiation of Brucella melitensis, B. abortus, B. suis, B. ovis, B.
canis, and B. neotomae Suitable for Both Conventional and Real-Time PCR Systems." Journal of
Microbiological Methods, 75(2): 375-378.
http://www.sciencedirect.com/science/article/pii/S0167701208002522
7.2.3 Burkholderia mallei [Glanders] - BSL-3 and Burkholderia pseudomallei
[Melioidosis] - BSL-3
Remediation Phase
Site Characterization
Post Decontamination
Analytical Technique
Real-Time PCR
Real-Time PCR/lmmunoassay
Culture and Real-Time PCR
Section
7.2.3.1
7.1. 41
7.2.3.2
1 Standardized procedures, reagents and agent-specific algorithms are available to LRN member
laboratories.
7.2.3.1 Site Characterization Sample Analysis (Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
SAM 2012
168
July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
Sample Preparation: Participate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction by bead beating and purification for all sample types should be performed
according to procedures within the EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use real-time PCR (Literature reference for Burkholderia mallei
[Clinical Chemistry, 2006, 52(2): 307-310]; Literature reference for Burkholderiapseudomallei
[Journal of Clinical Microbiology, 2006, 44(1): 85-90 and 2006, 44(8): 3028-3030]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), the target nucleic acid should be extracted, purified (EPA BA
Protocol, Section 9.2), and analyzed using the referenced target-specific PCR primers and probes
and assay parameters (Clinical Chemistry, 2006, 52(2): 307-310; Journal of Clinical
Microbiology, 2006, 44(1): 85-90 and 2006, 44(8): 3028-3030). The use of real-time PCR
analyses directly on samples (e.g., no culture component) allows for rapid detection of
Burkholderia mallei and Burkholderia pseudomallei.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control (purified nucleic acid), negative control, external inhibition 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's Quality
Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on
Environmental Samples (EPA 815-B-04-001) document at: http://www.epa.gov/sam/EPA-
QAQC-PCR.pdf. or consult the points of contact identified in Section 4.
Special Considerations: Burkholderia mallei and Burkholderia 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 BMBL, 5th Edition (CDC 2009)].
http://www.cdc.gov/biosafety/publications/bmbl5/
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
SAM 2012 169 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http: //oaspub .epa. gov/eims/eimscomm. getfile ?p_download_id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
Tomaso, H., Scholz, H.C., Al Dahouk, S., Eickhoff, M., Treu, T.M., Wernery, R., Wernery, U.
and Neubauer, H. 2006. "Development of a 5'-Nuclease Real-Time PCR Assay Targeting fliP for
the Rapid Identification ofBurkholderia mallei in Clinical Samples." Clinical Chemistry, 52(2):
307-310. http://www.clinchem.org/content/52/2/307.full.pdf+html
Novak, R.T., Glass, M.B., Gee, J.E., Gal, D., Mayo, M.J., Currie, B.J. and Wilkins, P.P. 2006.
"Development and Evaluation of a Real-Time PCR Assay Targeting the Type III Secretion
System of Burkholderia pseudomallei " Journal of Clinical Microbiology, 44(1): 85-90.
http://jcm.asm.org/content/44/l/85.full.pdf+html
Meumann, E.M., Novak, R.T., Gal, D., Kaestli, M.E., Mayo, M., Hanson,J.P., Spencer, E.,
Glass, M.B., Gee, J. E., Wilkins, P. P. and Currie, B.J. 2006. "Clinical Evaluation of a Type III
Secretion System Real-Time PCR Assay for Diagnosing Melioidosis." Journal of Clinical
Microbiology, 44(8): 3028-3030. http://icm.asm.org/content/44/8/3028.full.pdf+html
7.2.3.2 Post Decontamination (Culture and Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction and purification should be performed according to procedures within the
EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use culture (Sentinel Level Clinical Microbiology Laboratory
Guidelines for Suspected Agents of Bioterrorism and Emerging Infectious Diseases:
Burkholderia mallei and B. pseudo-mallei} and real-time PCR (Literature reference for
Burkholderia mallei [Clinical Chemistry, 2006, 52(2): 307-310]; Literature reference for
Burkholderiapseudomallei [Journal of Clinical Microbiology, 2006, 44(1): 85-90 and 2006,
44(8): 3028-3030]).
SAM 2012 170 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), samples are plated directly on sheep blood agar and incubated at
35 ฐC - 37 ฐC for 48 hours. Confirmation is performed using real-time PCR. Target nucleic acid
should be extracted, purified (EPA BA Protocol, Section 9.2), and analyzed using the referenced
target-specific PCR primers and probes and assay parameters (Clinical Chemistry, 2006, 52(2):
307-310; Journal of Clinical Microbiology, 2006, 44(1): 85-90 and 2006, 44(8): 3028-3030).
The use of real-time PCR analyses directly on isolates (e.g., no biochemical/serological
component) allows for rapid confirmation of Burkholderia mallei and Burkholderia
pseudomallei.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Special Considerations: Burkholderia mallei and Burkholderia 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 BMBL, 5th Edition (CDC 2009)].
http://www.cdc.gov/biosafety/publications/bmbl5/
Some laboratories may not have access to a positive control for this agent for culture analyses.
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
ASM. 2008. "Sentinel Level Clinical Microbiology Laboratory Guidelines for Suspected Agents
of Bioterrorism and Emerging Infectious Diseases: Burkholderia mallei and B. pseudomallei"
http://www.asm.org/images/pdf/Clinical/Protocols/bpseudomallei2008.pdf
Tomaso, H., Scholz, H.C., Al Dahouk, S., Eickhoff, M., Treu, T.M., Wernery, R, Wernery,U.
and Neubauer, H. 2006. "Development of a 5'-Nuclease Real-Time PCR Assay Targeting fliP for
the Rapid Identification of Burkholderia mallei in Clinical Samples." Clinical Chemistry, 52(2):
307-310. http://www.clinchem.org/content/52/2/307.full.pdf+html
SAM2012 111 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
Novak, R.T., Glass, M.B., Gee, J.E., Gal, D., Mayo, M.J., Currie, B.J. and Wilkins, P.P. 2006.
"Development and Evaluation of a Real-Time PCR Assay Targeting the Type III Secretion
System of Burkholderia pseudomallei " Journal of Clinical Microbiology, 44(1): 85-90.
http://icm.asm.org/content/44/l/85.full.pdf+html
Meumann, E.M., Novak, R.T., Gal, D., Kaestli, M.E., Mayo, M., Hanson,J.P., Spencer, E.,
Glass, M.B., Gee, J. E., Wilkins, P. P. and Currie, B.J. 2006. "Clinical Evaluation of a Type III
Secretion System Real-Time PCR Assay for Diagnosing Melioidosis." Journal of Clinical
Microbiology, 44(8): 3028-3030.
http://icm.asm.org/content/44/8/3028.full.pdf+html
7.2.4 Campylobacterjejuni [Campylobacteriosis] - BSL-2
Remediation Phase
Site Characterization
Post Decontamination
Analytical Technique
Real-Time PCR
Culture and Real-Time PCR
Section
7.2.4.1
7.2.4.2
7.2.4.1 Site Characterization Sample Analyses (Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction by bead beating and purification for all sample types should be performed
according to procedures within the EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use real-time PCR (Literature reference for Campylobacterjejuni
[Journal of Clinical Microbiology, 2010, 48(8): 2929-2933]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), the target nucleic acid should be extracted, purified (EPA BA
Protocol, Section 9.2), and analyzed using the referenced target-specific PCR primers and probes
and assay parameters (Journal of Clinical Microbiology, 2010, 48(8): 2929-2933). The use of
real-time PCR analyses directly on samples (e.g., no culture component) allows for rapid
detection of Campylobacterjejuni.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition control and blank. Ongoing
SAM 2012 172 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
analysis of QC samples to ensure reliability of the analytical results should also be performed.
PCR QC checks should be performed according to EPA's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-OAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http: //oaspub .epa. gov/eims/eimscomm. getfile ?p_download_id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
Cunningham, S.A., Sloan, L.M., Nyre, L.M., Vetter, E.A., Mandrekar, J. and Patel, R 2010.
"Three-Hour Molecular Detection of Campylobacter, Salmonella, Yersinia, and Shigella Species
in Feces With Accuracy as High as That of Culture." Journal of Clinical Microbiology, 48(8):
2929-2933. http://icm.asm.org/content/48/8/2929.full.pdf+html
7.2.4.2 Post Decontamination Sample Analyses (Culture and Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges et al. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose et al. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction and purification should be performed according to procedures within the
EPA BA Protocol (Protocol Section 9.2).
SAM 2012 173 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
Analytical Technique: Use culture ["ISO 17795: Water quality - Detection and enumeration of
thermotolerant Campylobacter species" (ISO 2005)] and real-time PCR (Literature reference for
Campylobacter jejuni [Journal of Clinical Microbiology, 2010, 48(8): 2929-2933]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), samples are inoculated into broth media and incubated, and then
plated onto selective agar. Confirmation is performed using real-time PCR. Target nucleic acid
should be extracted, purified (EPA BA Protocol, Section 9.2), and analyzed using the referenced
target-specific PCR primers and probes and assay parameters (Journal of Clinical Microbiology,
2010, 48(8): 2929-2933). The use of real-time PCR analyses directly on isolates (e.g., no
biochemical/serological component) allows for rapid confirmation of Campylobacter jejuni.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-OAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http: //oaspub .epa. gov/eims/eimscomm. getfile ?p_download_id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
ISO. 2005. ISO 17795: Water quality - Detection and Enumeration of Thermotolerant
Campylobacter Species, 2005.
http://www.iso.org/iso/iso catalogue/catalogue tc/catalogue detail.htm?csnumber=42082
Cunningham, S.A., Sloan, L.M., Nyre, L.M., Vetter, E.A., Mandrekar, J. and Patel, R 2010.
"Three-Hour Molecular Detection of Campylobacter, Salmonella, Yersinia, and Shigella Species
in Feces With Accuracy as High as That of Culture." Journal of Clinical Microbiology, 48(8):
2929-2933. http://icm.asm.org/content/48/8/2929.full.pdf+html
SAM 2012 174 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
7.2.5 Chlamydophila psittaci [Psittacosis] (formerly known as Chlamydia psittaci) -
BSL-2; BSL-3 for Aerosols and Large Volumes
Remediation Phase
Site Characterization
Post Decontamination
Analytical Technique
PCR
Tissue Culture and PCR
Section
7.2.5.1
7.2.5.2
7.2.5.1 Site Characterization Sample Analyses (PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction by bead beating and purification for all sample types should be performed
according to procedures within the EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use real-time PCR (Literature reference for Chlamydophila psittaci
[Journal of Clinical Microbiology, 2000, 38(3): 1085-1093]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), the target nucleic acid should be extracted, purified (EPA BA
Protocol, Section 9.2), and analyzed using the referenced target-specific PCR primers and probes
and assay parameters (Journal of Clinical Microbiology, 2000, 38(3): 1085-1093). The use of
real-time PCR analyses directly on samples (e.g., no culture component) allows for rapid
detection of Chlamydophila psittaci.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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 EPA's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-OAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
SAM 2012
175
July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
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://icm.asm.Org/content/38/3/1085.full.pdf+html
7.2.5.2 Post Decontamination Sample Analyses (Tissue Culture and PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Analytical Technique: Use tissue culture and real-time PCR (Literature Reference for
Chlamydophila psittaci [Journal of Clinical Microbiology, 2000, 38(3): 1085-1093]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), samples are inoculated onto buffalo green monkey kidney
(BGMK) cells to increase sensitivity. Target nucleic acid should be extracted, purified (EPA BA
Protocol, Section 9.2), and analyzed using the referenced target-specific PCR primers and probes
and assay parameters (Journal of Clinical Microbiology, 2000, 38(3): 1085-1093). The use of
SAM 2012 176 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
real-time PCR analyses directly on isolates allows for rapid confirmation of Chlamydophila
psittaci.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http: //oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
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://icm.asm.Org/content/38/3/1085.full.pdf4litml
7.2.6 Coxiella burnetii [Q-fever] - BSL- 3
Remediation Phase
Site Characterizaion
Post Decontamination
Analytical Technique
Real-Time PCR
Real-Time PCR/lmmunoassay
Tissue Culture and Real-Time PCR
Section
7.2.6.1
7.1.41
7.2.6.2
1 Standardized procedures, reagents and agent-specific algorithms are available to LRN member
laboratories.
7.2.6.1 Site Characterization Sample Analyses (Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
SAM2012 111 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction by bead beating and purification for all sample types should be performed
according to procedures within the EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use real-time PCR (Literature reference for Coxietta burnetii [BMC
Microbiology, 2008, 8:77]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), the target nucleic acid should be extracted, purified (EPA BA
Protocol, Section 9.2), and analyzed using the referenced target-specific PCR primers and probes
and assay parameters (BMC Microbiology, 2008, 8:77). The use of real-time PCR analyses
directly on samples (e.g., no culture component) allows for rapid detection ofCoxiella burnetii.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control (purified nucleic acid), negative control, external inhibition 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's Quality
Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on
Environmental Samples (EPA 815-B-04-001) document at: http://www.epa.gov/sam/EPA-
QAQC-PCR.pdf. or consult the points of contact identified in Section 4.
Special Considerations: Coxiella burnetii 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 BMBL, 5th Edition (CDC 2009)].
http://www.cdc.gov/biosafety/publications/bmbl5/
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
SAM 2012 178 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http: //oaspub .epa. gov/eims/eimscomm. getfile ?p_download_id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
Panning, M., Kilwinski, J., Greiner-Fischer, S., Peters, M., Kramme, S., Frangoulidis, D., Meyer,
H., Henning, K. and Drosten, C. 2008. "Fiigh Throughput Detection ofCoxiella burnetii by Real-
Time PCR With Internal Control System and Automated DNA Preparation." BMC Microbiology,
8:77. http://www.biomedcentral.com/content/pdf/1471-2180-8-77.pdf
7.2.6.2 Post Decontamination Sample Analyses (Tissue Culture and Real-Time
PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose et al. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Analytical Technique: Use tissue culture (Literature reference for Coxiella burnetii
[Antimicrobial Agents and Chemotherapy, 1991, 35(10): 2070-2077]) and real-time PCR
(Literature reference for Coxiella burnetii [BMC Microbiology, 2008, 8:77]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), samples are inoculated onto human erythroleukemia cells. Target
nucleic acid should be extracted, purified (EPA BA Protocol, Section 9.2), and analyzed using the
referenced target-specific PCR primers and probes and assay parameters (BMC Microbiology,
2008, 8:77). The use of real-time PCR analyses directly on isolates allows for rapid confirmation
ofCoxiella burnetii.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-OAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Special Considerations: Coxiella burnetii is a select agent requiring regulatory compliance (42
CFR parts 72 and 73, and 9 CFRpart 121); appropriate safety and BSL requirements should also
SAM 2012 179 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
be followed [see BMBL, 5th Edition (CDC 2009)].
http://www.cdc.gov/biosafety/publications/bmbl5/
Some laboratories may not have access to a positive control for this agent for culture analyses.
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
Raoult, D. Torres, H. and Drancourt, M. 1991. "Shell-Vial Assay: Evaluation of a New
Technique for Determining Antibiotic Susceptibility, Tested in 13 Isolates ofCoxiella bumetii"
Antimicrobial Agents and Chemotherapy, 35(10): 2070-2077.
http://aac.asm.org/content/35/10/2070.long
Panning, M., Kilwinski, J., Greiner-Fischer, S., Peters, M., Kramme, S., Frangoulidis, D., Meyer,
H., Henning, K. and Drosten, C. 2008. "High Throughput Detection ofCoxiella burnetii by Real-
Time PCR With Internal Control System and Automated DNA Preparation." BMC Microbiology.
8:77. http://www.biomedcentral.com/content/pdf/1471-2180-8-77.pdf
7.2.7 Escherichia coli O157:H7 - BSL-2
Remediation Phase
Site Characterization
Post Decontamination
Analytical Technique
Real-Time PCR
Culture and Real-Time PCR
Section
7.2.7.1
7.2.7.2
7.2.7.1 Site Characterization Sample Analyses (Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
SAM 2012 180 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "EPA Standard Analytical
Protocol for Escherichia coll O157:H7 in Water," EPA/600/R-10/056 (U.S. EPA 2010).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction by bead beating and purification for all sample types should be performed
according to procedures within the EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use real-time PCR (Literature reference for E. coli O157:H7
[Environmental Science and Technology, 2011, 45(7): 2250-2256]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), the target nucleic acid should be extracted, purified (EPA BA
Protocol, Section 9.2), and analyzed using the referenced target-specific PCR primers and probes
and assay parameters (Environmental Science and Technology, 2011, 45(7): 2250-2256). The
use of real-time PCR analyses directly on samples (e.g., no culture component) allows for rapid
detection of E. coli O157:H7.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub.epa.gov/eims/eimscomm.getfile7p download id=503892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
SAM 2012 181 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
U.S. EPA. September 2010. "Standard Analytical Protocol for Escherichia coll O157:H7 in
Water." EPA/600/R-10/056.
http: //oaspub .epa. gov/eims/eimscomm. getfile ?p_download_id=49 8 725
Sen, K., Sinclair, J.L., Boczek, L. and Rice, E.W. 2011. "Development of a Sensitive Detection
Method for Stressed E. coll O157:H7 in Source and Finished Drinking Water by Culture-qPCR."
Environmental Science and Technology, 45(6): 2250-2256.
http://pubs.acs.org/doi/abs/10.1021/esl03365b
7.2.7.2 Post Decontamination (Culture and Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "EPA Standard Analytical
Protocol for Escherichia coll O157:H7 in Water," EPA/600/R-10/056 (U.S. EPA 2010).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction and purification should be performed according to procedures within the
EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use culture [Standard Analytical Protocol for Escherichia coll O157:H7
in Water, EPA/600/R-10/056 (EPA 2010)] and real-time PCR (Literature reference for E. coll
O157:H7 [Environmental Science and Technology, 2011, 45(7): 2250-2256]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), samples are cultured using multiple media and immunomagnetic
separation (IMS). Typical isolates are then confirmed using biochemical and serological tests.
To expedite time to results, isolates should be confirmed using real-time PCR analyses. Target
nucleic acid should be extracted, purified (EPA BA Protocol, Section 9.2), and analyzed using the
referenced target-specific PCR primers and probes and assay parameters (Environmental Science
and Technology, 2011, 45(7): 2250-2256). The use of real-time PCR analyses allows for rapid
confirmation ofE. coll O157:H7.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
SAM 2012 182 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
U.S. EPA. September 2010. "Standard Analytical Protocol for Escherichia coll O157:H7 in
Water." EPA/600/R-10/056.
http: //oaspub .epa. gov/eims/eimscomm. getfile ?p_download_id=49 8 725
Sen, K., Sinclair, J.L., Boczek, L. and Rice, E.W. 2011. "Development of a Sensitive Detection
Method for Stressed E. coll O157:H7 in Source and Finished Drinking Water by Culture-qPCR."
Environmental Science and Technology, 45(7): 2250-2256.
http://pubs.acs.org/doi/abs/10.1021/esl03365b
7.2.8 Francisella tularensis [Tularemia] - BSL-3
Remediation Phase
Site Characterization
Post Decontamination
Analytical Technique
Real-Time PCR
Real-Time PCR/lmmunoassay
Culture and Real-Time PCR
Section
7.2.8.1
7.1.41
7.2.8.2
1 Standardized procedures, reagents and agent-specific algorithms are available to LRN member
laboratories.
7.2.8.1 Site Characterization Sample Analyses (Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
SAM 2012
183
July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction by bead beating and purification for all sample types should be performed
according to procedures within the EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use real-time PCR (Literature reference for Francisella tularensis
[Journal of Clinical Microbiology, 2003, 41(12): 5492-5499]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), the target nucleic acid should be extracted, purified (EPA BA
Protocol, Section 9.2), and analyzed using the referenced target-specific PCR primers and probes
and assay parameters (Journal of Clinical Microbiology, 2003, 41(12): 5492-5499). The use of
real-time PCR analyses directly on samples (e.g., no culture component) allows for rapid
detection of Francisella tularensis.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control (purified nucleic acid), negative control, external inhibition 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's Quality
Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on
Environmental Samples (EPA 815-B-04-001) document at: http://www.epa.gov/sam/EPA-
QAQC-PCR.pdf or consult the points of contact identified in Section 4.
Special Considerations: Francisella 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 BMBL, 5th Edition (CDC 2009)].
http://www.cdc.gov/biosafety/publications/bmbl5/
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
SAM 2012 184 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
Versage, J., Severin, D.D.M., Chu, M.C. and Petersen, J.M. 2003. "Development of a Multitarget
Real-Time TaqMan PCR Assay for Enhanced Detection ofFrancisella tularensis in Complex
Specimens." Journal of Clinical Microbiology, 41(12): 5492-5499.
http://icm.asm.org/content/41/12/5492.full.pdf+html
7.2.8.2 Post Decontamination Sample Analyses (Culture and Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) and "Use of Acid Treatment
and a Selective Medium to Enhance the Recovery ofFrancisella tularensis from Water"
(Humrighouse etal. 2011).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction and purification should be performed according to procedures within the
EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use culture ("Sentinel Level Clinical Microbiology Laboratory
Guidelines for Suspected Agents of Bioterrorism and Emerging Infectious Diseases: Francisella
tularensis" CDC et al. 2001 ) and real-time PCR (Literature reference for Francisella tularensis
[Journal of Clinical Microbiology, 2003, 41(12): 5492-5499]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), samples are plated directly onto selective media. Confirmation is
performed using real-time PCR. Target nucleic acid should be extracted, purified (EPA BA
Protocol, Section 9.2), and analyzed using the referenced target-specific PCR primers and probes
and assay parameters (Journal of Clinical Microbiology, 2003, 41(12): 5492-5499). The use of
real-time PCR analyses directly on isolates (e.g., no biochemical/serological component) allows
for rapid confirmation ofFrancisella tularensis.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-OAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Special Considerations: Francisella tularensis is a select agent requiring regulatory
compliance (42 CFR parts 72 and 73, and 9 CFRpart 121); appropriate safety and BSL
SAM 2012 185 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
requirements should also be followed [see BMBL, 5th Edition (CDC 2009)].
http://www.cdc.gov/biosafety/publications/bmbl5/
Some laboratories may not have access to a positive control for this agent for culture analyses.
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
Humrighouse, B.W., Adcock, N.J. and Rice, E.W., 2011. "Use of Acid Treatment and a Selective
Medium to Enhance the Recovery ofFrancisella tularensis from Water." Applied and
Environmental Microbiology, 77(18): 6729-6732.
http://aem.asm.org/content/77/18/6729.full.pdf+html
CDC, ASM, and APHL. 2001. "Sentinel Level Clinical Microbiology Laboratory Guidelines for
Suspected Agents of Bioterrorism and Emerging Infectious Diseases: Francisella tularensis."
http: //www. asm. org/images/pdf/Clinical/Protocols/tularemia.pdf
Versage, J.L., Severin, D.D, Chu, M.C. and Petersen, J.M. 2003. "Development of a Multitarget
Real-Time TaqMan PCR Assay for Enhanced Detection ofFrancisella tularensis in Complex
Specimens." Journal of Clinical Microbiology, 41(12): 5492-5499.
http://icm.asm.org/content/41/12/5492.full.pdf+html
7.2.9 Leptospira interrogans [Leptospirosis] - BSL-2
Remediation Phase
Site Characterization
Post Decontamination
Analytical Technique
Real-Time PCR
Culture and Real-Time PCR
Section
7.2.9.1
7.2.9.2
7.2.9.1 Site Characterization Sample Analyses (Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
SAM 2012 186 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction by bead beating and purification for all sample types should be performed
according to procedures within the EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use real-time PCR (Literature reference for Leptospira interrogans
[Molecular and Cellular Probes, 2005, 19(2): 111-117]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), the target nucleic acid should be extracted, purified (EPA BA
Protocol, Section 9.2), and analyzed using the referenced target-specific PCR primers and probes
and assay parameters (Molecular and Cellular Probes, 2005, 19(2): 111-117). The use of real-
time PCR analyses directly on samples (e.g., no culture component) allows for rapid detection of
Leptospira interrogans.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-OAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http: //oaspub .epa. gov/eims/eimscomm. getfile ?p_download_id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
SAM 2012 187 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
Palaniappan, R.U.M., Chang, Y.F., Chang, C., Pan, M.J., Yang, C.W., Harpending, P.,
McDonough, S.P., Dubovi, E., Divers, T., Qu, J. and Roe, B. 2005. "Evaluation of Lig-based
Conventional and Real Time PCR for the Detection of Pathogenic Leptospires." Molecular and
Cellular Probes, 19(2): 111-117.
http://www.sciencedirect.com/science/article/pii/S0890850804000970
7.2.9.2 Post Decontamination Sample Analyses (Culture and Real-time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction and purification should be performed according to procedures within the
EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use culture (Standard Method 9260 I: Leptospira) and real-time PCR
(Literature reference for Leptospira interrogans [Molecular and Cellular Probes, 2005, 19(2):
111-117]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), samples are inoculated into selective broth media and incubated
for up to six weeks at 30 ฐC. Confirmation is performed using real-time PCR. Target nucleic
acid should be extracted, purified (EPA BA Protocol, Section 9.2), and analyzed using the
referenced target-specific PCR primers and probes and assay parameters (Molecular and Cellular
Probes, 2005, 19(2): 111-117). The use of real-time PCR analyses directly on isolates (e.g., no
biochemical/serological component) allows for rapid confirmation of Leptospira interrogans.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
SAM 2012 188 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
APHA, AWWA and WEF. 2005. "Method 9260 I: Leptospira" Standard Methods for the
Examination of Water and Wastewater. 21st Edition, http://www.standardmethods.org/
Palaniappan, R.U.M., Chang, Y.F., Chang, C., Pan, M.J., Yang, C.W., Harpending, P.,
McDonough, S.P., Dubovi, E., Divers, T., Qu, J. and Roe, B. 2005. "Evaluation of Lig-based
Conventional and Real Time PCR for the Detection of Pathogenic Leptospires." Molecular and
Cellular Probes, 19(2): 111-117.
http://www.sciencedirect.com/science/article/pii/S0890850804000970
7.2.10 Listeria monocytogenes [Listeriosis] - BSL-2
Remediation Phase
Site Characterization
Post Decontamination
Analytical Technique
Real-Time PCR
Culture and Real-Time PCR
Section
7.2.10.1
7.2.10.2
7.2.10.1 Site Characterization Sample Analyses (Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011).
SAM 2012 189 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction by bead beating and purification for all sample types should be performed
according to procedures within the EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use real-time PCR [Microbiology Laboratory Guidebook - Chapter
MLG 8A.04 (USDA FSIS 2009)].
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), the target nucleic acid should be extracted, purified (EPA BA
Protocol, Section 9.2), and analyzed using the referenced target-specific PCR primers and probes
and assay parameters (Microbiology Laboratory Guidebook - Chapter MLG 8A.04, USDA,
FSIS, 2009). The use of real-time PCR analyses directly on samples (e.g., no culture component)
allows for rapid detection ofListeria monocytogenes.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http: //oaspub .epa. gov/eims/eimscomm. getfile ?p_download_id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
USDA, FSIS. 2009. "FSIS Procedure for the Use ofaListeria monocytogenes Polymerase Chain
Reaction (PCR) Screening Test." Chapter MLG 8A.04 m Microbiology Laboratory Guidebook..
http://www.fsis.usda.gov/PDF/MLG 8A 04.pdf
7.2.10.2 Post Decontamination Sample Analyses (Culture and Real-time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
SAM 2012 190 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction and purification should be performed according to procedures within the
EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use culture [BAM-Chapter 10 ( FDA CFSAN 2003)] and real-time PCR
[Microbiology Laboratory Guidebook - Chapter MLG 8A.04 (USDA FSIS 2007)].
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), samples are inoculated into broth media, incubated for 48 hours,
and then plated onto selective agar. Confirmation is performed using real-time PCR. Target
nucleic acid should be extracted, purified (EPA BA Protocol, Section 9.2), and analyzed using the
referenced target-specific PCR primers and probes and assay parameters (Microbiology
Laboratory Guidebook - Chapter MLG 8 A. 04 USDA, FSIS 2007). The use of real-time PCR
analyses directly on isolates (e.g., no biochemical/serological component) allows for rapid
confirmation ofListeria monocytogenes.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-OAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub.epa.gov/eims/eimscomm.getfile7p download id=503892
SAM 2012 191 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
Hitchins, A.D. and Jinneman, K. FDA, CFSAN. 2003. "Chapter 10 - Detection and Enumeration
of Listeria monocytogenes in Foods." Bacteriological Analytical Manual Online.
http://www.fda.gov/food/scienceresearch/laboratorymethods/bacteriologicalanalyticalmanualbam
/ucrnO? 1400.htm
USDA, FSIS. 2009. "FSIS Procedure for the Use ofaListeria monocytogenes Polymerase Chain
Reaction (PCR) Screening Test." Chapter MLG 8A.04 in Microbiology Laboratory Guidebook.
http://www.fsis.usda.gov/PDF/MLG 8A 04.pdf
7.2.11 Non-typhoidal Salmonella (Not applicable to S. Typhi) [Salmonellosis] - BSL-2
Remediation Phase
Site Characterization
Post Decontamination
Analytical Technique
Real-Time PCR
Culture and Real-Time PCR
Section
7.2.11.1
7.2.11.2
7.2.11.1 Site Characterization Sample Analyses (Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Analytical Protocol for
Non-Typhoidal Salmonella in Drinking Water and Surface Water" (U.S. EPA 2011).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction by bead beating and purification for all sample types should be performed
according to procedures within the EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use real-time PCR (Literature reference for non-typhoidal Salmonella
[Environmental Science and Technology, 2011, 45(20): 8996-9002]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), the target nucleic acid should be extracted, purified (EPA BA
Protocol, Section 9.2), and analyzed using the referenced target-specific PCR primers and probes
and assay parameters (Environmental Science and Technology, 2011, 45(20): 8996-9002). The
use of real-time PCR analyses directly on samples (e.g., no culture component) allows for rapid
detection of non-typhoidal Salmonella.
SAM 2012 192 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http: //oaspub .epa. gov/eims/eimscomm. getfile ?p_download_id=5 03 892
U.S. EPA. June 2011. "Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in
Drinking Water and Surface Water."
http://owpubauthor.epa.gov/infrastructure/watersecurity/wla/upload/epa817rl2004.pdf
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
Jyoti, A., Vajpayee, P., Singh, G., Patel, C.B., Gupta, K.C. and Shanker, R. 2011. "Identification
of Environmental Reservoirs of Nontyphoidal Salmonellosis: Aptamer-Assisted Bioconcentration
and Subsequent Detection of Salmonella Typhimurium by Quantitative Polymerase Chain
Reaction." Environmental Science and Technology, 45(20): 8996-9002.
http://pubs.acs.org/doi/abs/10.1021/es2018994
7.2.11.2 Post Decontamination Sample Analyses (Culture and Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
SAM 2012 193 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Method 1200: Analytical
Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water" (U.S. EPA 2011).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction and purification should be performed according to procedures within the
EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use culture [Method 1682 (U.S. EPA 2006)] or "Analytical Protocol for
Non-Typhoidal Salmonella in Drinking Water and Surface Water" (U.S. EPA 2011) and real-time
PCR (Literature Reference for Non-Typhoidal Salmonella [Environmental Science and
Technology, 2011, 45(20): 8996-9002]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), samples are inoculated into broth media, incubated for 24 hours,
and then plated onto multiple selective agars. Confirmation is performed using real-time PCR.
Target nucleic acid should be extracted, purified (EPA BA Protocol, Section 9.2), and analyzed
using the referenced target-specific PCR primers and probes and assay parameters
(Environmental Science and Technology. 45(20): 8996-9002). The use of real-time PCR
analyses directly on isolates (e.g., no biochemical/serological component) allows for rapid
confirmation of Non-Typhoidal Salmonella.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Special Considerations: This method will not detect Salmonella Typhi. MSRV and the
elevated incubation temperature (42 ฐC) are inhibitory for S. Typhi.
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub.epa.gov/eims/eimscomm.getfile7p download id=503892
SAM 2012 194 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
U.S. EPA. 2006. "Method 1682: Salmonella in Sewage Sludge (Biosolids) by Modified
Semisolid Rappaport-Vassiliadis (MSRV) Medium."
http://water.epa.gov/scitech/methods/cwa/bioindicators/upload/2008 11 25 methods method bi
ological 1682.pdf
U.S. EPA. 2011. "Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking
Water and Surface Water."
http://owpubauthor.epa.gov/infrastructure/watersecurity/wla/upload/epa817rl2004.pdf
Jyoti, A., Vajpayee, P., Singh, G., Patel, C.B., Gupta, K.C. and Shanker, R. 2011. "Identification
of Environmental Reservoirs of Nontyphoidal Salmonellosis: Aptamer-Assisted Bioconcentration
and Subsequent Detection of Salmonella Typhimurium by Quantitative Polymerase Chain
Reaction." Environmental Science and Technology, 45(20): 8996-9002.
http://pubs.acs.org/doi/abs/10.1021/es2018994
7.2.12 Salmonella Typhi [Typhoid fever] - BSL-2; BSL-3 for Aerosol Release
Remediation Phase
Site Characterization
Post Decontamination
Analytical Technique
Real-Time PCR
Culture and Real-Time PCR
Section
7.2.12.1
7.2.12.2
7.2.12.1 Site Characterization Sample Analyses (Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Standard Analytical
Protocol for Salmonella Typhi in Drinking Water" (U.S. EPA 2010).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction by bead beating and purification for all sample types should be performed
according to procedures within the EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use real-time PCR (CDC Laboratory Assay).
SAM 2012 195 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), the target nucleic acid should be extracted, purified (EPA BA
Protocol, Section 9.2), and analyzed using the referenced target-specific PCR primers and probes
and assay parameters (CDC Laboratory Assay). The use of real-time PCR analyses directly on
samples (e.g., no culture component) allows for rapid detection of Salmonella Typhi.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. 2010. "Standard Analytical Protocol for Salmonella Typhi in Drinking Water." EPA
600/R-10/133. http://oaspub.epa.gov/eims/eimscomm.getfile7p download id=499264
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
CDC Laboratory Assay. "Triplex PCR for Detection of S. Typhi Using SmartCyclerฎ." Contact:
Dr. Eija Trees, CDC.
7.2.12.2 Post Decontamination (Culture and Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
SAM 2012 196 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Standard Analytical
Protocol for Salmonella Typhi in Drinking Water" (U.S. EPA 2010).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction and purification should be performed according to procedures within the
EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use culture ("Standard Analytical Protocol for Salmonella Typhi in
Drinking Water," EPA 600/R-10/133 (U.S. EPA 2010) and real-time PCR (CDC Laboratory
Assay).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), samples are inoculated into broth media, incubated for 24 hours,
and then inoculated and plated onto multiple selective media. Confirmation is performed using
real-time PCR. Target nucleic acid should be extracted, purified (EPA BA Protocol, Section 9.2),
and analyzed using the referenced target-specific PCR primers and probes and assay parameters
(CDC Laboratory Assay). The use of real-time PCR analyses directly on isolates (e.g., no
biochemical/serological component) allows for rapid confirmation of Salmonella Typhi.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-OAQC-PCR.pdf or consult the points of
contact identified in Section 4.
Special Considerations: This method is not recommended for non-typhoidal Salmonella.
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
SAM 2012 197 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
U.S. EPA. 2010. "Standard Analytical Protocol for Salmonella Typhi in Drinking Water." EPA
600/R-10/133. http://oaspub.epa.gov/eims/eimscomm.getfile?p_download_id=499264
CDC Laboratory Assay. "Triplex PCR for Detection of S. Typhi Using SmartCyclerฎ." Contact:
Dr. Eija Trees, CDC.
7.2.13 Shigella spp. [Shigellosis] - BSL-2
Remediation Phase
Site Characterization
Post Decontamination
Analytical Technique
Real-Time PCR
Culture and Real-Time PCR
Section
7.2.13.1
7.2.13.2
7.2.13.1 Site Characterization Sample Analyses (Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction by bead beating and purification for all sample types should be performed
according to procedures within the EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use real-time PCR (Literature reference for Shigella spp. [Journal of
Clinical Microbiology, 2010, 48(8): 2929-2933]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), the target nucleic acid should be extracted, purified (EPA BA
Protocol, Section 9.2), and analyzed using the referenced target-specific PCR primers and probes
and assay parameters (Journal of Clinical Microbiology, 2010, 48(8): 2929-2933). The use of
real-time PCR analyses directly on samples (e.g., no culture component) allows for rapid
detection of Shigella spp.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-OAOC-PCR.pdf. or consult the points of
contact identified in Section 4.
SAM 2012 198 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
Cunningham, S.A., Sloan, L.M., Nyre, L.M., Vetter, E.A., Mandrekar, J. and Patel, R. 2010.
"Three-Hour Molecular Detection of Campylobacter, Salmonella, Yersinia, and Shigella Species
in Feces With Accuracy as High as That of Culture." Journal of Clinical Microbiology. 48(8):
2929-2933. http://icm.asm.org/content/48/8/2929.full.pdf+html
7.2.13.2 Post Decontamination Sample Analyses (Culture and Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction and purification should be performed according to procedures within the
EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use culture (Standard Method 9260 E: Shigella) and real-time PCR
(Literature reference for Shigella spp. [Journal of Clinical Microbiology, 2010, 48(8): 2929-
2933]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), samples are inoculated into broth media, incubated for 24 hours,
SAM 2012 199 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
and then plated onto multiple selective media. Confirmation is performed using real-time PCR.
Target nucleic acid should be extracted, purified (EPA BA Protocol, Section 9.2), and analyzed
using the referenced target-specific PCR primers and probes and assay parameters (Journal of
Clinical Microbiology, 2010, 48(8): 2929-2933). The use of real-time PCR analyses directly on
isolates (e.g., no biochemical/serological component) allows for rapid confirmation ofShigella
spp.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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 EPA's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http: //oaspub .epa. gov/eims/eimscomm. getfile ?p_download_id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
APHA, AWWA and WEF. 2005. "Method 9260 Detection of Pathogenic Bacteria E: Shigella"
Standard Methods for the Examination of Water and Wastewater. 21st Edition.
http: //www. standardmethods. org/
Cunningham, S.A., Sloan, L.M., Nyre, L.M., Vetter, E.A., Mandrekar, J., Patel, R 2010. "Three-
Hour Molecular Detection of Campylobacter, Salmonella, Yersinia, and Shigella Species in Feces
With Accuracy as High as That of Culture." Journal of Clinical Microbiology. 48(8): 2929-2933.
http://jcm.asm.org/content/48/8/2929.full.pdf+html
7.2.14 Staphylococcus aureus - BSL-2
Remediation Phase
Site Characterization
Post Decontamination
Analytical Technique
Real-Time PCR
Culture and Real-Time PCR
Section
7.2.14.1
7.2.14.2
SAM 2012 200 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
7.2.14.1 Site Characterization Sample Analyses (Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction by bead beating and purification for all sample types should be performed
according to procedures within the EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use real-time PCR (Literature reference for Staphylococcus aureus
[Journal of Food Protection, 2007, 70(12): 2855-2859]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), the target nucleic acid should be extracted, purified (EPA BA
Protocol, Section 9.2), and analyzed using the referenced target-specific PCR primers and probes
and assay parameters (Journal of Food Protection, 2007, 70(12): 2855-2859). The use of real-
time PCR analyses directly on samples (e.g., no culture component) allows for rapid detection of
Staphylococcus aureus.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-OAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
SAM 2012 201 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http: //oaspub .epa. gov/eims/eimscomm. getfile ?p_download_id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
Chiang, Y.C, Fan, C.M., Liao, W.W., Lin, C.K. and Tsen, H.Y. 2007. "Real-Time PCR Detection
of Staphylococcus aureus in Milk and Meat Using New Primers Designed From the Heat Shock
Protein Gene htrA Sequence." Journal of Food Protection, 70(12): 2855-2859.
http://www.ingentaconnect.com/content/iafp/ifp/2007/00000070/00000012/art00023
7.2.14.2 Post Decontamination Sample Analyses (Culture and Real-time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction and purification should be performed according to procedures within the
EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use culture (Standard Method 9213 B: Staphylococcus aureus) and real-
time PCR (Literature reference for Staphylococcus aureus [Journal of Food Protection, 2007,
70(12): 2855-2859]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), samples are inoculated into broth media, incubated for 24 hours,
and then plated onto selective media. Confirmation is performed using real-time PCR. Target
nucleic acid should be extracted, purified (EPA BA Protocol, Section 9.2), and analyzed using the
referenced target-specific PCR primers and probes and assay parameters (Journal of Food
Protection, 2007, 70(12): 2855-2859). The use of real-time PCR analyses directly on isolates
(e.g., no biochemical/serological component) allows for rapid confirmation of Staphylococcus
aureus.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition control and blank. Ongoing
analysis of QC samples to ensure reliability of the analytical results should also be performed.
SAM 2012 202 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
PCR QC checks should be performed according to EPA's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
Chiang, Y.C, Fan, C.M., Liao, W.W., Lin, C.K. and Tsen, H.Y. 2007. "Real-Time PCR Detection
of Staphylococcus aureus in Milk and Meat Using New Primers Designed From the Heat Shock
Protein Gene htrA Sequence." Journal of Food Protection. 70(12): 2855-2859.
http://www.ingentaconnect.com/content/iafb/ifb/2007/00000070/00000012/art00023
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
Remediation Phase
Site Characterization
Post Decontamination
Analytical Technique
Real-Time PCR
Culture and Real-Time PCR
Section
7.2.15.1
7.2.15.2
7.2.15.1 Site Characterization Sample Analyses (Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
SAM 2012 203 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Standard Analytical
Protocol for Vibrio cholerae Ol and O139 in Drinking Water and Surface Water" (U.S. EPA
2010).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction by bead beating and purification for all sample types should be performed
according to procedures within the EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use real-time PCR (Literature reference for Vibrio cholerae [(Journal of
Microbiological Methods, 2007, 68(2): 254-259]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), the target nucleic acid should be extracted, purified (EPA BA
Protocol, Section 9.2), and analyzed using the referenced target-specific PCR primers and probes
and assay parameters (Journal of Microbiological Methods, 2007, 68(2): 254-259). The use of
real-time PCR analyses directly on samples (e.g., no culture component) allows for rapid
detection of Vibrio cholerae.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. 2010. Standard Analytical Protocol for Vibrio cholerae Ol and O139 in Drinking
Water and Surface Water.
SAM 2012 204 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
Blackstone, G.M., Nordstrom, J.L., Bowen, M.D., Meyer, R.F., Imbro, P. and DePaola, A. 2007.
"Use of a Real Time PCR Assay for Detection of the ctxA Gene of Vibrio cholerae in an
Environmental Survey of Mobile Bay." Journal of Microbiological Methods, 68(2): 254-259.
http://www.sciencedirect.com/science/article/pii/S016770120600248X
7.2.15.2 Post Decontamination Sample Analyses (Culture and Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Standard Analytical
Protocol for Vibrio cholerae Ol and O139 in Drinking Water and Surface Water" (U.S. EPA
2010).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction and purification should be performed according to procedures within the
EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use culture (Standard Analytical Protocol for Vibrio cholerae Ol and
O139 in Drinking Water and Surface Water, U.S. EPA, 2010) and real-time PCR (Literature
reference for Vibrio cholerae [(Journal of Microbiological Methods, 2007, 68(2): 254-259]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), samples are inoculated into enrichment broth, incubated for 8
hours, and then plated onto selective media. Confirmation is performed using real-time PCR.
Target nucleic acid should be extracted, purified (EPA BA Protocol, Section 9.2), and analyzed
using the referenced target-specific PCR primers and probes and assay parameters (Journal of
Microbiological Methods, 2007, 68(2): 254-259). The use of real-time PCR analyses directly on
isolates (e.g., no biochemical/serological component) allows for rapid confirmation of Vibrio
cholerae.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
SAM2012 205 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
04-001) document at: http://www.epa.gov/sam/EPA-OAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http: //oaspub .epa. gov/eims/eimscomm. getfile ?p_download_id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
U.S. EPA. October 2010. "Standard Analytical Protocol for Vibrio cholerae Ol and O139 in
Drinking Water and Surface Water." EPA 600/R-10/139.
http://nepis.epa.gov/Adobe/PDF/P100978K.pdf
Blackstone, G.M., Nordstrom, J.L., Bowen, M.D., Meyer, R.F., Imbro, P. and DePaola, A. 2007.
"Use of a Real Time PCR Assay for Detection of the ctxA Gene of Vibrio cholerae in an
Environmental Survey of Mobile Bay." Journal of Microbiological Methods 68(2): 254-259.
http://www.sciencedirect.com/science/article/pii/S016770120600248X
7.2.16 Yersinia pestis [Plague] - BSL-3
Remediation Phase
Site Characterization
Post Decontamination
Analytical Technique
Real-Time PCR
Real-Time PCR/lmmunoassay
Culture and Real-Time PCR
Section
7.2.16.1
7.1.41
7.2.16.2
1 Standardized procedures, reagents and agent-specific algorithms are available to LRN member
laboratories.
7.2.16.1 Site Characterization Sample Analyses (Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
SAM 2012 206 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose et al. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction by bead beating and purification for all sample types should be performed
according to procedures within the EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use real-time PCR (Literature reference for Yersiniapestis [Diagnostic
Microbiology and Infectious Disease, 2006, 56(3): 261-268]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), the target nucleic acid should be extracted, purified (EPA BA
Protocol, Section 9.2), and analyzed using the referenced target-specific PCR primers and probes
and assay parameters (Diagnostic Microbiology and Infectious Disease, 2006, 56(3): 261-268).
The use of real-time PCR analyses directly on samples (e.g., no culture component) allows for
rapid detection of Yersinia pestis.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control (purified nucleic acid), negative control, external inhibition 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's Quality
Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on
Environmental Samples (EPA 815-B-04-001) document at: http://www.epa.gov/sam/EPA-
QAQC-PCR.pdf. or consult the points of contact identified in Section 4.
Special Considerations: Yersinia pestis 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 BMBL, 5th Edition (CDC 2009)].
http://www.cdc.gov/biosafety/publications/bmbl5/
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub.epa.gov/eims/eimscomm.getfile7p download id=503892
SAM 2012 207 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
Woron, A.M., Nazarian, E.J., Egan, C., McDonough, K.A., Cirino, N.M., Limberger, R.J. and
Musser, K.A. 2006. "Development and Evaluation of a 4-Target Multiplex Real-Time
Polymerase Chain Reaction Assay for the Detection and Characterization of Yersinia pestis''
Diagnostic Microbiology and Infectious Disease, 56(3): 261-268.
http://www.dmidiournal.com/article/S0732-8893(06)00232-X/fulltext
7.2.16.2 Post Decontamination Sample Analyses (Culture and Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction and purification should be performed according to procedures within the
EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use culture ("Sentinel Level Clinical Microbiology Laboratory
Guidelines for Suspected Agents of Bioterrorism and Emerging Infectious Diseases: Yersinia
pestis," ASM, 2010) and real-time PCR (Literature reference for Yersinia pestis [Diagnostic
Microbiology and Infectious Disease, 2006, 56(3): 261-268]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), samples can be inoculated into enrichment broth prior to plating
or plated directly on non-selective media and incubated for a minimum of three days.
Confirmation is performed using real-time PCR. Target nucleic acid should be extracted, purified
(EPA BA Protocol, Section 9.2), and analyzed using the referenced target-specific PCR primers
and probes and assay parameters (Diagnostic Microbiology and Infectious Disease, 2006, 56(3):
261-268). The use of real-time PCR analyses directly on isolates (e.g., no
biochemical/serological component) allows for rapid confirmation of Yersinia pestis.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
SAM2012 208 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
04-001) document at: http://www.epa.gov/sam/EPA-OAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Special Considerations: Yersiniapestis 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 BMBL, 5th Edition (CDC 2009)].
http://www.cdc.gov/biosafety/publications/bmbl5/
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
ASM. 2010. "Sentinel Level Clinical Microbiology Laboratory Guidelines for Suspected Agents
of Bioterrorism and Emerging Infectious Diseases: Yersinia pestis "
http://www.asm.org/images/pdf/Clinical/Protocols/ypestis06-ll-10.pdf
Woron, A.M., Nazarian, E.J., Egan, C., McDonough, K.A., Cirino, N.M., Limberger, R.J. and
Musser, K.A. 2006. "Development and Evaluation of a 4-Target Multiplex Real-Time
Polymerase Chain Reaction Assay for the Detection and Characterization of Yersinia pestis"
Diagnostic Microbiology and Infectious Disease, 56(3): 261-268.
http://www.dmidioumal.com/article/S0732-8893(06)00232-X/fulltext
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.
7.3.1 Adenoviruses: Enteric and Non-enteric (A-F) - BSL-2
Remediation Phase
Site Characterization
Post Decontamination
Analytical Technique
Real-Time PCR
Tissue Culture and Real-Time PCR
Section
7.3.1.1
7.3.1.2
SAM 2012 209 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
7.3.1.1 Site Characterization Sample Analyses (Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Method 1615: Enterovirus
and Norovirus Occurrence in Water by Culture and RT-qPCR" (U.S. EPA 2012). Note. Since the
concentration of adenovirus using the Nanoceramฎ filter has not been optimized, use of the
1MDS filter is recommended.
All sample types other than water should be processed as follows: (A) For virus recovery from
samples, follow the EPA BA Protocol (Section 11: sample processing and elution); and (B) for
virus concentration following recovery, follow relevant steps from Section 11.2.3 of Method
1615 (U.S. EPA 2012).
Nucleic acid extraction by bead beating and purification for all sample types should be performed
according to procedures within the EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use real-time PCR (Literature reference for adenoviruses [Applied and
Environmental Microbiology, 2005, 71(6): 3131-3136]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), the target nucleic acid should be extracted, purified (EPA BA
Protocol, Section 9.2), and analyzed using the referenced target-specific real-time PCR primers
and probes and assay parameters (Applied and Environmental Microbiology, 2005, 71(6): 3131-
3136). The use of real-time PCR analyses directly on samples (e.g., no tissue culture component)
allows for rapid detection of adenoviruses.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
SAM 2012 210 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. 2012. "Method 1615: Enterovirus andNorovirus Occurrence in Water by Culture and
RT-qPCR," EPA/600/R-10/181. http://www.epa.gov/nerlcwww/documents/Methodl615vl l.pdf
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
Jothikumar, N., Cromeans, T.L., Hill, V.R, Lu, X., Sobsey, M.D. and Erdman, D.D. 2005.
"Quantitative Real-Time PCR Assays for Detection of Human Adenoviruses and Identification of
Serotypes 40 and 41." Applied and Environmental Microbiology, 71(6): 3131-3136.
http://aem.asm.Org/content/71/6/313 l.full.pdf+html
7.3.1.2 Post Decontamination Sample Analyses (Tissue Culture and Real-Time
PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Method 1615: Enterovirus
and Norovirus Occurrence in Water by Culture and RT-qPCR" (U.S. EPA 2012). Note. Since the
concentration of adenovirus using the Nanoceramฎ filter has not been optimized, use of the
1MDS filter is recommended.
All sample types other than water should be processed as follows: (A) For virus recovery from
samples, follow the EPA BA Protocol (Section 11: sample processing and elution); and (B) for
virus concentration following recovery, follow relevant steps from Section 11.2.3 of Method
1615(U.S. EPA 2012).
Nucleic acid extraction and purification should be performed according to procedures within the
EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use tissue culture ["Method 1615: Enterovirus andNorovirus
Occurrence in Water by Culture and RT-qPCR" (U.S. EPA 2012)] and real-time PCR (Literature
reference for adenoviruses [Applied and Environmental Microbiology, 2005, 71(6): 3131-3136]).
SAM 2012 211 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), samples should be cultured to assess viability [Method 1615 (U.S.
EPA 2012)]. For confirmation, target nucleic acid should be extracted, purified (EPA BA
Protocol, Section 9.2), and analyzed using the referenced target-specific real-time PCR primers
and probes and assay parameters (Applied and Environmental Microbiology, 2005, 71(6): 3131-
3136).
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Special Considerations: For the viability assessment of adenoviruses 40 and 41, given that they
can be difficult to grow in culture, cell lines such as G293 or CaCo-2 may be considered when
these viruses are suspected to be present.
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http: //oaspub .epa. gov/eims/eimscomm. getfile ?p_download_id=5 03 892
U.S. EPA. 2012. "Method 1615: Enterovirus andNorovirus Occurrence in Water by Culture and
RT-qPCR," EPA/600/R-10/181. http ://www.epa.gov/nerlcwww/documents/Method 1615v 1 1 .pdf
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
Jothikumar, N., Cromeans, T.L., Hill, V.R., Lu, X., Sobsey, M.D. and Erdman, D.D. 2005.
"Quantitative Real-Time PCR Assays for Detection of Human Adenoviruses and Identification of
Serotypes 40 and 41." Applied and Environmental Microbiology, 71(6): 3131-3136.
http://aem.asm.Org/content/71/6/313 l.full.pdf+html
SAM 2012 212 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
7.3.2 Astroviruses - BSL not specified
Remediation Phase
Site Characterization
Post Decontamination
Analytical Technique
Real-Time Reverse Transcription-PCR
Integrated Cell Culture and Real-Time Reverse Transcription-PCR
Section
7.3.2.1
7.3.2.2
7.3.2.1 Site Characterization Sample Analyses (Real-Time Reverse
Transcription- PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Method 1615: Enterovirus
and Norovirus Occurrence in Water by Culture and RT-qPCR" (U.S. EPA 2012). Note. Since the
concentration of astrovirus using the Nanoceramฎ filter has not been optimized, use of the 1MDS
filter is recommended.
All sample types other than water should be processed as follows: (A) For virus recovery from
samples, follow the EPA BA Protocol (Section 11: sample processing and elution); and (B) for
virus concentration following recovery, follow relevant steps from Section 11.2.3 of Method
1615 (U.S. EPA 2012).
Nucleic acid extraction by bead beating and purification for all sample types should be performed
according to procedures within the EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use real-time reverse transcription-PCR (Literature reference for
astroviruses [Canadian Journal of Microbiology, 2004, 50(4): 269-278]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), the target nucleic acid should be extracted, purified (EPA BA
Protocol, Section 9.2), and analyzed using the referenced target-specific PCR primers and probes
and assay parameters (Canadian Journal of Microbiology, 2004, 50(4): 269-278). The use of
real-time reverse transcription-PCR analyses directly on samples (e.g., no culture component)
allows for rapid detection of astroviruses.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
SAM 2012 213 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
Special Considerations: Appropriate RNAse inhibitors should be included during sample
processing and analysis.
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http: //oaspub .epa. gov/eims/eimscomm. getfile ?p_download_id=5 03 892
U.S. EPA. 2012. "Method 1615: Enterovirus andNorovirus Occurrence in Water by Culture and
RT-qPCR," EPA/600/R-10/181. http://www.epa.gov/nerlcwww/documents/Methodl615vl_l.pdf
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
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://www.nrcresearchpress.com/doi/abs/10.1139/w04-012
7.3.2.2 Post Decontamination Sample Analyses (Integrated Cell Culture and
Real-Time Reverse Transcription-PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists these procedures for detection and viability assessment in
aerosols, surface wipes or swabs, drinking water and post decontamination waste water. Further
research is needed to develop comprehensive pathogen-specific procedures for different
environmental sample types included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Method 1615: Enterovirus
and Norovirus Occurrence in Water by Culture and RT-qPCR" (U.S. EPA 2012). Note. Since the
concentration of astrovirus using the Nanoceramฎ filter has not been optimized, use of the 1MDS
filter is recommended.
SAM 2012 214 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
All sample types other than water should be processed as follows: (A) For virus recovery from
samples, follow the EPA BA Protocol (Section 11: sample processing and elution); and (B) for
virus concentration following recovery, follow relevant steps from Section 11.2.3 of Method
1615 (U.S. EPA 2012).
Nucleic acid extraction and purification should be performed according to procedures within the
EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use integrated cell culture and real-time reverse transcription-PCR
(Literature Reference for Astroviruses [Canadian Journal of Microbiology, 2004, 50(4): 269-
278]).
Description of Method: The method is a real-time reverse transcription-PCR procedure that can
be integrated with cell culture (CaCo-2 cells) to enhance sensitivity. Following the appropriate
sample preparation procedure (see Sample Preparation Procedures above), concentrated samples
are analyzed directly or indirectly, after cell culture, by a two-step real-time reverse transcription-
PCR (i.e., reverse transcription followed by real-time PCR) assay using astrovirus-specific
primers and probes and assay parameters (Canadian Journal of Microbiology, 2004, 50(4): 269-
278).
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf, or consult the points of
contact identified in Section 4.
Special Considerations: Appropriate RNAse inhibitors should be included during sample
processing and analysis.
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods. 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology. 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub.epa.gov/eims/eimscomm.getfile ?p_download_id=503892
U.S. EPA. 2012. "Method 1615: Enterovirus and Norovirus Occurrence in Water by Culture and
RT-qPCR,"EPA/600/R-10/181.http://www.epa.gov/nerlcwww/documents/Methodl615vl_l.pdf
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
SAM 2012 215 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
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://www.nrcresearchpress.com/doi/abs/10.1139/w04-012
7.3.3 Caliciviruses: Noroviruses - BSL-2
Remediation Phase
Site Characterization
Post Decontamination
Analytical Technique
Real-Time Reverse Transcription-PCR
No method available to determine viable virus after
decontamination
Section
7.3.3.1
7.3.3.2
7.3.3.1 Site Characterization Sample Analyses (Real-Time Reverse
Transcription-PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for environmental sample types included in
SAM, other than water.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Method 1615: Enterovirus
and Norovirus Occurrence in Water by Culture and RT-qPCR" (U.S. EPA 2012).
All sample types other than water should be processed as follows: (A) For virus recovery from
samples, follow the EPA BA Protocol (Section 11: sample processing and elution); and (B) for
virus concentration following recovery, follow relevant steps from Section 11.2.3 of Method
1615 (U.S. EPA 2012).
Nucleic acid extraction by bead beating and purification for all sample types should be performed
according to procedures within the EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use real-time reverse transcription-PCR [EPA Method 1615 (U.S. EPA
2012)].
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), the target nucleic acid should be extracted, purified (EPA BA
Protocol, Section 9.2), and analyzed using the referenced target-specific PCR primers and probes
and assay parameters [EPA Method 1615 (U.S. EPA 2012)].
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
SAM2012 216 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
04-001) document at: http://www.epa.gov/sam/EPA-OAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Special Considerations: Appropriate RNAse inhibitors should be included during sample
processing and analysis.
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http: //oaspub .epa. gov/eims/eimscomm. getfile ?p_download_id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
U.S. EPA. 2012. "Method 1615: Enterovirus andNorovirus Occurrence in Water by Culture and
RT-qPCR," EPA/600/R-10/181. http ://www.epa.gov/nerlcwww/documents/Method 1615v 1 1 .pdf
7.3.3.2 Post Decontamination Sample Analyses
No method available to determine viable virus after decontamination.
7.3.4 Caliciviruses: Sapovirus - BSL-2
Remediation Phase
Site Characterization
Post Decontamination
Analytical Technique
Real-Time Reverse Transcription-PCR
Tissue Culture and Real-Time Reverse Transcription-PCR
Section
7.3.4.1
7.3.4.2
7.3.4.1 Site Characterization Sample Analyses (Real-Time Reverse
Transcription-PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
SAM 2012 217 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Method 1615: Enterovirus
and Norovirus Occurrence in Water by Culture and RT-qPCR" (U.S. EPA 2012).
All sample types other than water should be processed as follows: (A) For virus recovery from
samples, follow the EPA BA Protocol (Section 11: sample processing and elution); and (B) for
virus concentration following recovery, follow relevant steps from Section 11.2.3 of Method
1615 (U.S. EPA 2012).
Nucleic acid extraction by bead beating and purification for all sample types should be performed
according to procedures within the EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use real-time reverse transcription-PCR (Literature Reference for
sapoviruses [Journal of Medical Virology, 2006, 78(10): 1347-1353]).
Description of Method: The method is a TaqManฎ-based real-time reverse transcriptase PCR
assay that can detect four of the five distinct sapovirus genogroups (GI-GV) using a multiplex
assay. Following the appropriate sample preparation procedure (see Sample Preparation
Procedures above), the target nucleic acid should be extracted, purified (EPA BA Protocol,
Section 9.2), and analyzed using the referenced target-specific PCR primers and probes and assay
parameters (Journal of Medical Virology, 2006, 78(10): 1347-1353).
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Special Considerations: Appropriate RNAse inhibitors should be included during sample
processing and analysis.
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub.epa.gov/eims/eimscomm.getfile7p download id=503892
SAM 2012 218 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
U.S. EPA. 2012. "Method 1615: Enterovirus andNorovirus Occurrence in Water by Culture and
RT-qPCR," EPA/600/R-10/181. http://www.epa.gov/nerlcwww/documents/Methodl615vl_l.pdf
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
Oka, T., Katayama, K., Hansman, G.S., Kageyama, T., Ogawa, S., Wu, F.T., White, P.A. and
Takeda, N. 2006. "Detection of Human Sapovirus by Real-Time Reverse Transcription-
Polymerase Chain Reaction." Journal of Medical Virology, 78(10): 1347-1353.
http://cat.inist.fr/?aModele=afficheN&cpsidt=18099754
7.3.4.2 Post Decontamination Sample Analyses (Tissue Culture and Real-Time
Reverse Transcription-PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for environmental sample types included in
SAM, other than water.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Method 1615: Enterovirus
and Norovirus Occurrence in Water by Culture and RT-qPCR" (U.S. EPA 2012).
All sample types other than water should be processed as follows: (A) For virus recovery from
samples, follow the EPA BA Protocol (Section 11: sample processing and elution); and (B) for
virus concentration following recovery, follow relevant steps from Section 11.2.3 of Method
1615 (U.S. EPA 2012).
Nucleic acid extraction and purification should be performed according to procedures within the
EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use tissue culture (Literature reference for Sapovirus [Archives of
Virology, 1991, 120(1-2): 115-122]) and real-time reverse transcription-PCR (Literature
Reference for sapoviruses [Journal of Medical Virology, 2006, 78(10): 1347-1353]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), samples should be cultured using LL-PK cells supplemented with
intestinal contents preparation (ICP) to assess viability (Archives of Virology, 1991, 120(1-2):
115-122). For confirmation target nucleic acid should be extracted, purified (EPA BA Protocol,
Section 9.2), and analyzed using the referenced target-specific real-time PCR primers and probes
and assay parameters (Journal of Medical Virology, 2006, 78(10): 1347-1353).
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition control and blank. Ongoing
analysis of QC samples to ensure reliability of the analytical results should also be performed.
SAM 2012 219 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
PCR QC checks should be performed according to EPA's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Special Considerations: Appropriate RNAse inhibitors should be included during sample
processing and analysis. Culture procedure is for porcine sapovirus and may not be appropriate
for all strains of sapoviruses.
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http: //oaspub .epa. gov/eims/eimscomm. getfile ?p_download_id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
U.S. EPA. 2012. "Method 1615: Enterovirus andNorovirus Occurrence in Water by Culture and
RT-qPCR," EPA/600/R-10/181. http://www.epa.gov/nerlcwww/documents/Methodl615vl_l.pdf
Parwani, A.V., Flynn, W.T., Gadfield, K.L and Saif L.J. 1991. "Serial Propagation of Porcine
Enteric Calicivirus in a Continuous Cell Line. Effect of Medium Supplementation With Intestinal
Contents or Enzymes." Archives of Virology, 120(1-2): 115-122.
http://www.springerlink.com/content/u3v0041507k032hl/
Oka, T., Katayama, K., Hansman, G.S., Kageyama, T., Ogawa, S., Wu, F.T., White, P.A. and
Takeda, N. 2006. "Detection of Human Sapovirus by Real-Time Reverse Transcription-
Polymerase Chain Reaction." Journal of Medical Virology, 78(10): 1347-1353.
http://cat.inist.fr/?aModele=afficheN&cpsidt=18099754
7.3.5 Coronaviruses: Severe Acute Respiratory Syndrome (SARS) -associated Human
Coronavirus (HCoV) - BSL-2; BSL-3 for Propagation
Remediation Phase
Site Characterization
Post Decontamination
Analytical Technique
Reverse Transcription-PCR
Tissue Culture and Reverse Transcription-PCR
Section
7.3.5.1
7.3.5.2
SAM 2012 220 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
7.3.5.1 Site Characterization Sample Analyses (Reverse Transcription-PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011).
All sample types other than water should be processed as follows: (A) For virus recovery from
samples, follow the EPA BA Protocol (Section 11: sample processing and elution); and (B) for
virus concentration following recovery, follow relevant steps from Section 11.2.3 of Method
1615 (U.S. EPA 2012).
Nucleic acid extraction by bead beating and purification for all sample types should be performed
according to procedures within the EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use reverse transcription-PCR (Literature Reference for Coronavirus:
SARS [Journal of Virological Methods, 2004, 122(1): 29-36]).
Description of Method: This method uses a conventional single-tube reverse transcription-PCR
procedure using the Stratagene Robocyclerฎ. 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). Following the appropriate
sample preparation procedure (see Sample Preparation Procedures above), the target nucleic acid
should be extracted, purified (EPA BA Protocol, Section 9.2), and analyzed using the referenced
target-specific PCR primers and probes and assay parameters (Journal of Virological Methods,
2004, 122(1): 29-36).
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Special Considerations: Appropriate RNAse inhibitors should be included during sample
processing and analysis.
SAM 2012 221 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
U.S. EPA. 2012. "Method 1615: Enterovirus andNorovirus Occurrence in Water by Culture and
RT-qPCR," EPA/600/R-10/181. http ://www.epa.gov/nerlcwww/documents/Method 1615v 1 1 .pdf
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(1):
29-36. http://www.sciencedirect.com/science/article/pii/S0166093404002162
7.3.5.2 Post Decontamination Sample Analyses (Tissue Culture and Reverse
Transcription-PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011).
All sample types other than water should be processed as follows: (A) For virus recovery from
samples, follow the EPA BA Protocol (Section 11: sample processing and elution); and (B) for
virus concentration following recovery, follow relevant steps from Section 11.2.3 of Method
1615 (U.S. EPA 2012).
Nucleic acid extraction and purification should be performed according to procedures within the
EPA BA Protocol (Protocol Section 9.2).
SAM 2012 222 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
Analytical Technique: Use tissue culture (Literature reference for Coronavirus: SARS [Applied
Biosafety, 2007, 12(2): 100-108]) and reverse transcription-PCR (Literature Reference for
Coronavirus: SARS [Journal of Virological Methods, 2004, 122(1): 29-36]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), samples are inoculated onto Vero cell monolayers; the cells are
examined for cytopathic effects (CPE) to assess viability (Applied Biosafety, 2007, 12(2): 100-
108). For confirmation, target nucleic acid should be extracted, purified (EPA BA Protocol,
Section 9.2), and analyzed using the referenced target-specific PCR primers and probes and assay
parameters (Journal of Virological Methods, 2004, 122(1): 29-36).
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Special Considerations: Appropriate RNAse inhibitors should be included during sample
processing and analysis.
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. 2012. "Method 1615: Enterovirus andNorovirus Occurrence in Water by Culture and
RT-qPCR," EPA/600/R-10/181. http://www.epa.gov/nerlcwww/documents/Methodl615vl_l.pdf
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
Pagat, A., Seux-Goepfert, R., Lutsch, C., Lecouturier, V., Saluzzo, J. and Kusters, I.C. 2007.
"Evaluation of SARS-Coronavirus Decontamination Procedures." Applied Biosafety 12(2): 100-
108. http://www.absa.org/abi/abi/ABJ2007vl2n2.pdf
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(1):
29-36. http://www.sciencedirect.com/science/article/pii/S0166093404002162
SAM 2012 223 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
7.3.6 Hepatitis E Virus (HEV) - BSL-2
Remediation Phase
Site Characterization
Post Decontamination
Analytical Technique
Real-Time Reverse Transcription-PCR
Tissue Culture and Real-Time Transcription-PCR
Section
7.3.6.1
7.3.6.2
7.3.6.1 Site Characterization Sample Analyses (Real-Time Reverse
Transcription-PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for environmental sample types included in
SAM, other than water.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Method 1615: Enterovirus
and Norovirus Occurrence in Water by Culture and RT-qPCR" (U.S. EPA 2012). Note. Since the
concentration of hepatitis E virus using the Nanoceramฎ filter has not been evaluated, use of the
1MDS filter is recommended.
All sample types other than water should be processed as follows: (A) For virus recovery from
samples, follow the EPA BA Protocol (Section 11: sample processing and elution); and (B) for
virus concentration following recovery, follow relevant steps from Section 11.2.3 of Method
1615 (U.S. EPA 2012).
Nucleic acid extraction by bead beating and purification for all sample types should be performed
according to procedures within the EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use real-time reverse transcription-PCR (Literature Reference for
Hepatitis E Virus [Journal of Virological Methods, 2006, 131(1): 65-71]).
Description of Method: The method uses a TaqManฎ real-time reverse transcription-PCR assay
using the R.A.P.I.D.ฎ PCR systems to detect and quantitate all four major HEV genotypes.
Following the appropriate sample preparation procedure (see Sample Preparation Procedures
above), the target nucleic acid should be extracted, purified (EPA BA Protocol, Section 9.2), and
analyzed using the referenced target-specific PCR primers and probes and assay parameters
(Journal of Virological Methods, 2006, 131(1): 65-71).
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
SAM 2012 224 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
Special Considerations: Appropriate RNAse inhibitors should be included during sample
processing and analysis.
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http: //oaspub .epa. gov/eims/eimscomm. getfile ?p_download_id=5 03 892
U.S. EPA. 2012. "Method 1615: Enterovirus andNorovirus Occurrence in Water by Culture and
RT-qPCR," EPA/600/R-10/181. http://www.epa.gov/nerlcwww/documents/Methodl615vl_l.pdf
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
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= 173 673 5 7
7.3.6.2 Post Decontamination Sample Analyses (Tissue Culture and Real-Time
Reverse Transcription-PCR)
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges et al. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose et al. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Method 1615: Enterovirus
and Norovirus Occurrence in Water by Culture and RT-qPCR" (U.S. EPA 2012). Note: Since the
concentration of hepatitis E virus using the Nanoceramฎ filter has not been evaluated, use of the
1MDS filter is recommended.
All sample types other than water should be processed as follows: (A) For virus recovery from
samples, follow the EPA BA Protocol (Section 11: sample processing and elution); and (B) for
SAM 2012 225 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
virus concentration following recovery, follow relevant steps from Section 11.2.3 of Method
1615 (U.S. EPA 2012).
Nucleic acid extraction and purification should be performed according to procedures within the
EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use tissue culture (Literature reference for Hepatitis E Virus [FEMS
Immunology Medical Microbiology, 2009, 56(1): 73-79]) and real-time reverse transcription-
PCR (Literature Reference for Hepatitis E Virus [Journal of Virological Methods, 2006, 131(1):
65-71]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), samples are inoculated onto HPG11 cells; the cells are examined
for CPEs to assess viability [Federation of European Microbiological Societies (FEMS)
Immunology Medical Microbiology, 2009, 56(1): 73-79]. For confirmation target nucleic acid
should be extracted, purified (EPA BA Protocol, Section 9.2), and analyzed using the referenced
target-specific PCR primers and probes and assay parameters (Journal of Virological Methods,
2006, 131(1): 65-71).
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Special Considerations: Appropriate RNAse inhibitors should be included during sample
processing and analysis.
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http: //oaspub .epa. gov/eims/eimscomm. getfile ?p_download_id=5 03 892
U.S. EPA. 2012. "Method 1615: Enterovirus andNorovirus Occurrence in Water by Culture and
RT-qPCR," EPA/600/R-10/181. http://www.epa.gov/nerlcwww/documents/Methodl615vl_l.pdf
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
SAM 2012 226 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
Zaki, M., Foud, M.F. and Mohamed, A. F. 2009. "Value of Hepatitis E Virus Detection by Cell
Culture Compared With Nested PCR and Serological Studies by IgM and IgG." FEMS
Immunology Medical Microbiology, 56(1): 73-79.
http://onlinelibrary.wilev.eom/doi/10.llll/i.1574-695X.2009.00552.x/pdf
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= 173 673 5 7
7.3.7 Influenza H5N1 virus - BSL-3
Remediation Phase
Site Characterization
Post Decontamination
Analytical Technique
Real-Time Reverse Transcription-PCR
Isolation of H5N1 virus should not be performed
except at the Influenza Division, CDC.
Section
7.3.7.1
7.3.7.2
7.3.7.1 Site Characterization Sample Analyses (Real-Time Reverse
Transcription-PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011).
All sample types other than water should be processed as follows: (A) For virus recovery from
samples, follow the EPA BA Protocol (Section 11: sample processing and elution); and (B) for
virus concentration following recovery, follow relevant steps from Section 11.2.3 of Method
1615 (U.S. EPA 2012).
Nucleic acid extraction by bead beating and purification for all sample types should be performed
according to procedures within the EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use real-time reverse transcription-PCR (Literature Reference for
Influenza H5N1 [Emerging Infectious Diseases, 2005, 11(8): 1303-1305]).
Description of Method: This is a two-step, real-time reverse transcriptase-PCR multiplex assay.
The assay is specific for the H5 subtype. Note. Influenza H5N1 virus samples are to be handled
with BSL-3 containment and practices. Following the appropriate sample preparation procedure
(see Sample Preparation Procedures above), the target nucleic acid should be extracted, purified
(EPA BA Protocol, Section 9.2), and analyzed using the referenced target-specific PCR primers
and probes and assay parameters (Emerging Infectious Diseases, 2005, 11(8): 1303-1305).
SAM 2012 221 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Special Considerations: Appropriate RNAse inhibitors should be included during sample
processing and analysis.
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
U.S. EPA. 2012. "Method 1615: Enterovirus andNorovirus Occurrence in Water by Culture and
RT-qPCR," EPA/600/R-10/181. http://www.epa.gov/nerlcwww/documents/Methodl615vl l.pdf
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.ncbi.nlm.nih.gov/pubmed/16102326
ASM. 2008. "Sentinel Level Clinical Microbiology Laboratory Guidelines for Suspected Agents
of Bioterrorism and Emerging Infectious Diseases: Avian Influenza A H5N1."
http: //www. asm. org/images/pdf/Clinical/Protocols/avianiinfluenza 11 -200 8 .pdf
7.3.7.2 Post Decontamination Sample Analyses
Isolation ofH5Nl virus should not be performed except at the Influenza Division, CDC.
7.3.8 Picornaviruses: Enteroviruses - BSL-2
Remediation Phase
Site Characterization
Post Decontamination
Analytical Technique
Real-Time Reverse Transcription-PCR
Tissue Culture
Section
7.3.8.1
7.3.8.2
SAM 2012 228 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
7.3.8.1 Site Characterization Sample Analyses (Real-Time Reverse
Transcription-PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for environmental sample types included in
SAM, other than water.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Method 1615: Enterovirus
and Norovirus Occurrence in Water by Culture and RT-qPCR" (U.S. EPA 2012).
All sample types other than water should be processed as follows: (A) For virus recovery from
samples, follow the EPA BA Protocol (Section 11: sample processing and elution); and (B) for
virus concentration following recovery, follow relevant steps from Section 11.2.3 of Method
1615 (U.S. EPA 2012).
Analytical Technique: Use real-time reverse transcription-PCR ["Method 1615: Enterovirus and
Norovirus Occurrence in Water by Culture and RT-qPCR" (U.S. EPA 2012)].
Description of Method: The method uses a TaqManฎ real-time reverse transcriptase-PCR assay
to detect and quantify enteroviruses. Following the appropriate sample preparation procedure
(see Sample Preparation Procedures above), the target nucleic acid should be extracted, purified,
and analyzed using the referenced target-specific PCR primers and probes and assay parameters
(Method 1615, U.S. EPA 2012).
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Special Considerations: Appropriate RNAse inhibitors should be included during sample
processing and analysis.
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
SAM 2012 229 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
U.S. EPA. 2012. "Method 1615: Enterovirus andNorovirus Occurrence in Water by Culture and
RT-qPCR," EPA/600/R-10/181. http://www.epa.gov/nerlcwww/documents/Method 1615v 1 1 .pdf
7.3.8.2 Post Decontamination Sample Analyses (Tissue Culture)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for environmental sample types included in
SAM, other than water.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Method 1615: Enterovirus
and Norovirus Occurrence in Water by Culture and RT-qPCR" (U.S. EPA 2012).
All sample types other than water should be processed as follows: (A) For virus recovery from
samples, follow the EPA BA Protocol (Section 11: sample processing and elution); and (B) for
virus concentration following recovery, follow relevant steps from Section 11.2.3 of Method
1615 (U.S. EPA 2012).
Analytical Technique: Use tissue culture with serum neutralization ("Method 1615: Enterovirus
and Norovirus Occurrence in Water by Culture and RT-qPCR," U.S. EPA 2012).
Description of Method: This method describes procedures for determining infectivity and
quantifying enteroviruses using BGMK cells. Following the appropriate sample preparation
procedure (see Sample Preparation Procedures above), aliquots of the sample are used to
inoculate BGMK cells. Cell culture flasks are examined for evidence of CPE for a total of 14
days [Method 1615 (U.S. EPA 2012)].
At a minimum, the following QC checks should be performed and evaluated: positive control,
negative control and blank. Ongoing analysis of QC samples to ensure reliability of the analytical
results should also be performed.
SAM2012 230 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
U.S. EPA. 2012. "Method 1615: Enterovirus andNorovirus Occurrence in Water by Culture and
RT-qPCR," EPA/600/R-10/181. http ://www.epa.gov/nerlcwww/documents/Method 1615v 1 1 .pdf
7.3.9 Picornaviruses: Hepatitis A Virus (HAV) - BSL-2
Remediation Phase
Site Characterization
Post Decontamination
Analytical Technique
Real-Time Reverse Transcription-PCR
Integrated Cell Culture and Real-Time Reverse Transcription-PCR
Section
7.3.9.1
7.3.9.2
7.3.9.1 Site Characterization Sample Analyses (Real-Time Reverse
Transcription- PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Method 1615: Enterovirus
and Norovirus Occurrence in Water by Culture and RT-qPCR" (U.S. EPA 2012).
All sample types other than water should be processed as follows: (A) For virus recovery from
samples, follow the EPA BA Protocol (Section 11: sample processing and elution); and (B) for
SAM 2012 231 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
virus concentration following recovery, follow relevant steps from Section 11.2.3 of Method
1615 (U.S. EPA 2012).
Nucleic acid extraction by bead beating and purification for all sample types should be performed
according to procedures within the EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use real-time reverse transcription-PCR (Literature Reference for
Enteric Viruses [Journal of Food Protection, 2011, 74(10): 1756-1761]).
Description of Method: The method is a multiplex real-time reverse transcription-PCR
procedure optimized for the simultaneous detection of enteroviruses, HAV, reoviruses and
rotaviruses. Following the appropriate sample preparation procedure (see Sample Preparation
Procedures above), the target nucleic acid should be extracted, purified (EPA BA Protocol
Section 9.2), and analyzed using the referenced target-specific PCR primers and probes and assay
parameters (Journal of Food Protection, 2011, 74(10): 1756-1761).
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf or consult the points of
contact identified in Section 4.
Special Considerations: Appropriate RNAse inhibitors should be included during sample
processing and analysis.
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http: //oaspub .epa. gov/eims/eimscomm. getfile ?p_download_id=5 03 892
U.S. EPA. 2012. "Method 1615: Enterovirus andNorovirus Occurrence in Water by Culture and
RT-qPCR," EPA/600/R-10/181. http://www.epa.gov/nerlcwww/documents/Methodl615vl_l.pdf
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
Hyeon, J. Y, Chon, J.Y, Park, C, Lee, J.B., Choi, I.S., Kim, M.S. and Seo, K.H. 2011. "Rapid
Detection Method for Hepatitis A Virus from Lettuce by a Combination of Filtration and
SAM2012 232 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
Integrated Cell Culture-Real-Time Reverse Transcription PCR," Journal of Food Protection,
74(10): 1756-1761. http://www.ncbi.nlm.nih.gov/pubmed/22004827
7.3.9.2 Post Decontamination Sample Analyses (Integrated Cell Culture and
Real-Time Reverse Transcription-PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for environmental sample types included in
SAM, other than water.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose et al. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Method 1615: Enterovirus
and Norovirus Occurrence in Water by Culture and RT-qPCR" (U.S. EPA 2012).
All sample types other than water should be processed as follows: (A) For virus recovery from
samples, follow the EPA BA Protocol (Section 11: sample processing and elution); and (B) for
virus concentration following recovery, follow relevant steps from Section 11.2.3 of Method
1615 (U.S. EPA 2012).
Nucleic acid extraction and purification should be performed according to procedures within the
EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use integrated cell culture and real-time reverse transcription-PCR
(Literature Reference for Hepatitis A Virus [Journal of Food Protection, 2011, 74(10): 1756-
1761]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), samples are inoculated onto fetal rhesus monkey kidney (FRhK-
4) cells, and the cells are examined for CPE to assess viability. For confirmation, target nucleic
acid should be extracted, purified (EPA BA Protocol, Section 9.2), and analyzed using the
referenced target-specific PCR primers and probes and assay parameters (Journal of Food
Protection, 2011, 74(10): 1756-1761).
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-OAOC-PCR.pdf. or consult the points of
contact identified in Section 4.
Special Considerations: Appropriate RNAse inhibitors should be included during sample
processing and analysis.
SAM2012 233 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. 2012. "Method 1615: Enterovirus andNorovirus Occurrence in Water by Culture and
RT-qPCR," EPA/600/R-10/181. http://www.epa.gov/nerlcwww/documents/Methodl615vl l.pdf
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
Hyeon, J. Y, Chon, J.Y, Park, C, Lee, J.B., Choi, I.S., Kim, M.S. and Seo, K.H. 2011. "Rapid
Detection Method For Hepatitis A Virus From Lettuce by a Combination of Filtration and
Integrated Cell Culture-Real-Time Reverse Transcription PCR." Journal of Food Protection,
74(10): 1756-1761. http://www.ncbi.nlm.nih.gov/pubmed/22004827
7.3.10 Reoviruses: Rotavirus (Group A) - BSL-2
Remediation Phase
Site Characterization
Post Decontamination
Analytical Technique
Real-Time Reverse Transcription-PCR
Tissue Culture and Real-Time Reverse Transcription-PCR
Section
7.3.10.1
7.3.10.2
7.3.10.1 Site Characterization Sample Analyses (Real-Time Reverse
Transcription-PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Method 1615: Enterovirus
SAM2012 234 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
and Norovirus Occurrence in Water by Culture and RT-qPCR," U.S. EPA, 2012. Note: Since the
concentration of reoviruses using the Nanoceramฎ filter has not been optimized, use of the 1MDS
filter is recommended.
All sample types other than water should be processed as follows: (A) For virus recovery from
samples, follow the EPA BA Protocol (Section 11: sample processing and elution); and (B) for
virus concentration following recovery, follow relevant steps from Section 11.2.3 of Method
1615 (U.S. EPA 2012).
Nucleic acid extraction by bead beating and purification for all sample types should be performed
according to procedures within the EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use real-time reverse transcription-PCR (Literature Reference for
Rotavirus (Group A) [Journal of Virological Methods, 2009, 155(2): 126-131]).
Description of Method: The method is used to detect rotavirus using a one-step real-time
reverse-transcription PCR. Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), the target nucleic acid should be extracted, purified (EPA BA
Protocol, Section 9.2), and analyzed using the referenced target-specific PCR primers and probes
and assay parameters (Journal of Virological Methods, 2009, 155(2): 126-131).
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Special Considerations: Appropriate RNAse inhibitors should be included during sample
processing and analysis.
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. 2012. "Method 1615: Enterovirus and Norovirus Occurrence in Water by Culture and
RT-qPCR," EPA/600/R-10/181. http ://www.epa.gov/nerlcwww/documents/Method 1615v 1 1 .pdf
SAM2012 235 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
Jothikumar, N., Kang, G. and V.R. Hill. 2009. "Broadly Reactive TaqManฎ 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/article/pii/S0166093408003571
7.3.10.2 Post Decontamination Sample Analyses (Tissue Culture and Real-Time
Reverse Transcription-PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for environmental sample types included in
SAM, other than water.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Method 1615: Enterovirus
and Norovirus Occurrence in Water by Culture and RT-qPCR" (U.S. EPA 2012). Note. Since the
concentration of reoviruses using the Nanoceramฎ filter has not been optimized, use of the 1MDS
filter is recommended.
All sample types other than water should be processed as follows: (A) For virus recovery from
samples, follow the EPA BA Protocol (Section 11: sample processing and elution); and (B) for
virus concentration following recovery, follow relevant steps from Section 11.2.3 of Method
1615 (U.S. EPA 2012).
Nucleic acid extraction and purification should be performed according to procedures within the
EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use tissue culture [Method 1615: Enterovirus and Norovirus Occurrence
in Water by Culture and RT-qPCR (U.S. EPA 2012)] and real-time reverse transcription-PCR
(Literature Reference for Enteric Viruses (Journal of Virological Methods, 2009, 155(2): 126-
131).
Description of Method: This method describes procedures for determining infectivity and
quantifying enteroviruses using BGMK cells. Following the appropriate sample preparation
procedure (see Sample Preparation Procedures above), aliquots of the sample are used to
inoculate BGMK cells. Cell culture flasks are examined for evidence of CPE for a total of 14
days [Method 1615 (U.S. EPA 2012)]. For confirmation, target nucleic acid should be extracted,
purified (EPA BA Protocol, Section 9.2), and analyzed using the referenced target-specific PCR
primers and probes and assay parameters (Journal of Virological Methods, 2009, 155(2): 126-
131).
SAM2012 236 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Special Considerations: Appropriate RNAse inhibitors should be included during sample
processing and analysis.
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. 2012. "Method 1615: Enterovirus andNorovirus Occurrence in Water by Culture and
RT-qPCR," EPA/600/R-10/181. http ://www.epa.gov/nerlcwww/documents/Method 1615v 1 1 .pdf
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
Jothikumar, N., Kang, G. and V.R. Hill. 2009. "Broadly Reactive TaqManฎ 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/article/pii/S0166093408003571
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.
7.4.1 Cryptosporidium spp. [Cryptosporidiosis] - BSL-2
Remediation Phase
Site Characterization
Analytical Technique
Real-Time PCR
IMS/ Fluorescence assay
(FA)
IMS/FA
Section
7.4.1.1
7.4.1.2 1
7.4.1. 3 1
SAM 2012
237
July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
Remediation Phase
Post Decontamination
Analytical Technique
Cell Culture
Immunofluorescence (IFA)
Section
7.4.1.4
1 Methods 1622 and 1623 include the same sample processing and analytical procedures for
Cryptosporidiunr, either method could be used.
7.4.1.1 Site Characterization Sample Analyses (Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Real-Time PCR for
Quantification ofGiardia and Cryptosporidium in Environmental Water Samples and Sewage,"
(Guy et al. 2003) and "Development of Procedures for Direct Extraction of Cryptosporidium
DNA from Water Concentrates and for Relief of PCR Inhibitors" (Jiang et al. 2005).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Analytical Technique: Use real-time PCR (Literature References for Cryptosporidium spp.
[Applied and Environmental Microbiology, 2003, 69(9): 5178-5185 and Applied and
Environmental Microbiology, 2005, 71(3): 1135-1141]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), the target nucleic acid should be extracted, purified, and analyzed
using the referenced target-specific real-time PCR primers and probes and assay parameters
(Applied and Environmental Microbiology, 2003, 69(9): 5178-5185 and Applied and
Environmental Microbiology, 2005, 71(3): 1135-1141). The use of real-time PCR analyses
directly on samples (e.g., no culture component) allows for rapid detection of Cryptosporidium
spp.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
SAM2012 238 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
Guy, R.A., Payment, P., Krull, U.J. and Horgen, P.A. 2003. "Real-Time PCR for Quantification
ofGiardia and Cryptosporidium in Environmental Water Samples and Sewage." Applied and
Environmental Microbiology, 69(9): 5178-5185.
http://aem.asm.Org/content/69/9/5178.full.pdf+html
Jiang, J., Alderisio, K.A., Singh, A. and Xiao, L. 2005. "Development of Procedures for Direct
Extraction of Cryptosporidium DNA from Water Concentrates and for Relief of PCR Inhibitors."
Applied and Environmental Microbiology. 71(3): 1135-1141.
http://aem.asm.Org/content/71/3/1135.full.pdf+html
7.4.1.2 Site Characterization Sample Analyses (Immunomagnetic
Separation/Fluorescence Assay [IMS/FA])
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for detection in aerosols, surface wipes or swabs,
drinking water and post decontamination waste water. Further research is needed to develop
comprehensive pathogen-specific procedures for environmental sample types included in SAM,
other than water.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Method 1622:
Cryptosporidium in Water by Filtration/IMS/FA" (U.S. EPA 2005).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
SAM2012 239 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
Analytical Technique: Use immunomagnetic separation and fluorescence assay microscopy
("Method 1622: Cryptosporidium in Water by Filtration/IMS/FA," U.S. EPA 2005).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), samples are centrifuged to pellet the oocysts, and the supernatant
fluid is aspirated. A solution containing anti-Cryptosporidium antibodies conjugated to magnetic
beads is added to the pellet and mixed. The oocyst magnetic bead complex is separated from the
extraneous materials using a magnet, and the extraneous materials are discarded. The magnetic
bead complex is then detached from the oocysts. The oocysts are stained on well slides with
fluorescently labeled monoclonal antibodies (mAbs) and 4',6-diamidino-2-phenylindole (DAPI).
The stained sample is examined using fluorescence and differential interference contrast (DIG)
microscopy. Qualitative analysis is performed by scanning each slide well for objects that meet
the size, shape, and fluorescence characteristics of Cryptosporidium oocysts. Quantitative
analysis is performed by counting the total number of objects on the slide confirmed as oocysts.
This method is not intended to determine viability, species, or infectivity of the oocysts.
At a minimum, the following QC checks should be performed and evaluated: positive control,
negative control, matrix spike/matrix spike duplicate (MS/MSD) and blank. Ongoing analysis of
QC samples to ensure reliability of the analytical results should also be performed as stipulated in
the method.
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
U.S. EPA. 2005. "Method 1622: Cryptosporidium in Water by Filtration/IMS/FA." EPA 815-R-
05-001. http://www.epa.gov/sam/pdfs/EPA-1622.pdf
7.4.1.3 Site Characterization Sample Analyses (IMS/FA)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for detection in aerosols, surface wipes or swabs,
drinking water and post decontamination waste water. Further research is needed to develop
comprehensive pathogen-specific procedures for environmental sample types included in SAM,
other than water.
SAM 2012 240 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
Sample Preparation: Participate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Method 1623:
Cryptosporidium and Giardia in Water by Filtration/IMS/FA" (U.S. EPA 2005).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Analytical Technique: Use IMS and FA microscopy ["Method 1623: Cryptosporidium and
Giardia in Water by Filtration/IMS/FA" (U.S. EPA 2005)].
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), samples are centrifuged to pellet the oocysts and cysts, and the
supernatant fluid is aspirated. A solution containing wii-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.
At a minimum, the following QC checks should be performed and evaluated: positive control,
negative control, MS/MSD and blank. Ongoing analysis of QC samples to ensure reliability of
the analytical results should also be performed.
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http: //oaspub .epa. gov/eims/eimscomm. getfile ?p_download_id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
SAM 2012 241 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
U.S. EPA. 2005. "Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA."
EPA 815-R-05-002. http://www.epa.gov/sam/pdfs/EPA-1623.pdf
7.4.1.4 Post Decontamination Sample Analyses (Cell Culture
Immunofluorescence [IFA])
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Analytical Technique: Use cell culture IFA (Literature reference for Cryptosporidium spp.
[Canadian Journal of Microbiology, 2007, 53(5): 656-663]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), samples are used to inoculate HCT-8 monolayers and incubated.
Following incubation the monolayers are examined using IFA to determine the number of viable
oocysts present in the sample. The use of cell culture IFA analyses is a cost effective and
expedient alternative to mouse infectivity assays to determine in vitro infectivity of
Cryptosporidium oocysts.
At a minimum, the following QC checks should be performed and evaluated: positive control,
negative control and blank. Ongoing analysis of QC samples to ensure reliability of the analytical
results should also be performed.
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
SAM 2012 242 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http: //oaspub .epa. gov/eims/eimscomm. getfile ?p_download_id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
Bukhari, Z., Holt, D.M., Ware, M.W. and Schaefer III, F.W. 2007. "Blind Trials Evaluating In
Vitro Infectivity of Cryptosporidium Oocysts Using Cell Culture Immunofluorescence."
Canadian Journal of Microbiology, 53(5): 656-663.
http://www.nrcresearchpress.com/doi/abs/10.1139AV07-0327url ver=Z39.88-
2003&rfr id=ori:rid:crossref.org&rfr dat=cr pub%3dpubmed
7.4.2 Entamoeba histolytica - BSL-2
Remediation Phase
Site Characterization
Post Decontamination
Analytical Technique
Real-Time PCR
Cell Culture
Section
7.4.2.1
7.4.2.2
7.4.2.1 Site Characterization Sample Analyses (Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Analytical Technique: Use real-time PCR (Literature reference for Entamoeba histolytica
[Journal of Clinical Microbiology, 2005, 43(5): 2168-2172]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), the target nucleic acid should be extracted, purified, and analyzed
using the referenced target-specific real-time PCR primers and probes and assay parameters
(Journal of Clinical Microbiology, 2005, 43(5): 2168-2172). The use of real-time PCR analyses
directly on samples allows for rapid detection of Entamoeba histolytica.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition control and blank. Ongoing
SAM 2012 243 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
analysis of QC samples to ensure reliability of the analytical results should also be performed.
PCR QC checks should be performed according EPA's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-OAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http: //oaspub .epa. gov/eims/eimscomm. getfile ?p_download_id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
Roy, S., Kabir, M., Mondal, D., Ali, I.K.M., Petri Jr., W.A. and Haque, R 2005. "Real-Time-
PCR Assay for Diagnosis ofEntamoeba histolytica Infection." Journal of Clinical Microbiology,
43(5): 2168-2172. http://icm.asm.org/content/43/5/2168.full.pdf+html
7.4.2.2 Post Decontamination Sample Analyses (Cell Culture)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges et al. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose et al. 2011)..
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Analytical Technique: Use cell culture (Literature Reference for Entamoeba histolytica (Journal
of Parasitology, 1972, 58(2): 306-310).
SAM 2012 244 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), Entamoeba histolytica cysts are placed in a modified trypticase-
panmede liver digest-serum 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: positive control,
negative control and blank. Ongoing analysis of QC samples to ensure reliability of the analytical
results should also be performed.
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
Stringert, R.P. 1972. "New Bioassay System for Evaluating Percent Survival of Entamoeba
histolytica Cysts." The Journal of Parasitology, 58(2): 306-310.
http://www.istor.org/discover/10.2307/3278094?uid=3739704&uid=2129&uid=2&uid=70&uid=
4&uid=3739256&sid=47698759181407
7.4.3 Giardia spp. [Giardiasis] - BSL-2
Remediation Phase
Site Characterization
Post Decontamination
Analytical Technique
Real-Time PCR
IMS/FA
Cell Culture
Section
7.4.3.1
7.4.3.2
7.4.3.3
7.4.3.1 Site Characterization Sample Analyses (Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
SAM 2012 245 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
Sample Preparation: Participate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Real-Time PCR for
Quantification ofGiardia and Cryptosporidium in Environmental Water Samples and Sewage"
(Guy et al. 2003).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Analytical Technique: Use real-time PCR (Literature reference for Giardia [Applied and
Environmental Microbiology, 2003, 69(9): 5178-5185]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), the target nucleic acid should be extracted, purified, and analyzed
using the referenced target-specific real-time PCR primers and probes and assay parameters
(Applied and Environmental Microbiology, 2003, 69(9): 5178-5185). The use of real-time PCR
analyses directly on samples allows for rapid detection ofGiardia.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
SAM 2012 246 July 16, 2011
-------
SAM 2012 Section 7.0- Selected Pathogen Methods
Guy, R.A., Payment, P., Krull, U.J. and Horgen, P.A. 2003. "Real-Time PCR for Quantification
ofGiardia and Cryptosporidium in Environmental Water Samples and Sewage." Applied and
Environmental Microbiology, 69(9): 5178-5185.
http://aem.asm.Org/content/69/9/5178.full.pdf+html
7.4.3.2 Site Characterization Sample Analyses (IMS/FA)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Method 1623:
Cryptosporidium and Giardia in Water by Filtration/IMS/FA" (U.S. EPA 2005).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Analytical Technique: Use IMS and FA microscopy ["Method 1623: Cryptosporidium and
Giardia in Water by Filtration/IMS/FA" (U.S. EPA 2005)].
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), samples are centrifuged to pellet the oocysts and cysts, and the
supernatant fluid is aspirated. A solution containing wii-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.
At a minimum, the following QC checks should be performed and evaluated: positive control,
negative control, MS/MSD and blank. Ongoing analysis of QC samples to ensure reliability of
the analytical results should also be performed.
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
SAM 2012 247 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
U.S. EPA. 2005. "Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA."
EPA 815-R-05-002. http://www.epa.gov/sam/pdfs/EPA-1623.pdf
7.4.3.3 Post Decontamination Sample Analyses (Cell Culture)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Method 1623:
Cryptosporidium and Giardia in Water by Filtration/IMS/FA" (U.S. EPA 2005).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Analytical Technique: Use cell culture (Literature Reference for Giardia spp. [Transactions of
the Royal Society of Tropical Medicine and Hygiene, 1983, 77(4): 487-488]).
Description of Method: Procedures are described for analysis of cell culture samples and may
be adapted for assessment of aerosols, surface wipes or swabs and water samples (see Sample
Preparation Procedures above). Trypticase-yeast-iron-serum 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 ฐC in borosilicate glass tubes. The cells form a
dense, adherent monolayer on the surface of the glass or are observed swimming through the
liquid medium.
At a minimum, the following QC checks should be performed and evaluated: positive control,
negative control and blank. Ongoing analysis of QC samples to ensure reliability of the analytical
results should also be performed.
SAM 2012 248 July 16, 2011
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Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. 2005. "Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA."
EPA 815-R-05-002. http://www.epa.gov/sam/pdfs/EPA-1623.pdf
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
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/article/pii/0035920383901207
7.4.4 Toxoplasma gondii [Toxoplasmosis] - BSL-2
Remediation Phase
Site Characterization
Post Decontamination
Analytical Technique
Real-Time PCR
Cell Culture
Section
7.4.4.1
7.4.4.2
7.4.4.1 Site Characterization Sample Analyses (Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Method 1623:
Cryptosporidium and Giardia in Water by Filtration/IMS/FA" (U.S. EPA 2005).
SAM 2012 249 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Analytical Technique: Use real-time PCR (Literature reference for Toxoplasma gondii [Applied
and Environmental Microbiology, 2009, 75(11): 3477-3483]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), the target nucleic acid should be extracted, purified, and analyzed
using the referenced target-specific real-time PCR primers and probes and assay parameters
(Applied and Environmental Microbiology, 2009, 75(11): 3477-3483). The use of real-time PCR
analyses directly on samples allows for rapid detection of Toxoplasma gondii.
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-OAOC-PCR.pdf. or consult the points of
contact identified in Section 4.
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http://oaspub .epa.gov/eims/eimscomm.getfile ?p download id=5 03 892
U.S. EPA. 2005. "Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA."
EPA 815-R-05-002. http://www.epa.gov/sam/pdfs/EPA-1623.pdf
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
Yang, W., Lindquist, H.D. A., Cama, V., Schaefer III, F.W., Villegas, E., Payer, R, Lewis, E.J.,
Feng, Y. and Xiao, L. 2009. "Detection of Toxoplasma gondii Oocysts in Water Sample
Concentrates by Real-Time PCR." Applied and Environmental Microbiology, 75(11): 3477-
3483. http://aem.asm.Org/content/75/l l/3477.full.pdf+html
7.4.4.2 Post Decontamination Sample Analyses (Cell Culture)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
SAM 2012 250 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose et al. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Method 1623:
Cryptosporidium and Giardia in Water by Filtration/IMS/FA" (U.S. EPA 2005).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Analytical Technique: Use cell culture (Literature Reference for Toxoplasma gondii [Journal of
Microbiological Methods, 81(3): 219-225]).
Description of Method: Samples are subjected to a series of mechanical and chemical digestion
steps to release sporozoites from the Toxoplasma gondii oocysts and then inoculated onto
confluent fibroblast monolayers. Inoculated monolayers are then incubated undisturbed for ten
days to allow for plaque formation. After ten days, the monolayers are fixed, stained with crystal
violet, and examined for plaque formation. The literature reference also includes a qPCR
procedure to determine viability of Toxoplasma gondii oocysts, however, it may not be
appropriate depending on the type of disinfection used during remediation.
At a minimum, the following QC checks should be performed and evaluated: 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's
Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on
Environmental Samples (EPA 815-B-04-001) document at: http://www.epa.gov/sam/EPA-
QAQC-PCR.pdf. or consult the points of contact identified in Section 4.
Sources:
Hodges, L.R, Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
Villegas, E. N., Augustine, S.A., Villegas, L. F., Ware, M.W., See, M. J., Lindquist, H.D.A.,
Schaefer, III, F. W. and Dubey, J.P. 2010. "Using Quantitative Reverse Transcriptase PCR and
SAM 2012 251 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
Cell Culture Plaque Assays to Determine Resistance of Toxoplasma gondii Oocysts to Chemical
Sanitizers." Journal of Microbiological Methods, 81(3): 219-225.
http://www.sciencedirect.com/science/article/pii/S0167701210001107
7.5 Method Summaries for Helminths
Summaries of the analytical methods for helminths listed in Appendix C is provided in Section 7.5.1.
7.5.1 Baylisascaris procyonis [Raccoon roundworm fever] - BSL-2
Remediation Phase
Site Characterization
Post Decontamination
Analytical Technique
Real-Time PCR
Embryonation of Eggs and Microscopy
Section
7.5.1.1
7.5.1.2
7.5.1.1 Site Characterization Sample Analyses (Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges etal. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose etal. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Evaluation of a Molecular
Beacon Real-time PCR Assay for Detection of Baylisascaris procyonis in Different Soil Types
and Water Samples" (Gatcombe etal. 2010).
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction by bead beating and purification for all sample types should be performed
according to procedures within the EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use real-time PCR (Literature reference for Baylisascaris procyonis
[Parasitology Research, 2010, 106: 499-504]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), the target nucleic acid should be extracted, purified (EPA BA
Protocol, Section 9.2), and analyzed using the referenced target-specific real-time PCR primers
and probes and assay parameters (Parasitology Research, 2010, 106: 499-504). The use of real-
time PCR analyses directly on samples (e.g., no embryonation or microscopic examination)
allows for rapid detection of Baylisascaris procyonis.
SAM 2012 252 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
At a minimum, the following QC checks should be performed and evaluated when using this
protocol: positive control, negative control, external inhibition 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's Quality Assurance/Quality Control
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sam/EPA-QAQC-PCR.pdf. or consult the points of
contact identified in Section 4.
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." EPA 600/R-l 1/103.
http: //oaspub .epa. gov/eims/eimscomm. getfile ?p_download_id=5 03 892
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
Gatcombe, R.R., Jothikumar, N., Dangoudoubiyam, S., Kazacos, K.R. and Hill, V.R. 2010.
"Evaluation of a Molecular Beacon Real-time PCR Assay for Detection of Baylisascaris
procyonis in Different Soil Types and Water Samples," Parasitology Research, 106:499-504.
http://www.springerlink.com/content/k8t3581t07n82562
7.5.1.2 Post Decontamination Sample Analyses (Embryonation of Eggs and
Microscopy)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: SAM lists this method for analysis of aerosols, surface wipes or swabs,
drinking water and post decontamination waste water sample types. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types
included in SAM.
Sample Preparation: Particulate samples (surface swabs and wipes) should be prepared
according to "National Validation Study of a Swab Protocol for the Recovery of Bacillus
anthracis Spores From Surfaces" (Hodges et al. 2010) or "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces" (Rose et al. 2011).
Water samples should be processed according to "Comparison of Ultrafiltration Techniques for
Recovering Biothreat Agents in Water" (U.S. EPA and CDC 2011) or "Evaluation of a molecular
beacon real-time PCR assay for detection of Baylisascaris procyonis in different soil types and
water samples" (Gatcombe et al. 2010).
SAM2012 253 July 16, 2011
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SAM 2012 Section 7.0- Selected Pathogen Methods
All other environmental sample types should be processed according to procedures within the
EPA BA Protocol [U.S. EPA (anticipated publication October 2012)].
Nucleic acid extraction and purification should be performed according to procedures within the
EPA BA Protocol (Protocol Section 9.2).
Analytical Technique: Use microscopy and embryonation of eggs (U.S. EPA Environmental
Regulations and Technology, 2003, EPA/625/R-92/013).
Description of Method: The protocol describes procedures for analysis of solid and wastewater
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 eggs, 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
helminth eggs are fully embryonated. The concentrate is then microscopically examined for the
categories of helminth ova on a counting chamber.
At a minimum, the following QC checks should be performed and evaluated: positive control,
negative control and blank. Ongoing analysis of QC samples to ensure reliability of the analytical
results should also be performed.
Sources:
Hodges, L.R., Rose, L.J., O'Connell, H. and Arduino, M.J. 2010. "National Validation Study of a
Swab Protocol for the Recovery of Bacillus anthracis Spores From Surfaces." Journal of
Microbiological Methods, 81(2): 141-146.
http://www.sciencedirect.com/science/article/pii/S0167701210000692
Rose L.J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. "National Validation Study of a
Cellulose Sponge-Wipe Processing Method for Use After Sampling Bacillus anthracis Spores
From Surfaces." Applied Environmental Microbiology, 77(23): 8355-8359.
http://aem.asm.org/content/77/23/8355.full.pdf+html
U.S. EPA. [Anticipated publication October 2012] "Protocol for Detection of Bacillus anthracis
in Environmental Samples During the Remediation Phase of an Anthrax Event" (EPA BA
Protocol).
U.S. EPA. 2003. "Appendix I: Test Method for Detecting, Enumerating, and Determining the
Viability of Ascaris Ova in Sludge." U.S. EPA 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
SAM 2012 254 July 16, 2011
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SAM2012 Section 8.0- SelectedBiotoxinMethods
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 contamination incident. 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 as well as development
and testing 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
remediation 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.
Chemical Abstracts Service Registry Number (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; tests types are described below.
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.
Depending on site- and event-specific goals, a determination of whether contaminant concentrations are
above pre-existing levels may be necessary. Such determinations could involve investigations of
background levels at potentially uncontaminated locations in close proximity to the site, using methods
listed in Appendix D. Other means might include examination of historical information regarding
contaminant occurrence. For example, periodic episodes of some of the biotoxins (such as microcystins
and other algal toxins) have been detected and measured in surface waters throughout the United States
by methods similar to those in Appendix D (http://toxics.usgs.gov/highlights/algal_toxins/). When using
historical data, knowledge of the analytical methods and techniques used would be necessary, particularly
in terms of their similarity in performance and quality control (QC) to the methods listed in Appendix D.
SAM2012 255 July 16, 2011
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SAM2012 Section 8.0- SelectedBiotoxinMethods
The "analysis type" listed for each biotoxin method in Appendix D is intended to address: (1) the level of
certainty of results and (2) the remediation phase (e.g., site mapping, assessment, clearance). A tiered
approach (i.e., algorithm) may be used when implementing the analysis types, particularly when needed
to address a large number of samples. For example, methods identified as presumptive, which are
generally more rapid than confirmatory methods, might be used during the initial stages of remediation to
evaluate the extent of contamination once a contamination event and the type of contamination are
known. Presumptive methods also might be used to identify samples that should be analyzed using
confirmatory methods. In turn, the results of the confirmatory methods might be used to select samples to
be analyzed by applicable biological activity methods, which tend to be much slower and less available
than the confirmatory methods. Note that the use of the terms "presumptive" and "confirmatory" in this
document is not intended to redefine or supersede the Laboratory Response Network's (LRN) use of the
terms. The terms as used by the LRN are described in Section 8.1.4.
Many of the presumptive methods are immunoassays, which may be adapted for large-scale sample
processing while maintaining an appropriate level of analytical certainty. Confirmatory methods are
generally more time consuming and expensive and are intended to provide results with a high level of
certainty. Confirmatory methods should be considered for use when: (1) presumptive analysis indicates
the presence of the biotoxin, (2) a smaller subset of samples requires processing, or (3) as required for a
tiered approach to remediation. Methods that address biological activity tend to be even more time
consuming and expensive, and are intended to provide a high level of certainty in corroborating other
assay results. Depending on the goals of the remediation phase, biological activity methods may be
needed because biotoxins may be detectable but inactive; therefore, these assays may also provide
information about potential impact on human safety.
For small molecule biotoxins in Appendix D, the presence of intact compound structure is an indication
of biological activity; therefore, the confirmatory method listed for these biotoxins also serves as a
measure of biological activity. For protein biotoxins, biological activity may be determined directly using
in vivo (e.g., mouse bioassay) or in vitro (e.g., enzymatic activity) methods. However, biological
availability (i.e., biotoxin accessibility to site of action) and activity are both required to elicit toxicity and
some in vitro methods may not address both parameters.
Numerous analytical techniques using a variety of instrumentation (e.g., high performance liquid
chromatography-mass spectrometer [HPLC-MS], immunoassay [enzyme-linked immunosorbent assay
(ELISA)], immunoassay [lateral flow device (LFD)], electrochemiluminescence (ECL)-based, enzyme
immunoassay [EIA]) have been cited in Appendix D. It is recognized that new reports describing
advances in procedures for analysis of biotoxins frequently appear in the literature, and commercially
available equipment for these analyses are evolving rapidly. Accordingly, the individual techniques and
methods listed in Appendix D are to be regarded as a starting point. The biotoxin methods points of
contact listed in Section 4.0 of SAM should be consulted for additional information regarding currently
available methods.
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. Additional research on biotoxin contaminants is ongoing within EPA and includes impact of
disinfectants and preservatives.
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.
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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, Toxicology Data
Network (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.army.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 are found at the National Select Agent Registry
(http: //www. selectagents. gov/).
The Centers for Disease Control and Prevention (CDC) has additional information regarding select
agent toxins (http://www.cdc.gov/od/sap/sap/toxinamt.htm)
See Syracuse Research Corporation's (SRC) PHYSPROP and Chemfate, part of the Environmental
Fate Database supported by EPA (http ://srcinc.com/what-we-do/product.aspx?id= 133)
INCHEM contains both chemical and toxicity information(http: //www. inchem. org/)
The Registry of Toxic Effects of Chemical Substances (RTECS) database can be accessed via the
National Institute for Occupational Safety and Health (NIOSH) website 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
Federal Bureau of Investigation (FBI). See http://www.fbi.gov/about-us/lab/forensic-science-
communications
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: Selected 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 (aerosol, solid, particulate, liquid or drinking water) 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, voluntary
consensus standard bodies (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.
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Table 8-1. Sources of Biotoxin Methods
Name
Food and Drug Administration (FDA),
Bacteriological Analytical Manual
Online
Official Methods of Analysis of AOAC
International*
National Environmental Methods Index
(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*
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
Journal of Agricultural and Food
Chemistry*
Publisher
FDA
AOAC International
EPA, U.S. Geological
Survey (USGS)
Blackwell Synergy
Science Direct
Portland Press Ltd.
American Chemical Society
(ACS)
International Association for
Food Protection
Elsevier Science Publishers
John Wiley And Sons Ltd
National Institute of
Environmental Health
Sciences
Elsevier Science Publishers
Wiley-Blackwell
Elsevier Science Publishers
John Wiley And Sons Ltd.
AOAC International
Royal Society of Chemistry
Elsevier Science Publishers
American Society for
Microbiology (ASM)
John Wiley And Sons Ltd.
S. Tinsley Preston
Environmental Technology
Verification (ETV) Program
ACS Publications
Reference
http://www.fda.qov/Food/ScienceResearc
h/LaboratorvMethods/BacterioloqicalAnal
vticalManualBAM/default.htm
http://www.aoac.ora
http://www.nemi.qov/
http://www.blackwell-svnerav.com/loi/pto
http://www.sciencedirect.com/
http://www.biochemi.orq/
http://www.acs.ora/
http://www.foodprotection.orq/
http://www.elsevier.com/
http://www.wilev.com/
http://www.niehs.nih.aov/
http://www.iournals.elsevier.com/toxicon/
http://www.wilev.com/
http://www.elsevier.com/
http://www.wilev.com/
http://www.aoac.orq
http://www.rsc.ora/
http://www.elsevier.com/
http://www.asm.ora/
http://www.wilev.com/
http://www.iatox.com/
http://www.epa.aov/etv/
http://pubs.acs.ora/
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Name
Applied and Environmental Microbiology
(AEM)*
Journal of Chemical Health and Safety*
Publisher
ASM
Elsevier Science Publishers
Reference
http://aem.asm.orq/
http://www. elsevier.com/
' 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.10 Ensuring data quality also
requires that laboratory results are properly evaluated and the results of the data quality evaluation are
included within the data report when 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 (DQOs) 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, replicates) 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
Use of test kits and reagents prior to expiration date
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
Information regarding EPA's DQO process, considerations, and planning is available at:
http://www.epa.gov/QUALITY/dqos.html.
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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
> Matrix spike/matrix spike duplicates (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 quality assurance (QA)/QC procedures prior to sample analysis. These contacts will consult
with the EPA Environmental Response Laboratory Network (ERLN) and Water Laboratory Alliance
(WLA) coordinators 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.
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 chemical, biological and/or radiological (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
CDC. 2009. Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th Edition.
Available at: http://www.cdc.gov/OD/ohs/biosfty/bmbl5/bmbl5toc.htm
Fleming, D.O. and Hunt, D.L. (editors). 2006. Biological Safety: Principles and Practices, 4th Ed.
American Society for Microbiology (ASM) Press: Herndon, VA
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. Available at:
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. Available at: http://www.access.gpo.gov/nara/cfr/waisidx 07/40cfr270 07.html
Occupational Safety and Health Administration (OSHA) - 29 CFR part 1910.145 0. Occupational
Exposure to Hazardous Chemicals in Laboratories Available at:
http://www.access.gpo.gov/nara/cfr/waisidx 06/29cfrl910a 06.html
OSHA - 29 CFR part 1910.120. Hazardous Waste Operations and Emergency Response
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U.S. Department of Agriculture (USDA) - 9 CFRpart 121. Possession, Use, and Transfer of Select
Agents and Toxins
Please note that the Electronic Code of Federal Regulations (e-CFR) is available at
http://ecfr.gpoaccess.gov/.
8.1.4 Laboratory Response Network (LRN)
The LRN was created in accordance with Presidential Decision 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.
Many of the biotoxins listed in SAM are select agents. Additional information on select agents and
regulations may be obtained at the National Select Agent Registry at: http://www.selectagents.gov/.
Relevant to the purposes of analytical methods, SAM users should note that some of these agents are not
regulated if the amount under the control of a principal investigator does not exceed, at any time, the
amounts indicated in the table at: http://www.selectagents.gov/Permissible%20Toxin%20Amounts.html.
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)
CDC
1600 Clifton Road NE, Mailstop C-18
Atlanta, GA 30333
Telephone: (866) 576-5227
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 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
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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, 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
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.
Abrine - CAS RN: 526-31-8
Description: Small molecule, indole alkaloid marker for abrin.
Method
Journal of Food Protection. 2008. 71(9): 1868-1874
Journal of Agricultural and Food Chemistry. 2008.
56(23): 11139-11143
Pharmacology & Toxicology. 2001. 88(5): 255-260
Analytical Biochemistry. 2008. 378: 87-89
Analytical Technique
Immunoassay (ELISA, ECL-based)
LC-MS-MS
Ribosome inactivation assay
Enzyme activity
Section
8.2.1.1
8.2.1.2
8.2.1.3
8.2.1.4
8.2.1.1 Literature Reference for Abrin (Journal of Food Protection. 2008. 71(9):
1868-1874)
Analysis Purpose: Presumptive
Analytical Technique: Immunoassay (ELISA, ECL-based)
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 adapt and verify the
procedures for environmental sample types.
Description of Method: Procedures are described for using mouse monoclonal antibodies
(mAbs) and rabbit-derived polyclonal antibodies prepared against a mixture of abrin isozymes for
three separate ELISA and 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.E. 2008. "Rapid Detection of Ricin in Cosmetics and Elimination
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of Artifacts Associated With Wheat Lectin." Journal of Immunological Methods. 336(2): 251-
254). http://www.sciencedirect.com/science/journal/00221759
Source: Garber, E.A.E., Walker, J.L. and O'Brien, T.W. 2008. "Detection of Abrin in Food
Using Enzyme-Linked Immunosorbent Assay and Electrochemiluminescence Technologies."
Journal of Food Protection. 71(9): 1868-1874.
http://www.ingentaconnect.com/content/iafb/ifb/2008/00000071/00000009/art00015
8.2.1.2 Literature Reference for Abrin by Abrine Detection (Journal of
Agricultural and Food Chemistry. 2008. 56(23): 11139-11143)
Analysis Purpose: Complementary presumptive for abrin
Analytical Technique: LC-MS-MS
Method Developed for: Abrine in beverages
Method Selected for: SAM lists these procedures for complementary presumptive analysis of
abrin by abrine detection in aerosol, solid, particulate, liquid and water samples. Abrine, an
alkaloid present in equal concentrations with abrin in rosary peas (Abrus precatorius L.), is found
in crude preparations of abrin and may be an indicator of abrin contamination. Further research is
needed to adapt and verify the procedures for environmental sample types.
Description of Method: Procedures are described for sample extraction by solid-phase
extraction (SPE) or liquid-liquid extraction, followed by tandem mass spectrometry. The method
was verified in beverages (bottled water, cola, juice drink, 1% low fat milk, bottled tea) spiked
with abrine at either 0.5(ig/mL or 0.05(ig/mL. These samples were prepared for LC-MS-MS by
either an optimized SPE procedure or a liquid-liquid extraction procedure. For SPE, optimal
abrine recoveries were achieved with sample pH adjusted to 2 - 6 with formic acid, inclusion of a
water/methanol (95/5, v/v) washing step prior to elution, and use of a Strata-X SPE cartridge.
Liquid-liquid extraction was with an equal volume (2 mL) of acetonitrile/water (75/25, v/v).
Differences in recovery between the two extraction methods were determined using the two-sided
Student's ^test, assuming equal variance. At 0.5 (ig/mL, recovery of abrine by SPE was
significantly higher (P < 0.01) for water and juice drink as compared to liquid-liquid extraction,
but no significant differences were observed for cola and tea. At 0.05 (ig/mL, the differences in
recovery of abrine in water, tea, cola and juice drink were highly statistically different (P <
0.001), with better recoveries for the optimized SPE procedure. The method had a MDL of 0.025
(ig/mL and LOQ of 0.05 (ig/mL. Storage stability was also tested for abine at 10 (ig/mL in a
water/methanol stock solution (90/10, v/v) at three temperatures (0 ฐC, 4 ฐC and 23 ฐC). Aliquots
were analyzed in triplicate at 0, 1,7 and 21 days after sample preparation. There was no
statistically significant difference between abrine standards stored at the three temperatures at 21
days and no loss of abrine concentration.
Special Considerations: The biotoxin methods points of contact listed in Section 4.0 of SAM
should be consulted for additional information regarding water and drinking water analyses.
Source: Owens, J. and Koester, C. 2008. "Quantitation of Abrine, an Indole Alkaloid Marker of
the Toxic Glycoproteins Abrin, by Liquid Chromatography/Tandem Mass Spectrometry When
Spiked into Various Beverages." Journal of Agriculture and Food Chemistry. 56(23): 11139
11143. http://pubs.acs.org/doi/abs/10.1021/if802471y
8.2.1.3 Literature Reference for Abrin and Ricin (Analytical Biochemistry. 2008.
378(1): 87-89)
Analysis Purpose: Biological activity
Analytical Technique: Enzyme activity
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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 adapt and verify the
procedures for environmental sample types.
Description of Method: This in vitro assay is a ribonucleic acid (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 ฐC. 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 ฑ 0.7 mg/mL total protein and 4.1 ฑ 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 ฑ
0.6 mg/mL total protein and 3.7 ฑ 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 ฑ 0.2 mg/mL activity
equivalents. In contrast, the undiluted jequirity seed extract contained a calculated level of 740 ฑ
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): 8789.
http://www.sciencedirect.com/science/journal/00032697
8.2.1.4 Literature Reference for Abrin, Shiga Toxin, 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 adapt and verify 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 nanomolar (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.
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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.wilev.com/iournal/120703798/abstract
8.2.2 Botulinum Neurotoxins (Serotypes A, B, E, F)
Botulinum neurotoxins - Description: Protein composed of-100 kDa heavy chain and ~50
kDa light chain; can be complexed with hemagglutinin and non-hemagglutinin components for
total molecular weight (MW) of-900 kDa.
SNAP-25 - Description: Synaptosomal-associated protein 25; 25 kDa membrane-associated
protein cleaved by botulinum neurotoxin Serotypes A, C and E
VAMP 2 - Description: Vesicle-associated membrane protein 2 (also known as synaptobreven
2); cleaved by botulinum neurotoxin Serotypes B, D, F and G
Method
LRN
FDA, Bacteriological Analytical Manual Online, January
2001, Chapter 17, Clostridium botulinum
Journal of Chemical Health and Safety. 2008. 15(6):
14-19
Lateral Flow Device Immunoassay Kits
Analytical Technique
Immunoassay, Immunoassay
(ELISA) and Mouse bioassay
Immunoassay (ELISA) and
Mouse bioassay
Endopep-MS
Immunoassay (LFD)
Section
8.1.4
8.2.2.1
8.2.2.2
8.2.2.3
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 adapt and verify 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
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prepared by centrirugation 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 forms of toxin.
Source: FDA, Center for Food Safety and Applied Nutrition (CFSAN). 2001. "Chapter 17 -
Clostridium botulinum'' Bacteriological Analytical Manual Online.
http: //www. epa.gov/sam/pdfs/FDA-BAM-Chap 17 .pdf
8.2.2.2 Literature Reference for Botulinum Neurotoxins by SNAP-25 and VAMP 2
Cleavage Product Detection (Journal of Chemical Health and Safety.
2008. 15(6): 14-19)
Analysis Purpose: Complementary presumptive for botulinum neurtotoxins
Analytical Technique: LC-MS
Method Developed for: Botulinum neurotoxins Serotypes A, B, E and F in clinical samples
(stool, serum)
Method Selected for: SAM lists these procedures for complementary presumptive analysis of
botulinum neurotoxins by SNAP-25 and VAMP 2 cleavage product detection in aerosol samples.
SNAP-25 and VAMP 2 function as substrates for botulinum neurotoxins and may be an indicator
of botulinum neurotoxin contamination. Further research is needed to adapt and verify the
procedures for environmental sample types.
Description of Method: Procedures are described for antibody-based sample extraction,
followed by synthetic peptide cleavage and high resolution matrix-assisted laser-desorption
ionization (MALDI) time-of-flight (TOF)-MS. The method is verified for stool and serum
clinical samples obtained from an exposed individual. Botulinum neurotoxin Serotypes A, B, D
and E are obtained from Metabiologics (Madison, Wisconsin) and used as positive controls.
Rabbit polyclonal antibodies specific for Serotypes A, B, E and F are also obtained from
Metabiologics and are coupled to Dynabeadsฎ Protein G beads. A cocktail of protease inhibitors
and 20 (iL of beads are added to 100 (iL of stool sample. The mixture is incubated for two hours
at 37 ฐC, washed in buffer, followed by a water wash. A 500-(iL serum sample is added to 100
(iL of beads and similarly incubated and washed. Protease inhibitors are not required for serum
samples. After antibody isolation, the bead-extracted sample is incubated in a reaction buffer
with synthetic peptide substrates specific for Serotypes A, B, E and F. Samples are incubated at
37 ฐC for four hours. A 2-(iL aliquot of the reaction mixture supernatant is mixed with 18 (iL of
a matrix solution and 0.5 (iL of the resultant mixture is placed on a 192-spot MALDI plate. Mass
spectra are collected from 650 to 4500 m/z in the positive ion reflector mode on either an Applied
Biosystems 4700 Proteomics Analyzer or an Applied Biosystems 4800 TOF/TOF. Cleavage
product peaks specific for Serotypes A, B, E and F can be for observed for the positive controls
and positive stool and serum samples. Negative controls do not show these peaks.
Special Considerations: Additional detector platforms are available such as described in
"Development of an In Vitro Activity Assay as an Alternative to the Mouse Bioassay for
Clostridium botulinum Neurotoxin Type A," 2008. Applied and Environmental Microbiology.
74(14): 4309-4313. (http://aem.asm.org/content/74/14/4309.short). Fluorescence resonance
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energy transfer (FRET) based assays are also available as commercial products
(http: //www .biosentinelpharma. com/products .php).
Source: Barr, J.R., Kalb, S.R., Moura, H. and Pirkle, J.L. 2008. "Biological Monitoring of
Exposure to Botulinum Neurotoxins." Journal of Chemical Health and Safety. 15(6): 14-19.
http://www.sciencedirect.com/science/article/pii/S1871553208000418
8.2.2.3 EPA Environmental Technology Verification (ETV) Program Reports -
Lateral Flow Immunoassay Kits
Analysis Purpose: Presumptive
Analytical Technique: Immunoassay (LFD)
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 adapt and verify 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
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 immunochromatographic LFD 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.
LFD immunoassay kits have been evaluated by the EPA ETV Program using BADD and
BioThreat Alertฎ test strips for the detection of botulinum neurotoxins Types A and B and ricin.
Reports and information associated with these evaluations are available at:
http://www.epa.gov/sam/pdfs/ETV-BADD091904.pdf (BADD test strips) and
http://www.epa.gov/sam/pdfs/ETV-BioThreat092104.pdf (BioThreat Alertฎ test strips).
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, J., Bertocchi, 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/article/pii/S0022175908001671
Source: ETV. 2006. http://www.epa.gov/etv/
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8.2.3 Ricin (Ricinine)
Ricin-CASRN: 9009-86-3.
Description: 60 kDa glycoprotein consisting of a deadenylase (-32 kDa A chain) and lectin
(-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, alkaloid marker for ricin.
Method
LRN
Analytical Biochemistry. 2008. 378: 87-89
LFD 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 (LFD)
Immunoassay (ECL)
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 (ECL)
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 adapt and verify the
procedures for environmental sample types.
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://aoac.publisher.ingentaconnect.com/content/aoac/iaoac/2008/00000091/00000002/art00016
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
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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 adapt and verify 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 LOD 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 ฐC for 1 hour and
when stored at 25 ฐC and 5 ฐC for 3 weeks.
Special Considerations: The following updated literature reference adds the analyte abrine for
detection of select agent abrin: Rudolph C. Johnson, Yingtao Zhou, Ram Jain, Sharon W. Lemire,
Shannon Fox, Pat Sabourin and John R. Barr. 2009. "Quantification of L-Abrine in Human and
Rat Urine: A Biomarker for the Toxin Abrin." Journal of Analytical Toxicology, 33, (2), 77-84.
Source: Johnson, R.C., Lemire, S.W., Woolfitt, A.R., 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 ://j at.oxfordj ournals .org/content/29/3/149 .full .pdf+html
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. 1997. 35 (8): 2051 -2054
Analytical Technique
Ribosome inactivation assay
Immunoassay (ELISA)
Immunoassay (EIA)
Section
8.2.1.4
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 adapt and verify the
procedures for environmental samples.
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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 Toxins (Journal of Clinical
Microbiology. 1997. 35 (8): 2051 -2054)
Analysis Purpose: Presumptive
Analytical Technique: Immunoassay (EIA)
Method Developed for: Shiga toxin in clinical samples
Method Selected for: SAM lists these procedures for presumptive analysis in aerosol, solid,
particulate, liquid and water samples. Further research is needed to adapt and verify the
procedures for environmental sample types.
Description of Method: Procedures are described for a rapid EIA for the detection of Stx-1 and
Stx-2 using a commercially available kit. Fecal samples are assayed for Shiga toxin using the
EIA kit with overnight enrichment (500 specimens) and using a sorbitol-MacConkey's (sMac)
culture method (474 specimens). Samples producing positive results by EIA kit and/or sMac
culture are confirmed by Vero cell cytotoxicity assay using a 96-well format. Samples positive
by EIA kit and negative by sMac culture are recultured by the mitomycin immunoblot procedure
to isolate organisms and retested for confirmation by Vero cell cytotoxicity assay. The sMac
culture method had a sensitivity and specificity of 60% and 100%, respectively, for detection of
Shiga toxin producing E. coli O157:H7. EIA kit sensitivity and specificity are 100% and 99.7%,
respectively. The EIA kit is also capable of detecting Shiga toxin in cultures negative for the E.
coli O157:H7 serotype (e.g., E. coli serotypes O26:NM and 6:H-).
Source: Kehl, K.S., Havens, P., Behnke, C.E. and Acheson, D.W.K. 1997. "Evaluation of the
Premier EHEC Assay for Detection of Shiga Toxin-Producing Escherichia coli'' Journal of
Clinical Microbiology. 35(8): 2051-2054. http://jcm.asm.Org/cgi/reprint/35/8/2051 .pdf
8.2.5 Staphylococcal Enterotoxins (SEA, SEB, SEC)
CAS RNs: 37337-57-8 (SEA), 39424-53-8 (SEB), 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
Applied and Environmental Microbiology. 1997. 63(6):
2361-2365
Analytical Technique
Immunoassay
Immunoassay (EIA)
T-cell proliferation assay
Section
8.1.4
8.2.5.1
8.2.5.2
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8.2.5.1 AOAC Official Method 993.06: Staphylococcal Enterotoxins in Selected
Foods
Analysis Purpose: Presumptive
Analytical Technique: Immunoassay (EIA)
Method Developed for: Staphylococcal enterotoxins in selected foods
Method Selected for: SAM lists this method for presumptive analysis of Staphylococcal
enterotoxins Type B (SEE) in aerosol samples, and Types A (SEA) and C (SEC) in aerosol, solid,
particulate, liquid and water samples. Further research is needed to adapt and verify the
procedures for environmental sample types.
Description of Method: This method is an EIA using a mixture of high-affinity capture
antibodies for identification of toxin(s) in food samples. Samples are prepared by dilution in Tris
buffer, centrifugation, and filtration of the supernatant through a syringe, with adjustment to a
final pH of 7.0 to 8.0. Samples are incubated in 96-well plates with the mixture of antibodies
conjugated to horseradish peroxidase (HRP), and visualized with a peroxidase substrate. Assay
results are determined visually or using a microtiter plate reader. Test is considered positive for
Staphylococcal enterotoxins if absorbance is >0.200 and is considered negative if absorbance is
<0.200. Specific toxin serotypes are not differentiated. This method detects from 1.3 to 3.3
ng/mL Staphylococcal enterotoxin in extracts prepared from food containing 4 to 10 ng/mL
Staphylococcal enterotoxin.
Source: AOAC International. 1994. "Method 993.06: Staphylococcal Enterotoxins in Selected
Foods." Official Methods of Analysis of AOAC International. 16th Edition. 4th Revision; Vol. I.
http://www.aoac.org/
8.2.5.2 Literature Reference for Staphylococcal Enterotoxins Types A, B, and C
(Applied and Environmental Microbiology. 1997. 63(6): 2361-2365)
Analysis Purpose: Biological activity
Analytical Technique: T-cell proliferation assay
Method Developed for: Staphylococcal enterotoxin Type A (SEA) in selected foods
Method Selected for: SAM lists this method for biological activity assessment 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 adapt and verify the
procedures for environmental sample types.
Description of Method: This method is a T-cell proliferation assay using lymphocytes in a 96-
well plate format for identification of Staphylococcal enterotoxin(s) in food samples.
Lymphocytes are prepared from heparinized Lewis rat blood or human blood using Ficoll-
Paque. Cells are divided into aliquots at 0.5 x 105 to 1.0 x 105 cell per well in 100 (iL culture
medium into a U-bottomed 96-well tissue culture plate. Food samples (potato salad, canned
mushrooms, hot dogs, dry milk) are homogenized in PBS (1:1, wt/wt), centrifuged, the
supernatants diluted 1:10 in PBS, and added directly to sample wells containing lymphocytes.
Varying concentrations of SEA can be used as a standard curve. The treated samples are added to
the lymphocytes and incubated for two to five days at 37 ฐC. On the last day either 1 (iCi of
[methyl-3H] thymidine or 20 \\L of Alamar blue is added to the well. After 24 hours, supernatant
is either harvested onto glass fiber filters and the beta-radioactivity counted or the color reaction
of the Alamar blue treated wells is read on a plate reader at 570 nm. Both human and rat
lymphocytes produce strong T-cell proliferation in response to SEA. The radioactive assay
shows a significant level of proliferation (P < 0.05) as compared to control medium at levels as
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low as 0.1 pg SEA per well. The Alamar blue assay detects SEA at 1 ng per well. Diluted food
samples without SEA do not induce T-cell proliferation.
Special Considerations: This method was developed for SEA in selected foods and has not
been tested with SEE and SEC or in other sample types. However, because the T-cell
proliferation assay is not antigen specific, the method may be appropriate for SEB and SEC, both
of which have superantigen T-cell proliferation activity. This assay cannot identify the specific
superantigen nor can it assess emetic activity; additional testing to determine specificity and
assess toxin activity should be performed.
Source: Rasooly, L., Rose, N.R., Shah, D.B. and Rasooly, A. 1997. "In Vitro Assay of
Staphylococcus aureus Enterotoxin A Activity in Food." Applied and Environmental
Microbiology. 63(6): 2361-2365. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC168529/
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.
8.3.1 Aflatoxin (Type B1)
CASRN: 27261-02-5
Method
AOAC Official Method 991.31
Analytical Technique
Immunoassay (column) 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 (column) and high performance liquid chromatography-
fluorescence (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 adapt and
verify 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.
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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
CASRN: 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 (ELISA)
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
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 adapt and verify 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 (ELISA)
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 adapt
and verify the procedures for environmental sample types.
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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 ฐC. 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.E., 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/iafb/ifb/2005/00000068/00000006/art00027
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 adapt and verify 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% trifluoroacetic acid (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. wilev .com/i ournal/185 62/abstract
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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 (ELISA)
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 (ELISA)
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 adapt and verify 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
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.ncbi.nlm.nih.gov/pmc/articles/PMC1240733/
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 spectrometry
(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 adapt and verify 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 ds 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.
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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 (Solution phase
binding assay)
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 (solution phase binding assay)
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 adapt and verify the
procedures for environmental sample types.
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 adapt and verify the
procedures for environmental sample types.
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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% TFA, with the insoluble portion
of the sample removed by centrifugation. A portion of the sample extract is fractionated by size-
exclusion chromatography in order to prepare a sample containing small peptides in the range of
1000 to 2500 Da. 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
8.3.6 Cylindrospermopsin
CASRN: 143545-90-8
Method
FEMS Microbiology Letters. 2002. 216: 159-164
ELISA Kits for Cylindrospermopsin
Analytical Technique
HPLC-PDA
Immunoassay (ELISA)
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: Fligh 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 adapt and verify 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
Cis column with a linear gradient of 1 to 12% (v/v) methanol/water over 24 minutes at 40 ฐC,
with monitoring at 262 nm. The use of ds columns for environmental waters is suggested for
removal of the large number of organic compounds that may be present. This method detects and
recovers Cylindrospermopsin from spiked environmental water samples at 1 ug/L.
Source: Metcalf, J.S., Beattie, K.A., Saker, M.L. and Codd, G.A. 2002. "Effects of Organic
Solvents on the High Performance Liquid Chromatographic Analysis of the Cyanobacterial Toxin
Cylindrospermopsin and Its Recovery from Environmental Eutrophic Waters by Solid Phase
Extraction." FEMS Microbiology Letters. 216(2): 159-164.
http://cat.inist.fr/?aModele=afficheN&cpsidt=14002569
8.3.6.2 ELISA Kits for Cylindrospermopsin
Analysis Purpose: Presumptive
Analytical Technique: Immunoassay (ELISA)
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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 adapt and verify 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 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 (ig/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)
CASRN: 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 (ELISA)
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 (ELISA)
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 adapt and verify 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/article/pii/0168160588900797
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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 adapt and verify the
procedures for environmental sample types.
Description of Method: A LC/APCI-MS procedure based on TOF-MS, with a real-time
reference mass correction, is used for simultaneous determination ofFusarium 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.
http://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 (ELISA)
/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 (ELISA)/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 adapt and verify 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 centrirugation 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.
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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
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 adapt and verify 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 Ci8 SPE cartridge, followed by identification and
determination by HPLC-PDA. This procedure can detect microcystin concentrations as low as
250 ng/L and is the basis of the World Health Organization (WHO) method for the detection of
microcystins.
Source: Lawton, L.A., Edwards, C. and Codd, G.A. 1994. "Extraction and High-Performance
Liquid Chromatographic Method for the Determination of Microcystins in Raw and Untreated
Waters." Analyst. 119(7): 1525-1530.
http://www.rsc.org/Publishing/Journals/AN/article.asp?doi=AN9941901525
8.3.9 Picrotoxin
CASRN: 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 adapt and verify 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 ซ-hexane, followed by extraction with
chloroform. The chloroform is evaporated and the sample is reconstituted in acetonitrile-1 mM
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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/article/pii/0731708589801049
8.3.10 Saxitoxins
CAS RNs: 35523-89-8 (STX) and various congeners
Method
Journal of AOAC International. 1995. 78: 528-
532
ELISA Kits for Saxitoxin
Analytical Technique
HPLC-FL (post column
derivatization)
Immunoassay (ELISA)
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 adapt and verify 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 fmol are achieved as a result of reduced band broadening and optimized reaction
conditions. This method, when applied to low-toxicity shellfish, gives higher values than the
standard mouse bioassay.
Special Considerations: AOAC is in the process of publishing multi-laboratory tested alternate
procedures for high through-put saxitoxin analysis that may be available in the near future. For
single laboratory tested procedures and more details, see Van Dolah et. al:. Journal of AOAC
International. Vol. 92, No. 6. 2009. 1705
Source: Oshima, Y. 1995. "Postcolumn Derivatization Liquid Chromatographic Method for
Paralytic Shellfish Toxins." Journal of AOAC International. 78(2): 528-532.
http://cat.inist.fr/?aModele=afficheN&cpsidt=3469391
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8.3.10.2 ELISA Kits for Saxitoxins
Analysis Purpose: Presumptive
Analytical Technique: Immunoassay (ELISA)
Method Developed for: STX in water and solid samples (e.g., shellfish)
Method Selected for: SAM lists these procedures for presumptive analysis in aerosol, solid,
particulate, liquid and water samples. Further research is needed to adapt and verify 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
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: 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) may provide an indication that they are present.
The vendor of this kit indicates that it provides "screening results... positive samples requiring
some action should be confirmed by an alternative method."
AOAC is in the process of publishing multi-laboratory tested alternate procedures for high
through-put saxitoxin analysis that may be available in the near future. For single laboratory
tested procedures and more details, see Van Dolah et. al:. Journal of AOAC International. Vol.
92. No. 6. 2009. 1705.
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
CASRN: 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 (ELISA)
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 (ELISA) and LC/APCI-MS
procedures for T-2 Mycotoxin.
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8.3.12 Tetrodotoxin
CASRN: 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 [competitive
inhibition enzyme
immunoassay (CIEIA)]
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 adapt and verify 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 selective ion monitoring (SIM) mode is estimated
to be 0.7 pmol, with a signal to noise ratio of 2:1.
Source: Shoji, Y., Yotsu-Yamashita, M., Miyazawa, T. and Yasumoto, T. 2001. "Electrospray
lonization Mass Spectrometry of Tetrodotoxin and its Analogs: Liquid Chromatography/Mass
Spectrometry, Tandem Mass Spectrometry, and Liquid Chromatography/Tandem Mass
Spectrometry." Analytical Biochemistry. 290(1): 10-17.
http://www.sciencedirect.com/science/article/pii/S0003269700949534
8.3.12.2 Literature Reference for Tetrodotoxin (Journal of Clinical Laboratory
Analysis. 1992. 6(2): 65-72)
Analysis Purpose: Presumptive
Analytical Technique: Immunoassay (CIEIA)
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 adapt and verify the
procedures for environmental sample types.
Description of Method: Procedures are described for a 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 2012
283
July 16, 2011
-------
SAM 2012 Section 9.0- Conclusions
Section 9.0: Conclusions
SAM is intended for use by EPA and EPA-contracted and -subcontracted laboratories; it also can be used
by other agencies and laboratory networks, such as the Integrated Consortium of Laboratory Networks
(ICLN). The information also can be found on the SAM website (www.epa.gov/sam), which provides
searchable links to supporting information based on SAM analytes and the analytical methods listed.
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 an intentional or
unintentional contamination event.
The primary objective of this document is to identify appropriate methods that represent a balance
between providing existing, documented, 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 by a group of technical experts in each
appropriate field. The selected methods are subject to change following further research to improve
methods or following the development of new methods. The contacts listed in Section 4.0 encourage the
scientific community to inform them of any such method improvements.
Since publication of SAM Revision 1.0 in September 2004, NHSRC has continued to convene technical
work groups to evaluate and, if necessary, update the analytes and methods that are listed. Details
regarding changes that have been incorporated into each revision of SAM are provided in Attachment 1.
SAM 2012 also reflects a title change agreed to by stakeholders (i.e., EPA's NHSRC, WSD and WLA,
Office of Emergency Response and ERLN, ORIA and Regional Offices) during a 2010 SAM Summit, to
belter reflect SAM's focus on providing selected analytical methods for use across multiple laboratories
during environmental remediation and recovery. This current revision (SAM 2012) includes the addition
of vegetation as a sample type under the radiochemistry sections, the addition of method applicability
tiers to Appendix A (Selected Chemical Methods), several new methods added or replaced for currently
listed chemical analytes, clarification of immunoassay methods listed for biotoxin analytes, the addition
of restructured pathogen sections to more clearly define scope and application.
SAM 2012 284 July 16, 2011
-------
Appendix A - Selected Chemical Methods
Appendix A: Selected Chemical Methods
SAM 2012 - Appendix A July 16, 2012
-------
SAM 2012 Appendix A: Selected Chemical Methods
The fitness of a method for an intended use is related to site-specific data quality objectives (DQOs) for a particular environmental remediation activity. These selected chemical methods have been assigned the tiers (below) to indicate a
level of method usability for the specific analyte and sample type. The assigned tiers reflect the conservative view for DQOs involving timely implementation of methods for analysis of a high number of samples (such that multiple
laboratories are necessary), low limits of identification and quantification, and appropriate quality control.
Tier I: Analyte/sample type is a target of the method(s). Data are available for all aspects of method performance and quality control measures supporting its use for
a nalysis of environmental samples following a contamination event. Evaluation and/or use of the method(s) in multiple laboratories indicate that the method can be
i mplemented with no additional modifications for the analyte/sample type.
Tier II: (1) The analyte/sample type is a target of the method(s) and the method(s) has been evaluated for the analyte/sample type by one or more laboratories, or (2) the
a nalyte/sample type is not a target of the method(s), but the method has been used by laboratories to address the analyte/sample type. In either case, available data
a nd/or information indicate that modifications will likely be needed for use of the method(s) to address the analyte/sample type.
Tier III: The analyte/sample type is not a target of the method(s), and/or no reliable data supporting the method's fitness for its intended use are available. Data from other
a nalytes or sample types, however, suggest that the method(s), with significant modification, may be applicable.
Analyte(s)
Acephate
Acrylamide
Acrylonitrile
Aldicarb (Temik)
Aldicarb sulfone
Aldicarb sulfoxide
CASRN
79-06-1
107-13-1
116-06-3
1646-88-4
1646-87-3
Determinative
Technique
HPLC
HPLC
HPLC
HPLC
Method Type
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Solid Samples
Adapted from J.
Chromatogr. A,
(2007) 11 54(1): 3-25
Water extraction
8316
(EPA SW-846)
5035A
(EPA SW-846)
8260C
(EPA SW-846)
831 8A
(EPA SW-846)
8318A
(EPA SW-846)
8318A
(EPA SW-846)
II
III
II
II
II
III
Aqueous Liquid Samples
Adapted from
Chromatographia,
63(5/6): 233-237
8316
524.2
(EPA OW)
D7645-1 0
(ASTM)
D7645-1 0
(ASTM)
D7645-10
(ASTM)
II
II
II
II
II
II
Drinking Water Samples
538
(EPA OW)
8316
(EPA SW-846)
524.2
(EPA OW)
531.2
(EPA OW)
531.2
(EPA OW)
531.2
(EPA OW)
I
II
II
I
I
I
Air Samples
Adapted from J.
Chromatogr. A,
(2007) 11 54(1): 3-25
PV2004
(OSHA)
PV2004
(OSHA)
5601
(NIOSH)
5601
(NIOSH)
5601
(NIOSH)
III
I
III
I
III
III
Wipes
Adapted from J.
Chromatogr. A,
(2007) 1154(1): 3-25
3570/8290A
Appendix A
(EPA SW-846)
8316
(EPA SW-846)
3570/8290A
Appendix A
(EPA SW-846)
8260C
(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)
III
III
III
III
III
III
SAM 2012-Appendix A
A-l
July 16, 2012
-------
Analyte(s)
Allyl alcohol
Ammonia
Ammonium metavanadate
(analyze as total vanadium)
Arsenic, Total
Arsenic trioxide
(analyze as total arsenic)
Arsine
(analyze as total arsenic in non-air
samples)
Asbestos
Boron trifluoride
Brodifacoum
CASRN
504-24-5
7664-41-7
7803-55-6
7440-38-2
1327-53-3
7784-42-1
1332-21-4
7637-07-2
56073-10-0
Determinative
Technique
HPLC
Visible
spectro photometry
ICP-AES/ICP-MS
ICP-AES / ICP-MS
ICP-AES/ICP-MS
GFAA / ICP-AES /
ICP-MS
TEM
ISE
HPLC
Method Type
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Solid Samples
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)
3050B
(EPA SW-846)
6010C/6020A
(EPA SW-846)
3050B
(EPA SW-846)
601 OC/6020A
(EPA SW-846)
D5755-03 (soft
surfaces-microvac)
(ASTM)
Not of concern
3541/3545A
(EPA SW-846)
8321 B
(EPA SW-846)
II
III
NA
I
I
I
I
III
NA
III
Aqueous Liquid Samples
5030C
(EPA SW-846)
8260C
(EPA SW-846)
3535A/8330B
(EPA SW-846)
8330B
(EPA SW-846)
4500-NH3 B
(SM)
4500- NH3 G
(SM)
200.7/200.8
(EPA OW)
200.7/200.8
(EPA OW)
200.7/200.8
(EPA OW)
200.7/200.8
(EPA OW)
Not of concern
Not of concern
D7644-10
(ASTM)
II
III
I
I
I
I
I
NA
NA
II
Drinking Water Samples
5030C
(EPA SW-846)
8260C
(EPA SW-846)
3535A/8330B
(EPA SW-846)
8330B
(EPA SW-846)
350.1
(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)
Not of concern
Not of concern
D7644-10
(ASTM)
II
III
I
I
I
I
I
NA
NA
II
Air Samples
TO-151
(EPA ORD)
Not of concern
6015
(NIOSH)
IO-3.1
(EPA ORD)
IO-3.4/IO-3.5
(EPA ORD)
IO-3.1
(EPA ORD)
IO-3.4/IO-3.5
(EPA ORD)
IO-3.1
(EPA ORD)
IO-3.4/IO-3.5
(EPA ORD)
6001
(NIOSH)
10312:1995
(ISO)
ID216SG
(OSHA)
Not of concern
III
NA
I
I
I
I
I
I
I
NA
Wipes
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)
9102
(NIOSH)
6010C/6020A
(EPA SW-846)
9102
(NIOSH)
601 OC/6020A
(EPA SW-846)
D6480-05
(hard surfaces-wipes)
(ASTM)
Not of concern
3570/8290A
Appendix A
(EPA SW-846)
8321 B
(EPA SW-846)
NA
III
NA
I
I
I
I
I
NA
III
SAM 2012-Appendix A
A-;
July 16, 2012
-------
Analyte(s)
Bromadiolone
BZ [Quinuclidinyl benzilate]
Calcium arsenate
(analyze as total arsenic)
Carbofuran (Furadan)
Carbon disulfide
Carfentanil
Chlorfenvinphos
Chlorine
2-Chloroethanol
CASRN
28772-56-7
6581-06-2
7778-44-1
1563-66-2
59708-52-0
7782-50-5
107-07-3
Determinative
Technique
HPLC
ICP-AES / ICP-MS
HPLC/LC-MS-MS
HPLC
Visible
spectrophotometry
GC-MS/GC-FID
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
Solid Samples
3541 73545 A
(EPA SW-846)
8321 B
(EPA SW-846)
3541 73545 A
(EPA SW-846)
8321 B
(EPA SW-846)
3050B
(EPA SW-846)
6010C/6020A
(EPA SW-846)
831 8A
(EPA SW-846)
5035A
(EPA SW-846)
8260C
(EPA SW-846)
3541 73545 A
(EPA SW-846)
8321 B
(EPA SW-846)
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
5035A
(EPA SW-846)
8260C
(EPA SW-846)
III
III
I
II
I
III
I
NA
II
Aqueous Liquid Samples
D7644-10
(ASTM)
3520C/3535A
(EPA SW-846)
8321 B
(EPA SW-846)
200.7/200.8
(EPA OW)
D7645-10
(ASTM)
5030C
(EPA SW-846)
8260C
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8321 B
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
4500-CI G
(SM)
5030C
(EPA SW-846)
8260C
(EPA SW-846)
II
II
I
II
I
III
I
I
II
Drinking Water Samples
D7644-1 0
(ASTM)
3520C/3535A
(EPA SW-846)
8321 B
(EPA SW-846)
200.7/200.8
(EPA OW)
531.2
(EPA OW)
524.2
(EPA OW)
3520C/3535A
(EPA SW-846)
8321 B
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
4500-CI G
(SM)
5030C
(EPA SW-846)
8260C
(EPA SW-846)
II
II
I
I
I
III
I
I
II
Air Samples
Not of concern
TO-10A
(EPA ORD)
IO-3.1
(EPA ORD)
IO-3.4/IO-3.5
(EPA ORD)
5601
(NIOSH)
TO-15
(EPA ORD)
Not of concern
TO-10A
(EPA ORD)
Adapted from Analyst
(1999) 124(12):
1853-1857
4500-CI G
(SM)
2513
(NIOSH)
NA
III
I
I
I
NA
II
II
I
Wipes
3570/8290A
Appendix A
(EPA SW-846)
8321 B
(EPA SW-846)
3570/8290A
Appendix A
(EPA SW-846)
8321 B
(EPA SW-846)
9102
(NIOSH)
6010C/6020A
(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)
8321 B
(EPA SW-846)
3570/8290A
Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
Not of concern
III
III
I
III
NA
III
II
NA
NA
SAM 2012-Appendix A
A-3
July 16, 2012
-------
Analyte(s)
Chloropicrin
Chlorosarin
Chlorosoman
2-Chlorovinylarsonous acid
(2-CVAA) (degradation product of
Lewisite) (analyze as total arsenic)
Chlorpyrifos
Chlorpyrifos oxon
Crimidine
Cyanide, Amenable to chlorination
Cyanide, Total
CASRN
76-06-2
7040-57-5
85090-33-1
2921-88-2
5598-15-2
NA
Determinative
Technique
GC-MS/GC-ECD
ICP-AES / ICP-MS
GC-MS
GC-MS
Visible
spectrophotometry
Visible
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
Solid Samples
Adapted from Eur. J.
Lipid Sci. Technol.
(2011)113:345-355
3570
(EPA SW-846)
8270D3
(EPA SW-846)
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
3050B
(EPA SW-846)
6010C/6020A
(EPA SW-846)
3570
(EPA SW-846)
8270D
(EPA SW-846)
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
3570
(EPA SW-846)
8270D4
(EPA SW-846)
3135.21
(EPA RLAB)
ISM01.3CN
(EPA CLP)
II
II
III13
III13
I
II
III
II
I
I
Aqueous Liquid Samples
Adapted from J.
Chromatogr. A
(2000) 866: 65-77
551.1
(EPA OW)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
200.7/200.8
(EPA OW)
3511
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3511
(EPA SW-846)
8270D4
(EPA SW-846)
3135.21
(EPA RLAB)
ISM01.3CN
(EPA CLP)
II
I
III13
III13
I
II
III
II
I
I
Drinking Water Samples
Adapted from J.
Chromatogr. A
(2000) 866: 65-77
551.1
(EPA OW)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
200.7/200.8
(EPA OW)
525.2
(EPA OW)
525.2
(EPA OW)
3511
(EPA SW-846)
8270D4
(EPA SW-846)
3135.21
(EPA RLAB)
335.4
(EPA OW)
II
I
III13
III13
I
II
III
II
I
I
Air Samples
TO-10A2
(EPA ORD)
PV2103(OSHA)
TO-10A2
(EPA ORD)
TO-10A2
(EPA ORD)
IO-3.1
(EPA ORD)
IO-3.4/IO-3.5
(EPA ORD)
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
Not of concern
Not of concern
6010
(NIOSH)
III
I
III13
III13
I
I
III
NA
NA
I
Wipes
Adapted from Eur. J.
Lipid Sci. Technol.
(2011) 113:345-355
3570/8290A
Appendix A
(EPA SW-846)
8270D3
(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)
8270D
(EPA SW-846)
3570/8290A
Appendix A
(EPA SW-846)
8270D4
(EPA SW-846)
3135.21
(EPA RLAB)
ISM01.3CN
(EPA CLP)
III
II
III13
III13
I
II
III
II
III
III
SAM 2012-Appendix A
A-4
July 16, 2012
-------
Analyte(s)
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)
CASRN
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
Determinative
Technique
GC-MS/GC-ECD
GC-MS
GC-MS
GC-MS
GC-FID
HPLC
Method Type
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Solid Samples
Adapted from
Encyclopedia of Anal.
Chem. (2006)
DOI: 10.1002/9780
470027318.30809
CWA Protocol
(EPA NHSRC)
5035A
(EPA SW-846)
8260C
(EPA SW-846)
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
3541 73545 A
(EPA SW-846)
801 5C
(EPA SW-846)
E2866-12
(ASTM)
3570
(EPA SW-846)
8270D
(EPA SW-846)
3541 73545 A
(EPA SW-846)
8321 B
(EPA SW-846)
II
.
I
I
I
I
II
II
III
Aqueous Liquid Samples
Adapted from
Encyclopedia of Anal.
Chem. (2006)
DOI: 10.1002/9780
470027318.30809
CWA Protocol
(EPA NHSRC)
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)
D7597-09
(ASTM)
Not of concern
3535A
(EPA SW-846)
8321 B
(EPA SW-846)
II
.
I
I
I
I
II
NA
III
Drinking Water Samples
Adapted from
Encyclopedia of Anal.
Chem. (2006)
DOI: 10.1002/9780
470027318.30809
CWA Protocol
(EPA NHSRC)
524.2
(EPA OW)
525.2
(EPA OW)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
801 5C
(EPA SW-846)
538
(EPA OW)
Not of concern
3535A
(EPA SW-846)
8321 B
(EPA SW-846)
II
.
I
I
I
I
I
NA
III
Air Samples
TO-15
(EPA ORD)
CWA Protocol
(EPA NHSRC)
TO-15
(EPA ORD)
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
Not of concern
TO-10A2
(EPA ORD)
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
III
.
I
I
I
NA
III
II
III
Wipes
Not of concern
CWA Protocol
(EPA NHSRC)
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)
8321 B
(EPA SW-846)
3570/8290A
Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
3570/8290A
Appendix A
(EPA SW-846)
8321 B
(EPA SW-846)
NA
.
NA
II
II
I
II
II
III
SAM 2012-Appendix A
A-5
July 16, 2012
-------
Analyte(s)
Diphacinone
Disulfoton
Disulfoton sulfone oxon5
Disulfoton sulfoxide
Disulfoton sulfoxide oxon5
1,4-Dithiane
(degradation product of HD)
EA2192[S-2-(diisopropylamino)ethyl
methylphosphonothioic acid]
(hydrolysis product of VX)
Ethyl methylphosphonic acid (EM PA)
(degradation product of VX)
Ethyldichloroarsine (ED)
CASRN
82-66-6
298-04-4
2496-91-5
2497-07-6
2496-92-6
505-29-3
73207-98-4
1832-53-7
Determinative
Technique
HPLC
GC-MS/GC-FPD
GC-MS/GC-FPD
GC-MS
HPLC
Method Type
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Solid Samples
3541 73545 A
(EPA SW-846)
8321 B
(EPA SW-846)
3570
(EPA SW-846)
8270D
(EPA SW-846)
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
3570
(EPA SW-846)
8270D
(EPA SW-846)
3541 73545 A
(EPA SW-846)
8321 B
(EPA SW-846)
E2866-12
(ASTM)
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
III
II
III
III
III
II
III
II
III
Aqueous Liquid Samples
D7644-1 0
(ASTM)
525.2
(EPA OW)
525.2
(EPA OW)
525.2
(EPA OW)
525.2
(EPA OW)
3511
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
8321 B
(EPA SW-846)
D7597-09
(ASTM)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
II
II
III
II
III
II
III
II
III
Drinking Water Samples
D7644-10
(ASTM)
525.2
(EPA OW)
525.2
(EPA OW)
525.2
(EPA OW)
525.2
(EPA OW)
3511
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
8321 B
(EPA SW-846)
D7597-09
(ASTM)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
II
II
III
II
III
II
III
III
III
Air Samples
Not of concern
5600
(NIOSH)
5600
(NIOSH)
5600
(NIOSH)
5600
(NIOSH)
Not of concern
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
TO-15
(EPA ORD)
NA
I
III
III
III
NA
III
III
III
Wipes
3570/8290A
Appendix A
(EPA SW-846)
8321 B
(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)
3570/8290A
Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
3570/8290A
Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
3570/8290A
Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
3570/8290A
Appendix A
(EPA SW-846)
8321 B
(EPA SW-846)
3570/8290A
Appendix A
(EPA SW-846)
8321 B
(EPA SW-846)
9102
(NIOSH)
8270D
(EPA SW-846)
III
II
III
III
III
II
III
II
III
SAM 2012-Appendix A
A-6
July 16, 2012
-------
Analyte(s)
N-Ethyldiethanolamine (EDEA)
(degradation product of HN-1)
Ethylene oxide
Fenamiphos
Fentanyl
Fluoride
Fluoroacetamide
Fluoroacetic acid and fluoroacetate salts
(analyze as fluoroacetate ion)
Formaldehyde
Gasoline range organics
CASRN
22224-92-6
437-38-7
16984-48-8
NA
50-00-0
NA
Determinative
Technique
HPLC
IC-conductivity
detection
LC-MS
FGC-ECD / HPLC
Method Type
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Solid Samples
3541 73545 A
(EPA SW-846)
8321 B
(EPA SW-846)
5035A
(EPA SW-846)
8260C
(EPA SW-846)
3570
(EPA SW-846)
8270D
(EPA SW-846)
3541 73545 A
(EPA SW-846)
8321 B
(EPA SW-846)
Not of concern
Adapted from J.
Chromatogr. B (2008)
876(1): 103-108
Adapted from J.
Chromatogr. A (2007)
1139: 271-278
5035A
(EPA SW-846)
8260C
(EPA SW-846)
831 5A
(EPA SW-846)
5035A
(EPA SW-846)
801 5C
(EPA SW-846)
III
II
II
III
NA
II
III
III
I
I
Aqueous Liquid Samples
D7599-09
(ASTM)
5030C
(EPA SW-846)
8260C
(EPA SW-846)
3511
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8321 B
(EPA SW-846)
300.1, Rev 1.0
(EPA OW)
Adapted from J.
Chromatogr. B (2008)
876(1): 103-108
Adapted from J.
Chromatogr. B(2010)
878: 1045-1050
5030C
(EPA SW-846)
8260C
(EPA SW-846)
831 5A
(EPA SW-846)
5030C
(EPA SW-846)
801 5C
(EPA SW-846)
II
II
II
II
I
II
III
III
I
I
Drinking Water Samples
D7599-09
(ASTM)
5030C
(EPA SW-846)
8260C
(EPA SW-846)
525.2
(EPA OW)
3520C/3535A
(EPA SW-846)
8321 B
(EPA SW-846)
300.1, Rev 1.0
(EPA OW)
Adapted from J.
Chromatogr. B (2008)
876(1): 103-108
Adapted from J.
Chromatogr. B (2010)
878: 1045-1050
5030C
(EPA SW-846)
8260C
(EPA SW-846)
556.1
(EPA OW)
5030C
(EPA SW-846)
801 5C
(EPA SW-846)
III
II
I
II
I
II
III
III
I
I
Air Samples
TO-10A
(EPA ORD)
TO-15
(EPA ORD)
TO-10A
(EPA ORD)
Not of concern
Not of concern
Adapted from J.
Chromatogr. B (2008)
876(1): 103-108
S301-1
(NIOSH)
Adapted from J.
Chromatogr. A (2007)
1139:271-278
2513
(NIOSH)
2016
(NIOSH)
Not of concern
III
I
II
NA
NA
III
III
III
I
NA
Wipes
EPA600/R-11/143
(EPA/NIOSH)
Not of concern
3570/8290A
Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
3570/8290A
Appendix A
(EPA SW-846)
8321 B
(EPA SW-846)
Not of concern
Adapted from J.
Chromatogr. B (2008)
876(1): 103-108
Adapted from J.
Chromatogr. A (2007)
1139: 271-278
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)
II
NA
II
III
NA
III
III
NA
III
I
SAM 2012-Appendix A
A-7
July 16, 2012
-------
Analyte(s)
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)
(degradation product of GB)
Kerosene
Lead arsenate
(analyze as total arsenic)
CASRN
283-66-9
10035-10-6
74-90-8
7783-06-4
1832-54-8
64742-81-0
Determinative
Technique
HPLC
LC-MS
IC-conductivity
detection
IC-conductivity
detection
Visible
spectrophotometry
IC-conductivity
detection
IC-conductivity
detection
HPLC/LC-MS-MS
GC-FID
Method Type
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Solid Samples
8330B
(EPA SW-846)
8330B
(EPA SW-846)
Adapted from Analyst
(2001) 126:1689-
1693
Not of concern
Not of concern
Not of concern
Not of concern
Not of concern
E2866-12
(ASTM)
5035A
(EPA SW-846)
801 5C
(EPA SW-846)
3050B
(EPA SW-846)
6010C/6020A
(EPA SW-846)
I
II
NA
NA
NA
NA
NA
II
I
I
Aqueous Liquid Samples
3535A/8330B
(EPA SW-846)
8330B
(EPA SW-846)
3535A/8330B
(EPA SW-846)
Adapted from Analyst
(2001)126:1689-
1693
Not of concern
Not of concern
Not of concern
Not of concern
Not of concern
D7597-09
(ASTM)
5030C
(EPA SW-846)
801 5C
(EPA SW-846)
200.7/200.8
(EPA OW)
I
II
NA
NA
NA
NA
NA
II
I
I
Drinking Water Samples
3535A/8330B
(EPA SW-846)
8330B
(EPA SW-846)
3535A/8330B
(EPA SW-846)
Adapted from Analyst
(2001) 126:1689-
1693
Not of concern
Not of concern
Not of concern
Not of concern
Not of concern
D7597-09
(ASTM)
5030C
(EPA SW-846)
8015C
(EPA SW-846)
200.7/200.8
(EPA OW)
I
II
NA
NA
NA
NA
NA
III
I
I
Air Samples
Not of concern
Not of concern
7903
(NIOSH)
7903
(NIOSH)
6010
(NIOSH)
79036
(NIOSH)
6013
(NIOSH)
TO-10A
(EPA ORD)
Not of concern
10-3.1
(EPA ORD)
IO-3.4/IO-3.5
(EPA ORD)
NA
NA
I
I
I
I
I
III
NA
I
Wipes
3570/8290A
Appendix A
(EPA SW-846)
8330B
(EPA SW-846)
3570/8290A
Appendix A
(EPA SW-846)
Adapted from Analyst
(2001) 126:1689-1693
Not of concern
Not of concern
Not of concern
Not of concern
Not of concern
3570/8290A
Appendix A
(EPA SW-846)
8321 B
(EPA SW-846)
3570/8290A
Appendix A
(EPA SW-846)
801 5C
(EPA SW-846)
9102
(NIOSH)
6010C/6020A
(EPA SW-846)
I
III
NA
NA
NA
NA
NA
II
I
I
SAM 2012-Appendix A
July 16, 2012
-------
Analyte(s)
Lewisite 1 (L-1) 7
[2-chlorovinyldichloroarsine]
(analyze as total arsenic)
Lewisite 2 (L-2)
[bis(2-chlorovinyl)chloroarsine]
(analyze as total arsenic)
Lewisite 3 (L-3)
[tris(2-chlorovinyl)arsine]
(analyze as total arsenic)
Lewisite oxide
(degradation product of Lewisite) (analyze
as total arsenic)
Mercuric chloride (analyze as total
mercury)
Mercury, Total
Methamidophos
Methomyl
Methoxyethylmercuric acetate
(analyze as total mercury)
Methyl acrylonitrile
CASRN
7439-97-6
16752-77-5
126-98-7
Determinative
Technique
Visible
spectrophotometry /
CVAA / CVAFS
Visible
spectrophotometry /
CVAA / CVAFS
HPLC/LC-MS-MS
Visible
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
Solid Samples
3050B
(EPA SW-846)
6010C/6020A
(EPA SW-846)
3050B
(EPA SW-846)
6010C/6020A
(EPA SW-846)
3050B
(EPA SW-846)
6010C/6020A
(EPA SW-846)
3050B
(EPA SW-846)
6010C/6020A
(EPA SW-846)
7473 8
(EPA SW-846)
7473 8
(EPA SW-846)
Adapted from J.
Chromatogr. A (2007)
1154(1): 3-25
831 8A
(EPA SW-846)
7473ฐ
(EPA SW-846)
5035A
(EPA SW-846)
8260C
(EPA SW-846)
I
I
I
I
I
I
II
II
I
II
Aqueous Liquid Samples
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. 19
(EPA OW)
245. 19
(EPA OW)
Adapted from
Chromatographia
(2006) 63(5/6):
233-237
D7645-10
(ASTM)
245. 19
(EPA OW)
524.2
(EPA OW)
I
I
I
I
I
I
II
II
I
II
Drinking Water Samples
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
(EPA OW)
245.1
(EPA OW)
538
(EPA OW)
531.2
(EPA OW)
245.1
(EPA OW)
524.2
(EPA OW)
I
I
I
I
I
I
I
I
I
II
Air Samples
10-3.1
(EPA ORD)
IO-3.4/IO-3.5
(EPA ORD)
10-3.1
(EPA ORD)
IO-3.4/IO-3.5
(EPA ORD)
10-3.1
(EPA ORD)
IO-3.4/IO-3.5
(EPA ORD)
10-3.1
(EPA ORD)
IO-3.4/IO-3.5
(EPA ORD)
Not of concern
IO-5
(EPA ORD)
Adapted from J.
Chromatogr. A (2007)
11 54(1): 3-25
5601
(NIOSH)
IO-5
(EPA ORD)
PV2004
(OSHA)
I
I
I
I
NA
I
III
I
I
III
Wipes
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)
7473s
(EPA SW-846)
9102
(NIOSH)
7473s
(EPA SW-846)
Adapted from J.
Chromatogr. A (2007)
11 54(1): 3-25
3570/8290A
Appendix A
(EPA SW-846)
831 8A
(EPA SW-846)
9102
(NIOSH)
7473s
(EPA SW-846)
3570/8290A
Appendix A
(EPA SW-846)
8260C
(EPA SW-846)
I
I
I
I
I
I
III
III
I
III
SAM 2012-Appendix A
A-9
July 16, 2012
-------
Analyte(s)
Methyl fluoroacetate
(analyze as 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)
CASRN
453-18-9
60-34-4
950-35-6
105-59-9
993-13-5
Determinative
Technique
LC-MS
Visible
spectrophotometry/
HPLC-UV
HPLC
HPLC
HPLC/LC-MS-MS
HPLC
Method Type
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Solid Samples
J. Chromatogr. A
(2007) 1139: 271-278
3541 73545 A
(EPA SW-846)
J. Chromatogr.
(1993)617: 157-162
Not of concern
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
3541 73545 A
(EPA SW-846)
8321 B
(EPA SW-846)
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
E2866-12
(ASTM)
III
III
NA
III
I
NA
III
III13
II
Aqueous Liquid Samples
J. Chromatogr. B
(2010)878:
1045-1050
J. Chromatogr. (1993)
617: 157-162
Not of concern
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
D7599-09
(ASTM)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
D7597-09
(ASTM)
III
II
NA
III
I
NA
II
III13
II
Drinking Water Samples
J. Chromatogr. B
(2010)878:
1045-1050
J. Chromatogr. (1993)
617: 157-162
Not of concern
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
D7599-09
(ASTM)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
D7597-09
(ASTM)
III
II
NA
III
I
NA
III
III13
III
Air Samples
S301-1
(NIOSH)
J. Chromatogr. A
(2007)1139: 271-278
3510
(NIOSH)
OSHA 54
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
OSHA 40
TO-10A
(EPA ORD)
TO-10A2
(EPA ORD)
TO-10A
(EPA ORD)
III
I
I
III
I
I
III
III13
III
Wipes
J. Chromatogr. A
(2007) 1139: 271-278
3570/8290A
Appendix A
(EPA SW-846)
J. Chromatogr.
(1993)617: 157-162
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)
Not of concern
EPA
600/R-11/143
(EPA/NIOSH)
3570/8290A
Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
3570/8290A
Appendix A
(EPA SW-846)
8321 B
(EPA SW-846)
III
III
NA
III
II
NA
II
III13
II
SAM 2012-Appendix A
A-10
July 16, 2012
-------
Analyte(s)
Mevinphos
Monocrotophos
Mustard, nitrogen (HN-1)
[bis(2-chloroethyl)ethylamine]
Mustard, nitrogen (HN-2)
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)
Osmium tetroxide
(analyze as total osmium)
CASRN
7786-34-7
6923-22-4
538-07-8
505-60-2
20816-12-0
Determinative
Technique
GC-MS
GC-MS
HPLC
ICP-AES
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
Solid Samples
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
CWA Protocol
(EPA NHSRC)
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
8330B
(EPA SW-846)
3050B
(EPA SW-846)
601 OC
(EPA SW-846)
I
I
III13
III13
III13
.
II
I
II
Aqueous Liquid Samples
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
CWA Protocol
(EPA NHSRC)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A/8330B
(EPA SW-846)
8330B
(EPA SW-846)
200.7/200.8
(EPA OW)
I
I
III13
III13
III13
.
II
I
II
Drinking Water Samples
525.2
(EPA OW)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
CWA Protocol
(EPA NHSRC)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A/8330B
(EPA SW-846)
8330B
(EPA SW-846)
200.7/200.8
(EPA OW)
I
I
III13
III13
III13
.
II
I
II
Air Samples
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
CWA Protocol
(EPA NHSRC)
Not of concern
Not of concern
IO-3.1
(EPA ORD)
IO-3.4
(EPA ORD)
II
III
III13
III13
III13
.
NA
NA
II
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)
8270D
(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)
CWA Protocol
(EPA NHSRC)
3570/8290A
Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
3570/8290A
Appendix A
(EPA SW-846)
8330B
(EPA SW-846)
9102
(NIOSH)
601 OC
(EPA SW-846)
II
III
III13
III13
III13
.
II
I
III
SAM 2012-Appendix A
A-ll
July 16, 2012
-------
Analyte(s)
Oxamyl
Paraoxon
Paraquat
Parathion
Pentaerythritol tetranitrate (PETN)
Phencyclidine
Phorate
Phorate sulfone
Phorate sulfone oxon5
CASRN
23135-22-0
311-45-5
78-11-5
77-10-1
2588-06-9
Determinative
Technique
GC-MS
HPLC-UV/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
Solid Samples
831 8A
(EPA SW-846)
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
Adapted from J.
Chromatogr. A (2008)
1196-1197:
110-116
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
8330B
(EPA SW-846)
3570
(EPA SW-846)
8270D
(EPA SW-846)
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
II
III
II
I
I
II
I
III
III
Aqueous Liquid Samples
D7645-1 0
(ASTM)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
549.2
(EPA OW)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A/8330B
(EPA SW-846)
8330B
(EPA SW-846)
3511
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
II
III
I
I
I
II
I
III
III
Drinking Water Samples
531.2
(EPA OW)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
549.2
(EPA OW)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A/8330B
(EPA SW-846)
8330B
(EPA SW-846)
3511
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
I
III
I
I
I
II
I
III
III
Air Samples
5601
(NIOSH)
TO-10A
(EPA ORD)
Not of concern
TO-10A
(EPA ORD)
Not of concern
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
I
III
NA
III
NA
II
II
III
III
Wipes
3570/8290A
Appendix A
(EPA SW-846)
831 8A
(EPA SW-846)
3570/8290A
Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
Adapted from J.
Chromatogr. A (2008)
1196-1197:
110-116
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)
8270D
(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)
III
III
III
II
I
II
II
III
III
SAM 2012-Appendix A
A-12
July 16, 2012
-------
Analyte(s)
Phorate sulfoxide
Phorate sulfoxide oxon5
Phosgene
Phosphamidon
Phosphine
Phosphorus trichloride
Pinacolyl methyl phosphonic acid (PMPA)
(degradation product of GD)
Propylene oxide
R 33 (VR)
[methylphosphonothioic acid, S-[2-
ester]
Sarin (GB)
CASRN
2588-03-6
2588-05-8
75-44-5
7719-12-2
616-52-4
75-56-9
159939-87-4
107-44-8
Determinative
Technique
GC-MS
GC-NPD
Visible
spectrophotometry
Visible
spectrophotometry
HPLC/LC-MS-MS
GC-MS /GC-FID
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
Solid Samples
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
Not of concern
E2866-12
(ASTM)
5035A
(EPA SW-846)
8260C
(EPA SW-846)
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
CWA Protocol
(EPA NHSRC)
III
III
NA
I
NA
NA
II
II
III13
.
Aqueous Liquid Samples
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
Not of concern
D7597-09
(ASTM)
5030C
(EPA SW-846)
8260C
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
CWA Protocol
(EPA NHSRC)
III
III
NA
I
NA
NA
II
II
III13
.
Drinking Water Samples
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
Not of concern
D7597-09
(ASTM)
5030C
(EPA SW-846)
8260C
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
CWA Protocol
(EPA NHSRC)
III
III
NA
I
NA
NA
III
II
III13
.
Air Samples
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
OSHA 61
TO-10A
(EPA ORD)
6002
(NIOSH)
6402
(NIOSH)
TO-10A
(EPA ORD)
1612
(NIOSH)
TO-10A
(EPA ORD)
CWA Protocol
(EPA NHSRC)
III
III
I
II
I
I
III
I
III13
.
Wipes
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
3570/8290A
Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
Not of concern
Not of concern
3570/8290A
Appendix A
(EPA SW-846)
8321 B
(EPA SW-846)
Not of concern
3570/8290A
Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
CWA Protocol
(EPA NHSRC)
III
III
NA
II
NA
NA
II
NA
III13
.
SAM 2012-Appendix A
A-13
July 16, 2012
-------
Analyte(s)
Sodium arsenite
(analyze as total arsenic)
Sodium azide
(analyze as azide ion)
So man (GD)
Strychnine
Tabun (GA)
Tetraethyl pyrophosphate (TEPP)
Tetramethylenedisulfotetramine (TETS)
Thallium sulfate
(analyze as total thallium)
Thiodiglycol (TDG)
(degradation product of HD)
Thiofanox
CASRN
26628-22-8
107-49-3
80-12-6
39196-18-4
Determinative
Technique
1C CD
GC-MS
GC-MS
HPLC/LC-MS-MS
HPLC
Method Type
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Solid Samples
3050B
(EPA SW-846)
6010C/6020A
(EPA SW-846)
Adapted from J.
Forensic Sci. (1998)
43(1): 200-202 10
300.1, RevlO11
(EPA OW)
CWA Protocol
(EPA NHSRC)
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
3570
(EPA SW-846)
8270D
(EPA SW-846)
3570
(EPA SW-846)
8270D3
(EPA SW-846)
3050B
(EPA SW-846)
6010C/6020A
(EPA SW-846)
E2787-11
(ASTM)
3541 73545 A
(EPA SW-846)
8321 B
(EPA SW-846)
I
II
.
I
III13
II
II
I
II
III
Aqueous Liquid Samples
200.7/200.8
(EPA OW)
Adapted from J.
Forensic Sci. (1998)
43(1): 200-202 10
300.1, Revl.O11
(EPA OW)
CWA Protocol
(EPA NHSRC)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3511
(EPA SW-846)
8270D
(EPA SW-846)
3511
(EPA SW-846)
8270D3
(EPA SW-846)
200.7/200.8
(EPA OW)
D7598-09
(ASTM)
D7645-1 0
(ASTM)
I
II
.
I
III13
II
II
I
II
II
Drinking Water Samples
200.7/200.8
(EPA OW)
Adapted from J.
Forensic Sci. (1998)
43(1): 200-202 10
300.1, Rev 1.011
(EPA OW)
CWA Protocol
(EPA NHSRC)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3535A
(EPA SW-846)
8270D
(EPA SW-846)
3511
(EPA SW-846)
8270D
(EPA SW-846)
EPA 600/R-1 1/091
(EPA/CDC)
200.7/200.8
(EPA OW)
D7598-09
(ASTM)
538
(EPA OW)
I
II
.
I
III13
II
II
I
III
I
Air Samples
10-3.1
(EPA ORD)
IO-3.4/IO-3.5
(EPA ORD)
ID-211 (OSHA)
CWA Protocol
(EPA NHSRC)
Not of concern
TO-10A
(EPA ORD)
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)
I
I
.
NA
III13
II
II
I
III
III
Wipes
9102
(NIOSH)
6010C/6020A
(EPA SW-846)
CWA Protocol
(EPA NHSRC)
3570/8290A
Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
3570/8290A
Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
3570/8290A
Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
3570/8290A
Appendix A
(EPA SW-846)
8270D3
(EPA SW-846)
9102
(NIOSH)
6020A/6010C
(EPA SW-846)
E2838-1 1
(ASTM)
3570/8290A
Appendix A
(EPA SW-846)
8321 B
(EPA SW-846)
I
I
.
II
III13
II
II
I
II
III
SAM 2012-Appendix A
A-14
July 16, 2012
-------
Analyte(s)
1,4-Thioxane
(degradation product of HD)
Titanium tetrachloride
(analyze as 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 as total vanadium)
VE [phosphonothioic acid, ethyl-, S-(2-
(diethylamino)ethyl) O-ethyl ester]
VG [phosphonothioic acid, S-(2-
CASRN
15980-15-1
7550-45-0
102-71-6
121-45-9
99-35-4
118-96-7
78-53-5
Determinative
Technique
ICP-AES/ICP-MS
HPLC/LC-MS-MS
GC-MS
HPLC
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
Solid Samples
3570
(EPA SW-846)
8270D12
(EPA SW-846)
3050B
(EPA SW-846)
601 OC/6020A
(EPA SW-846)
3541/3545A
(EPA SW-846)
8321 B
(EPA SW-846)
3541/3545A
(EPA SW-846)
8270D3
(EPA SW-846)
8330B
(EPA SW-846)
8330B
(EPA SW-846)
3050B
(EPA SW-846)
6010C/6020A
(EPA SW-846)
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
II
I
III
III
I
I
I
III13
III13
Aqueous Liquid Samples
3511
(EPA SW-846)
8270D12
(EPA SW-846)
Not of concern
D7599-09
(ASTM)
Not of concern
3535A/8330B
(EPA SW-846)
8330B
(EPA SW-846)
3535A/8330B
(EPA SW-846)
8330B
(EPA SW-846)
200.7/200.8
(EPA OW)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
II
NA
II
NA
I
I
I
III13
III13
Drinking Water Samples
3511
(EPA SW-846)
8270D12
(EPA SW-846)
Not of concern
D7599-09
(ASTM)
Not of concern
3535A/8330B
(EPA SW-846)
8330B
(EPA SW-846)
3535A/8330B
(EPA SW-846)
8330B
(EPA SW-846)
200.7/200.8
(EPA OW)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
II
NA
III
NA
I
I
I
III13
III13
Air Samples
Not of concern
Not of concern
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
Not of concern
Not of concern
10-3.1
(EPA ORD)
IO-3.4/IO-3.5
(EPA ORD)
TO-10A
(EPA ORD)
TO-10A
(EPA ORD)
NA
NA
III
III
NA
NA
I
III13
III13
Wipes
3570/8290A
Appendix A
(EPA SW-846)
8270D12
(EPA SW-846)
9102
(NIOSH)
601 OC/6020A
(EPA SW-846)
(EPA/NIOSH)
3570/8290A
Appendix A
(EPA SW-846)
8270D3
(EPA SW-846)
3570/8290A
Appendix A
(EPA SW-846)
8330B
(EPA SW-846)
3570/8290A
Appendix A
(EPA SW-846)
8330B
(EPA SW-846)
9102
(NIOSH)
6010C/6020A
(EPA SW-846)
3570/8290A
Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
3570/8290A
Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
II
III
II
III
I
I
I
III13
III13
SAM 2012-Appendix A
A-15
July 16, 2012
-------
Analyte(s)
VM [phosphonothioic acid,
ester]
VX [0-ethyl-S-(2-
diisopropylaminoethyl)methyl-
phosphonothiolate]
White phosphorus
CASRN
50782-69-9
12185-10-3
Determinative
Technique
GC-NPD / GC-FPD
Method Type
Sample Prep
Determinative
Sample Prep
Determinative
Sample Prep
Determinative
Solid Samples
3541 73545 A
(EPA SW-846)
8270D
(EPA SW-846)
CWA Protocol
(EPA NHSRC)
7580
(EPA SW-846)
III13
.
I
Aqueous Liquid Samples
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
CWA Protocol
(EPA NHSRC)
7580
(EPA SW-846)
III13
.
I
Drinking Water Samples
3520C/3535A
(EPA SW-846)
8270D
(EPA SW-846)
CWA Protocol
(EPA NHSRC)
7580
(EPA SW-846)
III13
.
I
Air Samples
TO-10A
(EPA ORD)
CWA Protocol
(EPA NHSRC)
7905
(NIOSH)
III13
.
I
Wipes
3570/8290A
Appendix A
(EPA SW-846)
8270D
(EPA SW-846)
CWA Protocol
(EPA NHSRC)
3570/8290A
Appendix A
(EPA SW-846)
7580
(EPA SW-846)
III13
.
Ill
* Only laboratories approved under the ERLN umbrella are designated for handling the CWA standards needed for this method. For access to the nearest ERLN laboratory specially trained and equipped for CWA analysis,
contact the EPA Headquarters Emergency Operations Center (EPA/HQ-EOC) at 202-564-3850.
Footnotes
1 If problems occur when using this method, TO-10A should be used.
2 If problems occur when using this method, the canister Method TO-15 should be used.
3 If problems occur with analyses, lower the injection temperature.
4 If problems occur when using this method, SW-846 Method 8321B should be used as the Determinative method. Sample preparation methods should remain the same.
5 If problems occur during measurement of oxon compounds, analysts should consider use of procedures included in Kamal, A. ef a/., "Oxidation of selected organophosphate pesticides during chlorination of simulated
drinking water." Water Research. 2009. 43(2): 522-534. http://www.sciencedirect.com/science/iournal/00431354.
6 If problems occur when using this method, NIOSH Method 7906 should be used.
7 Laboratory testing is currently under way for speciation of Lewisite 1 using GC-MS techniques.
8 If equipment is not available or problems occur when analyzing solid and wipe samples, use CVAA Method 7471B (EPA SW-846).
9 If problems occur when using EPA Method 245.1 for these analytes during preparation and analysis of aqueous liquid samples, refer to EPA Method 7470A (SW-846).
10 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.
11 If analyses are problematic, refer to column manufacturer for alternate conditions.
12 If problems occur when using this method, SW-846 Method 8260C and appropriate corresponding sample preparation procedures (i.e., 5035A for solid samples, and 5030C for aqueous liquid and drinking water samples) should
be used
13 Data are not available for this analyte/sample type using this method. However, the referenced SW-846 method or the CWA Protocol may be applicable.
SAM 2012-Appendix A
A-16
July 16, 2012
-------
SAM 2012
July 16, 2011
-------
Appendix B - Selected Radiochemical Methods
Appendix B: Selected Radiochemical Methods
SAM2012 -AppendixB July 16, 2012
-------
SAM 2012 Appendix B: Selected Radiochemical Methods
Analyte Class
Gross Alpha
Gross Beta
Gamma
Select Mixed Fission Products1
Total Activity Screening
Determinative
Technique
Alpha / Beta
counting
Alpha / Beta
counting
Gamma
spectrometry
Gamma
spectrometry
Liquid
scintillation
Drinking Water Samples
900.0
(EPA)
900.0
(EPA)
901.1
(EPA)
901.1
(EPA)
Preparation of Samples for
Total Activity Screening
(Y-12)
Aqueous and Liquid Phase
Samples
7110B
(SM)
7110B
(SM)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Preparation of Samples for
Total Activity Screening
(Y-12)
Soil and Sediment Samples
AP1
(ORISE)
AP1
(ORISE)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Preparation of Samples for
Total Activity Screening
(Y-12)
Surface Wipes
FRMAC, Vol 2, pg. 33
(DOE)
FRMAC, Vol 2, pg. 33
(DOE)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Preparation of Samples for
Total Activity Screening
(Y-12)
Air Filters
FRMAC, Vol 2, pg. 33
(DOE)
FRMAC, Vol 2, pg. 33
(DOE)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Preparation of Samples for
Total Activity Screening
(Y-12)
Vegetation Samples
AP1
(ORISE)
AP1
(ORISE)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Preparation of Samples for
Total Activity Screening
(Y-12)
Analyte(s)
Americium-2414
Californium-2524
Cesium-137
Cobalt-60
Curium-2444
Europium-154
lodine-125
lodine-131
lridium-192
Molybdenum-99
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
14119-15-4
Determinative
Technique
Alpha
spectrometry
Gamma
spectrometry
Alpha
spectrometry
Gamma
spectrometry
Gamma
spectrometry
Alpha
spectrometry
Gamma
spectrometry
Gamma
spectrometry
Gamma
spectrometry
Gamma
spectrometry
Gamma
spectrometry
Drinking Water Samples
Qualitative
Determination2
Rapid
Radiochemical
Method for
Am-2413
(EPA)
901.1
(EPA)
D3084-05
(ASTM)
901.1
(EPA)
901.1
(EPA)
D3084-05
(ASTM)
901.1
(EPA)
Procedure #9
(ORISE)
901.1
(EPA)
901.1
(EPA)
901.1
(EPA)
Confirmatory
Am-04-RC
(HASL-300)
901.1
(EPA)
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)
901.1
(EPA)
Aqueous and Liquid Phase
Samples
Qualitative
Determination2
D3084-05
(ASTM)
7120
(SM)
D3084-05
(ASTM)
7120
(SM)
7120
(SM)
D3084-05
(ASTM)
7120
(SM)
Procedure #9
(ORISE)
Ga-01-R
(HASL-300)
7120
(SM)
Ga-01-R
(HASL-300)
Confirmatory
Am-04-RC
(HASL-300)
7120
(SM)
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)
Ga-01-R
(HASL-300)
Soil and Sediment Samples
Qualitative
Determination2
Actinides and
Sr-89/90 in
Soil Samples
(DOE SRS)
Ga-01-R
(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)
Ga-01-R
(HASL-300)
Confirmatory
Am-01-RC5
(HASL-300)
Ga-01-R
(HASL-300)
Am-01-RC5
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Am-01-RC5
(HASL-300)
Ga-01-R
(HASL-300)
Procedure #9
(ORISE)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Surface Wipes
Qualitative
Determination2
Rapid methods*
for acid or fusion
digestion
(EPA)
Ga-01-R
(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)
Ga-01-R
(HASL-300)
Confirmatory
Am-04-RC
(HASL-300)
Ga-01-R
(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)
Ga-01-R
(HASL-300)
Air Filters
Qualitative
Determination2
Rapid methods*
for acid or fusion
digestion
(EPA)
Ga-01-R
(HASL-300)
D3084-05
(ASTM)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
D3084-05
(ASTM)
Ga-01-R
(HASL-300)
Procedure #96
(ORISE)
Ga-01-R6
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Confirmatory
Am-04-RC
(HASL-300)
Ga-01-R
(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 #96
(ORISE)
Ga-01-R6
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Vegetation Samples
Qualitative
Determination2
Actinides and
Sr-89/90 in
Vegetation
(DOE SRS)
Ga-01-R
(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)
Ga-01-R
(HASL-300)
Confirmatory
Am-06-RC
(HASL-300)
Ga-01-R
(HASL-300)
Am-06-RC
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Am-06-RC
(HASL-300)
Ga-01-R
(HASL-300)
Procedure #9
(ORISE)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
SAM2012 -AppendixB
-------
Analyte(s)
Phosphorus-32
Plutonium-2384
Plutonium-2394
Polonium-210
Radium-226
Ruthenium-103
Ruthenium-106
Selenium-75
Strontium-89
Strontium-90
Technetium-99
Tritium
(Hydrogen-3)
Uranium-2344
CASRN
14596-37-3
13981-16-3
15117-48-3
13981-52-7
13982-63-3
13968-53-1
13967-48-1
14265-71-5
14158-27-1
10098-97-2
14133-76-7
10028-17-8
13966-29-5
Determinative
Technique
Liquid
scintillation /
Beta counting
Alpha
spectrometry
Alpha
spectrometry
Alpha
spectrometry
Alpha
spectrometry /
Radon
emanation
Gamma
spectrometry
Gamma
spectrometry
Gamma
spectrometry
Beta counting
Beta counting
Liquid
scintillation /
beta counting
Liquid
scintillation
Alpha
spectrometry
Drinking Water Samples
Qualitative
Determination2
Rapid
Radiochemical
Method for P-32
in water3
(EPA)
Rapid
Radiochemical
Method for
Pu3
(EPA)
Rapid
Radiochemical
Method for
Pu3
(EPA)
Po-02-RC
(HASL-300)
Rapid
Radiochemical
Method for
Ra-2263
(EPA)
901.1
(EPA)
901.1
(EPA)
901.1
(EPA)
905.0
(EPA)
Rapid
Radiochemical
Methods for
Sr-903
(EPA)
Tc-02-RC
(HASL-300)
906.0
(EPA)
Rapid
Radiochemical
Method for U3
(EPA)
Confirmatory
R4-73-014
(EPA)
EMSL-33
(EPA)
EMSL-33
(EPA)
Po-02-RC
(HASL-300)
903.1
(EPA)
901.1
(EPA)
901.1
(EPA)
901.1
(EPA)
905.0
(EPA)
905.0
(EPA)
Tc-02-RC
(HASL-300)
906.0
(EPA)
D3972-02
(ASTM)
Aqueous and Liquid Phase
Samples
Qualitative
Determination2
R4-73-014
(EPA)
D3084-05
(ASTM)
D3084-05
(ASTM)
Po-02-RC
(HASL-300)
7500-Ra B
(SM)
7120
(SM)
7120
(SM)
7120
(SM)
905.0
(EPA)
D5811-08
(ASTM)
D7 168-05
(ASTM)
906.0
(EPA)
7500-U B8
(SM)
Confirmatory
R4-73-014
(EPA)
EMSL-33
(EPA)
EMSL-33
(EPA)
Po-02-RC
(HASL-300)
7500-Ra C
(SM)
7120
(SM)
7120
(SM)
7120
(SM)
905.0
(EPA)
D5811-08
(ASTM)
D7 168-05
(ASTM)
906.0
(EPA)
7500-U C
(SM)
Soil and Sediment Samples
Qualitative
Determination2
RESL P-2
(DOE)
Actinides and
Sr-89/90 in
Soil Samples
(DOE SRS)
Actinides and
Sr-89/90 in
Soil Samples
(DOE SRS)
Po-02-RC
(HASL-300)
D3084-05
(ASTM)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Actinides and
Sr-89/90 in
Soil Samples
(DOE SRS)
Actinides and
Sr-89/90 in
Soil Samples
(DOE SRS)
APS
(ORISE)
AP2
(ORISE)
Actinides and
Sr-89/90 in
Soil Samples
(DOE SRS)
Confirmatory
RESL P-2
(DOE)
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)
Strontium in
Food and
Bioenvironmental
Samples
(EPA)
Sr-03-RC
(HASL-300)
APS
(ORISE)
AP2
(ORISE)
EMSL-33
(EPA)
Surface Wipes
Qualitative
Determination2
RESL P-2
(DOE)
Rapid methods*
for acid or fusion
digestion
(EPA)
Rapid methods*
for acid or fusion
digestion
(EPA)
Method 1 1 1
(EPA)
Rapid methods*
for acid or fusion
digestion
(EPA)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Confirmatory
RESL P-2
(DOE)
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)
Strontium in Food and
Bioenvironmental Samples
(EPA)
Rapid methods*
for acid or fusion
digestion
(EPA)
APS
(ORISE)
AP2
(ORISE)
Rapid methods*
for acid or fusion
digestion
(EPA)
Sr-03-RC
(HASL-300)
APS
(ORISE)
AP2
(ORISE)
EMSL-33
(EPA)
Air Filters
Qualitative
Determination2
RESL P-2
(DOE)
Rapid methods*
for acid or fusion
digestion
(EPA)
Rapid methods*
for acid or fusion
digestion
(EPA)
Method 1 1 1
(EPA)
Rapid methods*
for acid or fusion
digestion
(EPA)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Confirmatory
RESL P-2
(DOE)
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)
Strontium in Food and
Bioenvironmental Samples
(EPA)
Rapid methods*
for acid or fusion
digestion
(EPA)
APS
(ORISE)
Not
applicable7
Rapid methods*
for acid or fusion
digestion
(EPA)
Sr-03-RC
(HASL-300)
APS
(ORISE)
Not applicable7
EMSL-33
(EPA)
Vegetation Samples
Qualitative
Determination2
RESL P-2
(DOE)
Actinides and
Sr-89/90 in
Vegetation
(DOE SRS)
Actinides and
Sr-89/90 in
Vegetation
(DOE SRS)
Po-02-RC
(HASL-300)
Ra-03-RC
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Actinides and
Sr-89/90 in
Vegetation
(DOE SRS)
Actinides and
Sr-89/90 in
Vegetation
(DOE SRS)
APS
(ORISE)
AP2
(ORISE)
Actinides and
Sr-89/90 in
Vegetation
(DOE SRS)
Confirmatory
RESL P-2
(DOE)
Am-06-RC
(HASL-300)
Am-06-RC
(HASL-300)
Po-02-RC
(HASL-300)
Ra-03-RC
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Strontium in Food
and
Bioenvironmental
Samples
(EPA)
Sr-03-RC
(HASL-300)
Tc-01-RC
(HASL-300)
AP2
(ORISE)
U-02-RC
(HASL-300)
SAM2012 -AppendixB
-------
Analyte(s)
Uranium-2354
Uranium-2384
CASRN
15117-96-1
7440-61-1
Determinative
Technique
Alpha
spectrometry
Alpha
spectrometry
Drinking Water Samples
Qualitative
Determination2
Rapid
Radiochemical
Method for U3
(EPA)
Rapid
Radiochemical
Method for U3
(EPA)
Confirmatory
D3972-02
(ASTM)
D3972-02
(ASTM)
Aqueous and Liquid Phase
Samples
Qualitative
Determination2
7500-U B8
(SM)
7500-U B8
(SM)
Confirmatory
7500-U C
(SM)
7500-U C
(SM)
Soil and Sediment Samples
Qualitative
Determination2
Actinides and
Sr-89/90 in Soil
Samples
(DOE SRS)
Actinides and
Sr-89/90 in
Soil Samples
(DOE SRS)
Confirmatory
EMSL-33
(EPA)
EMSL-33
(EPA)
Surface Wipes
Qualitative
Determination2
Rapid methods*
for acid or fusion
digestion
(EPA)
Rapid methods*
for acid or fusion
digestion
(EPA)
Confirmatory
EMSL-33
(EPA)
EMSL-33
(EPA)
Air Filters
Qualitative
Determination2
Rapid methods*
for acid or fusion
digestion
(EPA)
Rapid methods*
for acid or fusion
digestion
(EPA)
Confirmatory
EMSL-33
(EPA)
EMSL-33
(EPA)
Vegetation Samples
Qualitative
Determination2
Actinides and
Sr-89/90 in
Vegetation
(DOE SRS)
Actinides and
Sr-89/90 in
Vegetation
(DOE SRS)
Confirmatory
U-02-RC
(HASL-300)
U-02-RC
(HASL-300)
Footnotes
1 Please note that this category does not cover all fission products. In addition to the specific radionuclides listed in this appendix, gamma-rav spectometrv with a high resoution HP(Ge) detector will identify and guantifv fission products with gamma
rays in the energy range of 30 keV to 2000 keV. The sensitivity will be dependent on the detector efficiency and the gamma-ray emission probabilities (branching ratio) for the specific radionuclide.
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 SAM lists this method for rapid qualitative screening of drinking water samples. The method is not intended for use in compliance monitoring of drinking water.
4 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.
5 In cases where only small sample volumes (ฃ100 g) are available, use HASL-300 Method Pu-12-RC.
6 This procedure should be used only for filters specifically designed for iodine.
7 Because tritium is not sampled using traditional air filters, this matrix is not applicable.
8 This method was developed for measurement of total uranium and does not distinguish between uranium isotopes.
* These rapid methods describe wipe and air filter digestion procedures, and include references to the analyte-specific separation procedures listed for rapid analysis of drinking water samples, to be used to complete anlaysis of the digested samples.
SAM2012 -AppendixB
-------
Appendix C - Selected Pathogen Methods
Appendix C: Selected Pathogen Methods
SAM2012 -Appendix C July 16, 2011
-------
SAM 2012 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 an appropriate replacement 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., for post decontamination phase samples), a viability-based procedure (such as culture) should be used. Rapid analysis techniques (such as PCR,
immunoassays) without culture are preferred for determination of extent and magnitude of contamination (e.g., for site characterization phase samples). Please see Figure 7-1.
Pathogen(s)
[Disease]
Analytical
Technique
Method Type
Analytical Method
Aerosol
(growth media, filter, liquid)
Particulate
(swabs, wipes, Sponge-Sticks,
vacuum socks and filters)
_ . . . ,, . Post Decontamination Waste
Drinking Water Wa(er
Bacteria
Bacillus anthracis (BA)
[Anthrax]
Brucella spp.
(B. abortus, B. melitensis,
B. suis)
[Brucellosis]
Culture
Real-time PCR/
RV-PCR
Culture
Real-time PCR
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
EPA BA Protocol (Anticipated
publication October 2012)
Hodges et a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03)
EPA BA Protocol (Anticipated publication October 2012)
EPA BA Protocol (Anticipated
publication October 2012)
Hodges et a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03)
EPA BA Protocol (Anticipated publication October 2012)
EPA BA Protocol (Anticipated
publication October 2012)
Hodges et a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03)
ASM Sentinel Level Clinical Microbiology Laboratory Guidelines for Suspected Agents ff Bioterrorism and Emerging Infectious Diseases: Brucella
species
EPA BA Protocol (Anticipated
publication October 2012)
Hodges et a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03)
Hinicefa/. 2008. J. Microbiol. Methods. 75(2): 375-378.
SAM 2012-Appendix C
C-1
July 16, 2012
-------
Pathogen(s)
[Disease]
Burkholdeha mallei
[Glanders] and
Burkholdeha pseudomallei
[Melioidosis]
Campylobacterjejuni
[Campylobacteriosis]
Chlamydophila psittaci
(formerly known as
Chlamydia psittaci)
[Psittacosis]
Analytical
Technique
Culture
Real-time PCR
Culture
Real-time PCR
Tissue culture
PCR
Method Type
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Analytical Method
Aerosol
(growth media, filter, liquid)
EPA BA Protocol (Anticipated
publication October 2012)
Particulate
(swabs, wipes, Sponge-Sticks,
vacuum socks and filters)
Hodges ef a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose ef
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
_ . . . ,., . Post Decontamination Waste
Drinking Water Wa(er
EPA Report (EPA 600/R-1 1/1 03)
ASM Sentinel Level Clinical Microbiology Laboratory Guidelines for Suspected Agents of Bioterrorism and Emerging Infectious Diseases: Burkholdeha
mallei and B. pseudomallei
EPA BA Protocol (Anticipated
publication October 2012)
Hodges ef a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose ef
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03)
Tomasoefa/. 2006. Clin. Chem. 52(2): 307-310, Novak ef a/. 2006. J. Clin. Microbiol. 44(1): 85-90, and Meumannefa/. 2006 J. Clin. Microbiol. 44(8):
3028-3030
EPA BA Protocol (Anticipated
publication October 2012)
Hodges ef a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose ef
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03)
ISO 17795
EPA BA Protocol (Anticipated
publication October 2012)
Hodges ef a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose ef
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03)
Cunningham ef a/. 2010. J. Clin. Microbiol. 48(8): 2929-2933.
EPA BA Protocol (Anticipated
publication October 2012)
Hodges ef a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose ef
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03)
Madicoefa/. 2000. J. Clin. Microbiol. 38(3): 1085-1093.
EPA BA Protocol (Anticipated
publication October 2012)
Hodges ef a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose ef
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03)
Madicoefa/. 2000. J. Clin. Microbiol. 38(3): 1085-1093.
SAM 2012- Appendix C
C-2
July 16, 2012
-------
Pathogen(s)
[Disease]
Coxiella burnetii
[Q-fever]
Escherichia coli O157:H7
Francisella tularensis
[Tularemia]
Analytical
Technique
Tissue Culture
Real-time PCR
Culture
Real-time PCR
Culture
Real-time PCR
Method Type
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Analytical Method
Aerosol
(growth media, filter, liquid)
EPA BA Protocol (Anticipated
publication October 2012)
Particulate
(swabs, wipes, Sponge-Sticks,
vacuum socks and filters)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
Drinking Water
Post Decontamination Waste
Water
EPA Report (EPA 600/R-1 1/1 03)
Raoultefa/. 1991. Antimicrob. Agents Chemother. 35(10): 2070-2077.
EPA BA Protocol (Anticipated
publication October 2012)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03)
Panning et al. 2008. BMC Microbiol. 8:77.
EPA BA Protocol (Anticipated
publication October 2012)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-11/103) or EPA Protocol (EPA/600/R-1 0/056)
EPA Protocol (EPA/600/R-1 0/056)
EPA BA Protocol (Anticipated
publication October 2012)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-11/103) or EPA Protocol (EPA/600/R-1 0/056)
Sen et al. 2011. Environ. Sci. Technol. 45(7): 2250-2256.
EPA BA Protocol (Anticipated
publication October 2012)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03) and Humrighouse et al. 201 1 . Appl.
Environ. Microbiol. 77(18): 6729-6732.
CDC, ASM and APHL: Sentinel Level Clinical Microbiology Laboratory Guidelines for Suspected Agents of Bioterrorism and Emerging Infectious
Diseases: Francisella tularensis
EPA BA Protocol (Anticipated
publication October 2012)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03)
Versageefa/. 2003. J. Clin. Microbiol. 41(12): 5492-5499.
SAM 2012- Appendix C
C-3
July 16, 2012
-------
Pathogen(s)
[Disease]
Leptospira
[Leptospirosis]
Listeria monocytogenes
[Listeriosis]
Non-typhoidal Salmonella
(Not applicable to
S. Typhi)
[Salmonellosis]
Analytical
Technique
Culture
Real-time PCR
Culture
Real-time PCR
Culture
Real-time PCR
Method Type
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Analytical Method
Aerosol
(growth media, filter, liquid)
EPA BA Protocol (Anticipated
publication October 2012)
Particulate
(swabs, wipes, Sponge-Sticks,
vacuum socks and filters)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al . 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
_ . . . ,., . Post Decontamination Waste
Drinking Water Wa(er
EPA Report (EPA 600/R-1 1/1 03)
Standard Method 9260 I: Leptospira
EPA BA Protocol (Anticipated
publication October 2012)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03)
Palaniappanefa/. 2005. Mol. Cell Probes. 19(2): 111-117.
EPA BA Protocol (Anticipated
publication October 2012)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03)
FDA, CFSAN. 2003. Bacteriological Analytical Manual Online.
EPA BA Protocol (Anticipated
publication October 2012)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03)
USDA, FSIS. 2009. Microbiology Laboratory Guidebook MLG 8A.04.
EPA BA Protocol (Anticipated
publication October 2012)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1 /1 03) and EPA Method 1 200 (EPA 81 7-R-1 2-
004)
EPA Method 1682 (EPA-821-R-06-14) or EPA Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
EPA BA Protocol (Anticipated
publication October 2012)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1 /1 03) and EPA Method 1 200 (EPA 81 7-R-1 2-
004)
Jyoti etal. Environ. Sci. Technol. 45(20): 8996-9002.
SAM 2012- Appendix C
C-4
July 16, 2012
-------
Pathogen(s)
[Disease]
Salmonella Typhi
[Typhoid fever]
Shigella spp.
[Shigellosis]
Staphylococcus aureus
Analytical
Technique
Culture
Real-time PCR
Culture
Real-time PCR
Culture
Real-time PCR
Method Type
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Analytical Method
Aerosol
(growth media, filter, liquid)
EPA BA Protocol (Anticipated
publication October 2012)
Particulate
(swabs, wipes, Sponge-Sticks,
vacuum socks and filters)
Hodges ef a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose ef
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
_ . . . ,., . Post Decontamination Waste
Drinking Water Wa(er
EPA Report (EPA 600/R-11/103) or EPA protocol (EPA 600/R-10/133)
EPA Protocol (EPA 600/R-1 0/1 33)
EPA BA Protocol (Anticipated
publication October 2012)
Hodges ef a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose ef
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-11/103) or EPA protocol (EPA 600/R-10/133)
CDC Laboratory Assay
EPA BA Protocol (Anticipated
publication October 2012)
Hodges ef a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose ef
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03)
Standard Method 9260 E: Shigella
EPA BA Protocol (Anticipated
publication October 2012)
Hodges ef a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose ef
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03)
Cunningham ef a/. 2010. J. Clin. Microbiol. 48(8): 2929-2933.
EPA BA Protocol (Anticipated
publication October 2012)
Hodges ef a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose ef
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03)
Standard Method 9213 B: Staphylococcus aureus
EPA BA Protocol (Anticipated
publication October 2012)
Hodges ef a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose ef
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03)
Chiang ef a/. 2007. J. Food Prot. 70(12): 2855-2859.
SAM 2012- Appendix C
C-5
July 16, 2012
-------
Pathogen(s)
[Disease]
Vibrio cholerae O1 and
0139
[Cholera]
Yersinia pestis
[Plague]
Analytical
Technique
Culture
Real-time PCR
Culture
Real-time PCR
Method Type
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Analytical Method
Aerosol
(growth media, filter, liquid)
EPA BA Protocol (Anticipated
publication October 2012)
Particulate
(swabs, wipes, Sponge-Sticks,
vacuum socks and filters)
Hodges ef a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose ef
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
Drinking Water
Post Decontamination Waste
Water
EPA Report (EPA 600/R-11/103) or EPA Protocol (EPA 600/R-1 0/1 39)
EPA Protocol (EPA 600/R-1 0/1 39)
EPA BA Protocol (Anticipated
publication October 2012)
Hodges ef a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose ef
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-11/103) or EPA Protocol (EPA 600/R-1 0/1 39)
Blackstoneefa/. 2007. J. Microbiol. Methods. 68(2): 254-259.
EPA BA Protocol (Anticipated
publication October 2012)
Hodges ef a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose ef
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03)
ASM Sentinel Level Clinical Microbiology Laboratory Guidelines for Suspected Agents of Bioterrorism and Emerging Infectious Diseases: Yersinia pestis
EPA BA Protocol (Anticipated
publication October 2012)
Hodges ef a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose ef
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03)
Woron etal. 2006. Diagn. Micr. Infec. Dis. 56(3): 261-268.
Viruses
Adenoviruses:
Enteric and
non-enteric (A-F)
Tissue Culture
Real-time PCR
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
EPA BA Protocol (Anticipated
publication October 2012)
Hodges ef a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose ef
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03) or EPA Method 1 61 5 (EPA/600/R-1 0/1 81 )
EPA Method 1615 (EPA/600/R-10/181)
EPA BA Protocol (Anticipated
publication October 2012)
Hodges ef a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose ef
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03) or EPA Method 1 61 5 (EPA/600/R-1 0/1 81 )
Jothikumarefa/. 2005. Appl. Environ. Microbiol. 71(6): 3131-3136
SAM 2012- Appendix C
C-6
July 16, 2012
-------
Pathogen(s)
[Disease]
Astrovi ruses
Caliciviruses: Noroviruses
Caliciviruses: Sapovirus
Analytical
Technique
Integrated Cell Culture
Real-time reverse
transcription-PCR
Real-time reverse
transcription-PCR
Tissue Culture
Real-time reverse
transcription-PCR
Method Type
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Analytical Method
Aerosol
(growth media, filter, liquid)
EPA BA Protocol (Anticipated
publication October 2012)
Particulate
(swabs, wipes, Sponge-Sticks,
vacuum socks and filters)
Hodges ef a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose ef
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
Drinking Water
Post Decontamination Waste
Water
EPA Report (EPA 600/R-1 1/1 03) or EPA Method 1 61 5 (EPA/600/R-1 0/1 81 )
Grimm etal. 2004. Can. J. Microbiol. 50(4): 269-278
EPA BA Protocol (Anticipated
publication October 2012)
Hodges ef a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose ef
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03) or EPA Method 1 61 5 (EPA/600/R-1 0/1 81 )
Grimm etal. 2004. Can. J. Microbiol. 50(4): 269-278
EPA BA Protocol (Anticipated
publication October 2012)
Hodges ef a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose ef
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03) or EPA Method 1 61 5 (EPA/600/R-1 0/1 81 )
EPA Method 1615 (EPA/600/R-10/181)
EPA BA Protocol (Anticipated
publication October 2012)
Hodges ef a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose ef
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03) or EPA Method 1 61 5 (EPA/600/R-1 0/1 81 )
Parwani etal. 1991. Arch. Virol. 120(1-2):115-122.
EPA BA Protocol (Anticipated
publication October 2012)
Hodges ef a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose ef
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03) or EPA Method 1 61 5 (EPA/600/R-1 0/1 81 )
Oka etal. 2006. J. Med. Virol. 78(10): 1347-1353
SAM 2012- Appendix C
C-7
July 16, 2012
-------
Pathogen(s)
[Disease]
Coronaviruses:
SARS-associated human
coronavirus
Hepatitis E virus (HEV)
Influenza H5N1 virus
Analytical
Technique
Tissue Culture
Reverse
transcription-PCR
Tissue Culture
Real-time reverse
transcription-PCR
Real-time reverse
transcription-PCR
Method Type
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Analytical Method
Aerosol
(growth media, filter, liquid)
EPA BA Protocol (Anticipated
publication October 2012)
Particulate
(swabs, wipes, Sponge-Sticks,
vacuum socks and filters)
Hodges ef a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose ef
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
_ . . . ,., . Post Decontamination Waste
Drinking Water Wa(er
EPA Report (EPA 600/R-1 1/1 03)
Pagat etal. 2007. Applied Biosafety 12(2): 100-108.
EPA BA Protocol (Anticipated
publication October 2012)
Hodges ef a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose ef
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03)
Adachi etal. 2004. J. Virol. Methods. 122(1): 29-36
EPA BA Protocol (Anticipated
publication October 2012)
Hodges ef a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose ef
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03) or EPA Method 1 61 5 (EPA/600/R-1 0/1 81 )
Zaki etal. 2009. FEMS Immunol. Med. Mic. 56: 73-79.
EPA BA Protocol (Anticipated
publication October 2012)
Hodges ef a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose ef
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03) or EPA Method 1 61 5 (EPA/600/R-1 0/1 81 )
Jothikumarefa/. 2006. J. Virol. Methods. 131(1): 65-71
EPA BA Protocol (Anticipated
publication October 2012)
Hodges ef a/. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose ef
a/. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03)
Ng etal. 2005. Emerg. Infect. Dis. 11(8): 1303-1305
SAM 2012- Appendix C
July 16, 2012
-------
Pathogen(s)
[Disease]
Picornaviruses:
Enteroviruses
Picornaviruses:
Hepatitis A virus (HAV)
Reoviruses:
Rotavirus (Group A)
Analytical
Technique
Tissue Culture
Reverse
transcription-PCR
Integrated Cell Culture
Real-time Reverse
Transcription-PCR
Tissue Culture
Real-time reverse
transcription-PCR
Method Type
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Analytical Method
Aerosol
(growth media, filter, liquid)
EPA BA Protocol (Anticipated
publication October 2012)
Particulate
(swabs, wipes, Sponge-Sticks,
vacuum socks and filters)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al . 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
Drinking Water
Post Decontamination Waste
Water
EPA Report (EPA 600/R-11/103) or EPA Method 1615 (EPA/600/R-10/181)
EPA Method 1615 (EPA/600/R-10/181)
EPA BA Protocol (Anticipated
publication October 2012)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-11/103) or EPA Method 1615 (EPA/600/R-10/181)
EPA Method 1615 (EPA/600/R-10/181)
EPA BA Protocol (Anticipated
publication October 2012)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-11/103) or EPA Method 1615 (EPA/600/R-10/181)
Hyeon etal. 2011. J. Food Prot. 74(10):1756-1761
EPA BA Protocol (Anticipated
publication October 2012)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-11/103) or EPA Method 1615 (EPA/600/R-10/181)
Hyeon etal. 2011. J. Food Prot. 74(10):1756-1761
EPA Protocol (Anticipated publication
October 201 2)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-11/103) or EPA Method 1615 (EPA/600/R-10/181)
EPA Method 1615 (EPA/600/R-10/181)
EPA BA Protocol (Anticipated
publication October 2012)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-11/103) or EPA Method 1615 (EPA/600/R-10/181)
Jothikumarefa/. 2009. J. Virol. Methods. 155(2): 126-131
SAM 2012- Appendix C
C-9
July 16, 2012
-------
Pathogen(s)
[Disease]
Analytical
Technique
Method Type
Analytical Method
Aerosol
(growth media, filter, liquid)
Particulate
(swabs, wipes, Sponge-Sticks,
vacuum socks and filters)
_ . . . ,, . Post Decontamination Waste
Drinking Water Wa(er
Protozoa
Cryptospohdium spp.
[Cryptosporidiosis]
Entamoeba histolytica
Cell Culture IFA
IMS/FA
Real-time PCR
Cell Culture
Real-time PCR
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
EPA BA Protocol (Anticipated
publication October 2012)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03)
Bukhari etal. 2007. Can. J. Microbiol. 53(5): 656-663.
EPA BA Protocol (Anticipated
publication October 2012)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1 /1 03) and EPA Method 1 622 (EPA 81 5-R-05-
001) or EPA Method 1623 (EPA 815-R-05-002)
EPA Method 1622 (EPA 815-R-05-001) or EPA Method 1623 (EPA 815-R-05-002)
EPA BA Protocol (Anticipated
publication October 2012)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-11/103) or Guy etal. 2003. Appl. Environ.
Microbiol. 69(9): 5178-5185, and Jiang et al. 2005. Appl. Environ.
Microbiol. 71(3): 1135-1141.
Guy etal. 2003. Appl. Environ. Microbiol. 69(9): 5178-5185 and Jiang etal. 2005. Appl. Environ. Microbiol. 71(3): 1135-1141.
EPA BA Protocol (Anticipated
publication October 2012)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03)
Stringert etal. 1972. J Parasitol. 58(2): 306-310.
EPA BA Protocol (Anticipated
publication October 2012)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-1 1/1 03)
Roy etal. 2005. J. Clin. Microbiol. 43(5): 2168-2172.
SAM 2012- Appendix C
C-10
July 16, 2012
-------
Pathogen(s)
[Disease]
Giardia spp.
[Giardiasis]
Tbxop/asma gone///
[Toxoplasmosis]
Analytical
Technique
Cell Culture
IMS/FA
Real-time PCR
Cell Culture
Real-time PCR
Method Type
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
Analytical Method
Aerosol
(growth media, filter, liquid)
EPA Protocol (Anticipated publication
October 201 2)
Particulate
(swabs, wipes, Sponge-Sticks,
vacuum socks and filters)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al . 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
_ . . . ,., . Post Decontamination Waste
Drinking Water Wa(er
EPA Report (EPA 600/R-11/103) or EPA Method 1623 (EPA 815-R-05-002)
Keisterefa/. 1983. T. Roy. Soc. Trap. Med. H. 77(4): 487-488.
EPA BA Protocol (Anticipated
publication October 2012)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-11/103) or EPA Method 1623 (EPA 815-R-05-002)
EPA Method 1623 (EPA 815-R-05-002)
EPA BA Protocol (Anticipated
publication October 2012)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-11/103) or Guy et al. 2003. Appl. Environ.
Microbiol. 69(9): 5178-5185.
Guyefa/. 2003. Appl. Environ. Microbiol. 69(9): 5178-5185.
EPA BA Protocol (Anticipated
publication October 2012)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
Villegasefa/. 2010. J. Microbiol. Methods 81(3): 219-225.
Villegasefa/. 2010. J. Microbiol. Methods 81(3): 219-225.
EPA BA Protocol (Anticipated
publication October 2012)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Method 1623 (EPA 815-R-05-002)
Yangefa/. 2009. Appl. Environ. Microbiology. 75(11): 3477-3483.
SAM 2012- Appendix C
C-11
July 16, 2012
-------
Pathogen(s)
[Disease]
Analytical
Technique
Method Type
Analytical Method
Aerosol
(growth media, filter, liquid)
Particulate
(swabs, wipes, Sponge-Sticks,
vacuum socks and filters)
_ . . . ,, . Post Decontamination Waste
Drinking Water Wa(er
Helminths
Baylisascaris procyonis
[Raccoon roundworm
infection]
Real-time PCR
Embryonation of Eggs and
Microscopy
Sample Preparation
Analytical Technique
Sample Preparation
Analytical Technique
EPA BA Protocol (Anticipated
publication October 2012)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-11/103) or Gatcombe et al. 2010. Parasitol. Res.
106:499-504.
Gatcombe etal. 2010. Parasitol. Res. 106:499-504.
EPA BA Protocol (Anticipated
publication October 2012)
Hodges et al. 2010. J. Microbiol.
Methods. 81(2):141-146, or Rose et
al. 2011. Appl. Environ. Microbiol.
77(23):8355-8359, or EPA BA
Protocol (Anticipated publication
October 201 2)
EPA Report (EPA 600/R-11/103) or Gatcombe et al. 2010. Parasitol. Res.
106:499-504.
EPA Document (EPA/625/R-92/013)
General Remediation Efficacy
Biological indicator (spore)
strips
Culture
Manufacturers' Instructions
SAM 2012- Appendix C
C-12
July 16, 2012
-------
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SAM 2012- Appendix C C - 13 July 16,2012
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U.S. EPA. 2005. "Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA." EPA 815-R-05-002. http://www.epa.gov/sam/pdfs/EPA-1623.pdf
U.S. EPA. 2003. "Appendix I: Test Method for Detecting, Enumerating, and Determining the Viability of Ascaris Ova in Sludge." U.S. EPA 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
U.S. EPA. 2005. "Method 1622: Cryptosporidium in Water by Filtration/IMS/FA." EPA815-R-05-001. http://www.epa.gov/sam/pdfs/EPA-1622.pdf
U.S. EPA. 2006. "Method 1682: Salmonella in Sewage Sludge (Biosolids) by Modified Semisolid Rappaport-Vassiliadis (MSRV) Medium." EPA-821-R-06-14. http://www.epa.gov/sam/pdfs/EPA-1682.pdf
U.S. EPA. June 2011. "Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water," http://owpubauthor.epa.gov/infrastructure/watersecurity/wla/upload/epa817r12004.pdf
U.S. EPA. October 2010. "Standard Analytical Protocol for Salmonella Typhi in Drinking Water." EPA 600/R-10/133. http://oaspub.epa.gov/eims/eimscomm.getfile?p_downloadjd=499264
U.S. EPA. October 2010. "Standard Analytical Protocol for Vibrio cholerae O1 and O139 in Drinking Water and Surface Water." EPA 600/R-10/139. http://nepis.epa.gov/Adobe/PDF/P100978K.pdf
U.S. EPA. September 2010. Standard Analytical Protocol for Escherichia coli O157:H7 in Water. EPA/600/R-10/056 http://oaspub.epa.gov/eims/eimscomm.getfile?p_downloadjd=498725
SAM 2012- Appendix C C - 14 July 16,2012
-------
References
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41(12): 5492-5499.
http://icm.asm.org/content/41/12/5492.full.pdf-i-html
Villegas, E. N., Augustine, S.A., Villegas, L. F., Ware, M.W., See, M. J., Lindquist, H.D.A., Schaefer, III, F. W. and Dubey, J.P. 2010. "Using Quantitative Reverse Transcriptase PCR and Cell Culture Plaque Assays to Determine
Resistance of Tbxop/asma gondii Oocyststo Chemical Sanitizers." Journal of Microbiological Methods, 81(3): 219-225. http://www.sciencedirect.com/science/article/pii/S0167701210001107
Woron, A.M., Nazarian, E.J., Egan, C., McDonough, K.A., Cirino, N.M., Limberger, R.J. and Musser, K.A. 2006. "Development and Evaluation of a 4-Target Multiplex Real-Time Polymerase Chain Reaction Assay for the Detection and
Characterization of Yersinia pestis." Diagnostic Microbiology and Infectious Disease, 56(3): 261-268.
http://www.dmidjournal.com/article/S0732-8893(06)00232-X/fulltext
Yang, W., Lindquist, H.D.A., Cama, V., Schaefer III, F.W., Villegas, E., Fayer, R., Lewis, E.J., Feng, Y. and Xiao, L. 2009. "Detection of Tbxop/asma gondii Oocysts in Water Sample Concentrates by Real-Time PCR." Applied and
Environmental Microbiology, 75(11): 3477-3483. http://aem.asm.org/content/75/11/3477.full.pdf-i-html
SAM 2012- Appendix C C - 15 July 16,2012
-------
Appendix D - Selected Biotoxin Methods
Appendix D: Selected Biotoxin Methods
SAM2012 -AppendixD July 16, 2011
-------
SAM 2012 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)
CAS RN /
Description
Analysis Type1
Analytical
Technique
Aerosol
(filter/cassette, liquid
impinger)
Solid
(soil, powder)
Particulate
(swabs, wipes, dust
socks)
Liquid Water
Drinking Water
Protein
Abrin
Botulinum neurotoxins
(Serotoypes A, B, E, F)
1393-62-0 (abrin)/
Glycoprotein consisting of
a deadenylase (25-32 kDa
A chain) and lectin (35
kDa B chain); an
agglutinin (A2B2) may be
present in crude
preparations
526-31-8 (abrine)/ small
molecule, abrin marker
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
SNAP-25, VAMP 2 /
botulinum neurotoxin
markers
Presumptive
Complementary
Presumptive
(abrine)
Confirmatory
Biological Activity
Presumptive
Complementary
Presumptive
(SNAP25, VAMP 2)
Confirmatory
Biological Activity
Immunoassay
(ELISA, ECL-
based)2
LC-MS-MS
Ribosome
inactivation assay
Enzyme activity3
Immunoassay
(LFD)4
LC-MS
Immunoassay4
(ELISA)
Mouse Bioassay
Adapted from Journal of
Food Protection 71 (9):
1868-1874
Adapted from Journal of
Agricultural and Food
Chemistry 56(23):
11139-11143
Adapted from
Pharmacology &
Toxicology
88(5): 255-260
Adapted from Analytical
Biochemistry
378(1): 87-89
Adapted from EPA
Environmental
Technology Verification
report
Adapted from Journal of
Chemical Health and
Safety
15(6): 14-19
Adapted from FDA
Bacteriological Analytical
Manual, Chapter 17
Adapted from FDA
Bacteriological Analytical
Manual, Chapter 17
Adapted from Journal of
Food Protection 71(9):
1868-1874
Adapted from Journal of
Agricultural and Food
Chemistry 56(23):
11139-11143
Adapted from
Pharmacology &
Toxicology
88(5): 255-260
Adapted from Analytical
Biochemistry
378(1): 87-89
Adapted from Journal of
Food Protection 71 (9):
1868-1874
Adapted from Journal of
Agricultural and Food
Chemistry 56(23):
11139-11143
Adapted from
Pharmacology &
Toxicology
88(5): 255-260
Adapted from Analytical
Biochemistry
378(1): 87-89
Adapted from Journal of
Food Protection 71(9):
1868-1874
Adapted from Journal of
Agricultural and Food
Chemistry 56(23):
11139-11143
Adapted from
Pharmacology &
Toxicology
88(5): 255-260
Adapted from Analytical
Biochemistry
378(1): 87-89
Adapted from Journal of
Food Protection 71 (9):
1868-1874
Adapted from Journal of
Agricultural and Food
Chemistry 56(23):
11139-11143
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.
SAM 2012- Appendix D
D-l
July 16, 2012
-------
Analyte(s)
Ricin
Shiga and Shiga-like Toxins
(Six, Stx-1, Stx-2)
Staphylococcal enterotoxins
(SEB)
Staphylococcal enterotoxins
(SEA, SEC)
CAS RN /
Description
9009-86-3 (ricin) /
60 kDa glycoprotein
composed of two subunits
(-32 kDa A chain and -34
kDa B chain); an
agglutinin of MW 120 kDa
may be present in crude
preparations
5254-40-3 (ricinine) /
small molecule, ricin
marker
75757-64-1 (Six) /
Protein composed of one
-32 kDa A chain and five
7.7 kDa B chains
39424-53-8 (SEB) /
Monomeric protein of
~ 28 kDa
39424-54-9 (SEC) /
Monomeric proteins of
-27-27. 5 kDa
Analysis Type1
Presumptive
Complementary
Presumptive
(ricinine)
Confirmatory
Biological Activity
Presumptive
Confirmatory
Biological Activity
Presumptive
Confirmatory
Biological Activity
Presumptive
Confirmatory
Biological Activity
Analytical
Technique
Immunoassay
(LFD)2
LC-MS
Immunoassay
(ECL)
Enzyme activity3
Enzyme
immunoassay
(EIA)
Immunoassay
(ELISA)
Ribosome
inactivation assay3
Enzyme
Immunoassay
(EIA)
TBD
T-cell proliferation
assay
Enzyme
Immunoassay
(EIA)
TBD
T-cell proliferation
assay
Aerosol
(filter/cassette, liquid
impinger)
Adapted from EPA
Environmental
Technology Verification
report
Adapted from Journal of
Analytical Toxicology
29(3): 149-155
Adapted from Journal of
AOAC International
91 (2): 376-382
Adapted from Analytical
Biochemistry
378(1): 87-89
Adapted from Journal of
Clinical Microbiology
35(8): 2051 -2054
Adapted from FDA
Bacteriological Analytical
Manual, Appendix 1
Adapted from
Pharmacology &
Toxicology
88(5): 255-260
Adapted from 993.06
(AOAC)
TBD
Adapted from Applied and
Environmental
Micro biology 63(6):
2361-2365
Adapted from 993.06
(AOAC)
TBD
Adapted from Applied and
Environmental
Micro biology 63(6):
2361-2365
Solid
(soil, powder)
Particulate
(swabs, wipes, dust
socks)
Liquid Water
Drinking Water
LRN
If analysis for this agent is required in solid, particulate, or liquid samples, contact
the LRN at (404) 639-2790 for information of the closest LRN laboratory capable
of receiving and processing the sample. The terms presumptive and confirmatory
as used for LRN methods are described in Section 8.1.4.
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
Clinical Microbiology
35(8): 2051-2054
Adapted from FDA
Bacteriological Analytical
Manual, Appendix 1
Adapted from
Pharmacology &
Toxicology
88(5): 255-260
Adapted from Journal of
Analytical Toxicology
29(3): 149-155
Adapted from Journal of
AOAC International
91 (2): 376-382
Adapted from Analytical
Biochemistry
378(1): 87-89
Adapted from Journal of
Clinical Microbiology
35(8): 2051 -2054
Adapted from FDA
Bacteriological Analytical
Manual, Appendix 1
Adapted from
Pharmacology &
Toxicology
88(5): 255-260
Adapted from Journal of
Analytical Toxicology
29(3): 149-155
Adapted from Journal of
AOAC International
91 (2): 376-382
Adapted from Analytical
Biochemistry
378(1): 87-89
Adapted from Journal of
Clinical Microbiology
35(8): 2051-2054
Adapted from FDA
Bacteriological Analytical
Manual, Appendix 1
Adapted from
Pharmacology &
Toxicology
88(5): 255-260
Adapted from Journal of
Analytical Toxicology
29(3): 149-155
Adapted from Journal of
AOAC International
91 (2): 376-382
Adapted from Analytical
Biochemistry
378(1): 87-89
Adapted from Journal of
Clinical Microbiology
35(8): 2051 -2054
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
Adapted from Applied and
Environmental
Microbiology 63(6):
2361-2365
Adapted from 993.06
(AOAC)
TBD
Adapted from Applied and
Environmental
Microbiology 63(6): 2361-
2365
TBD
Adapted from Applied and
Environmental
Micro biology 63(6):
2361-2365
Adapted from 993.06
(AOAC)
TBD
Adapted from Applied and
Environmental
Micro biology 63(6):
2361-2365
TBD
Adapted from Applied and
Environmental
Microbiology 63(6):
2361-2365
Adapted from 993.06
(AOAC)
TBD
Adapted from Applied and
Environmental
Microbiology 63(6):
2361-2365
TBD
Adapted from Applied and
Environmental
Micro biology 63(6):
2361-2365
Adapted from 993.06
(AOAC)
TBD
Adapted from Applied and
Environmental
Micro biology 63(6):
2361-2365
SAM 2012- Appendix D
D-2
July 16, 2012
-------
Analyte(s)
Small Molecule
Aflatoxin
(TypeBI)
a-Amanitin
Anatoxin-a
Brevetoxins
(Bform)
a-Conotoxin
CAS RN /
Description
23109-05-9
64285-06-9
79580-28-2
1 56467-85-5
1 43545 90 8
Analysis Type1
Presumptive
Confirmatory
Presumptive
Confirmatory
Presumptive
Confirmatory
Presumptive
Confirmatory
Presumptive
Confirmatory
Presumptive
Confirmatory
Presumptive
Confirmatory
Analytical
Technique
Immunoassay
(column)
HPLC-FL
Immunoassay
(ELISA)
HPLC
ampero metric
detection
TBD
HPLC-FL
(precolumn
derivatization)
Immunoassay
(ELISA)
HPLC-MS-MS
Immunoassay
(solution phase
binding assay)
HPLC- MS
Immunoassay
(ELISA)
HPLC-PDA
Immunoassay
(ELISA)
LC/APCI-MS
Aerosol
(filter/cassette, liquid
Implnger)
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): 299-311
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
Solid
(soil, powder)
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): 299-311
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
Partlculate
(swabs, wipes, dust
socks)
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): 299-311
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
Liquid Water
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): 299-311
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
Drinking Water
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): 299-311
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
SAM 2012- Appendix D
D-3
July 16, 2012
-------
Analyte(s)
Microcystins
Principal isoforms: LA, LR, LW,
RR, YR
Picrotoxin
Saxitoxins
Principal isoforms:
Saxitoxin (SIX)
Neosaxitoxin (NEOSTX)
Gonyautoxin (GTX)
Decarbamoylgonyautoxin
(dcGTX)
Decarbamoylsaxitoxin (dcSTX)
T-2 Mycotoxin
Tetrodotoxin
CAS RN /
Description
961 80-79-9 (LA)
101 043-37-2 (LR)
157622-02-1 (LW)
101 064-48-6 (YR)
1 24-87-8
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 Type1
Presumptive
Confirmatory
Presumptive
Confirmatory
Presumptive
Confirmatory
Presumptive
Confirmatory
Presumptive
Confirmatory
Analytical
Technique
Immunoassay
(ELISA)/
Phosphatase
assay
HPLC-PDA
Immunoassay
HPLC
Immunoassay
(ELISA)
HPLC-FL
(post column
derivatization)
Immunoassay
(ELISA)
LC/APCI-MS
Immunoassay
(CIEIA)
LC/ESI-MS
Aerosol
(filter/cassette, liquid
impinger)
Adapted from Journal of
AOAC International
84(4): 1035-1044
Adapted from Analyst
119(7): 1525-1530
TBD
Adapted from Journal of
Pharmaceutical and
Biomedical Analysis
7(3): 369-375
Adapted from ELISA kits
for Saxitoxins
Adapted from Journal of
AOAC International
78(2): 528-532
Adapted from Journal of
Food Protection 68(6):
1294-1301
Adapted from Rapid
Communications in Mass
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 Journal of
AOAC International
84(4): 1035-1044
Adapted from Analyst
119(7): 1525-1530
TBD
Adapted from Journal of
Pharmaceutical and
Biomedical Analysis
7(3): 369-375
Adapted from ELISA kits
for Saxitoxins
Adapted from Journal of
AOAC International
78(2): 528-532
Adapted from Journal of
Food Protection 68(6):
1294-1301
Adapted from Rapid
Communications in Mass
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 Journal of
AOAC International
84(4): 1035-1044
Adapted from Analyst
119(7): 1525-1530
TBD
Adapted from Journal of
Pharmaceutical and
Biomedical Analysis
7(3): 369-375
Adapted from ELISA kits
for Saxitoxins
Adapted from Journal of
AOAC International
78(2): 528-532
Adapted from Journal of
Food Protection 68(6):
1294-1301
Adapted from Rapid
Communications in Mass
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 Journal of
AOAC International
84(4): 1035-1044
Adapted from Analyst
119(7): 1525-1530
TBD
Adapted from Journal of
Pharmaceutical and
Biomedical Analysis
7(3): 369-375
Adapted from ELISA kits
for Saxitoxins
Adapted from Journal of
AOAC International
78(2): 528-532
Adapted from Journal of
Food Protection 68(6):
1294-1301
Adapted from Rapid
Communications in Mass
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 Journal of
AOAC International
84(4): 1035-1044
Adapted from Analyst
119(7): 1525-1530
TBD
Adapted from Journal of
Pharmaceutical and
Biomedical Analysis
7(3): 369-375
Adapted from ELISA kits
for Saxitoxins
Adapted from Journal of
AOAC International
78(2): 528-532
Adapted from Journal of
Food Protection 68(6):
1294-1301
Adapted from Rapid
Communications in Mass
Spectrometry
20(9): 1422-1428
Adapted from Journal of
Clinical Laboratory
Analysis 6(2): 65-72
Adapted from Analytical
Biochemistry
290(1): 10-17
Descriptions for presumptive, confirmatory and biological activity assays are provided in Section 8.0.
2 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.
3 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.
4 Immunoassays may produce variable results with uncomplexed form of toxin.
SAM 2012- Appendix D
D-4
July 16, 2012
-------
SAM 2012
July 16, 2011
-------
Attachment 1 - SAM Supporting Documents
Attachment 1:
SAM Revisions and Supporting Documents
SAM is updated periodically to incorporate revisions to the list of target analytes and environmental
sample types, and to provide the most recent analytical methods and procedures. The table below
provides information regarding additional changes that were incorporated into each revision of SAM,
since publication of SAM Revision 1.0 in September 2004.
SAM Revisions Tracking Table
SAM Revision
SAM Revision 1.0
SAM Revision 2.0
SAM Revision 3.0
SAM Revision 3.1
SAM Revision 4.0
SAM Revision 5.0
SAM 20 10
(Revision 6.0)
SAM 20 12
Publication Date
September 2004
September 2005
February 2007
November 2007
September 2008
September 2009
October 20 10
July 20 12
Changes incorporated summary
Standardized Analytical Methods for Use During Homeland
Security Events
Included chemical and biological contaminants
Added:
Radiochemicals
Several persistent CWA degradation products
Separate drinking water sample type for chemicals and
radiochemicals
Viability determination methods for pathogens
Separate section for biotoxins
Added explosive chemicals
Combined identification and viability methods for pathogens
Added drinking water sample type for pathogens
Title changed to: Standardized Analytical Methods for
Environmental Restoration Following Homeland Security
Events (SAM)
Developed a SAM website, to provide the SAM document and
a format for searching and linking to SAM methods by analyte
and sample type.
Added:
Wipe samples for chemistry analytes
Added PCR methods for pathogens
Added separate drinking water sample type to biotoxins
Removed non-aqueous liquid sample type from chemistry
Temporary removal of pathogens
Changed title to: Selected Analytical Methods for
Environmental Remediation and Recovery (SAM) - 2012
Added vegetation sample type, newly available rapid
methods, and total activity screening procedure for
radiochemistry analytes
Re-introduced pathogen methods with restructuring to
clarify method applications for site characterization and
post remediation
Assigned applicability tiers to chemistry methods to
indicate the extent of data available that support use of each
method for analyte/sample type pairs
SAM 2012
Attachment 1-1
July 16, 2011
-------
Attachment 1 - SAM Supporting Documents
The following documents and tools have been developed by EPA to provide information regarding a
contamination event. The information included in the documents is intended to be complementary to
information provided in the analytical methods listed in SAM. As additional documents containing
similar complementary information become available, they will be added to the list contained in this
Attachment.
Searchable SAM Web site at: www.epa.gov/sam/
"Guidelines for Development of Sample Collection Plans for Radiochemical Analytes in
Environmental Matrices Following Homeland Security Events," EPA/600/R-08/128, February 2009.
http://www.epa.gov/nhsrc/pubs/600r08128.pdf
"Sample Collection Procedures for Radiochemistry Analytes in Environmental Matrices,"
EPA/600/S-07/001, December 2006. http://www.epa.gov/nhsrc/pubs/600s07001 .pdf
"Sample Collection Information Document - Companion to SAM Revision 5.0," EPA/600/R-09/074,
June 2010. http://www.epa.gov/nhsrc/pubs/600r09074.pdf
"Field Screening Equipment Information Document - Companion to SAM Revision 5.0,"
EPA/600/R-10/091, September 2010.
http://www.epa.gov/sam/Field Screening Equipment Guide.pdf
"Rapid Screening and Preliminary Identification Techniques and Methods - Companion to SAM
Revision 5.0," EPA/600/R-10/090, September 2010.
http://www.epa.gov/sam/Rapid Screening and PreID.pdf
"Laboratory Environmental Sample Disposal Information Document - Companion to SAM Revision
5.0," EPA/600/R-10/092, September 2010. http://www.epa.gov/sam/LESDID.pdf
SAM 2012 Attachment 1-2 July 16, 2011
-------
-------
&EPA
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
ice of Research and Development
urity Research Center
National Homeland Security
Cincinnati, OH 45268
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